Quantitative assessment of the influence of common variations (rs8034191 and rs1051730) at 15q25 and lung cancer risk.

Several genome-wide association studies on lung cancer (LC) have reported similar findings of a new susceptibility locus, 15q25. After that, a number of studies reported that rs8034191 and rs1051730 polymorphisms at chromosome 15q25 have been implicated in LC risk. However, studies have yielded contradictory results. To derive a more precise estimation of the relationship, a meta-analysis of 43,742 LC cases and 58,967 controls from 17 published case-control studies was performed. Overall, significantly elevated LC risk was associated with rs8034191-C (OR = 1.26, 95% CI 1.22-1.31, P < 10(-5)) and rs105173-A variant (OR = 1.28, 95% CI 1.20-1.36, P < 10(-5)) when all studies were pooled into the meta-analysis. In the subgroup analysis by ethnicity, significantly increased risks were found for rs8034191 and rs105173 polymorphisms among Caucasians and African American, while no significant associations were observed for the two polymorphisms in East Asians. In addition, we found that rs8034191 and rs105173 confer risk, for both adenocarcinoma and squamous cell carcinoma when stratified by histological types of LC. Furthermore, our results on stratified analysis according to smoking status showed an increased LC risk in ever-smokers, while no associations were detected among never-smokers for the two polymorphisms. In conclusion, this meta-analysis demonstrated that the two common variations (rs8034191 and rs1051730) at 15q25 are a risk factor associated with increased LC susceptibility, but these associations vary in different ethnic populations.

Non-synonymous single nucleotide polymorphisms in the P2X receptor genes: association with diseases, impact on receptor functions and potential use as diagnosis biomarkers.

P2X receptors are Ca2+-permeable cationic channels in the cell membranes, where they play an important role in mediating a diversity of physiological and pathophysiological functions of extracellular ATP. Mammalian cells express seven P2X receptor genes. Single nucleotide polymorphisms (SNPs) are widespread in the P2RX genes encoding the human P2X receptors, particularly the human P2X7 receptor. This article will provide an overview of the non-synonymous SNPs (NS-SNPs) that have been associated with or implicated in altering the susceptibility to pathologies or disease conditions, and discuss the consequences of the mutations resulting from such NS-SNPs on the receptor functions. Disease-associated NS-SNPs in the P2RX genes have been valuable in understanding the disease etiology and the receptor function, and are promising as biomarkers to be used for the diagnosis and development of stratified therapeutics.

Stem Cell-Derived Exosomes: A New Method for Reversing Skin Aging.

Senescence is an inevitable natural life process that involves structural and functional degeneration of tissues and organs. Recently, the process of skin aging has attracted much attention. Determining a means to delay or even reverse skin aging has become a research hotspot in medical cosmetology and anti-aging. Dysfunction in the epidermis and fibroblasts and changes in the composition and content of the extracellular matrix are common pathophysiological manifestations of skin aging. Reactive oxygen species and matrix metalloproteinases play essential roles in this process. Stem cells are pluripotent cells that possess self-replication abilities and can differentiate into multiple functional cells under certain conditions. These cells also possess a strong ability to facilitate tissue repair and regeneration. Stem cell transplantation has the potential for application in anti-aging therapy. Increasing studies have demonstrated that stem cells perform functions through paracrine processes, particularly those involving exosomes. Exosomes are nano-vesicular substances secreted by stem cells that participate in cell-to-cell communication by transporting their contents into target cells. In this chapter, the biological characteristics of exosomes were reviewed, including their effects on extracellular matrix formation, epidermal cell function, fibroblast function and antioxidation. Exosomes derived from stem cells may provide a new means to reverse skin aging.

Identification of key residues coordinating functional inhibition of P2X7 receptors by zinc and copper.

P2X(7) receptors are distinct from other ATP-gated P2X receptors in that they are potently inhibited by submicromolar concentrations of zinc and copper. The molecular basis for the strong functional inhibition by zinc and copper at this purinergic ionotropic receptor is controversial. We hypothesized that it involves a direct interaction of zinc and copper with residues in the ectodomain of the P2X(7) receptor. Fourteen potential metal interacting residues are conserved in the ectodomain of all mammalian P2X(7) receptors, none of which is homologous to previously identified sites in other P2X receptors shown to be important for functional potentiation by zinc. We introduced alanine substitutions into each of these residues, expressed wild-type and mutated receptors in human embryonic kidney 293 cells, and recorded resulting ATP and BzATP-evoked membrane currents. Agonist concentration-response curves were similar for all 12 functional mutant receptors. Alanine substitution at His(62) or Asp(197) strongly attenuated both zinc and copper inhibition, and the double mutant [H62A/D197A] mutant receptor was virtually insensitive to inhibition by zinc or copper. Thus, we conclude that zinc and copper inhibition is due to a direct interaction of these divalent cations with ectodomain residues of the P2X(7) receptor, primarily involving combined interaction with His(62) and Asp(197) residues.

Monitoring microarray-based gene expression profile changes in hepatocellular carcinoma.

AIM: To find out key genes responsible for hepatocarcinogenesis and to further understand the underlying molecular mechanism through investigating the differential gene expression between human normal liver tissue and hepatocellular carcinoma (HCC). METHODS: DNA microarray was prepared by spotting PCR products of 1 000 human genes including 445 novel genes, 540 known genes as well as 12 positive (housekeeping) and 3 negative controls (plant gene) onto treated glass slides. cDNA probes were prepared by labeling normal liver tissue mRNA and cancer liver tissue mRNA with Cy3-dUTP and Cy5-dUTP separately through reverse transcription. The arrays were hybridized against the cDNA probe and the fluorescent signals were scanned. The data obtained from repeated experiments were analyzed. RESULTS: Among the 20 couple samples investigated (from cancerous liver tissue and normal liver tissue), 38 genes including 21 novel genes and 17 known genes exhibited different expressions. CONCLUSION: cDNA microarray technique is powerful to identify candidate target genes that may play important roles in human carcinogenesis. Further analysis of the obtained genes is helpful to understand the molecular changes in HCC progression and ultimately may lead to the identification of new targets for HCC diagnosis and intervention.

[Rapid detection of the genotyping of hepatitis C virus using DNA chip with coloration methods].

OBJECTIVE: To develop a new DNA chip with coloration, which can be used for rapid and economical detection of the genotyping of hepatitis C virus (HCV). METHODS: Probes and primers were designed according to the sequence of HCV 5' non-coding region (5' NCR) to fabricate DNA chip. Experimental group consisted of 60 positive serum samples and control group consisted of 20 negative serum samples. To obtain the aimed gene, then they were hybridized with DNA chip. Finally, the results showed in a nylon film. The results of DNA sequencing of samples were used as the control in double blind experimental. RESULTS: Using DNA chip, HCV was detected in positive of all serum specimens of experimental group and negative in control group. The determination of HCV genotype by DNA chip showed corresponding rate of 96.7% with those by sequence assay. CONCLUSION: It showed higher specialty and sensitivity using DNA chip to detect the genotype of HCV. It would be valuable for the clinical genotyping of HCV

Highly sensitive enumeration of circulating tumor cells in lung cancer patients using a size-based filtration microfluidic chip.

Circulating tumor cells (CTCs) in the peripheral blood could serve as a surrogate marker for the diagnosis of cancer metastasis and for therapeutic evaluation. However, the separation and characterization of CTCs is technically challenging owing to the extremely low number of CTCs present. Here we developed a size-based and high-throughput microfluidic chip, which exploits filtration microchannels to isolate the relatively larger CTCs from the rest of the blood constituents. High isolation efficiency of our microfluidic chip was demonstrated with three lung cancer cell lines spiked in blood samples at an optimal flow rate of 0.4 mL/h. The average recovery rates of 96%, 95% and 92% were obtained for A549, SK-MES-1, and H446, respectively. To clinically validate the chip, we also employed it to isolate CTCs from 59 lung cancer patients. CTCs were detected in 96.7% of patients with the mean number of 18.6 cells/mL, which was significantly higher than normal controls (P<0.05). The work here indicates that the size-based microfluidic platform with the advantage of capturing tumor cells without reliance on cell surface expression markers can provide a novel, inexpensive and effective tool for CTC detection and evaluation of cancer status.

Photoluminescence properties of Re(I) complex doped composite submicron fibers prepared by electrospinning.

In this paper, a Re(I) complex of Re(CO)3(Bphen)Br, where Bphen=4,7-diphenyl-1,10-phenanthroline is synthesized, and doped into poly(vinylpyrrolidone) submicron fibers through electrospinning technique. Their morphology, absorption, and emission spectra are investigated in detail. The composite fibers exhibit smooth and uniform morphology on the substrate, with an average diameter of ∼800 nm. A bright yellow emission peaking at 543 nm is observed from these composite fibers, and this emission is attributed to the triplet emissive state of Re(CO)3(Bphen)Br. When doped into poly(vinylpyrrolidone) matrix, the emission shows a blue-shift tendency compared with that of bulk sample, correspondingly, the photostability is also largely improved. Detailed analysis suggests that Re(CO)3(Bphen)Br occupies a homogeneous site within poly(vinylpyrrolidone) matrix, and the matrix provides a rigid environment for Re(CO)3(Bphen)Br.

Identification of pore residues engaged in determining divalent cationic permeation in transient receptor potential melastatin subtype channel 2.

The molecular basis for divalent cationic permeability in transient receptor potential melastatin subtype (TRPM) channels is not fully understood. Here we studied the roles of all eight acidic residues, glutamate or aspartate, and also the glutamine residue between pore helix and selectivity filter in the pore of TRPM2 channel. Mutants with alanine substitution in each of the acidic residues, except Glu-960 and Asp-987, formed functional channels. These channels exhibited similar Ca(2+) and Mg(2+) permeability to wild type channel, with the exception of the E1022A mutant, which displayed increased Mg(2+) permeability. More conservative E960Q, E960D, and D987N mutations also led to loss of function. The D987E mutant was functional and showed greater Ca(2+) permeability along with concentration-dependent inhibition of Na(+)-carrying currents by Ca(2+). Incorporation of negative charge in place of Gln-981 between the pore helix and selectivity filter by changing it to glutamate, which is present in the more Ca(2+)-permeable TRPM channels, substantially increased Ca(2+) permeability. Expression of concatemers linking wild type and E960D mutant subunits resulted in functional channels that exhibited reduced Ca(2+) permeability. These data taken together suggest that Glu-960, Gln-981, Asp-987, and Glu-1022 residues are engaged in determining divalent cationic permeation properties of the TRPM2 channel.

Conserved cysteine residues in the pore region are obligatory for human TRPM2 channel function.

TRPM2 proteins belong to the melastatin-related transient receptor potential or TRPM subfamily and form Ca(2+)-permeable cationic channels activated by intracellular adenosine diphosphoribose (ADPR). The TRPM2 channel subunit, like all its close relatives, is structurally homologous to the well-characterized voltage-gated potassium channel subunits, each containing six transmembrane segments and a putative pore loop between the fifth and sixth segments. Nevertheless, the structural elements determining the TRPM2 channel functions are still not well understood. In this study, we investigated the functional role of two conserved cysteine residues (at positions 996 and 1008) in the putative pore region of the human TRPM2 by site-directed mutagenesis, combined with electrophysiological and biochemical approaches. Expression of wild-type hTRPM2 channels in human embryonic kidney (HEK-293) cells resulted in robust ADPR-evoked currents. Substitution of cysteine with alanine or serine generated mutant channels that failed to be activated by ADPR. Furthermore, experiments done by Western blot analysis, immunocytochemistry, biotin labeling, and coimmunoprecipitation techniques showed no obvious changes in protein expression, trafficking or membrane localization, and the ability to interact with neighboring subunits that is required for channel assembly. Coexpression of wild-type and mutant subunits significantly reduced the ADPR-evoked currents; for the combination of wild-type and C996S mutant subunits, the reduction was approximately 95%, indicating that incorporation of one or more nonfunctional C996S subunits leads to the loss of channel function. These results taken together suggest that the cysteine residues in the pore region are obligatory for TRPM2 channel function.

[Establishing DNA chip technique for detecting hepatitis C virus genotypes and primary application].

OBJECTIVE: To establish a new method based DNA chip technique for detecting HCV genotypes. METHODS: Genotyping probes were designed according to the sequence of HCV 5' NCR to generate DNA chip. The probes on DNA chip contains 5 major genotypes and 8 subtypes. The DNA fragment amplified by labeling Cy5 fluorescence was hybridized with DNA chip. RESULTS: Fifty-five out of 65 isolates detected by DNA chip belonged to 1b- DNA sequencing of form a part of the isolates was used as the control. The results of both were completely consistent. CONCLUSION: The method is simple and rapid with high specificity and sensitivity. It can be applied in detection of HCV RNA and genotypes.