bluuebunny/crossref_metadata_2025 huggingface dataset:
Integration of Student Centered Physiology learning into related high school Science courses
Authors: Anjur Sowmya
Published: June 2021

Student centered teaching was previously found to enhance student understanding and even improve performance of students in other courses taken in the same semester in high school. In an effort to improve student performance in related science classes, the same student centered methods used in my Physiology and Disease class were implemented in a Biophysics class this semester. Initial student surveys showed that students preferred diverse teaching methods and hands on experiences to help them understand and transfer material better. Students also felt that integrating physiology concepts into Biophysics helped them understand the material from two very different perspectives, giving them a “more complete picture”. Further research and statistical evaluations of student test scores at the end of the semester are planned. These preliminary results suggest that student centered physiology learning can extend a positive influence when integrated into related science classes.
A biomedical instrumentation lab to accompany an advanced physiology course
Authors: Latman Neal S.
Published: June 2021

To understand physiology, it is important to understand the measurement of physiological functions. The purpose of this lab is to expose students to selected principles and methods of physiological measurements, historical significance of the developments those methods and measurements, and the hands‐on, practical use of selected biomedical instruments.

The lab topics include: measurement validity, sensitivity, specificity, accuracy, and reliability; normal clinical ranges; human factors; and a primer on electricity. Specific biomedical instrumentation and technology included: microfluidics/nanotechnology, electromyography, peripheral nerve sensory testing, medical nebulizers, biometric identification (fingerprints, vascular patterns, and irises), bioelectrical impedance analysis of body composition, self‐monitoring of blood glucose, volatile organic compounds in breath, pulse oximetry, clinical thermometry, structural determinations (ophthalmoscope/otoscope), ultra‐violet radiation monitoring, biomedical telemetry.

During the 6 years of use, the lab has been well received by the students with a mean rating of 6.1 on a scale of 1 (atrocious) to 7 (excellent) with no ratings below 4 (okay). 85% of the students rated the labs 6 (very good) or 7 (excellent).
Developing Simple Virtual Microscopic slides in developing Countries: Impact and attitudes of Medical students
Authors: Anyaehie Ugochukwu Bond , Anyanwu Emeka
Published: June 2021

Most of the problems predominant in developing countries today have long been solved in developed nations. The challenges of the use of glass slides in the teaching and learning has been well noted, but is even further compounded in Nigeria by large class sizes. The adoption of virtual microscopy in most Nigerian medical schools have been constrained by funding gaps and this creates additional problem attaining the learning objectives of Laboratory exposures. In an attempt to meet the learning goals of our undergraduate students, we designed simple low technology‐driven version of a virtual microscope and compared the impact on learning with the conventional microscopy presently in use. Review of the perceptions and grades of 265 students of the College of Medicine, University of Nigeria show a significant preference for virtual slides and grades were also significantly increased even among visually impaired students. The time and cost implications of both teaching and conducting examinations were also reduced by over 500%. The results re‐emphasize the need for design and adoption of simple sustainable technological innovations in developing countries to bridge gaps in purposeful learning environments.
Using “clicker cases” online: an effective strategy for engaging undergraduate students in physiology case studies
Authors: Knabb Maureen T
Published: June 2021

As institutions respond to the need for greater flexibility in access to higher education, there has been increasing emphasis in offering distance education courses. Accordingly, online classes (semimonthly) were presented within a traditional upper‐level undergraduate human physiology course (n = 21) to evaluate the effectiveness of online instruction. Only 32% of the students had previous experience with distance education courses. The online classes were in the format of a “clicker case” in which a case study with embedded questions was linked to an audio file and online quiz. Case studies were presented both in class and online to encourage application of physiology knowledge to a problem. Survey results to gauge student perceptions of this approach indicate that 94% liked the online case study format and 64% felt that they learned more from the online case studies compared to in class lecture. Students performed significantly better when comparing results for multiple‐choice exam questions for online versus in class case studies (78.6 ± 6.6 vs. 56.0 ± 20.7%, p = 0.03). Thus, this online approach was a more effective method for presenting case study material and led to higher exam scores.
Migration of Jurkat cells in response to CypA
Authors: Jurjus Rosalyn A , Pushkarsky Tatiana , Bukrinsky Michael
Published: June 2021

Cyclophilins (CyPs) are ubiquitously distributed proteins, identified initially as the main binding target for cyclosporine A (CsA). A potential contribution of cyclophilin in inflammatory diseases is suggested from several studies. The activities of secreted cyclophilins are related to interactions with CD147, a transmembrane glycoprotein that plays a major role in lymphocyte migration and activation and is a binding partner to the β1 integrin. Our hypothesis is that the CypA/CD147/β1 interaction is important in mediating cellular migration in inflammation. Using Jurkat cells as our model, we first started by optimizing Jurkat cells adhesion, spreading and migration.

In timelapse migration studies, Jurkat cells replated on fibronectin‐collagen I and poly‐lysine showed an increase in velocity by 3 folds when CypA was added to the media. The velocity of these cells significantly decreased when CD147 antibody (Ab) was added, compared to the corresponding isotype control. However, some element of CypA activation remained in the presence of CD147 Ab and it was not concentration dependent. Adding the CD147 Ab without prior CypA exposure did not have a significant effect. This suggests that the CypA‐CD147 activation might involve the β1 integrin. A β1 integrin blocking Ab combined with CD147 Ab is expected to completely prevent the CypA activation and the cell migration.

Grant Funding Source: R56AI081152
Identification of genes essential for Francisella invasion of non‐phagocytic cells
Authors: Lo Karen Y , Nano Francis E , Guttman Julian A
Published: June 2021

The intracellular bacterium Francisella tularensis subspecies tularensis (F. tularensis) is the causative agent of tularemia, a disease that can result in 30–35% mortality if left untreated. F. tularensis host cell invasion is crucial for their pathogenesis as without invasion disease does not occur. Upon host entry, the bacteria colonize both macrophages and epithelial cells. We hypothesize F. tularensis possesses unique virulence factors specific for the invasion of epithelial cells. To identify F. tularensis genes involved in this process, we utilized both in vitro and in vivo experimental approaches using a biosafety level 2 bacterial surrogate, F. tularensis subspecies novicida (F. novicida). A F. novicida mutant library was screened for bacteria with reduced invasion and intracellular growth using gentamycin‐based survival assays in hepatocytes in vitro. Select candidate mutants were tested in mice for their ability to colonize murine livers. We discovered that bacteria containing mutations in at least 6 previously uncharacterized genes had significantly reduced bacterial loads, resulting in mice that survived for extended durations as compared to wild‐type infections. Our findings demonstrate that novel bacterial proteins are crucial for Francisella virulence and these findings open a new door to the discovery of novel targets for the development of therapeutics and prophylactics.

Grant Funding Source : NSERC and CIHR
The spectrin cytoskeleton is required for efficient intestinal bacterial infections
Authors: Guttman Julian Andrew , Ruetz Tyson Joel
Published: June 2021

Crucial aspects of bacterial pathogenesis such as the invasion of host cells and cell‐to‐cell transmission require the hijacking of host cell cytoskeletal systems. Although much work has examined the role of the actin cytoskeleton during numerous bacterial infections, the involvement of the spectrin cytoskeleton has remained overlooked. The spectrin cytoskeleton is required for many cellular processes including cell motility, organization of the plasma membrane and epithelial cell polarization. To determine whether this cytoskeletal system influenced microbial infections we tested the hypothesis that bacterial pathogens control the spectrin cytoskeleton as part of their disease processes. To test this hypothesis we utilized cell biological techniques including the immunolocalization of spectrin cytoskeletal components and RNA interference, and coupled these analyses with microbiological techniques such as wild‐type and mutant bacterial infections, invasion assays and colonization experiments. We found that the spectrin cytoskeletal network was integral for Salmonella, Listeria, Shigella and pathogenic E. coli infections. We showed that host cell invasion and the generation of bacterial motility structures during infections required spectrin cytoskeletal components and knockdown of specific spectrin‐associated proteins haulted Listeria, Shigella and Salmonella internalization as well as pathogenic E. coli attachment to host cells, thus impeding the infections. These findings demonstrate that the spectrin cytoskeleton is essential for bacterial pathogenesis and could be used as a target for future therapeutics. This work was funded through NSERC and the CIHR.

Grant Funding Source: CIHR and NSERC
Alpha II‐Spectrin Interacting Partners in the Inner Ear Serve as Pointers to the Protein Composition of the Striated Organelle
Authors: Chidavaenzi Robstein Lovejoy , Lysakowski Anna
Published: June 2021

Inner ear hair cells sense sound and head movements via apically‐placed stereocilia. These stereocilia are comprised of paracrystalline arrays of parallel actin filaments that pack more densely at the base to form stereociliar rootlets. The later, generally described in the literature as anchored in the cuticular plate, have been observed by our lab to extend well into the cell body where they associate with an underlying cytoskeletal structure called the striated organelle (SO). Made up of alternating thin (~10nm) and thick (~35nm) filaments, the function of this organelle remains unknown. To ascertain its function, we are attempting to determine its protein composition. So far, we have been using α‐II‐spectrin, an actin crosslinking and molecular scaffold protein, as a biological marker for the SO since we localized it there with confocal fluorescent and EM immunogold studies (Vranceanu et al., submitted). Further employing α‐II‐spectrin as bait, we pulled down interacting partners in vestibular endorgan tissue using co‐immunoprecipitation and liquid chromatography mass spectroscopy. Here we identify some of these interacting proteins and present evidence that they also localize to the SO. Based on this putative protein composition, we can now speculate as to probable function for the organelle in inner ear electro‐mechanotransduction.

Grant Funding Source: American Hearing Research Foundation
Is BMP‐2 a component of the particulate matrix that is responsible for epithelial/mesenchymal transformation during heart development
Authors: Abd-Elhamid Tarek H. , Conway Marianne L. , Sinning Allan R.
Published: June 2021

Early cardiac development involves an epithelial/mesenchymal transformation (EMT) of endothelial cells in the atrioventricular (AV) canal and outflow tract (OT) regions of the heart. It has been shown that the myocardium secrets factors, collectively called particulate matrix that induce this process. Previous reports from our laboratory showed that, only myocardial conditioned media (MyoCM) or EDTA extract can replace the myocardium and drive mesenchyme formation. Reports from other laboratories suggest that bone morphogenetic protein 2 (BMP‐2) has a role in EMT. So, we asked whether BMP‐2 is a part of the particulate matrix. Initial experiments using immunostaining of stage 15–16 chick embryo sections with anti BMP‐2 antibody revealed that BMP‐2 signals were detected within the myocardium of the cushion forming regions and in a group of cells in the endocardial layer of these regions in the heart, but not in the extracellular matrix. To confirm these results, whole heart lysate, EDTA extract from stage 15–16 embryos and soybean agglutinin (SBA) binding proteins from MyoCM will be examined for the presence of BMP‐2 by western blotting analysis.

Grant Funding Source : none
A successful method for engaging students in active learning using prosected cadavers
Authors: Khirallah Samar , Walker James J
Published: June 2021

Over the past several years, we have taught a graduate level gross anatomy course using prosected cadavers. The laboratory component of the course constituted instructor‐led demonstrations of the anatomical structures. To create a more engaging and active learning environment, we developed a new teaching strategy that allowed students to construct their own anatomical knowledge through cooperative discovery‐ learning exercises. In the new method, instructors tagged structures on the cadavers prior to the laboratory sessions and then students worked in small groups to identify the tagged structures. We then compared the two methods by assessing both students’ learning and attitude. Four traditional demonstration sessions were alternated with 4 active learning sessions. Each lab session was followed by a short practical identification quiz. A survey was administered to evaluate students’ attitudes. Quiz scores were significantly higher following active learning sessions (mean score 54 vs 38) and continued to improve as students became more effective learners. The survey clearly showed a unanimous preference of the improved design. Structured exercises that emphasized kinesthetic manipulation of the anatomical specimens in conjunction with a cooperative learning environment advanced the learning process significantly and provided the motivation for advanced self‐learning.

Grant Funding Source: none
Getting crafty in the classroom: Reinforcing students’ knowledge of muscular attachments and positions with fabric muscles
Authors: Bee Mary Tracy , Montante James M , Grabowski Greg , Forbes William C
Published: June 2021

Helping students to visualize the origin, insertion, position and relationships of muscles is a challenge. We present a visual, interactive exercise to reinforce student understanding in this area. Students were asked to place precut two‐dimensional felt models of the muscles on a life sized skeleton or disarticulated bones matching the correct bony origins and insertions.

In order to assess the impact of this approach on student learning, the students were split into two groups. The first group spent 15 minutes utilizing the felt leg muscles while the second group spent 15 minutes reading their books to study the muscular attachments of the leg muscles. A pre‐test and a post‐test covering the material were administered to both groups. Students who worked with the felt muscles showed a greater improvement relative to the students who studied independently (p<0.05). The groups switched activities when studying the foot muscles and similar results were found. Student satisfaction was assessed with a survey administered after each unit. The majority of students indicated that the activity was fun and felt that the activity was an effective way to learn the material. We advocate the use of this type of activity to engage the students and to provide an opportunity for the students to reinforce their understanding of muscular attachments.
Beneficial Aspect of Cadaver Dissection – Medical Students Perspective
Authors: Kraszpulska Barbara
Published: June 2021

The aim of the study was to assess the beneficial aspect of cadaver dissection in the process of learning anatomy. The objectives of the study were: to determine medical students’ perspectives regarding if and how cadaver dissection was beneficial, and to determine the degree of respect toward the donors.

An anonymous survey was completed by 103 first‐year medical students (42 female; 61 male) at WSU at the end of the anatomy course (involving complete cadaver dissection). Ninety five percent of the students found the dissection to be beneficial. They listed as the most valuable aspects of doing dissection: seeing 3D orientation of anatomical structures (53%), helping with assigned material (40%), and understanding relationships in the human body (21%). All students (100%) declared appropriate respect and appreciation for the donors, although less than half (48% male; 34% female) would consider donating their own body. Medical students themselves were generally in favor of organs donations ‐ 90% male and 74% female. Ninety‐five percent of students disagreed with the idea of completely replacing cadaver dissection with computer‐assisted program.

These results suggest that medical students regard cadaver dissection as an effective time learning experience, which reinforces lecture and textbook material and provides a better understanding of the complicated relationships in the human body.
Accurate Accountability of Anatomical Donors and Material Permits Quality Control for the Medical School and Maintains Public Trust
Authors: McGillicuddy Michael P. , Caceres Noel , Marquez Samuel
Published: June 2021

SUNY Downstate recently purchased a state‐of‐the‐art accountability program system from SRS Computing (SRSC) for tracking donors in our school. The program was used originally for the Cremation Industry. The SRSC program offers a robust managing system that permits the direct application for medical school education. It allows the tracking of donors from the moment of receipt by assigning a number in the form of a bar code that is synchronized to the Case Management Tracking System, which then monitors the donors throughout the duration of the course. The donor is logged at the time of arrival to Downstate and is constantly monitored each & every step the donor is being used. If pro‐sections are taken from the donor for the purposes of medical research, that section is labeled with a bar code tag permitting tracking of the specimen until its return to the donor at the end of the study. This system allows Downstate to return the trust given to the school from each of the donor families that all Donors & Donor materials are returned for their intended disposition. The system allows selected data to be readily available for review such as date of death and age including the donor's family request for the disposal of cremains at the end of the study or course. In the past there have been allegations of body parts and materials being sold for profit, this tracking system ensures the location of any and all donors and donor materials in real time.

Grant Funding Source: None
Hypermethylation‐Mediated Silencing of p14ARF in Fibroblasts from Idiopathic Pulmonary Fibrosis (IPF)
Authors: Selman Moises , Cisneros Jose , Hagood James , Checa Marco , Ortiz-Quintero Blanca , Negreros Miguel , Herrera Iliana , Ramos Carlos , Pardo Annie
Published: June 2021

IPF is a lethal lung disease characterized by the formation and persistence of fibroblast foci and matrix accumulation. Evidence suggests that fibroblasts acquire an anti‐apoptotic phenotype and we hypothesized that epigenetic silencing of the pro‐apoptotic gene p14ARF may be involved. p14ARF expression was analyzed by RT‐PCR and Western blot in IPF and normal human lung fibroblasts. p14ARF gene promoter methylation was determined by methylation specific PCR (MS‐PCR) and by DNA digestion with endonuclease McrBc. Apoptosis was evaluated by annexin‐V and nuclear staining. p14ARF expression was significantly decreased in 4 of 8 IPF fibroblasts lines, which was restored after 5‐aza‐2′‐deoxycytidine treatment. MS‐PCR of bisulfite‐treated genomic DNA showed a correlation between the reduced expression of p14ARF and the presence of hypermethylated promoter. No amplification was observed in the DNA treated with McrBc confirming promoter hypermethylation. p14ARF hypermethylated IPF fibroblasts were more resistant to staurosporine‐induced apoptosis compared with normal and non‐methylated IPF fibroblasts (p<0.01) and showed reduced levels of p53. Apoptosis resistance was induced in fibroblasts inhibiting p14ARF expression by siRNA (p<0.05). These findings reveal that many IPF fibroblasts have reduced expression of p14ARF indicating that epigenetic mechanisms may cause their resistance to apoptosis.
Epigenetic Regulation of Natriuretic Peptide Receptor‐A Gene by Cooperative Interaction of Retinoic acid and Sodium Butyrate via Histone Modifications in Vascular Smooth Muscle Cells
Authors: Kumar Prerna , Nguyen Vickie , Pandey Kailash N
Published: June 2021

Activation of guanylyl cyclase/natriuretic peptide receptor‐A (GC‐A/NPRA) by cardiac hormones atrial and brain natriuretic peptides (ANP and BNP) produces the second messenger cGMP, which plays a pivotal role in maintaining blood pressure and cardiovascular homeostasis. The objective of the present study was to gain insight into the epigenetic mechanisms involved in all‐trans retinoic acid (ATRA) and histone deacetylase (HDAC) inhibitor, sodium butyrate (NaBu)‐induced Npr1 gene (coding for GC‐A/NPRA) expression. The studies were carried out in rat thoracic aortic smooth muscle cells cultured in DMEM containing 10% FBS and treated with ATRA and NaBu for 24 h. The results showed that ATRA and NaBu induced Npr1 promoter activity by 12‐fold and mRNA levels by 5‐fold in an additive manner. Simultaneous treatment with ATRA and NaBu significantly increased the levels of histones H3K9/14ac, H4K12ac, and H3K4me3 (p<0.001). Overexpression of class I HDACs (HDAC 1 and HDAC 2) reduced Npr1 promoter activity by 50%. ATRA and NaBu inhibited HDAC activity by 50%. Moreover, ATRA and NaBu cooperatively enhanced histone acetyltransferse activity. The results demonstrate that ATRA and NaBu regulate Npr1 gene expression by modulation of histone modifications. The identification of epigenetic signaling as a regulator of Npr1 gene will have important implications in prevention of hypertension and cardiac remodeling.
Hu antigen R and Tristetraprolin: Regulates Claudin 1 mRNA Stability in Colon Cancer
Authors: Sharma Ashok , Dhawan Punita
Published: June 2021

RNA binding proteins (RBPs) play a crucial role in Posttranscriptional gene regulation but the mechanism of most RBPs is stil unknown. The human RBP HuR/ELAVL1 is a conserved mRNA stability regulator. ELAV like RNA‐binding proteins Hu antigen R (HuR) and tristetraprolin (TTP) associate with labile mRNAs bearing AU‐rich elements located in the 3′ untranslated regions (3′‐UTRs) and modulate their stability. We have identified putative HUR–binding sites in the proximal region of the claudin‐1, 3′‐UTR and demonstrate that HuR and TTP interacts with these AU‐rich elements. Claudin‐1, a tight junction protein, expression is dysregulated in the majority of human colorectal carcinomas. We used a ribonomic and site‐directed mutagenesis approach to test the binding of HuR and TTP to claudin‐1, 3′UTR and their role in stabilization/destabilization of claudin‐1 mRNA in the presence of HDAC inhibitor. We found that Claudin‐1 mRNA was stabilized by activated HUR in both SW480 and SW620 cells, whereas overexpression of TTP inhibited it; limited TTP expression antagonized HuR‐mediated claudin 1 mRNA stabilization. On treatment with TSA the binding of HuR to claudin‐1 3′UTR decreases while the binding of TTP increases correlating well with the rapid decay of claudin‐1 mRNA in presence of TSA in colon cancer only. Thus, our data suggest that claudin‐1 is regulated via mRNA stability and a novel regulatory feedback loop between HuR and TTP in colon specific tumorigenesis.
The Molecular Mechanism of Bacteriophage T4 Rad50/Mre11 DNA Repair Complex
Authors: Herdendorf Timothy , Nelson Scott
Published: June 2021

Spontaneous double‐strand breaks (DSBs) are one of the most deleterious forms of DNA damage. If not properly repaired, DSBs can cause gross chromosomal rearrangements, leading to cellular dysfunction, tumorigenesis, and the development of cancer. In eukaryotes, archaea, and some bacteriophage, a complex made up of Mre11 and Rad50, which are a nuclease and ATPase, respectively, is involved in the initial processing of DSBs. Due to the lack of a tractable in vitro system, the biochemistry and mechanism of the MR complex is poorly understood. We have recently developed a robust in vitro system and our preliminary studies have indicated that the bacteriophage T4 homologs of Mre11 and Rad50 make an ideal model system for the mechanistic investigation of this complex. The wild‐type complex has been functionally evaluated under steady state and pre‐steady state conditions. The rate‐limiting step for the ATPase reaction has been determined for Rad50 and the MR‐D complex. Rad50 active site residues have been mutated and evaluated to determine their role in ATP binding, catalysis and allosteric communication between Rad50 active sites and Mre11/DNA.
Physical and Functional Interaction Between Fanconi Anemia Group J Helicase and MRE11 Nuclease
Authors: Brosh Robert Michael , Sommers Joshua , Muniandy Parameswary , Coulombe Yan , Masson Jean-Yves , Seidman Michael , Suhasini Avvaru
Published: June 2021

FANCJ mutations are linked to the chromosomal instability disorder Fanconi anemia (FA) and increase breast cancer risk. FANCJ encodes a DNA helicase implicated in homologous recombination (HR) repair of double‐strand breaks (DSBs) and interstrand cross‐links (ICLs), but its mechanism of action is not well understood. Live cell imaging demonstrates that FANCJ recruitment to laser‐induced DSBs, but not psoralen‐ICLs, is dependent on nuclease‐active MRE11 and independent of BRCA1. Inhibition of MRE11 nuclease by Mirin adversely affected FANCJ localization to laser‐induced DSBs, but not psoralen‐ICLs. FANCJ is required for timely recruitment of Bloom's syndrome helicase and CtIP to DSBs in vivo. FANCJ directly interacts with MRE11 and inhibits its exonuclease activity in a specific manner, suggesting that FANCJ harnesses MRE11 nuclease to facilitate DSB processing and appropriate end resection. Spontaneous or IR‐induced chromosomal instability in FANCJ‐depleted cells is suppressed when MRE11 is depleted. Interplay between FANCJ and MRE11 insures a normal response to IR‐induced DSBs, whereas FANCJ involvement in ICL repair is regulated by MLH1 and FANCA, but not FANCD2. Thus, MRE11 helps to recruit FANCJ to DSBs and FANCJ can modulate MRE11 nuclease activity at DNA ends, consistent with a collaborative role of FANCJ and BLM helicases with MRE11 in strand resection.
Scc1 sumoylation by Mms21 promotes sister chromatid recombination through counteracting Wapl
Authors: Wu Nan , Kong Xiangduo , Ji Zhejian , Yokomori Kyoko , Potts Patrick Ryan , Yu Hongtao
Published: June 2021

DNA double‐strand break (DSB) is the most deleterious form of DNA damage and can cause chromosome translocations that drive cancer formation. Homologous recombination (HR) is a key DSB repair pathway. To maintain heterozygosity of the genome, sister‐chromatid homologous recombination (SCR) is preferred to HR between two homologous chromosomes. In human cells, two related structural maintenance of chromosomes (Smc) complexes, cohesin and Smc5/6, facilitate SCR. We have previously shown that the Smc5/6 subunit Mms21 functions as a SUMO ligase. In this study, we demonstrate that Mms21 sumoylates the cohesin subunit Scc1 at multiple sites. SUMO2/3 is accumulated on laser‐induced DNA damages, and this accumulation is dependent on Smc5/6 and cohesin during the S/G2 phase when DSBs are primarily repaired through SCR. Human cells expressing a non‐sumoylatable Scc1 mutant (15KR) maintain proper sister‐chromatid cohesion during mitosis, but are defective in SCR. These results show that Scc1 sumoylation by Mms21 is essential for SCR. On the other hand, Scc1 15KR is still recruited to laser‐induced DNA damage sites. Depletion of Wapl, the negative cohesin regulator, rescues SCR defects and IR sensitivity of Mms21‐deficient or Scc1 15KR‐expressing cells. Therefore, our results suggest that Scc1 sumoylation by Mms21 promotes SCR by antagonizing Wapl at a step after the initial cohesin loading at DSBs in human cells.
A naturally‐occurring flavonoid quercetin inhibits autophagy in human rhabdomyosarcoma cell line
Authors: Dokladny Karol , Moseley Pope
Published: June 2021

Eukaryotes employ an evolutionarily conserved catabolic process of protein degradation called autophagy. It is a dynamic process in which large protein aggregates, entire organelles, and bulk of cytoplasm is sequestered within double membrane vacuoles and delivered for degradation to lysosomes. Quercetin is a naturally occurred flavonoid. Although the antioxidant properties of quercetin have been demonstrated in numerous studies, its effect on autophagy regulation is not known. The purpose of this study was to investigate the effect of quercetin on autophagy regulation in cell culture model (human rhabdomyosarcoma cell line). We found that quercetin (50 μM and 100 μM) inhibited autophagy demonstrated by Western blot analysis of the LC3 lipidation. Autophagy is a highly controlled process and Akt has been shown to negatively regulate autophagy. In our studies, quercetin‐induced down‐regulation of autophagy was associated with an increase in Akt activation demonstrated by increased level of Akt phosphorylation. We conclude that quercetin appears to be a potent autophagy modulator. Further studies are needed to fully determine cellular and molecular mechanisms of quercetin on autophagy regulation.
Downstream Effects of the Homophilic PECAM‐1 Interaction in Neutrophils
Authors: Sieracki Nathan Anthony , Komarova Yulia , Malik Asrar B.
Published: June 2021

Transmigration of neutrophils across the endothelial border and out of the blood vessel into tissues is essential to their ability to fight infection. We are interested in signaling pathways triggered in neutrophils by interaction with specific and spatially oriented components of the endothelial border milieu. The neutrophil/endothelial PECAM‐1 homophilic interaction has been shown critical for successful transmigration with many chemokines. Using microfabrication techniques, we have covalently immobilized the extracellular domain of junctional glycoprotein PECAM‐1 (Platelet/Endothelial Cell Adhesion Molecule) to a glass coverslide in a gradient fashion, and over biorelevant distances, in an effort to mimic traversal across the endothelial cell‐cell border. We demonstrate that neutrophil‐like HL‐60 cells and murine bone marrow neutrophils showed cell polarization when plated on PECAM‐1 coated glass slides but not on control slides, as evidenced by change in cell shape and localization of filamentous actin to the leading edge of the cell. PECAM‐1 is co‐localized with filamentous actin, as evidenced by a neutrophil‐specific antibody to PECAM‐1. These findings suggest an actin polymerization event triggered by an upstream PECAM‐1 homophilic interaction, and suggests that this homophilic interaction plays a role in cell‐shape change that may be necessary for transmigration.
Crystallization of Escherichia coli 70S Ribosome in complex with tRNAs
Authors: Young Brandon , Steitz Thomas A. , Innis Axel
Published: June 2021

The prokaryotic ribosome is responsible for translation, the process of creating polypeptides from amino acids. Specifically, ribosomes translate the codons in mRNAs into polypeptide chains. The prokaryotic ribosome is made up of a large subunit and a small subunit, which are known as the 50S (Svedberg units) and 30S subunits, respectively. The larger subunit catalyzes peptide bond formation within the peptidyl transferase center, while the smaller subunit mediates decoding of the mRNA. Together they make up the 70S prokaryotic ribosome. Studies have shown that certain nascent polypeptides can bind to the exit tunnel, resulting in inhibition of peptide bond formation which is referred to as ribosome stalling. Through the use of X‐Ray crystallography we will crystallize the 70S ribosome in complex with tRNAs and a nascent chain within the exit tunnel producing a structure. Although, no structure has been produced yet, we have been able to narrow down conditions that will optimize the growth of these crystals. This optimization will allow for production of the structure which will result in us being able to further explore the process of nascent chain mediated‐translational arrest in the Escherichia coli (E. coli) 70S ribosome.

This research was supported in part by a grant from the Howard Hughes Medical Institute.
Cyclical C7‐CoA esters derived from calcium levulinate, a pro‐drug of abuse
Authors: Harris Stephanie Rae , Zhang Guo-Fang , Sadhukhan Sushabhan , Puchowicz Michelle A , Anderson Vernon E. , Tochtrop Gregory P , Brunengraber Henri
Published: June 2021

Calcium levulinate (4‐ketopentanoate, LEV) is a dietary calcium supplement. We previously showed that (i) LEV is reduced in liver to γ‐hydroxypentanoate (GHP), a drug of abuse, analog of γ‐hydroxybutyrate, (ii) the reduction of LEV to GHP is enhanced by ethanol oxidation, and (iii) the metabolism of LEV and GHP result in substantial accumulation of LEV‐derived C5‐acyl‐CoAs in liver and brain, leading to CoA trapping. We also identified new C7‐CoA esters in rat livers perfused with LEV and in liver and brain of rats gavaged with Ca‐LEV. The C7‐CoA esters derive from the elongation of LEV‐CoA by acetyl‐CoA to 3,6‐diketoheptanoyl‐CoA, a γ‐diketo compound. The latter is converted to two cyclical CoA esters (one with a cyclopentane ring, and one with a pyrrole ring). We traced the origin of the C7‐CoA esters with labeled substrates. Both cyclical C7‐CoAs include five 13C from [13C5]LEV and two 13C from [13C2]acetyl‐CoA (derived from LEV or other [13C]substrates). The pyrrolic C7‐CoA ester contains one N derived from the N on C‐6 of [6‐15N]lysine. The C7‐CoA esters were identified in rat livers and hearts perfused with 6‐ketoheptanoate, the beta‐oxidation of which passes by 3,6‐diketoheptanoyl‐CoA. The formation of cyclical C7‐CoA esters may contribute to the toxicity of LEV by binding to the ε‐N of lysine residues of proteins in liver, heart or brain. Supported by NIDDK RoadMap + training grant, and by NIEHS.
Improvement of defective Cystic fibrosis airway epithelial repair after CFTR rescue
Authors: Trinh Nguyen Thu Ngan , Adam Damien , Bardou Olivier , Anik Privé , Maillé Émilie , Lingée Sarah , Ferraro Pasquale , Desrosiers Martin , Coraux Christelle , Brochiero Emmanuelle
Published: June 2021

Airway damage and remodelling is an important component of the progression of lung cystic fibrosis (CF) pathology. Our histological analyses of bronchial tissues from CF patients revealed several ultrastructural anomalies, including epithelial desquamation and tissue remodelling. Although some areas were in the process of epithelial regeneration, the repair mechanisms seemed obviously insufficient in maintaining epithelial integrity in CF airways. Our aim was thus to study the CFTR function in airway repair processes. Wound‐healing assays were performed to study the early phase of epithelial repair, in particular cell migration, in human CF and non‐CF airway cells. Our experiments showed that airway cells from CF patients repaired slower than non‐CF cells. In addition, CFTR inhibition or silencing in non‐CF primary airway cell monolayers partially inhibited wound‐closure. Interestingly, wt‐CFTR transduction in CF airway cell lines or CFTR correction with VRT‐325 in CFBE‐ΔF508 cells and primary CF bronchial monolayers significantly improved wound‐healing. Altogether our results demonstrated that functional CFTR play a critical role in wound‐repair, and CFTR correction may represent a novel strategy to promote the airway repair processes in CF.

Project funded by CFC (Cystic Fibrosis Canada).
MITOCHONDRIA‐TARGETED TREATMENT ATTENUATES VASCULAR OXIDATIVE STRESS, ENDOTHELIAL DYSFUNCTION AND HYPERTENSION
Authors: Dikalov Sergey , Nazarewicz Rafal , Bikineyeva Alfiya , Dikalova Anna
Published: June 2021

Mitochondrial reactive oxygen species (ROS) have been recently implicated in the development and progression of cardiovascular diseases. We have suggested that mitochondria targeted antioxidants can be beneficial for treatment of endothelial dysfunction, hypetension other parthological conditions. In this work we have studied specific molecular mechanisms responsible for stimulation of mitochondrial O2• and its downstream targets using cultured human aortic endothelial cells and angiotensin II‐induced hypertension. Depletion of cytoplasmic Nox2 isoform of NADPH oxidase inhibited stimulation of mitochondrial ROS by angiotensin II. Meanwhile, treatment with the mitochondria targeted superoxide dismutase mimetic mitoTEMPO or SOD2 overexpression inhibited production of mitochondrial O2•, reduced activity of redox dependent c‐Src and cytoplasmic NADPH oxidase, restored vascular NO in angiotensin II stimulated cells and reduced blood pressure in mice with angiotensin II and DOCA‐salt induced hypertension. We have suggested that mitochondrial ROS stimulates cytoplasmic oxidative stress via release of H2O2. Indeed, scavenging of mitochondrial H2O2 with mitochondria targeted glutathione peroxidase mimetic mitoEbselen inhibited production of cytoplasmic O2• by NADPH oxidase and reduced blood pressure in angiotensin II induced hypertension. Production of angiotensin II‐induced mitochondrial ROS was inhibited by specific PKCε peptide antagonist, ATP‐sensitive potassium channel blocker 5‐hydroxydecanoate, inhibitors of complex I rotenone, inhibitors of complex II malonate or malate. These studies show that mitochondrial ROS play an important role in feed‐forward stimulation of oxidative stress by activating mitochondrial reverse electron transfer which can be treated with mitochondria‐targeted antioxidants.

This work has been funded by National Institutes of Health HL094469.
Dysregulation of mitochondrial sphingolipid metabolism after traumatic brain injury
Authors: Novgorodov Sergei A. , Riley Christopher , Fedarovich Gleb , Yu Jin , Obeid Lina M. , Kindy Mark S. , Gudz Tatyana I.
Published: June 2021

To investigate mechanisms underlying a traumatic brain injury (TBI), we used a computer‐controlled cortical impact injury model in mice. The mouse brain injury was characterized by progressive cortical damage along with ablation of the grey and white matter demonstrated by magnetic resonance imaging and behavioral analysis. At the molecular level, TBI instigated a profound dysregulation of sphingolipid metabolism in brain. Sphingolipid analysis revealed increases in sphingomyelin and sphingosine that appear to result from activation of de novo biosynthesis of ceramide. Thus, the major metabolites of de novo ceramide biosynthetic pathway were up‐regulated after TBI. Remarkably, ceramide species were somewhat reduced concurrently with accumulation of sphingosine, possibly, due to enhanced ceramide degradation by ceramidase. Investigation of intracellular sites of sphingosine accumulation revealed the elevation of sphingosine in mitochondria. Furthermore, sphingosine accumulation appears to cause mitochondrial respiratory chain damage at the level of cytochrome oxidase that could be mimicked by exogenously added sphingosine to mitochondria. These studies highlight a novel mechanism of TBI injury involving dysregulation of sphingolipid metabolism in mitochondria and identify the sphingolipid responsible for mitochondrial dysfunction after TBI. Supported by VA Merit awards from RR&D and BLR&D Service.
Ultrastructural Modifications in the Mitochondria of Hypoxia‐ Adapted Drosophila melanogaster
Authors: Zhou Dan , Perkins Guy , Yin Songyue , Xue Jin , Liu Siqi , Ellisman Mark H , Haddad Gabriel G
Published: June 2021

Chronic hypoxia occurs under certain physiological or pathological conditions. As mitochondria are the predominant oxygen‐consuming organelles that utilize oxygen to generate ATP through oxidative phosphorylation in cells, their responses to hypoxia play an important role in the overall functional adaptation to hypoxia. Here, we report the adaptive mitochondrial ultrastructural modifications and the functional impacts in a hypoxia‐adapted Drosophila melanogaster strain that survives severe, otherwise lethal, hypoxic conditions. Using electron tomography, we discovered increased cristae fragmentation and a unique “honeycomb‐like” structure in the mitochondria of hypoxia‐adapted flies. The homeostatic levels of adenylate and energy charge were similar between hypoxia‐adapted and naïve control flies and the hypoxia‐adapted flies remained active under severe hypoxia and quantified by geotaxis behavior. The equilibrium ATP level was lower in hypoxia‐adapted flies than those of the naïve controls, tested under severe hypoxia that inhibited the motion of control flies. Our results suggest that the structural rearrangement in the mitochondria of hypoxia‐adapted flies may be an important adaptive mechanism that plays a critical role in preserving adenylate homeostasis and metabolism as well as muscle function under chronic hypoxic conditions.
Anfepramone effectsˈs evaluation over the expression profile of NLRP3 inflammasome, TXNIP, TLR4 and LEP in peripheral blood mononuclear cells of Mexican obese subjects
Authors: Sanchez Dulce Maria Lopez , de los Angeles Martinez Godinez Maria , Lopez Aaron Dominguez , Padilla Eleazar Lara , Hernandez Aldo Ahumada , Garcia Angel Miliar
Published: June 2021

Subjects (n = 42) with a body mass index (BMI) ≥30 kg/m2 who received Anfepramone 75 mg/day for six months were studied. Relative gene expression was quantified by real‐time PCR. Anthropometric and biochemical parameters were also measured. NLRP3, TXNIP, TLR4 and LEP expression levels increased after Anfepramone treatment (p ≤0.05). Parameters such as BMI and glucose concentration decreased after the period of treatment (p ≤0.01), whereas cholesterol and LDL cholesterol concentrations increased (p ≤0.01). Anfepramone treatment was effective for weight loss; however lipid profile was perturbed. Expression changes might be due to the raised on free fatty acids (FFA) which produce reactive oxygen species (ROS). ROS activate NLRP3 per se and through TXNIP, while FFA activate TLR4 leading to priming process. On the other hand the data highlight LEP high expression level being that the mechanism described is by central nervous system stimulation. Here we show the first results on expression of inflammatory genes after an obesity pharmacologic treatment; this knowledge will be helpful to improve drug management and to avoid secondary effects. We acknowledge Instituto Politécnico Nacional for its support to 20111112 project. We thank Productos Medix S.A. de C.V. for providing the Anfepramone treatment.
Increased protease activity in obese rats: implications on LDL and insulin receptors
Authors: Mazor Rafi , Alsaigh Tom , Schmid-Schonbein Geert
Published: June 2021

Obesity leads to increased morbidity and mortality from cardiovascular complications. Among other characteristics of the metabolic syndrome, obesity is a major risk factor for Insulin resistance and high levels of LDL, although the mechanisms are not completely understood. We hypothesize that calorie over consumption can lead to increased proteolytic activity which in turn cleave extracellular domains of key receptors e.g. the insulin and LDL receptors, therefore impairing their uptake and contributing to their high levels in the circulation. Wister rats were fed high fat diet (HD) for 16 weeks. A control group was provided with regular chow (ND). Enzymatic activity was measured in vivo on the mesentery microcirculation using intravital fluorescent microscope. Levels of the extracellular domain of the insulin and LDL receptors were measured by immunohistochemistry on isolated leukocytes. Glucose and LDL uptake were measured by fluorescent imaging. HF rats had significantly higher enzymatic activity in the mesentery microcirculation compared to controls. This activity was accompanied with reduced levels of the extracellular domain of both insulin and LDL receptors. Leukocytes of obese rats uptake glucose and LDL in a slower rate compared to controls. Our results suggest that calorie over‐consumption can promote proteolytic activity and cleavage of key receptors thus promoting insulin resistance and high circulating levels of LDL in obesity.
Cloning and expression of Cryptochrome proteins from Arabidopsis thaliana
Authors: Velez Julian D. , Dyer James H.
Published: June 2021

Two cryptochrome proteins, CRY1 and CRY2, occur in plants and animals. CRY1 regulates the circadian clock in a light‐dependent fashion in insects and plants. Also in plants, blue light photoreception can be used to cue developmental signals. Despite much research on the topic, the specific way that the pterin and flavin chromophores function in these proteins in Arabidopsis thaliana is still poorly understood. The goal of this research was to clone and express the Arabidopsis CRY1 and CRY2 proteins in an E. coli expression system. The constructs were cloned into a pBAD‐His vector and purified using Ni‐NTA affinity chromatography. Purified proteins will be used for structural and functional analysis.
Effects of storage conditions on soil molecular biology for forensic science purposes
Authors: Larson Sabreena A , Patel Niraj , Freeman Victoria , Hustedt Jason , Drijber Rhae , Carter David , Bailey Cheryl
Published: June 2021

The object of this project is to identify changes in soil microbial community DNA and Fatty Acid profiles caused by storage, using capillary electrophoresis single‐strand conformation polymorphism (CE‐SSCP) and fatty acid methyl ester (FAME) analysis.

There were six different storage treatments tested (−80°C, −20°C, 4°C, freeze dried, air dried, oven dried) on soil samples collected from 4 different locations in Nebraska. Samples were taken at a depth of 0.0 cm to 5.0 cm from three biological replicates at each collection site. After collection, soils were sieved and subsamples were placed into each storage treatment for five weeks then analyzed, with the exception of one subsample (fresh) which had DNA and fatty acids extracted within 36 hours of collection. Additional fresh samples were collected and processed two weeks after initial collection and seasonally.

The CE‐SSCP and FAME analysis will illustrate the microbial community and its diversity for an individual soil sample through the number of peaks, molecular size of peaks and relative peak heights. Using statistical analysis it can be determined which storage treatments altered the soil microbial community profile when compared to the fresh subsample. Results will be presented to show the effectiveness of these two methods to detect small variations in the soil microbial community. Funded by National Institute of Justice (25‐6228‐0159‐001).