Fluorescence In Situ Hybridization and Rehybridization Using Bacterial Artificial Chromosome Probes.

Fluorescence in situ hybridization (FISH) method enables in situ genetic analysis of both metaphase and interphase cells from different types of material, including cell lines, cell smears, and fresh and paraffin-embedded tissue. Despite the growing number of commercially available FISH probes, still for large number of gene loci or chromosomal regions commercial probes are not available. Here we describe a simple method for generating FISH probes using bacterial artificial chromosomes (BAC). Due to genome-wide coverage of BAC clones, there are almost unlimited possibilities for the analysis of any genomic regions using BAC FISH probes.

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.].

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.

Microbial Transformation of 14-Anhydrodigoxigenin by Alternaria alternata.

The microbial transformation of 14-anhydrodigoxigenin (1) by Alternaria alternata CGMCC 3.577 led to the production of seven new metabolites, 2-8. Their structures were determined by extensive spectroscopic (CD, IR, 1D- and 2D-NMR, and HR-ESI-MS) data analyses. The reactions in the bioprocess exhibited diversity, including specific oxidation, hydroxylation, reduction, epoxidation, and dehydration. In addition, a hypothetical biocatalytic pathway is proposed.

In situ hybridization on whole-mount zebrafish embryos and young larvae.

The in situ hybridization uses a labeled complementary RNA strand to localize a specific mRNA sequence in a tissue. This method is widely used to describe the spatial and temporal expression patterns of developmentally regulated genes. Here we describe a technique that employs in vitro synthesized RNA tagged with digoxigenin uridine-5'-triphosphate (UTP) to determine expression of genes on whole-mount zebrafish embryos and young larvae. Following hybridization, the localization of the specific transcript is visualized immunohistochemically using an anti-digoxigenin antibody conjugated to alkaline phosphatase that hydrolyzes the 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to 5-bromo-4-chloro-3-indole and inorganic phosphate. 5-Bromo-4-chloro-3-indole can be oxidized by nitro blue tetrazolium (NBT), which forms an insoluble dark blue diformazan precipitate after reduction.This protocol has been used for performing large-scale analyses of the spatial and temporal expression of the zebrafish genome, resulting in the description of more than 8,400 expression patterns that are available at the zebrafish information network (ZFIN.org) in the gene expression section.

Unintentional lethal overdose with metildigoxin in a 36-week-old infant--post mortem tissue distribution of metildigoxin and its metabolites by liquid chromatography tandem mass spectrometry.

A massive lethal overdose with beta-metildigoxin in a 36-week-old infant is presented. Determination of beta-metildigoxin and its metabolites digoxin, digoxigenin and digoxigenin-monodigitoxosid is achieved by a liquid chromatographic mass spectrometric (LC-MS/MS) method. Measured concentrations for beta-metildigoxin and digoxin in peripheral blood were 40.2 ng/ml and 25.6 ng/ml, respectively. Tissue distribution showed highest concentrations in kidney tissue and gastric content. The metabolite digoxigenin-monodigitoxosid could be detected in heart blood, duodenal content, gastric content and fat tissue while the metabolite digoxigenin could only be detected in gastric content since the drug was given by a stomach tube.

Probing the regiospecificity of enzyme-catalyzed steroid glycosylation.

The potential of a uniquely permissive engineered glycosyltransferase (OleD ASP) as a catalyst for steroid glycosylation is highlighted. The ability of OleD ASP to glucosylate a range of cardenolides and bufadienolides was assessed using a rapid LC-UV/MS-SPE-NMR analytical platform. While a bias toward OleD-catalyzed C3 monoglucosylation was observed, subtle alterations of the steroidal architecture, in some cases, invoked diglucosylation or, in one case (digoxigenin), C12 glucosylation. This latter case represents the first, and highly efficient, synthesis of digoxigenin 12-O-ß-D-glucoside.

A novel phytochemical, digoxigenin-3-O-rutin in the amelioration of isoproterenol-induced myocardial infarction in rat: a comparison with digoxin.

INTRODUCTION: The commonly used cardiac glycoside, digoxin (DIG), has a narrow therapeutic window. Although some investigations were made to counteract its toxic effects, no alternate phytochemical is available till date that is more potent and safer than DIG. AIMS: Our main aim was to isolate a novel cardenolide from the seeds of Trigonella foenum graceium and to evaluate its relative potential in comparison to that of DIG. EXPERIMENTAL DESIGN: In one experiment effects of the isolated compound at 2.5, 5.0, and 10 mg/kg (p.o.) were evaluated in isoproterenol (ISO)-induced cardiovascular problems in rats. As the test drug (TDR) reversed most of the ISO-induced changes, it was subjected to the phytochemical analyses and was identified as digoxigenin-3-O-rutin. In another experiment effects of DIG and rutin (Rtn) were compared with those of TDR or DIG alone. The hydroxyl radical scavenging activity was also measured by electron spin resonance (EPR). RESULTS: digoxigenin-3-O-rutin at 10 mg/kg markedly reduced the ISO-induced increase in cardiac lipid peroxidation and in the levels of serum creatinine phosphokinase-MB, glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, lactate dehydrogenase, and creatinine. It also reversed the ISO-induced changes in the cardiac histomorphology. Interestingly TDR appeared to be more effective than DIG alone or DIG and Rtn in combination. CONCLUSION: The newly isolated digoxigenin-3-O-rutin appears to be more potent and safe than digoxin. Its higher efficacy could be due to its structural specificity and might have been mediated through its better free radical scavenging action.

Stretching single DNA molecules to demonstrate high-force capabilities of holographic optical tweezers.

The well calibrated force-extension behaviour of single double-stranded DNA molecules was used as a standard to investigate the performance of phase-only holographic optical tweezers at high forces. Specifically, the characteristic overstretch transition at 65 pN was found to appear where expected, demonstrating (1) that holographic optical trap calibration using thermal fluctuation methods is valid to high forces; (2) that the holographic optical traps are harmonic out to >250 nm of 2.1 mum particle displacement; and (3) that temporal modulations in traps induced by the spatial light modulator (SLM) do not affect the ability of optical traps to hold and steer particles against high forces. These studies demonstrate a new high-force capability for holographic optical traps achievable by SLM technologies.

In vivo labeling and specific magnetic bead separation of RNA for biofilm characterization and stress-induced gene expression analysis in bacteria.

The method of in vivo labeling and separation of bacterial RNA was developed as an approach to elucidating the stress response of natural bacterial populations. This technique is based on the incorporation of digoxigenin-11-uridine-5'-triphosphate (DIG-11-UTP) in the RNA of active bacteria. The digoxigenin fulfills a dual role as a label of de novo synthesized RNA and a target for magnetic bead separation from a total RNA extract. Depending on the growth conditions and the population's composition, the assembly rate of DIG-11-UTP ranged from 1.2% to 12.5% of the total RNA in gram-positive and gram-negative reference bacteria as well as in natural biofilms from drinking water, surface water, and lake sediment. Separation of DIG-RNA from total RNA extracts was performed with a biotinylated anti-digoxigenin antibody and streptavidin-functionalized magnetic particles. The average separation yield from total RNA extracts was about 95% of labeled RNA. The unspecific bindings of non-labeled nucleic acids were smaller than 0.2%, as was evaluated by spiking experiments with an unmarked DNA amplicon. Applicability of the method developed was demonstrated by rRNA-directed PCR-DGGE population analysis of natural biofilms and expression profiling of two stress-induced genes (vanA and rpoS) in reference bacteria.

Non-radioactive labeled probe preparation for hbs gene detection.

Among some Bacillus species, a protein highly homologous to HU, classified HB and coded by hbs gene. According to the recent studies, the sequence of hbs gene just in one strain of Bacillus subtilis exists in gene bank (ATCC 23857). In this study, DNA from Bacillus subtilis ATCC 6633 was extracted and investigated by PCR. The PCR product was sequenced and shown to differ in just one nucleotide from B. subtilis ATCC 23857. Hence, it was chosen as reference and for the first time, used for non-radioactive labeled probe preparation. The PCR product in Bacillus subtilis with ATCC 6633 was labeled using non-radioactive DIG-labeled nucleotides and conditions of probe preparation and hybridization were optimized and checked it by Southern blotting.

Use of DNA combing to study DNA replication in Xenopus and human cell-free systems.

The Xenopus egg extract has become the gold standard for in vitro studies of metazoan DNA replication. We have used this system to study the mechanisms that ensure rapid and complete DNA replication despite random initiation during Xenopus early development. To this end we adapted the DNA combing technique to investigate the distribution of replication bubbles along single DNA molecules. DNA replicating in egg extracts is labelled by addition of digoxigenin-11-dUTP and/or biotin-16-dUTP at precise times. These two dTTP analogues are efficiently incorporated into DNA during replication in the extract. After DNA purification and combing the DNA is visualized with appropriate fluorescent antibody/streptavidin molecules. Replicated DNA appears as green or red tracts whose pattern reveals how each molecule was replicated, allowing to follow the dynamics of DNA replication through S phase. We describe (a) the preparation and use of egg extracts and demembranated sperm chromatin templates; (b) a simple method for preparing silanized glass coverslips suitable for DNA combing and fluorescence detection; (c) two alternative replicative DNA labelling schemes and their respective advantages; and (d) a protocol for combining replicative labelling with detection of specific DNA sequences by fluorescent in situ hybridization (FISH). Although most observations made in Xenopus egg extracts are applicable to other eukaryotes, there are differences in cell-cycle regulation between mammalian somatic cells and embryonic amphibian cells, which led to the development of human cell-free systems that can initiate semi-conservative chromosomal DNA replication under cell-cycle control. We have employed the knowledge gained with Xenopus extracts to characterize DNA replication intermediates generated in human cell-free systems using DNA combing. We describe here (a) the preparation and use of human cell-free extracts and initiation-competent template nuclei for DNA combing studies; (b) an optimized labelling scheme for DNA replication intermediates by molecular combing and fluorescence microscopy.

COMU: a safer and more effective replacement for benzotriazole-based uronium coupling reagents.

We describe a new family of uronium-type coupling reagents that differ in their iminium moieties and leaving groups. The presence of the morpholino group in conjunction with an oxime derivative--especially ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma)--had a marked influence on the solubilities, stabilities, and reactivities of the reagents. Finally, the new uronium salt derived from Oxyma (COMU) performed extremely well in the presence of only 1 equiv of base, thereby confirming the effect of the hydrogen bond acceptor in the reaction. COMU also showed a less hazardous safety profile than the benzotriazole-based HDMA and HDMB, which exhibited unpredictable autocatalytic decompositions. Furthermore, the Oxyma moiety contained in COMU suggests a lower risk of explosion than in the case of the benzotriazole derivatives.

Unstabilized DNA breaks in lymphocytes of patients with different subsets of systemic sclerosis.

The clastogenic effects on DNA, proven by the presence of micronuclei (MN) and the protective cellular mechanisms normally used to stabilize DNA breaks were investigated in three subsets of patients with systemic sclerosis (SSc). The frequency of MN found in cultures of peripheral lymphocytes in patients with anticentromere and antitopoisomerase I antibodies was significantly higher than that in the control group. The group with anticentromere antibody showed a significantly higher frequency of MN than did the subjects with antitopoisomerase antibody (4.22% versus 2.34%, P < 0.001). Patients with anti-RNA polymerase III, instead, had a low prevalence of typical micronucleated cells (0.98%), not significantly different from that of the healthy controls (0.82%). Moreover, when MN was characterized for the presence or absence of DNA fragments with free 3'-OH ends by digoxigenin-dUTP (DIG-dUTP) using terminal deoxynucleotidil transferase, its frequency was found to be increased in the groups with anticentromere and antitopoisomerase I antibodies with respect to that in the controls. The increase was significantly higher in the lymphocytes of the patients with anticentromere than in those with antitopoisomerase I antibody (35% versus 20.08%, P < 0.001). Nonetheless, the prevalence of unstable DNA fragments in patients with anti-RNA polymerase III antibody was low (2.05%) and not significantly different from that of the control group (1.18%). Our results indicate that there is a clastogenic effect on DNA and an interference in the protective cellular mechanisms normally stabilizing DNA breaks only in some subsets of SSc patients.

Detection of the intranuclear microsporidium Nucleospora salmonis in naturally and experimentally exposed Chinook salmon Oncorhynchus tshawytscha by in situ hybridization.

Nucleospora salmonis, an intranuclear microsporidian parasite of salmonid fish, is often difficult to observe in histological sections or wet mount preparations from lightly infected tissues because of its small size and location within the nuclei of lymphoblast-type cells. Diagnosis of infections by conventional light microscopy is directly dependent upon distinguishing different stages of the parasite from host cell nuclear material or vacuoles. To assist detection of stages of the parasite in tissues of its primary host, the Chinook salmon (Oncorhynchus tshawytscha), we developed a nonradioactive in situ hybridization (ISH) method. The new method was then used to detect N. salmonis among Chinook salmon after both natural and experimental exposures to the parasite. Probes derived from the small subunit ribosomal DNA (ssu-rDNA) sequence of the microsporidium were labeled with digoxigenin deoxyuridine triphosphate (DIG-dUTP) and hybridized to parasite DNA present in infected tissues. The ISH procedure effectively identified merogonic and spore stages of N. salmonis in paraffin-embedded tissues of clinically and subclinically infected fish. A Nucleospora-like microsporidium was also detected by ISH in tissues of a nonsalmonid fish, the English sole (Pleuronectes vetulus), using probes designed to a region of the ssu-rDNA of N. salmonis.

Production of anti-digoxigenin antibody HRP conjugate for PCR-ELISA DIG detection system.

There are several methods used to visualize the end product of polymerase chain reactions. One of these methods is an ELISA-based detection system (PCR-ELISA) which is very sensitive and can be used to measure the PCR products quantitatively by a colorimetric method. According to this technique, copies of DNA segments from genomic DNA are amplified by PCR with incorporation of digoxigenin-11-dUTP. Samples are analyzed in a microtiter plate format by alkaline denaturation and are hybridized to biotinylated allele-specific capture probes bound to streptavidin coated plates. Use of the produced anti-digoxigenin antibody horseradish peroxidase conjugate and the substrate 2,2'-azino-di-3-ethylbenzthiazolinsulfonate (ABTS) detected the hybridized DNA. One of the key components in this procedure is the anti-digoxigenin antibody HRP conjugate. Described here is the preparation, purification, and characterization of anti-digoxigenin antibody HRP conjugate for use in the PCR-ELISA DIG detection system. Several biochemical protocols and modifications were applied to increase the sensitivity and specificity of this conjugate for an efficient and cost-effective product.

Characterization of eastern oyster (Crassostrea virginica Gmelin) chromosomes by fluorescence in situ hybridization with bacteriophage P1 clones.

Chromosome identification is an essential step in genomic research, which so far has not been possible in oysters. We tested bacteriophage P1 clones for chromosomal identification in the eastern oyster Crassostrea virginica, using fluorescence in situ hybridization (FISH). P1 clones were labeled with digoxigenin-11-dUTP using nick translation. Hybridization was detected with fluorescein-isothiocyanate-labeled anti-digoxigenin antibodies and amplified with 2 layers of antibodies. Nine of the 21 P1 clones tested produced clear and consistent FISH signals when Cot-1 DNA was used as a blocking agent against repetitive sequences. Karyotypic analysis and cohybridization positively assigned the 9 P1 clones to 7 chromosomes. The remaining 3 chromosomes can be separated by size and arm ratio. Five of the 9 P1 clones were sequenced at both ends, providing sequence-tagged sites that can be used to integrate linkage and cytogenetic maps. One sequence is part of the bone morphogenetic protein type 1b receptor, a member of the transforming growth factor superfamily, and mapped to the telomeric region of the long arm of chromosome 2. This study shows that large-insert clones such as P1 are useful as chromosome-specific FISH probes and for gene mapping in oysters.

Cloth-based hybridization array system for the detection of multiple antibiotic resistance genes in Salmonella enterica subsp. enterica serotype Typhimurium DT104.

AIMS: A simple DNA macroarray system was developed for detection of antibiotic resistance and other marker genes associated with the multidrug-resistant food pathogen Salmonella enterica subsp. enterica serotype Typhimurium DT104. METHODS AND RESULTS: A multiplex polymerase chain reaction (PCR) incorporating digoxigenin-dUTP was used to simultaneously amplify seven marker sequences, with subsequent rapid detection of the amplicons by hybridization with an array of probes immobilized on polyester cloth and immunoenzymatic assay of the bound label. This system provided sensitive detection of the different genetic markers in the S. Typhimurium DT104 genome, giving positive reactions with as few as 10 CFU, and the hybridizations were highly specific, with no reactions of amplicons with heterologous probes on the array. CONCLUSIONS: This cloth-based hybridization array system (CHAS) provides a simple, cost-effective tool for monitoring S. Typhimurium DT104 in foods and their production environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The CHAS is a simple and cost-effective tool for the simultaneous detection of amplicons generated in a multiplex PCR, and the concept is broadly applicable to the detection and characterization of food pathogens.

[Oligonucleotide probes for the detection of Thermoanaerobacter]. / Oligonukleotidnye zondy dlia detektsii predstavitelei roda Thermoanaerobacter.

Based on the analysis of 16S rRNA nucleotide sequences, oligonucleotide probes were designed for the detection and identification of representatives of the genus Thermoanaerobacter. To increase the specificity level of detection, the genus Thermoanaerobacter was divided into three groups. The probe Tab 827 (5'-GCTTCCGCDYCCCACACCTA-3') detected all known representatives of the genus Thermoanaerobacter; the probe Tab_1 844 (5'-TTAACTACGGCACGRAATGCTTC-3') was specific for the first group of the species of the genus (T. wiegelii, T. siderophilus, T. sulfurophilus, T. brockii, T. kivui, T. ethanolicus, T. acetoethylicus, and T. thermohydrosulfuricus); the probe Tab_2 424 (5'-CACTAMYGGGGTTTACAACC-3') targeted the second group (T. thermocopriae, T. mathranii, and T. italicus); and the probe Tab_3 184 (5'-TC-CTCCATCAGGATGCCCTA-3') was specific for the third group (T. tengcongensis, T. yonseiensis, T. subterraneus, and Carboxydibrachium pacificum, an organism related to the genus Thermoanaerobacter according to its 16S rRNA sequence). The oligonucleotide probes were labeled with Dig-11-dUTP. Hybridization with the probes showed the affiliation with Thermoanaerobacter of several pure cultures that were morphologically similar to representatives of this genus but possessed metabolic features unusual for it (capacity for agarose hydrolysis, anaerobic oxidation of CO, growth at low pH values) or were isolated from habitats previously unknown for Thermoanaerobacter (deep-sea hydrothermal vents).

[Detection of hyperthermophilic archaea of the genus Desulforococcus by hybridization with oligonucleotide probes]. / Detektsiia gipertermofil'nykh arkhei roda Desulfurococcus putem gibridizatsii s oligonukleotidnymi zondami.

Based on the analysis of nucleotide sequences of 16S rRNA, oligonucleotide probes were designed for the detection and identification of representatives of the genus Desulfurococcus (kingdom Crenarchaeota of the domain Archaea). The detection procedure included obtaining of PCR products on DNA isolated from pure cultures, enrichments, or natural samples with a Crenarchaeota-specific primer pair designed: Cren 7F (5'-TTCCGGTTGATCCYGCCGGACC-3') and Cren 518R (5'-GCTGGTWTTACCGCGGCGGCTGA-3'). The PCR products were hybridized with Dig-11-dUTP-labeled oligonucleotide probes targeting the genus Desulfurococcus (Dco 198, 5'-CGTTAACYCCYGCCACACC-3) and its species D. mobilis (Dco_mob 198, 5'-CGTTAACCCCTGCCACACC-3') and D. amylolyticus (Dco_amy 198, 5'-CGTTAACCCCCGCCACACC-3'). With the use of these primers and probes, four new strains isolated from hydrotherms of Kamchatka and Kunashir Island were identified as members of the species Desulfurococcus amylolyticus. Desulfurococcus representatives were detected in several natural samples, including a sample taken from a marine hydrotherm at the Kunashir Island; this demonstrates that representatives of this genus occur not only in terrestrial but also in marine environments.