Detection of apoptosis and matrical degeneration within the intervertebral discs of rats due to passive cigarette smoking.

Although low-back pain is considered to be associated with cigarette smoking, the influence of cigarette smoking on the intervertebral discs (IVD) has not been confirmed. We established a rat model of passive cigarette smoking-induced IVD degeneration, and investigated the cytohistological changes in the IVD and the accompanying changes in gene expression. IVD from rats exposed to 8 weeks of passive cigarette smoking were stained with Elastica van Gieson, and exhibited marked destruction of the supportive structure of the reticular matrix in the nucleus pulposus (NP). Positive signals on safranin O, alcian blue, type II collagen and aggrecan staining were decreased in the destroyed structure. Safranin O and type II collagen signals were also decreased in the cartilage end-plate (CEP) after 4- and 8-weeks of cigarette smoking. In the CEP, the potential for apoptosis was increased significantly, as demonstrated by staining for single-strand DNA. However, there were no signs of apoptosis in the NP or annulus fibrosus cells. Based on these findings, we hypothesized that passive cigarette smoking-induced stress stimuli first affect the CEP through blood flow due to the histological proximity, thereby stimulating chondrocyte apoptosis and reduction of the extracellular matrix (ECM). This leads to reduction of the ECM in the NP, destroying the NP matrix, which can then progress to IVD degeneration

Monoclonal Antibodies to the Hypervariable Region 1 of Hepatitis C Virus Capture Virus and Inhibit Virus Adsorption to Susceptible Cells in Vitro

AbstractTo analyze the neutralizing-related activity of antibodies against the hypervariable region 1 (HVR1) of hepatitis C virus (HCV) in more detail, monoclonal antibodies (mAbs) against HVR1 were raised by immunizing various strains of mice with one of two synthetic HVR1 peptides that had been derived from two isolates of HCV. The epitope specificity of all six mAbs could be assigned by the use of a series of linear peptides in competitive ELISA. It seems that most subregions in the amino acid sequence of HVR1 can induce a humoral immune response in mice. All three mAbs specific to HVR1-6-1 had the ability to capture homologous HCV-6 and inhibit its absorption to susceptible cells in vitro despite the fact that the epitope of each mAb was at a different location in HVR1, whereas the other three mAbs specific to HVR1-7 could not capture HCV-6 nor inhibit the absorption of HCV-6 to susceptible cells. The data in this study suggest that mAbs against HVR1 can prevent the infectivity of HCV in an isolate-specific and epitope position-independent manner

Pitfalls of DNA Quantification Using DNA-Binding Fluorescent Dyes and Suggested Solutions

<div><p>The Qubit fluorometer is a DNA quantification device based on the fluorescence intensity of fluorescent dye binding to double-stranded DNA (dsDNA). Qubit is generally considered useful for checking DNA quality before next-generation sequencing because it measures intact dsDNA. To examine the most accurate and suitable methods for quantifying DNA for quality assessment, we compared three quantification methods: NanoDrop, which measures UV absorbance; Qubit; and quantitative PCR (qPCR), which measures the abundance of a target gene. For the comparison, we used three types of DNA: 1) DNA extracted from fresh frozen liver tissues (Frozen-DNA); 2) DNA extracted from formalin-fixed, paraffin-embedded liver tissues comparable to those used for Frozen-DNA (FFPE-DNA); and 3) DNA extracted from the remaining fractions after RNA extraction with Trizol reagent (Trizol-DNA). These DNAs were serially diluted with distilled water and measured using three quantification methods. For Frozen-DNA, the Qubit values were not proportional to the dilution ratio, in contrast with the NanoDrop and qPCR values. This non-proportional decrease in Qubit values was dependent on a lower salt concentration, and over 1 mM NaCl in the DNA solution was required for the Qubit measurement. For FFPE-DNA, the Qubit values were proportional to the dilution ratio and were lower than the NanoDrop values. However, electrophoresis revealed that qPCR reflected the degree of DNA fragmentation more accurately than Qubit. Thus, qPCR is superior to Qubit for checking the quality of FFPE-DNA. For Trizol-DNA, the Qubit values were proportional to the dilution ratio and were consistently lower than the NanoDrop values, similar to FFPE-DNA. However, the qPCR values were higher than the NanoDrop values. Electrophoresis with SYBR Green I and single-stranded DNA (ssDNA) quantification demonstrated that Trizol-DNA consisted mostly of non-fragmented ssDNA. Therefore, Qubit is not always the most accurate method for quantifying DNA available for PCR.</p></div

Recommended quantification methods for DNA available for PCR.

<p>Recommended quantification methods for DNA available for PCR.</p

Dilution curves of Frozen-DNA diluted with distilled water as determined by NanoDrop, Qubit and qPCR.

<p>Each Frozen-DNA sample was serially diluted with distilled water, and the concentration of each diluent was measured by NanoDrop (circles), BR-Qubit (squares), HS-Qubit (diamonds) and qPCR (triangles). The broken line shows the expected NanoDrop value. The concentration (ng/μl) of each original DNA solution measured by NanoDrop is shown at the top right: dilution ratio = 1. Two additional concentrations are also shown in each graph. The detection limits of NanoDrop, BR-Qubit, HS-Qubit and qPCR are 2 ng/μl, 2 ng/μl, 0.2 ng/μl and 1 pg/μl, respectively.</p

Electrophoresis of DNAs by Agilent 2200 TapeStation.

<p>DNAs (100 ng/lane) were electrophoresed on an Agilent 2200 TapeStation. The DNA integrity number (DIN) indicates the fragmentation of genomic DNA on a scale from 1 to 10. A high DIN indicates highly intact DNA, and a low DIN indicates strongly degraded DNA. Lane 1, Frozen-H1; lane 2, Frozen-H2; lane 3, Frozen-H3; lane 4, FFPE-H1; lane 5, FFPE-H2; lane 6, FFPE-H3; lane 7, Trizol-h1; lane 8, Trizol-h2; lane 9, Trizol-h4; lane 10, Trizol-h6; and lane 11, Trizol-h7.</p