Food Fingerprinting: Using a Two-Tiered approach to Monitor and Mitigate Food Fraud in Rice.

BACKGROUND: Rice is an important staple food that is consumed around the world. Like many foods, the price of rice varies considerably, from very inexpensive for a low-quality product to premium pricing for highly prized varieties from specific locations. Therefore, like other foods it is vulnerable to economically motivated adulteration through substitution or misrepresentation of inferior-quality rice for more expensive varieties. OBJECTIVE: In this article we describe results of a research project focused on addressing potential food fraud issues related to rice supplies in China, India, Vietnam, and Ghana. Rice fraud manifests differently in each country; therefore, tailored solutions were required. METHOD: Here we describe a two-tiered testing regime of rapid screening using portable Near Infrared technology supported by second tier testing using mass spectrometry-based analysis of suspicious samples. RESULTS: Portable Near Infrared spectroscopy models and laboratory-based Gas Chromatography-Mass Spectrometry methods were developed to differentiate between: high-value Basmati rice varieties and their potential adulterants; six Geographic Indicated protected rice varieties from specific regions within China; various qualities of rice in Ghana and Vietnam; and locally produced and imported rice in Ghana. Furthermore, an Inductively Coupled Plasma-Mass Spectrometry method was developed to support the Chinese rice varieties methods as well as a Liquid Chromatography Quadrupole Time-of-Flight Mass Spectrometry method for quality differentiation in Vietnam. CONCLUSIONS/HIGHLIGHTS: This two-tier approach can provide a substantially increased level of testing through rapid screening outside of the laboratory with the reassurance of corroborating mass spectrometry-based laboratory analysis to support decision making.

The opposing roles of the mTOR signaling pathway in different phases of human umbilical cord blood-derived CD34+ cell erythropoiesis.

As an indispensable, even lifesaving practice, red blood cell (RBC) transfusion is challenging due to several issues, including supply shortage, immune incompatibility, and blood-borne infections since donated blood is the only source of RBCs. Although large-scale in vitro production of functional RBCs from human stem cells is a promising alternative, so far, no such system has been reported to produce clinically transfusable RBCs due to the poor understanding of mechanisms of human erythropoiesis, which is essential for the optimization of in vitro erythrocyte generation system. We previously reported that inhibition of mammalian target of rapamycin (mTOR) signaling significantly decreased the percentage of erythroid progenitor cells in the bone marrow of wild-type mice. In contrast, rapamycin treatment remarkably improved terminal maturation of erythroblasts and anemia in a mouse model of ß-thalassemia. In the present study, we investigated the effect of mTOR inhibition with rapamycin from different time points on human umbilical cord blood-derived CD34+ cell erythropoiesis in vitro and the underlying mechanisms. Our data showed that rapamycin treatment significantly suppressed erythroid colony formation in the commitment/proliferation phase of erythropoiesis through inhibition of cell-cycle progression and proliferation. In contrast, during the maturation phase of erythropoiesis, mTOR inhibition dramatically promoted enucleation and mitochondrial clearance by enhancing autophagy. Collectively, our results suggest contrasting roles for mTOR in regulating different phases of human erythropoiesis.

Development of a Sensitive and Reliable UHPLC-MS/MS Method for the Determination of Multiple Urinary Biomarkers of Mycotoxin Exposure.

A variety of mycotoxins from different sources frequently contaminate farm products, presenting a potential toxicological concern for animals and human. Mycotoxin exposure has been the focus of attention for governments around the world. To date, biomarkers are used to monitor mycotoxin exposure and promote new understanding of their role in chronic diseases. The goal of this research was to develop and validate a sensitive UHPLC-MS/MS method using isotopically-labeled internal standards suitable for accurate determination of 18 mycotoxin biomarkers, including fumonisins, ochratoxins, Alternaria and emerging Fusarium mycotoxins (fumonisin B1, B2, and B3, hydrolyzed fumonisin B1 and B2, ochratoxin A, B, and alpha, alternariol, alternariol monomethyl ether, altenuene, tentoxin, tenuazonic acid, beauvericin, enniatin A, A1, B, and B1) in human urine. After enzymatic digestion with ß-glucuronidase, human urine samples were cleaned up using HLB solid phase extraction cartridges prior to instrument analysis. The multi-mycotoxin and analyte-specific method was validated in-house, providing satisfactory results. The method provided good linearity in the tested concentration range (from LOQ up to 25-500 ng/mL for different analytes), with R2 from 0.997 to 0.999. The limits of quantitation varied from 0.0002 to 0.5 ng/mL for all analytes in urine. The recoveries for spiked samples were between 74.0% and 133%, with intra-day precision of 0.5%-8.7% and inter-day precision of 2.4%-13.4%. This method was applied to 60 urine samples collected from healthy volunteers in Beijing, and 10 biomarkers were found. At least one biomarker was found in all but one of the samples. The high sensitivity and accuracy of this method make it practical for human biomonitoring and mycotoxin exposure assessment.

Effective expansion of forkhead box P3⁺ regulatory T cells via early secreted antigenic target 6 and antigen 85 complex B from Mycobacterium tuberculosis.

The expansion of CD4+ CD25+ forkhead box (FOX)P3+ regulatory T (Treg) cells has been observed in patients with Mycobacterium (M.) tuberculosis; however, the mechanism of expansion remains to be elucidated. The aim of the present study was to examine the role of the early secreted antigenic target 6(ESAT­6) and antigen 85 complex B (Ag85B) from M. tuberculosis on Treg cell expansion. To investigate the sensitivity of peripheral blood cultures to the M. tuberculosis ESAT­6 and Ag85B antigens, the proportion of circulating CD4+ CD25+ FOXP3+ Treg cells was determined using flow cytometry and the levels of FOXP3 mRNA were determined using reverse transcription quantitative polymerase chain reaction. The mRNA levels of FOXP3 and the proportion of circulating CD4+ CD25+ FOXP3+ Treg cells were increased in multiplicitous drug­resistant tuberculosis patients compared with those in healthy controls and patients with latent tuberculosis (TB) infection (LTBI) (P<0.001). The mycobacterial antigens ESAT­6 and Ag85B increased the expansion of the CD4+ CD25+ FOXP3+ Treg cells and the mRNA levels of FOXP3 in healthy controls and LTBI patients compared with the effect of Bacillus Calmette­Guerin (P<0.05). Additionally, the mRNA levels of FOXP3 were elevated in the LTBI patients following stimulations with the mycobacterial antigens (P=0.012). Therefore, the M. tuberculosis antigens ESAT­6 and Ag85B induced CD4+ CD25+ FOXP3+ Treg­cell expansion, particularly in patients with LTBI. These findings indicated that CD4+ CD25+ FOXP3+ Treg cells may have a primary role in the failure of the host immune system to eradicate M. tuberculosis.

Epigallocatechin-3-gallate inhibits proliferation of human aortic smooth muscle cells via up-regulating expression of mitofusin 2.

Previous studies have shown that epigallocatechin-3-gallate (EGCG) inhibits the proliferation of vascular smooth muscle cells (VSMCs) via the extracellular-signal-regulated kinase (ERK1/2) and mitogen activated protein kinases (MAPKs) pathway. Mitofusin 2 (Mfn-2) also suppresses VSMC proliferation through Ras-Raf-ERK/MAPK, suggesting a possible link between EGCG, Mfn-2 and ERK/MAPK. However, the effect of EGCG on Mfn-2 remains unknown. In this study, we investigated the role of Mfn-2 in the regulation of VSMC proliferation by EGCG, and assessed the underlying mechanisms. The effects of EGCG on the proliferation of cultured human aortic smooth muscle cells (HASMCs) were observed by 5-ethynl-2-deoxyuridine (EdU) incorporation assay. Mfn-2 gene and protein levels, and Ras, p-c-Raf and p-ERK1/2 protein levels were determined by quantitative real-time polymerase chain reaction and western blotting, respectively. Mfn-2 gene silencing was achieved by RNA interference. EGCG 50 µmol/L profoundly inhibited the proliferation of HASMCs in culture, up-regulated Mfn-2, and down-regulated the expression of p-c-Raf and p-ERK1/2. Furthermore, RNA interference-mediated gene knockdown of Mfn-2 antagonized EGCG-induced anti-proliferation and down-regulation of Ras, p-c-Raf and p-ERK1/2. These results suggest that EGCG inhibits the proliferation of HASMCs in vitro largely via Mfn-2-mediated suppression of the Ras-Raf-ERK/MAPK signaling pathway.

Effects of urotensin II on functional activity of late endothelial progenitor cells.

Urotensin II (UII) is a potent vasoactive cyclic peptide which has multiple effects on the cardiovascular system. However, the effects of UII on late endothelial progenitor cells (EPCs) are still unclear. The aim of the present study is to investigate whether UII influences the functional activity of late EPCs. Late EPCs were isolated from human umbilical cord blood by Ficoll density gradient centrifugation and treated with UII (10(-10), 10(-9), 10(-8), 10(-7) and 10(-6)M), or vehicle control. Expression of urotensin II receptor (UT) in late EPCs was confirmed by indirect immunofluorescence staining. Late EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, transwell chamber assay, and matrigel tube formation assay. Late EPCs adhesive assay was performed by replating cells on fibronectin-coated dishes, and then adherent cells were counted. Incubation with UII increased the migratory, adhesive and in vitro vasculogenesis capacity and inhibited the proliferative activity of late EPCs. Furthermore, these UII-mediated effects on late EPCs were attenuated by pretreatment with the UT antagonist urantide. These findings indicate that UII may exert multiple effects on functional activity of late EPCs through UT.

Osteopontin is involved in urotensin II-induced migration of rat aortic adventitial fibroblasts.

Recent studies suggest that both osteopontin and urotensin II (UII) play critical roles in vascular remodeling. We previously showed that UII could stimulate the migration of aortic adventitial fibroblasts. In this study, we examined whether osteopontin is involved in UII-induced migration of rat aortic adventitial fibroblasts and examined the effects and mechanisms of UII on osteopontin expression in adventitial fibroblasts. Migration of adventitial fibroblasts induced by UII could be inhibited significantly by osteopontin antisense oligonucleotide (P<0.01) but not sense or mismatch oligonucleotides (P>0.05). Moreover, UII dose- and time-dependently promoted osteopontin mRNA expression and protein secretion in the cells, with maximal effect at 10(-8)mol/l at 3h for mRNA expression or at 12h for protein secretion (both P<0.01). Furthermore, the UII effects were significantly inhibited by its receptor antagonist SB710411 (10(-6)mol/l), and Ca(2+) channel blocker nicardipine (10(-5)mol/l), protein kinase C (PKC) inhibitor H7 (10(-5)mol/l), calcineurin inhibitor cyclosporine A (10(-5)mol/l), mitogen-activated protein kinase (MAPK) inhibitor PD98059 (10(-5)mol/l) and Rho kinase inhibitor Y-27632 (10(-5)mol/l). Thus, osteopontin is involved in the UII-induced migration of adventitial fibroblasts, and UII could upregulate osteopontin gene expression and protein synthesis in rat aortic adventitial fibroblasts by activating its receptor and the Ca(2+) channel, PKC, calcineurin, MAPK and Rho kinase signal transduction pathways.

Nicotine improves the functional activity of late endothelial progenitor cells via nicotinic acetylcholine receptors.

The aim of this study is to investigate whether nicotinic acetylcholine receptors (nAChRs) are involved in the modulation of functional activity of late endothelial progenitor cells (EPCs) induced by nicotine. Total mononuclear cells (MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture plates. Late EPCs were positive for 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiI-acLDL) uptake and fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin lectin (UEA-1) binding. Expression of von Willbrand factor (vWF), kinase insert domain receptor (KDR), and α7 nAChR was detected by indirect immunofluorescence staining. Late EPCs of 3-5 passages were treated for 32 h with either vehicle or nicotine with or without pre-incubation of nAChR antagonism, mecamylamine, or α-bungarotoxin. The viability, migration, and in vitro vasculogenesis activity of late EPCs were assayed with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay, and in vitro angiogenesis assay, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated plates, and then adherent cells were counted. Incubation with 10 nmol/L nicotine enhanced viable, migratory, adhesive, and in vitro vasculogenesis capacity of late EPCs. The effect of nicotine on late EPCs can be attenuated by mecamylamine or α-bungarotoxin. In conclusion, nicotine improves the functional activity of late EPCs via nAChRs.

Effects of osteopontin on functional activity of late endothelial progenitor cells.

The aim of this study is to investigate the effect of osteopontin (OPN) on functional activity of late endothelial progenitor cells (EPCs). Total mononuclear cells (MNCs) were isolated from human umbilical cord blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture plates. Late EPCs were positive for both 1,1-dioctadecyl-3,3,3,3-tetramethylindocarbocyanine-labeled acetylated low-density lipoprotein (DiI-acLDL) and fluorescein-isothiocyanate-conjugated Ulex europaeus agglutinin lectin (UEA-1). Expression of von Willbrand factor (vWF) and kinase insert domain receptor (KDR) were detected by indirect immunofluorescence staining. Late EPCs of 3-5 passages were treated for 24 h with OPN (to make a series of final concentration: 0.005 µg/ml, 0.01 µg/ml, 0.05 µg/ml, 0.5 µg/ml, 2.5 µg/ml), or vehicle control. The proliferation, migration, and in vitro vasculogenesis activity of late EPCs were assayed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, modified Boyden chamber assay and an in vitro angiogenesis assay, respectively. Late EPCs adhesion assay was performed by replating cells on fibronectin-coated plates, and then adherent cells were counted. Incubation with OPN dose-dependently inhibited the proliferative, adhesive, and in vitro vasculogenesis capacity and increased migratory activity of late EPCs.

Transforming growth factor-ß1 involved in urotensin II-induced phenotypic differentiation of adventitial fibroblasts from rat aorta.

BACKGROUND: Urotensin II (UII) is a new vasoconstrictive peptide that may activate the adventitial fibroblasts. Transforming growth factor-ß1 (TGF-ß1) is an important factor that could induce the phenotypical transdifferentiation of adventitial fibroblasts. This study aimed to explore whether TGF-ß1 is involved in UII-induced phenotypic differentiation of adventitial fibroblasts from rat aorta. METHODS: Adventitial fibroblasts were prepared by the explant culture method. TGF-ß1 protein secretion from the cells was determined by enzyme-linked immunosorbent assay (ELISA). The mRNA and protein expression of α-smooth nuscle actin (α-SM-actin), the marker of phenotypic differentiation from fibroblasts to myofibroblasts, were determined using real-time quantitative RT-PCR (real-time RT-PCR) and Western blotting, respectively. RESULTS: UII stimulated the secretion of TGF-ß1 in cultured adventitial fibroblasts in a time-dependent manner. The secretion reached a peak at 24 hours, was higher by 69.8% (P < 0.01), than the control group. This effect was also concentration dependent. Maximal stimulation was reached at 10(-8) mol/L of UII (P < 0.01), which was increased by 59.9%, compared with in the control group (P < 0.01). The secretion of TGF-ß1 induced by UII was significantly blocked by SB-710411 (10(-7) mol/L), a specific antagonist of UII receptor. In addition, both UII (10(-8) mol/L) and TGF-ß1 significantly stimulated α-SM-actin mRNA and protein expression. Moreover, the α-SM-actin induced by UII was inhibited by the specific neutralizing antibody (20 µg/ml) of TGF-ß1, while the α-SM-actin expression stimulated by TGF-ß1 (20 ng/ml) was inhibited by SB-710411 (10(-7) mol/L), the UII receptor antagonist. CONCLUSION: This study suggests that UII could induce TGF-ß1 secretion in adventitial fibroblasts via UT activation, and TGF-ß1 might be involved in phenotypic differentiation from adventitial fibroblasts into myofibroblasts induced by UII, and TGF-ß1 signaling might be one of the important pathways by which UII is involved in vascular fibrosis.

In vivo anti-tumor effect of hybrid vaccine of dendritic cells and esophageal carcinoma cells on esophageal carcinoma cell line 109 in mice with severe combined immune deficiency.

AIM: To develop a fusion vaccine of esophageal carcinoma cells and dendritic cells (DC) and observe its protective and therapeutic effect against esophageal carcinoma cell line 109 (EC109). METHODS: The fusion vaccine was produced by fusing traditional polyethyleneglycol (PEG), inducing cytokine, sorting CD34+ magnetic microbead marker and magnetic cell system (MACS). The liver, spleen and lung were pathologically tested after injection of the fusion vaccine. To study the therapeutic and protective effect of the fusion vaccine against tumor EC109, mice were divided immune group and therapeutic group. The immune group was divided into P, E, D and ED subgroups, immunized by phosphate buffered solution (PBS), inactivated EC109, DC and the fusion vaccine respectively, and attacked by EC109 cells. The tumor size, weight, latent period and mouse survival period were recorded and statistically analyzed. The therapeutic group was divided into four subgroups: P, inactivated EC109, D and ED subgroups, which were attacked by EC109 and then treated with PBS, inactivated EC109, DC, and EC109-DC respectively. Pathology and flow cytometry were also used to study the therapeutic effect of the fusion vaccine against EC109 cells. RESULTS: Flow cytometry showed that the expression of folate receptor (FR), EC109 (C), DCs (D) in human nasopharyngeal carcinoma cell line (HNE1) (B) was 78.21%, 89.50%, and 0.18%, respectively. The fusion cells (C) were highly expressed. No tumor was found in the spleen, lung and liver after injection of the fusion vaccine. Human IgG was tested in peripheral blood lymphocytes (PBL). In the immune group, the latent period was longer in EC109-DC subgroup than in other subgroups, while the tumor size and weight were also smaller than those in ED subgroup. In the therapeutic group, the tumor size and weight were smaller in ED subgroup than in P, inactivated EC109 and DC subgroups. CONCLUSION: Fusion cells are highly expressed not only in FR but also in CD80. The fusion vaccine has a distinctive protective effect against tumor EC109 and can inhibit the growth of tumor in mice, and its immune protection against tumor attack is more significant.

Antitumor activity of a fusion of esophageal carcinoma cells with dendritic cells derived from cord blood.

The aim of this experiment was to develop a cytotoxic cancer vaccine (EC109-DC) prepared by fusions of esophageal carcinoma cells with dendritic cells derived from cord blood and to study the biological characteristics and resultant induction of antitumor immunity. CD34+ hematopoietic stem cells were isolated from cord blood using a CD34+ Progenitor Cell Isolation Kit by magnetic cell sorting system (MACS). CD34+ cells were incubated with rhGM-CSF, rhTNF-alpha and rhSCF for 2 weeks as DC (dendritic cells), and then by PEG-3600 to fuse with an esophageal carcinoma cell line. Selection with MACS marked with HLA-DR MicroBeads generated EC109-DC. Phenotypes and proliferation were analyzed by flow cytometry and cell culture in vitro. The lymphocyte proliferation reaction and CTL cytotoxicity were examined by MTT assay. The EC109-DC cells could proliferate slowly in vitro and highly expressed CD80, CD83 and CD86. The lymphocyte proliferation reaction and specific cytotoxicity against EC109 induced by EC109-DC cells were significantly higher than in control groups (p < 0.05). EC109-DC cells obtained by PEG fusion acquired the immuno-stimulating phenotype and could significantly stimulate the lymphocyte proliferation reaction and CTL activity. The results of this research provide the basis for materials to develop the DC-based vaccine against esophageal carcinoma.