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https://www.readbyqxmd.com/read/29770110/a-pcr-based-method-for-rna-probes-and-applications-in-neuroscience
#1
Ruifang Hua, Shanshan Yu, Mugen Liu, Haohong Li
In situ hybridization (ISH) is a powerful technique that is used to detect the localization of specific nucleic acid sequences for understanding the organization, regulation, and function of genes. However, in most cases, RNA probes are obtained by in vitro transcription from plasmids containing specific promoter elements and mRNA-specific cDNA. Probes originating from plasmid vectors are time-consuming and not suitable for the rapid gene mapping. Here, we introduce a simplified method to prepare digoxigenin (DIG)-labeled non-radioactive RNA probes based on polymerase chain reaction (PCR) amplification and applications in free-floating mouse brain sections...
2018: Frontiers in Neuroscience
https://www.readbyqxmd.com/read/29730973/development-of-in-situ-hybridization-for-detection-of-elephant-endotheliotropic-herpesvirus-in-asian-elephants
#2
Varankpicha Kochakul, Kittikorn Boonsri, Saruda Tiwananthagorn, Chalermchart Somgird, Chatchote Thitaram, Kidsadagon Pringproa
Elephant endotheliotropic herpesvirus (EEHV) is one of the most important viral infectious diseases affecting the elephant population worldwide, especially juveniles and young adults. We developed a chromogenic in situ hybridization (ISH) test for detection of EEHV in Asian elephants ( Elephas maximus). Digoxigenin (DIG) DNA probes from the polymerase and terminase genes of EEHV were synthesized using a PCR DIG-labeling method, and detection of hybridized probe to target EEHV DNA was carried out by anti-DIG immunolabeling...
May 1, 2018: Journal of Veterinary Diagnostic Investigation
https://www.readbyqxmd.com/read/29610365/chemiluminescence
#3
Larry J Kricka
Radioactive reagents have been gradually replaced by nonisotopic reagents for some tasks in molecular biology. Concern over laboratory safety and the economic and environmental aspects of radioactive waste disposal have been key factors in this change. Generally, the new nonisotopic systems have improved in terms of analytical sensitivity and the time required to obtain a result. The most prominent nonisotopic analytical methods exploit chemiluminescence, described here. This technique has been particularly effective when used in combination with an enzyme label, so that the amplifying properties of an enzyme label and the high sensitivity of a chemiluminescent detection reaction are combined to produce an ultrasensitive assay (e...
April 2, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29594544/voltammetric-determination-of-the-escherichia-coli-dna-using-a-screen-printed-carbon-electrode-modified-with-polyaniline-and-gold-nanoparticles
#4
Nahid Shoaie, Mehdi Forouzandeh, Kobra Omidfar
The authors describe an electrochemical assay for fast detection of Escherichia coli (E. coli). It is based on a dual signal amplification strategy and the use of a screen-printed carbon electrode (SPCE) whose surface was modified with a polyaniline (PANI) film and gold nanoparticles (AuNPs) via cyclic voltammetry (CV). In the next step, avidin was covalently immobilized on the PANI/AuNP composite on the SPCE surface. Subsequently, the biotinylated DNA capture probe was immobilized onto the PANI/AuNP/avidin-modified SPCE by biotin-avidin interaction...
March 12, 2018: Mikrochimica Acta
https://www.readbyqxmd.com/read/29509662/a-dna-based-assay-for-digoxin-detection
#5
Michael V Kjelstrup, Line D F Nielsen, Malthe Hansen-Bruhn, Kurt V Gothelf
The most common method for quantifying small-molecule drugs in blood samples is by liquid chromatography in combination with mass spectrometry. Few immuno-based assays are available for the detection of small-molecule drugs in blood. Here we report on a homogeneous assay that enables detection of the concentration of digoxin spiked into in a plasma sample. The assay is based on a shift in the equilibrium of a DNA strand displacement competition reaction, and can be performed in 30 min for concentrations above 10 nM...
March 6, 2018: Biosensors
https://www.readbyqxmd.com/read/29509251/enhanced-expression-of-non-coding-mir-92a-expression-is-implicated-in-the-development-of-lung-cancer
#6
Q Zhu, Q Zang, Z-M Jiang
OBJECTIVE: MicroRNAs (miRNAs/miRs) are small noncoding RNAs that primarily function in RNA silencing and gene regulation at the posttranscriptional level in animals, plants and certain viruses. They have been reported to play a vital role in development and progression of diseases such as cancer. The present study was undertaken to establish a correlation between a specific miRNA in this cluster, termed miR92a, and its association with nonsmall cell lung cancer (NSCLC). MATERIALS AND METHODS: Total RNA was isolated from the plasma using an RNeasy mini kit and cDNA was synthesized by TaqMan MicroRNA Reverse Transcription kit...
February 2018: European Review for Medical and Pharmacological Sciences
https://www.readbyqxmd.com/read/29496817/whole-mount-in-situ-hybridization-of-xenopus-oocytes
#7
Diana Bauermeister, Tomas Pieler
Whole-mount in situ hybridization (WMISH) is a common approach that is used to visualize spatial and temporal gene expression in embryos. In this process, digoxygenin-labeled antisense RNA is hybridized to the complementary transcript of interest and RNA hybrids are immunohistochemically detected using an alkaline phosphatase-conjugated antibody against digoxigenin. During Xenopus laevis oogenesis, certain RNAs localize to the animal or vegetal pole laying the foundation for germ cell development and germ layer formation of the future embryo...
March 1, 2018: Cold Spring Harbor Protocols
https://www.readbyqxmd.com/read/29466231/transcytosis-of-payloads-that-are-non-covalently-complexed-to-bispecific-antibodies-across-the-hcmec-d3-blood-brain-barrier-model
#8
Daniela Schmid, Annette Buntz, Thi Ngoc Hanh Phan, Klaus Mayer, Eike Hoffmann, Irmgard Thorey, Jens Niewöhner, Katrin Vasters, Ranjan Sircar, Olaf Mundigl, Roland E Kontermann, Ulrich Brinkmann
A transcellular shuttle system was generated for the delivery of non-covalently linked payloads across blood-brain barrier (BBB) endothelial cells. Transcytosis-enabling shuttles are composed of bispecific antibodies (bsAbs) that simultaneously bind transferrin receptor (TfR) and haptens such as digoxigenin or biocytinamide. Haptenylated payloads are attached to these vehicles via non-covalent hapten-antibody complexation. This enables targeting to and internalization into human BBB-derived microvascular endothelial hCMEC/D3 cells...
March 30, 2018: Biological Chemistry
https://www.readbyqxmd.com/read/29451211/electroacupuncture-preconditioning-protects-against-focal-cerebral-ischemia-reperfusion-injury-via-suppression-of-dynamin-related-protein-1
#9
Gao-Feng Zhang, Pei Yang, Zeng Yin, Huai-Long Chen, Fu-Guo Ma, Bin Wang, Li-Xin Sun, Yan-Lin Bi, Fei Shi, Ming-Shan Wang
Electroacupuncture preconditioning at acupoint Baihui (GV20) can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 (Drp1) can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 (depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day)...
January 2018: Neural Regeneration Research
https://www.readbyqxmd.com/read/29432776/establishment-of-cre-mediated-hbv-recombinant-cccdna-rcccdna-cell-line-for-cccdna-biology-and-antiviral-screening-assays
#10
Min Wu, Jin Li, Lei Yue, Lu Bai, Yaming Li, Jieliang Chen, Xiaonan Zhang, Zhenghong Yuan
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), existing in hepatocyte nuclei as a stable minichromosome, plays a central role in the life cycle of the virus and permits the persistence of infection. Despite being essential for HBV infection, little is known about the molecular mechanisms of cccDNA formation, regulation and degradation, and there is no therapeutic agents directly targeting cccDNA, fore mostly due to the lack of robust, reliable and quantifiable HBV cccDNA models. In this study, combined the Cre/loxP and sleeping beauty transposons system, we established HepG2-derived cell lines integrated with 2-60 copies of monomeric HBV genome flanked by loxP sites (HepG2-HBV/loxP)...
April 2018: Antiviral Research
https://www.readbyqxmd.com/read/29428908/molecular-cloning-and-expression-analysis-of-the-tyramine-br-receptor-genes-in-apis-cerana-cerana
#11
Li-Zhen Zhang, Yong Zhang, Jing-Hua Hu, Zi-Long Wang, Zhi-Jiang Zeng
Tyramine is a biological polyamine, which serves important functions as neurotransmitters, neuromodulators and neurohormone of the central nervous system. It participates in the regulation of various behavior and physiological processes in insects. For example, tyramine and its receptor genes are involved in the regulation of learning and memory in the animals. In this study, the full-length cDNA sequences of the tyramine receptor genes (Actyr1 and Actyr2) of the Chinese honeybee, Apis cerana cerana, were cloned and sequenced for the first time...
February 20, 2018: Yi Chuan, Hereditas
https://www.readbyqxmd.com/read/29428047/loss-of-hamster-leydig-cells-during-regression-after-exposure-to-a-short-photoperiod
#12
E Beltrán-Frutos, V Seco-Rovira, J Martínez-Hernández, C Ferrer, L M Pastor
The aim of the present study was to evaluate the changes that occur in hamster Leydig cells during regression. Animals were divided into control, mild regression (MR), strong regression (SR) and total regression (TR) groups. Leydig cells were characterised by light and electron microscopy. Terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) and proliferating cell nuclear antigen (PCNA) antibodies were used to detect apoptosis and proliferation respectively. Three types of Leydig cells (A, B and C) could be differentiated...
February 12, 2018: Reproduction, Fertility, and Development
https://www.readbyqxmd.com/read/29360689/resveratrol-protects-early-brain-injury-after-subarachnoid-hemorrhage-by-activating-autophagy-and-inhibiting-apoptosis-mediated-by-the-akt-mtor-pathway
#13
Dan Guo, Jiangtao Xie, Junjie Zhao, Tingqin Huang, Xiaoye Guo, Jinning Song
Early brain injury (EBI) plays a key role in determining the prognosis of patients suffering from subarachnoid hemorrhage (SAH). Resveratrol, a natural polyphenol, serves a neuroprotection function on EBI after SAH. However, the potential mechanism of resveratrol on EBI remains to be elucidated. Akt, also known as protein kinase B, and mammalian target of rapamycin (mTOR), the downstream protein of Akt, play key roles in cell survival and apoptosis, cell cycle regulation, and cellular protein homeostasis. In the present study, we examined the effect of resveratrol on EBI and their potential relationship with the Akt/mTOR pathway, autophagy, and apoptosis...
March 21, 2018: Neuroreport
https://www.readbyqxmd.com/read/29349574/pacific-ciguatoxin-induces-excitotoxicity-and-neurodegeneration-in-the-motor-cortex-via-caspase-3-activation-implication-for-irreversible-motor-deficit
#14
Pallavi Asthana, Ni Zhang, Gajendra Kumar, Virendra Bhagawan Chine, Kunal Kumar Singh, Yim Ling Mak, Leo Lai Chan, Paul Kwan Sing Lam, Chi Him Eddie Ma
Consumption of fish containing ciguatera toxins or ciguatoxins (CTXs) causes ciguatera fish poisoning (CFP). In some patients, CFP recurrence occurs even years after exposure related to CTXs accumulation. Pacific CTX-1 (P-CTX-1) is one of the most potent natural substances known that causes predominantly neurological symptoms in patients; however, the underlying pathogenies of CFP remain unknown. Using clinically relevant neurobehavioral tests and electromyography (EMG) to assess effects of P-CTX-1 during the 4 months after exposure, recurrent motor strength deficit occurred in mice exposed to P-CTX-1...
January 18, 2018: Molecular Neurobiology
https://www.readbyqxmd.com/read/29329000/synthesis-of-c-3-neoglycosides-of-digoxigenin-and-their-anticancer-activities
#15
Xiao-San Li, Yi-Chang Ren, Yu-Zhou Bao, Jie Liu, Xiao-Kun Zhang, You-Wei Zhang, Xue-Long Sun, Xin-Sheng Yao, Jin-Shan Tang
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...
February 10, 2018: European Journal of Medicinal Chemistry
https://www.readbyqxmd.com/read/29322446/nonradioactive-northern-blot-of-circrnas
#16
Xiaolin Wang, Ge Shan
Circular RNAs (circRNAs) are recognized as a special species of transcripts in metazoans with increasing studies, and northern blotting is a direct way to confirm the existence and to evaluate the size of individual circRNAs. Northern blotting probes can be radioactive isotope (32 P) labeled, which is not environment-friendly and sometimes inconvenient to use. Here, we describe a nonradioactive northern blot protocol with digoxigenin-labeled probe to detect circRNA.
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29177739/single-molecule-magnetic-tweezer-analysis-of-topoisomerases
#17
Kathryn H Gunn, John F Marko, Alfonso Mondragón
Magnetic tweezers (MT) provide a powerful single-molecule approach to study the mechanism of topoisomerases, giving the experimenter the ability to change and read out DNA topology in real time. By using diverse DNA substrates, one can study different aspects of topoisomerase function and arrive at a better mechanistic understanding of these fascinating enzymes. Here we describe methods for the creation of three different DNA substrates used in MT experiments with topoisomerases: double-stranded DNA (dsDNA) tethers, "braided" (intertwined or catenated) DNA tether pairs, and dsDNA tethers with single-stranded DNA (ssDNA) regions...
2018: Methods in Molecular Biology
https://www.readbyqxmd.com/read/29158675/transplantation-of-bone-mesenchymal-stem-cells-promotes-angiogenesis-and-improves-neurological-function-after-traumatic-brain-injury-in-mouse
#18
Shewei Guo, Yingwei Zhen, Anran Wang
Traumatic brain injury (TBI) has emerged as a leading cause of mortality and morbidity worldwide. Transplantation of bone mesenchymal stem cells (BMSCs) has emerged as a promising treatment for various central nervous system diseases. This study aims to evaluate the effect of BMSCs transplantation by intravenous injection on neurological function and angiogenesis of the TBI mice. C57BL/6 male mice were randomly divided into four groups: control, sham, TBI, and BMSC. Functional neurological evaluation was performed 1, 4, 7, 14, and 21 days after TBI using neurological severity scores...
2017: Neuropsychiatric Disease and Treatment
https://www.readbyqxmd.com/read/29140979/ginsenoside-rg3-inhibits-angiogenesis-in-a-rat-model-of-endometriosis-through-the-vegfr-2-mediated-pi3k-akt-mtor-signaling-pathway
#19
Yang Cao, Qing Ye, Mengfei Zhuang, Shuwu Xie, Ruihua Zhong, Jingang Cui, Jieyun Zhou, Yan Zhu, Tingting Zhang, Lin Cao
OBJECTIVE: This study aimed to investigate the link between the inhibitory effect of ginsenoside Rg3 on the ectopic endometrium growth and the VEGFR-2-mediated PI3K/Akt/mTOR signaling pathway, a mechanism known to inhibit angiogenesis and induce ectopic endometrial cell apoptosis. MATERIALS AND METHODS: A model of endometriosis was established by allotransplantation in rats. The rats were randomly divided into 5 groups: the ginsenoside Rg3 low-dose group (group A,5mg/kgBW/d of ginsenoside Rg3), the ginsenoside Rg3 high-dose group (group B, 10mg/kgBW/d of ginsenoside Rg3), the gestrinone group (group C, 0...
2017: PloS One
https://www.readbyqxmd.com/read/29069529/improved-free-energy-landscape-quantification-illustrated-with-a-computationally-designed-protein-ligand-interaction
#20
William J Van Patten, Robert Walder, Ayush Adhikari, Stephen R Okoniewski, Rashmi Ravichandran, Christine E Tinberg, David Baker, Thomas T Perkins
Quantifying the energy landscape underlying protein-ligand interactions leads to an enhanced understanding of molecular recognition. A powerful yet accessible single-molecule technique is atomic force microscopy (AFM)-based force spectroscopy, which generally yields the zero-force dissociation rate constant (koff ) and the distance to the transition state (Δx≠ ). Here, we introduce an enhanced AFM assay and apply it to probe the computationally designed protein DIG10.3 binding to its target ligand, digoxigenin...
January 5, 2018: Chemphyschem: a European Journal of Chemical Physics and Physical Chemistry
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