Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition

This is the final version of the article. Available from the publisher via the DOI in this record.Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.This work was financially supported by an NWO Vidi grant to SJJB (864.11.005). RNJ and BW are supported by the National Institutes of Health (GM 103500) and the Montana State University Agricultural Experimental Station. ES, KAD and KS are supported by an NIH grant RO1 GM10407, a program grant in Molecular and Cell Biology from Presidium of Russian Academy of Sciences and a Russian Foundation for Basic Research grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Food matrix and isoflavones bioavailability in early post menopausal women: A European clinical study

The estrogenic effects of soy isoflavones (IF) on symptoms of menopause are of particular interest. The aim of the present study was to improve compliance of IF in two IF-enriched foods providing the same IF circulating levels in postmenopausal women. Forty-two healthy postmenopausal women (mean age: 53.28 years) were recruited for a randomized, crossover, multicenter trial conducted in the Netherlands, Italy and France. Over 18 days, volunteers were assigned to two groups and supplemented with two different IF-enriched foods (100 mg IF aglycones/two servings). The first group had to eat two biscuits daily for three days. After a wash-out period (11 d), they received cereal bars for three days. The second group started with the cereal bars and finished with biscuits. After IF intake, plasma and urinary levels of genistein, daidzein, O desmethyl angolensin and equol significantly increased and returned to baseline level after the washout period. There was no difference between biscuits and cereals bars intake, as shown by group values at each end of experimental period (day 4 or day 18). Both matrixes are comparable in terms of IF-circulating levels and could be used independently

Design approaches in technology enhanced learning

Design is a critical to the successful development of any interactive learning environment (ILE). Moreover, in technology enhanced learning (TEL), the design process requires input from many diverse areas of expertise. As such, anyone undertaking tool development is required to directly address the design challenge from multiple perspectives. We provide a motivation and rationale for design approaches for learning technologies that draws upon Simon's seminal proposition of Design Science (Simon, 1969). We then review the application of Design Experiments (Brown, 1992) and Design Patterns (Alexander et al., 1977) and argue that a patterns approach has the potential to address many of the critical challenges faced by learning technologists

Valproic Acid Teratogenicity: A Toxicogenomics Approach

Embryonic development is a highly coordinated set of processes that depend on hierarchies of signaling and gene regulatory networks, and the disruption of such networks may underlie many cases of chemically induced birth defects. The antiepileptic drug valproic acid (VPA) is a potent inducer of neural tube defects (NTDs) in human and mouse embryos. As with many other developmental toxicants however, the mechanism of VPA teratogenicity is unknown. Using microarray analysis, we compared the global gene expression responses to VPA in mouse embryos during the critical stages of teratogen action in vivo with those in cultured P19 embryocarcinoma cells in vitro. Among the identified VPA-responsive genes, some have been associated previously with NTDs or VPA effects [vinculin, metallothioneins 1 and 2 (Mt1, Mt2), keratin 1-18 (Krt1-18)], whereas others provide novel putative VPA targets, some of which are associated with processes relevant to neural tube formation and closure [transgelin 2 (Tagln2), thyroid hormone receptor interacting protein 6, galectin-1 (Lgals1), inhibitor of DNA binding 1 (Idb1), fatty acid synthase (Fasn), annexins A5 and A11 (Anxa5, Anxa11)], or with VPA effects or known molecular actions of VPA (Lgals1, Mt1, Mt2, Id1, Fasn, Anxa5, Anxa11, Krt1-18). A subset of genes with a transcriptional response to VPA that is similar in embryos and the cell model can be evaluated as potential biomarkers for VPA-induced teratogenicity that could be exploited directly in P19 cell–based in vitro assays. As several of the identified genes may be activated or repressed through a pathway of histone deacetylase (HDAC) inhibition and specificity protein 1 activation, our data support a role of HDAC as an important molecular target of VPA action in vivo

Structural basis for CRISPR RNA-guided DNA recognition by Cascade

The CRISPR (clustered regularly interspaced short palindromic repeats) immune system in prokaryotes uses small guide RNAs to neutralize invading viruses and plasmids. In Escherichia coli, immunity depends on a ribonucleoprotein complex called Cascade. Here we present the composition and low-resolution structure of Cascade and show how it recognizes double-stranded DNA (dsDNA) targets in a sequence-specific manner. Cascade is a 405-kDa complex comprising five functionally essential CRISPR-associated (Cas) proteins (CasA1B2C6D1E1) and a 61-nucleotide CRISPR RNA (crRNA) with 5′-hydroxyl and 2′,3′-cyclic phosphate termini. The crRNA guides Cascade to dsDNA target sequences by forming base pairs with the complementary DNA strand while displacing the noncomplementary strand to form an R-loop. Cascade recognizes target DNA without consuming ATP, which suggests that continuous invader DNA surveillance takes place without energy investment. The structure of Cascade shows an unusual seahorse shape that undergoes conformational changes when it binds target DNA.

A rare missense mutation in <i>GJB3</i> (Cx31G45E) is associated with a unique cellular phenotype resulting in necrotic cell death

Erythrokeratodermia variabilis et progressiva (EKV-P) is caused by mutations in either the GJB3 (Cx31) or GJB4 genes (Cx30.3). We identified a rare GJB3 missense mutation, c.134G>A (p.G45E), in two unrelated patients and investigated its cellular characteristics. Expression of Cx31G45E-GFP caused previously undescribed changes within HeLa cells and HaCaT cells, a model human keratinocyte cell line. Cx31WT-GFP localised to the plasma membrane, but expression of Cx31G45E-GFP caused vacuolar expansion of the endoplasmic reticulum (ER), the mutant protein accumulated within the ER membrane and disassembly of the microtubular network occurred. No ER stress responses were evoked. Cx31WT-myc-myc-6xHis and Cx31G45E-GFP co-immunoprecipitated, indicative of heteromeric interaction, but co-expression with Cx31WT-mCherry, Cx26 or Cx30.3 did not mitigate the phenotype. Cx31 and Cx31G45E both co-immunoprecipitated with Cx43, indicating the ability to form heteromeric connexons. WT-Cx31 and Cx43 assembled into large gap junction plaques at points of cell-to-cell contact; Cx31G45E restricted the ability of Cx43 to reach the plasma membrane in both HaCaT cells and HeLa cells stably expressing Cx43 where the proteins strongly co-localised with the vacolourised ER. Cell viability assays identified an increase in cell death in cells expressing Cx31G45E-GFP, which FACS analysis determined was necrotic. Blocking connexin channel function with 18α-glycyrrhetinic acid did not completely rescue necrosis or prevent propidium iodide uptake, suggesting that expression of Cx31G45E-GFP damages the cellular membrane independent of its channel function. Our data suggest that entrapment of Cx43 and necrotic cell death in the epidermis could underlie the EKV skin phenotype

Verslag workshops Friese Veenweidevisie

Waterschap en provincie werken aan een Veenweidevisie voor Friesland. In de Veenweidevisie vormt de zorg over de toenemende snelheid van de maaivelddaling en het verlies van de veenbodem een centraal aandachtspunt. Voor het verkennen van de problemen en oplossingen zijn streekbijeenkomsten georganiseerd. Drie scenario’s zijn voor de toekomst van de veenweiden in Friesland beschreven. Deze drie scenario’s zijn voor drie voorbeeldgebieden, Hommerts, Grote Veenpolder en het Buitenveld, uitgewerkt in drie workshops

Case study: Treatment of oral and locomotory stereotypic behaviors in a mature sow

A 32-month-old female 225-kg nonpregnant cross-bred Newsham sow presented a 6-week history of stereotypic behaviors when housed in a laboratory research facility. A behavioral examination over 12 daylight hours revealed 3 main stereotypic motor patterns, namely (1) oral-nasal gate manipulation defined as placement of the snout between the bars of the pen gate with repetitive, forceful up and down movement; (2) head weaving defined as repetitive lateral head and snout movement toward the pen gates while rocking back and forth on her forequarters with hooves remaining on ground at all times; and (3) body weaving defined as repetitive shifting of body weight from one side to the other with front hooves lifting alternately off the ground. The sow performed the oral-nasal gate manipulation and head and body weaving 4.0%, 12.4%, and 6.8% of her total baseline time budget, respectively. The presumptive diagnosis was oral-nasal and locomotory stereotypies. Three treatments were used to mitigate the duration and frequency of these stereotypic behaviors. Treatment 1—Social treatment (change social stimuli by providing visual and nose-to-nose contact with different neighboring sows); Treatment 2—Forage treatment (change foraging substrates by providing peat moss as a rooting substrate); and Treatment 3—Space treatment (change pen configuration by increasing space). The sow performed the oral-nasal gate manipulation and head and body weaving 0%, 0.4%, and 0.1% of her total time budget, respectively; social treatment: the sow performed the oral-nasal gate manipulation and head and body weaving 0.9%, 15.3%, and 11.3% of her total time budget, respectively; and forage treatment: the sow performed the oral-nasal gate manipulation and head and body weaving 0.5%, 28.0%, and 15.5% of her total time budget, respectively. This study is one of the first reports to evaluate the treatment of established stereotypies in a mature sow. Results suggest the promise of environmental enrichment as an effective treatment strategy. Further research is needed to evaluate the persistence of these behavioral changes and relative importance of different environmental manipulations provided

Assessment of alternatives to correct inventory difference statistical treatment deficiencies

This document presents an analysis of alternatives to correct deficiencies in the statistical treatment of inventory differences in the NRC guidance documents and licensee practice. Pacific Northwest Laboratory's objective for this study was to assess alternatives developed by the NRC and a panel of safeguards statistical experts. Criteria were developed for the evaluation and the assessment was made considering the criteria. The results of this assessment are PNL recommendations, which are intended to provide NRC decision makers with a logical and statistically sound basis for correcting the deficiencies