Toxicities were compared to outputs from SBRs dosed with ionic/bulk analogs, undosed SBRs, and pristine ENMs. Pristine nanoAg and NZVI showed significant cytotoxicity to A549 cells in a dose-dependent manner from 1 to 67 μg/mL, while nanoTiO2 and nanoCeO2 only exerted cytotoxicity at 67 μg/mL. Only nanoAg induced a genotoxic response, at 9, 33 and 53 μg/mL. However, no significant cytotoxic or genotoxic effects of the SBR effluents or biosolids containing nanomaterials were observed.Here we characterize the toxicity of environmentally-relevant forms of engineered nanomaterials (ENMs), which can transform during wastewater treatment and persist in aqueous effluents and biosolids. In an aerosol exposure scenario, cytotoxicity and genotoxicity of effluents and biosolids from lab-scale sequencing batch reactors (SBRs) to A549 human lung epithelial cells were examined. The SBRs were dosed with nanoAg, nano zero-valent iron (NZVI), nanoTiO Toxicities were compared to outputs from SBRs dosed with ionic/bulk analogs , outputs from undosed SBRs, and pristine ENMs.This study examined cytotoxicity and genotoxicity of effluents and biosolids from lab-scale sequencing batch reactors (SBRs) receiving ENMs to A549 human lung epithelial cells. The SBRs were dosed with nanoAg, nano zero-valent iron (NZVI), nanoTiO22.12 , nanoCeO2  and their ionic/bulk analogs  were prepared/purchased and characterized as described in Ma et al. 2, or nanoCeO2; (2) SBRs dosed with Ag+, Fe2+, bulkTiO2, or bulkCeO2; and (3) undosed SBRs. The dosing was initiated at 0.1 mg/L and sequentially increased to 1, 10 and 20 mg/L. The SBRs were actively nitrifying at the time of this study; further details on SBR performance are reported in Ma et al. NanoAg , NZVI , nanoTiO2.22. Cells were seeded at a density of 1 × 104 in 100 μL culture medium in each well of 96-well plates. At 80% confluency, they were treated for 24 h either with pristine nanomaterials, ionic/bulk analogs, SBR aqueous effluents, or SBR biosolids as indicated. The exposure concentrations of the pristine materials (Table S1) varied from 1 to 67 μg/mL for the cytotoxicity assay to estimate IC50 values. Genotoxicity assays were carried out at key concentration values based on the results of the cytotoxicity assays (Table S1). The exposure concentrations of nanomaterials and ionic/bulk materials in SBR effluents and biosolids are shown in A549 human lung alveolar epithelial cells were obtained from ATCC . Cells were cultured in Dulbecco's Modified Eagle's Medium  containing 10% heat inactivated fetal bovine serum  and 1% penicillin–streptomycin , and were maintained in a humidified incubator at 37 °C and 5% CO2.32 Microplate Reader  at 440 nm with a reference wavelength of 660 nm. All treated A549 cells (a) were tested in triplicate in three independent experiments with three controls: (b) untreated A549 cells in culture medium; (c) samples in culture medium without A549 cells; and (d) culture medium only. In a single experiment, cell viability was calculated as percentage of the average absorbance derived from triplicate runs of treated cells relative to untreated control cells, with absorbances of corresponding controls subtracted out to address possible matrix interferences: fraction cell viability = [(a − c)/(b − d)].Cell viability was measured using the WST-1 assay  based on quantification of mitochondrial activity as an indicator of cytotoxicity. In viable cells, the tetrazolium salt WST-1 is converted to soluble formazan dye by mitochondrial succinate-tetrazolium reductase, which can be quantified by absorbance. After the A549 cells were treated with the samples for 24 h, WST-1 reagent was added to each well at 1/10 volume of the medium. The absorbance was quantified after incubating at 37 °C for 3 h using a Tecan Safire2.42O2 for 10 min were included as negative and positive controls, respectively. Images were analyzed using ImageJ 1.47 (http://rsbweb.nih.gov/ij/) with a macro designed to subtract background and count the number of foci within the defined nucleus masks.DNA damage in A549 cells was detected using immunofluorescent labeling of γH2AX foci as described elsewhere 2.5t test or pairwise t test was conducted in R-2.8.1 (http://cran.r-project.org/bin/windows/base/old/2.8.1/) to determine statistical differences between samples at a significance level of 0.05 (p < 0.05).The data were presented as mean ± standard deviation of three independent experiments. Student's 33.1+, NZVI and Fe2+ for 24 h decreased significantly (p < 0.05) in a dose-dependent manner . The IC50 values for these materials were estimated to be 53 ± 2, 21 ± 0.1, 38 ± 2 and 55 ± 2 μg/mL, respectively. In particular, the IC50 of Ag+ was significantly lower than that of nanoAg (p = 5 × 10−4), a result that is in agreement with other studies using A549 cell targets 2+ (p = 6 × 10−4). In another study, the cytotoxicty of NZVI (synthesized through reduction of FeCl3 by NaBH4 and coated with Pd) to human bronchial epithelial cells 16HBE14o was not significantly different from Fe2+versus 16HBE14o). The viability of cells exposed to nanoTiO2, nanoCeO2 and their bulk analogs only decreased at a concentration of 67 μg/mL, by 10–20% . But in a few other studies, no significant cytotoxicity of nanoTiO2 and nanoCeO2 to A549 cells was observed, even at concentrations up to 100 μg/mL Based on the WST-1 assay, the viability of cells exposed to nanoAg, Ag+ showed significantly higher numbers of γH2AX foci per cell  and greater percentages of cells containing γH2AX foci  (p < 0.05) relative to untreated control cells, suggesting genotoxicity resulting from DNA double strand breaks. The number of γH2AX foci per cell exposed to 9 μg/mL nanoAg was not significantly different from that of the control cells , but the percentage of cells containing γH2AX foci was significantly higher than for the control cells . Although IC50 concentrations of NZVI and Fe2+ , and nanoTiO2, nanoCeO2 and the bulk analogs at 67 μg/mL induced significant cytotoxicity to A549 cells, the number of γH2AX foci were not significantly different in cells treated with these materials . To the authors’ knowledge, genotoxicity of NZVI to human cells has not been investigated previously. Genotoxicities have been observed in A549 cells at concentrations of 2.5–15 μg/mL of nanoAg by 32P post-labeling of DNA adducts 2 by cytokinesis block micronucleus assay 2 by alkaline comet assay 2 or nanoCeO2 at 67 μg/mL. The distinct outcome of the nanoTiO2 in the present study could be attributed to the different genotoxicity assays employed. With respect to nanoCeO2, the prior study 2 with a size range of 16–22 nm, whereas the present study utilized commercial nanoCeO2 with an average particle size of 33 ± 12 nm.Only cells that were exposed to 33 and 53 μg/mL nanoAg and 21 μg/mL Ag3.2p > 0.05). The exposure concentration of nanoAg in this study (9.48 μg/L) was much higher than predicted concentrations in WWTP effluents (<0.5 μg/L) 2 (0.32 μg/L), and nanoCeO2 (0.08 μg/L) were within or lower than the lower bound concentrations predicted , except for biosolids containing NZVI (p = 0.08) (versus dosed SBRs (p > 0.05), indicating that the decrease in cell viability was not likely due to the nanomaterials or ionic/bulk materials, but probably instead to the high concentration of total solids. Cytotoxicity of biosolids was also examined, at 50 and 100 μg total solids/mL, and no significant effects were observed relative to control cells .The concentrations of nanomaterials in biosolids in this study  B. But, tp > 0.05), suggesting little or no DNA damage to A549 cells at concentrations exceeding most likely aerosol exposure levels.Genotoxicity of biosolids was examined at 200 μg total solids/mL. However, no significant differences were observed in terms of the number of γH2AX foci per cell or in the percentage of cells containing γH2AX foci in cells treated with biosolids relative to untreated control cells , cellular uptake, and intracellular processing pathways ex vivo and in vivo conditions.Uptake of pristine nanoAg, nanoTiOThe authors declare no conflicts of interest.


An F-box protein, called F-box only protein 7 (Fbxo7), acts as a cell cycle regulator by enhancing cyclin D-Cdk6 complex formation and stabilising levels of p27, a cyclin-dependent kinase inhibitor. We generated a murine model of reduced Fbxo7 expression to test its physiological role in multiple tissues and found that these mice displayed a pronounced thymic hypoplasia. Further analysis revealed that Fbxo7 differentially affected proliferation and apoptosis of thymocytes at various stages of differentiation in the thymus and also mature T-cell function and proliferation in the periphery. Paradoxically, Fbxo7-deficient immature thymocytes failed to undergo expansion in the thymus due to a lack of Cdk6 activity, while mature T cells showed enhanced proliferative capacity upon T-cell receptor engagement due to reduced p27 levels. Our studies reveal differential cell cycle regulation by Fbxo7 at different stages in T-cell development.GThe online version of this article (doi:10.1007/s00018-016-2427-3) contains supplementary material, which is available to authorized users. Fbxo7 ligase include the kinetochore protein HURP, NF-κB signalling regulators c-IAP and TRAF2, and NRAGE, a protein involved in apoptosis arising from nerve growth factor signalling  , indicating that these defects can be compensated for over time.To investigate the cellular deficiencies in the thymus, the total cell numbers at all T-cell developmental stages were quantified. Single cell suspensions from thymuses from WT and 8+) Fig. h, or for CD44+ DN, CD25+ C CD44+ DN, CD25+ C CD44+ DN, CD25+ C CD44+ DN, CD25+ C CD44+ DN, CD25+ C CD44+ DN, CD25+ C CD44+ DN, CD25+ C+ within the DN1 population (CD44+ CD25− CD4− and CD8−) cells. A significant reduction in the percentage (0.45-fold) and absolute number (0.76-fold) of ETPs was observed in Fbxo7LacZ/LacZ mice compared to WT  or CLPs (Lin− IL-7Rα+ and Flt3+) compared to WT [A possible cause for thymic hypoplasia might be insufficient seeding of the thymus or defects in the survival or development of ETPs after seeding. To address this, we quantified, by flow cytometry, the number and proportion of bone marrow (BM)-derived ETPs, identified as c-Kited to WT . These d+ cells at the other DP, CD4 SP, and CD8 SP stages . To further test this finding, immunoblotting of whole cell extracts of thymuses for cleaved caspase 3 showed that mice homozygous for the Fbxo7LacZ allele had the highest levels  and S/G2/M phases (>2 N DNA) were quantified and expressed as a percentage for each DN population. There was no statistically significant change in the percentage of cycling cells at the DN1 and DN2 stages. However, cycling cells (with >2 N DNA content) were significantly reduced by 29.5% in the DN3 population and by 59.5% in the DN4 population in mutant mice compared to WT  which suppress the immune response. Splenocytes were harvested and stained for CD4, CD25, and Foxp3. Although the number of CD4+ Foxp3+ cells was the not significantly different between WT and mutant mice, proportionally, there was a statistically significant 0.75-fold increase in CD4+ Foxp3+ TReg cells, due to the overall decrease in effector T cells in mutant mice  compared to WT at 24 h after TCR stimulation, . After 24 h, this number was largely unchanged in both cultures. However, by 72 h, the percentage of CD4+ T cells in WT cultures was 13.1% and comparable to the percentage of CD4 cells in mutant cultures of 11.4%. When the fold change in absolute cell number was calculated, mutant CD4+ T cells had expanded by 1.27- and 2.06-fold at 48 and 72 h compared to the starting number, whereas WT CD4+ T cells expanded 1.27 fold by 48 h but was back to day 0 numbers by 72 h .T-cell activation is followed by rapid increases in cell number, with cell doubling times reported to be as little as 4.5 h , and dif+ T cells. At day 0, there were significantly fewer CD8+ T cells in mutant cultures: 2.6% compared to 9.2% in WT samples . By 72 h, Fbxo7LacZ/LacZ and WT cultures contained 25.9 and 34.7% CD8+ T cells, respectively, showing that both WT and Fbxo7LacZ/LacZ CD8 cells expanded robustly. However, when the fold change in absolute cell number was calculated, Fbxo7LacZ/LacZ cultures had expanded to a much greater extent over 72 h . These results showed that activated mutant CD4+ T cells were capable of producing both Th1 and Th2 cytokines. The approximate twofold increase in the production of IL-2 and IFN-γ despite overall fewer cell numbers may be a due to the more rapid proliferation of mutant CD4 cells upon activation.Upon TCR activation, naive CD4+ T cells: 43.3% compared to 12.8% for WT cultures at 48 h, and 30.6% compared to 9.9% at 72 h, reflecting a 2.4 and 2.1 fold increase, respectively, in stimulated mutant CD4+ cells . At 72 h, both cultures had approximately 5% Annexin V+ cells  may contribute to the more limited expansion of CD4 cells relative to CD8 cells observed in culture .Since mutant T cells expanded more rapidly than WT cells after activation, we tested whether the capacity to undergo apoptosis was inhibited which might also contribute to the observed increased expansion upon activation. To determine the percentage of T cells undergoing apoptosis, WT and mutant splenocytes were stimulated as above. At 48 and 72 h of TCR stimulation, cells were removed and co-stained with antibodies to CD4 and CD8 along with Annexin V Fig. a, b. Intlls Fig. a. In con+ mutant CD4+ and CD8+ T cells. There was a 0.93-fold increase in the number of Annexin V+ cells: 27.2% mutant CD4+ cells compared to 14.1% in WT mice, and about a 0.4-fold increase in Annexin V+ mutant CD8+ cells: 54.9% compared to 39.4% in WT mice  in the cultures providing survival signals. For instance, IL-2 secreted by activated CD4+ T cells and to lesser extent by activated CD8+ T cells is a key regulator of T-cell apoptosis. IL-2 levels together with the intensity of antigenic stimulation influence the viability of activated T cells [Fbxo7LacZ/LacZ CD8+ T cells may also undergo increased levels of apoptosis, but the effect is offset by their high proliferation rate.Within the spleen, ll cells , 26, yetll cells . Howeverll cells . Both na T cells . A thirdFbxo7LacZ/LacZ mice show a dramatically reduced number of peripheral T cells which show delayed activation. Moreover, within their significantly reduced CD4 T-cell population, they show a proportional increase in TReg cells, which may limit their immune responses. However, once activated both CD4 and CD8 cells proliferate robustly, despite the higher levels of apoptosis of CD4 cells. In addition, activated Fbxo7-deficient CD4+ cells were capable of producing both Th1 and Th2 response cytokines and produced ~twofold more IL-2 and IFN-γ. Collectively, these attributes suggest that these mice may be compromised in their immune responses. In support of this idea, we noted the results of a Salmonella typhimurium challenge using 6-week-old C57BL/6-Fbxo7tm1a(EUCOMM)Wtsi mice performed by the Mouse Phenotyping Consortium. Their study showed that these mice had a significantly increased susceptibility to Salmonella-induced morbidity, with significantly higher bacterial counts in the spleen, liver, and caecal contents of Fbxo7-deficient mice compared to WT  [Overall, ication) .In addition to the extensive perturbations in the production and function of T cells caused by the loss of Fbxo7, our findings demonstrate the differential regulation of the G1 phase of the cell cycle in this cell lineage. These effects, which differ from the phenotypes of the single Cdk6 or p27 KO mouse models, reflect the capacity of Fbxo7 to act as a cell cycle regulator that directly impacts on both proteins that clearly provide non-redundant and critical functions at different stages in the development and functioning of T cells.Below is the link to the electronic supplementary material.Supplementary Fig. 1. (a–c) Gating strategies for flow cytometry used to detect CD4 and CD8 SP T cells in thymus (a) and spleen (b), as well as DN cells in thymus (c).  Representative flow cytometry plots showing the percentage of activated  CD4 (d) and CD8 (e) cells after stimulation with anti-CD3 and anti-CD28 antibodies, for 24 h (top panel) and 48 h (middle panel), or culture for 48 h with no activating antibodies (bottom panel)LacZ/LacZ (n = 10) thymuses. (b) Annexin V staining of DP, CD4 and CD8 SP populations of WT (n = 6) and Fbxo7LacZ/LacZ (n = 5) thymuses at 6–8 weeks of age.  Graphs showing the percentage of CD4+ (c) and CD8+ (d) T cells in mixed splenocytes cultures from WT and Fbxo7LacZ/LacZ mutant mice, at the indicated time points after CD3 and CD28 stimulation (n = 5).  Graphs showing the number of CD4+ (e) and CD8+ (f) T cells in mixed splenocytes cultures from WT and Fbxo7LacZ/LacZ mutant mice, at the indicated time points after CD3 and CD28 stimulation (n = 5). Data represented as mean ± SD, ***p < 0.001Supplementary material 1 (PPTX 609 kb)Supplementary Fig. 2. (a) Graph showing percentage of DN thymocyte population in mice older than 10 weeks in WT (n = 12) and Fbxo7


Tensin2 is a focal adhesion-localized multidomain protein expressed in various tissues, and its dysfunction leads to alterations in podocytes. However, these podocyte-related manifestations are dependent on murine strain. Tensin2 dysfunction results in susceptible strains developing podocyte foot process effacement and massive albuminuria, whereas podocytes in resistant strains remain almost intact. In our previous studies, quantitative trait loci analysis and congenic analysis using resistant C57BL/6J and susceptible ICGN mice identified a modifier locus associated with podocyte injury caused by tensin2 dysfunction on chromosome 2. However, the effect of this modifier locus on chromosome 2 is insufficient to explain the resistance of C57BL/6J mice to tensin2 dysfunction, indicating the existence of other modifier genes.Tns2nph and FVB.ICGN-Tns2nph, and detected a novel major modifier locus on chromosome 10. The combined effect of the C57BL/6J alleles of the two loci on chromosomes 2 and 10 reduced the urinary albumin excretion caused by tensin2 dysfunction to a level comparable to that of C57BL/6J mice.Whereas previous studies focused on the severity of chronic kidney disease, the present study focused on podocyte injury. We performed a genome-wide linkage analysis of backcrosses between two tensin2-deficient mouse strains, B6.ICGN-These data indicate that the resistance to podocyte injury caused by tensin2 dysfunction is mainly produced by the effects of the modifier genes on the two loci. The identification of these modifier genes is expected to help elucidate the mechanism underlying podocyte injury.The online version of this article (10.1186/s12863-018-0611-1) contains supplementary material, which is available to authorized users. Tns2 mRNA is detected in the brain, heart, kidney, skeletal muscle, liver, lung, colon, small intestine and other tissues [Tns2 knockdown enhances actin stress fiber formation and cell migration [Tensin2 (Tns2), a member of the tensin family that includes Tns1, Tns3 and Tns4, is a focal adhesion-localized multidomain protein that possesses protein tyrosine phosphatase (PTP) and Src homology 2 (SH2)-phosphotyrosine binding (PTB) domains [ tissues . Stable  tissues . In contigration . Taken tnph), showed normal glomerular development, whereas podocyte foot process effacement and abnormal thickening of the glomerular basement membrane (GBM) were observed by 14 days after birth [However, in vivo, Tns2 dysfunction only leads to apparent abnormalities in the kidney glomerulus , 8. Newber birth . GBM thier birth . As obseer birth , 11.nph mutation, albuminuria or podocyte foot process effacement was observed in FVB/NJ (FVB) and DBA/2J, but not in C57BL/6J (B6) and 129+Ter/SvJcl mice [nph mutation, Tpir, on chromosome 2 [Tpir showed alleviated podocyte foot process effacement, GBM thickening and albuminuria, although the degree of improvement was inferior to that in the B6 genetic background itself, which shows almost no podocyte alteration [The podocyte-related manifestations of Tns2 dysfunction also depend on murine strain. For example, in response to Jcl mice . This dimosome 2 , 18. ICGteration . A furthTns2nph (B6-Tns2nph) and FVB.ICGN-Tns2nph (FVB-Tns2nph) to identify modifier loci that may have been missed in previous studies.In this study, we focused on podocyte injury and undertook a genome-wide linkage analysis of backcrosses between two Tns2-deficient mouse strains, B6.ICGN-Tns2nph and FVB-Tns2nph congenic mice were generated as described previously [1 hybrid Tns2nph (F1) mice were obtained by mating female B6-Tns2nph mice to male FVB-Tns2nph mice. N2 backcross Tns2nph progenies were then obtained by mating female F1 mice to male FVB-Tns2nph mice -based method. Genomic deoxyribonucleic acid was extracted from ear tissue obtained during ear punching for identification. PCR was performed with the following thermal cycling: initial denaturation at 95 °C for 3 min; 10 cycles of 95 °C for 10 s, 65 °C for 30 s (reduced by − 1 °C/cycle), and 72 °C for 10 s; and 30 cycles of 95 °C for 10 s, 55 °C for 30 s, 72 °C for 10 s. Newly designed PCR primers are listed in the Additional file Genome-wide genotyping of the http://imagej.net/). Urinary creatinine was measured using a creatinine colorimetric assay kit  according to manufacturer’s instructions. The urinary albumin excretion was normalized against the urinary creatinine. Urine collection was performed twice over a three-day period, and the measured urinary albumin excretion was averaged for each mouse.Urine samples were collected from 8-week-old mice by gentle manual compression of the abdomen. To measure albumin, urine samples were diluted with sample buffer containing 2% sodium dodecyl sulfate (SDS), 5% ß-mercaptoethanol, 10% glycerol, 60 mM Tris-HCL (pH 6.8) and bromophenol blue, and heated at 95 °C for 5 min. Samples containing 1 μL of urine were applied to 10% SDS-polyacrylamide gel electrophoresis. As a positive control, 5 μg of bovine serum albumin was loaded simultaneously. The gel was fixed and stained with Coomassie brilliant blue  according to manufacturer’s instructions, and scanned using a standard commercial scanner. CBB-stained urinary albumin was quantified by the ImageJ gel analysis program  at 4 °C overnight. The PFA-fixed paraffin sections (2 μm thick) were subjected to normal histological processes and stained with periodic acid-Schiff (PAS) solution.Tns2nph and B6-Tns2nph mice, we defined the N2 mice with urinary albumin excretion larger than the minimum value in FVB-Tns2nph mice as the FVB type  and the N2 mice with urinary albumin excretion less than the maximum value in B6-Tns2nph mice as the B6 type . Another way of division was to define the upper 30% or 40% or 50% of N2 mice as the FVB type and the lower 30% or 40% or 50% of N2 mice as the B6 type. A chi-square test was applied to detect marker-trait associations.N2 mice were divided into two groups, affected and not affected, according to their urinary albumin excretion. With reference to the distribution of urinary albumin excretion observed in FVB-t-test and Dunnett’s multiple comparison test were used for comparisons of two or more than two independent groups, respectively. A p value < 0.05 was considered statistically significant. Dunnett’s multiple comparison test was carried out using GraphPad Prism 5 software .Data are expressed as means ± standard deviation. Student’s Tns2nph mice showed albuminuria  and severe glomerular injuries, such as marked expansion of the mesangial matrix, marked thickening of the GBM and abnormal dilation of capillary lumen, whereas B6-Tns2nph mice did not show either albuminuria  or glomerular injury, apart from a slight increase in the mesangial matrix in some glomeruli  and subsequent phenotyping for albuminuria. N2 mice showed a continuous spectrum of values for urinary albumin excretion and did not simply segregate into the B6- and FVB-Tns2nph types according to whether or not they were affected with albuminuria  and B6-Tns2nph mice (0.052–0.69 g/gCr), we defined the N2 mice with urinary albumin excretion larger than 3.1 g/gCr (59 of 174) as the FVB type (affected) and the N2 mice with urinary albumin excretion less than 0.7 g/gCr (89 of 174) as the B6 type (not affected). In the N2 mice as defined above, marker-trait associations were detected on chromosome 10. The genetic markers rs259766385 and rs239646663 on chromosome 10 showed a statistical significant relationship with albuminuria  was significantly lower than that in the group carrying FVB/FVB genotypes at the two markers (F/F group) , was approximately equal to those of the F/B and B/B groups  and appeared lower than that of the F/F group (2.4 g/gCr), although no significant difference was detected between the B/F and F/F groups . We assumed that this is because that the intended modifier effects are hindered due to a wide spectrum of values for urinary albumin excretion in affected mice themselves . B6 homozygous alleles of Tpir exerted a protective effect against podocyte alterations and reduced urinary albumin excretion in affected mice to approximately 50% [Tpir, and we designated this suggestive locus as Tpir1.Subsequently, F1 mice were backcrossed to FVB-see Fig. . TherefoTpir was insufficient in comparison with that of the B6 genetic background itself, our previous study predicted the existence of an as yet unidentified major modifier locus [Tpir1 and the significant locus exerted a protective effect on albuminuria at levels close to that of the B6 genetic background. We, therefore, designated the significant locus as Tpir2. No genes located around Tpir1 and Tpir2 are known to be involved in nephropathy.As the protective effect of the B6 allele of er locus , which ars259766385, the peak marker of Tpir2, consists of multiple syntenic regions on human chromosomes 12, 19, 21 and 22. It is notable that the syntenic regions on human 12q23.2 – q23.3 and 19p13.3, and 21q22.3 include the genetic markers associated with urinary albumin excretion  and glomerular filtration rate (rs2839235)


The phase identification, morphology, and elemental composition of products were characterized by X-ray diffraction (XRD), transmission electron microscopy (TEM), field- emission scanning electron microscopy (FE-SEM), energy dispersive X-ray (EDX) spectroscopy, and Fourier transform infrared spectroscopy (FTIR). The results revealed the orthorhombic phase of Sb2S3 single crystal-forming sheaf-like nanostructure, and a possible formation mechanism was proposed and discussed. Its direct band gap calculated from UV-visible absorption is 1.60 eV. In this research, the photocatalytic activities of Sb2S3 nanostructure were investigated through the degradation of methyl orange (MO) and methylene blue (MB) under visible light irradiation. The as-obtained 0.30 g HEC-added solution (0.3 HEC-Sb2S3) photocatalyst exhibited better photocatalytic activity than the other products, which degraded 91% of MO within 300 min and 90% of MB within 240 min under the Xe-lamp irradiation. The first-order plot was fitted with this experiment which the rate constant (k) of 0.3 HEC-Sb2S3 for MO and MB degradation are 0.0085 and 0.0098 min−1, respectively. Therefore, the new experience with a novel and simple synthetic procedure of Sb2S3 photocatalyst that exhibits the characteristics of a highly effective photocatalyst under visible light irradiation was discovered.Stibnite (Sb Presently, the photocatalytic activity of semiconductor nanomaterial has drawn considerable interest worldwide because they have significant potential for the degradation of dye pollutants in wastewater and elimination of environmental contaminations. A serious problem with toxic synthetic dyes was arisen from discharging the wastes of textile, paper, and plastic industries. Consequently, it is necessary to treat these dye pollutants in water before draining to natural resources.2 has been applied to environmental problems, due to it has traditionally formed as photocatalyst under irradiation for pollutant degradation by generating strong oxidants such as HO• radicals that can rapidly and non-selectively degrade organic compounds [2O3, CdS, ZnS, ZnO-SnO2, CeO2-TiO2, and Ag3PO4 composite were also used as photocatalyst because of their optimum optoelectrical, physical, and chemical properties [2 hollow spheres [2O3 hollow spheres [2O3 nanospindles, nanotubes, and nanotires [2-TiO2 nanobelt heterostructures [2 nanoparticles [2 nanoparticles [3PO4/CNFs/silica-fiber hybrid composites [Typically, a variety of procedures have been used for removal of synthetic dyes from water, including membrane separation, microbiological decomposition, adsorption, and photocatalysis –7. Amongompounds . Other toperties . A varieoperties , 11–15.  spheres , α-Fe2O3 spheres , α-Fe2O3anotires , and CeOructures ; microwaarticles ; and a c2 photocatalyst can only absorb ultraviolet light, due to its large band gap (3.0–3.2 eV) [Previously, the pure TiO–3.2 eV) . To solv–3.2 eV) –22.2S3 used for MB photocatalytic degradation under visible irradiation [2S3, and SnS2 quantum dots were studied for the photocatalytic degradation of MB [2S3 nanowires was investigated for the degradation of MO [Recently, chalcogenide photocatalysts can play an important role in the degradation of dyes, such as Inadiation ; SnS, Snon of MB ; the phoon of MO ; SnS micon of MO .2X3 , such as Sb2S3 or stibnite, have been studied intensively due to their numerous applications including optoelectronic, thermo-electric technologies, solar energy utilization, and semiconducting photocatalyst [2S3: refluxing [2S3. This method has a lot of benefits: very simple, effective, low cost, fast, and precise control of the reaction (mixing condition and size and shape controlling) [Herein, semiconducting photocatalysts of main group metal chalcogenide Acatalyst –25. Diffefluxing , spray pefluxing , hydrothefluxing . In thisrolling) –13, 27. 2S3, each of 2 mmol of antimony chloride  and 3 mmol of sodium thiosulfate  was dissolved in 30 mL propylene glycol . They were thoroughly mixed, followed by pH adjusting to 4 using 0.1 M HCl (diluted from concentrated HCl (37% v/v), Sigma-Aldrich), and stirred for 15 min. Then, 0.10, 0.20, and 0.30 g of HEC  were added to the mixed solutions and irradiated with microwave radiation at 300 W for 20 min to form 0.1 HEC-Sb2S3, 0.2 HEC-Sb2S3, and 0.3 HEC-Sb2S3 products, respectively. The as-obtained product appears as black precipitates which were separated by filtration, washed with absolute ethanol, and dried at the 70 °C for 24 h, for further characterization. For the present synthesis, different contents of hydroxyethyl cellulose (HEC) were also used as a template.All chemicals used in this experiment were analytical grade and used without further purification. To synthesize Sbα line in 2θ range of 10–60 deg. Shape, size, and composition of the precipitates were investigated by a field-emission scanning electron microscope  equipped with an energy dispersive X-ray (EDX) analyzer operating at 15 kV, a transmission electron microscope , a selected area electron diffractometer (SAED) operating at 200 kV, and Fourier transform infrared spectroscopy (FTIR). The optical property was studied by a UV-visible spectrometer  using a UV lamp with a resolution of 1 nm.Phase and purity were characterized by an X-ray diffractometer  operating at 20 kV and 15 mA and using Cu-K2S3 nanostructure were evaluated by MO (C14H14N3NaO3S) and MB (C16H18ClN3S) degradation under visible light irradiation. A 300-W Xe lamp equipped with a 420-nm cut-off filter was used as a visible light source. Practically, 30 mg of the photocatalyst was dispersed in a 100 mL of 11 mg L−1 MO solution and 100 mL of 10 mg L−1 MB solution, respectively. Prior to irradiation, the dye solution was continuously stirred for 1 h in the dark for adsorption-desorption equilibrium. Then, the visible light was turned on to start photocatalysis. During the process, 3 mL of suspension was withdrawn every a certain time interval and centrifuged to precipitate the photocatalyst. The absorbance of the clear solution or the change of MO and MB concentration were determined by UV-visible spectrophotometer  using the maximum absorbance at 464 and 665 nm, respectively. The photocatalytic efficiency of MO and MB degradation was calculated using the formulaC0 is the initial concentration of MO or MB concentration and C is the concentration of MO or MB after visible light irradiation within the elapsed time (t).The photocatalytic activities of Sb2S3 crystal were shown in Fig. 2S3 with Pbnm space group, in accordance with the JCPDS File no. 06-0474 [2S3 in PG is very viscous, and the delay of kinetic control can cause agglomeration due to inefficient physical mixing [The crystalline degree and phase purity of Sb 06-0474 . All the 06-0474 . In thisl mixing .Fig. 1XR2S3, Fig. 2S3 morphologies in different contents of HEC-added solutions. There are a number of nanorods with irregular shape in the HEC-free solution. When 0.10 g HEC was added to the mixed solution, it caused each nanorod to split more to be a bundle of nanorods. A bundle of nanorod was enlarged and more open at their end tips in 0.20 g HEC-added solution. Finally, the Sb2S3 morphology was changed into a sheaf-like structure and more beautiful in 0.30 g HEC-added solution. The present morphology is similar to the report by Ota et al. [To reveal the shape and size of the as-obtained Sba et al.  who stara et al. . But for2S3 synthesized in 0.30 g HEC-added solution (0.3 HEC-Sb2S3) are illustrated in Fig. 2S3 single crystal, corresponding to the diffraction pattern obtained by simulation [TEM image, SAED, and simulated patterns  of Sb2S32S3 product provides close to 2:3 atomic ratio of Sb:S—in good accordance with the stoichiometric composition of Sb2S3. It should be noted that other peaks were also detected—caused by the electronic transition of copper stub and carbon tape used for holding the analyzed sample [Quantification of the EDX spectrum Fig.  of 0.3 Hd sample .Fig. 4ED2S3 nanorods. The absorption was controlled by two photon energy (hν) ranges, corresponding to the high and low energies. When photon energy is greater than the energy band gap (Eg), the absorption was linearly increased with the increase in photon energy. This research shows the (αhν)2 and (hν) plot for direct allowed transition, where α is the total absorption coefficient, h is the Plank constant, and ν is the photonic frequency [2S3 is therefore estimated to be 1.60 eV, in accordance with 1.59–1.60 eV of Sb2S3 determined by Alonso et al. [2S3 nanorods determined by Ota et al. [2S3 calculated by Chate et al. [2S3 nanostructure has the potential to absorb visible light and can be used for solar-light-driven applications and optical nanodevices [Figure requency . The bano et al.  compare o et al. , 2.08 eVo et al. , and 2.2o et al. . The bana et al.  and 2.27e et al. . This anodevices , 35, 37.2S3 was controlled by hydrolytic decomposition of sodiumthiosulfate as a sulfur source. First, antimony (III) ions (Sb3+) in the solution formed complexes with thiosulfate. In the mixed solution containing Sb3+ and S2− at pH 4 as an acidic medium, thiosulfate ions slowly released sulfide ions to combine with antimony (III) ions, and colloidal complexes formed. Finally, the black precipitates of Sb2S3 were synthesized. Moreover, the concentration of Sb3+ and S2− ions in the solution also played the role in the formation rate of Sb2S3 [A possible formation mechanism of Sbof Sb2S3 , 38.2\do2S3 structure obtained by simulation. It shows that the infinite chains of stoichiometric composition of atoms run normal to the axis. The binding force of the chain in b direction is greatly weaker than the one along the chain (a- and c-axis) [Figure  c-axis) , 39. The c-axis) , 39, 40. c-axis) , the dou c-axis) , the dum c-axis) , and nan c-axis) , the pre c-axis) .Fig. 6Th2S3, 0.1 HEC-Sb2S3, 0.2 HEC-Sb2S3, and 0.3 HEC-Sb2S3 products  of 0.3 HEC-Sb2S3 for MO (0.0085 min−1) and MB (0.0098 min−1) solution were higher than the other samples—caused by the good crystallinity and the large surface area of the product [The absorption spectra of MB solution using Sbion site , 42. The product , 42.Fig.−1. As can be seen in Fig. −1 correspond to the bonds of metallic compounds and M–OH modes of product [−1 related to the stretching and bending vibration of O–H bond, respectively [−1 are in accordance with the C–H stretching band. Figure −1 of 0.3 HEC-Sb2S3 adsorbed by MO sample were attributed to the C–N vibrations, C–N aromatic stretching vibrations, the vibration of C–N bonds, and C–H in-plane bending vibration of benzene rings of MO molecules, respectively [−1 of 0.3 HEC-Sb2S3 adsorbed by MB was broadened compare to 0.3 HEC-Sb2S3—caused by the bonding with 0.3 HEC-Sb2S3 and MB molecules [−1 can be attributed to modes form as a result of H-bond of the type Nhet ··· HO (stretching heterocycle··· HO) [2S3 surfaces [−1 to decrease after adsorption [Figure  product –45. The ectively –46. The ectively , 14, 47.e··· HO) . These wsurfaces . These rsorption –48.Fig. 2S3 nanostructure. During the photocatalytic process, electrons and holes reacted with the adsorbed substances (O2 and OH−) on the surfaces of crystals to form oxidative radicals such as O2•− and •OH [2S3) [2O and CO2 molecules as the final products [Figure  and •OH –51 when OH [2S3) , 51. Theproducts , 52–54.F2S3 nanostructure composed of a number of nanorods act as the visible-light-driven photocatalyst was successfully synthesized by a simple microwave radiation. The purpose of this work was to gain new experience with a simple synthetic procedure of Sb2S3 photocatalyst that has a high degradation efficiency. The first-order plot was fitted with this experiment. The as-obtained 0.3 HEC-Sb2S3 photocatalyst exhibited better photocatalytic activity than the other products, which degraded 91% of MO within 300 min and 90% of MB within 240 min under the Xe-lamp irradiation. The 0.3 HEC-Sb2S3 product is beneficial to the photodegradation of the MO and MB dye. It may be mainly attributed to its band gap (Eg = 1.60 eV), which cover range of the solar spectrum and has strong absorption of the visible light. Consequently, these results suggest that the as-prepared 0.3 HEC-Sb2S3 product exhibits a good photocatalytic activity under the visible light irradiation and it is one of the promising photocatalytic materials for wastewater treatment.In summary, a sheaf-like Sb


Their ability to efficiently store energy acquired upon irradiation with X-rays was proven. The best TL performance was achieved for compositions containing 0.025%–0.05% of Pr and about 0.1% of Hf. Further enhancement of TL efficiency was attained by increasing the temperature of sintering of the ceramics up to 1700 °C and applying reducing atmosphere of forming gas. It was also proven that fast cooling after the sintering at 1700 °C significantly enhanced the storage phosphor performance. TL glow curve contained three components peaking around 130, 250 and 350 °C. Among them, the one at 250 °C contributed the most to the total TL.Lu In such compositions the excited electron after absorption of the incident photon gives no immediate emission but is immobilized in an excited state for quite a long time. Once irradiated, persistent phosphors continue releasing the acquired energy at room temperature for minutes or hours afterwards, while storage phosphors rather retain it for days or even years if no external stimulation—thermal or optical—is applied –3. The d2O4:Eu2+,Dy3+ [2O4:Eu2+,Nd3+ [3SiO5:Eu2+,Nd3+ [4(Si3O8)2:Eu2+,Dy3+ [3Al2O5Cl2:Eu2+,Tm3+ [2O4:Eu,Nd [2MgSi2O7:Eu,Dy [4Al14O25:Eu,Dy [2Si5N8:Eu,Tm [4:Ce3+,Ln3+  [4:Pr3+,Ln3+  [A significant number of new, efficient persistent phosphors have been discovered in last two decades as a consequence of Matsuzawa’s work on SrAlu2+,Dy3+ , and SrAu2+,Nd3+  showing u2+,Nd3+ ,7, Ba4 –15, YPO4 Ho, Dy) . This re Ho, Dy) ,18. His 1−xIx:Eu2+ [2+ [4:Dy [1−xIx:Eu2+ and CsBr:Eu2+ are proven to fulfill the requirements of digital radiography, though some of their parameters are rather far from “ideal” values. For example, both have quite low densities , which reduces their absorption coefficients for X-rays, especially the more energetic. Detailed comparisons of that parameter for different compositions can be easily made using the XCOM service delivered by NIST [4  have almost constant, and rather low absorption coefficients at higher energies, while the more dense materials, containing elements of higher Z numbers, show much higher, strongly increasing for lower energies, effectiveness to absorb γ- or X-rays. Consequently, this difference divides the storage phosphors into two groups: those of low densities, which are useful in personal dosimetry, and the high-density compositions that are more appropriated for imaging and whenever enhancement of probability of absorbing the incoming photons matters. [2O3 as the host are exceptional. The reason is the high effective Z number of lutetium oxide (67.3) and its very high density (9.42 g/cm3), which situate Lu2O3-based phosphors among the most efficient absorbers of γ- and X-rays.Research in the area of storage phosphors did not bring so spectacular results if new compositions are considered. Still, the mostly used storage phosphors are, discovered years ago, FBrxIx:Eu2+ ,20 and CEu2+ [2+ , CaSO4:D2+ [4:Dy , LiF:Mg,2+ [4:Dy ,23 and s by NIST . Thus, Lmatters. . Yet, otmatters. . Among t2O3-based compositions [2O3:Pr,Hf sintered ceramics.We have already published some data on energy storing in Luositions –29. In t2.2.1.2-H2 mixture using initial powders prepared in different manners: the Pechini technique, combustion synthesis or by mixing commercial oxides. The thermoluminescence (TL) glow curves of three ceramics made using such powders are given in We shall see later that efficient thermoluminescence (TL) requires preparation of the ceramics at temperature as high as 1700 °C (the highest temperature we can routinely apply in our furnaces). Therefore let us start comparing the TL glow curves of ceramics prepared at such a temperature and N2O3:Pr,Hf powders. This, we think, allows for much better uniformity of the starting powders even if the thermally stimulated diffusion of the material constituents is limited in the refractory oxide. Getting such an uniformity of the final material starting with commercial oxides would require diffusion of the various components over significant distances in the solid phase during its sintering. Evidently, from the data given in 2O3:Eu red emitting powder phosphor should be prepared by co-precipitation and subsequent thermal decomposition to achieve high performance. Solid state reaction reduced its efficacy by a factor of 2–3 [It is immediately seen that the TL efficiency of the materials made using the commercial oxides is by almost an order of magnitude lower than from the other two specimens. This effect was repetitively observed. The other two ceramics performed almost identically. We concluded that the powders for sintering should be prepared through procedure allowing for atomic mixing, as both in combustion and in Pechini synthesis the reactants are first dissolved in water, and then processed towards nanocrystalline Lur of 2–3 . Consequ2O3:Pr,Hf ceramics made at 1700 °C in reducing atmosphere and containing Pr and Hf at different concentrations. In 2O3:0.05%Pr,0.1%Hf and Lu2O3:0.025%Pr,0.1%Hf. Already for 0.1% of Pr the TL intensity gets reduced by a factor of about 4 and becomes even less efficient when the Pr concentration is yet doubled. Clearly, the efficient TL requires low Pr concentrations. We should note, however, that the TL glow curves of the two best samples—Lu2O3:0.05%Pr,0.1%Hf and Lu2O3:0.025%Pr,0.1%Hf—differ significantly especially in the high temperature range. When the Pr content is 0.05% the component around 250 °C dominates the glow curve, while for yet lower concentration of the activator, 0.025%, the most efficient TL appears around 330 °C. Still, for this composition the band around 250 °C comprises noticeable fraction of the total TL intensity. Apart from the high-temperature TL components, all samples give some thermoluminescence around 130 °C. Traps of such characteristics are able to free carriers even at room temperature [perature . Indeed,perature .2O3:Pr,Hf is tremendously susceptible to the concentration of Pr and less, though noticeably, sensitive to the content of Hf. Yet, both dopants are needed to form an efficient energy-trapping system in the lutetia host. In the next step, we shall try to determine the optimal sintering temperature and atmosphere of the Lu2O3:Pr,Hf storage phosphors.2-H2 mixture. Nevertheless, neither of the applied atmospheres totally annihilated the TL. This is the effectiveness of storing energy in the material, which was influenced by the atmosphere of fabrication.2O3:0.05%Pr,0.1%Hf annealed for 5 h at 1700 °C in a forming gas with different cooling rates. From the same batch of raw powder we made a ceramic with the regular cooling of 3 °C/min and another one applying the cooling rate of 7 °C/min in the most crucial range of 1700–1400 °C, while below 1400 °C the rate was again 3 °C/min. Comparison of the two TL glow curves is given in Finally, we decided to check if the cooling rate after sintering at 1700 °C may play a role. We prepared two ceramics: Lu2.1.1.2O3:0.05%Pr,0.1%Hf ceramics obtained at various sintering temperatures: 1200 °C  is assumed to be responsible for immobilization of electrons, while at the same time, Pr(III) may trap holes due to its ability to transform towards Pr(IV). Holes may be also trapped by the interstitial oxygen due to its negative net charge. Yet, its population is limited as, even at reducing atmosphere, the Hf(IV), with its positive net charge, may hinder (though not exclude) the generation of such a defect.Even in large single crystals, high-temperature treatment facilitates migration of constituent entities—atoms and defects. At high temperatures a formation of new defects, mostly vacancies, by expelling the atoms from surface to the vacuum takes place ,34. In c3.2 + N2 mixture (1:3 by volume), nitrogen or air atmospheres for 5 h. Sintering for 1 h gave samples with TL by a factor of 4–5 lower compared to specimens sintered for 5 h. Consequently, we present data only for materials sintered for 5 h. The typical heating and cooling rates were 3 °C/min. For comparison, one sample was prepared with the cooling rate of 7 °C/min in the range of 1700–1400 °C and 3 °C/min below 1400 °C in the reducing mixture.Samples were prepared as sintered ceramics. The starting powders of appropriate compositions were uniaxially pressed into 7 mm pellets under the load of 5 tons and were then sintered in a tube furnace at 1200, 1500, or 1700 °C in vacuum, H2 + N2 mixture) the influence of the Pr and Hf content on TL was systematically tested only for samples made at such conditions. The starting powders for sintering were typically prepared by the standard Pechini technique [2CH2COOH) combustion technique [2O3 (4N5), Pr6O11 (5N), HfO2 (3N), were sintered at 1700 °C in 25%H2 + 75%N2 mixture.Since the most powerful thermoluminescence was routinely observed for ceramics made at the highest temperature (1700 °C) and the most reducing conditions  was applied to examine the samples phase purity. The XRD patterns were recorded in the range of 2θ = 29–60 degree with the step of 2θ = 0.00857 degree and the counting time of 0.2 s per point. Micrographs were recorded with the use of a Hitachi S-3400N Scanning Electron Microscope (SEM) equipped with an energy dispersive X-ray spectroscopy (EDAX) analyzer. For the experiments freshly broken pellets of sintered ceramics were sputter coated with an ultrathin layer of gold.X-ray powder diffraction technique using Bruker D8 Advance diffractometer with Ni-filtered Cu K3+ red emission band around 630 nm. Since the spectrometer used CCD to record light, full spectrum in the range of 200–1100 nm was also taken every 1 s during heating. These spectra proved that only the red luminescence of Pr around 590–690 nm was produced. The typical heating rate was 4.8 °C/s and accuracy of the temperature is estimated to be ±2.5 °C. White X-Rays from a Cu X-ray tube were used for irradiation of the materials prior to thermoluminescence experiments.Thermoluminescence (TL) glow curves were recorded in the range of 20–500 °C using a custom-made temperature controller with linear heating rate and using Ocean Optics HR2000 CG spectrometer operating under the control of a Spectra Suit dedicated software. The thermoluminescent photons were collected with a 74-UV lens coupled to a QP600-1-SR waveguide. TL glow curves were recorded with the counting time of 1 s and monitoring the intensity of the Pr4.2O3:Pr,Hf ceramics were able to store energy when irradiated with X-rays. Efficiency of this process, as monitored by thermoluminescence intensity, was showed to strongly depend both on Pr and—to a slightly lesser degree—on Hf concentration. The optimal content of the dopants were found to be 0.025%–0.05% (Pr) and about 0.1% (Hf). It was also proven that high temperature of preparation (1700 °C) and reducing atmosphere of the forming gas were beneficial for the storage efficacy. Yet, each of the applied preparation atmospheres—forming gas, vacuum, nitrogen, air—allowed for energy storing in Lu2O3:Pr,Hf ceramics. Good stability of the TL signal (presented in [3 it assures very efficient absorption of ionizing radiation (gammas and X-rays) and thus it could be considered as a dosimetric material is some specific areas. We continue this research towards Lu2O3:Pr,Hf powder storage phosphor with low agglomeration to have some freedom in making larger screens, for example in the form of composed plates. This is a hard task, as the required high temperature of preparation naturally enhances aggregation of grains.In this paper we presented results showing that Luented in ) is an i


Slc2a4 gene, in L6 skeletal muscle cells.Obesity is strongly associated to insulin resistance, inflammation, and elevated plasma free fatty acids, but the mechanisms behind this association are not fully comprehended. Evidences suggest that endoplasmic reticulum (ER) stress may play a role in this complex pathophysiology. The aim of the present study was to investigate the involvement of inflammation and ER stress in the modulation of glucose transporter GLUT4, encoded by L6 cells were acutely (2 h) and chronically (6 and 12 h) exposed to palmitate, and the expression of several proteins involved in insulin resistance, ER stress and inflammation were analyzed.P < 0.01) and its mRNA  expression. Only acute palmitate treatment increased GRP78 , PERK , eIF-2A , IRE1a  and TRAF2  protein content, and PERK phosphorylation , but did not elicit eIF-2A, IKK phosphorylation or increased XBP1 nuclear content. Additionally, acute and chronic palmitate increased NFKB p65 nuclear content  and NFKB binding activity to Slc2a4 gene promoter .Chronic and acute palmitate exposure significantly reduced GLUT4 protein (~ 39%, Slc2a4/GLUT4 expression. This regulation involves activation of initial component of ER stress, such as the formation of a IRE1a-TRAF2-IKK complex, and converges to NFKB-induced repression of Slc2a4/GLUT4. These results link ER stress, inflammation and insulin resistance in L6 cells.Different pathways are activated in acute and chronic palmitate induced-repression of  Slc2a4 gene, which encodes GLUT4 protein, results in insulin resistance  until reach 70% of confluence. Cells were then differentiated for 6 days in differentiation medium . At seventh day, cells were incubated with FBS-free restriction/treatment medium  for 12 h before treatment. Palmitate was diluted in ethanol (vehicle) and added to a final concentration of 0.75 mM palmitate and 0.5% ethanol for 2, 6 and 12 h. In parallel, control plates were treated with a non-toxic final concentration of 0.5% ethanol [L6 cells were propagated and differentiated as previously reported . Briefly ethanol , 20.L6 cells were plated in 12-well plate and treated with 0.5% ethanol alone for 12 h or with 0.75 mM palmitate/0.5% ethanol for 2, 6 or 12 h. Cell viability was assessed using MTT reagent according to the manufacturer’s recommendations with some modifications. Briefly, after the incubation with the tested compounds, the medium was changed to a medium with 10% MTT (5 mg/ml) and incubated in dark for 4 h at 37 °C. The medium was removed and replaced with a solvent solution (0.04 N HCl in absolute isopropanol), and the plates were placed on a plate shaker for 15 min to solubilize the formazan crystals derived from MTT reduction. The absorbance of the converted dye was measured at 570 nm with a background subtraction at 690 nm using a microplate reader .Total membrane protein fraction for GLUT4 protein quantification was performed as previously reported . For RE Gapdh (#Rn99999916_s1) was used for normalization. The primer sequences for rat Slc2a4 were custom designed: 5 ′ GGCTGTGCCATCTTGATGAC-3′ (fw), 5′-CACGATGGACACATAACTCATGGA-3′ (rv) and FAM AACCCGCTCCAGCAGC MGB, as previously described [One microgram of total RNA was extracted with Trizol. cDNA, obtained by reverse transcription, was then amplified with Taqman system. Commercially available Taqman primers for rat escribed .Slc2a4 promoter (Slc2a4-NFKB) was 5`-GGGTTGGGGGCGTGGCCTTTTGG-3` [Nuclear proteins were extracted from L6 cells and subjected to EMSA as earlier described . The seqTTTGG-3` , 19.Data are expressed as means ± S.E.M. One-way analysis of variance (ANOVA), with Student–Newman–Keuls as a post hoc test was used for analysis of comparison, after comparison of the variances by the Bartelett’s test.The viability of L6 muscle cells incubated with 0.5% ethanol (vehicle) alone or with 0.75 mM palmitate was assessed with MTT assay. As shown in Fig. Slc2a4 gene expression was investigated over a time course of 2, 6 and 12 h in L6 myotubes. We have previously demonstrated that 0.75 mM palmitate for 20 h reduces GLUT4 protein content and glucose uptake in L6 cells [Slc2a4 gene transcript content , also known as growth arrest and DNA damage-inducible protein 34 (GADD34) was analyzed. Surprisingly, palmitate did not exert any effect in PR15A content in L6 cells Fig. .P < 0.01 versus C; Fig. The activation of PERK regulates numerous genes involved in apoptosis such as DDIT-3 (DNA damage-inducible transcript 3 protein), also known as growth arrest and DNA damage-inducible protein (GADD153) ; thus, tP < 0.05 versus C, Fig. Next, another pathway of UPR signaling was investigated. In parallel to the increased GRP78 protein content after 2 h-palmitate incubation, the content of protein kinase IRE1a also increased  and form a complex with inhibitor of nuclear factor kappa-B kinase (IKK) promoting activation of NFKB, independently of IKK activation . Here, iSlc2a4 gene by palmitate was investigated.Finally, the participation of NFKB in the modulation of Slc2a4 gene; in L6 muscle cells, the binding of NFKB dimers to a double-stranded oligonucleotide containing a NFKB site sequence inside the Slc2a4 promoter results in two complexes named a and b [a, P < 0.05 versus C; and, 68% for complex b; P < 0.01 versus C), and after 12 h . Additionally, palmitate increased the nuclear content of NFKB p65 in L6 cells (30, 23 and 32% after 2, 6 and 12 h, respectively; P < 0.05 versus C, Fig. Recently, we have reported that NFKB downregulates the  a and b . Accordi a and b , we confPalmitate is known to induce insulin resistance by imparing glucose uptake  and reduSlc2a4 gene. Actually, it has been reported that activation of UPR signaling represses the Slc2a4 gene in 3 T3-L1 adipocytes [Here we reported that palmitate reduced GLUT4 expression in L6 muscle cells; still, the correlation of mRNA and protein content was not consistent. Since post-transcriptional modulation has been reported to cause discrepancies between GLUT4 protein and its transcript abundancies , it was ipocytes . Also, iipocytes , pancreaipocytes , and C2Cipocytes . Up to dipocytes . The preipocytes . Here, wipocytes . Here weAcute palmitate exposure resulted in increased expression of PERK and eIF-2A; and surprisingly, while PERK activation was increased, eIF-2A phosphorylation was reduced. Some phosphatases such as PR15A can dephosphorylate eIF-2A, attenuating response to stress and promoting recovery of protein synthesis . In our We also demonstrated that acute palmitate even though was able to increase IRE1a expression, it did not result in its activation since phosphorylation of IRE1a and XBP1 nuclear content was not altered. However, our results strongly suggest that IRE1a can contribute to NFKB activation after acute palmitate exposure. It has been reported that basal IKK activity is sufficient to activate NFKB by IRE1a, and this occurs by physical interaction among IRE1a, TRAF2 and IKK . The forSlc2a4 gene promoter has a functional kB site to which NFKB binds and downregulates its transcription [Slc2a4 promoter, and consequently reduces Slc2a/GLUT4 expression. Consistent with these results, we have previously demonstrated that the same concentration of palmitate for 20 h reduces GLUT4 expression and glucose uptake; moreover, it induces tumor necrosis factor gene expression and NFKB accumulation in the nucleus in L6 cells [Slc2a4/GLUT4 in L6 cells, but did not increase NFKB binding in Slc2a4 gene promoter. Of note, a different pathway should be involved.Recently, it has been demonstrated the rat/mouse cription . Additiocription . Thus, NL6 cells . Our datSlc2a4/GLUT4 expression, in a ER- and inflammatory-stress mediated way; thus, contributing to induce insulin resistance (Fig. In summary, the data reveal that short-term exposition of muscle cells to palmitate can repress nce Fig. . ConsideSlc2a4/GLUT4 expression in L6 muscle cells. That is related to formation of a IRE1a-TRAF2-IKK complex, activation of NFKB and repression of Slc2a4 gene transcription. These findings indicate that ER stress may connect palmitate-induced inflammation and insulin resistance in L6 muscle cells.Palmitate reduces


However, the mechanisms behind the beneficial effects of reduced oxygen tension in embryogenesis remain unclear. The aim of this study was to evaluate the expression of oxygen related and antioxidant genes and mitochondrial function in mouse embryo cultured under hypoxic and normoxic conditions, to investigate the beneficial effect of low oxygen tension in preimplantation embryogenesis.In human IVF, embryos cultured with a lower OTwo-cell ICR mouse embryos were cultured to blastocysts under different oxygen tension (3% and 20%). The gene expression of oxygen-related proteins , HIF targets  and antioxidants  were assessed using quantitative RT-PCR and implantation-related protein  was validated by immunofluorescence. Apoptosis, mitochondrial membrane potential (MtMP) and ROS levels were measured by TUNEL, JC-1 and DCFDA assays, respectively.2 than in 20% O2. The transcription levels of MnSOD, PRDX5, VEGF and GLUT-3 also significantly increased in 3% O2 compared with 20% O2 (P < 0.05). Immunofluorescence showed that the intensity of staining for HIF-2α, MnSOD and LIFR were higher in 3% O2. Blastocysts cultured under 3% O2 exhibited significantly higher MtMP compared with 20% O2. ROS and Apoptosis levels were significantly higher in the 20% O2 group than in the 3% O2 group (P < 0.05).Blastocyst development rate (92.3% vs. 79.4%) and hatch rate (80% vs. 70.4%) were both higher in embryos cultured in 3% O2 tension may improve embryo viability by increasing expression of antioxidant enzymes and glucose transporter activities. It provides an environment conducive to viability by upregulation of LIFR/VEGF and increased MtMP which could enhance implantation potential and reduce apoptosis in mouse blastocyst. These effects may be initiated and regulated by HIF-2α, a key mediator in a hypoxic environment.Low O Therefore we chose 3% O2 concentration in our study to investigate the beneficial effect of low oxygen tension in preimplantation embryogenesis.Most mammals have a lower concentration of Ooviducts . The O2 2) affects embryo development and the intracellular redox balance [2 tension [2 tension (5%) during days 3–5 of in vitro culture, which gives higher cleavage, implantation, pregnancy, and birth rates in humans [2. These findings provide evidence that low O2 tension is more efficient and generates a higher number of and better quality embryos than atmospheric O2.Embryo culture systems are a vital component of assisted reproductive technology for the production of animal or human embryos, and many factors affect the development of mammalian preimplantation embryos in vitro. It is well known that environmental oxygen , which may play important roles in these events [2 tension during embryogenesis remain unclear.Low Oelopment . Many ste events . The ben2 conditions [2-dependent fashion at the level of the stability of the α-subunit protein [2 tension, HIFα protein is rapidly degraded because of O2-dependent hydroxylation by prolyl hydroxylase domain-containing proteins and a von Hippel–Lindau tumor suppressor protein-dependent ubiquitin–proteasome pathway [HIFs are recognized as the master transcriptional regulators of cellular hypoxic responses, and consist of two basic helix–loop–helix protein subunits (HIFα and HIFβ); the α subunit is only stable in cells under low Onditions , 13. HIF protein . At atmo pathway .2 environments [The effects of HIF are primarily mediated by a hypoxic transcriptional response through activation of the expression of a large number of genes involved in glycolysis, angiogenesis, proliferation, erythropoiesis, and hematopoiesis –18. Therronments . Moreoveronments , 21.2 species (ROS) are increased [2 concentrations is correlated with increased generation of ROS [During development from the 2-cell stage to the blastocyst stage, the embryo gradually increases its mitochondrial membrane potential, which relates to mitochondrial activity, to maintain rapid embryo growth and metabolism. At the same time, levels of reactive Oncreased . ROS, a ncreased . The redncreased . A numben of ROS –27.2 tension affects embryo development, HIF (HIF-1α and HIF-2α) gene expression profiles and the target gene of HIFs including, vascular endothelial growth factor (VEGF), a key of angiogenesis and implantation [2) and normoxic (20% O2) conditions during 2-cell to blastocyst formation were evaluated using TUNEL and JC-1 staining, respectively. We sought to determine whether low O2 tension can influence embryonic developmental competence, antioxidant capacity, and implantation potential during the preimplantation period through activation of the genes involved in these processes.Therefore by using RT-qPCR and immunofluorescence staining, the objective of this study was to examine whether Oantation , glucoseantation  and anti2, 37 °C in each experiment. The same type of incubator  was used for embryo culture. Embryo morphology and development were monitored daily until day 4 of the blastocyst stage (E3.5 ~ 4.5). Blastocyst development and hatch rates were assessed on daily basis by calculated proportion of embryos developed to the blastocyst stage  and hatching stage (trophectoderm has started to protrude through the zona) [This study was approved by the Animal Care and Use Committee of Taipei Medical University . Imprinting control region (ICR) mice (6–8 weeks old) were superovulated with 10 IU of pregnant-mare serum globulin (PMSG) , which was followed 48 h later by 10 IU human chorionic gonadotropin (HCG) (Sigma-Aldrich). 2-cell stage Embryos (E1.5) were collected from oviducts and ovaries 66 h after HCG treatment. About 30–50 Embryos were cultured per drop in human tubal fluid (HTF) medium  supplemented with 10% serum substitute (Irvine Scientific) and randomly assigned to either low (3%) or high (20%) oxygen tensions in a 5% COTotal RNA was extracted from pools of 50–65 expanded blastocysts from per treatment, using Tri Reagent (Sigma-Aldrich) according to the manufacturer’s instructions. The frozen blastocysts were thawed, vortexed for 20 s, and allowed to sit at room temperature for 5 min. Chloroform (Sigma-Aldrich) was added, samples were vigorously shaken, and then incubated at room temperature for 15 min. Samples were centrifuged at 13,000 rpm for 15 min at 4 °C and the upper aqueous phase was removed. Isopropanol (Sigma-Aldrich) was added and RNA was precipitated by overnight incubation at −80 °C, followed by centrifugation at 13,000 rpm for 30 min at 4 °C. RNA pellets were washed with 70% ethanol, air-dried, and redissolved in 10 mL of sterile water. The RNA solution was kept frozen at −80 °C until all samples were assembled for reverse transcription.2 group.The reverse transcription assay was carried out using a QuantiTect Whole Transcriptome Kit . The gene expression levels of (i) HIFs: HIF-1α and HIF-2α, (ii) HIF target genes: GLUT-3 and VEGF, and (iii) antioxidant enzyme genes: MnSOD and PRDX5 were analyzed by quantitative real-time PCR (RT-qPCR) performed in an Applied Biosystems 7300 Real-Time PCR System (Applied Biosystems) with SensiMix SYBR ® Hi-ROX kit . The procedure was as follows: 95 °C for 10 min; 40 cycles of 95 °C for 10 s and 60 °C for 30s; melting curve from 65 to 95 °C. A cycle threshold (Ct) was calculated for each sample using the GeneAmp 7300 software. The Primer sequences are listed in Table Blastocysts were fixed in 3.7% paraformaldehyde in PBS for 20 min at room temperature and washed twice in PBS containing BSA (BPBS). Embryos were permeabilized with 0.2% Triton X-100 in PBS for 30 min, then washed twice in BPBS. Embryos were incubated in blocking solution (0.1% Tween 20 in BPBS) for 1 h and then incubated with primary antibodies against HIF-2α  (1:100), MnSOD  (1:200), and LIFR  (1:200) overnight at 4 °C. On the following morning, embryos were rinsed three times in BPBS and incubated in the appropriate secondary antibody conjugated with Alexa 488–labeled goat antimouse IgG  (1:100) or Alexa 568–labeled goat antimouse IgG  (1:100) for 1 h in the dark. After washing in PBS, the stained embryos were mounted in Fluoroshield mounting medium with 4′,6-diamidino-2-phenylindole  and observed by confocal laser-scanning microscopy  to detect the fluorescence. For image analysis, the intensities of green fluorescence (Alexa 488) and red fluorescence (Alexa 568) were measured using Olympus Fluoview® software (FV10-ASW).2O2 generation in blastocysts by measuring the intensity of fluorescence. Blastocysts from each treatment group were incubated in HTF supplemented with 10 μM DCHFDA for 15 min at 37 °C. After incubation, blastocysts were washed three times with PBS containing 1 mg/mL polyvinyl pyrrolidone (PVP-PBS), mounted onto glass slides and visualized immediately under an epifluorescence microscope at 490 nm excitation and 525 nm emission.2′,7′-Dichlorofluorescein diacetate  was used to quantify HMtMP in blastocysts was measured by staining with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanide iodide (JC-1) . Embryos were incubated in HTF containing 5.0 mg/mL JC-1 for 15 min at 37 °C in the dark, washed with PBS, then observed at either 510 nm (green mitochondria/J-monomer) or 590 nm (red-to-orange mitochondria/J-aggregate) using a confocal microscope . Acquired images were analyzed using Image J® software (1.47v), which allowed for quantitation of the fluorescence signal intensity of the DCHFDA- or JC-1-stained embryos. The ratio of red (J-aggregate) to green (J-monomer) staining was determined for all sections of the embryo, from which an average ratio of J-aggregate to J-monomer staining for the entire embryo was determined. Both experiments were replicated three times with a group of 10–20 blastocysts in each replicate.Blastocysts were washed three times in PVP-PBS followed by fixation in 3.7% paraformaldehyde in PBS for 1 h. After fixation, the embryos were washed in PVP-PBS and permeabilized by incubation in 0.5% Triton X-100 for 1 h. The embryos were then washed twice in PVP-PBS and incubated with fluorescein-conjugated dUTP and terminal deoxynucleotidyl transferase  in the dark for 1 h at 37 °C. After counterstaining incubated embryos with 25 mL Hoechst 33,258 (Sigma-Aldrich) for 20 min in the dark to label all nuclei, embryos were washed in PVP-PBS, mounted with slight coverslip compression, and examined using a confocal laser scanning microscope .P values <0.05 was assumed statistically significant.Differences in ratios of embryo development were analyzed by Chi-square tests. After log transformed, Kolmogorov-Smirnov test was performed for normality test. The comparison of JC-1 staining, ROS and TUNEL assays were analyzed by student’s t-test. Differences in gene expression were analyzed by student’s t-test after log transformed. Data were expressed as mean ± SE. All analyses were performed using the SAS statistical package , and two-tailed 2 and 189 cultured in 20% O2 showed that embryos cultured in 3% O2 had significantly higher rates of blastocyst development (92.3% vs. 79.4%) and hatching (80% vs. 70.4%) . Analysis of expression of antioxidant genes in blastocysts showed that the transcription levels of MnSOD and PRDX5 genes were 8–10-fold higher in the 3% O2 group compared with the 20% O2 group.Expression of MnSOD and VEGF was significantly higher  in the 20% O2 group (6.57 ± 0.71) than in the 3% O2 group (4.81 ± 0.37)  percentage of apoptotic cells (8.0 ± 1.1%) compared with those from the 3% O2 group (4.7 ± 0.6%)  Fig. . In addi6%) Fig. .Fig. 3Bo2. After JC-1 staining, blastocysts fluoresced green at 488 nm, red at 543 nm is seen  compared with those cultured in 20% O2 (0.17 ± 0.04) (P < 0.001)  Fig. .Fig. 4Bl2 benefits the development of embryos from the 8-cell to blastocyst stage, which is consistent with studies showing low O2 tension in the uterus. Aside from follicular O2 tension, in humans several factors in the follicular microenvironment also potentially influence the oocyte competence and consequently the embryo competence [2 concentration in the oviduct and uterine environment is lower than the atmospheric O2 (20%) typically used for the culture of somatic cells. Time-lapse monitoring [2 tension has demonstrated that embryo development is delayed under atmospheric O2 tension compared with low O2 tension [2 tension (3%) during the preimplantation period can not only increase the rate of 2-cell to blastocyst development (P < 0.05), but can also promote the development of embryos as evidenced by a higher hatching rate (P < 0.05) that is similar to that of embryos in vivo. These results are consistent with similar mouse [2 is likely because of the effects of oxidative stress on the embryos associated with increased generation of ROS-induced DNA damage in cells. Some of this variation may relate to the strains of mice used, or to differences in culture media composition.Many studies suggest that lower Ompetence . The in nitoring , 33 of d tension . Our pre tension . The curar mouse , bovine ar mouse , and humar mouse  studies.2 concentration, and that the expression of HIF-2α was higher and predominantly localized to the nucleus in blastocysts cultured under 3% O2 compared with those cultured under 20% O2. HIF-2α shares 48% amino sequence identity with HIF-1α and therefore shares common functional domains and can activate genes encoding a hypoxia response element (HRE) [We found that HIF-1α mRNA expression was slightly increased by low Ont (HRE) . Howevernt (HRE) , 41.2. The O2-dependent regulation of HIF-1α and HIF-2α may be mediated by the same mechanisms. Some reports indicate that HIF-2α is relatively resistant to O2-dependent degradation [2 and has a short half-life [These results are consistent with those of a previous study  that fouradation , while Half-life . This mi2 concentration [Oxidative stress in in vitro culture is detrimental to embryos, causing retardation or arrest in development . In mammntration , 48. In −2), by-products of the electron transport chain, into hydrogen peroxide (H2O2) [2 had higher levels of MnSOD (P < 0.0001) and PRDX5 (P < 0.01) transcription than did blastocysts cultured in high O2. These results are consistent with those of a study in cattle [2 conditions can enhance the antioxidant-mediated defense against oxidative stress by scavenging the extracellular ROS generated, thereby providing embryos with a low oxidative stress environment. As expected, MnSOD was also higher in blastocysts cultured under 3% O2. These data provide mounting evidence that internal protection in embryos is activated by hypoxia through increased production of these antioxidant enzymes to limit ROS during subsequent development. Knockout animal studies revealed that MnSOD is a target of HIF-2α and is responsible for the maintenance of ROS as well as for mitochondrial homeostasis [2 corresponds with reductions in both ROS and the apoptosis index in our blastocysts.PRDX5, one of the six PRDX isoforms (PRDX1 to PRDX6) in mitochondria can improve blastocyst development by protecting cells from oxidative stress by regulation of ROS/reactive nitrogen species (RNS) homeostasis , 50. MnSe (H2O2) , 52. Blan cattle , suggesteostasis , 55. Imm2 group compared with the 20% O2 group (P < 0.05), which may increase the glucose uptake to generate sufficient amounts of ATP without producing excessive amounts of ROS, thereby leading to an elevation of the membrane potential in mitochondria. This upregulation of expression was similar to that observed by Kind et al. [2 tension.Many genes are known to contain the HIF binding site HRE, including glycolytic enzymes (GLUT-3) and angiogenesis factors (VEGF) . At the d et al. , and is d et al.  and mousd et al.  embryos 2 group than in the 20% O2 group (P < 0.0001), suggesting that the ability for subsequent angiogenesis during implantation was increased in embryos cultured under hypoxic conditions. Increasing the expression of GLUT-3 and VEGF may contribute to promoting embryonic development during early preimplantation under hypoxic conditions. However, although GLUT-3 and VEGF genes are well known to be targets of HIF, we found that low O2 did not affect the protein level of HIF-1α (data not shown). Hence, we propose that HIF-2α, but not HIF-1α, plays an important role in gene regulation during hypoxia.At implantation, VEGF plays an important role in endometrial vascular permeability by direct induction of angiogenesis, by recruiting endothelial cells, and by stimulating their proliferation , 61. VEG2 may enhance embryo attachment for subsequent implantation. Nevertheless, given the differences in implantation mechanism between mouse and human, the limitation of this study is based on mouse model and the applicability in human embryo requires further investigation. Combined with the VEGF findings reported here, this indicates that the mechanism involving hypoxia that modulates LIFR expression merits further investigation.The successful implantation of embryos depends on steroid hormones, growth factors, and cytokine interactions between the specific receptors in the developing embryo and the endometrium via paracrine or autocrine pathways . During Mitochondrial membrane potential is a key indicator of mitochondrial activity, because it reflects the process of electron transport and oxidative phosphorylation, the driving force behind ATP production . During 2 group had increased MtMP. Our previous GLUT-3 expression results indicate that this higher MtMP may be the result of increased glucose intake by cells during hypoxia. Alternatively, decreased MtMP could lead to lower ATP generation, which could cause embryo development to be arrested or be abnormal because of energy insufficiency [2 concentration is an exogenous factor that can enhance the generation of ROS by embryos [2 (20%), there is a subsequent increase in the production of ROS, which have toxic effects on the embryo through the induction of apoptosis. We demonstrated that the number of apoptotic cells in blastocysts cultured under 20% O2 was significantly increased, probably associated with increased ROS, resulting in embryonic oxidative stress injury. These findings are consistent with those of Yoon et al. [2 contain few apoptotic cells and undetectable DNA damage, explaining the better rate of development and hatching in embryos from the 3% O2 group.Our JC-1 staining results showed that embryos in the 3% Oficiency –71. ROS ficiency , 73, and embryos . When mo embryos  and bovi embryos  embryos n et al. . In cont2 concentrations.Overall, the immunofluorescence staining and TUNEL assay results support our hypothesis that hypoxic conditions can increase protection against apoptosis and increase activity in the mitochondria to improve embryo development following in vitro culture. Future studies will be aimed at investigating ATP production within blastocysts developed under low O2 environment and respond to it by changes in expression of O2-regulated (HIF-2α) and antioxidant genes. Optimal oxygen tension is a critical factor in improving embryo competence during in vitro culture. In mouse model, the embryos have to encounter a decreasing oxygen gradient as they develop (from oviduct to the uterus). Whereas, Nastri et al., observed in low O2 tension only a small improvement (~5%) in live birth and clinical pregnancy rates, the evidence was of very low quality [2 tension in human embryos. This study indicates that low O2 tension provides a more conducive environment for embryo culture at least in mouse model. This environment increases antioxidant and mitochondrial function and promotes embryonic glycolysis, angiogenesis and antiapoptotic effect which could enhance implantation and hatching. These effects may be critically mediated by HIF-2α in hypoxic responses (Fig. These data confirm that preimplantation embryos have the ability to detect a lower O quality . Hence wovement ~% in live


Acidianus manzaensis YN-25 consists of a chromosome of 2,687,463 bp, with a G+C content of 30.62% and 2,746 coding DNA sequences. This archaeon contains a series of specific genes involved in the oxidation of elemental sulfur and reduced inorganic sulfur compounds.The complete genome of  Acidianus manzaensis is a novel thermoacidophilic archaeon of the Acidianus genus which is capable of oxidizing elemental sulfur and reduced inorganic sulfur compounds (RISCs)  , 4. This (RISCs) –7. To inA. manzaensis YN-25 was isolated from an acid thermal spring in Tengchong, China (4)2SO4, 0.5 g/liter MgSO4·7H2O, 0.5 g/liter K2HPO4, 0.1 g/liter KCl, 0.01 g/liter Ca(NO3)2] added with 0.2 g/liter yeast extract as a growth factor. Elemental sulfur was used as the electron donor (170 rpm at 65°C). The genomic DNA of A. manzaensis was extracted by the TIANamp DNA kit  and sequenced using a combined strategy of the Illumina HiSeq 4000  and PacBio RSII  platforms , which makes up 0.2275% of the genome. The numbers of tRNAs and rRNAs are 42 and 2, respectively.The A. manzaensis YN-25 encodes several enzymes involved in the oxidation of sulfur and RISCs, including the sulfur oxygenase-reductase, thiosulfate:quinone oxidoreductase, and tetrathionate hydrolase. Electrons generated from sulfur and RISC oxidation enter the electron transport chain via quinone. Terminal quinol oxidases, like aa3 ubiquinol oxidase, receive electrons from quinone and transfer them to O2 coupled with ATP generation. Some electrons can be transmitted to the NADH complex to produce NADH by NADH-quinone oxidoreductase. The complete genome of A. manzaensis YN-25 provides novel insights into biologically catalyzed metal sulfide oxidation.A. manzaensis YN-25 has been deposited in GenBank under the accession number CP020477. The version described in this paper is the first version, CP020477.1.The complete genome sequence of


In this study, we assess the CK2α expression in human penile squamous cell carcinoma (SCC) and its clinical significance.A total of 157 human penile SCC tissue samples were immunohistochemically analyzed. In addition, 12 human penile SCC and adjacent normal tissues were examined for CK2α protein and mRNA expression by Western blotting and real-time quantitative PCR, respectively. Survival was analyzed using the Kaplan-Meier test and the log-rank test. Multivariate Cox proportional hazard regression analysis was performed to determine the impacts of CK2α expression and the clinicopathological features on patient disease-specific survival (DSS). Likelihood ratios (LRs), Akaike information criterion (AIC) values, and concordance indexes (C-indexes) were investigated to evaluate the accuracies of the factors. Bootstrap-corrected C-indexes were used for internal validation (with sampling 1000 times).P<0.001). CK2α expression was associated with the pathological T and N stages in the penile cancer tissues (P<0.001). High CK2α expression was with significantly poorer DSS compared with low expression one (P<0.001). Western blotting and real-time quantitative PCR also confirmed that CK2α expression was increased in the penile cancer tissues. In multivariate Cox regression analysis, CK2α overexpression still was one of independent prognostic factors for penile SCC (P=0.005). The predictive accuracy of CK2α was verified by analysis of the C-indexes.A significant difference in the distribution of CK2α was observed between the normal and penile carcinoma tissues (High protein kinase CK2α expression is associated with several prognostic factors and is thus a significant indicator of poor prognosis for penile cancer. Penile cancer is a rare tumors in the Europe and United States, with an incidence of less than 1.00 per 100,000 males , 3. The The stage, grade of differentiation and clinicopathological factors of a primary tumor are well known to be predictive factors for LNM and survival , 6, 7. HSaccharomyces cerevisiae genes encoding CK2 catalytic subunits is a lethal event, and it plays major roles in cell cycle progression, proliferation, and differentiation processes [Protein kinase CK2 is a highly conserved and ubiquitous serine/threonine kinase composing with two catalytic subunits and two regulatory subunits . CK2 is rocesses , 19. In rocesses –24. Thusrocesses . As we’vIn this pilot study, we evaluated CK2α expression in primary penile SCC. In addition, the relationships between CK2α expression and clinicopathologic features were examined, as well as the clinical prognostic value of CK2α in penile SCC.After a median time of 25 mo range 1–133 mo), 43 patients died of penile cancer. The patients’ clinical characteristics are shown in Table –133 mo, P<0.001). Additionally, quantitative real-time PCR results also proved that CK2α expression was significantly increased in the tumor tissues over the non-tumor ones  and N status (P < 0.001). However, there was no significant association between nuclear CK2α expression and age, body mass index (BMI), or tumor grade. The associations between CK2α immunoreactivity and the clinicopathologic features are shown in Table These results indicated that CK2α expression was significantly correlated with the T stage P=0.00 and N stP < 0.001). The patients with higher CK2α expression had 3-year and 5-year DSS rates of 57.6% (46.4%-68.8%) and 49.2% (36.3%-62.1%), compared with rates of 97.9% (93.8%-100.0%) and 94.0% (85.6%-100.0%) in the patients with low CK2α expression , tumor grade (P=0.022) and CK2α overexpression (P<0.001) were significantly correlated with DSS. Further, Cox multivariate analysis revealed that CK2α overexpression was an independent prognostic predictor . CK2α overexpression showed the best predictive accuracy for the LR, C-index and AIC among the basic factors. Accounting for CK2α expression, the predictive accuracy of the basic factors were with significantly increased  and pathologic nodal status (P<0.001)  at 25°C for 30 minutes. After 5-8 minutes incubation with 3,3′-diaminobenzidine, the sections were counterstained with hematoxylin and microscopic analysis.Mastadenoma and omission of the primary antibody were used as positive and negative control. 3% HTwo pathologists was independently scored immunoreactivity. Nuclear and cytoplasmic staining were both scored for the sake of the ubiquitous and nucleo-cytoplasmic distribution of CK2α. According to the percentage of positive staining area, the score were 0 (0%), 1 (1-20%), 2 (21-50%), 3 (51-75%), and 4 (76-100%). And the following scores were used: 0 = no staining; 1+ = weak staining; 2+ = moderate staining; and 3+ = strong staining. The sum of the intensity and extent scores range 0 to 7. For the final statistics, tumors with score of ≥6 were considered positive (+) for CK2α. All other tumors were considered negative (-).12 tumor and non-tumor pairs of colorectal tissues using for RNA with an RNA extraction kit . RNA quality was analyzed using an Agilent Stratagene Bioanalyzer . The RNA integrity numbers (RINs) of all 24 samples indicated that the RNA quality was good, and the samples were then used for cDNA synthesis, after which they were stored at -20°C until further use. The amplification mixture (25 μl) contained 0.030 ng cDNA (3 μl), 1× TaqMan Master Mix, and 1× TaqMan Gene Expression Assay Primer/Probe Mix . The thermal cycling parameters were as follows: 40 cycles at 95°C for 2 min, 95°C for 30 sec, 60°C for 30 sec, and 72°C for 30 s for pre-denaturation, denaturation, annealing and extension, respectively. The sequences of the primers used for PCR amplification designed according to GenBank mRNA sequences were TGTTCGTCATGGGTGTGAAC (forward) and ATGGCATGGACTGTGGTCAT (reverse).3VO4, 10 mM sodium pyrophosphate, 1 mM phenylmethylsulfonyl fluoride, 1 lg/ml aprotinin and 1 lg/ml leupeptin) were useing for protein extracts. Extracted protein should be centrifugated at 14,000 g to remove debris. Denatured proteins in the samples were subjected to 10% SDS-PAGE. Anti-CK2α polyclonal antibody (1:1500 dilution) and phosphate-buffered serum (PBS:5% milk +0.05% Tween-20) was using for blocking at 25°C room temperature overnight. GAPDH  was used as an internal control. Subsequently, goat anti-rabbit antibody (1:5000 dilution) was using for incubation for 1 hour at 25°C room temperature. Targeted proteins was enhanced with chemiluminescence reagents (Millipore). All experiments were conducted three times independently.The mixture of protease and phosphatase inhibitors , with statistical significance set at P<0.05.Receiver operating characteristic (ROC) curves were generated to identify the best point for prediction of patient prognosis. The correlations between CK2α expression and the clinicopathologic parameters were examined using Pearson's correlation analysis. DSS was calculated using the Kaplan-Meier method, and assessed by log-rank tests. Univariate and multivariate Cox proportional hazard regression analyses were performed to determine the impacts of the clinical and pathological factors on patient DSS with removal of nonsignificant factors in a stepwise backward fashion. Likelihood ratios (LRs), Akaike information criterion (AIC) values, and concordance indexes (C-indexes) were investigated to evaluate the accuracies of the factors. Bootstrap-corrected c-indexes were used for internal validation to better gauge expected future predictive accuracy (with sampling 1000 times). All data were analyzed using R2.11.1 (