A simple guide to de novo transcriptome assembly and annotation.

A transcriptome constructed from short-read RNA sequencing (RNA-seq) is an easily attainable proxy catalog of protein-coding genes when genome assembly is unnecessary, expensive or difficult. In the absence of a sequenced genome to guide the reconstruction process, the transcriptome must be assembled de novo using only the information available in the RNA-seq reads. Subsequently, the sequences must be annotated in order to identify sequence-intrinsic and evolutionary features in them (for example, protein-coding regions).

Comparing the copper binding features of alpha and beta synucleins.

Amyloid aggregation of α-synuclein (AS) is one of the hallmarks of Parkinson's disease (PD). Copper ions specifically bind at the N-terminus of AS, accelerating protein aggregation. Its protein homolog β-synuclein (BS) is also a copper binding protein, but it inhibits AS aggregation.

First-passage statistics of colloids on fractals: Theory and experimental realization.

In nature and technology, particle dynamics frequently occur in complex environments, for example in restricted geometries or crowded media. These dynamics have often been modeled invoking a fractal structure of the medium although the fractal structure was only indirectly inferred through the dynamics. Moreover, systematic studies have not yet been performed.

Distinct transcription kinetics of pluripotent cell states.

Mouse embryonic stem cells (mESCs) can adopt naïve, ground, and paused pluripotent states that give rise to unique transcriptomes. Here, we use transient transcriptome sequencing (TT-seq) to define both coding and non-coding transcription units (TUs) in these three pluripotent states and combine TT-seq with RNA polymerase II occupancy profiling to unravel the kinetics of RNA metabolism genome-wide. Compared to the naïve state (serum), RNA synthesis and turnover rates are globally reduced in the ground state (2i) and the paused state (mTORi).

Autophagy mediates the clearance of oligodendroglial SNCA/alpha-synuclein and TPPP/p25A in multiple system atrophy models.

Accumulation of the neuronal protein SNCA/alpha-synuclein and of the oligodendroglial phosphoprotein TPPP/p25A within the glial cytoplasmic inclusions (GCIs) represents the key histophathological hallmark of multiple system atrophy (MSA). Even though the levels/distribution of both oligodendroglial SNCA and TPPP/p25A proteins are critical for disease pathogenesis, the proteolytic mechanisms involved in their turnover in health and disease remain poorly understood. Herein, by pharmacological and molecular modulation of the autophagy-lysosome pathway (ALP) and the proteasome we demonstrate that the endogenous oligodendroglial SNCA and TPPP/p25A are degraded mainly by the ALP in murine primary oligodendrocytes and oligodendroglial cell lines under basal conditions.

A switch from α-helical to β-strand conformation during co-translational protein folding.

Cellular proteins begin to fold as they emerge from the ribosome. The folding landscape of nascent chains is not only shaped by their amino acid sequence but also by the interactions with the ribosome. Here, we combine biophysical methods with cryo-EM structure determination to show that folding of a β-barrel protein begins with formation of a dynamic α-helix inside the ribosome.

Study of antimycobacterial, cytotoxic, and mutagenic potential of polymeric nanoparticles of copper (II) complex.

This study aimed to encapsulate and characterise a potential anti-tuberculosis copper complex (CuCl(INH).HO:) into polymeric nanoparticles (PNs) of polymethacrylate copolymers (Eudragit®, Eu) developed by nanoprecipitation method. NE30D, S100 and, E100 polymers were tested.

Solution Structure and Conformational Flexibility of a Polyketide Synthase Module.

Polyketide synthases (PKSs) are versatile C-C bond-forming enzymes that are broadly distributed in bacteria and fungi. The polyketide compound family includes many clinically useful drugs such as the antibiotic erythromycin, the antineoplastic epothilone, and the cholesterol-lowering lovastatin. Harnessing PKSs for custom compound synthesis remains an open challenge, largely because of the lack of knowledge about key structural properties.

Defining the interactome of the human mitochondrial ribosome identifies SMIM4 and TMEM223 as respiratory chain assembly factors.

Human mitochondria express a genome that encodes thirteen core subunits of the oxidative phosphorylation system (OXPHOS). These proteins insert into the inner membrane co-translationally. Therefore, mitochondrial ribosomes engage with the OXA1L-insertase and membrane-associated proteins, which support membrane insertion of translation products and early assembly steps into OXPHOS complexes.

Decoupling of Spin Decoherence Paths near Zero Magnetic Field.

We demonstrate a method to quantify and manipulate nuclear spin decoherence mechanisms that are active in zero to ultralow magnetic fields. These include (i) nonadiabatic switching of spin quantization axis due to residual background fields and (ii) scalar pathways due to through-bond couplings between H and heteronuclear spin species, such as H used partially as an isotopic substitute for H. Under conditions of free evolution, scalar relaxation due to H can significantly limit nuclear spin polarization lifetimes and thus the scope of magnetic resonance procedures near zero field.

Kinetic control of nascent protein biogenesis by peptide deformylase.

Synthesis of bacterial proteins on the ribosome starts with a formylated methionine. Removal of the N-terminal formyl group is essential and is carried out by peptide deformylase (PDF). Deformylation occurs co-translationally, shortly after the nascent-chain emerges from the ribosomal exit tunnel, and is necessary to allow for further N-terminal processing.

Backbone Torsion Angle Determination Using Proton Detected Magic-Angle Spinning Nuclear Magnetic Resonance.

Protein torsion angles define the backbone secondary structure of proteins. Magic-angle spinning (MAS) NMR methods using carbon detection have been developed to measure torsion angles by determining the relative orientation between two anisotropic interactions─dipolar coupling or chemical shift anisotropy. Here we report a new proton-detection based method to determine the backbone torsion angle by recoupling NH and CH dipolar couplings within the HCANH pulse sequence, for protonated or partly deuterated samples.

Repositioning Food and Drug Administration-Approved Drugs for Inhibiting Biliverdin IXβ Reductase B as a Novel Thrombocytopenia Therapeutic Target.

Biliverdin IXβ reductase B (BLVRB) has recently been proposed as a novel therapeutic target for thrombocytopenia through its reactive oxygen species (ROS)-associated mechanism. Thus, we aim at repurposing drugs as new inhibitors of BLVRB. Based on IC (<5 μM), we have identified 20 compounds out of 1496 compounds from the Food and Drug Administration (FDA)-approved library and have clearly mapped their binding sites to the active site.

Sequestosome 1 Is Part of the Interaction Network of VAPB.

VAPB (Vesicle-Associated-membrane Protein-associated protein B) is a tail-anchored membrane protein of the endoplasmic reticulum that can also be detected at the inner nuclear membrane. As a component of many contact sites between the endoplasmic reticulum and other organelles, VAPB is engaged in multiple protein interactions with a plethora of binding partners. A mutant version of VAPB, P56S-VAPB, which results from a single point mutation, is involved in a familial form of amyotrophic lateral sclerosis (ALS8).

Chromatography-Free Purification Strategies for Large Biological Macromolecular Complexes Involving Fractionated PEG Precipitation and Density Gradients.

A complex interplay between several biological macromolecules maintains cellular homeostasis. Generally, the demanding chemical reactions which sustain life are not performed by individual macromolecules, but rather by several proteins that together form a macromolecular complex. Understanding the functional interactions amongst subunits of these macromolecular machines is fundamental to elucidate mechanisms by which they maintain homeostasis.

Small-sample limit of the Bennett acceptance ratio method and the variationally derived intermediates.

Free energy calculations based on atomistic Hamiltonians provide microscopic insight into the thermodynamic driving forces of biophysical or condensed matter systems. Many approaches use intermediate Hamiltonians interpolating between the two states for which the free energy difference is calculated. The Bennett acceptance ratio (BAR) and variationally derived intermediates (VI) methods are optimal estimator and intermediate states in that the mean-squared error of free energy calculations based on independent sampling is minimized.

Proteomic mapping of atrial and ventricular heart tissue in patients with aortic valve stenosis.

Aortic valve stenosis (AVS) is one of the most common valve diseases in the world. However, detailed biological understanding of the myocardial changes in AVS hearts on the proteome level is still lacking. Proteomic studies using high-resolution mass spectrometry of formalin-fixed and paraffin-embedded (FFPE) human myocardial tissue of AVS-patients are very rare due to methodical issues.

Bis-Rhodamines Bridged with a Diazoketone Linker: Synthesis, Structure, and Photolysis.

Two fluorophores bound with a short photoreactive bridge are fascinating structures and remained unexplored. To investigate the synthesis and photolysis of such dyes, we linked two rhodamine dyes via a diazoketone bridge (-COCN-) attached to position 5' or 6' of the pendant phenyl rings. For that, the mixture of 5'- or 6'-bromo derivatives of the parent dye was prepared, transformed into 1,2-diarylacetylenes, hydrated to 1,2-diarylethanones, and converted to diazoketones ArCOCNAr.

Unveiling the Coupling of Single Metallic Nanoparticles to Whispering-Gallery Microcavities.

Whispering-gallery mode resonators host multiple trapped narrow-band circulating optical resonances that find applications in quantum electrodynamics, optomechanics, and sensing. However, the spherical symmetry and low field leakage of dielectric microspheres make it difficult to probe their high-quality optical modes using far-field radiation. Even so, local field enhancement from metallic nanoparticles (MNPs) coupled to the resonators can interface the optical far field and the bounded cavity modes.

Tissue-specific regulation of translational readthrough tunes functions of the traffic jam transcription factor.

Translational readthrough (TR) occurs when the ribosome decodes a stop codon as a sense codon, resulting in two protein isoforms synthesized from the same mRNA. TR has been identified in several eukaryotic organisms; however, its biological significance and mechanism remain unclear. Here, we quantify TR of several candidate genes in Drosophila melanogaster and characterize the regulation of TR in the large Maf transcription factor Traffic jam (Tj).

Towards a native environment: structure and function of membrane proteins in lipid bilayers by NMR.

Solid-state NMR (ssNMR) is a versatile technique that can be used for the characterization of various materials, ranging from small molecules to biological samples, including membrane proteins. ssNMR can probe both the structure and dynamics of membrane proteins, revealing protein function in a near-native lipid bilayer environment. The main limitation of the method is spectral resolution and sensitivity, however recent developments in ssNMR hardware, including the commercialization of 28 T magnets (1.

Extracellular matrix remodeling through endocytosis and resurfacing of Tenascin-R.

The brain extracellular matrix (ECM) consists of extremely long-lived proteins that assemble around neurons and synapses, to stabilize them. The ECM is thought to change only rarely, in relation to neuronal plasticity, through ECM proteolysis and renewed protein synthesis. We report here an alternative ECM remodeling mechanism, based on the recycling of ECM molecules.