Pathogen Recognition and Innate Immunity

Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

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RNA-Guided Human Genome Engineering via Cas9

The discovery of Toll-like receptors (TLRs) as components that recognize conserved structures in pathogens has greatly advanced understanding of how the body senses pathogen invasion, triggers innate immune responses and primes antigen-specific adaptive immunity. Although TLRs are critical for host defense, it has become apparent that loss of negative regulation of TLR signaling, as well as recognition of self molecules by TLRs, are strongly associated with the pathogenesis of inflammatory and autoimmune diseases. Furthermore, it is now clear that the interaction between TLRs and recently identified cytosolic innate immune sensors is crucial for mounting effective immune responses. Here we describe the recent advances that have been made by research into the role of TLR biology in host defense and disease.

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The role of pattern-recognition receptors in innate immunity: update on Toll-like receptors

Pattern Recognition Receptors and Inflammation

The transdifferentiation of epithelial cells into motile mesenchymal cells, a process known as epithelial-mesenchymal transition (EMT), is integral in development, wound healing and stem cell behaviour, and contributes pathologically to fibrosis and cancer progression. This switch in cell differentiation and behaviour is mediated by key transcription factors, including SNAIL, zinc-finger E-box-binding (ZEB) and basic helix-loop-helix transcription factors, the functions of which are finely regulated at the transcriptional, translational and post-translational levels. The reprogramming of gene expression during EMT, as well as non-transcriptional changes, are initiated and controlled by signalling pathways that respond to extracellular cues. Among these, transforming growth factor-β (TGFβ) family signalling has a predominant role; however, the convergence of signalling pathways is essential for EMT.

Molecular mechanisms of epithelial–mesenchymal transition

Altered phospholipid molecular species and glycolipid composition in brain, liver and fibroblasts of Zellweger syndrome

Ribozymes are catalytic RNAs that can cleave RNAs at specific sites, thus they have been employed to degrade a target mRNA in vivo. Development of allosterically controllable ribozymes is of great current interest, but it remained difficult to furnish such functions to ribozymes in cultured cells or in animals. Recently, we designed allosterically controllable ribozymes termed maxizymes, which have sensor arms that recognize target mRNA sequences and, in the presence of such target sequences only, they form a cavity that can capture catalytically indispensable ions, cleaving the target. The maxizyme was applied to therapy for chronic myelogenous leukemia (CML). It cleaved specifically the chimeric BCR-ABL mRNA, which caused CML, without damaging the normal ABL or BCR mRNA in mammalian cells and also in mice, providing the first successful example for allosteric control of the activity of artificial ribozymes in vivo.

MAXIZYMEs: Allosterically controllable ribozymes with biosensor functions

PROBLEM TO BE SOLVED: To obtain a skin-lightening cosmetic which contains a substituted amino acid derivative, is safe and excellent in the effect of preventing the pigmentation of brown color and improving the same and causes no discoloration. SOLUTION: This cosmetic contains one or more of compounds of formula I or formula II (R1 is a (substituted) acyl; R2 is hydroxyl, amino, a lower alkoxy; the wavy line means that the absolute configuration of the carbon atom bonding to CO-R2 may be any of L, D or DL) or its salt as an active ingredient. In addition, one or more selected from skin-lightening agents and an ultraviolet absorbers, keatinization-improving agents and the like are formulated thereto whereby the objective cosmetic is increased in its melanin-inhibiting effect and can be used as a skin-lightening cosmetic having the effect of preventing skin-tanning. This cosmetic is effective for skin inflammation by ultraviolet rays, skin spots or stains, pigmentation after sun burn and the like with high safety almost without side-effect.

Skin lightening cosmetic

The phenomenon known as RNA interference (RNAi) by double-strand ed RNA (dsRNA) that was reported recently in the nematode Caenorhabditis elegans has been shown to operate by a mechanism that is widely conserved among species including plant cells. No quantitative analysis of the effects of RNAi on the expression of specific genes in plant cultured cells has been reported. An RNAi effect was observed 24 h after the introduction of dsRNA expression plasmids into tobacco BY-2 cells by electroporation. The simple system for suppression of specific genes in plant cells should be useful in attempts to elucidate the roles of individual genes in plant cells.

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A simple and rapid system for the quantitation of RNA interference in plant cultured cells.

RNA interference (RNAi) is an evolutionarily conserved phenomenon in which gene expression is silenced by double-stranded RNA (dsRNA) in a sequence-specific manner. This technology has the potential to affect all aspects of target discovery and validation. With the completion of the human genome, it is now possible to design small-interfering RNA (siRNA) libraries targeting every human gene. Specific siRNAs, libraries containing a pathway, gene family, or gene set of interest, are expected to unsecure new targets in pathways of therapeutic interest. Here, we highlight the potential of siRNA screens for target identification by using cell-based assays.

Makoto Miyagishi

Papers

Altered phospholipid molecular species and glycolipid composition in brain, liver and fibroblasts of Zellweger syndrome

MAXIZYMEs: Allosterically controllable ribozymes with biosensor functions

Skin lightening cosmetic

A simple and rapid system for the quantitation of RNA interference in plant cultured cells.

Genome-Wide Screening by Using Small-Interfering RNA-Expression Libraries