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1.  Rapid and sensitive detection of rare cancer cells by the coupling of immunomagnetic nanoparticle separation with ELISA analysis 
This study presents a rapid and sensitive method for detecting cancer cells occurring at low concentration. The method involves the simultaneous detection of two biomarkers of T helper cancer cells. One biomarker conjugates with immunofunctionalized magnetic nanoparticles (MNPs), enabling the separation of the T helper cells from a mixed population of cells. The other biomarker is used for detection during enzyme-linked immunosorbent assay (ELISA) analysis. The specific T helper cells can be quantified according to their ELISA absorbance values following magnetic separation. The experimental results demonstrate that immunofunctionalized MNPs can function as magnetic sensors and separate specific T helper cells from a mixed population with high efficiency and high specificity. Coupled with the ELISA technique, the immunofunctionalized MNPs can simultaneously detect rare cells. Results indicated increasing absorbance with increasing T cell number (from 10 to 106). The total detection time was less than 15 minutes, even at a low T cell count. The advantages of the proposed method for detecting specific cells at low concentration include ease of preparation, low cost, rapid detection, and high sensitivity. The proposed system can be adopted to detect circulating tumor cells in early tumor stages for diagnostic or prognostic purposes.
doi:10.2147/IJN.S32240
PMCID: PMC3390995  PMID: 22787392
ELISA; magnetic nanoparticles; immunoassay; cancer cell
2.  Antioxidant, Analgesic, Anti-Inflammatory, and Hepatoprotective Effects of the Ethanol Extract of Mahonia oiwakensis Stem 
The aim of this study was to evaluate pharmacological properties of ethanol extracted from Mahonia oiwakensis Hayata stems (MOSEtOH). The pharmacological properties included antioxidant, analgesic, anti-inflammatory and hepatoprotective effects. The protoberberine alkaloid content of the MOSEtOH was analyzed by high-performance liquid chromatography (HPLC). The results revealed that three alkaloids, berberine, palmatine and jatrorrhizine, could be identified. Moreover, the MOSEtOH exhibited antioxidative activity using the DPPH assay (IC50, 0.743 mg/mL). The DPPH radical scavenging activity of MOSEtOH was five times higher that that of vitamin C. MOSEtOH was also found to inhibit pain induced by acetic acid, formalin, and carrageenan inflammation. Treatment with MOSEtOH (100 and 500 mg/kg) or silymarin (200 mg/kg) decreased the serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels compared with the CCl4-treated group. Histological evaluation showed that MOSEtOH reduced the degree of liver injury, including vacuolization, inflammation and necrosis of hepatocytes. The anti-inflammatory and hepatoprotective effect of MOSEtOH were found to be related to the modulation of antioxidant enzyme activity in the liver and decreases in malondialdehyde (MDA) level and nitric oxide (NO) contents. Our findings suggest that MOSEtOH has analgesic, anti-inflammatory and hepatoprotective effects. These effects support the use of MOSEtOH for relieving pain and inflammation in folk medicine.
doi:10.3390/ijms14022928
PMCID: PMC3588023  PMID: 23364614
Mahonia oiwakensis Hayata; high-performance liquid chromatography; hepatoprotective effect; malondialdehyde
3.  Antidepressant-Like Activity of the Ethanolic Extract from Uncaria lanosa Wallich var. appendiculata Ridsd in the Forced Swimming Test and in the Tail Suspension Test in Mice 
This study investigated the antidepressant activity of ethanolic extract of U. lanosa Wallich var. appendiculata Ridsd (ULEtOH) for two-weeks administrations by using FST and TST on mice. In order to understand the probable mechanism of antidepressant-like activity of ULEtOH in FST and TST, the researchers measured the levels of monoamines and monoamine oxidase activities in mice brain, and combined the antidepressant drugs (fluoxetine, imipramine, maprotiline, clorgyline, bupropion and ketanserin). Lastly, the researchers analyzed the content of RHY in the ULEtOH. The results showed that ULEtOH exhibited antidepressant-like activity in FST and TST in mice. ULEtOH increased the levels of 5-HT and 5-HIAA in cortex, striatum, hippocampus, and hypothalamus, the levels of NE and MHPG in cortex and hippocampus, the level of NE in striatum, and the level of DOPAC in striatum. Two-week injection of IMI, CLO, FLU and KET enhanced the antidepressant-like activity of ULEtOH. ULEtOH inhibited the activity of MAO-A. The amount of RHY in ULEtOH was 17.12 mg/g extract. Our findings support the view that ULEtOH exerts antidepressant-like activity. The antidepressant-like mechanism of ULEtOH may be related to the increase in monoamines levels in the hippocampus, cortex, striatum, and hypothalamus of mice.
doi:10.1155/2012/497302
PMCID: PMC3332070  PMID: 22567032
4.  Gallic Acid Ameliorated Impaired Glucose and Lipid Homeostasis in High Fat Diet-Induced NAFLD Mice 
PLoS ONE  2014;9(6):e96969.
Gallic acid (GA), a naturally abundant plant phenolic compound in vegetables and fruits, has been shown to have potent anti-oxidative and anti-obesity activity. However, the effects of GA on nonalcoholic fatty liver disease (NAFLD) are poorly understood. In this study, we investigated the beneficial effects of GA administration on nutritional hepatosteatosis model by a more “holistic view” approach, namely 1H NMR-based metabolomics, in order to prove efficacy and to obtain information that might lead to a better understanding of the mode of action of GA. Male C57BL/6 mice were placed for 16 weeks on either a normal chow diet, a high fat diet (HFD, 60%), or a high fat diet supplemented with GA (50 and 100 mg/kg/day, orally). Liver histopathology and serum biochemical examinations indicated that the daily administration of GA protects against hepatic steatosis, obesity, hypercholesterolemia, and insulin resistance among the HFD-induced NAFLD mice. In addition, partial least squares discriminant analysis scores plots demonstrated that the cluster of HFD fed mice is clearly separated from the normal group mice plots, indicating that the metabolic characteristics of these two groups are distinctively different. Specifically, the GA-treated mice are located closer to the normal group of mice, indicating that the HFD-induced disturbances to the metabolic profile were partially reversed by GA treatment. Our results show that the hepatoprotective effect of GA occurs in part through a reversing of the HFD caused disturbances to a range of metabolic pathways, including lipid metabolism, glucose metabolism (glycolysis and gluconeogenesis), amino acids metabolism, choline metabolism and gut-microbiota-associated metabolism. Taken together, this study suggested that a 1H NMR-based metabolomics approach is a useful platform for natural product functional evaluation. The selected metabolites are potentially useful as preventive action biomarkers and could also be used to help our further understanding of the effect of GA in hepatosteatosis mice.
doi:10.1371/journal.pone.0096969
PMCID: PMC4053315  PMID: 24918580
5.  Efficient Production of an Engineered Apoptin from Chicken Anemia Virus in a Recombinant E. coli for Tumor Therapeutic Applications 
BMC Biotechnology  2012;12:27.
Background
Apoptin, a nonstructural protein encoded by the VP3 gene of chicken anemia virus (CAV), has been shown to not only induce apoptosis when introduced into the precursors of chicken thymocytes, but has been found to specifically kill human cancer cells, tumor cell and transformed cells without affecting the proliferation of normal cells. This tumor-specific apoptotic characteristic of the protein potentially may allow the development of a protein drug that has applications in tumor therapy. However, several major problems, which include poor expression and poor protein solubility, have hampered the production of apoptin in bacteria.
Results
Significantly increased expression of recombinant full-length apoptin that originated from chicken anemia virus was demonstrated using an E. coli expression system. The CAV VP3 gene was fused with a synthetic sequence containing a trans-acting activator of transcription (TAT) protein transduction domain (PTD). The resulting construct was cloned into various different expression vectors and these were then expressed in various E. coli strains. The expression of the TAT-Apoptin in E. coli was significantly increased when TAT-Apoptin was fused with GST-tag rather than a His-tag. When the various rare amino acid codons of apoptin were optimized, the expression level of the GST-TAT-Apoptinopt in E. coli BL21(DE3) was significantly further increased. The highest protein expression level obtained was 8.33 g/L per liter of bacterial culture after induction with 0.1 mM IPTG for 4 h at 25 °C. Moreover, approximately 90% of the expressed GST-TAT-Apoptinopt under these conditions was soluble. After purification by GST affinity chromatography, the purified recombinant TAT-Apoptinopt protein was used to evaluate the recombinant protein’s apoptotic activity on tumor cells. The results demonstrated that the E. coli-expressed GST-TAT-apoptinopt showed apoptotic activity and was able to induce human premyelocytic leukemia HL-60 cells to enter apoptosis.
Conclusions
On expression in E. coli, purified recombinant TAT-Apoptinopt that has been fused to a GST tag and had its codons optimized, was found to have great potential. This protein may in the future allow the development of a therapeutic protein that is able to specifically kill tumor cells.
doi:10.1186/1472-6750-12-27
PMCID: PMC3443062  PMID: 22672291

Results 1-5 (5)