The rockefeller university

Most natural product biosynthetic gene clusters identified in bacterial genomic and metagenomic sequencing efforts are silent under laboratory growth conditions. Here, we describe a scalable biosynthetic gene cluster activation method wherein the gene clusters are disassembled at interoperonic regions in vitro using CRISPR/Cas9 and then reassembled with PCR-amplified, short DNAs, carrying synthetic promoters, using transformation assisted recombination (TAR) in yeast. This simple, cost-effective, and scalable method allows for the simultaneous generation of combinatorial libraries of refactored gene clusters, eliminating the need to understand the transcriptional hierarchy of the silent genes. In two test cases, this in vitro disassembly-TAR reassembly method was used to create collections of promoter-replaced gene clusters that were tested in parallel to identify versions that enabled secondary metabolite production. Activation of the atolypene (ato) gene cluster led to the characterization of two unprecedented bacterial cyclic sesterterpenes, atolypene A (1) and B (2), which are moderately cytotoxic to human cancer cell lines. This streamlined in vitro disassembly-in vivo reassembly method offers a simplified approach for silent gene cluster refactoring that should facilitate the discovery of natural products from silent gene clusters cloned from either metagenomes or cultured bacteria.

Many cellular processes rely on precise and timely deformation of the

Background: IL-22 is potentially a pathogenic cytokine in patients with atopic dermatitis (AD), but the molecular effects of IL-22 antagonism have not been defined in human subjects. Objective: We sought to evaluate the cellular and molecular effects of IL-22 blockade in tissues from patients with moderate-to-severe AD. Methods: We assessed lesional and nonlesional skin from 59 patients with moderate-to-severe AD treated with anti-IL-22 (fezakinumab) versus placebo (2: 1) using transcriptomic and immunohistochemistry analyses. Results: Greater reversal of the AD genomic profile was seen with fezakinumab versus placebo, namely 25.3% versus 10.5% at 4 weeks (P = 1.7 x 10(-5)) and 65.5% versus 13.9% at 12 weeks (P = 9.5 3 10(-19)), respectively. Because IL-22 blockade showed clinical efficacy only in patients with severe AD, we used baseline median IL-22 mRNA expression to stratify for high (n = 30) and low (n = 29) IL-22 expression groups. Much stronger mean transcriptomic improvements were seen with fezakinumab in the IL-22-high drug-treated group (82.8% and 139.4% at 4 and 12 weeks, respectively) than in the respective IL-22-high placebo-treated group (39.6% and 56.3% at 4 and 12 weeks) or the IL-22-low groups. Significant downregulations of multiple immune pathways, including T(H)1/CXCL9, T(H)2/CCL18/CCL22, T(H)17/CCL20/DEFB4A, and T(H)22/IL22/S100A's, were restricted to the IL-22-high drug group (P <.05). Consistently, tissue predictors of clinical response were mostly genes involved in T-cell and dendritic cell activation and differentiation. Conclusions: This is the first report showing a profound effect of IL-22 blockade on multiple inflammatory pathways in AD. These data, supported by robust effects in patients with high IL-22 baseline expression, suggest a central role for IL-22 in AD, indicating the need for a precision medicine approach for improving therapeutic outcomes in patients with AD.

Many bacteria and archaea have the unique ability to heritably alter their genomes by incorporating small fragments of foreign DNA, called spacers, into CRISPR loci. Once transcribed and processed into individual CRISPR RNAs, spacer sequences guide Cas effector nucleases to destroy complementary, invading nucleic acids. Collectively, these two processes are known as the CRISPR-Cas immune response. In this Progress article, we review recent studies that have advanced our understanding of the molecular mechanisms underlying spacer acquisition and that have revealed a fundamental link between the two phases of CRISPR immunity that ensures optimal immunity from newly acquired spacers. Finally, we highlight important open questions and discuss the potential basic and applied impact of spacer acquisition research.

In November 2017, a formal debate on the role of bacteria in the pathogenesis of hidradenitis suppurativa (HS) was held at the 2nd Symposium on Hidradenitis Suppurativa Advances (SHSA) in Detroit, Michigan. In this report, we present both sides of the argument as debated at the SHSA meeting and then discuss the potential role of bacteria as classic infectious pathogens versus an alternative pathogenic role as activators of dysregulated commensal bacterial-host interactions. Although there was consensus that bacteria play a role in pathogenesis and thus are pathogenic, there was a compelling discussion about whether bacteria in HS incite an infectious disease as we classically understand it or whether bacteria might play a different role in HS pathogenesis.

RNA-binding proteins (RBPs) control and coordinate each stage in the life cycle of RNAs. Although in vivo binding sites of RBPs can now be determined genome-wide, most studies typically focused on individual RBPs. Here, we examined a large compendium of 114 high-quality transcriptome-wide in vivo RBP-RNA cross-linking interaction datasets generated by the same protocol in the same cell line and representing 64 distinct RBPs. Comparative analysis of categories of target RNA binding preference, sequence preference, and transcript region specificity was performed, and identified potential posttranscriptional regulatory modules, i.e. specific combinations of RBPs that bind to specific sets of RNAs and targeted regions. These regulatory modules represented functionally related proteins and exhibited distinct differences in RNA metabolism, expression variance, as well as subcellular localization. This integrative investigation of experimental RBP-RNA interaction evidence and RBP regulatory function in a human cell line will be a valuable resource for understanding the complexity of post-transcriptional regulation.

Octonionic representation of O(9, 1) with special emphasis placed on the analytic properties of octonionic functions is investigated.

New results are shown on the generalization of phenomenological hadronic supersymmetry based on QCD as an extension of spin-flavor SU(6) symmetry. A more general supersymmetric extension of the group SU(6/21) will be given with applications to pentaquark and tetraquark states for which there is a great deal of ongoing research.