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.

Extracellular Histones Induced Eryptotic Death in Human Erythrocytes.

BACKGROUND/AIMS: Circulating or extracellular histones (EHs) in the bloodstream act as a damage-associated-molecular-pattern (DAMP) agent that plays a critical role in the pathogenesis of many diseases such as sepsis and sterile inflammation. To date, not much information is available to describe the mechanistic relationship between human erythrocytes and the cytotoxicity of EHs, the protein members from a highly conserved histone family across species. The present study explored this key question with a hypothesis that EHs induce eryptosis. METHODS: Freshly isolated human red blood cells (RBCs) from healthy donors were treated with EHs or agents for positive controls in a physiological buffer for 3 or 24 h. After treatments, flow cytometry was employed to quantify surface phosphatidylserine (PS) exposure from annexin-V-RFP binding, cell shrinkage from flow cytometric forward scatter (FSC) analysis, Ca2+ rise by fluo-4, reactive oxygen species (ROS) production by H2DCFDA, and caspase-3 activation by FAM-DEVD-FMK measurement. Hemolysis and membarne permeabilization were estimated respectively from hemoglobin release into supernatant and calcein leakage from RBC ghosts. RESULTS: With positive controls for validation, EHs in the pathophsyiological range were found to accumulate annexin-V binding on cell surface, decrease FSC, upregulate ROS production, elevate Ca2+ influx and increase caspase-3 activity in a 3-h incubation. Of note, no RBC hemolysis and no calcein release from ghosts were obtained after EHs treatment for 24 h. Interestingly, external Ca2+ was not a prerequisite for the EHs-mediated ROS production and PS externalization. Also, the eryptotic hallmarks in the apoptotic RBCs were partially blocked by heparin and antibody (Ab) against Toll-like receptor 2 (TLR2). CONCLUSION: EHs act as a DAMP agent in the human RBCs that induces eryptosis. The cytotoxic effect is rapid as the hallmarks of eryptosis such as cell shrinkage, surface PS exposure, [Ca2+]i rise, ROS production and caspase-3 activation can be seen 3 h after treatment in a dose-dependent manner. The EHs' cytotoxic effects could be blocked by heparin and the Ab against TLR2.

A rapid sample-to-answer analytical detection of genetically modified papaya using loop-mediated isothermal amplification assay on lab-on-a-disc for field use.

With genetically modified (GM) food circulating on the market, a rapid transgenic food screening method is needed to protect consumer rights. The on-site screening efficiency of GM food testing is low. We report rapid sample-to-answer detection of GM papayas with loop-mediated isothermal amplification (LAMP) and a compact, portable, integrated microfluidic platform using microfluidic lab-on-a-disc (LOAD). GM samples were differentiated from non-GM papaya, based on the detection of a specific GM (P-35S (Cauliflower mosaic virus 35S promoter)) and non-GM DNA marker (papain) in 15 min. The detection limits for DNA and juice from papaya were 10 pg/µL and 0.02 µL, respectively. Our LOAD platform is a simple and robust solution for GM screening, which is anticipated to be a foundation for on-site testing of transgenic food.

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.

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.

Can Team Triage Improve Patient Flow in the Emergency Department? A Systematic Review and Meta-Analysis.

This systematic review was performed as a feasibility study for revamping the triage service of an emergency department (ED) in a district hospital. In view of the overcrowding problem that plagues EDs worldwide, we reviewed evidence from randomized controlled trials (RCTs) to determine whether ED team triage improves patient flow in comparison with single-nurse triage. We measured improvement in patient flow in terms of the reduction in length of stay (LOS) or wait time (WT) for all ED patients. Adopting the Cochrane methodology, we searched and evaluated data sources for RCTs comparing patients assessed by an ED triage team, with patients receiving single-nurse triage at the same site. The data extracted were independently reviewed by 2 authors for inclusion and quality assessment. As for risk of bias across studies, there was an overall assessment of every outcome across the included studies according to the GRADE (Grading of Recommendations Assessment, Development and Evaluation) criteria for RCTs. In total, 2,164 studies were identified and 2,106 were excluded on the basis of title/abstract, leaving 58 articles for full assessment. Four trials (all cluster RCTs) involving 14,772 patients (165 clusters) met the inclusion criteria. On the basis of our analysis, there was no statistically significant or clinically relevant reduction of LOS and WT for all patients in these studies. One study reported death as an outcome: Relative risk was 0.34 (95% CI [0.01, 8.24]), which suggested that team triage might reduce mortality. Overall, although we have found no conclusive evidence from RCTs to support the use of team triage for improving patient flow in the ED, the results need not deter nursing managers intending to introduce team triage for improving the morale of the triage nurse. However, they may need to consider economic and organizational factors, such as resource reallocation and staff receptiveness, in implementing the new practice.

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.

Allergy Testing and Drug Screening on an ITO-Coated Lab-on-a-Disc.

A lab-on-a-disc (LOAD) is a centrifugal microfluidic set-up based on centrifugal force without using micro-pumps to drive reagents and cells to various chambers through channels and valves for reactions. A LOAD coated with conductive transparent indium tin oxide (ITO) for thermal control was developed to screen allergy-blocking agents. When the acridine orange (AO)-loaded KU-812 human basophilic cells were activated in the LOAD by stimuli, AO trapped in the cytoplasmic granules was released externally as an allergic mediator mimetic to report degranulation. This response was monitored by fluorescence when the released AO in supernatant had been transferred, with a higher spinning speed, from the reaction chamber to detection chamber in the LOAD where AO reacted with exogenous DNA. We report here the principles of the system and an improved LOAD set-up with the ITO-coated glass resistive microheater to run assays at 37 °C. By using this platform, we demonstrate here for the first time that triptolide, an active ingredient from the Chinese medicine herb Tripterygium wilfordii Hook f., was able to suppress the fMLP-mediated degranulation in basophils. This serves as an example how LOADs can be used to screen agents to alleviate symptoms of allergy.

Allergen screening bioassays: recent developments in lab-on-a-chip and lab-on-a-disc systems.

Allergies occur when a person's immune system mounts an abnormal response with or without IgE to a normally harmless substance called an allergen. The standard skin-prick test introduces suspected allergens into the skin with lancets in order to trigger allergic reactions. This test is annoying and sometimes life threatening. New tools such as lab-on-a-chip and lab-on-a-disc, which rely on microfabrication, are designed for allergy testing. These systems provide benefits such as short analysis times, enhanced sensitivity, simplified procedures, minimal consumption of sample and reagents and low cost. This article gives a summary of these systems. In particular, a cell-based assay detecting both the IgE- and non-IgE-type triggers through the study of degranulation in a centrifugal microfluidic system is highlighted.

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.

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.