bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessing Social Phenotypes of M. xanthus Following Homologous Recombination of Arg‐Kinase Genes
Authors: Grady Mark , Fraga Dean , Kratt Shelby
Published: November 2023

In many eukaryotic cell systems, phosphagen kinases help modulate ATP levels during rapid energy consumption. In the past decade, more than two dozen bacterial species have been identified which express Arginine Kinases (AKs) that are homologous to those of invertebrate eukaryotes. Myxococcus xanthus is a model single‐celled myxosporulating organism which demonstrates social cooperation to acquire and conserve resources; this is exemplified by fruiting bodies which sustain spores, and rippling bodies which cover new territory and swarm around prey. The gene MXAN2252 has been identified in M. xanthus and Bragg et al (2012) determined that it expresses a protein “Ark” that can act to buffer arginine and ATP levels. While the deletion of MXAN2252 was nonfatal and did not impair vegetative growth in strain MS2252‐ΔAK, colonies are noticeably impaired at fruiting body formation when rapidly starved.

The main hypothesis of this work is that closely homologous AK genes will facilitate the social competence that is characteristic among Myxococcus strains. Homologous recombination has been used to insert the wildtype Ark gene (MXAN2252) and a homologous AK gene from invertebrate L. polyphemus. Strains with these recombined genes will then be tested to determine if and to what extent, relative to the wild type and the ΔAK strain, they are restored in developmental phenotypes of fruiting body formation and recovery from environmental stressors like acidity and osmolarity changes.

Support or Funding Information

This research was conducted with frequent collaboration with Dr Mitch Singer and expertise from his laboratory at UC Davis. Summer research in this subject was funded by the Copeland Fund for from the College of Wooster.
MiRNA‐21 mediates the antiangiogenic activity of metformin through targeting PTEN and SMAD7 expression and PI3K/AKT pathway
Authors: Luo Mao , Tan Xiao Yong , Wan Qin , Wu Jianbo
Published: November 2023

Background

Metformin, an anti‐diabetic drug commonly used for type 2 diabetes therapy, is associated with anti‐angiogenic effects in conditions beyond diabetes. miR‐21 has been reported to be involved in the process of angiogenesis. However, the precise regulatory mechanisms by which the metformin‐induced endothelial suppression and its effects on miR‐21‐dependent pathways are still unclear.

Methods

Bioinformatic analysis and identification of miR‐21 and its targets and their effects on metformin‐induced antiangiogenic activity were assessed using luciferase assays, quantitative real‐time PCR, western blots, scratch assays and CCK‐8 assays.

Results

In this study, miR‐21 was strikingly downregulated by metformin in a time‐ and dose‐dependent manner. miR‐21 directly targeted the 3′‐UTR of PTEN and SMAD7, and negatively regulated their expression. Overexpression of miR‐21 abrogated the metformin‐mediated inhibition of endothelial cells proliferation, migration, and the TGF‐β‐induced AKT/GSK‐3β, SMAD‐ and ERK‐dependent phosphorylations, and conversely, down‐regulation of miR‐21 aggravated metformin's action and revealed significant promotion effects.

Conclusions

Our study broadens our understanding of the regulatory mechanism of miR‐21 mediating metformin‐induced anti‐angiogenic effects, providing important implications regarding the design of novel miRNA‐based therapeutic strategies against angiogenesis.

Support or Funding Information

This work was supported by the American Heart Association Scientist Development Grant (10SDG2570037), National Natural Science Foundation of China Grant (81172050, 81570263)
mTORC1 balances cellular amino acid supply with demand for protein synthesis through post‐transcriptional control of ATF4
Authors: Thoreen Carson C , Park Yeonwoo
Published: November 2023

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth that is commonly deregulated in human diseases. Here we find that mTORC1 controls a transcriptional program encoding amino acid transporters and metabolic enzymes through a mechanism also used to regulate protein synthesis. Bioinformatic analysis of mTORC1‐responsive mRNAs identified a promoter element recognized by activating transcription factor 4 (ATF4), a key effector of the integrated stress response. ATF4 translation is normally induced by phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α) through a mechanism that requires upstream open reading frames (uORFs) in the ATF4 5′ UTR. mTORC1 also controls ATF4 translation through uORFs, but independent of changes in eIF2α phosphorylation. mTORC1 instead employs the 4E‐binding protein (4E‐BP) family of translation repressors. These results link mTORC1‐regulated demand for protein synthesis with an ATF4‐regulated transcriptional program that controls the supply of amino acids to the translation machinery.
Encoding the Human Phosphoproteome in an Engineered Bacterial System
Authors: Barber Karl , Rinehart Jesse
Published: November 2023

Phosphorylation can potently alter protein folding and function, such as by modulating enzymatic activity, exposing scaffolding domains, or directly recruiting interaction partners. Although serine phosphorylation is the most common post‐translational modification in eukaryotes, the significance of the vast majority of phosphorylation sites across the human proteome remains unknown. Using an orthogonal translation system for phosphoserine (SepOTS), we are able to co‐translationally incorporate phosphoserine as a non‐standard amino acid in E. coli. We demonstrate the ability to synthesize and observe phosphoproteome‐scale peptide libraries representing tens of thousands of human phosphoproteins in a single experiment. We then deploy this massive‐scale peptide library to identify physiologically‐relevant phosphobinding protein interaction partners. Our synthetic human phosphoproteome is uniquely poised to answer lingering questions regarding the functional role of phosphorylation in signal transduction pathways by simultaneously monitoring thousands of human phosphosites.

Support or Funding Information

K.W.B. is supported by the National Science Foundation Graduate Research Fellowship under Grant No. DGE‐1122492. J.R. is supported by NIH R01 GM117230.
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 Information

American 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 Information

This 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 Information

NIH/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.

NIH

Support or Funding Information

NIHGM68849
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 Information

Johns Hopkins University

National 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

Objectives

Cell 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.

Methods

The 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.

Results

We 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.

Conclusion

TM 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.
Structural and functional insights into the latrophilin3 ‐ FLRT3‐UNC5D complex that mediates glutamatergic synapse development
Authors: Ranaivoson Fanomezana Moutse , Ozgul Sinem , Kakehi Sumie , Jackson Verity , Martini Francesca , Bergami Francesco , von Daake Sventja , Seiradake Elena , Comoletti Davide
Published: November 2023

Latrophilins (LPHNs) are neuronal adhesion‐type G‐protein coupled receptors able to establish cell‐surface interactions with other proteins through their characteristically large ectodomain. Fibronectin leucine‐rich repeat transmembrane (FLRT) proteins constitute such endogenous ligands and the LPHN3‐FLRT3 trans‐cellular interaction has been shown to play an important role in glutamatergic synapse development in vitro and in vivo. With the use of a series of biochemical and biophysical techniques including analytical ultra‐centrifugation, deuterium‐exchange mass spectrometry, isothermal titration calorimetry and protein crystallography, we have performed an extensive characterization of the LPHN3‐FLRT3 interaction. In particular, 1) we have defined the binding domain of each partner to be the olfactomedin (OLF) domain in LPHN3 and the Leucin‐Rich Repeats (LRR) in FLRT3, 2) we have determined the crystal structure of the LPHN3‐OLF in two crystal forms, and 3) we have solved the crystal structure of the complex between LPHN3‐OLF and FLRT3‐LRR. In addition, 4) we have recently described higher‐ordered complexes formed by the LPHN‐FLRT sub‐complex and UNC5D, a receptor known to be implicated in axon guidance. Finally, 5) we are currently trying to identify novel ligands for some of these proteins. This work will enable us to understand, at the molecular level, the complex interplay of these proteins and their role in axon guidance and synapse formation.
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 Information

We acknowledge support from the North Carolina GlaxoSmithKline Foundation and C.D. Spangler Foundation, Lineberger Comprehensive Cancer Center, and UNC‐Asheville Undergraduate Research Program.
Identification of Endothelial Cell Specific RhoGAPs that Influence TCL Activation and Localization
Authors: Florke Rebecca R , Hamann Michael J
Published: November 2023

TCL (RhoJ) is a Rho family GTPase that is involved in cytoskeletal dynamics and angiogenesis. TCL is predominantly expressed in endothelial cells, where it contributes to the projection and lumenization of new vessels. TCL also has a unique role in the establishment and maintenance of stable tumor vasculature, which suggests that this GTPase could be a viable target in directed chemotherapy. However, the molecular pathways, particularly the GEFs and GAPs, regulating TCL activity are not well known. In order to identify GAPs that deactivate TCL, a bimolecular fluorescence complementation (BiFC) assay was designed using the fluorescent protein Venus. TCL and the GTPase binding portion of PAK were genetically fused to the two Venus fragments to detect GTP‐loaded TCL, and the assay was validated using known GAP/GTPase pairs. Initial experiments have identified BCR and MgcRacGAP as endothelial cell specific GAPs that decrease TCL activity. Additional experiments will examine how these GAPs influence TCL cellular localization. Identifying these RhoGAPs will help elucidate the molecular pathways involved in the regulation of TCL.
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 Information

NIH CA161882, AHA 14GRNT20460208 [RAF] and AHA 11SDG7580008 [JY]
δ‐Lactone Derivatives Induce Quorum Sensing Activity in Agrobacterium tumefaciens
Authors: Ewald Dawn A , Champion Ana K , Fry Michelle R
Published: November 2023

Previous work by Andersh et. al has shown that synthetic δ‐lactone derivatives exhibit bacteriostatic activity against Acinetobacter lwoffii and Bacillus subtilis. In this study, we probe the effects of the δ‐lactone derivatives on the process of quorum sensing due to the structural similarity of these compounds to homoserine lactones, quorum sensing autoinducers in Gram‐negative bacteria. Quorum sensing is under investigation in the area of antibiotic development, because quorum sensing modifiers may exhibit weaker evolutionary pressures on the bacteria to develop drug resistance. The quorum sensing activity of four chlorine‐substituted δ‐lactone derivatives were quantified using UV‐Vis absorbance on an engineered biodetector strain of Agrobacterium tumefaciens NTL4(pZLR4). Sub‐millimolar concentrations of the δ‐lactones were shown to enhance the quorum sensing activity of A. tumefaciens natural autoinducers, trigger this response in the absence of cognate autoinducer, and decrease bacterial growth. We are analyzing the effect of the δ‐lactone derivatives on the ability of A. tumefaciens to induce Crown Gall tumorigenesis in potato slices.
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 Information

Supported 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 Information

Penn State Behrend, 1,4‐Group
Characterization of a Double Deletion Mutant of Fusarium verticillioides Lacking Two Putative Trehalose‐6‐Phosphate Phosphatase Genes
Authors: McFarlin Brandon , Roberts Ethan S. , Remsen Edward E. , McGovern Christopher M. , Brown Daren W. , McQuade Kristi L.
Published: November 2023

Fusarium verticillioides is a fungal pathogen that commonly infects the stalk, ear, and kernels of corn and can produce fumonisins, a family of mycotoxins linked to disease in livestock and humans. Our goal is to characterize the role of the disaccharide trehalose in growth and stress response in F. verticillioides. Trehalose synthesis in fungi occurs via two steps, the first catalyzed by trehalose‐6‐phosphate synthase (TPS), and the second by trehalose‐6‐phosphate phosphatase (TPP). Previously, we found that trehalose synthesis is completely abolished in a strain lacking TPS, encoded by the TPS1 gene. We report here that mutant strains lacking either one (ΔTPS2) or both (ΔTPS2/ΔTPS3) of the putative TPP genes in F. verticillioides retain the ability to produce trehalose, albeit at reduced levels. The effects of osmotic and thermal stress on these mutants are being investigated.
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 Information

Michigan State University and the Great Lakes Bioenergy Research Center for funding the research project. The Great Lakes Bioenergy Research Center for travel funds.
Expression and Purification of a Novel Calcium Binding Protein Necessary for Phytopathogenesis in Xanthomonas strain
Authors: Margin Kayla Marie , Quay Juliana C , Minsavage Gerald V , Jones Jeffrey B , Hurlbert Jason C
Published: November 2023

Recently, we have identified a gene whose sequence is conserved in several species of Xanthomonas that, when expressed, elicits a hypersensitive response (HR) in tomato plants. Normally, HR elicitation is limited to very specific bacterial‐host pairings, but introduction and expression of this gene into Escherichai coli, a bacterial species that does not normally infect tomato plants, serves to elicit HR, indicating that the encoded protein is crucial to the infectious process. Bioinformatic analysis of the protein, which we have named EfhX (EF‐Hand containing protein from Xanthomonas) reveals that the protein is predicted to contain a single transmembrane α‐helix, spanning amino acids 60 to 81, and two calcium binding domains, termed EF‐Hands, in the carboxy‐terminal domain of the protein. In order to better understand the function of this novel protein, we have cloned the efhX gene from Xanthomonas aurantofolia and expressed it in Escherichia coli so as to obtain quantities of pure protein sufficient to grow protein crystals and determine the structure of the protein via x‐ray diffraction. We have successfully purified the protein to homogeneity (>95%) as determined by SDS‐PAGE and anti‐hexahistidine Western Blot. We have initiated crystallization trials to identify solution conditions suitable for growth of crystals for x‐ray diffraction experiments so that the structure of the protein can be determined.
The Characterization of Septoria lycopersici Pathogenicity in Micro Tom Tomatoes
Authors: Allen Katelyn E , Zwiesler‐Vollick Julie
Published: November 2023

Septoria lycopersici is a foliar fungal pathogen of tomatoes. This pathosystem has been well characterized with respect to the tomato antimicrobial secondary metabolite, alpha‐tomatine and the fungal detoxifying enzyme, tomatinase. However, there are many gaps in our knowledge of other aspects of the S. lycopersici pathogenicity. We know that fungal spores germinate on tomato leaves and grow biotrophically. The hyphae then invade the leaf apoplast via open stomata or wounds. At some point, S. lycopersici begins to damage the surrounding plant tissue. One of the aims of this project is to characterize the process of fungal infection with light, confocal fluorescence and scanning electron microscopy. This will provide the basis for further investigations of the key virulence factors present in S. lycopersici as well as potential defense mechanisms. We will also be assessing the ability of S. lycopersici to infect the dwarf tomato cultivar, Micro Tom. If Micro Tom tomatoes are susceptible, they could become the preferred host cultivar, due to their rapid life cycle. If Micro Tom tomatoes are not susceptible, this provides a source for possible resistance genes. In addition, the closely related wheat fungal pathogen, Mycosphaerella graminicola has been reported to be dimorphic with only the filamentous form able to infect plant hosts. The switch from yeast‐like to filamentous growth is regulated by a mitogen‐activated protein kinase. There are also reports that the switch to filamentous growth can be controlled by temperature. We will be characterizing the growth phase of S. lycopersici at various temperatures to determine if this fungal pathogen is also dimorphic.

Support or Funding Information

Lawrence Technological University Department of Natural Sciences
Enhanced Spare Respiratory Capacity in Extremophile Fish Following Exposure to Hydrogen Sulfide
Authors: Henpita Chathurika R , Tobler Michael , Shaw Jennifer H
Published: November 2023

Hydrogen sulfide (H2S) is well known as an environmental toxic gas produced by the decomposition of organic matter and geothermal sources. H2S is also produced endogenously in organisms as a result of cysteine catabolism. Exposure to H2S drives hormetic effects including the toxic inhibition of cytochrome c oxidase of the mitochondrial respiratory chain at high concentrations, and maintenance of normal vascular and neural functions at low concentrations. Physiological levels of H2S are necessary for blood pressure regulation, angiogenesis, and mitigating oxidative and inflammatory stress. Abnormal elevation of cellular H2S is correlated with vascular, metabolic and neurological dysfunction in most terrestrial and aquatic organisms, however there are some organisms such as the lugworm (Arenicola marina), tubeworms (Riftia pachyptila) and Atlantic molly (Poecilia mexicana) that thrive in extreme environments with high H2S concentrations. The mechanisms whereby organisms can tolerate extreme H2S are not fully understood. Since mitochondria are both the site of H2S toxicity as well as enzymatic detoxification, we sought to compare mitochondrial function in P. mexicana fish derived from two populations: sulfide‐tolerant and non‐tolerant, depending on the presence or absence of H2S in their habitat. We hypothesized that sulfide‐tolerant fish would reduce their oxygen consumption relative to the non‐tolerant population following H2S exposure. We further hypothesized that the wild captured sulfide‐tolerant population would maintain a greater degree of H2S tolerance compared to the lab‐reared sulfide‐tolerant population, which has not been exposed to H2S in several generations. Mitochondrial oxygen consumption rate (OCR) was measured in tolerant and non‐tolerant populations following seven doses of NaSH (H2S donor) using the Seahorse Biosciences XFe 96 coupling assay. Our key findings are that mitochondria from sulfide‐tolerant fish exhibited, i) a reduction in OCR under basal conditions and following exposure to low concentrations of H2S, ii) an increase in spare respiratory capacity following exposure to high concentrations of H2S and, iii) a greater degree of tolerance in the wild captured population, relative to lab‐reared.
Kinetic Characterization of 3‐Hydroxyisobutyrate Dehydrogenase from Arabidopsis thaliana
Authors: Binkley Christiana , McCool Mason , Maurer Taylor , Rouhier Kerry A
Published: November 2023

Branched‐chain amino acids (BCAAs) initially share a similar metabolic pathway. However, their paths diverge, resulting in unique catabolic pathways for each BCAA. This lab examined valine degradation, as it is the least characterized of all the BCAAs. During valine metabolism, the enzyme 3‐hydroxyisobutyrate dehydrogenase (HIBDH) catalyzes 3‐hydroxyisobutyric acid into methylmalonic semialdehyde as one of the concluding reactions. While HIBDH from Pseudomonas denitrificans had previously been cloned and expressed in Escherichia coli, the goal of this research was to examine the activity of HIBDH in plants. Thus, we cloned the HIBDH gene from Arabidopsis thaliana into E.coli, where its activity could be examined. Two HIBDH constructs were created in order to determine proper exclusion of the mitochondrial leader sequence; these constructs differ only by the length of their amino acid sequences. The expressed proteins were then purified by affinity chromatography and kinetically characterized. The construct composed of nucleotides 97–1044 (denoted At316) resulted in ~9 mg/mL of purified protein, while the construct generated from nucleotides 115–1044 (denoted At310) produced ~1 mg/mL from a similar volume and density of cells at the point of induction. Both constructs resulted in active protein using S‐HIBA (native substrate) and NAD, although very little activity was seen using construct At310. Construct At316 functioned optimally under alkaline conditions similar to HIBDH from P. denitrificans and Homo sapiens. Overall, the biochemical properties of HIBDH in A. thaliana were assessed, offering new insights into both the valine degradation pathway as a whole, as well as the biological and chemical parameters necessary for HIBDH function in plants.

Support or Funding Information

This project was funded by Kenyon College.
Subtle Defects of Four Homocystinuric Variants of Human Cystathionine β‐Synthase
Authors: Hines Judy P , Su Yang , Smith Aaron T. , Burstyn Judith N
Published: November 2023

In humans, CBS‐deficient homocystinuria (CBSDH) results from mutations in the gene encoding cystathionine β‐synthase (CBS), a heme‐containing PLP‐dependent enzyme that catalyzes condensation of serine and homocysteine to form cystathionine. The role of the heme in CBS is unknown; however, disruption of the native heme iron‐to‐cysteine thiolate bond eliminates enzyme activity even though the heme does not participate in catalysis. Four CBSDH‐associated single amino acid variants within the heme pocket, P49L, P78R, R125Q and R266K, were investigated through studies of their heme environments and catalytic properties. Electronic absorption, resonance Raman, and electron paramagnetic resonance spectroscopic studies revealed that there are subtle changes to the heme environment in these variants, consistent with changes in hydrogen bonding or local heme pocket structure. Most variants exhibited a less stable heme pocket, susceptible to thermal unfolding at lower temperatures than for the wild‐type protein. Variant proteins were often more stable to thermally mediated aggregation resulting from global protein unfolding. Kinetic characterization revealed that the allosteric activator S‐adenosylmethionine (AdoMet) lowered Km for both substrates but had little effect on kcat for the wild‐type protein, a result consistent with the current structural model for AdoMet activation. P49L, P78R and R266K exhibited subtle changes in their kinetic constants and were AdoMet responsive. In contrast, R125Q exhibited a significantly decreased kcat value and was unresponsive to AdoMet activation. The correlation between altered heme environment and catalytic activity suggests that the heme plays an important regulatory role in this protein.

Support or Funding Information

University of Wisconsin‐Madison