We evaluated SpCas9 activities at 12,832 target sequences using a high-throughput approach based on a human cell library containing single-guide RNA–encoding and target sequence pairs. Deep learning–based training on this large dataset of SpCas9-induced indel frequencies led to the development of a SpCas9 activity–predicting model named DeepSpCas9. When tested against independently generated datasets (our own and those published by other groups), DeepSpCas9 showed high generalization performance. DeepSpCas9 is available at http://deepcrispr.info/DeepSpCas9.
The functional rules for microRNA (miRNA) targeting remain controversial despite their biological importance because only a small fraction of distinct interactions, called site types, have been examined among an astronomical number of site types that can occur between miRNAs and their target mRNAs. To systematically discover functional site types and to evaluate the contradicting rules reported previously, we used large-scale transcriptome data and statistically examined whether each of approximately 2 billion site types is enriched in differentially downregulated mRNAs responding to overexpressed miRNAs. Accordingly, we identified seven non-canonical functional site types, most of which are novel, in addition to four canonical site types, while also removing numerous false positives reported by previous studies. Extensive experimental validation and significantly elevated 3' UTR sequence conservation indicate that these non-canonical site types may have biologically relevant roles. Our expanded catalog of functional site types suggests that the gene regulatory network controlled by miRNAs may be far more complex than currently understood.
Cryoprotective agents (CPAs) such as dimethyl sulfoxide (DMSO), glycerol, ethylene glycol, and propylene glycol have been used for the cryopreservation of cells and tissues. DMSO is the most effective CPA but shows high cytotoxicity and can effect differentiation. -Poly-L-lysine (PLL) derivatives show higher cryopreservation efficiency than the conventional CPAs. Culture medium solutions with 7.5 w/w% of PLL whose amino groups of more than 50 mol% were converted to carboxyl groups by succinic anhydride showed higher postthaw survival efficiency of L929 cells than those of current CPAs without the addition of any proteins. In addition, rat mesenchymal stem cells were cryopreserved more effectively than with DMSO and fully retained the potential for proliferation and differentiation. Furthermore, many kinds of cells could be cryopreserved with PLL having the appropriate ratio of COOH groups, regardless of the cell types, including adhesive and floating cells, human-and mouse-derived cells, primary cells, and established cell lines. The properties might be associated with the antifreeze protein properties. These results indicate that these polymeric extracellular CPAs may replace current CPAs and the high viability after thawing and nonnecessity of serum ensure that these CPAs may be used in various preservation fields.Key words: Cryopreservation; Poly-lysine; Dimethyl sulfoxide; Mesenchymal stem cell; Antifreeze protein INTRODUCTIONrequires designing optimal preservation protocols. Furthermore, current CPAs that penetrate the cell membrane are toxic themselves due to the high osmotic presCell transplantation is becoming a valuable tool in clinical medical care and many studies have focused on sure at the effective concentrations. DMSO is also toxic and affects the differentiation of many types of cells the development of effective methods of long-term preservation of living cells (8,11,21,22,33). The cryopreser-(12,31) and needs to be eliminated rapidly after thawing. In contrast, carbohydrate CPAs, such as trehalose and vation of cells is an important traditional approach of preservation of tissue for cell transplantation for various sucrose, cannot penetrate the cell membrane and have an exceptional ability to stabilize and preserve the memdiseases with recent advances in tissue engineering (4,15,23,24,28). Since Polge reported that a glycerol sobrane (3). In addition, some proteins and synthetic polymers have some degree of cryopreservative properties. lution could preserve cattle sperm at −79°C in 1949 (25), the rapid improvement of the science of cryobiolHowever, CPAs that cannot penetrate the cell membrane have weak cryoprotective properties and are insufficient ogy led to the discovery of the dimethyl sulfoxide (DMSO) as a cryoprotective agent (CPA) for other types for single application. Although many have reported that cryopreservation of stem cells with DMSO, glycerol, or of cells (18). Although many chemicals such as glycerol, ethylene glycol, propylene glycol (PG), and trehalose mixtures of CPAs, few s...
Crosstalk between cancer cells and carcinoma-associated fibroblasts (CAFs) has earned recognition as an interaction that plays a pivotal role in carcinogenesis. Thus, we attempted to clarify whether increase in the level of CAFs promotes cancer progression by proportionally enhancing the interaction between cancer cells and CAFs. We first analyzed clinical correlation between the levels of fibroblasts and cancer progression and found that the level of CAFs made a noticeable difference on the prognosis of patients with oral squamous cell carcinoma (OSCC). In vivo animal study also demonstrated that tumor volume depended on the dose of CAFs that was co-injected with OSCC cells. The same tendency was observed in an in vitro study. We also found that interleukin-1α (IL-1α) secreted from OSCC cells had dual effects on CAFs: IL-1α not only promoted the proliferation of CAFs but also upregulated the secretion of cytokines in CAFs such as CCL7, CXCL1, and IL-8. The induction activity of cytokine secretion by IL-1α surpassed that of proliferation in OSCC cells. In summary, we unraveled an important interactive mechanism of carcinogenesis: IL-1α released from carcinoma stimulates the proliferation of CAFs and the simultaneous increase in cytokine secretion from CAFs promotes cancer progression in human OSCC. On the basis of these findings, we propose that the level of CAFs is eligible for being selected as a prognostic factor that will be useful in routine diagnosis. We also propose that blockage of reciprocal interaction between cancer cells and CAFs will provide an insight for developing novel chemotherapeutic strategy.
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