bluuebunny/crossref_metadata_2025_split huggingface dataset:
Endotoxin‐Stimulated Hepatic Stellate Cells Increase Suppressive Potential of Regulatory T Cells via IDO‐Mediated AhR Activation: Therapeutic Implications
Authors: Kumar Sudhir , Dangi Anil , Gandhi Chandrashekhar R 
Published: November 2023

Due to adverse effects of the pharmacological drugs, use of immunosuppressive CD4+Foxp3+ regulatory T cells (Tregs) is an attractive and preferred approach to promote immunological tolerance. However, poor in vivo stability of the ex vivo expanded Tregs, and their conversion to injurious effector immune cells are major drawbacks. We reported earlier that hepatic stellate cells (HSCs) cause expansion and increase suppressive function of allogeneic Tregs; lipopolysaccharide (LPS) pretreatment of HSCs augment the expansion of Tregs. The aim of this study was to delineate the mechanisms underlying HSC‐induced expansion, increased potency and in vivo stability of Tregs. HSCs and Tregs were isolated from allogeneic mouse livers and spleens, respectively. Following co‐culture with untreated or LPS‐pretreated HSCs, proliferation of Tregs was measured by CFSE‐dilution method, and Foxp3 expression and its acetylation were determined via immunoprecipitation and Western blotting analyses. Expression of various genes was determined via quantitative RT‐PCR. Ex vivo HSC‐expanded Tregs were highly stable in vivo following adoptive transfer into mice and retained their increased suppressive property. LPS increased the expression and activity of immunoregulatory enzyme indoleamine 2,3‐dioxygenase 1 (IDO1) in HSCs, and LPS‐pretreated HSCs stimulated aryl hydrocarbon receptor (AhR) signaling in Tregs. Compared to WT HSCs, IDO1−/− HSCs were much inferior in stimulating expansion of Tregs and their Foxp3 expression. Pharmacologic blocking of IDO by 1‐methyl tryptophan in HSCs prevented AhR signaling, Foxp3 expression and expansion of Tregs. The HSC/IDO‐dependent increased acetylation of lysine residues of Foxp3 was associated with stability and competitive fitness of Tregs. Finally, HSCs stored frozen following 2–3 subcultures were much more potent than freshly isolated HSCs in expanding Tregs, thus providing a mode for clinical application. We conclude that HSCs cause expansion of allogeneic Tregs through IDO‐dependent AhR‐mediated signaling mechanism, and increase their stability through lysine‐acetylation of Foxp3. Importantly, availability of frozen HSCs for ex vivo expansion of Tregs provides an exciting therapeutic opportunity for immunological disorders.Support or Funding InformationSupported by a VA Merit Review Award (1IO1BX001174) and grants from NIH (PO1AIO81678 and R21AA020846) to CRG.
The Influence of Spatial Ability on Medical Student Performance in the Basic Sciences
Authors: Willis Anna , Edmondson Anna , Martin Charys 
Published: November 2023

BackgroundSpatial visualization ability (Vz) is associated with higher performance in anatomy; however, the influence of Vz on performance in the other basic sciences has not been established. This study aimed to assess the influence of Vz on academic performance in the first year basic science curriculum at the Medical College of Georgia. We hypothesized that students’ Vz will have a significant positive correlation with their performance in structural science components (anatomy, embryology, histology, and neuroscience) and the systems‐based modules focusing on structural science.MethodsFirst year medical students (n=83) in a systems‐based integrated curriculum completed the Mental Rotations Test (MRT) at the beginning of the year prior to the start of anatomy to establish Vz (pre‐Vz). Students completed the MRT again at the end of the year to reassess Vz (post‐Vz). Academic performance was assessed via grades in each basic science component (anatomy, biochemistry, embryology, histology, neuroscience, and physiology) and systems‐based module (Cellular and Molecular Basis of Medicine, Tissue and Musculoskeletal System, Cardiopulmonary System, Gastrointestinal and Urinary Systems, Endocrine‐Reproductive Systems, Head and Neck and Special Senses, and Medical Neuroscience).ResultsA two‐tailed T‐test determined that there was a significant difference between pre‐Vz and post‐Vz (12.9±4.72; 16.0±4.85 respectively, p≤0.05). ANCOVA analysis demonstrated significant positive correlations between pre‐Vz and performance in histology (r2=0.117), the Cardiopulmonary module (r2=0.114), and the Head and Neck and Special Senses module ((r2=0.092), p=<0.05). Significant positive correlations were found between post‐Vz and performance in anatomy (r2=0.106), histology (r2=0.130), neuroscience (r2=0.114), physiology (r2=0.095), the Tissue and Musculoskeletal System module (r2=0.086), the Cardiopulmonary module (r2=0.131), the Gastrointestinal and Urinary module (r2=0.094), the Head and Neck and Special Senses module (r2=0.158), and the Medical Neuroscience module ((r2=0.050), p≤0.05).ConclusionsIn summary, performance in one component and two modules were positively correlated with pre‐Vz scores while performance in four components and five modules were positively correlated with post‐Vz scores. These results suggest that acquired Vz, as opposed to Vz at the beginning of the year, is associated with improved performance in the basic sciences within the first year medical curriculum. This indicates that training medical students to improve their Vz may be beneficial for medical school performance. Future studies are required to assess how Vz changes throughout the first year curriculum to determine how a change in Vz correlates with student performance within the basic sciences.
Effect of Spatial Ability on Enhancing Neuroanatomy Education with an Interactive 3D E‐learning Module
Authors: Allen Lauren , Eagleson Roy , deRibaupierre Sandrine 
Published: November 2023

Neuroanatomy is one of the most challenging topics in anatomy with many medical students and novice residents reporting they have the least amount of knowledge in the clinical neurological sciences, consider it to be the most difficult subject area, and have the lowest confidence in their diagnosis of patients with neurological complaints. A main reason for the reported challenges is the difficulty in developing a clear understanding of highly‐complex spatial relationships that exist between structures in neuroanatomy. The implementation of interactive three‐dimensional (3D) models into neuroanatomy curricula may provide an effective solution for mitigating the difficulties in spatial knowledge encountered by students using conventional learning resources, however this has yet to be fully examined in the literature.An interactive 3D e‐learning module was developed to complement gross anatomy laboratory instruction. The module provided students the opportunity to manipulate a dynamic 3D model to view structures from any desired angle, view deep cortical structures at high magnification, and add interactive structural labels.Eighty‐seven participants completed the study, which utilized a cross‐over design, to divide participants into two groups. Each group completed baseline anatomy knowledge and spatial ability assessments, followed by access to either the 3D e‐learning module or gross anatomy laboratory. Participants completed the first post‐module anatomy knowledge assessment prior to switching to the other learning modality. A final post‐module knowledge assessment was administered following student exposure to the second learning modality.No baseline differences were observed between groups. Students who initially accessed the 3D module scored significantly higher on the first post‐module knowledge assessment than the students who initially accessed the gross anatomy laboratory. Students who accessed the 3D learning resources following gross anatomy resources, significantly improved on the final post‐module knowledge assessment. A negative correlation was observed between spatial ability and change in assessment score following access to the 3D module suggesting that students with low spatial ability experienced a greater positive effect on their learning of neuroanatomy following the use of the 3D learning module than students with higher spatial ability. Results of this study could be used to guide the effective development and implementations of efficacious 3D e‐learning resources to improve neuroanatomical instruction.Support or Funding InformationOntario Graduate Scholarship
Generation of a Foxc2 Gene Knockout Murine Melanoma Cell Line via CRISPR Cas9 Genome Editing Technology
Authors: Thompson Jefferson , Hargadon Kristian 
Published: November 2023

Melanoma is a leading cause of cancer‐related deaths each year, and murine model systems for the study of this disease are important tools for investigating factors that regulate the growth and metastasis of this cancer. The B16‐F1 melanoma is a well‐characterized murine cell line that is characterized by rapid growth and metastasis to local tumor‐draining lymph nodes. The D5.1G4 cell line is a mutated variant of B16 melanoma that grows slowly and does not invade regional lymph nodes. Recent whole genome expression microarray analysis of these cell lines revealed enhanced expression in B16‐F1 melanoma of the Foxc2 gene, which encodes a transcription factor that has been shown to regulate cell growth, proliferation, differentiation, and longevity. We hypothesized that the FOXC2 transcription factor might also promote lymph node metastasis by B16‐F1 melanoma. To investigate this possibility, we employed a CRISPR‐Cas9 genome editing approach to knock out the Foxc2 gene in B16‐F1 melanoma. The generation of the resulting B16‐F1DFoxc2 melanoma cell line and its utility as a model to study the role of FOXC2 in tumor progression and metastasis are described herein.Support or Funding InformationCommonwealth Health Research Board, Virginia Academy of Science Jeffress Memorial Fellowship, Virginia Foundation for Independent Colleges Undergraduate Science Research Fellowship, Hampden‐Sydney College Honors Council
Nuclear Extracts Enzymatically Convert M1dG to 6‐oxo‐M1dG in Duplex DNA
Authors: Mitchener Michelle M. , Wauchope Orrette R. , Marnett Lawrence J. 
Published: November 2023

Lipid and DNA peroxidation give rise to DNA adducts, the most abundant of which is 3‐(2‐deoxy‐β‐D‐erythro‐pentofuranosyl)pyrimido[1,2‐α]purin‐10(3H)‐one (M1dG). Increased levels of this adduct are positively correlated with a variety of physiological and pathophysiological conditions including aging, bacterial infection, and cancer. Recently, our laboratory reported the oxidative metabolism of M1dG to 6‐oxo‐M1dG in nuclear DNA isolated from multiple human and rodent cell lines, suggesting the presence of an activity responsible for conversion of M1dG to 6‐oxo‐M1dG in double‐stranded DNA. Nuclear extracts from RAW264.7 cells were prepared and incubated with double‐stranded oligonucleotides containing M1dG. Following incubation, the oligonucleotides were digested and analyzed for the presence of 6‐oxo‐M1dG. Heat‐denaturation or Proteinase K‐treatment of the nuclear lysates ablated conversion, indicating that the oxidation is an enzyme‐mediated process. We hypothesized that this enzyme might be an alpha‐ketoglutarate‐dependent dioxygenase, a class of iron‐dependent enzymes that is responsible for the oxidation of various alkylated nucleic acids and/or amino acids. Indeed, we found that preincubation of nuclear lysates with EDTA or metals known to displace ferrous ion from proteins, resulted in a decrease in 6‐oxo‐M1dG formation upon incubation of the lysates with the M1dG‐containing oligonucleotide. Conversely, preincubation with ferrous ion, but not ferric ion, increased the amount of 6‐oxo‐M1dG formed. Additionally, nuclear lysates preincubated with either N‐oxalylglycine or IOX1, two structurally distinct broad‐spectrum alpha‐ketoglutarate‐dependent dioxygenase inhibitors, showed reduced 6‐oxo‐M1dG formation compared to their control counterparts. These findings indicate the presence of a nuclear enzyme, conceivably an alpha‐ketoglutarate‐dependent dioxygenase, capable of converting M1dG to 6‐oxo‐M1dG in double‐stranded DNA.Support or Funding InformationThis work was supported by grants from the National Institutes of Health (R37 CA0878819 to L.J.M.) and US Public Health Services (T32 ES007028 to M.M.M.).
Heterologous Expression of the Human Polybromo‐1 Protein in the Methylotrophic Yeast Pichia pastoris
Authors: Hopson Sarah , Thompson Martin 
Published: November 2023

The human polybromo‐1 protein (BAF180) is known to be a driver mutation in clear cell renal cell carcinoma, where it is mutated in approximately 40% of cases. In its native form, BAF180 is the chromatin‐targeting subunit of the PBAF complex. The precise interactions of BAF180 with the nucleosome are unknown because the recombinant full‐length BAF180 protein has not been available for investigations; only phenomenological studies using BAF180 expressed in mammalian cells have been conducted. BAF180 (encoded by the PBRM1 gene) has nine domains: six bromodomains, two BAH (bromo‐adjacent homology) domains, and one HMG (high mobility group) box. Bromodomains are known to recognize acetylated‐lysines on histones; it is believed that these domains play a crucial role in nucleosome recognition. The BAH domains are critical for the ubiquitination of PCNA, a key regulator of DNA damage. The HMG box is known to be a DNA‐binding domain. Most often, the mutations seen in cancers are truncations, wherein one or more domains have been deleted. Deletion of the bromodomains might prevent recognition of a specific acetylated‐lysine on a histone, impeding the recruitment of the complete PBAF complex. The mutants without the BAH domains may be unable to ubiquitinate PCNA and so cannot overcome DNA damage. Deletion of the HMG box could prevent the recognition and/or binding of BAF180 to gene sites. It can therefore be inferred that mutations in BAF180, especially truncations, have a key role in the progression of cancer, possibly through the interactions noted above. Here we report the successful expression of the full‐length BAF180 protein using the GAP promoter in the heterologous host Pichia pastoris. The ability to express full‐length and mutated BAF180 will allow for biophysical binding studies. Knowledge of the binding interactions is critical for us to understand the role of BAF180 in cancer development and its progression.Support or Funding InformationMichigan Technological University
Single‐Molecule Analysis of Glucocorticoid Receptor and Cofactor Action in Living Cells
Authors: Paakinaho Ville , Presman Diego M , Ball David , Karpova Tatiana S , Schiltz R Louis , Levitt Peter , Morisaki Tatsuya , Mazza Davide , Lavis Luke D , Hager Gordon L 
Published: November 2023

The glucocorticoid receptor (GR) is a hormone‐regulated transcription factor (TF) that plays a central role in metabolic regulation and anti‐inflammatory activities. Population‐based assays such as ChIP‐seq have historically been utilized to investigate the interactions of TFs to DNA. However the dynamic binding behavior of these factors in living cells remains unclear. We have examined the behavior of GR and a number of its known cofactors; AP‐1, NF‐kB, GRIP1, and BRG1 at the single‐molecule level in living cells. These factors were fused with either Halo or Snap tags, and labeled with bright and stable fluorophore JF549. Single‐molecule tracking of these proteins (using HILO microscopy) reveals a highly dynamic binding behavior of GR and its cofactors. This supports transient rather than stable TF chromatin interactions. Specifically, two distinct binding populations have been observed for all factors tested; fast or slow stops. Fast stops have residence times of ~1 sec, whereas slow stops range between 6 and 14 sec. The majority of molecules remain unbound or exhibit fast stops, while only a small percentage exhibit slow stops. However, hormone activation of GR results in an increase in the percentage of molecules with slow stops compared to the unstimulated state. Additionally, the residence time of several cofactors is increased upon GR activation. Importantly, disrupting the interaction of GR and its cofactors decreases the factor's residence time, and proportions of molecules with slow stops. These data support the concept that slow stops represent functional binding events at specific response elements. Our results indicate that only a small proportion of GR and its cofactors are functionally bound at any given time. In conclusion, our single‐molecule observations affirm the general model that many TFs are highly dynamic in their chromatin binding activity.Support or Funding InformationThis research was supported by grants from the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research. Ville Paakinaho supported by the Sigrid Jusélius Foundation.
DNA‐binding Triggers the Tetramerization of the Glucocorticoid Receptor in Live Cells
Authors: Presman Diego M. , Ganguly Sourav , Schiltz R. Louis , Karpova Tatiana , Hager Gordon L. 
Published: November 2023

Transcription factors dynamically bind to chromatin and are essential for the regulation of genes. While a large percentage of these proteins appear to self‐associate to form dimers or higher‐order oligomers, the stoichiometry of DNA‐bound transcription factors has been poorly characterized in vivo. The glucocorticoid receptor (GR) is a ligand‐regulated transcription factor widely believed to act as a dimer. However, this paradigm has been established exclusively from in vitro studies. Using fluorescent imaging techniques and a cell line containing a tandem‐array of steroid response elements, we analyzed the oligomerization state of the GR in vivo. We show that while GR is mostly dimeric in the nucleoplasm, it presents higher oligomerization states, predominantly tetramers, when bound to the array. Consistent with these findings, a point mutation that mimics DNA‐induced conformational changes promotes higher oligomerization in the entire nucleoplasm. We therefore propose that DNA allosterically modulates the oligomerization state of GR, triggering a novel quaternary structure (i.e. a tetramer), which would be the truly active form of the receptor. Since GR's oligomerization state has been implicated in therapy outcome, our findings open new doors to the rational design of novel GR ligands.Support or Funding InformationThis research was supported by grants from the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.
Bioinformatics and Electrophoretic Mobility Shift Assays to Characterize Activators and Repressors of the Lupinus albus Formamidase Gene
Authors: Amadi Michael Chigoziem , Galang Nino Pierre , Sin David , Uhde‐Stone Claudia 
Published: November 2023

BackgroundLupinus albus (white lupin) is a legume adapted to grow under low phosphorous and nitrogen conditions. Phosphorous and nitrogen are important components of cellular molecules such as nucleic acids, amino acids, membrane phospholipids, and ATP. Reserves of the non‐renewable element phosphorus are on a steep decline due to over mining. Nitrogen is the most limiting nutrient of much of the world's agricultural land. We previously identified white lupin genes that showed induced expression under both phosphorus and nitrogen deficiency. Among these genes, a formamidase‐encoding gene in white lupin, named LaFmd, was induced under both nutrient deficiencies. Using promoter deletion analysis, we previously identified putative activator and inhibitor‐binding regions within the formamidase (LaFmd) promoter.MethodWhite lupin was grown hydroponically under phosphate deficiency, nitrogen deficiency, and as nutrient‐sufficient control. Protein was isolated from roots, using the P‐Per plant protein isolation kit (Thermo Fisher, Waltham, MA). Computational analysis (PlantPan 2.0 and PlantCARE) was performed to identify known regulatory DNA motifs. Biotin‐labeled DNA oligonucleotides of the hypothesized regulatory regions were ordered (ELIM Biopharm, Hayward, CA) to perform Electrophoretic Mobility Shift Assays.ResultsBioinformatics analysis identified putative regulatory elements within the hypothesized activator and inhibitor‐binding regions of the LaFmd promoter, including TFmatrixID_0051 and TFmatrixID_0130. Oligonucleotides spanning these regulatory elements were designed and ordered, and will be used for Electrophoretic Mobility Shift Assays. A band shift with protein from nutrient‐deficient conditions, but not nutrient‐sufficient control, would indicate binding of an activator. A band shift only under nutrient sufficiency would indicate binding of a repressor.ConclusionThe outlined approach will be used to identify transcription factors involved in regulating the formamidase gene under phosphorus and nitrogen deficiency.
A Genome Editing Approach to Studying Pmp22 Enhancer Functionality
Authors: Pantera Harrison , Moran John , Lopez‐Anido Camila , Svaren John 
Published: November 2023

The function of the peripheral nervous system is dependent on myelination of nerves by Schwann cells. This process is tightly governed by a network of transcription factors during development. Myelin stability is adversely affected in the most common form of the hereditary peripheral neuropathy called Charcot‐Marie‐Tooth Disease. This form, classified as CMT1a, is caused by a 1.4 Mb duplication on chromosome 17. This duplication includes the abundantly expressed myelin gene Peripheral Myelin Protein 22 (Pmp22), which is associated with multiple disease states in a gene dosage‐dependent manner. Previous studies have shown that reducing the expression of Pmp22 in rodent models of CMT1A results in amelioration of the neuropathy phenotype found in myelinating Schwann cells in the PNS. In recent work characterizing the mechanisms regulating Pmp22 transcription, we identified a cluster of enhancers approximately 100 kb upstream of the Pmp22 TSS. This upstream cluster was found within a smaller duplication identified in patients with CMT1A‐like symptoms, where the Pmp22 coding region itself was not part of the duplication. This cluster possesses multiple binding sites for factors that are required for Schwann cell development and myelination: Egr2 and Sox10. Previous studies in our lab implicate these sites as Pmp22 enhancers, but until now there has been an absence of evidence of functional importance in achieving the high endogenous levels of Pmp22 expression. Using genome editing tools including CRISPR/Cas9 in a modified rat Schwann cell line that expresses Pmp22 at near‐physiological levels, we have established clonal lines possessing deletions that include these upstream transcription factor‐binding sites. Preliminary data show an observed decrease in Pmp22 transcript expression ostensibly due to the loss of these sites. These data show for the first time the requirement of these upstream enhancers for full Pmp22 expression and alternate promoter usage.Support or Funding InformationThis work is supported by NIH grant R01 NS083841.