Brain Mechanisms of Social Threat Effects on Working Memory.

Social threat can have adverse effects on cognitive performance, but the brain mechanisms underlying its effects are poorly understood. We investigated the effects of social evaluative threat on working memory (WM), a core component of many important cognitive capabilities. Social threat impaired WM performance during an N-back task and produced widespread reductions in activation in lateral prefrontal cortex and intraparietal sulcus (IPS), among other regions. In addition, activity in frontal and parietal regions predicted WM performance, and mediation analyses identified regions in the bilateral IPS that mediated the performance-impairing effects of social threat. Social threat also decreased connectivity between the IPS and dorsolateral prefrontal cortex, while increasing connectivity between the IPS and the ventromedial prefrontal cortex, a region strongly implicated in the generation of autonomic and emotional responses. Finally, cortisol response to the stressor did not mediate WM impairment but was rather associated with protective effects. These results provide a basis for understanding interactions between social and cognitive processes at a neural systems level.

Esophageal tolerance to high-dose stereotactic ablative radiotherapy.

Dose-volume parameters are needed to guide the safe administration of stereotactic ablative radiotherapy (SABR). We report on esophageal tolerance to high-dose hypofractionated radiation in patients treated with SABR. Thirty-one patients with spine or lung tumors received single- or multiple-fraction SABR to targets less than 1 cm from the esophagus. End points evaluated include D(5cc) (minimum dose in Gy to 5 cm(3) of the esophagus receiving the highest dose), D(2cc) , D(1cc) , and D(max) (maximum dose to 0.01 cm(3) ). Multiple-fraction treatments were correlated using the linear quadratic and linear quadratic-linear/universal survival models. Three esophageal toxicity events occurred, including esophagitis (grade 2), tracheoesophageal fistula (grade 4-5), and esophageal perforation (grade 4-5). Chemotherapy was a cofactor in the high-grade events. The median time to development of esophageal toxicity was 4.1 months (range 0.6-6.1 months). Two of the three events occurred below a published D(5cc) threshold, all three were below a D(2cc) threshold, and one was below a D(max) threshold. We report a dosimetric analysis of incidental dose to the esophagus from SABR. High-dose hypofractionated radiotherapy led to a number of high-grade esophageal adverse events, suggesting that conservative parameters to protect the esophagus are necessary when SABR is used, especially in the setting of chemotherapy or prior radiotherapy.

Protocol for a randomized controlled trial examining multilevel prediction of response to behavioral activation and exposure-based therapy for generalized anxiety disorder.

BACKGROUND: Only 40-60% of patients with generalized anxiety disorder experience long-lasting improvement with gold standard psychosocial interventions. Identifying neurobehavioral factors that predict treatment success might provide specific targets for more individualized interventions, fostering more optimal outcomes and bringing us closer to the goal of "personalized medicine." Research suggests that reward and threat processing (approach/avoidance behavior) and cognitive control may be important for understanding anxiety and comorbid depressive disorders and may have relevance to treatment outcomes. This study was designed to determine whether approach-avoidance behaviors and associated neural responses moderate treatment response to exposure-based versus behavioral activation therapy for generalized anxiety disorder. METHODS/DESIGN: We are conducting a randomized controlled trial involving two 10-week group-based interventions: exposure-based therapy or behavioral activation therapy. These interventions focus on specific and unique aspects of threat and reward processing, respectively. Prior to and after treatment, participants are interviewed and undergo behavioral, biomarker, and neuroimaging assessments, with a focus on approach and avoidance processing and decision-making. Primary analyses will use mixed models to examine whether hypothesized approach, avoidance, and conflict arbitration behaviors and associated neural responses at baseline moderate symptom change with treatment, as assessed using the Generalized Anxiety Disorder-7 item scale. Exploratory analyses will examine additional potential treatment moderators and use data reduction and machine learning methods. DISCUSSION: This protocol provides a framework for how studies may be designed to move the field toward neuroscience-informed and personalized psychosocial treatments. The results of this trial will have implications for approach-avoidance processing in generalized anxiety disorder, relationships between levels of analysis (i.e., behavioral, neural), and predictors of behavioral therapy outcome. TRIAL REGISTRATION: The study was retrospectively registered within 21 days of first participant enrollment in accordance with FDAAA 801 with ClinicalTrials.gov, NCT02807480. Registered on June 21, 2016, before results.

The subnuclear localization of tRNA ligase in yeast.

Yeast tRNA ligase is an enzyme required for tRNA splicing. A study by indirect immune fluorescence shows that this enzyme is localized in the cell nucleus. At higher resolution, studies using indirect immune electron microscopy show this nuclear location to be primarily at the inner membrane of the nuclear envelope, most likely at the nuclear pore. There is a more diffuse, secondary location of ligase in a region of the nucleoplasm within 300 nm of the nuclear envelope. When the amount of ligase in the cell is increased, nuclear staining increases but staining of the nuclear envelope remains constant. This experiment indicates that there are a limited number of ligase sites at the nuclear envelope. Since the other tRNA splicing component, the endonuclease, has the characteristics of an integral membrane protein, we hypothesize that it constitutes the site for the interaction of ligase with the nuclear envelope.

SSB-1 of the yeast Saccharomyces cerevisiae is a nucleolar-specific, silver-binding protein that is associated with the snR10 and snR11 small nuclear RNAs.

SSB-1, the yeast single-strand RNA-binding protein, is demonstrated to be a yeast nucleolar-specific, silver-binding protein. In double-label immunofluorescence microscopy experiments antibodies to two other nucleolar proteins, RNA Pol I 190-kD and fibrillarin, were used to reveal the site of rRNA transcription; i.e., the fibrillar region of the nucleolus. SSB-1 colocalized with fibrillarin in a double-label immunofluorescence mapping experiment to the yeast nucleolus. SSB-1 is located, though, over a wider region of the nucleolus than the transcription site marker. Immunoprecipitations of yeast cell extracts with the SSB-1 antibody reveal that in 150 mM NaCl SSB-1 is bound to two small nuclear RNAs (snRNAs). These yeast snRNAs are snR10 and snR11, with snR10 being predominant. Since snR10 has been implicated in pre-rRNA processing, the association of SSB-1 and snR10 into a nucleolar snRNP particle indicates SSB-1 involvement in rRNA processing as well. Also, another yeast protein, SSB-36-kD, isolated by single-strand DNA chromatography, is shown to bind silver under the conditions used for nucleolar-specific staining. It is, most likely, another yeast nucleolar protein.

An early hierarchic role of U1 small nuclear ribonucleoprotein in spliceosome assembly.

Splicing of nuclear precursor messenger RNA (pre-mRNA) occurs on a large ribonucleoprotein complex, the spliceosome. Several small nuclear ribonucleoproteins (snRNP's) are subunits of this complex that assembles on the pre-mRNA. Although the U1 snRNP is known to recognize the 5' splice site, its roles in spliceosome formation and splice site alignment have been unclear. A new affinity purification method for the spliceosome is described which has provided insight into the very early stages of spliceosome formation in a yeast in vitro splicing system. Surprisingly, the U1 snRNP initially recognizes sequences at or near both splice junctions in the intron. This interaction must occur before the other snRNP's (U2, U4, U5, and U6) can join the complex. The results suggest that interaction of the two splice site regions occurs at an early stage of spliceosome formation and is probably mediated by U1 snRNP and perhaps other factors.

The "spliceosome": yeast pre-messenger RNA associates with a 40S complex in a splicing-dependent reaction.

The in vitro splicing reactions of pre-messenger RNA (pre-mRNA) in a yeast extract were analyzed by glycerol gradient centrifugation. Labeled pre-mRNA appears in a 40S peak only if the pre-mRNA undergoes the first of the two partial splicing reactions. RNA analysis after extraction of glycerol gradient fractions shows that lariat-form intermediates, molecules that occur only in mRNA splicing, are found almost exclusively in this 40S complex. Another reaction intermediate, cut 5' exon RNA, can also be found concentrated in this complex. The complex is stable even in 400 mM KCl, although at this salt concentration, it sediments at 35S and is clearly distinguishable from 40S ribosomal subunits. This complex, termed a "spliceosome," is thought to contain components necessary for mRNA splicing; its existence can explain how separated exons on pre-mRNA are brought into contact.

Two domains of yeast U6 small nuclear RNA required for both steps of nuclear precursor messenger RNA splicing.

U6 is one of the five small nuclear RNA's (snRNA's) that are required for splicing of nuclear precursor messenger RNA (pre-mRNA). The size and sequence of U6 RNA are conserved among organisms as diverse as yeast and man, and so it has been proposed that U6 RNA functions as a catalytic element in splicing. A procedure for in vitro reconstitution of functional yeast U6 small nuclear ribonucleoproteins (snRNP's) with synthetic U6 RNA was applied in an attempt to elucidate the function of yeast U6 RNA. Two domains in U6 RNA were identified, each of which is required for in vitro splicing. Single nucleotide substitutions in these two domains block splicing either at the first or the second step. Invariably, U6 RNA mutants that block the first step of splicing do not enter the spliceosome. On the other hand, those that block the second step of splicing form a spliceosome but block cleavage at the 3' splice site of the intron. In both domains, the positions of base changes that block the second step of splicing correspond exactly to the site of insertion of pre-mRNA-type introns into the U6 gene of two yeast species, providing a possible explanation for the mechanism of how these introns originated and adding further evidence for the proposed catalytic role of U6 RNA.

Evolution of a transfer RNA gene through a point mutation in the anticodon.

The transfer RNA (tRNA) multigene family comprises 20 amino acid-accepting groups, many of which contain isoacceptors. The addition of isoacceptors to the tRNA repertoire was critical to establishing the genetic code, yet the origin of isoacceptors remains largely unexplored. A model of tRNA evolution, termed "tRNA gene recruitment," was formulated. It proposes that a tRNA gene can be recruited from one isoaccepting group to another by a point mutation that concurrently changes tRNA amino acid identity and messenger RNA coupling capacity. A test of the model showed that an Escherichia coli strain, in which the essential tRNAUGUThr gene was inactivated, was rendered viable when a tRNAArg with a point mutation that changed its anticodon from UCU to UGU (threonine) was expressed. Insertion of threonine at threonine codons by the "recruited" tRNAArg was corroborated by in vitro aminoacylation assays showing that its specificity had been changed from arginine to threonine. Therefore, the recruitment model may account for the evolution of some tRNA genes.

Crystal structure and evolution of a transfer RNA splicing enzyme.

The splicing of transfer RNA precursors is similar in Eucarya and Archaea. In both kingdoms an endonuclease recognizes the splice sites and releases the intron, but the mechanism of splice site recognition is different in each kingdom. The crystal structure of the endonuclease from the archaeon Methanococcus jannaschii was determined to a resolution of 2.3 angstroms. The structure indicates that the cleavage reaction is similar to that of ribonuclease A and the arrangement of the active sites is conserved between the archaeal and eucaryal enzymes. These results suggest an evolutionary pathway for splice site recognition.

Cloning of yeast transfer RNA genes in Escherichia coli.

Four thousand Escherichia coli clones containing yeast DNA inserted into the plasmid pBR313 have been isolated. Of these, 175 clones were identified as carrying yeast transfer RNA genes. The initial analysis of the inserted transfer RNA genes via the colony hybridization technique with individual radioactive transfer RNA species is reported. The data indicate that yeast transfer RNA genes are not highly clustered, although some clustering exists. In addition, it was observed that the reiteration number of different transfer RNA genes may vary extensively.

The transfer RNA identity problem: a search for rules.

Correct recognition of transfer RNAs (tRNAs) by aminoacyl-tRNA synthetases is central to the maintenance of translational fidelity. The hypothesis that synthetases recognize anticodon nucleotides was proposed in 1964 and had considerable experimental support by the mid-1970s. Nevertheless, the idea was not widely accepted until relatively recently in part because the methodologies initially available for examining tRNA recognition proved hampering for adequately testing alternative hypotheses. Implementation of new technologies has led to a reasonably complete picture of how tRNAs are recognized. The anticodon is indeed important for 17 of the 20 Escherichia coli isoaccepting groups. For many of the isoaccepting groups, the acceptor stem or position 73 (or both) is important as well.

Cell-free circularization of viroid progeny RNA by an RNA ligase from wheat germ.

Linear, potato spindle tuber viroid RNA has been used as a substrate for an RNA ligase purified from wheat germ. Linear viroid molecules are efficiently converted to circular molecules (circles) which are indistinguishable by electrophoretic mobility and two-dimensional oligonucleotide pattern from viroid circles extracted from infected plants. In light of recent evidence for multimeric viroid replication intermediates, cleavage followed by RNA ligation by a cellular enzyme may (i) be a normal step in the viroid life cycle and (ii) may also reflect cellular events.

Directed deletion of a yeast transfer RNA intervening sequence.

Many eukaryotic genes contain intevening sequences, segments of DNA that interrupt the continuity of the gene. They are removed from RNA transcripts of the gene by a process known as splicing. The intervening sequence in a yeast tyrosine transfer RNA (tRNA Tyr) suppressor gene was deleted in order to test its role in the expression of the gene. The altered gene and its parent were introduced into yeast by transformation. Both genes exhibited suppressor function, showing that the intervening sequence is not absolutely essential for the expression of this gene.

Genetic regulation: the Lac control region.

The nucleotide sequence of the lac promoter-operator region has been determined. The 122 base pairs comprising this region include the recognition sites for RNA polymerase, the positive regulatory protein, CAP, and the negative regulatory protein, the repressor. Identification of mutant variants of the sequence combined with the in vitro biochemical studies of others has allowed us to tentatively identify the recognition site for each of these proteins, and to suggest how CAP might act at a distance to affect the interaction of RNA polymerase with the promoter.

Directed mutagenesis of dihydrofolate reductase.

Three mutations of the enzyme dihydrofolate reductase were constructed by oligonucleotide-directed mutagenesis of the cloned Escherichia coli gene. The mutations--at residue 27, aspartic acid replaced with asparagine; at residue 39, proline replaced with cysteine; and at residue 95, glycine replaced with alanine--were designed to answer questions about the relations between molecular structure and function that were raised by the x-ray crystal structures. Properties of the mutant proteins show that Asp-27 is important for catalysis and that perturbation of the local structure at a conserved cis peptide bond following Gly-95 abolishes activity. Substitution of cysteine for proline at residue 39 results in the appearance of new forms of the enzyme that correspond to various oxidation states of the cysteine. One of these forms probably represents a species cross-linked by an intrachain disulfide bridge between the cysteine at position 85 and the new cysteine at position 39.

Nicotinic receptor binding site probed with unnatural amino acid incorporation in intact cells.

The nonsense codon suppression method for unnatural amino acid incorporation has been applied to intact cells and combined with electrophysiological analysis to probe structure-function relations in the nicotinic acetylcholine receptor. Functional receptors were expressed in Xenopus oocytes when tyrosine and phenylalanine derivatives were incorporated at positions 93, 190, and 198 in the binding site of the alpha subunit. Subtle changes in the structure of an individual side chain produced readily detectable changes in the function of this large channel protein. At each position, distinct features of side chain structure dominated the dose-response relation, probably by governing the agonist-receptor binding.

Pre-mRNA splicing in yeast.

Splicing of introns from nuclear precursor messenger RNAs (pre-mRNAs) occurs in all eukaryotes. Two aspects of the splicing mechanism need to be understood: how intron sequences are recognized and aligned and how splicing is catalysed. Recent genetic and biochemical studies in the simple eukaryote Saccharomyces cerevisiae are revealing some of the features of the splicing mechanism.

PRP18, a protein required for the second reaction in pre-mRNA splicing.

We have investigated the role of a novel temperature-sensitive splicing mutation, prp18. We had previously demonstrated that an accumulation of the lariat intermediate of splicing occurred at the restrictive temperature in vivo. We have now used the yeast in vitro splicing system to show that extracts from this mutant strain are heat labile for the second reaction of splicing. The heat inactivation of prp18 extracts results from loss of activity of an exchangeable component. Inactivated prp18 extracts are complemented by heat-inactivated extracts from other mutants or by fractions from wild-type extracts. In heat-inactivated prp18 extracts, 40S splicing complexes containing lariat intermediate and exon 1 can assemble. The intermediates in this 40S complex can be chased to products by complementing extracts in the presence of ATP. Both complementation of extracts and chasing of the isolated prp18 spliceosomes takes place with micrococcal nuclease-treated extracts. Furthermore, the complementation profile with fractions of wild-type extracts indicates that the splicing defect results from a mutation in a previously designated factor required for the second step of splicing. The isolation of this mutant as temperature-sensitive lethal has also facilitated cloning of the wild-type allele by complementation.

PRP4: a protein of the yeast U4/U6 small nuclear ribonucleoprotein particle.

The Saccharomyces cerevisiae prp mutants (prp2 through prp11) are known to be defective in pre-mRNA splicing at nonpermissive temperatures. We have sequenced the PRP4 gene and shown that it encodes a 52-kilodalton protein. We obtained PRP4 protein-specific antibodies and found that they inhibited in vitro pre-mRNA splicing, which confirms the essential role of PRP4 in splicing. Moreover, we found that PRP4 is required early in the spliceosome assembly pathway. Immunoprecipitation experiments with anti-PRP4 antibodies were used to demonstrate that PRP4 is a protein of the U4/U6 small nuclear ribonucleoprotein particle (snRNP). Furthermore, the U5 snRNP could be immunoprecipitated through snRNP-snRNP interactions in the large U4/U5/U6 complex.