bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinically relevant issues in cardiac stem cell therapy
Authors: Wu Joseph C
Published: June 2021

Stem cells have been touted as the holy grail of medical therapy with promises to regenerate cardiac tissue, but it appears the jury is still out on this novel therapy. Recent studies have shown that that these cells do not survive nor engraft long‐term. In addition, only marginal benefit has been observed in large animal studies and human trials. Further application of advanced imaging technology will help scientists unravel the mysteries of stem cell therapy and address the clinical hurdles facing its routine implementation. In this talk, I will discuss how advanced imaging technology can help investigators better define the optimal delivery method, improve survival and engraftment, and evaluate efficacy and safety. Insights gained from this review may direct the development of future preclinical investigations and clinical trials. In addition, I will also discuss how molecular imaging has been used to address clinical hurdles of pluripotent stem cell therapy, focusing on issues such as differentiation, immunogenicity, and tumorigenicty.
In vitro models of heart disease and regeneration
Authors: Radisic Milica
Published: June 2021

The ability to generate human cardiomyocytes from either embryonic stem cells or induced pluripotent stem cells, provides an unprecedented opportunity to establish human in vitro models of cardiovascular disease as well as to develop replacement cardiac tissue for therapeutic applications. Here, we will present our recent efforts at generating a microarray of human cardiac microwires (CMW) using hESC derived cardiomyocytes (Hes2) and collagen gel compaction. Within this system, we recapitulated the basic microenvironment found in the heart, one which integrates cardiomyocytes, non‐myocytes, the extracellular matrix, and dynamic electromechanical forces. CMW are morphologically reproducible, maintain high cardiac protein expression in a highly dense, aligned, and suspended 3‐D extracellular matrix and can be cultured for at up to 30 days. A rapid, 7‐day maturation regimen that included electrical field stimulation yielded CMWs with a conduction velocity of 47.4 ± 12.4 cm/s, on par with healthy human heart tissue. The spontaneous beating rate in CMWs was amenable to pharmacological manipulation using lidocaine and norepinephrine while isoproterenol treatment caused hypertrophic response.
Post‐Termination Events in Eukaryotic Translation
Authors: Pestova Tatyana , Skabkin Maxim , Skabkina Olga , Hellen Christopher
Published: June 2021

Termination occurs when a stop codon enters the ribosomal A‐site. It consists of stop codon recognition followed by hydrolysis of the P‐site peptidyl‐tRNA. The hydrolysis step involves nucleophilic attack of water on peptidyl‐tRNA in the peptidyl transferase center of the large ribosomal subunit. In eukaryotes, termination results from the complex functional interplay between release factors eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples stop codon recognition and peptidyl‐tRNA hydrolysis by eRF1. Recycling of post‐termination complexes (post‐TCs) is promoted by the concerted action of ABCE1, an essential member of ATP‐binding cassette family of proteins, and eRF1, which remains associated with post‐TCs after peptide release. ABCE1 and eRF1 dissociate post‐TCs into free 60S and tRNA/mRNA‐bound 40S subunits. Subsequent tRNA/mRNA release from 40S subunits can be promoted either by initiation factors 1/1A/3, or by Ligatin. In some cases post‐TCs do not undergo complete recycling, and termination is instead followed by reinitiation on the same mRNA, usually downstream of the stop codon. This mostly occurs after translation of short open reading frames. To investigate the molecular mechanisms underlying reinitiation, we investigated different fates of ribosomal post‐termination complexes depending on the presence of initiation, as well as elongation, factors, and initiator or elongator tRNAs.
Structural Organization of HNF4alpha: A Master Transcription Factor of Hepatic and Pancreatic Genes
Authors: Rastinejad Fraydoon , Chandra Vikas , Huang Pengxiang , Potluri Nalini , Kim Youngchang
Published: June 2021

We have carried out a detailed crystallographic study on the intact Hepatocyte Nuclear Factor 4 alpha (HNF4alpha) receptor, a phospholipid associated transcriptional regulator of metabolic genes in liver and pancreatic cells. HNF4alpha has a phospholipid in its ligand‐bindng domain which does not regulate receptor activity. Instead, HNF4alpha responds to methylation and phosphorylation and possibly induced coactivators, to switch its transcriptional activity. Our structural characterization reveals a “nerve center” consisting of a tightly inter‐linked domain‐domain junction. This arrangement integrates the receptorˈs DNA‐binding and ligand‐binding domains. The methylation and phosphorylation sites map to the nerve center, allowing control the receptor's DNA binding ability from a distance –i.e. allosterically. We also found that several human MODY1 mutations similarly map to the domain‐domain junctions of the assembled receptor. The HNF4alpha‐DNA complex has a distinct quaternary organization when compared to our previously reported full‐length PPAR‐RXR crystal structure, despite the common DR+1 response element used by both receptor complexes. We identify new pockets and cavities, beyond the phospholipid binding site, in our HNF4alpha structure. These sites, in theory, could provide new opportunities for therapeutic targeting with allosteric modulators.
Protein export via the accessory SecA2 system of Mycobacterium tuberculosis
Authors: Braunstein Miriam , Sullivan Jonathan Tabb , Feltcher Meghan , Ligon Lauren
Published: June 2021

All bacteria have a Sec pathway that exports proteins from the bacterial cytoplasm to the cell envelope or the environment. SecA is an ATPase with a central role in the Sec pathway. Mycobacteria, including the pathogen Mycobacterium tuberculosis, possess two SecAs (SecA1 and SecA2). SecA1 is essential and the canonical SecA while SecA2 is a non‐essential accessory SecA that exports a limited set of proteins. The objective of our study is to understand the role of the SecA2 system in M. tuberculosis virulence. Using a M. tuberculosis secA2 mutant, we previously showed the SecA2 system to be required for intracellular growth of M. tuberculosis in macrophages. Here, we report our finding that the SecA2 system is required for M. tuberculosis to arrest phagosome maturation during macrophage infection. Phagosomes containing the secA2 mutant of M. tuberculosis are more acidified than phagosomes containing wild‐type M. tuberculosis. We further show that inhibitors of phagosome acidification rescue the attenuated phenotype of the secA2 mutant in macrophages, which demonstrates that the phagosome maturation defect of the secA2 mutant is responsible for the intracellular growth defect. These data suggest there are effectors of phagosome maturation that are exported via the accessory SecA2 system. Efforts are underway to identify these SecA2‐exported proteins. In parallel studies, we are using the exported proteins Ms1704 and Ms1712 of Mycobacterium smegmatis as model substrates of the SecA2 system for the purpose of identifying the features that define SecA2‐exported proteins. Supported by NIAID AI54540.
Lysine Methylation and Regulation of Gene Expression Programs
Authors: Gozani Or
Published: June 2021

My laboratory is interested in understanding the molecular mechanisms by which chromatin signaling networks effect nuclear and epigenetic programs, and how disruption in these mechanisms contribute to cancer and other pathologic states. Our work centers on the biology of protein lysine methylation, a principal chromatin‐regulatory mechanism thought to be involved in directing epigenetic processes. We study how lysine methylation events on histone and non‐histone nuclear factors are generated, sensed, and transduced, and how these chemical marks integrate with other modification and nuclear signaling systems to govern diverse functions. We previously demonstrated that the PHD finger (Plant Homeodomain), a signature chromatin‐associated domain, functions as a novel recognition module for histone H3 trimethylated at lysine 4. We linked this single histone species to multiple different functions via its recognition by discrete PHD finger nuclear proteins, including providing evidence that disrupting the read‐out of a histone modification can cause an inherited human disease. I will discuss new work focusing on an expression library containing the majority of predicted protein lysine methyltransferase (PKMT) enzymes present in the human genome, and studies aimed at elucidating the role of PKMTs in oncogenic programming. I will also present recent work employing protein microarray technologies for discovery and biological characterization of new methyllysine‐binding domains and methylation events.
Probing the dynamics of promoter proximally paused Pol II
Authors: Adelman Karen L
Published: June 2021

RNA polymerase II (Pol II) transcription in metazoans is punctuated by promoter‐proximal pausing, during which the enzyme begins elongation but pauses during synthesis of the first 100 nucleotides of RNA. This pausing of polymerase is increasingly recognized as an important checkpoint in transcriptional regulation of processes that include response to stimuli, development, and cell differentiation. However, many questions remain concerning the mechanisms underlying establishment vs. release of paused Pol II, and the functional consequences of this mode of regulation. I will discuss our progress towards addressing these questions, with a focus on our recent work investigating the dynamics and stability of paused Pol II.
The unusual enzyme chemistry in diphthamide biosynthesis
Authors: Lin Hening
Published: June 2021

Archaeal and eukaryotic translation elongation factor 2 contain a unique posttranslationally modified histidine residue called “diphthamide”, the target of diphtheria toxin. The biosynthesis of diphthamide were proposed to involve three steps, with the first step being the formation of a C‐C bond between the histidine residue and the 3‐amino‐3‐carboxypropyl group of S‐adenosylmethionine (SAM). However, details of the biosynthesis have remained unknown. Here we present structural and biochemical evidence showing that the first step of diphthamide biosynthesis in the archaeon Pyrococcus horikoshii uses a novel iron‐sulfur cluster enzyme, Dph2. Dph2 is a homodimer and each monomer contains a [4Fe‐4S] cluster. Biochemical data suggest that unlike the enzymes in the radical SAM superfamily, Dph2 does not form the canonical 5′‐deoxyadenosyl radical. Instead, it breaks the Cγ, Met‐S bond of SAM and generates a 3‐amino‐3‐carboxylpropyl radical. This work suggests that Pyrococcus horikoshii Dph2 represents a novel SAM‐dependent [4Fe‐4S]‐containing enzyme that catalyzes unprecedented chemistry.
Cellular Trafficking of Helical Rosette Nanotubes in Dendritic Cells
Authors: House Nicole , Fenniri Hicham , Singh Baljit
Published: June 2021

Self‐assembling helical rosette nanotubes (HRNs), composed of cytosine and guanine, have multiple applications. HRN interactions with dendritic cells (DCs), sentinels of the immune system, are key in determining HRN safety and viability. Splenic classical and plasmacytoid DCs were isolated from female C57/BL6 mice using magnetic cell sorting. HRN were conjugated with the RGD (arginine, glycine, aspartic acid) peptide and the fluorophore FITC in a ratio of 1:10μM respectively. Confocal microscopy was used to visualize the endocytic pathways used, HRN intracellular localization and the real‐time uptake process. HRNs used multiple pathways to take residence within clathrin and caveolin‐coated vesicles, early endosomes, and lysosomes. Able to recognize RGD, the integrin αVβ3 is a potential receptor for receptor‐mediated endocytosis. HRNs co‐localized with major histocompatibility complexes indicating possible direct interaction leading to antigen presentation. HRN exposure stimulated DC maturation as shown by dendrite formation and maturation marker (CD40, CD83) expression. Cell viability was confirmed through microscopy and caspase 3 and 9 expression. These data demonstrate that HRN engage αVβ3 to enter DCs through multiple, presumably redundant, pathways, and interact with antigen‐presenting molecules.

Grant Funding Source : National Sciences and Engineering Research Council of Canada
Inducible reexpression of HEXIM1 activates physiological rather than pathological responses in the adult heart
Authors: Watanabe Michiko , Doughman Yong Qui , Desjardins Candida , Hu Yanduan , Wang Connie , Hoit Brian , Chandler Margaret , Yu Xin , Montano Monica
Published: June 2021

The transcription factor Hexamethylene‐bis‐acetamide‐inducible protein 1 (HEXIM1) is a tumor suppressor and cyclin‐dependent kinase inhibitor that we and others have shown to be critical for cardiovascular development. To examine its function in the adult heart, we created and investigated a mouse line wherein HEXIM1 reexpression was induced in adult cardiomyocytes. These mouse hearts exhibited morphology, physiology, and gene expression resembling hearts that have undergone a physiological rather than a pathological response. We also discovered mechanistic aspects of HEXIM1 that may explain effects of HEXIM1 induction in adult hearts. HEXIM1 binds to and stabilizes Hypoxia Inducible Factor‐1 [alpha] that controls many genes involved in vasculogenesis and metabolism. This role of HEXIM1 may result in induction of angiogenesis essential for physiological hypertrophy and maintenance of cardiac function under stress. Thus, HEXIM1 plays a critical regulatory role in coordinating responses of the adult heart to stress and its reexpression in the adult may be useful in cardioprotection therapy.

This project was supported by NIH R01 CA92440 & AHA 0855543D (MMM), ARRA NIH R01 HL091171 (MW), NIH R01s HL73315 & HL86935 (X.Y.)
Do Lectures Matter? Lecture Attendance in Online and Face to Face Histology Courses
Authors: Barbeau Michele L , Rogers Kem A
Published: June 2021

We have developed an online histology course covering the same material as a Face to Face (F2F) course. Previously, we reported no significant differences among outcomes between the formats. Here we investigate differences in student attendance. The online course uses Wimba Classroom to facilitate lectures. Students can attend the lectures live, or view archived versions later and also access archives for additional review. Wimba classroom records each time a student enters the virtual classroom. F2F attendance was measured by passing attendance sheets though the class for each lecture. Data indicates that F2F attendance drops mid‐term and then recovers prior to testing. Similarly, online attendance also drops mid‐term; however, these students can view the archived lecture at a later time. Written exams for this course are multiple choice questions each based on the material covered in a specific lecture. There is a correlation (r2 = 0.211, p< 0.05) between attendance and exam grades for the F2F students. For the online students who can view the archives many times, there is also significant positive correlation between attendance and exam outcomes; however, this correlation becomes negative after two viewings per lecture. These results suggest that lecture attendance improves outcomes and that while repeated viewings of the lecture are beneficial, this benefit declines with more than two viewings.

Grant Funding Source : SSHRC
Enhancing science literacy in tertiary education: a real‐world exercise in literature research, scientific writing, and self and peer assessment
Authors: Filgueira Luis , Pauk Barbara , Meyer Jan
Published: June 2021

Science literacy, the ability to critically evaluate scientific outcomes and their presentation through self and peer assessment, is expected from scientists despite the fact that it is generally not taught. This project aimed to offer 75 undergraduate science students the opportunity to acquire and improve their skills in literature research, reading and writing of scientific papers, as well as self and peer review in simulations of real‐world processes of scientific publishing. This paper discusses the various assessment results, which were analyzed statistically, as well as students’ evaluation of their learning experience.

Students had to write a literature review on a topic related to the course content including at least 5 original journal articles published over the previous 10 years in leading international journals. Students were asked to review the manuscripts of five peers and then give an assessment of their own paper. They could use the online program “Turnitin.com” to check their manuscript for plagiarism. This program was also used for peer assessment. The students received feedback on their paper from 5 peers as well as from the academic teacher.

In summary, peer assessment was more severe than the teacher's or self assessment, but within a reasonable range. The students perceived the exercise as demanding, but also rewarding and as being a great learning experience.
The Downstate Video Atlas of Anatomy, a Project Powered by Dyad Pedagogy
Authors: Blumenberg Adam , Marquez Samuel
Published: June 2021

The medical school curriculum reform movement requires an effective method of eliciting leadership, independent research and productivity from each student. Dyad pedagogy has been demonstrated to answer this call. Working only in groups of two, a student worked with SUNY Downstate faculty members to create an expansive video atlas of anatomy. Production was labor intensive, ultimately generating 37 videos, each profiling an anatomical region. The application of dyad pedagogy allowed for constant feedback and refinement of the videos, optimizing them as a learning resource for the student body. Activity logs indicated that 38.6% of physician assistant and 25.5% of medical students (n=158 and 102 respectively) watched the videos 5 or more times. Usage increased up to 657% of average in the days before an exam. The significant percentage of students who repeatedly returned to the resource, as well as the usage peaks before a test day impy active utilization of the videos as a study tool. Informal surveys as well as unsolicited reviews from the class indicated enthusiasm and appreciation for the videos, confirming the success of the project and of dyad pedagogy in fueling the endeavor.

Grant Funding Source : SUNY Downstate College of Medicine
Faculty and Student perceptions of tablet‐based content in gross anatomy laboratory teaching
Authors: Smoot Betty J , Andler Carly , Clay Jensara , Mayfield Chandler , Ohara Peter , Topp Kimberly S
Published: June 2021

We describe an initial trial using a tablet‐based dissection manual (iPad) in teaching gross anatomy in a first year medical education program. One hundred and fifty students dissected in groups of 4 to a cadaver. Each group of 4 shared one tablet computer. The tablet dissection manual was generated by Faculty and 2nd year medical students based on the existing written lab guide, lecture content, syllabus content, cadaveric images and short videos of dissections. The content was designed to guide the students through the dissection and highlight key learning points. Students were surveyed after the first laboratory and after the seventh and final laboratory. Faculty (n=9) and Teaching assistants (n=12) were surveyed after the completion of the course. The surveys included questions comparing the value of the tablet content versus traditional assets (e.g. Netter's and Grant's Atlases), time spent using tablet versus traditional assets, changes in faculty teaching style, and faculty perception of the utility of the tablets. Student and faculty perceptions on the implementation of the tablet will be compared. Our findings will help inform the community of the benefits and limitations of tablet‐based instruction in early medical education.
A practical way to understand anatomical planes and directions in animals using vegetables
Authors: Tamayo-Arango Lynda Jhailu
Published: June 2021

In this work we use vegetables in representation of the animal body for teaching anatomical planes and directions in veterinary medicine. Banana represents the head and trunk, and runner bean represents the extremities, due to the difference in the anatomical planes and directions in these regions in animals. Students first determined in the banana the location of head and tail, and then they delimited with a marker pen the three main planes of the body (mean longitudinal, sagital, transversal and horizontal) and cut with a knife. After that, they marked the anatomical directions resulting of each plane, i.e. tranversal plane: cranial and caudal portions, horizontal plane: dorsal and ventral portions, and mean longitudinal plane: medial and lateral. They did the same with the runner bean, determining first the location of the extremity related to the trunk. In veterinary anatomy is also very much used the terms axial and abaxial in reference to the distal part of the extremities in animals with several digits (i.e. bovine, swine and carnivores). In horse are used for the proximal sesamoid bones. For the students to understand these concepts, they put together two runner beans and marked the corresponding directions. Finally, they had to draw everything they did. This method is a three‐dimensional reinforcement of concepts that most of the time are difficult to understand in a two‐dimensional way.

Grant Funding Source: n/a
Online versus on‐ground learning for anatomy undergraduates
Authors: McClure Jeff , Cook Amy
Published: June 2021

In this study students’ exam scores are compared between a traditional “on‐ground” anatomy course and an online anatomy course with the same learning objectives. Mean scores for lecture and lab exams were calculated for each cohort population. Exam scores of online students were significantly higher than exam scores of on‐ground students. The raw data of this study suggests that online learning is more effective than learning in the on‐ground traditional class room. Why is this the case? Current development of a hybrid anatomy course, marrying online learning with on‐ground learning, will help answer this question Further investigation of both population's demographics and previous education will also be necessary to eliminate any bias.
Use of video lab guides to supplement an undergraduate cadaver lab
Authors: Billings Heather J.
Published: June 2021

In our anatomy laboratory course for nursing majors, students use cadavers previously dissected in other courses. Faculty are the primary resource to locate and identify structures in the laboratory. To facilitate locating structures more independently, video lab guides were produced and tested for two exercises. Some structures were omitted from the videos and formally presented by faculty in the traditional manner. To assess efficacy of the videos, correct response rates to quiz questions were compared for two structures on the video, and two structures only presented by faculty. Similar percentages of students (n=136) correctly answered questions based on the videos as those presented by course faculty (62.1% vs. 64.3%; p=0.87). Student feedback about the videos was favorable, including requests they be made available on the course website for studying outside of the laboratory. Based on this preliminary analysis, video lab guides were at least as effective as faculty presentation of material for an undergraduate cadaver lab. As videos were no less effective that in‐person delivery of content, more videos are planned with refinements to further improve their efficacy.
Passing the Torch: Medical Student Near‐Peer Teachers Enhance Learning in Histology
Authors: Ko Huaising Cindy , Bassan Eric , Shaw Phyllis A. , Mak Ki M.
Published: June 2021

Near‐peer teaching is an integral part of the Histology course at Mount Sinai School of Medicine. To that end, 12 Histology teaching assistants (TAs) were selected from the second‐year students, based on their performance in the course and their interaction with fellow students and faculty. Among these, two were appointed as head TAs whose roles were to organize teaching assignments and serve as liaison between faculty and students. In total, TAs held eight 90‐minute reviews covering 2 to 3 topics that were taught that week, with emphasis on laboratory material. The sessions were conducted using PowerPoint images in a question‐and‐answer format to engage student participation. Images were then posted on the course website for students to review afterward. Prior to each of the 3 exams, TAs led mock practical sessions in which 28 images were shown and students were asked to identify specific structures and state their functions. At completion, answers were given with relevant discussion. An online survey revealed that 75% of students agreed or strongly agreed that the TAs were engaging teachers. Also, 70% of students stated that the reviews helped them prepare for the exam, while 80% said that the mock practical sessions enhanced their learning of the material.

Conclusion

Alongside the faculty, near‐peer teaching by medical student TAs contributes positively to student satisfaction and enhances learning in Histology.

Grant Funding Source: AAA
Three opportunities in gross anatomy for meeting the diverse needs of fourth year medical students based on their chosen specialty areas
Authors: Snow Mikel H , Winfield Michelle A.
Published: June 2021

Recent trends in medical education have led to reduced hours in first‐year gross anatomy courses as well as reduced dissection experiences for students. To address this issue, the KSOM offers two advanced electives and one teaching experience in gross anatomy as options for fourth year medical students who wish to enhance the anatomy most relevant to their residency training. The Advanced Anatomy & Surgical Skills Elective is a three‐week experience for 8–12 students per year who are going into general or orthopedic surgery. This elective integrates student dissection with clinical correlations by surgery faculty, hospital experiences, and clinical procedures/techniques carried out in the Surgical Skills Lab. The Independent Study in Anatomy Elective is a flexible experience tailored to the student's post‐graduate needs in anatomy by combining cadaveric dissection, radiology, cross‐sectional anatomy, and embryology with advanced readings. This elective, which can be from two to six weeks in length, is offered throughout the year and has accommodated 70 to 100 students per year for the past five years. An additional 12 to 18 fourth year medical students are selected each year to serve as lab instructors in the first year anatomy course. Based on their chosen specialty field, selected fourth year students are matched with one of five body regions (thorax, abdomen, pelvis, head & neck, limbs) for teaching in the first year course. Over the past five years, these three opportunities have provided advanced experiences in human anatomy for over 50% of the graduating classes.
How do block scheduling and integrated testing affect medical student performance?
Authors: Thompson Andrew R. , Braun Mark W. , OˈLoughlin Valerie Dean
Published: June 2021

The selection process of medical school often favors students who can recall a large amount of information for a brief period of time. This trait may persist in the early years of medical education as most courses are taught and tested independently. However, the ability to synthesize material from different courses both promotes a deeper level of learning and better prepares students to practice medicine.

This study examines how 2nd year medical students handled a transition to block course scheduling and integrated exams (pathology, pharmacology, and medicine at Indiana University School of Medicine). Specifically, comparison of similar pathology questions between the different exam formats was used to assess changes in student performance between the traditional and revised curricular years. Additionally, each pathology question was evaluated using Bloom's taxonomy to a) compare overall exam makeup, and b) determine if student performance differed based on taxonomic assignment.

Pathology scores on the first block exam were slightly lower than the previous year's single subject exam. However, on subsequent exams block scheduling students performed better than their previous year counterparts, despite integrated exams being longer and arguably more difficult. We suggest that an integrated testing format discourages “cramming” and promotes a deeper level of learning.
Characterization of the delta12‐Prostaglandin J2‐Coenzyme A interaction and its role in epigenetic modulation of gene expression
Authors: Farwell Sara Lynn Nicole , Narayan Vivek , Kodihalli Ravindra , Prabhu K. Sandeep
Published: June 2021

The histone acetyltransferase (HAT) p300 functions to coordinate transcription activation or repression and chromatin remodeling, which plays a pivotal role in embryonic development, tumorgenesis, asthma, chronic obstructive pulmonary disease, viral infections, and learning and memory disorders. Until now, inhibitors of p300 have been investigated with limited success. We have recently described the role of cyclopentenone prostaglandins metabolites as inhibitors of p300 HAT. In the present study, we demonstrate the ability of a stable cellular metabolite of PGJ2, Δ12‐ PGJ2, to form a covalend adduct with Coenzyme A using LC‐MS/MS. Here we describe the implications of such an adduct on p300 HAT activity and its likely role in cellular gene expression. NIH DK 077152
Downregulation of BRCA1‐Mediated Transcription by HP1γ via Modulation of Promoter Occupancy
Authors: Choi Jae Duk , Lee Jong-Soo
Published: June 2021

HP1γ is a chromatin protein involved in gene silencing. Here, we show that HP1γ regulates BRCA1‐mediated transcription via modulation of promoter occupancy and histone modification. BRCA1 interacts with both wild‐type HP1γ (HP1γ) and a mutant deficient in methylated‐histone (H3K9me) binding (HP1γV22A). HP1γ and HP1γV22A suppress BRCA1‐mediated transcription by causing BRCA1 disassembly from the BRCA1 target promoter and augmenting H3K9me. In contrast, another HP1γ mutant deficient in PXVXVL/I motif binding (HP1γW164A) fails to interact with BRCA1 and repress transcription. Consistently, HP1γ and HP1γV22A associate with the promoter in SUV39H1‐knockdown cells, suggesting that HP1γ binds to the BRCA1 target promoter and represses its transcription in a BRCA1‐HP1γ interaction‐dependent manner followed by BRCA1 disassembly. Time‐lapse studies on promoter association and histone methylation following DNA damage revealed that BRCA1 is initially assembled at the promoter, then HP1γ is recruited, and finally SUV39H1 is targeted. HP1γ/SUV39H1 assembly at the promoter results in the disassembly of BRCA1 and histone‐methylation leading to repression.
Multiple forms of the E. coli SOS response protein UmuD
Authors: Ollivierre Jaylene N. , Huang Qiuying , Beuning Penny J.
Published: June 2021

Escherichia coli SOS mutagenesis involves the induction of at least 57 genes in response to DNA damage, including the umuD gene products. UmuD2 is a homodimer of 139‐amino acid subunits that interacts with RecA:ssDNA resulting in cleavage of its N‐terminal 24‐amino acids, yielding UmuD’2. The goal of our research is to determine the conformation and dynamics of the UmuD proteins in order to understand its regulatory role in response to DNA damage. We have discovered an N‐terminally truncated version of the UmuD protein that is similar to full‐length UmuD2 in cleavage activity, but differs in its conformation. In vivo characterization reveals that this newly identified form is active in facilitating UV mutagenesis and displays a cold sensitive phenotype. This suggests that the length of the arms of the UmuD proteins is critical in regulating function. Indeed, we have shown by EPR that UmuD2 and UmuD’2 arm dynamics are different. Additionally, full‐length UmuD2 and cleaved UmuD’2 interact differentially with partner proteins resulting in dramatically different cellular outcomes. Our preliminary work points to yet another level of regulation in which the umuD gene products coordinate the DNA damage response. Research supported by NSF Career MCB‐0845033.
The role of atg18 in signal transduction pathways during Drosophila development
Authors: Boetefuer Erica Lee , Raden David L , Selva Erica M
Published: June 2021

8J16 and 9E6 are allelic mutations that disrupt Wnt/Wingless (Wg) or Hedgehog (Hh) signaling based upon their embryonic ‘lawn of denticles’ phenotype. Goals of this project were to identify the disrupted gene and determine its role in Drosophila developmental signaling. Complementation analysis revealed 8J16/9E6 are alleles of autophagy‐specific gene 18, atg18, which plays a role in autophagosome‐lysosome fusion. In yeast, atg18 negatively regulates endosome‐lysosome targeting. In Drosophila, endocytic machinery mutations that block lysosome targeting cause continuous signaling. Thus, atg18 mutants should show a buildup of lysosomes at the expense of endosomes and decrease signaling. To the contrary, Distalless, a long‐range Wg target, was enhanced in 8J16/9E6 mutant tissue in Drosophila wing discs. Analysis of 8J16/9E6 germline clone (glc) mutant embryos confirmed atg188J16, atg189E6, and atg18KG03090 are allelic. Immunofluorescent staining of glc embryos showed the segmentation defect is due to loss of Wg expression at embryonic stage 9. 8J16/9E6 glc embryos also display severe CNS defects not strictly the result of Wg loss and thus, are pleiotropic effects of these mutations. 8J16/9E6 are intronic point mutations not predicted to disrupt mRNA splicing and are temperature sensitive, yielding lethality at 25ºC, but are viable at 29ºC. Molecular explanations for this phenotype are explored. HHMI.
Soluble expression of chicken follistatin proteins in Escherichia coli
Authors: Choi Rocky , Kim Yong Soo
Published: June 2021

Follistatin (Fst), an autocrine glycoprotein, suppresses the activity of myostatin, a potent negative regulator of skeletal muscle growth. Fst treatment has shown to enhance muscle growth as well as ameliorate symptoms of muscular dystrophy in animal models, illustrating the potential of Fst as a therapeutic agent to enhance muscle growth in animal agriculture or to treat muscle wasting conditions or disease in humans. This study was designed to produce biologically active recombinant chicken Fst315 (chFST315) and chFST315 fused to Fc domain of chicken IgY (chFST315‐Fc) in soluble forms in E. coli. Fst contains multiple disulfide bonds, making it a challenge to express soluble Fst proteins in E. coli. Therefore, we employed either a periplasmic expression strategy or a genetically modified E. coli system (SHuffle®), which allows disulfide bond formation in the cytoplasm. In periplasmic expression of chFST315 and chFST315‐Fc using the pMAL‐p5x expression vector system, these recombinant proteins were expressed in insoluble forms in NEB Express E. coli. In contrast, cytoplasmic expression of chFST315 and chFST315‐Fc using the pMAL‐c5x expression vector system resulted in soluble expression of these proteins in SHuffle® E. coli. The recombinant Fst proteins produced in this study will be useful in investigating the potential of Fst to improve skeletal muscle growth of chicken.
A Newly Identified Protein Regulates Translation in Mammalian Mitochondria
Authors: CIMEN HUSEYIN , Koc Hasan , Koc Emine C.
Published: June 2021

Mitochondria are responsible for providing 90% of the energy in eukaryotic organisms by oxidative phosphorylation (OXPHOS). Mitochondrially encoded components of OXPHOS complexes are all essential and synthesized by a specialized translation system containing 55S ribosomes in mitochondria. In the reevaluation of mitochondrial ribosomal proteins (MRPs) and ribosome‐interacting proteins, we identified Gadd45GIP1 as a bona fide component in purified 55S ribosomes. Here, we report Gadd45GIP1 association with the large subunit of mammalian mitochondrial ribosomes and its role in the regulation of mitochondrial protein synthesis. In siRNA knock‐down studies, reduced expression of Gadd45GIP1 decreased de novo synthesis of the 13 mitochondrially encoded proteins of OXPHOS complexes in vivo. This indicates that the Gadd45GIP1 is an essential member of the mitochondrial ribosome, and its specific role in protein synthesis is currently under investigation.

Supported by NIH grant R01GM071034 (E.C.K.)
Metabolic changes in sialic acid synthesis pathway in GNE‐myopathy model mice with long‐term sialic acid treatment
Authors: Noguchi Satoru , Malicdan May Christine , Funato Fumiko , Nishino Ichizo
Published: June 2021

GNE‐myopathy is an autosomal recessive disorder characterized by muscle atrophy, distal muscle weakness, and accumulation of proteins and rimmed vacuoles in myofibers and is due to mutations in the GNE gene that encodes an essential enzyme in sialic acid (SA) biosynthesis. We recently showed that muscle atrophy and weakness were completely prevented in the GNE‐myopathy model mouse with SA (NeuAc and sialyllactose) and ManNAc. In this study, we aimed to clarify if long‐term SA treatment can influence SA pathway in the GNE‐myopathy model. Gene expression analysis of SA pathway enzymes/transporter in various organs of non‐treated wild mouse showed high expression of synthesis‐related genes in the liver and degradation‐related genes in the kidney, suggesting that liver is an anabolic organ and kidney is a catabolic organ for SA synthesis. Expression analysis of genes in tissues of GNE‐myopathy model mouse after long‐term treatment for more than 300 days showed further upregulation of genes for catabolic enzymes in kidney, and downregulation of genes for anabolic enzymes in skeletal muscle. The change in gene expression was, however, smaller in disease model compared to control. These results suggest that long‐term SA administration may somehow diminish the effects of treatment, and that the effect on the expression of SA‐metabolic genes are variable with compounds administered.
Development of photosensitizers for photodynamic therapy : R&D trend and patent analysis research
Authors: Sohn Eun-Soo , Sohn Bum-Soo , Kim Bok-Hwa , Sohn Eun-Hwa
Published: June 2021

Technology development associated with photodynamic therapy (PDT) comprised of photosensitizer, light source, and light delivery system becomes active recently by the development of photosensitizer and related technologies. Clinical applications of PDT are expanded gradually to the therapy for psoriasis, age‐related macular degeneration, diabetes, infectious disease and cancers occurring in various organs etc. Specifically advances in technology development of drug delivery system using nanotechnology have given photosensitizer with a high selectivity and therapeutic efficacy for target cell or tissue. This review is focused on studies investigating recent R&D trend and clinical applications of photosensitizer for photodynamic therapy and the understanding technology trend and position of major nations through paper reviews and patent analysis research.
Occupation of the electrophilic substrate‐binding site of glutathione S‐transferase M1‐1 enhances its function as an activator of 1‐cysteine peroxiredoxin
Authors: Ralat-Touset Luis Abel , Feinstein Sheldon I. , Fisher Aron B.
Published: June 2021

Glutathione S‐transferase mu (GST‐mu) can form an in vitro heterodimer with 1‐cysteine peroxiredoxin, peroxiredoxin 6 (Prdx6), in the presence of glutathione (GSH), but the peroxidase specific activity of this complex is only 1.0 μmol/min/mg Prdx6, which is ~20% of that of the complex between the pi isozyme of GST and Prdx6 under the same conditions [L.A. Ralat, Y. Manevich, A.B. Fisher, R.F. Colman, Biochemistry 45 (2006) 360–372]. These results suggested that the interactions are different between Prdx6 and GST isozymes, which share an identity of less than 30%. Here, we show that Prdx6 can reduce hydrogen peroxide at 4.7 ± 0.3 μmol/min/mg Prdx6, as assayed using the GSH reductase/GSH/NADPH‐coupled GSH‐peroxidase method, after a 1‐hour incubation with a 1:1 molar ratio of GST‐mu at pH 8, containing saturating amounts of GSH and an electrophilic GST substrate analog, 2,4‐dinitrophenol. Interestingly, after this same incubation, GST‐mu lost all of its activity with respect to monobromobimane, a substrate that occupies a distinguishable site from 2,4‐dinitrophenol. These results suggest that occupation of the electrophilic site of GST‐mu may render a conformational change which optimizes its interaction with Prdx6, allowing the transfer of GSH to the inactive Cys‐47−sulfenic acid form of Prdx6, regenerating an active Prdx6, while inhibiting GST‐mu activity. Supported by NIH/NHLBI R01554495.
N‐formyl peptide receptor‐1 is important for homeostasis of intestinal epithelial cells
Authors: Alam Ashfaqul , Leoni Giovanna , Wentworth Christy , Nusrat Asma , Neish Andrew
Published: June 2021

N‐formyl peptide receptors (FPRs), which in humans include FPR1, FPR2 and FPR3, are an important family of pattern recognition receptors that bind an array of bacterial as well as host derived peptide and nonpeptide ligands. FPRs are G protein coupled surface receptors, which are well described in mediating phagocyte functions. We have demonstrated FPRs on the apical surface of intestinal epithelial cells (IEC) and have shown that in IECs FPR induces ROS generation in response to bacterial fMLF (a formylated tripeptide), viable Lactobacillus rhamnosus GG, and endogenous annexin A1 mimetic (Ac 2‐26). Furthermore, we showed that FPR‐elicited ROS activates the noninflammatory ERK MAPK pathway and enhances migration of IECs. We hypothesize that FPR1, the high‐affinity receptor, is important for motility of intestinal epithelia. Mice deficient in FPR1 demonstrates normal intestinal tissue architecture; however, intestinal crypts of the FPR1 null mouse contain more proliferating cells, and show slower migration along the crypt‐villus axis. These data suggest that FPR1 is important in maintaining homeostasis of the intestinal epithelia.
A Giardia Cathepsin‐B‐like Protease Cleaves Interleukin‐8 From Intestinal Epithelial Cells
Authors: Cotton James , Bhargava Amol , Ferraz Jose , Hollenberg Morley , Beck Paul , Buret Andre
Published: June 2021

Interleukin‐8 (CXCL8) secretion from intestinal epithelial cells is an early event in acute intestinal inflammation. Infection with the non‐invasive, small intestinal protozoan parasite Giardia duodenalis rarely results in overt intestinal inflammation.

Objective

To determine if Giardia trophozoites modulate CXCL8 secretion from ex vivo human small intestinal biopsies or in vitro Caco‐2 monolayers administered interleukin (IL)‐1β or Salmonella typhimurium, and identify mechanisms involved.

Results

Co‐incubation of ex vivo human small intestinal biopsies or in vitro Caco‐2 monolayers with Giardia trophozoites significantly reduced supernatant levels of CXCL8 following stimulation with IL‐1β or S. typhimurium and involved parasite‐mediated cleavage of CXCL8. Giardia trophozoites secreted three cysteine proteases, one sensitive to the cathepsin‐B inhibitor Ca‐074Me. Inhibition of Giardia‐secreted cysteine proteases with a cysteine protease inhibitor (E‐64d) or Ca‐074Me prevented cleavage of CXCL8.

Conclusion

Giardia trophozoites secrete a cathepsin‐B‐like protease that cleaves CXCL8. Funding provided by NSERC, AIHS, and CCFC.
Targeted protein glycosylation (O‐GlcNAc) of mitochondrial proteins in rats selected for low running capacity
Authors: Johnsen Virginia Linnea , McConkey Marina , Hepple Russell T , Belke Darrell D , Koch Lauren G , Britton Steve L , Hittel Dustin S , Shearer Jane
Published: June 2021

Excess flux through the hexosamine biosynthesis pathway results in enhanced protein modification by O‐linked‐β‐N‐acetylglucosamine (O‐GlcNAc), glucotoxicity and insulin resistance. Contributing factors are complex and may include environment as well as genetics. Aims were to examine differences in O‐GlcNAc in rats artificially selected for either low (LCR) or high (HCR) running capacity. Reduced fitness is known to impair mitochondrial function, perpetuate O‐GlcNAc modification and contribute to reduced insulin sensitivity. Insulin sensitivity was assessed by a hyperinsulinemic‐euglycemic clamp in conscious animals while 2‐[14C]deoxyglucose tracked glucose disposal. Immunoblots of heart tissue examined O‐GlcNAc levels and enzymes that control its regulation (OGT, OGA). Artificial selection of rats to generation 19 resulted in 6‐fold greater running capacity and 63% reduction in fat mass in HCR compared to LCR (p<0.05). LCR rats were insulin resistant disposing of 65% less glucose than HCR (p<0.05). Tissue analysis revealed enhanced O‐GlcNAcylation of Complex I, Complex IV, VDAC and SERCA in LCR compared to HCR (p<0.05). Levels of OGT and OGA were not different between groups. Thus, increased O‐GlcNAc in LCR animals may contribute to mitochondrial dysfunction and the pathogenesis of insulin resistance.
Effect of proinflammatory cytokines on mitochondrial function in 3T3‐L1 adipocytes
Authors: Kuzmicic Jovan Paolo , Hahn Wendy , Burrill Joel , Lavandero Sergio , Bernlohr David
Published: June 2021

To assess the effects of proinflammatory cytokines on mitochondrial function 3T3‐L1 adipocytes were treated with TNFα, IL‐6 or IL‐1β (1 nM, 0–96 h) and evaluated for mitochondrial function as well as mRNA and protein expression. The treatment of 3T3‐L1 adipocytes with inflammatory cytokines induced mitochondrial dysfunction as evidenced by a significant reduction (p<0.05, n=5) in maximal respiratory capacity (uncoupled respiration), increased proton leak, decreased ATP synthesis‐driven respiration, respiratory control ratio and mitochondrial coupling efficiency. In addition, each of the cytokines reduced the NAD+/NADH ratio (p< 0.01, n=3). Each of the cytokines significantly reduced the mRNA expression of PGC1α and TNFα also decreased the mRNA levels of NRF1 and COXIV, suggesting impairment in mitochondrial biogenesis. TNFα significantly reduced (p<0.01, n=4) the level of the mitochondrial fusion protein OPA1 without affecting the mitochondrial fission proteins DRP1 or FIS1. These results indicate that inflammatory cytokines induce mitochondrial dysfunction and changes in mitochondrial biogenesis/dynamics pathways, possibly contributing to the development of insulin resistance and type 2 diabetes.

Supported by NIH DK084669 to DAB. JK holds a PhD scholarship from CONICYT Chile and a Fellowship from PABMB/ASBMB Promoting Research Opportunities for Latin American Biochemists.
Adipocytokines as potential biomarkers for breast and prostate cancers
Authors: ALOKAIL MAJED S , Al-Daghri Nasser M , Alkharfy KHALID M , ALATAS OMAR S , SABICO SHAUN , DRAZ HOSSAM M
Published: June 2021

The link between obesity and premenopausal women breast cancer and prostate cancer development has been recently attributed. Hence, the purpose of this study is to pave a way for development of a blood based gene expression tests and clinical biochemical tests for early detection of breast cancer in obese premenopausal women and prostate cancer in obese men. Fasting blood samples were tested and serum adipocytokines and inflammatory markers were measured using multiplex assays. We have determined the mRNA gene expression quantity of adiponectin, prostate specific antigen (PSA), parathyroid hormone‐related peptide (PTHrP), interleukin‐6 (IL‐6), epidermal growth factor (EGF), erbB‐2, tumour growth factor‐beta (TGF‐β), tumor necrosis factor‐beta (TNF‐β) and P53 genes in 1000 samples of controls, breast and prostate cancer in male and female subjects. The results showed significantly increased in gene expression levels for P53, PTHrP and IL‐6, and significantly decreased levels of adiponectin were found in obese group, while the levels of TNF‐alpha and TGF‐alpha were unaltered. In conclusion, this study revealed strong positive relation of P53, PTHrP, and interleukin 6 with the obesity and breast and prostate cancers development. This relation raises several questions for further experimentation concerning their exact role and physiological significance.