bluuebunny/crossref_metadata_2025 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 Information

Funded 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 Information

Supported by PAPIIT 201520
Disruption of Gankyrin‐Mediated Epithelial‐Mesenchymal Transition Due to Small Molecule Binding
Authors: Kabir Abbas Saifi , Muth Aaron
Published: April 2020

Introduction

Gankyrin 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.

Methods

In 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.

Discussion

The 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.

Conclusion

Gankyrin 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 Information

Funded 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 Information

NIH/NIDDK (1RO1 DK115424); AHA Southwest Affiliate (16GRNT27780043); Harry S. Moss Heart Trust Award
Identification of Two Pathogens in a Culture‐Negative Prosthetic Joint Infection Using Nanopore Sequencing
Authors: Petersen Lauren , Tsongalis Gregory J.
Published: April 2020

Periprosthetic joint infection (PJI) following total joint arthroplasty (TJA) is challenging to diagnose due to lower yield in pathogenic identification (~50%) using conventional microbiologic culture techniques. Here we present a case in which two pathogens were detected using nanopore sequencing from a 50 year‐old male with a suspected, yet culture‐negative, PJI. Three swabs were taken from multiple sites of drainage surrounding the suspected infected right knee joint following a routine arthroplasty (original TKA was done three years prior). DNA was extracted from the swabs using the MasterPure™ Gram Positive DNA Purification Kit (Lucigen). Sample libraries were prepared using the Rapid PCR Barcoding Kit (Oxford Nanopore Technologies) and sequenced on a v9.4.1 MinION flow cell. A total of 1,056,710 reads from all three swabs combined were aligned using a minimum Q score of 7.0 to the RefSeq database on the EPI2ME platform using the “What’s in my pot?” app. A total of 5,540 reads (0.52%) aligned to the bacteria Corynebacterium with the most abundant species being detected as Corynebacterium simulans. A total of 1,886 (0.18%) mapped to the fungi Malassezia globosa. Due to the patient having a visible arthrocutaneous fistula combined with visualized drainage from the joint using a methylene blue challenge test, the prosthetic knee was removed and an antibiotic spacer that contained 6 gm vancomycin, 7.2 gm tobramycin and 100 mg of amphotericin B was implanted. The patient also received a 6 week course of ceftriaxone infusions and has shown rapid clinical improvement. While our sequencing results were not used directly for patient care in this case, it demonstrates the promise that nanopore sequencing has for future use in infectious disease diagnostics.
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 Information

Providence 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 Information

NIH: R35GM119483, University of Minnesota
Differential Effects of Physiological Pyrophosphate on the Kinetics of Productive and Abortive Transcription Initiation
Authors: Evensen Claire Elizabeth , Henderson Kate , Palatnik Benjamin , Ishikuri Taka , Plaskon Dylan , Record M. Thomas
Published: April 2020

Transcription initiation is the first and most highly regulated step of gene expression, and a biophysical characterization of its mechanism is needed. Previous studies found two, roughly equal populations of DNA‐RNA open complexes (OCs) in transcription initiation: productive OC that escape from the promoter to make full length (FL) RNA and nonproductive OC that stall after synthesis of a short RNA, slowly release that RNA, and reinitiate (abortive initiation). The kinetics and mechanism of productive transcription initiation by E. coli RNA polymerase (RNAP) at the λPR promoter were recently determined without added pyrophosphate (PPi).1 Because each polymerization step of transcription releases PPi and total PPi concentrations in the cell are in the low mM range, it is important to determine the effects (e.g. product inhibition) of PPi on the catalytic and translocation steps of initiation, and compare with recent results for PPi effects on elongation.2 In the research reported here, we extend existing quantitative 32P‐detected initiation kinetic assays to physiological concentrations of PPi and assess PPi effects on both the amounts and rates of the steps of productive and abortive initiation. Using phosphorimager analysis of gel separations and one‐dimensional thin layer chromatography, we study single‐round initiation of FL RNA synthesis at RNAP‐λPR open complexes under conditions where overall forward rate constants for each step of NTP incorporation in hybrid extension up to and beyond the escape point (11bp for initiation from λPR promoter) were previously determined.1 This will allow determination of PPi effects on each step, and potentially a dissection into effects on translocation and catalytic steps. Results to date show distinct PPi effects on productive and nonproductive initiation. The level of FL RNA synthesis by productive complexes is significantly reduced, but the level of abortive initiation is increased, indicating that previously‐productive OC are stalled by PPi. An understanding of PPi effects on productive and abortive initiation is critical to future assessments of the regulation and fidelity of bacterial transcription initiation in vivo, and the in vivo significance of short abortive RNAs as regulators.
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

BACKGROUND

Peripheral 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.

METHODS

Engineered 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.

RESULTS

Comparative 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.

CONCLUSION

We 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 Information

Pelotonia 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 Information

This study has been supported in part by the New Jersey Health Foundation and PCOM Division of Research
Regulators Of G‐Protein Signaling 2 And 4 Differentially Regulate the Rewarding Effects of Nicotine and Cocaine
Authors: Emerson Nathaniel , Asswini Sara , Rose Madison D. , Seeley Sarah , Rorabaugh Boyd R. , DSouza Manoranjan S.
Published: April 2020

Addiction to nicotine and cocaine is a major cause of preventable premature morbidity and mortality. The reinforcing effects of nicotine and cocaine are primarily mediated by increasing dopamine in the mesolimbic reward pathway. The effects of neurotransmitters like dopamine in this pathway are mediated by G protein‐coupled receptors (GPCRs). Interestingly, regulator of G protein‐signaling (RGS) proteins negatively regulate the functioning of GPCRs. In this study we specifically focused on the role of RGS2 and RGS4, which are extensively distributed in the mesolimbic reward pathway. This study evaluated the effects of RGS2 and RGS4 in the rewarding effects of nicotine (0.5 mg/kg; s.c.; base) and cocaine (15 mg/kg; i.p.) using conditioned place preference procedure (CPP) in mice lacking either RGS2 (i.e. RGS2 KO) or RGS4 (RGS4 KO) and their wildtype counterparts. Because a role of sex has been reported in the effects mediated by different RGS proteins, both male and female mice lacking either RGS2 or RGS4 were studied while assessing the rewarding effects of cocaine and nicotine. Cocaine and nicotine‐induced CPP was observed in all wildtype mice irrespective of genotype and sex. Cocaine‐induced rewarding effects were attenuated in male RGS4 KO mice, but not male RGS2 KO mice compared to respective saline controls. In contrast, nicotine‐induced rewarding effects were attenuated in male RGS2 KO mice, but not in male RGS4 KO mice compared to respective saline controls. Interestingly, knockout of either RGS2 or RGS4 in female mice had no effect on nicotine or cocaine‐induced rewarding effects, respectively. Thus, these data suggest both genotype and sex‐dependent differential effects of RGS2 and RGS4 in the rewarding effects of nicotine and cocaine. Clinically, the data support a role for targeting RGS2 and RGS4 for the treatment nicotine and cocaine addiction, respectively.

Support or Funding Information

This work was supported by a Bower, Bennett and Bennett grant awarded to Dr. Manoranjan S. D’Souza by the Raabe College of Pharmacy, Ohio Northern University (ONU), Ada, Ohio.
Bone Morphogenetic Protein (BMP) Transcripts in Kidney Tissue of Cats with Renal Dysfunction
Authors: Panickar Kiran S. , Tavener Selena , Hollar Regina L. , Jewell Dennis E.
Published: April 2020

Bone morphogenetic proteins (BMPs) are growth factors that belong to the transforming growth factor‐β (TGF‐β) superfamily, and till date 15 BMPs have been described. BMPs, first described for their role in bone and cartilage formation, also play a role in renal fibrosis in chronic kidney disease (CKD). There is evidence to indicate that in rodent models of CKD, administration of recombinant BMP1–3 increases renal fibrosis whereas administration of a BMP1–3‐neutralizing antibody or BMP‐7 antibody reduces renal fibrosis and preserves renal function. The aim of the present study was to investigate changes in gene expression in the renal cortex obtained from cats with kidney disease or calcium oxalate stone formers (CaOx) at necropsy, to identify BMPs associated with renal dysfunction in cats and potential fibrosis. At time of death the circulating levels of creatinine as well as symmetric dimethyl arginine (SDMA), both markers of kidney decline in cats, were significantly higher in cats with renal disease (n=11) or stone‐forming cats (CaOx, n=12) when compared to controls (n=19). Using RNAseq in kidney tissue, we found a modest, but significant, increase in the expression of BMP‐1 in cats with kidney disease (2.48 fold) and stone formers (1.72 fold), compared to controls (both p<0.01). While the increase in BMP‐2 in CaOx cats was significant (1.46 fold; p<0.05 vs Con), the increase in cats with kidney disease was not (1.23 fold; NS). BMP2K, a BMP‐2 inducible kinase, was significantly increased in both kidney disease (1.43 fold) and CaOX (1.46 fold) (both p<0.05). In contrast, a significant decrease in BMP4 was observed in both groups (<2.2 fold and 1.68 fold in kidney disease and CaOx, respectively; both p<0.001 vs Con). A decrease was also seen in CRIM 1, a protein associated with podocyte filtration function and whose reduction is associated with fibrosis, in both groups. BMP‐7, whose potential therapeutic role in treating CKD and reversing fibrosis has been documented, was modestly decreased in both groups (both less than 1.5 fold) compared to controls. Given that there was an increase in all three forms of TGFβ (TGFβ1, TGFβ2, and TGFβ3), a potent initiator of renal fibrosis, in both groups, and a decline in BMP‐7, an endogenous inhibitor of TGFβ signaling in fibrosis, compared to controls, our results profile the BMPs potentially associated with renal fibrosis in cats that may contribute to kidney dysfunction. In summary, a nutritional therapy to slow the progression of kidney dysfunction may benefit from the inclusion of dietary ingredients that attenuate renal fibrosis in cats.

Support or Funding Information

This study was funded by Hill’s Pet Nutrition, Inc
Unraveling the B to A Conformational Transition in Duplex DNA at Single Base Pair Resolution
Authors: Jose Davis , Kalisz Michal , Lawson Kirsten
Published: April 2020

DNA shows conformational heterogeneity and the interest in non‐canonical DNA conformations is increasing as these conformers are found to be involved in many important biological process. Although triple helices, quadruple helices and supercoils are the most studied structures, the conformational transition of the standard B‐form DNA helix to A‐form DNA was the first observed one. The transition of B‐DNA double helix to A‐form is essential for biological functions as recognized by the presence of A‐form DNA in many protein‐DNA complexes. Even though it is known that the B to A conformational transition occurs, the specifics like where in the DNA it originates, how it propagates, and the detailed step‐by‐step mechanism involved is still unknown. By using site specifically positioned fluorescent oligonucleotides, we explored the local and global conformational changes in this transition. Our results showed that by using 2‐Aminopurine (2‐AP), a fluorescent analogue of Adenine, we could monitor the local and global conformational change simultaneously. As a next step our aim is to introduce spermine or spermidine to the system at different ethanol percentages and monitor the spectral changes at various intervals of this transition. This will help us to understand the fundamentals of the long known B to A DNA conformational transition.
Local application of FTY720 hydrogel to the carotid body area reversed hypertension induced by obesity and leptin infusion
Authors: Shin Mi Kyung , Kim Lenise J. , Tang Winnie Wan-yee , Yeung Bonnie Ho-yee , Gu Chenjuan , Mitrut Roxana , Sham James S. K. , Cui Honggang , Polotsky Vsevolod Y.
Published: April 2020

RATIONALE

Obesity increases the level of an adipocyte‐produced hormone leptin, which enhances the sympathetic activity leading to hypertension. However, the mechanism of the hypertensive effect of leptin in obesity is unclear. We have recently reported that acute leptin infusion induces hypertension via the Transient Receptor Potential Channel subfamily M member 7 (trpm7) in the carotid bodies (CB) (Shin et al., 2019). We hypothesize that this mechanism plays a role in hypertension when leptin levels are elevated chronically in diet induced obesity (DIO).

METHODS

We have developed a novel technique allowing continuous sustained release of the Trpm7 inhibitor FTY720 into the mouse CB using a hydrogel formulation. (1) The effect of FTY720 on blood pressure during chronic leptin infusion was monitored by telemetry implanted in the left femoral arteries of male lean C57BL/6J mice (n=14). FTY720 (N=8) or control hydrogel (N=6) was applied locally to the CB region. Blood pressure was recorded at baseline and during leptin infusion (120 μg/day for 3 days once a week via a SC pump) over a 3 week interval after hydrogel application. (2) The effect of FTY720 on blood pressure in mice with endogenously high leptin levels was determined similarly in DIO mice (DIO, N=16). Blood pressure was measured at baseline and after a single application of FTY720 (N=10) or control hydrogel (N=6) to the CB.

RESULTS

(1) In C57BL/6J mice, administration of leptin increased mean arterial pressure by 12.9 mmHg (p<0.0001) at baseline and throughout the control hydrogel treatment over the 3 week interval. FTY720 hydrogel completely abolished leptin‐induced hypertension (p < 0.05). (2) DIO mice showed elevated blood pressure levels, compared to lean mice at baseline (ΔMAP=+16.2 mmHg). FTY720 hydrogel decreased blood pressure (ΔMAP = −7.8 mmHg, p<0.005), whereas control hydrogel did not have an effect (ΔMAP = −1.7 mmHg, p=0.529).

CONCLUSIONS

We found that chronic hyperleptinemia induces hypertension, which can be treated by a single injection of an extended release preparation of FTY720 to the CB area. Our data (1) provide a new insight into the role of leptin signaling via Trpm7 in the CB in the pathogenesis of hypertension in obesity, and (2) suggest that injections of FTY720 hydrogel to the CB area may control blood pressure in obese patients with resistant hypertension.

Support or Funding Information

HL 133100, HL 128970, 19CDA34700025
Examining the Role of the chromatin remodeler CHD5 in Tumor Suppression and Neural Differentiation in Zebrafish
Authors: Denning Ellen , Sorlien Erin , Ogas Joseph
Published: April 2020

Gene expression and neurogenesis in vertebrates involves a wide variety of cellular machinery to coordinate the transcriptional changes that direct cellular differentiation. Critical aspects of this epigenetic machinery are chromatin remodeling factors. One such factor is CHD5, a vertebrate‐specific member of a family of ATP‐dependent chromatin remodeling proteins. CHD5 is expressed primarily in neural tissue where it is thought to contribute to neurogenesis and has been strongly linked to tumor suppression. Currently, little is known regarding the molecular mechanisms by which CHD5 contributes to neurogenesis or tumor suppression. We hypothesize that loss of CHD5 contributes to development of neuroblastoma because neural precursor cells lacking CHD5 fail to alter some aspect of their neural precursor transcriptome/epigenome. To study the biochemical properties and functional importance of CHD5 remodelers, we have developed a zebrafish model to study Chd5, the zebrafish homolog of human CHD5.

Using the CRISPR/Cas9 system, we have engineered chd5 knockout zebrafish embryos. These zebrafish did not display any overt developmental phenotypes, however next‐generation sequencing of brain tissue from zebrafish embryos reveals that loss of chd5 is associated with broad changes in gene expression. These genes are both up and down regulated, suggesting Chd5 is necessary for both activation and repression of genes during brain development in zebrafish. Analysis of these genes and of their possible contribution to Chd5‐dependent phenotypes is underway.

Support or Funding Information

Purdue Center for Cancer Research; Purdue College of Agriculture
Neuroprotective Potential of Dithiolethiones D3T And ACDT Against Iron Overload‐Induced Toxicity in Human Glioblastoma Cells
Authors: Kulkarni Neha , Betharia Swati
Published: April 2020

The redox potential of iron plays a dual role in which it serves as a vital co‐factor for numerous biological processes, but also catalyzes the production of reactive oxygen species (ROS) that can damage intracellular biomolecules. There is growing evidence of the involvement of iron overload‐induced oxidative stress in the pathogenesis of several neurodegenerative diseases. Various free radical scavengers and antioxidants such as N‐acetylcysteine (NAC) have shown some degree of neuroprotection against iron‐induced oxidative stress. Dithiolethiones 3H‐1,2‐dithiole‐3‐thione (D3T) and 5‐amino‐3‐thioxo‐3H‐(1,2) dithiole‐4‐carboxylic acid ethyl ester (ACDT) are organosulfur compounds that are known to induce production of the cellular antioxidant glutathione (GSH) and other phase‐II metabolic enzymes via activation of the Nrf2 transcription pathway. The present study was aimed to determine the neuroprotective ability of D3T and ACDT against iron overload‐mediated cellular toxicity in U‐87 MG human glioblastoma cells. Cells were exposed to an iron salt ferric ammonium citrate (FAC) for 24 hours with or without being pre‐treated with D3T or ACDT. The cells exposed to FAC alone showed significant decrease in cell viability in a concentration‐dependent manner as assessed using the CellTiter‐Glo® Assay. FAC exposure also depleted the levels of reduced form of GSH and significantly increased ROS generation, as determined by the GSH/GSSG‐GloTM Gluathione Assay and the Cellular ROS Assay kits respectively. Pre‐treatment with 50 μM of D3T or ACDT exhibited significant neuroprotective effects demonstrated by an increase in cell viability, increase in reduced GSH levels, and decrease in ROS production, all depicting a strong counteractive potential against iron overload‐induced neurotoxicity. In conclusion, these results indicate that both D3T and ACDT have neuroprotective ability against iron overload toxicity, the first such study in glial cells. Promisingly, the neuroprotective effects of these dithiolethiones were found to be higher than the positive control NAC. Further studies are currently underway to evaluate the effects of these dithiolethiones on specific cell death pathways induced by iron overload.
Efforts Toward GPR88 Deorphanization: Challenges and Alternative Approaches to Troublesome Cell‐Based Second Messenger Assays
Authors: Elayan Abedalrahman M. , Ruddick Kristie R. , Cole Judith A. , Baker Daniel L. , Parrill-Baker Abby L.
Published: April 2020

G protein‐coupled receptors (GPCR) comprise a family of over 800 integral membrane proteins involved in signal transduction and serve as the primary cellular sensors for chemical stimuli. About 35% of current drugs act on GPCR targets. In addition, of the 300 agents in current clinical trials, greater than 20% target novel GPCRs for which there are no approved drugs (orphan GPCRs). Ligand discovery and deorphanization efforts seeking to assign function to understudied GPCRs are not trivial. Notoriously complex in nature, GPCR‐dependent signaling centers around receptor‐ligand interactions that serve to stabilize conformational access to intracellular signaling partners, G proteins. In GPCR signaling, receptors and heterotrimeric G proteins (made of the Gα and Gβγ subunits) work together to transmit signals via downstream effectors and distinct pathways. The measurement of GPCR mediated second messengers such as cyclic AMP (cAMP), intracellular calcium (Ca2+) mobilization, and ERK activation (MAP kinase) are commonly used as tools to detect ligand induced GPCR activation. However, experimental pathway elucidation is complicated by the tendencies for pathway crosstalk, receptor desensitization, and receptor‐G protein interaction promiscuity. Cell‐based assays that monitor second messenger responses are often used as screening tools for potential GPCR agonists and antagonists, however, commonly used cell‐lines such as HEK‐293, express many endogenous GPCRs that can complicate data analysis. Many classes of Gα subunits exist: Gαs and Gα i/o G‐proteins modulate cyclic AMP (cAMP) through stimulation or inhibition of adenylate cyclase while Gαq subunits activate phospholipase C and a subsequent rapid release of intracellular Ca2+. Specifically, the orphan receptor, GPR88, has been linked with psychiatric diseases including schizophrenia and bipolar disorder. Lacking a validated endogenous agonist, the GPR88 signaling pathway remains poorly understood, however, in vivo knockout experiments and in vitro preclinical studies with small molecule synthetic agonists indicate that GPR88 couples to Gαi/o G‐proteins to inhibit adenylate cyclase activity and reduce intracellular cAMP in cell‐based assays. In this work, we describe our challenges in the study of the GPR88 signaling pathway using second messenger cell‐based assays and explain our decision to move to a real‐time NanoLuc‐based GPCR/G protein complementation assay.

Support or Funding Information

NIH grant 1 R15 MH109034
Isoprenaline‐induced myocardial injury in fertile and ovariectomized female Sprague Dawley rats
Authors: Borodzicz-Jażdżyk Sonia , Czarzasta Katarzyna , Wojciechowska Małgorzata , Główczyńska Renata , Kołodzińska Agnieszka , Puchalska Liana , Opolski Grzegorz , Cudnoch-Jędrzejewska Agnieszka
Published: April 2020

Background

One of the most widely used animal model of myocardial injury is induced by injection of isoprenaline (ISO), a non‐selective beta adrenoceptor agonist, which in high doses causes oxidative stress and coronary demand‐supply imbalance in cardiac tissue.

Purpose

The aim of the study was to assess the course and severity of acute ISO‐induced myocardial injury in fertile and ovariectomized female Sprague Dawley rats.

Methods

9‐weeks old Sprague Dawley rats were ovariectomized or sham operated, and after 3 weeks received an intraperitoneal injection with a single dose of 150 mg/kg b.w.t. ISO (ISOO and ISOP groups, respectively). Echocardiographic examination and blood collection were performed between 6 and 72 hours post ISO injection. Echocardiographic analysis included assessment of left ventricular (LV) dimensions, as well as systolic and diastolic function of LV.

Moreover, the plasma concentrations of troponin I (TnI) and N‐terminal pro‐B‐type natriuretic peptide (NT‐proBNP) were assessed.

Results

We observed significantly higher LV end diastolic diameter (p<0.01), LV end diastolic area (p<0.01) stroke volume and cardiac output in ISOO when compared to ISOP group.

Moreover, ISOO group showed significantly higher plasma concentrations of NT‐proBNP (p<0.001) and lower concentrations of TnI (p<0.001) in comparison to the ISOP group.

Conclusions

The course and severity of ISO‐induced myocardial injury differs significantly between fertile and ovariectomized female rats. Ovariectomy is associated with more pronounced left ventricular dilation and overload.

Support or Funding Information

Scientific work financed from budget funds for science in 2016–2019 as a research project of the Diamond Grant program (project No. DI2015 003045).