Eastern Staining: A Simple Recombinant Protein Detection Technology Using a Small Peptide Tag and Its Counter Partner Which is a Fluorescent Compound

Small peptide tags such as c-myc, HA, or FLAG tag have facilitated efficient Westernblotting of proteins of interest especially when specific antibodies for the proteins are not available. However, the conventional Western-blotting requires the multi-steps process taking at least several hours up to two days. With examples of various applications, here we show a convenient and time-saving method for protein detection which employs a fluorescent chemical BDED and its binding peptide RC-tag. And we propose “Estern staining”, as a standard term for protein detection method using fluorescent chemicals and their binding small peptide tags. Eastern staining may substitutes for the time-consuming “immuno-staining” in many versatile applications.22Yothe

A Novel sLRP6E1E2 Inhibits Canonical Wnt Signaling, Epithelial-to-Mesenchymal Transition, and Induces Mitochondria-Dependent Apoptosis in Lung Cancer

Aberrant activation of the Wnt pathway contributes to human cancer progression. Antagonists that interfere with Wnt ligand/receptor interactions can be useful in cancer treatments. In this study, we evaluated the therapeutic potential of a soluble Wnt receptor decoy in cancer gene therapy. We designed a Wnt antagonist sLRP6E1E2, and generated a replication-incompetent adenovirus (Ad), dE1-k35/sLRP6E1E2, and a replication-competent oncolytic Ad, RdB-k35/sLRP6E1E2, both expressing sLRP6E1E2. sLRP6E1E2 prevented Wnt-mediated stabilization of cytoplasmic β-catenin, decreased Wnt/β-catenin signaling and cell proliferation via the mitogen-activated protein kinase, and phosphatidylinositol 3-kinase pathways. sLRP6E1E2 induced apoptosis, cytochrome c release, and increased cleavage of PARP and caspase-3. sLRP6E1E2 suppressed growth of the human lung tumor xenograft, and reduced motility and invasion of cancer cells. In addition, sLRP6E1E2 upregulated expression of epithelial marker genes, while sLRP6E1E2 downregulated mesenchymal marker genes. Taken together, sLRP6E1E2, by inhibiting interaction between Wnt and its receptor, suppressed Wnt-induced cell proliferation and epithelial-to-mesenchymal transition

Titanium dioxide nanoparticles oral exposure to pregnant rats and its distribution

Background: Titanium dioxide (TiO2) nanoparticles are among the most manufactured nanomaterials in the industry, and are used in food products, toothpastes, cosmetics and paints. Pregnant women as well as their conceptuses may be exposed to TiO2 nanoparticles; however, the potential effects of these nanoparticles during pregnancy are controversial, and their internal distribution has not been investigated. Therefore, in this study, we investigated the potential effects of oral exposure to TiO2 nanoparticles and their distribution during pregnancy. TiO2 nanoparticles were orally administered to pregnant Sprague-Dawley rats (12 females per group) from gestation days (GDs) 6 to 19 at dosage levels of 0, 100, 300 and 1000 mg/kg/day, and then cesarean sections were conducted on GD 20. Results: In the maternal and embryo-fetal examinations, there were no marked toxicities in terms of general clinical signs, body weight, food consumption, organ weights, macroscopic findings, cesarean section parameters and fetal morphological examinations. In the distribution analysis, titanium contents were increased in the maternal liver, maternal brain and placenta after exposure to high doses of TiO2 nanoparticles. Conclusion: Oral exposure to TiO2 during pregnancy increased the titanium concentrations in the maternal liver, maternal brain and placenta, but these levels did not induce marked toxicities in maternal animals or affect embryo-fetal development. These results could be used to evaluate the human risk assessment of TiO2 nanoparticle oral exposure during pregnancy, and additional comprehensive toxicity studies are deemed necessary considering the possibility of complex exposure scenarios and the various sizes of TiO2 nanoparticles

European Society of Toxicologic Pathology (Pathology 2.0 Molecular Pathology Special Interest Group): Review of In Situ Hybridization Techniques for Drug Research and Development

In situ hybridization (ISH) is used for the localization of specific nucleic acid sequences in cells or tissues by complementary binding of a nucleotide probe to a specific target nucleic acid sequence. In the last years, the specificity and sensitivity of ISH assays were improved by innovative techniques like synthetic nucleic acids and tandem oligonucleotide probes combined with signal amplification methods like branched DNA, hybridization chain reaction and tyramide signal amplification. These improvements increased the application spectrum for ISH on formalin-fixed paraffin-embedded tissues. ISH is a powerful tool to investigate DNA, mRNA transcripts, regulatory noncoding RNA, and therapeutic oligonucleotides. ISH can be used to obtain spatial information of a cell type, subcellular localization, or expression levels of targets. Since immunohistochemistry and ISH share similar workflows, their combination can address simultaneous transcriptomics and proteomics questions. The goal of this review paper is to revisit the current state of the scientific approaches in ISH and its application in drug research and development

Long-Term Clinical Outcomes of Sirolimus- Versus Paclitaxel-Eluting Stents for Patients With Unprotected Left Main Coronary Artery Disease Analysis of the MAIN-COMPARE (Revascularization for Unprotected Left Main Coronary Artery Stenosis: Comparison of Percutaneous Coronary Angioplasty Versus Surgical Revascularization) Registry

ObjectivesThe aim of this study was to evaluate long-term clinical outcomes after implantation of sirolimus-eluting stents (SES) or paclitaxel-eluting stents (PES) among patients with unprotected left main coronary artery (LMCA) disease.BackgroundThere have been few comparisons of long-term outcomes among currently available drug-eluting stents (DES) for the treatment of LMCA disease.MethodsA total of 858 consecutive patients with unprotected LMCA stenosis were treated with SES (n = 669) or PES (n = 189) between May 2003 and June 2006. Primary outcome was the composite of death, myocardial infarction (MI), or target vessel revascularization (TVR).ResultsBaseline clinical and angiographic characteristics were similar in the 2 groups. During 3 years of follow-up, the adjusted risk of primary composite outcome was similar among the groups (SES vs. PES: 25.8% vs. 25.7%, hazard ratio [HR]: 0.95, 95% confidence interval [CI]: 0.64 to 1.41, p = 0.79). The 2 groups also showed a comparable adjusted rate of each component of outcome: death (9.1% vs. 11.0%, HR: 0.92, 95% CI: 0.47 to 1.80, p = 0.82), MI (8.1% vs. 8.0%, HR: 0.80, 95% CI: 0.43 to 1.48, p = 0.47), and TVR (12.1% vs. 10.6%, HR: 1.10, 95% CI: 0.53 to 2.29, p = 0.81). The 3-year rates of definite or probable stent thrombosis were 0.6% in the SES group and 1.6% in the PES group (adjusted p = 0.18).ConclusionsIn consecutive patients with unprotected LMCA disease undergoing DES implantation, SES and PES showed similar long-term clinical outcomes in terms of death, MI, repeat revascularization, and stent thrombosis

Scutellaria baicalensis

Antimycin A (AMA) damages mitochondria by inhibiting mitochondrial electron transport and can produce reactive oxygen species (ROS). ROS formation, aging, and reduction of mitochondrial biogenesis contribute to mitochondrial dysfunction. The present study sought to investigate extracts of Scutellaria baicalensis and its flavonoids (baicalin, baicalein, and wogonin), whether they could protect mitochondria against oxidative damage. The viability of L6 cells treated with AMA increased in the presence of flavonoids and extracts of S. baicalensis. ATP production decreased in the AMA treated group, but increased by 50% in cells treated with flavonoids (except wogonin) and extracts of S. baicalensis compared to AMA-treated group. AMA treatment caused a significant reduction (depolarized) in mitochondrial membrane potential (MMP), whereas flavonoid treatment induced a significant increase in MMP. Mitochondrial superoxide levels increased in AMA treated cells, whereas its levels decreased when cells were treated with flavonoids or extracts of S. baicalensis. L6 cells treated with flavonoids and extracts of S. baicalensis increased their levels of protein expression compared with AMA-treated cells, especially water extracts performed the highest levels of protein expression. These results suggest that the S. baicalensis extracts and flavonoids protect against AMA-induced mitochondrial dysfunction by increasing ATP production, upregulating MMP, and enhancing mitochondrial function

Incidence, Predictors, Treatment, and Long-Term Prognosis of Patients With Restenosis After Drug-Eluting Stent Implantation for Unprotected Left Main Coronary Artery Disease

ObjectivesThe aim of this study was to evaluate the incidence, predictors, and long-term outcomes of patients with in-stent restenosis (ISR) after percutaneous coronary intervention (PCI) with drug-eluting stents (DES) for unprotected left main coronary artery (LMCA) disease.BackgroundFew data on the clinical course and management of patients experiencing restenosis after DES treatment for unprotected LMCA disease have appeared.MethodsBetween February 2003 and November 2007, 509 consecutive patients with unprotected LMCA disease underwent DES implantation, with 402 (80.1%) undergoing routine surveillance or clinically driven angiographic follow-up. A major adverse cardiac event was defined as the composite of death, myocardial infarction (MI), or target-lesion revascularization.ResultsThe overall incidence of angiographic ISR in LMCA lesions was 17.6% (71 of 402 patients, 57 with focal-type and 14 with diffuse-type ISR. Forty patients (56.3%) underwent repeated PCI, 10 (14.1%) underwent bypass surgery, and 21 (29.6%) were treated medically. During long-term follow-up (a median of 31.7 months), there were no deaths, 1 (2.2%) MI, and 6 (9.5%) repeated target-lesion revascularization cases. The incidence of major adverse cardiac event was 14.4% in the medical group, 13.6% in the repeated PCI group, and 10.0% in the bypass surgery group (p = 0.91). Multivariate analysis showed that the occurrence of DES-ISR did not affect the risk of death or MI.ConclusionsThe incidence of ISR was 17.7% after DES stenting for LMCA. The long-term clinical prognosis of patients with DES-ISR associated with LMCA stenting might be benign, given that these patients were optimally treated with the clinical judgment of the treating physician