Ovariectomized (OVX) rat models and mouse embryonic fibroblasts cell models were used. E2 loss in OVX rats induced body weight gain and central abdominal fat accumulation, which were ameliorated by E2 treatment under chow and high-fat diet (HFD) conditions. E2 decreased the expression of the adipocyte marker genes PPARγ, aP2, C/EBPα, and S100A16. E2 inhibited adipogenesis. Overexpression of S100A16 reversed the E2-induced adipogenesis effect. A luciferase assay showed that E2 inhibited the expression of S100A16. E2 treatment decreased body weight gain and central abdominal fat accumulation under both chow and HFD conditions. Also, E2 suppressed adipogenesis by inhibiting S100A16 expression.The aim of this study is to determine the effects of E In postmenopausal women, estrogen loss is an independent risk factor for more severe menopausal symptoms, such as metabolic disease, insulin resistance, and type 2 diabetes  rats that received estrogen treatment or control treatment under chow and high-fat diet (HFD) conditions. We wanted to determine which physiological indicators of metabolic disease benefit from estrogen administration, and evaluate the effect of estrogen on n=16) received PBS injections. We measured body weight every week after the first injection.Fifty healthy female specific pathogen-free Sprague–Dawley (SD) rats 6 weeks of age  were purchased from and housed at the Experimental Animal Center of Nanjing Medical University . The experiments were approved by the Nanjing Medical University Ethical Committee. Animals were housed at 23±1 °C with a 12 h light:12 h darkness cycle and 45±5% humidity and allowed free access to normal chow diet and water. Thirty-two female SD rats underwent bilateral oophorectomy at the age of 8 weeks under general anesthesia induced with ketamine . After 2 weeks of recovery, the rats were allowed free access to water and were fed normal powder chow or HFD. The HFD was supplied by OpenSource DIETS . Sixteen OVX rats received s.c. injections (at the back of the neck) of 200 μg/kg 17β-estradiol  twice a week for 16 weeks. Control rats (n=9); OVX rats (n=8); and OVX rats given E2 treatment (n=8). There were also three groups of rats on HFD: normal control rats (n=9); OVX rats (n=8); and OVX rats given E2 treatment (n=8). All procedures were approved by the Experimental Animal Center of Nanjing Medical University.Three groups of rats were on the chow diet: normal control rats . All protocols involving the use of animals have been approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Nanjing Medical University. In addition, we investigated the morphology of visceral fat cells from all rats fed normal chow and HFD by using hematoxylin and eosin staining.To assess the effects of E2 treatment, IPGTT was conducted. All rats were starved for 12 h, and then their tail blood glucose concentrations (mM) were monitored using a handheld glucometer . Blood was collected from the inner canthus to detect insulin concentration. Next, all rats received i.p. injections of glucose (2 g/kg body weight); tail blood glucose concentrations were measured using the handheld glucometer and blood was collected from the inner canthus at 15, 30, 60, and 120 min. Blood samples were analyzed for insulin at every time point using an immunoradiometric assay kit (#KAP1251) supplied by DIAsource ImmounoAssays S.A .Fourteen weeks after the E2 treatment, all rats were anesthetized with ketamine (120 mg/kg i.p.), and blood was collected by cardiac puncture from the left ventricle into tubes precoated with potassium-EDTA, and centrifuged at 3000 g and 4 °C for plasma preparation. The plasma concentrations of E2 were quantified using immunoradiometric assay kits supplied by DIAsource ImmounoAssays S.A. (#KIP0629). The plasma levels of total cholesterol (TC), triglyceride (TG), LDL, HDL, alanine transaminase (ALT), lactate dehydrogenase (LDH), creatinine (Cr), urea, and uric acid (UA) were determined by the Medical Laboratory of Jiangsu Province Hospital, The First Affiliated Hospital of Nanjing Medical University. All assays were performed according to the manufacturer's instructions.At the end of 16 weeks of E2 and stored at −80 °C for the extraction of protein, and part of the samples was formalin fixed and paraffin embedded for the production of pathological sections. The same procedure was performed on the rest of the tissues.Visceral fat was removed rapidly; part of the samples was frozen in liquid NTg+/+ mice at 13.5 days post coitum. The construct was generated by inserting S100A16 cDNA into a vector with the PCAG promoter. We then obtained transgenic mice by the microinjection method. This F0 transgenic mouse was bred with the C57BL/6 mouse to obtain F1 transgenic mouse lines. S100A16-positive transgenic mice and their genotype were confirmed by PCR, and the expression of S100A16 was determined by qPCR and western blot. Embryos were chopped and incubated in a 0.25% trypsin and 0.01% EDTA solution at 37 °C for 10 min. The cells dispersed by pipetting were washed with PBS and then maintained in DMEM (Gibco) containing 10% fetal bovine serum (FBS) , 100 U/ml penicillin, and 0.1 mg/ml streptomycin in a 5% CO2 atmosphere at 37 °C. To induce adipocyte differentiation, 2-day post-confluent MEFs (designated day 0) were cultured with DMEM containing 10% FBS and MIX ). After 48 h of incubation (designated day 2), the medium was replaced with DMEM containing 10% FBS and 1 μg/ml insulin.Mouse embryonic fibroblasts (MEFs) were isolated from the embryos of C57BL/6 and S100A162O to remove unbound dye, visualized, and photographed under a microscope.Differentiated MEFs (day 10) were washed three times with PBS and stained with filtered Oil Red O solution  for 30 min at room temperature. The cells were then washed with ddHCellular TG content was determined using a TG GPO-POD assay kit (Sigma). MEFs were cultured and induced in a 10-cm well by MIX to differentiate into adipocytes (10 days), washed with PBS twice, scraped into 500 μl PBS, sonicated to homogenize the suspension, and then assayed for total TG content.Tissues and cells were washed twice with ice-cold PBS. Then, 100 mg tissue was lysed with 1 ml lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% (v/v) Nonidet-P40, 1 mM EDTA, 1 mM NaF, 10 μg/ml aprotinin, 10 μM leupeptin, and 1 mM phenylmethanesulfonyl fluoride). The cells were scraped into the lysis buffer, and then tissues and cells were allowed to stand on ice for 30 min. After centrifugation at 4 °C, the proteins in the supernatant were extracted and separated on SDS–polyacrylamide gels before being subjected to a standard western blot assay and imaged using Molecular Imager ChemiDoc XRS+ with the Image Lab Software .Luciferase reporter plasmid construction. The S100A16 promoter (1500 bp) was amplified by PCR using primers terminating in an XhoI and HindIII recognition sequence. The PCR product was digested and ligated into a pGL3-basic vector.Transient transfection and luciferase assay. The day before transfection, CHO cells were plated on 12-well cell culture plates at 3×105 cells/well. Transfection was performed using the X-tremeGENE HP DNA Transfection Reagent , following the manufacturer's protocol. Six hours later, cells were treated with E2 (400 pg/ml) and/or tamoxifen (0.05 μM) for 24 h. The Renilla luciferase reporter plasmid pRL-SV40 was used as an internal control. Luciferase activity was measured using the dual luciferase assay system  with a luminometer ). Luciferase assays were performed in triplicate and repeated at least three times to confirm their reproducibility.in vitro and in vivo results were analyzed using one-way ANOVA. P<0.05 was considered to indicate statistical significance. The results are presented as mean±s.d. of the values from three to six replicates per group.The 2 concentrations at the time the animals were killed. Under the chow conditions, in comparison to the control rats, E2 concentrations increased in the OVX rats treated with E2 (759.11±53.22 vs 77.09±5.21 pg/ml) and decreased in the OVX rats not treated with E2 (42.0±3.02 vs 77.09±5.21 pg/ml); also under the HFD conditions, E2 concentrations increased in the OVX rats treated with E2 (1209.08±198.02 vs 100.82±14.09 pg/ml) and decreased in the OVX rats not treated with E2 (34.0±3.15 vs 100.82±14.09 pg/ml) (We measured E9 pg/ml) .2 was more efficacious at lowering the percentage of body weight gain and visceral fat weight than control treatment. Under both diet conditions, low levels of E2 had the opposite effect. Under the chow conditions, the percentage of body weight gain was 5.32% in the high-E2 group, 61.40% in the low-E2 group, and 40.62% in the control group. Visceral fat weight was 4.7±0.91 g in the high-E2 group, 35.1±1.83 g in the low-E2 group, and 20.8±1.75 g in the control group . The blood glucose of the OVX rats treated with E2 dropped to close to the normal levels, at 20.4±5.7 nM at 120 min  showed a significant decrease in S100A16 expression in a dose-dependent manner  than the S100A16Tg+/+ cells . The S100A16 promoter has numerous transcription factor-binding sites, and also four estrogen receptor (ER)-binding sites . In CHO cells, EIn the present study, we studied the effect of different estrogen dosages on weight gain, insulin sensitivity, glucose and lipid metabolism, differentiation of MEFs, and liver and renal function in OVX and control female rats fed chow and HFD.2 loss induced glucose metabolism disorder and insulin resistance under different diet conditions  resulted in the initiation of signal transduction. E2 may exert the effect of inhibiting body weight gain via leptin-like effects (S100A16, related to obesity, which is a member of the S100 protein family (S100A16 within the 1500 bp by using AliBaba2 analysis (PPARγ, C/EBPα, and aP2 in the fat tissues from the different groups of rats mentioned earlier. The results showed that E2 treatment inhibited the expression of adipocyte marker genes, including PPARγ, C/EBPα, and aP2, and of S100A16 (2 acts via the regulation of S100A16 expression, MEFs were extracted from the S100A16Tg+/+ mice bred in our laboratory and incubated with E2 at different dosages and for different times, we found that the E2 inhibited S100A16 expression in a dose- and time-dependent manner (2 in the differentiation of MEFs extracted from S100A16Tg+/+ mice and control C57BL/6 mice by Oil Red O staining and TG determination. It was significant that S100A16 promoted the differentiation of MEFs and that E2 inhibited the effects of S100A16 (2, and this effect was ameliorated by the addition of tamoxifen (Little is known about the molecular mechanism underlying Eanalysis . Therefo S100A16 , which wt manner . At the  S100A16 . Resultsamoxifen .2 inhibited adipogenesis and S100A16 expression, which indicates that E2 might decrease S100A16 expression by inhibiting S100A16 gene transcription. Our study has revealed the interaction of S100A16 with estrogen, and will be applicable to clinical treatment involving estrogen.So we think that estrogen might regulate metabolism by mediating S100A16 expression. Though many studies must be processed to reveal the molecular mechanisms of the effects of estrogen on metabolism via the S100A16 pathway. Indeed, our data showed that ER Z, D S, and W Z conducted the molecular and the animal studies, carried out the data collection, and wrote this paper; Y X, D L, M L, Q H, and Y Z conducted the animal studies; A Z conducted part of the molecular studies; and Y L designed this study.


IDH1/2 and TERTp mutations, 1p/19q codeletion and EGFR amplification as to their impact on prognostication. Prognostic association of grading was independent of other prognostic variables including age, histological type, IDH1/2, 1p/19q and TERTp status. No single marker, including IDH1/2, superseded grading in prognostication, indicating grading was still a very important tool. Prognosis was most favorable in 31.7% of patients with IDH1/2 mutation and either 1p/19q codeletion or TERTp mutation (IDHmut-OT), intermediate in 45.8% of patients with IDH1/2 mutation only (IDHmut) and 16.9% of patients without any of the alterations (IDHwt), and poorest in 5.6% of patients with wild-type IDH1/2 and either TERTp mutation or EGFR amplification (IDHwt-ET). Our results suggested not all IDH1/2 wild-type lower-grade gliomas are aggressive and additional biomarkers are required to identify glioblastoma-equivalent tumors. Multivariate analysis revealed independent prognostic values of grading and genetic signature. Grade II IDHwt-ET gliomas exhibited shorter survival than IDH1/2 mutated grade III gliomas, suggesting combination genetic signature potentially superseded grading in prognostication. In summary, biomarker-based stratification is useful in the diagnosis and prognostication of lower-grade gliomas, and should be used together with grading.We studied if combination genetic signature potentially stratifies lower-grade gliomas better than histology by investigating 214 lower-grade gliomas for  Isocitrate dehydrogenase 1 (IDH1) mutation and chromosome 1p/19q codeletion represent the most important molecular markers with clinical implications in lower-grade gliomas. Both of the markers are strong favorable prognostic factors in lower-grade gliomas as shown in the literature  were used for PCR amplification and primer sequences were shown in IDH1 and IDH2, DNA amplification was conducted in a 10-μl reaction volume containing 1μl of cell lysate, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 2.5 mM MgCl2, 0.2 mM of each deoxyribonucleoside triphosphate, 0.4 μM of each primer and 0.2U of AmpliTaq Gold DNA polymerase , and was incubated at 95°C for 10 minutes, followed by 40–45 cycles of 95°C for 20 seconds, 60°C for 20 seconds and 72°C for 30 seconds, and a final extension of 72°C for 1 minute. For amplification of TERTp, PCR was performed in 10μl reaction mixture containing 1μl of cell lysate, 0.3 mM of each dNTP, 2.5 mM MgCl2, 0.3 μM of each primer and 0.2U of KAPA HiFi HotStart DNA Polymerase . The reaction mixture was incubated at 95°C for 5 minutes, followed by 40–45 cycles of 98°C for 20 seconds, 68°C for 15 seconds and 72°C for 30 seconds, and a final extension of 72°C for 1 minute. PCR products were then treated with exonuclease I and alkaline phosphatase  at 37°C for 15 minutes and then at 80°C for 15 minutes. Sequencing was performed using BigDye Terminator Cycle Sequencing kit v1.1 (Life Technologies). The products were resolved in Genetic Analyzer 3130xl and analyzed by Sequencing Analysis software.Mutations of eviously , 32, 33.P-value of <0.05 (two sided) was considered statistically significant.Statistical analysis was conducted using IBM SPSS Statistics 20 . Association between molecular markers and clinical parameters were examined by Chi square test or Fisher's exact test, whichever was appropriate. Overall survival (OS) was defined as the time between diagnosis and death or last follow-up. Survival curves were constructed by Kaplan-Meier method and survival distribution between groups was compared by Log-rank test. Multivariate analysis was performed by Cox proportional hazards model.


MTW1. This increase in protein failed to change protein recruitment, consistent with the hierarchical assembly model. However, we find that deletion of Psh1, a key ubiquitin ligase that is known to restrict inner kinetochore protein loading, does not increase levels of outer kinetochore proteins, thus breaking the normal kinetochore stoichiometry. This perturbation leads to chromosome segregation defects, which can be partially suppressed by mutation of Ubr2, a second ubiquitin ligase that normally restricts protein levels at the outer kinetochore. Together these data show that Psh1 and Ubr2 synergistically control the amount of proteins at the kinetochore.The accurate segregation of chromosomes during cell division is achieved by attachment of chromosomes to the mitotic spindle via the kinetochore, a large multi-protein complex that assembles on centromeres. The budding yeast kinetochore comprises more than 60 different proteins. Although the structure and function of many of these proteins has been investigated, we have little understanding of the steady state regulation of kinetochores. The primary model of kinetochore homeostasis suggests that kinetochores assemble hierarchically from the centromeric DNA via the inclusion of a centromere-specific histone into chromatin. We tested this model by trying to perturb kinetochore protein levels by overexpressing an outer kinetochore gene,  As cells divide, their replicated chromosomes must be correctly allocated to the two nascent daughter cells. This is achieved by the kinetochore, which provides a physical link between the chromosomes and the microtubules that drive their movement. If chromosome separation fails, the resulting cells have an abnormal number of chromosomes. This state is called aneuploidy and is a hallmark of cancer cells. The regulation of the kinetochore is therefore of critical importance in maintaining genome integrity. Since a number of cancer cells have over-active kinetochore genes, it has been proposed that an excess of kinetochore proteins can disrupt the normal assembly or maintenance of kinetochores. We tested this idea in yeast by increasing the amount of a specific kinetochore protein, but found no effect upon the normal loading of kinetochore proteins. Instead, we find that two ubiquitin ligases play a role in maintaining the normal balance of the different kinetochore proteins and that this correlates with correct segregation of the chromosomes. Accurate chromosome segregation is necessary for the equal distribution of genetic material between daughter cells during cell division and is achieved by kinetochores which link chromosomes to spindle microtubules . PerturbBudding yeast is a key model to study kinetochore composition and assembly because of its comparatively simple structure; there is only one microtubule attachment per chromosome and per kinetochore , 7. KineIn budding yeast the position of the centromeres is sequence specific. Cbf1 and the CBF3 complex associate to centromere DNA elements (CDE), CDEI and CDEIII, respectively –13. The The outer kinetochore mediates interactions with microtubules emanating from opposite spindle pole bodies. The yeast homologues of the KNL1/ MIS12/ NDC80 network (KNM) are the essential complexes SPC105, MIND and NDC80, respectively , 22. Botpsh1Δ cells [Although the centromeric DNA sequence (CEN) is essential to assemble kinetochores, protein degradation has been shown to be important to control cellular levels of various kinetochore proteins. The E3 ubiquitin ligase Psh1 restricts the localization of Cse4 to centromeres . Psh1 lo1Δ cells  and thes1Δ cells . More re1Δ cells . Thus ki1Δ cells . In this1Δ cells . These din vivo and how much flexibility exists in kinetochore composition. To investigate this, we tested how increased levels of kinetochore proteins affect kinetochore composition. We used fluorescence microscopy to quantify the levels of proteins at kinetochore foci. We found that Mtw1 levels at the kinetochore correlate with chromosome number and they are not transcriptionally controlled. Moreover, we found that psh1Δ mutants, in addition to the elevated Cse4 protein, have increased levels of inner kinetochore proteins but not outer kinetochore proteins. However, the levels of outer kinetochore proteins are increased in the psh1Δ ubr2Δ double mutant, in which both Cse4 and Dsn1 are unconstrained. Finally, we found that ubr2Δ suppresses psh1Δ mitotic and meiotic defects. These findings are consistent with multiple regulatory pathways acting independently on the different kinetochore complexes.Numerous studies in budding yeast have revealed the stoichiometry of the various protein sub-complexes forming the kinetochore , 33–37. MTW1. Mtw1 forms part of the essential MIND complex [CEN plasmid and its MTW1 gene is driven by a constitutively-active copper promoter (CUP1) [MTW1 plasmid produced significant ectopic expression as judged by loading of plasmid-encoded Mtw1 at the kinetochore  . We usedetochore . We quanetochore . Each baP signal . This ise signal . Again,  strain, . Finallytely 50% . We alson levels . Thus, tthe cell . Thus, wMTW1-YFP at either one or two of the endogenous MTW1 alleles. We find that diploid kinetochore Mtw1 levels are approximately double that of haploids and heterozygous mtw1Δ/MTW1-YFP strains compensate by loading equivalent Mtw1 as diploid strains  are both unaffected in psh1Δ cells ..psh1Δ cee poison  suggeststh psh1Δ . This ubpression , suggestdsn1-S240D/S250D did not increase Mtw1 kinetochore levels in wild type or psh1Δ cells, but only in psh1Δ ubr2Δ double mutant and slightly in ubr2Δ . Then, YFP (pHT5), CFP (pHT222), MTW1-YFP (pHT15) or MTW1-CFP (pHT223) were cloned downstream of the CUP1 promoter by gap repair. A sequence encoding four alanine residues was used as a linker between MTW1 and the fluorescent tags, and between SPC42 and RFP. Plasmids were transformed into appropriate strains by lithium acetate transformation and continuously selected in synthetic media lacking leucine.Yeast strains used in this study are either W303 or S288C background, as indicated in MTW1, PSH1 and UBR2 genes were disrupted by transforming with PCR products containing either MX6-KAN or MX6-NAT selection cassettes flanked with ~250 bp of sequences upstream and downstream the corresponding coding regions. Gene deletions were confirmed by PCR. Since MTW1 is an essential gene, it was disrupted in a haploid strain harbouring CUP1-pMTW1-YFP::LEU2 plasmid (pHT15). Transformants were selected in synthetic media lacking leucine and containing G418 and confirmed by PCR. Diploid strain MTW1-YFP/MTW1-CFP (PT11) was transformed using mtw1Δ::KANMX PCR to obtain heterozygous diploids MTW1-YFP/mtw1Δ::KANMX (PT69 and PT70). Loss of CFP or YFP kinetochore foci was tested by fluorescence microscopy and insertion of the KANMX cassette at one of the MTW1 locus was confirmed by PCR.600 0.3 and grown for 3 hours before imaging or protein extraction.For microscopy and western blot analysis cells were grown in synthetic complete (SC) or lacking leucine SC–LEU media supplemented with 100mg/ml of adenine . Yeast strains were grown overnight at 23°C. Cultures were diluted in fresh media to ≈ ODCells from log-phase cultures were mounted on microscope slides with 0.7% LMP agarose in SC +ADE or SC-LEU +ADE, and covered with 0.17 mm glass coverslips. Our microscope system uses a Zeiss AxioImager Z2 microscope, 63X Plan Apo, 1.4NA, oil immersion objective and a Hamamatsu CCD ORCAII camera . The resulting pixel size was 0.205 μm. Excitation light was provided by LED Colibri system (excitation band-pass filter): CFP 445 nm (445/25), YFP 505 nm (510/15), GFP 470 nm (474/28) and RFP 590 nm (585/35). Emission band-pass filters were as follows: CFP 47HE (480/40), YFP 46HE (535/30), GFP 38HE (525/50), and RFP 63HE (629/62). Exposure times were optimized for each fluorescent protein and ranged from 100 to 250ms. Z stacks consisted of 17 vertically separated slices with 0.4 μm spacing. The theoretical dynamic range of our system is ~3000 levels of brightness, however, in practice this will be somewhat lower.3). The background region was 2 pixels separated from the kinetochore box (23.51 μm3). Average intensity of the background was subtracted from average kinetochore intensity to obtain the final fluorescence value. Finally, fluorescence values were normalized to the average of wild type or control populations. For quantitation, only post-anaphase kinetochores of dividing cells were selected.A custom-made protocol in Volocity software was used to quantify fluorescence intensity at kinetochore foci. The protocol finds the brightest spots in the image. Spots within 3 pixels from x,y,z edges of the image were removed from the analysis. A 3D box was drawn concentric to the brightest pixels  and transferred to a fresh tube containing 0.5 mm glass beads. Cells were disrupted with a cell homogenizer. Cells extracts were harvested into a fresh tube and boiled for 5 minutes. Cells debris was pelleted and 20 μL of the protein extracts were loaded in a 12% acrylamide gel (Biorad). Proteins were transferred into a PVDF blotting membrane . The western blot was performed with monoclonal anti-GFP antibody (Roche), anti-PGK1 (Invitrogen), goat anti-mouse HRP antibody (Abcam), and ECL kit .600 = 1, serially diluted and spotted into YPD or selective media plates with 0.2% DMSO and 10–15 μg/ml benomyl. For testing effects of overexpression increasing concentrations of CuS04 were added to the media as indicated. Plates were incubated for 2 days at 30°C before images were captured.Yeast strains were grown o/n at 30°C in YPD or selective media. Cultures were adjusted to OD600 reached 0.6 (2X107 cells/ml). Cultures were washed once with water, resuspended in SPO media and incubated at 23°C for 3 days. Four independent cultures were tested for each genotype. To test spore viability, 22 tetrads per genotype were dissected in YPD and grown for 2 days at 30°C.Diploid strains were grown in YPD at 23°C for 24 hours. Then, cultures were diluted 100X in YEPA media and grown at 23°C until OD600 0.3 in YPA  and grown for 12–15 h at 30°C. Cells were then resuspended in sporulation media (1% potassium acetate pH7) at 23°C for 12 hours. Finally, 1μM β-estradiol (Sigma) was added to induce NDT80 expression. Cells were imaged every hour to follow meiotic divisions.Diploid strains were grown in YPD for 24 hours at 30°C. Cultures were diluted to ODMATa strains lacking the Bar1 protein were used to facilitate α-factor G1 synchronization. Strains were grown overnight at 30°C, diluted to OD600 = 0.3 and grown for 1 hour. The asynchronous sample was collected at this time, then α-factor was added and cells were incubated for additional for 2.5 hours. G1 arrest was confirmed by the presence of the characteristic ‘shmoo’ morphology. Cells were washed twice with water and resuspended in YPD with Pronase E. Samples were taken every 30 minutes until 180 minutes. Cells were prepared for flow cytometry as in [his3-/HIS1 strains were streaked on fresh YPD plates and grown for 2 days at 30°C. Five colonies of each strain were resuspended in YPD. 3x106 cells were mixed with 3x107 cells of log-phase cultures of haploid mating tester strains (HIS3/his1-). Cells were concentrated by gentle centrifugation and incubated overnight at 23°C. The next day these cells were plated on synthetic dropout plates and incubated for 3 days at 30°C to select for HIS+ mating products. For each colony, mating products originating from both mating type MATa and MATα tester strains were summed. For each strain, the median number of colonies from the 5 colonies was calculated.Diploid Strains with a tetracycline operator array, inserted at the URA3 locus of chromosome V and a tetracycline repressor linked to mRFP, were grown overnight in synthetic media at 23°C. The day after the culture was diluted and further grown until log phase. Cells were imaged as explain above. In each image, cells showing aberrant chromosome segregation were identified as containing two TetR-mFRP foci in G1 or S-MCEN plasmid with a selectable marker and grown for two days. 9 colonies were grown overnight in YPD and then plated in either YPD or selective media. The percentage of plasmid loss was calculated by subtracting the amount of cells growing in the selective media to the number of cells growing in YPD. The data is presented as the median of percentage plasmid loss of 9 colonies.Strains were transformed with a S1 FigMTW1 is also expressed from endogenous locus (green and blue lines and circles).(A-B) Quantitation of Mtw1-YFP kinetochore and background fluorescence intensity. Top panels and bottom panels display the distribution of intensities and the mean intensity ± standard deviation. Fluorescence intensity levels are normalised relative to the mean intensity of the endogenously tagged Mtw1-YFP strain (black line and circle). Using a background correction region further from kinetochore did not change quantitation of Mtw1-YFP. Strains ectopically expressing Mtw1-YFP have higher background when (TIF)Click here for additional data file.S2 FigMTW1-YFP/mtw1Δ are haplo-sufficient. Serial dilutions of cells grown in YPD were spotted onto YPD plates containing benomyl diluted in DMSO. Cells were grown for 2 days at 30°C. (C) MTW1 over-expression does not affect growth or benomyl sensitivity.(A) Total Mtw1 protein increases in haploid and diploid strains from (TIF)Click here for additional data file.S3 FigMTW1. (B) The median proportion of cells losing a copy of a CEN plasmid after overnight growth without selection was not significantly different between cells containing an empty plasmid (control) and those containing MTW1 (MTW1-OX) . (C) A tetracycline operator array, inserted at the URA3 locus of chromosome V, is marked with a tetracycline repressor linked to mRFP. Both normal and aberrant segregation of the chromosome V marker were seen in cells containing an empty plasmid (left panels) and MTW1 (right panels). Cell outlines are shown in the RFP image as dashed lines, arrowheads highlight aberrant segregation, the scale bar is 5μm. (D) The proportion of cells showing aberrant chromosome V segregation was not significantly different between cells containing an empty plasmid (control) and those containing MTW1 (MTW1-OX) . (E-F) Quantitation of Dsn1-GFP (E) and Ndc80-GFP (F) kinetochore levels in control (black), low MTW1-OX (light blue) and high MTW1-OX (dark blue). Fluorescence intensity levels are normalised relative to control mean intensity. Left panel and right panels display the mean intensity ± standard deviation and the distribution of intensities, respectively. Ectopic MTW1 was expressed from a CUP1p. No additional copper was added to the low MTW1-OX cells. 100 μM CuSO4 was added to the high MTW1-OX cells for 3 hours before imaging.(A) Cell cycle progression is not altered in cells expressing ectopic (TIF)Click here for additional data file.S4 Fig4. Cells spots were grown for 2 days at 30°C prior to imaging.Serial dilutions of cells were spotted into synthetic media lacking leucine to select for plasmid, with several concentrations of benomyl and CuSO(TIF)Click here for additional data file.S5 Figctf19Δ, csm1Δ, cbf1Δ, mam1Δ and cnn1Δ mutants. Fluorescence intensity levels are normalised relative to wild-type mean intensity. Top panels and bottom panels display the distribution of intensities and the mean intensity ± standard deviation, respectively.(A-B) Quantitation of Mtw1 kinetochore levels in (TIF)Click here for additional data file.S6 Figpsh1Δ (blue), ubr2Δ (green) and psh1Δ ubr2Δ (red) cells. Fluorescence peak height values and full width at half maximum (FHWM) values are normalised relative to wild-type mean intensity ± standard deviation. Top panels and bottom panels display the distribution of intensities and the mean intensity ± standard deviation, respectively. (A) Cse4-GFP. Peak height: wild type 1.00±0.15, psh1Δ 1.72±0.37***, ubr2Δ 0.96±0.14, psh1Δ ubr2Δ 1.91±0.48***. FHWM: wild type 1.00±0.26, psh1Δ 1.16±0.31***, ubr2Δ 0.98±0.25, psh1Δ ubr2Δ 1.17±0.28***. (B) Mif2-GFP. Peak height: wild type 1.00±0.24, psh1Δ 1.36±0.43***, ubr2Δ 1.16±0.28, psh1Δ ubr2Δ 1.28±0.30***. FHWM: wild type 1.00±0.25, psh1Δ 1.02±0.23, ubr2Δ 1.05±0.24, psh1Δ ubr2Δ 1.05±0.21. (C) Dsn1-GFP. Peak height: wild type 1.00±0.19, psh1Δ 1.12±0.21, ubr2Δ 1.06±0.23, psh1Δ ubr2Δ 1.18±0.28***. FHWM: wild type 1.00±0.21, psh1Δ 0.97±0.17, ubr2Δ 1.03±0.24, psh1Δ ubr2Δ 1.05±0.23 (D-E) Total Mtw1 and Dsn1 protein do not change in psh1Δ, ubr2Δ and psh1Δ ubr2Δ cells. Western blot of total cell extracts. Quantification of cellular levels of Mtw1-YFP/-CFP relative to Pgk1 is shown below.(A-C) Quantitation of size of kinetochore foci in wild type (black) (TIF)Click here for additional data file.S7 Figphs1Δ, ubr2Δ, and psh1Δ ubr2Δ. MATabar1Δ cells were synchronized in G1 with alpha-factor, and then released (Time 0). (B) Changes in the G1, S and G2/M populations during the course of the experiment.(A) Cell cycle profiles of wild type, (TIF)Click here for additional data file.S8 FigCUP1p DSN1-OX plasmid, with several concentrations of benomyl and CuSO4. Cells spots were grown for 2 days at 30°C prior to imaging.Serial dilutions of cells were spotted into YPD plates containing NAT to select for (TIF)Click here for additional data file.S1 Table(XLSX)Click here for additional data file.S2 Table(XLSX)Click here for additional data file.


Pycnoporus sanguineus RP15 grown in wheat bran and corncob under solid-state fermentation was optimized by response surface methodology using a Central Composite Rotational Design. A laccase (Lacps1) was purified and characterized and the potential of the pure Lacps1 and the crude culture extract for synthetic dye decolorization was evaluated. At optimal conditions  milled corncob, 0.8% (w/w) NH4Cl and 50 mmol·L−1 CuSO4, initial moisture 4.1 mL·g−1), the laccase activity reached 138.6 ± 13.2 U·g−1. Lacps1 was a monomeric glycoprotein . Optimum pH and temperature for the oxidation of 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) were 4.4 and 74.4 °C, respectively. Lacps1 was stable at pH 3.0–8.0, and after two hours at 55–60 °C, presenting high redox potential (0.747 V vs. NHE). ABTS was oxidized with an apparent affinity constant of 147.0 ± 6.4 μmol·L−1, maximum velocity of 413.4 ± 21.2 U·mg−1 and catalytic efficiency of 3140.1 ± 149.6 L·mmol−1·s−1. The maximum decolorization percentages of bromophenol blue (BPB), remazol brilliant blue R and reactive blue 4 (RB4), at 25 or 40 °C without redox mediators, reached 90%, 80% and 60%, respectively, using either pure Lacps1 or the crude extract. This is the first study of the decolorization of BPB and RB4 by a P. sanguineus laccase. The data suggested good potential for treatment of industrial dye-containing effluents.Laccase production by  Laccases  are multicopper oxidases broadly distributed in plants, insects, fungi and bacteria . Some yeE°) of the T1 copper site is characteristic of each enzyme, ranging from 0.4 to 0.8 V vs. normal hydrogen electrode (NHE). According to it, the laccases are classified into three groups: low- (0.4–0.5 V), medium- (0.5–0.6 V) and high- (0.7–0.8 V) redox potential  vs. the potential of the redox couple [Fe(dipyridyl)2Cl3/Fe(dipyridyl)2Cl2] vs. NHE in Volts, where A0 and A corresponded to the absorbances of the solution at 740 nm before and after the reaction equilibrium. The potential of the redox couple was calculated from the Nerst equation associated with the redox species involved (E = E° + 0.058 log {[Fe(dipyridyl)2Cl3]/[Fe(dipyridyl)2Cl2]}), in which the concentrations of each species were obtained from the initial values considering the concentration changes due to the interaction with the enzyme after the equilibrium.The value of the T1 P. sanguineus RP15 or the pure Lacps1 and three different dyes as substrates: the anthraquinone dyes RBBR and RB4, and the triphenylmethane dye BPB (Sigma-Aldrich Chem. Co.). The reaction mixtures contained the dye at final concentrations of 100 mg·L−1 (RBBR and RB4) or 25 mg·L−1 (BPB) in McIlvaine buffer, pH 4.5, in a final volume of 1 mL. The reactions were performed under magnetic stirring at 25 or 40 °C and initiated by the addition of the enzyme to the reaction media. The enzymatic load was varied from 0.1 to 5.0 U·mL−1 and reaction mixtures containing aliquots of boiled crude extract or pure enzyme were used as negative controls. Dye decolorization was followed continuously in a Carry 60 UV–Visible spectrophotometer (Agilent Technologies) equipped with thermostated cell holders by monitoring the decrease of the absorbance at the wavelength of maximum absorbance of each dye . The percentage of decolorization was calculated according to the equation: Decolorization (%) = [Ai − At/Ai] × 100, where Ai is the initial absorbance of the reaction mixture and At is the absorbance after incubation for t minutes [n = 3). The enzymatic assays were carried out in duplicate.Decolorization experiments were performed employing either the laccase-rich crude extract from  minutes . The exp


IL13 receptor α2 (IL13Rα2) is a high-affinity IL13 receptor overexpressed in a variety of human cancer types. In many of these cancers  the expression of this receptor is associated with poor prognosis .Because of the short cytoplasmic tail of IL13Rα2 (14 amino acids), it was initially regarded as a decoy, unable to transduce cell signaling. Recently, several articles described that the binding of IL13 to IL13Rα2 triggered the activation of several proteins, such as Src, AKT and AP-1 . By a prMoreover, we found that FAM120A was associated to proteins involved in vesicle trafficking, suggesting a role in protein transport that is common in scaffold proteins. In fact, the silencing of FAM120A provoked a partial reduction in IL13Rα2 expression in the cell membrane, whereas total IL13Rα2 expression remained unchanged. Moreover, FAM120A silencing caused a significant downregulation of other scaffold proteins and linkers to cytoskeleton, such as AHNAK, SH3PXD2B, SPTAN1, PDLIM7 and DSG2 as well as some proteins involved in protein transport such as MYO1D, S100A10 and ACTN4. Finally, FAM120A silencing down-regulated three proteins involved in RNA splicing , which may explain the previous link between FAM120A and RNA biology.In summary, FAM120A is a scaffold protein required for the proper IL13Rα2-triggered signaling, which is involved in colon cancer metastasis. FAM120A also modulates IL13Rα2 cell membrane location and promotes the expression of other scaffold proteins involved in signaling or transport. These results support the use of FAM120A as potential target for therapy and reinforce the therapeutic value of IL13Rα2 in colon cancer. In other cancer types, where IL13Rα2 has been described as a poor prognosis biomarker, FAM120A could also play an important role as regulator of IL13-triggered signaling. In fact, according to cBioPortal , FAM120A


The level of hydrogen peroxide, superoxide anion, and malondialdehyde was increased by 0.5, 1.0 and 1.5 mM La3+, and the activity of catalase and peroxidase was increased by 0.05 and 0.1 mM La3+. However, La3+ treatments stimulated superoxide dismutase activity in the roots of rice seedlings at all tested concentrations. In addition, the probe 2′,7′-dichlorofluorescein diacetate (H2DCF-DA) was used to investigate the instantaneous change of ROS in the root cells with the laser-scanning confocal microscopy. The result indicated that ROS level was declined after treated with 0.05 mM La3+. The results showed that the appropriate concentration of La3+ decreased the level of ROS, and hormetic effects on the antioxidant metabolism were found in the roots of rice exposed to 0.05, 0.1, 0.5, 1.0, and 1.5 mM La3+.In this study, the effects of La In China, REEs enriched fertilizers have been used since the 1970s2452O2), singlet oxygen (1O2), superoxide anion ·). In the plant cells, ROS could act as a signal molecule and trigger a series of cellular responses111314Reactive oxygen species (ROS) mainly include hydrogen peroxide .Hormesis is defined as a phenomenon that low doses of an otherwise harmful agent could result in stimulatory or beneficial effects2O2 was significantly increased by increasing solution La concentrations from 0.5 to 1.5 mM  was imaged in the cells of root and the regions of interest (ROI 1–3) were circled with Leica Confocal Software . The flu3+ ,4 and 5.3+. Compared to the control, the activity of SOD was significantly increased in roots after La3+ treatments .In summary, the probe HO. sativa L. cv Shengdao 16) were sterilized by soaking in 75% alcohol for 1 min, in 0.1% mercury chloride for 15 min, and in 1.0% sodium hypochlorite for 20 min. Then the seeds were rinsed five times with sterilized water, and germinated in half-strength Murashige and Skoog agar medium at pH 5.8 . Subsequently, La(NO3)3 was added to the basal medium before autoclaving to obtain La3+ concentrations of 0, 0.05, 0.1, 0.5, 1.0, and 1.5 mM. Plants were grown 13 days in the different concentration of La3+ at 25.0 ± 2 °C using a 14/10 h light/dark cycle under a light intensity of 200 μmol m−2 s−1 in a growth chamber.Seeds of rice  of roots was determined in 13 day-old seedlings. The roots biomass (0.5 g in fresh mass) was homogenized under ice-cold conditions in 5.0 ml of extraction buffer containing 50 mM phosphate buffer (pH 7.5), 1.0% polyvinylpyrrolidone (PVP), 0.5% Triton X-100 and 1 mM EDTA, centrifuged at 10,000 × g at 4 °C for 20 min to remove particulate plant debris. The supernatant was used for the assay of antioxidant enzyme activities. The SOD activity was determined at 550 nm, and one unit of SOD activity was the amount of enzyme that inhibits 50% nitrite formation2O2, 2O2 was determined at 405 nm based on the principle that H2O2 could form a stable complex with ammonium molybdateThe plant samples were obtained as the assay of antioxidant enzyme activities. The same supernatant was used for the determination of soluble proteins, H2DCF-DA, Molecular Probes). For assessment of ROS, the rice roots were immersed in 1.5 ml of 10 μM DCFH-DA  for 2 hr at 20 °C. Then the roots were rinsed with MES-KCl buffer  to remove DCFH-DA solution from the surface of roots.The production of ROS in the roots was visualized with 2′,7′-dichlorofluorescein diacetate  were circled with Leica Confocal Software, and the fluorescence intensity of ROI was acquired. Data were processed with a Leica TCS Image Browser, and transferred to Adobe Photoshop 6.0 for preparation of figures.A laser-scanning confocal microscope  with an argon-ion laser as the excitation source at 488 nm was used to view the sites of ROS in the root cells. The laser-scanning mode was XYt, 30 optical sections were acquired, and the time interval between two sections was 10.0 s. To investigate the instantaneous change of ROS in the root, MES-KCl buffer or 0.05 mM LaThe roots were fixed for 2 h at 4 °C in 2.5% (v/v) glutaraldehyde and 0.1 M phosphate buffer solution (pH 7.3), and postfixed in 1% (w/v) aqueous osmium tetraoxide for 2 h. Samples were dehydrated in a 50–100% ethanol series and embedded in Epon 812 resin. Ultra-thin sections of 70 nm thickness were cut with an Ultracut Eultramicrotome  and stained with uranyl acetate and lead citrate. Then the subcellular distribution of La was detected with a Hitachi H-600 transmission electron microscope (TEM).P < 0.05).The assays for oxidative stress and the soluble protein were carried out in three different experiments, and results are expressed as mean ± standard error (SE). Statistical comparisons were done with one-way ANOVA using SPSS 16.0 for Windows . Tukey test was performed for post hoc comparisons when the difference was significant .


The receptor for activated C-kinase 1 (RACK1) is a conserved protein belonging to the WD40 repeat family of proteins. It folds into a beta propeller with seven blades which allow interactions with many proteins. Thus it can serve as a scaffolding protein and have roles in several cellular processes.Dictyostelium discoideum gpbB gene as the Dictyostelium RACK1 homolog. The protein is mainly cytosolic but can also associate with cellular membranes. DdRACK1 binds to phosphoinositides (PIPs) in protein-lipid overlay and liposome-binding assays. The basis of this activity resides in a basic region located in the extended loop between blades 6 and 7 as revealed by mutational analysis. Similar to RACK1 proteins from other organisms DdRACK1 interacts with G protein subunits alpha, beta and gamma as shown by yeast two-hybrid, pulldown, and immunoprecipitation assays. Unlike the Saccharomyces cerevisiae and Cryptococcus neoformans RACK1 proteins it does not appear to take over Gβ function in D. discoideum as developmental and other defects were not rescued in Gβ null mutants overexpressing GFP-DdRACK1. Overexpression of GFP-tagged DdRACK1 and a mutant version (DdRACK1mut) which carried a charge-reversal mutation in the basic region in wild type cells led to changes during growth and development.We identified the product of the DdRACK1 interacts with heterotrimeric G proteins and can through these interactions impact on processes specifically regulated by these proteins. Every cell has the capability to detect extracellular signals, and then mounts an appropriate response to these signals. Specific stimuli include light, hormones, neurotransmitters, growth factors, and odorants. They are sensed by cell surface receptors which in case of G protein-coupled receptors interact with heterotrimeric guanine nucleotide binding proteins (G proteins), key intermediates in cellular signaling processes that link the receptors with intracellular effector proteins generating cellular responses ,2.D. discoideum harbors a single Gβ, one Gγ, and twelve Gα subunits [D. discoideum development is relatively simple as compared to higher eukaryotes. The roles of its individual Gα subunits, however, appear to be quite distinct with respect to developmental morphology and cellular differentiation as indicated by the phenotypes of gene disruption or overexpression mutants. Gα2 is required for adenylyl cyclase A (ACA), guanylyl cyclase (GC) and phospholipase C (PLC) activation. Gα2-null mutants do not aggregate and overexpression of wild-type Gα2 results in precocious activation of guanylyl cyclase by cAMP in vegetative cells [D. discoideum cells lacking functional G protein β subunit are severely defective in phagocytosis, chemotaxis, aggregation, and development [The heterotrimeric G proteins consist of α, β, and γ subunits. Upon binding of agonist to the receptor, a conformational change in the Gα subunit promotes the release of GDP and binding to GTP which then releases Gβγ . The libsubunits -13. All ve cells . Gα4 medve cells , and Gα8ve cells . D. discelopment ,12-21.S. cerevisiae cells lacking RACK1 are viable whereas in a mouse model RACK1 depletion causes lethality at gastrulation [RACK1 (Receptor for activated C kinase 1) is present in organisms from all eukaryotic kingdoms like plants, fungi and animals. rulation . The prorulation ,24. The rulation -28.S. cerevisiae and C. neoformans, which possess only one Gβγ subunit but multiple Gα subunits, RACK1 has been reported to interact with free Gα and Gγ; interactions with the heterotrimeric Gβγ subunits were shown as well [RACK1 structurally mimics a Gβ harboring seven WD repeats which build up the seven-bladed beta-propeller. Different from Gβ RACK1 lacks the typical N-terminal alpha helix which is necessary for the tight interaction of Gβ with the Gγ subunit. In  as well ,30. Furt as well ,32.D. discoideum GpbB (DDB0185122) which is described as a Gβ-like protein in the databases is a RACK1 homolog. We initially identified GbpB as a binding partner of RpkA, an unusual G protein coupled receptor (GPCR) which functions in phagocytosis and antibacterial defense in D. discoideum[Here we show that iscoideum. RpkA haD. discoideum genome and has 2 exons. The open reading frame encompasses 1136 bp which encodes a protein of 329 amino acids migrating as a 36 kDa protein on SDS polyacrylamide gels. Blast results showed that GpbB is highly related to the RACK1 family of proteins and the alignment of RACK1 sequences from diverse organisms such as H. sapiens, D. melanogaster, A. thaliana, D. discoideum and S. cerevisiae revealed significant sequence identity. The greatest difference is observed between propeller blades 6 and 7 where an extended loop of mainly basic amino acids is present in the D. discoideum and the A. thaliana RACK1 proteins  is located on chromosome 2 of the A. thaliana, Asc1p from S. cerevisiae, RACK1 from T. thermophila and RACK1 from human [1 and forms a knob-like projection from the upper face of the propeller  [D. discoideum protein as it is rich in lysine residues. The general features described for RACK1 proteins are also present in DdRACK1 when we modelled the DdRACK1 sequence to the crystal structure of S. cerevisiae RACK1 (Asc1p) which reveals a comparable structure  . This seWhen we expressed RFP-DdRACK1 in AX2 cells expressing the G protein beta-subunit as GFP-fusion protein for labeling the plasma membrane we found the protein present throughout the cytosol. A similar cytosolic RACK1 distribution was obtained when immunofluorescence studies were performed with AX2 cells stained with antibodies against DdRACK1 and actin for labeling the cell cortex  receptor by the Fyn kinase in the brain [It has been suggested that RACK1 can dimerize in vivo and this dimerization is required for specific processes including the regulation of the he brain -48,39,37Little is known about post-translational modifications of RACK1 apart from phosphorylation which is emerging as an important factor that modulates the binding of proteins to RACK1. Phosphorylation of specific tyrosine residues and their corresponding functions has been reported ,49-54. TThe mechanism of membrane association of DdRACK1 is not known. In general, membrane association of proteins can be achieved by various mechanisms. For instance, polybasic clusters as defined by arginine- and lysine-enriched amino acid sequences enable diverse transmembrane and cytosolic proteins to bind lipids . Also, pAlthough dot-blot overlay assays are convenient assays, they need to be supported by different methods as apparent specificities may be distorted and as they do not allow reliable quantification . We therA. thaliana and D. discoideum RACK1 proteins carry an insertion between propeller blades 6 and 7, which contains primarily basic amino acids, in case of DdRACK1 six lysine residues  P3, whereas binding to all other PIP variants was completely abolished  [C. neoformans the RACK1 orthologue Gib2 functions as Gβ for Gpa1 [D. discoideum both organisms have a single Gβ gene. To investigate DdRACK1-Gα interactions, yeast two-hybrid assays were performed using DdRACK1 fused to pACT2-AD. The Gα subunits Gα1, Gα2, Gα4, Gα5, Gα6, Gα7, Gα8, Gα9, Gα10, Gα11 and Gα12 were fused to pAS2-BD. In these assays, we detected stronger interactions between DdRACK1 and Gα2 as well as between DdRACK1 and Gα8, whereas a weak interaction was detected between DdRACK1 and Gα4 as concluded from the β-galactosidase staining assay  . Similarfor Gpa1 . Like D.gβ null mutant LW6 [K. aerogenes, they formed smooth plaques as observed for the mutant strain. Remarkably the plaque size was even further reduced when we compared the AX2, LW6 and LW6/GFP-RACK1 strains , 6.54 ± 3.53 μm/min (LW6) and 6.34 ± 1.95 μm/min (LW6/GFP-DdRACK1), respectively.D. discoideum knockout and/or knockdown mutants for RACK1 using different molecular biology techniques were not successful. Since RACK1 acts as a scaffold protein, interference with its levels might lead to cellular defects which give an indication about its involvement in critical cellular roles. Knowing fully well that an overexpression of RACK1 has effects on various cell types [Our attempts to generate either ll types -61, we tll types .7 cells/ml). However, once the cells had reached maximum density, AX2/GFP-DdRACK1 and AX2/GFP-DdRACK1mut cells did not stay in the stationary phase for long like AX2 as cell counts dropped rapidly  plates. In these assays we noticed an expanded growth zone containing AX2/GFP-DdRACK1 amoebae when they were grown on K. aerogenes when compared with AX2  is a member of the evolutionarily conserved family of WD40 repeat proteins which forms seven β-propeller blades. It was initially discovered through its ability to function as a scaffold protein, bringing in close proximity protein kinase C (PKC) and its substrates ,23. In tS. cerevisiae[A. thaliana[A. thaliana RACK1A. The major difference between the WD repeats is in the loops that provide the distinct features of each member of the WD family and distinguish RACK1 interactions from those of other WD proteins [A. thaliana protein was the first RACK1 orthologue to be structurally described [A. thaliana where RACK1 is expressed by three genes, DdRACK1 is expressed by only one gene, gpbB, as in metazoans. Two conserved surface regions of A. thaliana RACK1A have been proposed to represent protein-protein interaction sites [DdRACK1 is a WD40 repeat protein harboring a seven-bladed β-propeller that shares similarities with the heterotrimeric G protein β subunit. The modelled structure of DdRACK1 features the seven β-propeller architecture with each propeller blade arranged in sequential order and made up of four-stranded antiparallel β-sheets. Although differences exist, particularly in the extended loop that connects β-propeller blades 6 and 7, the structures of RACK1 from  thaliana and huma thaliana show sigproteins ,65. The escribed . Unlike on sites . The firAlthough DdRACK1 is mainly cytosolic as seen from live confocal microscopy pictures, immunofluorescence and fractionation studies, a portion of it was also found in the membrane fraction, buttressing localization to cellular membranes by proteins of the RACK1 family. Furthermore, DdRACK1 was detected at the cell periphery and the leading edge of highly polarized aggregation competent cells. This implies that RACK1 regulates signal transduction at the leading edge. RACK1 is essential for cell migration, and the protein binds to many components of the cell migration machinery including kinases, phosphatases and the cytoplasmic domains of cell surface receptors ,66. RACKRACK1 dimerizes both in vivo and in vitro ,41,46. TD. discoideum, in particular, PI  P3 gradients are formed within the plasma membrane [D. discoideum is mediated by the polybasic stretch. Whether RACK1 proteins from other species also interact with membrane lipids needs to be investigated.Phosphoinositides (PIPs) regulate fundamental biological processes including cell growth and survival, membrane trafficking and cytoskeletal dynamics . PIPs armembrane . They armembrane ,75. In tmembrane . HoweverDictyostelium[D. discoideum has twelve Gα subunits, one Gβ and one Gγ subunit. It is generally assumed that Gβ forms heterotrimers with the γ and all α subunits [G protein-linked signal transduction plays an essential role in the developmental program of yostelium-78. D. dsubunits . For RACsubunits -28. We dD. discoideum (DdRACK1) which has significant sequence identity with other previously studied RACK1 species and similar biochemical features as bona fide RACK1 proteins. Together with the Arabidopsis protein it contains an unusual polybasic region through which it can bind to cellular membranes uncovering a further mechanism how RACK1 can be targeted to membranes. At the biochemical level it interacts with several proteins among them ribosomal proteins, enzymes, cytoskeletal proteins and most notably heterotrimeric G proteins. Upon overexpression we observe phenotypes that imply changes in signaling pathways regulated by the interacting G proteins. In summary  or in a submerged culture at 21-23°C in axenic medium [7 cells which were washed twice with Soerensen phosphate buffer  on phosphate agar plates and monitored. Development was also followed for cells starved in Soerensen phosphate buffer in shaken suspension  or in petri dishes. Mutants were maintained in the presence of appropriate antibiotics (2–4 μg/ml G418) (Roche Applied Science) (or 3–5 μg/ml Blasticidin) . The following strains have been used; AX2-214 (wild type) [Cells were either grown on a lawn of c medium . Developld type) , AX2 expD. discoideum RACK1 as glutathione S transferase (GST) fusion protein in E. coli, a full-length cDNA was cloned into pGEX-4 T-1 vector . E. coli strain XL1 Blue was used for expression of the GST fusion protein. Induction of protein expression was with 0.25 mM isopropyl β-D-thio-galactoside (IPTG) when an OD600 of 0.8 was reached. Cells were further cultured at 30°C for 3 hours. They were harvested, lysed in 50 mM Tris/HCl, pH 7.4 to 8.0, 100 mM NaCl, supplemented with Protease inhibitors  and 1 mM DTT) with an EmulsiFlex cell homogenizer . Lysates were separated into soluble and insoluble fractions by centrifugation at 18,000 × g. The fusion proteins from the soluble fraction were purified using GST-Sepharose beads .For expression of recombinant For cleavage of proteins from GST-Sepharose beads, the GST fusion proteins were washed 5 times with cleavage buffer . Beads were then resuspended in cleavage buffer and 3–10 U thrombin/mg fusion protein were added to the beads and incubated with little agitation at room temperature overnight. As RACK1 was released from the beads together with some GST, we next performed an anion exchange chromatography step in order to separate the proteins. For this the protein solution was dialyzed against 20 mM Tris/HCl, pH 8.0, and 1 mM EDTA overnight before loading onto a DE-52 Sephadex column which had been calibrated with 50 mM Tris/HCl, pH 8.0, 1 mM EDTA. The protein was eluted with 1 M NaCl and the eluate dialyzed and analyzed by SDS-PAGE.For expression in AX2 the RACK1 cDNA was cloned into pBsr-N2-GFP vector  and expressed as GFP-RACK1 under control of the actin 15 promoter and also into mRFPmars plasmid  for RFP-RACK1 ,83. A PCPIP-strips supplied by Echelon Biosciences, Inc.  were used to perform phosphoinositide binding according to the supplied protocol. Briefly, GST and GST-fusion proteins were eluted from the glutathione agarose beads with elution buffer .The membranes were blocked with 0.1% ovalbumin (Sigma # A-5253) in TBS for one hour at room temperature. After discarding the blocking solution membranes were incubated with 1 mg/ml GST-fusion proteins in TBS-T  at room temperature for one hour. The protein solution was then discarded and the membranes were washed with TBS-T three times 10 minutes each. Bound protein was detected by western blot analysis with GST polyclonal antibodies as primary and anti-rabbit IgG-peroxidase (Sigma # A-6154) as secondary antibody followed by enhanced chemiluminescence.Phosphatidylserine (PS), phosphatidylcholine (PC), phosphatidylethanolamine (PE), PI (3) P, PI (4) P, PI (5) P, PI  P2, PI  P2, PI  P2, and PI  P3 were obtained from Sigma and diluted in chloroform. Liposome binding experiments were performed with a modified published liposome binding assay protocol . Lipid mBoth fractions were then analyzed by SDS-PAGE followed by Coomassie blue staining. ImageJ was used for quantification.D. discoideum G protein β-, γ-, α1-, α2-, α4-, α5-, α6-, α7-, α8-, α9-, α10-, α11- and α12-subunits were cloned in frame into the yeast pAS2-1 vector (Clontech), respectively, resulting in fusion to the GAL4-DNA-BD . Full-length cDNA of DdRACK1 was cloned into the yeast pACT2 vector (Clontech) resulting in a fusion to the GAL4-DNA-AD . Yeast Y190 strain was used for this assay.For the yeast two-hybrid screen, the full-length cDNAs of 2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgSO4, pH 7.0, 50 mM β-mercaptoethanol, X-Gal ) and incubated at 30°C and checked between 1 to 6 h, and after 24 h (for detection of weak interactions) for the appearance of blue colonies.Candidate colonies expressing interacting proteins were screened by plating on SD/-Leu/-Trp/-His/+3AT plates after which membrane colonies-lift β-galactosidase activity assay was performed according to the MATCHMAKER Y2H system manual. Briefly, colonies on SD/-Leu/-Trp/-His/+3AT selection plates were transferred to a Nitrocellulose membrane (Protran BA 85) by placing the membrane over colonies on selection plates for 20 min. The filter was carefully lifted off the agar plates and transferred (with colonies facing up) to a pool of liquid nitrogen for 10 sec. The frozen filter was then allowed to thaw at room temperature and placed on a Whatman filter paper presoaked in freshly prepared X-Gal solution (60 mM NaD. discoideum cells were lysed in 50 mM (10 mM for immunoprecipitation assay) Tris/HCl, pH 7.4, 150 mM NaCl, 0.5% NP40, supplemented with protease inhibitor cocktail (Sigma), 0.5 mM PMSF, 0.5 mM EDTA, and 1 mM Benzamidine by passing them through a 25G syringe (10–20 strokes) and incubated with agitation for 15 min at 4°C (to ensure complete cell lysis) followed by a centrifugation step at 16,000 rpm for 10 min. The supernatants were either incubated with GST and GST-fusion proteins, respectively, or with GFP-trap beads . After incubation for 3 h GST beads were washed three times with wash buffer , GFP-trap beads were washed with a different wash buffer . The beads were resuspended in SDS sample buffer, incubated at 95°C for 5 min and the proteins separated by SDS-PAGE and analyzed by western blot. The Gβ and Gγ subunits used in this study were previously cloned into GFP  and YFP  vectors, respectively [For pull down and immunoprecipitation experiments ectively ,13.Purified DdRACK1 was used for a multimerization experiment as previously described . BrieflySamples from immunoprecipitation experiments from GFP-DdRACK1 bound to GFP-trap beads in the presence or absence of phosphatase inhibitors were analyzed by western blots and probed with anti-phosphotyrosine monoclonal antibody (5E7) .Immunofluorescence study was performed as previously described . BrieflyThis analysis was done as previously described ,82. BrieCell fractionation of AX2 cells was done as previously described . AntibodFor generation of rabbit polyclonal antibodies against DdRACK1, the GST-part of GST-DdRACK1 was removed by thrombin cleavage and DdRACK1 was used to immunize rabbits . The antibodies specifically recognized the bacterially produced recombinant protein, the RFP- and GFP-tagged fusion proteins as well as the endogenous protein in western blots of whole cell lysates; they were used in immunoprecipitation experiments as well as for immunofluorescence studies. Monoclonal antibody K73-875-7 was generated against bacterially expressed mRFPmars .H. sapiens (P63244), D. melanogaster (O18640), A. thaliana (O24456), S. cerevisiae (P38011), and D. discoideum (P46800) were retrieved from Uniprot protein database and aligned using ClustalW program with Blosum 62 matrix. The aligned sequences were processed through EsPript for representation. The structural coordinates of S. cerevisiae RACK1 (Asc1p) was obtained from protein databank (PBD: 3FRX) . RACK1 was detected with polyclonal antibodies, actin with mAb act1-7. Nuclei were stained with DAPI. Scale bar, 5 μm. Figure S3. Localization of DdRACK1 in polarized cells. In this image, DdRACK1 was seen at the leading edge of polarized aggregation competent cells (arrow). Antibodies were as in Figure S2. Scale bar, 5 μm. Figure S4. Yeast two-hybrid analyses and β-galactosidase activity staining. Yeast Y190 strain that has lacZ and His3 reporter genes was co-transformed with DdRACK1 in pACT2 vector and the Gα5, Gα6, Gα7, Gα9 and Gα12 protein subunits in pAS2 vector, respectively. Colonies did not grow on selection plates. Figure S5. Growth on lawns of K. aerogenes of AX2/GFP strain. Images of AX2/GFP strain on K. aerogenes lawns were taken between days 4 and 6. Plaque expansion was similar to that of AX2. Scale bar, 1 mm. Figure S6. Development of AX2/GFP strain on petri dishes under phosphate buffer. 1 × 107 cells were starved on petri dishes and images taken at the indicated time points. The developmental behavior was similar to that of AX2. Scale bar, 250 μm.Distribution of endogenous DdRACK1 in AX2/GFP-Gβ cells. Some enrichment of RACK1 was seen at the cell periphery. Polyclonal RACK1 specific antibodies were used. Nuclei were stained with DAPI. Scale bar, 5 μm. Click here for file


Anelloviridae family. In swine, two genetically distinct species, Torque teno sus virus 1a (TTSuV1a) and 1b (TTSuV1b) are currently grouped into the genus Iotatorquevirus. More recently, a novel Torque teno sus virus species, named Torque teno sus virus k2b (TTSuVk2b), has been included with Torque teno sus virus k2a (TTSuVk2a) into the genus Kappatorquevirus. In the present study, TTSuV1 (TTSuV1a and TTSuV1b), TTSuVk2a and TTSuVk2b prevalence was evaluated in 721 serum samples of healthy pigs from Sardinian farms, insular Italy. This is the largest study to date on the presence of TTSuV in healthy pigs in Italy. The global prevalence of infection was 83.2% (600/721), being 62.3% (449/721), 60.6% (437/721), and 11.5% (83/721) the prevalence of TTSuV1, TTSuVk2a and TTSuVk2b, respectively. The rate of co-infection with two and/or three species was also calculated, and data show that co-infections were significantly more frequent than infections with single species, and that TTSuV1+TTSuVk2a double infection was the prevalent combination (35.4%). Quantitative results obtained using species-specific real time-qPCR evidenced the highest mean levels of viremia in the TTSuV1 subgroup, and the lowest in the TTSuVk2b subgroup. Interestingly, multiple infections with distinct TTSuV species seemed to significantly affect the DNA load and specifically, data highlighted that double infection with TTSuVk2a increased the viral titers of TTSuV1, likewise the co-infection with TTSuVk2b increased the titers of TTSuVk2a.Torque teno viruses (TTVs) are a large group of vertebrate-infecting small viruses with circular single-stranded DNA, classified in the  Anelloviridae, are a large group of non-enveloped viruses which contain a single-stranded, negative sense, circular DNA genome; because of their heterogeneity, AVs are divided into 11 genera, each one with various numbers of species . AVs are similar to circoviruses Anelloviruses (AVs), recently classified in the new family Torque teno sus virus 1a (TTSuV1a) and Torque teno sus virus 1b (TTSuV1b) are now classified in the genus IotatorquevirusTorque teno sus virus k2b (TTSuVk2b), classified with Torque teno sus virus k2a (TTSuVk2a) in the Kappatorquevirus genus of the family Anelloviridae, has been described in pig sera The observation that some pet animals (cats and dogs) and livestock  are infected with AVs identical or very similar to human AVs (human and sus TTV [TTSuV] exhibit nearly 50% nucleotide identity) has led to the hypothesis that interspecies transmission is a fairly common event. Recent molecular surveys carried out in different geographic areas, found 24 to 100% pigs positive for TTSuV So far, the presence of TTSuVs in Italian pig herds has been poorly investigated n = 133, females n = 588; animals under twelve months of age n = 113, animals over twelve months of age n = 608) were aliquots of a larger stored collection of sera previously analyzed within the regional monitoring plan for African swine fever (ASF) and Classical swine fever (CSF) enforced in Sardinia, in compliance with the European Community requirements. Sera had been collected between 2012 and 2013 from clinically healthy pigs by staff veterinarians of the Italian Public Health System, according to Decrees No. 9, 16.05.2007, No. 1567/decA/23, and No. 20/09.07.2013 of the “Assessorato dell’Igiene e Sanità e dell’Assistenza Sociale, Regione Autonoma della Sardegna” at farms located in central and southern Sardinia. No animal was sacrificed for the purpose of this study, and swine sera were collected according to the “Guida al prelievo e recapito dei campioni”, Istituto Zooprofilattico Sperimentale della Sardegna “G. Pegreffi”, Rev. 25.05.2012. Sera were stored at −80°C until use.Seven hundred and twenty one domestic swine sera  according to the manufacturer’s instructions. DNA was eluted in 100 µl of elution buffer and stored at −20°C until use. All DNA extraction procedures included a negative control, containing only PBS as extraction substrate.5′-CGGGTTCAGGAGGCTCAAT-3′) and TTSuV1_R (5′-GCCATTCGGAACTGCACTTACT-3′) 2 and 0.75 U of PolyTaq DNA polymerase (Polymed). After an initial denaturation step for 5 min at 94°C, the amplification was performed by 50 cycles of 15 s at 94°C, 20 s at 54°C, 30 s at 72°C followed by a final extension step for 5 min at 72°C. For TTSuVk2a detection, PCR reactions were carried out as described above with an annealing step of 20 s at 56°C using the primer pair TTSuV2_F (5′-TCATGACAGGGTTCACCGGA-3′) and TTSuV2_R (5′-CGTCTGCGCACTTACTTATATACTCTA-3′) 5′-CGAATGGCTGAGTTTATGCCGC-3′) and TTV3-361R (5′-TTCGCTGTGACTGGCGTCTC-3′) PCR assays specific for TTSuV species 1 (both genotypes TTSuV1a and TTSuV1b), k2a and k2b were used to detect the presence of viral DNA. Briefly, for TTSuV1 detection, 25 µl of the PCR reaction contained 3 µl of serum DNA, 10 pmol of each primer TTSuV1_F  assays were performed on positive samples to determine the viral DNA load. Briefly, 20 µl PCR mixture contained 10 µl of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), 8 pmol of each primer  6 to 10 copies prepared in duplicate by ten-fold serial dilution of plasmids TTV1_G26 (TTSuV1 isolate TTV1_G26), TTV2_GE9 2 (TTSuVk2a isolate TTV2_GE9), and TTSuVk2b (TTSuVk2b isolate 38E05), respectively 10 DNA copy numbers per ml of serum was used to compare data. Limit of detection was 10 copies per reaction for each qPCR assay.The copy number of TTSuV1, TTSuVk2a and TTSuVk2b DNA per ml of serum was determined in reference to a standard curve ranging from 10The specificity of reactions was confirmed by the sequence analysis of randomly selected amplicons (5 to 10 for each assay). PCR products were purified using QIAquick PCR purification kit (Qiagen). Sequencing reactions were bidirectionally carried out using BigDye Terminator v3.1 cycle sequencing kit (Applied Biosystems) and run using ABI Prism 3100 Genetic analyzer (Applied Biosystems). The nucleotide sequences were compared with reference sequences for each species available in GenBank using the online program NCBI BLAST.10 DNA copies per ml was normally distributed. Differences in mean viral titers among species were analyzed using the one-way analysis of variance (ANOVA) or unpaired Student’s t test when appropriate. Values of p<0.05 were accepted as significant. The statistical analyses were performed using Prism software (GraphPad).TTSuV prevalence-related data were analyzed using the Chi-square test. TTSuV viral load expressed as logTTSuV prevalence in Sardinian pig herds was evaluated by detection of TTSuV1, k2a and k2b DNA in 721 sera using species-specific PCR assays. Results are summarized in Considering the single infections, 37.9% (273/721) of the sera was found positive. Interestingly, the most prevalent species was TTSuV1 (20.4%), followed by TTSuVk2a (16%) and TTSuVk2b (1.5%). The prevalence was significantly different from each other (p<0.05). Instead, within gender and age groups, no significant difference was found between TTSuV1 and TTSuVk2a. Moreover, when comparing the prevalence of single infections between genders and between age groups, no differences were observed (p>0.05) (data not shown).Furthermore, our data indicate that the rate of co-infections  was significantly higher than that of single infections (p<0.005). In particular, the TTSuV1+TTSuVk2a co-infection was the prevalent combination (35.4%) followed by the TTSuV1+TTSuVk2a+TTSuVk2b (5.8%), TTSuVk2a+TTSuVk2b (3.5%), and TTSuV1+TTSuVk2b (0.7%) co-infections. The frequencies were significantly different from each other (p<0.05) .Thus, TTSuV1 and TTSuVk2a were mainly found in combination rather than in single infections or in association with TTSuVk2b. Likewise, TTSuVk2b was mostly detected in the TTSuV1+TTSuVk2a+TTSuVk2b triple infection rather than in single infections or with one of the two other species.The same observation was made within age groups and within genders, except that the TTSuV1 prevalence in single and co-infection with TTSuVk2a was not significantly different in younger animals and the TTSuVk2b prevalence in single and co-infections was not significantly different in males. Moreover, the triple infection frequency was significantly higher in younger (12.4%) than in older animals (4.6%) (p<0.005) (data not shown).10 copies per ml of serum for TTSuV1, 3.62–8.47 (mean 5.50±SD 0.94) log10 copies per ml of serum for TTSuVk2a, and 3.75–8.31 (mean 4.73±SD 0.83) log10 per ml of serum for TTSuVk2b, respectively. 7.6% of the TTSuVk2a-positive samples and 3.3% of the TTSuVk2b-positive samples were under the limit of quantification (<3.3 log10 per ml of serum). Differences in viral DNA load among the three species were statistically significant (p<0.001) (10 per ml of serum) and TTSuVk2a (mean 5.31±SD 0.66 log10 per ml of serum) viral load was not statistically significant , viral DNA load was in the range of 4.44–8.12 (mean 6.18±SD 0.83) logp<0.001) . On the nificant .10 per ml of serum) (p<0.05), whereas the TTSuVk2a titers did not differ (mean 5.31±SD 0.83 log10 per ml of serum). Noteworthy, the TTSuVk2a DNA load significantly increased in the TTSuVk2a+TTSuVk2b co-infection (mean 6.59±SD 1.23 log10 per ml of serum) (p<0.001), but not in the triple infection (mean 5.62±SD 0.76 log10 per ml of serum) (10 per ml of serum) TTSuV1 titers did not vary significantly.Interestingly, when comparing the viral load between single and co-infections, the TTSuV1 titers were significantly higher in the double infection with TTSuVk2a (mean 6.34±SD 0.79 logf serum) . With re10 per ml of serum) were significantly higher than that of TTSuVk2b (mean 4.75±SD 0.51 log10 per ml of serum) (p<0.001), but no significant difference was observed within the triple infection (p>0.05). TTSuVk2b DNA load did not differ significantly between TTSuVk2a+TTSuVk2b co-infection and triple infection (mean 4.90±SD 1.19 log10 per ml of serum) . Otherwise, within the triple infection the viral load of TTSuV1 and TTSuVk2a did not significantly differ (p>0.05). Similarly, within the TTSuVk2a+TTSuVk2b co-infection, the TTSuVk2a titers (mean 6.59±SD 1.23 logf serum) .10 per ml of serum) and TTSuVk2a (mean 5.91±SD 0.97 log10 per ml of serum) were not different among males (p>0.05) (10 per ml of serum) (p<0.05)  . No stat(p<0.05) .10 per ml of serum, and TTSuVk2a mean 5.82±SD 0.97 log10 per ml of serum) than in older swine  (p<0.05)  and older animals  (p>0.05).When analyzing the age groups, TTSuVs DNA titers were statistically different from each other . Interes(p<0.05) , while iTo our knowledge, this is the largest study ever published on the presence of TTSuV in healthy pigs in Italy. More than seven hundred non-consecutive sera were examined for TTSuV DNA using species-specific PCR, and quantified by real time-qPCR. Sardinia is an Italian region at intensive pig farming for commercial use; however, some of these animals are imported from other Italian regions, and some were born abroad (Europe) and imported after weaning. Therefore, our study was scarcely influenced by the ethnic and local origin of the animals, but it is nonetheless important as it well represents the national situation, for which little is known. To date there is only one paper that investigated TTVsuV natural infection in Italy, but it was carried out on a much smaller number of animals and using assays unable to discriminate all TTSuV species invitro system to investigate the possibility that the different viral species can compete each other.Quantitation of positive samples with species-specific real time-qPCR assays evidenced a wide range of viral titers within the species, with the highest levels in TTSuV1 subgroup, and the lowest in TTSuVk2b-positive samples, as previously reported Anelloviridae in human microbiome, and the increasing of viral load in plasma during immunosuppressive therapy could be used to predict and check the immune competence Dynamics of TTSuV DNA load in serum of healthy and sick pigs have been poorly studied until now, but it has been suggested that TTSuV infection could worsen pre-existing or subsequent infections with other pathogens, mainly with porcine circovirus type 2 (PCV2), the causative agent of post-weaning multisystemic wasting syndrome (PMWS) in pigs. In particular, a recent study showed that TTSuV2, but not TTSuV1 DNA load increased over time in animals affected by PMWS Finally, the contribution of animal AVs to the epidemiology of TTVs in humans remains a matter of debate, especially for swine AVs. For this reason, to investigate interspecies transmission of infection and the dynamics of genetic evolution of the virus in humans we started to collect and analyze samples of farms and slaughterhouses employees who have been in contact with the infected animals examined in this study.


The products were fully characterized by X-ray diffraction, scanning electron microscopy, transmission electron microscopy, energy-dispersive X-ray spectroscopy, and UV-vis absorption spectroscopy. It was found that the photocatalysis performance of the as-prepared samples could be enhanced by formation of ZnxCd1−xS solid solutions. In addition, their photocatalytic activities are dependent on the Zn/Cd molar ratios and nanostructures of ZnxCd1−xS solid solutions. Hollow Zn0.2Cd0.8S spheres exhibit extremely high photocatalytic activity and good re-usability, and the photocatalytic conversion of RhB reaches as high as 96% after 50 min of irradiation.Formation of solid solutions is a good strategy to acquire materials with special properties and bring forth new type of applications or enhance the performance of currently existing devices. In this study, hollow Zn Among them, using semiconductor photocatalysts to degrade organic dyes are extensively explored1−) and holes (h+) and the fact that it is easily corroded911131517xCd1−xS, a kind of ternary transition metal sulfides has fine and tunable absorption in the visible region of solar energy and excellent electrical conductibility, which has been recognized as compatible candidates for photocatalysts19xCd1−xS, such as quantum dots21xCd1−xS have not been reported so far. Hollow structured materials show many advantages including porous structure, large surface area, high light-harvesting efficiency and fast mobility of charge carriers, and have promising application in the field of photocatalytic processes. Therefore, the design and fabrication of ZnxCd1−xS hollow structure is one admirable strategy in photocatalysis.II-VI semiconductor CdS with an appropriate band gap (2.4 eV) is regarded as an outstanding visible-light responsive material for photocatalysts7xCd1−xS hollow spheres with the assistance of carboxymethyl cellulose (CMC) under hydrothermal conditions. This one-step route provides both the expected hollow structure and ternary chalcorenide ZnxCd1−xS semiconductors with different molar ratios. More importantly, the as-obtained hollow ZnxCd1−xS spheres exhibit great enhancement of photocatalytic activities for the degradation of Rhodamine B (RhB) under visible-light irradiation.Herein, we propose a simple straightfoward route to prepare ZnxCd1−xS hollow spheres were prepared with the assistance of carboxymethyl cellulose (CMC) and ammonia via a hydrothermal process, using zinc acetate, cadmium nitrate and l-cysteine as reactants. The molar ratios of Zn and Cd used were 10:0, 9:1, 8:2, 1:1, 2:8, 1:9 and 0:10, and the corresponding resulting products were labeled as ZnS, Zn0.9Cd0.1S, Zn0.8Cd0.2S, Zn0.5Cd0.5S, Zn0.2Cd0.8S, Zn0.1Cd0.9 S, and CdS, respectively. xCd1−xS and CdS samples. The diffraction peaks observed at 27.0°, 28.5° and 30.6° in 0.9Cd0.1S, Zn0.8Cd0.2S, Zn0.5Cd0.5S, Zn0.2Cd0.8S, Zn0.1Cd0.9S products, from the XRD patterns in 2+ is smaller than that of Cd2+, the increasing of Zn/Cd molar ratios leads to the shrinkage of the unit-cell volume of the ZnxCd1−xS samples. Therefore, all the diffraction peaks shift to the higher angles with increasing the Zn2+ concentration as shown in xCd1−xS solid solutions have been successfully prepared.A series of ZnxCd1−xS samples was investigated by scanning electron microscopy (SEM). 0.9Cd0.1S, Zn0.8Cd0.2S, Zn0.5Cd0.5S, Zn0.2Cd0.8S, Zn0.1Cd0.9S and ZnS samples. It can be clearly observed that similar to CdS sample, with the increase of Zn content in the solid solutions, the obtained samples are all hollow structures with diameters of about 300 nm and the shell of the hollow structures are composed of small nanoparticles. In the process of reaction, both ammonia and carboxymethyl cellulose (CMC) play key roles in the formation of the ZnxCd1−xS hollow structures. It is known that CMC is a kind of water soluble polyanionic compounds with a large number of carboxyl and hydroxyl groups. With the addition of CMC in the reaction system, the solution could be divided into numerous homogeneous “chambers”xCd1−xS during the growth process. Without the addition of CMC, only particles and irregular microspheres were obtained (xCd1−xS nanoparticles and the assembly function of CMC further assembles these nanoparticles into uniform spheres. Ammonia can reacts with Zn2+ and Cd2+ to form [Zn(NH3)4]2+ and [Cd(NH3)4]2+ complexes, respectively. Under the hydrothermal conditions, after ZnxCd1−xS spheres form, the existence of ammonia makes ZnxCd1−xS dissolve to form Zn[(NH3)4]2+ and [Cd(NH3)4]2+ complexes. So, in the reaction system dissolution–precipitation dynamic equilibrium is presented:The morphology of these Znobtained . With thobtained . Even wiobtained . The “chxCd1−xS particles dissolve and the surface nanoparticles grow bigger. With continuous dissolve of the core ZnxCd1−xS small particles, inner cavities eventually form, resulting in the formation of ZnxCd1−xS hollow nanospheres.Under the dissolution-precipitation dynamic balance, based on the Ostwald’s ripening process, smaller ZnxCd1−xS hollow spheres were further elucidated by energy dispersive X-ray spectroscopy (EDS) and transition electron microscopy (TEM). 0.2Cd0.8S nanospheres. From the EDS pattern in 0.2Cd0.8S and the hollow spheres is composed of small nanoparticles. The selected area electron diffraction (SAED) pattern inset in 0.2Cd0.8S spheres. 0.2Cd0.8S sample, from which the lattice fringes are clearly visible and the spacing is about 0.338 nm, which corresponds to (002) lattice plane of hexagonal Zn0.2Cd0.8S.The composition, morphology and structure of the as-prepared ZnxCd1−xS hollow spheres and solid Zn0.2Cd0.8S sphere, using the UV−vis diffuse reflectance spectra (DRS), were shown in xCd1−xS hollow spheres are shown in xCd1−xS solid solutions with the increase of x value shows a blue-shift in turn relative to those of the hollow CdS spheres in the visible-light region, which further confirms the formation of the solid solution and indicates that the hollow ZnxCd1−xS spheres have a bigger band gap than the CdS hollow spheres. Moreover, there is an enhanced light absorbance in the visible light region for Zn0.2Cd0.8S and Zn0.1Cd0.9S hollow spheres than CdS sample, which means a small x value would be beneficial to improve the absorption of visible light for ZnxCd1−xS solid solutions. Plots obtained through the Kubelka-Munk transformation from the UV−vis diffuse reflectance spectra of all the prepared nanostructures are shown in 0.1Cd0.9S, Zn0.2Cd0.8S, Zn0.5Cd0.5S, Zn0.8Cd0.2S and Zn0.9Cd0.1S, respectively. In 0.2Cd0.8S spheres’ DRS spectra were shown, which display that the absorption band edges are 534 and 520 nm for hollow and solid Zn0.2Cd0.8S sphere, respectively. The direct band-gap values of the hollow Zn0.2Cd0.8S spheres and solid Zn0.2Cd0.8S sphere can be estimated to be about 2.32 and 2.38 eV, respectively. Moreover, the visible light absorption of the hollow Zn0.2Cd0.8S spheres was clearly stronger than that of solid Zn0.2Cd0.8S spheres. The results show that both the Zn/Cd molar ratio and nanostructures of sample affect its optical properties and the formation of hollow solid solution would be beneficial to their photocatalytic performances.The optical property measurement of ZnxCd1−xS hollow spheres, the photocatalytic performances of products were evaluated by the photodegradation of RhB, one of the common dyes, in aqueous solution under visible light. 0.2Cd0.8S hollow spheres under exposure to visible light. At the beginning, RhB shows a major absorption band at 554 nm. With the irradiation time prolonging, the absorption peak at 554 nm is blue-shift and decreases quickly. The shift of the absorption is caused by de-ethylation of RhB because of the attack by one of the active oxygen species on the N-ethyl group and RhB is transformed to rhodamine. Rhodamine can be then further decomposed due to the further destruction of the conjugated. During this process, the measured absorbance (A) would be the sum of the abosorbances of remaining RhB and its decomposition product. After 50 min degradation, the absorbance of the dye solution decreases to 4% of the original absorbance, which means both RhB and rhodamine have been almost decomposed and indicates Zn0.2Cd0.8S hollow spheres exhibit high photocatalytic activities for the decomposition of RhB.In order to determine the potential applications of Zn0, calculated from A/A0) with different samples as photocatalysts under different irradiation times. It can be clearly found that Zn0.2Cd0.8S and Zn0.1Cd0.9S hollow spheres show high photocatalytic activities compared to other samples, and the photocatalytic conversion of RhB with hollow Zn0.2Cd0.8S spheres reaches as high as 96% after 50 min of irradiation. The degradation of RhB over the as-prepared catalysts with a 50 minute irradiation time decreased in the order of Zn0.2Cd0.8S > Zn0.1Cd0.9S > Zn0.5Cd0.5S > Zn0.8Cd0.2S ≈ CdS > Zn0.9Cd0.1S > ZnS. Because of the large band gap of ZnS (3.7 eV), ZnS show relatively low photocatalytic activity under visible light. But what is worthy noting that most of hollow ZnxCd1−xS spheres show enhanced activities than CdS hollow spheres, this result suggests that formation of ZnxCd1−xS solid solutions can improve the photoactivity of CdS. The Zn/Cd molar ratio has a great effect on the photocatalytic activity of ZnxCd1−xS solid solutions and Zn0.2Cd0.8S shows the best performances.xCd1−xS samples affects the photocatalytic activities of the composite, but nanostructures of ZnxCd1−xS also affect its photocatalytic performances. 0.2Cd0.8S nanostructures. The photocatalytic activity of hollow Zn0.2Cd0.8S sphere is clearly better than that of solid Zn0.2Cd0.8S spheres as shown in 0.2Cd0.8S sample. The adsorption of contaminant molecules, light irradiation absorption, charge transportation and separation are three crucial factors for the photocatalytic activities of the composite0.2Cd0.8S sphere have much stronger visible light adsorbance and a smaller band gap according to UV-vis DRS , further supporting the advantages of the hollow structure.Not only Zn/Cd molar ratios of Zn-vis DRS . Secondl0.2Cd0.8S sphere photocatalyst by collecting and reusing the photocatalyst for three cycles. 0.2Cd0.8S sphere has a small loss due to the loss of catalyst during each cycle of collecting and cleaning. These results indicate that Zn0.2Cd0.8S hollow sphere are photostable and reusable in the degradation of RhB.Stability and re-usability is an important standard to measure a photocatalyst. So, we further investigated the durability of hollow ZnxCd1−xS hollow spheres. The prepared ZnxCd1−xS hollow spheres show the enhanced photocatalytic activity for the degradation of RhB under visible light (λ > 420 nm) irradiation, and hollow Zn0.2Cd0.8S spheres were found to be highly efficient for RhB removal. Moreover, this hollow spherical ZnxCd1−xS catalyst showed improved stability and would have great application potential in water treatment.We demonstrated in this work an effective hydrothermal route for the synthesis of solid solution Zn3COO)2·2H2O and Cd(NO3)2·5H2O with the sum mole number of Zn2+and Cd2+ being 0.5 mmol were dissolved in a solution of sodium carboxymethyl cellulose  with magnetic stirring, then ammonia  and l-cysteine  were added under magnetic stirring. After stirring for 10 min, the mixture was transferred into a 40 mL autoclave. The autoclave was sealed, heated at 150 °C for 6 h, and cooled down to room temperature naturally. The precipitate was collected by centrifugation, washed with distilled water and ethanol several times, and then dried at 60 °C for 12 h. The same procedures were applied to synthesize CdS hollow spheres and ZnS sample, respectively.In a typical synthesis, the appropriate molar ratios of Zn(CHα radiation (λ = 1.5418 Ǻ). The morphology of the samples were characterized by scanning electron microscopy . Energy-dispersive X-ray spectroscopy (EDS) attached to the SEM instrument was used to analyze the composition of the ZnxCd1−xS samples. Transmission electron microscopy (TEM), high-resolution TEM (HRTEM) images, and the corresponding selected area electron diffraction (SAED) were obtained with a JEOL JEM-2100 instrument transmission electron microscope at an acceleration voltage of 200 KV. UV/Vis diffusion reflectance spectra (DRS) of the samples were studied with a UV-3600 spectrophotometer  and BaSO4 was used as the reference.Powder X-ray diffraction (XRD) patterns were collected by using a Bruker D8 ADVANCE diffractometer with CuK−5 M, 80 mL) in a beaker at ambient temperature. A 300 W xenon lamp was used as the visible-light source with a cutoff filter to cut off the light below 420 nm. Before irradiation, the suspension was continuously magnetically stirred in the dark for 60 min to achieve the adsorption–desorption equilibrium between the photocatalyst powder and the dye. Then, the photocatalytic reaction was initiated. At specific time intervals, 4 ml of the aqueous solution was taken and separated through centrifugation (10000 rpm) for the absorbance measurements, which were recorded with a Hitachi U-3900 spectrophotometer.The photocatalytic activities of the synthesized samples for the photocatalytic decolorization of an aqueous solution of Rhodamine B (RhB) were evaluated as follows: the as-prepared sample (20 mg) was suspended in a aqueous solution of RhB .