bluuebunny/crossref_metadata_2025_split huggingface dataset:
Selection of Mutations that Affect Herpesvirus Entry into Cells
Authors: Hippler Daniel P , Fan Qing , Longnecker Richard , Connolly Sarah A 
Published: April 2020

During virus entry into cells, herpesviruses rely on the coordinated action of multiple viral glycoproteins. For herpes simplex virus type 1 (HSV1), four glycoproteins (gD, gH, gL, and gB) are required to fuse the viral and host membranes. gB functions as a fusion protein and is conserved in the herpesvirus family. It mediates viral entry by undergoing a conformational change that results in fusion viral and host membrane. This conformational change is triggered by both gD, the receptor‐binding protein, and gHgL, a heterodimer that modulates fusion. Capturing a physical interaction among these glycoproteins has been challenging, most likely because the interactions are low affinity and/or transient. The goal of this study is to characterize the interactions between gB and other herpesvirus proteins using natural selection. We previously generated a fusion‐deficient gB mutant. By serially passaging HSV1 carrying this gB mutant, we are selecting for mutations that restore fusion function. To shift the selective pressure away from second‐site mutations within gB, we generated a virus that is deleted for the gB gene but carries the mutant gB protein in its membrane. After six rounds of passage, the virus showed enhanced replication and the viral genome was submitted for next‐generation sequencing. The sequence results will be analyzed to determine the locations of novel mutations. The impact of these mutations on virus entry will be examined by mapping them to existing structures and analyzing mutant function in cell‐cell fusion assays.Support or Funding InformationNIH 1R01AI148478‐01
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

BackgroundNeurological 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.MethodsWe 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+).ResultsNeurological 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.ConclusionsGlobal 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 InformationThe 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 InformationThe 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 InformationResearch 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 InformationSupported 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 InformationThis 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

IntroductionThe 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.MethodsAn 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.ResultsMany 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).ConclusionsLearners 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

BackgroundDoxorubicin (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.ObjectiveEvaluate the influence of pharmacological modulation of MasR/Ang‐(1‐7) axis on DOX‐induced cardiotoxicity in cardiomyoblast cells.MethodsMurine 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.ResultsMasR 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.ConclusionModulation 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 InformationCAPES, 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 InformationSupported by NIH Grant