Relationships between CYP2D6 phenotype, breast cancer and hot flushes in women at high risk of breast cancer receiving prophylactic tamoxifen: results from the IBIS-I trial

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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.ISSN:1520-6882ISSN:0003-270

Fate and state transitions during human blood vessel organoid development

Blood vessel organoids (BVOs) derived from human pluripotent stem cells have emerged as a novel system to understand human vascular development, model disorders, and develop regenerative therapies. However, it is unclear which molecular states constitute BVOs and how cells differentiate and self-organize within BVOs in vitro and after transplantation. Here we reconstruct BVO development over a time course using single-cell transcriptomics. We observe progenitor states that bifurcate into endothelial and mural fates, and find that BVOs do not acquire definitive arterio-venous endothelial identities in vitro. Chromatin accessibility profiling identifies gene regulatory network (GRN) features associated with endothelial and mural fate decisions, and transcriptome-coupled lineage recording reveals multipotent progenitor states within BVOs. We perform single-cell genetic perturbations within mosaic BVOs to dissect the impact of transcription factor (TF) and receptor depletion on cell differentiation, and highlight multiple TFs including MECOM and ETV2 as strong-effect regulators of human BVO development. We show that manipulation of VEGF and Notch signaling pathways alters BVO morphogenesis and endothelial GRNs, and induces arteriovenous-like state differentiation. We analyze matured BVOs after transplantation using scRNA-seq, and observe matured endothelium with clear arteriovenous specification. We also observe off-target cell fates with bone and adipocyte features, suggesting multipotent states reside within the BVOs in vitro that expand and diversify in less restrictive conditions. Finally, we map vascular disease associated genes to BVO cell states to highlight the potential of BVOs for disease modeling. Altogether, our data and analyses provide the first comprehensive cell state atlas of BVO development and illuminate both the power and limitation of BVOs for translational research

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 β-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

Sequence logo of the seven relatively strong promoter candidates from <i>B</i>. <i>subtilis</i>.

<p>A: Sequences of predicted -35 and -10 boxes and the spacers between the two elements. About half of the spacers between -35 and -10 boxes were around 17 base pairs. These three sequence logos were constructed using Weblogo3. B: Sequence logo of -10 hexamers; similar to cardinal “TATAAT” sequence. C: Sequence logo of -35 hexamer, not consistent with the classical “TTGACA” sequences. The -35 logo was very consistent with the consensus logo (5/6 positions were majoritarily correct). This was better than the -10 logo where only 4/6 positions were mostly the consensus base. D: Sequence logo of promoter candidates when the -10 box was fixed; the different spacing between the promoter elements may have contributed to difficulty in assigning the -35 box. This was presumably because the best-fit -10 sequences were used to make the logo. Since the varying lengths of the spacers have not been taken into account, there was no consensus -35 box.</p

Oligonucleotides used in this study.

<p>Oligonucleotides used in this study.</p

Comparison of P43 and P<i>trnQ</i> driving α-amylase in pMA5.

<p>A: Enzyme activity of α-amylase that secreted into the medium during fermentation in flasks for 72 h. Error bars represent standard deviations of biological triplicates. B: SDS-PAGE analysis of α-amylase in the supernatant secreted by <i>B</i>. <i>subtilis</i> 1A751. 10 μL of each supernatant sample was loaded on the gel. Lane M: molecular weight marker; Lane 1: pMA5 without α-amylase; Lane 2, 4, 6 represented pMA5 containing P43 after incubation for 36 h, 48 h, and 60 h, respectively. Lane 3, 5, 7 represent pMA5 containing P<i>trnQ</i> after incubation for 36 h, 48 h, and 60 h, respectively.</p