SUMMARY Small RNAs target invaders for silencing in the CRISPR-Cas pathways that protect bacteria and archaea from viruses and plasmids. The CRISPR RNAs (crRNAs) contain sequence elements acquired from invaders that guide CRISPR-associated (Cas) proteins back to the complementary invading DNA or RNA. Here, we have analyzed essential features of the crRNAs associated with the Cas RAMP module (Cmr) effector complex, which cleaves targeted RNAs. We show that Cmr crRNAs contain an 8-nucleotide 5’ sequence tag (also found on crRNAs associated with other CRISPR-Cas pathways) that is critical for crRNA function and can be used to engineer crRNAs that direct cleavage of novel targets. We also present data that indicates that the Cmr complex cleaves an endogenous complementary RNA in Pyrococcus furiosus, providing direct in vivo evidence of RNA targeting by the CRISPR-Cas system. Our findings indicate that the CRISPR RNA-Cmr protein pathway may be exploited to cleave RNAs of interest.

The present study demonstrates an eco-friendly and low cost protocol for synthesis of silver nanoparticles using the cell-free filtrate of Aspergillus flavus NJP08 when supplied with aqueous silver (Ag+) ions. Identification of the fungal isolate was based on nuclear ribosomal DNA internal transcribed spacer (ITS) identities. Transmission electron microscopy (TEM) and energy dispersive spectroscopy (EDS) revealed the formation of spherical metallic silver nanoparticles. The average particle size calculated using Dynamic Light Scattering measurements (DLS) was found to be 17±5.9 nm. UV-Visible and Fourier transform infrared (FTIR) spectroscopy confirmed the presence of extracellular proteins. SDS-PAGE profiles of the extracellular proteins showed the presence of two intense bands of 32 and 35 kDa, responsible for the synthesis and stability of silver nanoparticles, respectively. A probable mechanism behind the biosynthesis is discussed, which leads to the possibility of using the present protocol in future "nano-factories".

Anti-PD-1 therapy is used as a front-line treatment for many cancers, but mechanistic insight into this therapy resistance is still lacking. Here we generate a humanized (Hu)-mouse melanoma model by injecting fetal liver-derived CD34+ cells and implanting autologous thymus in immune-deficient NOD-scid IL2Rγnull (NSG) mice. Reconstituted Hu-mice are challenged with HLA-matched melanomas and treated with anti-PD-1, which results in restricted tumor growth but not complete regression. Tumor RNA-seq, multiplexed imaging and immunohistology staining show high expression of chemokines, as well as recruitment of FOXP3+ Treg and mast cells, in selective tumor regions. Reduced HLA-class I expression and CD8+/Granz B+ T cells homeostasis are observed in tumor regions where FOXP3+ Treg and mast cells co-localize, with such features associated with resistance to anti-PD-1 treatment. Combining anti-PD-1 with sunitinib or imatinib results in the depletion of mast cells and complete regression of tumors. Our results thus implicate mast cell depletion for improving the efficacy of anti-PD-1 therapy.

CD44 contributes to inflammation and fibrosis in response to injury. As fibroblast recruitment is critical to wound healing, we compared cytoskeletal architecture and migration of wild-type (CD44WT) and CD44-deficient (CD44KO) fibroblasts. CD44KO fibroblasts exhibited fewer stress fibers and focal adhesion complexes, and their migration was characterized by increased velocity but loss of directionality, compared with CD44WT fibroblasts. Mechanistically, we demonstrate that CD44WT cells generated more active TGFβ than CD44KO cells and that CD44 promotes the activation of TGFβ via an MMP-dependent mechanism. Reconstitution of CD44 expression completely rescued the phenotype of CD44KO cells whereas exposure of CD44KO cells to exogenous active TGFβ rescued the defect in stress fibers and migrational velocity, but was not sufficient to restore directionality of migration. These results resolve the TGFβ-mediated and TGFβ-independent effects of CD44 on fibroblast migration and suggest that CD44 may be critical for the recruitment of fibroblasts to sites of injury and the function of fibroblasts in tissue remodeling and fibrosis.

Highlights d Ectopic ELAV/Hu RBPs (Elav/Rbp9/Fne) induces global neural 3 0 UTR extensions d ELAV/Hu RBPs mediate bypass of proximal pA signals at the nascent transcript level d elav mutants induce an fne splicing switch to relocalize it from cytoplasm to nucleus d elav/fne double mutant larval CNS exhibit dramatic loss of neural 3 0 UTR extensions