bluuebunny/crossref_metadata_2025 huggingface dataset:
Do screen display systems improve anatomy student performance?
Authors: Bardales ‐ Cahua Marco Antonio , Ulloa ‐ Deza Delfin Ramel , Fernández‐Rodríguez Lissett Jeanette
Published: November 2023

Technology has provided various didactic tools for the anatomy classroom, including interactive screen display systems that supplement traditional cadaver‐based teaching methods. Our study investigates whether adding such display systems influences student performance. We followed a cohort of 623 students enrolled in an anatomy course over four semesters (2013–2015) that met inclusion criteria. The students enrolled in the first two semesters dissected cadavers only, while the students enrolled later used a SECTRA table display system and dissected cadavers. We find that grades in the anatomy course improved significantly (p<0.00) when the display system was implemented in the curriculum.

Support or Funding Information

School of Medicine Antenor Orrego University
A blended learning approach for human skeletal anatomy with undergraduate students
Authors: Wakeham Travis R. , Morin Brigitte E. , Durocher John J.
Published: November 2023

Consistent with current trends in higher education, we implemented blended learning activities into an introductory undergraduate anatomy and physiology course. The first phase of this project included developing self‐paced exam review videos and restructuring lab activities. The two laboratory modifications were designed to reinforce core concepts taught during the lecture. Specifically, we implemented: 1) online pre‐lab content pages with short readings and interactive animations; and 2) essay‐based laboratory quizzes that were graded by graduate teaching assistants through the mastery approach. The purpose of this study was to determine the effectiveness of these newly developed activities on skeletal anatomy learning. In order to do so, we compared student exam scores from the same course taught through traditional didactic lectures and laboratories last year to those of this year. The content of the skeletal anatomy laboratory learning module and lecture exam questions were the same for the two year analysis. A Wilcoxon rank‐sum test revealed that skeletal muscle exam scores significantly improved this year (P<0.01). Anonymous evaluations also revealed high satisfaction from students. We conclude that blended learning can be effectively implemented to improve student learning and satisfaction in regard to human skeletal anatomy.

Support or Funding Information

This project was supported by the blended learning grant program though the William G. Jackson Center for Teaching and Learning at Michigan Technological University.
Anatomists as Coaches for Developing Clinical Professionals
Authors: Rizzolo Lawrence J.
Published: November 2023

Anatomy can and should be part of a school‐wide culture that embodies the habits‐of‐mind of the clinical professional. To that end, Yale and a growing number of schools use techniques that foster self‐directed learning such as flipped curricula, team‐based‐learning, and group process. This trend moves students from consumers of knowledge to builders who construct their own conceptual framework. The trend also moves anatomy faculty from fonts of knowledge towards the less familiar role of coach. The executive coaching movement provides a roadmap to guide students and faculty through their respective processes of transformation. Unlike mentors or teachers, coaches strive to help coachees develop their innate talents via a collaborative process. There are three principal elements: 1) a course structure that gives students ample opportunity to exercise anatomical and clinical reasoning, professional responsibility and group process; 2) formative assessments that engage students in self‐reflection and self‐discovery; and 3) an attitude of collaboration between anatomists and students as equal partners. Each element presents challenges. The first can be developed with the aid of those clinicians next in line to train the student. The second is aided by the insights of positive psychology, appreciative inquiry, and other philosophies of coaching. The third is perhaps the most challenging, because of the inherent power differential between instructor and student. It requires creating a safe environment to openly explore ideas that is divorced from the grading system. Caring, compassionate instructors can meet these challenges. The result is effective instruction that is fun and transformational.
Effectiveness of a course utilizing cadaver‐based dissection in preparation for gross anatomy as perceived by undergraduate students upon entry to professional school
Authors: Dennis Jennifer , Barry Alla
Published: November 2023

In light of the demand required of gross anatomy in professional school, we installed a cadaver dissection suite on our campus with two goals: first, to incorporate prosected human specimens into our undergraduate anatomy courses, and second, to develop an advanced human dissection course for highly, motivated, pre‐professional students. For enrollment into the course, students must complete an application process and be selected by the Anatomy faculty at Missouri Southern; the course has been offered four semesters from Spring 2013 to Fall 2015. In this study, we examine the perceived effectiveness of BIO 475, Advanced Human Dissection, in preparing students as they transition into gross anatomy (or equivalent) during their first year of professional school. To address this, an electronic survey was distributed to graduates that had completed the BIO 475 course. The survey requested information about the importance of BIO 475 as it related to gross anatomy (or its equivalent), their preparedness as provided by their undergraduate training, and the perceived level of comfort with adapting to the increased demands in professional school. Additionally, we addressed the helpfulness of BIO 433, Histology, in preparing students for similar material at the professional level. A majority of the students reported that the BIO 475 course was beneficial to their professional studies, and that they felt more adequately prepared for the demands of gross anatomy at the professional level. Although many students reported that the structures included in the course were adequate, one area emphasized as important for inclusion was anatomical structures of the hand. Students who completed both BIO 475 and BIO 433 reported that both courses better prepared them for gross anatomy and/or histology at the professional level. The development of the Advanced Human Dissection course is a distinct benefit to students as they matriculate into their professional studies, and it is imperative that undergraduate faculty recognize the value of a human dissection course in adequately preparing students for this transition. As such, we have begun advising all pre‐professional students to complete both courses, and will continue to collect and analyze the perceived usefulness of the courses in the future.

Support or Funding Information

None to report.
Preliminary Multi‐Site Usage For The Online ‘Medical Imaging Solution For Teaching And Research’ (MISTR)
Authors: Carter Yasmin , Costa John , Burbridge Brent
Published: November 2023

INTRODUCTION

Clinical imaging is one of the most common ways medical professionals experience anatomy during their careers it is, however, only fractionally covered in the early years of medical school. This is related, in no small part, to the wholesale reduction in anatomical teaching time experienced by most programs. Additionally, there are concerns with lack of educator experience, and lack of representative images. Ideally, students need to study clinical imaging in a self‐directed manner on their own time. Anatomical teaching at UMass medical school is in the unique position of being under the purview of the Department of Radiology, allowing unprecedented access to trained educators and clinical imaging.

METHODS

Utilizing the previously described (Carter et al., 2015) ‘Medical Imaging Solution for Teaching and Research’ (MISTR), an online image education resource, created at the University of Saskatchewan, a multi‐site collaborative program was designed. This education tool included access to the MISTR‐Repository, a library of clinical image cases of both normal and variant anatomy, online case‐based education modules, and vitally, O.D.I.N. a unique online dicom image navigator that preserves image resolution and facilitates easy image access.

DISCUSSION

The key to MISTR's success is the modular nature of the program. Multiple instructors can utilize individual clinical images in multiple ways. New cases can be built utilizing the same images without disturbing other cases. Purpose built plugins for ‘Blackboard’ the learning management system used by both sites, allows a portal for all instructors to access all cases and to quickly allow instructors to associate images or cases with their coursework. The success of the program will continue to be tested with the release of the beta version in early 2016.

SIGNIFICANCE

Integrating clinical imaging earlier into the medical curriculum and closely associating it with gross anatomy allows students to better assimilate their understanding of anatomical structures as viewed in dissection and text books with that seen though different imaging modalities, long term we suggest this will enable easier comprehension of advanced clinical imaging and patient outcomes. Additionally, the creation of an online viewer allows students to work in a simulated clinical imaging environment at their own pace, in their own time.
Creation of a Learning Module for Healthcare Graduate Students on ALS
Authors: Diouf Johnna Rena , Pascoe Mike A. , Ringel Steven P.
Published: November 2023

Amyotrophic lateral sclerosis (ALS) is a commonly diagnosed neurodegenerative disease. Although recent research has elucidated potential causes of this disease, many allied health students do not have a deep knowledge of the disease, including its pathophysiology, genetic research and treatments. This can translate into a delayed diagnosis of ALS due to symptom overlap with other neurodegenerative diseases like multiple sclerosis. Some studies show that over 50% of ALS cases are given a different disease diagnosis at first. A rigorous literature search found no detailed self‐guided learning resources on ALS to address this potential gap in allied health student knowledge. Therefore, a learning module on ALS was created with the goal of providing a robust foundation of the various aspects of the disease, enabling a stronger knowledge foundation for clinical practice. The case‐based module was constructed using interactive presentation software (Articulate Storyline) that allowed users to explore the connections between pathophysiology, progression stages, research, and treatments of the disease. The module starts with a homepage that is linked to all other sections. All sections have a home button that returns to the homepage. Each section has informative text on its given subject (e.g., pathophysiology). Illustrations were provided along with the text to give visual context, providing a medium to aide visual learners. A clinical case scenario was integrated throughout the learning module to allow acquired knowledge to be applied to a real‐life example. Each section related to the progression of the disease in the clinical case. Also included in the module were 3‐dimensional renderings of neurons to demonstrate the varying effects of the disease dependent on the type of neuron that is affected (i.e., upper vs. lower motor neurons). Small quizzes were administered after each section to allow the student to assess their understanding of the material of a given section. The deployment of this module will add a much‐needed resource on neuropathology for medical and other health sciences students, especially those who plan to specialize in neurology, as it may improve their foundational knowledge and could be used as reference later in their careers. A future goal of the study will be to test the effectiveness of the module.
Integrating Clinical Medicine with the Basic Sciences; Pulmonology Learning Modules for Medical Students
Authors: Doroudi Majid , Hoomayon Babak , Cohen Richard
Published: November 2023

Many medical students find memorizing the detailed of their basic science courses difficult and daunting, especially if they do not know what the application of that subject is during their medical practice. The overall objective of this study was to encourage students to adopt a more anatomical approach to clinical skills and also to show explicitly the relevance of pulmonary anatomy and embryology, thus moving away from rote memorization of physical exam skills and anatomical knowledge.

In order to accomplish the goal of interactive case‐based modules we used the Articulate Engage software suite to create two modules that progress slide by slide in order to encourage a logical flow from patient presentation through to treatment. Using Articulate we were also able to add interactive labeled diagrams to review pulmonary anatomy, pictures to study clinical skills, and integrated quizzes to test student knowledge. The pulmonary modules were evaluated objectively from the survey questionnaire results and subjectively from the comments from the results. Before the modules 20% of students felt they knew the pulmonary materials well or very well. After the modules 58% of students felt they knew the pulmonary materials well or very well. Over 90% of students surveyed strongly or very strongly wanted to see more modules like these modules in their program.
Coordination of Gene Expression and Cell Cycle Progression in Response to Stress; a Novel Checkpoint in S Phase
Authors: Garriga Francesc Posas , Duch Alba , Canal Berta , de Nadal Eulàlia
Published: November 2023

Exposure of cells to increases in extracellular osmolarity results in the activation of the Hog1 stress‐activated protein kinase (SAPK). Activation of Hog1 is required to generate a set of osmoadaptive responses essential for survival under high osmolarity. Adaptation to osmostress requires the induction of a large number of genes, which indicates the necessity to regulate several aspects of the cell physiology. In addition to gene expression, the SAPK also controls cell cycle. Here, we showed that the SAPK is able to modulate cell cycle delay in different phases of the cell cycle including S phase. During S phase, Hog1 controls Mrc1, a protein of the replication complex, to control DNA replication (Duch et al., 2013). The control of replication through Mrc1 is required to coordinate stress‐responsive gene induction with duplication of DNA upon osmostress. Remarkably, we have recently found that Mrc1 is also important to delay replication in response to other environmental stresses suggesting a conserved mechanism of S phase regulation upon transcriptional outbursts.

Support or Funding Information

This work was supported by grants from the Spanish Government (BFU2012‐33503 to F.P., BFU2014 52333 to E.d.N.) and the Fundación Marcelino Botín to F.P. F.P. and E.d.N. are recipients of an ICREA Academia (Generalitat de Catalunya).
Small Molecule Ogg1 Activators Ameliorate mtDNA Oxidation and Promote Cell Health
Authors: Katchur Steven R , Davis Alicia M , Ellis Elizabeth , Pickard Benjamin , Pyne Susan , Rumsey William
Published: November 2023

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation, loss of the alveolar unit, and increased levels of oxidative damage to macromolecules, including DNA. 8‐oxoguanine (8‐OG) is the most common oxidative DNA lesion and its removal and repair is executed through the base excision repair pathway (BER). The purpose of this study was to determine whether enhancing the activity of the DNA glycosylase, Ogg1, would benefit epithelial cell health during an oxidative challenge. Paraquat, an herbicide that intercalates within the inner mitochondrial membrane was used in combination with A549 cells to produce excessive amounts of reactive oxygen species. Levels of 8‐OG were measured using immunofluorescence and single cell phenotypic analysis was undertaken using a high content imaging platform. Transduction of A549 cells with full length‐Ogg1 baculovirus lowered the maximal levels of 8‐OG in mtDNA by 200% as compared to null virus control cells. Conversely, administration of Ogg1 siRNA rendered the cells more vulnerable to paraquat, increasing 8‐OG content by almost 500%. Exemplars of small molecule Ogg1 activators identified through a high‐throughput‐screen, were shown to reduce paraquat‐induced 8‐OG formation by approximately 50%. Moreover, Ogg1 activators improved paraquat‐induced loss of mitochondrial membrane potential, while paraquat‐induced cytochrome c translocation to the nucleus was blocked. The paraquat‐stimulated decline in the cellular energy state, i.e., the ATP/ADP ratio, was prevented in the presence of the Ogg1 activators. Associated with the beneficial effects of the Ogg1 activators described above was the preservation of nuclear area. These data provide evidence of cytoprotection from oxidation of mtDNA when Ogg1 protein is increased or through allosteric activation of the protein, suggesting that the BER pathway may be a target for potential small molecule intervention in COPD.

Support or Funding Information

GlaxoSmithKline
Characterizing a New Role of Novel uup gene in E.coli during DNA Repair
Authors: Joung Bowon , Chen Stefanie H , Cox Michael M
Published: November 2023

Escherichia coli survival after DNA damage requires a highly coordinated suite of DNA repair mechanisms. A number of nonessential E.coli genes involved in DNA metabolism remain poorly characterized. Our recent screen identified a requirement for the uup gene in E.coli for survival after high doses of ionizing radiation. The uup gene encodes a cytosolic ATP‐binding cassette(ABC) ATPase that exhibits DNA‐binding activity in its C‐terminal domain. Null mutants of uup gene increase the frequency of transposon excision, implying more frequent replication slippage events. To further characterize the role of Uup in maintaining genomic stability, this study used a UV‐induced DNA damage assay involving other major DNA recombinational proteins, such as RecG and RecA, in combination with Uup. Under 50 kW of UV exposure, the ΔrecG single mutant displayed a loss of cell viability and increased filamentation. However, the ΔrecG Δuup double mutant increased the survival of ΔrecG single mutant by 10 fold in rich media. In minimal media, the ΔrecG Δuup double mutant exhibited an epistatic interaction for survival, relative to each ΔrecG and Δuup single mutant. The ΔrecA loses its capacity to repair double strand breaks via homologous recombination, leading to severe DNA damage sensitivity. Furthermore, the ΔrecA Δuup double mutant showed an additive decrease in the survival compared to ΔrecA single mutant. In conclusion, this study proposes a new role of the novel uup gene in DNA replication repair in order to ensure genomic integrity.
Tripartite DNA Lesion Recognition and Verification by XPC, TFIIH, and XPA in Nucleotide Excision Repair
Authors: Golebiowski Filip M. , Li Chia‐Lung , Onishi Yuki , Samara Nadine L. , Sugasawa Kaoru , Yang Wei
Published: November 2023

Transcription factor IIH (TFIIH) is essential for both transcription and nucleotide excision repair (NER). DNA lesions are initially detected by NER factors XPC and XPE or stalled RNA polymerases, but only bulky lesions are preferentially repaired by NER. To elucidate substrate specificity in NER, we have prepared homogeneous human ten‐subunit TFIIH and its seven‐subunit core (Core7) without the CAK module and show that bulky lesions in DNA inhibit the ATPase and helicase activities of both XPB and XPD in Core7 to promote NER, whereas non‐genuine NER substrates have no such effect. Moreover, the NER factor XPA activates unwinding of normal DNA by Core7, but inhibits the Core7 helicase activity in the presence of bulky lesions. Finally, the CAK module inhibits DNA binding by TFIIH and thereby enhances XPC‐dependent specific recruitment of TFIIH. Our results support a tripartite lesion verification mechanism involving XPC, TFIIH, and XPA for efficient NER.

Support or Funding Information

We thank Dr. Mikalai Lapkouski for help with negative staining electron microscopy; and Lindsay Wise for purified XPA protein. This work is funded by NIH intramural program (DK075037‐06), and Grants‐in‐Aid for Scientific Research from JSPS and MEXT, Japan.
Early replication stress leads to abnormal mitosis and genome rearrangement
Authors: Forsburg Susan
Published: November 2023

Yeast cell cycle genetics suggests that cells under replication stress generate damage signals that are sufficient to activate checkpoints and restrain mitosis. Yet in mammalian cancer cells, replication stress is not necessarily sufficient to block division. This can lead to abnormal mitotic structures including chromosome bridges, lagging chromosomes, ultrafine anaphase bridges, and micronuclei. This abnormal mitosis is a source for increased mutations including dramatic chromosome rearrangements including chromothripsis, resulting in characteristic genome instability. We have examined the response to different forms of replication stress in the fission yeast, S. pombe, employing visual methods and single‐cell live‐cell analysis.

First, we observe distinct patterns of single‐strand binding protein RPA and homologous recombination protein Rad52 depending on the timing and type of stress we induce. This creates a fingerprint of different S phase‐specific stress responses which we can correlate to distinct outcomes.

Second, we identify a novel stress‐associated phenotype in a temperature sensitive mutation of the Mcm4 helicase subunit. Despite under‐replicated DNA, the cells fail to arrest during temperature shift, and following release. Biochemical analysis suggests that they evade the checkpoint. Our dynamic pedigrees reveal surprising examples of division under stress including all the mitotic structures described above, including ultrafine anaphase bridges and apparent micronuclei. These abnormal mitoses are associated with genome instability, mutations, and chromosome rearrangement in the surviving cells.

Our study shows that a subset of cells within a whole population can continue to divide, despite significant negative effects on genome integrity, and establishes a genetic model to study mechanisms that allow division to occur despite stress.

Support or Funding Information

NIH R01 GM081418 &GM111040
Estrogen induces RAD51C expression and localization to sites of DNA damage
Authors: Holz Marina K.
Published: November 2023

Homologous recombination (HR) is a conserved process that maintains genome stability and cell survival by repairing DNA double‐strand breaks (DSBs). The RAD51‐related family of proteins is involved in repair of DSBs, consequently, deregulation of RAD51 in human cells causes chromosomal rearrangements and stimulates tumorigenesis. RAD51C in particular has been identified as a potential tumor suppressor and a breast and ovarian cancer susceptibility gene. Recent studies implicated estrogen as a DNA‐damaging agent that causes DSBs. We found that in estrogen receptor (ER)α‐positive breast cancer cells, estrogen transcriptionally regulates RAD51C expression in ERα‐dependent mechanism. Moreover, estrogen induces RAD51C assembly into nuclear foci at DSBs, which is a precursor to RAD51 complex recruitment to the nucleus. These findings provide insight into the mechanism of genomic instability in ERα‐positive breast cancer. We propose that in normal breast epithelia, estrogen‐induced DNA damage is compensated by estrogenic activation of expression of DNA repair proteins, such as RAD51C. Conversely, individuals with mutations in RAD51C would be more susceptible to accumulation of DNA damage, leading to tumorigenesis and cancer progression.

Support or Funding Information

NIH–CA151112; Atol Charitable Trust; LAM Foundation– 098P0113; American Cancer Society–RSG‐13‐287‐01‐TBE and National Cancer Center.
Epigenetics Impacts Copy Number Heterogeneity and Drug Resistant Gene Selection
Authors: Whetstine Johnathan R , Black Joshua C , Mishra Sweta , Sexton Brittany , Van Rechem Capucine , Murphy Sedona
Published: November 2023

Copy number heterogeneity is a feature of tumors and associates with acquired drug resistance. The mechanistic bases for copy number heterogeneity, especially selection of specific regions within the genome, are unknown. We have recently identified the histone tri‐demethylase KDM4A as the first enzyme capable of promoting site‐specific copy number changes. KDM4A overexpression as well as stabilization promotes localized copy gain without global chromosome instability. Tumors with increased KDM4A levels are enriched in copy gains for cytobands observed in cell culture models. We further demonstrate that these events are the result of rereplication. These copy gains occur during S phase and are removed as cells are exiting S phase. The cytoband gains are affiliated with drug resistant tumors; therefore, we asked whether targeting the copy gains through chemical inhibition of KDM4A was possible and whether a chemical screen could demonstrate that an active process is associated with elimination of copy gained regions. Our most recent data demonstrates the druggability of these processes. In fact, we have now identified small molecules that impact KDM4A and copy gain as well as the ability to remove gains during cell cycle progression. Lastly, a screen for environmental and chemical agents has uncovered input signals that are responsible for generating site‐specific gains through altering the chromatin environment. Taken together, we have identified genetic, epigenetic and environmental factors promoting copy number heterogeneity in tumors and established that these events are targetable through inhibition of chromatin regulators.

Support or Funding Information

NIH/NIGMS

Leukemia and Lymphoma Society

American Cancer Society

American Lung Association

Alex Lemonade Stand Foundation
Electrostatic and Hydrophobic Interactions Mediate Single‐Stranded DNA Recognition and Acta2 Repression by Purine‐Rich Element Binding Protein B
Authors: Ferris Lauren , Rumora Amy , Wheeler Tamar , Kelm Robert
Published: November 2023

Fibroblast to myofibroblast differentiation is characterized by increased expression of cytoskeletal smooth muscle α‐actin. In human and murine fibroblasts, the gene encoding smooth muscle α‐actin (Acta2) is tightly regulated by a network of transcription factors that either activate or repress the 5′ promoter in response to environmental cues signaling tissue repair and remodeling. Purine‐rich element binding protein B (Purβ) suppresses the expression of Acta2 by cooperatively interacting with the sense strand of a 5′ polypurine sequence containing an inverted muscle‐CAT (MCAT) motif required for gene activation. In this study, we evaluated the physical and chemical basis of nucleoprotein complex formation between the Purβ repressor and the purine‐rich strand of the MCAT cis‐regulatory element in the mouse Acta2 promoter. Quantitative single‐stranded DNA (ssDNA)‐binding assays conducted in the presence of increasing concentrations of monovalent salt or anionic detergent indicated that high affinity nucleoprotein complex assembly is driven by a combination of electrostatic and hydrophobic interactions. Results of pH titration analysis suggested that basic amino acid residues are likely involved in mediating the interaction of Purβ with ssDNA. Computational homology modeling was performed to identify positionally‐conserved cationic residues in each ssDNA‐binding subdomain of Purβ. Site‐directed mutagenesis confirmed that several solvent‐exposed lysine and arginine residues located in the intermolecular (R267) and intramolecular (K82, R159) subdomains are essential for the transcriptional repressor function of Purβ. In keeping with their stable expression but diminished Acta2 repressor activity in transfected fibroblasts, purified Purβ mutants R267A, R159A/R267A, and K82A/R159A/R267A were unaltered in terms of protein folding and quaternary structure but exhibited markedly reduced binding affinity for the purine‐rich strand of the Acta2 MCAT element. Moreover, the Purβ double and triple point mutants were also found to be defective in binding to the Acta2 corepressor protein, Y‐box binding protein 1. Collectively, these findings establish the repertoire of non‐covalent interactions that account for the unique structural and functional properties of the Acta2 repressor protein Purβ.

Support or Funding Information

AHA 09GRNT2170060 and NIH T32 HL007594
Activation of p21 following DNA damage transcriptionally controlled by a telomeric shelterin protein TRF2 in a telomere‐length dependent fashion
Authors: Chowdhury Shantanu
Published: November 2023

We report and unexpected finding ‐ TRF2 occupancy at the p21, as well as other promoters, depends on telomere‐length, even though the sites are as much as 62 Mb distal to the nearest telomere. And, p21 repression by TRF2 was telomere length‐dependent. Together our data are consistent with a model that involves partitioning of TRF2 between telomeres and interstitial sites, which influences gene regulation. DNA secondary structures called G‐quadruplexes were recently detected in‐vivo using antibodies. Further, it remains to be understood whether and how detected interstitial G‐quadruplexes function biologically. In support of the underlying mechanism of TRF2‐p21 regulation we find telomere repeat factor 2 (TRF2) associates with hundreds of potential interstitial G‐quadruplex sites, a significant number of which overlap with gene promoter regions. Detailed mutational and expression analysis of the cyclin‐dependent kinase CDKNIA (p21) promoter quadruplex site revealed TRF2 occupancy results in recruitment of the REST‐repressor protein complex and decreases p21 expression in response to drug induced DNA damage.

Support or Funding Information

Wellcome Trust ‐ Department of Biotechnology India Alliance and Council of Scientific and Industrial Research
Defining a Transcriptional Regulatory Network for Peroxisome Biogenesis
Authors: Paz Edwin A. , Gaasterland Terry , La Spada Albert
Published: November 2023

Peroxisomes are highly dynamic organelles that are involved in lipid metabolism and hydrogen peroxide break‐down. Dysregulation of peroxisomal processes are associated with a group of neurodevelopmental disorders including Zellweger syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease. Underlying these disorders are mutations in PEX genes resulting in faulty peroxin production and function. We hypothesize that important contributors to neurodegenerative diseases are dysregulated transcriptional networks affecting peroxisome biology. Using a bioinformatics approach, we have identified potential transcription factors and their consensus transcriptional factor binding site (TFBS) motifs, and are testing if these TFBSs are coordinately regulating peroxisome biogenesis and function. Identifying novel transcriptional regulators of peroxisome biogenesis may reveal novel targets that can be exploited for therapeutic intervention.

Support or Funding Information

R01 AG033082

K12 GM068524
Microarray expression profile analysis of long non‐coding RNAs in pulmonary tuberculosis with Traditional Chinese Medicine
Authors: Jiang Tingting , Shi Li‐Ying , Wei Li‐Liang , Chen Zhong‐Liang , Wang Chong , Liu Chang‐Ming , Li Zhong‐Jie , Li Ji‐Cheng
Published: November 2023

The present study aimed to explore the expression profile of long non‐coding RNAs (lncRNAs) and mRNAs in pulmonary tuberculosis (TB) patients with Traditional Chinese Medicine (TCM) by Arraystar Human LncRNA Microarray. TB patients were divided according to the TCM syndromes into pulmonary Yin deficiency (PYD) syndrome, hyperactivity of fire due to Yin deficiency (HFYD) syndrome, and deficiency of Qi and Yin (DQY) syndrome. Plasma samples were collected from 33 TB patients and 11 healthy controls. Total RNA was extracted and purified, and then hybridized with Arraystar Human LncRNA Microarray V3.0 containing 30586 lncRNAs probes. Bioinformatic analyses including gene ontology (GO), and kyoto encyclopedia of genes and genomes (KEGG) pathway were conducted to analyze the differentially expressed lncRNAs of TB with different TCM syndromes. The related clinical data was also analyzed.1523, 558, and 596 differentially expressed lncRNAs were found in PYD, HFYD, and DQY patients, respectively, compared with the healthy controls (ratios of >2, or <0.5, and P<0.05). Pathway analysis revealed that fatty acid metabolism (P=0.005067587), protein digestion and absorption (P=5.89017E‐05), and Hippo signaling pathway (P=0.000164) have higher enrichment score. The expression of 18 transcripts with zinc ion binding function, 10 transcripts related to the metabolic process and 9 transcripts involved in the apoptotic process were progressively increased from PYD→HFYD→DQY syndrome. Clinical analysis (143 controls, 90 PYD cases, 116 HFYD cases, and 85 DQY cases) showed that the lipid indexes of TB patients was abnormal, and the triglyceride concentration was significantly different among PYD, HFYD, and DQY patients (P=0.0352). Our study is the first to acquire the microarray expression profile of lncRNAs, and analyze the pathway enrichment and expression tendency of differentially expressed lncRNAs among PYD, HFYD, and DQY syndromes. Our results indicate that the analysis of expression profile of lncRNAs may be a novel method to explore the biological essence of TCM syndrome.

Support or Funding Information

This work was supported by grants from National Basic Research Program of China (No.2014CB543002), National Special Sci‐Tech Projects (No. 2012ZX10005001‐006), and National Natural Science Foundation of China (No.81273882, NO. 81573709).
Ascertaining a Role For Rnp1p in Post‐Transcriptional Gene Control in S. cerevisiae
Authors: Mackay Katherine E. , Segal Scott P.
Published: November 2023

Translational control plays a vital role in the response to such physiological stresses, as high cell density or glucose deprivation. When physiological stress is present, translation is repressed and mRNA becomes unbound to ribosomes. A number of proteins are involved in the process of removing mRNA from the translational pool, where they are, in turn, sent to stress granules (SGs) to be stored or P‐bodies to be degraded. One of these translational repression proteins is Sbp1p, which contains two RRMs that flank a central RGG box. Sbp1p has been shown to localize to P‐bodies and SG. However, its interactions with translational initiation factors through its RGG box, suggests it may play a greater role in mRNA storage.

RNP1, which is an SBP1 paralog, encodes a protein containing two RRMs but lacks the RGG box present in SBP1. Given these structural similarities, RNP1 may also play a role in translational repression and possible localization of mRNA to P‐bodies or SGs. To determine whether Rnp1p may play a role in these processes, Rnp1p was overexpressed, and assembly of P‐bodies and SGs were ascertained. Upon overexpression assembly of P‐bodies was induced in unstressed cells, whereas SGs were not. This suggested that Rnp1p plays a role in P‐body assembly directly, or through translational repression. To support this claim, physiologically stressed rnp1Δ cells had a significant reduction in P‐body assembly. Perhaps the lack of an RGG box may target Rnp1p to P‐bodies allowing it, in turn, to have a role in mRNA for degradation, rather than storage.

To further ascertain whether Rnp1p has a direct role in P‐body assembly or translational repression, future experiments will be conducted to determine if Rnp1p localizes to P‐bodies under physiological stress. Additionally, translation will be monitored, and mRNA half‐lives will be measured in strains over‐expressing and lacking Rnp1p.
Biochemical Characterization of the Interaction between Innate Immune Receptor Nod2 and its Chaperones
Authors: Wastyk Hannah C. , Mohanan Vishnu , Schaefer Amy , Hou Ching‐Wen , Lauro Mackenzie , Grimes Catherine L.
Published: November 2023

The human body houses over 10 trillion bacterial cells, of which our innate immune system must delineate between pathogenic and commensal. Disease can arise during misregulation of this bacteria, and one example is the debilitating inflammatory bowel disorder, Crohn's disease (CD). The molecular pathology of CD is known to be a mutation in Nod2, an innate immune receptor responsible for binding directly to bacterial cell wall fragment, muramyl dipeptide (MDP). MDP has recently been shown to stabilize Nod2 in a cell based assay, and after binding to MDP, Nod2 signaling proteins activate the NF‐κB pathway, a protective inflammatory response. In Crohn's variants, however, Nod2 is rapidly degraded, thereby the downstream signaling is disrupted. Our initial work identified Hsp70 as a chaperone molecule that binds to and stabilizes Nod2. The purpose of this study is to characterize this interaction and stabilization effect, such that the natural stabilization of Nod2 within the cell can be extrapolated to a possible treatment option that mimics Hsp70's activity on unstable Nod2 Crohn's variants. Hsp70 is a highly ubiquitous protein that is able to confer selective stability by tuning its specific activity via a number of different cochaperones. Our data shows that Hsp40 contributes to the chaperone complex, and through limited proteolysis, we show that both Hsp70 and Hsp40 mediate Nod2 stability in vitro, and that this stability is further enhanced with the addition of Nod2's ligand, MDP. Furthermore, we have biochemically characterized the importance of Hsp70's ATPase activity for Nod2's stability. Significance of this study extends beyond CD, as understanding Hsp70's action and determining a method of control for Nod2 could be extrapolated to stabilize a large number of disease‐relevant mutated proteins.

Support or Funding Information

The authors would like to thank the following sources of funding: the Chemistry‐Biology Interface program of NIH, the Howard Hughes Medical Institute, and the Hofmann Fund through Nucleus.
En'Cas'ing The Stress: Engineering a Human Cell Line Knockout of Heat Shock Response Coordinator Genes Using CRISPR Cas9 System
Authors: Canales Gabriela Isla‐Inez , Pani Bibhusita , Nudler Evgeny
Published: November 2023

The Heat Shock Response (HSR) is an evolutionarily conserved response to high temperatures and other stresses that controls adaptive proteostasis, and is primarily regulated by the factor, Heat shock transcription factor 1 (HSF1). In mammalian cells, HSF1 is converted from an inactive monomeric form to an active trimer in response to heat stress. A ribonucleoprotein complex comprising of eukaryotic translation elongation factor eEF1A1, and a long noncoding RNA HSR1 are the key components of HSF1 activation. Once activated, HSF1 is recruited to Heat Shock Protein (HSP) promoter regions, upregulating chaperone activity in the cell. Along with mediating initiation of HSR, eEF1A1 is also a vital component of protein synthesis machinery. Interestingly, another isoform of eEF1A, called eEF1A2, is expressed in some specialized terminally differentiated cells of skeletal muscle, heart, pancreatic islets and motor neurons, all of which are prone to protein aggregation. The two isoforms are 92% identical and are reciprocally regulated. To better understand the role of eEF1A1 and HSF1 proteins in humans, we use a CRISPR‐Cas9 nickase system to knockout HSF1 and eEF1A1 in a human cell line. We showed that the hTERT‐immortalized, normal diploid foreskin fibroblast cell line, BJ‐5ta, produces both eEF1A isoforms. This will allow us to perform eEF1A1 knockout in these cells. We hypothesize that HSF1 knockout cell line will survive under normal conditions but express very low thermotolerance. Conjointly, we hypothesize that the elimination of eEF1A1 may be compensated by the upregulation of eEF1A2. If viable, the eEF1A1 knockout cell line will be used for screening mutants of eEF1A2 restoring activation of HSR. Both HSF1 and eEF1A1 knockout lines will also be used for future studies to improve upon the current model of the HSR pathway and potentially reveal therapeutic targets for diseases like ALS, Alzheimer's disease, Parkinson's disease, type 2 diabetes, and amyloidosis.

Support or Funding Information

This investigation was supported in part by a MARC Undergraduate Student Training in Academic Research (U‐STAR) National Research Service Award (NRSA) Institutional Research Training Grant (2 T34 GM008663) from the National Institutes of Health, National Institute for General Medical Sciences.

This research was supported in part by a grant to UMBC from the Howard Hughes Medical Institute through the Precollege and Undergraduate Science Education Program.
Evaluation of ADAMTS Levels in Patients with Chronic and Aggressive Periodontitis
Authors: Demircan Kadir , Tayman Ayşe , Bayram Nezihe Aslı , Demir Berrin Tuğtağ , Karagöz Zehra Fırat , Fıstık Tuba , Kurga Şivge , Önder Canan , Günhan Meral
Published: November 2023

Introduction

ADAMTS is a 19‐membered gene family which was first discovered in colon cancer, is associated with inflammation. ADAMTS is a member of matrix metalloproteinases family and they are involved in degradation and repair of extracellular matrix. In recent studies, ADAMTSs are activated with lots of signaling pathways and regulated with lots of cytokines. They play an important role in pathogenesis of many diseases. Based on this information it has been hypothesized that ADAMTS levels would be increased in inflammatory diseases such as periodontal disease. Chronic periodontitis that is the most prevalent form of periodontitis, shows the characteristics of a slowly progressing inflammatory disease. Also, systemic and environmental factors (e.g., diabetes mellitus, smoking) may revise the host's immune response so that periodontal destruction becomes more progressive. The aim of this study is to investigate the presence of ADAMTS genes and proteins in chronic and aggressive periodontitis patients.

Methods

Fifteen aggressive and 15 chronic periodontitis patients that were systemically healthy were included in this study. Patients presented with chronic or aggressive periodontitis and needed tooth extractions were selected. Gingival biopsies containing epithelium and connective tissue were obtained during the extraction of periodontally involved teeth. Systemically healthy patients who need crown lengthening or gingivectomy procedure for aesthetic reasons were included to the control group. mRNA expression of ADAMTSs were evaluated by real‐time PCR method and position of the marked protein in the tissue is determined by Immunohistochemistry method.

Results and Conclusion

Differences were observed between the control and disease groups. According to results of the study, it can be concluded that ADAMTS expressions have a potential to be a new marker in pathogenesis of periodontal disease. Western‐blot studies are continuing to determine the protein levels.
Comparison Between hPEP and spPEP Inhibition When Varying Size and Position of Inhibitory Peptide of Casein α s1
Authors: Vishram Avleen Kaur , Clack Beatrice A
Published: November 2023

Eurygaster integriceps Puton (Sunn pest) is the most damaging insect pest of wheat in areas of North Africa, Eastern Europe and Western and Central Asia. Sunn pest injects an enzyme characterized as a prolyl endylprotease (spPEP) into wheat grains that digests the gluten protein (Darkoh et al., 2010). Previous studies have shown that casein peptides generated by Lactobacillus species have inhibitory effects on human (hPEP) and bacterial prolyl endoproteases (PEP) which makes casein a good natural candidate against spPEP. Developing biopesticides for food products from casein αs1 is complicated due to some allergenic amino acid sequences. Ruiter et al (2005) identifies a non‐allergenic peptide region on casein αs1 that includes a previously identified inhibitory sequence (LNENLLRFFVAPFPEVFG) to hPEP (Jeanneret, 2011). The Objective of this study was to produce 16 peptides derived from aS1 casein of varying lengths and varying size containing the hPEP inhibitory sequence. The inhibitory region is positioned at 0, 10, 20 and 30 amino acids from either N′ terminal or C‐terminal end. Each of the peptides were cloned into pET15b for expression with a His tag for purification. Peptides were incubated with either hPEP or spPEP in the presence of the substrate GPpNA. We report the inhibition of both enzymes based on size and placement of the inhibitory sequence.

Support or Funding Information

Funding: Office of Research and Sponsored Projects, SFASU and Ed and Gwen Cole, Nacogdoches, TX.
Deletion of nicastrin Leads to Hypomyelination and Transcriptional Changes in Juvenile Mice
Authors: Yamato Mizuho , Zhu Yi , Dries Daniel , Yu Gang
Published: November 2023

The γ‐secretase complex is a four‐subunit membrane‐bound protease complex that regulates a number of critical cellular functions through the cleavage of transmembrane substrate proteins. Of the four subunits, nicastrin is involved in substrate recruitment. Germline deletion of any of the four subunits leads to death during embryonic development. Previous work described a line of conditional knockout (cKO) mice in which nicastrin was knocked out of oligodendrocytes (OLs, the myelinating cells in the central nervous system). To examine the transcriptional changes that led to the pathological differences in juvenile mice, RNA‐Seq was performed. Here, we present the analysis of several genes with altered expression from the study using qPCR. Meanwhile, optic nerves were examined by electron microscopy (EM) to observe myelination during development. The results indicate that deletion of nicastrin from γ‐secretase leads to transcriptional changes and significantly affects myelination in the central nervous system of juvenile mice.

Support or Funding Information

This work was supported by the National Institute of Neurological Disorders and Stroke (R01 NS079796), the National Institute of Aging (AG029547, R01 AG023104 and F32 AG031625), the Welch Foundation (I‐1776), the Hartwell Foundation, and the Alzheimer's AssociationNational Institute of Neurological Disorders and Stroke (R01 NS079796), the National Institute of Aging (R01 AG023104 and F32 AG031625), the Welch Foundation (I‐1776), the Hartwell Foundation, and the Alzheimer's Association.
Protein Disulfide Isomerase Can Reverse the Aggregation of α‐Synuclein
Authors: Burress Helen , Cilenti Lucia , Serrano Albert , Taylor Michael , Tatulian Suren , Teter Ken
Published: November 2023

Neurotoxic amyloid fibrils of α‐synuclein contribute to the etiology of Parkinson's disease. Protein disulfide isomerase (PDI) provides a protective effect against neurodegeneration that is linked to its chaperone function and its ability to disrupt protein aggregation. Here, we report PDI can both prevent and reverse the aggregation of α‐synuclein. Assays using Thioflavin T fluorescence or transmission electron microscopy demonstrated PDI can inhibit the formation of α‐synuclein fibrils when added at the onset of aggregation and can reverse fibrillization when added to intermediate fibrils 30 h after the initiation of aggregation. The chaperone but not oxidoreductase activity of PDI was required to disrupt α‐synuclein fibrillization. Biophysical studies found that substrate binding leads to the partial unfolding of PDI, which provides a possible molecular basis for the dissolution of α‐synuclein fibrils: the structural expansion of unfolded PDI could push against two or more proteins in the fibril, thus acting as a wedge to displace individual proteins from the aggregate. These data establish a new functional property for the chaperone activity of PDI that may explain how it protects against neurodegeneration.