Highly efficient, in vivo optimized, archaeal endonuclease for controlled RNA splicing in mammalian cells.

ARCHAEA-ExPRESs is an mRNA modification technology that makes use of components derived from the Archaeon Methanocaldococcus jannaschii, namely the tRNA splicing endonuclease (MJ-EndA) and its natural substrate, the bulge-helix-bulge (BHB) structure (1). These components can perform both cis- and trans-splicing in cellular and animal models and may provide a convenient way to modulate gene expression using components independent of cellular regulatory networks. To use MJ-EndA in stable expression mammalian systems, we developed variants characterized by high efficiency and sustainable in vivo activity. The MJ-EndA variants were created by the introduction of proper localization signals followed by mutagenesis and direct selection in mammalian cells. Of note, enzyme selection used an in vivo selection method based on puromycin resistance conferred to cells by BHB-mediated intron splicing from an out-of-frame puromycin N-acetyl transferase (PAC) gene. This approach yielded several endonuclease variants, the best of which showed 40-fold higher activity compared to the parental enzyme and stable processing of 30% of the target mRNA. Notably, these variants showed complete compatibility with long-term expression in mammalian cells, suggesting that they may be usefully applied in functional genomics and genetically modified animal models.

ARCHAEA-ExPRESs targeting of alpha-tubulin 4 mRNA: a model for high-specificity trans-splicing.

Effectiveness of trans-splicing-mediated mRNA reprogramming depends on specificity and efficiency. We have previously developed a new strategy (ARCHAEA-ExPRESs) that uses a tRNA endonuclease derived from Archaea and its natural substrate, the bulge-helix-bulge (BHB) structure. ARCHAEA-ExPRESs provides increased specificity in functional targeting. In fact, this system is based on a double check, the base pairing and the formation of a BHB structure between the target mRNA and the targeting RNA. In this study, we demonstrate the high specificity of ARCHAEA-ExPRESs by tagging the endogenous alpha-tubulin 4 via trans-splicing. Alpha-tubulin 4 belongs to a gene family sharing high degree of nucleotide sequence homology. The formation of a perfect BHB structure between targeting RNAs and the isotype alpha-tubulin 4 enables selective trans-splicing. Most important, ARCHAEA-ExPRESs functionality is conserved in vivo following transient expression of archaeal tRNA endonuclease in mouse liver. Production of the recombinant protein is strictly dependent on the expression of the archaeal endonuclease, and the efficiency of the system depends on the relative amount of the target and targeting mRNAs. These data prove the effectiveness of ARCHAEA-ExPRESs in an endogenous highly demanding context and disclose the possibility to utilize this system in a variety of technological or therapeutic applications.

Relations of mindfulness facets with psychological symptoms among individuals with a diagnosis of obsessive-compulsive disorder, major depressive disorder, or borderline personality disorder.

OBJECTIVES: To explore differences in mindfulness facets among patients with a diagnosis of either obsessive-compulsive disorder (OCD), major depressive disorder (MDD), or borderline personality disorder (BPD), and healthy controls (HC), and their associations with clinical features. DESIGN AND METHOD: One hundred and fifty-three patients and 50 HC underwent a clinical assessment including measures of mindfulness (Five Facets Mindfulness Questionnaire - FFMQ), psychopathological symptoms (Symptom Check List-90-R), dissociation (Dissociative Experience Scale), alexithymia (Alexithymia Scale 20), and depression (Beck Depression Inventory-II). Analysis of variance (ANOVA) and analysis of covariance (ANCOVA) were performed to assess differences in mindfulness scores and their associations with clinical features. RESULTS: The three diagnostic groups scored lower on all mindfulness facets (apart from FFMQobserving) compared to the HC group. OCD group had a significant higher FFMQ total score (FFMQ-TS) and FFMQacting with awareness compared to the BPD group, and scored higher on FFMQdescribing compared to BPD and MDD groups. The scores in non-judging facet were significantly lower in all the three diagnostic groups compared to the HC group. Interestingly, higher FFMQ-TS was inversely related to all psychological measures, regardless of diagnostic group. CONCLUSIONS: Deficits in mindfulness skills were present in all diagnostic groups. Furthermore, we found disease-specific relationships between some mindfulness facets and specific psychological variables. Clinical implications are discussed. PRACTITIONER POINTS: The study showed deficits in mindfulness scores in all diagnostic groups compared to a healthy control group. Overall, mindfulness construct has a significantly negative association with indexes of global distress, dissociative symptoms, alexithymia, and depression. Mindfulness-based interventions in clinical settings should take into account different patterns of mindfulness skills and their impact on disease-specific maladaptive cognitive strategies or symptomatology.

An archaeal endoribonuclease catalyzes cis- and trans- nonspliceosomal splicing in mouse cells.

The tRNA endonuclease from the archaebacterium Methanococcus jannaschii (MJ endonuclease) can cleave RNAs forming specific bulge-helix-bulge (BHB) structures recognized by the enzyme. The resulting cleavage products are subsequently joined together by an endogenous ligase. We demonstrate the potential of using this strategy for repairing RNA in higher organisms by expressing the enzyme in mouse cells. Reporter target mRNAs modified with 17-nucleotide introns, flanked by sequences capable of forming BHB structures in cis, were expressed in mouse cells. RNA molecules that can form BHB substrates in trans with targeted mRNAs were also designed. Co-transfection of mouse cells with plasmids expressing these RNAs and the MJ endonuclease led to formation of RNA chimeras in which the target and exogenous RNA were recombined across the BHB. This technology is not limited to mRNA, but could in principle be used to destroy, modify or restore the function of a vast repertoire of RNA species or to join selectable tags to target RNAs.

Classification and class-modeling of "Riviera Ligure" extra-virgin olive oil using chemical-physical parameters.

The Protected Designation of Origin (PDO) "Riviera Ligure" for extra-virgin olive oils from Liguria specifies three additional geographical mentions corresponding to three different geographical areas. To obtain a complete characterization of this typical Italian product, 217 samples of olive oils produced in this North Italian region during 1998/99 and 1999/2000 were analyzed. For each sample 31 variables were determined by chemical-physical analyses, and the data were subjected to a multivariate statistical analysis. For the 1998/99 crop, characterized by favorable climatic conditions, class-models of the three geographical areas were obtained with good predictive ability, also considering the influence of the month of olive harvesting. The oil samples from the 1999/2000 crop were similarly studied, but bad climatic conditions and a widespread Dacus oleae infestation leveled out the peculiar features of the oils produced in the three areas.