bluuebunny/crossref_metadata_2025_split huggingface dataset:
G Protein‐Coupled Estrogen Receptor (GPER)‐Mediated Relaxation of Coronary Arteries is Mitigated by Phosphorylation of ERK1/2
Authors: Harlow Rebecca C. , Yu Xuan , Kotaki Bobak P. , Stallone John N. , Heaps Christine L. , Han Guichun 
Published: November 2023

GPER is a membrane‐bound estrogen receptor, distinct from ERα or ERβ, and exerts genomic and non‐genomic effects. GPER's effect on the cardiovascular system has been controversial; evidence indicate it relaxes arteries, whereas other findings suggest it contracts arteries. Our objective is to better understand the dual nature of GPER. Previously, our work demonstrated that G‐1 stimulates cAMP production. We hypothesize GPER mediates relaxation response through cAMP and constriction via ERK1/2. Isometric tension studies were used to measure GPER‐mediated coronary tone response in porcine coronary arteries. Western blots were applied to detect pERK1/2 in primary cell culture of smooth muscle cells. The identity of smooth muscle cells was validated by immunohistochemistry techniques using α actin as a marker. Under adenylate cyclase inhibition by SQ22536, G‐1 stimulated phosphorylation of ERK1/2. The effect of G‐1 was blocked by G36, a GPER inhibitor. A time course of G‐1 (1 nM) demonstrated that detection of pERK1/2 peaked at 2 and 5 min, decreased at 15 min, and returned below baseline by 30 min. Similar results were found for a time course of E2 (1 nM) detecting pERK1/2, however, under no adenylate cyclase inhibition. Tension studies demonstrated that G‐1 caused concentration‐dependent relaxation of PGF2α (1 μM) precontracted, endothelium denuded coronary arteries. PD98059, a MEK 44/42 inhibitor which blocks the phosphorylation of ERK1/2, led to further relaxation than G‐1 alone. We conclude that phosphorylation of ERK1/2 lessens the coronary artery relaxation caused by GPER.Support or Funding InformationAmerican Heart Association
Loss of MyoD transdifferentiate myoblasts into brown adipocytes
Authors: Wang Chao , Liu Weiyi , Nie Yaohui , Yue Feng , Kuang Shihuan 
Published: November 2023

Brown adipose tissue (BAT) represents a promising agent to ameliorate obesity and other metabolic disorders. However, the abundance of BAT decreases with age and BAT paucity is a common feature of obese subjects. As brown adipocytes and myoblasts share a common Myf5 lineage origin, elucidating the molecular mechanisms underlying the fate choices of brown adipocytes versus myoblasts may lead to novel approaches to expand BAT mass. Here we identify MyoD as a key negative regulator of brown adipocyte development. CRISPR/CAS9‐mediated deletion of MyoD in C2C12 myoblasts facilitates their adipogenic transdifferentiation. MyoD knockout downregulates miR‐133 and upregulates the miR‐133 target IGF1R, leading to amplification of PI3K–Akt signaling. Accordingly, inhibition of PI3K or Akt abolishes the adipogenic gene expression of MyoD null myoblasts. Strikingly, loss of MyoD converts satellite cell‐derived primary myoblasts to brown adipocytes through upregulation of Prdm16, a target of miR‐133 and key determinant of brown adipocyte fate. Conversely, forced expression of MyoD in brown preadipocytes blocks brown adipogenesis and upregulates the expression of myogenic genes. Importantly, miR‐133a knockout significantly blunts MyoD–induced myogenic conversion of brown preadipocytes. Our results establish MyoD as a negative regulator of brown adipocyte development by upregulating miR‐133 to suppress Akt and Prdm16.Support or Funding InformationThis work was supported by a grant from the US National Institutes of Health (R01AR060652 to S.K.) and Purdue incentive grant from Purdue University Office of Vice President for Research (OVPR) to S.K.
Skeletal Muscle‐derived Cytokines Regulate Myogenesis by Modulating Cell Cycle Withdrawal
Authors: Kim Dongwook , Singh Nilmani , Chen Jie 
Published: November 2023

Regenerative myogenesis after injury is a unique capacity of adult skeletal muscle to maintain normal tissue structure. This ability declines with aging or muscle diseases. Muscle regeneration consists of precisely organized processes such as cell cycle withdrawal followed by myocyte fusion. However, regulatory mechanisms of skeletal myogenesis remain incompletely understood. Interestingly, muscle cells express a variety of cytokines, which may play roles in skeletal myogenesis in an autocrine or paracrine manner. Previously, we performed an RNAi screen that revealed many cytokines as candidate regulators of myogenic differentiation. We have now identified several of those cytokines, including Tumor Necrosis Factor Superfamily Member 10 (Tnsfs10), Fas ligand (FasL) and C‐C Motif Chemokine Ligand 8 (Ccl8), as negative regulators of myogenesis. Strikingly, all those cytokines inhibit myogenic differentiation by preventing myoblasts from exiting the cell cycle. Together with our previous finding that Fms‐like tyrokine kinase 3 ligand (Flt3L) positively regulates myogenesis by promoting cell cycle exit of the myoblasts, our results have led us to propose that cell cycle withdrawal is a main checkpoint through which muscle‐expressed cytokines control myogenesis.Support or Funding InformationNIH/NIAMS, R01AR048914
Differential activation of class I phosphoinositide 3‐kinase by growth factors
Authors: Ghosh Dibbendu , Liu Shu‐Lin , Cho Wonhaw 
Published: November 2023

Growth factors bind to specific receptors on the cell surface, leading to the activation of diverse cellular processes. Many growth factors are known to activate Class I phosphoinositide 3‐kinase (PI3K), that generates of PtdIns(3,4,5)P3 in the plasma membrane (PM). Our ratiometric sensor for PtdIns(3,4,5)P3, which allows in situ quantification of PtdIns(3,4,5)P3 in the PM of mammalian cells, showed variable degrees of PtdIns(3,4,5)P3 increases in the PM upon stimulation by diverse growth factors. Some growth factors, such as fibroblast growth factor‐19, even caused a decrease in the basal level of PtdIns(3,4,5)P3 in the PM. Further quantitative lipid imaging by our orthogonal lipid sensors indicate that PtdIns(3,4,5)P3 and other lipids in the PM work in concert to modulate various downstream signaling pathways of growth factor‐induced Class I PI3K activation.NIHSupport or Funding InformationNIHGM68849
Endogenous Ligand for Orphan Nuclear Receptor NR2E3 Forms a Light‐Sensitive Retinal Transcription System
Authors: Connor Blaine , Lee Young‐Sam 
Published: November 2023

The orphan nuclear receptor NR2E3 (also known as PNR) is a transcription factor important for retinal photoreceptor development and maintenance. Mutations in NR2E3 cause retinal diseases and vision loss in human. However, despite the importance of ligand‐binding in the regulation of nuclear receptors, the endogenous NR2E3 ligand is not yet known. Identification of the endogenous NR2E3 ligand will provide a better understanding on how NR2E3 and retinal development are regulated. Here we report that biliverdin is an endogenous NR2E3 ligand and that the biliverdin‐NR2E3 complex forms a light‐sensitive retinal transcription system. Using gel‐filtration chromatography‐mass spectrometry, we identified biliverdin, a pigment from heme metabolism, as an endogenous NR2E3 ligand. Biliverdin bound to NR2E3 was irreversibly sensitive to red light in vitro, while the combination of biliverdin and red light together induced the expression of NR2E3‐regulated genes (rhodopsin and Rev‐ErbAa) in cultured retinoblastoma cells. This is the first case where a nuclear receptor senses light. The biliverdin‐NR2E3 complex might play a role of regulating retinal development in response to light.Support or Funding InformationJohns Hopkins UniversityNational Cancer Institute, National Institute of Health
Thrombomodulin Promotes Cell Adhesion and Migration and Enhances Angiogenesis through Interaction with Fibronectin
Authors: Wu Hua‐Lin , Hsu Yun‐Yan , Shi Guey‐Yueh 
Published: November 2023

ObjectivesCell surface adhesion molecules control cell adhesion and migration by interacting with the extracellular matrix (ECM). Angiogenesis, which triggers tumor growth and metastasis, requires endothelial cell‐ECM interactions. Thrombomodulin (TM) is an integral membrane protein expressed on the vascular endothelium. We investigated whether TM interacts with the ECM proteins and participates in tumor angiogenesis.MethodsThe solid‐phase binding assay was performed to analyze the interaction of recombinant TM lectin‐like domain (rTMD1) with the ECM. TM over‐expression and TM knockdown techniques were used to investigate the roles of TM in cell adhesion, migration, signaling, and endothelial tube formation. The proximity ligation assay was carried out to monitor TM‐fibronectin interactions on tumor blood vessels.ResultsWe demonstrated that rTMD1 mainly interacted with the ECM fibronectin. The rTMD1 binding to fibronectin was inhibited by the anti‐TM antibody. In addition, we identified the N‐terminal 70‐kDa domain of fibronectin as the TM‐binding site. Over‐expression of TM in TM‐deficient A2058 melanoma cells promoted cell adhesion and migration on fibronectin and invasion through Matrigel. Furthermore, TM over‐expression enhanced focal adhesion kinase (FAK) phosphorylation and matrix metalloproteinase‐9 production. In human umbilical vein endothelial cells (HUVECs), up‐regulation of TM by phorbol 12‐myristate 13‐acetate promoted endothelial cell adhesion and FAK tyrosine phosphorylation, whereas knockdown of TM by RNA interference attenuated cell adhesion and FAK tyrosine phosphorylation. Vascular endothelial growth factor, which is a tumor angiogenic factor, up‐regulated TM expression in HUVECs and enhanced cell adhesion and tube formation, while TM knockdown reduced these cellular behaviors. In subcutaneous B16F10 melanoma tumors of mice, immunofluorescence staining showed that TM was highly expressed and co‐localized with fibronectin on the tumor blood vessels. Moreover, the proximity ligation assay confirmed the interaction between TM and fibronectin on the tumor vasculature.ConclusionTM binds to fibronectin and promotes cell adhesion, migration, invasion, and FAK activation, thereby enhancing angiogenesis. TM may represent a new target for anti‐angiogenic therapy in tumors.
Bioluminescent Assay for GTPases Allows Measurement of GTPase, GAP and GEF Activities
Authors: Mondal Subhanjan , Hsiao Kevin , Goueli Said 
Published: November 2023

Small‐GTPases and heterotrimeric GTPases, play a major role in various cellular functions such as cell signaling, cell proliferation, cell differentiation, cytoskeleton modulation and cell motility. Deregulation or mutation of these proteins resulting in serious pathological conditions. Targeting GTPases and its regulators have been challenging due to lack of convenient assays. To overcome the challenges in analyzing activities of GTPases and their regulatory proteins GTPase Activating Proteins (GAP) and Guanine Nucleotide Exchange Factors (GEF) we have developed a homogenous bioluminescent assay system to analyze these proteins in a simple, convenient “add‐mix‐read” format.The assay consists of optimized reaction buffers that allow continuous progression of the GTPase cycle and hydrolysis of GTP. The assay relies on enzymatic conversion of GTP remaining after the GTPase reaction to ATP and bioluminescent detection of the ATP. We show that the assay is sensitive, and robust when analyzing for analyzing intrinsic GTPase‐activity, GAP‐stimulated GTPase‐activity, GAP‐activity and GEF‐activity for various small GTPases. Heterotrimeric GTPases (Ga) are involved in the GPCR signaling pathway, where GPCRs acts as GEFs and RGS family proteins acts as GAPs for Ga. We also use the assay to monitor activities of Ga proteins. The assay has minimal false hits when tested for compound interference using the library of pharmacologically active compounds indicative of the robustness in identifying small molecule modulators of GTPase using high throughput screening.
Determinants in the C‐terminal region of Gα12 and Gα13 allow distinct mechanisms of cell growth signaling
Authors: Fleming Todd L. , Meigs Thomas E. 
Published: November 2023

The G12/13 class of heterotrimeric G proteins, composed of the α subunits Gα12 and Gα13, is implicated in a variety of receptor‐mediated cellular responses that include cell proliferation and cytoskeletal rearrangements. Gα12 and Gα13 are well‐characterized activators of serum response element (SRE) mediated transcription, a pathway important in cell growth signaling. Invertebrate homologs of the G12/13 class harbor ability to drive cytoskeletal changes, but their signaling in cell growth has not been studied. We found the Drosophila and Caenorhabditis elegans α subunits, Concertina (Cta) and Gpa‐12 respectively, to lack ability to drive SRE signaling as mutationally activated forms expressed in cultured human cells. To identify determinants of growth signaling that evolved in Gα12 and Gα13, we assembled chimeras using regions of Cta and Gpa‐12 in place of corresponding sections of the mammalian α subunits. The C‐terminal regions of Gα12 and Gα13 were found to utilize distinct structural features for driving SRE‐mediated transcription. Insertion of a 45 amino acid region of Cta fully disrupted Gα12 signaling to SRE, whereas the same Cta substitution had no effect on Gα13 signaling. We identified key amino acids in this region required for Gα12 signaling to SRE, along with Gα12 target proteins that may require these residues for binding. We also generated Gα12 and Gα13 chimeras using regions of Gpa‐12, and found Gα13 harboring the Gpa‐12 C‐terminus to exhibit normal SRE signaling. However, insertion of Gpa‐12 sequence adjacent to the switch III region fully disrupted Gα13 signaling to SRE. These findings reveal key growth signaling determinants in the G12/13 C‐terminal regions, and indicate that Gα13 utilizes structural features distinct from Gα12 in driving SRE‐mediated transcription.Support or Funding InformationWe acknowledge support from the North Carolina GlaxoSmithKline Foundation and C.D. Spangler Foundation, Lineberger Comprehensive Cancer Center, and UNC‐Asheville Undergraduate Research Program.
Cardiovascular development defects produced by combined loss of RGS6 and oxidizable CaMKII due to defective Notch signaling
Authors: Chakravarti Bandana , Yang Jianqi , Ahlers Katelin , Luo Zili , Flaherty Heather A. , Meyerholz David , Anderson Mark E , Fisher Rory A. 
Published: November 2023

Congenital heart defects (CHDs) are the most common birth defects with nearly 40,000 infants born annually with a CHD in the U.S. alone. There is a critical lack of understanding of the etiology of CHDs and therefore, effective therapeutic strategies for this disease. Emerging evidence has implicated defective Notch signaling as the major cause of embryonic death of mice due to cardiovascular defects. RGS6 is abundantly expressed in the heart and mediates doxorubicin (Dox)‐ and alcohol‐induced myocardial cell apoptosis and cardiomyopathies by functioning as an upstream activator of NADPH oxidase (Nox) and consequent reactive oxygen species (ROS) generation. Oxidative activation of the Ca2+/calmodulin‐dependent protein kinase II δ (CaMKIIδ) on two key methionines in response to ROS generation in heart promotes cardiac disease including cardiomyopathy and cell death. While interrogating the relationship between RGS6 and CaMKII oxidation in heart we discovered a severe defect in embryonic cardiovascular development due to combined loss of RGS6 and ox‐CaMKII. To determine the function of RGS6 and ox‐CaMKII in embryonic cardiovascular development and the underlying mechanisms responsible for cardiovascular defects due to their combined loss, we developed double mutant (RGS6−/−/CaMKIIVV) mice by intercrossing of knock‐in mice expressing an oxidation resistant form of CaMKIIδ (CaMKIIVV) and RGS6−/− mice. Here, we show that RGS6−/−/CaMKIIVV mice undergo midgestational embryonic lethality associated with cardiac, hematopoietic, and vascular remodeling defects in placentas, yolk sacs, and embryos. Combined loss of RGS6 and ox‐CaMKII promoted endothelial cell apoptosis in hearts and decreased the expression of arterial markers and Notch signaling genes including Efnb2, Gja4, Gja5, Notch1, Jag1, Hey1, Hey2 and Hey1L in both yolk sacs and embryos. In conclusion, RGS6 and ox‐CaMKII are required together for cardiovascular development and function in parallel pathways as critical upstream modulators of Notch signaling.Support or Funding InformationNIH CA161882, AHA 14GRNT20460208 [RAF] and AHA 11SDG7580008 [JY]
Regulation of Inflammation, Innate Immunity and Intestinal Homeostasis by HOIL1
Authors: MacDuff Donna Alexandra , Reese Tiffany A , Baldridge Megan T , Kimmey Jacqueline M , Weiss Leslie A , Song Christina , Nice Timothy J , Carrero Javier , Colonna Marco , Edelson Brian T , Sibley David , Stallings Christina L , Casanova Jean‐Laurent , Iwai Kazuhiro , Virgin Skip 
Published: November 2023

The Linear Ubiquitin Chain Assembly Complex (LUBAC), composed of HOIL1, HOIP and SHARPIN, is an important regulator of NFκB activation in response to multiple stimuli. In humans, HOIL1 deficiency is associated with an immune disorder involving auto‐inflammation, immunodeficiency and inflammatory bowel disease (IBD)‐like symptoms. We found that barrier‐raised Hoil‐1−/− mice are profoundly immunodeficient after acute infection with Listeria monocytogenes, Toxoplasma gondii or Citrobacter rodentium. Increased susceptibility to Listeria was due to a role for Hoil‐1 in hematopoietic cells and macrophages in production of protective cytokines. In contrast, Hoil‐1−/− mice showed enhanced control of chronic Mycobacterium tuberculosis or murine γ‐herpesvirus 68 (MHV68), and these infections conferred a hyper‐inflammatory phenotype. Surprisingly, chronic infection with MHV68 complemented the immunodeficiency of Hoil‐1−/− and IL‐6−/− mice in response to Listeria. Thus chronic herpesvirus infection generates signs of auto‐inflammation and effectively complements genetic immunodeficiency in mutant mice, highlighting the importance of accounting for the virome in genotype‐phenotype studies.Furthermore, we found that susceptibility to enteric Citrobacter rodentium was due to a defect in innate immunity that resulted in spread of the bacteria to systemic sites early, and in ulceration of the distal colon late in the infection. However, induction of IL‐22, and other inflammatory cytokines in the distal colon, was not significantly impaired. Additionally, we found that Hoil1−/− mice exhibited defective control of persistent enteric murine norovirus (MNV) infection, similar to interferon‐λ (IFNλ) receptor‐deficient mice, and Hoil1 was essential for the induction of IFNλ and IFNβ in bone marrow‐derived dendritic cells in response to MNV infection. Finally, we found that specific pathogen‐free Hoil1−/− mice exhibited elevated Type 2 inflammatory cytokines in intestinal tissue, which drove morphological changes in intestinal epithelial cells in a STAT6‐dependent manner, and was partially ameliorated by antibiotics treatment. These data indicate that Hoil1 regulates inflammation and intestinal homeostasis in response to commensal microbes and enteric pathogens, and that HOIL1 disfunction may be an important contributor to inflammatory bowel disease pathology.Support or Funding InformationSupported by the Crohn's and Colitis Foundation Genetics Initiative.
Analysis of bacterial micro‐biome on potato tubers treated with sprout regulator 1,4‐Dimethylnaphthalene
Authors: Diaz Rene Alfonso , Patel Ravi Narendra , Campbell Michael A 
Published: November 2023

The compound 1,4 dimethyl naphthalene (DMN) has been shown to be an effective inhibitor of sprouting when applied to stored potato tubers. DMN has also been shown to be a growth inhibitor of bread yeast (Saccharomyces cerevisiae). Thus, we hypothesize that DMN would alter the micro‐biome that exists on the surface of stored potato tubers following treatment for sprout suppression. Potatoes were cut in half with one piece treated with DMN and the other piece exposed to an identical volume of sterile water as a control. DMN surface residues were determined after 48 hrs of treatment. The amount DMN residue deposited on the uncut surface was 4.2 ± 1.1ppm, which is similar to levels used for sprout inhibition. Deposition of DMN on the cut surfaces was much lower (1.54 ±0.65 ppm (n=33)), which may be a function of the low solubility of DMN in water. Periderm disks from the cut and uncut tuber surfaces were then frozen in liquid nitrogen and stored at −80°C. A Power Soil DNA Isolation Kit (mobio.com) was used to isolate DNA from the frozen samples. DNA was quantified and then used for PCR using primers specific to bacterial species (V3, V4). PCR products were quantified, checked by gel electrophoresis and then sequenced using Illumina technology. The total number of sequences generated was 3187447. The software program Mothur (v1.36) was used to determine the level of microbial diversity demonstrating that there were 20527 unique sequences that functioned as operational taxonomic units. Homogeneity of molecular variance (HOMOVA) was used to analyze the population of bacteria. There was no statistical difference between the cut and uncut surfaces of potato tubers (p=0.452). However, our alpha analysis for the skin of the potato did show a difference (p=0.017) between populations of uncut control and uncut DMN. Also, comparing alpha diversity between DMN cut and uncut, there appears to be a difference in population (p‐value=0.0001). We conclude that DMN does have an impact on the microbial diversity on potato tubers.Support or Funding InformationPenn State Behrend, 1,4‐Group
Isolation of Bio Energy Crop Phyllosphere Bacteria from Switchgrass
Authors: Sleda Melissa Ann , Grady Keara , O'Brien Keelyn , Bennett Allison , Shade Ashley 
Published: November 2023

The aerial surface of plants, known as the phyllosphere, represents the largest environmental surface area of microbial habitation on the planet, with an estimated surface of 108 km2 globally. The phyllosphere of bioenergy crops, such as switchgrass (Panicum virgatum), are of particular interest for bioprospecting for efficient microbial biomass degraders because of the potential for microbial adaptation to exploit leaf surfaces. The purpose of our study is to curate an isolate collection of switchgrass phyllosphere bacteria for genomic interrogation and future study of phyllosphere microbial interactions. We hypothesized that different cultivation techniques yield phylogenetically unique isolates that have specialized functions (and thus genetic pathways) to support their leaf lifestyle or have benefit for their host. These functions could include tolerance to dessication, radiation, and nutrient limitations which are important stressors on leaf surfaces. We collected leaves from switchgrass at the Great Lakes Bioenergy Center's intensive field plots (Kellogg Biological Station, Hickory Corners, MI). We washed cells from leaves and then used targeted plate‐based cultivation conditions, including several different types of media, to target phyllosphere microbial growth. We then sequenced the isolates using Sanger sequencing of the 16S rRNA gene, a marker of bacterial and archaeal phylogenetic diversity. We compared switchgrass phyllosphere gene sequences to 16S rRNA sequence databases to make taxonomic identifications of our isolates. To date, our collection includes sixty switchgrass isolates including genera belonging to Sphingomonas, Pseudomonas, Pantoea, Luteibacter, Arthrobacter, and Rhizobium. Some of our isolates can degrade cellulase. This data will complement a seasonal time course of cultivation‐independent assessment of phyllosphere diversity. As a result of this research, we expect to quantify the intra‐annual variability of switchgrass phyllosphere microbial communities and to identify core switchgrass phyllosphere members. This will help set the course for predicting and managing the dynamics of crop microbiota for the hosts' benefit.Support or Funding InformationMichigan State University and the Great Lakes Bioenergy Research Center for funding the research project. The Great Lakes Bioenergy Research Center for travel funds.