Tumor-derived exosomes are gaining attention as important factors that facilitate communication between neighboring cells and manipulate cellular processes associated with cancer development or progression. The conventional techniques for the isolation and detection of exosomes face several limitations, restricting their clinical applications. Hence, a highly efficient technique for the isolation and identification of exosomes from biological samples may provide critical information about exosomes as biomarkers and improve our understanding of their unique role in cancer research. Here, we describe the use of antibody cocktail-conjugated magnetic nanowires to isolate exosomes from plasma of breast and lung cancer patients.The isolated exosomes were characterized based on size and concentration using nanoparticle tracking analysis. Levels of exosomal proteins were measured by bicinchoninic acid assay and enzyme-linked immunosorbent assay. Morphology was visualized by transmission electron microscopy. Immunoblotting (Western blotting) was used to detect the presence of exosomal markers.The use of antibody cocktail-conjugated magnetic nanowires resulted in approximately threefold greater yield when compared to the conventional methods. The elongated feature of nanowires significantly improved the efficiency of exosome isolation, suggesting its potential to be translated in diverse clinical applications, including cancer diagnosis and treatment.The nanowire-based method allows rapid isolation of homogeneous population of exosomes with relatively high yield and purity from even small amounts of sample. These results suggest that this method has the potential for clinical applications requiring highly purified exosomes for the analysis of protein, lipid, mRNA, and miRNA.
Abstract The correlation between copy number variation (CNV) and the susceptibility to systemic lupus erythematosus (SLE) has been reported for various immunity-related genes. However, the contribution of CNVs to SLE susceptibility awaits more investigation. To evaluate the copy numbers in immunity-related genes such as TNFAIP3 , TNIP1 , IL12B , TBX21 ( T-bet ), TLR7 , C4A , C4B , CCL3L1, and CCL3L3 , the modified real competitive polymerase chain reaction (mrcPCR) assay was employed, and the association between the copy numbers and SLE susceptibility was analyzed in 334 SLE patients and 338 controls. CCL3L3 -null status was significantly associated with SLE susceptibility (OR > 18, P < 0.0001), which remained significant by Bonferroni’s correction (corrected P = 0.0007). However, the significant association between C4B low-copy status and SLE susceptibility (OR = 1.6051, P = 0.0331) became non-significant by Bonferroni’s correction (corrected P = 0.3938). Except for these results, no other significant association between SLE susceptibility and copy number status in other genes was observed. The CCL3L3 -null status may be a significant factor for SLE susceptibility.
To elucidate the epigenetic mechanisms of drug resistance, epigenetically reprogrammed H460 cancer cells (R-H460) were established by the transient introduction of reprogramming factors. Then, the R-H460 cells were induced to differentiate by the withdrawal of stem cell media for various durations, which resulted in differentiated R-H460 cells (dR-H460). Notably, dR-H460 cells differentiated for 13 days (13dR-H460 cells) formed a significantly greater number of colonies showing drug resistance to both cisplatin and paclitaxel, whereas the dR-H460 cells differentiated for 40 days (40dR-H460 cells) lost drug resistance; this suggests that 13dR-cancer cells present short-term resistance (less than a month). Similarly, increased drug resistance to both cisplatin and paclitaxel was observed in another R-cancer cell model prepared from N87 cells. The resistant phenotype of the cisplatin-resistant (CR) colonies obtained through cisplatin treatment was maintained for 2-3 months after drug treatment, suggesting that drug treatment transforms cells with short-term resistance into cells with medium-term resistance. In single-cell analyses, heterogeneity was not found to increase in 13dR-H460 cells, suggesting that cancer cells with short-term resistance, rather than heterogeneous cells, may confer epigenetically driven drug resistance in our reprogrammed cancer model. The epigenetically driven short-term and medium-term drug resistance mechanisms could provide new cancer-fighting strategies involving the control of cancer cells during epigenetic transition.
Abstract Although several high-fidelity SpCas9 variants have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper–Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences and developed DeepSniper, a deep learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.
The cost-effectiveness of whole exome sequencing (WES) remains controversial due to variant call variability, necessitating sensitivity and specificity evaluation. WES was performed by three companies (AA, BB, and CC) using reference standards composed of DNA from hydatidiform mole and individual blood at various ratios. Sensitivity was assessed by the detection rate of null–homozygote (N–H) alleles at expected variant allelic fractions, while false positive (FP) errors were counted for unexpected alleles. Sensitivity was approximately 20% for in-house results from BB and CC and around 5% for AA. Dynamic Read Analysis for GENomics (DRAGEN) analyses identified 1.34 to 1.71 times more variants, detecting over 96% of in-house variants, with sensitivity for common variants increasing to 5%. In-house FP errors varied significantly among companies (up to 13.97 times), while DRAGEN minimized this variation. Despite DRAGEN showing higher FP errors for BB and CC, the increased sensitivity highlights the importance of effective bioinformatic conditions. We also assessed the potential effects of target enrichment and proposed optimal cutoff values for the read depth and variant allele fraction in WES. Optimizing bioinformatic analysis based on sensitivity and specificity from reference standards can enhance variant detection and improve the clinical utility of WES.
Abstract Gliosarcoma is a rare glioblastoma variant characterized by a biphasic tissue pattern with alternating areas displaying glial (GFAP-positive) or mesenchymal (reticulin-positive) differentiation. Previous analyses showed identical genetic alterations in both glial and mesenchymal tumor areas, suggesting that gliosarcomas are genetically monoclonal, and mesenchymal differentiation was considered to reflect the elevated genomic instability of glioblastomas. In the present study, we compared genome-wide chromosomal imbalances using array CGH (105K) in glial and mesenchymal tumor areas of 13 gliosarcomas. Patterns of gain and loss were similar, except for gain at 13q13.3-q14.1 (log2 ratio ≤ 3), containing the STOML3, FREM2, and LHFP genes, which was restricted to the mesenchymal tumor area of a gliosarcoma. Further analyses of 64 cases of gliosarcoma using quantitative PCR showed amplification of the STOML3, FREM2 and LHFP genes in 14 (21%), 10 (15%), and 7 (11%) of mesenchymal tumor areas but not in glial tumor areas. Immunohistochemistry confirmed that STOML3 and FREM2 overexpression was more extensive in mesenchymal than in glial tumor areas. These results suggest that mesenchymal components in a small fraction of gliosarcomas may be derived from glial cells with additional genetic alterations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3401. doi:1538-7445.AM2012-3401
Abstract Although several high-fidelity SpCas9 variants that have reduced activities at mismatched target sequences have been reported, it has been observed that this increased specificity is associated with reduced on-target activity, limiting the applications of the high-fidelity variants when efficient genome editing is required. Here, we developed an improved version of Sniper-Cas9, Sniper2L, which represents an exception to this trade-off trend as it showed higher specificity with retained high activity. We evaluated Sniper2L activities at a large number of target sequences, and developed DeepSniper, a deep-learning model that can predict the activity of Sniper2L. We also confirmed that Sniper2L can induce highly efficient and specific editing at a large number of target sequences when it is delivered as a ribonucleoprotein complex. Mechanically, the high specificity of Sniper2L originates from its superior ability to avoid unwinding a target DNA containing even a single mismatch. We envision that Sniper2L will be useful when efficient and specific genome editing is required.
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