Our revisit of the complexation between anionic DNA and cationic polyethylenimine (PEI) in both water and phosphate buffered saline (PBS) by using a combination of laser light scattering (LLS) and ...gel electrophoresis confirms that nearly all the DNA chains are complexed with PEI to form polyplexes when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches ~
3, but the PEI/DNA polyplexes have a high
in-vitro gene transfection efficiency only when N:P
≥
10. Putting these two facts together, we not only conclude that this extra 7 portions of PEI chains are free in the solution mixture, but also confirmed that it is these free PEI chains that substantially promote the gene transfection no matter whether they are applied hours before or after the administration of the much less effective PEI/DNA polyplexes (N:P
=
3). The uptake kinetics measured by flow cytometry shows that the addition of free PEI leads to a faster and more efficient cellular internalization of polyplexes, but these free PEI chains mainly contribute to the subsequent intracellular trafficking. In contrast, the bound PEI chains mainly play a role in the DNA condensation and protection, leading to a different thinking in the development of non-viral vectors.
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Bone development is dynamically regulated by homeostasis, in which a balance between adipocytes and osteoblasts is maintained. Disruption of this differentiation balance leads to various bone-related ...metabolic diseases, including osteoporosis. In the present study, a primate-specific microRNA (miR-637) was found to be involved in the differentiation of human mesenchymal stem cells (hMSCs). Our preliminary data indicated that miR-637 suppressed the growth of hMSCs and induced S-phase arrest. Expression of miR-637 was increased during adipocyte differentiation (AD), whereas it was decreased during osteoblast differentiation (OS), which suggests miR-637 could act as a mediator of adipoosteogenic differentiation. Osterix (Osx), a significant transcription factor of osteoblasts, was shown to be a direct target of miR-637, which significantly enhanced AD and suppressed OS in hMSCs through direct suppression of Osx expression. Furthermore, miR-637 also significantly enhanced de novo adipogenesis in nude mice. In conclusion, our data indicated that the expression of miR-637 was indispensable for maintaining the balance of adipocytes and osteoblasts. Disruption of miR-637 expression patterns leads to irreversible damage to the balance of differentiation in bone marrow.
Our revisit of the complexation between DNA and polyethylenimine (PEI) by using a combination of laser light scattering and gel electrophoresis confirms that nearly all the DNA chains are complexed ...with PEI to form polyplexes when the molar ratio of nitrogen from PEI to phosphate from DNA (N:P) reaches ~
3, irrespective of the PEI chain length and solvent. Each solution mixture with N:P
>
3 contains two kinds of PEI chains: bound to DNA and free in the solution. It has been shown that it is those free PEI chains that play a vital role in promoting the gene transfection. The effects of the length of the bound and free chains on the gene transfection were respectively studied. Both short and long PEI chains are capable of condensing DNA completely at N:P
~
3 but long ones are ~
10
2-fold more effective in the gene transfection, apparently due to their fast endocytosis and intracellular trafficking. The cellular uptake kinetics studied by flow cytometry reveals that long free chains increase the uptake rate constant of the DNA/PEI complexes. In the intracellular pathway, they are able to prevent the development of the later endolysosomes, and facilitate the subsequent release of the polyplexes from the endosomes. Our result shows that the “proton sponge” effect is not dominant because the shut-down of the proton pump only partially attenuates the transfection efficiency. A possible mechanism is speculated and presented.
Effect of length and topology of free PEI chains on the gene transfection efficiency, where different free PEIs were added at 0, 2 or 4
h after the polyplex administration.
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MiR‐637 (microRNA‐637) is a primate‐specific miRNA belonging to the small noncoding RNA family, which represses gene regulation at the post‐transcriptional expression level. Although it was ...discovered approximately 5 years ago, its biomedical significance and regulatory mechanism remain obscure. Our preliminary data showed that miR‐637 was significantly suppressed in four HCC cell lines and, also, in most of the hepatocellular carcinoma (HCC) specimens, thereby suggesting that miR‐637 would be a tumor suppressor in HCC. Simultaneously, the enforced overexpression of miR‐637 dramatically inhibited cell growth and induced the apoptosis of HCC cells. The transcription factor, signal transducer and activator of transcription 3 (Stat3), is constitutively activated in multiple tumors, and aberrant Stat3 activation is linked to the promotion of growth and desensitization of apoptosis. Our study showed that Stat3 tyrosine 705 phosphorylation and several Stat3‐regulated antiapoptotic genes were down‐regulated in miR‐637 mimics‐transfected and Lv‐miR637‐infected HCC cells. In addition, miR‐637 overexpression negatively regulated Stat3 phosphorylation by suppressing autocrine leukemia inhibitory factor (LIF) expression and exogenous LIF‐triggered Stat3 activation and rescued cell growth in these cells. A nude mice model also demonstrated the above‐described results, which were obtained from the cell model. Furthermore, we found that LIF was highly expressed in a large proportion of HCC specimens, and its expression was inversely associated with miR‐637 expression. Conclusion: Our data indicate that miR‐637 acted as a tumor suppressor in HCC, and the suppressive effect was mediated, at least in part, by the disruption of Stat3 activation. (HEPATOLOGY 2011)
Mesenchymal stromal cells (MSCs) are multipotent cells that have great potential for regenerative medicine, tissue repair, and immunotherapy. Unfortunately, the outcomes of MSC-based research and ...therapies can be highly inconsistent and difficult to reproduce, largely due to the inherently significant heterogeneity in MSCs, which has not been well investigated. To quantify cell heterogeneity, a standard approach is to measure marker expression on the protein level via immunochemistry assays. Performing such measurements non-invasively and at scale has remained challenging as conventional methods such as flow cytometry and immunofluorescence microscopy typically require cell fixation and laborious sample preparation. Here, we developed an artificial intelligence (AI)-based method that converts transmitted light microscopy images of MSCs into quantitative measurements of protein expression levels. By training a U-Net+ conditional generative adversarial network (cGAN) model that accurately (mean Formula: see text = 0.77) predicts expression of 8 MSC-specific markers, we showed that expression of surface markers provides a heterogeneity characterization that is complementary to conventional cell-level morphological analyses. Using this label-free imaging method, we also observed a multi-marker temporal-spatial fluctuation of protein distributions in live MSCs. These demonstrations suggest that our AI-based microscopy can be utilized to perform quantitative, non-invasive, single-cell, and multi-marker characterizations of heterogeneous live MSC culture. Our method provides a foundational step toward the instant integrative assessment of MSC properties, which is critical for high-throughput screening and quality control in cellular therapies.
The commercially available branched polyethyleneimine (PEI) with a molar mass of 25 kD (PEI-25K) is an effective
in vitro vector to transfer genes, but its cytotoxicity limits its applications in ...bio-related research. To solve such an efficiency-versus-cytotoxicity catch-22 problem, the disulfide bond has been previously used to link less toxic short PEI chains (2 kD), but previous literature results are controversial. Recently, we found that it is vitally important to remove both carbon dioxide and water in the linking reaction as well as to control the structure of the resultant chains linked by dithiobis(succinimidyl propionate) (DSP). Under a programmable mixing of PEI and DSP, we can use laser light scattering (LLS) to in-situ monitor the linking reaction kinetics in DMSO in terms of the change of the average molar mass (
M
w
). Therefore, we were able to withdraw a series of linked PEI chains with different molar masses from one reaction mixture. Two such linked PEI samples (
M
w
~
7 kD, PEI-7K-L and ~
400 kD, PEI-400K-L) were used to illustrate the effect of the sample preparation and the chain structure on the
in vitro gene transfection and cytotoxicity. Our results reveal that PEI-7K-L is less cytotoxic and more effective in the gene transfection than both PEI-25K and Lipofectamine 2000 in the
in vitro gene transfection. However, PEI-400K-L has no gene transfection efficiency even though it is non-toxic.
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Highlights • CSCs driver angiogenesis via releasing proangiogenic factors and exosomes. • A vascular niche products instructive angiocrine factors to nourish CSCs. • This positive feedback loop ...contributes to the tumorigenesis and therapeutic resistance of HCC.
The human skin microbiome has important roles in skin health and disease. However, bacterial population structure and diversity at the strain level is poorly understood. We compared the skin ...microbiome at the strain level and genome level of Propionibacterium acnes, a dominant skin commensal, between 49 acne patients and 52 healthy individuals by sampling the pilosebaceous units on their noses. Metagenomic analysis demonstrated that although the relative abundances of P. acnes were similar, the strain population structures were significantly different in the two cohorts. Certain strains were highly associated with acne, and other strains were enriched in healthy skin. By sequencing 66 previously unreported P. acnes strains and comparing 71 P. acnes genomes, we identified potential genetic determinants of various P. acnes strains in association with acne or health. Our analysis suggests that acquired DNA sequences and bacterial immune elements may have roles in determining virulence properties of P. acnes strains, and some could be future targets for therapeutic interventions. This study demonstrates a previously unreported paradigm of commensal strain populations that could explain the pathogenesis of human diseases. It underscores the importance of strain-level analysis of the human microbiome to define the role of commensals in health and disease.
The mechanisms by which mucosal homeostasis is maintained are of central importance to inflammatory bowel disease. Critical to these processes is the intestinal epithelial cell (IEC), which regulates ...immune responses at the interface between the commensal microbiota and the host. CD1d presents self and microbial lipid antigens to natural killer T (NKT) cells, which are involved in the pathogenesis of colitis in animal models and human inflammatory bowel disease. As CD1d crosslinking on model IECs results in the production of the important regulatory cytokine interleukin (IL)-10 (ref. 9), decreased epithelial CD1d expression--as observed in inflammatory bowel disease--may contribute substantially to intestinal inflammation. Here we show in mice that whereas bone-marrow-derived CD1d signals contribute to NKT-cell-mediated intestinal inflammation, engagement of epithelial CD1d elicits protective effects through the activation of STAT3 and STAT3-dependent transcription of IL-10, heat shock protein 110 (HSP110; also known as HSP105), and CD1d itself. All of these epithelial elements are critically involved in controlling CD1d-mediated intestinal inflammation. This is demonstrated by severe NKT-cell-mediated colitis upon IEC-specific deletion of IL-10, CD1d, and its critical regulator microsomal triglyceride transfer protein (MTP), as well as deletion of HSP110 in the radioresistant compartment. Our studies thus uncover a novel pathway of IEC-dependent regulation of mucosal homeostasis and highlight a critical role of IL-10 in the intestinal epithelium, with broad implications for diseases such as inflammatory bowel disease.
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