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Immune systems must recognize and destroy different pathogens that threaten the host. CRISPR-Cas immune systems protect prokaryotes from viral and plasmid infection utilizing small CRISPR RNAs that are complementary to the invader's genome and specify the targets of RNA-guided Cas nucleases. Type III CRISPR-Cas immunity requires target transcription, and whereas genetic studies demonstrated DNA targeting, in vitro data have shown crRNA-guided RNA cleavage. The molecular mechanism behind these disparate activities is not known. Here, we show that transcription across the targets of the Staphylococcus epidermidis type III-A CRISPR-Cas system results in the cleavage of the target DNA and its transcripts, mediated by independent active sites within the Cas10-Csm ribonucleoprotein effector complex. Immunity against plasmids and DNA viruses requires DNA, but not RNA, cleavage activity. Our studies reveal a highly versatile mechanism of CRISPR immunity that can defend microorganisms against diverse DNA and RNA invaders.

Cryptococcus neoformans is an opportunistic yeast that kills over 625,000 people yearly through lethal meningitis. Host phagocytes serve as the first line of defense against this pathogen, but fungal engulfment and subsequent intracellular proliferation also correlate with poor patient outcome. Defining the interactions of this facultative intracellular pathogen with host phagocytes is key to understanding the latter's opposing roles in infection and how they contribute to fungal latency, dissemination, and virulence. We used high-content imaging and a human monocytic cell line to screen 1,201 fungal mutants for strains with altered host interactions and identified multiple genes that influence fungal adherence and phagocytosis. One of these genes was PFA4, which encodes a protein S-acyl transferase (PAT), one of a family of DHHC domain-containing proteins that catalyzes lipid modification of proteins. Deletion of PFA4 caused dramatic defects in cryptococcal morphology, stress tolerance, and virulence. Bioorthogonal palmitoylome-profiling identified Pfa4-specific protein substrates involved in cell wall synthesis, signal transduction, and membrane trafficking responsible for these phenotypic alterations. We demonstrate that a single PAT is responsible for the modification of a subset of proteins that are critical in cryptococcal pathogenesis. Since several of these palmitoylated substrates are conserved in other pathogenic fungi, protein palmitoylation represents a potential avenue for new antifungal therapeutics.

Herpes simplex virus 1 (HSV-1) is a common virus that can rarely invade the human central nervous system (CNS), causing devastating encephalitis. The permissiveness to HSV-1 of the various relevant cell types of the CNS, neurons, astrocytes, oligodendrocytes, and microglia cells, as well as their response to viral infection, has been extensively studied in humans and other animals. Nevertheless, human CNS cell-based models of anti-HSV-1 immunity are of particular importance, as responses to any given neurotropic virus may differ between humans and other animals. Human CNS neuron cell lines as well as primary human CNS neurons, astrocytes, and microglia cells cultured/isolated from embryos or cadavers, have enabled the study of cell-autonomous anti-HSV-1 immunity in vitro. However, the paucity of biological samples and their lack of purity have hindered progress in the field, which furthermore suffers from the absence of testable primary human oligodendrocytes. Recently, the authors have established a human induced pluripotent stem cells (hiPSCs)-based model of anti-HSV-1 immunity in neurons, oligodendrocyte precursor cells, astrocytes, and neural stem cells, which has widened the scope of possible in vitro studies while permitting in-depth explorations. This mini-review summarizes the available data on human primary and iPSC-derived CNS cells for anti-HSV-1 immunity. The hiPSC-mediated study of anti-viral immunity in both healthy individuals and patients with viral encephalitis will be a powerful tool in dissecting the disease pathogenesis of CNS infections with HSV-1 and other neurotropic viruses.

Background: Genomic profiling of lesional and nonlesional skin of patients with atopic dermatitis (AD) using microarrays has led to increased understanding of AD and identification of novel therapeutic targets. However, the limitations of microarrays might decrease detection of AD genes. These limitations might be lessened with next-generation RNA sequencing (RNA-seq). Objective: We sought to define the lesional AD transcriptome using RNA-seq and compare it using microarrays performed on the same cohort. Methods: RNA-seq and microarrays were performed to identify differentially expressed genes (criteria: fold change, >= 2.0; false discovery rate <= 0.05) in lesional versus nonlesional skin from 18 patients with moderate-to-severe AD, with real-time PCR (RT-PCR) and immunohistochemistry used for validation. Results: Both platforms showed robust disease transcriptomes and correlated well with RT-PCR. The common AD transcriptome identified by using both techniques contained 217 genes, including inflammatory (S100A8/A9/A12, CXCL1, and 2'-5'-oligoadenylate synthetase-like [OASL]) and barrier (MKi67, keratin 16 [K16], and claudin 8 [CLDN8]) AD-related genes. Although fold change estimates determined by using RNA-seq showed somewhat better agreement with RT-PCR (intraclass correlation coefficient, 0.57 and 0.70 for microarrays and RNA-seq vs RT-PCR, respectively), bias was not eliminated. Among genes uniquely identified by using RNA-seq were triggering receptor expressed on myeloid cells 1 (TREM-1) signaling (eg, CCL2, CCL3, and single immunoglobulin domain IL1R1 related [SIGIRR]) and IL-36 isoform genes. TREM-1 is a surface receptor implicated in innate and adaptive immunity that amplifies infection-related inflammation. Conclusions: This is the first report of a lesional AD phenotype using RNA-seq and the first direct comparison between platforms in this disease. Both platforms robustly characterize the AD transcriptome. Through RNA-seq, we unraveled novel disease pathology, including increased expression of the novel TREM-1 pathway and the IL-36 cytokine in patients with AD.

This study explored whether different ratios of fructose (F) and glucose (G) in sugar can engender significant differences in self-administration and associated neurobiological and physiological responses in male Sprague-Dawley rats. In Experiment 1, animals self-administered pellets containing 55% F + 45% G or 30% F + 70% G, and Fos immunoreactivity was assessed in hypothalamic regions regulating food intake and reward. In Experiment 2, rats self-administered solutions of 55% F + 42% G (high fructose corn syrup (HFCS)), 50% F + 50% G (sucrose) or saccharin, and mRNA of the dopamine 2 (D2R) and mu-opioid (MOR) receptor genes were assessed in striatal regions involved in addictive behaviors. Finally, in Experiment 3, rats self-administered HFCS and sucrose in their home cages, and hepatic fatty acids were quantified. It was found that higher fructose ratios engendered lower self-administration, lower Fos expression in the lateral hypothalamus/arcuate nucleus, reduced D2R and increased MOR mRNA in the dorsal striatum and nucleus accumbens core, respectively, as well as elevated omega-6 polyunsaturated fatty acids in the liver. These data indicate that a higher ratio of fructose may enhance the reinforcing effects of sugar and possibly lead to neurobiological and physiological alterations associated with addictive and metabolic disorders.

Translocating proteins across the double membrane of Gram-negative bacteria, type III secretion systems (T3SS) occur in two evolutionarily related forms: injectisomes, delivering virulence factors into host cells, and the flagellar system, secreting the polymeric filament used for motility. While both systems share related elements of a cytoplasmic sorting platform that facilitates the hierarchical secretion of protein substrates, its assembly and regulation remain unclear. Here we describe a module mediating the assembly of the sorting platform in both secretion systems, and elucidate the structural basis for segregation of homologous components among these divergent T3SS subtypes sharing a common cytoplasmic milieu. These results provide a foundation for the subtype-specific assembly of T3SS sorting platforms and will support further mechanistic analysis and anti-virulence drug design.

We measure exclusive pi(+)pi(-) production in proton-antiproton collisions at center-of-mass energies root s = 0.9 and 1.96 TeV in the Collider Detector at Fermilab. We select events with two oppositely charged particles, assumed to be pions, with pseudorapidity vertical bar eta vertical bar < 1.3 and with no other particles detected in vertical bar eta vertical bar < 5.9. We require the pi(+)pi(-) system to have rapidity vertical bar y vertical bar < 1.0. The production mechanism of these events is expected to be dominated by double pomeron exchange, which constrains the quantum numbers of the central state. The data are potentially valuable for isoscalar meson spectroscopy and for understanding the pomeron in a region of transition between nonperturbative and perturbative quantum chromodynamics. The data extend up to dipion mass M(pi(+)pi(-)) = 5000 MeV/c(2) and show resonance structures attributed to f(0) and f(2)(1270) mesons. From the pi(+)pi(-) and K+K- spectra, we place upper limits on exclusive chi(c0)(3415) production.

Adult stem cells occur in niches that balance self-renewal with lineage selection and progression during tissue homeostasis. Following injury, culture or transplantation, stem cells outside their niche often display fate flexibility(1-4). Here we show that super-enhancers(5) underlie the identity, lineage commitment and plasticity of adult stem cells in vivo. Using hair follicle as a model, we map the global chromatin domains of hair follicle stem cells and their committed progenitors in their native microenvironments. We show that super-enhancers and their dense clusters ('epicentres') of transcription factor binding sites undergo remodelling upon lineage progression. New fate is acquired by decommissioning old and establishing new super-enhancers and/or epicentres, an auto-regulatory process that abates one master regulator subset while enhancing another. We further show that when outside their niche, either in vitro or in wound-repair, hair follicle stem cells dynamically remodel super-enhancers in response to changes in their microenvironment. Intriguingly, some key super-enhancers shift epicentres, enabling their genes to remain active and maintain a transitional state in an ever-changing transcriptional landscape. Finally, we identify SOX9 as a crucial chromatin rheostat of hair follicle stem cell super-enhancers, and provide functional evidence that super-enhancers are dynamic, dense transcription-factor-binding platforms which are acutely sensitive to pioneer master regulators whose levels define not only spatial and temporal features of lineage-status but also stemness, plasticity in transitional states and differentiation.

Motivation: RNA-Seq is a method for profiling transcription using high-throughput sequencing and is an important component of many research projects that wish to study transcript isoforms, condition specific expression and transcriptional structure. The methods, tools and technologies used to perform RNA-Seq analysis continue to change, creating a bioinformatics challenge for researchers who wish to exploit these data. Resources that bring together genomic data, analysis tools, educational material and computational infrastructure can minimize the overhead required of life science researchers. Results: RNA-Rocket is a free service that provides access to RNA-Seq and ChIP-Seq analysis tools for studying infectious diseases. The site makes available thousands of pre-indexed genomes, their annotations and the ability to stream results to the bioinformatics resources VectorBase, EuPathDB and PATRIC. The site also provides a combination of experimental data and metadata, examples of pre-computed analysis, step-by-step guides and a user interface designed to enable both novice and experienced users of RNA-Seq data.