Single-Cell Profiling Reveals the Origin of Phenotypic Variability in Adipogenesis

Phenotypic heterogeneity in a clonal cell population is a well-observed but poorly understood phenomenon. Here, a single-cell approach is employed to investigate non-mutative causes of phenotypic heterogeneity during the differentiation of 3T3-L1 cells into fat cells. Using coherent anti-Stokes Raman scattering microscopy and flow cytometry, adipogenic gene expression, insulin signaling, and glucose import are visualized simultaneously with lipid droplet accumulation in single cells. Expression of adipogenic genes PPARγ, C/EBPα, aP2, LP2 suggests a commitment to fat cell differentiation in all cells. However, the lack of lipid droplet in many differentiating cells suggests adipogenic gene expression is insufficient for lipid droplet formation. Instead, cell-to-cell variability in lipid droplet formation is dependent on the cascade responses of an insulin signaling pathway which includes insulin sensitivity, kinase activity, glucose import, expression of an insulin degradation enzyme, and insulin degradation rate. Increased and prolonged insulin stimulation promotes lipid droplet accumulation in all differentiating cells. Single-cell profiling reveals the kinetics of an insulin signaling cascade as the origin of phenotypic variability in drug-inducible adipogenesis

Real-Time CARS Imaging Reveals a Calpain-Dependent Pathway for Paranodal Myelin Retraction during High-Frequency Stimulation

High-frequency electrical stimulation is becoming a promising therapy for neurological disorders, however the response of the central nervous system to stimulation remains poorly understood. The current work investigates the response of myelin to electrical stimulation by laser-scanning coherent anti-Stokes Raman scattering (CARS) imaging of myelin in live spinal tissues in real time. Paranodal myelin retraction at the nodes of Ranvier was observed during 200 Hz electrical stimulation. Retraction was seen to begin minutes after the onset of stimulation and continue for up to 10 min after stimulation was ceased, but was found to reverse after a 2 h recovery period. The myelin retraction resulted in exposure of Kv 1.2 potassium channels visualized by immunofluorescence. Accordingly, treating the stimulated tissue with a potassium channel blocker, 4-aminopyridine, led to the appearance of a shoulder peak in the compound action potential curve. Label-free CARS imaging of myelin coupled with multiphoton fluorescence imaging of immuno-labeled proteins at the nodes of Ranvier revealed that high-frequency stimulation induced paranodal myelin retraction via pathologic calcium influx into axons, calpain activation, and cytoskeleton degradation through spectrin break-down

The proline-rich domain of tau plays a role in interactions with actin

<p>Abstract</p> <p>Background</p> <p>The microtubule-associated protein tau is able to interact with actin and serves as a cross-linker between the microtubule and actin networks. The microtubule-binding domain of tau is known to be involved in its interaction with actin. Here, we address the question of whether the other domains of tau also interact with actin.</p> <p>Results</p> <p>Several tau truncation and deletion mutants were constructed, namely N-terminal region (tauN), proline-rich domain (tauPRD), microtubule binding domain (tauMTBD) and C-terminal region (tauC) truncation mutants, and microtubule binding domain (tauΔMTBD) and proline-rich domain/microtubule binding domain (tauΔPRD&MTBD) deletion mutants. The proline-rich domain truncation mutant (tauPRD) and the microtubule binding domain deletion mutant (tauΔMTBD) promoted the formation of actin filaments. However, actin assembly was not observed in the presence of the N-terminal and C-terminal truncation mutants. These results indicate that the proline-rich domain is involved in the association of tau with G-actin. Furthermore, results from co-sedimentation, solid phase assays and electron microscopy showed that the proline-rich domain is also capable of binding to F-actin and inducing F-actin bundles. Using solid phase assays to analyze apparent dissociation constants for the binding of tau and its mutants to F-actin resulted in a sequence of affinity for F-actin: tau >> microtubule binding domain > proline-rich domain. Moreover, we observed that the proline-rich domain was able to associate with and bundle F-actin at physiological ionic strength.</p> <p>Conclusion</p> <p>The proline-rich domain is a functional structure playing a role in the association of tau with actin. This suggests that the proline-rich domain and the microtubule-binding domain of tau are both involved in binding to and bundling F-actin.</p

Conjunctival Reconstruction with Progenitor Cell-Derived Autologous Epidermal Sheets in Rhesus Monkey

Severe ocular surface diseases are some of the most challenging problems that the clinician faces today. Conventional management is generally unsatisfactory, and the long-term ocular consequences of these conditions are devastating. It is significantly important to find a substitute for conjunctival epithelial cells. This study was to explore the possibility of progenitor cell-derived epidermal sheets on denuded amniotic membrane to reconstruct ocular surface of conjunctiva damaged monkeys. We isolated epidermal progenitor cells of rhesus monkeys by type IV collagen adhesion, and then expanded progenitor cell-derived epidermal sheets on denuded amniotic membrane ex vivo. At 3 weeks after the conjunctiva injury, the damaged ocular surface of four monkeys was surgically reconstructed by transplanting the autologous cultivated epidermal progenitor cells. At 2 weeks after surgery, transplants were removed and examined with Hematoxylin-eosin staining, Periodic acid Schiff staining, immunofluorescent staining, scanning and transmission electron microscopy. Histological examination of transplanted sheets revealed that the cell sheets were healthy alive, adhered well to the denuded amniotic membrane, and had several layers of epithelial cells. Electron microscopy showed that the epithelial cells were very similar in appearance to those of normal conjunctival epithelium, even without goblet cell detected. Epithelial cells of transplants had numerous desmosomal junctions and were attached to the amniotic membrane with hemidesmosomes. Immunohistochemistry confirmed the presence of the conjunctival specific markers, mucin 4 and keratin 4, in the transplanted epidermal progenitor cells. In conclusion, our present study successfully reconstructed conjunctiva with autologous transplantation of progenitor cell-derived epidermal sheets on denuded AM in conjunctival damaged monkeys, which is the first step toward assessing the use of autologous transplantation of progenitor cells of nonocular surface origin. Epidermal progenitor cells could be provided as a new substitute for conjunctival epithelial cells to overcome the problems of autologous conjunctiva shortage

RNA-Seq Analyses Generate Comprehensive Transcriptomic Landscape and Reveal Complex Transcript Patterns in Hepatocellular Carcinoma

RNA-seq is a powerful tool for comprehensive characterization of whole transcriptome at both gene and exon levels and with a unique ability of identifying novel splicing variants. To date, RNA-seq analysis of HBV-related hepatocellular carcinoma (HCC) has not been reported. In this study, we performed transcriptome analyses for 10 matched pairs of cancer and non-cancerous tissues from HCC patients on Solexa/Illumina GAII platform. On average, about 21.6 million sequencing reads and 10.6 million aligned reads were obtained for samples sequenced on each lane, which was able to identify >50% of all the annotated genes for each sample. Furthermore, we identified 1,378 significantly differently expressed genes (DEGs) and 24, 338 differentially expressed exons (DEEs). Comprehensive function analyses indicated that cell growth-related, metabolism-related and immune-related pathways were most significantly enriched by DEGs, pointing to a complex mechanism for HCC carcinogenesis. Positional gene enrichment analysis showed that DEGs were most significantly enriched at chromosome 8q21.3–24.3. The most interesting findings were from the analysis at exon levels where we characterized three major patterns of expression changes between gene and exon levels, implying a much complex landscape of transcript-specific differential expressions in HCC. Finally, we identified a novel highly up-regulated exon-exon junction in ATAD2 gene in HCC tissues. Overall, to our best knowledge, our study represents the most comprehensive characterization of HBV-related HCC transcriptome including exon level expression changes and novel splicing variants, which illustrated the power of RNA-seq and provided important clues for understanding the molecular mechanisms of HCC pathogenesis at system-wide levels

Risk factors of chronic kidney diseases in Chinese adults with type 2 diabetes

201901 bcrcVersion of RecordPublishe