Huntingtin Co-Isolates with Small Extracellular Vesicles from Blood Plasma of TgHD and KI-HD Pig Models of Huntington's Disease and Human Blood Plasma.

(1) Background: Huntington's disease (HD) is rare incurable hereditary neurodegenerative disorder caused by CAG repeat expansion in the gene coding for the protein huntingtin (HTT). Mutated huntingtin (mHTT) undergoes fragmentation and accumulation, affecting cellular functions and leading to neuronal cell death. Porcine models of HD are used in preclinical testing of currently emerging disease modifying therapies. Such therapies are aimed at reducing mHTT expression, postpone the disease onset, slow down the progression, and point out the need of biomarkers to monitor disease development and therapy efficacy. Recently, extracellular vesicles (EVs), particularly exosomes, gained attention as possible carriers of disease biomarkers. We aimed to characterize HTT and mHTT forms/fragments in blood plasma derived EVs in transgenic (TgHD) and knock-in (KI-HD) porcine models, as well as in HD patients' plasma. (2) Methods: Small EVs were isolated by ultracentrifugation and HTT forms were visualized by western blotting. (3) Results: The full length 360 kDa HTT co-isolated with EVs from both the pig model and HD patient plasma. In addition, a ~70 kDa mutant HTT fragment was specific for TgHD pigs. Elevated total huntingtin levels in EVs from plasma of HD groups compared to controls were observed in both pig models and HD patients, however only in TgHD were they significant (p = 0.02). (4) Conclusions: Our study represents a valuable initial step towards the characterization of EV content in the search for HD biomarkers.

Miro proteins and their role in mitochondrial transfer in cancer and beyond.

Mitochondria are organelles essential for tumor cell proliferation and metastasis. Although their main cellular function, generation of energy in the form of ATP is dispensable for cancer cells, their capability to drive their adaptation to stress originating from tumor microenvironment makes them a plausible therapeutic target. Recent research has revealed that cancer cells with damaged oxidative phosphorylation import healthy (functional) mitochondria from surrounding stromal cells to drive pyrimidine synthesis and cell proliferation. Furthermore, it has been shown that energetically competent mitochondria are fundamental for tumor cell migration, invasion and metastasis. The spatial positioning and transport of mitochondria involves Miro proteins from a subfamily of small GTPases, localized in outer mitochondrial membrane. Miro proteins are involved in the structure of the MICOS complex, connecting outer and inner-mitochondrial membrane; in mitochondria-ER communication; Ca2+ metabolism; and in the recycling of damaged organelles via mitophagy. The most important role of Miro is regulation of mitochondrial movement and distribution within (and between) cells, acting as an adaptor linking organelles to cytoskeleton-associated motor proteins. In this review, we discuss the function of Miro proteins in various modes of intercellular mitochondrial transfer, emphasizing the structure and dynamics of tunneling nanotubes, the most common transfer modality. We summarize the evidence for and propose possible roles of Miro proteins in nanotube-mediated transfer as well as in cancer cell migration and metastasis, both processes being tightly connected to cytoskeleton-driven mitochondrial movement and positioning.

Proteomic Analysis of Human Neural Stem Cell Differentiation by SWATH-MS.

The unique properties of stem cells to self-renew and differentiate hold great promise in disease modelling and regenerative medicine. However, more information about basic stem cell biology and thorough characterization of available stem cell lines is needed. This is especially essential to ensure safety before any possible clinical use of stem cells or partially committed cell lines. As proteins are the key effector molecules in the cell, the proteomic characterization of cell lines, cell compartments or cell secretome and microenvironment is highly beneficial to answer above mentioned questions. Nowadays, method of choice for large-scale discovery-based proteomic analysis is mass spectrometry (MS) with data-independent acquisition (DIA). DIA is a robust, highly reproducible, high-throughput quantitative MS approach that enables relative quantification of thousands of proteins in one sample. In the current protocol, we describe a specific variant of DIA known as SWATH-MS for characterization of neural stem cell differentiation. The protocol covers the whole process from cell culture, sample preparation for MS analysis, the SWATH-MS data acquisition on TTOF 5600, the complete SWATH-MS data processing and quality control using Skyline software and the basic statistical analysis in R and MSstats package. The protocol for SWATH-MS data acquisition and analysis can be easily adapted to other samples amenable to MS-based proteomics.

Determination of OH groups in humic acids using methylation with dimethylsulfate.

The methylation of humic acids (HA) with dimethylsulfate in acetone and methanol followed by the iodometric determination of the methoxy groups (Zeisel reaction) were applied to determine the contents of -OH groups in solid samples of HA of different origins. For the coal- and peat-derived HA samples, the contents of -OH groups determined after methylation in acetone ranged from 6.6 to 8.7 mmol/g, whereas the contents of -OH groups determined after methylation in methanol ranged from 4.0 to 5.0 mmol/g. These differences may be related to the content of carboxylic groups in the HA molecule that were not methylated in methanol, as confirmed by a comparison with results of conventional titrimetric determinations. Observed differences were interpreted as results of different polarity of both solvents and alkalinity of the reaction mixture during the methylation. The contents of alcoholic groups as well as some other minor -OH groups can be estimated using the -OH group contents obtained after methylation in both solvents together with the results of the conventional determinations of acidic functional groups. A repeatability of the -OH groups determination as estimated from a series of triplicate analyses of different HA samples (n=7) was in range of 0.15-0.73 mmol/g and 0.08-1.06 mmol/g (standard deviations) for methylation in acetone and methanol, respectively. Thus, the average repeatability of the -OH groups determination was estimated to be 0.38 and 0.50 mmol/g for methylation in acetone and methanol, respectively.