Suspension culture strategies to enrich colon cancer stem cells.

How to efficiently obtain high-purity cancer stem cells (CSCs) has been the basis of CSC research, but the optimal conditions for serum-free suspension culture of CSCs are still unclear. The present study aimed to define the optimal culture medium composition and culture time for the enrichment of colon CSCs via suspension culture. Suspension cell cultures of colon cancer DLD-1 cells were prepared using serum-free medium (SFM) containing variable concentrations of epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to produce spheroids. Culture times were set at 10, 20 and 30 days. A total of nine different concentrations of EGF and bFGF were added to SFM to generate nine experimental groups. The proportions of CD44+, CD133+, and CD44+CD133+ double-positive spheroid cells were detected via flow cytometry. mRNA expression of stemness-, epithelial-mesenchymal transition- and Wnt/ß-catenin pathway-associated genes was determined via reverse transcription-quantitative PCR. Self-renewal ability was evaluated by a sphere-forming assay. Tumorigenesis was studied in vitro using a colony formation assay and in vivo via subcutaneous cell injection in nude mice. It was found that the highest expression proportions of CD133+ and CD44+ spheroid cells were observed in group (G)9 (20 ng/ml EGF + 20 ng/ml bFGF) at 30 days (F=123.554 and 99.528, respectively, P<0.001), CD133+CD44+ cells were also observed in G9 at 30 days (and at 10 days in G3 and 20 days in G6; F=57.897, P<0.001). G9 at 30 days also displayed the highest expression of Krüppel-like factor 4, leucine-rich repeat-containing G protein-coupled receptor 5, CD44, CD133, Vimentin and Wnt-3a (F=22.682, 25.401, 3.272, 7.852, 13.331 and 17.445, respectively, P<0.001) and the lowest expression of E-cadherin (F=10.851, P<0.001). G9 at 30 days produced the highest yield of cell spheroids, as determined by a sphere forming assay (F=19.147, P<0.001); colony formation assays also exhibited the greatest number of colonies derived from G9 spheroids at 30 days (F=60.767, P<0.01), which also generated the largest mean tumor volume in the subcutaneous tumorigenesis xenograft model (F=12.539, P<0.01). In conclusion, 20 ng/ml EGF + 20 ng/ml bFGF effectively enriched colon CSCs when added to suspension culture for 30 days, and conferred the highest efficiency compared with other combinations.

Correlation analysis of serum miR-21 and miR-210 with hs-CRP, TNF-α, IL-6, and ICAM-1 in patients with sepsis after burns.

BACKGROUND: The aim of this study was to explore expression levels and clinical values of miR-21 and miR-210 in patients with sepsis after burns. METHODS: One hundred and twenty-eight burn patients who were treated in Binzhou Medical University Hospital were selected as research objects, among which 69 complicated with sepsis were in an observation group and 59 complicated with infection were in a control group. MiR-21 and miR-210 expression in the patients' serum was detected by RT-PCR. Serum inflammatory cytokines were detected by an enzyme-linked immunosorbent assay (ELISA). Correlation analysis was used for the correlations of the miR-21 and miR-210 with the cytokines. Receiver operating characteristic (ROC) curves were plotted to analyze the diagnostic values of the miR-21 and miR-210 for sepsis after burns and their predictive values for the poor prognosis of sepsis patients. RESULTS: MiR-21 expression reduced remarkably and miR-210 expression rose remarkably in the serum of patients with sepsis after burns. According to the analysis of the ROC curves, both of the miR-21 and miR-210 had relatively high diagnostic sensitivity for the disease, but the diagnostic value of their combined detection was higher. Contents of hs-CRP, TNF-α, IL-6, and ICAM-1 in serum were remarkably higher in the observation group than those in the control group. According to the correlation analysis, miR-21 was negatively correlated with the expression of the four cytokines, while miR-210 was positively correlated with that. The predictive value of miR-21 for the prognosis of sepsis patients was not high, but miR-210 had a certain predictive value, and their combined detection had a higher value. CONCLUSION: In serum of patients with sepsis after burns, miR-21 expression reduces remarkably and miR-210 expression rises. The miR-21 and miR-210 are related to the degree of inflammatory responses in septic patients, and their combined detection has a certain value for diagnosing the disease and predicting its prognosis.

Intraoperative cell salvage is associated with reduced allogeneic blood requirements and has no significant impairment on coagulation function in patients undergoing cesarean delivery: a retrospective study.

OBJECTIVE: The aim of the study is to examine the association between Intraoperative cell salvage (ICS), allogeneic blood transfusion (ABT) and coagulation function in obstetrics. METHODS: A total of 486 pregnant women undergoing cesarean delivery, of whom 157 were enrolled in this retrospective study. Patients were divided into ICS group (n = 101, ICS used during operation) and control group (n = 56, ICS not used during operation). Clinical data, including plasma prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (Fib) and thrombin time (TT) levels, were collected from all patients preoperatively (within 12-24 h) and postoperatively (within 6-12 h) and analyzed by t test, two-way repeated-measures ANOVA and Spearman's correlation. RESULTS: The use of ICS is associated with lower requirement rate for ABT (P < .001), while the blood loss was similar between the two groups (P = .990). Mean volume of ICS transfusion was 432.65 mL. Compared to preoperative values, the postoperative PT and APTT levels were significantly increased, while Fib was decreased in the two groups (all P < .01). No significant difference in coagulation function was observed between groups in preoperative and postoperative phase (P > .05). Furthermore, PT, APTT and TT after surgery were not correlated with the transfused volume of salvaged blood (P > .05) while the levels of Fib were negatively correlated with the volume (P < .01). In addition, there were no transfusion reactions in both two groups. CONCLUSIONS: Intraoperative cell salvage is correlated with reduced allogeneic blood requirements but did not impair blood coagulation significantly in patients undergoing cesarean delivery.

Krüppel-like factor 4 regulates stemness and mesenchymal properties of colorectal cancer stem cells through the TGF-ß1/Smad/snail pathway.

Krüppel-like factor 4 (KLF4) was closely associated with epithelial-mesenchymal transition and stemness in colorectal cancer stem cells (CSCs)-enriched spheroid cells. Nonetheless, the underlying molecular mechanism is unclear. This study showed that KLF4 overexpression was accompanied with stemness and mesenchymal features in Lgr5+ CD44+ EpCAM+ colorectal CSCs. KLF4 knockdown suppressed stemness, mesenchymal features and activation of the TGF-ß1 pathway, whereas enforced KLF4 overexpression activated TGF-ß1, phosphorylation of Smad 2/3 and Snail expression, and restored stemness and mesenchymal phenotypes. Furthermore, TGF-ß1 pathway inhibition invalidated KLF4-facilitated stemness and mesenchymal features without affecting KLF4 expression. The data from the current study are the first to demonstrate that KLF4 maintains stemness and mesenchymal properties through the TGF-ß1/Smad/Snail pathway in Lgr5+ CD44+ EpCAM+ colorectal CSCs.

Experimental Study on Bond-Slip Behavior between Corroded I-Shaped Steel and Concrete in Subsea Tunnel.

Degradation of the bond between I-shaped steel and concrete due to the corrosion of I-shaped steel significantly affects the durability of steel reinforced concrete (SRC) structures. This study carried out the accelerated corrosion test and push-out test to study the bond-slip behavior and characteristics considering the corrosion of I-shaped steel, and test results indicated that: (1) The performance degradation of the bond-slip accelerated when the corrosion ratio reached 12%. (2) The corrosion failure pattern of SRC experienced slip phase and destruction phase in the rising stage. (3) Based on the principle of minimum potential energy, the bond stress was obtained only with the load and the displacement in the free end and the loading end. (4) Meanwhile, a new bond-slip degradation model was developed using the interface damage theory. Finally, the proposed model agreed with the experimental results.

Anthropometric indices to predict insulin resistance in women with polycystic ovary syndrome in China.

RESEARCH QUESTION: Which anthropometric index (waist-to-height ratio, waist circumference, waist-to-hip ratio and body mass index) is the best in predicting insulin resistance among Chinese women with polycystic ovary syndrome? DESIGN: A total of 1124 patients with polycystic ovary syndrome at the Reproductive Endocrinology Division of West China Second University Hospital of Sichuan University were enrolled in this study. Identification of insulin resistance was based on homeostasis model assessment of insulin resistance scores 2.77 or over. Receiver operator characteristic analysis was carried out using the four anthropometric indices as the continuous variables and insulin resistance as the categorical variable to obtain the areas under the curve. RESULT: The area under the curve for the waist-to-height ratio (0.748 ± 0.019) was greater than those for waist circumference (0.739 ± 0.019), body mass index (0.738 ± 0.017), and waist-to-hip ratio (0.659 ± 0.020) in the prediction of insulin resistance. The waist-to-height ratio also had the highest Youden indices compared with those of waist circumference, body mass index, and waist-to-hip ratio; the waist-to-height ratio cut-off was 0.49. CONCLUSION: The waist-to-height ratio with a cut-off of 0.49 was the most accurate anthropometric indicator for predicting insulin resistance among Chinese women with polycystic ovary syndrome.

Long noncoding RNA ROR promotes breast cancer by regulating the TGF-ß pathway.

BACKGROUND: Breast cancer is the leading cause of oncological mortality among women. Efficient detection of cancer cells in an early stage and potent therapeutic agents targeting metastatic tumors are highly needed to improve survival rates. Emerging evidence indicates that lncRNAs (long noncoding RNAs) are critical regulators of fundamental cellular processes in a variety of tumors including breast cancer. The functional details of these regulatory elements, however, remain largely unexplored. METHODS: In this study, lncRNA ROR (linc-ROR) was examined by real-time PCR in different breast cancer cell lines and breast tumor tissues/non-tumor tissues were collected from both breast cancer patients and healthy controls. Linc-ROR was knockdown in breast cancer cell lines and the effects on cell proliferation, migration and invasion were tested both in vitro and in vivo tumor model. Effects of linc-ROR knockdown on TGF-ß signaling pathway were investigated by Western blot. RESULTS: Our studies have suggested that linc-ROR, a critical factor for embryonic stem cell maintenance, probably acts as an oncogenic factor in breast cancer cells, causing poor prognostic outcomes. Overexpression of linc-ROR seems to be responsible for promoting proliferation and invasion of cancer cells as well as tumor growth in nude mice. The regulatory action of linc-ROR can affect the activity of the TGF-ß signaling pathway, which has been proven critical for mammary development and breast cancer. CONCLUSIONS: The results have highlighted the potential importance of linc-ROR in the progression of advanced breast cancer, and thus will stimulate efforts in the development of novel diagnostic and therapeutic strategies.

Pathway­based detection of idiopathic pulmonary fibrosis at an early stage.

Idiopathic pulmonary fibrosis (IPF) is the most common interstitial pneumonia and the most aggressive interstitial lung disease. Usually, IPF is confirmed by the histopathological pattern of typical interstitial pneumonia and requires an integrated multidisciplinary approach from pulmonologists, radiologists and pathologists. However, these diagnoses are performed at an advanced stage of IPF. At present, pathway­based detection requires investigation, as it can be performed at an early stage of the disease. The aim of the present study was to find an effective method of diagnosing IPF at an early stage. Microarray data forE­GEOD­33566 were downloaded from the ArrayExpress database. Human pathways were downloaded from Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. An individual pathway­based method to diagnose IPF at an early stage was introduced. Pathway statistics were analyzed with an individualized pathway aberrance score. P­values were obtained with different methods, including the Wilcoxon test, linear models for microarray data (Limma) test and attract methods, generating three pathway groups. Support vector machines (SVM) were used to identify the best group for diagnosing IPF at an early stage. There were 106 differential pathways in Wilcoxon­based KEGG Pathway (n>5) group, 100 in the Limma­based KEGG Pathway (n>5) group, and seven in the attract­based KEGG Pathway (n>5) group. The pathway statistics of these differential pathways in three groups were analyzed with linear SVM. The results demonstrated that the Wilcoxon­based KEGG Pathway (n>5) group performed best in diagnosing IPF.

Effects of CYP3A4 polymorphisms on efficiency of general anesthesia combined with epidural block in patients undergoing cardiac valve replacement.

This study aims to investigate the effects of CYP3A4 polymorphisms (*4, *5 and *6) on efficiency of general anesthesia (GA) combined with epidural block (EB) in patients undergoing cardiac valve replacement. From January 2014 to October 2015, a total of 511 patients undergoing cardiac valve replacement (case group) and 503 healthy individuals (control group) were selected for the study. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was applied for genotyping of CYP3A4 gene. Central venous pressure (CVP), mean arterial pressure (MAP), heart rate (HR), pulse oximetry (SPO2), extubation and duration of intensive care unit (ICU) stay during the surgery were observed and recorded. A nine-month follow-up was conducted. Genotype and allele frequency of CYP3A4*4 were significantly different between the case and control groups (all P < 0.05). Compared with wild-type *1*1 patients with heterozygous *1*4 of CYP3A4*4 showed significant difference in HR, MAP, SPO2 and CVP and in the time of extubation and ICU stay. CYP3A4*4 polymorphism may be associated with the effect of GA combined with EB in cardiac surgery. These results demonstrate that CYP3A4*4 polymorphism is correlated with the effects of GA combined with EB in cardiac surgery. CYP3A4 polymorphisms increase the risk of GA combined with EB among patients undergoing cardiac valve replacement.

Multivariate analysis of the volatile components in tobacco based on infrared-assisted extraction coupled to headspace solid-phase microextraction and gas chromatography-mass spectrometry.

A novel infrared-assisted extraction coupled to headspace solid-phase microextraction followed by gas chromatography with mass spectrometry method has been developed for the rapid determination of the volatile components in tobacco. The optimal extraction conditions for maximizing the extraction efficiency were as follows: 65 µm polydimethylsiloxane-divinylbenzene fiber, extraction time of 20 min, infrared power of 175 W, and distance between the infrared lamp and the headspace vial of 2 cm. Under the optimum conditions, 50 components were found to exist in all ten tobacco samples from different geographical origins. Compared with conventional water-bath heating and nonheating extraction methods, the extraction efficiency of infrared-assisted extraction was greatly improved. Furthermore, multivariate analysis including principal component analysis, hierarchical cluster analysis, and similarity analysis were performed to evaluate the chemical information of these samples and divided them into three classifications, including rich, moderate, and fresh flavors. The above-mentioned classification results were consistent with the sensory evaluation, which was pivotal and meaningful for tobacco discrimination. As a simple, fast, cost-effective, and highly efficient method, the infrared-assisted extraction coupled to headspace solid-phase microextraction technique is powerful and promising for distinguishing the geographical origins of the tobacco samples coupled to suitable chemometrics.

Chemical constituents of tobacco smoke induce the production of interleukin-8 in human bronchial epithelium, 16HBE cells.

BACKGROUND: Interleukin-8 (IL-8) functions as a major chemoattractant and plays pivotal roles in the initiation and development of chronic obstructive pulmonary disease (COPD), and tobacco smoke is a most risk factor contributing to the development of COPD. Hence, we have screened some of the tobacco smoke-derived chemical compounds that potentially induce the production of IL-8 in human bronchial epithelium, 16HBE cells. METHODS: Twenty-eight hazardous smoke components belonging to 9 classes including nicotine, ammonia, aromatic amines, polycyclic aromatic hydrocarbons, phenols, carbonyls, hydrocyanic acid, nitrosamines and other volatile organics were used in the experiments. Proliferation of 16HBE cells was determined by cell counting kit-8 kit, luciferase activity was measured in IL-8 reporter gene-expressing 16HBE cells, and IL-8 levels in culture supernatants were quantified by enzyme-linked immunosorbent assay. RESULTS: At the non-toxic dosages, chemical compounds belonging to nicotine, aromatic amines, benzopyrene, phenols, aldehydes, and some other volatile organics dose-dependently increased IL-8 reporter gene expression. Consistently, the representative compounds belonging to nicotine, aromatic amines, benzopyrene, phenols, aldehydes, and some other volatile organics significantly and dose-dependently increased IL-8 levels in the culture supernatants of 16HBE cells, among these compounds, benzopyrene is a most potent stimulator for inducing IL-8 production. CONCLUSIONS: The present study has identified particular tobacco smoke constituents responsible for inducing the IL-8 production in human bronchial epithelium, which might help shed light on the pathogenesis of tobacco smoke-induced COPD.

Ginkgolide B functions as a determinant constituent of Ginkgolides in alleviating lipopolysaccharide-induced lung injury.

Ginkgolides are the major bioactive components of Ginkgo biloba extracts, however, the exact constituents of Ginkgolides contributing to their pharmacological effects remain unknown. Herein, we have determined the anti-inflammatory effects of Ginkgolide B (GB) and Ginkgolides mixture (GM) at equivalent dosages against lipopolysaccharide (LPS)-induced inflammation. RAW 264.7 cell culture model and mouse model of LPS-induced lung injury were used to evaluate in vitro and in vivo effects of GB and GM, respectively. In RAW 264.7 cells, GB and GM at equivalent dosages exhibit an identical capacity to attenuate LPS-induced inducible nitric oxide synthase mRNA and protein expression and subsequent NO production. Likewise, GB and GM possess almost the same potency in attenuating LPS-induced expression and activation of nuclear factor kappa B (p65) and subsequent increases in tumor necrosis factor-α mRNA levels. In LPS-induced pulmonary injury, GB and GM at the equivalent dosages have equal efficiency in attenuating the accumulation of inflammatory cells, including neutrophils, lymphocytes, and macrophages, and in improving the histological damage of lungs. Moreover, GB and GM at equivalent dosages decrease the exudation of plasma protein to the same degree, whereas GM is superior to GB in alleviating myeloperoxidase activities. Finally, though GB and GM at equivalent dosages appear to reduce LPS-induced IL-1ß mRNA and protein levels and IL-10 protein levels to the same degree, GM is more potent than GB to attenuate the IL-10 mRNA levels. Taken together, this study demonstrates that GB functions as the determinant constituent of Ginkgolides in alleviating LPS-induced lung injury.

Rapid determination of the volatile components in tobacco by ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction with gas chromatography-mass spectrometry.

An ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction was first employed to determine the volatile components in tobacco samples. The method combined the advantages of ultrasound, microwave, and headspace solid-phase microextraction. The extraction, separation, and enrichment were performed in a single step, which could greatly simplify the operation and reduce the whole pretreatment time. In the developed method, several experimental parameters, such as fiber type, ultrasound power, and irradiation time, were optimized to improve sampling efficiency. Under the optimal conditions, there were 37, 36, 34, and 36 components identified in tobacco from Guizhou, Hunan, Yunnan, and Zimbabwe, respectively, including esters, heterocycles, alkanes, ketones, terpenoids, acids, phenols, and alcohols. The compound types were roughly the same while the contents were varied from different origins due to the disparity of their growing conditions, such as soil, water, and climate. In addition, the ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction method was compared with the microwave-assisted extraction coupled to headspace solid-phase microextraction and headspace solid-phase microextraction methods. More types of volatile components were obtained by using the ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction method, moreover, the contents were high. The results indicated that the ultrasound-microwave synergistic extraction coupled to headspace solid-phase microextraction technique was a simple, time-saving and highly efficient approach, which was especially suitable for analysis of the volatile components in tobacco.

[Cigarette smoking in different manners induces acute lung injury in rats].

Objective: To investigate the effects of cigarette smoking in different manners on acute lung injury in rats. Methods: The commercially available cigarettes with tar of 1,5, 11 mg were smoked in Canada depth smoking (health canada method, HCM) manner, and those with tar of 11 mg were also smoked in international standard (ISO) smoking manner. Rats were fixed and exposed to mainstream in a manner of nose-mouth exposure. After 28 days, the bronchoalveolar lavage fluids from left lung were collected for counting and classification of inflammatory cells and determination of pro-inflammatory cytokines IL-1ß and TNF-α. The right lungs were subjected to histological examination and determination of myeloperoxidase (MPO) and superoxide dismutase (SOD) activities and glutathione, reactive oxygen species (ROS) and malondialdehyde (MDA) levels. Results: In both HCM and ISO manners, the degree of lung injury was closely related to the tar content of cigarettes, and significant decrease in the body weight of rats was observed after smoking for one week. In a HCM manner, smoking with cigarette of 11 mg tar resulted in robust infiltration of macrophages, lymphocytes and neutrophils into lungs, significant increase in IL-1ß and TNF-α levels and MPO activities, and significant decrease in GSH levels and SOD activities and increase in ROS and MDA levels (all P<0.05). Smoking with cigarette of 5 mg tar led to moderate increase in IL-1ß and TNF-α levels, and MPO activities (all P<0.05), and moderate decrease in GSH levels and SOD activities and increase of ROS and MDA levels (all P<0.05). However, smoking with cigarette of 1 mg tar affected neither inflammatory cell infiltration nor IL-1ß and TNF-α levels. Conclusion: Cigarette smoking in nose-mouth exposure manner can induce acute lung injury in rats; and the degree of lung injury is closely related to the content of tar and other hazards in cigarettes.

Characteristic fragmentation behavior of tobacco-specific N-nitrosamines using electrospray ionization multistage tandem mass spectrometry incorporating deuterium labeling.

RATIONALE: Tobacco-specific N-nitrosamines (TSNAs) mainly include 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), N-nitrosonornicotine (NNN), N-nitrosoanabasine (NAB) and N-nitrosoanatabine (NAT) that are formed from tobacco alkaloids during the curing process and contained in tobacco and tobacco smoke. They are linked with carcinogenesis. Analytical methods for quality control of products and determination of their metabolites are therefore of great importance. METHODS: The characteristic fragmentation behaviors of tobacco-specific TSNAs have been studied by electrospray ionization multistage tandem mass spectrometry. The deutero-labeled TSNA compounds have also been employed to clarify the fragmentation mechanism, which further confirms the proposed fragmentation patterns. RESULTS: Detailed analysis of the resultant fragments shows there are two different kinds of fragmentation patterns with the general fragment backbone of pyrrolidine or piperidine rings. In one route, pyrrolidine or piperidine rings undergo direct fragmentation and form some stable intermediates without affecting the parent rings. The other, however, involves ring opening and then ring closure at the pyridine-2 carbon atom to form multi-membered rings. CONCLUSIONS: This characteristic fragmentation behavior therefore provides useful information on identification of TSNAs that may be used to monitor such kinds of compound in the biological metabolism.

Post-harvest induced production of salvianolic acids and significant promotion of antioxidant properties in roots of Salvia miltiorrhiza (Danshen).

Danshen, the dried roots of Salvia miltiorrhiza, is an extremely valued Traditional Chinese Medicine. Previously, we have demonstrated that salvianolic acid B (SaB), the important bioactive ingredient in this herb, was a post-harvest product. Here, we further reported that all salvianolic acids (SAs) in the roots were post-harvest products of the drying process. In addition, the results of various radical scavenging activity assays, including lipid peroxidation (1), DPPH (2), hydroxyl (3) and superoxide (4), were significantly increased along with the accumulation of total salvianolic acids in the process. The contents of chemical targets and antioxidant activities both reached the highest value under thermal treatment at 130 °C for 80 min. In this dehydration period, contents of SaB, and sum of nine SAs increased from 0.01% to 5.51%, and 0.20% to 6.61%; and IC50 of antioxidant activity decreased from 4.85 to 2.69 (1); 7.75 to 0.43 (2); 2.57 to 1.13 (3) and 17.25 to 1.10 mg/mL. These results further supported the hypothesis that the newly harvested plant roots were still physiologically active and the secondary metabolites might be produced due to dehydration stress after harvest. Our findings supplied an important and useful theoretical basis for promoting the quality of Danshen and other medicinal plant materials.

A rapid and sensitive method for hydroxyl radical detection on a microfluidic chip using an N-doped porous carbon nanofiber modified pencil graphite electrode.

Hydroxyl radicals (˙OH) play an important role in human diseases. Traditional detection methods are time consuming and require expensive instruments. Here, we present a simple and sensitive method for the detection of hydroxyl radicals on a microfluidic chip using an electrochemical technique. Aniline monomer is electrochemically polymerized on the surface of a pencil graphite electrode and carbonized at 800 °C. The resulting N-doped porous carbon nanofiber-modified pencil graphite electrode is embedded into a microfluidic chip directly as a working electrode. 4-Hydroxybenzoic acid (4-HBA) is selected as the trapping agent owing to its unique 3,4-DHBA product and high trapping efficiency. A low detection limit of 1.0 × 10(-6) M is achieved on the microfluidic chip. As a demonstration, the microfluidic chip is successfully utilized for the detection of ˙OH in cigarette smoke. The strong π-π stacking and hydrophobic interactions between the nitrogen-doped carbon materials and the pencil graphite make the modified electrode well-suited for the microfluidic chip.

Core-shell Ag@SiO(2) nanoparticles concentrated on a micro/nanofluidic device for surface plasmon resonance-enhanced fluorescent detection of highly reactive oxygen species.

A micro/nanofluidic device integrating a nanochannel in a microfluidic chip was developed for sensitive fluorescent determination of highly reactive oxygen species (hROS) enhanced by surface plasmon resonance-enhanced fluorescence (SPREF). The nanochannel was simply fabricated by polyaniline nanostructures modified on a glass slide. Core-shell Ag@SiO2 nanoparticles were concentrated in front of the nanochannel for fluorescence enhancement based on the SPREF effect. As a demonstration, hROS in the mainstream of cigarette smoke (CS) were detected by the present micro/nanofluidic device. The fluorescent probe for trapping hROS in puffs of CS employed a microcolumn that was loaded with a composite of DNA (conjugated fluorophores, FAM) and Au membrane (coated on cellulose acetate). With a laser-induced fluorescence detection device, hROS was determined on the basis of the amount of FAM groups generated by DNA cleavage. With the optimization of the trapping efficiency, we detected about 4.91 pmol of hROS/puff in the mainstream CS. This micro/nanofluidic-SPREF system promises a simple, rapid, and highly sensitive approach for determination of hROS in CS and other practical systems.

Sensitive determination of reactive oxygen species in cigarette smoke using microchip electrophoresis-localized surface plasmon resonance enhanced fluorescence detection.

A sensitive approach to the determination of reactive oxygen species (ROS) in puffs of cigarette smoke (CS) has been developed. The experimental system consists of a microfluidic chip electrophoresis and a laser induced fluorescence (LIF) device enhanced by localized surface plasmon resonance. Core-shell Ag@SiO2 nanoparticles were prepared and then immobilized on the surface of the microchannel to increase the fluorescence intensity based on localized surface plasmon resonance-enhanced fluorescence (LSPREF) effect. The ROS in puffs of CS were trapped via the oxidation of 2',7'-dichlorodihydrofluorescein (DCHF) that had been loaded on polyacrylonitrile (PAN) nanofibers in a micro-column. Determination of ROS was based on the amount of 2',7'-dichlorofluorescein (DCF), which is the sole product from DCHF oxidation. With the optimization of the trapping efficiency, we detected about 8.0 pmol of ROS per puff in the mainstream CS. This microchip electrophoresis-SPREF system enables sensitive quantitation of ROS in CS with low consumption of reagent, material, and analysis time.

Genetic variation in alkaloid accumulation in leaves of Nicotiana.

Alkaloids are plant secondary metabolites that are widely distributed in Nicotiana species and contribute greatly to the quality of tobacco leaves. Some alkaloids, such as nornicotine and myosmine, have adverse effects on human health. To reduce the content of harmful alkaloids in tobacco leaves through conventional breeding, a genetic study of the alkaloid variation among different genotypes is required. In this study, alkaloid profiles in leaves of five Nicotiana tabacum cultivars and Nicotiana tomentosiformis were investigated. Six alkaloids were identified from all six genotypes via gas chromatograph-mass spectrometry (GC-MS). Significant differences in alkaloid content were observed both among different leaf positions and among cultivars. The contents of nornicotine and myosmine were positively and significantly correlated (R(2)=0.881), and were also separated from those of other alkaloids by clustering. Thus, the genotype plays a major role in alkaloid accumulation, indicating a high potential for manipulation of alkaloid content through traditional breeding.