bluuebunny/crossref_metadata_2025_split huggingface dataset:
Synthesis and Evaluation of Photoswitchable c‐Raf Inhibitor
Authors: Weiblen Emilee Ann , Ranger Marah Noel , Streu Craig Nolan 
Published: June 2021

Cancer is one of the leading causes of death in the United States. As a result of its staggering impact, it is unsurprising that scientists have been working diligently to find treatments. Chemotherapeutics and other anticancer drugs have come a long way since their discovery, however the vast majority of them still cause unintended side effects. The main issue with many cancer medications at the moment is the high rate at which healthy, noncancerous cells are damaged along alongside the cancer cells. Highly desirable new treatments for cancer are ones that can selectively target cancerous cells while excluding healthy cells. One way that we can address the problems with the current therapies is by using photoisomerizable compounds, more specifically azo‐stilbenes.Photoisomerizable compounds are molecules that are isomerized by light. In other words, their structure changes when exposed to specific light wavelengths. The azo‐stilbenes, in particular, undergo a trans to cis isomerization when hit with blue or UV light. This isomerization is easily reversible and can be accomplished quickly by irradiating the molecule with a longer wavelength of light (typically >500 nm) or more slowly at room temperature in the absence of light.Photoisomerizable compounds like the azo‐stilbene group have become widely used in many fields, such as functional materials, yet these azo‐compounds have more recently found utility in cancer research due to their ability to be isomerized. These azo‐compounds take one conformation in their trans form and when hit with light isomerize into the cis conformation, thus taking a very different shape. Given that shape complementarity is critical for many drug‐target interactions, this isomerization results in a change in the bioactivity of these molecules. When in their trans form, the azo‐compounds cannot bind to the same target that they can in their cis form. Judicious application of the appropriate wavelengths of light can be used as a switch that will allow for selectivity in cytotoxic treatments. With the use of azo‐stilbenes, off‐target side effects could be greatly reduced and potentially eradicated.One example of a potential target for photo‐switchable therapeutics is c‐Raf. This kinase plays a key role in a pathway that regulates the cellular cycle including the production of new cells and apoptosis, which is dysregulated in some cancers. This work will describe the design, completed synthesis, photokinetic analysis, and preliminary bioactivity measurements for two lead molecules with potential as photoswitchable drugs based upon a previously reported c‐Raf inhibitor.Support or Funding InformationAlbion College Foundation for Undergraduate Research Scholarship and Creativity (FURSCA)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Pharmacological activities of Nepenthes miranda extracts
Authors: Lien Yi 
Published: June 2021

Carnivorous Nepenthes plants exhibit many ethnobotanical uses and their extracts possess many pharmacological activities. In this study, we prepared different extracts from leaves, stems, pitchers, and flowers of N. miranda (Nm) and investigated the antioxidant, antibacterial, anticancer, and anti‐inflammatory properties. To evaluate extraction efficiency, different solvents such as methanol, ethanol, acetone, and distilled water were used individually for Nm extract preparations. Through DPPH radical scavenging assay, Folin‐Ciocalteu method, and aluminium chloride calorimetric analysis, the stems extract obtained by 100% acetone (Nm‐s‐a) showed the highest total antioxidant activity, total phenolic content, and total flavonoid content, respectively. In addition, we found that the same extract also exhibited the highest antibacterial activity against three pathogens, namely, Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. The inhibition zones of Nm‐s‐a determined by agar‐well diffusion assay ranged from 9–29 mm and were in the following order: E. coli > S. aureus > P. aeruginosa. To demonstrate the cytotoxicity effect of Nm‐s‐a, human embryonic kidney 293 cells (HEK 293) and melanoma cells were cultured with various concentrations of Nm‐s‐a. Furthermore, inhibition of human neutrophil elastase by Nm‐s‐a was also found, suggesting the anti‐inflammatory activity. These results indicated that N. miranda may have some medicinal potentials. To further elucidate the structure‐activity relationship, our laboratory is currently using LC‐MS to isolate and identify the active compounds from Nm‐s‐a.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Development of a reference panel as a way to harmonize data of genotyped Sclerotinia sclerotiorum
Authors: Persson Rachel 
Published: June 2021

Several population studies of S. sclerotiorum have been conducted and resulted in important publications furthering our knowledge of this pathogen. Genotype data from those studies has been published with the notion that this data could be re‐used by other scientists. However, amplicon sizes were known to vary across labs due to primer modifications for fluorophore labeling and the instrument used to perform fragment analysis. Thus, it is necessary to create a reference panel of DNA that represents allele sizes known to be present within the population, which is a technique used in human DNA forensics for the same type of genetic marker. Given recent completion of genotyping of more than 366 S. sclerotiorum isolates from across the U.S., development of a DNA Reference Panel that represents most alleles within S. sclerotiorum loci isis now possible. Here, we identified 22 S. sclerotiorum isolates that were collected and genotyped previously and represented all known possible alleles among the 366 S. sclerotiorum. Then, DNA was isolated from those 22 S. sclerotiorum strains and genotyping was performed using 11 previously developed SSR markers. PCR used a 3‐primer indirect fluorescent labeling system that was slightly different from the system used to genotype the 366 isolates, which used direct primer labeling. Amplicons were sized using capillary electrophoresis and raw chromatograms imported into R for sizing and analysis. This data was used to determine differences in allele sizes and create an R function to automatically re‐classify observed data so that it could be seamlessly integrated into our 366 genotype database. Using results of 22 isolates, harmonization of data from two previous studies that genotyped 95 isolates mainly from Brazil and 366 isolates from U.S. took place. From this data, estimation of population genetic statistics, such as genetic/genotypic diversity, evenness, and richness, and test for evidence of sexual recombination was done. Taken together, the DNA reference panel represents most alleles within S. sclerotiorum and allows for integration of previously published population genetic data of this plant pathogen.Support or Funding InformationSupported by Margarita Marroquin‐Guzman and Sydney E. Everhart. Funded by Undergraduate Creative Activities and Research Experience at UNLThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The landscape of aging and maturation signatures of mouse oocyte development
Authors: Lee Tin Lap , Qian Yan , Cao Qin , Liao Jingyue , Lee Annie Wing Tung , Yip Yuk Lap , Leung Tak Yeung , Zhao Ming Peng , Chan David YL , Lee Tin Chiu , Chan Wai Yee 
Published: June 2021

Female fertility declines dramatically with age, and childbearing at advanced maternal age is a big health concern due to greater risk of early pregnancy‐related complications such as miscarriage, genetic disorders, and ectopic pregnancy. The negative impact on fertility caused by advanced maternal aging is also implicated in assisted reproduction technologies. A major factor that accounts for fertility deterioration in older women is poor oocyte quality. By applying RNA‐seq, we compared transcriptome profiles of GV and MII oocytes from the young and old group. We identified 4613 maturation‐related DEGs at the young age and 4223 maturation‐related DEGs at old age. Then, we compared the maturation‐related dynamics at the young age against the maturation‐related dynamics at old age. These DEGs showed high Pearson's correlation (r = 0.8181), suggesting that most of them shared the same maturational expression pattern. To characterize DEGs whose maturational expression changes were affected by age, we established a linear regression model and used a statistical cutoff (95% prediction interval) to define 260 DMRs. The signatures will contribute to novel knowledge essential to the development of assays and treatments for female reproductive aging.Support or Funding InformationHong Kong Collaborative Research Fund C4054‐16GThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Advantages of proteomics using meter‐long monolithic columns with small inner diameter
Authors: Ohtani Yuta , Aburaya Shunsuke , Minakuchi Hiroyoshi , Miura Natsuko , Aoki Wataru , Ueda Mitsuyoshi 
Published: June 2021

BackgroundTargeted proteomics by selected reaction monitoring (SRM) with high signal‐noise ratio enables to quantify hundreds of proteins with high sensitivity. However, since the lowest number of proteins is less than 10 copies in a single cell, further improvement from the aspects of sensitivity is necessary to detect these ones.MethodsUnlike conventional particle‐packed columns used in liquid chromatography (LC), monolithic columns are characterized by an orderly 3D‐network structure [1]. With the monolithic columns, high‐performance separation with a thin skeleton and back pressure can be achieved. Further improvement in detection‐sensitivity is achieved by reducing column inner diameter (I.D.), because small elution volume concentrates analytes and efficient desolvation increases ionization efficiency. Hence, we developed meter‐long monolithic columns with small I.D. to SRM‐based proteomics and aimed to improve detection‐sensitivity of mass spectrometry (MS) to increase peak intensity in peptide levels. BSA tryptic digests were directly injected into monolithic columns with different length (500 mm or 1000 mm) and I.D. (100 μm or 75 μm), and peak capacity and peak area were evaluated.Results & DiscussionSeparation efficiency increased 1.9 times by lengthening a monolithic column from 500 mm to 1000 mm. Detection‐sensitivity increased 2.3 times in peptide level by reducing column I.D. from 100 μm to 75 μm. Furthermore, even when column I.D. was reduced, there were no differences in the coefficient of variation values of the peak area. These results showed reduction of I.D. of meter‐long monolithic columns increased detection‐sensitivity in SRM‐based proteomics [2].Support or Funding InformationThis work was supported by JST CREST (Grant Number JPMJCR16G2), Japan.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of G protein‐coupled receptor kinase 6 in Regulation of Platelet Activation through Selective GPCR Desensitization
Authors: Kim Soochong , Chaudhary Preeti Kumari 
Published: June 2021

Many platelet agonists mediate their cellular effects through GPCRs to induce platelet activation. Considering the crucial roles of GPCR kinases (GRKs) in GPCR functions, little is known regarding the regulation of GPCR signaling by GRKs in platelets. In this study, we identify the molecular basis for regulation of specific GPCR desensitization by GRK6 in platelets. We used mice lacking GRK6 to evaluate the functional role of GRK6 in platelet activation. Platelet aggregation, dense‐ and α‐granule secretion, and fibrinogen receptor activation induced by ADP, U46619, thrombin, and AYPGKF were significantly potentiated in GRK6 −/− platelets compared to WT platelets. However, CRP‐induced platelet aggregation and secretion were not affected in GRK6 −/− platelets. Interestingly, platelet aggregation induced by co‐stimulation of serotonin and epinephrine which activate Gq‐coupled 5HT2A and Gz‐coupled α2A adrenergic receptor, respectively, was not affected in GRK6 −/− platelets, suggesting GRK6 is involved in specific GPCR regulation. In addition, platelet aggregation was restored in response to second challenge of ADP, U46619, thrombin, and AYPGKF in GRK6 −/− platelets whereas re‐stimulation of agonist failed to induce aggregation in WT platelets, indicating GRK6 contributes to P2Y1, P2Y12, thromboxane A2, and PAR receptor desensitization. The surface expression level of PAR4 upon AYPGKF stimulation in GRK6 −/− platelets was higher than WT platelets, indicating a crucial role of GRK6 in internalization of activated PAR4 receptor. Furthermore, AYPGKF‐induced Akt, ERK, and PKCδ phosphorylations were significantly potentiated and sustained in GRK6 −/− platelets. Finally, GRK6 −/− mice show an enhanced and stable thrombus formation after FeCl3 injury to the carotid artery, indicating GRK6 is critical for thrombus formation in vivo. We conclude that GRK6 plays an important role in regulating platelet functional responses through selective GPCR desensitization.Support or Funding InformationThis work was supported by the National Research Foundation of Korea (NRF) Grant of the Korean government (NRF‐2016R1D1A1B01010310) and the Global Research and Development Center (GRDC) Program through the NRF funded by the Ministry of Education, Science and Technology (2017K1A4A3014959).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Scube3 deficiency impairs postnatal skeletal development and repair
Authors: Yuh‐Charn Lin , Ruey‐Bing Yang 
Published: June 2021

Signal peptide‐CUB‐EGF domain‐containing protein 3 (SCUBE3) is the third and last member of a secreted and membrane‐tethered SCUBE protein family acting as co‐receptors to facilitate cellular signaling elicited by transforming growth factor (TGF)‐β superfamily including TGF‐β1 and bone morphogenetic proteins (BMP2 and 4). Although Scube3 is highly expressed in developing endochondral tissues, Scube3 inactivation results in no overt skeletal phenotype in mouse embryos. However, whether Scube3 plays a critical role in adult bone tissue homeostasis remains largely unknown. In this study, we used gain and loss‐of‐function studies to show that SCUBE3 postnatally exerted osteogenic roles by promoting BMP‐stimulated osteoblast differentiation in the mouse mesenchymal progenitor cells or the bone‐marrow derived mesenchymal stromal‐cell cultures. Furthermore, molecular analyses revealed that membrane SCUBE3 indeed functions as a BMP co‐receptor by augmenting the interactions between BMP2/4 ligand proteins with BMP type I receptors, thus its downstream Smad1/5/8 signaling. In agreement with these in vitro studies and the prominent roles of BMP2/4 in adult bone formation and regeneration, genetic ablation of Scube3 caused defected postnatal endochondral bone growth and impaired BMP‐mediated chondrocyte proliferation and differentiation as well as compromised fracture healing at the initial stages. Our data demonstrate that SCUBE3 is an important postnatal factor in modulating BMP‐dependent endochondral bone formation and repair and might serve as a therapeutic target for skeletal trauma and disease.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Role of Gβγ G Protein Subunit in the Dopamine D2 Signaling Pathway on Ciliary Activity of Gill Lateral Cells in Crassostrea virginica
Authors: Baxter Kameca , Carroll Margaret A. , Catapane Edward J. 
Published: June 2021

Gill lateral cell (GLC) cilia of Crassostrea virginica are controlled by serotonin‐dopamine (DA) innervations. The gill is innervated by the branchial nerve which originates in the visceral ganglia. The DA nerves are inhibitory and slow down GLC cilia beating rates. The serotonin nerves are excitatory and speed them up. Previous work of our lab showed the post‐synaptic DA receptors in GLC are D2 type (D2R). The D2R signaling pathway involves inhibition of adenylyl cyclase and the splitting of the G protein into the Gβγ and Gαi/o subunits. Gβγ activates PLC (phospholipase C). Gβγ has not been well studied in bivalves with respect to GLC cilia beating. We hypothesize that the Gβγ component of the D2R signaling pathway is involved in slowing down the beating rate of GLC cilia. To test this we determined the effects of a Gβγ activator and inhibitor on GLC cilia beating rates using stroboscopic microscopy. We conducted acute experiments testing the effects of the Gβγ activator and inhibitor, with and without the presence of DA on excised gill. Ciliary activity of gill lateral cells was measured by stroboscopic microscopy and expressed as beats/min ± sem. We found that applying DA (10−6 – 10−3M) to gill is cilio‐inhibitory, causing a dose dependent decrease of beating rates to 0. Applying guanosine 5′‐O‐(3‐thiotriphosphate) (GTP), a Gβγ activator, had a slight inhibitory effect on GLC cilia beating over the range of 10−7 – 10−3M. Adding DA prior to GTP did not significantly alter the actions of GTP. Applying the Gβγ inhibitor gallein caused an increase in GLC cilia beating rates when applied after DA. The study shows Gβγ does play a role in effecting the beating of GLC cilia. When Gβγ is activated it activates PLC and caused a cilio‐inhibitory response. When Gβγ is inhibited, the cilia beating rates sped up, even in the presence of DA. In light of the fact that the D2R signaling pathway involves activation of PLC as well as inhibition of the adenylyl cyclase, it appears that the PLC path contributes to the cilio‐inhibition. This study provides new knowledge of the actions of Gβγ in the D2R pathway in bivalve gill. Because the D2R pathway is significantly involved in the actions of antidepressants, neuroleptics, and drugs of abuse, as well as being implicated in neuropsychiatric and neurodegenerative disorders, further investigations of the physiological role of D2R using this gill system should be helpful to understand the D2R signaling pathway.Support or Funding InformationThis work was supported in part by grant 2R25GM06003 of the Bridge Program of NIGMS, NIH grant K12GM093854‐07A1 IRACDA Program of Rutgers University and 604060048 of PSC‐CUNY.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Deletion and Characterization of Two Signaling Genes in Streptomyces
Authors: Bowman Bridget , Bennett Jennifer 
Published: June 2021

Streptomyces coelicolor is a filamentous, high G‐C (guanine‐cytosine), gram‐positive bacterium that produces an aerial mycelium. The genome consists of a 8,667,507 base pair linear chromosome for this organism and contains the largest number of genes so far discovered in a bacterium. It belongs to the phylum Actinobacteria. In addition, streptomycetes are used to produce the majority of antibiotics in the medical field. This study focuses on the effect of the deletion of Sco5218, which encodes a putative integral membrane protein and Sco5219, which encodes a putative lipoprotein in Streptomyces coelicolor. Both of these genes are found in a potential operon. The goal of this research is to construct the double mutant and discern differences in phenotype compared to the single deletion mutants of each gene. We hypothesize that the double mutant will have an enhanced effect on the sporulation defect of Streptomyces. Sco5218 is believed to contain an HD‐GYP domain through bioinformatics analysis and is highly conserved throughout the Streptomyces genus. The HD‐GYP domain is typically associated with cyclic di‐GMP specific phosphodiesterase activity. Following REDIRECT protocol, the Streptomyces coelicolor cosmid clone was introduced into E. coli via electroporation. PCR was then utilized for the deletion of Sco5218 and Sco5219. This PCR product was run onto a gel and purified. This pure PCR product was then transformed into the plasmid vector which will then be mated into Streptomyces coelicolor. The relevance of this research allows us to see the importance of the HD‐GYP domain in bacteria. We witnessed a slight delay in sporulation with the Sco5218 mutant deletion. With the added deletion of Sco5219 we hypothesize that there will be an enhanced delay in sporulation. If we witness a delay in the sporulation with the double mutant in Streptomyces we can deduce that both genes play a role in bacterial development. These findings will allow us to further delve into the study of other bacteria and have a basis for other orthologs containing the HD‐GYP domain.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The PH Domain of ASAP1 Binds N terminus of Arf1 in Presence of PIP2 for Efficient GTPase‐activating Protein Activity
Authors: ROY NEELADRI SEKHAR , Zhai Peng , Jian Xiaoying , Jenkins Lisa , Luo Ruibai , Yohe Marielle E. , Randazzo Paul A. 
Published: June 2021

ASAP1 is a multi‐domain GTPase‐activating protein (GAP) for ADP‐ribosylation factor (Arf) guanine nucleotide binding proteins. ASAP1 has been found to affect cell behaviors dependent on adhesions and actin, including the proliferation, invasion and metastasis of cancer cells. The cellular function of ASAP1 depends on its highly regulated and robust Arf GAP activity, which refers to the enzymatic activity of converting Arf•GTP to Arf•GDP. Arf GAP activity requires the PH domain together with the catalytic Arf GAP domain and is modulated 4 orders of magnitude by the membrane phospholipid phosphatidylinositol 4,5‐ bisphosphate (PIP2) binding to the PH domain. However, the mechanistic basis of PIP2‐stimulated GAP activity is not completely understood. Here we found that the N terminal extension of the substrate Arf1 is necessary for the PIP2‐stimulated activity of ASAP1 in GTP hydrolysis assays. A peptide 2–17 from N terminal Arf1 inhibited GAP activity with full length Arf1 as substrate. Using fluorescence anisotropy and Forster resonance energy transfer (FRET), we found binding between the N‐terminal 2–17 amino acids of Arf1 and ASAP1. Binding was confined to the ASAP1 PH domain and depended on presence of PIP2. Limited proteolysis and crosslinking experiments indicate conformational changes in ASAP1 PH domain on binding PIP2. Mutational studies involving deletion of the putative interacting residues on the PH domain results in loss of binding to N terminal Arf1 as well as ASAP1 GAP activity. We propose that PIP2 binding to PH domain brings conformational changes that facilitate binding to N terminal Arf1 and then the later interaction favorably orients Arf GAP domain with the substrate Arf1 enhancing GAP activity of ASAP1.Support or Funding InformationThis work is supported by NIH Intramural Program (Project BC 007365).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.