bluuebunny/crossref_metadata_2025_split huggingface dataset:
Depletion of Cranial Suture Stem Cells by Pharmacological Exposures May Precipitate Premature Suture Fusion
Authors: Durham Emily , Howie R. Nicole , LaRue Amanda , Cray James 
Published: June 2021

The CDC National Birth Defects Prevention Study has published data suggesting that “environmental” exposures including maternal thyroid diseases, use of selective serotonin reuptake inhibitors (SSRIs), and maternal nicotine use may increase the incidence and or severity of craniofacial anomalies including craniosynostosis. Craniosynostosis is defined as the premature fusion of the suture(s) of the skull occurring in 1:1800–2500 births. A proposed mechanism of craniosynostosis is the disruption of proliferation and differentiation of stem cells in the perisutural area. Here, we hypothesize that the aforementioned teratogenic exposures can cause a depletion of stem cells within the suture resulting in premature suture fusion. To determine if in utero pharmacological exposures deplete stem cells within the calvarial sutures, we exposed pregnant wild‐type mice to levothyroxine, citalopram (SSRI), and nicotine and investigated suture fusion and the presence of stem cell markers ex vivo. Additionally, we exposed primary calvarial suture derived cells to clinically relevant doses levothyroxine, citalopram, and nicotine in vitro and assessed the presence of stem markers via flow cytometry. Micro‐CT assessment of coronal and posterior interfrontal suture fusion revealed that in utero exposure to nicotine and citalopram increased the risk of premature posterior interfrontal suture fusion and exposure to citalopram resulted in an increased likelihood for coronal suture fusion at post‐natal day 15. Further, we confirmed a reduction in Gli1+ cells ex vivo in correlation with in utero teratogen exposure. Our in vitro analysis also indicates a depletion of stem cell populations with teratogen exposure via flow cytometry. Teratogenic exposures including maternal thyroid disorder, maternal use of SSRIs and maternal nicotine use may target calvarial stem cell populations for depletion precipitating an increased risk for craniosynostosis. Investigating the newly defined stem cell niche of the calvarial sutures and its relationship to suture maintenance will provide insight into future manipulation of these cells for therapeutic benefit.Support or Funding InformationNational Institute of Dental and Craniofacial Research [F31DE026684 to ELD; R03DE023350A and R03DE02619‐01A to JC; 5T32DE017551 to MUSC]Cleft Palate Foundation Cleft/Craniofacial Anomalies Grant Award to JCPlastic Surgery Education Foundation Pilot Award to JCNational Institute on Aging (NIA) 1P01AG036675 (Augusta University)National Institutes of Health National Institute of General Medicine [P30GM103331]South Carolina Clinical and Translational Research Institute (NIH/NCATS NIH/NCATS UL1TR000062)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Critical role of claudin‐7 in maintaining intestinal crypt stem cell functions
Authors: Xing Tiaosi , Sabu Stephiya , Benderman Lesley Jasmine , Chen Yanghua 
Published: June 2021

Intestinal epithelial integrity and self‐renewal are two main features that maintain intestinal homeostasis. Tight junctions control epithelial integrity while intestinal crypt stem cells fuel epithelial self‐renewal. Claudin‐7 is a tight junction membrane protein present along the entire intestinal epithelium. Using our global (gCldn7−/−) and inducible intestinal‐specific (cCldn7−/−) claudin‐7 knockout mouse models, we found that claudin‐7 deletion induces rapid crypt stem cell loss and increases cell proliferation. We hypothesized that claudin‐7 regulates intestinal epithelial self‐renewal. To elucidate the underlying mechanism, we employed ex vivo organoid culture using Cldn7+/+ and gCldn7−/− small intestines. We found that claudin‐7 deletion not only decreases the survival of organoids and crypt stem cells, but also induces the formation of spheroids without villi compared to normal organoids with villi derived from Cldn7+/+ small intestines. In addition, immunostaining of Cldn7+/+ and gCldn7−/− organoids demonstrated that claudin‐7 deletion increases the proliferative epithelial cells and decreases the number of enterocytes, Paneth cells, enteroendocrine cells, and tuft cells. Interestingly, the number of goblet cells was unaffected which comports with our in vivo findings. Western blotting analysis revealed that β‐catenin (a key molecule in the Wnt signaling pathway) is upregulated in claudin‐7‐deficient intestines. Furthermore, immunostaining revealed that β‐catenin localizes predominately to the epithelial cell cytoplasm. More importantly, we found that activation of the Wnt signaling pathway increases the size of the claudin‐7‐deficient spheroids compared to claudin‐7‐positive organoids. These findings suggest that claudin‐7 plays a novel, but as yet unidentified function in the regulation of epithelial cell renewal in intestines. Claudin‐7 is required for regeneration of intestinal epithelium, survival of crypt stem cells, and differentiation of intestinal epithelial cells. Claudin‐7 also maintains the balance of epithelial cell proliferation and suppresses Wnt signaling pathway during normal intestinal epithelial renewal.Support or Funding InformationThis study is supported by the National Institute of Health grant DK103166.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Endosomal Escape of ASOs Internalized by Stabilin Receptors Is Regulated by Rab5C and EEA1.
Authors: Miller Colton , Harris Edward N. , Seth Punit P. 
Published: June 2021

One of the major drug classes that has been gaining traction in the pharmacological world has been the emergence of Antisense oligonucleotides (ASOs). Increased modifications of these drugs have led to major breakthroughs leading to a boost in this drug class being adapted to clinical trials. Second generation (Gen 2) Antisense oligonucleotides show increased nuclease stability and affinity for their RNA targets which has translated to improved potency and therapeutic index in the clinic. Gen 2 ASOs are typically modified using the phosphorothioate (PS) backbone modification which enhances ASO interactions with plasma, cell surface and intracellular proteins. This facilitates ASO distribution to peripheral tissues and also promotes cellular uptake after injection into animals. Recent work of ours has shown that a group of class H Stabilin receptors specifically internalize PS‐ASOs in the sinusoidal cells of the liver and the spleen. This work identifies a breakthrough candidate for the determination of a molecular pathway for these drugs to reach their desired target in the cell. Further exploration of the endocytotic uptake by these proteins has led to a rudimentary understanding of this pathway. By modulating expression of specific proteins involved in the trafficking and maturation of the endo‐lysosomal compartments, we show that the proteins Rab5C and EEA1 play a direct role in the early endosomal pathway. These proteins, along with Rab7A and LBPA (lyso‐lipid) in the late endosomal pathway, are important for trafficking of PS‐ASOs and facilitate their escape from endo‐lysosomal compartments after Stabilin‐mediated internalization. In conclusion, this research identifies key interacting partners involved in ASO‐containing endosomal maturation, as well as late endosomal partners involved in PS‐ASO endosomal release. This work also identifies the limiting proteins in the pathway for PS‐ASO translocation and escape from the endosome.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Use of gradient gel filtration as a refolding, buffer exchange and fine‐tuning step in denaturing protein purification
Authors: Tovar Carmel N. , Nguyen Samantha K. , Odunuga Odutayo O. 
Published: June 2021

Purification of recombinant proteins in denaturing conditions has many practical advantages. Chemical denaturants such as urea and guanidine hydrochloride are used to solubilize recombinant proteins from inclusion bodies. Proteins purified in denaturing conditions generally contain fewer contaminants compared to their counterparts purified in native buffers; thus reducing the number of steps in the purification scheme. Chemically denatured proteins can be stored for long time in buffer containing the denaturant and then refolded as required. Refolding is commonly performed by equilibrium dialysis and sometimes on metal affinity columns by diluting out the denaturant and allowing the protein to fold back into its native state. The refolding steps often result in significant loss of protein through aggregation. Using a modified gel filtration technique, we successfully refolded a spectrum of proteins that are either highly aggregation‐prone or contain several disulfide bonds or both, back to their native states. Some of the proteins are well known to precipitate significantly during refolding. However, using this technique, precipitation was either minimal or non‐existent. Secondary and tertiary structural analysis using circular dichroism and fluorescence microscopy coupled with functional assays confirmed that these proteins were correctly refolded and biologically active. This strategy provides a robust approach to refolding denatured proteins that can also be used simultaneously as buffer exchange and additional purification steps.Support or Funding InformationWelch Foundation Grant # AN‐0008This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A Novel Monomodular and Multifunctional Processive β‐1,4‐Endoglucanase Has Been Identified and Characterized from Porcine Gut Microbiome
Authors: Wang Weijun , Archbold Tania , Lam Joseph S. , Kimber Matthew S. , Fan Ming Z. 
Published: June 2021

Cellulose‐degrading enzymes play an important role in dietary fibre digestion of humans and animals, as well as in multiple industrial applications. Herein, a plasmid metagenomic expression library was constructed for mining cellulose‐degrading enzymes from the pig hindgut microbiota that was modulated by feeding a purified crystalline cellulose supplemented diet (Journal of Animal Science, 2012, 90 (Suppl. 4):400–402). One of the cellulase genes screened from this library was localized to a 2949‐base pair insert fragment p4818. Within the sequence preceding this enzyme‐coding region, ‐10 and ‐35 promoter elements were identified along with three transcription‐factor binding sites. The enzyme possesses a catalytic domain consistent with those of the glycoside hydrolase family 5 subfamily 2 (GH5_2), thus named as p4818Cel5_2A. The protein was overexpressed, purified, and characterized; and it displayed optimal enzymatic activity at pH 6.0 and 50 °C. The enzyme also exhibited resilience to trypsin digestion, where >70 % activity was retained after 6.0 h incubation with trypsin at 37 °C. p4818Cel5_2A exhibited a broad substrate profile with the highest specific activity towards β‐glucan, carboxymethyl cellulose (CMC), followed by hydroxyethyl‐cellulose and regenerated amorphous cellulose (RAC). The enzyme was also active on other substrates including crystalline cellulose substrates such as Avicel and Solkafloc, as well as the hemicelluloses of glucomannan and xyloglucan. Furthermore, based on viscosity and hydrolysis product analyses, the enzyme behaved like a typical endo‐β‐1,4‐glucanase, and displayed processivity towards RAC. In summary, p4818Cel5_2A exhibited novel enzyme properties for potential industrial applications.Support or Funding InformationRelated research activities have been supported by grants (to M.Z.F.) from the Ontario Ministry of Agriculture, Food and Rural Affairs (OMAFRA) New Directions & Alternative Renewable Fuels Research Program; and the University of Guelph Gryphon's LAAIR (Leading to Accelerated Adoption of Innovative Research) program.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
A molecular validation of serotonin signaling systems association with major depressive disorder (MDD) in South Indian population
Authors: Rashmi Subramanian , Sangilimuthu Alagaryadav , Srinivasan Dinesh Kumar , Balachandar Vellingiri 
Published: June 2021

Major depressive disorder (MDD) is a multi factorial disease that affects morbidity, mortality and quality of life. It is chronic and recurrent phenotype of complex etiological relationship between genetic and environmental factors. The serotonergic system plays an important role in the modulation of brain function. Tryptophan hydroxylase (TPH) converts tryptophan in the brain to 5‐hydroxytryptophan (5‐HTP). TPH is the rate‐limiting enzyme in 5‐HT synthesis. Most tissues synthesize 5‐HT, a relatively unstable substance at low levels. Minor changes in TPH abundance or the activity can dramatically alter 5‐HT content and serotonergic function. With regard to MDD, there is a strong evidence for serotonergic abnormalities. The present study was performed to validate the association of single nucleotide polymorphisms (SNPs) in several functional variants of serotonergic genes [serotonin‐transporter‐linked polymorphic region (5HTTLPR), TPH1 and TPH2] with MDD in South Indian population. We have analyzed genotypic polymorphism in well characterized sample of MDD patients and controls with PCR‐RFLP methods. We identified a significant difference in the frequency of 5HTTLPR, TPH1 and TPH2 gene alleles between patients and controls. In addition, there was a significant difference in genotypic distribution of 5HTTLPR and TPH2 alleles between these two groups. The distribution of 5HTTLPR genotype frequencies SS, SL, LL and TPH2 GG, TT, GT and SS, GG allele showed strong association with MDD. To our knowledge, the present study reveals for the first time that polymorphism in 5HTTLPR and TPH2 genes may play a role in susceptibility to MDD in South Indian population.Support or Funding InformationThis work was supported by research grant from the Karpagam Research Foundation, Karpagam Faculty of Medical Sciences and Research, Coimbatore, India.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
“Regulator of G‐protein Signaling 10 (RGS10) inhibits NF‐κB signaling, COX‐2 and inflammatory cytokine production in ovarian cancer cells”
Authors: Almutairi Faris , Hooks Shelley B. 
Published: June 2021

Epithelial Ovarian Cancer (EOC) is the fifth leading cause of cancer‐related deaths among females in the United States. NF‐κB signaling and production of inflammatory mediators have been strongly linked to ovarian cancer progression and chemoresistance. Regulator of G‐protein Signaling 10 (RGS10) regulates ovarian cancer survival and chemoresistance, and has been linked to NF‐κB signaling in immune cells. The goal of the current study was to determine the ability of RGS10 to inhibit NF‐κB signaling and subsequent inflammatory effects in ovarian cancer. RGS10 is a negative regulator of Gαi signaling and multiple Gi coupled GPCRs are implicated in ovarian cancer. Therefore, we predicted that RGS10 regulates NF‐κB signaling and inflammatory cytokines downstream of Gαi mediated‐pathway. We found that knock‐down of RGS10 enhanced NF‐κB phosphorylation and produced significantly higher levels of COX‐2 and TNF‐α mRNA compared to cells transfected with control siRNA in SKOV‐3 EOC cells. Surprisingly, we found that Pertussis toxin (ptx), which is a Gαi inhibitor, had no effects on upregulation of p‐NF‐κB, COX‐2 and TNF‐α expressions‐mediated by RGS10 knock‐down. These results provide insight in the mechanism that RGS10 utilizes to regulate ovarian cancer survival and chemoresistance.Support or Funding Information

National Institutes of Health
Rivkin Center for Ovarian Cancer.
This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Characterizing The Effects of Ascorbic Acid and Hyperbaric Oxygen on Glioblastoma Cells
Authors: DeBlasi Janine , Poff Angela , Ward Nathan , Koutnik Andrew , Rogers Christopher , Diamond David , Moss Sara , D'Agostino Dominic 
Published: June 2021

There is emerging evidence supporting the use of intravenous ascorbic acid (AA) as a pro‐oxidative, non‐toxic adjuvant therapy. Before AA is implicated in the clinic, however, more research needs to be conducted to better understand AA's mechanisms of action, and to determine what cancers would be most amenable to this adjunctive therapy. When administered intravenously, AA can achieve pharmacological, millimolar concentrations (~0.3–20 mM) and act as a pro‐oxidant in tumorous tissue, contrary to its antioxidant effects observed with oral administration, which produces micromolar, physiological concentrations (≤0.1 mM). Our study aims to evaluate the effects of pharmacological AA on glioblastoma cells (VM‐M3 and U87), and to see if another pro‐oxidative therapy, hyperbaric oxygen (HBOT), can sensitize cells to treatment. We hypothesize that 1‐hour treatment with AA will decrease VM‐M3 and U87 cell viability, and that co‐treatment with antioxidant N‐Acetylcysteine (NAC) will attenuate cell death. We also hypothesize that hydrogen peroxide mediates AA‐induced VM‐M3 and U87 cell death, and thus that exogenous catalase treatment will attenuate this effect. It was found that 1‐hour treatment with pharmacological AA decreased viability in both VM‐M3 and U87 cells, with VM‐M3 cells exhibiting greater sensitivity to treatment (assessed with crystal violet staining). HBOT did not enhance AA‐induced cytotoxicity in U87 cells; however, HBOT did sensitize VM‐M3 cells to AA treatment. Additionally, 1‐hour co‐treatment with AA and NAC, as well as AA and catalase, rescued cell death in U87 and VM‐M3 cells, suggesting that AA‐induced oxidative stress is mediated by hydrogen peroxide. This study also investigates the effects of AA and HBOT on HIF‐1 alpha, catalase, and transferrin receptor protein expression, yielding further insight into the pro‐oxidative role of AA in cancer.Support or Funding InformationWork supported by: American Physiological Society Undergraduate Research Excellence Fellowship (APS UGREF); Foundation (501c3); Metabolic Therapy and Cancer Research Account (#250244); Scivation: Florida High Tech Corridor Funding (#MED052‐0061361).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Citrullinated Histone 3 Contributes to Endothelial Barrier Dysfunction During Sepsis
Authors: Meegan Jamie , Yang Xiaoyuan (Sherry) , Jannaway Melanie , Chatterjee Victor , Yuan Sarah Y. 
Published: June 2021

Circulating extracellular histones are known to contribute to tissue injury in systemic inflammatory responses to infection, trauma, and burns. The mechanisms of histone modifications and their effects on inflammation are currently being investigated. One such modification, namely citrullination of histone 3, is catalyzed by the peptidylarginine deiminase 4 (PAD4) enzyme and is thought to initiate the process of NETosis (the release of neutrophil extracellular traps) in response to infection. NETs are composed of a chromatin web with attached proteases and histones, including citrullinated histone 3 (citH3). This study aimed to identify the effects of citH3 on endothelial barrier dysfunction during inflammation. We identified increased citH3 in the tissues and plasma of mice subjected to cecal ligation and puncture, a clinically relevant animal model of polymicrobial sepsis. In vivo, stimulation of mesenteric microvessels with citH3 elicited plasma protein leakage into the surrounding tissue. In cultured endothelial cell monolayers, treatment with citH3 induced barrier dysfunction as indicated by reduced transendothelial electrical resistance. Endothelial monolayer dysfunction partially resulted from disrupted adherens junction proteins VE‐cadherin and β‐catenin, as well as disturbed actin cytoskeletal homeostasis, as demonstrated by immunofluorescence with confocal microscopy. Taken together, the data suggest citH3 induces endothelial barrier dysfunction, which can exacerbate the inflammatory response during sepsis.Support or Funding InformationThis work is supported by NIH R01s HL070752 and HL126646.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Improved methods for characterizing ion channel expression in smooth muscle, endothelial and adventitial cells from collecting lymphatic vessels
Authors: Gui Peichun , Zawieja Scott , Li Min , Castorena‐Gonzale Jorge , Davis Michael J. 
Published: June 2021

Lymph is propelled centrally by collecting lymphatic vessels though active, twitch‐like contractions, which are initiated by a pacemaker that generates action potentials to drive calcium influx through voltage‐gated calcium channels in lymphatic smooth muscle cells (LMCs). Whether the pacemaker is intrinsic to LMCs or to another cell type in the vessel wall is not yet established. Both the frequency and amplitude of lymphatic contractions are regulated in turn by a number of additional factors, including the transmural pressure difference, wall shear stress, neurotransmitters, locally‐ and remotely‐generated vasoactive factors, and endothelial‐derived products. Difficulties in identifying the complex interactions of the various cell types within the vessel wall limit discernment of the mechanisms underlying lymphatic pacemaking and the factors that modulate it. Cells within the wall include LMCs, lymphatic endothelial cells (LECs), dendritic cells, macrophages, mast cells, fibroblasts, nerve endings and possibly glia. We developed methods for purifying specific cell populations and determining the expression and function of important ion channels with a focus on identifying the ion channels that control and modulate lymphatic function. Our strategy was to utilize fluorescent reporter mice (SMMHCGFP, Prox1GFP and PDGFRaGFP, respectively) to 1) identify the distribution of LMCs, LECs and a population of adventitial fibroblast‐like cells in mouse inguinal‐axillary lymphatic (IALs) vessels; 2) enzymatically dissociate IALs and utilize fluorescent activated cell sorting of GFP+ cells to obtain pure cell populations for transcriptional analysis of key ion channels; 3) confirm the presence and functionality of identified ion channels through whole cell patch clamp of GFP+ cells. LMCs expressed message for SMA, TMEM16A, SK3, BK and Cav1.2 but were negative for eNOS, Prox1 and VE‐caderin. LECs were positive for VE‐cadherin and eNOS but negative for SMA, SMMHC, PDGFRa, Cav1.2, TMEM16A and all SK‐IK‐BK channels. PDGFRa+ cells were positive for VE‐cadherin, SMA, SMMHC, TMEM16A, Cav1.2 and SK1, but negative for eNOS and all other SK‐IK‐BK channels. Single GFP+ cells used for patch clamping revealed spindle‐shaped LMCs expressing functional TMEM16A, Cav1.2, Cav3 and variable BK currents, LECs (also spindle‐shaped) that did not exhibit inward cation current or SK‐IK current, and rounded PDGFRa+ cells with SK currents. Our techniques improve on whole‐vessel and explant analyses, align with the results of recent functional studies, and show the feasibility of using these methods to more accurately determine the functional roles of the various cell populations in the lymphatic wall.Support or Funding InformationNIH HL122578This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.