bluuebunny/crossref_metadata_2025 huggingface dataset:
Development of oligonucleotide‐based contrast agent with higher relaxivity
Authors: Heidarian Mohammad , Kadeer Elshad , Halim Marlin
Published: June 2021

Gadolinium‐based contrast agents (GBCAs) is at present, the most widely used class of contrast agents in clinical magnetic resonance imaging (MRI). Although generally deemed to be safe, in some patients, accumulation of toxic gadolinium(III), Gd3+ ions leading to the development of nephrogenic systemic fibrosis may occur.

Our project aims to develop a GBCA with higher efficacy that can potentially result in lower toxicity. Most current clinical GBCAs are complexes composed of Gd3+ ion chelated in an organic ligand molecule with an overall molecular weight of ~0.5 kDa. Since the efficacy (as measured by the parameter called relaxivity) of the agent is dependent on its size, we herein proposed a GBCA with significantly increased size and weight through the use of 30‐ to 40‐base long oligonucleotides as a ligand for the Gd3+ complex, in lieu of the conventional small organic molecule. The chelation of the complex will result in an agent of ~10 kDa, which will exhibit markedly different relaxivity from that of the significantly smaller GBCAs. Using an in vitro selection procedure, an oligonucleotide strand with a high binding affinity to the complex is isolated. The relaxivity of this supramolecular construct is subsequently measured on a nuclear magnetic resonance spectrometer. Overall, this design may allow for a reduced dosage of the GBCAs which will lower the chances of Gd3+ accumulation in tissues.

Support or Funding Information

California State University, East Bay Office of Research and Sponsored Programs Faculty Support Grant

California State University, East Bay Center for Student Research Scholarship

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NGS‐Integrator: A Tool for Combining Information from Multiple Genome‐Wide NGS Data Tracks Using Minimum Bayes Factors
Authors: Jung Hyun Jun , Wen Bronte , Chen Lihe , Saeed Fahad , Knepper Mark A
Published: June 2021

Genome‐wide studies that generate multiple high‐throughput next‐generation sequencing (NGS) datasets to identify genomic DNA elements for gene transcription require data integration methods to minimize complexity and false‐positive findings. This can involve integration of multiple genome‐wide data generated from same type or different types of high‐throughput NGS techniques. Since several strategies to integrate multiple genome‐wide NGS datasets based on peak calling tools have been developed, these conventional methods are typically applied to individual replicates and not the aggregate data from multiple data.

NGS‐integrator, a Java‐based tool, integrates genome‐wide NGS datasets into a single data track for a genome browser based on minimum Bayes Factor (MBF) calculated from the signal‐to‐noise ratio. NGS‐integrator consists of two elements, “Calculator” and “Integrator”. The “Calculator” element calculates the complement of MBF at each genomic position, estimating the median of the noise values across a sliding window straddling the position at which signal value is being calculated. This calculation assumes that the features being detected are relatively sparsely represented along the genome, allowing the median to be representative of the true noise level. The “Integrator” element calculates the joint probability between the multiple NGS data tracks based on the complement of MBF calculated with ‘Calculator’. We used NGS‐integrator to integrate data from three types of data generated in our laboratory in cultured mpkCCD kidney cells, viz. ChIP‐Seq for the transcription factor ELF1, ATAC‐Seq, and ChIP‐Seq for the histone modification H3K27Ac and RNA polymerase II.

Support or Funding Information

The work was supported by the Division of Intramural Research, National Heart, Lung, and Blood Institute projects ZIA‐HL001285 (to MAK) and ZIA‐HL006129 (to MAK). BW was supported by the NHLBI Summer Internship Program (June–August 2018).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Reconstruction of biological subsystems using bottom‐up genetics
Authors: Aoki Wataru , MOTONE Keisuke , UEDA Mitsuyoshi
Published: June 2021

Background

One of the purposes of life science researches is to elucidate the relationships between genotypes and phenotypes. However, it is often difficult to fill in information gaps in a target biological subsystem, and tremendous amounts of time and effort are required to reconstruct a subsystem's functions. Hence, we have proposed “bottom‐up genetics” as a novel methodology that enables high‐throughput determination of sets of genes required for the functioning of any biological subsystem [1, 2].

Methods

Bottom‐up genetics utilizes artificial cells, including a randomly introduced whole gene library, PURE system (reconstituted in vitro transcription and translation), and a reporter that fluoresces only when a particular function of a target biological subsystem is active. The set of genes necessary for the target biological subsystem can be identified by isolating fluorescent cells and multiplex NGS analysis of genes contained in these cells. The importance of this methodology is that screening for the set of genes involved in a target biological subsystem and reconstituting the entire subsystem can be done simultaneously.

Results & Discussion

We tried to reconstruct β‐galactoside hydrolysis subsystem of Escherichia coli using bottom‐up genetics. We prepared an artificial cell library that contained the PURE system solution, 100 μM CMFDG (fluorescence reporter), and 5 nM E. coli ORF library. We isolated fluorescent and non‐fluorescent cells by FACS, and the genes included in them were determined by single‐cell NGS analysis. As a result, we succeeded in determining necessary and sufficient gene for β‐galactoside hydrolysis. This result indicated successful high‐throughput reconstruction of the β‐galactoside hydrolysis subsystem from the E. coli ORF library by bottom‐up genetics.

Support or Funding Information

This work was supported by JSPS KAKENHI (Grant No. 26830139).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mRNA and miRNA Expression Analysis in Multiple Brain Regions Following Soman Exposure in Rats
Authors: Gautam Aarti , Naidu Lalith , Getnet Derese , Kumar Raina , Dimitrov George , Sowe Bintu , Rossetti Franco , Meyerhoff James L , Hammamieh Rasha , Lumley Lucille A , Jett Marti
Published: June 2021

Acute and chronic effects of exposure to the potent organophosphate acetylcholinesterase inhibitor soman are an increasing concern for military and civilian populations. High dose exposure results in increased levels of acetylcholine and cholinergic crisis. This cholinergic crisis induces convulsions and seizures and can result in death if left untreated. Some individuals do not experience seizures on high dose exposure although they experience long‐term neurological injury. Understanding the downstream effects of soman at the molecular level is vital to developing a methods for prognosis and treatment and for explaining inter‐individual differences in response. We performed an integrated analysis of miRNA and mRNA expression in brain regions associated with soman exposure and resultant seizure activity in rats. Adult male Sprague‐Dawley rats were exposed subcutaneously to soman. Samples from seizing and non‐seizing animals were collected from the heart, kidney, liver, lungs, spleen, and brain, including the amygdala, hippocampus, hypothalamus, piriform, medial prefrontal cortex, parietal cortex, and thalamus at 72 hrs and 90 days following exposure. Initial data from the piriform cortex demonstrates significant differences in expression patterns between animals that seized and those that did not. The observed miRNA expression differences across brain tissue types indicate the hypothalamus has a distinct response from other regions. Functional annotation analysis of miRNA‐mRNA interaction pairs identified many that are likely to be involved in the seizing response.

Ongoing work will fully characterize the gene networks and pathways altered by soman exposure and associated with soman‐induced seizure responses across different brain regions and should lead to useful assays for diagnosis, prognosis, or exposure surveillance.

Disclaimers:

Research was conducted in compliance with the Animal Welfare Act, and all other Federal requirements. The views expressed are those of the authors and do not constitute endorsement by the U.S. Army.

Support or Funding Information

Support was provided by interagency agreements between BARDA, the Geneva Foundation (ARO agreement no. W911NF‐13‐1‐0376), and the U.S. Army Medical Research Institute of Chemical Defense (USAMRICD) as well as a memorandum of agreement between USAMRICD and the U.S. Army Center of Environmental Health (USACEHR).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Delineating the Neuro‐Immune Regulatory Network of Acute Kidney Injury by Systems Biology Approach
Authors: Gupta Aprajita , Puri Sanjeev , Puri Veena
Published: June 2021

Acute Kidney injury (AKI) is one of the major health concerns with high rate of morbidity and mortality across the globe. AKI is a heterogeneous clinical syndrome which is associated with inflammatory cascade and modulation in immune circuit which gradually lead to stress, apoptosis, hormonal change, pain and intonation in the nervous system. This is mediated by production of cytokines such as TNF‐α and other neuro‐molecules and their infiltration into different systems, pathways and organs. TNF‐α reticulates to interact with other biomolecules and mediates the neuro‐inflammation in AKI.

We have employed an in‐silico approach to retrieve the neuro‐inflammatory molecules of AKI. The genes involved in pain and neuro‐inflammation of AKI and their respective signalling pathways were retrieved by PANTHER, GENOMATIX and Target Explorer. The selected genes CGRP, TNF‐a, TRPV1 and PTGER were taken for in‐vitro study. The study was performed on male Balb/c mice by inducing AKI through the intraperitoneal injection of folic acid (250 mg/kg). The detection and modulation of selected genes were observed in harvested kidneys and brains by quantitative real‐time PCR analysis. The protein expression of CGRP and TNF‐a was estimated in kidney and brain by immune‐histology. ELISA was employed to estimate the protein content of TNF‐α and CGRP in serum.

The in‐silico approach retrieved a list of 49 genes which participate in neuro‐immune process of AKI. The STRING‐CYTOSCAPE combined with PANTHER pointed towards the role of CGRP, TRPV1 and PTGER4 in TNF‐α mediated AKI pathways. The mRNA expression by qPCR suggested modulatory expressions of CGRP, TNF‐α, TRPV1 and PTGER4 with the progression of injury in kidney and brain tissues. The variation was also reflected by immuno‐histology and ELISA.

This study unravels the neuro‐inflammatory molecules of AKI that affect the kidney and brain. While TNF‐a and CGRP emerge as the leaders of this regulation, the work also unveils the route of the communication between the two. It is observed that most of these intermediaries signal through the calcium‐mediated pathways pointing towards one of the pilot routes between them.

Support or Funding Information

1. DST‐PURSE, New‐Delhi, India 2. Centre for Systems Biology and Bioinformatics, Panjab University, Chandigarh, India. 3. Centre for Stem Cell and Tissue Engineering, Panjab University, Chandigarh, India

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Functional characterization of isl2/nr2f1b activation target stap2b in vascular development
Authors: Wang Yi‐Shan , Wu Chang‐Yi
Published: June 2021

Vascular formation is regulated by complicated molecular mechanism that remain to be fully elucidated. Our previous study identified the transcription factors Islet2 (isl2) and nr2flb regulated by Notch signaling required for vein specification and intersegmental vessels (ISV) growth in zebrafish. We performed microarray experiments to identify the potential downstream targets regulated by isl2/nr2flb. In this study, we selectively assay the function relevance of one activation target, signal‐transducing adaptor protein 2b (stap2b), and showed this gene function in zebrafish vascular development during early embryonic stages

STAP2 is a recently identified as an adaptor protein that has been showed functions in many cellular signal pathways in immune system. stap2b is the STAP2 orthologue gene on zebrafish, however, there is no description of stap2b functioning in vascular development. Our in‐situ hybridization results showed stap2b expressed in developing vessels, and knockdown this gene by morpholino injection caused vascular defects in ISV and caudal vein plexus (CVP), suggesting the role of stap2b in vascularization. We showed that vascular defects do not caused by cell death, but due to the impairment of migration and/or proliferation. Decreased expression of vascular marker flk, stabilin, ephrinb2, and flt4 were found in stap2b morphants. Furthermore, overexpression of stap2b enhance the growth of ISV and promoted endothelial cell migration and/or proliferation. These data suggest that stap2b is necessary and sufficient to promote vascular development.

We examine the interaction between stap2b and Notch signals, and showed stap2b is regulated by Notch signals to control ISV growth. In addition, stap2b may interact with BMP signals to function in CVP patterning. We also conclude that our microarray results identify stap2b acting downstream of isl2/nr2flb. Taken together, we showed that stap2b plays an important role in the vascular development of 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.
Investigation of the role of RCAN1 Gene in apoptosis
Authors: Fausto Alejandra , Medh Rheem
Published: June 2021

Our lab is interested in understanding the molecular basis of chemotherapeutic evoked apoptosis and resistance in leukemic lymphoblasts. The long‐term goal of our research is to improve the therapeutic effectiveness of chemotherapeutic agents. Antileukemic agents and signaling pathway inhibitors induce apoptosis by regulating gene expression; a drawback of this treatment is the development of resistance to these agents. The RCAN1 (Regulator of Calcineurin 1) gene is located on chromosome 21 and is one of many genes implicated in apoptosis. Upregulation of RCAN1 correlates with sensitivity to antileukemic agents and signaling pathway inhibitors. The protein product of this gene, RCAN1, binds to the protein phosphatase calcineurin, inhibiting its activity, and altering cellular responses to survival and death triggers. We are testing the hypothesis that if the protein function of the RCAN1 is knocked out, cells will be protected from apoptosis. We are screening a series of antileukemic agents and signaling pathway inhibitors, utilizing paired wild‐type and RCAN1 knockout HAP1 cell lines, to determine which agents might mediate an apoptotic response via RCAN1 involvement. HAP1 is a human near‐haploid cell line derived from the male chronic myelogenous leukemia (CML) cell line KBM‐7, allowing for efficient CRISPR‐mediated genome editing. Viability of cells exposed to various agents was evaluated by MTT assays and transcript levels were monitored via reverse‐transcription‐PCR analysis of RNA extracted from treated cells. MTT assays show that RCAN1 knockout offered the greatest protection from apoptosis caused by the AKT inhibitor MK‐2206, when compared to wildtype cells. Upregulation of BIM, a proapoptotic protein, by MK‐2206 was reduced in RCAN1 knockout HAP1 cells compared to the wild‐type HAP1 cells. Our results suggest that RCAN1 may influence the AKT pathway to mediate apoptosis.

Support or Funding Information

This research is supported by GM081099 awarded to Rheem Medh and NIH GM008395 for student support of Alejandra Fausto.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Olfactory Receptor Family 7 Subfamily C Member 1 as a potential prognostic marker for pediatric T‐cell acute lymphoblastic leukemia patients
Authors: Ahmed Haidar , Espinoza Juan Silva , Jeon Dayoung , Jauregui Alexia , Bill Charles A. , Parada Zachary , Paz David , Hernandez Alice , Kirken Robert A. , Bill Colin A. , Vines Charlotte M.
Published: June 2021

A recent study showed that self‐renewing colorectal cancer‐stem cells/cancer‐initiating cells ectopically express the olfactory receptor family 7 subfamily C member 1 (OR7C1). OR7C1, which is a G protein‐coupled receptor (GPCR) was associated with the expression of stem‐cell markers and decreased survival of colon cancer patients. We study T‐cell acute lymphoblastic leukemia (T‐ALL), a neoplasm of immature T‐cell precursors that predominately affect children with an incidence of ~20% of the overall acute lymphoblastic leukemia (ALL) cases. In a screen of 9 pediatric T‐ALL patients, we identified a single nucleotide polymorphism (SNP) in the OR7C1 gene in isolated T‐cells from all 9 patients with T‐ALL that was not present in control T‐cells. Based on published 3D structures of GPCRs we have used in silico modeling to predict the structural effects of the OR7C1 point mutation located in the second extracellular loop, which we expect will regulate downstream signaling. We hypothesize that the SNP form of OR7C1 contributes to the proliferation and aggressiveness of T‐ALL. We identified OR7C1 expression in human T‐ALL cell lines by reverse transcription PCR and western blot analysis with no expression in control peripheral blood mononuclear cells. Sequencing of amplified DNA from T‐ALL cell lines determined the same OR7C1 SNP in two of the human T‐ALL cell lines that we observed in pediatric T‐ALL patients. Using an antibody to OR7C1, we have isolated OR7C1 expressing cells by fluorescence‐activated cell sorting in T‐ALL cell line, HuT78. Currently, we are determining the stemness characteristics of our enriched OR7C1 expressing HuT78 population with the aim of defining the role of OR7C1 in T‐ALL cell proliferation and invasiveness.

Support or Funding Information

Research reported in this poster was supported by the National Institute of General Medical Sciences of the National Institutes of Health under linked Award Numbers SC1GM111172, RL5GM118969, TL4GM118971, UL1GM118970, and 5G12MD007592.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Targeting Calcium Channels to prevent EMT
Authors: Hermanson Kole J , Bhattacharya Atrayee , Dhasarathy Archana J
Published: June 2021

Breast cancer is the most common cancer among women worldwide, with many breast cancer‐associated deaths being attributed to metastasis of the primary tumor. In a process known as Epithelial to Mesenchymal transition (EMT), normal epithelial cells lose their cell‐to‐cell adhesions, become elongated and migratory, resulting in the mesenchymal phenotype. The SNAIL protein is known to be a master regulator of EMT, serving to repress expression of genes associated with cellular adhesion and activate genes involved in chemoresistance. In vivo and in vitro studies have both indicated that high levels of SNAIL correlate with poor prognosis, increased chemoresistance, and greater probability of recurrence of the tumor.

Calcium signaling in the cell is important for several biological functions, and was implicated in EMT and breast cancer. To determine which calcium channels were involved in SNAIL upregulation during TGF‐β induced EMT, we blocked store‐operated calcium entry (SOCE) with 2‐aminoethoxydiphenylborane (2APB). This reduced cell migration but, paradoxically, increased the level of TGF‐β dependent SNAIL gene activation. We determined that this increased SNAIL transcription involved signaling through the AKT pathway and subsequent binding of NF‐κB (p65) at the SNAIL promoter in response to TGF‐β. We also demonstrated that the calcium channel protein ORAI3 and the stromal interaction molecule 1 (STIM1) are required for TGF‐β dependent SNAIL transcription. These results suggest that calcium channels differentially regulate cell migration and SNAIL transcription, indicating that each of these steps could be targeted to ensure complete blockade of cancer progression. We are currently testing whether this increase in SNAIL contributes to chemoresistance, and if blockade of ORAI3 can inhibit chemoresistance.

Support or Funding Information

This work was funded by grant support from the National Institutes of Health grants P20‐GM104360 to Archana Dhasarathy and UND School of Medicine and Health Sciences.

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 effect of hypoxia and oxygen supplementation on gynecological cancer cells
Authors: Kraus Aileen Kathryn , Higgins Jillian Lee , Arruda Dominic T. , Guo Hao , Lautato Matthew , Lautato Christopher , DaCunha Eliana , Chen Ashley , Yao Ricky , Li Xaiofei , Yang Dongqin , Wan Yinsheng
Published: June 2021

Existing data have shown that cobalt chloride or CoCl2, as well as 2% O2, introduces hypoxia and activates transcription factor HIF1‐α in ovarian cancer cells (CaOV3 cell line). We hypothesize that this transcription factor is also activated in HeLa cells in response to CoCl2. Initially, we analyzed the morphology of cultured HeLa cells with the addition of various concentrations of CoCl2 in the cultured medium. The results revealed that cells with as low as 8 μM of CoCl2 treatment differ from the untreated cells because they are less confluent and irregularly shaped. We further investigated the CoCl2 effects through Western blot analysis and confocal imaging. We found that high concentration of CoCl2 directly induces hypoxia leading to the degradation of the cytoskeleton and nuclear membrane. As the concentration of CoCl2 increases, the activities of HIF1‐α and phosphorylated S6 decrease. The cell viability with MTT assay showed that there is no significant effect of CoCl2 on HeLa cells, except at the highest concentration tested. We then tested the effect of oxygen supplement by PDMS‐CaO2 disks on HeLa cells with MTT assay as well. The data showed that the viability of HeLa cells is lower when treated with these disks as opposed to CoCl2 treatment. Finally, we analyzed the mitochondrial activity of cultured CaOV3 cells with CoCl2 as well as control and PDMS‐CaO2 disks using MitoTraker. It was clear that 400 μM CoCl2 induces maximal mitochondrial activity as Mitotraker signal is highly concentrated in the surrounding cytoskeleton of the cells. PDMS‐CaO2 disks notably reduce mitochondrial activity in CaOV3 cells, as they appear dull and darker in comparison to the untreated, control cells. Taken all together, our preliminary data suggest that the manipulation of oxygen level in gynecological cancer cells may provide insights into the understanding of the role of oxygen in solid tumors and eventually offer clinic oxygen therapy.

Support or Funding Information

The RI‐INBRE grant funded this research in addition to the Providence College Biology Department.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
γ‐Tocotrienol and α‐Tocopheryloxyacetic Acid Increase the Effectiveness of Docetaxel Treatment of PC‐3 Prostate Cancer Cells and Docetaxel‐resistant PC‐3 Cells
Authors: Asay Spencer , Graham Andrew , Oblad Richard , Kenealey Jason
Published: June 2021

Prostate cancer is the second most commonly diagnosed cancer in men, and metastatic prostate cancer is currently incurable. Prostate cancer frequently becomes resistant to standard of care treatments such as docetaxel. Chemotherapeutic resistance is one of the greatest challenges facing successful cancer treatment, but can be ameliorated with combination chemotherapy. Vitamin E analogs, such as γ‐tocotrienol (γ‐T3) and α‐tocopheryloxyacetic acid (α‐TEA), have been proposed and studied as potential chemotherapeutics. We modeled combination therapy using mixture design response surface methodology (MDRSM), a statistical technique designed to optimize mixture compositions, to see if combinations of three chemotherapeutic agents (γ‐T3, α‐TEA, and docetaxel) would be better than docetaxel alone in the treatment of PC‐3 human prostate cancer cells. Response surfaces were generated for cell viability and apoptotic responses and the optimal treatment combination for each response was calculated. We found that a combination of γ‐tocotrienol, α‐TEA, and docetaxel was more effective than docetaxel alone in the treatment of PC‐3 cells, and docetaxel resistant PC‐3 cells.

Support or Funding Information

This work was funded by a generous donation from the Bryant Adams 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.
Myeloid cell‐derived IL‐6 in Chronic Lymphocytic Leukemia
Authors: McManus Sheighlah Dallas , Bertilaccio Maria Teresa Sabrina
Published: June 2021

First‐line therapies, like chemo immunotherapy have limitation that lead patients to build resistance to mainstream treatments for chronic lymphocytic leukemia. Traditionally, the focus has been on leukemic cells. Conversely, little attention has been brought to the influence myeloid cells have on CLL progression. This study focused on the production of IL‐6 specifically by monocytes, macrophages, and myeloid‐derived suppressor cells (MDSCs) in the microenvironment of CLL cells. Experimentation revealed an increase of myeloid cell‐derived IL‐6 production in the peripheral blood and lymphoid tissues of Eμ‐TCL1 transgenic mice. Ultimately, these findings can be the basis for exploration of new treatments that target myeloid cells, and can be presented as an alternative for patients who have acquired a resistance to current therapies for CLL.

Support or Funding Information

SM was supported by the CPRIT Research Training Program RP170067

This study was supported by CLL Global Research Foundation (to MTSB)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Colonic Epithelial Cells Express an Oncolytic Bispecific IgA/IgG heterodimer
Authors: Nair Padmanabhan Padmanabhan
Published: June 2021

The gastrointestinal epithelium sheds about 10(12) cells per day. Contrary to conventional wisdom a majority of these cells (>90%) are excreted into the fecal stream intact. We first demonstrated the isolation of these cells in a viable state (1991) and showed the feasibility of studying the expression of various bomarkers in health and disease. During the course of our studies mapping the expression of surface markers using flow cytometry we discovered that these cells had the ability to destroy a variety of cancer cells. These colonic cells expressed among other markers (COX‐2 during an inflammatory condition) IgA and IgG along with a subpopulation of cells co‐expressing IgA/IgG. Blocking the IgG component with anti‐IgG antibody discharged the oncolytic property of these cells. We also observed that these cells expressed Lgr5 and Musashi1, the putative markers of gastrointestinal progenitor stem cells. Long term tissue culture experiments revealed the persistence of the heterodimer. In a cohort of 80 random human samples we observed the absence of this hybrid IgA/IgG antibody in two subjects of African American origin. Lineage directed differentiation of these cells generated a progeny that secreted this chimeric antibody. This antibody was shown to arrest the growth of human colon cancer in xenotransplants in nude mice. From these observations It was concluded that this is a novel native antibody that may be absent in certain individuals as a result of a genetic aberration.

Support or Funding Information

SBIR grants R43 & R44 DK‐56567 and R‐43 & R‐44 CA‐81799 from the NIDDK and NCI of the National Institutes of Health.

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 Effect of Notch, IL‐1 and Leptin (NILCO) Inhibition in Xenograft Colorectal Cancer
Authors: Ozyurt Rumeysa , Erkasap Nilufer , Ozkurt Mete , Erkasap Serdar , Ulukaya Engin , Dimas Kostantinos
Published: June 2021

The aim of this study was to investigate the effects of inhibition of Notch, IL‐1 and leptin systems on tumorigenesis and angiogenesis in xenograft colorectal cancer model. This study was conducted with the approval of the EU/2010/63 directive and the Greek Presidential Decree 56/2013 (Approval No. 5542/228006/1). In each group, 40 scid (NOD/SCID) mice with 6–8 weeks of age were studied in 5 groups. For tumor formation 1 × 107 human colon cancer cells HCT‐15 were injected subcutaneously into the dorsal flank of the mice. When the tumor size was 100–250 mm3, treatment was started. Group 1: Leptin receptor antagonist ALLO‐aca was injected with 1mg / kg / day subcutaneously (s.c) for 15 days (n: 8). Group 2: γ‐secretase inhibitor DAPT was injected with 10 mg / kg / day subcutaneously (s.c) for 15 days (n: 8). Group 3: Recombinant human IL‐1 receptor antagonist (IL‐1Ra) was injected with 1mg / kg / day subcutaneously (s.c) for 15 days (n: 8). Group 4: 4% DMSO/Safflower Oil (DAPT solvent) was injected subcutaneously (s.c) for 15 days. (n: 8). Group 5: 10% DMSO/0,9%NaCl (Allo aca and IL‐1 iınhibitor solvent) was injected subcutaneously (s.c) for 15 days. (n: 8). After 15 days, tissues were collected and gene expression experiments were performed. mRNA abundance of leptin, leptin OB‐RL receptor, Notch1 receptor, JAGGED1 ligand, IL‐1β, IL‐1R tI, VEGF‐A, VEGFR2 conducted by qRT‐PCR. According to PCR results, leptin, its receptor Ob‐R, Notch1, JAGGED1, VEGF‐A and VEGFR1 mRNA abundance were decreased only in the DAPT group (respectively; p<0.05, p<0.05, p<0.05, p<0.01, p<0.05, p<0.01). The mRNA abundance of VEGFR2 was decreased only in the IL‐1 inhibitor group (p<0.05). According to the results, DAPT at 10 mg / kg / day dose Notch receptor and its ligand was reduced as well as decreased mRNA abundance of leptin and VEGF systems that pro‐angiogenogenic factors. The results of our study support the oncogenic role of Notch, IL‐1 and Leptin crosstalk in colorectal cancer. Inhibition of NILCO signaling could lead to the development of new adjuvant therapies to reduce or eliminate tumor angiogenesis.

Support or Funding Information

This study is supported by a grant from Eskisehir Osmangazi University Scientific Research Projects (Project no:2017‐11028)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Synergistic effect of epigenetic inhibitors decitabine and suberoylanilide hydroxamic acid on colorectal cancer in vitro
Authors: Rizk Sandra , Najem Sonia Abou , Hodroj Mohammad Hassan , Khawaja Ghada
Published: June 2021

Colorectal cancer (CRC) is major public health burden and a common cause of oncological deaths worldwide. Alterations of the epigenetic landscape are a well‐documented hallmark of CRC phenotype. Accumulation of aberrant DNA methylation and histone acetylation orchestrates to modify gene activity and drive tumor onset, progression and metastasis. In this study we evaluated the effect of suberoylanilide hydroxamic acid (SAHA), a pan histone deacetylase inhibitor, and decitabine (DAC), a DNA methyltransferase inhibitor, either alone or in combination, on Caco‐2 human colon cancer cell line in vitro. Our results showed that SAHA and DAC, separately, significantly decreased cell proliferation, induced apoptosis and cell cycle arrest of Caco‐2 cell line. On the other hand, the sequential treatment of Caco‐2 cells, first with DAC and then with SAHA, induced a synergistic anti‐tumor effect; the sequential treatment significantly enhanced growth inhibition and apoptosis of Caco‐2 cell line in comparison to cells treated with either drug alone. Furthermore, the combination therapy upregulates protein expression levels of pro‐apoptotic proteins Bax, p53 and cytochrome c, down regulates the expression of anti‐apoptotic Bcl‐2 protein and increases the cleavage of procaspases 8 and 9; this suggests that the combination activates apoptosis via both the intrinsic and extrinsic pathways. Mechanistically, we demonstrated that the synergistic anti‐neoplastic activity of combined SAHA and DAC involves an effect on PI3K/AKT and Wnt/β‐catenin signaling pathways as it down regulates p‐PI3K, p‐AKT, Wnt1, p‐GSK3, cyclin A1+A2, cyclin E1 and up regulates p‐β‐catenin protein expression levels. In conclusion, our results provide evidence for the profound anti‐tumorigenic effect of sequentially combined SAHA and DAC in CRC cell line and offer new insights into the corresponding underlined molecular mechanism.

Support or Funding Information

This work was funded by SRDC, School of Arts and Sciences, Lebanese American University

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Linking microbial metabolism to antibiotic tolerance in the microbiome
Authors: Belenky Peter A
Published: June 2021

Empirical antimicrobial therapy has saved billions of lives in the past 70 years but has also led to the development of the antimicrobial resistance crisis. Additionally, we now know that each antibiotic exposure has the potential to eliminate trillions of beneficial microbes and lead to profound complications. Thus, as we improve antibiotic therapy we must understand why and how antibiotics kill members of the microbiome. Previous data indicate that active and oxidative metabolism of bacteria is linked to antibiotic efficacy in vitro. Here we utilize a metagenomic/metatranscriptomic approach to profile the impacts of microbial metabolism on antibiotic susceptibility in the total microbiome in human subjects and murine models. We found that reduce metabolic activity of specific taxa before and during treatment is a key determinant of susceptibility to broad‐spectrum antibiotics. We found that taxa within the microbiome that direct metabolism to anabolic and fermentative pathways survive whereas taxa that utilize oxidative catabolism are eliminated. Utilizing metatranscriptomic RNAseq, at a species resolution, within our microbiome we describe the transcriptional response of the 12 most abundant species such as Akkermansia muciniphila, Bacteroides thetaiotaomicron and others to antibiotics. In the case of B. thetaiotaomicron we found that it upregulates fiber fermentation (including the sus operon) in response to amoxicillin and that polysaccharide supplementation protects this bacterium in vitro and in the microbiome. Based on these experiments we implement and report a murine study to profile the impacts of an array of purified fibers on antibiotic‐induced dysbiosis. This work indicates that host diet modulation and metabolic state may provide an important intervention to protect microbiome homeostasis and may lead to the identification of prebiotic therapy to protect the microbiome from antibiotics.

Support or Funding Information

This work was supported by funding received from the COBRE Center for Computational Biology of Human Disease (NIH P20 GM109035) and from the National Science Foundation Graduate Research Fellowship (Grant No. 1644760).

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Identifying Microbial Resistance of the Apis mellifera Melanization Immune Response
Authors: Knier Rochelle
Published: June 2021

Apis mellifera, one of the most common species of honey bee, has been suffering disease and colony collapse. While there has been effort in identification and treatment of the pathogens, the study of their insufficient immunoresponse has gone under‐researched. Melanization is a common form of immune response in insects and has been seen in Apis mellifera. The goal of this study is to isolate melanin from hemolymph and conduct multiple experiments with known pathogens against it with the purpose of identifying microbial resistance and further inferring the mechanism of immunity. With this knowledge, a method of improving the Apis mellifera immune response may be found.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Elucidating the effect of antibiotics on Pal's release from Escherichia coli
Authors: Bauer Morgan , Gallardo Leslie , Jackson Niaya , Michel Lea
Published: June 2021

The clinical condition of sepsis often results from a systemic bacterial infection, followed by an over‐exuberant immune response, which leads to widespread inflammation. Severe sepsis can result in organ failure or death. Past studies have proposed a role for bacterial Peptidoglycan‐Associated Lipoprotein (Pal) in the pathogenesis of Gram‐negative sepsis. In this study, we confirmed the ability of Escherichia coli (E. coli) to release Pal under certain conditions, and we employed both in vitro and in vivo (mouse) studies and protein detection methods to determine the effect of antibiotics on Pal's release from E. coli. Results from our studies suggest that antibiotics that target peptidoglycan may enhance Pal's release more so than other antibiotics.

Support or Funding Information

Rochester Institute of Technology

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Oxidative stress‐induced protein packaging in Enterotoxigenic Escherichia coli outer membrane vesicles and its functional impact on host‐pathogen interactions
Authors: Orench‐Rivera Nichole , Kuehn Meta
Published: June 2021

Enterotoxigenic Escherichia coli is the leading cause of traveler's diarrhea worldwide and it remains an important causal agent of diarrheal disease, especially among children of lower income countries. Because it must adjust to different ecological niches such as fruit and vegetable surfaces, water, and its animal or human host; its cell envelope must undergo remodeling. In Gram‐negative bacteria, the cell envelope is the first barrier against environmental stress and studies have shown that the process of outer membrane vesiculation is essential for bacterial growth and survival during stressful conditions. Recent studies in our lab revealed that outer membrane vesicles (OMVs) can contain varying ratios of lipopolysaccharide (LPS) types during cell envelope remodeling under stress in Salmonella enterica, suggesting a role for vesicles in membrane remodeling. In this study, outer membrane vesicles and outer membrane fractions from ETEC were isolated from bacterial cultures, purified via density centrifugation, and submitted for proteomic analysis using quantitative one‐dimensional liquid chromatography, tandem mass spectrometry (1D‐LC‐MS/MS) to quantify differences in protein expression in vesicles and membranes during oxidizing and non‐oxidizing conditions (plus or minus Hydrogen Peroxide). Protein packaging into vesicles was determined by calculating the log‐2 of the ratio of protein expression in OMVs/outer membranes for both treatment conditions. Results indicated that two distinct sets of proteins were shown to be differentially packaged into OMVs as a function of peroxide treatment. Upon further analysis implementing a Bayesian hierarchical model, lipoproteins were observed to be preferentially exported during stress, in contrast to integral proteins, which were preferentially retained in the outer membrane. Additionally, OMVs derived from stressed cultures showed less cytotoxicity towards Y‐1 adrenal cells in a cell rounding assay. These results allow us to speculate about potential roles for OMVs in membrane remodeling of pathogenic bacteria undergoing environmental shifts both outside and inside the human host.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Intestinal epithelial O‐GlcNAc signaling is indispensable for anti‐helminth type 2 immunity
Authors: Zhao Ming , Ren Kaiqun , Ruan Hai‐Bin
Published: June 2021

More than 1 billion people worldwide are infected with helminths, which may nutritionally and physically impair infected people, particularly children. Tuft cells lying in the intestinal epithelium have been recently proposed to sense parasitic worms in the lumen and initiate type 2 immunity, which in turn remodels the intestinal epithelium and expulses parasites. However, little is known regarding the molecular mechanisms underlying tuft cell hyperplasia. O‐linked N‐Acetylglucosamine (O‐GlcNAc), a posttranslational modification that decorates a single sugar moiety onto intracellular proteins at serine and threonine residues, plays a central role in many biological processes such as gene transcription, protein stability, and cell signaling. Dysregulated O‐GlcNAc signaling has been associated with a plethora of human diseases including cardiovascular disease, diabetes, neurodegeneration, and inflammation; however, the role of O‐GlcNAcylation in intestinal homeostasis has not been fully elucidated. Here, we found that O‐GlcNAc transferase (OGT), the enzyme catalyzing O‐GlcNAcylation, was essential for homeostatic tuft cell development in mice. The deficiency in intestinal epithelial O‐GlcNAcylation impaired tuft cell hyperplasia and type 2 responses to H. polygyrus and N. brasiliensis infections. Intriguingly, such defects were not observed in mice where OGT was specifically deleted in committed tuft cells, suggesting that OGT is required for the differentiation of intestinal progenitors toward the tuft cell lineage. Indeed, STAT6, a type 2 cytokine‐responsive transcriptional factor essential for tuft cell development, is O‐GlcNAcylated. O‐GlcNAcylation of STAT6 enhances its tyrosine phosphorylation and transcriptional activity, while O‐GlcNAc‐deficient STAT6 blunted tuft cell differentiation. Taken together, our data demonstrate that protein O‐GlcNAcylation, by modifying STAT6 signaling, is indispensable for epithelial remodeling and tuft cell‐initiated type 2 immune responses to helminth infections.

Support or Funding Information

American Diabetes Association (18‐IBS‐167)

NIAID (R21AI140109)

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Parasite‐Derived Vesicular‐Mediated Protein Export by the Human Pathogen Babesia microti
Authors: Ben Mamoun Choukri , Thekkiniath Jose , Kilian Nicole , Lawres Lauren , Gewirtz Meital , Abraham Amanah , Graham Morven , Liu Xinran , Ledizet Michel
Published: June 2021

Babesia microti is the primary agent of human babesiosis, a potentially fatal malaria‐like illness with worldwide geographic distribution. Like its Plasmodium relatives, B. microti intraerythrocytic development is heavily dependent on its successful interaction with the host. While available data indicate that the parasite exports a large number of proteins into the host, the mechanism by which these proteins are exported remains completely unknown. Unlike malaria parasites, B. microti lacks a parasitophorous vacuole, and its secreted proteins are devoid of canonical motifs for trafficking into the erythrocyte cytoplasm or cell environment. Here we show that following invasion of the host erythrocyte, B. microti undergoes a major morphogenetic change during which it produces an interlacement of vesicles (the IOV system) that extends from the plasma membrane of the parasite into the cytoplasm of the infected erythrocyte. Using cell fractionation and confocal and immunoelectron microscopy analyses, we demonstrate that the IOV system serves as a major export system of the major immunodominant antigens of the parasite. The IOV system of B. microti represents a new mechanism for delivery of parasite factors into the host in the phylum Apicomplexa.

Support or Funding Information

This work was funded by NIH grants AI123321 and R43AI136118 and in part supported by the NIH grant S10 OD020142.

This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Evidence for the Novel Metabolic Pathway of Ralstonia eutropha H16 to Metabolize L‐ascorbate Ryan Joyce, Tyler Stack, Michael Carter
Authors: Joyce Ryan Patrick
Published: June 2021

L‐Ascorbate is an antioxidant produced by many eukaryotes and is available for microbial organisms in eukaryotic detritus. Ralstonia eutropha H16, a soil bacterium that is studied due to its extensive metabolism and bioplastic producing capabilities, can use L‐ascorbate via a metabolic pathway that is different from the known pathways of Escherichia coli and plants. Transcript levels were measured in L‐ascorbate‐grown Ralstonia eutropha H16 and compared to transcript levels of succinate‐ and fructose‐grown Ralstonia eutropha H16. Genes with a larger than 50‐fold upregulation during L‐ascorbate growth were investigated. A BLAST search suggested that the proteins encoded by the genes of interest in Ralstonia eutropha H16 differ from enzymes of the Escherichia coli L‐ascorbate metabolic pathway. Ralstonia eutropha H16 does not convert L‐ascorbate into L‐threonate and oxalate, as plants do. Genes of interest, h16_rs15015 and h16_rs19815, were deleted. The resultant mutants do not grow with L‐ascorbate. The deletion of specific metabolic genes allows us to better understand their function and importance to the cell's metabolism, furthering our understanding of how different enzymatic metabolic pathways function in nature.

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