bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transport and synthesis of a bacterial natural product
Authors: Dassama Laura M. K. , Kenney Grace E. , Rosenzweig Amy C. 
Published: June 2021

Nature is replete with natural products that are pharmacological agents, secondary metabolites, and chemical agents, among others. A common class of natural products is those that bind metals and are taken into cells to fulfill the cell's metal needs. A large majority of these metallo‐natural products bind iron and are referred to as siderophores (iron “carriers”). Similar molecules that bind copper rather than iron have been discovered recently. These molecules were initially isolated from the growth media of methanotrophic bacteria (bacteria that metabolize methane), which have an elevated copper requirement. These “chalkophores” (copper “carriers”) have been termed methanobactins (Mbns). Mbns are secreted when methanotrophic bacteria are cultured under conditions of limiting copper. Mbns acquire copper from the environment and are taken up into the cell. Mbns have a high affinity for copper, and this has sparked interest in their potential use for the treatment of diseases of toxic copper overload. Since its discovery over a decade ago, the mechanisms of Mbn synthesis, transport and its contribution(s) to the overall metal homeostasis of methanotrophic bacteria have been the focus of intensive investigations. This work describes the enzyme machinery responsible for its transport and biosynthesis.Support or Funding InformationNIH grants GM118035 (A.C.R) and F32GM110934 (L.M.K.D.), NSF grant MCB0842366 (A.C.R.), AHA 14PRE20460104 (G.E.K), and a Burroughs Wellcome Fund PDEP award (L.M.K.D.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Altering the Specificity Properties of 2‐(2′‐hydroxyphenyl)benzenesulfinate desulfinase from N. asteroides A3H1
Authors: Smith Emily Elizabeth , Hoang Dylan , Cromwell Daniel , Watkins Linette 
Published: June 2021

Biodesulfurization is a metabolically important process that occurs naturally in bacterial cells found near oil and coal deposits. In sulfur‐specific biodesulfurization, the dsz pathway, enzymes selectively remove the organic sulfur found in crude oil without disrupting the carbon backbone. The rate‐limiting step within this metabolic pathway is the conversion of 2‐(2′‐hydroxyphenyl)benzenesulfinate (HPBS) to 2‐hydroxybiphenyl using HPBS desulfinase (DszB). The DszB enzyme from Nocardia asteroides A3H1 was overexpressed in E. coli, purified and characterized kinetically. Homology studies identified an active site loop with conserved amino acids that could play a role in the specificity for different organosulfur compounds found in crude oil. Point mutations were generated to study the importance of amino acids 195 and 200 on the specificity of the enzyme. Three mutant enzymes (A195R, A200R and A195/200R) prepared using codon‐optimized gene constructs were co‐expressed with GroESL and purified from E. coli using Ni2+‐affinity chromatography. Substrate specificity experiments were performed to define the role of the active site loop on specificity. Kinetic measurements indicate a decreased specificity for HPBS. Fixed‐time sulfite assays indicate an increased preference for smaller ringed substrates, demonstrating the importance of amino acids 195 and 200 and defining the specificity of the enzyme.Support or Funding InformationSupported by NSF grant, CHE 1461175, and James Madison University Department of Chemistry and BiochemistryThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
1. A light inducible gene activation system toward controllable cell‐based therapeutics
Authors: Huang Ziliang , Wu Yiqian , Pan Yijia , Allen Molly , Chang Ya‐Ju , Chien Shu , Wang Yingxiao 
Published: June 2021

T cells engineered to express chimeric antigen receptors (CARs) on cell surface can recognize and engage with target cancer cells with redirected specificity. This CAR T cell‐mediated immunotherapy is under intensive investigation as a revolutionary therapy for cancer. However, adverse effects have been reported, including on‐target off‐tumor cytotoxicity, cytokine release syndrome and tumor lysis syndrome. One of the solutions is to engineer on/off‐switchable cells to achieve high‐precision control over their activation. Optogenetics utilizing genetically encoded dimerizers has been applied to remotely activate gene expression. Here, we developed a new blue light‐mediated light‐inducible nuclear translocation and dimerization (LINTAD) system for gene regulation to control cell activation, by integrating a LOV2‐based light‐inducible nuclear localization signal with CRY2‐CIB1 dimerization. We demonstrated light‐controllable gene expression and functional modulation in HEK 293T and Jurkat T cell lines. We further applied the LINTAD system in primary human CAR T cells and showed that the light‐stimulated cells possessed a significantly stronger cytotoxicity against target cancer cells than the non‐stimulated ones. We also extended the LINTAD system to activate the expression of the TNF‐related apoptosis‐inducing ligand (TRAIL) in HEK 293T cells, and observed a gain of cytotoxicity upon light stimulation. Therefore, our newly developed LINTAD system can serve as an efficient and general tool to remotely and non‐invasively control gene activation in live cells for therapeutic applications.Support or Funding InformationThis work is supported by grants from NIH HL121365, GM125379 (S.C., Y. Wang), CA204704, CA209629, NSF CBET1360341, DMS1361421 (Y. Wang), and UC San Diego.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biosynthesis of acyl‐CoAs sustains prostate cancer progression
Authors: Cai Houjian , Ma Yongjie 
Published: June 2021

Objective of the studyFatty acid metabolism is essential for the biogenesis of cellular components and ATP production to sustain proliferation of cancer cells. Bioconversion of exogenous or de novo synthesized fatty acids to their corresponding fatty acyl‐CoAs, which is required for the fatty acids to participate in metabolic processes, is catalyzed by long chain fatty acyl‐CoA synthetases (ACSLs). However, the influence of individual ACSL isoforms on the progression of prostate cancer remains unknown. The goal of this study is 1) to analyze how ACSLs regulates the biosynthesis of acyl‐CoAs, thereby prostate cancer progression.Statement of MethodsLentivirus carrying shRNA of ACSL1 was constructed and produced in house. Oxygen Consumption Rate (OCR) and Extracellular Acidification Rate (ECAR) were measured by Seahorse. The amount of protein was evaluated by immunoblotting. The amount of acyl‐CoAs was analyzed by LC‐MS/MS. The amount of triglyceride and neutral lipid were measured by gas Chromatography and Oil red O staining, respectively. TCGA database was used for analysis of ACSL1 expression levels in different stages of prostate cancer. Additionally, inhibition of cell proliferation, migration, invasion assays and cell cycle analysis were analyzed in numerous cancer cells. Prostate xenograft tumor models were applied for in vivo studies. Prostate tumor tissue array was utilized for the analysis of ACSL1 expression.Summary of the resultsIn this study, the correlation of ACSL expression levels and the amount of intracellular acyl‐CoAs was systematically analyzed. We demonstrate that expression levels of ACSL1 were significantly associated with the levels of a variety of acyl‐CoAs, and were elevated in human prostate tumors. ACSL1 increased the biosynthesis of fatty acyl‐CoAs including C16:0−, C18:0−, C18:1− and C18:2‐CoA, triglycerides and lipid accumulation in cancer cells. In addition, ACSL1 modulated mitochondrial respiration through regulation of carnitine palmitoyltransferase 1A (CPT1A) activity. Knockdown of ACSL1 expression inhibited the cell cycle, and suppressed the proliferation and migration of prostate cancer cells in vitro, and growth of prostate xenograft tumors in vivo.ConclusionOur study demonstrates ACSL1 as playing a role in prostate tumor progression. Our results provide a therapeutic strategy of targeting fatty acid metabolism for the treatment of prostate cancer.Support or Funding InformationNIH (R01CA172495) and DOD (W81XWH‐15‐1‐0507)
Characterizing the physiologic effects of exogenous ketone supplements – an alternative or adjuvant to the ketogenic diet
Authors: Poff Angela , Koutnik Andrew , Deblasi Janine , Rogers Christopher , Kesl Shannon , Ward Nathan , D'Agostino Dominic 
Published: June 2021

The ketogenic diet (KD) is a high fat, adequate protein, low carbohydrate diet that is used clinically for refractory pediatric epilepsy, and is under investigation as a treatment for a wide variety of disorders, including many neurological diseases, cancer, obesity, and diabetes. Studies suggest that many of the beneficial effects associated with the KD are mechanistically attributable to the ketone bodies, leading our lab and others to develop and test exogenous ketone supplements (KS). KS elevate blood ketones without the need for dietary restriction of carbohydrate. In order to further our understanding of this new technology, we sought to characterize the metabolic effects, safety, and toxicity of KS in healthy rats. KS tested include the R,S‐1,3‐butanediol acetoacetate diester, 1,3‐butanediol, medium chain triglyceride oil, caprylic acid, and beta‐hydroxybutyrate mineral salts, alone and in various combinations. Healthy rats were administered KS by oral gavage or in their food in an acute (once, or 7 days), sub‐chronic (1 month) or chronic (4 months) fashion and analyzed for a variety of metabolic and physiologic biomarkers. KS rapidly elevate blood ketones to therapeutic levels (2–5mM), and simultaneously decrease blood glucose. Some types of KS induce a mild calorie restriction effect, and preliminary evidence suggests ketone ester treatment during CR may preserve lean muscle mass in this catabolic state via a non‐insulin dependent mechanism. Satiety may be increased by KS as demonstrated by an elevation in leptin following KE administration, even when animals were self calorie‐restricting. KS significantly alters the global metabolome, indicating a shift from carbohydrate‐based to fat‐based metabolism. Some markers of this were an elevation in medium chain fatty acids, an increase in acetyl coA and acetylcarnitine suggestive of enhanced mitochondrial LCFA oxidation, and an increase in Kreb's cycle intermediates such as citrate, succinate, and fumarate. Inflammatory profiling following chronic, but not acute, KS administration revealed decreases in many pro‐inflammatory cytokines, including IL‐1β, IL‐6, IFN‐γ, MCP‐1, and RANTES. KS appears safe as there were no signs of toxicity or adverse changes in total cholesterol, HDL, LDL, triglycerides, or markers of liver/kidney function over the chronic treatment protocol. KS may be a useful alternative or adjuvant to the KD for disorders where nutritional ketosis is therapeutic.Support or Funding InformationOffice of Naval Research, Scivation Inc, Disruptive Nutrition, Epigenix FoundationThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Parathyroid Hormone Shows Novel Calcium Sensing Ability in Binding to Parathyroid Hormone 1 Receptor
Authors: Culhane Kelly J. , Yan Elsa C.Y. 
Published: June 2021

Family B G protein coupled receptors (GPCRs) are important drug targets for metabolic diseases, such as diabetes and osteoporosis. Despite this importance, the structure and function of family B GPCRs remain poorly understood. We have developed a nanodisc purification protocol to purify family B GPCRs for molecular studies. Here, we focus on parathyroid hormone 1 receptor (PTH1R). PTH1R regulates homeostasis of Ca2+ and phosphate and is a drug target for osteoporosis. It has two native ligands, parathyroid hormone (PTH) and parathyroid hormone related peptide (PTHrP). Truncated versions of both ligands, PTH(1–34) and PTHrP(1–36), can fully activate the receptor and PTH(1–34) is clinically used for osteoporosis treatments. In this study, we purified PTH1R in nanodiscs and used fluorescence anisotropy to examine ligand binding. We found that the affinity increases more than 5‐fold in the presence of 15 mM Ca2+ for PTH(1–34) but not PTHrP(1–36). This result carries physiological significance because PTH(1–34) but not PTHrP(1–36) is involved in bone remodeling process, in which Ca2+ concentrations can fluctuate up to 40 mM. We hypothesize that the Ca2+‐dependent binding is mediated by non‐conserved, negatively charged residues of both PTH(1–34) and PTH1R. In PTH(1–34), mutations of Glu19 and Glu22 that are not conserved in PTHrP(1–36) largely abolished the observed Ca2+ effect. In the receptor, mutations in residues Glu258, Glu259 and Glu260 in the extracellular loop 1 also largely abolish the Ca2+ effect. The results not only support the hypothesis but also demonstrate the unique ability of PTH(1–34) in sensing Ca2+ while activating PTH1R. This dual function of a peptide hormone is a novel observation in endocrine signaling. Understanding it can potentially reveal the complex role of PTH signaling in bone remodeling and improve the PTH(1–34) treatment for osteoporosis. Further investigations of the differences between PTH(1–34) and PTHrP(1–36) in response to Ca2+ during PTH1R activation will provide insights into molecular mechanism of ligand‐biased signaling process and allosteric interactions of family B GPCRs.Support or Funding InformationThis work was supported by an NSF career award to ECYY (MCB‐0955407) and KJC is supported by T32 GM008283‐27This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Calcium Dependent Properties of Ig 80–83 of N2A Titin
Authors: Thompson Blair Marie , Dutta Samrat , Monroy Jenna , Nishikawa Kiisa 
Published: June 2021

Titin is a giant elastic protein responsible for passive force generation in muscles. In addition, numerous studies have implicated a role for titin in active muscle. Recent work on muscles from mdm mice, suggests that during muscle activation, the N2A region of titin binds to actin thereby increasing titin and active muscle stiffness. Atomic force microscopy (AFM) can be used to measure the elastic properties of a titin construct, specifically by unfolding and refolding of its constituent immunoglobulin (Ig) domains. We hypothesized that the Ig domains from the N2A region of titin (Ig 80–83) exhibit different elastic properties in the presence and absence of calcium. To test this hypothesis, we stretched a N2A‐like construct consisting of Ig 80–83 and a Halo‐tag domain at different pulling velocities, in the presence and absence of calcium. Our results suggest a non‐linear unfolding energy landscape for serially linked Ig 80–83 domains in the presence of calcium. We also found, while calcium increased the persistence length of unfolded Ig domains in the N2A‐like construct, its contour length did not change. Increase in persistence length indicates an increase in the stiffness of the N2A domain, which supports the hypothesis that titin stiffness increases in the presence of calcium. These results differ from previous studies without calcium and indicate unique properties of Ig domains specific to the N2A region of titin.Support or Funding InformationAPS IOSP Fellowship Awarded to B. Thompson, NSF IOS‐1451650 Awarded to J. MonroyThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Insulin/IGF‐1 Enhances Intestinal Epithelial Crypt Proliferation through PI3K/Akt, and Not ERK Signaling in Obese Humans
Authors: Zhou Weinan , Rowitz Blair M. , Dailey Megan J. 
Published: June 2021

The intestinal epithelium plays important roles in nutrient absorption, satiety hormone release and immune barrier function. In order to ensure proper tissue actions are maintained, the cells are continuously regenerated through proliferation and differentiation of stem cells located in the intestinal crypts. Obesity affects this process and results in greater stem cell proliferation and abnormal tissue growth and function. Obesity‐induced high levels of insulin/insulin‐like growth factor‐1 (IGF‐1) in the stem cell niche are found to impact the changes in proliferation in rodents indicating that insulin/IGF‐1 receptors may play a role in modulating intestinal epithelial stem cell proliferation. Insulin receptor resistance is a hallmark of obesity that can be driven by an imbalance between or abnormal signaling within two downstream signaling pathways, PI3K and ERK, in non‐intestinal epithelial tissues. To determine whether insulin/IGF‐1 can induce proliferation in human intestinal epithelial stem cells and if PI3K or ERK pathways are involved, we used primary small intestinal epithelial crypts and investigated 1) the effect of insulin/IGF‐1 on crypt proliferation, and 2) the effect of insulin/IGF‐1 signaling inhibitors (i.e. PI3K/Akt pathway inhibitor Wortmannin and ERK pathway inhibitor PD98059) on insulin/IGF‐1‐induced crypt proliferation. We found that insulin/IGF‐1 enhanced crypt proliferation in obese humans. Inhibition of PI3K/Akt pathway attenuated insulin/IGF‐1‐induced crypt proliferation, but inhibition of ERK pathway had no effect. These results suggest that the classical metabolic PI3K pathway and not the canonical proliferation ERK pathway is involved in insulin/IGF‐1‐induced increase in crypt proliferation in obese humans. Pathway‐specific impairments in insulin/IGF‐1 signaling may contribute to reciprocal relationships between insulin/IGF‐1 resistance and intestinal epithelial stem cell proliferation and differentiation that fosters tissue dysfunction.Support or Funding InformationThis work was supported by USDA Hatch (to MJD) ILLU‐538‐926.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The Role of Sphingosine‐1‐Phosphate Activity in Liver Preservation Injury
Authors: Fyffe‐Freil Ria Christian , Limkemann Ashley , Lindell Susanne L. , Mangino Mangino J. 
Published: June 2021

Lipid signaling molecules, such as lysophosphatidic acid (LPA) and sphingosine‐1‐phosphate (S1P), act as growth and survival factors, and may be involved in liver preservation injury. Recent studies have proposed that there is a cytoskeletal component of preservation injury involving cytoskeletal linker proteins called ERM proteins. In preservation injury, moesin (the predominant ERM protein in the liver) is dysregulated, and found in its inactive, closed conformation. We hypothesize that serum‐derived growth factors (like LPA and/or S1P) may control known upstream regulators of moesin, RhoA and Rho kinase (ROK), which ultimately control moesin functionality and cell adhesion to the basement membrane. To begin testing this hypothesis, we demonstrated that inhibition of RhoA and ROK potentiated preservation injury in hepatocyte models of hypothermic ischemia. Preliminary data suggests that adding exogenous LPA to hepatocyte culture conditions prior to hypothermic ischemia induction can rescue cell viability from preservation injury. We also observed cytotoxicity with low concentrations (0.1%) of bovine serum albumin (BSA), which is known to avidly bind and hold these lipid growth factors. Inhibition of S1P synthesis via selective pharmacological inhibition of sphingosine kinase 2 (SphK2) produced striking results (figure 1). In vitro, cell viability was significantly decreased upon SphK2 inhibition, and in vivo, rodents receiving a liver transplant after that liver was flushed with a solution containing a SphK2 inhibitor had 100% mortality, relative to only 20% control mortality. These data support the hypothesis that serum‐derived lipid signaling molecules, such as LPA and S1P, are protective during hypothermic ischemia, and removing these at the time of organ preservation and flushing may be harmful.Support or Funding InformationNIH R01 DK087737DoD W81XWH‐16‐2‐0040DoD W80XWH‐17‐1‐0602This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The mammalian blastema and induced regeneration in mice.
Authors: Muneoka Ken 
Published: June 2021

Fingertips of humans and toe tips of mice possess the capacity to regenerate following amputation injury. The mouse toe tip has been developed as a regeneration competent model to investigate the regeneration process, and digit amputation at a more proximal level serves as a non‐regenerative wound model to test strategies for enhanced regeneration. The process of toe tip regeneration is divided into a series of overlapping events beginning with an inflammatory response, a bone degeneration response, epidermal closure, migration and proliferation of progenitor stem cells, blastema formation and redifferentiation. These stages are distinct from wound healing of a non‐regenerative amputation that results in truncated skeletal elements covered with scar tissue. BMP signaling has been shown to be essential for toe tip regeneration and BMP treatment is effective in enhancing the regenerative capacity of proximal level amputations. The ability to induce a regeneration response from non‐regenerative amputation wound identifies an unrealized regenerative potential of cells present at traumatic wounds in mammals. To date, we have determined that the regeneration of bone, cartilage, synovial cavity, tendon and ligament can be induced from a non‐regenerative amputation wound with different regimens of targeted growth factor treatments. These studies provide a road map for developing strategies to control regeneration events in mammals and serve as a new direction for the field of regenerative medicine.Support or Funding InformationDARPA (W911NF‐09‐1‐0305)US Army Research Laboratory (W911NF‐09‐1‐0305)Texas A&M UniversityThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.