bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Background

Targeted 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.

Methods

Unlike 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 & Discussion

Separation 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 Information

This 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 Information

This 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 Information

This 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 Information

This 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.
Involvement of Endothelial Small GTPase RhoA in Angiogenesis In Vitro and In Vivo
Authors: Zahra Fatema Tuz , Sajib Md Sanaullah , Ichiyama Yusuke , Akwii Racheal , Tullar Paul , Kubota Yoshiaki , Mikelis Constantinos M
Published: June 2021

Angiogenesis, the formation of new blood vessels, is a highly orchestrated process requiring a well‐regulated balance between pro‐angiogenic and anti‐angiogenic factors and corresponding signaling networks. It is an essential process during development and in specific adult conditions such as wound healing and ovulation. Imbalanced angiogenesis is a characteristic of several diseases such as cancer, inflammation, rheumatoid arthritis, osteoarthritis and age‐related macular degeneration. The Rho family of small GTPases plays a central role in a variety of cellular processes involving cytoskeletal rearrangement, cell movement, microtubule dynamics, signal transduction, gene expression which are necessary for vascular development and angiogenesis. Among the small GTPases of the Rho family, RhoA, Rac1 and Cdc42 are the best characterized. Although the role of endothelial Rac1 and Cdc42 in embryonic vascular development and retinal angiogenesis has been studied recently, the role of endothelial RhoA is yet to be explored. In this study, we aim to identify the involvement of endothelial RhoA in angiogenesis in vitro and in vivo. In vitro, RhoA seems to be involved in endothelial cell proliferation, cell migration, invasion and sprouting triggered by important angiogenesis inducers, such as Vascular Endothelial Growth Factor (VEGF) and Sphingosine‐1 Phosphate (S1P). To study the involvement of endothelial RhoA in vascular development in vivo, we generated endothelial‐specific RhoA deficiency through the Tie2‐Cre and Cdh5‐CreERT2 promoters. In vivo deletion of RhoA in endothelial cells during embryogenesis leads to decreased survival of endothelial RhoA‐deficient mice. However, the few mutant survivors do not present morphological or behavioral abnormalities. In line with that, inducible RhoA deficiency in the retinal vessels in different developmental days did not affect the radial growth, the number of filopodia per area and the area with deep vascular plexus. Ongoing experiments aim to clarify the compensation mechanisms of RhoA deficiency in physiological angiogenesis in vivo versus in vitro models, providing a better understanding of the role of endothelial small GTPase RhoA in normal physiology and human diseases.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Study the function of GTPase related genes wasf1 and ect2 in zebrafish vascular development
Authors: Chiu Yan‐Hui , Wu Chang‐Yi
Published: June 2021

Vascular development is important in vertebrates and regulated by complex signaling pathways. In the process of forming vessels are mainly divided into vasculogenesis and angiogenesis. Zebrafish is a powerful vertebrate model organism to study vascular development. Our previous study in zebrafish identified the transcription factors nr2f1b/islet2 required for blood vessels, mainly focus on the patterning of intersegmental vessels (ISV) and caudal vein plexus (CVP). The further microarray results showed that the expression of several GTPase related genes was regulated by nr2f1b/islet2, and we are interested in how those GTPase related genes contribute to vascular patterning in zebrafish.

In this study, we reported two novel GTPase related genes wasf1 and ect2 that investigated their significance for the vascular development. In‐situ hybridization show that ect2 and wasf1 mRNA is expressed in developing vessels. Knockdown of ect2 and wasf1 by morpholino microinjection cause the defect in blood vessel formation. TUNEL assay and AO staining data show that vascular defects do not caused by cell death, but likely due to the impairment of proliferation and/or migration. Consistent with the defect in vascularture, loss of ect2 or wasf1 reduced the expression of the vascular markers. We further perform Western blotting to examine the relations between wasf1/ ect2 and major angiogentic VEGF/ BMP signals. We showed that GTPase related proteins wasf1 and ect2 play an important roles of vascular development mediated by VEGF/ BMP signalings in zebrafish.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Changes in the expression of ERK, JNK and p38 in small intestine related to prolonged intake of commercial sweeteners and their relationship with periodontal state in BALB/c mice
Authors: Sánchez Carlos Manuel Abascal Sherwell , Estrada José Antonio , Contreras Irazú
Published: June 2021

Introduction

Obesity is a public health problem associated with increased consumption of high‐calorie diets, which predisposes the development of non‐communicable diseases such as obesity and diabetes mellitus type II. A common strategy for their prevention is the addition of non‐nutritive sweeteners in diet. The need for evidence on the molecular interactions between sweeteners and mucous membranes after chronic consumption of these compounds has generated hypotheses that establish a relationship between sucralose intake and high prevalence of inflammatory bowel disease in developed countries.

Objectives

To establish a profile of protein expression in the intestinal epithelium and cellular infiltration in periodontal tissue after chronic consumption of non‐nutritive sweeteners in BALB/c mice.

Material and Methods

8‐week‐old BALB/c mice were supplemented with non‐nutritive sweeteners (sucralose and steviol glycosides) for 6 weeks. Expression of total and phosphorylated forms of ERK, JNK and p38 in intestinal tissue were evaluated by western‐blot. Jaws were analyzed to determine histopathological changes in oral mucose membranes related to chronic sweetener consumption

Results

Densitometry analysis of protein bands showed a increased expression of ERK, JNK and p38 phosphorylated proteins in mice supplemented with sucralose and steviol glycosides, compared to control groups; however, these differences were not statistically significant. Similarly, histopathological study of the oral cavity showed increased cellularity, vascularity and cell clustering in gingivae in the same groups.

Conclusions

The present study provides evidence that frequent consumption of non‐nutritive sweeteners may generate pro‐inflammatory changes in oral cavity and intestinal mucosae, based on higher expression of phosphorylated ERK, JNK and p38 proteins and increased cellular recruitment at the periodontal level.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterization of KPC‐2 Backbone Dynamics Upon Avibactam Binding via NMR Spectroscopic Methods
Authors: Shurina Ben , VanPelt Jamie , Ramelot Theresa , Page Richard
Published: June 2021

Antibiotic resistance is a continual threat in the clinic. One of the resistance modes bacteria employ is the expression of the beta‐lactamases that target most of the clinically relevant antibiotics, such as penicillin. The enzyme hydrolyzes the β‐lactam bond, which renders the antibiotic ineffective. Targeting β‐lactamases with small molecule inhibitors can restore efficacy of current generation antibiotics. Klebsiella pneumoniae carbapenemase (KPC) presents a global threat to public health as it confers multidrug resistance, including resistance to last resort antibiotics. Avibactam is the only FDA approved inhibitor for KPC are on the market, however some variants of KPC are showing increased resistance to avibactam. Futhermore, avibactam can be slowly degraded by KPC‐2 through a de‐sulfonation mechanism. A conserved omega‐loop near the active site of KPC‐2 has been implicated in substrate binding and is of particular interest as a target for future inhibitors. We report the first backbone dynamic characterization of a Class A carbapenemase in the presence and absence of an inhibitor. Backbone dynamic parameters for KPC‐2 obtained through the use of 3D HNCO based T1, T2, and heteronuclear NOE relaxation experiments. Chemical shift perturbations were obtained by comparing avibactam bound and apo states. H/D exchange experiments were also conducted to examine relative rates of exchange in avibactam bound and apo KPC‐2 in order to estimate slow timescale dynamics. The binding of avibactam caused an increase in R1 relaxation rates, suggesting avibactam can increase the dynamic flexibility of KPC‐2. The changes in the R2 relaxation rates suggest that the binding of avibactam might make the Ω‐loop slightly more rigid. These results could be used as a benchmark in measuring interactions with the Ω‐loop, interactions with KPC‐2, or interactions with other β‐lactamases.

Support or Funding Information

NIH R35 GM128595

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Gut dysbiosis with decreased Akkermansia muciniphila and differential expression of tumor‐associated genes contribute to a tumor‐preventive microenvironment in intestinal epithelial Pten‐deficient mice
Authors: Rhee Sang Hoon , Howe Cody , Kim Su Jin , Mitchell Jonathon , Im Eunok
Published: June 2021

Phosphatase and tensin homolog (Pten) antagonizes PI3K‐Akt signaling; therefore, Pten impairment causes tumorigenesis. However, the correlation between Pten deficiency and colon cancer has remained elusive due to numerous opposite observations. To study this correlation, we examined whether Pten deficiency in intestinal epithelial cells (IECs) induces tumorigenesis.

With mucosal biopsies of human colon cancer and normal colon, Pten mRNA was evaluated by quantitative PCR. Using IEC‐specific Pten knockout mice (PtenΔIEC/ΔIEC), we examined the mitotic activity of IECs; and PtenΔIEC/ΔIEC; Apcmin/+ mice were generated by combining PtenΔIEC/ΔIEC with Apcmin/+ mice. Tumor‐associated gene was evaluated by micro‐array analysis. Fecal microbiome was analyzed through 16S rRNA gene sequencing.

We found that Pten mRNA level was reduced in human colon cancer relative to normal tissues. Augmented chromatids, increased Ki‐67 and PCNA expression, and enhanced Akt activation were identified in IECs of PtenΔIEC/ΔIEC mice compared to Pten+/+ littermate. Combining PtenΔIEC/ΔIEC with Apcmin/+ condition caused rapid and aggressive intestinal tumorigenesis. However, PtenΔIEC/ΔIEC mice did not develop any tumors. While maintaining the tumor‐driving potential, these data indicated that IEC‐Pten deficiency alone did not induce tumorigenesis in mice. Furthermore, the expression of tumor‐promoting and tumor‐suppressing genes was decreased and increased, respectively, in the intestine of PtenΔIEC/ΔIEC mice compared to controls. The abundance of Akkermansia muciniphila, capable of inducing chronic intestinal inflammation, was diminished in PtenΔIEC/ΔIEC mice compared to controls.

These findings suggested that altered tumor‐associated gene expression and changed gut microbiota shape a tumor‐preventive microenvironment to counteract the tumor‐driving potential, leading to the tumor prevention in PtenΔIEC/ΔIEC mice.

Support or Funding Information

This research was supported by a grant from Oakland University and the National Institutes of Health (DK079015, S.H.R).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Fungistatic Effects of 1,4‐dimethylnaphthalene on Pythium ultimum and Aspergillus niger
Authors: Stefanick Veronica , Dobson Kara , Campbell Michael
Published: June 2021

The compound 1,4‐dimethylnaphthalene (DMN) was originally isolated from dormant potatoes and is currently being used as a commercial sprout inhibitor. In order to determine how the microbiome of potatoes is affected by the compound, two fungal species were grown and quantified under control and DMN conditions: Pythium ultimum and Aspergillus niger. These two fungal species are commonly found on the surface of potatoes and can result, respectively, in Pythium leak and black rot on onions. Fungal cultures were obtained from ATCC (P. ultimum: 58811, A. niger: 16888) and grown on potato dextrose agar (PDA). Mycelial cultures were grown in 9.5 Liter BBL GasPak chambers, exposed to either mineral oil (control) or DMN for two days. The mycelial mats exhibited reduced growth under DMN conditions in comparison to controls. DMN treatment of mature A. niger cultures also prevented sporulation. This indicates that DMN displays fungistatic properties but does not function as a fungicide towards P. ultimum and A. niger. In order to determine how DMN affects the gene expression of these fungi, RNA sequences will be collected and gene expression will be observed between control and DMN treatments.

Support or Funding Information

Supported in part by a grant from the 1,4‐Group of Meridian, ID and the Behrend College Undergraduate Research Fund

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Understanding Commensal Bacteria Influence Host Heme Metabolism
Authors: DuBois Jennifer Lynn , Coe Genevieve , Celis Arianna I. , Walk Seth T.
Published: June 2021

Heme serves as a critical source of iron for human pathogens that can access O2 and the abundant heme stores in red blood cells. The lack of a well‐defined cellular heme transporter in the intestinal lumen cells suggests a possible role for microbes in metabolizing the tetrapyrrole on behalf of themselves and the host. A fully anaerobic pathway for heme breakdown (chuA chuW chuS) was recently discovered in E. coli, which could render heme iron bioavailable to the host, or minimize the toxic or inflammatory effects of heme for the microbiome and host, respectively. We tested these hypotheses using a chu‐containing commensal E. coli strain an isogenic DchuW knock out, and mice with defined microbiota. We provide evidence that the microbiome may serve as a missing link in rendering heme iron bioavailable to animal hosts.

Support or Funding Information

This work was supported by the National Institutes of Health, grant R21DK114607.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Comparative Study of Equine Gut Microbiomes Using 16S and 18S rRNA Sequencing
Authors: Jorgenson Jillian K. , Bennett Jennifer A. , Burk Steffanie V.
Published: June 2021

The gut microbiome of domestic horses (Equus ferus caballus) can be broken down into three primary divisions: (1) bacterial composition, (2) fungal composition, and (3) eukaryotic parasite communities. While each has been the subject of various scientific explorations, in this study we analyze the potential interconnectedness of these three categories. To determine the extent to which each component of the gut microbiome influences the other, we extracted and amplified DNA from equine fecal samples and utilized a combination of 16S and 18S rRNA Illumina Sequencing to illustrate the bacterial and fungal compositions taxonomically with the assistance of QIIME2 software. Meanwhile, fecal egg counts for each sample were completed using the modified Stoll method to estimate the parasitic load for each horse. The identification of relationships between bacterial communities, fungal communities, and parasite load could have clinical applications to future studies of equine health. Additionally, comparisons could be made between domestic horses and exotic hoofstock as a means to improve our understanding of gut microbiome health in lesser studied species.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Molecular mechanism of cereblon‐dependent down regulation of AMP‐activated protein kinase
Authors: Yang Seung‐Joo , Jeon Seung Je , Han Hyemin , Park Chul‐Seung
Published: June 2021

Cereblon (CRBN), initially identified as a target protein of human mental retardation, is a substrate receptor of cullin‐ring E3 ubiquitin ligase (CRL) and a primary target of thalidomide‐induced teratogenicity. We previously reported that CRBN can negatively regulate the functional activity of AMP‐activated protein kinase (AMPK) by binding to the a subunit of the AMPK complex. We also noticed that the exogenous expression of CRBN reduced the content of g subunit in AMPK complex. However, the molecular mechanism for the CRBN‐dependent reduction of g subunit from AMPK complex was still unclear. Thus, we investigated the molecular mechanism of CRBN‐dependent inhibition of AMPK. We noticed that the amount of g subunit in AMPK complex was increased in Crbn‐knockout (KO) MEFs and the g subunit was degraded more rapidly in wild‐type MEFs than in Crbn‐KO MEFs. After treating MG132, a proteasome inhibitor, the g subunit is accumulated more rapidly in wild‐type MEFs compared to Crbn‐KO MEFs. Moreover, ectopic expression of CRBN promoted the degradation of g subunit in Crbn‐knockout MEFs only in the presence of a subunit. We further found that AMPK g subunit was ubiqutinated by CRBN and ubiquitination persisted in the presence of IMiDs. These results strongly suggest that CRBN negatively regulates the AMPK activity by recruiting AMPK complex to CRL and degrading its g subunit.

Support or Funding Information

This work was supported by GIST Research Institute (GRI) grant funded by the GIST in 2019

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Transcriptional regulation of the yeast TGL3‐encoded triacylglycerol lipase
Authors: Lu Chieh , Ye Bo‐Hong , Su Wen‐Min
Published: June 2021

The yeast TGL3‐encoded triacylglycerol (TAG) lipase catalyzes the hydrolytic reaction of TAG yielding fatty acid and diacylglycerol (DAG). The reaction products DAG and fatty acid not only can be used as building blocks for the synthesis of phospholipids, but also serve as lipid signaling molecules. In addition, fatty acid can undergo β‐oxidation to generate ATP. During growth resumption, the stored TAG in lipid droplet is rapidly hydrolyzed by Tgl3 to provide precursors for membrane phospholipids and to generate lipid signaling molecules that are essential for normal cell growth and cell cycle progression at the G1/S phase. However, excessed fatty acid and DAG are also detrimental to cell growth as they exhibit detergent‐like properties that can disrupt membrane structure and cause defect in cell integrity. Thus, Tgl3 lipase activity must be tightly controlled and mechanisms of regulation need to be elucidated. In this study, using β‐galactosidase reporter assay, we found a sharp induction of TGL3 expression after cells were transferred into fresh medium from the stationary phase. Maximum expression was observed at first hour where the β‐galactosidase activity was 62% greater than the activity at the stationary phase. Moreover, the TGL3 promoter was shown to interact with the purified Reb1 transcriptional factor in the electrophoretic mobility shift assay. A Reb1 consensus recognition sequence (GGGTAA, −362 to −357) in TGL3 promoter is responsible for its specific binding to the Reb1 protein. The effects of Reb1‐mediated TGL3 regulation on the function of Tgl3 as well as lipid metabolism are currently under investigation using the TGL3 Reb1‐binding site mutations.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comparative Analysis of Healthcare Providers and Patient Perspectives on Natural Remedies as Alternative Medicine in Two Communities in Peru
Authors: Simental Brianna N , Loaiza Anaiz
Published: June 2021

More often than not there is a disconnect between healthcare providers (HCPs) and their patients regarding the use of natural remedies as an alternative medicine. Lack of knowledge and communication between healthcare providers and patients can change the efficacy of the medication and can be a safety hazard for the patient. Examining the relationship between healthcare providers' view on the use of natural remedies is important, especially in developing countries like Peru. Due to unequal access to healthcare people may rely more heavily on alternative medicine. Healthcare providers and patients will have a different perspective on natural remedy use and their effectiveness. This miscommunication could affect patient care and treatment due to uninformed guidance and lack of knowledge of natural remedies. To analyze the perspectives of HCPs and patients about natural remedy use, we designed a cross‐sectional study that was conducted over two summers, among healthcare providers and women in two communities in Peru. Two individual surveys were distributed and used for data collection. Our preliminary results suggest that there is a disconnect between the percentage of patients that use natural remedies reported by healthcare providers and the percentage of women in the community that reported their use of natural remedies. There was also a divide between the perspective of HCP's on whether their patients feel comfortable addressing their health concerns and the perspectives of the patients who felt comfortable addressing their health concerns but did not trust their HCP. Further analysis is necessary to examine other barriers that prevent open communication between healthcare providers and their patients.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Crystallization of SARS Coronavirus 3CL Protease to Identify Inhibitor Targets
Authors: Fernandes Samantha Zinnia , Anson Brandon , Mesecar Andrew
Published: June 2021

The SARS coronavirus has been identified as the pathogen responsible for the 2003 outbreak of severe acute respiratory syndrome (SARS). This highly contagious virus, which most likely originated in Southern China, has since been found in over 32 countries, affecting about 8,500 people, and killed over 900 patients. Due to SARS ease of transmission through aerosols and the virus's high mortality rate (~10%), its reemergence is recognized as a looming threat to global health.

However, neither a vaccine nor effective treatment exists to combat the illness in the event of another outbreak. The virus's 3 Chymotrypsin‐like main protease, is a major target for inhibitor studies, as it is crucial to viral replication through its proteolytic processing of polyprotein 1ab (pp1ab) at 11 different cleavage sites.

This project worked to optimize the crystallization conditions for the SARS 3CL protease for use in X‐ray fragment based screening to identify possible inhibitors. The results of this step in the drug discovery study for the SARS‐CoV can be used as a basis for pioneering new scaffolds for inhibitor studies in other coronaviruses.

Support or Funding Information

Funding from NSF DBI “REU Site: Molecular and Biochemical Analysis of Proteins” to Purdue University, Purdue College of Biochemistry, and the Purdue College of Agriculture

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dual Expression of Neuropilin‐2 in Tumor and Stroma in Oral Dysplasia and Oral Squamous Cell Carcinoma
Authors: Almazyad Asma , Shahrabi‐Farahani Shokoufeh , Gartung Allison , Panigraphy Dipak , Bielenberg Diane R.
Published: June 2021

The majority of oral malignancies (90%) are squamous cell carcinoma (OSCC), and two‐thirds of OSCC patients present at diagnosis with disseminated disease. OSCC can develop from pre‐cancerous lesions known as oral dysplasia. Early detection of these lesions leads to better management and outcome. We investigated the role of the Neuropilin‐2 (NRP2) cell surface receptor in oral dysplasia and OSCC. NRP2 is regarded as an important mediator of angiogenesis and lymphangiogenesis, but its role in epithelial cells is less understood. NRP2 can bind VEGFA in a complex with VEGFR2 or VEGFC in a complex with VEGFR3 to stimulate the sprouting of blood or lymphatic vessels during development but its role in adult tumor‐associated neovascularization has not been clearly identified. Interestingly, NRP2 also binds Semaphorin‐3F (SEMA3F) in a competitive fashion with VEGFA/C, and SEMA3F is reported to inhibit lymphangiogenesis. Here, we hypothesize that oral dysplasia and OSCC express NRP2 in transformed epithelial cells as well as in tumor‐associated lymphatic endothelium, and that this expression pattern renders them amenable to SEMA3F therapy. Our results indicate that adult human and mice normal tongue do not express NRP2 in the epithelium or in quiescent lymphatic vessels. However, NRP2 is significantly upregulated in moderate or severe oral dysplasia and OSCC in epithelial cells and lesion‐associated lymphatic vessels. Two different human OSCC cell lines injected orthotopically in immunocompromised mice showed high NRP2 expression. Immunocytostaining also confirmed NRP2 protein expression in two different cell lines isolated from 4NQO‐induced OSCC in mice. Additionally, a panel of different human and mice OSCC cell lines showed high expression of NRP2 protein using western blot. Lastly, SEMA3F, in a dose‐dependent manner, inhibited migration of Nrp2‐expressing mouse OSCC cells in vitro. In summary, the upregulation of NRP2 in early stages of cancer progression in OSCC may point to important signaling pathways not previously realized in disease. Targeting NRP2 with neutralizing antibodies or with its inhibitory SEMA3F ligand may a promising new paradigm to treat or prevent progression and metastasis of OSCC.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
RAGE targeting ameliorates responses to antenatal SHS exposure
Authors: Hirschi K M , Tsai K YF , Clark C , Hutchinson T , Hall P , Arroyo J A , Reynolds P R
Published: June 2021

Secondhand tobacco smoke (SHS) exposure during pregnancy is associated with increased health complications to both mother and fetus. Exposure can lead to anomalous organ development, increased blood pressure, and decreased metabolism. Receptors for advanced glycation end‐products (RAGE) are pro‐inflammatory transmembrane receptors increased by SHS. Inhibition of RAGE signaling by semisynthetic glycosaminoglycan ethers (SAGEs) has been shown to reduce inflammation in the adult and improve fetal birthweight. We tested the hypothesis that in vivo inhibition of RAGE by SAGEs during antenatal SHS exposure improves postnatal health of the offspring. Pregnant Balb/C mice were exposed to SHS alone or SHS + SAGEs from embryonic day (E) 14 to E17. RAGE expression and general measures of development were assessed in the offspring at four and twelve weeks of age. Statistical analysis was completed using nonparametric Mann‐Whitney testing. Antenatal SHS resulted in the following at four weeks of age: 1) increased body weight (p =0.0118) which was reduced to basal levels when treated with SAGEs; 2) decreased heart (p=0.0017) and kidney (p=0.0001) weights and improvement with concurrent SAGEs; 3) increased neonatal blood pressure and further increases with SAGEs; 4) increased expression of RAGE in kidney (p=0.0090), heart (p=.044), and lung (p=0.0022) with significant recovery with SAGEs; 5) mild reduction in alveolar surface area via MLI assessment and airspace normalization with SAGE treatment. No significant differences were apparent for any metric at 12 weeks of age. We conclude that inhibition of RAGE protects against fetal health complications following antenatal SHS exposure. Our results further suggest that there is a correlation between RAGE expression and a plausible role for RAGE signaling in key organs following maternal SHS exposure. These studies provide insight into tobacco‐mediated heath compromise and implicate possible avenues worth pursuing further in the alleviation of symptoms of antenatal SHS exposure.

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 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
IGF1 is Developmentally Regulated in the Neonatal Intestine and Protects Neonatal Mice against Necrotizing Enterocolitis by Preserving the VEGF/VEGFR2 Signaling Pathway in an Animal Model
Authors: Yan Xiaocai , Managlia Elizabeth , De Plaen Isabelle
Published: June 2021

Necrotizing enterocolitis (NEC) is a common gastrointestinal disease affecting premature infants and associated with high morbidity and mortality. Its pathogenesis remains poorly understood. Decreased levels of serum IGF1 has been shown to be associated with a higher incidence of NEC in premature infants. We previously found that neonatal pups are most susceptible to NEC in the first few days of life. Here we hypothesize that IGF‐1 levels are low during the first days of life, and that exogenous IGF1 is protective against NEC via promoting intestinal VEGF/VEGFR2 signaling. To test our hypotheses, we first assessed the developmental expression of IGF1 in the serum and the intestinal tissues in neonatal mice during the first 3 weeks of life, and determined whether exposure to a NEC protocol affects serum and intestinal IGF1 levels. Secondly, we examined whether exogenous IGF1 protects against NEC and affects intestinal VEGF production both in vivo and in vitro. We found that: Serum IGF1 level is low during the first 2 days of life, and gradually increases over 3 weeks. In contrast, IGF1 expression in the intestinal tissue is low at birth, peaks between 3 days and 2 weeks of age, before decreasing to low level by 3 weeks. In pups exposed to a NEC protocol compared to DF controls, serum IGF1concentration was significantly decreased at 12 hours and 24 hours (by 35%, p<0.05; and 59%, p<0.0001 respectively ). Intestinal tissue IGF1 expression decreased as early as 6 hours into NEC, which occurred before the decrease in serum IGF1. Exogenous IGF1 treatment decreased mortality in pups exposed to NEC protocol (41% vs. 68%, p<0.05), decreased the incidence of severe NEC (histological score ≥ 2) (χ2=4.03, p<0.05) and increased intestinal VEGF/VEGFR2 protein expression. Neonatal intestinal endothelial cells treated with IGF1 in vitro had increased VEGF mRNA transcription and protein expression (p<0.05 for both). We conclude that the beneficial effect of IGF1 on experimental NEC may be due to protection of small intestinal microvasculature development through preserving VEGF/VEGFR2 signaling pathway.

Support or Funding Information

Shire

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Effect of Pin1/DAPK1 inhibition on IKK‐mediated cell death in ischemic stroke mice model
Authors: Kang Hyunbon , Hong Yonggeun
Published: June 2021

Peptidyl prolyl isomerase (Pin1) and Death‐associated protein kinase 1 (DAPK1) have been investigated as a key factor in cell death of cerebral ischemia. But, Pin1 and DAPK1 signaling pathway have not been clearly elucidated in case of ischemia/reperfusion. Cerebral ischemia/reperfusion injury is known to be induced by imbalanced inflammation and oxidative stress. Also, recent articles identified the Ikappa B kinase (IKK), which is a main mediator of inflammation signal‐induced neuronal cell death in ischemic stroke. Here, we examined the close relationship among DAPK1, Pin1 and IKK in ischemia/reperfusion (I/R). To investigate the effect of Pin1/DAPK1/IKK on I/R neuronal cell death, MCAo (Middle cerebral artery occlusion) surgery was performed to experimental animals, and they were injected with either Pin1 inhibitor or DAPK1 inhibitor. The experimental animals were randomly divided into six groups; vehicle‐day1, Pin1 inhibitor‐day1, DAPK1 inhibitor‐day1, vehicle‐day3, Pin1 inhibitor‐day3, DAPK1 inhibitor‐day3. No significant changes were found in the body weight and NDSS scores among groups. However, the inhibition groups exhibited decrease in infarct size and neuronal cell damage after cerebral ischemia/reperfusion. In addition, IKK expression was decreased in inhibition groups. Taken together, regulation of Pin1/DAPK1/IKK interaction would exert neuroprotective effect against I/R injury.

Support or Funding Information

NRF‐2013R1A2A2A01067169, NRF‐2017R1A2A2A01067169, KRIBB‐KGM4611714

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.