bluuebunny/crossref_metadata_2025 huggingface dataset:
In situ observation of the deterioration process of sulfide-based solid electrolytes using airtight and air-flow TEM systems
Authors: Tsukasaki Hirofumi , Igarashi Keisuke , Wakui Akiko , Yaguchi Toshie , Nakajima Hiroshi , Kimura Takuya , Sakuda Atsushi , Tatsumisago Masahiro , Hayashi Akitoshi , Mori Shigeo
Published: June 2021

Sulfide-based solid electrolytes (SEs) exhibiting high ionic conductivity are indispensable battery materials for next-generation all-solid-state batteries. However, sulfide-based SEs have a major drawback in their low chemical stability in air. When exposed to H2O or O2 gas, toxic H2S is generated, and their ionic conductivity considerably declines. However, their degradation mechanism caused by air exposure has not been understood yet. To clarify the degradation process, in this study, we developed a transmission electron microscope (TEM) system to evaluate the air stability of battery materials. Using a vacuum transfer double-tilt TEM holder with a gas-flow system, the in situ observation of the degradation process was conducted for a sulfide-based Li4SnS4 glass ceramic under an air-flow environment. Consequently, electron diffraction (ED) patterns and TEM images could clearly capture morphological changes and the amorphization process caused by air exposure. Moreover, based on the analysis of ED patterns, it is observed that Li4SnS4 is likely to decompose because of the reaction with H2O in air. Therefore, this airtight and air-flow TEM system should be effective in clarifying the process of the deterioration of sulfur-based SEs during exposure to air.
Gabapentinoids Associated With Lower Explantation Rate in 203 Patients With Spinal Cord Stimulation for Failed Back Surgery Syndrome
Authors: Nissen Mette , Ikäheimo Tiina-Mari , Huttunen Jukka , Leinonen Ville , Jyrkkänen Henna-Kaisa , von und zu Fraunberg Mikael
Published: June 2021

BACKGROUND

Spinal cord stimulation (SCS) is an effective treatment in failed back surgery syndrome (FBSS). The effect of neuropathic pain medication use on SCS outcome is poorly understood.

OBJECTIVE

To study the effect of gabapentinoid use on SCS outcome measured by trial success, explantation rate and opioid dose reduction during a 2-yr follow-up.

METHODS

The study cohort included 203 consecutive FBSS patients who underwent SCS in a single tertiary center during January 1997 to March 2014. Purchase data of gabapentinoids, opioids, tricyclic antidepressants, serotonin and noradrenaline reuptake inhibitors, and benzodiazepines during January 1995 to March 2016 were retrieved from national registries.

RESULTS

In multivariate Cox regression analysis, patients using gabapentinoids had significantly fewer explantations during the 2-yr follow-up (hazard ratio [HR] 0.2, 95% CI 0.04-0.81, P = .03). In contrast, patients with opioid use of >40 morphine milligram equivalent before implantation had significantly more explantations (HR 6.7, 95% CI 2.5-18, P < .01). In bivariate logistic regression analysis adjusted for patient specific factors, year of SCS implantation, use of neuropathic pain medication, opioids, and benzodiazepines, patients using gabapentinoids significantly more often discontinued opioids or reduced their dose by more than 50% during the 2-yr follow-up (odds ratio 5.7, 95% CI 1.4-23, P = .015).

CONCLUSION

The use of gabapentinoids was associated with a significantly lower spinal cord stimulator explantation rate and a higher chance of opioid discontinuation or >50% dose reduction. This indicates that patients with SCS could benefit from concomitant use of gabapentinoids. Prospective randomized trials are warranted to verify this hypothesis.
Frequencies and Variations of Magnaporthe oryzae Avirulence Genes in Hunan Province, China
Authors: Peng Zhirong , Li Ling , Wu Shenghai , Chen Xiaolin , Shi Yinfeng , He Qiang , Shu Fu , Zhang Wuhan , Sun Pingyong , Deng Huafeng , Xing Junjie
Published: June 2021

Rice blast caused by Magnaporthe oryzae poses significant threaten to rice production. For breeding and deploying resistant rice varieties, it is essential to understand the frequencies and genetic variations of avirulence (AVR) genes in the pathogen populations. In this study, 444 isolates were collected from Hunan Province, China in 2012, 2015, and 2016, and their pathogenicity was evaluated by testing them on monogenic rice lines carrying resistance genes Pita, Pizt, Pikm, Pib, or Pi9. The frequencies of corresponding AVR genes AVRPizt, AVRPikm, AVRPib, AVRPi9, and AVRPita were characterized by amplification and sequencing these genes in the isolates. Both Pi9 and Pikm conferred resistance to >75% of the tested isolates, while Pizt, Pita, and Pib were effective against 55.63, 15.31, and 3.15% of the isolates, respectively. AVRPikm and AVRPi9 were detected in 90% of the isolates and AVRPita, AVRPizt, and AVRPib were present in 26.12, 66.22, and 79% of the isolates, respectively. Sequencing of AVR genes showed that most mutations were single nucleotide polymorphisms, transposon insertions, and insertion mutations. The variable sites of AVRPikm and AVRPita were mainly located in the coding sequence regions (CDS), and most were synonymous mutations. A 494-bp Pot2 transposon sequence insertion was found at the 87 bp position upstream of the start codon in AVRPib. Noteworthy, although no mutations were found in CDS of AVRPi9, a GC-rich inserted sequence of ∼200 bp was found at the 1,272 bp position upstream of the start codon in three virulent isolates. As AVRPikm and AVRPi9 were widely distributed with low genetic variation in the pathogen population, Pikm and Pi9 should be promising genes for breeding rice cultivars with blast resistance in Hunan.
Modulation of Nicotinic Receptors by Cannabinoids
Authors: Rodriguez‐Tirado Carla Sofia , Colon‐Saez Jose Orlando , Lasalde‐Dominicci Jose
Published: June 2021

The legalization of medicinal and recreational use of marijuana by many states in the USA, has resulted in an increase in its use. The two most abundant phytocannabinoids in marijuana Δ9‐Tetrahydrocannabinol (THC) and cannabidiol (CBD) have been shown to have tremendous therapeutic potential in many neurological disorders such as anxiety, seizures, post‐traumatic stress disorder and pain. One study showed that CBD use reduces cigarette consumption by 40%. However, little is known about the effects that these phytocannabinoids have on the function of nicotinic acetylcholine receptors (nAChRs). Published results showed that CBD reduces the response of the α7 nAChR to acetylcholine (ACh). Through this project we are characterizing the effects of CBD and THC on the function of the α7 nAChR. Our results indicate that CBD reduces the response of the α7 nAChR to ACh by 49% (p value=0.0003); and that CBD alone does not activate the receptor. Interestingly, THC seems to have the opposite effect, it appears to increase the response of the α7 nicotinic receptor to ACh by a 128% (pvalue=0.44). When both drugs were combined at a 1:1 ratio, the observed effect was an inhibition. The effect was intermediate to the effect seem with either drug alone, the observed response was 62% of the normal response (pvalue=0.007). The response of the α7 nicotinic receptor to CBD and THC seems to be independent of the concentration of ACh applied. Which suggests that neither CBD nor THC compete with ACh for the binding site. Dissecting the effects of phytocannabinoids found in marijuana on the nervous system is critical to increase our chances of using it successfully in the clinic.

Support or Funding Information

NIH‐Maximizing Access to Research Careers (Grant # 5T34GM007821‐38)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
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 Information

This 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 Information

Emily 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 Information

This 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 Information

Deanship 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 Objective

CCN5/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.

Methods

Effect 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 Conclusion

EGCG 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 Information

Supported 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 Information

This 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 Information

This 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.
Anti‐oxidative activity of db mice treated with glycosaminoglycan of cricket, Gryllus bimaculatus
Authors: Ahn Mi Young , Hwang Jae Sam
Published: June 2021

Gryllus bimaculatus (Gb, cricket) is newly emerged as an edible insect in several countries. Edible or medicinal insects have been employed by purification and identification of potent active substances as food or drugs. Anti‐diabetic activities of G. bimaculatus glycosaminoglycan (GbG) or dung beetle (Catharsius molossus) glycosaminoglycan (CaG) were evaluated in diabetic mice based on their abilities to reduce glucose, ALT, AST, LDL‐cholesterol, and BUN levels. Db mice were assigned into the following groups: 1) Db heterozygous (PBS, normal control), 2) Db homozygous (PBS, diabetes control), 3) CaG (5 mg/kg), 4) GbG (5 mg/kg), and 5) metformin (10 mg/kg). They were orally administered with PBS or CaG or GbG or metformin for one month. Sero‐biochemical levels were evaluated to determine their anti‐diabetic, and anti‐oxidative activities after one month of CaG or GbG treatment in Db mice. Blood glucose level was decreased after treatment with CaG or GbG. GbG produced meaningful anti‐diabetic effect. It inhibited LDL‐cholesterol and alkaline phosphatase levels. Regarding oxidative damage, levels of hepatocellular biomarkers levels and protein carbonyl content were reduced in db mice treated with CaG or GbG. Anti‐oxidative activities of catalase, superoxide dismutase, and glutathione peroxidase were significantly increased in GbG treated group compared to those in the control. Therefore, G. bimaculatus glycosaminoglycan could be used as a therapeutic agent with anti‐diabetic effect and anti‐oxidative activity for diabetic mice.

Support or Funding Information

This work was supported by Rural Development Administration basic Research project, PJ011853.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evaluation of Plasma Cell‐Free Circulating DNA Integrity in Patients with Prostate Cancer in Jamaica
Authors: Condappa Andrew , Aiken William , McLaughlin Wayne , McGrowder Donovan , Gossell‐Williams Maxine
Published: June 2021

Cell free circulating DNA (cfcDNA) is a promising diagnostic tool for prostate cancer (PCa). The study aimed to measure the cfcDNA concentration and cfcDNA integrity in prostate cancer patients using quantitative polymerase chain reaction (qPCR) analysis on plasma samples. The study also assessed correlation between cfcDNA concentration and integrity with baseline PSA, Gleason score, total prostate volume, age and the number of presenting comorbidities. Eleven patients with PCa and 9 patients with Benign Prostatic Hyperplasia (BPH) were enrolled. Plasma samples were collected before prostate biopsy and quantified by qPCR amplification of ALU115 DNA sequence, and the quantitative ratio of ALU 247 to ALU 115 reflected the integrity of cfcDNA. There were no significant differences in median (IQR) plasma cfcDNA concentration or its integrity between the patients with PCa [47.9 (214.93) ng/mL, 0.61 (0.49)] and patients with BPH [41.5 (55.13) ng/mL, 0.67 (0.45); p = 0.382, p = 0.342]. There was no correlation between cfcDNA and cfcDNA integrity with baseline PSA, Gleason score, total prostate volume, age and the number of presenting comorbidities.

Conclusion

The small sample size of the current study did not allow for the differentiation of PCa from BPH using cfcDNA concentration and integrity. There was also no correlation with cfcDNA concentration and integrity with the selected clinical and pathological variables.

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Acetylcholine signaling pathway: A novel target for lung cancer in smokers
Authors: Dasgupta Piyali , Lau Jamie K. , Brown Kathleen C. , Bow Eric , Robateau Zachary R. , Rollyson William D. , Stover Cody A. , Rimoldi John M. , Cutler Stephen , Hardman W. Elaine , Carpenter A. Betts , Chen Yi C.
Published: June 2021

The clinicopathological features of lung cancer in smokers is completely different from never‐smokers. Whereas lung cancer patients (who are never smokers) respond well to targeted therapies involving EGFR inhibitors and ALK kinase inhibitors, the response of these drugs in lung cancer patients who are smokers is poor. Nicotine is the addictive component of cigarette smoke. Nicotine (at concentrations present in the plasma of moderate smokers) accelerates the growth, angiogenesis and metastasis of lung cancers. The biological activity of nicotine is mediated by nicotinic acetylcholine receptors (nAChRs). The endogenous ligand for nAChRs in the lung is the neurotransmitter acetylcholine (ACh). ACh is known to be an autocrine growth factor for both SCLC and NSCLC. One of the ways that nicotine and cigarette smoke promote the growth of lung cancers is by promoting the production of ACh, which binds to its cognate receptor (on lung cancer cells) in an autocrine manner and induces the proliferation of lung cancer cells. The objective of the present study is to investigate the whether the ACh‐mitogenic signaling pathway are altered in human lung cancer. We show that levels three acetylcholine‐signaling proteins namely ChAT, VAChT and AChE are significantly altered in lung cancer occurring in active smokers. Small‐molecule regulators of these proteins suppress the growth of human lung cancer cells. Our studies will facilitate the identification of targeted therapies for the majority of lung cancer patients who are exposed to tobacco smoke.

Support or Funding Information

Funding for our study was supported by an NIH R15‐AREA Grant (2R15CA161491‐02) and the West Virginia IDeA Network of Biomedical Research Excellence (WV‐INBRE) grant GM103434 (PI: Dr. G. Rankin). Furthermore, this study was supported in part by an Institutional Development grant (IDeA) Grant number P20GM104932 from the National Institute of General Medical Sciences (NIGMS) and the Research Core B of COBRE, a component of the National Institutes of Health (NIH)

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Regulation of Trophoblast Invasion by Pyruvate Kinase Isozyme M2 (PKM2)
Authors: Mejia Juan F. , Hall Parker , Hirschi Kelsey M. , Reynolds Paul R. , Arroyo Juan A.
Published: June 2021

The Pyruvate kinase isozymes M2 (PKM2) protein is a metabolic enzyme that regulates the final step of glycolysis. This enzyme is present in highly proliferating cells such as those present in embryonic tissues, cancer and in the placental trophoblast. We recently studied PKM2 protein in the human placenta at term. Our results demonstrated upregulated placental PKM2 during preeclampsia (PE) where trophoblast invasion is decreased and hypoxia is present. Our objective was to determine the cytosolic and nuclear PKM2 expression pattern and determine PKM2 regulation of trophoblast cell invasion. Trophoblast cells were cultured in normoxic and hypoxic conditions and real time cell invasion was determined during activation (Fructose‐6‐bisphosphate) or inhibition (Shikonin) of PKM2. Treated cells were lysed for western blot analysis of PKM2. During normoxia, we observed: 1) 2‐fold increase (p<0.002) in trophoblast invasion when PKM2 was activated; 2) a 68‐fold reduction (p<0.002) in trophoblast invasion when PKM2 activation was inhibited; 3) a 1.4‐fold induction (p<0.03) of nuclear PKM2 in the trophoblast when PKM2 was activated; 4) a 1.6‐fold induction (p<0.04) of nuclear PKM2 after PKM2 inactivation; and 5) a 1.4‐fold decrease (p<0.03) in cytosolic PKM2 after PKM2 inactivation. During hypoxia we observed: 1) decreased cell invasion (~3.0‐fold; p<0.005) in treated cells; 2) decreased cytosolic PKM2 expression (~1.6‐fold; p<0.03) in treated cells; and 3) no significant differences in the expression of cytosolic PKM2. We conclude that PKM2 regulates trophoblast cell invasion depending of its cellular localization. Furthermore, our results suggest that PKM2 regulation is affected by hypoxia. These results suggest that PKM2 could be a mediator of trophoblast cell invasion and its abundance influences the development of complicated pregnancies like PE.

Support or Funding Information

This work was supported by a grant from the Flight Attendant's Medical Research Institute (FAMRI, PRR and JAA) and a BYU Mentoring Environment Grant (JAA and PRR).

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identification of AQP5‐regulating miRNAs and their role in breast cancer cell migration
Authors: PARK EUI‐JUNG , Jung Hyun Jun , Choi Hyo‐Jung , Kwon Tae‐Hwan
Published: June 2021

Background

We previously reported that AQP5 expression is critical for proliferation and migration of human breast cancer cells. However, the regulatory machinery for AQP5 expression in human breast cancer cells is unclear. The present study aimed to identify the AQP5‐regulating miRNAs, as one of regulatory mechanisms for AQP5 expression, and to examine roles of these miRNAs in human breast cancer cell migration.

Methods

1) To predict putative miRNAs interacting with AQP5 3′‐UTR of mRNA, TargetScan, Diana‐tools, miRDB and miRanda programs were exploited; 2) to identify AQP5‐regulating miRNAs, miRNA mimic was transfected in MCF‐7 and MDA‐MB‐231 cells and immunoblotting and luciferase reporter assay was performed; 3) to verify the effect of selected miRNAs in cell migration, migration assay and live cell imaging were performed; and 4) to identify putative target genes of the selected miRNAs, DIANA‐mirPath program was used.

Results

In silico analysis revealed that 19 miRNAs were identified as a putative regulator of AQP5 and 6 of 19 miRNAs were commonly identified from more than 3 different programs. Among them, selected miRNAs (miR‐185‐3p, miR‐1226‐3p, miR‐19a‐3p and miR‐19b‐3p mimic) were transfected in MCF‐7 and MDA‐MB‐231 cell. Immunoblotting demonstrated that miR‐1226‐3p, miR‐19a and miR‐19b significantly reduced AQP5 protein expression in MDA‐MB‐231 cells. In MDA‐MB‐231 cells, cell migration assay showed a significant decrease when cells were transfected with miR‐1226‐3p mimic (75 ± 6% of control), miR‐19a‐3p mimic (59 ± 6%) or miR‐19b‐3p mimic (39 ± 5%). The miRNA‐induced decreased cell migration was also observed by live cell imaging when cell migration was stimulated by LDL treatment. DIANA‐mirPath program showed that three miRNAs (miR‐1226‐3p, miR‐19a‐3p and miR‐19b‐3p) could be involved in 54 different signaling pathways including MAPK and PI3K/Akt signaling pathways.

Conclusion

miR‐1226‐3p, miR‐19a‐3p and miR‐19b‐3p regulate AQP5 protein expression and decrease cell migration of MDA‐MB‐231 cells. Further studies will be needed to examine their involvement in AQP5 regulation and cell migration in an animal model of breast cancer for demonstrating the miRNA‐mediated AQP5 regulation as a potential therapeutic target for breast cancer.

Support or Funding Information

This study was supported by the National Research Foundation of Korea Grants 2014R1A5A2009242 and 2016R1A2B4009365 funded by the Ministry of Science, ICT and Future Planning, Korea

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Influence of Cigarette Smoking on Myocardial Infarction Induced Renal Damage
Authors: Habeichi Nada , Abidi Emna , Alawasi Hiam , El‐Yazbi Ahmed , Zouein Fouad
Published: June 2021

Both, cigarette smoking (CS) and myocardial infarction (MI) remain a major public health concern and a leading cause of cardio‐renal morbidity in both males and females. In addition to nicotine, cigarette smoke contains thousands of carcinogenic and non‐carcinogenic chemicals that play a key role in reducing cell viability in renal proximal tubular epithelial cells through acute or chronic pathological mechanisms. In this study, the impact of CS on renal remodeling in both genders was investigated in a mouse model of MI using 2 groups: MI as a control, and MI + CS. Histological analysis of MI group showed alterations in the kidney structure including patchy glomerular retraction which was heightened by two‐fold post‐CS in both genders. As well, molecular analysis revealed that reactive oxygen species production was significantly accentuated in MI+CS compared to MI group similarly in both genders. These findings indicate that CS aggravates MI‐induced renal damage without an exhibition of gender based differences. In order to get more conclusive results, further molecular analysis is under way to study the role of inflammation, and fibrosis on the observed renal changes

Support or Funding Information

Supported by AUB Seed fund #10041

This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
GNAS‐PKA Oncosignaling Network in Colorectal Cancer
Authors: Steffen Dana J. , Amornphimoltham Panomwat , Valera Juan Luis Callejas , Taylor Susan , Hunter Tony , Tamayo Pablo , Gutkind J. Silvio
Published: June 2021

G protein coupled receptors (GPCRs) represent the largest class of drug target among currently approved small molecules. However, there is a dearth of oncology drugs targeting GPCRs. Analysis of cancer genomes reveals that G proteins often harbor activating mutations, inherently making their cognate receptor resistant to modulation. In particular, activated GNAS mutants, encoding a constitutively active, GTPase defective Gαs, can be identified in approximately 5% of all sequenced tumors, including 4% of colorectal cancers (CRCs). By stimulating adenylyl cyclases, GNAS activation leads to the intracellular accumulation of cAMP, a central second messenger, which in turn, activates protein kinase A (PKA). Similarly, 10% of CRC patients harbor gene amplification of GNAS, and another 45% have genetic alterations in PKA subunits, adenylyl cyclases, or phosphodiesterases (which inhibit cAMP accumulation). These findings highlight the importance of understanding oncogenic signaling downstream of GPCRs and the potential role of GNAS‐PKA signaling in CRC. The aim of our study is to define the contribution of the GNAS‐PKA pathway in CRCs and explore the cancer‐associated GNAS‐PKA signaling network. We hypothesize that increased PKA activity downstream of GNAS mutations, or in response to persistent GPCR activation, plays a central role in the growth and progression of CRCs. Preliminary results demonstrate that PKA inhibition significantly reduces tumor burden in xenograft mouse models. Interestingly, activation of GNAS alone in both transgenic and chemical carcinogen mouse models is insufficient to induce tumorigenesis. However, upon the co‐activation of GNAS and RAS, mice show rapid development of squamous cell carcinoma. This finding is strongly supported by bioinformatic analysis revealing a co‐occurrence of GNAS and KRAS mutations clinically. We are currently profiling the GNAS‐PKA pathway by large scale transcriptomic and proteomic experiments to understand signaling circuitries and mechanisms of tumorigenesis. Emerging results from our studies will be presented.

Support or Funding Information

Dana Steffen was supported by the National Science Foundation Graduate Research Fellowship Program (NSF GRFP) and the UCSD Graduate Training Program in Cellular and Molecular Pharmacology through an institutional training grant from the National Institute of General Medical Sciences (T32 GM007752). This study is supported in part by the San Diego NCI Cancer Centers Council (C3) Collaborative Translational Cancer Research Pilot 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.