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61.
62.
《Biomarkers》2013,18(7):616-628
A competitive enzyme-linked immunosorbent assay (ELISA) for detection of a type I collagen fragment generated by matrix metalloproteinases (MMP) -2, -9 and -13, was developed (CO1-764 or C1M). The biomarker was evaluated in two preclinical rat models of liver fibrosis: bile duct ligation (BDL) and carbon tetra chloride (CCL4)-treated rats. The assay was further evaluated in a clinical study of prostate-, lung- and breast-cancer patients stratified according to skeletal metastases. A technically robust ELISA assay specific for a MMP-2, -9 and -13 neo-epitope was produced and seen to be statistically elevated in BDL rats compared to baseline levels as well as significantly elevated in CCL4 rats stratified according to the amount of total collagen in the livers. CO1-764 levels also correlated significantly with total liver collagen and type I collagen mRNA expression in the livers. Finally, the CO1-764 marker was not correlated with skeletal involvement or number of bone metastases. This ELISA has the potential to assess the degree of liver fibrosis in a non-invasive manner. 相似文献
63.
Rangaraj M. Rangayyan Shantanu Banik J.E. Leo Desautels 《Journal of visualized experiments : JoVE》2013,(78)
We demonstrate methods for the detection of architectural distortion in prior mammograms of interval-cancer cases based on analysis of the orientation of breast tissue patterns in mammograms. We hypothesize that architectural distortion modifies the normal orientation of breast tissue patterns in mammographic images before the formation of masses or tumors. In the initial steps of our methods, the oriented structures in a given mammogram are analyzed using Gabor filters and phase portraits to detect node-like sites of radiating or intersecting tissue patterns. Each detected site is then characterized using the node value, fractal dimension, and a measure of angular dispersion specifically designed to represent spiculating patterns associated with architectural distortion.Our methods were tested with a database of 106 prior mammograms of 56 interval-cancer cases and 52 mammograms of 13 normal cases using the features developed for the characterization of architectural distortion, pattern classification via quadratic discriminant analysis, and validation with the leave-one-patient out procedure. According to the results of free-response receiver operating characteristic analysis, our methods have demonstrated the capability to detect architectural distortion in prior mammograms, taken 15 months (on the average) before clinical diagnosis of breast cancer, with a sensitivity of 80% at about five false positives per patient. 相似文献
64.
《Epigenetics》2013,8(1):106-112
The methylated DNA immunoprecipitation method (MeDIP) is a genome-wide, high-resolution approach that detects DNA methylation with oligonucleotide tiling arrays or high throughput sequencing platforms. A simplified high-throughput MeDIP assay will enable translational research studies in clinics and populations, which will greatly enhance our understanding of the human methylome. We compared three commercial kits, MagMeDIP Kit TM (Diagenode), Methylated-DNA IP Kit (Zymo Research) and Methylamp? Methylated DNA Capture Kit (Epigentek), in order to identify which one has better reliability and sensitivity for genomic DNA enrichment. Each kit was used to enrich two samples, one from fresh tissue and one from a cell line, with two different DNA amounts. The enrichment efficiency of each kit was evaluated by agarose gel band intensity after Nco I digestion and by reaction yield of methylated DNA. A successful enrichment is expected to have a 1:4 to 10:1 conversion ratio and a yield of 80% or higher. We also evaluated the hybridization efficiency to genome-wide methylation arrays in a separate cohort of tissue samples. We observed that the MagMeDIP kit had the highest yield for the two DNA amounts and for both the tissue and cell line samples, as well as for the positive control. In addition, the DNA was successfully enriched from a 1:4 to 10:1 ratio. Therefore, the MagMeDIP kit is a useful research tool that will enable clinical and public health genome-wide DNA methylation studies. 相似文献
65.
66.
En-Wei Tao Wing Yin Cheng Wei-Lin Li Jun Yu Qin-Yan Gao 《Journal of cellular physiology》2020,235(2):683-690
tRNA-derived stress-induced RNAs (tiRNAs), important components of tRNA-derived fragments, are gaining popularity for their functions as small noncoding RNAs involved in cancer progression. Under cellular stress, tiRNAs are generated when mature tRNA is specifically cleaved by angiogenin and suggested to act as transducers or effectors involved in cellular stress responses. tiRNAs facilitate cells to respond to stresses mainly via reprogramming translation, inhibiting apoptosis, degrading mRNA, and generating stress granules. This review introduces the cellular biogenesis, molecular mechanisms, and biological roles of tiRNAs in stress response and disease regulation. A better understanding of their roles in regulating cancer may provide novel biomarkers or therapeutic targets for diagnosis and treatment. 相似文献
67.
Noelia Blanco Ambrose J. Williams Danming Tang Dejin Zhan Shahram Misaghi Robert F. Kelley Laura C. Simmons 《Biotechnology and bioengineering》2020,117(7):1946-1960
Optimal production of bispecific antibodies (bsAb) requires efficient and tailored co-expression and assembly of two distinct heavy and two distinct light chains. Here, we describe a novel technology to modulate the translational strength of antibody chains via Kozak sequence variants to produce bsAb in a single cell line. In this study, we designed and screened a large Kozak sequence library to identify 10 independent variants that can modulate protein expression levels from approximately 0.2 to 1.3-fold compared with the wild-type sequence in transient transfection. We used a combination of several of these variants, covering a wide range of translational strength, to develop stable single cell Chinese hamster ovary bispecific cell lines and compared the results with those obtained from the wild-type sequence. A significant increase in bispecific antibody assembly with a concomitant reduction in the level of product-related impurities was observed. Our findings suggest that for production of bsAb it can be advantageous to modify translational strength for selected protein chains to improve overall yield and product quality. By extension, tuning of translational strength can also be applied to improving the production of a wide variety of heterologous proteins. 相似文献
68.
Lekha E. Manjunath Anumeha Singh Sarthak Sahoo Ashutosh Mishra Jinsha Padmarajan Chaithanya G. Basavaraju Sandeep M. Eswarappa 《The Journal of biological chemistry》2020,295(50):17009
Stop codon read-through (SCR) is a process of continuation of translation beyond a stop codon. This phenomenon, which occurs only in certain mRNAs under specific conditions, leads to a longer isoform with properties different from that of the canonical isoform. MTCH2, which encodes a mitochondrial protein that regulates mitochondrial metabolism, was selected as a potential read-through candidate based on evolutionary conservation observed in the proximal region of its 3′ UTR. Here, we demonstrate translational read-through across two evolutionarily conserved, in-frame stop codons of MTCH2 using luminescence- and fluorescence-based assays, and by analyzing ribosome-profiling and mass spectrometry (MS) data. This phenomenon generates two isoforms, MTCH2x and MTCH2xx (single- and double-SCR products, respectively), in addition to the canonical isoform MTCH2, from the same mRNA. Our experiments revealed that a cis-acting 12-nucleotide sequence in the proximal 3′ UTR of MTCH2 is the necessary signal for SCR. Functional characterization showed that MTCH2 and MTCH2x were localized to mitochondria with a long t1/2 (>36 h). However, MTCH2xx was found predominantly in the cytoplasm. This mislocalization and its unique C terminus led to increased degradation, as shown by greatly reduced t1/2 (<1 h). MTCH2 read-through–deficient cells, generated using CRISPR-Cas9, showed increased MTCH2 expression and, consistent with this, decreased mitochondrial membrane potential. Thus, double-SCR of MTCH2 regulates its own expression levels contributing toward the maintenance of normal mitochondrial membrane potential. 相似文献
69.
70.
Jean-Numa Gillet 《Journal of biomolecular structure & dynamics》2013,31(4):690-705
Cell milking is a 100% renewable green energy for CO2 by extraction of biofuels inside the cytosol of photosynthetic micro-organisms as microalgae and bacteria. The cells are exposed to a hydrophobic solvent forming holes and cracks through their membranes from which the biofuels can leak out. In protein folding, the goal would be to find pathways to the unique functional protein conformer. However, in the lipid-bilayer interaction with the solvent for milking, the objective is to block the pathways for damaged membrane conformations of low free energy with undesired nanostructures, using the solvent properties, as shown with an ab initio structural bioinformatic model. Statistical thermodynamics is used to compute the free energy (including entropy) from the molecular dynamics trajectory of the biomolecular system with many conformational changes. This model can be extended to the general problem of biomolecules folding as for proteins and nucleic acids. Using an adaptation of the Einstein diffusion law, the conformational change dynamics of the lipid bilayer depends on the two diffusion coefficients of the solvent: D1 before the irreversible folding transition time and the much smaller D2 thereafter. In contrast to the n-hexane and n-heptane hydrocarbons of smaller size, the residual D2?=?4.7?×?10?7?cm2/s of the n-decane solvent, with the highest partition coefficient among the three extractors, is the only to present a D2 value that is significantly below the critical threshold of 10?6?cm2/s. Therefore, the membrane would resist to long hydrocarbons and the exposed cells would remain viable for milking. 相似文献