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A major goal of methodologies related to large scale gene expression analyses is to initiate comprehensive information on transcript signatures in single cells within the tissue's anatomy. Until now, this could be achieved in a stepwise experimental approach: (1) identify the majority of transcripts in a single cell (single cell transcriptome); (2) provide information on transcripts on multiple cell subtypes in a complex sample (cell heterogeneity); and (3) give information on each cell's spatial location within the tissue (zonation transcriptomics). Such genetic information will allow construction of functionally relevant gene expression maps of single cells of a given anatomically defined tissue compartment and thus pave the way for subsequent analyses, including their epigenetic modifications. Until today these aims have not been achieved in the area of cardiovascular disease research though steps toward these goals become apparent: laser capture microdissection (LCM)-based mRNA expression microarrays of atherosclerotic plaques were applied to gain information on local gene expression changes during disease progression, providing limited spatial resolution. Moreover, while LCM-derived tissue RNA extracts have been shown to be highly sensitive and covers a range of 10-16,000 genes per array/small amount of RNA, its original promise to isolate single cells from a tissue section turned out not to be practicable because of the inherent contamination of the cell's RNA of interest with RNA from neighboring cells. Many shortcomings of LCM-based analyses have been overcome using single-cell RNA sequencing (scRNA-seq) technologies though scRNA-seq also has several limitations including low numbers of transcripts/cell and the complete loss of spatial information. Here, we describe a protocol toward combining advantages of both techniques while avoiding their flaws.

Additional file 1: Table S1. The NRF1 downstream genes with a cell cycle association were identified by DAVID, KOBAS and Metascape online databases. Table S2. The overlapping NRF1 target genes in GO and Reactome analyses are detailed in the table. Table S3. The putative specific loci and the scoring of NRF1 in the promoter proximal regions of E2F1 were analyzed by the open-access database JASPAR.

Recent advances in cardiovascular research have led to a more comprehensive understanding of molecular mechanisms of atherosclerosis. It has become apparent that the disease involves three layers of the arterial wall: the intima, the media, and a connective tissue coat termed the adventitia. It is also now appreciated that arteries are surrounded by adipose and neuronal tissues. In addition, adjacent to and within the adventitia, arteries are embedded in a loose connective tissue containing blood vessels (vasa vasora) and lymph vessels, artery-draining lymph nodes and components of the peripheral nervous system, including periarterial nerves and ganglia. During atherogenesis, each of these tissues undergoes marked structural and cellular alterations. We propose that a better understanding of these cell-cell and cell-tissue interactions may considerably advance our understanding of cardiovascular disease pathogenesis. Methods to acquire subcellular optical access to the intact tissues surrounding healthy and diseased arteries are urgently needed to achieve these aims. Tissue clearing is a landmark next-generation, three-dimensional (3D) microscopy technique that allows to image large-scale hitherto inaccessible intact deep tissue compartments. It allows for detailed reconstructions of arteries by a combination of labelling, clearing, advanced microscopies and other imaging and data-analysis tools. Here, we describe two distinct tissue clearing protocols; solvent-based modified three-dimensional imaging of solvent-cleared organs (3DISCO) clearing and another using aqueous-based 2,2'-thiodiethanol (TDE) clearing, both of which complement each other.

This study investigated the molecular mechanism by which bronchoalveolar lavage fluid exosomes (BALF-exo) alleviated acute lung injury (ALI). BALF-exo was isolated from mice. LPS was used to induce inflammation in rat alveolar macrophages (NR8383). NR8383 cell models were treated with BALF-exo or BALF-exo loaded with miR-223-3p mimics/inhibitors, or STK39 was overexpressed in NR8383 cells before LPS and BALF-exo treatment. These cells were subjected to apoptosis, autophagy, and inflammation assays. RNA immunoprecipitation and dual-luciferase reporter assay were conducted to verify the binding between miR-223-3p and STK39. LPS-induced ALI mouse models were treated with BALF-exo loaded with miR-223-3p mimics. miR-223-3p was lowly expressed in BALF-exo from LPS-treated mice. BALF-exo loaded with miR-223-3p mimics increased viability and autophagy and decreased apoptosis and inflammation in NR8383 cell models. Inhibition of miR-223-3p in BALF-exo or overexpression of STK39 in NR8383 cells repressed the therapeutic effect of BALF-exo in LPS-treated NR8383 cells. STK39 was a target gene of miR-223-3p. miR-223-3p shuttled by BALF-exo negatively regulated the expression of STK39 in NR8383 cells. BALF-exo loaded with miR-223-3p mimics also reduced lung injuries in ALI mice. In conclusion, miR-223-3p loaded in BALF-exo alleviates ALI by targeting STK39 in alveolar macrophages.

This paper analyzes the crosstalk issues for a closed-loop active insulated gate bipolar transistor (IGBT) gate driver (AGD) based on di/dt and dv/dt detection and presents a crosstalk suppression (CTS) circuit. Closed-loop active gate drivers for IGBTs are beneficial in switching transient control by generating extra gate control signals to regulate the gate drive voltage or current with di/dt and dv/dt feedback. AGDs achieve the compensation of the IGBT nonlinearities and are independent of operating points. In a phase-leg configuration, however, AGDs produce additional crosstalk interference besides that due to reverse transfer capacitance. These two kinds of crosstalk interferences together lead to an oscillation in the IGBT gate voltage, and then a shoot-through fault may occur. An equivalent circuit model of the feedback circuit is presented to analyze and evaluate the crosstalk issues. The design and realization of a CTS circuit are presented to eliminate the crosstalk and extra gate drive losses caused by crosstalk. The experimental results substantiate the validity of the theoretical crosstalk analysis and the effectiveness of the proposed CTS circuit.

Myopia is very prevalent worldwide, especially among Asian populations. Orthokeratology is a proven intervention to reduce myopia progression. The current study investigated association between baseline corneal biomechanics and orthokeratology responses, and changes of corneal biomechanics from long-term orthokeratology. We fitted 59 adult subjects having myopia between −4.00D to −5.00D with overnight orthokeratology. Corneal biomechanics was measured through dynamic bidirectional corneal applanation (in terms of corneal hysteresis, CH and corneal resistance factor, CRF) and corneal indentation (in terms of corneal stiffness, S and tangent modulus, E). Subjects with poor orthokeratology responses had lower E (mean 0.474 MPa) than subjects with good orthokeratology responses (mean 0.536 MPa). Successful orthokeratology for 6 months resulted in reducing CH (reduced by 5.8%) and CRF (reduced by 8.7%). Corneal stiffness was stable, but E showed an increasing trend. Among subjects with successful orthokeratology, a higher baseline S resulted in greater myopia reduction (Pearson correlation coefficient, r = 0.381, p = 0.02).