bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Publicly Guided Effort to Rapidly Produce and Disseminate 100 Intersectional Genetic Alleles for the Study of Nervous System Circuit Development and Function in the Mouse
Authors: Martinez Vena , Sun Jenny , McKinney Andrew , Jiang Xiaolong , Ray Russell 
Published: June 2021

The goal of this project is to build and distribute a community library of more than 100 mouse intersectional genetic alleles to facilitate the developmental, anatomical, molecular, and functional characterization of neural circuit organization in behavior and physiology based on public input. Even within narrowly defined cell types, significant diversity is found at multiple levels including genetic and molecular signatures, activity patterns, and synaptic connectivity. Current challenges now center on tools to identify, access, and study cell populations with increasing precision. Intersectional genetics offers exceptionally high resolution to consistently delineate distinct cell types in the embryo and adult mouse for functional, molecular and anatomical studies. Intersectional genetics utilizes a ubiquitously expressed conditional allele that is activated by Cre and Flp site specific recombinases. Upon activation, the intersectional genetic alleles may express any number of Genetically Encodable Effector Molecules (GEEMS), such as channelrhodopsins for neural activity perturbations or an L10‐GFP ribotrap fusion to affinity purify translating mRNAs. The intersectional allele is activated by overlapping expression of both Flp and Cre recombinases in the same cell. Traditionally, these recombinases have been deployed as gene knock‐in or transgenic alleles that are designed to express in a cell or gene specific fashion. The use of two selectors (Cre and Flp) to define a cellular population enables high specificity and modularity in combining any set of Cre, Flp and intersectional alleles to fit an experiment. The number of Cre and Flp recombinase mouse lines are constantly growing, giving greater access to increasing numbers of cell types. Additionally, these recombinases are the focus of multiple efforts to deploy them in ways that select cells based on other unique properties such as neuronal activity and synaptic connectivity. To make this powerful genetic approach more accessible to as many research laboratories as possible, we are building a production pipeline to create a suite of resources for anyone to easily make their own intersectional mouse line, to produce over 100 targeted ES cell lines that can be developed into mouse lines, as well as 10–15 high demand mouse lines all for public distribution. Priority for the GEEMS to be intersectionally deployed will be based on a public input collected at mouseintersectionalgenetics.org. All plasmids, cell lines and mouse lines developed under this effort will be distributed without restriction to non‐profit research organizations.Support or Funding InformationMcNair Medical InstituteR01HL130249R01HL130249‐02S1This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Neuroinflammatory responses in a mouse model of acute organophosphate intoxication
Authors: Calsbeek Jonas , Guignet Michelle , Dawson Mallory , Bruun Donald , Lein Pamela 
Published: June 2021

Organophosphates are a class of synthetic, neurotoxic compounds that include nerve agents and pesticides that can cause convulsions, status epilepticus (SE), and death following acute intoxication. The current standard of care for OP intoxication can prevent mortality; however, survivors often exhibit persistent neuropathologic changes and cognitive deficits. These observations underscore the need for improved therapies. Currently available preclinical models of acute OP intoxication almost exclusively use rats or guinea pigs, two species for which there currently are limited numbers of available transgenic lines. To overcome this limitation, we have developed a mouse model of acute OP intoxication using the OP pesticide diisopropylfluorophosphate (DFP). Within minutes after a single injection of DFP, adult male C57BL/6 mice displayed robust behavioral seizures consistent with the induction SE. At 1, 7, 14, and 28 days post‐intoxication, animals were euthanized, and the brains were removed for FluoroJade C labeling to quantify neurodegeneration, and GFAP/S100β or IBA1/CD68 immunohistochemistry to quantify reactive astrogliosis and microglial activation, respectively. A significant increase in neurodegeneration, reactive astrogliosis, and activation of microglia was observed in the somatosensory cortex, hippocampus, and piriform cortex of DFP mice relative to vehicle controls. These findings identify mice as a sensitive platform for the investigation of DFP‐induced neurological consequences that recapitulate the patterns of injury observed in widely used preclinical models. The availability of genetically modified mouse strains will provide unique opportunities to elucidate the molecular mechanisms contributing to the pathophysiology of acute OP intoxication, which will provide insight regarding more effective targets for therapeutic intervention.Support or Funding InformationThis work supported by the NINDS CounterACT Program (grant # U54 NS079202).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Stress hormones induce DNA damage in ovarian cancer cells
Authors: de Lis Lamboy Caraballo Rocio , Armaiz Guillermo 
Published: June 2021

Activation of the sympathetic nervous system (SNS) is associated with cancer progression. Recent data suggest that adrenergic signaling alters the genetic integrity of cancer cells by damaging the DNA and/or influencing the DNA‐maintenance machinery composed of the DNA Damage Response Pathway and DNA Repair Pathways. Consequently, accumulation of genomic alterations can lead to genomic instability and the acquisition of other cancer hallmarks that drive tumor progression. Still, the exact mechanisms of how sustained adrenergic signaling influences genomic stability is an area that needs to be further studied. We hypothesize that increased adrenergic stimuli will induce significant levels of DNA damage. To assess the extent of DNA damage after catecholamine exposure, we determined p‐H2AX (sensitive marker for double‐stranded DNA damage) expression by western blot and immunofluorescence analyses in SKOV3 and other high‐grade serous ovarian cancer cell lines. Neutral Comet Assays were performed to assess double‐stranded DNA damage after catecholamine exposure. SKOV3 cells that were exposed to epinephrine (Epi) and norepinephrine (Ne) at 1h, 3h, 6h, 12h, and 24h displayed increased expression of p‐H2AX. Moreover, immunofluorescence analyses for p‐H2AX in SKOV3 and OV90 showed increased foci formation (reflecting 1:1 foci/double‐strand break) when compared to the control group. Lastly, Neutral Comet Assay analyses in catecholamine‐treated SKOV3 and COV362 cells displayed higher levels of double‐stranded DNA breaks in comparison to untreated cells. Lastly, to determine if DNA damage is due to adrenergic stimuli, cells were pre‐treated with propranolol (non‐specific β‐blocker) and results showed a significant reduction in the levels of DNA damage. These results suggest that catecholamines can induce double‐stranded DNA damage in ovarian cancer cells.Support or Funding InformationNIMHHD (G12MD007579), NIGMS‐RISE R25GM082406, NCI (U54CA163071 and U54CA163068), U54CA163071‐06, and Ponce Research Institute.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 and Global Effect of C‐terminus of Hfq in Small RNA Regulation
Authors: Kavita Kumari , Zhang Aixia , Tai Chin‐Hsien , Storz Gisela , Gottesman Susan 
Published: June 2021

Hfq, a homohexameric RNA chaperone, is known for stabilizing small noncoding RNAs (sRNAs) and facilitating riboregulation by promoting sRNA base pairing with target mRNAs. The proteins encoded by these target mRNAs are often involved in virulence and stress responses. Hfq has a conserved N‐terminal region sufficient for most of its function and a poorly conserved disordered C‐terminus (CT). The length of the C‐terminus varies among bacteria; it is 37 aa in E. coli. Since the role of C‐terminus of Hfq has been poorly understood we have investigated its role in vivo in small RNA stability and function. Mutant derivatives of Hfq were expressed from the chromosomal locus, allowing in vivo comparisons with the wild‐type (WT) Hfq, expressed under similar conditions and at physiological levels. While deletion of the CT (Hfq65) had modest phenotypes, combinations of this deletion with a weak mutation (K31A) in the distal face of the protein was fully defective for function in different assays. This strong synergistic phenotype of CT deletion with K31A allowed us to further dissect the CT. Loss of the region between 65–72aa, the best conserved part of the CT, was critical for the synergistic defect. These results suggest that the initial region of the CT supports the function of the distal face, where many mRNAs and a subset of sRNAs are known to bind. To understand the global effect of CT on interacting RNAs, we compared RNA seq data of Hfq immunoprecipitation for WT and Hfq65. Overall, Hfq binding RNA patterns were not much affected. However, consistent with previous findings, and with the cooperation of the CT with the distal face of Hfq, Hfq65 was most defective in the accumulation of the sRNAs (Class II) that bind the proximal and distal faces of Hfq. Some class I sRNAs, which bind the proximal and rim face of Hfq, were also modestly depleted in Hfq65, suggesting the possibility of a broader role for the CT. The mechanistic aspects of CT collaboration with the Hfq core are under investigation. Our results suggest that the C‐terminus collaborates with the Hfq distal face, reinforcing the specificity and strength of RNA binding to Hfq.Support or Funding InformationThis research was supported by the Intramural Research Program of the NIH, National Cancer Institute, Center for Cancer Research.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Point Mutation of Aquaporin 4 Impairs its Binding in Neuromyelitis Optica
Authors: Replogle Jessica Beth Sakash , Calderon Irene , Cutforth Siena , Devine Erin , Fraley James , Lamarre Thomas , Moran Elizabeth , Valencia Luis , Suder Karen Cruse 
Published: June 2021

Aquaporin 4 (AQP4) is the principal aquaporin expressed in astrocytes throughout the central nervous system. An autoantibody, IgG, that binds to AQP4 leads to a rare disease called neuromyelitis optica (NMO), which is characterized by paralysis and loss of vision. Current treatments for NMO are few and involve non‐specific immunosuppression. The Summit Country Day School MSOE Center for BioMolecular Modeling MAPS Team used 3D modeling and printing technology to examine structure‐function relationships of the AQP4 epitope for NMO‐IgG. Understanding the molecular structure of the AQP4 epitope is an important step in explaining its binding to NMO‐IgG. NMO‐IgG primarily interacts with the accessible AQP4 extracellular loops A, C, and E. These loops change their conformation when AQP4 arrange into orthogonal arrays of particles, thus enabling attachment of NMO‐IgG and facilitating complement binding. Studies on the point mutation of aspartate in position 69 (D69) revealed conformational changes such as 1) reorientation of threonine in position 62, 2) weakened hydrogen bond interaction between leucine in position 53 and threonine in position 56, 3) increased mobility of loop A, and 4) disruption of epitope reorganization of AQP4. Overall, D69 substitution and the subsequent conformational changes in loop A leads to impairment of binding between AQP4 and the NMO‐IgG. Determining the role of extracellular loop A between and within AQP4 monomers will aid in understanding how it contributes to antigen recognition. Prevention of the binding of AQP4 by NMO‐IgG is an important strategy for developing targeted therapy in NMO.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 Pre‐N Domain is a Distinct Feature of Grp94 that is Essential for Client Maturation and Regulation
Authors: Huck Jack D. , Que Nanette L.S. , Hong Feng , Li Zihai , Gewirth Daniel T. 
Published: June 2021

The chaperone cycle of hsp90s involves a series of large conformational shifts between open and closed dimer states that are driven by the binding of ATP to the N‐terminal domain (NTD). Unlike its cytosolic counterpart, the ER hsp90 paralog, Grp94, does not possess a network of co‐chaperones to regulate dimer closure and modulate the rate of ATP hydrolysis. Although hsp90s are highly homologous with each other, regions of divergence may lead to the functional differences that are key to Grp94's adaptation to the ER. One such region, the Pre‐N domain, precedes the NTD and varies substantially in length and sequence among hsp90s, with the Grp94 Pre‐N domain (50 a.a.) dwarfing the Pre‐N domains of the other paralogs (Trap‐1: 24 aa; Hsp90a: 16 aa; Hsp90b: 11 aa). For Grp94, the Pre‐N domain slows the rate of ATP hydrolysis but the mechanism by which it does so and its role in client maturation are not understood. Here we describe crystal structures of Grp94 containing the Pre‐N domain in open and closed dimer states. Surprisingly for a significant regulatory element, the Pre‐N domain makes only minimal stabilizing contacts with the opposite NTD in the closed dimer state. In the open dimer state, however, the Pre‐N domain adopts a novel trajectory and secondary structure that may regulate the ATP‐bound conformation through allosteric auto‐inhibition of the NTD. Our functional analysis further revealed that both the length and sequence composition of the Pre‐N domain are required for client proteins to reach the cell surface. We are currently investigating the interplay between the Pre‐N domain and other ER folding factors, such as post‐translational modifications, to understand the role of the Pre‐N domain in client maturation. Taken together, these results imply a functional role for the Grp94 Pre‐N domain that mimics that of the cytoplasmic Hsp90 co‐chaperones, which are missing in the ER.Support or Funding InformationResearch in the Gewirth lab is supported by NIH grants R01‐CA095130 and P01‐CA186866, a grant from the Richard and Mae Stone Goode Foundation of Buffalo, and grant 02‐2016‐079 from the Avon Foundation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Modulation of gap junctional intercellular communication in HaCaT cells by xenobiotics
Authors: Shipman Richard David , Jost Jannika , Pollet Dieter , Waldmann Petra 
Published: June 2021

Gap junctions are clusters of transmembrane proteins which enable direct communication between adjacent cells. Gap junctions allow intercellular diffusion of small molecules like metabolites (such as glucose), second messengers (such as cAMP, ATP) and ions (such as Mg2+, K+). That process, called gap junctional intercellular communication (GJIC), allows coordination of many essential functions in multicellular organisms, such as directed cell differentiation, cell growth, cellular and tissue homeostasis. Moreover, dysfunctional GJIC has been linked to a variety of human diseases.The final aim of this project is to elucidate whether estrogenic acting chemicals play a role in GJIC. As a first step, two industrial chemicals, bisphenol A (BPA) and 4‐nonylphenol (4‐NP), were selected and assessed as modulators of GJIC in the human epidermal keratinocyte line HaCaT.To study GJIC of HaCaT cells, the scrape loading/dye transfer (SL/DT) assay was applied to HaCaT cultures grown at standard conditions with serial dilution treatments of either BPA or 4‐NP versus ‐a vehicle treatment (< 1% ethanol) for 24 hours. The SL/DT assay entails cutting a cell monolayer with a scalpel to allow for the incorporation of a fluorescent dye (Lucifer Yellow) into the ‘scraped’ cells. The dye transfer via opened gap junctions into adjacent cells is then investigated by fluorescence microscopy and image analysis. The number of Lucifer Yellow positive cells is a quantitative descriptor of GJIC activity in treated cells versus untreated cells. In addition to the results provided by the SL/DT functional assay, quantitative PCR was performed in parallel to assess the mRNA levels for GJIC protein connexins (Cx26, Cx36, and Cx43).The results of the SL/DT assays showed that both BPA and 4‐NP reduce GJIC in the human epidermal keratinocyte line in a dose range of 10 down to 0.1 μM. Currently qPCR experiments are being conducted to elucidate whether this is due to closed gap junctions or to modulation in expression of connexin encoding genes.With this research, these properties alongside qPCR may be relevant to answer the question of whether the observed modulation of GJIC in skin cells is due to an estrogenic activity. Furthermore, a better understanding of GJIC modulation may clarify the role of GJIC in disease and make it a future target for pharmacological, and particularly, dermatopharmacological approaches.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Super‐SILAC based quantitative phosphoproteomics reveals the potential biomarkers in lung cancer
Authors: Hsiao Ting‐Feng , Wang Chih‐Liang , Wu Yi‐Cheng , Wu Chia‐Chun , Yu Chia‐Jung 
Published: June 2021

Lung adenocarcinoma (ADC) is the most common histological subtype of lung cancer, the leading cause of cancer‐related deaths worldwide. Epidermal growth factor receptor (EGFR) mutations strikingly target lung ADC in East Asia. EGFR tyrosine kinase inhibitor (EGFR‐TKI) has been developed as a targeted therapy for treatment of advanced lung ADC with activating EGFR mutations. Unfortunately, neither druggable targets nor useful biomarkers can be used to benefit ADC patients with wild‐type (WT) EGFR. We herein aimed to identify the differentially expressed phosphoproteins and kinases in ADC tissues with EGFR‐WT. We applied super‐SILAC combined with TiO2 phosphopeptide enrichment and liquid chromatography‐tandem mass spectrometry to establish the stage‐related phosphoproteome with 20244 phosphopeptides in ADC tissues. The bioinformatics analyses revealed the stage‐dependent phosphorylation profile, upstream signaling and the kinase cascade in ADC tissues. We also observed that the impact of EGF, PTK2B, JAK2, ICAM1, or VEGF on pathogenesis of the early‐ and late‐stage cancers was indeed different. To explore the clinical applications of these dysregulated phosphoproteins in lung ADC, the potential markers were selected for further verifications/validations via western blotting and immunohistochemistry staining.Support or Funding InformationMinistry of Science and Technology, Taiwan, R.O.C. (105‐2320‐B‐182‐035‐MY3) and Chang Gung Medical Research Fund (CMRPD1H0081, CMRPD1H0082 and BMRP894).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
mTOR inhibition promotes LPS‐induced acute lung injury by inducing endothelial hyperpermeability
Authors: Chen Xiaolin , Fan Xing , Sun Chongxiu 
Published: June 2021

Although mTOR inhibitors are widely used to treat various disorders in clinic, a considerable number of side effects have been described, including pneumonitis. The intrinsic mechanisms are not completely understood. This study aimed to elucidate the role of endothelial barrier dysfunction in the pathogenesis of mTOR inhibition‐induced lung injury.Measurement of TER (transendothelial electrical resistance) and transendothelial BSA flux indicated that inhibition of mTOR either through transfection with siRNA, or treatment with inhibitors rapamycin or torin 1, increased endothelial permeability, accompanied by F‐actin remodeling as visualized by immunofluorescent. Consistently, mice with specific knockout of Mtor in endothelial cells exhibited significantly aggravated lung injury, neutrophil infiltration and lung edema in response to LPS exposure.Studies with Rho‐associated kinase (ROCK) inhibitor (Y‐27632) and myosin light chain (MLC) kinase inhibitor (ML‐7) indicated that the hyperpermeability response was dependent on MLC phosphorylation, which is regulated by PKC activity. PKC contributed to mTOR inhibition‐induced MLC phosphorylation and endothelial hyperpermeability through P38/NF‐κB pathway to upregulate MLC kinase expression and ROCK/MYPT1 pathway to negatively control MLC phosphatase activity.Taken together, our results demonstrate that inhibition of mTOR promotes LPS‐induced acute lung injury, at least partly through inducing endothelial barrier dysfunction.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Dietary Complexation of Peanut Protein to Polyphenolic Extracts Reduces Peanut Specific Plasma IgE levels in Peanut Sensitized C3H/HeJ mice
Authors: Randolph Priscilla D , Bansode Rishipal D , Williams Leonard L , Plundrich Nathalie , Lila Mary Ann 
Published: June 2021

Peanut Allergy is a common food allergy that can cause severe anaphylaxis reactions in children and adults. Treatment strategies that are utilized to combat peanut allergy includes but not limited to strict avoidance and rescue medication upon exposure to peanut. Oral immunotherapy is another strategies used to increase the threshold of peanut sensitivity in individuals who are allergic to peanut. Cross‐linking of dietary proteins by polyphenols can change their structural and immunological properties and can affect the protein's, thermal and morphological properties. Thus the purpose of this study was to determine whether the peanut proteins complexes to polyphenols extract would reduce allergic response in C3H/HeJ mice. Five‐week‐old C3H/HeJ mice were purchased. Before exposing mice to protein‐polyphenols aggregates of either low (15%: w/w) or high (40%; w/w) complexation ratios of blueberry (BB‐Low and BB‐High) and cranberry (CB‐Low and CB‐High) extract, mice were sensitized to peanut flour. Peanut sensitivity was also established by measuring the increase in peanut specific plasma IgE and IgG levels before treatment. Once the final peanut challenge, animals were scored for anaphylactic reactions, sacrificed, and blood samples were collected. Tissues samples aliquot from the liver, adipose, pancreas, spleen, and small intestine were collected and frozen at −80oC. Western blot analysis of spleen lysates revealed CD63/ant‐IgE protein expression was reduced by 35% and 44% in CB‐low and CB‐high diet groups, respectively, compared to the control. The reduction in CD63/anti‐IgE protein expression was 22% and 53% in BB‐low and BB‐high diet, respectively when compared to the control group. Therefore, our results demonstrate that the complexation for polyphenols to peanut flour can potentially lower plasma IgE of peanut‐sensitized C3H/HeJ mice.Support or Funding InformationUSDA NIFA: 240792This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.