A high spatial resolution osteogenic differentiation in human mesenchymal stem cells induced by femtosecond laser.

A variety of physical and chemical methods have been developed in research laboratories for the induction of stem cell differentiation. However, the use of exogenous chemicals and materials may limit their widespread utility in clinics. To develop a clean and precise induction approach with minimal invasion, we reported here that 1-second stimulation by a tightly focused femtosecond laser (fsL) (140 mW/µm2 , 200 fs) can modulate the signaling systems in human mesenchymal cells, such as intracellular calcium and reactive oxygen species. Upon stimulation on an automatic platform, hMSCs were found to express osteoblastic markers and form calcium-rich deposits. Moreover, tissue mineralization was observed when the fsL-illuminated hMSCs were ectopically transplanted into nude mice. Collectively, we described a novel and non-contact optical stimulation method for cell differentiation with high spatiotemporal resolution.

Mitochondrial Fuel Dependence on Glutamine Drives Chemo-Resistance in the Cancer Stem Cells of Hepatocellular Carcinoma.

Chemo-resistance hinders treatment of patients with hepatocellular carcinoma. Although there are many models that can be found in the literature, the root mechanism to explain chemo-resistance is still not fully understood. To gain a better understanding of this phenomenon, a chemo-resistant line, R-HepG2, was developed from a chemo-sensitive HepG2 line through an exposure of doxorubicin (DOX). The R-HepG2 exhibited a cancer stem cell (CSC) phenotype with an over-expression of P-glycoprotein (P-gp), conferring it a significant enhancement in drug efflux and survival. With these observations, we hypothesize that metabolic alteration in this drug-resistant CSC is the root cause of chemo-resistance. Our results show that, unlike other metabolic-reprogrammed CSCs that exhibit glycolytic phenotype described by the "Warburg effect", the R-HepG2 was metabolically quiescent with glucose independence, high metabolic plasticity, and relied on glutamine metabolism via the mitochondria for its chemo-resistance Intriguingly, drug efflux by P-gp in R-HepG2 depended on the mitochondrial ATP fueled by glutamine instead of glycolytic ATP. Armed with these observations, we blocked the glutamine metabolism in the R-HepG2 and a significant reduction of DOX efflux was obtained. We exploited this metabolic vulnerability using a combination of DOX and metformin in a glutamine-free condition to target the R-HepG2, resulting in a significant DOX sensitization. In conclusion, our findings highlight the metabolic modulation of chemo-resistance in CSCs. We delineate the altered metabolism that drives chemo-resistance and offer a new approach to target this CSC through metabolic interventions.

Technological Advances in Multiscale Analysis of Single Cells in Biomedicine.

Single-cell analysis has recently received significant attention in biomedicine. With the advances in super-resolution microscopy, fluorescence labeling, and nanoscale biosensing, new information may be obtained for the design of cancer diagnosis and therapeutic interventions. The discovery of cellular heterogeneity further stresses the importance of single-cell analysis to improve our understanding of disease mechanism and to develop new strategies for disease treatment. To this end, many studies are exploited at the single-cell level for high throughput, highly parallel, and quantitative analysis. Technically, microfluidics are also designed to facilitate single-cell isolation and enrichment for downstream detection and manipulation in a robust, sensitive, and automated manner. Further achievements are made possible by consolidating optically label-free, electrical, and molecular sensing techniques. Moreover, these technologies are coupled with computing algorithms for high throughput and automated quantitative analysis with a short turnaround time. To reflect on how the technological developments have advanced single-cell analysis, this mini-review is aimed to offer readers an introduction to single-cell analysis with a brief historical development and the recent progresses that have enabled multiscale analysis of single-cells in the last decade. The challenges and future trends are also discussed with the view to inspire forthcoming technical developments.

Development of peptide-based chemiluminescence enzyme immunoassay (CLEIA) for diagnosis of dengue virus infection in human.

Dengue is the most prevalent mosquito-borne viral disease in tropical and subtropical regions worldwide. Since its clinical symptoms are non-specific and easily mistaken as other kinds of infection, laboratory diagnosis is required to confirm dengue infections. In this study, ten peptides (E1-E10) from the envelope protein of dengue virus (DENV) were first identified using bioinformatic tool. The screened peptides were then synthesized for the peptide-based chemiluminescence enzyme immunoassay (CLEIA). Two peptides, E1 and E7, were found as the best candidate antigen and therefore used as downstream application in the development of low-cost peptide-based anti-DENV immunoglobulin M antibodies (IgM) indirect CLEIA. 176 serum samples were used to study the presence of anti-DENV IgM antibodies to evaluate the diagnostic ability of IgM-CLEIA. Receiver operating characteristic curve (ROC) was used to estimate the diagnostic cut-off value. The sensitivity and the specificity reached 82.5% and 94.6% respectively when peptide E1 was used, but declined to 79.2% and 92.9% respectively when peptide E7 was used. Therefore, the combination of E1 and E7 was used to improve the sensitivity and the specificity to 85.0% and 96.4% respectively in 1.5 h assay time, providing a potentially practical use for the diagnosis of DENV infections in patients' serum.

The second generation tyrosine kinase inhibitor dasatinib induced eryptosis in human erythrocytes-An in vitro study.

Dasatinib, a new tyrosine kinase inhibitor, is used clinically to kill chronic myelogenous leukemia and acute lymphoblastic leukemia through apoptosis. Obviously, anemia is developed in many patients receiving dasatinib for treatment. Until now, the mechanism for the cytotoxic effects of dasatinib in human erythrocytes is not fully understood. As many tyrosine kinases are found in human erythrocytes, it is therefore logical to hypothesize that dasatinib is able to induce apoptosis (or eryptosis) in human erythrocytes. True to our expectation, dasatinib inhibited tyrosine kinase and induced eryptosis in human erythrocytes with early denature of esterase, cell shrinkage, loss of membrane integrity with inside-out phosphatidylserine, increase in the cytosolic Ca2+ ion concentration ([Ca2+]i), caspase-3 activation and change in cellular redox state. Mechanistically, the rise of [Ca2+]i seems to be a key mediator in the dasatinib-mediated eryptosis because depletion of external Ca2+ could suppress the eryptotic effects. Also, dasatinib was able to reduce membrane fluidity in human RBCs. For the direct action on membrane, dasatinib permeabilized RBC ghosts in a way similar to digitonin. Taken together, we report here for the first time that dasatinib inhibited tyrosine kinase and induced eryptosis in human erythrocytes through Ca2+ loading and membrane permeabilization.

An Aptamer Bio-barCode (ABC) assay using SPR, RNase H, and probes with RNA and gold-nanorods for anti-cancer drug screening.

With modifications to an ultra-sensitive bio-barcode (BBC) assay, we have developed a next generation aptamer-based bio-barcode (ABC) assay to detect cytochrome-c (Cyto-c), a cell death marker released from cancer cells, for anti-cancer drug screening. An aptamer is a short single-stranded DNA selected from a synthetic DNA library that is capable of binding to its target with high affinity and specificity based on its unique DNA sequence and 3D structure after folding. Similar to the BBC assay, Cyto-c is captured by a micro-magnetic particle (MMP) coated with capturing antibodies (Ab) and an aptamer specifically against Cyto-c to form sandwich structures ([MMP-Ab]-[Cyto-c]-[Aptamer]). After washing and melting, our aptamers, acting as a DNA bio-barcode, are released from the sandwiches and hybridized with the probes specially designed for RNase H for surface plasmon resonance (SPR) sensing. In an aptamer-probe duplex, RNase H digests the RNA in the probe and releases the intact aptamer for another round of hybridization and digestion. With signal enhancement effects from gold-nanorods (Au-NRs) on probes for SPR sensing, the detection limit was found to be 1 nM for the aptamer and 80 pM for Cyto-c. Without the time-consuming DNA amplification steps by PCR, the detection process of this new ABC assay can be completed within three hours. As a proof-of-concept, phenylarsine oxide was found to be a potent agent to kill liver cancer cells with multi-drug resistance at the nano-molar level. This approach thus provides a fast, sensitive and robust tool for anti-cancer drug screening.

Osthole Enhances Osteogenesis in Osteoblasts by Elevating Transcription Factor Osterix via cAMP/CREB Signaling In Vitro and In Vivo.

Anabolic anti-osteoporotic agents are desirable for treatment and prevention of osteoporosis and fragility fractures. Osthole is a coumarin derivative extracted from the medicinal herbs Cnidium monnieri (L.) Cusson and Angelica pubescens Maxim.f. Osthole has been reported with osteogenic and anti-osteoporotic properties, whereas the underlying mechanism of its benefit still remains unclear. The objective of the present study was to investigate the osteopromotive action of osthole on mouse osteoblastic MC3T3-E1 cells and on mouse femoral fracture repair, and to explore the interaction between osthole-induced osteopromotive effect and cyclic adenosine monophosphate (cAMP) elevating effect. Osthole treatment promoted osteogenesis in osteoblasts by enhancing alkaline phosphatase (ALP) activity and mineralization. Oral gavage of osthole enhanced fracture repair and increased bone strength. Mechanistic study showed osthole triggered the cAMP/CREB pathway through the elevation of the intracellular cAMP level and activation of the phosphorylation of the cAMP response element-binding protein (CREB). Blockage of cAMP/CREB downstream signals with protein kinase A (PKA) inhibitor KT5720 partially suppressed osthole-mediated osteogenesis by inhibiting the elevation of transcription factor, osterix. In conclusion, osthole shows osteopromotive effect on osteoblasts in vitro and in vivo. Osthole-mediated osteogenesis is related to activation of the cAMP/CREB signaling pathway and downstream osterix expression.

An Assay Using Localized Surface Plasmon Resonance and Gold Nanorods Functionalized with Aptamers to Sense the Cytochrome-c Released from Apoptotic Cancer Cells for Anti-Cancer Drug Effect Determination.

To determine the degree of cancer cell killing after treatment with chemotherapeutic drugs, we have developed a sensitive platform using localized surface plasmon resonance (LSPR) and aptamers to detect the extracellular cytochrome-c (cyto-c), a mitochondrial protein released from cancer cells for the induction of apoptosis after treatment, to evaluate the effectiveness of cancer therapy. In this assay, a short single-stranded 76-mer DNA aptamer with a unique DNA sequence, which binds towards the cyto-c like an antibody with a high binding affinity and specificity, was conjugated to gold nanorods (AuNR) for LSPR sensing. Practically, cyto-c was first grabbed by a capturing antibody functionalized on the surface of micro-magnetic particles (MMPs). Subsequently, the AuNR-conjugated aptamer was added to form a complex sandwich structure with cyto-c (i.e., (MMP-Ab)-(cyto-c)-(AuNR-aptamer)) after washing away the non-target impurities, such as serum residues and intracellular contents, in a microfluidic chip. The sandwich complex led to formation of AuNR aggregates, which changed the LSPR signals in relation to the amount of cyto-c. With the LSPR signal enhancement effects from the AuNRs, the detection limit of cyto-c, sparked in human serum or culture medium, was found to be 0.1 ng/mL in our platform and the whole sensing process could be completed within two hours. Moreover, we have applied this assay to monitor the apoptosis in leukemia cancer cells induced by a potential anti-cancer agent phenylarsine oxide.

Identification of microRNAs in Throat Swab as the Biomarkers for Diagnosis of Influenza.

BACKGROUND: Influenza is a serious worldwide disease that captures global attention in the past few years after outbreaks. The recent discoveries of microRNA (miRNA) and its unique expression profile in influenza patients have offered a new method for early influenza diagnosis. The aim of this study was to examine the utility of miRNAs for the diagnosis of influenza. METHODS: Thirteen selected miRNAs were investigated with the hosts' throat swabs (25 H1N1, 20 H3N2, 20 influenza B and 21 healthy controls) by real-time quantitative polymerase chain reaction (RT-qPCR) using U6 snRNA as endogenous control for normalization, and receiver operating characteristic (ROC) curve/Area under curve (AUC) for analysis. RESULTS: miR-29a-3p, miR-30c-5p, miR-34c-3p and miR-181a-5p are useful biomarkers for influenza A detection; and miR-30c-5p, miR-34b-5p, miR-205-5p and miR-449b-5p for influenza B detection. Also, use of both miR-30c-5p and miR-34c-3p (AUC=0.879); and miR-30c-5p and miR-449b-5p (AUC=0.901) are better than using one miRNA to confirm influenza A and influenza B infection, respectively. CONCLUSIONS: Given its simplicity, non-invasiveness and specificity, we found that the throat swab-derived miRNAs miR-29a-3p, miR-30c-5p, miR-34b-5p, miR-34c-3p, miR-181a-5p, miR-205-5p and miR-449b-5p are a useful tool for influenza diagnosis on influenza A and B.

Cytotoxic and sublethal effects of silver nanoparticles on tendon-derived stem cells - implications for tendon engineering.

Tendon injuries occur commonly in sports and workplace. Tendon-derived stem cells (TDSCs) have great potential for tendon healing because they can differentiate into functional tenocytes. To grow TDSCs properly in vivo, a scaffold is needed. Silver nanoparticles (AgNPs) have been used in a range of biomedical applications for their anti-bacterial and -inflammatory effects. AgNPs are therefore expected to be a good scaffolding coating material for tendon engineering. Yet, their cytotoxicity in TDSCs remains unknown. Moreover, their sublethal effects were mysterious in TDSCs. In our study, decahedral AgNPs (43.5 nm in diameter) coated with polyvinylpyrrolidone (PVP) caused a decrease in TDSCs' viability beginning at 37.5 µg ml-1 but showed non-cytotoxic effects at concentrations below 18.8 µg ml-1. Apoptosis was observed in the TDSCs when higher doses of AgNPs (75-150 µg ml-1) were used. Mechanistically, AgNPs induced reactive oxygen species (ROS) formation and mitochondrial membrane potential (MMP) depolarization, resulting in apoptosis. Interestingly, treating TDSCs with N-acetyl-l-cysteine (NAC) antioxidant significantly antagonized the ROS formation, MMP depolarization and apoptosis indicating that ROS accumulation was a prominent mediator in the AgNP-induced cytotoxicity. On the other hand, AgNPs inhibited the tendon markers' mRNA expression (0-15 µg ml-1), proliferation and clonogenicity (0-15 µg ml-1) in TDSCs under non-cytotoxic concentrations. Taken together, we have reported here for the first time that the decahedral AgNPs are cytotoxic to rat TDSCs and their sublethal effects are also detrimental to stem cells' proliferation and tenogenic differentiation. Therefore, AgNPs are not a good scaffolding coating material for tendon engineering.

In vitro vitamin K(2) and 1α,25-dihydroxyvitamin D(3) combination enhances osteoblasts anabolism of diabetic mice.

In this study, we evaluated the anabolic effect and the underlying cellular mechanisms involved of vitamin K2 (10 nM) and 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) (10 nM), alone and in combination, on primary osteoblasts harvested from the iliac crests of C57BL/KsJ lean (+/+) and obese/diabetic (db/db) mice. A lower alkaline phosphatase (ALP) activity plus a reduced expression of bone anabolic markers and bone formation transcription factors (osteocalcin, Runx2, Dlx5, ATF4 and OSX) were consistently detected in osteoblasts of db/db mice compared to lean mice. A significantly higher calcium deposits formation in osteoblasts was observed in lean mice when compared to db/db mice. Co-administration of vitamin K2 (10 nM) and 1,25(OH)2D3 (10 nM) caused an enhancement of calcium deposits in osteoblasts in both strains of mice. Vitamins K2 and 1,25(OH)2D3 co-administration time-dependently (7, 14 and 21 days) increased the levels of bone anabolic markers and bone formation transcription factors, with a greater magnitude of increase observed in osteoblasts of db/db mice. Combined vitamins K2 plus 1,25(OH)2D3 treatment significantly enhanced migration and the re-appearance of surface microvilli and ruffles of osteoblasts of db/db mice. Thus, our results illustrate that vitamins K2 plus D3 combination could be a novel therapeutic strategy in treating diabetes-associated osteoporosis.

Immunogenicity and escape mechanisms of allogeneic tendon-derived stem cells.

The immunogenicity of tendon-derived stem cells (TDSCs) has implications for their clinical use for the promotion of tendon repair. The immunogenicity and escape mechanisms of rat patellar TDSCs were examined after allogeneic transplantation. Our results showed that TDSCs exhibited low immunogenicity as evidenced by the following: (i) the incubation of target TDSCs with immunized serum did not show antibody recognition and did not induce the complement-dependent cytotoxicity; (ii) target TDSCs elicited a very low level of lymphocyte proliferation and did not exhibit host lymphocyte-mediated cytotoxicity; and (iii) target TDSCs dose dependently suppressed the phorbol 12-myristate 13-acetate (PMA)- and ionomycin-induced host lymphocyte proliferation. For the mechanistic studies, TDSCs expressed major histocompatibility complex (MHC)-I but a very low level of MHC-II, CD86 and CD80 for the induction of T-cell response. Also, TDSCs were found to express intracellular Fas and FasL. γ-IFN pretreatment did not increase the level of MHC-II and CD86 for the upregulation of immune response. Moreover, the immunosuppressive mediators indoleamine 2,3-dioxygenase (IDO) and transforming growth factor-beta 1 (TGF-ß1) were found not to be involved in the escape mechanism of target TDSCs from host lymphocyte attack. In conclusion, allogeneic TDSCs exhibited low immunogenicity. Allogeneic TDSCs might be used for transplantation.

Allogeneic tendon-derived stem cells promote tendon healing and suppress immunoreactions in hosts: in vivo model.

The medium- to long-term healing effect and infiltration of inflammatory cells, after transplantation of allogeneic tendon-derived stem cell (TDSC) to the rat patellar tendon window wound, were examined. Allogeneic patellar TDSCs derived from a green fluorescent protein rat were used. The outcome of tendon healing and the infiltration of inflammatory cells were examined by histology and immunohistochemistry up to week 16 postinjury. The fate of the transplanted cells was examined by ex vivo fluorescent imaging and immunohistochemistry. Our results showed that the transplantation of allogeneic TDSCs promoted tendon healing with no increased risk of ectopic chondro-ossification up to week 16. A low infiltration of T cells, ED1 macrophages, ED2 macrophages, and mast cells in the window wound was obtained. The transplanted TDSCs were found in the window wound at week 1 and 2, but were absent after week 4 postinjury. In conclusion, allogeneic TDSCs promoted tendon repair in the medium to long term and exhibited weak immunoreactions and anti-inflammatory effects in the hosts after transplantation in a rat model. There was no increased risk of ectopic chondro-ossification after TDSC transplantation. The decrease in the number of transplanted cells with time suggested that allogeneic TDSCs did not promote tendon repair through direct differentiation.

Ferutinin induces in vitro eryptosis/erythroptosis in human erythrocytes through membrane permeabilization and calcium influx.

Ferutinin, isolated from the root of Ferula hermonis and proposed to be used as an antiosteoporosis phytoestrogen, has death promoting activities in a number of cancer cells. However, the effect of ferutinin on the induction of apoptosis in human red blood cells (RBCs), also known as eryptosis or erythroptosis, remains unclear. Given that ferutinin is a small molecule that can induce apoptosis in the cancer cells by opening the mitochondrial permeability transition pores, we therefore hypothesized that the effect of ferutinin to elicit apoptosis in human RBCs devoid of mitochondria would be minimal. This study tried to determine the in vitro effect of ferutinin on the induction of apoptosis in human RBCs. Eryptosis/erythroptosis after ferutinin treatment was examined for phosphatidylserine (PS) externalization, calcein leakage, and other apoptotic feature events by flow cytometry and confocal microscopy. Contrary to our prediction, ferutinin caused eryptosis/erythroptosis in human RBCs and simultaneously increased caspase-3 activity and the cytosolic free Ca(2+) ion level ([Ca(2+)]i). Yet, Ca(2+) seems not to be the sole mediator in ferutinin-mediated eryptosis/erythroptosis because depletion of the external Ca(2+) could not eliminate the apoptotic effect from ferutinin. Subsequent replenishment of the external Ca(2+) was able to promote PS externalization, caspase-3 activation, and rise of [Ca(2+)]i. Also, ferutinin at high dose (40 µM or above) was able to permeabilize the membrane of RBC ghosts in a way similar to that of digitonin. At low dose, ferutinin activated the P- and L-type Ca(2+) channels as the ferutinin-mediated [Ca(2+)]i rise was suppressed by the P-type (ω-agatoxin IVA) and L-type (verapamil and diltiazem) Ca(2+) channel blockers. Taken together, we report here for the first time that ferutinin induces in vitro apoptosis in human RBCs. Mechanistically, eryptosis/erythroptosis is mediated by membrane permeabilization and upregulation of [Ca(2+)]i with the activation of caspase-3.

Folate-conjugated Fe3O4@SiO2@gold nanorods@mesoporous SiO2 hybrid nanomaterial: a theranostic agent for magnetic resonance imaging and photothermal therapy.

In this paper, we investigated the functional imaging and targeted therapeutic properties of core@multi-shell nanoparticles composed of a superparamagnetic iron oxide (SPIO) core and gold nanorods (GNRs) in the mesoporous silica shells functionalized with folic acid (Fe3O4@SiO2@GNRs@mSiO2-FA). The as-synthesized five-component hybrid nanocomposite was revealed to have insignificant cytotoxicity. Intracellular uptake of the nanoparticles was studied in the folate receptor over-expressing human epidermoid carcinoma of the nasopharynx (KB) cells. Due to their magnetic/optical properties as well as the folate targeting potential, compared with Fe3O4@SiO2@GNRs@mSiO2 nanoparticles, higher cellular uptake efficiency was observed for Fe3O4@SiO2@GNRs@mSiO2-FA nanoparticles in KB cells. Characterizations were achieved using both dark field and magnetic resonance (MR) imaging techniques. The hyperthermia induced by Fe3O4@SiO2@GNRs@mSiO2-FA nanoparticles resulted in a higher cytotoxicity in KB cells. Thus, the Fe3O4@SiO2@GNRs@mSiO2-FA hybrid nanomaterial is an effective and promising MR imaging and photothermal therapy agent for folate-receptor over-expressing cancer cells.

Tendon-derived stem cells (TDSCs) promote tendon repair in a rat patellar tendon window defect model.

Injured tendons heal slowly and often result in the formation of mechanically and functionally inferior fibrotic scar tissue or fibrous adhesions. This study investigated the use of tendon-derived stem cells (TDSCs) for tendon repair in a rat patellar tendon window defect model. Fibrin glue constructs with or without GFP-TDSCs were transplanted into the window defect. The patellar tendons were harvested for histology, ex vivo fluorescent imaging and biomechanical test at various time points up to week 4. Our results showed that TDSCs significantly enhanced tendon healing as indicated by the increase in collagen production as shown by hematolxylin stain-ability of the tissue, improvement of cell alignment, collagen fiber alignment and collagen birefringence typical of tendon. The labeled cells were observed at weeks 1 and 2 and became almost undetectable at week 4. Both the ultimate stress and Young's modulus were significantly higher in the TDSCs group compared to those in the fibrin glue group at week 4. In conclusion, TDSCs promoted earlier and better repair in a rat patellar tendon window defect model.

Polyphyllin D, a steroidal saponin from Paris polyphylla, inhibits endothelial cell functions in vitro and angiogenesis in zebrafish embryos in vivo.

ETHNOPHARMACOLOGICAL RELEVANCE: Angiogenesis, the process of blood vessel formation, is critical to tumour growth. The importance of angiogenesis in tumour development has lead to the development of anti-angiogenic strategies to inhibit tumour growth. In this study, polyphyllin D (PD), an active component in Chinese herb, Paris polyphylla, was evaluated for its potential anti-angiogenic effects. MATERIALS AND METHODS: The inhibitory effects of PD on three important processes involved in angiogenesis, i.e. proliferation, migration and differentiation were examined using human microvascular endothelial cell line HMEC-1 by MTT assay, scratch assay and tube formation assay, respectively. Using zebrafish embryos as an animal model of angiogenesis, the anti-angiogenic effect of PD was further verified in vivo. RESULTS: PD suppressed the growth of HMEC-1 cells at 0.1-0.4 µM without toxic effects. At 0.3 µM and 0.4 µM, PD significantly inhibited endothelial cell migration and capillary tube formation. About 70% of the zebrafish embryos showed defects in intersegmental vessel formation upon treatment with PD at concentrations of 0.156 µM and 0.313 µM. CONCLUSION: The anti-angiogenic effects of PD have been explored in the study which implied a potential therapeutic development of PD in cancer treatment.

P-glycoprotein enhances radiation-induced apoptotic cell death through the regulation of miR-16 and Bcl-2 expressions in hepatocellular carcinoma cells.

P-glycoprotein (Pgp), an efflux pump, was confirmed the first time to regulate the expressions of miR/gene in cells. Pgp is known to be associated with multidrug resistance. RHepG2 cells, the multidrug resistant subline of human hepatocellular carcinoma HepG2 cells, expressed higher levels of Pgp as well as miR-16, and lower level of Bcl-2 than the parental cells. In addition, RHepG2 cells were more radiation sensitive and showed more pronounced radiation-induced apoptotic cell death than the parental cells. Mechanistic analysis revealed that transfection with mdr1 specific antisense oligos suppressed radiation-induced apoptosis in HepG2 cells. On the other hand, ectopic mdr1 expression enhanced radiation-induced apoptosis in HepG2 cells, SK-HEP-1 cells, MiHa cells, and furthermore, induced miR-16 and suppressed its target gene Bcl-2 in HepG2 cells. Moreover, the enhancement effects of Pgp and miR-16 on radiation-induced apoptosis were counteracted by overexpression of Bcl-2. The Pgp effect on miR-16/Bcl-2 was suppressed by Pgp blocker verapamil indicating the importance of the efflux of Pgp substrates. The present study is the first to reveal the role of Pgp in regulation of miRNA/gene expressions. The findings may provide new perspective in understanding the biological function of Pgp.

Immunoassays using polypeptide conjugate binders with tuned affinity.

Evaluation of: Albrecht C, Fechner P, Honcharenko D et al. A new assay design for clinical diagnostics based on alternative recognition elements. Biosens. Bioelectron. 25(10), 2302-2308 (2010). This article focuses on a novel concept regarding the use of polypeptide conjugates as high-affinity binders to recognize targets in immunoassays. The polypeptide conjugate binder is a hybrid molecule in which a small recognition ligand is covalently linked to a synthetic polypeptide, which binds to a protein target of interest with high affinity and selectivity. Affinity of the polypeptide conjugate to its target can be tuned by changing the length of the aliphatic spacer in between the small recognition ligand and the polypeptide. Other pros and cons of this new approach for protein recognition and sensing are compared with other immunoassays such as ELISA, the bio-barcode assay and the aptamer-based barcode assay. It is anticipated that there will be more innovation and integration using new materials and technologies to provide novel ways to detect disease markers.

Mitochondrial monoamine oxidase-A-mediated hydrogen peroxide generation enhances 5-hydroxytryptamine-induced contraction of rat basilar artery.

BACKGROUND AND PURPOSE: We evaluated the role(s) of monoamine oxidase (MAO)-mediated H2O2 generation on 5-hydroxytryptamine (5-HT)-induced tension development of isolated basilar artery of spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. EXPERIMENTAL APPROACH: Basilar artery (endothelium-denuded) was isolated for tension measurement and Western blots. Enzymically dissociated single myocytes from basilar arteries were used for patch-clamp electrophysiological and confocal microscopic studies. KEY RESULTS: Under resting tension, 5-HT elicited a concentration-dependent tension development with a greater sensitivity (with unchanged maximum tension development) in SHR compared with WKY (EC(50) : 28.4 ± 4.1 nM vs. 98.2 ± 9.4 nM). The exaggerated component of 5-HT-induced tension development in SHR was eradicated by polyethylene glycol-catalase, clorgyline and citalopram whereas exogenously applied H2O2 enhanced the 5-HT-elicited tension development in WKY. A greater protein expression of MAO-A was detected in basilar arteries from SHR than in those from WKY. In single myocytes and the entire basilar artery, 5-HT generated (clorgyline-sensitive) a greater amount of H2O2 in SHR compared with WKY. Whole-cell iberiotoxin-sensitive Ca(2+) -activated K(+) (BK(Ca) ) amplitude measured in myocytes of SHR was approximately threefold greater than that in WKY (at +60 mV: 7.61 ± 0.89 pA·pF(-1) vs. 2.61 ± 0.66 pA·pF(-1) ). In SHR myocytes, 5-HT caused a greater inhibition (clorgyline-, polyethylene glycol-catalase- and reduced glutathione-sensitive) of BK(Ca) amplitude than in those from WKY. CONCLUSIONS AND IMPLICATIONS: 5-HT caused an increased generation of mitochondrial H2O2 via MAO-A-mediated 5-HT metabolism, which caused a greater inhibition of BK(Ca) gating in basilar artery myocytes, leading to exaggerated basilar artery tension development in SHR.