Non-small cell lung carcinoma (NSCLC) is the most common malignancy with the highest morbidity and mortality. In this study, we found that tripartite motif containing 47 (TRIM47) expression level was higher in tumor tissues than in normal adjacent tissues. Overexpression of TRIM47 closely correlated with poor prognosis in patients with NSCLC. Multivariate Cox regression analyses showed that TRIM47 overexpression could be considered an independent prognostic factor for NSCLC. TRIM47 depletion significantly inhibited cell proliferation and induced G1phase arrest in A549 and H358 cell lines. Moreover, TRIM47 silencing remarkably inhibited cell migration, cell invasion, and tumorigenicity in nude mice. Gene set enrichment analysis (GSEA) revealed that cancer-related process and pathways, including p53-cell cycle and NFκB-epithelial mesenchymal transition (EMT) pathway, were significantly correlated with TRIM47 expression. Real-time PCR and Western blot analysis revealed that TRIM47 exerts an inhibitory effect on p53 and an facilitatory effect on NF-κB, thereby promoting tumor proliferation and metastasis. Taken together, TRIM47 acts as a tumor oncogene in NSCLC. Our data provide insight into the possible biological mechanism of TRIM47 in the progression of NSCLC and highlight its usefulness as a potential therapeutic target. Lung cancer is a primary cause of cancer-related morbidity and mortality worldwide, accounting for nearly one-fifth of cancer deaths due to invasion, metastases and drug resistance , 2. NSCLThe TRIM family proteins are evolutionarily conserved proteins, composed of a ring finger domain, one or two B-box domains, and an associated coiled-coil domain in the amino-terminal region . TRIM prIn our current study, we explored the expression level, biological functions and potential molecular mechanisms of TRIM47 in NSCLC. TRIM47 mRNA was significantly overexpressed in tumor tissues compared to normal adjacent tissues. Then, we found that TRIM47 silencing inhibited cell proliferation, migration, invasion and xenograft tumor growth. Furthermore, gene set enrichment analysis (GSEA) on the RNA-sequencing data from The Cancer Genome Atlas (TCGA) indicated that high expression of TRIM47 was related with p53-cell cycle and NFκB-EMT signaling pathways, which were further identified in NSCLC cells. Collectively, our data suggest that TRIM47 is a potent prognostic factor and a potential target for the treatment of NSCLC.We first investigated TRIM47 expression level in 45 NSCLC tissues and normal adjacent tissues to determine its significance in NSCLC by real-time PCR. As shown in Figure P = 0.0054, Figure P < 0.0001, Figure Next, by analyzing the immunohistochemical results, we found that TRIM47 expression in the NSCLC samples was negative in 36 cases and positive in 54 cases. Based on the statistical results, TRIM47 expression was notably less prevalent in the normal adjacent tissues than in the NSCLC tissues Figure . Our datP = 0.011), TNM stage (P = 0.002), lymph node metastasis (P = 0.003), and tumor size (P = 0.016). We got the same results on TRIM47 mRNA level in 45 NSCLC patients (P = 0.017) could be considered an independent prognostic factor for NSCLC patients  by Western blot and real-time PCR. As shown in Figure A CCK-8 assay was used to evaluate the effects of TRIM47 silencing on the proliferation of NSCLC cells. The proliferation was significantly suppressed by shRNA-TRIM47 compared with the NC group Figure . These dPI staining and flow cytometry analysis were used to evaluate the effects of TRIM47 knockdown on the cell cycle. TRIM47-shRNA infection caused a significant increase of G0/G1 phase cells and a decrease of S and G2/M phase cells compared with the NC group Figure .The migration and invasion ability of NSCLC cells were assessed to investigate the function of TRIM47 in the metastasis of cancer cells. In sharp contrast to control cells, suppressing TRIM47 expression showed remarkable reduced migration and invasion ability . The weight of TRIM47-depleted tumors was less than that of control tumors. Collectively, our in vitro and in vivo observations suggest that TRIM47 functions as an oncogene in NSCLC.To verify the positive role of TRIM47 on tumorigenicity As described above, how TRIM47 promotes proliferation and metastasis remained unknown. High throughput RNA-sequencing data of the NSCLC cohort from TCGA and GEO were used to perform GSEA to probe the TRIM47-associated pathways in an unbiased manner. The result showed that high expression of TRIM47 was related to p53-cell cycle and NFκB-EMT signaling pathway, which were associated with proliferation and metastasis Figure .The protein and mRNA levels of p53, p21, cyclin D1, CDK4, and CDK6, which are closely related to p53-cell cycle pathway, were detected by Western blot and real-time PCR. We found that the p53 protein level increased after TRIM47 knockdown Figure . Both thTo investigate the function of TRIM47 in metastasis, the expression levels of genes related to the NFκB-EMT pathway were evaluated. The protein expression level of P-p65 was down-regulated after TRIM47 knockdown, whereas the overexpression of TRIM47 did not affect the p65 protein amount Figure . MeanwhiP = 0.017) could be considered an independent prognostic factor for NSCLC , Shanghai Jiao Tong University School of Medicine.According to ATCC protocols, NSCLC cell lines  and HEK293T cells  were cultured in Roswell Park Memorial Institute-1640  and DMEM (Life Technologies), respectively, at 37°C in a humidified incubator under 5% CO2 conditions. Fetal bovine serum (10%)  and 1% penicillin/streptomycin (Life Technologies) were supplemented into the culture media.To detect the TRIM47 expression level in NSCLC tissues and normal adjacent tissues, streptavidin peroxidase immunohistochemistry was used. The sections were incubated at 4°C overnight with rabbit anti-human TRIM47 antibody  after deparaffinization and antigen retrieval. Then, the sections were incubated with a drop of biotin-labeled secondary antibody for 30 minutes at 37°C. We subsequently added a drop of horseradish peroxidase-conjugated streptomycin working solution to the sections and incubated them for 30 minutes at 37°C. Visualization was performed with 3,3′-diaminobenzidine (DAB), and sections were counterstained with hematoxylin.TRIM47 expression was assessed with regard to the percentage of positive cells and the staining intensity. The staining intensity was scored as follows: 0 for no intensity, 1 for no or very weak staining, 2 for moderate intensity, and 3 for strong intensity. The percentage of positive cells was rated as follows: 0 for < 10%, 1 for 10% to < 25%, 2 for 25% to < 50%, and 3 for > 50%. Total histological score = staining intensity + percentage of positive cells. The patients could be classified into two groups: negative expression group (0–2) and positive expression group (3–6).Two shRNA sequences  and a non-specific scramble shRNA sequence were synthesized by Genepharm Technologies . The shRNA were inserted into pLVTHM vector , then, the lentiviral packaging plasmid was transfected into HEK293T cells with Lipofectamine 2000 . Lentiviruses were collected at 48 hours or 72 hours after transfection, and we used the lentiviruses to infect A549 and H358 cells.Total RNA of specimens and cells harvested and washed with PBS were extracted with TRIzol reagent (Invitrogen). cDNA was then reverse transcribed with oligodT using M-MLV reverse transcriptase . Quantitative real-time PCR was performed with Maxima SYBR Green qPCR Master Mixes (Thermo Fisher Scientific) in an ABI 7300 system . The relative expression levels of genes of interest were determined using the 2−ΔΔCt method, GAPDH served as an internal control. The primers used are listed in RIPA lysis buffer (Thermo Fisher) with protease inhibitor (Sigma) was used to extract the total protein. The same amounts of total proteins were separated via SDS-PAGE gel and then transferred to polyvinylidene fluoride  membranes. After blocking with 5% skim milk, the membranes were incubated with the primary antibodies overnight at 4°C and then with the horseradish peroxidase-conjugated secondary antibody . Signals were detected with enhanced chemiluminescence system .4 cells/well and then incubated for 24, 48, or 72 hours. Following the manufacturer's instructions, cell viability was assessed using a Cell Counting Kit-8 . A microplate reader (Bio-Rad) was used to measure the optical density values of each well at a wavelength of 450 nm.A549 and H358 cells were seeded in 96-well plates at a density of 1 × 10Treated and untreated cells were harvested by trypsinization, washed in PBS and fixed with ice-cold 70% ethanol at −20°C for at least 2 hours. After washing with PBS, cells were incubated with ribonuclease (Sigma) at 37°C for 15 min and then incubated with PI  in the dark for 30 min at room temperature. Finally, a flow cytometer (BD Biosciences) was used to analyze DNA content.Tumor cell migration and invasion assays were performed using Boyden chambers  containing polycarbonate filters . Cells transfected with the indicated virus were serum starved for 24 h and seeded in the upper chamber; then, medium containing 20% FBS was added in the lower chamber. After 24 h of incubation, cells on the upper side of the filter were completely removed. The remaining cells were fixed in 4% paraformaldehyde and stained with 0.05% crystal violet after washing with PBS. The cells on the lower surface of the filters were defined as migrating cells and counted under the microscope .Invasion assays were also performed in Boyden chambers with polycarbonate filters coated with Matrigel (BD Biosciences) on the upper side. The rest was performed as migration assay.6 cells) in a total volume of 100 μl were injected subcutaneously into the right flanks of nude mice. Tumor length and width were measured and recorded every 4 days starting 2 weeks after inoculation. Tumor volume was calculated as 1/2×length×width2.The experimental procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University School of Medicine. Male BALB/c-nu mice  were purchased from Shanghai Laboratory Animal Company . For each cell line, cell suspensions (2 × 10http://www.broad.mit.edu/gsea/msigdb/index.jsp) were used to run GSEA and 1000 permutations were used to calculate the P-value; the “permutation type” was set to “gene set”. All other parameters were set to default, except that the gene set should represent at least 15 genes.The TCGA dataset of NSCLC was analyzed using GSEA2-2.2.2, as previously described . The gent-test was used to evaluate differences between groups. The relationship between the clinicopathological features and the expression of TRIM47 was evaluated by chi-square tests. Overall survival analyses were assessed by the Kaplan–Meier method with the log-rank test and Cox regression analysis. Receiver operating characteristic (ROC) analysis was used to calculated the sensitivity and specificity of TRIM47 as a diagnosis biomarker. P < 0.05 was regarded as significant.The statistical analyses were performed using SPSS version 16 software , and graphical representations were generated with GraphPad Prism 5 software . The data are presented as the means ± S.D., and Student's


CreBrn3b knock-in allele reduced axonal midline crossing at the optic chiasm and optic tract fasciculation. In contrast, Tie2-Cre-mediated endothelial NRP1 ablation induced axon exclusion zones in the optic tracts without impairing axon crossing. Similar defects were observed in 120/120Vegfa and 188/188Vegfa mice, which have vascular defects as a result of their expression of single VEGF-A isoforms. Ectopic midline vascularisation in endothelial Nrp1 and 188/188Vegfa mutants caused additional axonal exclusion zones within the chiasm. As in vitro and in vivo assays demonstrated that vessels do not repel axons, abnormally large or ectopically positioned vessels are likely to present physical obstacles to axon growth. We conclude that proper axonal wiring during brain development depends on the precise molecular control of neurovascular co-patterning.Visual information is relayed from the eye to the brain via retinal ganglion cell (RGC) axons. Mice lacking NRP1 or NRP1-binding VEGF-A isoforms have defective RGC axon organisation alongside brain vascular defects. It is not known whether axonal defects are caused exclusively by defective VEGF-A signalling in RGCs or are exacerbated by abnormal vascular morphology. Targeted NRP1 ablation in RGCs with a  Summary: NRP1 plays a dual role in retinal ganglion cells and in vascular endothelial cells to organise axons along the optic pathway between the mouse retina and diencephalon. The coordinated development of neuronal and vascular networks is a prerequisite for normal nervous system architecture and function. Beginning around embryonic day (E) 10 in mice, vessels sprout from a perineural vascular plexus into the brain and spinal cord and extend towards the ventricular surface, where they branch and anastomose to form a subventricular vascular plexus . This va164 and VEGF188, but not VEGF120, also bind with high affinity to neuropilin 1 (NRP1)  . VEGF-A 1 (NRP1) , which i1 (NRP1) .In vascular endothelial cells, NRP1 acts as a VEGF-A receptor to promote arteriogenesis and postnatal angiogenesis . Additio164 and VEGF188 or as a receptor for class 3 semaphorins . Additionally, the optic tracts split into two bundles each in a subset of Nrp1-null mutants (6/9) . NRP1 loss therefore causes several different defects in optic pathway organisation. Blood vessels bordering the optic pathway in Nrp1 null mutants also were mispatterned  A. The opatterned D. As obsatterned , neighboatterned D. AccordNrp1 null (floxed) alleles in RGCs with Six3-Cre or Nes-Cre resulted in only partial NRP1 targeting , we investigated whether a CreBrn3b knock-in allele could selectively ablate NRP1 in RGCs without affecting blood vessel expression. Brn3b (Pou4f2) is expressed in RGCs during or shortly after their terminal cell division , but spared endothelial cells in and around the eye and at the optic chiasm . In agreement, immunolabelling with an antibody specific for NRP1 . In contrast, immunolabelling confirmed effective NRP1 knockdown in RGC axons of fl/−;Brn3b+/CreNrp1 mice, both in the eye and at the optic chiasm . These findings demonstrate that CreBrn3b is a suitable tool to ablate NRP1 in RGCs.The for NRP1  demonstrNrp1 null mutants . Double labelling for YFP and NRP1 confirmed that Nrp1 deletion in endothelial cells of the eye and around the optic chiasm was successful in fl/;Tie2-CreNrp1 mutants, whilst NRP1 expression in RGCs was maintained, albeit at slightly lower levels due to the +/−Nrp1 allele .To generate endothelial reporter . Tie2-Cre embryo , includifl/−;Tie2-CreNrp1 mutants lacking endothelial NRP1  of fl/−;Tie2-CreNrp1 mutants, but not in control embryos, the ipsilateral optic tract had split into two distinct bundles  are expressed dorsally to the developing optic chiasm . These observations suggest that endothelial cells secrete factors that in sum have a growth-promoting effect on retinal axon outgrowth. In addition, we found no evidence for contact-dependent repulsion of retinal axons by endothelial cells in co-cultures of retinal explants with aortic rings. Thus, retinal axons grew amongst the vessels extending from the aortic rings, with some axons growing along and across the surface of the vessels  and UK Home Office guidelines. Mice were mated and the morning of vaginal plug formation counted as E0.5. Embryos were fixed overnight in 4% formaldehyde in PBS or used as donors for tissue culture experiments. We used the following mouse strains: wild-type C57BL/6J mice; mice carrying a to E15.5 ; Nrp1fl/Tie2-Cre , Six3-CrSix3-Cre  or Nes-C. The ipsilateral index was calculated as the ratio of fluorescence intensity in the ipsilateral optic tract relative to the sum of the fluorescence intensity in the ipsilateral and contralateral optic tracts . Quantification of the number of optic pathway holes was performed using non-saturated images in a 300 µm-wide region at the midline of the ventral diencephalon and proximal 900 µm of the contralateral optic tract .Anterograde DiI labelling and calculation of the ipsilateral index was performed using fixed tissue as described previously . BrieflyRetrograde labelling was performed as described previously . BrieflyNrp1 null tissue . Blood vessels were detected using biotinylated IB4 (Sigma-Aldrich) followed by Alexa-Fluor633- or Alexa-Fluor488- conjugated streptavidin (Life Technologies). For combined DiI and IB4 labelling, detergents were omitted from all solutions. Images were captured using a Nikon SMZ1500 microscope, Zeiss Axiophot microscope or Zeiss LSM 510 or 710 confocal microscopes. Three-dimensional surface rendering of confocal z-stacks  was performed with Imaris (Bitplane). Quantification of vessel morphology was performed using ImageJ in confocal z-stacks. Analyses were performed blind to genotype.Formaldehyde-fixed tissue was cryopreserved in 30% sucrose in PBS and embedded in OCT before snap freezing for cryosectioning at 15 µm. Alternatively, fixed tissue was embedded in 3% agarose and sectioned at 100 µm on a vibratome. Staining was performed as described , 2006b. In situ hybridisation was performed on 100 µm vibratome sections as described (Vegfa, Flt1, Slit1 or Slit2 (escribed , 2006a uor Slit2 , 2000. IPeripheral regions of E14.5 wild-type mouse retinas were cultured in a 1:1 mixture of bovine dermal collagen and rat tail collagen as described , 2000, ez-stacks were captured using a Zeiss LSM 710 confocal microscope and 3D reconstructions of the z-stacks generated using Imaris.Aortic rings were prepared as described  and cultt-test, and comparisons of more than two groups by one-way ANOVA with Tukey post-hoc analysis. For data that did not appear normally distributed, comparisons were made using Kruskal–Wallis rank sum test with Tukey post-hoc analysis. The specific tests used are indicated in the figure legends. Because of background stain differences, direct statistical comparisons were not made between mutations on different genetic backgrounds.All data sets were analysed using the Shapiro–Wilk normality test. For normally distributed data, comparisons of two groups were made using Student's unpaired, two-tailed


The complex architecture of adult brain derives from tightly regulated migration and differentiation of precursor cells generated during embryonic neurogenesis. Changes at transcriptional level of genes that regulate migration and differentiation may lead to neurodevelopmental disorders. Androgen receptor (AR) is a transcription factor that is already expressed during early embryonic days. However, AR role in the regulation of gene expression at early embryonic stage is yet to be determinate. Long non-coding RNA (lncRNA) Sox2 overlapping transcript (Sox2OT) plays a crucial role in gene expression control during development but its transcriptional regulation is still to be clearly defined. Here, using Bicalutamide in order to pharmacologically inactivated AR, we investigated whether AR participates in the regulation of the transcription of the lncRNASox2OTat early embryonic stage. We identified a new DNA binding region upstream of Sox2 locus containing three androgen response elements (ARE), and found that AR binds such a sequence in embryonic neural stem cells and in mouse embryonic brain. Our data suggest that through this binding, AR can promote the RNA polymerase II dependent transcription of Sox2OT. Our findings also suggest that AR participates in embryonic neurogenesis through transcriptional control of the long non-coding RNA Sox2OT. In the developing telencephalon, a large number of neurons originate from neuroepithelial cells and migrate across telencephalic regions. This occurs at approximately between embryonic day (E) E10.5 and E12 in mice when neuroepithelial cells transform into radial glia cells that possess neural stem cells (NSCs)/progenitor cells features . This prAndrogen receptor (AR) is a ligand dependent nuclear transcription factor  that binSox2OT is a long non-coding RNA, characterized by high degree of evolutionarily conservation , that acAlthough Sox2OT is dynamically regulated in the mammalian embryogenesis, and it is expressed in mouse and human brain, little is known about its transcriptional regulation mechanisms.We tested here the hypothesis that AR plays a transcriptional role in the expression of Sox2OT in early mouse neurodevelopmental stages. To block the transcriptional regulation of androgen responsive genes we used a pharmacological approach based on Bicalutamide treatment. Bicalutamide is an FDA-approved non-steroidal AR pure antagonist ,22, thatAnimal studies were approved by the Ethics Committee of the Carlo Besta Neurological Institute, and were conducted in accordance with the guidelines of the Italian Ministry of Health. The use and care of animals followed Italian law DL 116/1992 and EU directive 2010/63/EU. C57BL6J wild-type littermate mice (Charles River Laboratories) were used for all experiments.The animals were housed in our pathogen free facility under 12 h light/12 h dark conditions. Mice were given water and diet ad libitum. Mice were monitored daily by members of the laboratory and by animal health technicians.Bicalutamide(N-[4-cyano-3-(trifluoromethyl)phenyl]-3-(4-fluorophenyl)sulfonyl- 2-hydroxy-2-methylpropanamide) is non-steroidal AR pure antagonist that binds to cytoplasmic AR triggering its rapid degradation,22. AccoEmbryonic NSCs: the optimal concentration for all in vitro experiments was 1μM for 12h supplemented after 3 days of embryonic NSCs culture. Treatments were applied daily without media change. Control cells were cultured in appropriate culture media with 0.01% of DMSO.Mouse treatments: according to the literature, AR activation in rats embryos depends on mother testosterone serum levels –31; at tPregnant female were sacrificed by cervical dislocation under anesthesia at gestational E14 stage, embryos were dissected out of the amniotic sacs and the meninges were removed from the telencephalon. The forebrains were triturated and dissociated into single-cell suspensions by flushing through a p200 pipette tip and washed twice in Dulbecco's modified Eagle's medium (DMEM):F12 supplemented with 2% B27 (Invitrogen). Cells were plated at a density of 20 cells/μl in the embryonic NSCs medium: DMEM:F12 Glutamax supplemented with 2% B27 (Invitrogen), 2 μg/mL heparin and 20 ng/mL EGF and 10 ng/mL FGF-2 (Peprotec). Cultures were incubated at 37°C in a humidified atmosphere containing 5% CO2. Four days after plating, neurospheres were dissociated and subcultured as following typical neurosphere growth protocols . All con6 embryonic NSCs and E12.0 forebrains using the RNeasy microkit (Qiagen) according to the manufacturer’s instructions. cDNA was synthesized using 1 μg of total RNA and the first-strand cDNA synthesis kit (Biorad) according to the manufacturer’s instructions. Real-time PCR was performed using iTaq SYBR Green Supermix (Bio-Rad) using CFX 96 Real Time System (Bio-Rad). All real time PCR reactions were performed in triplicate.Total RNA was isolated from 1 × 10Primer efficiencies was close to 100% for both target and reference gene. RT-qPCR was performed using CFX 96 Real Time System (Bio-Rad) and melting curve analysis was always performed at the end of each PCR assay to control specificity. The delta Ct (ΔCt) method was performed to determine relative concentrations using the average of the Ct of mouse GAPDH as normalizing value. High ΔCt values represent low levels of expression and vice versa. the primers used in RT-qPCR were: mouse GAPDH 5’-aactttggcattgtggaagg-3’ and 5’-acacattgggggtaggaaca-3’; ar 5’-ttgcaagagagctgcatcagtt-3’ and 5’-actgtgtgtggaaatagatgggc-3’; Sox2OT 5’-tgctacaagacaacaccctga-3' and 5'-ccaaagccatcaaccagatt-3' .6 embryonic NSCs s and 8 E12.0 forebrains were lysed with 500 μl ice-cold RIPA buffer , and placed on ice for 10 minutes. Upon incubation on ice, samples were then centrifuged at 13000 rpm for 10 minutes at 4°C, aliquots of each sample, containing equal amount of proteins (500μg), were separated by SDS-PAGE and transferred onto PVDF membranes, were probed with antibody against rabbit anti-androgen receptor (Thermo scientific) or mouse anti-androgen receptor. To avoid blurring of specific signals resulting from a possible cross-detection of precipitating antibodies with secondary antibodies of the Western blot, we employed antibodies to rabbit or mouse IgG light-chain as secondary antibodies (Santa Cruz). Proteins of interest were visualized with the Pierce ECL Western blotting. Densitometry analysis wasdone by ImageJ software (NIH) by measuring levels of the protein of interest relative to the internal control (β-actin), as previously described [1 × 10escribed . The conImmunocytochemistry: Cell preparations for analysis of embryonic NSCs were performed as described in Ferri et al.,  and FavaProcedure was adapted from Schaeren-Wiemers and Gerfin-Moser  and Henr7 of embryonic NSCs and 10 E12.0 embryos freshly dissected mouse forebrains, were using for each DHS assay adapted from Ling and Waxman [Approximately 2 × 10d Waxman  and Lingd Waxman  for embrd Waxman .We calcu7 of embryonic NSCs at passages between 2–4 and 10 E12.0 forebrains for each assay (n = 1).ChIP was performed using approximately 2 × 10The pellet was resuspended in 5 ml PBS containing 1% final concentration formaldehyde and incubated for 10 min at room temperature with rotation. Cross-linking reactions were stopped with 0.125 M glycine for 8 min at room temperature and washed twice with ice-cold PBS.5’-ctattttcccctcgcttaacctc-3’ and 5’-tctgggtctaaagtgggcat -3’; primers for non target sequence (negative control) were 5'-attaagacacaaaggagagaggtcc-3' and 5'-tgtcatgtatcaagtttccaaaacc-3'. These non target sequence primers were previously used in ChiP-seqexperiments aimed at mapping genome-wide AR binding site in mouse caput epididymis[Mouse embryos were harvested from timed pregnant females (Charles River) at E12.0. The forebrains were dissected in cold PBS and batches of eight forebrains each were collected in a tube (n = 1), washed twice, cut to <1mm size and crosslinked with 1% formaldehyde for 10min stopped with 0.125 M glycine for 10 min at room temperature. Chromatins from forebrain tissue and embryonic NSCs were isolated following this procedure: Nuclei were isolated and chromatin was sheared to approximately 600 bp using a sonicator (Bandeline SONOPLUS). Cross-linked chromatin was immunoprecipitated using ChIP-grade antibodies: 5 μg anti Androgen Receptor (C-19 Santa Cruz Biotechnology), 3 μg anti Phospho-Rpb1 CTD pSer2+5 (Thermo scientific) and 2 μg IgG control antibodies (Pierce) overnight at 4°C. immunoprecipitation and DNA recovery were obtained according to the manufacturer's protocol pierce magnetic chip kit (pierce). Aliquots of the purified DNA were diluted and analyzed by PCR with the following primer pairs: arso-Sox2OT pididymis. The pri7 of embryonic NSCs and 10 E12.0 freshly dissected mouse forebrains was using for each RIP assay performed as previously described [Approximately 1 × 10escribed . Total RAll statistical analyses were performed using GraphPad Prism 7.0 Software. Data were analyzed by Student's two-tailed, paired t-test and results were expressed as the mean value ± SEM. All experiments were carried out on a minimum of 4 occasions unless stated otherwise (n = number of independent experiments). The asterisks in each graph indicate statistically significant changes:* p < 0.05, ** p < 0.01, *** p <0.001.AR-ctGAPDH):Δct control = 11.46±0.46, ΔctBicalutamide = 14.97±0.19; t-test p = 0.0004;2-ΔΔCtcontrol = 1.00, 2-ΔΔCt Bicalutamide = 0.09; Fig 1A). To confirm this data we analyzed AR protein levels. We found detectable AR protein levels in E12.0 mouse forebrains; Bicalutamide significantly reduced AR protein levels . Since in E12.0 forebrains embryonic neural stem cells (embryonic NSCs) are the primary progenitor cells that initiate lineages leading to the formation of differentiated neurons [AR-ctGAPDH):Δct control = 13.09±0.19; Δct Bicalutamide = 16.17±0.17, t-test p = 0.0005; 2-ΔΔCt control = 1.00, 2-ΔΔCt Bicalutamide = 0.12.Normalized mean value ± SEM AR/actin: control = 1.00±0.02; Bicalutamide = 0.57±0.009 t-test p = 6.4 x10-7; This result agrees with previous data showing that AR is expressed in rodent adult NSCs [Previous data suggested that AR mRNA in CNS is detectable at mouse embryonic day 12.5 (E12.5) shortly before embryonic hormone production . To conf neurons ,44, we hult NSCs . The preult NSCs .Sox2OT-ctGAPDH): for E12.0:Δct control = 4.26±0.20; Δct Bicalutamide = 6.51±0.32, t-test p = 0.010; 2-ΔΔCtcontrol = 1.00,2-ΔΔCtBicalutamide = 0.21; for eNSCs control = 4.51±0.18; Δct Bicalutamide = 6.00±0.16, t-test p = 0.0009;2-ΔΔCt control = 1.00, 2-ΔΔCtBicalutamide = 0.36; Fig 1E). To confirm this result, we analyzed AR by immunofluorescence and Sox2OT mRNA by in situ hybridization in embryonic NSCs and found that Bicalutamide treatment decreased both AR and Sox2OT signals . Hence, these results show that AR and Sox2OT are expressed in E12.0 forebrains and in embryonic NSCs and that AR downregulation elicits Sox2OT downregulation. This result suggested that AR is directly involved in the transcriptional regulation of Sox2OT in embryonic NSCs and E12.0 forebrains.Sox2OT is a lncRNA transcribed in the same orientation of Sox2 that can act as an enhancer during brain development and participate in Sox2 transcriptional regulation ,18. To t(a) Real-time quantitative RT-qPCR analysis of AR mRNA in E12.0 forebrains. E12 embryos were exposed daily to Bicalutamide 200μg/Kg from E10.0 onwards. AR expression was higher in control samples than in Bicalutamide treated samples. Data are expressed as mean value ± SEM of delta ct : Δct control = 11.46±0.46; ΔctBicalutamide = 14.97±0.19; two-tailed t-test p = 0.0004, n = 4 samples analyzed for each condition.(b) Representative western blot showing AR expression in control E12.0 forebrains and E12.0 forebrains treated with Bicalutamide. Bicalutamide significantly decreased AR protein levels. The histograms represent the mean of densitometry calculations for western blot data. Data were normalized to β-actin expression. Values are expressed as normalized mean value ± SEM of triplicate experiments: control = 1.00±0.05; Bicalutamide = 0.71±0.06; p = 0.0079 two-tailed t-test.(c) AR expression in eNSCs analyzed by real-time PCR. Bicalutamide significantly decreased AR expression. Values are expressed as mean±SEM of Δct (ctAR-ctGAPDH). Δct control = 13.09±0.19, ΔctBicalutamide = 16.17±0.17; two-tailed t-test p = 0.0005; n = 5 samples for each condition.(d) Representative western blot showing AR expression in control eNSCs and eNSCs treated with Bicalutamide. Data are normalized to β-actin protein. Values are expressed as normalized mean value±SEM of triplicate experiments, n = 5 samples for each condition. AR/actin: control = 1.00±0.02; Bicalutamide = 0.57±0.01, two-tailed t-test p = 6.4 x10-7.(e) Sox2OT mRNA expressions analyzed by Real-time quantitative RT-PCR in E12.0 brains And eNSCs treated or untreated with Bicalutamide . For E12.0 brains: Δct control = 4.26±0.20; ΔctBicalutamide = 6.51±0.32, p = 0.010; for eNSC: control = 4.51±0.18; ΔctBicalutamide = 6.00±0.16, p = 0.0009; n = 4 samples analyzed for each condition, two-tailed t-test.(f) Representative images showing AR protein signal (immunofluorescence with AR antibody in red) and Sox2OT mRNA (in situ hybridization for Sox2OT in green) in eNSCs. The histograms show the percentage of Sox2OT positive cells normalized to total nuclei counted for each neurosphere. Bicalutamide decreases both AR protein levels and the number of Sox2OT positive cells. Values are expressed as mean±SEM of %Sox2ot positive cells: control = 54.87±1.69, Bicalutamide = 2.19± 0.70, two-tailed t-test p = 5.9 x10-11; n = 5 samples analyzed for each condition. Scale bar is 50 μm.Fig 1E and 1F) led us to hypothesize that AR may interact with chromatin of Sox2OT gene. To test this hypothesis we first analyzed the presence of androgen response elements (AREs) in the 5000 bps upstream of the Sox2 translational start site (ATG) that encompass the Sox2OT locus. High density hits with a score over 0.80 and elevated conservation (over 50% of identity) of the candidate AREs sites were considered. By JASPAR motifs, we identified a sequence located -2356 bps upstream to Sox2 ATG  containing 3 neighboring putative ARE sites including 5’-tagtacaccccgatt-3’ at site -1995, 5’-gagaaaacaatgctt-3’at position -1977 and 3’-aaggacttatagaaa-5’ at site -1933 . Accordingly, comparative genomic analysis using the VISTA Browser (http://genome.lbl.gov/vista/index.shtml), showed high conservation among the species for ARSO-Sox2OT sequence . Data from http://genome.ucsc.edu/ENCODE/) focusing on whole brain, cortex and cerebrum tissues of E14.5, E18.5 and adult mice (8 weeks) . The results obtained by ENCODE Open Chromatin by DNase-Duke University and Open Chromatin by DNase-Washington University showed that ARSO-Sox2OT is a region of chromatin highly sensitive to cleavage by DNase I . Because DNase I hypersensitive sites (DHS) are structural landmarks indicative of regulatory chromatin regions involved in the cell-type-specific regulation [Fig 1E and 1F), we hypothesized that AR modulates the DNase I hypersensitivity of ARSO-Sox2OT. To test this hypothesis, we performed a DNase hypersensitivity assays in embryonic NSCs and E12.0 forebrains in basal conditions and after Bicalutamide treatment. The size distribution of DNA short fragments released by DNase I digestion of isolated nuclei was determined by DNA electrophoresis  followed by PCR amplification with sequence specific primers. We found that ARSO-Sox2OT chromatin region in basal condition is highly sensitive to cleavage by DNase I both in embryonic NSCs and E12.0 forebrains . This result confirmed the data obtained from epigenetic analysis . We also found that Bicalutamide treatment significantly decreased the hypersensitivity to DNase I .The evidence showing that AR downregulation elicits Sox2OT downregulation . AR occupancy was readily detected at ARSO-Sox2OT region in concert with RNA polymerase II which transcribes the Sox2OT gene . In tissues and cells treated with Bicalutamide neither AR nor RNA polymerase II were enriched in ChIPs .To further confirm that AR mediates Sox2OT transcription, we assessed whether AR interact with Sox2OT transcript. We conducted AR- and RNA Polymerase II-directed RNA immunoprecipitation (RIP) coupled with qPCR using primer sets targeting the ARE elements at the sense Sox2OT RNA  located at Sox2OT gene and show that AR binds the Sox2OT transcript in E12.0 forebrains and embryonic NSCs.Our evidence for AR expression in embryonic NSCs and E12.0 forebrains agrees with previous papers showing AR expression in the neurogenic territories of most vertebrate groups –57,45 suin vivo inactivate AR include the in vivo delivery of small interfering RNA(siRNA) and the use of AR knock out mice. siRNAs are very efficient tools for in vitro mRNA silencing, however siRNA use in mammalian adult central nervous system is limited, because in vivo delivery technology such as intravenously and intraperitoneally administration of synthetic siRNAs is not fully safe and efficient. Additional important limitations exist when siRNA have to be delivered to both pregnant mice and their embryos. Regarding the possibility to perform experiments on mouse models of AR deficiency, the brain-specific models, nestinCre ARKO [To induce AR inactivation we used Bicalutamide treatment. Although this pharmacological approach have intrinsic limitations, it is an established methods to decrease AR protein and mRNA levels. Indeed, Bicalutamide triggers rapid AR degradation at a nuclear location and promotes AR turnover . The mecCre ARKO and synapCre ARKO  are usefCre ARKO ,70, howeHence, we choose a model for pharmacological inactivation of AR using because this compound has high specificity for AR. Indeed, it was used in many studies aimed at inactivate AR ,71,72. Min silico screening and Chip-seq analysis. These very same analyses prompted in the past to the identification of ARE sites in the genome of various human cells type [The second novelty of our manuscript involvesthe AR binding on Sox2OT locus.AR binding to specific DNA motifs in the promoters –78 and ills type . At ARE lls type . We showlls type  and thatlls type –84. We clls type . This hylls type  and thatlls type –89.Our RIP results show that AR interacts with Sox2OT mRNA. This data agrees with previous studies demonstrating that AR regulates non-coding RNAs by means of mechanisms common to protein-coding transcripts. Sheflin and colleagues  demonstrOur findings support the hypothesis that AR has a pivotal role in controlling the transcription of Sox2OT during mouse early neurodevelopmental stages. The physiological significance of the regulation of Sox2OT by AR remains open. AR-regulated Sox2OT in NSCs may regulate stemness, cell proliferation, differentiation, cell fate (into neurons or glia).Because Sox2OT is transcribed in the same orientation of Sox2, Sox2OTcan act as an enhancer during brain development and participates in Sox2 transcriptional regulation ,18, we cS1 FigWhite box, Sox2 gene; gray boxes, region tested by ChIP with ChIP-grade antibodies; vertical red lines, AR consensus sites (ARE). The direction of Sox2 gene transcription is shown with arrow. The phylogenetic conservation (CNS) Mouse-human of ARSO-Sox2OTsequence is shown below the gene diagram (derived from the VISTA browser). High sequence conservation of ARE (UCSC browser) at aligned sites across the species (shown at the left). Identical nucleotides are shown in the color pink.(TIF)Click here for additional data file.S2 FigIntegration at ARSO-Sox2OT site of ENCODE high-throughput experiments performed in mouse cerebral tissues. Respect to Sox2 locus at top, vertical rectangular shows the ARSO-Sox2OTchromatin region. From top to bottom, row reports the genomic coverage obtained by DNAse-Seq and ChIP-Seq against different histone modifications. Letters on the right column indicate the tissues analyzed, row by row. Sample information isreported at bottom. Top panel shows results obtained by ENCODE Open Chromatin by DNase-Duke University and Open Chromatin by DNase-Washington University. Is evident a peak in the ARSO-Sox2OT sequence, which is more present in embryonic than adult tissues.H3K4me3/1, H3K36me3, and H3K27ac are modifications associated with active chromatin and H3K27me3 and H3K9me3 for silenced heterochromatic region. The image was generated using Wash U Epigenome Browser.(TIF)Click here for additional data file.


Meteorological factors have been shown to affect the physiology, distribution, and amounts of inhaled allergens. The aim of this study was to develop a model to predict the trends for onset of allergic rhinitis (AR) patients. A total of 10,914 consecutive AR outpatients were assessed for the number of daily patient visits over a period of 4 years. Meteorological data were used to assess the relationship between meteorological factors and AR incidence by time-series data and regression analysis. Predictive models for incidence of AR were established in pollen-, dust mite- and mould-sensitive groups of patients, and the predictive performances of meteorological factors on the incidence of AR were estimated using root mean squared errors (RMSEs). The incidence of pollen-, dust mites- and mould-sensitive AR patients was significantly correlated with minimum temperature, vapour pressure, and sea-level pressure, respectively. The correlation between comprehensive meteorological parametric (CMP) and incidence of AR was higher than the correlation between the individual meteorological parameters and AR incidence. CMP had higher performance than individual meteorological parameters for predicting the incidence of AR patients. These findings suggest that the incidence of pollen-, dust mites- and mould-sensitive AR can be predicted employing models based on prevailing meteorological conditions. AR is characterized by sneezing, rhinorrhea, nasal congestion and nasal pruritus, which are often accompanied by ocular pruritus, redness and/or lacrimation in 60–70% of patients. Prevalence of AR has increased over the last few decades, with about 25% of the worldwide population affected3, including in China1. AR significantly impacts the quality of life of affected individuals, affecting work and social life, causing multiple comorbidities, and requiring considerable healthcare resources6. Several studies have demonstrated that inhaled allergens, including pollen, mould, and dust mites, as well as exposure to cockroach and animal wool in the environment, may trigger the exacerbation of AR attacks9. The main inhaled allergens of AR in Beijing are from pollen, dust mite and mold3. Other studies have demonstratedmeteorological factors such as temperature, irradiance, and relative humidity toaffect the physiology, distribution, and amounts of these allergens12, suggesting that meteorological factors are associated with allergic disease outbreaks and severity. Hjelmroos and colleagues12 demonstrated that birch trees growing at higher temperatures produced pollen with an increased content of major allergen, which also had higher allergenicity. Furthermore, for pollen allergens, the positioning of the branches also influenced the antigenic proteins and allergens produced; with the greatest amounts of antigenic proteins and allergens being extracted from pollen present in the south-facing branches and allergens decreasing progressively in pollen from west- through east- to north-facing branches12. Similarly, several studies have reported that heavy rainfalls and thunderstorms are associated with increased number and severity of asthma attacks, both in adults and children16. It has been suggested that under wet conditions or during thunderstorms, pollen grains may rupture by osmotic shock and release into the atmosphere respirable, allergen-carrying cytoplasmic starch granules (0.5–2.5 µm) or other paucimicronic components that can reach the lower airways and induce asthma reactions in pollinosispatients14. However, appropriate models for predictingincidence of AR are scarce, and urgently needed especially in China with its increasing population of AR patients.Allergic rhinitis (AR) is an inflammatory disease of the nasal mucosa, induced by an immunoglobulin E (IgE)-mediated reaction in allergen-sensitized subjectsThe aim of this study was thus to develop a model for predicting the trends for onset of AR patients, based on correlation between meteorological factors and incidence of AR in patients sensitized to pollen, mould and house dust mite allergens.17, were subsequently recruited to the study on the same day. Repeating patients were identified according to the department’s computerised patient database and were excluded. The vast majority of the patients  were residents of Beijing, with the remaining subjects  from other cities. These patients were included in the study, because they had resided in Beijing in the recent three years, when they were recruited into the study.The study population comprised 10,914 consecutive outpatients (aged 6 to 60 years) recruited from the Allergic Rhinitis Treatment Centre of Beijing TongRen Hospital, during the period from February 4, 2007 to November 22, 2010. The patients who had clinical symptoms of AR underwent SPT for a diagnosis of AR. All patients with a confirmed diagnosis of AR, based on medical history and positive skin prick tests (SPTs) to specified allergensTo be included into the study, all patients had to demonstrate clinical symptoms of AR, such as sneezing, itchy and runny nose, and nasal congestion; and positive SPT results to relevant allergens. The patients were also required to complete a standardized questionnaire  under the guidance of doctors and not have taken any drug, which might affect SPT results , for at least one week prior to SPT. The onset of AR was determined according to the questionnaire. Fifty seven AR subjects had failed to fill in the questionnaire appropriately and were therefore excluded from the study.The study protocol was approved by the Ethics Committee of Beijing TongRen Hospital and performed in accordance with the guidelines of the World Medical Association’s Declaration of Helsinki. All patients gave written informed consent prior to inclusion in the study and all patient information was anonymized prior to analysis.et al.18. Skin tests were graded after 20 minutes in comparison to the positive control’s wheal diameter.SPT was performed according to standardized procedures on the day of visit for diagnosis of allergy, using 21 inhalant allergens . Histamine diphosphate (1 mg/mL) and PBS (1 mg/mL) were used as positive and negative controls, respectively. According to international guidelines, positive reaction was defined as a mean wheal diameter exceeding by 3 mm or more than the negative control value. Reactions were graded from +1 to +3 level  according to Stytis 1.AR patients were divided into 5 groups based on their SPT results for the most predominant sensitizing allergens; namely (i) pollen , (ii) dust mite, (iii) mold , (iv) cockroach, and (v) animal wool allergen groups. For the purpose of this study we have primarily investigated only the first three groups because these allergens are the main causes of AR in Beijing and show some degree of regularity in their annual peaks19.Meteorological data included daily maximum, average and minimum temperatures, relative humidity, vapour pressure, precipitation wind speed, sea-level pressure, and dew point temperature. The data were recorded daily at the meteorological observatory of Dongcheng District of Beijing sited in the centre of the city and reported by the Beijing Meteorological Administration. Daily mean values were used in the analysesSimilarly, data for pollen were collected from a monitoring station situated in the vicinity of the meteorological monitoring station. The pollen stations are managed by Beijing Meteorological Administration. Pollen grains were collected daily using a Durham sampler  and then counted under a microscope by reading the slides. Mean daily counts of tree pollen were used for analysis.AR incidence was assessed as the number of different individual AR patients visiting our hospital from 2007 to 2010. In this regard data on “AR incidence”, were collected 92 times from 2007 to 2010 (once every two weeks) and were used in further analyses to develop the model predicting the incidence of allergic rhinitis based on meteorological factors, as shown in Fig. AR incidence was first assessed over time, using autocorrelation function (ACF) and partial autocorrelation function (PACF) combined with Bayesian information criteria (BIC) to determine the stationarity of data and fit model’s order. Autoregressive moving average (ARMA) model was considered to estimate the temporal trend of the data. White noise test was performed on the residuals to demonstrate the significance for the fitting model.th average vapour pressure, “TDi” represents the ith average minimum temperature, “Pi” represents the ith average sea-level pressure, and “i” various recording times.Correlation between incidence of AR symptoms and meteorological factors was analyzed and P values < 0.05 were considered to be significant. Comprehensive meteorological parametric (CMP) equations were established to summarize the complex collective effects of all prevailing meteorological factors on AR incidence Equation ,1\documeThe predictive performances of meteorological factors and the CMP on the incidence of AR were estimated by root mean squared errors (RMSEs). The predictive models were built on about 70% of all samples randomly selected from the whole dataset, and were then validated on the rest 30% samples. The AR cases were divided into 4 grades according tothe probability distribution of AR (data not shown). The classification criteria were defined as follows: level I: AR cases ≤ 16; level II: AR cases = 17–52; level III: AR cases = 53–104; level IV: AR cases ≥ 105. The predictions of AR incidence were performed according to daily meteorological factors and the diagnostic values of the models. Statistical analyses were carried out using SAS 9.2  and OriginPro 9.0  and P < 0.05 was considered to be statistically significant.The demographic characteristics of the participants are shown in Table εt is white noise and xt is predicted trend of AR incidence; t (time unit) is bi-weekly.AR incidence in patients sensitive to pollen, dust mite, and mould allergens were assessed over time, using the respective regression equations as shown below:Figure Correlation between AR incidence and meteorological factors are presented in Table CMP is a comprehensive parameter of above-mentioned meteorological factors. AR incidence in patients sensitive to pollen, dust mite, and mould allergens were significantly correlated with CMP P < 0.05), and the correlation between CMP and incidence of AR was higher than the correlation between the individual meteorological parameters and AR incidence for the same period Table  and 5.Fi, and the1, y2, y3 are predicted values for AR incidence in pollen-, dust mite- and mould-sensitized groups, respectively; and “x” represents comprehensive meteorological parameters.When evaluating the predictive performance of individual meteorological factors and CMP, 67 samples (72.8%) were selected from the whole dataset randomly. The remaining 25 samples were used to validate the predictive models built on the 67 samples. When predicting the number of patients sensitive to pollen and mould, CMP had the highest performance , while having the second highest performance (RMSE: 4.25) for predicting the number of patients sensitive to dust mites Table . These r3and because they all show onset peaks between August and September  and to a smaller extent from neighbouring cities 3.1%). AR patients sensitized to pollen, dust mite and mould were investigated primarily because these allergens are the main causes of AR in Beijing ber Fig. , which s.1%. AR p21. In the current study, Beijing’s pollen monitoring data showed that air pollen concentrations peaked during the periods from August to September and from April to May. August and September constitute the blooming period for weeds, such as Artemisia and Humulus, whereas April and May is important for woody plants, such as birch, Cypress and Salicaceae3. However, as Beijing has a temperate climate, with the environment having higher humidity and temperature in August and September, these conditions are also suitable for dust mite and mould growth. Thus, the peaks of AR incidence noted in August to September and in May in the current study are consistent with the peaks of “allergenic protein” noted during these periods in the year.Exposure to allergens may trigger the exacerbation of AR attacks; with increased concentrations of inhaled allergens being responsible for increased incidence and severity of allergic diseasesConsistent with previous findings, our study has also demonstrated that the incidence of AR patients in the three groups significantly correlate with meteorological factors, minimum temperature, vapour pressure and sea-level pressure Table . Indeed,22, potentially affecting AR incidence. Climate change is a constant and ongoing process postulated to be mainly driven by human activities23. It was recently shown that climate change increases the prevalence of certain ailments, including respiratory diseases and allergic responses, affecting the most vulnerable populations i.e. the elderly, children, and less socioeconomically fortunate24. Although the potential of meteorological factors as predictors for incidence of AR symptoms may be useful in the development of correlation prediction models, meteorological parameters are only one area of influencing factors and also help raise awareness for the role of other factors such as extreme environmental pollution and possibly other less obvious factors on human health, especially AR. This is particularly so for air pollution , that is especially severe during May and the September - October period when anticyclonic weather situations occur most frequently over Beijing area, and substantially contribute to the higher occurrence of AR cases. However, this study is somewhat limited in that it is a single centre study with a relatively small cohort, and all meteorological parameters were assessed locally. The study is also somewhat limited in that it does not account for the effects of meteorological parameters on air pollution and the incidence of AR or the impact of the meteorological parameters on incidence of AR in the very young, elderly and socioeconomically disadvantaged subjects. Thus, further large multicentre studies assessing the various meteorological factors in several locations, and taking into account the presence of particularly air pollution levels at the time of monitoring, as well as the age and socioeconomic status of the subjects are needed to substantiate this model in the future.The finding from the present study that AR incidence peaks in the three groups of patients were well synchronized with the CMP peak, suggests that changes in meteorological conditions can result in plant, mould, and dust mite growth, and increased allergen release25. Despite the differences in the sampling techniques, longitudinal studies by Crispen and colleagues26 and Kishikawa and colleagues27, have demonstrated that both techniques were equally suitable and comparable in monitoring airborne pollen under local conditions. Indeed, the study by Crispen and colleagues25 compared local pollen counts in Missoula, Montana in 1978 determined using a Durham sampler with those determined in 2006 using a Burkard sampler and demonstrated that the two sampling methods were comparable when reporting relative frequency of occurrence. Furthermore, Kishikawa and colleagues27 indicated that Durham’s technique was not inferior to Burkard’s technique, but rather defined the start of pollen dispersion more distinctly.In the present study, pollen was measured using a Durham sampler, which collects airborne pollen by a gravimetric method. Although, this technique is relatively simple and inexpensive, pollen sampling is influenced by wind speed, orientation of the sampler with respect to wind direction, as well as concentration of pollen in the air. In comparison, the Burkard sampler is comparatively new and collects airborne pollen by a volumetric method, which involves drawing ambient air into the sampler at 10 l/min. Although sampling efficiency is comparatively high, the wind speed and the particle size shape and density have been reported to affect sampling efficiency of the Burkhard samplerIn conclusion, to our knowledge this is the first model, which provides a sound methodological basis for the prediction of the incidence of AR based on meteorological factors in Beijing (China). In addition to meteorological factors, other parameters affecting the onset time of AR; for example environmental pollution, which is an extreme risk for respiratory diseases in megacities such as Beijing, and personal health conditions should be further assessed to confirm the feasibility of the prediction model described here.


Our results thus suggest that pretreatment with AR inhibitor prepares the monocytes against hyperglycemic stress by overexpressing the Nrf2-dependent antioxidative proteins.We have shown earlier that pretreatment of cultured cells with aldose reductase (AR) inhibitors prevents hyperglycemia-induced mitogenic and proinflammatory responses. However, the effects of AR inhibitors on Nrf2-mediated anti-inflammatory responses have not been elucidated yet. We have investigated how AR inhibitor fidarestat protects high glucose- (HG-) induced cell viability changes by increasing the expression of Nrf2 and its dependent phase II antioxidant enzymes. Fidarestat pretreatment prevents HG (25 mM)-induced Thp1 monocyte viability. Further, treatment of Thp1 monocytes with fidarestat caused a time-dependent increase in the expression as well as the DNA-binding activity of Nrf2. In addition, fidarestat augmented the HG-induced Nrf2 expression and activity and also upregulated the expression of Nrf2-dependent proteins such as hemeoxygenase-1 (HO1) and NQO1 in Thp1 cells. Similarly, treatment with AR inhibitor also induced the expression of Nrf2 and HO1 in STZ-induced diabetic mice heart and kidney tissues. Further, AR inhibition increased the HG-induced expression of antioxidant enzymes such as SOD and catalase and activation of AMPK- The cytytokines . Our recytokines , 4 by pr signals , 5. We h signals –8. Whileγ-glutamylcysteine synthetase (GCS), glutathione S-transferases (GSTs), and AR [Previous studies indicate that transcription factor nuclear factor-erythroid-2-related factor 2 (Nrf2) regulates a battery of cytoprotective genes which maintain cellular redox homeostasis. Nrf2 binds to the antioxidant response element (ARE) and transcriptionally regulates the gene expression of several antioxidant and phase II detoxifying enzymes including hemeoxygenase 1 (HO1), NAD(P)H-quinone dehydrogenase 1 (NQO1), , and AR . General, and AR .κB-dependent inflammatory signals, it is not known how AR inhibition increases the resistance of cells to withstand oxidative stress stimuli initiated by hyperglycemia. Therefore, in this study, we examined our hypothesis that AR inhibition promotes the activation of Nrf2-mediated cytoprotective pathways that protect cells against hyperglycemic stress. Our results suggest that AR inhibitor fidarestat increases the expression as well as the DNA-binding activity of Nrf2 in Thp1 monocytes. In addition, fidarestat increased the expression of Nrf2 downstream target proteins such as HO1 and NQO1 in Thp1 cells and heart and kidney tissues of STZ-induced diabetic mice. AR inhibitor also prevented the expression of antioxidant enzymes such as SOD and catalase. Collectively, our results demonstrate that AR inhibition protects the cells against hyperglycemia-induced changes in the cell viability by activating Nrf2/HO1-mediated antioxidative pathway, which could also account for the anti-inflammatory effects of AR inhibitors in diabetes.Although our previous studies indicate that AR inhibition prevents hyperglycemia-induced NF-α1, AMPK-α1, GAPDH, and histone H3 were purchased from Cell Signal Inc. Fidarestat was obtained from Livwel Therapeutics Inc., USA. 3--2,5-diphenyltetrazolium-bromide (MTT) and other reagents used in Western blot analysis were obtained from Sigma. HO1 assay kit was purchased from Abcam. Nrf2 transcription factor binding assay and superoxide dismutase activity assay kits were purchased from Cayman Chemicals. Catalase activity was determined spectrophotometrically using hydrogen peroxide (30%) (Sigma-Aldrich).RPMI-1640, penicillin/streptomycin, and fetal bovine serum (FBS) were purchased from Invitrogen. Antibodies against Nrf2 were obtained from Santa Cruz. Antibodies against KEAP1, HO1, NQO1, phospho-AMPK-2. Prior to treatment, cells were serum starved in the respective medium containing 0.1% FBS ± fidarestat (10 μM) for 14 h and further stimulated with high glucose  for different time intervals.Human Thp1 monocytic cells were obtained from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 medium supplemented with 10% FBS and penicillin/streptomycin at 37°C in a humidified atmosphere of 5% COμM) for overnight (14 h) at37°C followed by incubation with HG (25 mM) for another 48 h. At the end of incubation period, cells were incubated with 10 μl MTT reagent (5 mg/ml) for 4 h at 37°C. The formazan crystals formed by the viable cells were solubilized by the addition of 100 μl DMSO. Absorbance was measured at 570 nm using a microplate reader. Cell viability was also examined by counting the live and dead cells using a hemocytometer [Cell viability was determined using a standard MTT assay . Brieflyytometer . AR actiytometer . Nrf2 DNNuclear and cytoplasmic proteins from the treated cells were isolated using a nuclear extraction kit . Protein concentration in the extracts was measured with Bradford reagent (Bio-Rad). Equal amount of proteins were subjected to 12% SDS-PAGE electrophoresis followed by Western blot analysis using specific antibodies against Nrf2, Keap1, HO1, NQO1, AMPK, histone H3, and GAPDH. The antigen-antibody complexes were detected by enhanced Super Signal West Pico Chemiluminescent Substrate (Thermo Scientific). Membranes were stripped with Restore TM PLUS stripping buffer (Thermo Scientific) and used for reprobing with other antibodies or loading controls.2 incubator for 48 h at 37°C. Silencing of Nrf2 was determined by Western blotting.Thp1 cells cultured in RPMI 1640 medium containing 10% FBS were incubated with Nrf2-siRNA (120 nM) or siRNA negative control with HiPerFect Transfection Reagent as per the manufacturer's instructions . The cells were incubated in a humidified COμl of cDNA using the iTaq Universal SYBR Green Supermix (Bio-Rad). Housekeeping gene GAPDH was used as a normalizer. ABI Prism 7500 Sequence detection system using forward: 5′-CGGGCCAGCAACAAAGTG-3′, and reverse: 5′-CCAGAAAGCTGAGTGTAAGGACC-3′ was used for qPCR analysis of HO1 gene.Total RNA was isolated from the treated cells using TRIzol reagent and was quantified by using a nanodrop spectrophotometer (NanoDrop Technologies). TaqMan reverse transcription reagents kit was used for the synthesis of cDNA from total RNA (Life Technologies). Q-PCR amplifications (performed in triplicate) were performed by using 1 Seven-week-old C57BL/6 male mice were purchased from Envigo. Diabetes was induced in mice by injecting a single dose of streptozotocin  and blood glucose levels were measured by a glucometer (True Metrix). The mice with blood glucose levels >400 mg/dl were selected and randomly divided into diabetic and diabetic + fidarestat groups. Fidarestat  was administered to diabetic mice, and the animals were euthanized on day 3.p values were determined using the unpaired Student's t-test (GraphPad Prism software) and a p value of <0.05 considered as statistically significant.Data are presented as mean ± SD. The The effect of AR inhibition on HG-induced Thp1 cells viability was examined by measuring the live and dead cell counts as well as MTT absorbance. The data shown in the To examine how pretreatment of cells with AR inhibitor prevents HG-induced decrease in Thp1 cell viability, we examined the effect of AR inhibitor on the expression of Nrf2. Pretreatment of Thp1 cells with fidarestat alone or HG alone induced Nrf2 expression in a time-dependent manner. Further, preincubation of cells with fidarestat followed by incubation with HG significantly augmented the HG-induced increase in the expression of Nrf2 . SimilarHO1 increased significantly in cells treated with HG in the presence of fidarestat as compared to HG- or fidarestat-treated cells  significantly increased cell death of Thp1 cells when compared to that of control cells . FurtherWe next examined the effect of fidarestat on the expression of antioxidant proteins, HO1, and Nrf2 in STZ-induced diabetic mouse heart and kidney tissues. Similar to data shown in the Thp1 cells, AR-inhibitor (fidarestat) also augmented the STZ-induced increase in the expression of Nrf2, HO1, and NQO1 in the heart and kidney homogenates of mice .α1 activation has been shown to activate Nrf2 signals, we next investigated the effect of fidarestat on HG-induced AMPK-α1 activation in Thp1 cells. Our results shown in α1 in Thp1 cells. Further, to investigate the effect of Nrf2 on AMPK-α1 activation, Nrf2-siRNA-transfected Thp1 cells were stimulated with HG ± fidarestat and examined AMPK-α1 activation. The results shown in α1. These results suggest that by regulating the Nrf2-mediated AMPK-α1, AR inhibitor could modulate hyperglycemic stress in Thp1 cells.Since AMPK-κB in various cellular studies [κB-mediated proinflammatory pathways in vitro and in vivo models of hyperglycemia [We have shown previously that pretreatment with AR inhibitors prevents cytokine-, chemokine-, HG-, and LPS-induced inflammatory response mediated by NF- studies –15. Furtglycemia , 16. HowSOD, catalase, GSTs, AR, HO1, NQO1, and so forth [Nrf2 transcription factor regulates the expression of a number of cytoprotective antioxidative genes including so forth , 10. Sevso forth , 18. Furso forth –23. In aso forth . Anotherso forth , 26. Simso forth –29. Thesα1 in Thp1 cells. Our results suggest that pretreatment with fidarestat stimulates phosphorylation of AMPK-α1 in HG-induced Thp1 cells. However, fidarestat pretreatment has no effect on the AMPK-α1 activation in Nrf2-ablated cells.Increased expression of Nrf2 leads to increased expression of enzymes linked to antioxidative  and anti-inflammatory  functions that counteract against oxidative insults , 30. HO1α1. This suggests that AR inhibition prevents hyperglycemia-induced complications by upregulating anti-inflammatory Nrf2 pathway in addition to downregulating the proinflammatory NF-κB pathway.In conclusion, we have shown that AR inhibitor fidarestat prevents HG-induced Thp1 cell death by induction of Nrf2 expression; DNA binding activity; and expression of HO1, NQO1, SOD, and catalase via activation of AMPK-


The segregation of the readily releasable pool of synaptic vesicles (RRP) in sub-pools that are differentially poised for exocytosis shapes short-term plasticity. However, the frequency-dependent mobilization of these sub-pools is poorly understood. Using slice recordings and modeling of synaptic activity at cerebellar granule cell to Purkinje cell synapses of mice, we describe two sub-pools in the RRP that can be differentially recruited upon ultrafast changes in the stimulation frequency. We show that at low-frequency stimulations, a first sub-pool is gradually silenced, leading to full blockage of synaptic transmission. Conversely, a second pool of synaptic vesicles that cannot be released by a single stimulus is recruited within milliseconds by high-frequency stimulation and support an ultrafast recovery of neurotransmitter release after low-frequency depression. This frequency-dependent mobilization or silencing of sub-pools in the RRP in terminals of granule cells may play a role in the filtering of sensorimotor information in the cerebellum. To date, the deciphering of cellular mechanisms underlying synaptic efficacy has been challenged by the contentious definition of the RRP  into corresponding changes of neurotransmitter release. During trains of APs, the immediate tuning of synaptic efficacy is determined by the combination of multiple parameters including AP frequency, past firing activity, number of release-competent synaptic vesicles (SVs), also referred as the readily-releasable pool (RRP), vesicular release probability  was observed.In order to better understand the mechanisms underlying the strong facilitation of glutamate release during repetitive stimulations of the GC-PC synapse, we first measured synaptic transmission over a broad range of frequencies using transverse cerebellar slices prepared from young mice (P18 to P25). To focus on presynaptic mechanisms and to rule out postsynaptic contributions, we pharmacologically blocked the induction of postsynaptic long-term plasticity (LTP and LTD), NMDA-dependent plasticity , and endn = 9 compared to 40.14 ± 7.50 at 100 Hz, n = 27, p=0.82, t-test, At many synapses, asynchronous release of quanta can be triggered by repeated stimulation . Since an = 35) (n = 32) with a mean time constant of 15.9 s ± 1.2 s. A lack of LFD was observed for ~12% of PCs recorded in the vermis (5 cells out of 39 cells). These cells were excluded from the statistical analysis.Conversely, at 2 Hz, the synaptic responses did not facilitate during the first stimuli  and deprn = 35) . We name+ accumulation in the extracellular space  . Neverthar space . To testar space . The lacar space  suggesteWhile the onset and the plateau of LFD varied among PCs , the depIn vivo recordings have shown that GCs fire spontaneously in a range of 0.4 to 10 Hz . TherefoN should not restore glutamate release after LFD. LFD was induced by 300 stimuli at 2 Hz, and a train of 50 stimuli at 100 Hz was applied immediately after the last stimulus  instead of the 300 single pulses at 2 Hz (LFD). The total number of stimuli was the same in LFDtriplet and LFD. The triplet frequency was changed from 10 Hz to 200 Hz during the experiment. Since LFD protocols can be repeated several times after a recovery time  and during recovery by 100 Hz train belong to the same pool (the reluctant pool), then the number of quanta released during LFD and recovery train or during LFDtriplet and recovery train should be the same. We next applied LFD and LFDtriplet (triplet at 100 Hz) successively in the same cells followed by the application of a 100 Hz recovery train (triplet +recovery train) was estimated  a frequency-dependent ultrafast replenishment of the RRP and/or by (2) the recruitment of a reluctant pool of SVs that cannot be mobilized at low frequency as suggested in ery time , we systmulation . For exary train . Cumulatstimated . The norPhysiologically, the duration of spontaneous activity of GCs preceding high-frequency bursts is variable. Therefore, the recruitment of the reluctant pool may occur regardless of the status of the fully-releasable pool. This latter pool may be fully available, partially silenced or fully silenced. Since strong paired-pulse facilitation indicates recruitment of the reluctant pool . These s2+-independent replenishment, a sequential two-pool model with Ca2+-dependent recruitment  and the second one to a Ca2+-independent filling step (R1→V). In this case, R0+R1 and V correspond to the reluctant and the fully-releasable pool, respectively, as observed in our experiments. In the simulations, release was triggered by Gaussian-shaped Ca2+ signals with amplitudes of ~22 µM , indicating a single-step process. Recovery following LFDtriplet also followed a single exponential time course  and LFDtriplet  (triplet (~90 s) may reflect the time required to reconstitute the reluctant pool. To test this possibility, the following paradigm was designed: SVs from both the reluctant and fully-releasable pools were depleted by LFDtriplet (triplets at 100 Hz), then a 100 Hz test train was applied after a variable resting period  . The delr.p First, we studied the properties of LFD and recovery by 100 Hz train upon an increase in rp. Based on our previous studies, we estimated that an increase of [Ca2+]e from 2.5 mM to 4 mM can increase rp from 0.25 to 0.67 for SVs in the fully-releasable pool .2, 5% CO2) and containing (in mM) 120 NaCl, 3 KCl, 26 NaHCO3, 1.25 NaH2PO4, 2.5 CaCl2, 2 MgCl2, 10 glucose and 0.05 minocyclin. Slices were then prepared  in an ice-cold solution containing (in mM) 93 N-Methyl-D-Glucamine, 2.5 KCl, 0.5 CaCl2, 10 MgSO4, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 3 Na-Pyruvate, 2 Thiourea, 5 Na-ascorbate, 25 D-Glucose and 1 Kynurenic acid  at 34°C until further use.Acute horizontal cerebellar slices were prepared from male C57Bl/6 mice aged 18–25 days. Mice were anesthetized by isoflurane inhalation and decapitated. The cerebellum was dissected out in ice-cold ACSF bubbled with carbogen -methyl]amino]propyl(diethoxymethyl)phosphinic acid), 100 µM D-AP5 (D-(-)−2-Amino-5-phosphonopentanoic acid) and 1 µM AM251 −5-(4-iodophenyl)−4-methyl-N-(piperidin-1-yl)−1H-pyrazole-3-carboxamide) and 2 µM JNJ16259685 -(cis-4-methoxycyclohexyl)-methanone). Recordings were made at 34°C in PCs located in the vermis. PCs were visualized using infrared contrast optics on an Olympus BX51WI upright microscope. Whole-cell patch-clamp recordings were obtained using a Multiclamp 700A amplifier . Pipette (2.5–3 MΩresistance) capacitance was cancelled and series resistance (Rs) between 5 and 8 mΩ was compensated at 80%. Rs was monitored regularly during the experiment and the recording was stopped when Rs changed significantly (>20%). The membrane potential was held at −60 mV. The intracellular solution for voltage-clamp recording contained (in mM): 140 CsCH3SO3, 10 Phosphocreatine, 10 HEPES, 5 QX314-Cl, 10 BAPTA, 4 Na-ATP and 0.3 Na-GTP. Parallel fibers were stimulated extracellularly using a monopolar glass electrode filled with ACSF, positioned at least 100 µm away from the PC to ensure a clear separation between the stimulus artifact and EPSCs. Pulse train and low-frequency stimulation were generated using an Isostim A320 isolated constant current stimulator  controlled by WinWCP freeware . The synaptic currents evoked in PCs were low-pass filtered at 2 KHz and sampled at 20 to 50 KHz .After at least 1 hr of recovery at 34°C, a slice was transferred to a recording chamber. In order to block inhibitory transmission, postsynaptic plasticity and retrograde signaling, GABA2+-dependent SV fusion and replenishment  were similar to those given by 2+-dependent priming and unpriming were kprim = 8*108 M−1s−1 and kunprim = 120 s−1, respectively. Ca2+-independent filling and unfilling rates were kfill = 200 s−1 and kunfil = 150 s−1, respectively. Ninety percent of released SVs were recycled into the unprimed pool, supported by a slow Ca2+-independent filling rate of kbasal = 0.002 s−1, reflecting filling from a reserve pool. Recovery from low-frequency depression  was simulated by restarting the model at high frequency with the size of the releasable pool set to the value at the end of the 2 Hz train, and the unprimed pool (R0) fully recovered. The one-pool model  and half-width (5 µs) of the estimated action potential-mediated Ca2+ signal at the release sensor of PF synapses  or the signed rank test were used in all other cases. Statistical tests were performed using SigmaPlot 11 (Systat Software). The levels of significance are indicated as ns (not significant) when p>0.05, * when p≤0.05, ** when p≤0.01 and *** when p≤0.001.Data were acquired using WinWCP 4.2.x freeware . Analyses were performed using PClamp9 , Igor (6.22A) graphing and analysis environment . Error bars in figures show SEMs. Student’s  In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Eve Marder as the Senior Editor.Thank you for submitting your article "A frequency-dependent mobilization of heterogeneous pools of synaptic vesicles shapes presynaptic plasticity" for consideration by The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.Summary:Doussau et al. examined the organization of the synaptic vesicle pool mediating glutamate release and plasticity at the granule cell to Purkinje cell synapse. They found that a long-lasting 2 Hz stimulation depletes a fully-releasable SV pool and that subsequent high frequency stimulation instantaneously recruited a second, reluctant SV pool. This reluctant pool could not be recruited during the 2 Hz single action potential stimulation, yet high frequency triplets applied at 2 Hz recruited both pools. Thus, the authors describe a mechanism that can shift from full depression to maximal strength within a few stimuli at high frequency. This constitutes an efficient mechanism to enhance the signal to noise ratio of distinct sensory inputs.Although easily releasable and reluctantly releasable SV pools are conceptually not new, the present study beautifully presents a novel and exciting presynaptic mechanism of information processing that could be of general relevance. The activity-dependent recruitment of various pools could act as second layer that can potentially code relevant information about the previous firing history of the cell. This synapse shows remarkable dichotomy in synaptic plasticity, their plastic properties change in an activity-dependent manner. The question of how this happens is intriguing. The authors' suggestion that this phenomenon can be explained by a two-pool vesicle model is solidly built on previous publication, it is logical and carefully assessed in the manuscript. The first part of the manuscript provides detailed experimental data on the parameters of the bidirectional presynaptic plasticity. These experiments are well thought out and carefully executed. The idea of using triplets in While all the reviewers congratulate the authors on high-quality data and on combination of experiments and modelling, there are some points that should be addressed before publication of the manuscript:Essential revisions:At what frequency range can LFD be triggered in this synapse? This might be important because Jörntell and Ekerot show a range of 5-10 Hz for spontaneous activity.Does the recruitment of the reluctant pool also occur after 25-30 stimuli at 2 Hz, when depression has started to set in but is not yet fully expressed? This might be important when considering the results published by Jörntell and Ekerot. The increase in signal to noise may not be as strong in this case as compared to full depression after 300 stimuli.How can the authors be sure that they indeed record from a constant and stable set of terminals throughout their experiments? Given a q of 8 pA at a single varicosity Schmidt et al. (2013), the large EPSCs recorded by the authors suggest that in the order of 100 varicosities are typically stimulated, if one PF forms several varicosities per PC and considering multiple AZs , the number of stimulated PFs could be as low as 20-30. With these assumptions, it is actually a bit surprising that GC stimulation yielded almost the same EPSC amplitudes than PF stimulation. Also, the conclusions with regard to vesicle pools are more complicated because theoretically one group of PFs could have only "fully-releasable" vesicles and another group could have only "reluctant" vesicles. In this case, the overall conclusions would be different, at least when considering a single GC-PC connection rather than an ensemble of such connections. The authors should cover these issues in the Discussion (and rather shorten the discussion of molecular mechanisms).2+ entry”: "This indicates that.part of the reluctant pool was actually recruited during LFD upon increase in pr." Consumption of the reluctant pool during LFD is one interpretation. However, the higher Ca2+ could also have a direct effect on recruiting the reluctant pool during the 100 Hz train, assuming that it does not get partially consumed during LFD. If the reluctant pool gets consumed, the total number of quanta released during LFD should be increased when comparing high and low Ca2+ conditions. Did the authors observe that?Subsection “Reluctant SVS can be recruited by increasing Ca2+ entry”: This paragraph should end with a conclusion. My take is that buffering Ca2+ decreases the recruitment of the reluctant pool. However, increasing external Ca2+ also decreased the recruitment of the reluctant pool (see 8A/B). How can this discrepancy be explained?Subsection “Reluctant SVS can be recruited by increasing Ca2+ binding kinetics it may not be able to respond within 10-30 ms . Please clarify this in the Discussion.The authors claim that Synaptotagmin-7 a good candidate for mediating reluctant release. However, due to its slow Ca2+ sensitivity […]" The evidence for this seems to be somewhat indirect. This could be explicitly discussed or addressed experimentally.Discussion, subsection “Mechanism of recruitment of reluctant SVs”: "Fully-releasable and reluctant SV differ by their CaThe discussion of molecular mechanisms could be more focused. Any of the proposed mechanisms would have to be differentially implemented for the two pools within the same active zone. Essential revisions:At what frequency range can LFD be triggered in this synapse? This might be important because Jörntell and Ekerot show a range of 5-10 Hz for spontaneous activity.We agree that the frequency range that can trigger LFD is an important issue for cerebellar physiology. Since we hypothesize that LFD may increase the signal to noise ratio, it is important to test whether physiological values of GC spontaneous firing rate (i.e. in absence of sensorimotor inputs) may trigger LFD. in vivo GC recordings was first performed in crus I and IIa by Chadderton et al.  who found a mean value of 0.4 Hz ± 0.2 Hz. Jörntell and Ekerot  found different values and notice that “generally, strong peripheral activation was associated with low spontaneous activity [of GCs] and vice versa”. As mentioned in the Discussion, frequencies chosen in our work for triggering LFD and for the recovery are in the range of the firing frequencies found by Jörntell and Ekerot recorded in GCs responding strongly or moderately to joint movement inputs. However, as mentioned by the reviewers, these GCs fire spontaneously at frequencies higher than 2 Hz .Accordingly, we performed a new set of experiments in which sustained low-frequencies stimulation at 0.5 Hz, 2 Hz and 5 Hz were successively applied on the same beam of parallel fibers (PFs) and on the same Purkinje cells (PCs). As now shown in the Does the recruitment of the reluctant pool also occur after 25-30 stimuli at 2 Hz, when depression has started to set in but is not yet fully expressed? This might be important when considering the results published by Jörntell and Ekerot. The increase in signal to noise may not be as strong in this case as compared to full depression after 300 stimuli.The recruitment of the reluctant pool in function of the status of the synapse  is, indeed, an important issue to understand how information are processed at GC-PC synapses. We therefore designed a specific experiment to address this point.N (number of release sites) underlies these high PPR values . We then considered high PPR values as a signature of the recruitment of the reluctant pool . To test whether the recruitment of the reluctant pool can occur at any time during LFD, we elicited random paired-pulse stimulation at high frequencies during the course of the LFD protocol at 2Hz or 5Hz . As shown in Figure 4—figure supplement 2, extra stimulation leading to paired-pulse high frequency stimulations were associated with unexpectedly high PPR values . These high PPR values could be elicited at any time point during the course of LFD protocols and were not correlated to the percentage of inhibition. These new results indicate that the recruitment of the reluctant pool can occur whatever was the state of the fully-releasable pool (available or silenced).The strong paired-pulse ratio facilitation during paired-pulse stimulation elicited at high frequencies is one of the most striking particularities of the behavior of the release machinery in GC boutons. As demonstrated in previous publication, an ultrafast increase in How can the authors be sure that they indeed record from a constant and stable set of terminals throughout their experiments? Given a q of 8 pA at a single varicosity Schmidt et al. (2013), the large EPSCs recorded by the authors suggest that in the order of 100 varicosities are typically stimulated, if one PF forms several varicosities per PC and considering multiple AZs , the number of stimulated PFs could be as low as 20-30. With these assumptions, it is actually a bit surprising that GC stimulation yielded almost the same EPSC amplitudes than PF stimulation. Also, the conclusions with regard to vesicle pools are more complicated because theoretically one group of PFs could have only "fully-releasable" vesicles and another group could have only "reluctant" vesicles. In this case, the overall conclusions would be different, at least when considering a single GC-PC connection rather than an ensemble of such connections. The authors should cover these issues in the Discussion (and rather shorten the discussion of molecular mechanisms).We agree that our calculation can only give a rough estimation of the number of quanta released during a given paradigm of stimulation notably because ~10% of PFs establish more than one contact on a single PC . Hence, our calculation overestimate the number of PF recruited by external stimulations and therefore underestimate the number of quanta released by varicosity during a given paradigm of stimulation. The percentage of PF establishing more than one active zone per Purkinje is based on observation made on only 39 PFs  and it is not known whether this percentage vary in function of the position of PFs in the molecular layer. Also, 80% of synaptic contact established by PFs on PC are silent  and again, it is not known whether 2 active zones made by a single PF on a same PC are both functional. Anyhow, we added a caveat to mention putative error that can be made in the estimation of the number of quanta released.GCL stimulation may lead to large EPSC since numerous GC somata are stimulated by extracellular stimulations. This point was not clear in the previous version of the manuscript and we modified The segregation of GC terminal in groups having only the fully-releasable pool one or only the reluctant one is an interesting hypothesis. This hypothesis is mentioned in this new version of the Discussion (see first paragraph of the Discussion). Nevertheless, we have previously showed that the properties of the PPR (paired-pulse ratio) are identical between pair recordings (that is at unitary GC-PC synapses) or compound stimulations . Since the ultrafast recruitment of the reluctant pool underlies part of the large paired-pulse facilitation observed at GC synapses , we believe that most of the GC terminals are equipped with both pools.The text mentioning GC stimulation and the corresponding schematic  were proWe cannot exclude errors due to changes in GC or PF excitability. The fact that values of PPR are identical between unitary GC-PC synapse  and at PF-PC synapses , suggest that GC and PF excitability are not affected by paired-pulse facilitation. However, one can argue that changes in GC or PF excitability may underlie part of the depression during LFD. Because of differences in membrane properties between the soma and axon , changes in membrane excitability following repetitive activity should be strongly different in both compartments. The fact that the kinetics of LFD after stimulating GC somata or PFs were not significantly different suggests that, change in excitability, if any, weakly contributes to LFD.triplet. It appears that after LFDtriplet, the recovery of glutamate release by high-frequency trains can be completely abolished  that leads to a higher recruitment of the fully releasable pool and ii) the ultra-fast recruitment or the reluctant pool. At the end of the LFD protocol, the fully releasable pool has been silenced, then it cannot participate to the first responses of 100 Hz train. In other words, differences in the first responses of 100 Hz train at rest or after LFD are due to differences in the availability of the fully-releasable pool after LFD.At rest, the first responses at 100 Hz (i.e. the second response of the train) rely both on the fully releasable pool and on the reluctant pool. The high value of facilitation observed at 100 Hz is the consequence of i) the well-described increase in 2+ entry”: "This indicates that.part of the reluctant pool was actually recruited during LFD upon increase in pr." Consumption of the reluctant pool during LFD is one interpretation. However, the higher Ca2+ could also have a direct effect on recruiting the reluctant pool during the 100 Hz train, assuming that it does not get partially consumed during LFD. If the reluctant pool gets consumed, the total number of quanta released during LFD should be increased when comparing high and low Ca2+ conditions. Did the authors observe that? Subsection “Reluctant SVS can be recruited by increasing Ca2+ entry”: This paragraph should end with a conclusion. My take is that buffering Ca2+ decreases the recruitment of the reluctant pool. However, increasing external Ca2+ also decreased the recruitment of the reluctant pool (see 8A/B). How can this discrepancy be explained?Subsection “Reluctant SVS can be recruited by increasing Ca2+]e from 2 mM to 4 mM, we classically observed a strong increase in the basal level of neurotransmitter release (~2 fold increase in EPSC peak amplitudes). Strikingly, this increase in rp did not affect the properties of LFD when EPSCs were scaled to initial amplitude . Hence, the number of quanta released during LFD upon high [Ca2+]e condition was indeed strongly increased; the previous version of 2+]e probably reflects a recruitment and a consumption of a substantial fraction of the reluctant pool. Simulations suggest that the fully-releasable SVs can transit back to the reluctant pool during LFD. Potentially, these “newly” reluctant SVs would be recruited by a 100 Hz train but experiments using LFDtriplet or high [Ca2+]e clearly showed that it was not the case. As mentioned in the Discussion, LFD also involves an “a posteriori” mechanism that might impede the high-frequency dependent recruitment of SVs used during LFD.In a given presynaptic cterminal, the total number of releasable SVs is supposed to be constant. By increasing extracellular " The evidence for this seems to be somewhat indirect. This could be explicitly discussed or addressed experimentally.Discussion, subsection “Mechanism of recruitment of reluctant SVs”: "Fully-releasable and reluctant SV differ by their Ca2+-dependent. We agree that this does not necessarily mean that the two pools differ by their Ca2+ sensitivity. Since this hypothesis cannot be drawn from our experiments, we removed it.Our model and experiments show that the recruitment of the reluctant pool is Ca


Podgorsak, PhD. in the JACMP Vol 5, Issue 3, 2004. A slightly revised version of this 695 page book is available without cost to anyone with web access. This new IAEA version is available on the web site: It is with great pleasure I welcome as a Guest Editorial the following biography of Moses Greenfield by Dr. Amos Norman. This Fall 2005 issue of the JACMP is dedicated to Dr. Greenfield, and contains contributions from several of his former students.Michael D. Mills, PhDMoses Greenfield was born in New York City on 8 March 1915. He earned a B.S. degree in physics from City College of New York where he also was awarded the Belden Medal in mathematics. In 1937 he received an M.S. degree in physics from New York University. and completed there all the requirements for the Ph.D. except the dissertation, He accepted an appointment as a Research Physicist during World War II at the Bureau of Ships, United States Navy, in Washington D.C. where, with others, he showed that a faulty design was responsible for the tendency of Liberty Ships to suddenly split in two. While working there he also was able, with the guidance of George Gamow, to write his Ph.D. dissertation on “The Problem of Energy Production in Red Giants” and was awarded the degree in 1941.He left the Navy in 1946 for California and a position as a consultant for North American Aviation in Stellar Space Research. Then in 1948 he accepted an appointment as Associate Professor in the Department of Radiology in the new medical school under construction at the University of California, Los Angeles. He spent his entire career at UCLA since then, moving up in due course to Professor and Professor Emeritus. Early in his career of teaching, research and public service, in 1956, he founded with Amos Norman a graduate program in Medical Physics and served for many years as its director. This program, was later renamed the Interdepartmental Graduate Program in Biomedical Physics to reflect its large expansion to include the study and application of the many new devices – CT and Ultrasound Scanners, PET, SPECT and MRI – and procedures – IMRT, for example – made possible by the rapid evolution of computers.This program, which to date has granted some 266 M.S. and Ph.D. degrees, is Professor Greenfield's major contribution to the Medical Physics Profession. Undoubtedly, this contribution, together with his many published papers in the field and, notably, the work with his graduate students and post docs on the measurement of bone density, influenced the American Association of Physicists in Medicine to give him its highest praise, the William D. Coolidge Award. There is every reason to expect that the next several decades will witness as many innovations in the practice of medical physics as in the past half century during which the slide rule was replaced by the computer. A reasonable guess is that it will witness more machine analysis of images and the incorporation of biological factors unique to each patient into radiation therapy treatment planning. The Biomedical Physics Graduate Program at UCLA is likely to flourish, and remain an enduring tribute to Professor Greenfield's leadership.At 90 Moe Greenfield continues to attend and actively participate in meetings of the Board of the Emeriti Association and of the Faculty of the Biomedical Physics Program. He can be found frequently dining with his wife Bella, a mathematician, and friends at the UCLA Faculty Center where the conversation often turns to discussion of his children, stepchildren and 10 grandchildren (so far). I look forward to celebrating his one hundredth birthday in 2015 with his family, friends and colleagues.Amos Norman