bluuebunny/crossref_metadata_2025 huggingface dataset:
End binding protein 3 promotes IP3‐gated calcium release and permeability of the endothelial barrier (893.7)
Authors: Geyer Melissa , Huang Fei , Thambiayya Kalidasan , Vogel Steve , Malik Asar B. , Komarova Yulia
Published: June 2021

VE‐cadherin, the major endothelial adhesion molecule of AJs, forms hemophilic adhesion and induces re‐organization of actin and microtubule (MT) cytoskeleton. In the current study, we have addressed the role of MT end binding (EB) protein 3 in regulating stability of VE‐cadherin adhesion and lung tissue‐fluid homeostasis. Depletion of EB3 significantly attenuated inositol 1,4,5‐thriphosphate (IP3)‐gated release of calcium from intracellular stores and cell shape change in response to serine protease thrombin. Using acceptor‐photobleaching FRET and pulldown experiments we demonstrated that EB3 interacted with inositol 1,4,5‐thriphosphate receptor type 3 (IP3R3) both in cells and in cell‐free system. Immunofluorescent staining of endogenous IP3R3 and live‐cell imaging of exogenously‐expressed IP3R3‐GFP in EB3 depleted cells showed that the interaction between EB3 and IP3R3 at the MT tip is required for IP3R3 clustering. Rescue with EB3 mutant lacking the last four amino acids did not restored IP3R3 clustering suggesting that presence of acidic tail is critical for interaction with IP3R3. Pre‐treatment of cells with membrane permeant variant of IP3R3‐derivative peptide (IPRP), containing the EBs consensus motif, significantly inhibited the interaction between EB3 and IP3R3 as well as attenuated thrombin‐induced release of calcium from intracellular stores. Furthermore, infusion of IPRP in lung microvasculature significantly inhibited increase in lung vascular liquid permeability (Kf,c) in response to activation of Protease‐activated receptors‐1 with an agonist peptide. These results identify a novel function of EB3 in regulating calcium signaling and tissue‐fluid homeostasis in lung.
Aspirin and salicylate are solutes of both sodium monocarboxylate transporters (SMCT1/Slc5a8 and SMCT2/Slc5a12) (896.9)
Authors: Plata Consuelo , Gavi‐Maza Marisol , Vázquez Norma , Romero Michael , Gamba Gerardo
Published: June 2021

The Na+/monocarboxylate transporters (SMCT1 and SMCT2) are expressed several organ between them intestine and kidney, suggesting that its main physiological function are the absorption of fuel molecules involved in cellular metabolism and have only one ‐COOH group in its chemical structure; (lactate, propionate). Drugs with similar chemical properties also are translocated by SMCTs (B3 vitamin, pyrazinoate). However some propionate‐derivative NSAIDs (ibuprofen) reduce the activity of mammalian SMCT1. Using 22Na+ uptake in Xenopus oocytes injected with zebrafish SMCT’s (zSMCT1, zSMCT2) or human SMCT1 cRNA, we evaluated the effect of aspirin and salicylate on the function SMCT’s. Our results indicate a 22Na+ uptake that is aspirin concentration dependent in all SMCT’s. Similar affinity by aspirin was observed between hSMCT1 (Km ~2.4mM) and zSMCT1 (Km~2.5 mM) in contrast to a lower affinity of zSMCT2 (Km ~5mM). Also the difference of substrate affinity between zSMCT2 and hSMCT1 was evident by salicylate (hSMCT1Km ~4mM vs zSMCT1Km ~30 mM). Interestingly, although zSMCT1 and hSMCT1 shares a similar affinity and transport capacity for several substrates, the salicylate transport by zSMCT1 was not detected and only reduced a small percentage propionate transport induced by zSMCT1 (~10%) in contrast of ibuprofen which drops to 80%. The clear difference of salicylate transporter between SMCT1 could be explained by changes in the structure of proteins because the amino acids sequences are approximately 50% different, so the salicylate could be candidate for studies of structural‐function between SMCT1.

Grant Funding Source: CONACYT48709MJ(CP)
FGF‐10 protects the intestinal epithelium from LPS‐induced injury (902.14)
Authors: Schall Kathy , Danopoulos Soula , Frey Mark , Ford Henri , Grikscheit Tracy , Al Alam Denise
Published: June 2021

Necrotizing enterocolitis (NEC) is a major cause of death among premature infants and is characterized by epithelial cell death, barrier damage and inflammation. There is no proven therapeutic approach identified for NEC. Fibroblast Growth Factor 10 (FGF10) is a soluble growth factor that enhances epithelial cell proliferation and survival during colon development. We hypothesized that FGF10 enhances epithelial cell survival and decreases inflammation following insults in the small intestine. We tested this hypothesis with an in vivo model in which rtTA Tet(O)Fgf10 and rtTA mice were fed doxycycline containing diet for 16 hours to induce Fgf10, and then injected with LPS for 90 minutes to induce acute enteritis and epithelial apoptosis. To define FGF10’s role specifically in intestinal epithelial cells, we challenged IEC‐6 rat ileal crypt cells with LPS +/‐ recombinant FGF10 and measured cell death. In vivo, Fgf10 overexpression blocked LPS‐induced loss of villus integrity and decreased the number of caspase‐3 positive cells. The inflammatory mediators Tnf, Il‐6, Tlr2 and Tlr4 were elevated in the mice challenged with LPS, a response that was inhibited by Fgf10 overexpression. In vitro, FGF10 reduced LPS‐induced IEC‐6 cell death. In conclusion, FGF10 increases cell survival and decreases inflammatory mediators, which may be useful in future therapeutic interventions for NEC.
Adiponectin and adiponection receptor 1 enhance colon inflammation (902.8)
Authors: Peng Yu‐Ju , Liu Bing‐Hsien , Mersmann Harry , Ding Shih‐Torng
Published: June 2021

Adiponectin (ADN) is an adipokine derived from adipocytes. It binds to adiponectin recetor1 and 2 (AdipoR1 and 2) to exert into function regulating in whole‐body energy homeostasis, anti‐cardiovascular disease and inflammatory response. The role of ADN in inflammation response is likely multifaceted. We established the murine colitis model. After administration of 2% DSS in drinking water for 7 days, AdipoR1‐transgenic mice developed more severe colitis than wild‐type mice, the symptoms including body weight loss, bloody‐diarrhea, short colon length, crypt disruption and immunity cell infiltration. We also administrated ADN to THP‐1 cell (monocyte cell line) and HT29 cell (colon epithelial cell line). In colon epithelial cell, ADN does not enhance the expression of pro‐inflammatory factor (TNFα, IL‐1 and IL‐6) and inducible cyclooxygenase 2/cox2) but chemokine (IL‐8). However, ADN increased cox2 and IL‐8 expression in the inflammatory state. In macrophage ADN increased the pro‐inflammatory cytokine (TNFα) and chemokine (IL‐8). ADN increase the expression of IL‐1 in cell inflammatory state. In macrophage, ADN does not affect cox2 expression. ADN decreased cox2 expression and increase IL‐1 expression in the inflammatory state. Our data show that ADN may play a pro‐inflammatory role in colitis by increasing pro‐inflammatory cytokine and inducing immune cell infiltration.
Potassium channel KCNJ15 plays a critical role in gastric acid secretion (904.1)
Authors: Liu Wensheng , Yuan Jianye , Karvar Serhan , Baker Susan , Baker Robert , Ji Guang , Xie Jianqun , Zhu Lixin
Published: June 2021

Gastric acid secretion is mediated by a K+ dependent proton pump, which requires a K+ channel to continuously supply K+ at the luminal side of the apical membrane. However, the identity of potassium channel coupled with gastric acid secretion is still elusive. We aimed to investigate the role of potassium channel KCNJ15 in acid secretion. A recombinant adenovirus expressing CFP tagged KCNJ15 was used to infect parietal cells and live cell imaging was conducted to record the pathway of KCNJ15‐CFP in stimulated acid secretion. KCNJ15 molecules are scattered all over in resting cells, but exhibited apical localization in histamine‐stimulated live cells. Further, the effect of knocking down KCNJ15 on acid secretion was analyzed. KCNJ15 shRNA was delivered into rabbit primary parietal cells using a recombinant adenoviral vector. The knockdown of KCNJ15 was confirmed by western blotting. Histamine stimulated acid production was monitored by [14C] aminopyrine uptake assays. Knocking down KCNJ15 abolished histamine stimulated acid secretion, indicating that KCNJ15 is critical for stimulated acid secretion. In addition to rabbit, we also detected KCNJ15 proteins in human and mouse gastric mucosa, suggesting KCNJ15 could play a similar role in acid secretion in other species. These results further support that KCNJ15 plays a critical role in gastric acid secretion.
Characterization of sodium‐independent Pi uptake in Caco2 cells (908.3)
Authors: Candeal Eduardo , Caldas Yupanqui , Guillén Natalia , Levi Moshe , Sorribas Victor
Published: June 2021

While inorganic phosphate (Pi) uptake in epithelia has both Na‐dependent and Na‐independent components, only Na‐dependent transporters have been identified to date. In this work we have characterized the Na‐independent Pi uptake in the intestinal Caco2BBe cell line, as a preliminary step in the molecular identification of novel intestinal Pi transporters.

Caco2BBe cells grown in DMEM were incubated overnight in the presence of 1 or 4mM Pi to mimic the intestinal adaptation to high Pi diet recently described. Only Na‐independent Pi uptake was observed in these cells, which was dramatically increased in cells preincubated with 4mM Pi. Uptake in 1mM Pi adapted cells decreased with alkaline pH, but it increased with the pH at 4mM Pi. The increase in Pi transport at 4mM Pi was completely inhibited by Pi, phosphonoformic acid and oxalate, while DIDS, SITS, SO4=, and CO3= did not. Km values were 0.06 and 0.2 mM after adaptation to 1 and 4 mM Pi, respectively.

In conclusion, two different Na‐independent Pi transports are expressed in Caco2BBe cells, one of them being overexpressed when preincubated with high Pi medium. The molecular identity of the transporters are still unkown.
Determining the downstream signaling pathways through which prostaglandin E2 inhibits inflammatory mediator output in in human myometrial cells (911.6)
Authors: Levinson Hayley , Wood Stephen , Slater Donna
Published: June 2021

Objective: Prostaglandin E2 (PGE2) represses interleukin‐1β (IL‐1β) induced inflammatory mediator output in human myometrial cells via EP2/4 receptors. Downstream EP2/4 signaling involves upregulation of intracellular cAMP which may activate multiple pathways, including protein kinase A (PKA) and exchange proteins activated by cAMP (Epacs).

Methods: Myometrial biopsies from term elective Caesarean deliveries prior to labour onset (n=5, 37‐40 weeks gestation) were used to isolate myometrial cells. PKA and Epac gene expression was assessed by RT‐PCR. Myometrial cells were either non‐treated or stimulated with IL‐1β +/‐ co‐treatment with cAMP analogs to selectively activate PKAI, PKAII, or Epacs. Interleukin‐8 (CXCL8) output was measured by ELISA. Statistical analysis was performed by repeated measures ANOVA followed by a Bonferroni test.

Results: Expression of both Epac isoforms and six of seven PKA subunits were identified in isolated myometrial cells. IL‐1β increased CXCL8 output in human myometrial cells (p蠄0.001). Co‐treatment with PKAI or PKAII selective cAMP analogs repressed this response (p蠄0.001). No significant repression was observed when cells were co‐treated with the Epac selective cAMP analog.

Conclusions: PGE2 mediated repression of IL‐1β induced inflammatory output in human myometrial cells is mediated in part by PKA, but not Epac, signaling pathways.

Grant Funding Source: Supported by Canadian Institutes of Health Research and Alberta Innovates‐Health Solutions
Histological characterization of soft‐embalmed porcine tendon and muscle (914.1)
Authors: Martyniuk Amanda , Easteal Ron , Ellis Randy
Published: June 2021

Embalmed material is used in many fields because of its benefits over fresh materials; however, typical formalin‐fixation results in materials that are vastly different from their original state. Soft‐embalming has received recognition as a viable alternative to fresh or formalin‐fixed materials in education and research, especially in orthopedics. Soft‐embalmed tissue has mainly been characterized by gross features, with minimal investigation into the microscopic features of different types of soft‐embalmed tissues.

Deep flexor muscle and tendon were harvested from porcine forelimbs. Samples were soft‐embalmed using one of three different methods: phenol‐based, phenoxyethanol‐based, and Thiel embalming. The samples were visualized using Masson’s trichrome staining immediately following embalming and three weeks later.

All types of soft‐embalming displayed typical tendon structural characteristics at both time points. The staining of the tendons ranged from almost entirely blue, as would be expected, to almost entirely red, which was unexpected, depending on the treatment. All soft‐embalmed muscle samples exhibited typical colouring, but there were structural differences. At both time points, muscle of phenol‐based and phenoxyethanol‐based embalming generally exhibited normal muscle structure, while Thiel‐embalmed muscle appeared to have suffered degradation.

This histological analysis is a step forward in understanding the effects of different soft‐embalming methods on different tissues, which can aid in determining their efficacy.

Grant Funding Source: This research was supported in part by NSERC and CIHR
Human embryonic larynx morphogenesis: three‐dimensional and multiplanar study (918.26)
Authors: Labrouse Marc , Tonnelet Romain , Renard Yohann , Micard Emilien , Duparc Fabrice , Delmas Vincent , Braun Marc
Published: June 2021

The purpose of this study was to describe the human larynx embryology, using three‐dimensional reconstructions from seven human embryos histological sections, and to compare our results with literature.

Seven human embryos from two historic French collections (Paris‐Descartes University's Delmas‐Rouvière collection and Rouen University's Tardif collection) stained with hematoxylin & eosin or Masson's trichrome were reclassified according to the Carnegie Atlas criteria (CS (Carnegie stages) 17, 18, 19, 21 and 22).

A local made computer software developed by IADI lab (INSERM U947, Nancy) allowed automatic slices selection, automatic rigid registration, and dust filtering. Once registration had been completed, DICOM files were used with ORS Visual software (v1.5, Montreal), which allowed multiple display and post‐processing possibilities (3D VR; orthogonal/curved multiplanar reformation). Due to large size files, image processing was performed on PC with Windows 7 (64 bit) and 20 GB RAM.

Three‐dimensional and multiplanar study of the larynx morphogenesis was possible with appropriate thresholding. In the first stages of this study, the early laryngeal aditus was visible. Two posterior elevations, the arytenoid swellings, one anterior, the epiglottis, and the vocal folds were seen in the last stages.

Human embryonic larynx morphogenesis works are very rare because of the smallness of this anatomical structure. Our study, using historic human embryos histological sections has shown the larynx growth. This demonstration was completely novel and had never been published previously, to our knowledge. Indeed, our method is fully respectful towards the microscopic structure unlike methods using Amira© segmentation and triangulation process. The precise knowledge of this morphogenesis will help to better understand congenital malformations.
Phenotypic similarities and dissimilarities in a Paleoamerican sample: what are the implications in modeling the first migrations into the New World (918.4)
Authors: Alsup Kathleen
Published: June 2021

Researchers can interpret craniometric phenotypic similarities and dissimilarities in very different ways. This can have important implications in developing theories and models used to interpret many anthropological questions. The purpose of this poster is to investigate the craniometric relationships of a Paleoamerican sample and discuss the implications of these relationships between the crania.

A sample of 33 crania, with published dates of 7000 years BP or older, were investigated. 18 cranial measurements were utilized. Cranial variation was analyzed through the calculation of Mahalanobis distances between each pair of crania. A principal coordinate analysis was used to visualize the general phenotypic patterns among the crania.

The distance relationships among the Paleoamerican crania suggest a separation between the South American and North and Central American specimens. There does not appear to be distinct differences among the South American specimens from different areas and sites.

Further discussion of the results include the implications of heterogeneity of the complete Paleoamerican sample, the distinctness of many North American and Central American crania and the relative homogeneity of the South American subsample. Further analysis of these points may be of great interest in modeling the first migration(s) into the Americas.
Anatomical diversity of the avian feeding system: preliminary analysis of the hyolingual apparatus (918.9)
Authors: Heiner Elsa , Witmer Lawrence
Published: June 2021

Birds are extremely diverse and have a comparably broad range of feeding strategies. Different feeding methods vary in the anatomy of the tongue, jaw muscles, beak, and oral cavity. As a preliminary analysis, the hyolingual apparatuses of three species, the wild turkey (Meleagris gallopavo), the ostrich (Struthio camelus), and the pileated woodpecker (Dryocopus pileatus), were studied. MicroCT scanning was used to visualize the bony structures of the feeding apparatuses, using the software program Avizo. The specimens were then analyzed with Avizo after contrast‐enhancement by soaking in Lugol’s iodine, followed by a second microCT scan in order to visualize the soft tissues, such as the hyoid musculature. Gross dissection was then used to validate the imaging studies. These imaging techniques revealed that turkeys have a fairly generalized avian tongue with the full complement of well‐ossified bony elements. Ostriches have a short, broad fleshy tongue that is reduced and primitive in some ways in that only the ceratobrancial element typically fully ossifies and other elements remain cartilaginous. Woodpeckers have a specialized probing tongue with a very long and muscular hyoid. Different functional categories of tongue function correlate well with structure and are traceable both with dissection and 3D imaging. Future directions include expanding the sample to other species with divergent feeding modes.
The body mass estimation from human talus: inductive approach and 3D morphometric study (919.1)
Authors: Jung Go‐Un , Lee U‐Young , Kim Dong‐Ho , Kwak Dai‐Soon , Ahn Yong‐Woo , Kim Yi‐Suk
Published: June 2021

The body mass (BM) that is gauging physical characteristics of individuals is still challengeable information to acquire from the skeletal remains in the fields of biological anthropology. The aim of this study is to develop the BM‐predicting equations from the talus using 3D morphometrics and investigate whether the estimated BM can be used as a biological profile in forensic anthropology.The right‐side talus in type of 3D models obtained from 76 Koreans (32 males and 44 females) of known stature, bi‐iliac breadth, and femoral head diameter were used. In this study, the inductive approach is needed in order to construct information about the weight for the CT‐based Digital Korean Collection, applying morphometric and mechanical method for assuming the weight of the individuals from the collection. For developing BM‐predicting equations, nine variables of 3D talus were measured through Mimics software (Materialise NV, Leuven, Belgium) and statistical analyses were performed using SPSS 20.0 (SPSS Inc., Chicago, IL). Overall this study, analysis of covariance found no statistically significant differences between sexes in most talar measurements except that talar length is just not. The linear regression analyses revealed that talar height was the most highly correlated measurement with the BM (R2 = 0.702, SEE = 3.73), followed by talar length (R2 = 0.701, SEE = 3.74) and talar width (R2 = 0.631, SEE = 4.15). Through the multiple regression analysis, four measurements of talar height, talar length, trochlear breadth and head height were selected as a BM‐predicting equations by stepwise selection with increasing the coefficient of determination (Adjusted R2 = 0.779, SEE = 3.19). In conclusion, the BM‐predicting equations were developed and expected this study do role as a stimulus of further study for assessment the effectiveness of anthropological body mass estimation serving as a biological profile.

Grant Funding Source: Supported by the National Research Foundation of Korea (NRF‐2013R1A1A2009306)
Microscopic analysis of characteristic surface features of a set of retrieved total hip implants (922.13)
Authors: Burbano Maria , Roy Diana , Russell Robert , Huo Michael , Rodrigues Danieli
Published: June 2021

Introduction: Total Hip Arthroplasty (THA) is considered one of the most successful orthopaedic interventions used for joint replacement. The purpose of this study was to characterize the particular surface features common to retrieved THA implants, in order to point out principal characteristics that lead to their failure.

Methods: A set of fourteen retrieved implants was studied, their designs varying from metal‐on‐polyethylene (MoP), ceramic‐on‐polyethylene (CoP) and metal‐on‐metal (MoM). All specimens were analyzed using digital microscopy (Keyence VHX2000). Particularly corroded and scratched areas were further analyzed under Scanning Electron Microscopy (SEM, JEOL 5600), and Energy Dispersive X‐ray Spectrometer (EDS, JEOL 5600).

Results: The surface analysis revealed certain patterns of features. The implants were divided in their components (cup, liner, head and stem), each of which was studied for common characteristics. Large amount of scratching was found throughout all of the heads and cups, followed by biological deposition on the surfaces, pitting and finally one single case of corrosion.

Conclusion: The high level of scratching observed on the bearing surfaces of the implants leads to the belief that mechanical events induced failure of the implants. This study encourages surgeons to place higher importance of implant placement and material choice when applying THA.

Grant Funding Source: Supported by the University of Texas at Dallas startup funds (Dr. Rodrigues)
A half‐sized écorché by Talrich (19th century), description of an anatomical model in plaster on an anterior view (923.4)
Authors: Le Floch‐Prigent Patrice , Gillot Jean‐Baptiste
Published: June 2021

During the 19th century, the anatomical models in plaster were frequent but were still in balance between the expressionism of the previous centuries and the cold reality of the anatomical dissections. This half‐sized, anterior view of a model of Jules Talrich who was an official molder in the “Faculté de Médecine de Paris” in this time (1856) was exactly between this two extremes with a tendancy to a complete respect of the representation of the anatomical structures. The écorché was in a supine position near the anatomical position of reference with the exception of a 15° degree of each lower limb and a 30° extension of the head. The different layers of the represented muscles are described in each main region. Differences were noticeable for the lower limbs. The anatomical veracity of the model was very good and the artistic rendering limited. A metallic plate embedded in the plaster authentified its author as Talrich. The model was 95cm high, 36cm wide and 12cm thick. It was posteriorly hollowed and not colored.
Evidence for the hypothesis that two forms of human DNA polymerase δ participate in DNA replication (925.2)
Authors: Lin Szu Hua Sharon , Zhang Sufang , Lee Ernest , Zhang Zhongtao
Published: June 2021

Eukaryotic DNA polymerase δ (Pol δ) is the polymerase primarily involved in lagging strand DNA synthesis. Human Pol δ is a heterotetramer (Pol δ4) composed of four subunits. The p12 subunit is degraded in response to DNA damage, resulting in the in vivo conversion of Pol δ4 to Pol δ3, the trimer lacking p12. Thus, Pol δ3 is the form that is involved in DNA repair. The hypothesis that Pol δ3 is also capable of performing lagging strand DNA synthesis was tested in a reconstituted system for Okazaki fragment processing using purified enzymes and model oligonucleotide substrates. In the combined Pol δ3/Fen1 reactions, the products formed by removal of the downstream blocking primer were almost exclusively mononucleotides, showing that the reaction proceeded by nick translation, and was not affected by Fen1 concentrations. With Pol δ4/Fen1, the products were 1‐10 nts at low Fen1 concentrations, showing that the reaction proceeded by the short flap pathway. As Fen1 concentrations were increased the product size was shifted to mono‐nucleotides, i.e.,. to a nick translation mode. These studies show that Pol δ3 is ideally suited for Okazaki fragment processing. In parallel studies, we have shown that p12 is degraded during the G1/S transition so that a mixture of Pol δ4 and Pol δ3 is present in S phase, under the control of CRL4Cdt2, which also regulates the degradation of the licensing factors Cdt1, Set8 and p21.

Grant Funding Source: Supported by GM31973 and ES14737
Tpz1 switches telomeres between ON and OFF states (926.3)
Authors: Qiao Feng
Published: June 2021

Telomeres are nucleoprotein complexes comprising telomeric DNA repeats bound by multiprotein shelterin complex. A dynamic binary switch between telomerase‐extendible and ‐nonextendible telomeric states determines telomere length homeostasis. However, the molecular nature of the two states is largely unknown. Here, we show that, in fission yeast, Tpz1 (the ortholog of human TPP1)‐mediated complete linkage within the shelterin complex, bridging telomeric dsDNA to ssDNA, controls telomerase‐nonextendible state. Disruption of this linkage leads to unregulated telomere elongation while still retaining the shelterin components on telomeres. Therefore, the linkage within the shelterin components, rather than the individual shelterin components per se, defines the telomerase‐nonextendible state. Furthermore, epistasis analyses reveal that Tpz1 also participates in the activation of telomeres to the extendible state via its interaction with Ccq1. Tpz1‐Ccq1 interaction triggers the protein processing of shelterin components and causes the conformational change of the sheltering complex, which in turn switches the telomere from the nonextendible to the extendible state. Our results reveal Tpz1 as the key regulator that switches telomeres between ON and OFF states.

Grant Funding Source: Supported by R01‐GM098943
The architecture of yeast DNA polymerase zeta (927.2)
Authors: Malik Radhika , Llorente Yacob , Jain Rinku , Choudhary Jayati , Johnson Robert , Prakash Louise , Prakash Satya , Ubarretxena‐Belandia Iban , Aggarwal Aneel
Published: June 2021

DNA polymerase ζ (Polζ) is a multi‐subunit enzyme that is specialized for the extension step of translesion DNA synthesis (TLS). Despite its central role in maintaining genome integrity, little is known about its overall architecture. Initially identified as a homodimer of the catalytic subunit Rev3 and the accessory subunit Rev7, yeast Polζ has recently been shown to also form a stable four‐subunit enzyme (Polζ‐d) upon incorporation of Pol31 and Pol32, the accessory subunits of yeast Polδ. To understand the 3‐D architecture and assembly of Polζ and Polζ‐d we employed electron microscopy (EM). We show here how the catalytic and accessory subunits of Polζ and Polζ‐d are organized relative to each other. In particular, we show that Polζ‐d has a bilobal architecture resembling the replicative polymerases, and that Pol32 lies in proximity to Rev7. Collectively, our study provides the first views of Polζ and Polζ‐d, and a structural framework for understanding their roles in DNA damage bypass.
Mechanism of hRev1 polymerase action on G‐quadruplex DNA (927.3)
Authors: Eddy Sarah , Zafar Maroof , Ketkar Amit , Maddukuri Leena , Eoff Robert
Published: June 2021

Faithful replication of genomic information is crucial for maintenance of cellular function and integrity. DNA can adopt non‐B‐form structures in vivo, such as stable G‐quadruplex DNA (QDNA). The accurate replication of QDNA is important, as these structures regulate transcription of oncogenes and telomere dynamics. Recent studies have implicated Rev1 polymerase in successful QDNA replication. We hypothesized that human Rev1 might help maintain fork progress at QDNA sites because of its unique protein template‐directed mechanism of DNA replication and sought to investigate the hRev1 biochemical mechanism of action on QDNA substrates. We find that hRev1 preferentially binds to QDNA substrates. hRev1 is able to catalyze dCMP insertion, but the efficiency of nucleotidyl transfer is impaired opposite tetrad‐guanines. Results from dimethyl sulfate footprinting and stopped‐flow fluorescence assays are consistent with the notion that hRev1 can disrupt QDNA structures without performing nucleotidyl transfer. The specific molecular features that allow hRev1 to bind and disrupt QDNA are the subject of ongoing experiments. In conclusion, our study has identified new properties related to the functional and mechanistic role assumed by hRev1 in QDNA replication.

Grant Funding Source: Supported in part by USPHS R00 GM084460 (R.L.E.)
Examining the temporal expression and regulation of type I MAGE proteins in spermatogenesis (930.4)
Authors: Quintana Mercedes , Fon Tacer Klementina , Hao Yi‐Heng , Potts Ryan
Published: June 2021

Background: Type I Melanoma Antigen Gene (MAGE) proteins are normally expressed in the trophoblast and germline cell lineages, but are aberrantly expressed in various human cancers. Recently, MAGEs were shown to enhance the activity of E3 RING ligases, however the physiological function of MAGEs in the testis and tumorigenesis is yet unknown. With these questions in mind the aim of the project was to confirm germline specific expression of MAGE genes, and assess whether the temporal pattern of MAGE gene expression is associated with specific developmental stages of spermatogenesis.

Methods Testes were collected from C57BI/6 mice at different times during postnatal development. Spermatogenic cells were isolated and enriched using velocity sedimentation in 2‐4% BSA gradient. Expression of type I MAGEs was measured from collected spermatogenic cells using RT‐QPCR. Interaction of type‐I human MAGEs and human ZBTB16/PLZF stem cell marker protein was explored by co‐immunoprecipiration experiments in HeLa cells using exogenously expressed tagged forms of the proteins.

Results MAGE‐B4 was expressed in spermatogonia during mitosis. MAGE‐As were expressed in spermatogonia during mitosis and prepachytene spermatogonia before entering meiosis. MAGE‐B1, ‐Al1, and ‐Al2 were expressed in haploid spermatids in spermiogenesis. MAGE‐B3, ‐B16, ‐B18 and ‐Al3 were expressed in all germ cells and somatic cells during spermatogenesis. Additionally, human MAGE‐A11 and ‐A12 were shown to interact with human ZBTB16/PLZF.

Conclusion Type I MAGEs are expressed in different developmental stages of spermatogenesis, suggesting diverse functions. Future investigation is necessary to determine if MAGE‐A genes regulate the activity of ZBTB16/PLZF.

Grant Funding Source: CPRIT R117
MAML1 mediates MEF2C degradation independent of ubiquitination (930.5)
Authors: Zein Ivan , White Brandon
Published: June 2021

Myocyte Enhancer Factor 2C (MEF2C), a member of a family of transcription factors involved in muscle cell differentiation and proliferation, is known to interact with Mastermind‐like 1 (MAML1). MAML1 transcriptional coactivator was originally identified in the Notch pathway and is also involved in the beta‐catenin, p53, and MEF2C signaling pathways. MAML1 was previously shown to interact with MEF2C and is required for muscle development in the absence of Notch. Since MAML1 was shown to act as a coactivator of Notch while also to induce degradation of the Notch Intracellular Domain, we speculated whether or not MAML1 could also serve a similar function with MEF2C. Our results indicate that MEF2C is degraded in the presence of MAML1. However, this degradation is significantly reduced when using a MAML1 deletion mutant (75‐300). Furthermore, treatment with lactacystin, a proteasome inhibitor, did not inhibit MEF2C degradation while the use of lysosome inhibitors significantly attenuated MEF2C turnover. Our findings suggest that MEF2C degradation is mediated by MAML1 through a lysosomal degradation pathway, independent of the ubiquitin‐proteasomal pathway.

Grant Funding Source: Supported by NSF‐RUI Grant#1052039 to JBW
A second degradation signal within the short‐lived transcription factor MATalpha2 (937.1)
Authors: Hickey Christopher , Hochstrasser Mark
Published: June 2021

The yeast transcriptional repressor MATalpha2 (alpha2) is a short‐lived protein known to be ubiquitylated by at least two distinct pathways, one involving the ubiquitin ligase (E3) Doa10 and the other operating with the E3 Slx5/Slx8. Simultaneous perturbation of both pathways leads to considerable stabilization of alpha2 (approximately 6‐fold); however, deletion of either pathway alone only leads to moderate stabilization of alpha2 (approximately 2‐fold). Although a degradation signal within alpha2, known as Deg1, is well established for Doa10‐dependent degradation, little is known about the determinants within alpha2 that direct its Doa10‐independent degradation. Here we report that alpha2 contains an additional degradation signal, termed Deg2, that is non‐overlapping with Deg1 and leads to ubiquitin‐dependent degradation when fused to normally long‐lived proteins. We also report numerous mutations within Deg2 that result in stabilization of alpha2, including several mutations in the homeodomain that impair the ability of alpha2 to interact with DNA. These data further our understanding of one of the best‐studied substrates of the ubiquitin‐proteasome system and highlight a previously encountered link between the ability of a transcription factor to function and its ubiquitin‐dependent degradation.

Grant Funding Source: Supported by the U.S. National Institute of General Medical Sciences
Structural basis of Rifampin resistance in bacterial RNA polymerase (939.1)
Authors: Murakami Katsuhiko
Published: June 2021

For over four decades, Rifampin (Rif), a semi‐synthetic derivative of Rifamycin, has been used as a first line antibiotic treatment of tuberculosis and is the cornerstone of current short‐term tuberculosis treatment. The mode of action involves tight Rif binding to a beta subunit of bacterial RNA polymerase (RNAP) to inhibit RNA transcription. Although many Rif resistant (RifR) strains with mutations in the Rif‐binding pocket can be isolated in bacterial culture, only three specific RifR mutations account for over 80 % of Mycobacterium tuberculosis (MTB) RifR strains in clinical isolates due to RifR associated fitness costs.

In this study, we have determined the X‐ray crystal structures of the E. coli RNAP RifR mutants each having one of three major RifR mutations found in clinical isolates. Each RifR RNAP structure shows a unique conformation of the Rif binding pocket and their structural deviations from the wild‐type Rif binding pocket are consistent with their Rif resistances suggesting that the RifR results from alternating the shape complementary between the Rif binding pocket and Rif rather than disrupting their hydrophilic and hydrophobic interactions. This study provides an important step for understanding the structure−activity relationship of Rif against RifR RNAP inhibition and toward developing superior Rif analogues against RifR MTB.

Grant Funding Source: Supported by NIH
Development and testing of affinity tag purification vectors for a metal‐dependent regulator protein from Thermobifida fusca (946.14)
Authors: Sticher Zachary , Fenton Jordan , Novak Walter
Published: June 2021

Metal‐dependent regulators are transcriptional regulator proteins that are used by a variety of pathogenic bacterial cells to maintain appropriate iron levels in the cell. The overarching goal of our research is to identify the function of the N‐terminus in DNA and metal‐binding in a metal‐dependent regulator from Thermobifida fusca. Previously, we demonstrated the metal‐dependent regulator protein from T. fusca could be overexpressed and purified to greater than 93% purity using nickel affinity and size‐exclusion chromatography. Although this technique works well for the wild‐type protein, we found that some mutations severely reduce the affinity of the protein for the nickel affinity column. Here we describe the creation of cleavable and non‐cleavable C‐terminal 6×His tagged fusion proteins. Initial results using the non‐cleavable tag suggest that the protein aggregates and also exhibits reduced affinity for the DNA operator.

Grant Funding Source: Supported by the Wabash College Haines Biochemistry Fund.
The QA repeat domain of TCERG1 mediates its inhibitory effect towards C/EBPα and the ability of TCERG1 to be relocalized in the nucleus (946.6)
Authors: Miller Nick , Hsiao Sheng‐Pin , Timchenko Nikolai , Roesler William
Published: June 2021

Previously we showed that TCERG1 inhibits the growth arrest and transactivation functions of C/EBPα, through a mechanism that requires the relocalization of TCERG1 to sites of C/EBPα occupancy in the nucleus. TCERG1 contains a unique QA repeat domain, for which no function has been assigned, as well as 3 WW domains within the N‐terminal half of TCERG1 which has been shown previously to mediate its relocalization. These domains were explored for their roles in mediating the inhibitory and relocalization activities of TCERG1. While mutations of the WW domains were without effect, deletion of the QA repeat domain in TCERG1 prevented its relocalization in the nucleus (assessed by confocal microscopy), and abrogated its ability to repress both the growth arrest and transactivation functions of C/EBPα. Reducing the number of QA repeats from 38 to 20 had no impact on the activities of TCERG1, while further reduction to 10 resulted in a significant loss of relocalization ability and inhibitory activity towards C/EBPα. Interestingly, deletion of the QA repeat domain did not alter the ability of TCERG1 to interact with C/EBPα in a co‐IP assay. Based on these data, we speculate that one function of the QA repeat domain is to allow relocalization of TCERG1 from its nuclear speckle compartment. (Note ‐ the first two authors contributed equally to this work)
Febrile temperature decreases the cell‐surface expression of the human ether‐a‐go‐go‐related gene (hERG) channel by facilitating channel degradation (949.3)
Authors: Zhao Yan , Koichopolos Jennifer , Wang Tingzhong , Zhang Shetuan
Published: June 2021

The human‐ether‐a‐go‐go‐related gene (hERG) encoded IKr and KvLQT1+minK encoded IKs are primary K+ currents responsible for cardiac repolarization in many species including humans. Dysfunction of either IKr or IKs can lead to long QT syndrome (LQTS), which predisposes the affected individuals to arrhythmias, syncope, or sudden death. Recent studies have reported that fever may serve as a trigger for QT interval prolongation and arrhythmias in patients. In the present study, we investigated the effect of febrile temperature on hERG and KvLQT1+minK channels using Western blot, patch clamp, and immunocytochemistry. Our data show that culturing cells in febrile temperature (40°C) reduced the expression and current amplitude of hERG channels stably expressed in HEK cells as well as IKr in neonatal rat cardiomyocytes. However, this manipulation did not reduce KvLQT1+minK current. Our data further show that accelerated protein degradation is responsible for the reduced plasma abundance of hERG channels. We have previously shown that hypokalemia accelerates hERG degradation and reduces hERG expression at the plasma membrane. Our data revealed that a reduction in K+ greatly exaggerated, and K+ insensitive mutation S624T completely abolished febrile temperature ‐induced hERG degradation. We conclude that febrile temperature may facilitate the development of LQTS via accelerating hERG degradation.

Supported by the Canadian Institutes of Health Research (CIHR).

Grant Funding Source: Supported by Canadian Institutes of Health Research
Mutation of serine 32 to threonine in peroxiredoxin 6 preserves enzymatic activity but prevents trafficking to lamellar bodies in vivo (950.1)
Authors: Sorokina Elena , Dodia Chandra , Tao Jian‐Qin , Feinstein Sheldon , Fisher Aron
Published: June 2021

Peroxiredoxin 6 (Prdx6), a peroxidase that also expresses phospholipase A2 (aiPLA2) activity, protects lungs from oxidative stress and participates in lung surfactant phospholipid (PL) turnover. Prdx6 has been localized to both cytosolic and acidic (lamellar body [LB] and lysosomal) compartments in lung epithelium. We propose that Prdx6 LB targeting facilitates its role in the degradation and remodeling of lung surfactant phosphatidylcholine. Although, we have previously shown that mutation of serine 32 (S32) to alanine abolishes Prdx6 aiPLA2 activity, we now show that serine to threonine mutation has no effect on either Prdx6 peroxidase or aiPLA2 activity. Since S32 is a necessary component of the amino acid motif that mediates Prdx6 LB targeting, we studied the effect of the S32T mutation on Prdx6 LB localization. In a “knock‐in” mouse model carrying an S32T mutation in the Prdx6 gene, there was phospholipid (PL) accumulation in the lung and a marked decrease in aiPLA2 activity in LB. Prdx6 was absent from LB of mutant mice by both western blot and immunofluorescence analysis despite normal protein expression in lung epithelial cells. Thus, the S32T mutation preserves the PLA2 activity of Prdx6 but does not support its targeting to LB, which results in PL accumulation, despite the normal Prdx6 PLA2 activity in lung tissue.

Grant Funding Source: HL102016 and HL105509
Small molecule modulation of DLD diaphorase activity to decrease reactive oxygen species production in the mitochondria (953.1)
Authors: Mesquitta Makeda , Berkhowlz Donald , Walters Michael , Isaya Grazia , Thompson James
Published: June 2021

Oxidative stress and damage is considered to be a significant causative factor in aging and neurodegenerative diseases. Concurrent with the clinical phenotypes associated with these diseases is mitochondrial dysfunction in the form of biochemical malfunctions that create or potentiate reactive oxygen species (ROS) production. Dihydrolipoamide dehydrogenase (DLD) is a mitochondrial multifunctional enzyme with a homodimeric structure, resolved as two monomers bridged by an interface domain. In its dimeric form, DLD plays a critical role in energy metabolism and has been suggested to have a destructive role as a generator of ROS in its monomeric form. The latter activity has been confirmed in prior studies in both mouse and yeast models. Decreased superoxide activity has been measured in DLD knockout (‐/‐) mice vs. WT mice (Starkov et al., 2004) and higher diaphorase activity with increased hydrogen peroxide levels (Vaubel et al., 2011) has been shown in DLD mutants expressed in yeast systems. Our lab using virtual screening and computational docking has designed a small molecule library containing 40 synthetic compounds that putatively bind to a pocket within the interface domain of DLD with the aim of stabilizing the dimer. Primary screening of the compound library using the diaphorase assay is in progress. Validation of hits will be performed using Differential Scanning Fluorimetry (DSF), X‐ray crystallography and other techniques including in‐vivo analyses. We hope that these studies will enhance our understanding of the physiological role of diaphorase activity and facilitate the development of therapeutics for pathologies in which diaphorase activity has a suggested damaging function.
Mitochondrial membrane lipidome defines yeast longevity (956.2)
Authors: Titorenko Vladimir , Beach Adam , Richard Vincent , Leonov Anna , Piano Amanda , Feldman Rachel
Published: June 2021

Lithocholic bile acid (LCA) is a potent anti‐aging natural compound that in yeast cultured under longevity‐extending caloric restriction (CR) conditions acts in synergy with CR to enable a significant increase in chronological lifespan. We investigated a mechanism underlying the longevity‐extending effect of LCA under CR. We found that exogenously added LCA enters yeast cells, is sorted to mitochondria, resides mainly in the inner mitochondrial membrane, and also associates with the outer mitochondrial membrane. LCA elicits an age‐related remodeling of phospholipid synthesis and movement within both mitochondrial membranes, thereby altering mitochondrial membrane lipidome and triggering major changes in mitochondrial size, number and morphology. In synergy, these changes in the membrane lipidome and morphology of mitochondria alter the age‐related chronology of mitochondrial respiration, membrane potential, ATP synthesis and reactive oxygen species homeostasis. The LCA‐driven alterations in these vital mitochondrial processes extend yeast longevity. In sum, our findings suggest a mechanism underlying the ability of LCA to delay chronological aging in yeast by accumulating in both mitochondrial membranes and altering their phospholipid compositions. We concluded that mitochondrial membrane lipidome plays an essential role in defining yeast longevity. Supported by NSERC of Canada.
Attenuation of carbon tetrachloride toxicity in acute hepatosteatosis induced by intake of a high carbohydrate diet after food deprivation (959.1)
Authors: Ahn Chul , Kwon Do , Lee Jung , Choi Yoo , Kim Young
Published: June 2021

Hepatic fat accumulation constitutes a major component in the pathogenesis of liver injury. We determined the impact of lipid deposition in liver on cytochrome P‐450 (CYP) system and CCl4‐induced hepatotoxicity. Rats were fasted for 48 h, and then provided with a high‐carbohydrate/fat‐free diet (FH) or a normal diet (FN) for 48 h. Hepatic triglyceride level was not different between the normal control and the FN group, but 4 times greater in the FH group, which was consistent with the histopathological observation. FN intake resulted in a small increase in microsomal p‐nitrophenol hydroxylase, p‐nitroanisole O‐demethylase, and erythromycin N‐demethylase activities, but aminopyrine N‐demethylase was decreased. The microsomal enzyme activities determined were all inhibited markedly by FH intake. Immunoblotting analysis showed that proteins of CYP2E1, 1A, 3A, and 2B1/2 were decreased in the rats fed FH. Expression of CYP mRNAs was also down‐regulated. A dose of CCl4 was administered to the rats fed the different diets. The resulting liver injury was significantly lower in the FH group than in the FN group as determined by elevation of serum enzyme activities and histopathological examination. The results indicate that acute hepatosteatosis can precipitate erratic responses to various endogenous and exogenous substances via alterations in hepatic CYP‐mediated metabolizing capacity.