bluuebunny/crossref_metadata_2025_split huggingface dataset:
GRAPES: A Versatile Tool for Analyzing Structural Variation From Whole‐Genome and Targeted DNA Sequencing Data
Authors: del Olmo Bernat , Matés Jesús , Mademont‐Soler Irene , Pérez‐Serra Alexandra , Coll Monica , Allegue Catarina , Puigmulé Marta , del Olmo Garcia‐Bassets Ivan , Pascali Vincenzo , Oliva Antonio , Brugada Ramon 
Published: June 2021

The annotation of human genomic diversity is nowadays at an unprecedented rapid pace, but arguably still limited by the lack of computational approaches that integrate the analysis of multiple forms of genetic variation and are suitable for the analysis of both, whole‐genome sequencing and targeted sequencing strategies. For the analysis of structural variations (SVs), for example, which include >50 bp deletions, duplications, copy‐number variations (CNVs), inversions, mobile‐element insertions, and translocations, only a few computational tools integrate breakpoint signatures and read‐depth information in a single approach. And, none of them makes use, in the analysis of targeted sequencing, of breakpoint information to filter false‐positive CNVs when small adjacent introns are completely captured despite that targeted sequencing is still more popular than whole‐genome sequencing (e.g. exome‐sequencing and many common approaches of genetic‐testing are forms of targeted sequencing). Here, we present the development of GRAPES (Genomic Rearrangement Analyzer for Paired‐End Sequencing), which is as a versatile tool that allows the analysis of SVs on whole‐genome and targeted DNA sequencing data by integrating discordant‐pairs, split‐reads, read‐depth, and local assembly. In the case of targeted sequencing, GRAPES uses on‐ and off‐ target read depth coupled with breakpoint analysis to provide accurate predictions. We have validated GRAPES using the accurately annotated NA12878 genome, the ICR96 CNV exon validation series, and a panel of simulated datasets, and it achieves higher rates of sensitivity and specificity than previously developed approaches. We have tested GRAPES to annotate SVs from targeted sequencing data of patients affected with inherited cardiac disorders, uncovering complex SVs that were unresolved by using other algorithms. In sum, GRAPES not only is a comprehensive tool for the analysis of SVs from whole‐genome sequencing and targeted sequencing data, but also allows the annotation of SVs overlooked by current single‐approach approaches.Support or Funding InformationInternational Doctorate Grant in Biomedical Research and Public health of the Università Cattolica del Sacro CuoreThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Population Genetics of Scaevola on Culebra, Puerto Rico.
Authors: Hodkinson Lauren , Witherup Susan 
Published: June 2021

On the islands of Puerto Rico, a native species of coastal shrub, Scaevola plumieri, co‐occurs with an invasive species, Scaevola taccada, introduced from the indo‐pacific islands. We are interested exploring the genetic diversity of both S. plumieri and S. taccada on the island of Culebra, PR in order to characterize the differences in genetic variation of an endemic species versus an invasive species. We hypothesize that the S. plumieri will have a greater amount of genetic diversity than the invading species due to S. taccada's more recent establishment on the island. Previous studies in our lab have shown that the alleles for two microsatellite regions were distributed across all populations on Culebra and had no correlation between distance and genetic diversity. To improve our characterization of diversity, we developed novel species‐specific microsatellite primers for S. plumieri using whole genome sequencing data. Six pooled samples from different beaches across Culebra were sent for Illumina MiSeq paired‐end sequencing and a Python computer code was written to identify potential microsatellites from the raw sequencing data. Leaf samples from both S. plumieri and S. taccada populations were collected from Culebra during January of 2015 and March of 2017, dried with silica gel, and the genomic DNA from each leaf was extracted. Microsatellites in the S. plumieri samples were amplified using the newly developed primers whereas microsatellites in S. taccada were amplified using previously developed primers. Fragment peaks from analysis by capillary gel electrophoresis were investigated using the program Geneious and peaks representing the fragment size of the microsatellite alleles were identified and recorded for each individual. These data were then evaluated through the program Arlequin which implements a series of tests to determine molecular variance (AMOVA), identify deviations from Hardy Weinberg Equilibrium and correlate geographic distance and genetic variation. In the future we plan to further expand our analysis using these novel S. plumieri primers to incorporate plant samples form Vieques, PR and mainland PR for a complete characterization of Scaevola diversity across all islands of Puerto Rico.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Temporal Regulation and Functional Impact of ERK Activity Near the Plasma Membrane
Authors: Keyes Jeremiah , Ganesan Ambhighainath , Zhang Jin 
Published: June 2021

The ERK1/2 pathway plays critical roles in eukaryotic biology by transducing extracellular signals into cell‐fate decisions. One conundrum is in understanding how disparate signals induce specific responses through a common, ERK‐dependent kinase cascade. One major mechanism to control ERK‐signaling to induce proper cellular responses is through precise regulation over the temporal dynamics of ERK activity. For example, in the pheochromocytoma PC‐12 cell line, ERK exhibits transient activity in response to Epidermal Growth Factor (EGF), ultimately resulting in proliferation. On the other hand, PC‐12 cells respond to Nerve growth factor (NGF) with sustained ERK phosphorylation and eventual differentiation into neuronal‐like cells. An additional mechanism of control over ERK is through spatial regulation of activity; however, it has been difficult to accurately examine differences in activity among specific subcellular locations. To address this issue, we have expanded the toolbox of FRET‐based ERK biosensors by creating a series of improved biosensors that are specifically targeted to various subcellular regions to measure spatiotemporal changes in ERK enzymatic activity in real‐time in living cells. Using these sensors, we have tested the hypothesis that a single extracellular signal can induce different temporal dynamics between subcellular compartments in PC‐12 cells. We report that while EGF induces transient ERK activity in the cytosol and nucleus, EGF simultaneously induces sustained ERK activity near the plasma membrane of PC‐12 cells. Furthermore, we report that PKA and cAMP play an integral role in the regulation of ERK at the plasma membrane. Specifically, we observe that increasing basal cAMP prior to EGF induces a more transient activity at the plasma membrane in a PKA‐dependent manner. However, our results also indicate that PKA activity post‐EGF is necessary for sustained ERK activity near the plasma membrane, revealing the importance of understanding the temporal dynamics of pathway crosstalk. Furthermore, we report that selective inhibition of ERK activity near the plasma membrane drastically alters PC‐12 cell morphology and adhesion. These data further our fundamental understanding of how the spatiotemporal dynamics and native biochemistry of ERK in living cells modulates cell behavior.Support or Funding InformationIRACDA fellowship through NIGMS/NIH award K12GM068524, R01 DK073368, R01 MH111516, R35 CA7622 from NIHThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of the Spc105 Complex in Organization and Microtubule‐Binding Activity of the Budding Yeast Kinetochore
Authors: Kim Jae ook , Zelter Alex , Johnson Richard , MacCoss Michael J. , Asbury Charles L. , Davis Trisha N. 
Published: June 2021

Equally partitioning duplicated chromosomes during mitosis is of paramount importance; failure can cause aneuploidy, which predisposes individuals to cancer and birth defects. Chromosome segregation is orchestrated by kinetochores, a super‐complex of proteins that assemble onto centromeric DNA. Kinetochores must strongly couple chromosomes to spindle microtubules and they must sense and correct aberrant microtubule attachments. The Spc105 complex is an essential part of the budding yeast kinetochore, as are its homologues found widely across eukaryotes; however, it has yet to be completely defined. Spc105 complex plays an important role in the mitotic checkpoint, which produces a soluble ‘wait’ signal to delay anaphase in the absence of proper kinetochore‐microtubule attachments. The Spc105 complex is also proposed to contribute directly to kinetochore‐microtubule coupling. However, the extent to which the Spc105 complex and its homologues in other organisms contribute to microtubule coupling is unclear because it has been extremely difficult to study the purified complex in vitro owing to its biochemical instability. Molecular understanding of the binding of Spc105 complex to other complexes within the kinetochore is also lacking. We purified the recombinant Spc105 complex for the first time through co‐expression of its two protein components, Spc105p and Kre28p. Chemical cross‐linking and mass spectrometry (XL/MS) reveal that Spc105p and Kre28p interact through a region of about 200 amino acids. Additional XL/MS and pulldown experiments suggest regions through which the Spc105 complex interacts with the microtubule‐binding Ndc80 complex. Total internal reflection fluorescence (TIRF) microscopy shows that the Spc105‐GFP complex binds directly on microtubules. The ability to recombinantly purify the Spc105 complex will allow molecular details of its organization at the kinetochore and its contribution to the kinetochore function to be studied directly.Support or Funding InformationNational Institutes of Health (R01 GM040506) ‐ Trisha N DavisThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Temporal control of growth factor‐mediated signaling pathways during cell differentiation and Xenopus embryonic development
Authors: Mondal Payel , Krishnamurthy Vishnu Vardhan , Khamo John , Sharum Savanna , Zhang Kai 
Published: June 2021

Growth factor‐mediated signaling pathways regulate a wide‐spectrum of cellular functions such as survival, proliferation, differentiation, and apoptosis. Different growth factors typically trigger the activation of the same set of downstream signaling pathways which includes the mitogen activated protein kinase (MAPK), phosphoinositide 3‐kinase (PI3K)‐AKT, and phospholipase C (PLC) pathways Evidence suggests that precise spatiotemporal regulation of these signaling pathways is involved in generating diverse cellular outcomes. Although conventional genetic and pharmacological approaches have helped us understand different aspects of signaling pathways, a quantitative delineation of their signaling kinetics is pending due to a lack of tools that allow for precise temporal control of these pathways. Non‐neuronal optogenetics, an emerging technique that utilizes light to control intracellular signaling pathways, is a remarkable tool to help achieve this goal. Here, we introduce a bicistronic optogenetic system which we recently developed in our laboratory to achieve temporal control of the MAPK pathway. This tool uses the photoactivatable protein cryptochrome 2 (CRY2) and the N‐terminal domain of cryptochrome‐interacting basic‐helix‐loop‐helix (CIBN) from Arabidopsis thaliana. This novel system allows reversible, light‐mediated activation of the MAPK signaling pathway in intact mammalian cells as well as in developing Xenopus laevis embryos. In PC12 neuronal cells, light‐controlled, intermittent MAPK activity reveals a memory effect in light‐induced neurite outgrowth. In Xenopus embryos, developmental stage‐specific MAPK activation further reveals that this pathway can reprogram cell fate after a crucial time window when germ layers are specified. Our strategy can be generalized to control other kinase pathways with a similar activation mechanism. Results from our research will help resolve intracellular mechanisms of growth factor‐mediated signal transduction during cell differentiation and embryonic development.Support or Funding InformationKai Zhang, Department of Biochemistry, University of Illinois Urbana‐ChampaignThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Wnt16 regulates chondrocyte differentiation through Wnt/ planar cell polarity (PCP) pathway
Authors: ZENG Yelin , YUENG Wai , MAK King Lun , Zhao Hui 
Published: June 2021

Wnt signaling, a highly conserved signaling pathway, plays important roles in endochondral ossification which is a key process for skeletal development and bone repair. Wnt16, as one of the nineteen Wnt ligands, is reported to repress osteoclastogenesis, prevent cortical bone fragility fractures and to be upregulated in osteoarthritis. But how Wnt16 mediates chondrocyte differentiation during endochondral ossification is still unclear. Here, we investigate the roles of Wnt16 specifically in chondrocytes during endochondral ossification. First, we generated Col2a1‐Wnt16 transgenic mice in which Wnt16 was overexpressed in chondrocytes under the control of Col2a1 promoter and enhancer. The transgenic mice showed a great reduction of tissue mineralization during embryonic development. We also genetically knocked out Wnt16 by generating Wnt16Loxp/Loxp;Col2a1‐Cre mutant mice to examine whether Wnt16 is required for skeletal development. The mutant mice showed no severe phenotype in early skeletal development. However, after 2‐month‐old, the mutant mice displayed a smaller body size and lower bone mass as compared to that of control littermates. In vitro, our studies showed that Wnt16 delays chondrocyte hypertrophy and subsequent maturation. Mechanistically, we found that Wnt16 mainly activates the planar cell polarity (PCP) pathway through activation of JNK in primary chondrocyte. After treated chondroprogenitor cell line ATDC5 with SP600125, a JNK specific inhibitor, Wnt16‐induced delay of chondrocyte hypertrophy is eliminated. In addition, our data suggest that Wnt16 mainly interacts with Ror2 or CD146, co‐receptors of PCP pathway, but not Vangl2 or Ryk. Collectively, our current study provides evidence that Wnt16 delays chondrocyte hypertrophy through PCP pathway partially by binding to Ror2 and CD146. Our findings deepen the understanding of chondrocyte differentiation during endochondral ossification.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
DDX3 induces neural crest through activation of an Akt‐Wnt signaling axis
Authors: Perfetto Mark , Li Jiejing , Wei Shuo 
Published: June 2021

DDX3 is a DEAD‐box RNA helicase critical for normal development. Recently, mutations in DDX3 were found to be associated with multiple abnormalities including cleft lip/palate, phenotypes that are often caused by aberrant neural crest development. Here we show that knockdown of DDX3 inhibits neural crest induction in Xenopus embryos. This is accompanied by reduction in Akt activity and canonical Wnt signaling. Blocking Akt activity phenocopies DDX3 loss of function in inhibiting Wnt signaling and neural crest induction, and a constitutively active mutant of Akt rescues the neural crest induction phenotypes caused by DDX3 knockdown. These results show for the first time that a DDX3‐Akt‐Wnt axis is required for neural crest induction.Support or Funding InformationNIH Grant GM114105This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation and Functional Studies of Tribbles homolog 2 (TRIB2) in Ovarian Granulosa Cells
Authors: Warma Aly , Price Christopher A. , Silversides David W. , Lussier Jacques G. , Ndiaye Kalidou 
Published: June 2021

Tribbles homolog (TRIB) 1, 2 and 3 represent atypical members of the serine/threonine kinase superfamily and are homologs of Drosophila tribbles. TRIB2 mRNA is rapidly induced by mitogens, has a short half‐life, and is expressed in a cell‐specific manner. We previously identified TRIB2 as a differentially expressed gene in granulosa cells (GC) of bovine preovulatory follicles. This study aimed to further investigate TRIB2 mRNA and protein regulation, to identify its binding partners, and study its function in GC of bovine dominant follicles. Granulosa cells were obtained from follicles at different developmental stages: small follicles (SF: 2–4 mm), dominant follicles (DF) at day 5 of the estrous cycle (day 0 = day of the estrous), ovulatory follicles (OF) 24 h following injection of an ovulatory dose of hCG, and corpus luteum (CL) at day 5 of the estrous cycle. RT‐qPCR analyses showed greatest expression of TRIB2 in GC of DF, while the weakest expression was in OF (P < 0.0001). Temporal expression of TRIB2 mRNA was further studied in follicular walls (granulosa and theca cells) obtained from ovulatory follicles recovered at 0, 6, 12, 18 and 24 h after hCG injection. There was a 20‐ and 166‐fold reduction of TRIB2 steady‐state mRNA levels in follicular walls, respectively at 6 and 24 hours post‐hCG as compared to 0 hour (P < 0.001). Additionally, we have generated specific anti‐TRIB2 polyclonal antibodies that confirmed, in western blot analyses, TRIB2 downregulation by LH/hCG at the protein level. Yeast two‐hybrid screening of a DF‐cDNA library as well as functional studies using the CRISPR‐Cas9 approach are currently being used to identify TRIB2 binding partners and its function in GC. These results support a physiologically relevant role of TRIB2 in follicular development and GC function.Support or Funding InformationResearch supported by internal funds from Université de Montréal to KN and NSERC of Canada grant # 104199 to JGL.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optimization and Initiation of a Genome‐Wide Forward Genetic Death Screen for the Lacritin Receptor Complex using the Brunello CRISPR/Cas 9 sgRNA Library
Authors: Teixeira Karina Luiza Dias , Mao Leran , Doench John , Laurie Gordon W. 
Published: June 2021

IntroductionOcular tears refract 80% of light and are essential for epithelial homeostasis. 7% of the world's population suffer from tear insufficiency associated with compromised visual acuity and chronic ocular surface inflammation. Lacritin, a glycoprotein constituent of tears, restores homeostasis in a mouse dry eye model, and in human corneal epithelial cells (HCET) subjected to dry eye‐like IFN‐g and TNF‐a stress. It does so by transiently stimulating autophagy and restoring oxidative phosphorylation. Lacritin also promotes basal tearing by increasing the sensitivity of ion channel TRPM8 on corneal sensory nerves to tear drying. Less well understood is the receptor complex, likely inclusive of a GPCR ‐ based on pertussis toxin and MK912 sensitivity, active N‐24, but not inactive C‐25, lacritin truncation mutant inhibition (64%) of 125 I‐iodoclonidine binding to purified alpha‐2C adrenergic receptor (ADRA2C), and function loss after pooled siRNA knockdown of ADRA2C. ADRA2C is known to complex with ADRA2A, which in turn can modulate TRPM8. Yet, lacritin is unlike any other ADRA2C agonist, requires prior binding to heparanase modified syndecan‐1 (SDC1), failed to inhibit 125 Iiodoclonidine binding to ADRA2C and did not activate a beta‐annexin based ADRA2C Tango assay.GoalOur goal is to identify the lacritin GPCR(s) using a forward genome‐wide CRISPR/Cas9 screen in HCET cells stressed with interferon‐gamma (IFNG) and tumor necrosis factor (TNF).MethodsInitial experiments took advantage of GFP expressing pRosetta to establish optimal HCET viral infection and polybrene concentration. Next, we optimized HCET infection with the Brunello human CRISPR/Cas9 lentiviral library (Addgene #73179) and selection with puromycin, followed by INFG/TNF. For the latter, Alamar Blue and Colony Assays such to identify INFG/TNF levels that killed all cells yet made possible recovery by lacritin using the N‐94 synthetic peptide representing lacritin's C‐terminal active domain. Deep sgRNA sequencing will be performed on genomic DNA from cells stressed with INFG/TNF without or with N‐94.ResultsFor HCET cells, the optimal polybrene concentration was 4 mg/ml that yielded an infection efficiency of ~97% as determined by FACS. The optimal volume of Brunello lentiviral library to obtain 30–50% of infection efficiency was 200 μl. 2 μg/ml was best for puromycin, 1000 U/ml for INFG, 100 ng/ml for TNF and 1000 nM for N‐94 recovery of ~50% of cells. The library consists of 76,441 sgRNAs targeting 19,114 human genes (including ADRA2C, ADRA2A, TRPM8 and SDC1) plus 1000 non‐targeting sgRNA controls. The forward screen is now underway.Support or Funding InformationNIH R01 EY024327NIH R01 EY026171Uva Pinn Scholar AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.