bluuebunny/crossref_metadata_2025_split huggingface dataset:
Isolation of Intricate α‐Synuclein Assemblies Across the Dynamic Landscape
Authors: Lucas Heather R. 
Published: April 2020

Cardinal features of Parkinson’s disease (PD) include the development of granular inclusions known as Lewy bodies that are enriched with aggregates of α‐synuclein (αS). αS has long been established as an intrinsically disordered protein, and the array of its physiologically relevant oligomeric conformations is ever‐expanding. In recent years, a higher order conformation was identified as a helical tetramer that is tailored by buried hydrophobic interactions and is distinctively aggregation resistant. The monomer and tetramer reside in a dynamic coexistence within cells, yet the canonical mechanism by which the tetramer assembles remains elusive due to the various folding patterns and low energy barriers involved in αS structural transitions. A shift from metastable tetramers to the monomeric form likely serves as a mechanism for disease initiation. The underlying pathology causing this shift remains unknown, but the importance of understanding tetramer stability and disassembly has therapeutic potential that cannot be overemphasized. Hence, a platform for investigating analytical approaches to stabilize tetrameric αS is warranted, as are methods to discriminate αS conformational states. Isolation of tetrameric αS is complicated by its dynamic nature, thus thorough bioanalytical studies on this conformer have been hampered by accessibility issues. We recently developed a robust recombinant expression platform that enables purification of native tetrameric αS without any detergents, crosslinkers, or other structure‐modifying additives by the sequential combination of anion exchange and size exclusion chromatography steps. The successful isolation of this intricate αS conformer is confirmed through various analytical methods, including advanced light scattering techniques, mass spectrometry, immunoblotting, and circular dichroism spectroscopy for secondary structural analyses. The development of multifaceted and innovative approaches that are necessary to tease apart the dynamic landscape of αS quaternary structure will be presented. Moreover, novel methodology to facilitate localized surface plasmon resonance analysis of this intricate conformer will be highlighted as well as the effects of PD‐relevant structural modifiers present in cellular milieu, including metals and/or liposomes, strengthening the application of this platform to advance tetrameric αS as a therapeutic target. The talk will conclude with new insights on strategies to shift the conformational equilibrium towards tetramer in mixed populations as a means to thwart αS misfolding and aggregation.Support or Funding InformationSupported by NIH R01 GM134015, NCATS UL1TR002649 & CCTR Endowment Fund, VCoA ARDRAF Award No. 18‐7, and NIA L‐13046.
Identification of Proteins that Interact with Otoferlin to build a ‘Otoferlin Interactome’
Authors: Manchanda Aayushi , Bonventre Josephine , Johnson Colin 
Published: April 2020

Otoferlin is a C2‐domain transmembrane vesicular protein that has been associated with congenital deafness. It is majorly responsible for facilitating synaptic vesicle exocytosis in cochlear hair cells. Synaptotagmin and Otoferlin have previously been compared to one another for their calcium sensor functions, but hair cells exocytosis is not as well‐understood as neuronal exocytosis. In case of hair cell synapses, we still lack complete knowledge of the SNARE proteins or their equivalent that aid in the fusion of vesicle membranes to the presynaptic membranes, thereby leading to exocytosis. Currently, a major bottleneck in our understanding of Otoferlin’s role in hearing and deafness can be attributed to the lack of knowledge about its mechanism of functioning. Using brain extracts from mouse, we conducted coimmunoprecipitation experiments to find interaction partners for Otoferlin so as to build a Otoferlin interactome, that can shed some light on the mechanism by which hair cell exocytosis actually occurs. The proteins interacting with Otoferlin were identified by Mass spectroscopy and consisted largely of neuronal proteins that share functions with SNARE proteins or had other neurotransmission‐related functions. We use Human Embryonic Kidney (HEK) cells and a zebrafish model to validate the direct interactors of Otoferlin and conduct further assays to determine their role in hair cell exocytosis and/or endocytosis. This knowledge of Otoferlin interactome leads us to a better understanding of the mechanism whereby neurotransmission is carried out in the inner hair cells.Support or Funding Information1R01DC014588 (C.P. Johnson) 4/2016‐3/2021 NIDCD (NIH). In vivo and in vitro studies of the deafness associated protein otoferlin
Modulation of TRPC store‐operated channels by Orai and STIM proteins
Authors: Kolesnikov Dmitrii , Glushankova Liubov , Skopin Anton , Gusev Konstantin , Shuyskiy Leonid , Sozonova Svetlana , Kaznacheyeva Elena , Shalygin Alexey 
Published: April 2020

Store‐operated calcium entry is significant pathway of calcium concentration rise in cytoplasm. The main players in this process are Orai store‐operated channels, which are activated directly by STIM calcium sensors. Data on participation of TRPC channels in store‐operated calcium entry is controversial.Study was focused on interaction between STIM and Orai proteins with endogenous TRPC and its role in pathology. We investigated activity of TRPC channels in a model object — HEK293 cells and in the podocytes of the rat model of diabetic nephropathy, using single channel recording patch camp technique.To eliminate Orai channels activity in HEK293 cells we used several experimental approaches: overexpression of dominant‐negative Orai mutant, Orai genes knockout and knockdown via CRISPR‐Cas 9 system and shRNA. Endogenous TRPC1 channels remain active in cells without functional Orai proteins and become insensitive to store depletion.In experiments with different extracellular calcium concentration we confirmed that calcium entry is needed for TRPC1 activation by store depletion.In experiments with calcium, barium or sodium as a charge carrier we observed similarities in dependence of open channel probability versus membrane potential between TRPC1 and Orai channels.The role of STIM calcium sensors in TRPC1 channels activation was investigated in cells with different expression level of STIM1 and STIM2 proteins.The findings allow us to propose that STIM proteins don’t activate the endogenous TRPC channels directly. TRPC channels are activated downstream to calcium entry through the Orai channels.Support or Funding InformationThe study was supported by grant of Russian science foundation № 19‐14‐00114 (Glushankova L., Skopin A., Kaznacheyeva E.), and by grant of Russian foundation for basic research № 19‐315‐90065 (Shalygin A., Kolesnikov D.).
Decitabine Reduces Kidney Growth in an ARPKD Mouse Model
Authors: Ginsparg Ari Benjamin , Shan Dan , Kim Harrison , Mrug Michal 
Published: April 2020

Autosomal recessive polycystic kidney disease (ARPKD) is a progressive disorder affecting especially kidneys and liver. Using the Cys1cpk/cpk mouse, a commonly used ARPKD phenocopy, we observed an increased association between the rate of renal disease progression and kidney DNA methylation. To test the functional relevance of this association in vivo, among FDA‐approved demethylating drugs we prioritized decitabine based on its cystogenesis‐inhibiting effects and lowest toxicity in our cell‐based model. We treated time‐bred Cys1cpk/cpk mice from postnatal day 7 (P7) to P14 either with a vehicle (DMSO) or decitabine (1mg/kg intraperitoneally one, four, or six days per week). All doses of decitabine reduced MRI‐derived total kidney volume (TKV)‐based indices of cystic disease severity. However, four‐ and six‐day per week dosing was associated with reduced body weight gain. Together, our studies point to decitabine, dosed once per week, as a promising renal cystogenesis‐inhibiting strategy.Support or Funding InformationThe project was funded under NIH R25 DK 112731, NIH/NIDDK 1R01DK097423, NIH/NIDDK P30 DK074038, and VA I01BX002298
Heat Waves Produce Ecologically Relevant Expression Profiles for Temperature‐responsive Genes
Authors: Marroquin-Flores Rosario A. , Mortimer Nathan T. , Paitz Ryan T. , Bowden Rachel M. 
Published: April 2020

The red‐eared slider turtle (Trachemys scripta elegans), exhibits temperature‐dependent sex determination (TSD), where sex‐specific genetic profiles are produced in response to incubation temperature. Despite over 50 years of study on this topic, we still do not fully understand how temperature affects the molecular mechanisms underlying TSD, and prior studies aimed at characterizing genes in the sex‐determining pathway have primarily used constant incubation temperatures. Such studies fail to capture the thermal variability that developing embryos naturally experience, but have been used to identify potential candidate genes in the sex‐determining pathway. One candidate, cold‐inducible RNA‐binding protein (Cirp), has sex‐specific expression in several species of turtles and other reptiles under constant incubation conditions. We characterized Cirp expression in the gonads of T. s. elegans embryos exposed to temperatures that are a better mimic of natural nest conditions. All eggs were incubated for the first 24 days at male‐producing temperatures (MPT; 25.0 ± 3°C) before a subset were shifted to female‐producing temperatures (FPT; 29.5 ± 3°C) to induce ovarian development via a simulated heatwave. Embryonic gonads were sampled on day 24, then sampled from both FPT and MPT for five consecutive days. In contrast to results from constant temperature studies, we found that Cirp expression did not differ in embryos at MPT and FPT. We then submitted a subset of our samples for RNAseq to identify genes exhibiting differential expression (DE) in the gonads of embryos at FPT and MPT. A preliminary analysis found 74 genes to be upregulated and 102 genes to be downregulated by day 5 of the heatwave. Similar work using constant temperature incubation found 1887 DE genes in the gonads of embryos at the same stage of development. Only 25 DE genes were shared between the constant and fluctuating temperature datasets. Our initial comparison of gene expression under constant and fluctuating conditions suggests that while constant incubation conditions result in many more DE genes, many of these genes may not be differentially expressed under more natural incubation conditions. Further, these findings highlight the importance of using ecologically relevant conditions to identify genes expressed during early gonadogenesis in TSD species.Support or Funding InformationAmerican Physiological Society William Townsend Porter Pre‐Doctoral Fellowship, Society for Integrative and Comparative Biology Grants‐in‐Aid‐of‐Research, Beta Lambda Phi Sigma Biological Honors Society Weigel Grant
3D Printed Microfluidics for Studying Sleep Pathways in Caenorhabditis elegans
Authors: Honer Madison , Fingerut Jonathan , Nelson Matthew D. 
Published: April 2020

Despite sleep being essential and highly conserved at the genetic level, a thorough understanding of the mechanisms of sleep regulation and its core function are lacking. Caenorhabditis elegans, a genetically tractable nematode worm, is a powerful model system for addressing these questions. Conserved neurochemistry and a compact 302‐celled nervous system allow for the identification and rapid characterization of novel sleep‐regulating genes of relevance to more complex animals. Orcokinin neuropeptides, predominantly found in ecdysozoan animals, have been implicated during the regulation of circadian rhythms and molting, however, a role during sleep regulation has not been described. In C. elegans the genes nlp‐14 and ‐15 code for peptides predicted to be orcokinin orthologs. We sought to determine if these peptides control a behavior called stress‐induced sleep (SIS), which occurs following incidents of noxious stimuli that damages their cells. Using a combination of loss‐of‐function and gain‐of‐function approaches, we find that nlp‐14 and nlp‐15 play a central role during SIS, regulating unique aspects of behavioral quiescence. To identify an orcokinin receptor, we have been conducting forward genetic screens to isolate mutants that suppress a strong gain‐of‐function sleep phenotype of nlp‐14. In the past, we employed a manual screening protocol that required an abundance of time and resulted in a high false‐positive rate. To improve upon this, we fabricated screening devices using 3D printing, which allows for higher throughput and selectivity. These microfluidic devices use gravity‐based sorting chambers which are both cheap and easily employed. In these chambers we place mutagenized animals, induced to fall asleep by nlp‐14 over‐expression, in a lower sorting chamber. Sleeping animals are trapped in the lower chamber, while suppressor mutants display gravitaxis behavior, the ability to swim against gravity, thus, swimming to an upper collection chamber. These rare suppressor mutants are then isolated for whole‐genome sequencing in order to reveal the essential genes involved in these sleep‐regulating pathways.Support or Funding InformationNSF Award #1845020 (PI Nelson)
AKR1B1‐Induced epithelial‐mesenchymal transision Mediated by RAGE‐ Oxidative Stress in the Lens of DM Cataracts
Authors: Cheng Pei-Wen , Chen Ying-Ying , Wu Tsung-Tien 
Published: April 2020

Background and purposeCataracts in patients with diabetes are a major cause of blindness in developed and developing countries. It’s been known that lens epithelial cells (LECs) would undergo epithelial‐mesenchymal transision (EMT) that formed to cataract. Great deals of studies have demonstrated that overproduction of AKR1B1 and RAGE played a central role in the pathogenesis of diabetic cataract. The aim of this study was to examine whether EMT formation in the DM cataract through an AKR1B1 enhanced AGE and ROS generation in LECs from DM patients.MethodsFluorescence was used to estimate the AKR1B1, RAGE, AMPK and EMT biomarkers in the human lens epithelial sections from DM or non‐DM cataracts. All data were expressed as the mean ± SEM (n=12 for each group). A one‐way analysis of variance (ANOVA) with Scheffe’s post hoc comparison was employed to compare the group differences.ResultsImmunohistochemistry staining founding that the pathologic phases and N‐cadherin expression levels were significantly enhanced in the human lens epithelial sections from DM (+) than DM (−) cataract patients. Immunofluorescent staining also demonstrated that AKR1B1 and RAGE were significantly enhanced in the human lens epithelial sections from DM (+) than DM (−) cataract patients. Interestingly, AcSOD2 levels were significantly higher in the LECs of the patients with DM, not in non‐DM cataracts, however, the phosphorylation level of AMPKT172 was has reverse effect. These results indicated that AMPKT172 may be required for reduced superoxide generation and the pathogenesis of diabetic cataract formation.ConclusionsCollectively, our results revealed that AKR1B1 overexpression would abolish AMPK activation, thereby increasing AcSOD2 and RAGE‐induced EMT formation in the LECs of diabetic cataracts as potentially important elements in the pathogenesis of diabetic cataract.Support or Funding Information108‐2320‐B‐075B‐002 ‐
The Effect of Magnolol on a Polycystic Ovary Syndrome Rat Model with Insulin Resistance
Authors: Kanaan Amjad , Khachab Maha , Nassar Aline , Zaatiti Hayat 
Published: April 2020

Polycystic ovary syndrome (PCOS) is a prevalent reproductive and metabolic disorder. Insulin resistance (IR) is highly associated with and aggravates the symptoms of PCOS. The Insulin sensitizing agents, Thiazolidinediones, are PPARγ agonists that improve many of the symptoms of PCOS. Magnolol is a natural PPARγ agonist that improves insulin sensitivity in experimental models. Using a dehydroepiandrosterone (DHEA)‐induced rat model of PCOS and IR, we aim to explore both the potential beneficial effect and the molecular mechanisms of action of Magnolol. Post‐pubertal female Sprague Dawley rats, injected subcutaneously with DHEA (6mg/100g body weight) for 28 days, exhibited a significant increase in body weight (242.5 ± 5.4 grams) as compared to age‐ and weight‐matched controls (226.8 ± 4.7 grams). The DHEA‐injected rats developed an increased number of cystically dilated follicles with thicker granulosa as compared to control. PCOS rats showed statistically significant elevated serum levels of Luteinizing hormone (LH) (10.0 ± 2.7 ng/ml) and fasting insulin (2.18 ± 0.9 ng/ml); with an increased Homeostatic Model Assessment Index of insulin resistance (HOMA‐IR) (9.98 ± 4.9) as compared to controls (6.3 ± 0.2 ng/ml; 0.6 ± 0.3 ng/ml; 2.7 ± 3.9 respectively). Compared to control, PCOS rats had a significant lower ovarian protein expression of peroxisome proliferator activated receptor gamma (PPARγ), insulin receptor substrate 1 (IRS1), and protein kinase B (Akt) by Western Blot. Conversely, the PCOS group showed an increased mammalian target of rapamycin (mTOR) pathway activity as evident by an increase protein expression of phosphorylated mTOR as compared to control. When treated for 28 days with oral Magnolol (500mg/kg), the DHEA‐induced PCOS rats showed a statistically significant decrease in body weight (231.6 ± 13 grams) and serum LH levels (3.9 ± 1.8 ng/ml) as compared to the non‐treated PCOS rats (250.3 ± 9.7 grams; 9.5 ± 5.7 ng/ml respectively). The number of cystically dilated follicles in the Magnolol‐treated PCOS rats was significantly reduced compared to the non‐treated PCOS rats. In the Magnolol‐treated PCOS rats the ovarian protein expression of PPARγ, IRS1 and Akt was significantly increased, while the mTOR expression was decreased compared to the non‐treated PCOS group. In conclusion, Magnolol ameliorates the DHEA‐induced PCOS phenotype histologically, hormonally and metabolically. Fundamentally, this work explores the elusive pathophysiologic association between IR and PCOS, by targeting pathways common to both disorders.Support or Funding InformationThis work is supported by the University of Balamand, Faculty of Medicine and Medical Sciences
Investigating L1 promoter activity during mouse development using LacZ transgenes
Authors: Saha Partha S. , LaCanne Trenton , An Wenfeng 
Published: April 2020

Long interspersed elements type 1 (L1s) are highly abundant retrotransposons in the human genome. To fully understand the role of L1s during development and disease, it is important to track L1 expression and epigenetic regulation at individual L1 loci throughout development at the cellular level. However, it is extremely technically challenging to monitor transcriptional activities of individual endogenous L1s due to high sequence homology. Toward this goal, we created independent single‐copy LacZ transgenic mouse lines, in which the LacZ reporter is regulated by a copy of the endogenous mouse L1 promoter. To control for the effect of gene‐body DNA methylation on L1 promoter activity, we also generated independent mouse lines carrying the CpG‐less version of the LacZ, termed LacG. After mapping of the transgene locus in each line, mouse tissues were stained with X‐gal to visualize L1 promoter activity at single cell level. QuPath was used to quantify X‐gal positive cells in whole‐slide scanned brain sections. We found that the LacG reporter supported robust expression as compared to the LacZ reporter. However, there were substantial variations among the LacG reporter lines, in which the transgene is integrated in random genomic loci. Intraline variations of expression in the same organ might be dictated by generation and age, which is yet to be established statistically. Orientation dependent regulation of L1 promoter was also studied by targeting the LacG transgene in a specific intronic locus in either sense or antisense orientation. Interestingly, antisense orientation showed augmented expression than its counterpart. Ongoing effort aims to reveal the pattern of these signals in different anatomical regions of brain and also in other major organs, which might support L1 retrotransposition in different developmental period in these mouse models. The goal is to provide a comprehensive mapping of L1 promoter activity in a cell dependent and locus dependent manner. Such information will be instrumental in our understanding of L1’s involvement in physiological and pathological conditions, including embryogenesis, gametogenesis, and under environmental stress.Support or Funding InformationSupported by NIH R21HD080143 and R21OD017965.
Maternal FUT2 genotype in relation to risk of acute infections and rotavirus vaccine shedding in infancy
Authors: Adkins Grace E. , Thorman Alexander , Conrey Shannon C. , Cline Allison R. , Staat Mary A. , Payne Daniel C. , Piasecki Alexandra M. , Burke Rachel M. , Bowen Michael D. , Morrow Ardythe L. 
Published: April 2020

The human FUT2 gene codes fucosyltransferase2, an enzyme that catalyzes the production and secretion of H‐antigen in bodily fluids and tissues. FUT2 status has been associated with risk of infection for some pathogens. In addition, mothers who are homozygous positive or heterozygous for the FUT2 gene produce a class of oligosaccharides in their milk that can act as decoy receptors for some selected pathogens. The link between maternal FUT2 status and illnesses in breast‐fed infants is not clear.The Pediatric Respiratory & Enteric Virus Acquisition and Immunogenesis Longitudinal (PREVAIL) Cohort study of mother‐infant pairs in Cincinnati, OH assessed whether an epidemiologic association may exist between maternal FUT2 genotype and incidence of acute gastroenteritis (AGE), acute respiratory infection (ARI), and rotavirus vaccine shedding in their infants. We expected that the exclusively breastfed children of FUT2+ mothers would have lower incidences of infection due to the unique breastmilk oligosaccharide composition.We included all 75 mother‐infant pairs who exclusively breastfed their infants, which provided a total of 1,243 child‐weeks of follow‐up from birth to 6 months. All mothers were genotyped for FUT2 by PCR. Of these, 40 (53%) infants were vaccinated against rotavirus and had ≥1 weekly stool sample collected within 8 weeks after vaccination and tested by real time RT‐PCR assays to determine vaccine shedding. Infant infection data came from weekly maternal reports of AGE and ARI that were collected by using an automated text messaging system.Infants of FUT2+ mothers were found to have longer duration of rotavirus shedding post‐vaccination than babies of FUT2− mothers (p=0.007). Additionally, infants of FUT2+ mothers had more AGE symptomatic study weeks (5.6%) than children of FUT2− mothers, who had no symptomatic AGE weeks (p<0.001). Similarly, infants of FUT2+ mothers had ARI with fever for approximately 2.7% of study weeks, whereas babies of FUT2− mothers had no cases of ARI with fever (p=0.006). In this ongoing study, we plan to continue our analysis to include the infant’s own FUT2 status and medically‐attended illness.Support or Funding InformationCDC grant number U191P001059, IP‐16‐004