bluuebunny/crossref_metadata_2025_split huggingface dataset:
Ectodomain shedding in the downregulation of cellular ACE‐2 by ER stress in lung cells (716.3)
Authors: Dang Vinh , Uhal Bruce 
Published: June 2021

Previous work from this laboratory has demonstrated that angiotensin (ANG) converting enzyme‐2 (ACE‐2) protects against lung fibrosis by limiting the local accumulation of the profibrotic peptide, ANGII by converting ANGII to angiotensin 1‐7 (ANG 1‐7). However, this protective enzyme is down‐regulated in human and experimental lung fibrosis. We currently know of three ways that ACE‐2 is down‐regulated in lung cells: 1) exposure to hyperoxia in fetal lung fibroblasts, 2) treatment with proteasome inhibitors, MG132 or Clasto‐Lactacystin β‐lactone (CLBL) in alveolar epithelial cells (AECs), and 3) expressing the surfactant protein C mutation G100S (which causes lung fibrosis in people). In all of these cases, ACE‐2 was only measured in cell lysates, but not in the cell culture medium to assess “shedding”. We hypothesize that the down‐regulation of cellular ACE‐2 may be mediated by ectodomain shedding by ADAM17/TACE. Western blots of cell lysates revealed a significant decrease in immunoreactive cellular ACE‐2 with CLBL (p= < 0.01) and a potential recovery in ACE‐2 with TAPI‐2, an inhibitor of ADAM17/TACE. Contrary to our hypothesis, a similar effect was also observed in serum‐free cell culture media (p= < 0.05). From this, we can speculate that ADAMS17/TACE may play a partial role in down‐regulating ACE‐2 by degrading it, in addition to other enzymes. With this model, further research can be done to investigate alternative mechanisms that lead to the down‐regulation of cellular ACE‐2 in lung cells.Grant Funding Source: Supported by APS STRIDE Fellowship and PHS HL ‐ 45136
Expression of functional light‐activated ion channels in embryonic chick muscle (731.1)
Authors: Whitaker Jessica , Sharp Andrew 
Published: June 2021

Optogenetic tools allow for fine control of targeted populations of neurons. Muscle cells are an electrically excitable cell type that could be targeted for regulation by optogenes. Our lab studies the effects of altered embryonic movement and sensory feedback on the neural development of chick embryos. Previously, we have used electroporation of a modified transposon‐based expression system into the neural tube of embryos to introduce excitatory and inhibitory light‐activated ion channels into neural tissue (Sharp and Fromherz, 2011). Similar to neurons, muscle cells can be induced to contract in response to changes in membrane potential. Therefore, we have transformed muscle precursor cells in the somites of embryonic day (E) 3 chick embryos with the goal of being able to control the activity of muscles in ovo. We have observed expression of ChR2 in muscle of intact embryos and sectioned tissue via a fluorescent reporter molecule as late in development as E16. Sectioned muscle tissue reveals patterns of fluorescence consistent with membrane expression of ChR2. Further, we have demonstrated the ability to cause hip flexion when ChR2 is expressed in the sartorius muscle. Upon exposure to pulses of blue light, the hip flexed with and average onset latency of 0.137 s. Hip flexions stopped after five or six consecutive pulses. This approach will allow for the modulation of muscle activity during embryogenesis.Grant Funding Source: Supported in part by a Research‐Enriched Academic Challenge Award to J.W. from SIUC.
Myofiber damage precedes macrophage infiltration after contraction‐induced injury in dysferlin‐null muscle (731.3)
Authors: Roche Joseph , Tulapurkar Mohan 
Published: June 2021

Mutations in the DYSF gene that encodes the protein, dysferlin, lead to late‐onset, human muscular dystrophies known as dysferlinopathies. The A/J mouse spontaneously develops a mild dysferlinopathy after 6 months of age and at younger ages models the subclinical phase of the human disease. We subjected 3‐4 month old A/J mice to in vivo large‐strain lengthening injury (LSI) from lengthening contractions and studied the role of inflammation in the onset of muscle damage and recovery thereafter. We show that post‐LSI, myofiber damage in the tibialis anterior muscle occurs prior to inflammatory cell infiltration. Indeed, most edema and inflammation, monitored by T2‐weighted MRI and by immunofluorescence labeling of neutrophils and macrophages, respectively, develops 24‐72 hr after LSI, well after the appearance of damaged myofibers. The cytokines IL‐1A, MCP‐1, and MCSF are elevated several‐fold at 72 hr after injury, consistent with extensive macrophage infiltration. Control A/WySnJ mice show much less myofiber damage, much less inflammation and much lower cytokine levels after LSI than A/J. Depletion of monocytes and macrophages by systemic clodronate administration, fails to suppress myofiber damage or to accelerate functional recovery in A/J mice. Our studies suggest that, although macrophage infiltration is prominent in A/J muscle following LSI, it is the consequence and not the primary cause of progressive myofiber damage.$graphic_F38BE44F‐E3E9‐4CCD‐9D37‐186DE40DCC8F$Grant Funding Source: Supported by a grant to JAR from the Jain Foundation Inc.
The effect of N‐acetyl cysteine on myofibroblast phenotype expression by immortalized keratinocytes, Ker‐CT‐Ras (731.5)
Authors: Webb Jessica , Vaughan Melville , Black Morgan , Gainer Sonnie 
Published: June 2021

Recent research activity has focused on the tumor stroma. Tumor stroma are typically connective tissues containing fibroblasts and myofibroblasts. These cells are required for the wound healing processes of the body. There is evidence that myofibroblast presence in tumor stroma leads to poor prognosis. Mechanical tension, one of three key factors, enhances differentiation of myofibroblasts.In vitro carcinomas can form through a pathway which involves the up‐regulation of the ras protein. Precancerous keratinocytes lead to two types of carcinomas. They take on properties of fibroblasts and metastasize, spreading into the dermis. Fibroblasts generate tension in the dermis during the wound healing process. Our experiment focuses on precancerous keratinocytes and their journey into the dermis, which we model using an in vitro stress‐relaxed collagen matrix.We set up Ker‐CT‐Ras lattices void of fibroblasts and generated contraction data. With Ker‐CT‐Ras as with fibroblasts, transforming growth factor beta (TGF‐beta) increases tension generating ability and up‐regulates production of stress fibers. N‐acetyl cysteine (NAC) in the lab was shown to block tgf‐beta signaling in fibroblasts. Here we focus on the effect of NAC on Ker‐CT‐Ras. Additional data presents the comparative tension‐generating ability of fibroblast lattices (DP‐147‐H‐Tert) and co‐culture lattices comprised of the two. Data taken from a monolayer (coverslip) model visually describes the structural properties of the myofibroblast phenotype. Overall, we found that response of Ker‐CT‐Ras to these signals is similar to that of fibroblasts.Grant Funding Source: Supported by CURE‐S‐STEM, OK‐LSAMP, and RCSA grants
Encapsulation of antibiotics in poly(ethylene glycol)‐block‐poly(L‐lactide) for delivery in dentin tubules during root canal treatment (733.4)
Authors: Haseeb Ridwan , Lau Michael , Rodriguez Lucas , Montagner Franciso , Palmer Kelli , Stefan Mihaela , Rodrigues Danieli 
Published: June 2021

Aim: To encapsulate an antibiotic in oral applications with PEG/PLA‐block copolymer into microparticles for infected dentin tubules. To maintain drug release for extended periods to prevent bacterial regrowth after root canal treatment.Methodology: Drug encapsulation was carried out through an oil‐water emulsion‐solvent evaporation method. In summary, the PEG/PLA copolymer and the oral drug were dissolved in an oil phase (dichloromethane), which was combined with an aqueous (polyvinyl alcohol and de‐ionized water). The solution was stirred, centrifuged, washed, and lyophilized. Particle size was determined using digital microscopy. Antimicrobial effectiveness was assessed in vitro by placing small amounts of encapsulated particles on bacterial agar plate cultures and monitoring growth inhibition.Results: Encapsulated particles ranged in size from 300 nm to 1.0 m. A 2 week bacterial inhibition test showed that the particles exhibited zones of inhibition between 3 mm and 5 mm.Conclusion: The proposed method with the PEG/PLA copolymer encapsulates the oral antibiotic producing particles with size distribution that may penetrate the dentinal tubules (2.5 m in diameter). Bacterial inhibition tests showed that the particles inhibited bacterial growth after 2 weeks. Ongoing bacterial inhibition tests will determine the release profile of the microparticles.
Mechanism of mycobacterial recombination mediator proteins function in homologous recombination and strand annealing (735.12)
Authors: Korolev Sergey , Ryzhikov Mikhail , Gupta Richa , Glickman Michael 
Published: June 2021

Recombination mediator proteins (RMPs) are important for genome stability in all organisms and support two reactions: the recombinase binding to single‐stranded (ss)DNA to initiate homologous recombination (HR) and strand annealing. Both reactions are inhibited by ssDNA binding proteins (e.g. gp32, SSB, RPA). RMPs promote DNA transfer from SSB to recombinase or to anneal DNA. In spite of two ubiquitous functions, RMPs lack sequence and structural homology. Previously, we demonstrated that E. coli RMPs, RecOR, do not dismiss SSB from ssDNA, but form the complex with SSB altering the conformation of bound ssDNA. RecO binds SSB and the complex promotes DNA annealing or RecA loading in the absence or presence of RecR, respectively. Surprisingly, mycobacterial RecO does not interact with SSB. Yet, both proteins form complex on the shared ssDNA substrate in both pathways.In mycobacteria, RecFOR deletion reduces clastogens resistance to the same degree as a deletion of the major homologous recombinase RecA. Chromosomal DNA break repair assay surprisingly revealed that all three proteins are involved in both pathways: HR and annealing. Here we will describe biochemical studies of DNA annealing and strand exchange reactions supported by mycobacterial RecFOR. The selection of one or the other pathway is dictated by the availability of recombinase or complimentary strand and the stoichiometry of the DNA‐protein complexes. We believe that the intermediate complex of RMP with SSB and ssDNA represents a common critical step in reactions supported by all RMPs during HR and strand annealing DNA repair pathways in all organisms.
RNA binding protein hnRNP‐U coordinates repair of radiation‐induced damage clusters in human genome via its Ser59 phosphorylation (736.8)
Authors: Dutta Arijit , Mitra Sankar , Hegde Muralidhar 
Published: June 2021

Ionizing radiation (IR), widely used in diagnostic procedures and cancer therapy, induces complex clusters of damage in the genome that include DNA double‐strand breaks (DSB) and much higher levels of closely spaced oxidized bases and single strand‐breaks (SSB). While overt DSBs are primarily repaired via non‐homologous end joining (NHEJ), oxidized bases and SSBs are repaired via base excision repair (BER)/ SSB repair pathway. However, how these multiple repair pathways are co‐ordinated at IR‐induced damage clusters is poorly understood. Here we demonstrate temporal control of NHEJ vs. BER, co‐ordinated by RNA binding protein hnRNP‐U, which was recently implicated in regulating BER for enhancing the activities of NEIL1/NEIL2 glycosylases. We found Ku, a key NHEJ protein inhibits early BER proteins (NEILs, OGG1 & APE1) at the damage clusters, preventing repair of oxidized bases at close proximity. hnRNP‐U, also recruited at the repair site cannot overcome Ku inhibition initially due to its phosphorylated state (at S59 by DNA‐PK in response to IR). Subsequently, hnRNP‐U dephosphorylated after NHEJ completion relieves Ku inhibition and facilitates BER initiation. This differential ability of unmodified vs. S59‐P hnRNP‐U is due to its phosphorylation‐dependent decrease in affinity for the DNA glycosylases. These studies provide a molecular mechanism for sequential repair of IR‐induced DSBs followed by BER of proximal oxidized bases, where hnRNP‐U acts as a ‘molecular switch’. We propose that such hierarchical repair of IR‐induced damage is important in maintaining genomic fidelity.Grant Funding Source: Supported by USPHS grant R01 CA158910 and HMRI
The roles of differential branches of ER stress signaling in determining cell fate of autophagic hepatoma cells (737.2)
Authors: Ye Hui‐Lan , Chen Xing‐Zhou , Zhang Guo , Yin Xiu‐Xiu , Chen Li , Zhang Fa‐Can 
Published: June 2021

Autophagy is a conserved lysosomal degradative pathway in eukaryotic cells and its dual effects on cell fate are controversial. The present study was designed to clarify the switching effect of IRE1 activity on ER stress‐induced autophagic cell fate and monitor the dynamic changes of autophagic processing. Firstly, by Micro Cellular Imaging and Analysis System, the fluorescent signals were dynamically recorded in HepG2 cells which were pretransfected with mRFP‐EGFP‐LC3 plasmid. With this system, cytosolic yellow puncta indicated the formation of autophagosome, while the red fluorescence puncta indicated the turnover of autolysosome in these cells. In the presence of tunicamycin (Tm) (5µg/mL) for 24h, it was found that the number of yellow puncta was markedly elevated in HepG2 cells. Interestingly, not only the intracellular yellow puncta but also red puncta were significantly increased in these cells exposured to thapsigargin (Tg) (500nM). Secondly, by western blotting, both Tm and Tg evidently induced an increment of IRE1 protein, an early attenuated branch of ER stress. However, another critical ER stress signaling molecule PERK was found to persistently increase in these cells in response to Tg, but not Tm. Thirdly, inhibiting the IRE1 activity by siRNA substantially decreased the LC3II conversion, and dramatically promoted the Tg‐induced apoptosis of HepG2 cells with about 40%. Consistently, Tg‐induced red puncta signal was apparently enhanced with IRE1 siRNA treatment. Taken together, these results suggested that IRE1 as well as PERK, the hallmarks of ER stress signaling, importantly govern the autophagic cellular outcome, which may serve as a novel potential therapeutic target for HCC.Grant Funding Source: Supported by NSFC Grants 881172260, 30630145, 81360077 & 2010GXNSFC013020 to G.Z.
Impaired lysosome function can induce autophagy via suppression of mTOR activity (738.1)
Authors: Yang Zuolong , Guo Ne , Khambu Bilon , Zhang Hao , Liu Peiqing , Yin Xiao‐ming , Li Min 
Published: June 2021

Autophagy is an evolutionarily conserved mechanism by which cytoplasmic material is degraded in the lysosomal compartment to fuel starving cells and eliminates intracellular pathogens. Autophagy can be activated via MTORC1 downregulation by amino acid deprivation and by certain chemicals. Lysosome is the degrading machine for autophagy, but has also been linked to MTORC1 activation through the Rag GTPase pathway. In this study, classical lysosome inhibitors, such as chloroquine, E64d and pepstatin A, were found to be able to inhibit mTORC1 in a Rag‐dependent manner. All these lysosome inhibitors were able to activate events associated with early autophagy biogenesis represented by ATG16L1 and ATG12 puncta formation, indicating a consequence of autophagy activation following MTORC1 suppression. Our data suggested a general link from lysosome function to the Rag‐MTORC1 signaling axis and autophagy activation. Thus the lysosome is not only required for autophagic degradation, but also affects autophagy activation. Lysosomal inhibitors can have a dual effect in suppressing autophagy degradation and in initiating autophagy.Grant Funding Source: Supported by Guandong Provincial Natural Science Foundation, China (S2013010015876)
Effects of O‐GlcNAcylation on insulin signaling on the level of insulin receptor substrate 1 (739.13)
Authors: Hu Ping , Kim Min‐Sik , Pandey Akhilesh , Hart Gerald 
Published: June 2021

β‐O‐Linked N‐acetylglucosamine(O‐GlcNAc) modification (O‐GlcNAcylation) is a dynamic posttranslational modification on Ser/Thr residues and increased O‐GlcNAc is associated with insulin resistance. Insulin receptor substrate 1(IRS1) acts as an adaptor protein transferring signals from insulin to downstream PI3K and Akt. It is O‐GlcNAcylated and highly phosphorylated. Here we mainly focused on how O‐GlcNAcylation affects insulin signaling on IRS1 level. We found that insulin signaling is regulated through pharmacologically modifying the two key enzymes’ activity: O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA). In addition, we showed phosphorylation status of IRS1 changed accordingly by vertical isoelectrical focusing and two‐dimensional gel electrophoresis. We are performing Stable Isotopic Labeling by Amino Acids in Cell Culture (SILAC) on 3T3‐L1 to detect phosphorylation changes on IRS1 as well as other signaling proteins. In addition, We mapped two O‐GlcNAc sites by CID/ETD‐MS/MS, Ser635 and Ser1001. Ser635 is a main phosphorylation site associated with type II diabetes. We found Cycling rate of O‐GlcNAc on Ser635 is fast, phosphorylation and O‐GlcNAcylation compete at Ser635 in an insulin‐responsive manner. Ongoing studies are testing how O‐GlcNAc on Ser635 affects insulin signaling in 3T3‐L1 and whether O‐GlcNAc of Ser635 affects phosphorylation level of nearby sites. These data suggests O‐GlcNAcylation on IRS1 plays an important role in regulating insulin signaling.Grant Funding Source: NIH R01CA42486, R01DK61671; N01‐HV‐00240; P01HL107153, R24DK084949
Characterization of Celf1 function in mouse lens cell lines (746.1)
Authors: Dang Christine , Siddam Archana , Lachke Salil 
Published: June 2021

Although the human genome encodes ~1500 RNA binding protein (RBP) encoding genes, mutations in only ~20 are associated with human disease. We used a novel bioinformatics approach iSyTE (integrated Systems Tool for Eye gene discovery) to identify a new RBP, Celf1 (CUGBP Elav (embryonic lethal, abnormal vision)‐Like Family member 1), in the lens that is potentially associated with cataract. Celf1 contains three conserved RNA Regulatory Motifs that function in sequence‐specific binding to target RNAs. To develop a new resource to study Celf1 function in lens cells, we generated and characterized Celf1‐knockdown (KD) mouse lens epithelial cell lines. We first selected the mouse lens epithelial cell line 21EM15 as it expresses several critical lens‐expressed genes and thus recapitulates important aspects of lens biology. We infected 21EM15 cells with lentiviral particles carrying inhibitory short hairpin (sh)RNAs against mouse Celf1 mRNA and isolated infected cells based on their acquired resistance to puromycin. Celf1‐KD was confirmed by reduced Celf1 expression in Western blotting. We are presently characterizing the effect of Celf1‐KD on distinct classes of RNA granules (e.g. Stress granules and Processing bodies) involved in post‐transcriptional regulation. In sum, we describe development of a new Celf1‐KD lens cell line as a unique resource for gaining novel functional insights of an RBP in lens biology.Grant Funding Source: Supported by Pew Charitable Trusts
Binding of RNase R to ribosomes: comparison of non‐stop mRNA and REP sequence‐containing message (748.2)
Authors: Liang Wenxing , Deutscher Murray 
Published: June 2021

RNase R, a 3' to 5' exoribonuclease able to act on most RNAs including those with extensive secondary structure, is subject to a complex regulatory mechanism. Most RNase R in exponential phase cells is sequestered on ribosomes, where it is stable. In contrast, the free form of the enzyme is turned over rapidly, presumably because it is deleterious to cells due to its promiscuous action on essential RNA molecules. We previously showed that RNase R binding to ribosomes is stimulated by non‐stop mRNA leading to its participation in trans‐translation. We show here that RNase R binding to ribosomes is also stimulated by messages containing intergenic REP sequences suggesting that degradation of these messages utilizes a similar mechanism to non‐stop mRNA decay. Interestingly, the stimulation of RNase R binding is lost when the REP sequence is moved further away from the translation termination codon and the amount of protein produced from the message is elevated. These data show that as with non‐stop mRNA, other messages which result in ribosome stalling prior to normal termination also lead to RNase R binding and likely the induction of trans‐translation.Grant Funding Source: Supported by National Institutes of Health Grant GM16317
Post‐transcriptional regulation of interleukin‐3: roles of AREs, HuR and p38 (749.5)
Authors: Hernandez Marimar 
Published: June 2021

Interleukin‐3 (IL‐3) is a pro‐inflammatory cytokine that is secreted by T‐cells upon stimulation, although its mRNA is constitutively expressed, suggesting that is regulated at this level. Our group has shown that the IL‐3 3’UTR is responsible for repressing its expression, being the Adenosine/Uridine‐Rich Elements (ARE) region mainly responsible for this repression. Chimeric luciferase constructs harboring either wild‐type IL‐3 3’ UTR or different mutations on its ARE region were transfected on Jurkat cells and the reporter activity was measured upon T‐cell stimulation. We observed that when the nonamer ARE is interrupted, the 3’UTR is not able to elicit the same response as the wild type 3’UTR. We carried out EMSAs using protein extracts from Jurkat cells at 6, 12 and 24hrs after activation and identified HuR as an ARE‐Binding Protein able to bind the IL‐3 3’UTR AREs. HeLa cells were co‐transfected with plasmids over‐expressing HuR and the luciferase construct which harbors the IL‐3 3’UTR. Luciferase activity and mRNA levels decreased when HuR was over‐expressed. Since p38 has been shown to increase pro‐inflammatory cytokine secretion and is known to phosphorylate HuR, we assessed its possible role in the regulation of IL‐3. After pretreatment of Jurkat cells with the p38 inhibitor SB202190, IL‐3 mRNA levels were significantly decreased upon T‐cell stimulation suggesting that p38 is indeed involved in the post‐transcriptional regulation of IL‐3.Grant Funding Source: Supported by U54 CA96297; NIGMS MBRS RISE R25GM061838
Core‐spanning hydrophilic residues lend stability and preserve functionality in β‐helical protein HpmA265 (752.5)
Authors: Glasgow Evan 
Published: June 2021

In the present work, the bacterial β helical hemotoxin HpmA has been cloned and expressed in truncated form (HpmA265) and subject to several structural experiments. Folded (active) HpmA265 can rescue the hemolytic function of denatured, full‐length HpmA in vitro by direct interaction, and this phenomenon is quantified with a hemolytic assay. In attempt to correlate functionality with structure, circular dichroism spectroscopy has been used in conjunction with guanidine hydrochloride titrations to assess the structural/functional contributions of key hydrophilic residues in the protein’s core. Native HpmA265 was found to have a folding energy of ∆Gº = ‐50.2 ± 2.7 kJ/mol. Ala or Phe substitutions were made either at Q125 or Y134, and mutant folding energies were ‐25.0 ± 2.4 kJ/mol (Q125A), ‐48.0 ± 6.5 kJ/mol (Y134A), ‐32.4 kJ/mol (Q125F), and ‐45.4 ± 4.5 kJ/mol (Y134F). The data suggest more substantial perturbation by mutation of Q125 than of Y134. This idea complements the graphically‐predicted contribution of Q125 versus Y134, in that Q125 coordinates with an embedded water molecule in a core‐spanning hydrogen bond network and appears to be held in the proper orientation by the Y134 hydroxyl group. Additional networked residues will be assessed in this way. A greater understanding of the β helix fold may contribute to understanding of similar proteins, such as β amyloid and bacterial adhesion factors.Grant Funding Source: Supported by NSF‐RUI 105435 (to TW)