[Effects of paraquat on the proliferation of human neural stem cells via DNA methylation].

Objective: To investigate the effects of Paraquat on neural stem cell proliferation in vitro and explore the its mechanism based on DNA methylation pathway. Methods: Nestin, ß-tubulin III, and glial fibrillary acidic protein (GFAP) were detected by indirect immunofluorescence assay to evaluate self renewal and differentiation potentia of ReNcell CX human neural stem. The cells were treated with terminal concentrations of 0, 5, 25, 50, and 100µmol/L PQ for 24 hours, and the cells were induced by 50 µmol/L PQ for different time (6, 12, 24, 48 h). Cell viability was determined by MTT assay. The proliferation of neural stem cells was evaluated using Sox2/Brdu and Nestin/Brdu double immunofluorescence staining. The global DNA methylation level was assayed by MethyflashTM methylated DNA Quantification kit. The expression levels of Dnmts mRNA and protein were analyzed by quantitative reverse transcription polymerase chain reaction(qRT-PCR) and Western blot, respectively. Results: Immunofluorescence showed that nestin was primarily expressed in proliferative neural stem cell and peotein biomarkers (ß-tubulin III, GFAP) for neuron and astrocyte were expressed in differentiated cells. MTT assay showed PQ induced cell survival rate decrease in a time and dose dependent manner. Double immunfluorescence staining of cells showed colocalization of Sox2 and Brdu. The percentage of Brdu/Sox2 positive cells was significantly lower in the PQ-exposed (25, 50, 100µmol/L PQ treatment) groups compared to control (P<0.05); Meanwhile, The percentage of Brdu/Nestin positive cells was also significantly lower in the PQ-exposed(50,100µmol/L PQ treatment) groups compared to control (P<0.05). The results of global DNA methylation revealed a significant decrease in PQ-exposed groups (P<0.05). Western blot showed that compared with control group, the protein and mRNA levels of Dnmt1, Dnmt3a in PQ-exposed group were significantly decreased (P<0.05), but there was a significant increase in expression level of Dnmt3b in 50, 100 µmol/L PQ-treated group(P<0.05). Conclusion: Paraquat could inhibite the proliferation of human neural stem cells through reducing the level of DNA methylation reaction by suppressing the protein expression and transcription of DNA methylated transferase(Dnmts).

[Study on the mechanism of brain damage based on structural covariant network to evaluate the brain structure of nicotine addicts].

Objective: To explore the neuromechanism of nicotine dependence, structural covariation networks (SCNs) based on voxel-based morphometry (VBM) were used to study the synergistic changes in gray matter volume in different cerebral cortices of nicotine dependent individuals. Methods: During the period from August 2016 to February 2018, a total of 118 long-term smokers and 57 non-smoking healthy controls (both 18-55 male volunteers) through online platforms and leaflets were recruited. The subjects were scanned with SIEMENS Skyro 3.0T magnetic resonance scanner and underwent routine MRI sequence (preliminary elimination of intracranial lesions) and 3D-T1 (3D-mprage) sequence structure. Two imaging experts used Matlab software platform to carry on segmentation by using SPM8, and to find out the differences between the two groups of brain regions, and differences in brain regions as region of interest (ROI) structure association network analysis. Results: The gray matter volume (GMV) of the right anterior central gyrus and the left inferior parietal lobe in the smoking group decreased(voxels size were 55 and 284, respectively), and no gray matter volume (GMV) area increased. The network structure of covariant analysis found that when the inferior parietal lobe as the seed points, the smoking group showed a rising trend in left parietal lobe and left temporal pole, bilateral middle temporal gyrus, left postcentral gyrus and right middle frontal gyrus gray matter volume, and a downtrend in the right side of the left medial frontal gyrus, superior parietal lobe, bilateral temporal gyrus, left cingulate gyrus and left cerebellum (central) compared with the control group. Conclusion: In long-term smokers, there is a volume change of gray matter in the brain structure. Abnormal changes in the structure covariant network of the inferior parietal lobe can lead to impaired brain function in nicotine dependent patients.

[Role of MAPK signaling pathway in epithelial-mesenchymal transition of type II alveolar epithelial cells induced by Paraquat].

Objective: To investigate the roles of p38 mitogen-activated protein kinases (p38 MAPK) , extracellular regulated protein kinases (ERK) and c-Jun N-tenninal kinases (JNK) of MAPK signaling pathway in Paraquat-induced epithelial to mesenchymal transition (EMT) of type II alveolarepithelial cells. Methods: RLE-6NT cells were incubated with different concentrations of PQ (0, 25, 50, 100µmol/L) for 6, 12 and 24 h. Cell morphology alteration was observed under phase-contrast microscopy. Cell viability was determined using an MTT assay. Cell migration ability was detected using scratch wound assay. Protein expression of P-p38 MAP, P-Erk1/2, P-JNK, E-cad, ZO-1, Vimentin and а-SMA were detected by western blot. The level of genes related to fibrosis (COL-I, COL-III, FN and FSP-1) were analyzed via quantitative real-time RT-PCR. Results: Cell morphology started to undergo EMT changes with a phenotype characteristic of mesenchymal cells, including an elongated shape and a lack of tight cell-cell adhesions induced by 100µmol/L PQ treatment in a time-dependent manner. MTT showed that cell viability decreased with increasing PQ concentration (50、100、200、300 µmol/L PQ treatment for 24 h) and increasing treatment time (200 µmol/L PQ treatment for 6, 12, 24, 36, 48 h) . Compared to control group, the expressions of the epithelial phenotype marker E-cad and ZO-1 significantly decreased with PQ treatment (50, 100µmol/L) in a time-dependent manner (P<0.05) . Additionally, the level of the mesenchymal marker (a-SMA, vimentin) dramatically increased with PQ treatment in the same concentration-and time-dependent manner (P<0.05) . Cell migration ability was markedly increased after 24 h of 100 µmol/L PQ treatment compared to control (P<0.05) . The phosphorylated forms of p38 MAPK, Erk1/2, and JNK were increased at 24 h after stimulation with PQ (P<0.05) . This PQ induced (100 µmol/L) phosphorylation was markedly attenuated in the presence of the p38 MAPK, ERK and JNK inhibitors (SB-203580, SP-600125 and PD98059) respectively. Furthermore, RT-PCR showed that PQ significantly induced the upregulation expression of COL I and III mRNA, Fn, and FSP-1 mRNA (P<0.05) . Conclusion: PQ-induced pulmonary fibrosis occurs via EMT, which is mediated by the MAPK pathway.

[The regulation of tight junction protein via PKCα/ß for abnormal permeability of brain microvascular endothelial cells exposed to paraquat].

Objective: To explore if conventional protein kinase C (cPKC: PKCα and PKCß) contributes to paraquat (PQ) -induced abnormal permeability of mouse brain microvascular endothelial cells (BMECs) via the regulation of tight junction (TJ) proteins. Methods: The immortalized mouse brain endothelial cell line (bEnd.3) was used to establish a monolayer blood-brain barrier (BBB) model. In order to evaluate the function of the in vitro BBB model, the transendothelial electrical resistance (TEER) and permeability were measured by a Millicell-ERS volt-ohmmeter and sodium fluorescent (Na-FLU) , respectively. MTT assay was used to determine the relative survival rate of cells. The dose-response relationship was determined by treating cells with 0, 50, 100, 200, and 300 µmol/L PQ for 24 hours. The time-response relationship was determined by treating cells with 200 µmol/L PQ for 6, 12, 24, 48, and 72 hours. After the treatment of cells with 0, 100, 200, and 300 µmol/L PQ for 24 hours, the protein and mRNA expression levels of ZO-1, Occludin, and Claudin-5 were measured by immunofluorescence (IF) and quantitative real-time RT-PCR (qRT-PCR) , respectively; the expression of PKCα, PKCß, phosphorylated (p) -PKCα, and p-PKCß was determined by Western blot. After the treatment of cells with 200? mol/L PQ for 24 hours following the pretreatment with a classical PKC inhibitor (Go 6983, 1 µmol/L) for 1 hour, the protein expression of ZO-1, Occludin, Claudin-5, p-PKCα, and p-PKCß was measured by Western blot. Results: The TEER of the bEnd. 3 cells increased gradually with the cell culture time, and reached a peak value of 114.3±6.9 Ω·cm(2) on day 6. According to the permeability analysis by Na-FLU, cell permeability gradually decreased with the cell culture time, and reached 1.7±0.2 cm/min on day 6, suggesting a well-behaved barrier function of cells. Compared with the control group, the survival rates of the bEnd.3 cells were significantly reduced after exposure to 100, 200, or 300 µmol/L PQ for 24 hours (P<0.05) , or after exposure to 200 µmol/L PQ for 6, 12, 24, 48, or 72 hours (P <0.05) , indicating a dose-and time-dependent relationship. The IF and qRT-PCR results showed that the protein and mRNA expression levels of ZO-1, Occludin, and Claudin-5 were significantly reduced with the increase in the concentration of PQ (P<0.05) . The Western blot analysis showed that compared with the control group, cells exposed to PQ had significantly higher protein expression of p-PKCα and p-PKCß and significantly lower protein expression of ZO-1, Occludin, and Claudin-5 (P<0.05) . Compared with the PQ treatment group, the Go 6983 intervention group had significantly higher protein expression of ZO-1, Occludin, and Claudin-5 and significantly lower protein expression of p-PKCα and p-PKCß (P<0.05) . Conclusion: By activation of cPKC (PKCα and PKCß) , PQ reduces the protein and mRNA expression of TJ proteins and enhances the permeability of murine BMECs.

[Structural changes of brain gray matter in male long-term smokers under magnetic resonance imaging].

Objective: To evaluate gray matter structure changes in long-term male smokers by voxel-based morphological method. Methods: Fifty long-term smokers and 37 non-smoking healthy volunteers were scanned with Siemens Skyro 3.0T magnetic resonance scanner from August 2014 to August 2016. The subjects underwent routine MRI (excluding intracranial lesions) sequences and 3D-T1 structural sequences (3D-mprage). SPM8 pretreatment based on Matlab was used to analyze the structural data. All of the data were analyzed by SPM8 software. The data were compared between groups with independent sample t test. Spearman correlation analysis was used to analyze the relationship between gray matter volume (GMV) and smoking data of two groups. Results: The gray matter volume of bilateral thalamic, right supramarginal gyrus, left supramarginal gyrus and left putamen of smoking group were (0.55±0.07), (0.40±0.05), (0.48±0.07) and (0.14±0.04) voxels, respectively, and the gray matter volume of the corresponding gyri in control group were (0.61±0.09), (0.43±0.06), (0.54±0.07) and (0.16±0.03) voxels, respectively; and the gray matter volume of smoking group were all lower than those in control group (t=-3.81, -3.51, -3.86, -2.33, all P<0.05), family wise error (FWE) correction (P<0.05). The gray matter volume of bilateral thalamus, right supramarginal gyrus and left putamen was negatively correlated with smoking index (r=-0.368, -0.189, -0.274, all P<0.05), and also negatively correlated with smoking years (r=-0.391, -0.221, -0.355, all P<0.05), and bilateral thalamus gray matter volume was negatively correlated with daily cigarette smoking (r=-0.186, P<0.05). Conclusion: The changes of brain structure of smokers mainly occur on reward-related pathways and marginal systems, and related to accumulation of cigarette smoking.

Integrative Profiling of Bee Communities from Habitats of Tropical Southern Yunnan (China).

Understanding and managing pollination service is hindered by taxonomic impediments and paucity of data, particularly in the tropics. Herein we apply integrative species delineation and taxonomy to test impacts of land use on the diversity of bee communities within Xishuangbanna (Yunnan, south China), a highly biodiverse tropical region which has undergone extensive land conversion to rubber plantation. 128 Operational Taxonomic Units (OTU) were inferred by an iterative and integrative approach. Bee activity differed significantly across land use samples, although community composition corresponded more to level of vegetation density, when accounting for spatial structure. Species diversity was high in young rubber plantations, although composition overlapped with other species-rich habitats (natural forest edge and river banks), and older plantations (>8 years) showed very low diversity under all measures. Community structures were similar between the natural forest interior and edge, although analysis indicated contrasting drivers of diversity, with clustering in the interior and overdispersion in the forest edge. Further, phylogenetic diversity and derived indices were underestimated when reference data were omitted from analysis. The description of bee communities herein permits more informed choices in land management with respect to ensuring continuation of essential services by bees.

Effects of upregulation of Id3 in human lung adenocarcinoma cells on proliferation, apoptosis, mobility and tumorigenicity.

The inhibitor of DNA-binding/differentiation 3 (Id3) protein is a helix-loop-helix transcription factor and may have an important role in cell proliferation and differentiation. This study was to evaluate the effects of upregulation of Id3 in human lung adenocarcinoma cells on proliferation, apoptosis, mobility and tumorigenicity. Short interference RNA suppression of Id3 (miRId3) in A549 cells was used to investigate the functional role(s) of Id3. Next, we used in vitro wound-healing assay and trans-well assay to study the effects of overexpressed Id3 on migration and invasion of A549 cells. Furthermore, to explore the influence of overexpressed Id3 on in vivo tumorigenesis, adenoviruses containing Id3 gene (Ad-Id3) and empty vector (Ad-LacZ) were generated. Co-transfection of pcDNA/miRId3 and pEGFP/Id3 into A549 cells reversed the Id3-induced cell proliferation inhibition and apoptosis. Upon Id3 transfection, A549 cells displayed decreased migratory and invasive capabilities, however, co-transfection of miRId3 and Id3 into A549 cells reversed the Id3-induced inhibitions of migratory and invasive capabilities. Three groups of nude mice were inoculated with Ad-LacZ, Ad-Id3 transfectants and untransfected A549 cells, respectively. Twenty-eight days after inoculation, tumors induced by Ad-Id3 transfectants grew much more slowly compared with Ad-LacZ transfectants and control group. This study provides for the first time both in vitro and in vivo proofs that forced expression of Id3 in lung adenocarcinoma cells reduces tumor growth rate and may be a potential target for tumor suppression.

Complete sequence of the mitochondrial genome of the Japanese buff-tip moth, Phalera flavescens (Lepidoptera: Notodontidae).

We sequenced the complete mitochondrial genome of Phalera flavescens. The mitogenome is 15,659 bp in length, including 13 protein-coding genes (atp6, atp8, cox1-3, nad1-6, nad4L, cob), two ribosomal RNAs (rrnS and rrnL), 22 transfer RNAs and an AT-rich region, a putative control region (D-loop). Gene order and orientation were found to be identical to those of other completely sequenced lepidopteran mitogenomes. All 13 protein-coding genes start with the common codon ATN, except for the cox1 gene, which uses CGA as the initial codon. Nine of the 13 protein-coding genes stop with codon TAA, while the cox1, cox2, nad5, and nad4 genes stop with the single nucleotide T. All tRNA genes can be folded into canonical cloverleaf secondary structure, except for trnS1, which loses the ''DHU'' arm. Six overlapping sequences totaling 20 bp (1-8 bp for each sequence) and 16 intergenic spacer sequences, totaling 276 bp (1-58 bp for each sequence) are scattered throughout the genome; the largest intergenic spacer is located between the trnQ and nad2 genes. A microsatellite-like structure (AT)(6)ACC(AT)(6) and 16-bp poly-T elements preceded by the ATTTA motif are present in the D-loop region. Additionally, unexpectedly, an extra 190-bp insertion, with unknown function, was found in the small subunit rRNA gene (rrnS); this gene is the longest known (1020 bp) among all of the Lepidoptera.

A fuzzy-set-theory-based approach to analyse species membership in DNA barcoding.

Reliable assignment of an unknown query sequence to its correct species remains a methodological problem for the growing field of DNA barcoding. While great advances have been achieved recently, species identification from barcodes can still be unreliable if the relevant biodiversity has been insufficiently sampled. We here propose a new notion of species membership for DNA barcoding-fuzzy membership, based on fuzzy set theory-and illustrate its successful application to four real data sets (bats, fishes, butterflies and flies) with more than 5000 random simulations. Two of the data sets comprise especially dense species/population-level samples. In comparison with current DNA barcoding methods, the newly proposed minimum distance (MD) plus fuzzy set approach, and another computationally simple method, 'best close match', outperform two computationally sophisticated Bayesian and BootstrapNJ methods. The new method proposed here has great power in reducing false-positive species identification compared with other methods when conspecifics of the query are absent from the reference database.

Overexpression of Id3 induces apoptosis of A549 human lung adenocarcinoma cells.

OBJECTIVES: Inhibitor of differentiation 3 (Id3) protein has been implicated in the control of multiple cell death signalling pathways and in aetiology of numerous diseases. The aims of this study were to construct a recombinant eukaryotic expression vector (pEGFP/Id3), containing human Id3 (hId3) fused with enhanced green fluorescent protein (EGFP), and to determine effects of ectopic Id3 overexpression, on human lung adenocarcinoma cell (A549) proliferation. MATERIALS AND METHODS: Human Id3 cDNA was inserted into pEGFP-N1 vector to yield the recombinant eukaryotic expression vector pEGFP/Id3. Cells were transfected with pEGFP or pEGFP/Id3, and proliferation of EGFP-expressing cells was monitored by flow cytometry (FCM) and confocal fluorescence microscopy. RT-PCR, immunoblotting and immunocytochemistry were used to assess Id3 mRNA transcription and protein expression. Apoptosis was evaluated by Annexin V/7-AAD staining and FCM, while nuclear morphology of apoptotic cells was examined using Hoechst 33258 staining. RESULTS: Over 4 days transfection with pEGFP, the proportion of EGFP-positive A549 cells peaked at approximately 60% by 48 h and remained stable over the next 48 h. In contrast, the proportion of EGFP-positive cells in cultures transfected with pEGFP/Id3 decreased from a peak of 60% at 48 h to <5% at 96 h, suggesting that Id3 expression inhibited cell proliferation or survival. Annexin V/7-AAD and Hoechst 33258 staining revealed significantly higher rates of apoptosis in pEGFP/Id3-transfected cells. CONCLUSION: Overexpression of Id3 triggered apoptosis in A549 human lung adenocarcinoma cells, implicating Id3 in negative control of tumour growth. These Id3-induced pro-apoptotic signalling pathways require further study, but this preliminary investigation suggests that Id3 regulation could be exploited in anti-tumour therapies.

Estimating sample sizes for DNA barcoding.

Sample size has long been one of the basic issues since the start of the DNA barcoding initiative and the global biodiversity investigation. As a contribution to resolving this problem, we propose a simple resampling approach to estimate several key sampling sizes for a DNA barcoding project. We illustrate our approach using both structured populations simulated under coalescent and real species of skipper butterflies. We found that sample sizes widely used in DNA barcoding are insufficient to assess the genetic diversity of a species, population structure impacts the estimation of the sample sizes, and hence will bias the species identification potentially.

Genetic analysis of four porcine avian influenza viruses isolated from Shandong, China.

A Bayesian phylogenetic analysis of eight separate gene segments indicated A/Swine/Shandong/2/2003 (H5N1), A/Swine/Shandong/na/2003 (H9N2), A/Swine/Shandong/nb/2003 (H9N2) and A/Swine/Shandong/nc/2005 (H9N2) probably represent two multiple reassortant lineages, that had not been described before, with genes coming from H5N1, H9N2 and other lineages from poultry in Asia. Amino acid motifs within the haemagglutinin sequence of A/Swine/Shandong/nb/2003 suggested it may be able to infect people, whereas the sequences of the other three isolates suggested they would not have had that capability. Our analysis emphasizes the need for a comprehensive study of the interactions between H5N1 and H9N2 viruses in Asia that includes sequencing and phylogenetic investigation.