Colorimetric Whole-Mount In Situ Hybridization in Planarians.

Whole-mount in situ hybridization (WISH) is an extremely useful technique for visualizing specific mRNA targets and solving many biological questions. In planarians, this method is really valuable, for example, for determining gene expression profiles during whole-body regeneration and analyzing the effects of silencing any gene to determine their functions. In this chapter, we present in detail the WISH protocol routinely used in our lab, using a digoxigenin-labelled RNA probe and developing with NBT-BCIP. This protocol is basically that already described in Currie et al. (EvoDevo 7:7, 2016), which put together several modifications developed from several laboratories in recent years that improved the original protocol developed in the laboratory of Kiyokazu Agata in 1997. Although this protocol, or slight modifications of it, is the most common protocol in the planarian field for NBT-BCIP WISH, our results show that key steps such as the use and time of NAC treatment to remove the mucus need to be taken into account depending on the nature of the gene analyzed, especially for the epidermal markers.

Association of Neutrophil Extracellular Traps with Plaque Instability in Patient with Carotid Artery Stenosis.

BACKGROUND: Vulnerable carotid plaques are related to cerebral thromboembolic and ischemic events. Neutrophil extracellular traps (NETs) can induce endothelial dysfunction and induce inflammation and coagulation. The aim of the present study was to investigate NETs in patients with carotid artery plaques. METHODS: Carotid plaques were collected by carotid endarterectomy (CEA) from 26 symptomatic and 8 asymptomatic patients between August 2017 and January 2021. The specimens were stained with hematoxylin-eosin and Elastica-van Gieson. Immunohistochemistry was performed staining by CD31 for identifying endothelial cells. NETs were detected by digoxigenin-labeled antihistone H3 (HH3) (citrulline R2+R8+R17). The relationships between the presence of NETs and patient profile and histopathological findings were assessed. RESULTS: HH3-positive cells were detected in 17 (asymptomatic = 2 symptomatic = 15) of 34 carotid plaques (median = 9.7/mm). The number of NETs was correlated with the number of diffusion-weighted imaging high-intensity lesions [P = 0.01], plaque rupture [P = 0.001], intraplaque hemorrhage [P = 0.02], intra luminal thrombus [P = 0.001], and thin fibrous cap [P = 0.001]. CONCLUSIONS: The presence of NETs was associated with the instability of carotid plaques, intraluminal thrombus, which may lead to subsequent cerebral infarction. Clarifying the roles of NETs in carotid plaques may improve the treatment of carotid artery disease.

RNF168 E3 ligase participates in ubiquitin signaling and recruitment of SLX4 during DNA crosslink repair.

SLX4/FANCP is a key Fanconi anemia (FA) protein and a DNA repair scaffold for incision around a DNA interstrand crosslink (ICL) by its partner XPF nuclease. The tandem UBZ4 ubiquitin-binding domains of SLX4 are critical for the recruitment of SLX4 to damage sites, likely by binding to K63-linked polyubiquitin chains. However, the identity of the ubiquitin E3 ligase that mediates SLX4 recruitment remains unknown. Using small interfering RNA (siRNA) screening with a GFP-tagged N-terminal half of SLX4 (termed SLX4-N), we identify the RNF168 E3 ligase as a critical factor for mitomycin C (MMC)-induced SLX4 foci formation. RNF168 and GFP-SLX4-N colocalize in MMC-induced ubiquitin foci. Accumulation of SLX4-N at psoralen-laser ICL tracks or of endogenous SLX4 at Digoxigenin-psoralen/UVA ICL is dependent on RNF168. Finally, we find that RNF168 is epistatic with SLX4 in promoting MMC tolerance. We conclude that RNF168 is a critical component of the signal transduction that recruits SLX4 to ICL damage.

Analysis of mRNA, miRNA, and DNA in Bone Cells by RT-qPCR and In Situ Hybridization.

The aim of this chapter is to describe a method used to evaluate gene expression and microRNAs (miRNAs) in bone cells or tissue using Reverse transcription and quantitative Polymerase Chain Reaction (RT-qPCR), and a method to assess chromogenic in situ hybridization (CISH) on Formalin Fixed Paraffin Embedded (FFPE ) mouse bone tissue to detect both DNA and mRNA transcripts using the double digoxigenin (DIG) locked nucleic acid (LNA™) probes .

Engineered Biosensors from Dimeric Ligand-Binding Domains.

Biosensors are important components of many synthetic biology and metabolic engineering applications. Here, we report a second generation of Saccharomyces cerevisiae digoxigenin and progesterone biosensors based on destabilized dimeric ligand-binding domains that undergo ligand-induced stabilization. The biosensors, comprising one ligand-binding domain monomer fused to a DNA-binding domain and another fused to a transcriptional activation domain, activate reporter gene expression in response to steroid binding and receptor dimerization. The introduction of a destabilizing mutation to the dimer interface increased biosensor dynamic range by an order of magnitude. Computational redesign of the dimer interface and functional selections were used to create heterodimeric pairs with further improved dynamic range. A heterodimeric biosensor built from the digoxigenin and progesterone ligand-binding domains functioned as a synthetic "AND"-gate, with 20-fold stronger response to the two ligands in combination than to either one alone. We also identified mutations that increase the sensitivity or selectivity of the biosensors to chemically similar ligands. These dimerizing biosensors provide additional flexibility for the construction of logic gates and other applications.

Revisiting the binding kinetics and inhibitory potency of cardiac glycosides on Na+,K+-ATPase (α1ß1): Methodological considerations.

INTRODUCTION: Ouabain and digoxin are classical inhibitors of the Na+,K+-ATPase. In addition to their conventional uses as therapeutic agents or experimental tools there is renewed interest due to evidence suggesting they could be endogenous hormones. Somewhat surprisingly, different publications show large discrepancies in potency for inhibiting Na+,K+-ATPase activity (IC50), particularly for the slow binding inhibitors, ouabain and digoxin. METHODS: Using purified pig kidney Na+,K+-ATPase (α1ß1FXYD2) and purified detergent-soluble recombinant human Na+,K+-ATPase (α1ß1FXYD1) we have re-evaluated binding and inhibition kinetics and effects of K+ concentration for ouabain, digoxin, ouabagenin and digoxigenin. RESULTS: We demonstrate unequivocally that for slow binding inhibitors, ouabain and digoxin, long incubation times (≥60 min at 37 °C) are required to avoid under-estimation of potency and correctly determine inhibition (IC50 around 100-200 nM at 5 mM K+) contrary to what occurs when pre-incubation of the drugs without ATP is followed by a short incubation time. By contrast, for the rapidly bound inhibitors, ouabagenin and digoxigenin, short incubation times suffice (<10 min). The strong reduction of inhibitory potency observed at high un-physiological K+ concentrations (≥5 mM) also explained the low potency reported by some authors. DISCUSSION: The data resolve discrepancies in the literature attributable to sub-optimal assay conditions. Similar IC50 values are obtained for pig kidney and recombinant human Na+,K+-ATPase, showing that inhibitory potencies are not determined by the species difference (pig versus human) or environment (membrane-bound versus detergent-soluble) of the Na+,K+-ATPase. The present methodological considerations are especially relevant for drug development of slow binding inhibitors.

The cardenolides strophanthidin, digoxigenin and dihydroouabain act as activators of the human RORγ/RORγT receptors.

Two isoforms of a ligand-activated nuclear receptor, RORγ and RORγT, have been implicated in various physiological functions, including energy metabolism, circadian rhythm and immune system development. Using a stably transfected reporter cell line, we screened two chemical libraries and identified three cardenolides (natural, plant-derived pesticides) as activators of RORγ-dependent transcription. These compounds increased G6PC and NPAS2 expression in HepG2 cells, accompanied by increased occupancy of RORγ within the promoters of these genes. Further, strophanthidin, digoxigenin and dihydroouabain upregulated IL17A and IL17F expression and enhanced IL17 secretion in Th17 human lymphocytes. Molecular docking analyses of these compounds to the RORγ LBD showed favorable docking scores, suggesting that cardenolides may act as agonists of the receptor. Thus, our results provide new chemical structures for further development of RORγ-selective modulators with virtual therapeutic potential.

Wound Healing, Inflammation, and Corneal Ultrastructure After SMILE and Femtosecond Laser-Assisted LASIK: A Human Ex Vivo Study.

PURPOSE: To assess the wound healing, inflammation, and tissue ultrastructure in the human corneal stroma after small incision lenticule extraction (SMILE) and femtosecond laser-assisted LASIK (FS-LASIK). METHODS: Sixteen corneoscleral discs of 16 human donors unsuitable for corneal transplantation were obtained from an eye bank. Eight eyes underwent SMILE with -5.00 diopters (D) of myopic correction; in 3 of them the lenticule was not extracted. Further 5 donor corneas were subjected to FS-LASIK with -5.00 D ablation, and 3 eyes served as the control group without surgical intervention. Postoperatively, specimens were incubated in organ culture medium for 72 hours before being subjected to immunofluorescence staining for CD11b, Ki67, fibronectin, terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick-end labelling assay, and high-magnification scanning electron microscopy. RESULTS: Keratocyte apoptosis, keratocyte proliferation, and infiltration of immune cells were generally mild and comparable between FS-LASIK and SMILE (irrespective of surgical lenticule extraction). By staining for fibronectin, we observed a trend toward milder fibrotic response in the corneal stroma after SMILE than after FS-LASIK. On the contrary, scanning electron microscopy analysis revealed a smoother, more regular ultrastructural appearance of the residual corneal bed after FS-LASIK. CONCLUSIONS: Corneal stromal wound healing after SMILE and FS-LASIK was virtually identical with respect to keratocyte proliferation and apoptosis in the human donor eye model. Although reactive fibrosis adjacent to the laser application site appeared less marked after SMILE, the stromal bed after LASIK exhibited a smoother surface texture. [J Refract Surg. 2018;34(6):393-399.].

Synthesis of C3-Neoglycosides of digoxigenin and their anticancer activities.

Cardiac glycosides exhibit significant anticancer effects and the glycosyl substitution at C3 position of digoxigenin is pivotal for their biological activity. In order to study the structure-activity relationship (SAR) of cardiac glycosides toward cancers and explore more potent anticancer agents, a series of C3-O-neoglycosides and C3-MeON-neoglycosides of digoxigenin were synthesized by the Koenigs-Knorr and neoglycosylation method, respectively. In addition, digoxigenin bisdigitoxoside and monodigitoxoside were prepared from digoxin by sodium periodate (NaIO4) oxidation and 6-aminocaproic acid hydrolysis. The SAR analysis revealed that C3-O-neoglycosides of digoxigenin exhibited stronger cytotoxicity and induction of Nur77 expression of tumor cells than C3-MeON-neoglycosides. Also, 3ß-O-glycosides exhibited stronger anticancer effects than 3α-O-glycosides. Among them, 3ß-O-(ß-l-fucopyranosyl)-digoxigenin (3i) showed the highest activity on induction of Nur77 expression and translocation from the nucleus to cytoplasm, leading to cancer cell apoptosis.

Accurate Electrochemistry Analysis of Circulating Methylated DNA from Clinical Plasma Based on Paired-End Tagging and Amplifications.

Circulating methylated DNA has been a new kind of cancer biomarker, yet its small fraction of trace total DNA from clinical samples impairs the accurate analysis. Though fluorescence methods based on quantitative methylation specific PCR (qMSP) have been adopted routinely, yet alternative electrochemistry assay of such DNA from clinical samples remains a great challenge. Herein, we report accurate electrochemistry analysis of circulating methylated DNA from clinical plasma samples based on a paired-end tagging and amplifications strategy. Two DNA primers each labeled with digoxigenin (Dig) and biotin are designed for the recognition and amplification of methylated DNA. Paired-end tagging amplicons and avidin-HRP molecules are successively captured on the electrode modified with Anti-Dig. Then HRP executes catalytic reaction to generate amplified signal. The design of paired-end tagging can readily integrate downstream electrochemical amplified reaction, and two heterogeneous amplifications enable high assay sensitivity. As little as 40 pg of methylated genomic DNA (∼10 genomic equivalents) is well identified, and our strategy can even distinguish as low as 1% methylation level. Tumor-specific methylated DNA is clearly detected in the plasma of 10 of 11 NSCLC patients. The high clinical sensitivity of 91% (10/11) indicates the good consistency with clinical diagnosis. Excellent spatial control of electrochemistry allows simpler detection of more methylation patterns compared to fluorescence methods. The developed electrochemical assay is a promising liquid biopsy tool for the analysis of tumor-specific circulating DNA.

Synthesis of MeON-neoglycosides of digoxigenin with 6-deoxy- and 2,6-dideoxy-d-glucose derivatives and their anticancer activity.

Cardiac glycosides show anticancer activities and their deoxy-sugar chains are vital for their anticancer effects. In order to study the structure-activity relationship (SAR) of cardiac glycosides toward cancers and get more potent anticancer agents, a series of MeON-neoglycosides of digoxigenin was synthesized and evaluated. First, ten 6-deoxy- and 2,6-dideoxy-d-glucopyranosyl donors were synthesized starting from methyl α-d-glucopyranoside and 2-deoxy-d-glucose. Meanwhile, the digoxigenin was obtained by acidic hydrolysis of commercially available digoxin as glycosyl acceptor. Then, a 22-member MeON-neoglycoside library of digoxigenin was successfully synthesized by neoglycosylation method. Finally, the induction of Nur77 expression and its translocation from the nucleus to cytoplasm together with cytotoxicity of these MeON-neoglycosides were evaluated. The SAR analysis revealed that C3 glycosylation is required for their induction of Nur77 expression. Moreover, some MeON-neoglycosides (2b and 8b) could significant induce the expression of Nur77 and its translocation from the nucleus to cytoplasm. However, these compounds showed no inhibitory effects on the proliferation of cancer cells, suggesting that they may not induce apoptosis of NIH-H460 cancer cells and their underlying potential and application toward cancer cells deserves future study.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization.

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Generation of digoxigenin-incorporated probes to enhance DNA detection sensitivity.

Telomere length in humans has been correlated with cancer and age-related diseases. The standard method to measure telomere length relies on Southern blot analysis with radioactively or non-radioactively labeled probes containing several telomeric DNA repeats. However, this approach requires relatively large amounts of genomic DNA, making it difficult to measure telomere length when a limited amount of sample is available. Here, we describe a non-radioactive labeling method that uses 3' fill-in combined with lambda exonuclease digestion to incorporate one or more digoxigenin (DIG) molecules into bridged nucleic acid (BNA)-containing oligonucleotides (ONTs). Using our method, we were able to generate probes to detect both C- and G-rich telomeric DNA strands. Compared with commercially available DIG-labeled telomere probes, probes generated using this new approach significantly enhance the sensitivity of telomere length measurements.

Autophagy-Induced Apoptosis in Lung Cancer Cells by a Novel Digitoxin Analog.

We have synthesized a novel derivative of Digitoxin, termed "MonoD", which demonstrates cytotoxic effects in lung cancer cells with much higher potency as compared to Digitoxin. Our data show that within 1 h of MonoD treatment, H460 cells showed increased oxidative stress, increased formation of autophagic vacuoles, and increased expression of pro-autophagic markers Beclin-1 and LC3-II. Cells pretreated with MnTBAP, a superoxide scavenger not only lowered superoxide production, but also had lower levels of LC3-II and Beclin-1. Prolonged treatment with MonoD-induced apoptosis in lung cancer cells. We investigated MonoD-dependent regulation of Akt and Bcl2, proteins that are known regulators of both autophagy and apoptosis. Molecular and pharmacologic inhibitors of Bcl2 and Akt, when combined with MonoD, led to higher expression of LC3-II and Beclin-1 as compared to MonoD alone, suggesting a repressive effect for these proteins in MonoD-dependent autophagy. Pretreatment of cells with an autophagy inhibitor repressed the apoptotic potential of MonoD, confirming that early autophagic flux is important to drive apoptosis. Therapeutic entities such as MonoD that target multiple pathways such as autophagy and apoptosis may prove advantageous over current therapies that have unimodal basis for action and may drive sustained tumor regression, which is highly desirable. J. Cell. Physiol. 231: 817-828, 2016. © 2015 Wiley Periodicals, Inc.

A novel semi-automated in situ hybridisation protocol for microRNA detection in paraffin embedded tissue sections.

MicroRNAs (miRNAs) are small non-coding RNAs that modulate gene expression by binding to complementary sequences on target messenger RNA transcripts. In situ hybridisation (ISH) methods have been applied to the study of miRNA in tissue samples in order to understand which is the source of the miRNA of interest. In this paper, the authors describe a novel semi-automated bright field ISH method to visualise miRNAs in formalin fixed paraffin embedded tissue sections. The relevance of this work resides in the use of 3,3'-diaminobenzidine and peroxidase as the detection method, which provides a good defined deposition within tissues This method, which reveals the cells of origin of specific miRNAs, will enable investigators to further explore the biological role of miRNAs.

Cardenolide aglycones inhibit tumor necrosis factor α-induced expression of intercellular adhesion molecule-1 at the translation step by blocking Na⁺/K⁺-ATPase.

Cardiac glycosides, which are inhibitors of Na(+)/K(+)-ATPase, are classified into cardenolides and bufadienolides. We have recently shown that two cardenolide glycosides, ouabain and odoroside A, inhibit Na(+)/K(+)-ATPase, thereby preventing nuclear factor κB-inducible protein expression by blocking Na(+)-dependent amino acid transport. In this study, we investigated the mechanism of action of cardenolide aglycones in tumor necrosis factor α (TNF-α)-induced gene expression. Ouabagenin, digitoxigenin, and digoxigenin were found to inhibit the TNF-α-induced cell-surface expression of intercellular adhesion molecule-1 (ICAM-1) in human lung carcinoma A549 cells. Those cardenolide aglycones did not inhibit the TNF-α-induced expression of ICAM-1 mRNA, but strongly inhibited the TNF-α-induced expression of ICAM-1 as translation product. The inhibition of the TNF-α-induced ICAM-1 expression by ouabagenin, digitoxigenin, and digoxigenin was significantly reversed by the ectopic expression of ouabain-resistant rat Na(+)/K(+)-ATPase α1 isoform. Moreover, knockdown of Na(+)/K(+)-ATPase α1 isoform augmented the inhibition of the TNF-α-induced ICAM-1 expression by ouabagenin or ouabain. These results clearly indicate that cardenolide aglycones inhibit the TNF-α-induced ICAM-1 expression at the translation step by blocking Na(+)/K(+)-ATPase.

Integrating genomics and proteomics data to predict drug effects using binary linear programming.

The Library of Integrated Network-Based Cellular Signatures (LINCS) project aims to create a network-based understanding of biology by cataloging changes in gene expression and signal transduction that occur when cells are exposed to a variety of perturbations. It is helpful for understanding cell pathways and facilitating drug discovery. Here, we developed a novel approach to infer cell-specific pathways and identify a compound's effects using gene expression and phosphoproteomics data under treatments with different compounds. Gene expression data were employed to infer potential targets of compounds and create a generic pathway map. Binary linear programming (BLP) was then developed to optimize the generic pathway topology based on the mid-stage signaling response of phosphorylation. To demonstrate effectiveness of this approach, we built a generic pathway map for the MCF7 breast cancer cell line and inferred the cell-specific pathways by BLP. The first group of 11 compounds was utilized to optimize the generic pathways, and then 4 compounds were used to identify effects based on the inferred cell-specific pathways. Cross-validation indicated that the cell-specific pathways reliably predicted a compound's effects. Finally, we applied BLP to re-optimize the cell-specific pathways to predict the effects of 4 compounds (trichostatin A, MS-275, staurosporine, and digoxigenin) according to compound-induced topological alterations. Trichostatin A and MS-275 (both HDAC inhibitors) inhibited the downstream pathway of HDAC1 and caused cell growth arrest via activation of p53 and p21; the effects of digoxigenin were totally opposite. Staurosporine blocked the cell cycle via p53 and p21, but also promoted cell growth via activated HDAC1 and its downstream pathway. Our approach was also applied to the PC3 prostate cancer cell line, and the cross-validation analysis showed very good accuracy in predicting effects of 4 compounds. In summary, our computational model can be used to elucidate potential mechanisms of a compound's efficacy.

Analysis of RNA transport in Xenopus oocytes and mammalian cells.

In eukaryotes, many RNA species are transcribed, processed in the nucleus, and exported to the cytoplasm, where they are destined to function or to be further matured. Some RNAs are even reimported to the nucleus. In addition, many RNAs are localized at specific nuclear bodies before their export and/or after their nuclear reimport. To understand how RNAs are transported, Xenopus oocytes are extremely useful cells, thanks to their large size. RNA transport can be easily examined by microinjecting radioactively or fluorescently labeled RNAs into Xenopus oocytes. Mammalian cultured cells are sometimes useful by virtue of RNA-FISH technique. Here, we describe methods to analyze RNA localization and export using these cells.

Identification of ocular dominance domains in New World owl monkeys by immediate-early gene expression.

Ocular dominance columns (ODCs) have been well studied in the striate cortex (V1) of macaques, as well defined arrays of columnar structure that receive inputs from one eye or the other, whereas ODC expression seems more obscure in some New World primate species. ODCs have been identified by means of eye injections of transneuronal transporters and examination of cytochrome oxidase (CO) activity patterns after monocular enucleation. More recently, live-imaging techniques have been used to reveal ODCs. Here, we used the expression of immediate-early genes (IEGs), protooncogene, c-Fos, and zinc finger protein, Zif268, after monocular inactivation (MI) to identify ODCs in V1 of New World owl monkeys. Because IEG expression is more sensitive to activity changes than CO expression, it is capable of revealing activity maps in all layers throughout V1 and demonstrating brief activity changes within a couple of hours. Using IEGs, we not only revealed apparent ODCs in owl monkeys but also discovered a number of unique features of their ODCs. Distinct from those in macaques, these ODCs sometimes bridged to other columns in layer 4 (Brodmann layer 4C). CO blobs straddled ODC borders in the central visual field, whereas they centered ODC patches in the peripheral visual field. In one case, the ODC pattern continued into V2. Finally, an elevation of IEG expression in layer 4 (4C) was observed along ODC borders after only brief MI. Our data provide insights into the structure and variability of ODCs in primates and revive debate over the functions and development of ODCs.

Prognostic significance of let-7b expression in breast cancer and correlation to its target gene of BSG expression.

Let-7 microRNAs (miRNAs) are found in a wide range of species, and alterations of let-7 miRNA family member expression levels in humans are associated with various types of cancer. However, few researchers have reported alterations in let-7b levels in breast cancer (BC). Specifically, the use of altered let-7 expression as a prognostic biomarker is of particular interest and significance. The aim of this study was to investigate whether let-7b could be used as a biomarker of tumor progression and patient prognosis in BC and to determine the target gene of let-7b. We retrospectively analyzed the clinical pathological characteristics of 80 BC. We utilized digoxigenin-labeled locked nucleic acid-miRNA probes to detect let-7b expression in 80 BC and 22 benign breast disease (BBD) histologic specimens by in situ hybridization, and also detect the expression of BSG-a potential target gene of let-7b-by immunohistochemistry. We observed that the levels of let-7b expression in BBD were higher than in BC specimens (P < 0.05), indicating that let-7b could inhibit growth and facilitate differentiation of BBD. Also, loss of let-7b expression on BC tissue specimens raised the possibility that let-7b could play a crucial role in the pathogenesis of BC. Furthermore, let-7b expression in breast cancer patients was inversely associated with tumor lymph node metastasis (P = 0.001), patient overall survival (P = 0.027), relapse-free survival (P = 0.016), and BSG protein expression (P = 0.001). Breast cancer patients with low let-7b expression had poor prognoses, indicating let-7b might act as cancer suppressor gene in BC development and progression by inhibiting the expression of BSG.