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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
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
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
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 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...
December 20, 2017: Biological Chemistry
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
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)...
February 9, 2018: Antiviral Research
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
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
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...
January 22, 2018: Neuroreport
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
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...
January 2, 2018: European Journal of Medicinal Chemistry
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 (32P) 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
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
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
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
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
Andrea B Eberle
Chromatin immunoprecipitation (ChIP) is a widely used method to determine the occupancy of specific proteins within the genome, helping to unravel the function and activity of specific genomic regions. In ChIP experiments, normalization of the obtained data by a suitable internal reference is crucial. However, particularly when comparing differently treated samples, such a reference is difficult to identify. Here, a simple method to improve the accuracy and reliability of ChIP experiments by the help of an external reference is described...
2018: Methods in Molecular Biology
Weikang Guo, Zongli Diao, Wenhu Liu
Cardiovascular disease is the leading cause of mortality in patients with chronic kidney disease. Endothelial cell injury and apoptosis may promote atherosclerosis and cardiovascular disease. The present study investigated the potential mechanisms of asymmetric dimethylarginine (ADMA)‑induced apoptosis in human umbilical vein endothelial cells (HUVECs). It was demonstrated that ADMA decreased B‑cell lymphoma‑2 expression and increased cleaved‑caspase‑3 expression. Furthermore, terminal deoxynucleotidyl transferase (TdT)‑mediated‑digoxigenin‑11‑dUTP nick end labeling results indicated that ADMA induced apoptosis in HUVECs...
November 2017: Molecular Medicine Reports
Kai Lin Cham, Tomoko Soga, Ishwar S Parhar
Really interesting new gene (RING) finger protein is a type of zinc-binding motif found in a large family of functionally distinct proteins. RING finger proteins are involved in diverse cellular processes including apoptosis, DNA repair, cell cycle, signal transduction, tumour suppressor, vesicular transport, and peroxisomal biogenesis. RING finger protein 38 (RNF38) is a member of the family whose functions remain unknown. To gain insight into the putative effects of RNF38 in the central nervous system, we localised its expression...
2017: Frontiers in Neuroanatomy
Ghazaleh Bagheri, Jeremy D Lehner, Jianmin Zhong
Ixodes pacificus is a host of many bacteria including Rickettsia species phylotypes G021 and G022. As part of the overall goal of understanding interactions of phylotypes with their tick host, this study focused on molecular detection of rickettsiae in ovarian and midgut tissue of I. pacificus by fluorescent in situ hybridization (FISH), PCR, and ultrastructural analysis. Of three embedding media (Technovit 8100, Unicryl, and paraffin) tested for generating thin sections, tissues embedded in paraffin resulted in the visualization of bacteria with low autofluorescence in FISH...
October 2017: Ticks and Tick-borne Diseases
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