bluuebunny/crossref_metadata_2025_split huggingface dataset:
Chemically‐controlled orthogonal regulation of multiple endogenous genes
Authors: Nomura Wataru , Matsumoto Daisuke , Sugii Taisuke , Kobayakawa Takuya , Tamamura Hirokazu 
Published: June 2021

Genome editing platforms are powerful tools for gene manipulation. These platforms offer a wide variety of promising applications in medical science and biotechnology. Induction of protein domain associations by stimuli such as light or chemicals has been widely used for the temporal control of biomolecular function such as enzymatic activity. Further, the DNA binding specificity of genome editing tools can be applied in gene regulation systems by combining them with transcription‐activator or transcription‐repressor domains. Targeting the gene promoter sequence using multiple artificial transcription factors enables synergistic and efficient regulation of gene expression. In addition, the use of these platforms in combination can expand the scope of targetable sequences by avoiding DNA‐binding issues, such as PAM recognition for CRISPR‐Cas9, 5′‐T recognition for TALE domain, and complementary strand recognition for ZF domain. Furthermore, the guide RNA for Cas9 cannot distinguish alleles because of the commonly used gRNA sequence recognized by Cas9, thereby limiting the utility of this approach for orthogonal regulation of multiple genes. In this study, the rapamycin‐ (R‐) or gibberellin‐ (G‐) inducible system was applied for gene regulation using multiple TALEs and dCas9 to enable orthogonally inducible regulation of endogenous genes. For the R‐inducible system, FKBP‐tagged TALEs, and dCas9, the effector domains with FRB, VP64 for activation and SID×4 for repression, were constructed. GID‐tagged TALEs and dCas9 as well as GAI‐fused VP64 were constructed for the G‐inducible system. The fold change in gene expression was evaluated by luciferase reporter assays. The results showed that gene expression was regulated by addition of chemicals, indicating rapamycin‐induced FKBP‐FRB association and gibberellin‐induced GID‐GAI association. The systems were further utilized for controlling endogenous gene expression in 293A cells. The results revealed that the expression of two genes could be differentially regulated by rapamycin and gibberellin in a time‐dependent manner. This system could achieve the control of gene function in cellular phenomena owing to the programmable timing of expression and differential expression of multiple genes.Support or Funding InformationThis work was supported in part by the New Energy and Industrial Technique Development Organization (NEDO) of Japan, JSPS KAKENHI (25410171), Grant‐in‐Aid for Scientific Research on Innovative Areas (24119506 and 26119703) to W. N.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Protein homology suggests similarities in histone mRNA processing between humans and slime molds
Authors: Roth Stefen Paul , Lackey Patrick 
Published: June 2021

Histone mRNA is the only metazoan mRNA that is not polyadenylated. Instead, it ends with a conserved 3′ stem loop that, along with its protein binding partner the stem‐loop binding protein (SLBP), is necessary for processing, export into the cytoplasm, and degradation. These mechanisms are well‐studied in humans, Drosophila, and Xenopus, but they are completely absent in fungi like Saccharomyces cerevisiae. Instead, the histone mRNA in these eukaryotes have traditional 3′ poly(A) tails. Because of this, the evolutionary history of the mechanism of stem‐loop control is not well understood. Recently, bioinformatics work has identified histone mRNA with stem‐loops and proteins that closely resemble SLBP in very simple eukaryotes like Dictyostelium discoideum and Chlamydomonas reinhardtii. This presents us with an opportunity to better understand this evolutionary history. The stem loop and SLBP also play a key role in the recruitment of key processing and cleavage factors including, NPAT, FLASH, the U7 snRNP complex (via the snRNP subunits LSM10 and LSM11), and the histone cleavage complex (HCC) made up of symplekin, CPSF 73 and CPSF 100. Together, these proteins work to efficiently process histone pre – mRNA in lower through higher eukaryotes. We have identified homologs of FLASH, NPAT, symplekin, ZFP100 and LSM11 in Dictyostelium. Together with the previously identified binding domains and well understood polyadenylation proteins, this creates a proto‐HCC complex, through which we can begin to understand how the histone processing and cleavage mechanism in these lower organisms compares to the better‐understood mechanism in metazoans.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fluorescence lifetime imaging of compartmental pH dynamics using red fluorescent protein sensors in live cells
Authors: Haynes Emily , Rajendran Megha , Lyon Angeline , Noinaj Nicholas , Day Richard , Tantama Mathew 
Published: June 2021

pH regulation plays a crucial role in protein function, cell metabolism, intracellular degradation, and a wide range of other physiological processes. Monitoring perturbations in local pH environments due to cellular activities or diseases, such as increased neuron activity, receptor trafficking, or extracellular acidosis in a tumor microenvironment, would better our understanding of disease pathology. Live‐cell imaging with fluorescent protein‐based sensors allows us to monitor broad pH changes; however, there are currently limited options to quantitatively measure pH dynamics with subcellular resolution in live specimens. Concentration and intensity dependence of the fluorophore, sample thickness, and photo‐bleaching are limiting factors, and additionally, most of the current genetically encoded pH sensors are generated from green fluorescent protein, limiting our ability to perform multiplex imaging to monitor pH simultaneously in different compartments. As an intrinsic property of fluorophores, fluorescence lifetime does not rely on protein concentration, method of measurement or fluorescence intensity. Utilizing this property, we can monitor discrete compartmental changes in pH using fluorescence lifetime imaging microscopy (FLIM), while opening up a wider selection of potential fluorescent sensors for multi‐channel imaging. It is the goal of this project to develop genetically‐encoded red fluorescent pH sensors to quantitatively measure micro‐environmental pH changes using fluorescence lifetime. Here we present in vitro and live‐cell characterization; in proof‐of‐concept studies we have utilized FLIM to measure the pH of different subcellular compartments simultaneously in real‐time. Our results demonstrate our progress towards understanding the nuances of subcellular pH and aberrant cell behavior, and how the role of pH is affected in both healthy and disease states.Support or Funding InformationEmily Haynes is supported by her Charles H. Viol Chemistry Fellowship. We also gratefully acknowledge support from the Showalter Fund and from National Institutes of Health grants R21 NS092010 and R21 EY026425.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Phosphorylation of G protein γ subunit Ste18 and the Ste5 scaffold form a braking system that governs pathway activation kinetics and switch‐like signaling in yeast
Authors: Torres Matthew , Choudhury Shilpa , Baradaran‐mashinchi Parastoo 
Published: June 2021

Heterotrimeric G proteins (Gαβγ) play a critical role as transducers in G protein signaling systems responsible for activating a wide range of physiological processes including neurotransmitter and hormone signaling. In yeast, G protein signaling differentially activates two distinct mitogen‐activated protein kinases (MAPKs) – Fus3 and Kss1 – a phenomenon that requires conformational switching of the scaffold protein Ste5. Here we show that rapid receptor‐activated phosphorylation of the intrinsically disordered N‐terminal tail of the yeast Gγ subunit (Ste18), in combination with MAPK phosphorylation of Ste5, serves as a brake system that controls the rate and stability of scaffold association at the plasma membrane. Disabling the brake by precise point mutation of Ste18 phosphorylation sites or a MAPK docking site on Ste5 differentially governs the kinetics and amplitude of MAPK activation as well as the switch‐like morphological response to the stimulus. Complete elimination of the brake results in a response that is 5 times faster and ~4 times amplified over the response in wild type cells. Finally, we show that phosphorylation of Ste18 appeared late in evolutionary history ‐ at the same time as the MAPK docking site on Ste5, suggesting the possibility that both sides of the brake co‐evolved. Since phosphorylated intrinsically disordered tails are structurally conserved elements found in all Gγ subunits, including mammals, these results provide compelling evidence that such tails may function as intrinsic regulators of G protein signaling by altering the interactions of Gβγ subunits with their binding partners.Support or Funding InformationThis work was supported by funding from NIH grant GM117400 to M.T.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Thermal Manipulation During Brolier Chicken Embryogenesis Increases the Basal mRNA levels of antioxidant factors and Alters Their Production Dynamics during Thermal Stress
Authors: Al‐Zghoul Moh'd Borhan 
Published: June 2021

This study investigated the effects of thermal manipulation during embryonic days 12–18 on the kinetics of the liver mRNA levels of antioxidant factors (Catalase, Glutathione (GSH), Superoxide Dismutases (SOD)) during thermal stress. One thousand fertile chicken eggs were randomly divided into four groups: Hubbard Control group (37.8°C), Hubbard TM (39°C for 18 h), Cobb Control group (37.8°C), Cobb TM (39°C for 18 h). On post‐hatch days 14 and 28 of age, 30 randomly selected chicks from each group were thermally stressed at 41.0°C for 6 h while another 30 randomly selected chicks from each group were kept under thermo‐neutral conditions. The Tc of TM chicks was only numerically lower than that of the control during the study period. However, during TS at days 14 and 28 of age, the Tc of TM chicks was significantly lower than that of the control. On post‐hatch days 14 and 28, the basal mRNA levels of antioxidant factors were significantly higher than those of the control. Furthermore, during TS, rapid increases in the mRNA levels of antioxidant factors in the TM groups were observed. These results indicate that, as well as altering the basal mRNA levels of antioxidant factors during the first week post‐hatch, TM also altered the dynamics of the mRNA expression of antioxidant factors, which was associated with improved acquisition of thermotolerance during thermal stress.Support or Funding InformationDeanship of research/Jordan University of Science and Technology (Project#: 125/2017)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
EGCG promotes cell growth inhibition and reprograms mesenchymal‐epithelial transition by restoring CCN5/WISP2 in triple negative breast cancer cells in vitro and in vivo
Authors: Ghosh Arnab , Das Amlan , Banerjee Snigdha , Baltezor Michael , Zeng Liang , Banerjee Sushanta K. 
Published: June 2021

Background and ObjectiveCCN5/WISP‐2, a matricellular secretory protein that is localized in both cytoplasm and nucleus, is mainly expressed in luminal cells rather than basal cell types in breast cancer tumors. Endogenous CCN5 plays critical role in promoting growth arrest and reprograming Mesenchymal to Epithelial Transition(MET), inhibition of migration and invasion, overall inducing the invasive to non‐invasive phenotypes. Thus, we hypothesize that targeting endogenous CCN5 restoration in Triple‐negative‐breast cancer (TNBC) cells could be an ideal strategy to prevent the progression of TNBC. To test the hypothesis, we set out to identify a molecule that has the capability to restore CCN5 in TNBC cells. Here we report that CCN5, which is normally absent in TNBC cells can be restored by a common dietary supplement epigallocatechin‐3‐gallate (EGCG), an ester of epigallocatechin and gallic acid, and is a type of catechin, to promote growth arrest and reverse EMT.MethodsEffect of EGCG on breast cancer cells were characterized by in vitro protein and gene expression profilings, cytotoxic assays, colony formation assays, migration assays, anchorage independent growth assays. In vivo effect of EGCG on tumorigenesis was investigated in xenograft model of immunosuppressive mice.Result and ConclusionEGCG treatment in breast cancer cells significantly elevated and restored CCN5 expression in TNBC cells along with inhibition of cell proliferation, migration and colony formation and induction of apoptosis. Reprogramming of mesenchymal to epithelial transition(MET) was also observed in EGCG‐treated TNBC cells. Oral administration of EGCG resulted in drastic inhibition of tumor growth and propagation in immunosuppressive mouse xenografts of MDA‐MB‐231 cells, along with restoration of CCN5 expression. Altogether, these studies clearly indicate that EGCG is an ideal molecule to restore CCN5 in TNBC cells in preventing the TNBC growth, progression and possibly make these cells susceptible to anti‐estrogen treatment, as restoration of CCN5 causes estrogen receptor(ER)‐α in TNBC cells. Moreover, it is reasonable to speculate that the CCN5 pathways probably play an important part in the anti‐tumorigenic role of EGCG in TNBC cells.Support or Funding InformationSupported by Midwest Biomedical Research Foundation and VA Merit Grant.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Recombinant MS2 L Is Biologically Functional
Authors: Hoffer Ashley L. , Streff Kimberly J. , Piefer Andrew J. 
Published: June 2021

The single stranded RNA bacteriophage MS2 attacks its host, Escherichia coli, with the help of its lysis protein, L, rather than lytic enzymes. The L protein consists of 75 amino acids, with a hydrophilic N‐terminal that contains mainly basic residues and a hydrophobic C‐terminal end. Prior studies have shown that the L protein is necessary for the cell to lyse, however it is not known if L is sufficient for cell lysis. This study may lead to identifying whether or not the L protein in MS2 is the singular factor in cell lysis. Tests have been conducted using a previously engineered plasmid that contains MS2 L under the control of the arabinose inducible pBAD promoter. Transformed E. coli cultures were induced with arabinose and compared via spectrophotometry. The results show a decrease in the amount of cells within 40 minutes after induction compared with non‐induced and GFP producing controls demonstrating that the L protein is indeed functional and triggering E. coli lysis. The question still remains as to whether or not L is functioning independently or in concert with other E. coli proteins. In order to explore this possibility, a GST‐tag will be cloned in‐frame with the L ORF. After determining whether the GST‐L is biologically functional, we will perform pull‐down experiments to determine whether host proteins are associated with L.Support or Funding InformationThis work was funded by the Hartwick College Chemistry Department as well as a Hartwick College Faculty Research Grant awarded to AJP.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
An Integrated System Approach Identified the Human Proteasome as a Conserved Critical Machinery for ZIKV and DENV Replication
Authors: Song Guang , Lee Emily , Pan Jianbo , Xu Miao , Rho Hee‐Sool , Cheng Yichen , Whitt Nadia , Yang Shu , Kouznetsova Jennifer , Klumpp‐Thomas Carleen , Michael Samuel G. , Simeonov Anton , Huang Wenwei , Xia Menghang , Huang Ruili , Lal‐Nag Madhu , Zhu Heng , Tang Hengli , Zheng Wei , Qian Jiang , Song Hongjun 
Published: June 2021

Zika virus (ZIKV) and dengue virus (DENV) are two related flaviviruses that share similar replication process but exhibit distinct clinical outcomes. Systematic understanding of virus‐host cell interaction networks can reveal cellular pathways not only critical for viral replication but also for pathogenesis. Here we employed a protein array approach to comprehensively identify host proteins interacted by both ZIKV and DENV proteins. The results uncovered multiple conserved host pathways and cellular protein complexes that are engaged by both viruses, such as proteasome complex and spliceosomal complex, involving in flavivirus infections. An RNAi screening of 10,415 druggable genes found that the proteins such as these related to proteasome significantly reduced ZIKV infection. Additionally, a high‐throughput screening of bioactive compound collections identified 134 compounds with anti‐ZIKV activity. Among these compounds, eight proteasome inhibitors suppressed both ZIKV and DENV replication. This system biology approach integrating the orthogonal pathways and protein‐protein interactions has revealed the proteasome as a critical host machinery for ZIKV replication. This study provides rich and valuable datasets for further studies of flavivirus‐host interactions, disease pathogenesis, and new drug targets.Support or Funding InformationThis work was supported by the Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health (W.Z.) and partially supported by NIH grants R01GM111514 (to H.Z & J.Q.), U19AI131130 (to G‐l.M, & H.T.), R35NS097370 (to G‐l.M.), R37NS047344 (to H‐j.S.), and FSU Zika seed funding (to H.T.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
FOXP3 O‐GlcNAcylation Controls Regulatory T Cell Homeostasis and Suppressive Function
Authors: LIU BING , Barrero Oscar Salgado , Sahu Chinmayi , Ball Lauren E. , Hogquist Kristin A. , Ruan Hai‐Bin 
Published: June 2021

Regulatory T (Treg) cells are a distinct lineage of T lymphocytes that control immunological self‐tolerance and homeostasis. The use of Treg cells for immunotherapy has been challenging, partially because of the functional heterogeneity and instability of Treg cells. The transcription factor FOXP3 is crucial for Treg lineage specification during development and is continuously required for the suppressive function of mature Treg cells. FOXP3 can be regulated by various post‐translational modifications, which enable transient regulation of FOXP3 functionality in response to environmental cues. Here, we investigate the novel O‐linked N‐Acetylglucosamine (O‐GlcNAc) modification on FOXP3 in Treg cell function and stability.Protein O‐GlcNAcylation is enriched in induced Treg cells, compared to naïve T cells. Chemical augmentation of O‐GlcNAcylation promotes the induction of Treg cell signature genes. O‐GlcNAcylation, controlled by O‐GlcNAc cycling enzymes O‐GlcNAc transferase (OGT) and O‐GlcNAcase (OGA), modifies and stabilizes FOXP3 by counteracting with ubiquitination. Treg cell‐specific OGT deletion in mice does not affect Treg cell lineage specification, but results in fewer suppressive effector Treg cells and the development of an aggressive autoimmune syndrome. O‐GlcNAc‐deficient Treg cells show reduced FOXP3 protein expression and increased effector T‐cell signatures. In summary, we demonstrate that protein O‐GlcNAcylation, by modulating FOXP3, is indispensable for Treg cell suppressive function and lineage stability. Manipulating O‐GlcNAc levels may represent a novel approach to translate Treg cell immunotherapy into clinical practice.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Expanded Exploration of FUT8 Substrate Specificity Towards a Variety of Its Less Preferred Substrates
Authors: Zhang Roushu , Yang Qiang , Wang Lai‐Xi 
Published: June 2021

Core‐fucosylation, the attachment of fucose to the asparage‐linked GlcNAc of N‐glycan in an α(1,6) linkage, is an important modification that modulates the functionality and activity of glycoproteins. The changes of core‐fucosylation level on glycoprotein biomarkers are often detected in chronic diseases and cancer development. The α(1,6)‐fucosyltransferase (FUT8) is the sole enzyme catalyzing core‐fucosylation in mammalian systems. Exploring the enzymatic activity and substrate specificity of FUT8 is treasurable for both fundamental and therapeutic purposes. Previously, intensive in vitro studies illustrated that addition of a GlcNAc to the α(1,3) mannose arm is a prerequisite for the FUT8‐catalyzed core‐fucosylation, using either glycans or small glycopeptides as the substrates. In contrast, we recently demonstrated that within a proper protein or peptide context, it is feasible to core‐fucosylate Man5GlcNAc2 (Man5) by FUT8 both in vivo and in vitro. More impressively, the erythropoietin with Man5 glycosylation (EPO‐Man5) showed complete transfer after an over‐night reaction in vitro. Our findings suggested that FUT8 may have much broader substrate specificity than previously thought. Therefore, we further tested this enzyme using other glycoforms such as high‐mannose (HM, Man5–9GlcNAc2) glycans, paucimannose (PM, Man1–4GlcNAc2) glycans, and complex‐type (CT) glycans under its preferred context in vitro. Interestingly, the majority of the glycoforms we chose to analyze can be core‐fucosylated by FUT8; even though the transferring efficiency varies. For instance, decent transfer efficiency was observed on EPO‐HM and EPO‐PM glycoproteins in vitro. To our knowledge, this is the first reported synthesis of core‐fucosylated Man7–9 by FUT8. Moreover, enzymatic activity was also detected towards Fmoc‐labeled highly‐branched CT glycans purified from ovalbumin. In conclusion, our results indicate that an appropriate structural or peptide context can facilitate the enzymatic activity of FUT8 towards a broad range of its less preferred acceptor substrates, suggesting that FUT8 has a more relaxed substrate specificity that previously implicated.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.