An Innovative Method for Exosome Quantification and Size Measurement

Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology.Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).     

Plaque2.0—A High-Throughput Analysis Framework to Score Virus-Cell Transmission and Clonal Cell Expansion

Classical plaque assay measures the propagation of infectious agents across a monolayer of cells. It is dependent on cell lysis, and limited by user-specific settings and low throughput. Here, we developed Plaque2.0, a broadly applicable, fluorescence microscopy-based high-throughput method to mine patho-biological clonal cell features. Plaque2.0 is an open source framework to extract information from chemically fixed cells by immuno-histochemistry or RNA in situ hybridization, or from live cells expressing GFP transgene. Multi-parametric measurements include infection density, intensity, area, shape or location information at single plaque or population levels. Plaque2.0 distinguishes lytic and non-lytic spread of a variety of DNA and RNA viruses, including vaccinia virus, adenovirus and rhinovirus, and can be used to visualize simultaneous plaque formation from co-infecting viruses. Plaque2.0 also analyzes clonal growth of cancer cells, which is relevant for cell migration and metastatic invasion studies. Plaque2.0 is suitable to quantitatively analyze virus infections, vector properties, or cancer cell phenotypes.     

Reprint of PSII Manganese Cluster: Protonation of W2, O5, O4 and His337 in the S1 state explored by combined quantum chemical and electrostatic energy computations

Photosystem II (PSII) is a membrane-bound protein complex that oxidizes water to produce energized protons, which are used to built up a proton gradient across the thylakoidal membrane in the leafs of plants. This light-driven reaction is catalyzed by withdrawing electrons from the Mn₄CaO₅-cluster (Mn-cluster) in four discrete oxidation steps [S₁ − (S₄ / S₀)] characterized in the Kok-cycle. In order to understand in detail the proton release events and the subsequent translocation of such energized protons, the protonation pattern of the Mn-cluster need to be elucidated. The new high-resolution PSII crystal structure from Umena, Kawakami, Shen, and Kamiya is an excellent basis to make progress in solving this problem. Following our previous work on oxidation and protonation states of the Mn-cluster, in this work, quantum chemical/electrostatic calculations were performed in order to estimate the pKa of different protons of relevant groups and atoms of the Mn-cluster such as W2, O4, O5 and His337. In broad agreement with previous experimental and theoretical work, our data suggest that W2 and His337 are likely to be in hydroxyl and neutral form, respectively, O5 and O4 to be unprotonated. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: Keys to Produce Clean Energy.     

An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications

We proposed a modification the procedure of genotyping based in labeled universal primer and tailed primer. In the standard protocol, three primers are used in the same PCR reaction, a forward primer with tail added at the 5′ end of the identical sequence to labeled universal primer with dye-fluorescent and a reverse primer. Unfortunately, the choice of a labeled primer characterized by a large number of complementary sequences in target genomes (which is more probable in larger genomes) result in unspecific amplifications (false positive) can cause absence or decrease amplification of the locus of interest and also false interpretation of the analysis. However, identification of possible homologies between the primer chosen for labelling and the genome is rarely possible from the available DNA data bases. In our approach, cycling is interrupted for the addition of the labeled primer only during the final cycles, thus minimizing unspecific amplification and competition between primers, resulting in the more fidelity amplification of the target regions.     

Pectolytic Bacteria Associated with Soft Rot of Calla Lily (Zantedeschia spp.) Tubers

Soft rot is the most important disease on calla lily in Poland. The isolation of the presumptive pathogen from symptomatic tubers on nutrient agar yielded bacteria with different colony morphology. Of 41 isolates collected, 10 showed pectolytic activity on crystal violet pectate medium and caused soft rot on potato slices. All pectolytic bacteria appeared to be Gram‐negative rods producing typical soft rot on inoculated leaf petioles of calla lily. Bacteria with colonies which morphologically resembled those used for inoculation were re‐isolated from diseased petioles. Their identification was based on phenotypic characters and sequence of the gene fragment coding 16S rRNA. It was found that, in addition to Pectobacterium carotovorum subsp. carotovorum, soft rot of calla lily can be caused by Pectobacterium carotovorum subsp. atrosepticum, Pseudomonas marginalis, Pseudomonas veronii and Chryseobacterium indologenes. The latter two are described for the first time as plant pathogens. The pectolytic activity of all identified bacteria, except that of P. carotovorum subsp. atrosepticum, was lower than that of P. carotovorum subsp. carotovorum, but strains of P. veronii showed a higher activity than P. marginalisand C. indologenes species.     

Biodegradation of epoxyconazole and piraclostrobin fungicides by Klebsiella sp. from soil

Three bacterial strains have been isolated from soil in which soybean had been continuously cropped and treated with Opera^®, a fungicide containing epoxyconazole and pyraclostrobin. The three strains (1,805, 2,801 and 3,803), obtained from soil at 80–100 cm depth, were selected on medium containing 0.03% Opera^®. Morphological examination revealed that the strains were Gram-negative, and two of them (1,805 and 2,801) exhibited polymorphism. The growth profiles demonstrated that 1,805 and 3,803 were more efficient growing in the presence of Opera^® than 2,801. Maximum growth was reached between 24 and 48 h, however, 2,801 was not able to survive after this period. The total protein content produced by 1,805, 2,801 and 3,803 in liquid selective medium containing Opera^® were 111.0 ± 0.02, 80.0 ± 0.05 and 130.5 ± 0.07 μg/ml, respectively. According to its biochemical and molecular features, strain 1,805 was identified as Klebsiella sp. On the basis of the characteristics presented (facultative anaerobic nature, polymorphic character and capacity of growing in the presence of Opera^®) strain 1,805 seems to be able to degrade the epoxyconazole and pyraclostrobin.     

In Vivo Formation and Binding of SeHg Complexes to the Erythrocyte Surface

The in vivo dynamics of selenium (Se) and mercury (Hg) interaction was studied in mouse tissues using direct visualization of individual Se, Hg, and SeHg particles on the surface of circulating erythrocytes. This high-resolution detection of Se and Hg was obtained by scanning electron microscopy coupled to X-ray microanalysis. BALB/c mice were injected in the peritoneal cavity with Se and Hg salts, and the animals were sacrificed 3 min after the Hg injection. Only a minority (9%) of the metal dots seen on mouse liver erythrocytes were SeHg complexes when Se and Hg salts were mixed together before injection. In contrast, the majority (73%) of metal dots on liver erythrocytes were SeHg complexes if Se was injected at least 5 min before Hg injection. All metal dots on liver erythrocytes were of SeHg complexes if Se was injected 9 or 12 min before the Hg injection. We conclude that the formation of stable in vivo SeHg complexes requires preliminary interaction of Se with a putative serum factor before complexes between Se and Hg are formed and are bound to the erythrocyte cell surface.