bluuebunny/crossref_metadata_2025 huggingface dataset:
Fructose transporter knockdown S. aureus reveals critical role of the airway epithelium in limiting ASL sugar accumulation and bacterial growth (897.4)
Authors: Garnett James , Braun Daniela , Lindsay Jodi , Baker Emma , Baines Deborah
Published: June 2021

Sugars promote the growth of S. aureus, a key bacterium in respiratory infections. We used an airway epithelial‐bacterial co‐culture model to examine the effect raising basolateral sugar concentration on the apical growth of S. aureus and the role of the epithelium in limiting this effect.

S. aureus addition on the apical surface of H441 monolayers led to enhanced paracellular 14C‐L‐glucose flux across the epithelium. S. aureus growth was significantly increased by elevating basolateral glucose or fructose concentrations (p < 0.05, n = 5). A S. aureus strain in which the fructose specific permease (fruA) had been disrupted (NE768), exhibited significantly reduced growth in the presence of fructose compared to glucose, in both isolated culture (p < 0.001, n = 3) and in epithelial‐bacterial co‐culture (after 7h; p < 0.05, n = 5). S. aureus JE2 (parent strain) growth in co‐culture was greater in fructose than glucose, despite similar growth rates in isolated culture. This correlated with the reduced rate of epithelial fructose uptake compared to glucose (p < 0.05, n = 5) which we predict results in increased availability of fructose for apical bacterial growth. Pre‐treatment of H441 monolayers with metformin (1 mM, 18h), significantly inhibited both glucose‐ and fructose‐induced bacterial growth (p < 0.05, n = 4), which corresponded to reduced paracellular flux of sugar across metformin‐treated monolayers (p < 0.001, n = 8).

These data provide new evidence that fructose and glucose diffuse across the airway epithelium and are utilised by S. aureus to support its growth and highlights the importance of the airway epithelium in restricting S. aureus growth by limiting airway surface liquid (ASL) sugar accumulation through paracellular permeability and uptake.

Grant Funding Source: Supported by the Medical Research Council
Harmful effects of ‘fast foods’ on enteric neurons (903.1)
Authors: Rivera Leni , Leung Christopher , Pustovit Ruslan , Angus Peter , Furness John
Published: June 2021

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. Fats and compounds known as advanced glycation end products (AGEs) exacerbate liver injury. AGEs are complexes of reducing sugars and proteins formed when proteins are overheated in the presence of fats and sugars (e.g., by deep frying). AGEs are low in traditional diets, but are common when diets include so‐called fast foods. Impaired gastrointestinal function has been implicated in the development of NAFLD, but there is very little known about the effects AGEs and fats on enteric neurons in the context of NAFLD. This study aimed to assess damage to myenteric neurons within the ileum, caecum, and colon of C57bl/6 mice with NAFLD induced by high fat high cholesterol (HFHC), and HFHC baked diets (which increases AGEs content fivefold) for 20 weeks. There was a significant decrease in the number of Hu and nitric oxide synthase (NOS) immunoreactive neurons, as well as a loss of neurofilament immunoreactive fibres in the HFHC and HFHC baked groups compared to control. In addition, there was a significant increase in Hu translocation to the nucleus, granular Hu deposits in the cytoplasm, and the presence of cytoplasmic vacuoles (indices of damage) in the HFHC and HFHC baked groups compared to control. This work indicates that HFHC and HFHC baked diets result in enteric neuropathy that may underlie impaired gastrointestinal function in NAFLD.

Grant Funding Source: National Health and Medical Research Council
EGF‐induced intestinal cell proliferation is dependent on loss of FOXO3 activity and increased lipid droplet density (906.1)
Authors: Chadalawada Gita , Chen Sharon , Cabe Maleen , Crawford Susan , Savkovic Suzana
Published: June 2021

The proliferation of intestinal cells, critical for homeostasis, wound healing, and sustained tumor growth, is in part controlled by EGF. The source of cellular energy required for the high metabolic needs of proliferative intestinal cells remains unclear. Triacylglycerols are stored in lipid droplets (LDs), and these neutral lipids can be a rich source of energy for various cell functions. We investigated whether intestinal epithelial cells increased intracellular energy stores by inactivating FOXO3 and altering EGF receptor (EGFR) signaling targets. Human intestinal HT29 cells, treated with EGF (10 ng/ml) with or without pharmacological inhibitors of various EGFR pathways were assessed for LD status and proliferation. In HT29 cells, EGF stimulated proliferation is associated with increased LD density while LDs depletion significantly lowers EGFR and proliferation. Moreover, depleting LDs prevent EGF mediated FOXO3 inactivation supporting dependency of LD density on FOXO3. Inhibition of EGFR reduced increased LD density, suggesting the involvement of its downstream pathways. Indeed, inhibition of phosphoinositide 3‐kinase, mTOR, and prostaglandin E2 diminishes EGF stimulated LD density. The novel mechanism of intestinal cell proliferation mediated by EGFR dependent energy from increased LD density could serve as a new therapeutic approach for intestinal wound healing or cancer treatment.

Grant Funding Source: Supported by NIH ‐CA160809
Influence of Hfe on neurobehavior in 1‐year‐old mice (907.5)
Authors: Chang JuOae , Kim Jonghan
Published: June 2021

Influence of Hfe on neurobehavior in 1‐year‐old mice JuOae Chang, Jonghan Kim Department of Pharmaceutical Sciences, Bouve College of Health Sciences, Northeastern University, Boston, MA, USA Mutations in HFE (hemochromatosis) gene are associated with neurodegenerative diseases with age. To better understand the effect of HFE on the age‐related neurobehavioral dysfunction, we conducted a series of behavior tests using 1‐yr‐old mice. Hfe‐deficient (Hfe‐/‐) and control wild‐type (Hfe+/+) mice showed similar performances in marble‐burying, elevated plus maze and rotarod, suggesting that Hfe deficiency does not affect emotional behavior or motor function over age. In contrast, Hfe‐/‐ mice spent less time around the target hole than wild‐type mice (5 vs 12 s; p=0.048) in the Barnes maze test, representing reduced spatial memory function upon Hfe deficiency. Since memory loss is associated with disrupted iron homeostasis in the brain, we determined metal concentrations using ICP‐MS. Compared with controls, Hfe‐/‐ mice had similar levels of Fe and other metals (Zn, Cu, Mn) in the brain, despite elevated non‐heme iron levels in other tissues, such as serum (70% increase), liver (170%), and kidney (40%; p<0.05 for all; n=7‐8/group). These results indicate that Hfe deficiency alters systemic iron status and not brain iron. Taken together, loss of Hfe function attenuates spatial memory capacity without altering iron in the brain, suggesting a possible role of Hfe in the memory process by an iron‐independent manner.

Grant Funding Source: R00 ES017781
The effect of iron overload on spatial memory and acetylcholine receptor expression in the hippocampus of rats (907.8)
Authors: Han Murui , Kim Jonghan
Published: June 2021

Title: Effect of iron overload on spatial memory and acetylcholine receptor expression in the hippocampus of rats Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA Author: Murui Han, Jonghan Kim Iron plays an essential role in cognition and memory. While iron deficiency is associated with reduced memory capacity, we have previously demonstrated that iron overload improved a recognition memory performance in growing rats. We here characterized the effect of iron overload on special memory and explored molecular mechanisms involved in memory. Barnes maze test was conducted using rats fed iron overload diet (10,000 mg iron/kg diet) or control diet (50 mg/kg) for 4 weeks. Compared with controls, iron overload (IO) rats spent less time (57% decrease; p<0.001) and poked less errors (13% decrease; p=0.042) to find the target hole during the first training day (n=8/group), which indicates a better spatial memory upon iron loading. Western blotting was employed to examine memory‐related proteins in the hippocampus. IO rats up‐regulated nicotinic acetylcholine receptor alpha‐7 (nAChR; p=0.045; n=3‐4/group). Activities of superoxide dismutase (anti‐oxidant enzyme) or levels of malondialdehyde (lipid peroxidation marker) were not altered in IO rats. In addition, ICP‐MS results showed that brain metal levels (iron, zinc, manganese) were not altered in IO rats, indicating no evidence that IO promotes oxidative stress. Collectively, increasing memory performance under iron loading in growing rats could be due to up‐regulation of nAChR with brain metals unaffected. Supported by NIH R00 ES017781.

Grant Funding Source: NIH R00 ES017781
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
The role of melatonin on uteroplacental antioxidant enzymes activities and fetal growth retardation in diabetic rats (910.1)
Authors: Olayaki Luqman
Published: June 2021

Melatonin is an endogenous substance produced by the mammalian pineal gland and other organs. It is known to have antioxidant, antiaging and anticancer effects. The aim of the study was to investigate the effect of oral melatonin on fetal growth retardation, uteroplacental antioxidant enzymes activities and lipid peroxidation in experimental diabetic rats. Twenty pregnant rats were divided into four groups of five rats each. Diabetes mellitus was induced by intraperitoneal administration of 120mg/kg body weight of alloxan in groups 2, 3 and 4. From gestational day 1 to 19, 1mg/kg and 10mg/kg of oral melatonin were administered to the rats in groups 3 and 4. On the 19th day of gestation, the rats were sacrificed by cervical dislocation and placental, fetal and uterine tissues were harvested. In the harvested tissues, glutathione peroxidase (GPx), Superoxide dismutase (SOD), Catalase (CAT) and malondialdehyde (MDA) were estimated. In addition, fetal weight, fetal size and plasma glucose were determined. Our results showed that, in diabetic rats, fetal growth retardation was associated with a significant reduction in placental and uterine antioxidant enzymes (GPx, SOD and CAT) activities (P less than 0.01) and an increase in lipid peroxidation, MDA concentration (P less than 0.01). Treatment with oral melatonin improved the fetal weight, placental and uterine antioxidant enzymes activities and reduced lipid peroxidation, without affecting the degree of hyperglycemia. Our study has shown that reduced uteroplacental antioxidant enzyme activities and increased lipid peroxidation could be other sources of fetal growth retardation in diabetic rats. In conclusion, oral administration of melatonin to pregnant diabetic rats improved fetal growth. This effect seems to depend on the ability of melatonin to improve uteroplacental antioxidant enzymes activities and reduced lipid peroxidation.
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.
Morphometrics of the human lumbar spine (915.5)
Authors: Jelaca‐Tavakoli Maria
Published: June 2021

Morphometrics of human lumbar spine is of interest to diverse fields of study such as anthropology, orthopedics and bioengineering. The aim of this study is to deliver detailed tabulated morphometrics of the lumbar spine as user‐friendly cross‐disciplinary comprehensible data. The sample used in this study was comprised of 701 skeletonized black (n = 428) and white (n = 273) adults (440 males and 261 females) with healthy spines between 16 and 96 years of age from two North American and two South African fully documented anatomical collections. A total of fifty‐five dimensions were collected via digital caliper from individual skeletonized lumbar spine. Each lumbar vertebra provided eleven morphometric sites. Compilation of the lumbar morphometric data tabulated as means, range and standard deviations divided by sex and ethnic backgrounds are presented. Diverse applications of the resulting data include but are not confined to applications in orthopedic spinal surgeries, bioengineering of 3D vertebra and various subfields of anthropology.
The effects of eggshell removal on embryonic skeletal development in the American alligator (Alligator mississippiensis) (921.7)
Authors: Membreno N , Elsey R , Owerkowicz T
Published: June 2021

Embryonic archosaurs (crocodilians and birds) are known to sequester calcium from their heavily mineralized eggshell and use it to mineralise their developing skeleton. In order to test the importance of eggshell calcium to embryonic growth, we studied growth of embryos and hatchlings of the American alligator with and without the calcareous eggshell. The outer eggshell layer was manually removed early in incubation, and embryos (n=6) were sampled at weekly intervals until hatching. The experimental and sham‐handled control eggs were incubated at 30ºC and 100% relative humidity. Results show that experimental (“shell‐less”) embryos were significantly smaller in body mass and length than their clutch‐sibling controls. Under identical rearing conditions, experimental hatchlings did not demonstrate catch‐up growth despite ad infinitum diet and remained significantly smaller than control hatchlings. Observation of biting behavior revealed that experimental hatchlings had more compliant lower jaws and generated weaker bite forces. Our results suggest that yolk calcium reservoir is sufficient for healthy development, but eggshell calcium is required for normal embryonic growth. We posit that increased calcium absorption during development allows the embryo to hatch at larger size, which ultimately allows greater force development during locomotion and predation.
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
V‐ATPase‐mediated activation of cathepsins in breast cancer cells: investigating the role of vacuolar H+‐ATPases in breast cancer metastasis (934.8)
Authors: Uhlman Andrew , Hinton Ayana
Published: June 2021

V‐ATPases are multi‐subunit H+ pumps that acidify intracellular compartments and are essential for bone degradation synaptic signaling, etc. They are at the cell surface of the highly metastatic breast cancer cell line MDA‐MB231, and may contribute to its metastatic phenotype. We hypothesize that V‐ATPases create a low pH environment that promotes tumor cell invasion by activating secreted cathepsins. Cathepsins cleave extracellular matrix proteins and activate other proteases, facilitating local tumor invasion. We are studying the role of subunit the V‐ATPase in secretion and activation of cathepsins B and L. Inhibition of V‐ATPases with concanamycin A (a general V‐ATPase inhibitor) leads to a large reduction in the activity of intracellular cathepsin B. In addition, inhibition of V‐ATPases leads to a large reduction in the activity of extracellular cathepsin L. Thus it seems that V‐ATPases are playing a role in activation of both intracellular and extracellular cathepsins. It is known that V‐ATPases are required for activation of proteases such as cathepsins within lysosomes, however, not as much is known about the activation of secreted cathepsins. Our findings support the hypothesis that cell surface V‐ATPases are important for activation of cathepsins in the extracellular space. Since concanamycin can penetrate the cell membrane we cannot rule out the alternative hypothesis that V‐ATPases within the cell are activating pools of cathepsin L within secretory vesicles. Further experiments with immunoblotting and cathepsin activity assays are being used to differentiate between the role V‐ATPases are playing in activation of cathepsin L within secretory vesicles versus activation of cathepsin L in the extracellular space. This study increases our understanding of the role V‐ATPases are playing in the activation of secreted cathepsins in the tumor microenvironment of MB231 cells.
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
Identification of critical residues in spliceosomal protein Dib1 (939.6)
Authors: Lucas Amber , Whitten Steven , Maeder Corina
Published: June 2021

Pre‐messenger RNA in eukaryotic cells must have its introns removed in order to be able toproduce functional proteins. This removal of introns is facilitated by a macromolecule known as the spliceosome. The spliceosome is a large complex of five snRNA and ~80 proteins that orients the pre‐mRNA such that two transesterification reactions are catalyzed resulting in the removal of introns from pre‐mRNA resulting in a mature mRNA. The mRNA can then be translated in to a functional protein. Mutations in the spliceosome and the proteins that regulate the spliceosome can have drastic effects and lead to diseases such as Retinitis Pigmentosa. Dib1 is an evolutionarily conserved protein in the spliceosome associated with the U5 snRNP in the spliceosome and is required for cell viability. Although the human homolog, Dim1, has been crystalized its role still remains unknown. In order to identify Dib1’s function, we have created point mutations in the protein and analyzed their effects on cell viability. We have identified a variety of temperature sensitive and lethal dib1 mutants in Saccharomyces cerevisiae. We are characterizing these mutants to determine the effect these mutations have on Dib1 protein structure and their overall impact on splicing activity. This study will identify the role of critical residues in Dib1 in pre‐mRNA splicing.
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)
Features of the RAP1A and RAP1B core promoters: recombinant approaches to testing regulatory element function in expression control (946.10)
Authors: Augustin Ryan , Painter Mark , Niemela Amy , Cruise Jennifer
Published: June 2021

The RAP1 genes, closely related to the RAS oncogenes, have been shown to have numerous functions, yet little is known about the expression regulation of these genes in human cells. Our lab has constructed plasmids containing putative promoter regions of human RAP1A and RAP1B, driving a gene for green fluorescent protein (GFP). We have transiently transfected these plasmids into a wide‐range of human cell types, and analyzed GFP expression via fluorescence microscopy, qRT‐PCR, and western blotting. By comparing the efficacy of each putative core promoter segment, some distinct regulatory regions have been revealed. Additionally, comparing promoter‐driven expression to the actual native expression of the RAP1B genes in specific cell types has suggested an important regulatory role for enhancer elements not included in the core promoter regions. Various sub‐cloning techniques, including inverse fusion PCR cloning (IFPC), have been employed to add putative regulatory regions to existing GFP core promoter plasmids. These should allow us to further characterize the expression control of the RAP1 genes, and to understand the cell‐type differences observed in native RAP1 expression.
Cell‐free system for membrane protein production from cultured insect cells (948.1)
Authors: Uozumi Nobuyuki , Nanatani Kei , Soma Satoshi , Sato Yoko , Aizawa Keishi , Ezure Toru , Ando Eiji
Published: June 2021

Membrane proteins constitute almost one third of all gene products in any types of organism. Overproduction of the proteins in the living cells often shows cellular toxicity to host cells, ending up to failure of amount of proteins needed for performing the experiments. In contrast, cell‐free (= in vitro) synthesis of membrane proteins has been recognized as a powerful technique for studying membrane proteins. Ezure et al. has developed the cell‐free translation system from cultured insect cells, Sf21 (Spodoptera frugiperda) cells as a commercial kit, Transdirect (Shimadzu, Japan). Here we have tested the possibility of ER membrane from the insect culture cells as this new translocation system for membrane protein synthesis. This system consists of ER microsome isolated from Sf21 cells. We evaluated the production of polytopic membrane proteins and tail‐anchor proteins from bacteria, plant cells and animal cells in an insect cell‐free system combined with the Transdirct and the translocation system. The modification of membrane proteins as post‐translational modification occurred in this system. This indicates that the developed insect cell‐free translocation method to synthesis of membrane proteins is likely to be general techniques to help the understanding of structure and function of membrane proteins.
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).
Altered intracellular trafficking of EGFR by extracellular glucose concentration in squamous carcinoma cells (948.2)
Authors: Yoshimoto Shohei , Morita Hiromitsu , Nagano Koki , Sugiyama Goro , Hiroshi Takeuchi Hiroshi , Nakamura Seiji , Hirata Masato
Published: June 2021

Cancer cells usually exhibit the increased uptake of glucose than normal cells via glucose transporters, and pathogenesis and progression of squamous cell carcinoma are closely related to the expression of epidermal growth factor receptor (EGFR). To connect these two events, we here examined whether intracellular translocation of EGFR is regulated by glucose concentration. Human gingival, tongue and skin squamous carcinoma cells (HSC‐2, HSC‐3 and A431) were grown in D‐MEM in the presence of either high or low concentration of glucose (25mM or 5.5mM: HG or LG). When cancer cells were maintained in HG medium, the expression of EGFR with a molecular size of 170kDa was observed as assessed by Western blot analysis. In contrast, cells cultured in LG medium showed the major positive band to anti‐EGFR antibody, with 140kDa. We assumed the size difference is caused by glycosylation, as the treatment of cell extract with 170kDa band by N‐glycosidase produced the band with 140kDa. We further examined the effect of tunicamycin (10μM) to inhibit glycosylation process, resulting in that EGFR turned into 140kDa even if cells were cultured in HG medium. Biotinylation assay revealed that the only 170kDa protein was detected, indicating that the 170kDa bands are present at the plasma membrane. These results suggest that glucose concentration regulates glycosylation of EGFR and subsequent translocation to the plasma membrane.
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
Systems architecture of a bacterial cell cycle (95.1)
Authors: Shapiro Lucy
Published: June 2021

Understanding of the cell cycle control logic in Caulobacter has progressed to the point where we now have an integrated view of the operation of the entire bacterial cell cycle system functioning as a state machine. Oscillating levels of a small number of temporally‐controlled master regulators drive cell cycle progression. Cell cycle regulation is, to a striking degree, a whole cell phenomenon with transcriptional circuitry interwoven with the 3‐D deployment of phospho‐signaling proteins and proteases. The phospho‐signaling system monitors the topology of the cell and is central to establishing asymmetric cell division. The overall regulatory ‘wiring diagram’ incorporates changes in DNA methylation state that enhance system robustness.
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
The genetic basis of mitochondrial DNA depletion syndrome (958.2)
Authors: Venkataraman Chantel , Field Martha , Stover Patrick
Published: June 2021

Mutations in DGUOK, a gene encoding an enzyme in the mitochondrial nucleotide synthesis salvage pathway, and MPV17, a gene encoding an inner mitochondrial membrane protein of unknown function, have been shown to cause the hepatocerebral form of mitochondrial DNA depletion syndrome. MPV17 has been postulated to function as a nucleotide transporter. Transient HeLa cell culture models using siRNA and stable HeLa cell culture models using shRNA were created. The models facilitated studies addressing the effects of nucleotide availability on mtDNA copy number. It was observed that mtDNA copy number modestly decreased in MPV17 siRNA cells cultured in nucleotide replete medium. Supplementation with nucleotides individually indicates that pyrimidine availability increases mtDNA copy number in MPV17 siRNA‐treated cells as much as 10‐fold. Further experiments to determine the quality of the mtDNA through determination of uracil content are currently being conducted.

Grant Funding Source: Supported by NIH R37DK58144