Next-generation DNA sequencing of Panax samples revealed new genotypes: Burrows-Wheeler Aligner, Python-based abundance and clustering analysis.

Background: There are two major species of the Panax genus, namely Panax ginseng and Panax quinquefolius. Other than the nucleic acid test and nucleic acid amplification test, DNA sequencing can be used to authenticate the species of ginseng samples, especially when their physical forms cannot be used for differentiation. Method: In this work, next generation sequencing was used to obtain millions of reads from fourteen ginseng samples (root, powder, and granule). Then Gaussian Mixture clustering analysis was applied to analyze the reads from each sample. Results and Discussion: A new genotype has been revealed in this study. Two samples have been authenticated with certainty, while the others may be hybrid in nature as revealed by the clustering results.

Development of organic three-phase laminar flow microfluidic chip for extraction of ginsenosides from Panax ginseng.

BACKGROUND: Herbal extracts contain multiple active constituents, so the sample preparation based on the liquid-liquid extraction (LLE) is demanding, especially when a study subsequent to extraction is needed. Since the laminar flow occurring in microchannels can be formed between two miscible organic phases, a new method of extracting polar compounds from the crude extract of Panax ginseng Meyer in aqueous ethanol by pure n-butanol in the three-phase laminar flow microfluidic chip was established. METHODS: A new chip consisting of long microchannels with a guide structure was employed to improve the extraction efficiency caused by the low diffusion ability of saponins. The method was evaluated by using the extraction yields and purities of ginsenosides Rg1, Re and Rb1 as the indicators, and extraction conditions such as flow rate, temperature and other governing factors were optimized. RESULTS: Using the new chip method, the extraction efficiencies of ginsenoside Rg1, Re and Rb1 were 63.1%, 69.5% and 71.6%, respectively, which are higher than the 26% achieved in a previous report. The extraction yields of 1.53, 0.51, 0.90 mg/g were also higher than those obtained previously by the successive laminar flow microchip method. CONCLUSION: The proposed new microfluidic chip method has simplified the sample pretreatment steps to improve the yield of ginsenoside extraction from ginseng samples.

Cytosolic Calcium Measurement Utilizing a Single-Cell Biochip to Study the Effect of Curcumin and Resveratrol on a Single Glioma Cell.

A microfluidic method has been developed for real-time measurement of the effects of curcumin on the intracellular calcium concentration in a single glioma cell (U87-MG). This method is based on quantitative fluorescence measurement of intracellular calcium in a cell selected in a single-cell biochip. This biochip consists of three reservoirs, three channels, and a V-shaped cell retention structure. Because of the adherent nature of glioma cells, a single cell can adhere within the aforementioned V-shaped structure. The single-cell calcium measurement will minimize cell damage caused by conventional cell calcium assay methods. Previous studies have shown that curcumin increased cytosolic calcium in glioma cells using the fluorescent dye: Fluo-4. So in this study, the effects of 5 µM and 10 µM solutions of curcumin on the increases of cytosolic calcium in a single glioma cell have been measured. Moreover, the effects of 100 µM and 200 µM of resveratrol are measured. At the final stage of the experiments, ionomycin was used to increase the intracellular calcium to the highest possible level due to dye saturation. It has been demonstrated that microfluidic cell calcium measurement is a real-time cytosolic assay that requires small quantities of reagent, which will have potential uses for drug discovery.

Nucleic acid amplification test (NAAT) conducted in a microfluidic chip to differentiate between various ginseng species.

Panax ginseng and Panax quinquefolius have different medicinal properties and market values; however, they can be difficult to distinguish from one another based on physical appearances alone. Therefore, a molecular test that can be performed in commercial settings is needed to overcome this difficulty. A locus that contains a single nucleotide polymorphism (SNP) site to differentiate between P. ginseng and P. quinquefolius has been selected. An isothermal nucleic acid amplification test (NAAT) has been developed for use in a microfluidic chip; this NAAT method, which is based on lesion-induced DNA amplification (LIDA), amplifies the extracted plant genomic samples and enhances the detection of specific SNPs. This NAAT method was used to authenticate five ginseng root samples which indicated that two of the five samples appear to be mislabeled. These authentication results were consistent with those obtained from next generation sequencing (NGS) although this molecular test is more affordable and faster than NGS.

Centrifugal dynamic hybridization conducted in a microfluidic chip for signal enhancement in nucleic acid tests.

A rotating platform has been developed using a centrifugal chip holder to mount the standard chips for liquid delivery achieved by centrifugal pumping. This platform allows for dynamic hybridization to be performed in the microchannels constructed in the standard chips made using the 50 mm × 75 mm glass slides which allows for fast hybridization reactions. The results show that when the oligonucleotide-oligonucleotide hybridization is performed, there is good differentiation between perfectly complementary strands over 1 bp-mismatching counterparts when the long target strand is firstly immobilized and the short probe is secondly hybridized (Method 2), but not when the short probe is first immobilized, and the long target is subsequently hybridized (Method 1). When the differentiation between immobilized ginseng PCR product strands is performed, the correct result is achievable by Method 1, after signal enhancement and addition of formamide. The use of Method 2 is successful only when the PCR strand is captured, but not immobilized. In both methods, proper differentiation is achievable using the N1Q probe, un-achievable without centrifugal hybridization.

The genetic authentication of Panax ginseng and Panax quinquefolius based on using single nucleotide polymorphism (SNP) conducted in a nucleic acid test chip.

Panax ginseng and Panax quinquefolius, which are commonly called Chinese ginseng and American ginseng respectively, have different medicinal properties and market values; however, these samples can be difficult to differentiate from one another based on physical appearances of the samples especially when they are in powdery or granular forms. A molecular technique is thus needed to overcome this difficulty; this technique is based on the nucleic acid test (NAT) conducted on the microfluidic chip surface. Three single nucleotide polymorphism (SNP) sites (i.e. N1, N2, N3) on the Panax genome that differ between P. ginseng (G) and P. quinquefolius (Q) have been selected to design probes for the NAT. Primers were designed to amplify the antisense strands by asymmetric PCR. We have developed three different NAT methodologies involving surface immobilization and subsequent (stop flow or dynamic) hybridization of probes (i.e. N1G, N1Q, N2G, N2Q, N3Q) to the antisense strands. These NAT methods consist of two steps, namely immobilization and hybridization, and each method is distinguished by what is immobilized on the microfluidic chip surface in the first step (i.e. probe, target or capture strand). These three NATs developed are called probe-target method 1, target-probe method 2 and three-strand complex method 3. Out of the three methods, it was found that the capture strand-target-probe method 3 provided the best differentiation of the ginseng species, in which a 3' NH2 capture strand is first immobilized and the antisense PCR strand is then bound, while N2G and N3Q probes are used for detection of P. ginseng (G) and P. quinquefolius (Q) respectively.

Speleological and environmental history of Lida Ajer cave, western Sumatra.

Some of the earliest evidence for the presence of modern humans in rainforests has come from the fossil deposits of Lida Ajer in Sumatra. Two human teeth from this cave were estimated to be 73-63 thousand years old, which is significantly older than some estimates of modern human migration out of Africa based on genetic data. The deposits were interpreted as being associated with a rainforest environment based largely on the presence of abundant orangutan fossils. As well as the main fossil-bearing chamber, fossil-bearing passages are present below a sinkhole, although the relationship between the different fossil deposits has only been tenuously established. Here, we provide significant new sedimentological, geochronological and palaeoecological data aimed at reconstructing the speleological and environmental history of the cave and the clastic and fossil deposits therein. Our data suggest that the Lida Ajer fossils were deposited during Marine Isotope Stage 4, with fossils from the lower passages older than the main fossil chamber. Our use of stable carbon and oxygen isotope analyses of mammalian tooth enamel demonstrates that early humans probably occupied a closed-canopy forest very similar to those present in the region today, although the fossil orangutans may have occupied a slightly different niche. This article is part of the theme issue 'Tropical forests in the deep human past'.

Screening of high-efficiency and low-toxicity antitumor active components in Macleaya cordata seeds based on the competitive effect of drugs on double targets by a new laminar flow chip.

It is urgent to obtain targeted drugs that selectively bind to pathological targets rather than physiological targets in the early stage of drug screening. G-Quadruplex has become one of the important targets in the development of anti-tumor drugs. However, drugs that target quadruplexes may also bind to dsDNA, which may lead to adverse reactions. In this study, a new three-phase laminar flow chip was constructed to enable the multi-components of a traditional Chinese medicine extract to dynamically and competitively bind with G-quadruplex DNA (on target) and double-stranded DNA (off target), so as to select high-efficiency and low-toxicity anti-tumor drugs. The results showed that there were five compounds in the extracts of Macleaya cordata seeds that exhibited obvious differences in binding to the two targets. Furthermore, the binding constants and modes of four identified alkaloids as they bound to two DNA targets were verified by fluorescence spectra and molecular docking methods. The toxicity to HepG2 and LO2 cells from the four alkaloids was also compared. The results showed that sanguinarine and chelerythrine could be used as candidate drugs with stronger binding to HT24 than DNA26. The chip can also be used for other types of double-target screening of other traditional Chinese medicine extracts or compound libraries.

Safety, tolerability, pharmacokinetics, and pharmacodynamics of HBM9161, a novel FcRn inhibitor, in a phase I study for healthy Chinese volunteers.

Blockade of the binding between neonatal Fc receptor and IgG-Fc reduces circulating IgG, and thus emerges as a potential therapy for IgG-mediated autoimmune conditions. This was a double blind, randomized, single ascending dose study to evaluate the safety, pharmacokinetics, and pharmacodynamics of HBM9161 (a fully humanized Fc receptor monoclonal antibody) in healthy Chinese volunteers. Subjects were randomized to receive a single s.c. dose of HBM9161 or placebo in a 3:1 ratio in 3 dosing cohorts (340 mg, 510 mg, or 680 mg, respectively), and then followed up for 85 days. Study end points included incidence of adverse event (AE), serum drug concentration, IgG and its subclasses, and anti-drug antibodies (ADAs). Twenty-four subjects were randomized. Dose-dependent reduction of total IgG occurred rapidly from baseline to reach nadir at day 11, then recovered steadily from day 11 to day 85. The mean maximum percentage reductions from baseline total IgG were 21.0 ± 9.3%, 39.8 ± 5.13%, and 41.2 ± 10.4% for subjects receiving HBM9161 340 mg, 510 mg, and 680 mg, respectively. The exposure of HBM9161 (areas under the curve [AUCs] and peak plasma concentration [Cmax ]) increased in a more than dose-proportional manner at the dose examined. All reported AEs were mild in severity. The most reported AEs in the HBM9161 groups were influenza-like illness and rash. Two subjects developed ADA during the study period. A single s.c. dose of HBM9161 results in sustained and dose-dependent IgG reduction, and was well-tolerated at a dose up to 680 mg in Chinese subjects. The data warrant further investigation of its effects in IgG-mediated autoimmune disorders.

Presence of an EML4-ALK gene fusion detected by microfluidic chip DNA hybridization.

Non-small cell lung cancer (NSCLC) accounts for ∼80-85% of all lung cancer cases, and the EML4-ALK fusion oncogene is a well-known contributor to NSCLC cases. Expensive methods such as FISH, IHC, and NGS have been used to detect the EML4-ALK fusion oncogene. Here, a cost-effective and facile method of detecting and differentiating an EML4-ALK fusion oncogene from the wild-type gene has been accomplished by DNA hybridization using the microfluidic biochip. First, oligonucleotide probes were confirmed for successful detection of immobilized sense strands. Second, capture of the sense PCR product strands (fusion and WT) and their subsequent detection and differentiation were accomplished. Our proof-of-concept study shows the ability to detect 1% fusion products, among WT ones.

Simultaneous determination of free and total paclitaxel in blood in a three-phase laminar flow microchip.

In this study, a three-phase laminar flow microfluidic chip (TPL chip) combined with HPLC was developed for monitoring free and total concentrations of paclitaxel (PTX) in blood simultaneously. A diluted whole blood sample (aqueous phase) was introduced into the chip, ethyl acetate (organic phase) was introduced into the chip for extraction, and an interphase was used to prevent the blood sample from coming into direct contact with the organic phase. Because only free drug can quantitatively diffuse into the organic extraction phase and the free drug fraction has a linear relationship with the dilution factor of blood, both the free and total drug concentrations can be obtained by detecting the concentration of paclitaxel in the organic extraction phase. The governing factor such as flow rate for extraction was optimized. Docetaxel was used as an internal standard. The reliability of the quantitative diffusion of molecules in the TPL chip was proved by the methodological investigation of PTX in PBS sample, which showed a good linearity in the concentration range of 0.5 - 100 µg/mL and a detection limit of 7 ng/mL. Good repeatibilities for retention time (RSD of PTX is 1.23%, docetaxel is 1.14%, n = 5) and peak area ratio of PTX to docetaxel (RSD is 4.38%) were obtained. For blood sample analysis, only 100 µL of sample was needed and whole pretreatment was finished in 35 min, and a recovery of 94~117% were obtained. The provided method showed advantages in fast analysis speed, minimum sample handing, and potential ability of automation, and integration.

Construction of an Array of Photonic Crystal Films for Visual Differentiation of Water/Ethanol Mixtures.

A photonic crystal film (PCF) which consists of a porous layered structure with a highly ordered periodic arrangement of nanopores has been used to differentiate between various mixtures of water and ethanol (EtOH). The refractive index difference between the wall (silica) of the empty nanopore and air which occupies it results in the structural color of the PCF. This color disappears when the nanopores are infiltrated by a liquid with a similar refractive index to silica (or silicon dioxide). The disappearance of the structural color provides a means to construct a colorimetric sensor to differentiate between various water/EtOH mixtures based on their wettability of the nanopores in the PCF. In this study, an array of silica-based PCFs was synthesized on a silicon substrate with a precise control of nanopore properties using the co-assembly/sedimentation method. Using this method, we benefitted from having different PCFs on a single substrate. Chemical coatings, neck angles, and film thicknesses on each PCF were the three factors used to adjust the wettability of the pores. Nanopore wetting by water/EtOH mixtures was studied in a systematic manner based on the three factors, and the findings were used to develop a sensor for visual differentiation of various water/EtOH mixtures. The final developed sensor consisting of an array of six PCFs was able to differentiate between seven different water/EtOH mixtures: W10, W20, W30, W40, W50, W60, and W70, in which W10 means 10% of water in EtOH.

The microfluidic capture of single breast cancer cells for multi-drug resistance assays.

Utilizing the microfluidic single-cell technique enables us to study the inhibition of multidrug resistance due to drug efflux on a single triple-negative breast cancer cell. This method examines drug efflux inhibition on a single cell in a microfluidic chip using a conventional optical detection system constructed from an inverted microscope and a microphotometer. More importantly, the integration of single-cell selection, dye and drug loading, and fluorescence measurement for intracellular drug accumulation is all conducted on a single microfluidic chip. By using a microfluidic chip and the adherent nature of the cancer cell lines, a single breast cancer cell could be selected and retained near the cell retention structure in the chip. This enabled us to detect dye accumulation in the MDR breast cells in the presence of cyclosporine A (CsA). CsA and rhodamine 123 (Rh123) were used as the P-glycoprotein (P-gp) inhibitor and fluorescent dye, respectively. Furthermore, Paclitaxel, a commonly known chemotherapeutic used in breast cancer patients, was administered in the presence of both reagents. During the entirety of the experiment fluorescence measurement was used to monitor the fluctuating levels of intracellular Rh123 levels, and an optical imaging system was used to monitor the shape and size of the cell. The results showed that the Rh123 fluorescence signal in a single-cell increased dramatically over its same-cell control due to the competitive inhibition of paclitaxel and the non-competitive inhibition subjected by CsA.

Dielectrophoretic trapping of single leukemic cells using the conventional and compact optical measurement systems.

Here, we report a microfluidic same-single-cell analysis to study the inhibition of multidrug resistance due to drug efflux on single leukemic cells. Drug efflux inhibition was investigated in the microfluidic chip using two different fluorescence detection systems, namely, a compact single-cell bioanalyzer and the conventional optical detection system constructed from an inverted microscope and a microphotometer. More importantly, a compact signal generator was used to conduct dielectrophoretic cell trapping together with the compact SCB. By using the DEP force, a single acute myeloid leukemia cell was trapped in the cell retention structure of the chip. This allowed us to detect dye accumulation in the MDR leukemic cells in the presence of cyclosporine A (CsA). CsA and rhodamine 123 were used as the P-glycoprotein inhibitor and fluorescent dye, respectively. The result showed that the Rh123 fluorescence signal in a single-cell increased dramatically over its same-cell control on both fluorescence detection systems due to the inhibition by CsA.

Millifluidic chip with a modular design used as a sample pretreatment cartridge for flour and flour food products.

The integration of sample pretreatment remains one of the hurdles towards a rapid, automated micro total analytical system (µ-TAS) for real samples. In this paper, a modular design, which was used for sample preparation, has been developed as the polydimethylsiloxane (PDMS) millifluidic chips with channels at a millimeter level. Multiple functional units, including extraction, filtration, mixing and solid phase extraction (SPE), for sample pretreatment were integrated in one chip. In this chip, each functional unit was connected by pump tubings and one-way valves in series to form a fully automated system. Based on the modular design, multiple functional units have been combined in different sequences according to practical needs. In addition, the proposed system has characteristics of miniaturization, portability, and real-time application. Herein, spiked benzoyl peroxide (BPO) in flour samples was used as a model compound to study the system's performances. With a portable integrated Raman spectrometer for detection, the detection limit of BPO was 0.017gkg-1, with a linear relationship from 0.025 to 0.5gkg-1. This modular design was demonstrated to be effective and it can be expanded for pretreatment of other food samples.

Integration of laminar flow extraction and capillary electrophoretic separation in one microfluidic chip for detection of plant alkaloids in blood samples.

The design, construction and testing for integration of liquid-liquid extraction (EX) and capillary electrophoretic (CE) separation on one glass microchip was reported. In this EX-CE chip, a 1.5 cm-long and 200 µm-wide EX channel was used for extraction based on the two-phase laminar flow, followed by a single-cross CE unit for on-line analysis without any auxiliary devices. One side of the EX channel surface for the organic solvent phase was selectively modified to be hydrophobic while the surface of the other side for the aqueous phase remained hydrophilic, and the extraction product reservoir is also used as the sample reservoir for the subsequent chip separation in the CE channel. With the surface-directed liquid flow behavior and liquid level adjustment in various reservoirs of the EX-CE chip, no disturbance occurred between the extraction (EX) and capillary electrophoretic (CE) units. A small heating block was placed under the chip to accelerate solvent evaporation after liquid-liquid extraction. Sanguinarine (SAN), a plant alkaloid, was used as a model analyte to evaluate the performance of the EX-CE chip. The influences of organic solvent type and liquid flow speed on the extraction efficiency were investigated. Rhodamine 123 (Rh123) was used as an internal standard for quantification of Sanguinarine (SAN) in a physiological buffer (e.g. PBS) or blood samples. A good linearity in the concentration range of 0.05 µg mL-1 to 0.1 mg mL-1 for SAN in PBS was obtained, with the detection limit of 0.5 ng mL-1. Good repeatibilities for migration times (RSD of SAN is 0.63%, Rh123 is 0.91%, n = 5) and peak area ratio of SAN to Rh123 (RSD is 1.3%, n = 5) were obtained. For blood sample analysis, only 20 µL of sample was needed, and the whole analysis was finished in 17 min. In addition to the advantages in fast analysis speed, minimum sample handling, potential automation, the reported method showed an on-line sample pre-concentration capability.

Epstein-Barr virus latent membrane protein-1 up-regulates cytokines and correlates with older age and poorer prognosis in Hodgkin lymphoma.

AIMS: Previously, we reported an association between Epstein-Barr virus (EBV)-positive Hodgkin lymphoma (HL), older age, and poorer prognosis. The aim of this study was to investigate the mechanisms underlying this association. METHODS AND RESULTS: Transfection of HL cell lines with EBV latent membrane protein-1 (LMP1) resulted in up-regulation of many cytokine genes as assessed by the use of oligonucleotide microarrays. The up-regulation of cytokines was validated by using an inflammatory cytokine protein array: macrophage inflammatory protein (MIP)-1α, MIP-1ß, and interleukin (IL)-13. Immunostaining of HL samples (n = 104) showed that expression of MIP-1α, MIP-1ß and IL-13 correlated with EBV infection and LMP1 expression. Combined expression of these cytokines was more common in patients aged >60 years (P < 0.001), and was associated with a poorer prognosis (P = 0.042). In another cohort, serum levels of MIP-1α, MIP-1ß and IL-13 were increased in HL patients (n = 53) and highest in EBV-positive HL patients as compared with healthy controls (n = 40). Xenograft mice injected with EBV-positive HL cells had higher serum levels of MIP-1α, MIP-1ß and IL-13 than mice injected with EBV-negative HL cells, although there was no difference in growth. CONCLUSIONS: EBV infection appears to promote the release of cytokines in HL patients, and negatively impacts on patient survival. Physiological immunosenescence probably explains the association between EBV infection and older age. Cytokine modulation is a potential therapeutic target for EBV-positive HL patients.

Dielectrophoretic Microfluidic Chip Enables Single-Cell Measurements for Multidrug Resistance in Heterogeneous Acute Myeloid Leukemia Patient Samples.

The front-line treatment for adult acute myeloid leukemia (AML) is anthracycline-based combination chemotherapy. However, treatment outcomes remain suboptimal with relapses frequently observed. Among the mechanisms of treatment failure is multidrug resistance (MDR) mediated by the ABCB1, ABCC1, and ABCG2 drug-efflux transporters. Although genetic and phenotypic heterogeneity between leukemic blast cells is a well-recognized phenomenon, there remains minimal data on differences in MDR activity at the individual cell level. Specifically, functional assays that can distinguish the variability in MDR activity between individual leukemic blasts are lacking. Here, we outline a new dielectrophoretic (DEP) chip-based assay. This assay permits measurement of drug accumulation in single cells, termed same-single-cell analysis in the accumulation mode (SASCA-A). Initially, the assay was optimized in pretherapy samples from 20 adults with AML whose leukemic blasts had MDR activity against the anthracyline daunorubicin (DNR) tested using multiple MDR inhibitors. Parameters tested were initial drug accumulation, time to achieve signal saturation, fold-increase of DNR accumulation with MDR inhibition, ease of cell trapping, and ease of maintaining the trapped cells stationary. This enabled categorization into leukemic blast cells with MDR activity (MDR(+)) and leukemic blast cells without MDR activity (MDR(-ve)). Leukemic blasts could also be distinguished from benign white blood cells (notably these also lacked MDR activity). MDR(-ve) blasts were observed to be enriched in samples taken from patients who went on to enter complete remission (CR), whereas MDR(+) blasts were frequently observed in patients who failed to achieve CR following front-line chemotherapy. However, pronounced variability in functional MDR activity between leukemic blasts was observed, with MDR(+) cells not infrequently seen in some patients that went on to achieve CR. Next, we tested MDR activity in two paired AML patient samples. Pretherapy samples taken from patients that achieved CR to front-line chemotherapy were compared with samples taken at time of subsequent relapse. MDR(+) cells were frequently observed in leukemic blast cells in both pretherapy and relapsed samples, consistent with MDR as a mechanism of relapse in these patients. We demonstrate the ability of a new DEP microfluidic chip-based assay to identify heterogeneity in MDR activity in leukemic blasts. The test provides a platform for future studies to characterize the mechanistic basis for heterogeneity in MDR activity at the individual cell level.

Improvement in High-Grade Osteosarcoma Survival: Results from 202 Patients Treated at a Single Institution in Taiwan.

The aim of this study was to compare survival before and after 2004 and define the prognostic factors for high-grade osteosarcomas beyond those of typical young patients with localized extremity disease. Few studies have reported the long-term treatment outcomes of high-grade osteosarcoma in Taiwan. A total of 202 patients with primary high-grade osteosarcoma who received primary chemotherapy at Taipei Veterans General Hospital between January 1995 and December 2011 were retrospectively evaluated and compared by period (1995-2003 vs 2004-2011). Patients of all ages and tumor sites and those following or not following controlled protocols were included in analysis of demographic, tumor-related, and treatment-related variables and survival. Overall survival and progression-free survival at 5 years were, respectively, 67.7% and 48% for all patients (n = 202), 77.3% and 57.1% for patients without metastasis (n = 157), and 33.9% and 14.8% for patients with metastasis (n = 45). The survival rates of patients treated after 2004 were significantly higher (by 13%-16%) compared with those of patients treated before 2004, with an accompanying 30% increase in histological good response rate (P = .002). Factors significantly contributing to inferior survival in univariate and multivariate analyses were diagnosis before 2004, metastasis at diagnosis, and being a noncandidate for a controlled treatment protocol. By comparison with the regimens used at our institution before 2004, the current results support the effectiveness of the post-2004 regimens, which consisted of substantially reduced cycles of high-dose methotrexate and a higher dosage of ifosfamide per cycle, cisplatin, and doxorubicin, for treating high-grade osteosarcoma in Asian patients.