Functioning of the Drosophila Wilms'-tumor-1-associated protein homolog, Fl(2)d, in Sex-lethal-dependent alternative splicing

fl(2)d, the Drosophila homolog of Wilms'-tumor-1-associated protein (WTAP), regulates the alternative splicing of Sex-lethal (Sxl), transformer (tra), and Ultrabithorax (Ubx). Although WTAP has been found in functional human spliceosomes, exactly how it contributes to the splicing process remains unknown. Here we attempt to identify factors that interact genetically and physically with fl(2)d. We begin by analyzing the Sxl-Fl(2)d protein-protein interaction in detail and present evidence suggesting that the female-specific fl(2)d(1) allele is antimorphic with respect to the process of sex determination. Next we show that fl(2)d interacts genetically with early acting general splicing regulators and that Fl(2)d is present in immunoprecipitable complexes with Snf, U2AF50, U2AF38, and U1-70K. By contrast, we could not detect Fl(2)d complexes containing the U5 snRNP protein U5-40K or with a protein that associates with the activated B spliceosomal complex SKIP. Significantly, the genetic and molecular interactions observed for Sxl are quite similar to those detected for fl(2)d. Taken together, our findings suggest that Sxl and fl(2)d function to alter splice-site selection at an early step in spliceosome assembly.     

Biochemistry without oxygen

Published procedures for experimentation under anoxic conditions generally involve specialized apparatus that hinders the easy manipulation of experimental samples. We describe here some procedures that rapidly remove oxygen from experimental solutions, maintain anoxia with simple equipment for long periods of time, and do not interfere with normal sample addition and removal, spectrometric measurements, chromatographic manipulations, and the like. Anoxia can be achieved and maintained by the use of an enzyme system (glucose oxidase, glucose, catalase), or an inorganic oxygen-reducing system (ferrous pyrophosphate), or dithionite. Physical isolation of experimental samples from atmospheric oxygen can be maintained by continuous flushing with treated argon gas and/or by an overlay of heavy mineral oil.     

Microstructural abnormalities in subcortical reward circuitry of subjects with major depressive disorder

Background

Previous studies of major depressive disorder (MDD) have focused on abnormalities in the prefrontal cortex and medial temporal regions. There has been little investigation in MDD of midbrain and subcortical regions central to reward/aversion function, such as the ventral tegmental area/substantia nigra (VTA/SN), and medial forebrain bundle (MFB).

Methodology/Principal Findings

We investigated the microstructural integrity of this circuitry using diffusion tensor imaging (DTI) in 22 MDD subjects and compared them with 22 matched healthy control subjects. Fractional anisotropy (FA) values were increased in the right VT and reduced in dorsolateral prefrontal white matter in MDD subjects. Follow-up analysis suggested two distinct subgroups of MDD patients, which exhibited non-overlapping abnormalities in reward/aversion circuitry. The MDD subgroup with abnormal FA values in VT exhibited significantly greater trait anxiety than the subgroup with normal FA values in VT, but the subgroups did not differ in levels of anhedonia, sadness, or overall depression severity.

Conclusions/Significance

These findings suggest that MDD may be associated with abnormal microstructure in brain reward/aversion regions, and that there may be at least two subtypes of microstructural abnormalities which each impact core symptoms of depression.     

Higher Dimensional Meta-State Analysis Reveals Reduced Resting fMRI Connectivity Dynamism in Schizophrenia Patients

Resting-state functional brain imaging studies of network connectivity have long assumed that functional connections are stationary on the timescale of a typical scan. Interest in moving beyond this simplifying assumption has emerged only recently. The great hope is that training the right lens on time-varying properties of whole-brain network connectivity will shed additional light on previously concealed brain activation patterns characteristic of serious neurological or psychiatric disorders. We present evidence that multiple explicitly dynamical properties of time-varying whole-brain network connectivity are strongly associated with schizophrenia, a complex mental illness whose symptomatic presentation can vary enormously across subjects. As with so much brain-imaging research, a central challenge for dynamic network connectivity lies in determining transformations of the data that both reduce its dimensionality and expose features that are strongly predictive of important population characteristics. Our paper introduces an elegant, simple method of reducing and organizing data around which a large constellation of mutually informative and intuitive dynamical analyses can be performed. This framework combines a discrete multidimensional data-driven representation of connectivity space with four core dynamism measures computed from large-scale properties of each subject’s trajectory, ie., properties not identifiable with any specific moment in time and therefore reasonable to employ in settings lacking inter-subject time-alignment, such as resting-state functional imaging studies. Our analysis exposes pronounced differences between schizophrenia patients (N_sz = 151) and healthy controls (N_hc = 163). Time-varying whole-brain network connectivity patterns are found to be markedly less dynamically active in schizophrenia patients, an effect that is even more pronounced in patients with high levels of hallucinatory behavior. To the best of our knowledge this is the first demonstration that high-level dynamic properties of whole-brain connectivity, generic enough to be commensurable under many decompositions of time-varying connectivity data, exhibit robust and systematic differences between schizophrenia patients and healthy controls.     

Identification of the Cysteine Residue Responsible for Disulfide Linkage of Na+ Channel α and β2 Subunits

Voltage-gated Na⁺ channels in the brain are composed of a single pore-forming α subunit, one non-covalently linked β subunit (β1 or β3), and one disulfide-linked β subunit (β2 or β4). The final step in Na⁺ channel biosynthesis in central neurons is concomitant α-β2 disulfide linkage and insertion into the plasma membrane. Consistent with this, Scn2b (encoding β2) null mice have reduced Na⁺ channel cell surface expression in neurons, and action potential conduction is compromised. Here we generated a series of mutant β2 cDNA constructs to investigate the cysteine residue(s) responsible for α-β2 subunit covalent linkage. We demonstrate that a single cysteine-to-alanine substitution at extracellular residue Cys-26, located within the immunoglobulin (Ig) domain, abolishes the covalent linkage between α and β2 subunits. Loss of α-β2 covalent complex formation disrupts the targeting of β2 to nodes of Ranvier in a myelinating co-culture system and to the axon initial segment in primary hippocampal neurons, suggesting that linkage with α is required for normal β2 subcellular localization in vivo. WT β2 subunits are resistant to live cell Triton X-100 detergent extraction from the hippocampal axon initial segment, whereas mutant β2 subunits, which cannot form disulfide bonds with α, are removed by detergent. Taken together, our results demonstrate that α-β2 covalent association via a single, extracellular disulfide bond is required for β2 targeting to specialized neuronal subcellular domains and for β2 association with the neuronal cytoskeleton within those domains.     

Genetic testing for hemochromatosis: attitudes and acceptability among young and older adults

Hemochromatosis is a genetic disorder of iron overload common in persons of northern European descent. We examined attitudes about testing for hemochromatosis in 118 young adults (YA) (19.7 years +/- 1.9) and 50 older adults (OA) (58.5 years +/- 13.7). Participants read about hemochromatosis and two related tests: transferrin saturation measurement (iron test) and HFE genotyping (HFE test). Interest in each test and attitudes about genetic testing were assessed. More than 80% of all participants were willing to undergo either test, if offered. A majority preferred the iron test because of the information it provides about current health. A majority of participants identified at least one benefit of genetic testing, with improved health through early detection/prevention being most common. YA were more likely to report disadvantages of genetic testing (p < 0.001) and were more concerned about potential negative psychological effects (p < 0.005). OA were more concerned about potential discrimination (p < 0.0001). These findings suggest that young and older adults view genetic testing as beneficial and would accept HFE testing were it offered as part of a screening program.     

Deletion mapping of the ilvGOEDAC genes of Escherichia coli K-12.

Summary A set of dilv phage has been examined that carry overlapping segments of isoleucine-valine structural and regulatory genes derived from the ilv cluster at 83 min on the Escherichia coli K-12 chromosome. The ilv genes present in these phage, and their order, have been determined by transduction of auxotrophs, escape synthesis, and deletion mapping. The order of ilv genes in the phage, and hence the order in the host chromosome, was found to be ilvG-ilvO-ilvEDA-ilvC. Lysogens containing dilv phage were constructed for dominance analysis of regulatory mutations in the ilvO and ilvA genes. The ilvO671 allele is cis-dominant to ilvO ⁺, while the ilvA538 allele is trans-recessive to ilvA ⁺. Thus, the ilvO gene, that is identified by cis-dominant regulatory mutations that result in increased ilvG and ilvEDA expression, is situated between and may be contiguous with ilvG and ilvEDA.