Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2-driven and idiopathic Parkinson's disease.

Activating leucine-rich repeat kinase 2 (LRRK2) mutations cause Parkinson's and phosphorylation of Rab10 by pathogenic LRRK2 blocks primary ciliogenesis in cultured cells. In the mouse brain, LRRK2 blockade of primary cilia is highly cell type specific: For example, cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2-pathway mutant mice. We show here that the cell type specificity of LRRK2-mediated cilia loss is also seen in human postmortem striatum from patients with LRRK2 pathway mutations and idiopathic Parkinson's. Single nucleus RNA sequencing shows that cilia loss in mouse cholinergic interneurons is accompanied by decreased glial-derived neurotrophic factor transcription, decreasing neuroprotection for dopamine neurons. Nevertheless, LRRK2 expression differences cannot explain the unique vulnerability of cholinergic neurons to LRRK2 kinase as much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. These data strongly suggest that loss of cilia in specific striatal cell types decreases neuroprotection for dopamine neurons in mice and human Parkinson's.

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.

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.

Loss of primary cilia and dopaminergic neuroprotection in pathogenic LRRK2-driven and idiopathic Parkinson's disease.

Activating LRRK2 mutations cause Parkinson's disease. Previously, we showed that cholinergic interneurons and astrocytes but not medium spiny neurons of the dorsal striatum lose primary cilia in LRRK2 mutant mice. Single nucleus RNA sequencing shows that cilia loss in cholinergic interneurons correlates with higher LRRK2 expression and decreased glial derived neurotrophic factor transcription. Nevertheless, much higher LRRK2 expression is seen in medium spiny neurons that have normal cilia in mice and humans. In parallel with decreased striatal dopaminergic neurite density, LRRK2 G2019S neurons show increased autism-linked CNTN5 adhesion protein expression; glial cells show significant loss of ferritin heavy chain. Human striatal tissue from LRRK2 pathway mutation carriers and idiopathic Parkinson's disease show similar cilia loss in cholinergic interneurons and astrocytes and overall loss of such neurons. These data strongly suggest that loss of cilia in specific striatal cell types decreases neuroprotection for dopamine neurons in mice and human Parkinson's disease.

Quantification of Protein Secretion from Circulating Tumor Cells in Microfluidic Chambers.

Cancer cells can be released from a cancerous lesion and migrate into the circulatory system, from whereon they may form metastases at distant sites. Today, it is possible to infer cancer progression and treatment efficacy by determining the number of circulating tumor cells (CTCs) in the patient's blood at multiple time points; further valuable information about CTC phenotypes remains inaccessible. In this article, a microfluidic method for integrated capture, isolation, and analysis of membrane markers as well as quantification of proteins secreted by single CTCs and CTC clusters is introduced. CTCs are isolated from whole blood with extraordinary efficiencies above 95% using dedicated trapping structures that allow co-capture of functionalized magnetic beads to assess protein secretion. The patform is tested with multiple breast cancer cell lines spiked into human blood and mouse-model-derived CTCs. In addition to immunostaining, the secretion level of granulocyte growth stimulating factor (G-CSF), which is shown to be involved in neutrophil recruitment, is quantified The bead-based assay provides a limit of detection of 1.5 ng mL-1 or less than 3700 molecules per cell. Employing barcoded magnetic beads, this platform can be adapted for multiplexed analysis and can enable comprehensive functional CTC profiling in the future.

Single-cell protein profiling in microchambers with barcoded beads.

Single-cell profiling provides insights into cellular behaviour that macroscale cell cultures and bulk measurements cannot reveal. In the context of personalized cancer treatment, the profiling of individual tumour cells may lead to higher success rates for therapies by rapidly selecting the most efficacious drugs. Currently, genomic analysis at the single-cell level is available through highly sensitive sequencing approaches. However, the identification and quantification of intracellular or secreted proteins or metabolites remains challenging. Here, we introduce a microfluidic method that facilitates capture, automated data acquisition and the multiplexed quantification of proteins from individual cells. The microfluidic platform comprises 1026 chambers with a volume of 152 pL each, in which single cells and barcoded beads are co-immobilized. We demonstrated multiplexed single-cell protein quantification with three different mammalian cell lines, including two model breast cancer cell lines. We established on-chip immunoassays for glyceraldehyde-3-phosphate dehydrogenase (GAPDH), galectin-3 (Gal-3) and galectin-3 binding protein (Gal-3bp) with detection limits as low as 7.0 × 104, 2.3 × 105 and 1.8 × 103 molecules per cell, respectively. The three investigated cell types had high cytosolic levels of GAPDH and could be clearly differentiated by their expression levels of Gal-3 and Gal-3bp, which are important factors that contribute to cancer metastasis. Because it employed commercially available barcoded beads for this study, our platform could be easily used for the single-cell protein profiling of several hundred different targets. Moreover, this versatile method is applicable to the analysis of bacteria, yeast and mammalian cells and nanometre-sized lipid vesicles.

Evaluation of Container Closure System Integrity for Frozen Storage Drug Products.

Sometimes, drug product for parenteral administration is stored in a frozen state (e.g., -20 °C or -80 °C), particularly during early stages of development of some biotech molecules in order to provide sufficient stability. Shipment of frozen product could potentially be performed in the frozen state, yet possibly at different temperatures, for example, using dry ice (-80 °C). Container closure systems of drug products usually consist of a glass vial, rubber stopper, and an aluminum crimped cap. In the frozen state, the glass transition temperature (Tg) of commonly used rubber stoppers is between -55 and -65 °C. Below their Tg, rubber stoppers are known to lose their elastic properties and become brittle, and thus potentially fail to maintain container closure integrity in the frozen state. Leaks during frozen temperature storage and transportation are likely to be transient, yet, can possibly risk container closure integrity and lead to microbial contamination. After thawing, the rubber stopper is supposed to re-seal the container closure system. Given the transient nature of the possible impact on container closure integrity in the frozen state, typical container closure integrity testing methods (used at room temperature conditions) are unable to evaluate and thus confirm container closure integrity in the frozen state. Here we present the development of a novel method (thermal physical container closure integrity) for direct assessment of container closure integrity by a physical method (physical container closure integrity) at frozen conditions, using a modified He leakage test. In this study, different container closure systems were evaluated with regard to physical container closure integrity in the frozen state to assess the suitability of vial/stopper combinations and were compared to a gas headspace method. In summary, the thermal physical container closure integrity He leakage method was more sensitive in detecting physical container closure integrity impact than gas headspace and aided identification of an unsuitable container closure system. LAY ABSTRACT: Sometimes, drug product for parenteral administration is stored in a frozen state (e.g., -20 °C or -80 °C), particularly during early stages of development of some biotech molecules in order to provide sufficient stability. Container closure systems for drug products usually consist of a glass vial, rubber stopper, and an aluminum crimped cap. In the frozen state, the glass transition temperature (Tg) of commonly used rubber stoppers is between -55 and -65 °C. Leaks during frozen temperature storage and transportation are likely to be transient, yet they can possibly risk container closure integrity and lead to microbial contamination and sterility breach. After thawing, the rubber stopper is expected to re-seal the container closure system. Given the transient nature of the possible impact on container closure integrity in the frozen state, typical container closure integrity testing methods (used at room temperature conditions) are unable to evaluate and thus confirm container closure integrity in the frozen state. Here we present the development of a novel method (thermal container closure integrity) for direct measurement of container closure integrity by a physical method (physical container closure integrity) at frozen conditions, using a modified He leakage test. In this study, we found that the thermal container closure integrity He leakage method was more sensitive in detecting physical container closure integrity impact than gas headspace and aided identification of an unsuitable container closure system.

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.