Facile production of tagless membrane scaffold protein for nanodiscs.

The initial step in the preparation of nanodiscs is to express and purify the membrane scaffold protein (MSP) to homogeneity. Current methods used for the isolation and purification of MSP utilize nickel affinity chromatography. However, the presence of a polyhistidine tag on the MSP often interferes with downstream steps where nanodiscs reconstituted with protein need to be isolated from empty ones. Therefore, one must engage in the finicky process of removing the polyhistidine tag from the MSP using a protease before the formation of nanodiscs. Herein, we describe a robust streamlined approach to produce tagless MSP by expression as inclusion bodies followed by cleavage with cyanogen bromide, and purification by gel filtration chromatography. In addition, the MSP prepared is devoid of tryptophan residues which facilitates tryptophan-based spectroscopic studies of reconstituted proteins. Dynamic light scattering and transmission electron microscopy showed that the tagless MSP produced was competent to produce nanodiscs.

Rapid preparation of nanodiscs for biophysical studies.

Nanodiscs, which are disc-shaped entities that contain a central lipid bilayer encased by an annulus of amphipathic helices, have emerged as a leading native-like membrane mimic. The current approach for the formation of nanodiscs involves the creation of a mixed-micellar solution containing membrane scaffold protein, lipid, and detergent followed by a time consuming process (3-12 h) of dialysis and/or incubation with sorptive beads to remove the detergent molecules from the sample. In contrast, the methodology described herein provides a facile and rapid procedure for the preparation of nanodiscs in a matter of minutes (<15 min) using Sephadex® G-25 resin to remove the detergent from the sample. A panoply of biophysical techniques including analytical ultracentrifugation, dynamic light scattering, gel filtration chromatography, circular dichroism spectroscopy, and cryogenic electron microscopy were employed to unequivocally confirm that aggregates formed by this method are indeed nanodiscs. We believe that this method will be attractive for time-sensitive and high-throughput experiments.

Molecular Visualization of Neuronal TDP43 Pathology In Situ.

Nuclear exclusion and cytoplasmic accumulation of the RNA-binding protein TDP43 are characteristic of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Despite this, the origin and ultrastructure of cytosolic TDP43 deposits remain unknown. Accumulating evidence suggests that abnormal RNA homeostasis can drive pathological TDP43 mislocalization, enhancing RNA misprocessing due to loss of nuclear TDP43 and engendering a cycle that ends in cell death. Here, we show that adding small monovalent oligonucleotides successfully recapitulates pathological TDP43 mislocalization and aggregation in iPSC-derived neurons (iNeurons). By employing a multimodal in situ cryo-correlative light and electron microscopy pipeline, we examine how RNA influences the localization and aggregation of TDP43 in near-native conditions. We find that mislocalized TDP43 forms ordered fibrils within lysosomes and autophagosomes in iNeurons as well as in patient tissue, and provide the first high-resolution snapshots of TDP43 aggregates in situ. In so doing, we provide a cellular model for studying initial pathogenic events underlying ALS, FTLD, and related TDP43-proteinopathies.

[Psychoactive drugs use in patients with panic disorder]. / Consumo de sustancias psicoactivas en pacientes con trastorno de pánico.

INTRODUCTION: The objectives of this study were to evaluate the prevalence of drug use in out-patients with panic disorder and their influence in evolution and therapeutic response of panic disorder. MATERIAL AND METHODS: The sample was made up of 79 out-patients diagnosed of panic disorder or agoraphobia with panic disorder according to the ICD-10 criteria and 83 controls from the same center with other psychiatric disorders. Subjects were followed-up for six months. RESULTS: Prevalence of regular lifetime drug use was: 13 % for alcohol, 52 % for nicotine and 47 % for caffeine. No other drug use was observed. Patients with panic disorder used less caffeine than controls, there being no differences in other drug use. Caffeine use was associated with higher antidepressant dosages. CONCLUSIONS: Thus, prevalence of regular drug use in panic disorder during the lifetime of out-patients with panic disorder was: 13 % for alcohol, 47 % for caffeine use and 52 % for nicotine use. Those with panic disorder use less caffeine than other psychiatric patients, but there were no differences in other drug use. Presence of agoraphobia has no repercussion on consumption. There were no differences in clinical manifestations and treatment responses between users and non-users, but drug use may modify antidepressant dosages.