bluuebunny/crossref_metadata_2025_split huggingface dataset:
Effect of Maternal Age on Offspring Growth and Craniofacial Development After Early Prenatal Alcohol Exposure in a Mouse Model
Authors: Draghici Diana , Hardy Daniel B. , Allman Brian , Willmore Katherine 
Published: April 2020

Prenatal alcohol exposure (PAE) can result in fetal alcohol spectrum disorder (FASD), defined as a continuum of developmental defects including growth restriction, cognitive‐behavioral deficits, and craniofacial anomalies. Despite the known consequences of PAE, there is great variation in craniofacial and behavioral outcomes of affected children even when considering timing and quantity of exposure; phenotypic variation which could arise from maternal differences. In the present study, we asked: Does maternal age influence the nature and extent of growth restriction and craniofacial defects of PAE in offspring? To address this question, we used a C57BL/6 mouse model, as it enables precise control of dosing, gestational timing and maternal age. Dams were divided into four groups: 1. ethanol‐treated young dams (6–10 weeks); 2. control young dams; 3. ethanol‐treated old dams (6–7 months); and 4. control old dams. All dams were injected with either ethanol (treatment groups) or vehicle solution (control groups) on gestational day 7.5. Offspring growth restriction was analyzed by measures of body mass, liver mass, and body‐to‐liver mass ratio at birth. Micro‐CT scans of newborn soft tissue and skeletal craniofacial features underwent 3D analyses of shape and size to compare craniofacial phenotypes among treatment groups. Initial results of young ethanol‐treated and control groups demonstrated that ethanol‐treated offspring were born prematurely and showed significant growth restriction. Both soft tissue and skeletal craniofacial features were altered in the ethanol‐treated group, consistent with the FASD phenotype. Surprisingly, craniofacial shape of ethanol‐treated offspring was less variable than controls. Data collection for the old dam groups is ongoing, and we predict that offspring of old dams will have more severe growth and phenotypic consequences from PAE, exhibiting greater phenotypic variation compared to offspring of young dams. As many women are delaying childbirth, there is an increasing need to carryout preclinical studies with appropriate experimental controls to better understand the relative risk of PAE and maternal age, and ultimately contribute to public health policy for pregnant women.Support or Funding InformationFunded by Children’s Health Research Institute, Internal Research Grant Fund to KEW “The Effects of Moderate Prenatal Alcohol Exposure on Craniofacial Morphology and Development” R5211A04
ZNF365 is overexpressed in Pulmonary Fibrosis
Authors: Urista Juan Manuel , Maldonado Mariel , Calyeca Jazmín , Ramírez Remedios , Ortíz-Quintero Blanca , Becerril Carina , Selman Moisés , Pardo Annie 
Published: April 2020

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease of unknown etiology characterized by the expansion of fibroblast population and excessive extracellular matrix (ECM) deposition. Transforming growth factor‐beta 1 (TGF‐β1) is one of the most potent mediators of the fibrotic response and induces fibroblast proliferation, and their differentiation to myofibroblasts, cells characterized by increased contractility, apoptosis resistance and the production of multiple ECM components (mainly collagens).Global gene expression of human lung fibroblasts stimulated with TGFβ performed in our laboratory demonstrated the overexpression of ZNF365, a zinc finger protein that belongs to the C2H2 domain family that has been poorly described in the literature. The purpose of this study is to evaluate ZNF365 expression in IPF lung tissue, the expression of its ortholog Zfp365 in mice lung tissue and its functional effects on human lung fibroblasts and epithelial cells in vitro.Expression of ZNF365 and Zfp365 were evaluated by qPCR and WB in human and mice samples, as well as in fibroblasts and epithelial cells stimulated with TGFβ. Localization of the protein was performed by immunohistochemistry in IPF lungs and in bleomycin‐induced lung fibrosis in mice. For silencing experiments, specific siRNAs and scrambled controls designed for ZNF365 were used. For overexpression, cells were stably transfected with ZNF365. Growth rate and proliferation were analyzed using WST‐1 and CyQUANT reagent, and apoptosis was measured by flow cytometry.We found that ZNF365 is overexpressed in IPF lung tissues and localized in alveolar and bronchial epithelial cells and in fibroblasts/myofibroblasts. The orthologous Zfp365 is overexpressed in lung tissue from bleomycin instilled mice, and the immunoreactive protein was observed in alveolar and airway epithelium. In vitro studies confirmed that ZNF365 is overexpressed in control and IPF fibroblasts stimulated with TGFβ. Likewise, TGFβ induced the upregulation of ZNF365 in a human alveolar epithelial cell line. Functional assays revealed that ZNF365 silencing in human lung fibroblasts and alveolar epithelial cells induced a significant reduction of growth rate and proliferation compared with the scrambled and WT controls. Preliminary data show that silencing of this protein increases senescence‐asociated β‐galactosidase activity and increases expression of the histone variant γH2A.X, another senescence marker. These results demonstrate that ZNF365 is upregulated in IPF and experimental lung fibrosis and may be involved in the regulation of proliferation and senescence of human lung cells.Support or Funding InformationSupported by PAPIIT 201520
Disruption of Gankyrin‐Mediated Epithelial‐Mesenchymal Transition Due to Small Molecule Binding
Authors: Kabir Abbas Saifi , Muth Aaron 
Published: April 2020

IntroductionGankyrin is a 25 kDa protein that plays a crucial role in cellular growth, proliferation, metastasis, and epithelial‐mesenchymal transition (EMT). It is a highly conserved protein which has 7 ankyrin repeats that binds to the S6 ATPase subunit of the 26S proteasome. It has been shown that gankyrin exerts its oncogenic activities by interacting with a variety of tumor suppressor proteins (TSPs) such as p53 and retinoblastoma protein (Rb). The interaction between gankyrin and certain TSPs promotes their proteasomal degradation. Specifically, the degradation of Rb appears to play a role in gankyrin‐mediated EMT which also contributes towards the highly metastatic nature of gankyrin‐overexpressing cancers. Recently, a small molecule binder of gankyrin has shown to inhibit gankyrin’s ability to aid in the degradation of certain TSPs. It has been previously suggested that small molecule binding of gankyrin induces a conformational change in gankyrin’s structure, which may disrupt its interactions with various TSPs, including Rb. Therefore, our work seeks to show that a small molecule can induce a conformational change which can ultimately affect gankyrin’s ability to induce EMT in certain cancers. Our work is supported by results from fluorescence experiments, western blotting, EMT assays and thermal shift assays.MethodsIn this research, we hypothesize that the conformational change brought about by the small molecule, not only disrupts gankyrin’s binding with the TSPs, but also with proteins that are involved in gankyrin mediated EMT in certain gankyrin overexpressing cell lines. The activity of the proteins involved in EMT, such as STAT3, is also regulated by certain TSPs such as Rb. To test our hypothesis, we performed a series of cell based assays to determine the dose‐dependent effect of a small molecule on TGF‐β induced EMT in gankyrin overexpressing cell lines. We will also perform a series of western blotting and co‐immunoprecipitation (co‐IP) assays to observe the effects of the small molecule on the levels and activity of specific EMT‐related proteins, such as E‐cadherin. We are also seeking to observe the effects of small molecule binding to gankyrin on its interaction with the proteasome, certain TSPs and specific EMT proteins.DiscussionThe cell based assay showed a dose dependent decrease in the TGF‐β induced EMT in gankyrin overexpressing cell lines, in presence of a small molecule. Fluorescence‐based and thermal shift assays strongly suggest that small molecule binding of gankyrin induces a conformational change. Western blot and co‐IP experiments are currently underway.ConclusionGankyrin plays the role of a key middle link which mediates both cancer cell proliferation and metastasis. It achieves this by interacting with and regulating both TSPs and EMT‐associated proteins.
Raphe‐Derived Neuromodulators Within the Retrotrapezoid Nucleus (RTN) Modulate Ventilatory Chemoreflexes In Vivo.
Authors: Dillard Matt , Duffy Erin , Cook-Snyder Denise , Hodges Matt 
Published: April 2020

Brain chemoreceptors, including serotonergic (5‐HT) neurons in the medullary raphe (MR), play a vital role in the control of breathing through feedback control of blood gas homeostasis and modulating breathing to match metabolic demand. Recent in vitro studies have highlighted important contributions of raphe‐derived neuromodulators (via 5‐HT2 and/or 5‐HT7 receptors) to the chemosensitivity of additional putative brain chemoreceptors, the Phox2b‐expressing retrotrapezoid nucleus (RTN) neurons. However, the importance of raphe‐derived neuromodulation of the RTN in the hypoxic and hypercapnic ventilatory chemoreflexes has not been tested in vivo. We hypothesized that unilateral ablation of serotonergic terminals within the RTN will attenuate the ventilatory chemoreflexes in vivo. To test this hypothesis, 8‐week‐old Sprague‐Dawley rats received unilateral stereotaxic injections of the 5‐HT pre‐synaptic terminal toxin 5,7‐Dihydroxytryptamine (5,7‐DHT; 2 mg/ml, 2 μl vol.) targeted to the RTN under isoflurane anesthesia 30 minutes after pretreatment with desipramine (30mg/kg; IP; to limit off target effects on other aminergic neurons/terminals). A control group received an equal volume injection of vehicle. Breathing was measured via whole body plethysmography while exposed to room air, hypercapnia (7% CO2 challenge), or hypoxia (12% O2 challenge) before and 3, 5, 7, 10, and 14 days post‐lesion. Three days after injection the lesion group (n=6) showed significant decreases in the hypoxic (−44%) and hypercapnic (−43%) ventilatory chemoreflexes. The attenuation of both chemoreflexes was still present 7 days post‐lesion but returned to baseline by 14 days post‐lesion. The control group (n=6) showed a similar pattern, with a significant decrease in both the hypoxic and hypercapnic ventilatory chemoreflexes at 3 days post injection but returned to baseline by 7 days post‐injection. Despite both groups showing decreased responses 3 days following surgery, the 5,7‐DHT group showed a 23% lower response to hypoxia compared to control injections (p = .01), and a 20% lower response to hypercapnia than control injections at 3 days (p = .03), indicating a greater effect from neurotoxic lesions than surgery per se. In ongoing studies, fluorescent microbeads were added to injected solutions to mark/confirm injection location, and immunofluorescent staining will be used to determine if: 1) control injections created a lesion, and 2) if 5‐HT terminals are decreased at 3 and 7 days but return by 14 days post‐DHT lesion. Our preliminary data suggest that serotonergic pre‐synaptic terminals within the RTN facilitate both hypoxic and hypercapnic ventilatory chemoreflexes in vivo.Support or Funding InformationFunded by NIH HL122358.
Inhibitor of MyoD family A increased fibronectin production by mesangial cells
Authors: Shotorbani Parisa Yazdizadeh , Chaudhari Sarika , Tao Yu , Tsiokas Leonidas , Ma Rong 
Published: April 2020

Overproduction of extracellular matrix proteins, including fibronectin by glomerular mesangial cells (MCs) contributes to the development of diabetic nephropathy. Inhibitor of myogenic differentiation family isoform a (I‐mfa) is a multifunctional cytosolic protein, functioning as a transcription modulator or plasma membrane channel protein regulator. However, its renal effects are unknown. The aim of the present study was to determine if I‐mfa regulated fibronectin production by MCs and the mechanisms involved. In cultured human MCs, I‐mfa expression levels were upregulated and downregulated by transiently transfecting I‐mfa expression plasmids and siRNA against I‐mfa, respectively. Western blot analysis was conducted to determine the abundance of fibronectin protein in human MCs with different treatments and in kidney tissues from rats with and without diabetic nephropathy. RT‐PCR analysis was performed to assess mRNA levels of I‐mfa and fibronectin in human MCs with various treatments and in glomeruli from mice with and without diabetic nephropathy, respectively. We found that overexpression of I‐mfa significantly increased fibronectin protein abundance in human MCs. Silencing I‐mfa significantly reduced the expression level of fibronectin mRNA and blunted TGF‐β1‐stimulated production of fibronectin protein. We further found that high glucose, a central pathogenic stimulus in diabetes increased I‐mfa protein content in a time (≥ 48 h) and concentration (≥ 25 mM) dependent manner. Although high glucose exposure increased fibronectin at the protein level, it did not significantly elevate transcript levels of fibronectin in MCs. In agreement with the data from cultured cells, the abundance of I‐mfa protein was significantly greater in the renal cortex of rats with diabetic nephropathy compared to non‐diabetic rats. However, there was no significant difference in the level of glomerular I‐mfa mRNA between the mice with and without diabetic nephropathy. Moreover, hydrogen peroxide significantly increased I‐mfa protein abundance in a dose‐dependent manner (≥ 100 μM) in cultured human MCs. Antioxidants polyethylene glycol‐catalase, ammonium pyrrolidithiocarbamate and N‐acetyl cysteine significantly blunted high glucose‐induced increase of I‐mfa protein abundance. Taken together, our study suggests that an increased level of I‐mfa induced by high glucose/diabetes through the production of reactive oxygen species stimulates fibronectin production by MCs.Support or Funding InformationNIH/NIDDK (1RO1 DK115424); AHA Southwest Affiliate (16GRNT27780043); Harry S. Moss Heart Trust Award
Studying Cardiovascular Disease Using Human Stem Cell‐derived 3D Cardiomyocytes
Authors: Dash Nicholas , Bohl Veronica , Toth Charles 
Published: April 2020

Heart disease is the leading cause of death in both men and women in the United States. This experiment provides insight on how using metabolites derived naturally from the microbiome can influence cardiac inflammation. 3D cardiomyocytes were derived from human induced pluripotent stem cells through the utilization of small molecules and growth factors. Quantitative PCR was performed to verify that the organoids expressed genes associated with cardiomyocyte cells. Primers for the genes PGK1, LDHA, BNIP3, and c‐JUN were used for the qPCR, as these genes are expressed more prominently in cardiomyocyte cells than the undifferentiated iPSCs. Confocal microscopy was performed to make sure that specific cardiac markers are being expressed in the 3D cultures. Cardiovascular disease was modeled by inducing inflammation in the organoids using lipopolysaccharide (LPS), a gram‐negative bacteria membrane product associated with inflammation and septic shock, indoxyl‐3‐sulfate (I3S), which is representative of uremic toxins build‐up, and poly (I:C), which simulates a viral infection. Gene expression of pro‐inflammatory genes in the organoids was studied using qPCR and TNF‐α and IL‐6 secretion was determined by ELISA. Cell viability was analyzed by a luminescence assay. The goal of the experiment was to understand how metabolites naturally derived from the microbiome affect the inflammatory responses occurring in cardiovascular disease.Support or Funding InformationProvidence College Undergraduate Research Grant Committee and Gift from Dr. John Mullen
Uncovering and Exploiting HUH‐endonuclease ssDNA Recognition for Protein‐DNA Bioconjugation
Authors: Tompkins Kassidy , Nelson Andrew , Litzau Lauren , Everett Blake , Houtti Mohamed , Pornschloegl Leland , Lemmex Andrew , Swanson Lidia , Aird Eric , Evans Robert , Knights Dan , Gordon Wendy 
Published: April 2020

HUH‐endonucleases (histidine‐hydrophobic‐histidine) create nicks in hairpin loops at the origin of replication during viral rolling circle replication and bacterial plasmid conjugation. Intriguingly, these metal‐dependent enzymes can form rapid, covalent, and specific linkages with single‐stranded DNA (ssDNA) under physiologic conditions. In recent years, we have developed these nucleases as a multiplexable, protein‐DNA bioconjugation (HUH‐tags) technology for live cell imaging, force spectroscopy, genome editing, cell targeting, etc. Though viral HUH‐tags bear valuable biophysical properties for bioconjugation, it would seem multiplexing the viral HUH‐tags we explored would be difficult because they target a highly conserved nonamer sequence (5′‐TA(A/G)TATT*AC‐3′). Interestingly, we noticed varying degrees of promiscuity in sequence recognition between these HUH‐tags. Therefore, we hypothesized that we could exploit or engineer sequence orthogonality using variants of the target sequence. To this end, we generated an unbiased ssDNA library containing all permutations of the target sequence 5′ of the cleavage site (16,384 sequences) and developed a next‐generation sequencing approach that assesses the degree of HUH‐tag cleavage of all sequences contained in the library. Strikingly, we found robust sequence orthogonality and can now multiplex several of these viral HUH‐tags. Our ultimate aim is to engineer the target sequence recognition to create a vast toolkit of multiplexable, viral HUH‐tags. However, relatively few structural and mechanistic insights into the ssDNA recognition have limited our ability to rationally engineer HUH‐tags for this purpose. Addressing this bottleneck, we solved the first ever structure of a HUH‐tag derived from a viral replication initiator protein from wheat dwarf virus bound to its ssDNA target generating an atomic resolution map of protein‐DNA contacts. Together, our structural insights and sequence cleavage screening method may allow us to rationally develop and engineer a powerful toolkit of fusion tags for multiplexable protein‐DNA bioconjugation.Support or Funding InformationNIH: R35GM119483, University of Minnesota
Differential Contribution of Neuronal Uptake Transporters to Oxaliplatin Peripheral Neurotoxicity
Authors: Huang Kevin M. , Leblanc Alix F. , Alberti Paola , Sprowl Jason A. , Wang Jing , Lustberg Maryam B. , Cavaletti Guido , Hu Shuiying , Sparreboom Alex 
Published: April 2020

BACKGROUNDPeripheral neurotoxicity afflicts up to 90% of patients with colorectal cancer receiving oxaliplatin therapy. Oxaliplatin preferentially accumulates in and causes toxicity to peripheral sensory neurons present in the dorsal root ganglia (DRG), but mechanistic details of the transporters involved in this process remain uncertain. In the present study, we set out to unambiguously identify these transporter(s) to support the notion that oxaliplatin accumulation in DRGs is a key trigger for the development of peripheral neurotoxicity.METHODSEngineered mouse models with transporter deficiency were used to assess peripheral neurotoxicity by the Von Frey Hair test. Immunofluorescence and primary ex vivo culture of DRGs were performed to determine localization, expression, and function. Pertinent findings were validated in Sprague‐Dawley rats as a secondary model organism, while the translational feasibility of using transport inhibitors to mitigate oxaliplatin‐induced peripheral neurotoxicity was demonstrated in preclinical models of colorectal cancer.RESULTSComparative in vitro and in vivo association studies revealed that deficiency of OCT2 confers significant protection against acute and chronic peripheral neurotoxicity, but this was not observed for the putative oxaliplatin transporters OCT3, OCTN1, OATP1B2, OATP2B1, and MATE1. Within DRGs, OCT2 was localized to satellite glial cells (SGCs), and accumulation of oxaliplatin in ex vivo cultures of SGCs was prevented by pretreatment with the OCT2 inhibitor, dasatinib. The importance of this transport system was verified in rats, where pretreatment with dasatinib in wild‐type rats recapitulated paw sensitivity phenotypes observed in OCT2‐deficient rats. Dasatinib did not influence the systemic clearance of oxaliplatin, indicating that the observed neuroprotection is directly mediated by inhibition of neuronal transport. At the applied dose and schedule that afford neuroprotection, we found that dasatinib did not negatively affect the antitumor properties of oxaliplatin against colorectal cancer cells in vitro and in vivo.CONCLUSIONWe identified a previously unrecognized, SGC‐specific pathway of oxaliplatin‐induced neurological injury that is mediated by the organic cationic transporter, OCT2, which can be targeted pharmacologically without compromising the anticancer properties of oxaliplatin. These findings not only shed light on the etiology of oxaliplatin‐induced neurotoxicity, but also provide a rationale for the development of targeted interventions using transporter inhibitors to mitigate a debilitating side effect associated with conventional chemotherapy.Support or Funding InformationPelotonia Graduate Fellowship and NIH grants: R01CA187176 and R01CA215802
Human Primary Osteoarthritic Chondrocytes Reared in Long‐term, Serum‐free, Three‐dimensional Alginate Culture Remain Metabolically Stable: A Nucleic Acid Study
Authors: Healy Andrea , Morris Gregory Philip , Popper Hannah , Mattioli Patrisia , Laird Patrick , Milton LaBraya , Pollard Mark , Miller Lawrence , Behling Kathryn C. , D’Angelo Marina 
Published: April 2020

The pathogenic process that defines osteoarthritis (OA) is the imbalance between excess degradation of the extracellular matrix (ECM) in articular cartilage and a decreased ability of chondrocytes to remodel the matrix. There are many factors that contribute to this disease including unchecked actions of matrix metalloproteinase 13 (MMP‐13), an enzyme specific for the breakdown of collagen type II which is the main component of cartilage extracellular matrix. We have developed a three‐dimensional, serum‐free culture system to rear primary, human OA chondrocytes (HOACs). However, we had noted that the ECM phenotype of these cells seemed to be changing when cultured past 5 days. Primary human articular chondrocytes were isolated from patients who underwent surgery for total knee arthroplasty (TKA). The medial and lateral aspects of the joint samples were kept separated and labeled as greatest or least pathology. The number of live cells were determined and plated at 1.8 × 106 cells/0.5ml alginate per well of a 12‐well plate. Cells were cultured in complete serum‐free media for 11 days with RNA and DNA collected at days 2, 5, 8 and 11. Total RNA and DNA were isolated via Trizol reagent protocol. Quantitation of nucleic acids was accomplished with the NanoDrop system. Over the eleven days in culture there was no statistically significant difference in the total amount of RNA isolated either in the least pathology samples (day 2=4.3ugRNA/culture versus day 11=2.8ugRNA/culture) or the greatest pathology samples (day 2=5.3ugRNA/culture versus day 11=6.2ugRNA/culture). The same was true for the total DNA isolated from the least pathology samples (day 2=1.9ugDNA/culture versus day 11=1.6ugDNA/culture) or the greatest pathology samples (day 2=8ugDNA/culture versus day 11=1.7ugDNA/culture). Future studies include quantitative, real‐time PCR to measure expression of metabolic agents including MMPs and collagens present in OA chondrocytes. This study is the first of its kind to isolate RNA and DNA from completely serum‐free, three‐dimensional cultures of primary human OA chondrocytes.Support or Funding InformationThis study has been supported in part by the New Jersey Health Foundation and PCOM Division of Research