bluuebunny/crossref_metadata_2025_split huggingface dataset:
Deciphering the mechanism of action of cholesterol analogs as inhibitors of Mycobacterium tuberculosis
Authors: Carrillo‐Alvarado Miguel Angel , Pardo Andrew , Alsaihati Laila , Noveron Juan C , Ouellet Hugues 
Published: June 2021

Drug‐resistance continues to be a public health problem, with nearly half million people infected by multidrug‐resistant strain of Mycobacterium tuberculosis (Mtb) worldwide, urging the need to develop new antitubercular drugs. Cholesterol plays a critical role in phagocytosis by host macrophages. Mtb also utilizes cholesterol as a source of carbon and energy during infection and degrades it through three catabolic pathways acting on AB‐ and CD‐rings, and on the aliphatic side‐chain. These pathways produce acetyl‐ and propionyl‐CoA intermediates, which are then incorporated into cellular lipids and virulence factors. Cholesterol analogs with degradation‐resistant alkyl side‐chains, including 3β,16β,26‐trihydroxy‐cholesterol (16OHC), have been recently shown to effectively inhibit Mtb growth, but the mechanism of action remains unclear. In this study, we combined the use of synthetic cholesterol analogs with genetic and mass spectrometry approaches to demonstrate whether cholesterol side‐chain catabolic activities could be used as potential drug targets. Metabolomic profiling of Mtb wild‐type and mutant strains with decreased cholesterol side‐chain degrading activities fed with 16OHC revealed that this cholesterol analog is metabolized by all strains, except by Dcyp125 cells, to produce a metabolite that we identified as 3‐oxo‐chola‐1,4‐dien‐16β‐ol‐24‐oate methyl ester. Unexpectedly, our data showed that 16OHC is not an inhibitor of the cholesterol side‐chain degradation pathway, but rather an inhibitor of the AB‐ring degradation pathway. The inhibitory effect of 16OHC on selected recombinant Mtb AB‐ring degradation enzymes is being tested and data will be presented.Support or Funding InformationFinancial support from the Ralph & Kathleen Ponce de Leon Endowment at UTEP, NIH BBRC 5G12MD007592 and SC1 AI116567‐01A1 (NIH/NIAID/NIGMS) are gratefully acknowledged.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
SOD2 Overexpression Affects Mouse Hepatic OXPHOS Complex Integrity
Authors: Harris Tyler J , Steyls Samantha , Christian Brooke 
Published: June 2021

Reactive oxygen species (ROS), such as superoxide and hydrogen peroxide, are produced as byproducts of cellular respiration and can damage DNA, proteins, and lipids within a cell. To combat the production of superoxide, cells express superoxide dismutase 2 (SOD2), an antioxidant enzyme which converts superoxide into hydrogen peroxide. Mice that overexpress SOD2 were used to examine tissue‐specific ROS‐responsive genes. Microarray data indicates a disruption in genes encoding proteins that function in oxidative phosphorylation in SOD2 mice. BN‐PAGE analysis confirms the defect, but kinetic analysis of complexes 2 and 5 show no significant difference. We propose that the overexpression of SOD2 may destabilize assembled OXPHOS complexes, rendering them more sensitive to detergent. Current research is being conducted to test the stability of SOD2 oxidative phosphorylation complexes against oxidative stress, as well during aging.Support or Funding InformationASU Office of Student ResearchASU Department of ChemistryThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
4D cell biology: Big data image analytics and lattice light‐sheet imaging reveal dynamics of clathrin‐mediated endocytosis in stem cell‐derived intestinal organoids
Authors: Schöneberg Johannes , Dambournet Daphné , Liu Tsung‐Li , Forster Ryan , Hockemeyer Dirk , Betzig Eric , Drubin David 
Published: June 2021

New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue‐scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high‐content imaging data. We expressed fluorescent protein fusions of clathrin and dyna‐ min2 at endogenous levels in genome‐edited human embryonic stem cells, which were differentiated into hESC‐derived intestinal epithelial organoids. Lattice light‐sheet imaging with adaptive optics (AO‐LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 μm xyz) at 5.7 s/frame. We developed an open‐source data analysis package termed pyLattice to process the resulting large (□60 GB) movie data sets and to track clathrin‐mediated endocytosis (CME) events. CME tracks could be recorded from □35 cells at a time, resulting in □4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO‐LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues.Support or Funding InformationJ.S. acknowledges a Moore/Sloan Data Science Fellowship from the Berkeley Institute for Data Science and a Siebel Fellowship from the Siebel Stem Cell Institute–UC Berkeley. This work was supported by National Institutes of Health (NIH) Grant R35GM118149 to D.G.D. D.H. is a Pew–Stewart Scholar for Cancer Research supported by the Pew Charitable Trusts and the Alexander and Margaret Stewart Trust. D.H. is supported by the Siebel Stem Cell Institute and NIH R01‐CA196884.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Short Variant of Mitochondrial Dynamin OPA1 Renders Improved Cell Survival under Stress Conditions
Authors: Lee Hakjoo , Yoon Yisang 
Published: June 2021

The protein optic atrophy 1 (OPA1) is a dynamin‐related membrane remodeling protein associated with the inner mitochondrial membrane. OPA1 is a critical factor for mitochondrial structure and function by mediating mitochondrial inner membrane fusion and maintaining cristae structure. Under cellular stress associated with mitochondrial dysfunction, the inner membrane‐anchored long OPA1 (L‐OPA1) undergoes proteolytic cleavage to form short soluble OPA1 (S‐OPA1) lacking the transmembrane domain. The prevalent thought has been that S‐OPA1 is a functionally insignificant proteolytic product of L‐OPA1. Hence, the loss of L‐OPA1 and accumulation of S‐OPA1 by OPA1 cleavage under stress conditions is thought to be detrimental to cell survival. However, we recently demonstrated that S‐OPA1 is fully competent for maintaining energetics and cristae structure. Therefore, in this study, we used cells exclusively expressing L‐, S‐OPA1, or both, and tested the capacity of L‐ and S‐OPA1 in supporting cell survival under stress conditions. Under hydrogen peroxide‐mediated oxidant stress, we found that S‐OPA1‐expressing cells have better survival than L‐OPA1 cells. Interestingly, this difference in cell survival under oxidant stress was only observed in glucose‐free OXPHOS media but not in the presence of glucose, suggesting that S‐OPA1 may confer higher mitochondrial energetic activity compared with L‐OPA1. We also observed better survival of S‐OPA1‐expressing cells under nutrient starvation. Under apoptotic stress by treating cells with the transcription inhibitor actinomycin D, S‐OPA1 cells showed greatly decreased cytochrome c release compared to L‐OPA1 cells. These observations suggest that S‐OPA1 is more protective during apoptosis than L‐OPA1. Mechanistically, we found that S‐OPA1 cells have higher mitochondrial electron transport activity in nutrient depletion. Furthermore, by testing mitochondrial calcium retention capacity, we found that S‐OPA1 cells show increased resistance to mitochondrial permeability transition induced by mitochondrial calcium overload, compared with L‐OPA1 only cells. These data indicate that S‐OPA1 confers more efficient energy metabolism and more resistance to mitochondrial stress, allowing prolonged cell survival under stress.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Redox Regulation of TRPML1 Channel Activity and Lysosome Trafficking in Podocytes
Authors: Li Guangbi , Li Pin‐Lan 
Published: June 2021

Recent studies have indicated that redox signaling is importantly involved in the control of exosome secretion from a variety of cells. However, the mechanism by which reactive oxygen species (ROS) regulates exosome release remains unknown. In the present study, we hypothesized that ROS attenuate TRPML1 channel‐mediated Ca2+ release in podocytes through inhibition of acid ceramidase (AC) and thereby reduce lysosome trafficking, leading to prolongation of MVB fate to promote exosome secretion. Using GCaMP3 Ca2+ imaging technique, it was found that ML‐SA1‐induced Ca2+ release from lysosomes in podocytes was almost completely blocked by H2O2. However, sphingosine as an AC product of ceramide hydrolysis increased TRPML1 channel‐mediated Ca2+ release from lysosome of podocytes, which was not prevented by pre‐treatment with H2O2. Given the fact that Hcy induces NADPH oxidase‐derived ROS production in podocytes, we tested whether Hcy‐induced endogenous ROS production inhibits TRPML1 channel activity in these cells. It was found that pre‐treatment of podocytes with Hcy for 24 hours completely blocked TRPML1 channel‐mediated Ca2+ release. In the presence of catalase, Hcy‐induced blockade of lysosomal TRPML1 channel activation was prevented. Moreover, inhibition of NADPH oxidase by gp91 ds‐tat peptide or diphenyleneiodonium also abolished the inhibitory effects of Hcy on ML‐SA1‐induced TRPML1 channel‐mediated Ca2+ release. By confocal microscopy, we observed that ML‐SA1 enhanced the lysosome‐MVB interaction in podocytes, which was diminished by pre‐treatment with H2O2. However, when podocytes were pretreated with sphingosine, ML‐SA1 remained to enhance lysosome‐MVB interaction in the presence or absence of H2O2. Functionally, ML‐SA1 was found to enhance lysosome trafficking in podocytes towards the perinuclear region, which was prevented by pre‐treatment of these cells with H2O2. From these results, we conclude that both exogenous and endogenous ROS inhibit TRPML1 channel activity in lysosome of podocytes through inhibition of AC, which may cause derangement of lysosome trafficking and lysosome‐MVB interaction, leading to increased exosome secretion.Support or Funding InformationThis study was supported by NIH grants DK54927, DK120491, and HL057244.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Overexpression of a Novel E3 Ligase leads to a Skeletal Muscle Myopathy through Alterations in Cytoskeleton Proteins and Enhanced Protein Degradation
Authors: Hughes David C. , Baehr Leslie M. , Driscoll Julia , Waddell David S. , Bodine Sue C 
Published: June 2021

Multiple E3 ubiquitin ligases have been identified to be transcriptionally increased in skeletal muscle under atrophy‐inducing conditions. We have recently observed a novel E3 ligase, F‐box and Leucine‐rich protein 22 (FbxL22), to be elevated during denervation‐induced muscle atrophy. Little is known about the role of FbxL22 in skeletal muscle, with recent research highlighting its impact on cardiac muscle function and sarcomeric protein turnover (Spaich et al., 2012).Subsequently, male C57BL/6 mice (12‐week old) were subjected to in vivo electroporation with a mouse specific FbxL22 plasmid (20ug) in the tibialis anterior (TA) muscle with the contralateral leg serving as an empty vector control (pcDNA3.1). After 14 days, TA muscles were sectioned for H&E stain and GFP visualisation to confirm plasmid incorporation. Muscle mass and cross‐sectional area (CSA) were determined. Western blotting was performed to determine intermediate filament, autophagy and poly‐ubiquitin protein levels in the transfected TA muscles.Interestingly, we observed no difference in muscle mass after 14 days, but we did observe increased numbers of large and small fibers in the FbxL22 transfected muscle. At the protein level, there was a 75% reduction in alpha‐actinin‐2, which was accompanied by 4‐fold increase in desmin and vimentin. We additionally observed a 2‐fold increase in polyubquitination, and 1.5‐fold increase in autophagy markers (p62 and LCB II).Our data highlights the powerful impact of FbxL22 on skeletal muscle structure. Future studies will seek to investigate the chronic effect of FbxL22 on skeletal muscle and characterize its role during the process of muscle atrophy.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mTOR is a Mechanistic Target of Muscle and Cancer Cross‐Talk with Exercise
Authors: Goodenough Chelsea G , Davis Amanda R , Deaver J William , Riechman Steven E , Fluckey James D 
Published: June 2021

INTRODUCTIONThe mTOR signaling pathway regulates growth and cellular proliferation, which is vital for normal cell function. However, when this pathway is dysregulated, such as in cancer, it can lead to hyperactivation of signals which promote cell proliferation and protect against cell death. DEPTOR, a potent endogenous mTOR inhibitor, generally has low expression in many cancers. We have unique preliminary findings that myokines released during electrically‐stimulated muscle contraction of hemicorpus‐hindlimb perfused rats affect mTOR signaling and DEPTOR protein content in breast cancer cells. When MCF‐7 cancer cells were treated with perfusate collected during muscle contraction, a significant inhibition of proliferation was noted alongside diminished mTOR activity and elevated DEPTOR expression.PURPOSETo investigate changes in anabolic signaling and resultant protein synthesis rates in MCF7 human breast cancer cells exposed to pharmacologically administered mTOR inhibitors.METHODSMCF7 cells were cultured in 10cm plates until 60% confluence in standard Growth Media (GM). Cells were treated with either Rapamycin or Torin1 inhibitors (100nM or 250nM, respectively) in 10 ml of GM. Cells for protein synthesis analysis were additionally treated with heavy water (2H2O) to yield a 4% final volume. All groups were incubated in respective media for 24hrs and prepped for western blot or rates of protein synthesis analysis.RESULTSDEPTOR protein expression increased in both rapamycin and Torin1 treated cells (2‐fold & 7‐fold, respectively). Both Rapamycin and Torin 1 treated cells show decreased daily rates of protein synthesis compared to cell control (14.41, 9.09 vs 20.46 %/d, respectively).CONCLUSIONOur results suggest that diminished DEPTOR protein content and concomitant mTOR hyperactivity are important contributors to MCF7 cellular proliferation, in culture. Pharmacological administration of mTOR inhibitors suppresses protein synthesis and consequent anabolic signaling, with a rescue of DEPTOR protein content, which, in part, is consistent with our previous findings using exercise perfusate.Support or Funding InformationThis work was funded in part by the College of Health and Education and the Huffines Institute for Sports Medicine at Texas A&M University.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Biostimulating Effect of 685 nm Low‐Intensity Laser Radiation on Cell Viability in Multicellular Spheroids Culture
Authors: Gastaldi Gabriela Gomes Cardoso , de Oliveira Juliana Paula , de Andréa Dernowsek Janaína , Rezende Rodrigo Alvarenga , da Silva Jorge Vicente Lopes , Parizotto Nivaldo Antônio , Amaral Creusa Sayuri Tahara , Amaral André Capaldo 
Published: June 2021

Recent research evidences substantial morphological and physiological differences between cells maintained in vitro in two‐dimensional (2D) and three‐dimensional (3D) culture conditions. The current consensus is that 3D culture is better because it precisely mimics the cellular microenvironment in vivo. The biomodulatory effect of low‐intensity laser radiation (LILR) on cells in 2D cultures is already well established. On the other hand, the effect of this radiation on cells in 3D cultures has not yet been widely investigated. The aim of this work was to analyze the biomodulatory effect of LILR, at the wavelength (ƛ) of 685 nm, on the viability of osteogenic precursor cells (OPC) cultured as multicellular spheroids (MS). Agarose molds containning wells (300 μm diameter) were used for MS formation using the non‐adherent surface culture concept. A cell count (2×105) of the OPC lineage (MC3T3) was seeded in each mold and maintained under ideal growth conditions (Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum/37°C/95% humidified atmospheric air/5% CO2). The molds were irradiated for 5 consecutive days at doses of 0.5, 1.0 or 1.5 J/cm2 (triplicates), and the first irradiation was performed immediately after seeding. After this period, the cultures were submitted to cell viability assay and the results were expressed as a percentage of non‐irradiated control cultures (ANOVA ONE‐WAY, p≤0.05). The results showed that LILR, at ƛ 685 nm, provided a 79% increase in cell viability at the dose of 1J/cm2. The remaining doses exhibited similar viability rates that the control. These results confirm the dose‐dependent biostimulatory effect of LILR at ƛ 685 nm on MS culture and contribute to the understanding of the real influence of this radiation on cells under ideal conditions for in vitro simulation studies.Support or Funding InformationCoordenação de Aperfeiçoamento de Pessoal de Nível Superior ‐ CAPES ‐ BrazilThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcriptional Upregulation of CaMKIIδ through the JAK/STAT3 Pathway is Necessary for the IL‐6‐Dependent Increase in Endothelial Cell Migration
Authors: O'Brien Brendan Joseph , Singer Harold , Adam Alejandro , Ginnan Roman 
Published: June 2021

Multifunctional Ca2+/calmodulin‐dependent protein kinase II (CaMKII) is a well‐characterized family of proteins comprised of four separate isoforms: α, β, δ, and γ. These isoforms are differentially expressed in a tissue‐specific manner and contribute to diverse physiological and pathophysiological conditions. However, little is known regarding CaMKII isoform expression and function in the endothelium. Previous data from our lab has shown that CaMKIIδ6 is the primary isoform expressed in human umbilical vein endothelial cells (HUVECs) and promote thrombin‐induced loss of barrier function. Interleukin 6 (IL‐6) is a proinflammatory cytokine that induces loss of barrier function in HUVECs through a JAK/STAT3 signaling cascade. The purpose of this study was to test if CaMKIIδ mediates IL‐6‐induced increase in endothelial barrier permeability. Using qPCR, we determined that CaMKIIδ is the most abundant isoform of the enzyme present in HUVECs. Upon treatment with IL‐6, significant increases in both CaMKIIδ transcription and protein expression were observed. Treatment of HUVECs with JAK inhibitor ruxolitinib and siRNA for STAT3 decreased CaMKIIδ expression. However, adenoviral overexpression of various mutant CaMKII isoforms or siRNA knock down of CaMKIIδ did not affect IL‐6‐dependent loss of barrier function as measured by electric cell‐substrate impedence sensing (ECIS). It has been reported that IL‐6 signaling not only induces a loss of endothelial barrier function, but also increases endothelial cell migration. Indeed, siRNA knock down of CaMKIIδ attenuated IL‐6‐induced endothelial cell migration in a scratch‐wound assay. For the first time, our findings thus far have identified transcriptional induction of CaMKIIδ by IL‐6 that is necessary for the subsequent increase in endothelial cell migration.Support or Funding InformationRO1HL49426‐21 to Harold A. SingerThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Polarized Expression of Kir7.1 Channels in a 3D Organoid Culture Model
Authors: Beverley Katie , Shahi Pawan , Pattnaik Bikash 
Published: June 2021

BackgroundLeber Congenital Amaurosis (LCA) is an autosomal recessive genetic condition of the retina that leads to blindness in infants and young children. Previous data indicate that mutations in Kir7.1, an inwardly rectifying potassium channel, located in the retinal pigmented epithelium apical membrane contributes to a LCA blindness subtype. The specific localization of the Kir7.1 in the polarized RPE cell is critical as it contributes to retina function. Using standard cell culture models it is impossible to reproduce Kir7.1 distribution, molecular interaction and hence function. Culturing cells in 3D‐organoids will likely provide an appropriate in vitro system to study Kir7.1 disease mechanism and develop therapeutics.HypothesisWe hypothesize that Kir7.1 protein will have a polarized distribution in differentiated Mardin Darby Canine Kidney (MDCK) 3D Cell Culture Model that allows us to study channel biology.MethodsWe isolated mRNA from cultured MDCK cells that were either transfected with Kir7.1 or non‐transfected. We then utilized PCR and ran the PCR products on an agarose gel. We have optimized a 3D cell culture model of MDCK cells. About 3×104 cells/ml were grown in a collagen cocktail (24 mM glutamine, 2.8 mM NaHCO3, 1X MEM alpha, 20 mM HEPES, 2 mg/ml Collagen I) for 5 days in a compartmentalized chamber coverslip. On Day 5 or when organoids were noticed, we transduced with GFP Kir7.1 through lentiviral particles (viral titer 3.54×109) 500 MOI. Approximately one week after transduction, we stained the organoids with Hoescht and WGA‐Alexa 594 for nucleus and membrane staining respectively. Using Nikon C2‐confocal microscope, z‐stack images were acquired and subjected to off‐line analysis.ResultsOur PCR results indicate that MDCK cells do not natively express Kir7.1 as it was only expressed in the transfected cells. After 5 days in culture, we began to observe well developed spherical organization of mature MDCK cells. A single layer of Hoescht positive cells could be visualized. Kir7.1 expression was mostly detected by GFP positive signal in the membrane lining the inner surface of the sphere. WGA‐Alexa 594, however, lined the outer membrane of the organoids.ConclusionThe development of the MDCK 3D cell culture model allows for the study of polarized epithelium. Kir7.1 localization to the inner membrane of the organoid confirms that the basolateral membrane localization in MDCK cells corresponds with apical distribution in the RPE cells. Our data also indicates that MDCK cells are a good model for studying Kir7.1 because they do not natively express the protein. The experimental set up is thus robust and time efficient to study Kir7.1 channel biology in its most native setting.Support or Funding InformationNIH T32HD041921 NIH EY024995This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.