A vortex pump-based optically-transparent microfluidic platform for biotech and medical applications.

This paper reports an automated polymer based microfluidic analysis system integrated with a surface plasmon resonance (SPR) biosensor that demonstrates the detection of specific binding of biomolecules and that qualitatively monitors cell adhesion on the sensor surface. Micropumps, microchannels, and an SPR biosensor were integrated into a single polymer (PMMA) based microfluidic system. The integrated system has been studied for its potential applications in bio-molecules detection and drugs discovery. Two experiments, (1) monitoring the reaction between the BSA-BSA antibody, and (2) monitoring the activities of living cells in the presence or absence of trypsin in a RPMI-1640 medium, were conducted to show the biomedical application capability. Because SPR based bio-detection requires optically transparent substrates, PMMA is a potential replacement for glass and silicon-glass in microfluidic systems, if bio-compatibility and low-cost are desired. Hence, our work has shown the feasibility of commercializing an SPR based bio-medical/chemical analysis system in the near future.

Polyphyllin D is a potent apoptosis inducer in drug-resistant HepG2 cells.

In a search for new anticancer agents, we identified a novel compound polyphyllin D (PD) (diosgenyl alpha-L-rhamnopyranosyl-(1-->2)-(alpha-L-arabinofuranosyl)-(1-->4)]-[beta-D-glucopyranoside) that induced DNA fragmentation and phosphatidyl-serine (PS) externalization in a hepatocellular carcinoma cell line HepG2 derivative with drug resistance (R-HepG2). PD is a saponin originally found in a tradition Chinese medicinal herb Paris polyphylla. It has been used to treat liver cancer in China for many years. We evaluated the cell-killing mechanisms of this compound in R-HepG2 and its parental cells. The mitochondrial apoptotic pathway was found to be involved in the PD-induced apoptosis because PD elicited depolarization of mitochondrial transmembrane potential (DeltaPsim), generation of H2O2, as well as release of cytochrome c and apoptosis-inducing factor in a dose- and time-dependent manner. In conclusion, we show for the first time that PD is a potent anticancer agent that can overcome drug resistance in R-HepG2 cells and elicit programmed cell death via mitochondrial dysfunction.

microRNA-199a-3p, DNMT3A, and aberrant DNA methylation in testicular cancer.

It was previously demonstrated that miR-199a was downregulated in testicular germ cell tumor (TGCT), probably due to hypermethylation of its promoter. Further study found that re-expression of miR-199a in testicular cancer cells (NT2) led to suppression of cell growth, cancer migration, invasion and metastasis. More detailed analyses showed that these properties of miR-199a could be assigned to miR-199a-5p, one of its two derivatives. The biological role of the other derivative, miR-199a-3p in TGCT, remains largely uncharacterized. In this report, we identified DNA (cytosine-5)-methyltransferase 3A (DNMT3A), the de novo methyltransferase, as a direct target of miR-199a-3p using a 3'-UTR reporter assay. Transient expression of miR-199a-3p in NT2 cells led to decrease, while knocking down of miR-199a-3p in a normal human testicular cell line (HT) led to elevation, of DNMT3A2 (DNMT3A gene isoform 2) mRNA and protein levels. In clinical samples, DNMT3A2 was significantly overexpressed in malignant testicular tumor, and the expression of DNMT3A2 was inversely correlated with the expression of miR-199a-3p. However, DNMT3A did not affect miR-199a expression in NT2 cells. Further characterization of miR-199a-3p revealed that it negatively regulated DNA methylation, partly through targeting DNMT3A. Overexpression of miR-199a-3p restored the expression of APC and MGMT tumor-suppressor genes in NT2 cells by affecting DNA methylation of their promoter regions. Our studies demonstrated the deregulation of miR-199a-3p expression in TGCT may provide novel mechanistic insights into TGCT carcinogenesis and suggested a potentially therapeutic use of synthetic miR-199a-3p oligonucleotides as effective hypomethylating compounds in the treatment of TGCT.

A miR-199a/miR-214 self-regulatory network via PSMD10, TP53 and DNMT1 in testicular germ cell tumor.

It was previously demonstrated that microRNA-199a (miR-199a) was down-regulated in testicular germ cell tumor (TGCT) partially caused by hypermethylation of its promoter. miR-199a is encoded by two loci in the human genome, miR-199a-1 on chromosome (Chr) 19 and miR-199a-2 on Chr 1. Both loci encode the same miR-199a. Another microRNA, microRNA-214 (miR-214), also locates on Chr 1. Previous study revealed that it is co-transcribed with miR-199a-2. However, the biological significance of the co-expression of miR-199a and miR-214 remains largely unknown. In this study, we determined that miR-199a and miR-214 were concordantly expressed in NT2 cells and TGCT patient tissues. After 5-aza treatment, miR-199-3p/5p and miR-214 expression was significantly increased. Silencing of DNMT1with siRNA restored the expression of miR-199a and miR-214, accompanied by de-methylation of the promoters of miR-199a-1/2. TP53 down-regulated the expression of DNMT1 in NT2 cells and overexpression of TP53 restored the expression of miR-199-3p/5p and miR-214. In addition, silencing of PSMD10 up-regulated the expression of TP53, while miR-214 over-expression resulted in PSMD10 down-regulation and TP53 up-regulation. Collectively, our findings highlighted a miR-199a/miR-214/PSMD10/TP53/DNMT1 self-regulatory network, which might be a potential therapeutic target in the treatment of TGCT.

Targeted photoporation and transfection in human HepG2 cells by a fiber femtosecond laser at 1554 nm.

We report the transfection of human hepatocarcinoma (HepG2) cells by femtosecond (fs) laser pulses at 1554 nm. It was found that HepG2 cells could be perforated transiently to admit propidium iodide by that laser. This photoporation was safe, as the membrane resealed itself within a short time and no mitochondrial depolarization was detected. The cells were next photoporated in the presence of DNA plasmids for the expression of green fluorescence protein, and about 80% of the exposed cells showed green fluorescence 24 h later. Thus it could be concluded that it is safe and efficient to use fs laser at 1554 nm to transfect foreign molecules into cells under a standard microscope.

The 3a Protein of SARS-coronavirus Induces Apoptosis in Vero E6 Cells.

An outbreak of severe acute respiratory syndrome (SARS) occurred in China and the first case emerged in mid November 2002. The etiologic agent of this disease was found to be a previously unknown coronavirus, SARS-CoV. The detailed pathology of SARS-CoV infection and the host response to the viral infection are still not known. The 3a gene encodes a non-structural viral protein which is predicted to be a transmembrane protein. In this study, we showed that the 3a protein was localized to the endoplasmic reticulum (ER) in 3a-transfected monkey kidney Vero E6 cells. In vitro experiments of chromatin condensation and DNA fragmentation suggest that the 3a protein may trigger apoptosis. Our data show that over-expression of a single SARS-CoV protein can induce apoptosis in vitro. Thus GFP-3a fusion protein could also be used as a biosensor for monitoring the cytopathic features of SARS infection, e.g. lymphopenia, in animal model systems, similar to nucleocapsid and 7a proteins.

The 3a protein of severe acute respiratory syndrome-associated coronavirus induces apoptosis in Vero E6 cells.

An outbreak of severe acute respiratory syndrome (SARS) occurred in China and the first case emerged in mid-November 2002. The aetiological agent of this disease was found to be a previously unknown coronavirus, SARS-associated coronavirus (SARS-CoV). The detailed pathology of SARS-CoV infection and the host response to the viral infection are still not known. The 3a gene encodes a non-structural viral protein, which is predicted to be a transmembrane protein. In this study, it was shown that the 3a protein was expressed in the lungs and intestinal tissues of SARS patients and that the protein localized to the endoplasmic reticulum in 3a-transfected monkey kidney Vero E6 cells. In vitro experiments of chromatin condensation and DNA fragmentation suggested that the 3a protein may trigger apoptosis. These data showed that overexpression of a single SARS-CoV protein can induce apoptosis in vitro.