bluuebunny/crossref_metadata_2025 huggingface dataset:
Determining the effectiveness of the Hybrid teaching method in a Biology course at a small liberal arts college
Authors: Resendes Karen K , Mroz Kevin
Published: June 2021

Hybrid courses are designed to maximize learning efficiency by having students experience a normal lecture style combined with a web‐based mode of teaching. These courses allow students to develop independent learning habits, but also reinforce learning by incorporating regular lectures. In our study, we will attempt to determine the effectiveness of the hybrid method in the small classroom setting, using a introductory level biology course for non biology majors in the Spring of 2012 at Westminster College, a small liberal arts college We will compare our results to a previous study that demonstrated that a non‐hybrid version of this course at Westminster displayed similar student retention rates to a large lecture hybrid course at San Diego State University. We intend to determine if a hybrid course is not only efficient at a small college, but also whether or not it provides an advantage over the hybrid course style at a large university. We will compare retention rates between sections of material taught using the hybrid method with that of material presented in the traditional style, using pre and post‐tests. Hybrid components will include pre‐class readings and quizzes along with a variety of interactive online activities performed after material is taught in class. We will also examine the influence of the hybrid style on the students’ interest levels in biology and their impressions on the usefulness of the course.
Assessment of online course design for a lecture‐only pathophysiology course
Authors: Walton Kristen LW
Published: June 2021

BIO 375 Pathophysiology at Missouri Western State University is a 4‐credit‐hour, upper division, lecture‐only course primarily serving pre‐nursing and other allied health majors. In response to demand from this student population, the course was offered in an online format for the first time in Summer 2011. It was designed to parallel the face‐to‐face version as closely as possible. Video lectures, abbreviated versions of lecture slides, study guides, and other materials were posted online. Quizzes and assignments were completed electronically, but exams were given in a proctored, written format. Of the 34 students who enrolled in the course, 31 finished, with an overall course GPA of 2.65. This is similar to the course GPA from the prior summer offering (Summer 2010 GPA = 2.53, n=19) and the average GPA across four regular academic year semester offerings (average GPA = 2.61, n=319). Students were surveyed at the end of the course for positive and negative aspects of the online course format. Positive elements of the course included flexibility of schedule and the ability to re‐watch video lectures (both selected by 77% of respondents), while 42% selected the requirement for proctored exams and 38% selected “less opportunity to interact with the instructor than in a traditional class” as negatives. These data will be used to refine the course structure for future online course offerings.
The use of novel Camtasia videos to improve performance of at‐risk students
Authors: Miller Cynthia Jayne
Published: June 2021

Students in undergraduate Physiology courses have difficulty understanding complex, multi‐step processes, and these concepts consume a large portion of class time. We hypothesized that online multimedia resources may improve student performance and reduce the in‐class workload. A narrated Camtasia video was created using PowerPoint slides of a cardiomyocyte action potential. Historically, student performance on an exam essay for this topic was very poor following a traditional didactic lecture (class avg. of 64.43%). Replacement of lecture with the online video improved the class avg. by 6.45% and the class median by 8.33% (n=198, 9 course sections). Furthermore, the video appeared to have the largest influence on at‐risk students, reducing the percentage of failing students from 38.2% in past semesters to 24.07% with access to the video. Anonymous polling of the students indicated the video was helpful in visualizing the process and allowing repetition of material. While creation of multimedia resources may require significant instructor preparation time, these results suggest that the time invested is advantageous to student success, particularly for at‐risk students.
Francisella enters host cells by Clathrin‐mediated endocytosis at a cholesterol rich domain
Authors: Lin Ann E , Kim Youra , Law HT , Nano Francis E , Guttman Julian A
Published: June 2021

Francisella tularensis are highly infectious bacterial pathogens that cause a type of acute debilitating febrile illness called tularemia. While Francisella reportedly colonize and establish infection in multiple organs, the liver remains as the primary site of infections. Although these infections are responsible for the mortality of 35% of infected individuals if left untreated, the strategies these pathogens use to invade non‐phagocytic cells remain elusive. Therefore, our objective is to examine the subcellular mechanisms these bacteria utilize to infect their hosts. We generated the hypothesis that Francisella co‐opt normal human endocytic machinery to internalize into host epithelial cells. To test this, we developed two in vitro Francisella‐based infection models using murine hepatocyte epithelial cells. By using a combination of pharmacological inhibitors, RNA interference‐mediated protein knock down and protein immunolocalization, we showed that Francisella exploit clathrin and cholesterol dependent mechanisms to gain entry into hepatocytes. We conclude that this is the first study demonstrating the importance and significance of clathrin‐mediated endocytosis to facilitate efficient Francisella invasion into non‐phagocytic cells.

Grant Funding Source: CIHR
Morphological changes in the radial spokes of two Chlamydomonas mutants expressing various truncated RSP3
Authors: Meek Bill , Sivadas Priyanka , Yang Pinfen
Published: June 2021

Various cell types utilize motile flagella and cilia to propel through the surrounding fluid or move the fluid over their surface. The machinery for movement is built around a 9 + 2 axoneme. The axonemes of flagella and cilia are microtubule‐based complexes that are constructed from hundreds of different polypeptides. Some of these polypeptides are arranged into macromolecular complexes and include the dynein motors, radial spokes (RS), and the central pair of microtubules or apparatus (CP). The T‐shaped radial spokes regulate flagellar beating. The CP positioning and orientation of the RS propagate different bends along the axoneme, and this is associated with stroke force (Satir and Christensen, 2007). It was hypothesized that the radial spoke protein 3 (RSP3) is a multi‐modular scaffold that anchors RIIa and DPY‐30 domains that tether multiple effector mechanisms for the spoke complex; a single scaffold will provide the rigidity necessary for mechanical feedback (Sivadas and Yang, 2011). Using the Chlamydomonas radial spoke complex model, truncation mutants of RSP3, missing the two RIIa‐containing RSPs along with subsets of spoke stalk proteins and mutants missing two DPY‐30 RSPs and the bulbous spokehead modules were generated. The morphological test of the hypothesis that RSP3 forms the scaffold of the radial spoke to which other RSPs bind was done at the ultrastructural level.
Novel imaging of the implantation of left‐sided pacing leads within reanimated swine hearts
Authors: Eggum Julianne Hana , Iaizzo Paul
Published: June 2021

Objective

To obtain unique images of left‐sided pacing lead implantations for the educational benefits of both physicians and medical device design engineers.

Methods

We employed Visible Heart® methodologies to obtain simultaneous videoscopic (internal and external) and flouroscopic footage of the device‐tissue interfaces during left‐sided pacing lead (Medtronic 4196 4F dual electrode lead) implantations within reanimated swine hearts. We targeted the lateral cardiac veins via delivery through the coronary sinus. We used an isolated cardiopleged heart from Yorkshire‐cross swines; the University of Minnesota's Animal Care and Use Committee approved all experimental protocols involved in this study.

Results

Figure 1 displays typical images obtained during the implantation of left‐sided leads: (1) fluoroscopic and videoscopic images of the initial cannulation of the coronary sinus; (2) fluoroscopic and fiberscopic images of the subselection of a target lateral vein; and (3) fluoroscopic, videoscopic, fiberscopic, and external images of a subsequently implanted left‐sided lead.

Conclusion

Using Visible Heart ® methodologies, we were able to obtain novel footage of left‐sided lead implantations that are of educational value for students, patients, physicians, and left‐sided lead device design engineers.

Research Support Source: The University of Minnesota's Institute for Engineering in Medicine and Medtronic Inc. (Minneapolis, MN)
The type I TGFβ receptor ALK‐1 functions in TGFβ‐mediated signaling in angiogenesis
Authors: DUFFY IAIN , Watters January , HAWKER JAMES
Published: June 2021

Activin‐Like Kinase 1 (ALK1, TSR1), a type I transforming growth factor (TGFβ) family receptor found in endothelial cells, is involved in angiogenesis (new blood vessel formation). Control of angiogenesis is critical in diseases such as cancer, heart disease, or wound repair. We show that expression of ALK‐1 but not ALK 5 type I receptor increases dramatically in endothelial cells after tube formation stimulated by TGFβ + fibroblast growth factor (FGF). FGF‐2 + TGFβ also upregulate ALK 1 and downregulate ALK5 cell surface expression in human endothelial cells. Further, we show that TGFβ signals through a complex of ALK 1, the ALK signaling protein Smad 1, and the co‐receptor endoglin. Three ligands, TGFβ, activin, and bone morphogenetic protein 9 (BMP‐9), have been shown to bind ALK‐1. We show that endogenous activin binds ALK 1 and can be displaced by exogenous TGFβ. This suggests it may function to regulate control of ALK 1 activation. Antisense RNA that targets ALK 1 and also ALK 5 disrupts tube formation. This data suggests a critical role for the ALK‐1 receptor in endothelial tube formation. We are generating inactive mutants of ALK‐1, ALK‐5, and endoglin to further test their roles in angiogenesis and signal transduction. We hope to dissect the role of ALK‐1 and TGFβ family ligands in angiogenesis of tumors. Research support was from AHA‐TX and Dept Veteran's Affairs grants (JH) and funds from Saint Leo University (ID).
Fibrin Promotes Survival and Differentiation of Endothelial Progenitor Cells after Transplantation
Authors: Wang Hai-Jie , Quan Zhe , Wang Qiang-Li , Tan Yu-Zhen
Published: June 2021

Transplantation of endothelial progenitor cells (EPCs) is a potential therapy for angiogenesis and improving microcirculation of the local tissue in ischemic diseases. However, survival and differentiation of the transplanted EPCs decrease so that efficiency of EPC transplantation is undesirable. This investigation was designed to investigate cytoprotective effects of fibrin in ischemic microenvironment. VEGFR‐2+ cells were sorted from mononuclear cells in bone marrow of male SD rats. Fibrin glue was prepared by mixing fibrinogen and thrombin. The cells were seeded in fibrin or fibrin supplemented with VEGF. The cells in fibrin glue may migrate well. Under hypoxia and withdraw of serum and glucose, apoptotic and necrotic cells in fibrin group were less than that in control group. After incubation in fibrin glue for two weeks, the cells differentiated into endothelial cells and organized into capillary‐like structures. After transplantation of fibrin‐delivered cells into the infracted myocardium of female rats, angiogenesis and cardiac regeneration increased, cardiac functions were improved significantly. These results demonstrate that fibrin has protective effects for survival and differentiation of EPCs. Delivery with fibrin in EPC transplantation is effective for repairing the infracted myocardium.
Liver‐specific Deletion of Protein Tyrosine Phosphatase (PTP) 1B Improves Endothelial Dysfunction and Cardiovascular Alterations Associated with Obesity in mice
Authors: Agouni Abdelali , Tual-Chalot Simon , Duluc Lucie , Martinez Carmen Maria , Mody Nimesh , Andriantsitohaina Ramaroson , Delibegovic Mirela
Published: June 2021

Obesity is tightly associated with insulin resistance (IR) and endothelial dysfunction. It is suggested that hepatic IR is sufficient to promote progression to cardiovascular disease. We showed recently that specific deletion of liver PTP1B (L‐PTP1B), a negative regulator of insulin receptor, improves whole body glucose and lipid homeostasis. Thus, we propose to investigate the impact of these improvements in L‐PTP1B−/− mice, on cardiovascular function in the context of diet‐induced obesity.

We analyzed glucose tolerance, blood pressure, cardiac and vascular function, in L‐PTP1B−/− and control mice, fed a chow or high fat diet (HFD).

Compared to control littermates, L‐PTP1B−/− mice had improved glucose and lipid homeostasis without changes in body mass. HFD feeding increased systolic blood pressure in both mouse groups; however, it was lower in L‐PTP1B−/− mice. Structure and function analysis of the left ventricle showed that HFD‐feeding decreased cardiac index in control mice, while L‐PTP1B−/− mice were fully protected. HFD feeding significantly impaired endothelium‐dependent relaxation in response to acetylcholine in aortas taken from control mice compared to chow diet‐fed mice, while L‐PTP1B−/− mice were protected.

Altogether, these data indicate that targeting L‐PTP1B may be useful to reduce obesity‐associated cardiovascular risk in addition to diabetes.
Collaborative Learning Utilizing Case‐based Problems
Authors: Hilvano Nestor T , Mathis Karen M
Published: June 2021

Engaging students in discussion and creating high impact teaching and learning practices is a challenging aspect in every classroom. Small group discussion and poster presentations were used to solve case‐based problems to highlight issues for the learner and to allow each student to demonstrate understanding and application of theory to real life examples through open‐ended, focused questions. This study consisted of students enrolled in Anatomy and Physiology course sequence. Assessment is based on group goal and individual accountability. Rubrics for evaluation were developed for self and peer assessment of group's dynamic to ensure that every group member participates. A poster session at the University of Cincinnati Clermont College provides our students with an opportunity to explain their work to an audience, as well as generate active discussion and peer evaluations. The assessment results showed a positive significant (p value of 0.0001–0.0025) in terms of students’ knowledge, attitude and psychomotor skills. In conclusion by engaging students to work cooperatively on case‐based problems, our students had significant improvement in critical thinking skills and academic achievement, and a significant positive impact on attitude, motivation, self‐esteem, and interpersonal skills.
Plastinated prosections in a systems‐integrated gross anatomy curriculum
Authors: Anstrom John , Danelisen Igor , Wyeth Richard
Published: June 2021

Human gross anatomy courses for medical students have undergone significant changes over the past two decades. At the Via College of Osteopathic Medicine gross anatomy dissection has been reformatted from a regional to a systems‐integrated course with major organ systems covered in separate dedicated “blocks”. Years 1 and 2 comprise 8 consecutive blocks. Students begin gross anatomy in block 2, entitled “musculoskeletal”, and progress through blocks 3–6, neuro, cardiopulmonary, gastrointestinal, and genito‐urinary, respectively. The extended schedule results in an overlap of year 1 and 2 students in the gross anatomy laboratory, as blocks 2 and 6 run concurrently. Logistically it is impractical to have two classes share cadavers or to double cadaver numbers present in the lab. To resolve this problem, auxiliary tools are being developed to teach block 6, genitourinary anatomy. One major effort has been the production of prosected, plastinated pelvises as substitutes for cadavers. Initial assessments indicate that students comprehend relevant anatomy using this modality but express a preference for cadaver dissection. Additional plastinated pelvic specimens, prosected to highlight important clinically significant structural relationships, are being developed to further facilitate student learning and create greater student acceptance. Supported by a grant from the Harvey W. Peters Foundation.
BROADENING THE EDUCATION OF ANATOMY WITH ART
Authors: Babin Kathryn P. , Koba Alexandra M.
Published: June 2021

A series of anatomical drawings were created in the framework of illustrating a book titled, “Art and Science of Anatomy: Special Dissections of the Head and Neck”. A survey of the undergraduate anatomy students proved the interpretation and construction of the visual material to be an effective method in improving the students comprehension, retention, and overall quality of the undergraduate anatomy education. Hand drawn illustrations are clear, beautiful, and appeal to the sense of intrigue, when shown the medical illustrations creative learners beg to know more. The drawings themselves were created in an effort to assist in teaching a regional approach to human anatomy, as well as to complement and enhance the material for both the visual right and left‐brain pre‐clinical thinkers.

Grant Funding Source : GIAR, Grant in Aide of Research administered by Sigma‐Xi, The Scientific Research Society
Computer‐assisted instruction increased students’ ability to interpret 3D relationships and retain essential course material in undergraduate human anatomy and A&P courses
Authors: Tallitsch Robert B. , Abdel-Malek Karim , Krippel Jaynee , Beck Allison , Croll Paul , Fenwick Shirley , Kelley Kirk , Peters Brenda
Published: June 2021

Studies indicate that computer‐assisted instruction (CAI) may improve learning in many subject areas. Little is known about the effect of anatomical software upon learning in undergraduate Human Anatomy and A&P courses. Results from a three‐year study investigating the effect of CAI in undergraduate human anatomy and combined A&P courses demonstrated significant, positive effects upon student (n=710). (1) The Purdue Visualization of Rotations Test was administered to one‐half of the participants the first week of class each term and the remaining one‐half at the completion of the class. Statistically significant differences were seen between control (no CAI) and experimental (CAI) data. (2) Two 10‐minute, online questionnaires were administered the first week of class and at the end of the class to determine changes in students’ interest in the course. Additional data will need to be collected in year three to more fully determine whether CAI enhances student interest. (3) A Test of Essential Material was administered upon completion of the course to all participating students. Statistically significant differences were seen between no CAI and CAI data. These data demonstrate that CAI with Cyber‐Anatomy® increases student's ability to interpret 3D relationships and retain essential course material. (All research protocols were approved by the respective institutions’ IRB.)
An interactive 3D model of the cranial nerve and brainstem nuclei for enhanced learning of neuroanatomy
Authors: Pedersen Kelly Lynn , de Ribaupierre Sandrine , Wilson Timothy D
Published: June 2021

Neuroanatomy is a complex sub‐discipline of anatomy that requires abstract thinking and strong spatial reasoning. Traditional methods of learning include dissection, diagrams, and histology. This pedagogical approach requires students to formulate three‐dimensional (3D) mental images from two‐dimensional (2D) cross‐sections. Previous studies demonstrate students with lower spatial abilities have difficulty learning the anatomy of the brainstem nuclei partly due to their inability to conceptualize topography. The purpose of this study was to design and implement a 3D model of the cranial nerve and brainstem nuclei into an online learning tool that highlights their spatial relationships. The second purpose was to test the learning tool against traditional methods. This tool was compared to a classical approach using a randomized, cross‐over design. It is hypothesized that while subject to the same learning objectives, students learning with the 3D tool demonstrate enhanced knowledge of the spatial relationships of the nuclei compared to the students who learned through the classical approach. A standardized test and an open‐ended questionnaire were used to measure efficacy and student preferences. Information from this study will help guide the formation of new e‐learning tools that are becoming pervasive in anatomical sciences.

Grant Funding Source: none
The Educational Value of Virtual 3D Skeletal Model Rendered from CAT Scan in Anatomy Education
Authors: Balzano Vincent Mario , Lee Lisa MJ
Published: June 2021

The objective of the study was to evaluate the educational value of a 3D virtual model of the human skeletal system, rendered from a CAT scan of a cadaver. With the current trend of decreasing student‐instructor contact hours in basic science curricula in professional schools, ancillary resources are becoming increasingly important. As dissection of the cadaver involves a superficial to deep approach with emphasis on soft tissues, anatomy of the skeletal system is often taught using disarticulated bones or complete skeletal models which are scarce in most institutions. Limited study of skeleton also affects students’ understanding of soft tissue structures. To compensate for deficiencies, an interactive 3D virtual skeletal model of the human torso was created from a series of DICOM images of a cadaver and was made available to the first‐year dental students in a human anatomy course at The Ohio State University. Pre‐ and post‐quizzes were conducted to evaluate whether learning took place from the simple interaction with the virtual model. A survey was conducted to evaluate students’ perception of the resource with respect to its educational value and students’ preference of using such a resource in the future.

Grant Funding Source: N/A
To Implement and determine the effectiveness of a multi‐modal teaching strategy in a Caribbean Medical School
Authors: singh keerti , Richardson Priscilla , Hennis Heather , Branday Joseph Michael
Published: June 2021

Objectives

1) To introduce and implement a web based multi‐modal module in Embryology based on student learning preference 2) To assess student retention of embryology using multimedia imaging approaches in short and long term.

Design and methods

A plan for organising teaching strategies based on topics in embryology will be rolled out with the assistance of a curriculum specialist. These will include web‐based videos and 3‐D animations, as well as didactic and lab exercises. Students will be presented with surveys and questionnaires to assess their perception of learning strategies strengths and weaknesses. We propose to do this study for the current cohort and compare it with students in the next academic year. The course includes 6 lectures in all, three will be taught with the conventional methods and three lectures will be taught incorporating videos and animations in the lectures. Quizzes ( 20–25 mins) will be uploaded on the moodle course website on the same evening after the lecture. Each quiz will comprise with equal number of questions from lectures, slides, models and videos. Students scores will be evaluated and compared from the day of the lecture, mid term and end of term exams. Students will be asked to complete a questionnaire and give their feedback on their perceived learning. A statistical analysis of the data will be done. This will give an insight whether the multi‐modal teaching aid benefits lerners on a long term or a short term basis and finally developing a web based module for the same.

Conclusions

Implementing a web‐based strategy in embryology will fulfill the faculty's education goal of utilizing technology more effectively, and align our curriculum with international best standards in medical education. Thereby impact student learning positively in both the short and long term

Grant Funding Source: University of West Indies
NPM1 and APE1: nucleolar teamwork in controlling base excision DNA repair
Authors: Lirussi Lisa , Antoniali Giulia , Vascotto Carlo , DˈAmbrosio Chiara , Poletto Mattia , Romanello Milena , Marasco Daniela , Demple Bruce , Bhakat Kishor , Colombo Emanuela , Scaloni Andrea , Tell Gianluca
Published: June 2021

Apurinic/apyrimidinic endonuclease 1 (APE1), the main abasic endonuclease in base excision repair (BER) acting on oxidative and alkylation DNA lesions, is a promising target for anticancer therapy. In nucleoli, APE1 interacts through N‐terminal lysine residues with nucleophosmin 1 (NPM1). We used a reconstitution strategy to address the impact of the NPM1‐APE1 interaction on resistance to genotoxic damage. Lysine‐mutated APE1 proteins were expressed while endogenous APE1 was silenced by siRNA to address the role of APE1 acetylation during cell response to DNA damage and a NPM1 knock‐out system to determine its role in modulating APE1 in BER pathway. We demonstrated that APE1 acetylation at lysines 27, 31, 32 and 35, enhanced by genotoxic stress, inhibited binding to NPM1 and promoted re‐localization of APE1 from nucleoli to the nucleoplasm. Interestingly, NPM1 stimulated APE1 BER activity in vivo, since cells lacking NPM1 were more sensitive to bleomycin or alkylating agents and displayed impaired DNA repair. These results highlight an unexpected role for NPM1 and the nucleolus in coordinating APE1 and BER pathways, and point to acetylation at critical lysine residues for controlling subnuclear protein trafficking during DNA repair.

This work is supported by grants from AIRC and FIRB.
Polyamine Effects on β‐Lactam Antibiotics in Pseudomonas aeruginosa
Authors: Lewis Martha , Kwon Don
Published: June 2021

Pseudomonas aeruginosa is a human pathogen causing a variety of diseases. Treatment of P. aeruginosa infections use β‐lactam and aminoglycoside antibiotics; however, the treatment is difficult due to multidrug resistant P. aeruginosa. Polyamines (spermidine and spermine) are cationic compounds and known as enhancing antibiotic susceptibility. In this study, polyamine effects on growth of P. aeruginosa and β‐lactam susceptibility were examined. Results showed that polyamines were non‐toxic on growth of P. aeruginosa and significantly enhanced β‐lactam susceptibility, suggesting polyamines may be used to treat multidrug resistant P. aeruginosa infections in combining with existing β‐lactam antibiotics such as Aztreonam, Carbenicillin, Ceftriaxone, and Ticarcillin.
Engagement of ICAM‐1 mediates neutrophil crawling on the luminal surface of the intestinal epithelium and signals to regulate barrier function
Authors: Sumagin Ronen , Parkos Charles A
Published: June 2021

A hallmark of inflammatory mucosal diseases is transepithelial migration (TEM) of neutrophils (PMN) and subsequent accumulation in a lumenal space. TEM has been extensively modeled, but PMN interactions with apical epithelial ligands and resulting effects on epithelial barrier function remain poorly understood. We previously showed that interferon γ (IFNγ) treatment of T84 intestinal epithelial cells enhanced ICAM‐1 expression on the apical epithelial cell membrane, leading to increased PMN adhesion. Here we show that, after TEM, 59.3±7.6% of apically‐associated PMN exhibit crawling behavior. There was a 33% increase in PMN crawling on IFNγ treated T84 monolayers along with decreased crawling distance and velocity. These effects were ICAM‐1 specific, as they were reversible by treatment with anti‐ICAM‐1 antibody (Ab). Experiments modeling functional engagement of epithelial ICAM‐1 by Ab‐mediated crosslinking significantly impaired epithelial barrier, as determined by decreased transepithelial resistance, increased flux of FITC‐dextran (~2.5‐fold) and increased PMN TEM (~1.5‐fold). These findings were paralleled by actin rearrangement and decreased cell surface JAM‐A and E‐Cadherin. These results suggest that after TEM, PMN engagement of apically expressed epithelial ICAM‐1 results in signaling to alter barrier and subsequent recruitment of leukocytes.
The Role of Tissue Transglutaminase 2 in the Modulation of Polymorphonuclear Leukocyte Function
Authors: Lebel David Phillip , Lai Thung S. , Anderson Meghan K , Reaves Titus A.
Published: June 2021

Inflammatory Bowel Disease is mostly represented by Crohn's Disease and Ulcerative Colitis. The disease presents as a series of inflammatory disorders that involves dysregulated neutrophil (PMN) migration across the intestinal epithelium. Tissue Transglutaminase (TGM) 2 is an enzyme that catalyzes calcium‐dependent crosslinks of amino groups and has been implicated in a variety of cellular functions including cell growth and wound healing, which involves GTPase mediated activities (shown to affect PMN adhesion). TGM 2 antibodies are present in the small intestine of patients with Celiac Disease (diet induced inflammation in the small intestine) and a majority of Crohn's Disease patients also have Celiac Disease. Despite these observations, the role of TGM 2 in PMN activities has not been widely explored. We determined that TGM 2 has an extracellular and intracellular localization in PMN and appears to inhibit translocation of CD11b (activating integrin of PMN) to the surface of PMN. TGM 2 can inhibit PMN attachment to fibrinogen (a ligand for CD11b) by approximately 50% and can inhibit fMLP‐directed PMN migration by approximately 50%, which strongly suggests that TGM 2 may be connected to CD11b mediated events. The fMLP receptor is a G‐protein coupled (7TM spanning) receptor that regulates PMN response by an up‐regulation of adhesion and migration receptors. Using Flow Cytometry, we determined that TGM 2 affects PMN adhesion and migration through the PMN fMLP receptor. These data highlight TGM 2 as a potential target in the treatment of mucosal inflammation involving aberrant PMN migration.
Leukocyte CD47 plays a critical role in T‐cell recruitment in vivo through affinity regulation of LFA‐1 and VLA‐4 integrins
Authors: Azcutia Veronica , Routledge Matthew , Newton Gail , Manica Andre , Croce Kevin , Luscinskas Francis William
Published: June 2021

Host defense critically depends on effector T‐cell adhesive interaction with the vascular endothelium. CD47 is broadly expressed and participates in leukocyte recruitment and immune functions. CD47 (Integrin Associated Protein) functionally associates with multiple integrins. Here we examined the hypothesis that CD47 regulates LFA‐1 and VLA‐4 adhesive function in T‐cell recruitment. Intravital microscopy studies revealed that transferred CD47ko Th1 cells have reduced adhesion to WT TNFα‐inflamed cremaster microvessels versus WT Th1 cells. Studies in an in vitro flow model showed that CD47ko Th1 cells, as compared to WT cells, also have reduced adhesion and diapedesis of TNFα‐activated endothelium, and bind less to immobilized ICAM‐1 and VCAM‐1. Similarly, human Jurkat T‐cells lacking CD47 bind less to TNFα‐activated endothelium and to immobilized ICAM‐1 and VCAM‐1. Finally, Jurkat CD47 null cells have a reduction in Mn+2‐induced binding of mAb 24 (67%↓) and KIM127 (51%↓), which detect activated conformations of β2 integrins. However, the absence of CD47 does not affect their β2 integrin dependent “adhesion strengthening” to ICAM‐1 as determined by a cell detachment assay. Taken together, our results indicate CD47 primarily affects T‐cell VLA‐4 and LFA‐1 affinity regulation, and this function is necessary for T‐cell adhesion and diapedesis. Supported by NIH HL‐36028 and AHA fellowship 11POST7730055.
The Shortest Nascent Peptide That Can Direct Ribosome Stalling
Authors: sothiselvam shanmugapriya , Ramu Haripriya , Vazquez-Laslop Nora , Mankin Alexander
Published: June 2021

An intrinsic property of the ribosome, an RNA machine, is to sense and functionally respond to the peptide being synthesized. In extreme cases, the ribosome halts translation while polymerizing specific peptides, forming a stalled complex at a precise codon on the translated ORF. Regulation of expression of inducible antibiotic resistance genes depends on such programmed translation arrest, controlled by the nascent peptide (generally 7–10 amino acids long) and an antibiotic bound to the ribosome. Macrolide resistance gene ermD is preceded by regulatory ORF ermDL encoding the peptide ‘MTHSMRLRFPTLNQ’ where stalling occurs at the Leu‐7 codon. By systematically shortening the ermDL ORF, we found that antibiotic‐promoted stalling can still be supported by the nascent peptide as short as ‘MRL’ with the peptidyl‐tRNA positioned in the P‐site. The identities of both the second and the A‐site codons of the shortened ORF are critical for stalling. We are determining the spectrum of inducing antibiotics and identity of the ribosomal RNA sensors that operate with the mini‐stalling peptide ‘MRL’. These data provide insights into the basic mechanisms of nascent peptide recognition and programmed translation arrest. This shortest version of the stalling peptide opens new venues for structural analysis of the stalled ribosome complexes.
Candida albicans exhibits a pepstatin A‐insensitive secreted aspartic protease as a virulence factor
Authors: Aoki Wataru , Kitahara Nao , Miura Natsuko , Morisaka Hironobu , Yamamoto Yoshihiro , Kuroda Kouichi , Ueda Mitsuyoshi
Published: June 2021

Candida albicans exists in most people, but opportunistically causes severe diseases. One of the major virulence factors is secreted aspartic protease family, encoded by 10 SAP genes (1). Sap isozymes are suggested to contribute to many virulence processes. However, there is a controversy whether Sap family is really a major virulence factor or not, because use of pepstatin A showed little protective effect. In this study, we found Sap7 was a pepstatin A‐insensitive protease and tried to determine the mechanism of pepstatin A‐insensitivity. SAP7 gene was cloned from C. albicans and heterogeneously produced by Pichia pastoris. Proteolytic activity of wild type Sap7 and alanine‐substituted mutants was measured using FRETS‐25Ala library. As a result, we found that Sap7 was pepstatin A‐insensitive and that the M242A and T467A mutants had sensitivity to pepstatin A. M242 and T467 were located in the entrance to an active site, and the substitution of alanine made the entrance wider. This alteration might enable pepstatin A accessibility to the center (2). Grants; JSPS for Young Scientists and Regional Innovation Creation R&D Programs.
Ezrin functionality and ischemia‐reperfusion (I/R) injury in transplantation
Authors: Mangino Martin J. , Tian Tao , Lindell Susanne Lyra
Published: June 2021

Organ transplantation is limited by organ donation and the inevitable I/R injury associated with organ preservation. To date, little clinical advancements in I/R injury have been made because the basic molecular mechanisms remain poorly understood. The objective was to characterize the role of ezrin in preservation injury since it is hypothesized that the sub‐lamellar cytoskeletal system is an important mediator of ischemic injury. Ezrin dissociates from the cytoskeletal structures during cold ischemia in renal tubules. In LLC‐PK1 cells, preservation injury is exacerbated with ezrin knock‐down (siRNA) and attenuated with ezrin over expression. Point mutations of ezrin at T567A potentiated whereas T567D attenuated preservation injury when expressed in LLC‐PK1 cells, implicating the binding functionality of ezrin as important for protection. Specific ezrin binding sites were also identified on purified mitochondria from renal tubules and exogenous recombinant ezrin attenuated the sensitivity of the mitochondrial permeability transition pore to calcium. Finally, dissociation of the ezrin analog moesin in liver and sinusoidal endothelial cells was also observed with preservation. In conclusion, ezrin plays an important causal role in I/R injury via its linker functionality and possibly by a novel mitochondrial binding role. Supported by NIH R01 DK087737.
The structure of a ternary complex of porcine sarcosine dehydrogenase with glycine and folic acid
Authors: Kim Jung-Ja , Yong Wei , Misra Ila , Fu Zhuji
Published: June 2021

We have determined the crystal structure of porcine sarcosine dehydrogenase (SDH) complexed with glycine and folic acid. SDH is a mitochondrial bifunctional flavoenzyme that catalyzes the oxidative demethylation of sarcosine to produce glycine, and the formation of 5,10‐methylene tetrahydrofolate by transferring the oxidized methyl group to tetrahydrofolate. Electron transfer flavoprotein is the physiological electron acceptor, linking sarcosine oxidation to the mitochondrial electron tansport system. Similar to the structure of dimethylglycine oxidase from Arthrobacter globiformis, the SDH molecule is comprised of two distinct, approximately equal sized domains. The N‐terminal domain contains a covalently‐bound FAD (C8α of FAD to N3 of His108), and the C‐terminal domain binds folate. The structure also shows that a glycine molecule (product) is bound at the re‐face of the flavin ring and its nitrogen atom makes hydrogen bonds with His293 and Tyr318, suggesting that these two residues play a role in catalysis. The pterin ring of folic acid is found ~40 Å away from the glycine binding site, and the two binding sites are connected by a large irregular shaped channel. This work was supported by National Institutes of Health Grant GM29076.
Thioesterase Superfamily Member 2 (Them2) Promotes the Hepatic Oxidation of Plasma Free Fatty Acids
Authors: Kang Hye Won , Han Shuxin , Cohen David E.
Published: June 2021

Them2 is a mitochondria‐associated long chain acyl‐CoA thioesterase that hydrolyzes fatty acyl‐CoAs to free fatty acids and CoA in vitro. However, the metabolic role of Them2 in liver remains unclear. Here we utilized chow and high fat fed Them2+/+ and Them2−/− mice to explore regulation of hepatic lipid metabolism by Them2. The absence of Them2 expression reduced total and mitochondrial fatty acyl‐CoA thioesterase activities in liver. This led to a 28% increase in fatty acyl‐CoA concentrations, which was mainly attributable to increases in C16:0, C18:0 and C18:3‐fatty acyl CoA molecular species. Reduced hepatic FGF21 mRNA levels along with decreased plasma β‐hydroxybutyrate concentrations implied that fatty acid oxidation was reduced in livers of Them2−/− mice. Whereas high fat fed Them2+/+ mice developed hepatic steatosis, livers of Them2−/− mice did not accumulate excess triglycerides. In the absence of changes in lipogenic gene expression, this was attributable to increases in hepatic triglyceride secretion rates and decreases in free fatty acid uptake into the liver, as reflected by lower CD36 mRNA levels. Collectively, these findings suggest that the fatty acyl‐CoA thioesterase activity of Them2 in liver promotes oxidation of plasma free fatty acids and reduces their hepatic secretion as triglycerides. Supported by the NIH and AHA.
Curcumin analogue as a novel 11βHSD1 modulator to treat glucocorticoid excess diseases
Authors: Xiao Jian , Zhang Hongyu , Wang Zhouguang , Shi Hongxue , Wu Fenzan , Lin Beibei , Li Xiaokun
Published: June 2021

Background

Glucocorticoid (GC) excess is associated with metabolic syndrome and hypogonadism. 11β‐hydroxysteroid dehydrogenase 1 (11βHSD1) regulates intracellular GC concentration thus being a therapeutic target.

Methods

A new curcumin analogue, (1E,4E)‐1,5‐bis(2‐bromophenyl)penta‐1,4‐dien‐3‐one (GL63) was synthesized, and we compared the effect of GL63 and curcumin in 11β‐HSD1 oxidase activity and reductase activity in primary rat Leydig cells, CHOP cell lines transfected human 11βHSD1 gene in vitro, and in vivo effects in GC‐excessive model: acute stress and high fat diet (HFD) model.

Results

The IC50 of inhibiting 11βHSD1 reductase activity was 3 ± 0.2 μM and 100 nM, and the EC50 of stimulating oxidase activity was 2.04 ± 0.2 μM and 86 ± 0.2 nM by curcumin and GL63, respectively, in rat Leydig cells. Curcumin inhibited 11βHSD2 with IC50 of 13 μM for rat kidney, while GL63 did not. Oral administration of curcumin (100 to 200 mg/kg/day) or GL63 (1–4 mg/kg/day) for 3 d completely prevented the decreases of serum testosterone levels induced by restraint stress in male rats, and oral administration of curcumin (200 mg/kg/day) and GL63 (2 and 4 mg/kg/day) for 2 months improved lipid profiles induced by HFD.

Conclusion

A novel 11β‐HSD1 modulator, GL63, has been developed and effectively prevented GC excess diseases such as stress‐induced hypogonadism or HFD‐induced fatty liver.
Visualization of ferritin in Anopheles gambiae 4a3b cells
Authors: Conley Zachary R. , Geiser Dawn L. , Winzerling Joy J.
Published: June 2021

Ferritin is a 24‐subunit molecule, made up of heavy and light chain proteins, that stores and controls the release of dietary iron in mammalian, plant, and insect systems. Previous work in mosquitoes demonstrates an important function of the dietary iron consumed in a blood meal and subsequently stored in ferritin in the process of egg development. One approach to limit the spread of arthropod borne diseases, such as yellow fever, West Nile virus and malaria, is to reduce the number of potential vectors by interfering with mosquito fecundity to decrease transmission rates. The localization of ferritin subunits inside mosquito cells under various treatment conditions is important to further elucidate the regulation of iron metabolism in this disease vector and to observe the dynamics of intracellular ferritin subunits following iron administration. Deconvolution microscopy was used to capture 3D fluorescent images of mosquito cells to visualize both ferritin subunits in multiple focal planes. Fluorescent probes illuminate cell organelles (i.e. Golgi apparatus, lysosomes, endoplasmic reticulum, nuclei, etc.) while secondary probes for both ferritin subunits demonstrate abundance and co‐localization within organelles. These images will help to develop a model for the biochemical regulation of ferritin and perhaps advance novel hypotheses for the crucial role of iron in mosquito reproduction. Research support from the Agricultural Experiment Station and the College of Agriculture and Life Sciences, the Undergraduate Biology Research Program and the Honors College at the University of Arizona.
Exploring the Role of an Atypical Kinase in Ubiquinone Biosynthesis
Authors: Barber Grant Eric , Stefely Jon , Pagliarini David J.
Published: June 2021

Defects in Coenzyme Q (Q) biosynthesis cause human disease and may have a significant impact on aging. Particularly, patients with mutations in an atypical human mitochondrial kinase develop Q deficiency and progressive cerebellar ataxia. However, the mechanisms by which this kinase affects Q biosynthesis and ataxia are unknown. Identifying the endogenous substrates of this atypical kinase would represent a key advance in our understanding of its function. Toward this end, we performed multiple in vitro experiments. Radiolabeling of mouse mitochondrial lysates with 32P‐ATP and the atypical kinase revealed lipid phosphorylation. We are now testing specific lipids as substrates using a high‐throughput in vitro kinase assay (ADP‐Glo) we developed for measuring this kinase's activity. We have also created analog‐sensitive mutants of this kinase as tools to selectively manipulate its activity and are testing their activity. Our work toward discovering substrates of this atypical kinase represents important progress in characterizing its biological role in coenzyme Q biosynthesis and cellular aging.
Uncovering the biological function of UQCRB, a terpestacin‐binding mitochondrial protein, implies its pro‐angiogenic activity in vitro and in vivo
Authors: Chang Junghwa , Jung Hye Jin , Lee Sang-Kyu , Cho Seung-Woo , Kwon Ho Jeong
Published: June 2021

Recently, we identified ubiquinol‐cytochrome c reductase binding protein (UQCRB), one of subunits of mitochondrial Complex III, as a target protein of the anti‐angiogenic natural small molecule, terpestacin. The Complex III of mitochondrial respiratory chain has been considered as a key regulator in hypoxia‐induced angiogenesis through mitochondria‐derived reactive oxygen species (ROS) involved in O2 sensing. Here, cell permeable UQCRB protein is generated using protein transduction domain (PTD), a small peptide transferring its binding complex into the cell, to reveal the biological function of UQCRB. Consequently, intracellular delivery of PTD‐UQCRB fusion protein enhances generation of mitochondrial ROS and HIF‐1α stability. Also, PTD‐UQCRB transduction induces vascular endothelial growth factor (VEGF) expression and invasion of human umbilical vascular endothelial cells (HUVECs) in vitro. Furthermore, trans‐membrane delivery of PTD‐UQCRB enhances wound healing in vivo. These results provide new insights into the function of PTD‐UQCRB in angiogenesis via mitochondria mediated ROS generation and also open new basis on application of PTD‐UQCRB as a pro‐angiogenic agent via regulating mitochondrial function. This study is supported by self‐research funding.
Effects of Ionizing Radiation on Mitochondria in Human Cell Lines
Authors: Sweet Erik , Kwiatkowski David , Reynods Jacquelyn , O'Donnell Robert , Pogozelski Wendy
Published: June 2021

We have previously reported that levels of the common 4977‐bp deletion in human mitochondrial DNA increases when human cell lines are exposed to ionizing radiation. Here we expand these studies, using a variety of techniques to show the effects of ionizing radiation on mitochondrial DNA structure.
Histone H4 acetylation at K16 residue and mitochondrial activity in neuronal cells
Authors: Dmitriev Ruslan I , Zhdanov Alexander V , Papkovsky Dmitri B
Published: June 2021

The role of acetylation of histone H4 tail at residue K16 (H4K16Ac) in epigenetic regulation is widely recognized as the mark of nucleosome de‐compactization and activation of gene expression. The enzymes responsible for this modification are histone acetyl transferases (HATs) which use acetyl‐CoA (ACA) as a substrate. Recently it has been shown that mitochondria can supply ~50% of the total pool of ACA for this modification suggesting their role in the regulation of H4K16Ac. We hypothesized that this may occur under certain stress conditions such as ischemia‐reperfusion. Neuronal‐like PC12 cells were exposed to oxygen‐glucose deprivation (OGD) for different time intervals. A dramatic decrease in H4K16Ac has been found during the OGD, which could be reversed to the original levels by reperfusion. On the other hand, gene expression analysis did not show any changes in the expression of known H4K16Ac targets, namely, the UCP2, calreticulin and HIP1. The activity of HAT MYST1 (KAT8) may involve the acetylation of p53 with following activation of BAX and PUMA genes; however we failed to demonstrate any changes in their expression in samples exposed to OGD. This concludes that the decrease of H4K16Ac and possible acetylation of other proteins during ischemia‐reperfusion treatment may not related to regulation of UCP2, calreticulin and HIP2 genes and needs further investigation. Supported by SFI.