A Systemic Approach to Isolate, Retrieve, and Characterize Trophoblasts from the Maternal Circulation Using a Centrifugal Microfluidic Disc and a Multiple Single-Cell Retrieval Strategy.

Rare cells in the blood often have rich clinical significance. Although their isolation is highly desirable, this goal remains elusive due to their rarity. This paper presents a systemic approach to isolate and characterize trophoblasts from the maternal circulation. A microfluidic rare cell disc assay (RaCDA) was designed to process an extremely large volume of up to 15 mL of blood in 30 min, depleting red blood cells (RBCs) and RBC-bound white blood cells (WBC) while isolating trophoblasts in the collection chip. To minimize cell loss, on-disc labeling of cells with fluorescent immuno-staining identified the trophoblasts. Retrieval of trophoblasts utilized an optimized strategy in which multiple single cells were retrieved within the same micropipette column, with each cell encapsulated in a fluid volume (50 nL) separated by an air pocket (10 nL). Further, whole-genome amplification (WGA) amplified contents from a few retrieved cells, followed by quality control (QC) on the success of WGA via housekeeping genes. For definitive confirmation of trophoblasts, short-tandem repeat (STR) of the WGA-amplified content was compared against STR from maternal WBC and amniocytes from amniocentesis. Results showed a mean recovery rate (capture efficiency) of 91.0% for spiked cells with a WBC depletion rate of 99.91%. The retrieval efficiency of single target cells of 100% was achieved for up to four single cells retrieved per micropipette column. Comparison of STR signatures revealed that the RaCDA can retrieve trophoblasts from the maternal circulation.

Exosomes-Potential for Blood-Based Marker in Alzheimer's Disease.

Exosomes are believed to be secreted from multivesicular endosomes and containing proteins and nucleic acids, including mRNA and microRNAs, which have been implicated to play a role in neurodegenerative diseases. Neuron-derived exosomes at the circulation provide a unique potential as biomarkers towards assessment of Alzheimer's disease (AD), even at the pre-clinical stage. This review briefly discusses their biogenesis and transport, exosomal protein verses soluble protein, evidence for their role in AD, isolation of exosomes, and challenges and future directions to realize reliable blood-based biomarkers to meet phenomenal unmet clinical and pre-clinical need of AD.

Improving the Purity of Extracellular Vesicles by Removal of Lipoproteins from Size Exclusion Chromatography- and Ultracentrifugation-Processed Samples Using Glycosaminoglycan-Functionalized Magnetic Beads.

Extracellular vesicles (EVs) are present in blood at much lower concentrations (5-6 orders of magnitude) compared to lipoprotein particles (LP). Because LP and EV overlap in size and density, isolating high-purity EVs is a significant challenge. While the current two-step sequential EV isolation process using size-expression chromatography (SEC) followed by a density gradient (DG) achieves high purity, the time-consuming ultracentrifugation (UC) step in DG hinders workflow efficiency. This paper introduces an optimized magnetic bead reagent, LipoMin, functionalized with glycosaminoglycans (GAGs), as a rapid alternative for LP removal during the second-step process in about 10 minutes. We evaluated LipoMin's efficacy on two sample types: (a) EV fractions isolated by size exclusion chromatography (SEC + LipoMin) and (b) the pellet obtained from ultracentrifugation (UC + LipoMin). The workflow is remarkably simple, involving a 10 min incubation with LipoMin followed by magnetic separation of the LP-depleted EV-containing supernatant. Results from enzyme-linked immunosorbent assay (ELISA) revealed that LipoMin removes 98.2% ApoB from SEC EV fractions, comparable to the LP removal ability of DG in the SEC + DG two-step process. Importantly, the EV yield (CD81 ELISA) remained at 93.0% and Western blot analysis confirmed that key EV markers, flotillin and CD81, were not compromised. Recombinant EV (rEV), an EV reference standard, was spiked into SEC EV fractions and recovered 89% of CD81 protein. For UC + LipoMin, ApoA1 decreased by 76.5% while retaining 90.7% of CD81. Notably, both colorectal cancer (CRC) and Alzheimer's disease (AD) samples processed by SEC + LipoMin and UC + LipoMin displayed clear expression of relevant EV and clinical markers. With a 10 min workflow (resulting in a 96% time saving compared to the traditional method), the LipoMin reagent offers a rapid and efficient alternative to DG for LP depletion, paving the way for a streamlined SEC + LipoMin two-step EV isolation process.

Characterization of markers, functional properties, and microbiome composition in human gut-derived bacterial extracellular vesicles.

Past studies have confirmed the etiologies of bacterial extracellular vesicles (BEVs) in various diseases, including inflammatory bowel disease (IBD) and colorectal cancer (CRC). This study aimed to investigate the characteristics of stool-derived bacterial extracellular vesicles (stBEVs) and discuss their association with stool bacteria. First, three culture models - gram-positive (G+)BcBEVs (from B.coagulans), gram-negative (G-)EcBEVs (from E.coli), and eukaryotic cell-derived EVs (EEV, from Colo205 cell line) - were used to benchmark various fractions of stEVs separated from optimized density gradient approach (DG). As such, WB, TEM, NTA, and functional assays, were utilized to analyze properties and distribution of EVs in cultured and stool samples. Stool samples from healthy individuals were interrogated using the approaches developed. Results demonstrated successful separation of most stBEVs (within DG fractions 8&9) from stEEVs (within DG fractions 5&6). Data also suggest the presence of stBEV DNA within vesicles after extraction of BEV DNA and DNase treatment. Metagenomic analysis from full-length (FL) region sequencing results confirmed significant differences between stool bacteria and stBEVs. Significantly, F8&9 and the pooled sample (F5-F9) exhibited a similar microbial composition, indicating that F8&9 were enriched in most stBEV species, primarily dominated by Firmicutes (89.6%). However, F5&6 and F7 still held low-density BEVs with a significantly higher proportion of Proteobacteria (20.5% and 40.7%, respectively) and Bacteroidetes (24% and 13.7%, respectively), considerably exceeding the proportions in stool and F8&9. Importantly, among five healthy individuals, significant variations were observed in the gut microbiota composition of their respective stBEVs, indicating the potential of stBEVs as a target for personalized medicine and research.

Author Correction: Immunofluorescence can assess the efficacy of mTOR pathway therapeutic agent Everolimus in breast cancer models.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Immunofluorescence can assess the efficacy of mTOR pathway therapeutic agent Everolimus in breast cancer models.

When breast cancer patients start to exhibit resistance to hormonal therapy or chemotherapy, the mTOR inhibitor everolimus can be considered as an alternative therapeutic agent. Everolimus can deregulate the PI3K/AKT/mTOR pathway and affect a range of cellular functions. In some patients, the agent does not exhibit the desired efficacy and, even worse, not without the associated side effects. This study assessed the use of immunofluorescence (IF) as a modality to fill this unmet need of predicting the efficacy of everolimus prior to administration. Cell viability and MTT assays based on IF intensities of pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 on breast cancer cells (Hs578T, MCF7, BT474, MDA-MB-231) and patient-derived cell culture from metastatic sites (ABC-82T and ABC-16TX1) were interrogated. Results show that independent pho-4EBP1 Thr37/46 and pho-S6K1 Ser424 IF expressions can classify data into different groups: everolimus sensitive and resistant. The combined IF baseline intensity of these proteins is predictive of the efficacy of everolimus, and their intensities change dynamically when cells are resistant to everolimus. Furthermore, mTOR resistance is not only consequence of the AKT/mTOR pathway but also through the LKB1 or MAPK/ERK pathway. The LKB1 and pho-GSK3ß may also be potential predictive markers for everolimus.