bluuebunny/crossref_metadata_2025_split huggingface dataset:
Hydrophobic residues and amino terminal phosphorylation site are essential for urate/urate exchange mediated by human glucose transporter 9, hSLC2A9 (908.10)
Authors: Long Wentong , Witkowska Kate , O'Neill Debbie , Wong Kenneth , Cheeseman Chris 
Published: June 2021

Human urate homeostasis is achieved primarily in liver and kidney, where urate is significantly regulated by human SLC2A9a and 9b. These transporters show trans‐acceleration between urate and hexoses, and urate/urate; however, the mechanism has not been identified.Our preliminary data suggested that phosphorylation of hSLC2A9 is an important process for urate transport regulation and serine 9 has been postulated to be an important phosphorylation site in the N‐terminus of 9a. Objective:To investigate urate/urate trans‐acceleration mechanism mediated by9a,9b and their mutants. Methods: hSLC2A9 isoforms and mutants were over expressed in X. laevis oocytes. Urate/urate trans‐stimulation activities were determined by radiolabelled urate uptake and efflux measurements. Membrane protein expression levels were determined by Biotinylation and Western Blot analysis. Results:Urate trans‐stimulated urate flux and efflux in wild type hSLC2A9a and 9b and most of their mutants, except S9A and W110A in the predicted N‐terminal phosphorylation site and TM7 of 9a, respectively. This result suggests that substitution of alanine at S9 and W110, removes the asymmetry of the transporter, which normally allows for trans‐acceleration. These finding advance our understanding of how trans‐stimulation contributes to the regulation of urate and hexoses by hSLC2A9.Grant Funding Source: CIHR
Effects of selective pituitary hormone deficiencies on macrophage phagocytic activity in rat (911.2)
Authors: Viñuela‐Berni Verónica , Quintanar‐Stephano Andrés , Carrasco‐Esparza Norma , Organista‐Esparza Alejandro , Tinajero‐Ruelas Manuel , Berczi Istvan , Kovacs Kalman 
Published: June 2021

We showed that neurointermediate pituitary lobectomy (NIL) decreases whereas anterior pituitary lobectomy (AL) increases humoral and cell‐mediated immune responses. Studies indicate that PRL and GH are immune stimulatory/modulatory hormones. Macrophages (Mθ) have receptors for AVP, OXT, PRL and GH. In order to obtain a deeper insight into the role of anterior and posterior pituitary hormones on innate immunity, Mθ phagocytic activity was assessed in NIL, AL, HYPOX, sham operated (SHAM) and Control rats. Carbon black (CB) blood clearance, phagocytic index, CB blood half‐life time (T1/2), and histologic changes in liver, spleen and lung were also studied. Results. CB clearance was faster in SHAM > Control > NIL > HYPOX > AL rats. Differences were noted only in AL with slower clearance, suggesting that adenohypophysial but not neurohypophysial hormones are responsible for the faster CB clearance. Phagocytic index was decreased in NIL rats, indicating that AVP and OXT are required to regulate phagocytic activity. Blood CB T1/2 was 10 minutes in Control and SHAM rats, and 20, 17 and 18 minutes in AL, HYPOX and NIL rats. The longer T1/2 suggests that all pituitary hormones participate in CB clearance. Liver, spleen and lung histology showed Mθ markedly loaded with CB. These findings indicate that adenohypophysial and neurohypophysial hormones participate differently in the regulation of Mθ phagocytic activity.Grant Funding Source: CONACYT MEXICO 62317
Surgical anatomy of the carotid‐recurrent recess (913.8)
Authors: Hojaij Flávio , Akamatsu Flávia , Itezerote Ana , Ribeiro João , Andrade Mauro , Jacomo Alfredo 
Published: June 2021

Background: Recurrence of thyroid carcinoma in the central compartment of the neck is relatively frequent. Most commonly it occurs at the right side of the neck, lateral to the inferior laryngeal nerve and medial to the common carotid artery. We investigated the anatomy of this region in order to establish whether some anatomical characteristic may influence recurrence.Methods and results: Ten cervical regions of adult cadavers were carefully dissected and lymph nodes were consistently found into a recess which shape resembled a polyedrical area limited superiorly by the inferior thyroid artery, medial and inferiorly by the inferior laryngeal nerve and brachiocephalic artery and posteriorly by the prevertebral fascia and right parietal pleura and laterally by the common carotid artery.Conclusions: We found a polyedrical space on the right side of the neck that we called carotid‐recurrent recess that invariably harbored lymph nodes that can be reached by metastasis from thyroid carcinoma. Anatomoclinical characteristics of this recess suggest that it should be systematically explored during neck dissections of central compartment in order to avoid unnoticed lymph node metastasis.
Extracellular matrix protection factor: a novel class of post‐traumatic osteoarthritis therapeutic (922.11)
Authors: Cho Ellen , Chmielewski Sarah , Nolt Jason , Klunk James , Youngwirth Jonathan , Palumbo Dante , Maugle Tyson , Lukashova Lyudmila , Belogorodsky Dimitry , Holmes Tiffany , Althauser Samuel , Pinkney Nathan , Selim Abdulhafez , D'Angelo Marina 
Published: June 2021

Injury‐related, post‐traumatic osteoarthritis (PTOA) is a disease of the joints caused by an imbalance between extracellular matrix destruction and production. We have developed an innovative disease modifying therapeutic technology to treat PTOA. Extracellular matrix protection factor (ECPF‐1) is a novel, safe and effective intra‐articular injection that reduces the pain and damage caused by OA. Utilizing the peptide as an early intervention therapeutic, we have assessed its effects on the progression of PTOA. Peptide or control saline was injected into the injured knee joint for four consecutive weeks. Endpoint assessment of: toxicity, measured by CBC and serum chemistry; joint space narrowing, measured by Xray; joint functionality, measured by stride test; and tissue pathology, measured by micro computed tomography and histology were completed. Intra‐articular injections of ECPF‐1 in a rat model of PTOA demonstrated no cellular toxicity, normal serum chemistry following 4 weekly injections, diminished tissue destruction and increased animal mobility. All data indicates that ECPF‐1 is non‐toxic and diminishes the pathology associated with OA.Grant Funding Source: Supported by intramural funds
Hrq1, a homolog of the human RecQ4 helicase, acts catalytically and structurally to promote genome integrity (924.1)
Authors: Bochman Matthew , van Kessel Katherine , Paeschke Katrin 
Published: June 2021

Mutations in human RecQ4 are associated with cancer and premature aging. However, RecQ4 research is difficult because it is a fusion between a helicase and an essential DNA replication factor. Previously, we characterized Saccharomyces cerevisiae Hrq1, an enzyme homologous to the disease‐linked helicase domain of RecQ4, and discovered that Hrq1 has two unusual activities: it uses its helicase activity to protect against DNA inter‐strand crosslinks (ICLs) and acts structurally to protect against telomere addition (TA). Thus, HRQ1 joins PSO2 as the only yeast genes whose mutation specifically results in ICL sensitivity, and Hrq1 joins Pif1 as the only known TA inhibitors. In vitro, purified Hrq1 and hRecQ4 are robust DNA‐stimulated ATPases and 3’‐5’ helicases with similar unwinding kinetics. Unlike hRecQ4, however, Hrq1 exists in solution as two oligomeric forms: a monomer/dimer and a ring‐shaped heptamer. Work is ongoing to characterize which oligomeric species is responsible for the observed biochemical activities of Hrq1. Additionally, an in vitro system is being developed to determine the mechanism by which Hrq1 inhibits telomerase and if hRecQ4 functions in a similar manner.Grant Funding Source: Supported by the NIH, ACS, NJCCR, and the DFG
Nucleosome acidic patch promotes H2AX and H2A ubiquitination and DNA damage signaling (925.1)
Authors: Miller Kyle 
Published: June 2021

Post‐translational modifications of histones, including ubiquitination, participate in the chromatin response to DNA damage. Indeed, RNF168 and RING1B/BMI1 function in the DNA damage response by ubiquitinating H2A/H2AX on Lys‐13/15 and Lys‐118/119, respectively. How the ubiquitin pathway engages chromatin to provide site‐specificity between histone residues is undefined. Here we identify the nucleosome acid patch as a critical chromatin mediator of H2A/H2AX ubiquitination. The acidic patch is required for RNF168‐ and RING1B/BMI1‐dependent H2A/H2AXub in vivo. The acidic patch functions within the nucleosome as nucleosomes containing a mutated acidic patch exhibit defective H2A/H2AXub by RNF168 and RING1B/BMI1 in vitro. Furthermore, direct perturbation of the nucleosome acidic patch in vivo by the expression of an acidic patch interacting viral peptide, LANA, results in defective H2AXub and DNA damage responses. The acidic patch therefore is a critical nucleosome feature that may serve as a scaffold to integrate multiple ubiquitin signals on chromatin to compose selective ubiquitinations on histones for DNA damage signaling and repair. We aim to further our understanding of the molecular mechanisms by which the nucleosome acidic patch mediates histone modifications to orchestrate DNA damage signaling.Grant Funding Source: Supported by CPRIT (R1116).
Conjugation of ubiquitin and ThiS in an archaeal system (931.2)
Authors: Hepowit Nathaniel , Berganini Alyssa , Maupin‐Furlow Julie 
Published: June 2021

Ubiquitin (Ub) conjugation in eukaryotes and ThiS thiocarboxylation in bacteria require their cognate activating enzyme, E1 and ThiF, respectively. Conversely in archaea, the E1/ThiF analog, UbaA is required for the conjugation of the Ub/ThiS‐like protein SAMP2 and in SAMP2‐mediated sulfur relay for tRNA thiolation. To further corroborate our current model about the role of UbaA at the crossroad of protein conjugation and sulfur transfer, we in trans‐express the human Ub and E. coli ThiS in the haloarchaeon Haloferax volcanii. Interestingly, both Ub and ThiS covalently linked onto archaeal cytosolic proteins in an UbaA‐dependent manner, demonstrating that UbaA has a broad specificity in activating Ub‐fold family of proteins. This substrate promiscuity supports our hypothesis that the mechanism of UbaA‐mediated SAMP2 activation in archaea provides an evolutionary link that differentiates isopeptide‐based conjugation shared among eukaryotes and sulfur mobilization shared among bacteria.Grant Funding Source: Supported by NIH
Resveratrol, a novel natural proteasome inhibitor (935.3)
Authors: Qureshi Nilofer , Reis Julia , Qureshi Asaf 
Published: June 2021

We have previously established that the cellular organelle proteasome is a central mediator of inflammation and it regulates multiple signaling pathways. Furthermore, we have also established that the chymotrypsin‐like activity of the proteasome is most important in the regulation of lipopolysaccharide (LPS)‐induced inflammation. We have recently identified resveratrol as natural inhibitor of this activity. Resveratrol is a plant‐derived natural product phytoalexin found in red grapes and other fruits, and has been reported to manifest a number of positive pharmaco‐therapeutic benefits in humans, including anti‐cancer, anti‐aging, anti‐ischemic and anti‐inflammatory properties. The mechanisms responsible for these benefits have not been studied in detail. We hypothesized that treatment of macrophages in vitro with resveratrol will block LPS‐induced, Toll‐like receptor (TLR) 4‐mediated signaling by inhibiting the activity of some proteasomal proteases, thereby inhibiting the inflammatory response. We now conclude that pretreatment of mouse macrophages in vitro with resveratrol disrupts several major TLR4‐dependent signaling pathways triggered by LPS, leading to markedly reduced secretion of several proinflammatory cytokines and nitric oxide synthase generation. Further, pretreatment of mice with resveratrol results in similar anti‐inflammatory effects in vivo. We have also shown an altered cytokine expression pattern in LPS‐stimulated human monocytes pretreated with resveratrol. Thus one of the primary mechanism(s) responsible for these reported pleiotropic effects of resveratrol on multiple cell types, and its effects on the host inflammatory system may be due to the inhibition of the proteasomal proteases. These findings will provide new opportunities for therapeutic intervention in the treatment of diseases that are based on the proteasome and have a strong inflammatory component.Grant Funding Source: Supported by NIH
Multiple mechanisms for transcription fidelity maintenance in vivo (939.2)
Authors: Huang Ingold , Kireeva Maria , Irvin Jordan , Gotte Deanna , Schafer Brenda , Strathern Jeffrey , Kashlev Mikhail 
Published: June 2021

Transcription errors have been proposed to contribute to tumorigenesis and neurodegenerative diseases. An experimental validation of the biological impact of transcription errors faces major challenges, including the transient nature of mRNAs and the lack of a reliable methodology to distinguish transcription errors from translation errors. Here we report a genetic screen using Saccharomyces cerevisiae in which a transient transcription error is converted to a selectable genetic change. This assay can detect a transcription error rate as low as 1 × 10‐6. In this screen, we identified nine mutations in the catalytic subunit of RNA polymerase II (Pol II) that increased transcription errors in vivo. Functional in vitro analysis revealed that these mutations promoted misincorporation, improved mismatch extension, or decreased Pol II sensitivity to the proofreading factor TFIIS. Thus, we identify multiple molecular mechanisms that ensure that DNA is accurately transcribed in vivo. The error‐prone Pol II mutants will be used to elucidate the consequences of reduced transcription fidelity in yeast and mammalian cells and their role in human diseases.Grant Funding Source: Supported by NIH Intramural Research Program
SAGA‐mediated acetylation interacts with Prp5 ATPase activity in pre‐mRNA splicing (940.1)
Authors: Merkhofer Evan , Johnson Tracy 
Published: June 2021

Regulation of gene expression occurs at multiple levels, including the removal of non‐coding sequences from protein‐coding pre‐messenger RNA transcripts, commonly known as RNA splicing. Correct gene expression requires high fidelity in pre‐mRNA splicing, as up to 15% of mutations leading to disease result in aberrant pre‐mRNA splicing. Though previously thought of as biologically distinct processes, recent studies have shown there is a complex interplay between transcription and pre‐mRNA splicing, however, the regulation underlying this coordination remains unclear. Using the budding yeast Saccharomyces cerevisiae, we have identified interactions between components of the SAGA (Spt/Ada/Gcn5/Ada) transcriptional coactivator complex and the ATP‐dependent spliceosomal DEAD‐box protein Prp5. Prp5 plays a critical role in mediating ATP‐dependent RNA remodeling in splicing, though its precise role in transcription or chromatin remodeling is unclear. Deletion of genes encoding components of the SAGA complex required for histone acetylation, including the histone acetyltransferase (HAT) gcn5, bypasses the Prp5 ATPase requirement in splicing. This regulation is specific to HAT components of the SAGA complex: deletion of non‐HAT SAGA components, or non‐SAGA histone acetyltransferases does not rescue the Prp5 ATPase mutant phenotype. The interaction of Prp5 ATPase activity with Gcn5 also involves chromatin, as deletion of histone targets acetylated by Gcn5 also bypasses the Prp5 ATPase requirement. Analysis of gene expression using splicing‐specific microarrays reveals that deletion of gcn5 results in improved splicing of a subset of genes dependent on Prp5 ATPase activity for splicing. These data suggest that Prp5 may coordinate co‐transcriptional splicing through its interaction with chromatin and chromatin‐interacting proteins, and indicates a possible role of chromatin and chromatin‐interacting proteins in regulating U2 snRNP assembly or structure.Grant Funding Source: Supported by NIH GM05474 and NIH K12 GM068524‐10
A novel chemical‐genomic approach for the dissection of the CTD code (940.2)
Authors: Rodriguez‐Molina Juan , Tseng Sandra , Ansari Aseem 
Published: June 2021

The C‐terminal domain (CTD) of the largest subunit of RNA Pol II serves as a central hub for the regulation of transcription and its coordination with co‐transcriptional events. The CTD is composed of a highly conserved heptapeptide (Y1S2P3T4S5P6S7) repeated in tandem 26 times in the yeast Saccharomyces cerevisiae, and serves as a scaffold for an array of combinatorial post‐translational modifications which establish a CTD code. In transcription initiation, RNA Pol II and general transcription factors assemble at promoter regions prior to transcription elongation. The final factor to be recruited, TFIIH and its associated kinase, Kin28, phosphorylates the Ser5 and Ser7 residues of CTD. The co‐occurrence of Ser5 phosphorylation and transcription initiation led to the hypothesis that Ser5 phosphorylation by Kin28 is required for transcription initiation and RNA Pol II promoter release.Our initial chemical genomic analysis revealed that although Ser5 and Ser7 phosphorylation levels decrease upon inhibition of Kin28, RNA Pol II is still able to initiate transcription and engage in active elongation. However, because residual kinase activity may still be present once the inhibitor reaches equilibrium, we set out to re‐explore the role of Kin28 in general transcription through a more rigorous method. To compellingly dispel any doubt of our initial findings, we have applied a novel strategy in which we specifically and irreversibly block Kin28 kinase activity in vivo. Our results show that inhibition of Kin28 causes an arrest in cell growth consistent with its essential role in cellular processes. Additionally, chromatin immunoprecipitation and genome‐wide analyses reveal a dramatic decrease in Ser5 across the genome, while RNA Pol II remains at high levels across all transcribed genes. Our results suggest that in fact Kin28 is not required for general Pol II transcription and that targeted irreversible inhibition of kinases in vivo can be extended to other kinases.Grant Funding Source: Supported by NHGRI Training Grant to the Genomic Sciences Training Program (5T32HG002760)
Novel intracellular interactors of lysyl oxidase (948.11)
Authors: Okkelman Irina , Sukaeva Aliya , Kirukhina Katerina , Pestov Nikolay 
Published: June 2021

Lysyl oxidases (LOX, LOXL1‐4) is a family of copper‐dependent amine oxidases that catalyze oxidative deamination of lysine in different extracellular matrix and nuclear proteins. Lysyl oxidases are involved in regulation of cell motility, epithelial‐mesenchymal transition (EMT) and maintenance of extracellular matrix structure. The catalytic domain of lysyl oxidases is known to interact with different extracellular matrix proteins. On the other hand, intracellular and intranuclear interactome of lysyl oxidase remains poorly studied.We performed a yeast two hybrid screening of a universal normalized human cDNA library using mature form of LOX as bait in order to find new partners of lysyl oxidases. Among more than 200 clones sequenced we identified several interesting proteins, including nuclear residents, as putative partners of LOX: guanine nucleotide exchange factor SOS2, mediator of cell motility MEMO1, growth factor EBP1 and transcriptional repressor p66β. Using pull‐down experiments, we showed that catalytic domains of lysyl oxidases LOX, LOXL1 and LOXL3 interact with the full‐length p66β. The LOX interaction site is located within the GATA‐type zinc‐finger domain containing region of p66β. Importantly, co‐expression of mLOX and p66β in vivo lead to nuclear translocation of LOX. Thus p66β/LOX interaction may be involved in nuclear transport of LOX, which does not contain a nuclear localization signal. We hypothesize that p66β/LOX interaction may affect EMT mediator function of lysyl oxidases through the transcription repressor activity of p66β, a component of the repressor complex NuRD.Grant Funding Source: Supported by RFBR (Grants 13‐04‐01413 and 12‐04‐31374).
Cross‐talk between Connexin 43 and CFTR in ER quality control (949.4)
Authors: Molina Samuel , McCarty Nael , Koval Michael 
Published: June 2021

An endoplasmic reticulum (ER) resident chaperone, ERp29, was previously identified to promote the quality control of cystic fibrosis transmembrane conductance regulator protein (CFTR). ERp29 also regulates the trafficking and oligomerization of a gap junction protein, connexin43 (Cx43). The ERp29/Cx43 complex regulates Cx43 oligomerization in the ER allowing for the formation hexameric hemichannels only in the TGN. Here we examined the ability of HeLa cells stably expressing Cx43 to form Cx43 membrane channels in the presence of the wild type and the most common mutant form of CFTR, ΔF508, in order to assess chaperone cross‐talk. CFTR expression is controlled by a TetOn system where CFTR mRNA and protein expression is regulated to mRNA and protein levels observed in primary airway cells. With a TetOn expression system we are able to probe cellular stress conditions in the presence and absence of transiently expressed CFTR. We assessed ER stress by using a luciferase‐based transcription factor assay, qRT‐PCR for ER stress markers and chaperones, and we also analyzed the effect of Δ508‐CFTR on gap junction intracellular communication via dye transfer studies. Mutant ΔF508‐CFTR can induce trafficking errors through ERp29 leading to decreased Cx43 protein trafficking and increased overall cellular sensitization to ER stress in a HeLa cell model which mimics cystic fibrosis airway cell protein expression levels.Grant Funding Source: Supported by: Emory+Children's CCFADR, Emory URC, CFF, NIH‐NIAAA, NIH‐NHLBI
Reactive oxygen species/neddylation is a novel pathway of transforming growth factor‐β‐induced smooth muscle cell differentiation (952.2)
Authors: Fei Jia , Chen Shiyou 
Published: June 2021

Smooth muscle cell (SMC) differentiation is an essential process in cardiovascular development. SMCs can be differentiated from pluripotent mesenchymal progenitors by transforming Growth Factor‐β (TGF‐β) while ubiquitin‐mediated protein degradation blocks SMC differentiation. Neural precursor cell‐Expressed Developmentally Down‐regulated 8 (NEDD8), a member in ubiquitin family, promotes protein stability and prevents protein degradation by conjugating to target proteins. We found that NEDD8/neddylation is essential for TGF‐β‐induced SMC differentiation. NEDD8 protein is increased during TGF‐β‐induced SMC differentiation and conjugates to smooth muscle α‐actin (α‐SMA). Blockade of neddylation by neddylation inhibitor (MLN‐4924) or NEDD8 short hairpin interfering RNA significantly inhibited the expression of α‐SMA and calponin (CNN) in TGF‐β‐treated cells. We found that reactive oxygen species (ROS) attenuation by SOD2 overexpression blocked TGF‐β‐induced NEDD8 conjugation to α‐SMA. SOD2 appeared to block neddylation through inhibiting expression of Casitas B‐lineage lymphoma (c‐Cbl), suggesting that ROS/c‐Cbl mediates TGF‐β‐mediated neddylation of α‐SMA and CNN. NEDD8/neddylation appears to enhance α‐SMA and CNN stability and prevent proteasome‐mediated degradation because administration of proteasome inhibitor (MG‐132) significantly inhibited α‐SMA and CNN protein degradation in neddylation‐depleted cells. Taken together, our study demonstrates that NEDD8‐mediated neddylation through ROS/c‐Cbl is a novel molecular event critical for TGF‐β‐induced SMC differentiation.Grant Funding Source: NIH‐R01