VAP-A and its binding partner CERT drive biogenesis of RNA-containing extracellular vesicles at ER membrane contact sites

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ImShot: An Open-Source Software for Probabilistic Identification of Proteins In Situ and Visualization of Proteomics Data

Imaging mass spectrometry (IMS) has developed into a powerful tool allowing label-free detection of numerous biomolecules in situ. In contrast to shotgun proteomics, proteins/peptides can be detected directly from biological tissues and correlated to its morphology leading to a gain of crucial clinical information. However, direct identification of the detected molecules is currently challenging for MALDI–IMS, thereby compelling researchers to use complementary techniques and resource intensive experimental setups. Despite these strategies, sufficient information could not be extracted because of lack of an optimum data combination strategy/software. Here, we introduce a new open-source software ImShot that aims at identifying peptides obtained in MALDI–IMS. This is achieved by combining information from IMS and shotgun proteomics (LC–MS) measurements of serial sections of the same tissue. The software takes advantage of a two-group comparison to determine the search space of IMS masses after deisotoping the corresponding spectra. Ambiguity in annotations of IMS peptides is eliminated by introduction of a novel scoring system that identifies the most likely parent protein of a detected peptide in the corresponding IMS dataset. Thanks to its modular structure, the software can also handle LC–MS data separately and display interactive enrichment plots and enriched Gene Ontology terms or cellular pathways. The software has been built as a desktop application with a conveniently designed graphic user interface to provide users with a seamless experience in data analysis. ImShot can run on all the three major desktop operating systems and is freely available under Massachusetts Institute of Technology license.     

Production of reactive oxygen species by withaferin A causes loss of type collagen expression and COX-2 expression through the PI3K/Akt,p38, and JNK pathways in rabbit articular chondrocytes

Withaferin A (WFA) is a major chemical constituent of Withania somnifera, also known as Indian ginseng. Many recent reports have provided evidence of its anti-tumor, anti-inflammation, anti-oxidant, and immune modulatory activities. Although the compound appears to have a large number of effects, its defined mechanisms of action have not yet been determined.     

In-depth Profiling and Quantification of the Lysine Acetylome in Hepatocellular Carcinoma with a Trapped Ion Mobility Mass Spectrometer

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide with limited therapeutic options. Comprehensive investigation of protein posttranslational modifications in HCC is still limited. Lysine acetylation is one of the most common types of posttranslational modification involved in many cellular processes and plays crucial roles in the regulation of cancer. In this study, we analyzed the proteome and K-acetylome in eight pairs of HCC tumors and normal adjacent tissues using a timsTOF Pro instrument. As a result, we identified 9219 K-acetylation sites in 2625 proteins, of which 1003 sites exhibited differential acetylation levels between tumors and normal adjacent tissues. Interestingly, many novel tumor-specific K-acetylation sites were characterized, for example, filamin A (K865), filamin B (K697), and cofilin (K19), suggesting altered activities of these cytoskeleton-modulating molecules, which may contribute to tumor metastasis. In addition, we observed an overall suppression of protein K-acetylation in HCC tumors, especially for enzymes from various metabolic pathways, for example, glycolysis, tricarboxylic acid cycle, and fatty acid metabolism. Moreover, the expression of deacetylase sirtuin 2 (SIRT2) was upregulated in HCC tumors, and its role of deacetylation in HCC cells was further explored by examining the impact of SIRT2 overexpression on the proteome and K-acetylome in Huh7 HCC cells. SIRT2 overexpression reduced K-acetylation of proteins involved in a wide range of cellular processes, including energy metabolism. Furthermore, cellular assays showed that overexpression of SIRT2 in HCC cells inhibited both glycolysis and oxidative phosphorylation. Taken together, our findings provide valuable information to better understand the roles of K-acetylation in HCC and to treat this disease by correcting the aberrant acetylation patterns.     

Monoclonal anti-β1-adrenergic receptor antibodies activate G protein signaling in the absence of β-arrestin recruitment

Thermostabilized G protein-coupled receptors used as antigens for in vivo immunization have resulted in the generation of functional agonistic anti-β₁-adrenergic (β₁AR) receptor monoclonal antibodies (mAbs). The focus of this study was to examine the pharmacology of these antibodies to evaluate their mechanistic activity at β₁AR. Immunization with the β₁AR stabilized receptor yielded five stable hybridoma clones, four of which expressed functional IgG, as determined in cell-based assays used to evaluate cAMP stimulation. The antibodies bind diverse epitopes associated with low nanomolar agonist activity at β₁AR, and they appeared to show some degree of biased signaling as they were inactive in an assay measuring signaling through β-arrestin. In vitro characterization also verified different antibody-receptor interactions reflecting the different epitopes on the extracellular surface of β₁AR to which the mAbs bind. The anti-β₁AR mAbs only demonstrated agonist activity when in dimeric antibody format, but not as the monomeric Fab format, suggesting that agonist activation may be mediated through promoting receptor dimerization. Finally, we have also shown that at least one of these antibodies exhibits in vivo functional activity at a therapeutically-relevant dose producing an increase in heart rate consistent with β₁AR agonism.     

Lidoflazine Combined with Nucleotide Precursors Increases ATP Content and Adenosine Production in Cardiomyocytes

We have previously identified that the nucleoside transport blocker dipyridamole increases adenosine production but may cause depletion of the nucleotide pool in cardiomyocytes during extended exposure and that this effect was abolished by co-administration of adenine and ribose. The present study aimed to establish whether lidoflazine, a newer generation of nucleoside transport inhibitor with calcium antagonist properties, would cause a similar effect. We conclude that lidoflazine did not affect the nucleotide pool while the combined application of lidoflazine with precursors of nucleotide resynthesis increased ATP concentration and further enhanced adenosine production.     

Végétation marine de l'Ile de Port-Cros (Parc National). XII: Sur Acrochaetium molinieri sp. nov. et Lophosiphonia cristata Falkenberg

Abstract

Marine vegetation of Port-Cros Island (National park). XII: on Acrochaetium molinieri sp. nov. and Lophosiphonia cristata Falkenberg. – Acrochaetium molinieri sp. nov. is characterized by its bicellular base; only one of these cells originates an erect filament. Lophosiphonia cristata, rarely mentioned from the Mediterranean, is richly illustrated and described in detail     

The still uncertain identity of the channel-forming unit(s) of the mitochondrial permeability transition pore

Mitochondria from different organisms can undergo a sudden process of inner membrane unselective leakiness to molecules known as the mitochondrial permeability transition (MPT). This process has been studied for nearly four decades and several proteins have been claimed to constitute, or at least regulate the usually inactive pore responsible for this transition. However, no protein candidate proposed as the actual pore-forming unit has passed rigorous gain- or loss-of-function genetic tests. Here we review evidence for -and against- putative channel-forming components of the MPT pore. We conclude that the structure of the MPT pore still remains largely undefined and suggest that future studies should follow established technical considerations to unambiguously consolidate the channel forming constituent(s) of the MPT pore.