Microfluidic Platform for Profiling of Extracellular Vesicles from Single Breast Cancer Cells.

Extracellular vesicles (EVs) are considered as valuable biomarkers to discriminate healthy from diseased cells such as cancer. Passing cytosolic and plasma membranes before their release, EVs inherit the biochemical properties of the cell. Here, we determine protein profiles of single EVs to understand how much they represent their cell of origin. We use a microfluidic platform which allows to immobilize EVs from completely isolated single cells, reducing heterogeneity of EVs as strongly seen in cell populations. After immunostaining, we employ four-color total internal reflection fluorescence microscopy to enumerate EVs and determine their biochemical fingerprint encoded in membranous or cytosolic proteins. Analyzing single cells derived from pleural effusions of two different human adenocarcinoma as well as from human embryonic kidney (SkBr3, MCF-7 and HEK293, respectively), we observed that a single cell secretes enough EVs to extract the respective tissue fingerprint. We show that overexpressed integral plasma membrane proteins are also found in EV membranes, which together with populations of colocalized proteins, provide a cell-specific, characteristic pattern. Our method highlights the potential of EVs as a diagnostic marker and can be directly employed for fundamental studies of EV biogenesis.

Identifying extracellular vesicle populations from single cells.

Extracellular vesicles (EVs) are constantly secreted from both eukaryotic and prokaryotic cells. EVs, including those referred to as exosomes, may have an impact on cell signaling and an incidence in diseased cells. In this manuscript, a platform to capture, quantify, and phenotypically classify the EVs secreted from single cells is introduced. Microfluidic chambers of about 300 pL are employed to trap and isolate individual cells. The EVs secreted within these chambers are then captured by surface-immobilized monoclonal antibodies (mAbs), irrespective of their intracellular origin. Immunostaining against both plasma membrane and cytosolic proteins was combined with highly sensitive, multicolor total internal reflection fluorescence microscopy to characterize the immobilized vesicles. The data analysis of high-resolution images allowed the assignment of each detected EV to one of 15 unique populations and demonstrated the presence of highly heterogeneous phenotypes even at the single-cell level. The analysis also revealed that each mAb isolates phenotypically different EVs and that more vesicles were effectively immobilized when CD63 was targeted instead of CD81. Finally, we demonstrate how a heterogeneous suppression in the secreted vesicles is obtained when the enzyme neutral sphingomyelinase is inhibited.

Promoter Screening from Bacillus subtilis in Various Conditions Hunting for Synthetic Biology and Industrial Applications.

The use of Bacillus subtilis in synthetic biology and metabolic engineering is highly desirable to take advantage of the unique metabolic pathways present in this organism. To do this, an evaluation of B. subtilis' intrinsic biological parts is required to determine the best strategies to accurately regulate metabolic circuits and expression of target proteins. The strengths of promoter candidates were evaluated by measuring relative fluorescence units of a green fluorescent protein reporter, integrated into B. subtilis' chromosome. A total of 84 predicted promoter sequences located upstream of different classes of proteins including heat shock proteins, cell-envelope proteins, and proteins resistant against toxic metals (based on similarity) and other kinds of genes were tested. The expression levels measured ranged from 0.0023 to 4.53-fold of the activity of the well-characterized strong promoter P43. No significant shifts were observed when strains, carrying different promoter candidates, were cultured at high temperature or in media with ethanol, but some strains showed increased activity when cultured under high osmotic pressure. Randomly selected promoter candidates were tested and found to activate transcription of thermostable ß-galactosidase (bgaB) at a similar level, implying the ability of these sequences to function as promoter elements in multiple genetic contexts. In addition, selected promoters elevated the final production of both cytoplasmic bgaB and secreted protein α-amylase to about fourfold and twofold, respectively. The generated data allows a deeper understanding of B. subtilis' metabolism and will facilitate future work to develop this organism for synthetic biology.

Improving Protein Production on the Level of Regulation of both Expression and Secretion Pathways in Bacillus subtilis.

The well-characterized gram-positive bacterium Bacillus subtilis is an outstanding industrial candidate for protein expression owing to its single membrane and high capacity of secretion, simplifying the downstream processing of secretory proteins. During the last few years, there has been continuous progress in the illustration of secretion mechanisms and application of this robust host in various fields of life science, such as enzyme production, feed additives, and food and pharmaceutical industries. Here, we review the developments of Bacillus subtilis as a highly promising expression system illuminating strong chemical- and temperatureinducible and other types of promoters, strategies for ribosome-binding-site utilization, and the novel approach of signal peptide selection. Furthermore, we outline the main steps of the Sec pathway and the relevant elements as well as their interactions. In addition, we introduce the latest discoveries of Tat-related complex structures and functions and the countless applications of this full-folded protein secretion pathway. This review also lists some of the current understandings of ATP-binding cassette transporters. According to the extensive knowledge on the genetic modification strategies and molecular biology of Bacillus subtilis, we propose some suggestions and strategies for improving the yield of intended productions. We expect this to promote striking future developments in the optimization and application of this bacterium.