E.coli cells into small protoplast-like components, which may, at least in part, explain the effectiveness of this approach. This work demonstrates that one-step NID minipreps are a robust method to generate high quality plasmid DNA, and NID approaches can also isolate bacteriophage lambda particles, outperforming current standard alkali-based minipreps.Plasmid DNA minipreps are fundamental techniques in molecular biology. Current plasmid DNA minipreps use alkali and the anionic detergent SDS in a three-solution format. In addition, alkali minipreps usually require additional column-based purification steps and cannot isolate other extra-chromosomal elements, such as bacteriophages. Non-ionic detergents (NIDs) have been used occasionally as components of multiple-solution plasmid DNA minipreps, but a one-step approach has not been developed. Here, we have established a one-tube, one-solution NID plasmid DNA miniprep, and we show that this approach also isolates bacteriophage lambda particles. NID minipreps are more time-efficient than alkali minipreps, and NID plasmid DNA performs better than alkali DNA in many downstream applications. In fact, NID crude lysate DNA is sufficiently pure to be used in digestion and sequencing reactions. Microscopic analysis showed that the NID procedure fragments  Escherichia coli (E. coli) cell walls. These procedures can be classified according to the disruptive factor used. Alkali procedures use NaOH with SDS E. coli cells using lysozyme and the NID Brij-58 Plasmid DNA minipreps are essential for molecular biology research and rely on disrupting NID-based plasmid DNA isolation procedures have been used in a multiple-solution format, but they have not gained significant popularity E.coli cells into small protoplast-like components.Here, we describe a robust and time-efficient procedure using NIDs that provides a one-tube, one-solution approach to isolate plasmid DNA. Interestingly, a slightly modified NID procedure also isolates bacteriophage lambda particles. Unlike the complete cell disintegration occurring in alkali minipreps, NID plasmid minipreps lead to fragmentation of rd Edition, Nov 2005, pg. 12), was kindly provided by NCI-Frederick, MD (http://web.ncifcrf.gov/research/brb/ productDataSheets/recombineering/plasmid.aspx). E.coli cells UB-61 lysogenic for a heat-inducible bacteriophage lambda (cI857 ind 1 Sam7) was kindly provided by S. Casjens (University of Utah). All enzymes were purchased from New England Biolabs , except PstI (Promega), and SstI and XhoI (Invitrogen). Enzymatic reactions were performed at 37°C for 1 hour if not otherwise specified.NIDs, salts and lysozyme were purchased from Sigma–Aldrich  or Mallinckrodt Baker . RNase A was purchased from 5 PRIME . Antibiotics were purchased from USB , Sigma–Aldrich, and EMD/Calbiochem . Bacterial media were obtained from Quality Biological . DH5α cells were supplied by Invitrogen . The following high copy number plasmids were used: 1) pUC19 (2.7 Kb), purchased from Invitrogen ; 2) pCYPAC3 (18.8 Kb) DNA was quantified using a two wavelength spectrophotometric method on nanodrop spectrophotometer .Alkali isolation was performed according to the original description by Birnboim and Doly 1.5-2 ml of bacterial cultures were pelleted at 6000-7000 rpm for 1 min.After drawing 150 µl extraction buffer into a pipette tip, the pellet was loosened off the tube wall with the tip without releasing the buffer. Then the extraction buffer was added and the pellet resuspended.The bacterial suspension was incubated at 65°C for 5 min.Suspensions were centrifuged at maximum rpm for 10 min or until a tight bacterial pellet was formed. The pellet was removed with a toothpick.100-120 µl isopropanol was added, followed by mixing and centrifugation of the solution at 7000 rpm for 10 min at RT.DNA usually forms film-like precipitates that adhere well to tube walls and are invisible in isopropanol solutions. After discarding the supernatant, the DNA was centrifuged after adding 70% ethanol. Ethanol was removed, and the DNA pellet was dissolved in 20-50 µl TE buffer.4Cl, 0.5% IGEPAL CA-630 (or Triton X-100), lysozyme 100 µg/ml, and RNase A 25 µg/ml. Addition of 20–50 mM CaCl2 to the extraction buffer reduces extraction of chromosomal DNA and large plasmids, but greatly facilitates formation of cellular debris during sedimentation. A 100x enzyme stock containing 10 mg/ml lysozyme and 2.5 mg/ml of RNase A prepared in 50% glycerol and 50 mM Tris pH 8 was stored at −20°C and used repeatedly.The composition of the extraction buffer was: 5% sucrose, 20–50 mM EDTA, 50 mM Tris pH 8, 0.75 M NHImage densitometry/gel quantification analysis was performed using Image J  AAA CGA CGG CCA GT) for sequencing B256 plasmid, and r pLTM330_3617 (GCT GGT TCT TTC CGC CTC A). The sequence fragments were detected on an ABI 3130XL Genetic Analyzer. Samples were then analyzed and base-called by Applied Biosystems DNA Sequencing Analysis Software V5.2 .DNA sequencing was performed using the ABI BigDye Terminator Cycle Sequencing Kit v1.1 (NID DNA) or v3.1 (NID crude lysates) according to the manufacturer's instructions on a Gene Amp 9700 PCR machine. The primers used were M13f (GTAtot) were considered as the sum of the operational time  and the preparation/working equipment times . Ttot =  Top + Tid. Top is a product of k  by n (number of samples). Top =  kn. Thus, Ttot =  kn + Tid where Tid is a constant (c). Ttot/Top =  1 + c/kn, showing that as the number of samples increases Ttot/Top approaches 1. In other words, as n increases, Ttot and Top can be used interchangeably.To estimate time-efficiency of the NID relative to the alkali procedure, the total NID and alkali procedure completion times  using the data presented in E.coli UB-61 cells were grown at 30°C until OD600 =  1. The culture was divided into 12–15 ml aliquots and heat-shocked for 10 min at 42–43°C, then re-combined and incubated at 37°C for 7–8 hours. One or two 5 ml aliquots were lysed with chloroform (full lysis sample). The other 5 ml aliquots were spun down and the cells were resuspended in the following lambda NID extraction buffer: 5% sucrose, 50 mM Tris pH 8, 2 M NH4Cl, 50 mM CaCl2, lysozyme 120 µg/ml and 0.5% Tween 80. No EDTA was included in this buffer. Bacteria were incubated at 40°C for 45 min with occasional mixing to prevent bacterial sedimentation, and centrifuged at 12–15,000 rpm for 5–10 min. Easy cell sedimentation and absence of viscous pellets suggested no lysis of bacteria. The supernatant was precipitated with 1∶1 volume 1.25 M NH4Cl and 20% PEG 8000 after 1 hour incubation in ice.Lysogenic The full lysis samples were precipitated with the following solution: 1 M NaCl and 10% PEG 8000 added as powder.2) containing >5 Kunitz units/ml DNaseI and incubated at 37°C for 1 hour. Subsequently, 15 mM EDTA was added. DNA was extracted with phenol/chloroform and before isopropanol precipitation the aqueous phase was mixed with 20 µg linear polyacrylamide carrier to ensure quantitative DNA recovery The phage containing pellets were processed according to Sambrook et al. with minor modifications 4 from the original lytic mixture and increased the last incubation time from 2 to 10 min. to increase the amount of plasmid DNA extracted. The mixture was then centrifuged at 5000 RPM (3000 ×g) for 5 min, and the cleared lysate was precipitated with 150 µl isopropanol. DNA pellets were resuspended in 35 µl TE buffer containing 30 µg/µl RNase A. The insoluble material was spun down and the cleared DNA solution was loaded on a gel.Bacterial cells from 2 ml cultures were resuspended in 60 µl Godson's sucrose solution (25% w/v sucrose in 10 mM Tris pH 8.1). Cells were kept on ice throughout the procedure. We mixed in advance 7.5 µl 16 mM EDTA and 7.5 µl 0.85 mg/ml lysozyme in 250 mM Tris 8.1. This solution was mixed with the bacterial suspension and incubated on ice for 1 min. The bacterial suspension was then transferred to a lytic mixture containing 15 µl 5% Brij-58 in 10 mM Tris pH 7.4 and 60 µl deionized water and incubated on ice for 10 min. We excluded 10 mM MgSOBacterial cells from 2 ml cultures were resuspended in 47 µl Clewell's sucrose solution . Cells were kept on ice throughout the procedure. Subsequently, 10 µl 5 mg/ml lysozyme in 250 mM Tris pH 8 was added, and the solution was kept on ice for 5 min. 19 µl 0.25 M EDTA was added and incubation continued for another 5 min. 76 µl detergent mixture  was then added to the 76 µl bacterial suspension and kept on ice for 5–10 min. The mixture was then centrifuged at 48,000g at 2°C for 25 min. The cleared lysate was precipitated with 150 µl isopropanol, and the DNA pellets were resuspended in 35 µl TE buffer containing 30 µg/µl RNase A. The insoluble material was spun down and the cleared DNA solution was loaded on a gel.Bacterial cells from 2 ml cultures were resuspended in 60 µl DEPC/Summerton's sucrose solution . The cells were incubated on ice for 5 min. Subsequently, 15 µl lysozyme (6 mg/ml in water) was added and incubation continued for 20 min in ice. An equal volume (75 µl) ice cold salt-detergent solution  was added, gently mixed and incubated at 25°C for 20 min without mixing. The mixture was then centrifuged at 40,000 g for 30 min at 0°C. The cleared lysate was precipitated with 150 µl isopropanol, and the DNA pellets were resuspended in 35 µl TE buffer containing 30 µg/µl RNase A. The insoluble material was spun down and the cleared DNA solution was loaded on a gel.20–40 µl bacterial cell lysates from the various isolation methods were placed on slides and mounted with cover slips. Images were recorded using either an Olympus IX81/Metamorph v6.2r6 microscope (100x objective in oil) or a Leica DMR A/Metamorph v6.3r1 (63x objective in oil).E.coli were exposed to NIDs with HLBs <15  for 2 hours at 4°C (4Cl and NaAc) to the NID solutions decreased the required NID extraction time down to 5 minutes for all NIDs studied (4Ac and NH4HCO3) were effective but differed in their propensity to extract chromosomal DNA and precipitate DNA in aqueous isopropanol solutions (data not shown). These data demonstrate that osmolytes and elevated temperatures enhance the activities of a variety of NIDs, allowing rapid and efficient extraction of plasmid DNA in a single solution format.To evaluate how different NIDs perform in plasmid DNA extraction, we used various NIDs mixed with lysozyme-treated DH5α cells harboring pUC19. Plasmid DNA was extracted effectively when s at 4°C , lane 8.s at 4°C , lane 9.We next compared performance of the pLTM330 plasmid DNA obtained by either NID or alkali miniprep in digestion, ligation and sequencing reactions. The NID and alkali minipreps produced similar amounts and molecular forms of DNA, except for some “irreversibly denatured”  DNA forms in the alkali method , and dissolved the DNA in 20 µl TE buffer (vs. 40 µl in high copy number plasmids). As the amount of DNA was enough for only two digestions, we combined DNA from three independent samples. We used the salt-sensitive restriction endonuclease KpnI, with expected digestion products of 1.5 and 3.5 Kb. We found that 3u KpnI are sufficient to achieve almost complete digestion . These dCrude lysates of alkali minipreps cannot be used for downstream applications because of their low DNA concentration, SDS content and acidic pH. NID minipreps are free of these shortcomings, leading us to examine whether NID crude lysates could be directly used in downstream applications, such as digestion and sequencing reactions. We assessed how NID plasmid DNA from crude lysates  performed in digestion reactions compared to isopropanol precipitated-NID plasmid DNA . All tested restriction endonucleases except XhoI digested crude lysate DNA well . DNA molAlkali minipreps require three solutions for completion of plasmid DNA isolation, but NID minipreps require only one. To confirm that NID minipreps are more time-efficient than alkali minipreps, we compared completion time up to thirty samples of alkali and NID minipreps. The time required to complete one miniprep was similar, although the NID miniprep required relatively less operator-dependent time . As the alk =  aTnid+b, where a and b are constants). Thus, the ratio of the variables Talk/Tnid =  a+b/Tnid =  a+b/nk  and 1.20±0.05 min. (r2 = 0.991) for the alkali and NID minipreps, respectively. Thus, at high sample numbers , NID minipreps are 69% more time-efficient compared to alkali minipreps (2.03/1.20 – 1×100). Overall, these data demonstrate that NID minipreps save significant time compared to alkali minipreps, and suggest that NID minipreps are particularly suitable for high-throughput applications.Claiming a time advantage of a procedure over previous ones is often difficult because it is hard to evaluate the data and extrapolate the conclusions to any sample number. To address these issues, we developed a linear regression mathematical model. When the same operator completes both the alkali and NID procedure, the total procedures times are dependent variables as shown in b/nk see . When th2 in the extraction buffer decreased phage leakage  in vivo host cell lysis is required. Products of two lambda phage genes S, and R are necessary for cell lysis. Holin, the S gene product, causes lesions (holes) in the cytoplasmic membrane at precise times of the vegetative cycle just before host lysis. The R gene product, endolysin, is a transglycosylase with murein-degrading activity. It has no secretory signal sequence and thus needs the function of S to reach the murein sacculus. Sam7, an amber mutation (Trp56UAG) of the S gene causes accumulation of intracellular virions and endolysin to very high levels. Adding chloroform, which functionally compensates holin, to an induced S-R+ lysogen (such as UB-61 cells) results in almost instantaneous lysis +R- lysogens are resistant to chloroform treatment, suggesting that some R gene product is required for chloroform-induced lysis of S-R+ lysogens The phage lambda is a temperate double-stranded DNA bacteriophage harboring about 50 Kb DNA with an isometric head about 50 nm in diameter, and a flexible tail about 150 nm long. The phage linear size is approximately 10 times smaller than Current phage DNA extraction procedures usually require a purification step because DNA isolation from complete cell lysates leads to heavy contamination of phage particles with cell debris. Phenol-extracted DNA from full lysis samples not subjected to additional purification of either phage particles or phage DNA often degrades or aggregates with impurities upon storage, and it is almost completely resistant to HindIII digestion . However, lambda DNA isolated with phenol from NID extracted phage particles shows no degradation or aggregation after 2 month storage at 4°C . Thus, NID procedures do not require any specific structure, size or gene expression of extrachrosomal elements, and can simplify phage DNA purification because they do not produce complete cell lysis and NID crude lysates are relatively pure .E.coli cell membrane proteins compared to Tween 20 (HLB 16.7) We used 0.75 M salt in the extraction buffer of the NID miniprep procedure because numerous insoluble particles appeared in the DNA solution when we used 0.25 M salt  generate DNA of sufficiently high quality for common downstream applications. The timed saved by the NID miniprep increases rapidly at higher sample numbers, suggesting that NID minipreps are particularly suitable for high-throughput applications. Overall, the NID miniprep outperforms current alkali plasmid DNA isolation methods, and represents a new standard in plasmid DNA minipreps.Figure S1NID crude lysates can be used in sequencing reactions. NID crude lysates of XL1-Blue cells harboring B256 plasmid were prepared as described in Materials and Methods but using 5 mM EDTA in the extraction buffer. Lysate EDTA was chelated with 5 mM MgSO4 before the sequencing reaction. 12 µl primer/crude lysate mix containing 4 pmoles M13f primer was diluted by adding 15 µl water. 6 µl of this diluted mix and 4 µl of Big Dye mix made up the sequencing reaction mixes. (A): 1 µl crude lysate. (B): 3 µl crude lysates. (C): 4 µl crude lysates.(TIF)Click here for additional data file.


CRABP2), a member of a family of specific carrier proteins for Vitamin A, belongs to a family of small cytosolic lipid binding proteins. Our previous study suggested that CRABP2 was involved in skeletal muscle development; however, the molecular function and regulatory mechanism of CRABP2 in myogenesis remained unclear. In this study, we found that the expression of the CRABP2 gene was upregulated during C2C12 differentiation. An over-expression assay revealed that CRABP2 promotes myogenic transformation by regulating the cell cycle during C2C12 differentiation. The region from −459 to −4 bp was identified as the core promoter and contains a TATA box, a GC box and binding sites for the transcription factors MyoD and Sp1. Over-expression, site-directed mutagenesis and EMSA assays indicated that the transcription factors MyoD and Sp1 regulate CRABP2 expression and promote myoblast differentiation in C2C12 cells.Cellular retinoic acid binding protein 2 ( Myogenesis can be regarded as a two-step process consisting of the determination, in which the satellite cells commit to the mature muscle lineage, and subsequent differentiation of mononuclear myoblasts to multinuclear myotubes Sp1 belongs to a family of zinc-finger transcription factors involved in the early development of an organism CRABP2 is a low molecular mass (15 kDa) protein that belongs to the multi-gene family of cellular retinoic acid binding proteins CRABP2 was expressed in many organs http://symatlas.gnf.org/SymAtlas/), which demonstrated that CRABP2 mRNA was upregulated from day 6.5 to day 10.5. These data indicate that the CRABP2 gene may play a vital role during embryonic development.Vitamin A and its related molecules  specifically bind many distinct cytoplasmic proteins and play essential roles in vision, growth, reproduction and cell differentiation CRABP2 gene was unknown. Our recent LongSAGE analysis indicated that the CRABP2 gene contributes to prenatal skeletal muscle development in pigs CRABP2 gene plays an important role in myogenesis. In this study, we analysed the biological function and regulatory mechanism of the CRABP2 gene in C2C12 cells.The precise biological function and regulatory mechanism of the CRABP1, CRABP2, CRBP1, CRBP2) expressing carrier proteins for Vitamin A and related molecules by RT-PCR during C2C12 differentiation. The results shown reveal expression levels of CRABP1, CRABP2, CRBP1, CRBP2 observed. We found that the CRABP1 and CRBP2 genes were not detected and the mRNA expression of the CRBP1 gene was unchanged, but the CRABP2 gene was markedly upregulated  was performed. The results suggested that CRABP2 was upregulated from day 0 to day 4 during myogenic differentiation in C2C12 cells   . It has CRABP2 promoter, we inserted a 2.4 kb fragment into the promoter-less vector pGL3-basic. The resulting plasmid (pGL3-A) was transfected into C2C12 cells, and the luciferase activity measured. Compared with cells transfected with pGL3-basic, the cells transfected with the pGL3-A plasmid had significantly increased luciferase activity, revealing that the 2.4 kb fragment was a functional promoter fragment. To further identify the precise promoter region, the four promoter luciferase reporter plasmids  were constructed by further deletion using pMD18 T-A as a template. As shown by transient transfection results, the pGL3-E plasmid contained the basal promoter activity  were constructed by further deletion of pGL3-E. The results indicated that the pGL3-E plasmid with the −459 bp to −4 bp fragment contained the minimal core promoter region  were carried out to analyse binding capabilities in cell nuclear extracts.To further confirm the function of CRABP2 expression in C2C12 cells at different times during differentiation.Oligonucleotides were synthesized and BIO-labelled and then incubated with nuclear extracts from C2C12 cells. Specific DNA-protein complexes were identified from C2C12 nuclear extracts . The binCRABPs were involved in skeletal muscle development in the pig throughout embryonic development.23 Therefore, the aim of this study was to investigate the function of CRABPs during the process of differentiation of C2C12 cells into myotubes. The RT-PCR and real time PCR analyses suggested that CRABP2 was increasingly expressed during C2C12 cell differentiation. Interestingly, we found that CRABP2 over-expression accelerated the process of C2C12 differentiation into myotubes in vitro. These observations suggest that CRABP2 contributes to the process of C2C12 myoblast differentiation into myotubes.Our previous LongSAGE analysis suggested that CRABP2 gene. The EMSA results indicated that the predicted MyoD and Sp1 binding sites bound to the MyoD and Sp1 proteins, respectively, in nuclear extracts from C2C12 cells. Additionally, excess amounts of unlabelled oligonucleotides inhibited the binding activities in C2C12 cells. This result further proved that the MyoD and Sp1 binding sites were functional regulators for the CRABP2 gene. These results demonstrated that the putative MyoD and Sp1 binding sites in the core promoter of CRABP2 may be responsible for transcriptional regulation in C2C12 cells.We then carried out a promoter analysis through a step-by-step deletion process to identify the core promoter region. Bioinformatics analyses showed that the promoter region contained a TATA box, a GC box, and MyoD and Sp1 transcription factor binding sites. We constructed a MyoD over-expression vector and an Sp1 site-directed mutation vector. Co-transfection was performed to demonstrate the effects of MyoD and Sp1 transcription factors on the CRABP2 contains a CANNTG sequence. The MyoD gene family forms a very complex regulatory network by interacting with a number of positive and negative factors to regulate CRABP2 gene transcription, controlling cell growth and cell differentiation. The Sp1 transcription factor was involved in regulating cell growth and controlling morphogenetic pathways both in mammals and in invertebrates CRABP2 gene. Therefore, MyoD and Sp1 are important regulators for the CRABP2 gene and directly bind to the promoter region to influence CRABP2 expression. CRABP2 is in turn involved in regulating C2C12 cell differentiation.Transcription factors are trans-regulatory factors that exert biological function by interaction with cis-regulatory elements of structural genes. The MyoD family recognizes and binds the consensus sequence CANNTG (N for any nucleotide), and the promoter region of CRABP2 expression was upregulated from day 0 to day 4 at the mRNA level in differentiating C2C12 cells. The over-expression of CRABP2 led to cell cycle changes in C2C12 cells in vitro. We have identified a biological function for CRABP2 in C2C12 differentiation and found that MyoD and Sp1 play a regulatory role in CRABP2 gene transcription by binding in the core promoter region between −459 bp and −4 bp upstream of the CRABP2 gene. These results give new insight into the biological function and regulatory mechanism of the CRABP2 gene.In summary, 2. The C2C12 cells were seeded in six-well plates. After approximately 12–16 h, when cell confluence reached approximately 60%–70%, the differentiation of C2C12 myoblasts into myotubes was induced by the addition of differentiation medium (DMEM containing 2% horse serum instead of 10% FBS).C2C12 cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) supplemented with 10% FBS, 2 mM glutamine, 100 units/ml penicillin and 100 µg/ml streptomycin and were maintained at 37°C in 5% COStarting at the beginning of differentiation, the C2C12 cells were cultured in six-well plates and harvested for RNA extraction at 0, 1, 2, 3 and 4 days. Total RNA samples were extracted using TRIZOL  according to the manufacturer's instructions. The cDNA samples were obtained by reverse transcription from 1 µg RNA using oligo(dT) and M-MLV reverse transcriptase .2+), 100 µmol/L of each dNTP, 0.5 µmol/L of each PCR primer, 1.0 U Taq DNA polymerase  and 0.5 µl of cDNA template. The primers were designed and synthesized as shown in The RT-PCR (Reverse Transcription-Polymerase Chain Reaction) was performed in a volume of 10 µl containing 1 µl of 10× PCR buffer . The PCR reaction contained 50× ROX Reference DyeII, 2× SYBR Green Realtime PCR Master Mix , 10 µM primers and 1.0 µl of cDNA template. Pure water was added for a total volume of 20.0 µl. Gene amplification was performed under the following conditions: 95°C for 30 s, followed by 40 cycles of 95°C for 5 s, 60°C for 20 s and 72°C for 34 s. The PCR was performed in biological triplicate for each condition. The target gene data was analysed and normalized to CRABP2 was constructed to study its function in C2C12 cells. The cDNA fragment was isolated and ligated into the pGC-FU vector linearized by cutting with Age I restriction enzyme, purchased from GeneChem Co., Ltd, Shanghai, China. The expression plasmid containing the fusion protein was extracted using ultrapure endotoxin-free extraction kits . The transfection of 293T cells was carried out using Lipofectamine 2000 according to the manufacturer's instructions. After a 48 h incubation, the cell supernatant containing virus-like particles was collected. After concentration, the viral titres were determined in the 293T cells.The vector overexpressing 2. When cell confluence reached approximately 60–70%, lentivirus of a specific quantity was added to cells. After 12 h, the transfection medium was removed and replaced with normal growth medium.The C2C12 cells were plated on 6-well plates and cultured overnight at 37°C in 5% COAfter 3–4 days, the GFP expression was tested by fluorescence microscopy to determine transfection efficiency. The cells were then harvested and used for the cell cycle assay and RNA extraction. All transfections were performed in triplicate for each plasmid.The C2C12 cells were collected and centrifuged at 1200 rpm for 5 min. The cells were washed twice in 4°C pre-chilled PBS , fixed with 4°C pre-chilled 70% ethanol, centrifuged at 1500 rpm for 5 min to remove stationary phase cells and resuspended in PBS. The cells were filtered once through 400 mesh and centrifuged at 1200 rpm for 5 min, and after the PBS supernatant was removed, the cells were then stained with 1 ml of PI at 4°C in darkness for 30 min. Finally, the cells were analysed by flow cytometry .CRABP2 gene was amplified from mouse genomic DNA and then inserted into the pMD18-T vector. It was named pMD18 T-A.The 5′-upstream 2.4 kb promoter fragment of the 3-A). Fragments of different lengths from the 5′-upstream sequence were amplified using pMD18T-A as a template and then ligated into linearized pGL3-basic vector . The plasmids pGL3-A, pGL3-B, pGL3-C, pGL3-D and pGL3-E were constructed to contain −2292 bp to +115 bp, −1646 bp to +115 bp, −1111 bp to +115 bp, −719 bp to +115 bp and −459 bp to +115 bp of the CRABP2 gene, respectively.The construction of the luciferase reporter gene plasmid was carried out as follows: the pMD18 T-A vector was cut using MluI and NcoI restriction enzymes and then ligated into linearized basic vector , pGL3-b (−178 bp to +115 bp), pGL3-c (−4 bp to +115 bp) and pGL3-d (+44 bp to +115 bp), were constructed using the above methods. Each promoter luciferase plasmid was constructed using a sequence-specific forward primer and a common reverse primer . The core promoter region was identified by the Transcription Element Search System (http://www.cbil.upenn.edu/cgi-bin/tess/tess), the Berkeley Drosophila Genome Project programs (http://www.fruitfly.org/seq_tools/promoter.html) and TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html).The sequence of the 5′-upstream 2.4 kb promoter fragment of the All experiments were performed in triplicate, and the data were presented as mean ± SD. Student's t-test was used to determine statistical significance.The C2C12 cells were plated on 24-well plates and cultured overnight at 37°C to ensure approximately 60%-70% confluence. Co-transfections were performed using 2.0 µl of Lipofectamine 2000 reagent , 0.8 µg of the firefly luciferase plasmid DNA and 0.008 µg of pRL-TK plasmid DNA  as an internal control. The transfection medium was removed and replaced with growth medium after 5 h.The Firefly and Renilla luciferase activities were measured at 24 h after transient transfection using the Dual-Glo Luciferase assay system  and a TD20/20 luminometer (Turner Designs) according to the manufacturer's instructions. Each plasmid was tested in three independent experiments. The luciferase activity was normalized using the Renilla luciferase activity levels and expressed as relative luciferase unites (RLU) to reflect the gene promoter activity.MyoD gene was amplified using mouse muscle mRNA with the primers listed in The cDNA sequence of the CRABP2 gene in C2C12 cells, we constructed a site-directed mutation vector of Sp1. The site-directed mutagenesis of the Sp1 binding site was obtained by a two-step PCR protocol using pMD18 T-A as a template. The original GGC site was mutated to TTA. The mutagenic primers were 5′ AGGTGGTGTGGAAGGCGGTTAGGGGGCGGGGCCGCCTCATGCACCAGCT 3′ (forward primer) and 5′ CCTAACCGCCTTCCACACCACCT 3′ (reverse primer). Thermocycler parameters were set at 95°C for 5 min followed by 27 cycles of 95°C for 30 s, 60°C for 30 s and 72°C for 30 s with a final extension time of 5 min at 72°C. The final PCR product was cloned into pGL3-basic and named pGL3-muE.To identify Sp1 as a regulatory factor for the Nuclear extracts from C2C12 myoblasts and myotubes were prepared according to the instruction manual of the NProtein Extraction kit . The proteins were measured using a BCA Protein Assay Kit . The probes for MyoD and Sp1 were synthesized by Integrated DNA Technologies .The binding reactions were performed in a 10 µl mixture containing 1.0 µl of 10× Binding Buffer, 1.0 µl of Poly (dI∶dC), 2 µg of nuclear extract and 0.5 µl of Bio-labelled probes. For the competition experiments, unlabelled probes were added to the binding reaction mixture and co-incubated. Subsequently, DNA-protein complexes were separated by 6.5% non-denaturing polyacrylamide gel electrophoresis in Tris-Boric acid (TBE) buffer. Electroblotting and chemiluminescence detection were performed according to the instructions of the BiotinLight™ EMSA Kit . Results were observed by chemiluminescence imager.


In vitro functional assays demonstrate that forced overexpression of Six1 significantly enhances the growth rate and proliferation ability of pancreatic cancer cells. Knockdown of endogenous Six1 decreases the proliferation of these cells dramatically. Furthermore, Six1 promotes the growth of pancreatic cancer cells in a xenograft assay. We also show that the gene encoding cyclin D1 is a direct transcriptional target of Six1 in pancreatic cancer cells. Overexpression of Six1 upregulates cyclin D1 mRNA and protein, and significantly enhances the activity of the cyclin D1 promoter in PANC-1 cells. We demonstrate that Six1 promotes cell cycle progression and proliferation by upregulation of cyclin D1. These data suggest that Six1 is overexpressed in pancreatic cancer and may contribute to the increased cell proliferation through upregulation of cyclin D1.Six1 is one of the transcription factors that act as master regulators of development and are frequently dysregulated in cancers. However, the role of Six1 in pancreatic cancer is not clear. Here we show that the relative expression of Six1 mRNA is increased in pancreatic cancer and correlated with advanced tumor stage.  Pancreatic cancer is the fourth leading cause of cancer-related mortality across the globe Six1 is a mammalian homolog of the Drosophila sine oculis gene and is highly conserved from Drosophila to humans in vitro and in vivo functional assays, we show that forced overexpression of Six1 aberrantly promotes proliferation, contributing to pancreatic tumorigenesis. We also demonstrate that the gene encoding cyclin D1 is a direct transcriptional target of Six1 in pancreatic cancer cells. Furthermore, we show that Six1 promotes cell cycle progression and proliferation by upregulation of cyclin D1.However, the role of Six1 in pancreatic cancer is unknown. The critical role of Six1 in the initiation and progression of numerous cancers impelled us to study the function of Six1 in pancreatic cancer. In this study, we demonstrate that Six1 is overexpressed in pancreatic cancer and correlated with advanced tumor stage. Using The collection of tissue samples was approved and supervised by the Research Ethics Committee of Zhengzhou University. Written informed consents were obtained from all patients who provided samples. Animal studies and xenograft tumor model were approved and supervised by Research Ethics Committee of Zhengzhou University.2 atmosphere at 37°C.Human pancreatic carcinoma cell lines PANC-1 (CRL-1469) and MIA PaCa-2 (CRL-1420) were obtained from American Type Culture Collection ATCC . All cell lines were cultured in Dulbecco’s modified Eagle’s medium  supplemented with 10% fetal bovine serum , 100 units/ml penicillin, and 0.1 mg/ml streptomycin  in 5% COPrimary pancreatic tumor (n = 51) and adjacent non-tumor tissues (n = 13) were collected between 2005 and 2010 from patients with resected primary pancreatic ductal adenocarcinomas at the First Affiliated Hospital of Zhengzhou University. The adjacent non-tumor tissue was obtained from a segment of the resected specimens that was the farthest from the tumor (more than 5 cm). Tissues were flash frozen immediately after surgery. No previous local or systemic treatment had been conducted on these patients before the operation. All data including age, gender, tumor size, location, stage, differentiation, perineural invasion and lymph node status were obtained from original pathology reports. Pathologic staging was updated according to current American Joint Committee on Cancer guidelines. Gene expression for Six1 was obtained for each pancreatic tumor, and all samples were mean centered. Samples were then divided into two groups for further analysis: samples in which Six1 expression was above the mean (Six1 “high”), and the remaining samples (Six1 “low”). Written informed consents were obtained from all patients and this study was approved by the Research Ethics Committee of Zhengzhou University .Human Six1 cDNA was amplified from human skeletal muscle by Reverse transcription polymerase chain reaction (RT-PCR). Human skeletal muscle tissue was obtained from the normal surrounding tissue margin of sarcoma resection specimens. The detailed reaction condition of PCR was performed according to previous report 5′-GGGAGAACACCGAAAACAA-3′; the sequence of the cyclin D1 targeted siRNA is 5′-GTTCATTTCCAATCCGCCC-3′ or 5′-GCCGAGAAGUUGUGCAUCUUU-3′. PANC-1 and MIA PaCa-2 cells were seeded in 6-well plates, grown to 50% confluency and transfected with siRNA or scramble control siRNA  duplexes using lipofectamine 2000  according to the manufacturer’s recommendations. 20 nM siRNAs was used per well and cells were incubated for another 48 to 72 hours before being harvested for protein extraction and immunoblot analysis.For small interfering RNA (siRNA)-mediated down regulation of Six1 or cyclin D1, the following siRNA sequences were purchased from Ribobio Company, Guangzhou, P.R.China). The sequences of the Six1 targeted siRNA are 5′-AGAACGAGAGCGUACUCAA-3′ or 20 (50 µM), 1 µL of 10 mM dNTP mix and DEPC-treated water to a final volume of 10 µL. The mixture was then incubated at 85°C for 5 min and chilled on ice for at least 1 minute before adding 10 µl of cDNA Synthesis Mix . The 20 µl reverse transcription reaction was incubated at 50°C for 30 min, followed by 85°C for 5 min. RNA template was removed by adding 1 µl of RNase H and incubating at 37°C for 20 min. Quantitative PCR was done using SYBR Green dye on an Applied Biosystems 7300 Real-time PCR system . The quantitative PCR was performed with 12.5 µL of 2×SYBR Green PCR mastermix , 1 µL of template cDNA, 200 nM each amplification primer and DEPC-treated water to a final volume of 25 µL. The reactions were incubated in a 96-well optical plate at 95°C for 5 min, followed by 40 cycles of 94°C for 15 s and 60°C for 30 s. The threshold cycle (CT) data was determinate using default threshold settings. The CT is defined as the fractional cycle number at which the fluorescence passes the fixed threshold. The real-time PCR data was analysed by comparative CT method 5′-GGAGCGAGATCCCTCCAAAAT-3′ and 5′-GGCTG TTGTCATACTTCTCATGG-3′; for Six1, 5′-AAGGAGAAGTCGAGGGG TGT-3′ and 5′-TGCTTGTTGGAGGAGGAGTT-3′; for cyclin D1, 5′-GCTGCGAAGT GGAAACCATC-3′ and 5′-CCTCCTTCTGCACACATTTGAA-3′.Total RNA was isolated using the RNeasy mini kit according to the manufacturer’s instructions . The quantity and purity of the isolated RNA was quantified with a NanoDrop 1000 spectrophotometer  at wavelengths of 230, 260, and 280 nm. Only RNA samples with a 260/280 ratio from 1.9 to 2.1 and a 260/230 ratio from 2.0 to 2.5 were acceptable for further analysis. The integrity of total RNA was assessed by visualization of the 18S and 28S ribosomal RNA patterns in a denaturing agarose gel . And the sharp, clear 28S and 18S rRNA bands and a 28S/18S ratio of two are considered to be good quality RNA. The first-strand cDNA was synthesized using the SuperScript® III First-Strand Synthesis System according to the manufacturer’s instructions . Quantitative PCR was done using SYBR Green dye on an Applied Biosystems 7300 Real-time PCR system . Briefly, 10 µL of RNA/primer mixture was prepared in a sterile 0.2 mL tube as follows: 0.5 µg of total RNA, 1 µL of Oligo(dT)http://www.oncomine.org) were used to study gene expression of Six1 in human pancreatic cancer samples as we previously described http://www.ncbi.nlm.nih.gov/geo) Oncomine Cancer Microarray database 2, 0.5% Triton X-100, phosphatase inhibitor mixture , and a protease inhibitor mixture ]. Lysates were clarifed by centrifugation at 10,000×g for 20 min. Proteins (10–25 µg) were resolved on SDS-PAGE, transferred onto nitrocellulose membranes . The membrane was blocked in TBS-T buffer  containing 5% (w/v) non-fat milk at room temperature for 1 hour and then probed with antibodies for Six1, cyclin A, cyclin B1, cyclin E, GAPDH , Phospho-Rb , Rb  and cyclin D1  at 4°C overnight. Detection was performed with the SuperSignal West Femto Maximum Sensitivity Substrate Trial Kit . The band images were digitally captured and quantified with a FluorChem FC2 imaging system .Western blot analysis was performed as we previously described One thousand cells were seeded into 96-well culture plates and incubated at 37°C for different periods of time. Then the 20 µL of MTT  was added to each well and incubated for 4 h at 37°C. The culture medium was removed and 150 µL of DMSO was added to solubilize the crystals for 20 min at room temperature and the absorbance at 570 nm was read by an ELISA plate reader . For cell counting, cells plated in 6-well plates were incubated at 37°C for different periods of time and then removed by trypsinization, and the number of viable cells was counted in a hemocytometer with the use of trypan blue staining. Every sample was measured in triplicate and repeated three times.Cells were exposed to 10 µM BrdU  for 30 min and fixed in 70% ethanol, and then washed with PBS, resuspended, and incubated with 4 N HCL and 0.5% Triton X-100 for 30 min at room temperature. After washing with PBS, cells were neutralized with 0.1 M sodium borate before being labeled with FITC-conjugated BrdU antibody  and incubated with 50 µg/ml propidium iodide  according to the manufacturer’s protocol before being analyzed by a Becton Dickinson FACStar Plus flow cytometer.Six hundred cells were seeded into a 6-well plate. After 14 days, cells were stained by 0.5% crystal violet  in methanol for 10 min. Colonies (more than 50 µm diameter) were counted directly on the plate. Statistical significance was calculated from at least three independent experiments.Panc-1 cells were plated in 24-well plates at 100,000 cells/well and allowed to grow under normal culture conditions. At 80% conﬂuency, cells were transfected with expression vectors (Six1 or empty vector), the full-length human cyclin D1 promoter  7 cells) were fixed with 1% formaldehyde for 10 min at room temperature, followed by washing twice with 20 ml of cold 1×PBS and harvested by scraping. After centrifugation, cell pellets were lysed in 1 mL of SDS Lysis Buffer containing Protease Inhibitor Cocktail II. Cell lysate was sonicated on wet ice four times for 15 seconds each time at intervals of 15 seconds to obtain chromatin fragments of about 200–1000 bp nucleotides. Centrifuged at 12,000×g at 4°C for 10 min to remove insoluble material, and then removed supernatant to fresh microfuge tubes in 100 µL aliquots. Each 100 µL supernatants were diluted with 900 µL of ChIP dilution buffer and preincubated with Protein G agarose at 4°C for 1 hour, and then pelleted agarose by brief centrifugation and removed 10 µL (1%) of the supernatant as Input. Supernatants were incubated at 4°C overnight with 5 µg of Six1 , RNA Polymerase II antibody  and normal IgG,  incubated overnight at 4°C with rotation, and then added 60 µL of Protein G Agarose to each tube and incubated for 1 hour at 4°C with rotation. After centrifugation, removed supernatant and washed the Protein G Agarose-antibody/chromatin complex by resuspending the beads in 1 mL each of Low Salt/High Salt/LiCl Immune Complex Wash Bufferwash buffer for one time, followed by washing twice with TE buffer. After the final washing, the immunoprecipitates were eluted and reverse cross-linked by incubation overnight at 65°C in elution buffer. DNA was then purified with a PCR purification kit . Immunoprecipitated DNAs were analyzed by quantitative real-time PCR using the following primers: cyclin D1 promoter (−1189 to −985), 5′-AGGAACCTT CGGTGGTCTTG-3′ and 5′-CCTTGACCAGTCGGTCCTTG-3′; cyclin D1 promoter (−348 to −190), 5′-CTCAGGGATGGCTTTTGGGC-3′ and 5′-ACTCCCCTGTAGT CCGTGTG-3′; the positive control GAPDH gene proximal promoter, 5′-TACTAGC GGTTTTACGGGCG-3′ and 5′-TCGAACAGGAGGAGCAG AGAGCGA-3′.The ChIP assays were performed by using a ChIP assay kit  as suggested by the manufacturer. Brifely, cells  was injected with PANC-1 cells which was stablely transfected with pcDNA4/TO; group 2 (Six1) was injected with PANC-1 cells which was stablely transfected with pcDNA4/TO-Six1. When the needle was injected below the skin, held the needle parallel to the animal’s body to avoid puncturing underlying structures and aspirated the syringe to ensure that the needle has not entered a blood vessel. The tumor size was measured every 5 days with calipers. The tumor volume was calculated with the formula: (Length × Width2)/2. Five weeks following implantation, a tumor began to appear at the site of implantation with 400 to 800 mm3 in volume. Mice were euthanized by asphyxiation in a CO2 chamber and tumors excised using standard forceps, scissors, and surgical blades. All procedures were conducted in accordance to Animal Care and Use Committee guidelines of Zhengzhou University.Female BALB/c (6–8 weeks old) athymic nude mice were purchased from Experimental Animal Center of Henan province . The mice were housed at five/cage in microisolator units under humidity and temperature controlled conditions with 12-hour light-dark cycles. There were fed with a standard sterile laboratory diet  at least three days before use. Animal health was examined prior to tumor implantation and randomization to ensure that only animals without any symptoms of disease were selected to enter testing procedures. During the experiments, animals were monitored twice daily regarding tumor burden, general condition, food and water supply. The mice were randomly distributed into two groups and subcutaneously injected in the flank regions with 5.0×10P<0.05 was considered significant.All data were expressed as mean ± s.e.m. Between groups and among groups comparisons were conducted with Student t test and ANOVA, respectively. Mann-Whitney U test is used for nonparametric variables. The association of Six1 mRNA expression and clinicopathological characteristics including age, gender, tumor size, location, stage, differentiation, perineural invasion and lymph node status was analysed by either the Chi-square or Fisher’s two-tailed exact test. The Spearman rank correlation test was assessed to verify the association between expression levels of Six1 and cyclin D1 on log-transformed values. Statistical analysis was performed using GraphPad Prism software version 4.0 (PRISM4) , and P<0.01)  . We furtP<0.01) . It may P = 0.018). About 67% (18 of 27) of advanced stage (III & IV) of pancreatic ductal adenocarcinomas were found to overexpress Six1. There is no significant association between Six1 and age, gender, tumor size, location, differentiation, perineural invasion and lymph node status . As expected, the tumor growth curve showed that tumors derived from Six1 group grew more rapidly than those from the control group. Five weeks after injection, the tumor volume of Six1 group was 0.47±0.26 cm3, whereas the tumor volume of control group was 0.08±0.06 cm3  and percentage of BrdU positive cells (E) of Six1 siRNA and scramble control siRNA group in MIA PaCa-2 cells. F, Percentage of BrdU positive cells of Six1 siRNA and scramble control siRNA group in PANC-1 cells. All the experiments were performed in triplicate; bars, s.e.m.; *P<0.05.(TIF)Click here for additional data file.Figure S2The effect of Six1 siRNAs on the endogenous expression levels of Six1 was examined in PANC-1 and MIA PaCa-2 cells by quantitative PCR (A and B) and western blot analyses (C and D). GAPDH was used as an internal control. All the experiments were performed in triplicate; bars, s.e.m.; *P<0.05.(TIF)Click here for additional data file.Figure S3Six1 correlates with cyclin D1 in the xenograft tumors from PANC-1 cells stably transfected with either Six1 or control plasmids as determined by western blot and then quantified by Fluorchem computer analysis .(TIF)Click here for additional data file.Figure S4Analyses of expression of various cyclins by Western blot in two stable cell lines ectopically overexpressing Six1: PANC-1 (A) and MIA PaCa-2 (B) cells. GAPDH was used as an internal control.(TIF)Click here for additional data file.


However, it has not been reported so far whether Pb2+ plays a role in the pathology of Alzheimer disease through interaction with human Tau protein and thereby mediates Tau filament formation. In this study, we have investigated the effect of Pb2+ on fibril formation of recombinant human Tau fragment Tau244–372 and its mutants at physiological pH.Neurofibrillary tangles, mainly consisted of bundles of filaments formed by the microtubule-associated protein Tau, are a hallmark of Alzheimer disease. Lead is a potent neurotoxin for human being especially for the developing children, and Pb2+ significantly accelerates the exposure of hydrophobic region and filament formation of wild-type Tau244–372 on the investigated time scale. As evidenced by circular dichroism and Fourier transform infrared spectroscopy, fibrils formed by wild-type Tau244–372 in the presence of 5–40 µM Pb2+ contain more β-sheet structure than the same amount of fibrils formed by the protein in the absence of Pb2+. However, unlike wild-type Tau244–372, the presence of 5–40 µM Pb2+ has no obvious effects on fibrillization kinetics of single mutants H330A and H362A and double mutant H330A/H362A, and fibrils formed by such mutants in the absence and in the presence of Pb2+ contain similar amounts of β-sheet structure. The results from isothermal titration calorimetry show that one Pb2+ binds to one Tau monomer via interaction with His-330 and His-362, with sub-micromolar affinity.As revealed by thioflavin T and 8-anilino-1-naphthalene sulfonic acid fluorescence, the addition of 5–40 µM Pbvia interaction with His-330 and His-362. Our results suggest the possible involvement of Pb2+ in the pathogenesis of Alzheimer disease and provide critical insights into the mechanism of lead toxicity.We demonstrate for the first time that the fibrillization of human Tau protein is accelerated by exposure to lead  Neurofibrillary tangles are mainly consisted of bundles of filaments formed by the microtubule-associated protein Tau bona fide paired helical filaments in vitro244–372 consisting of the four-repeat microtubule binding domain for studying kinetics of Tau fibril formation. In this study, we investigated the effect of Pb2+ on fibril formation of recombinant Tau244–372 and its mutants at physiological pH by using several biophysical methods, such as thioflavin T (ThT) binding, far-UV circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, transmission electron microscopy (TEM), and isothermal titration calorimetry (ITC). We demonstrated for the first time that the fibrillization of human Tau protein was accelerated by exposure to 5–40 µM Pb2+via interaction with His-330 and His-362, with sub-micromolar affinity. Our results suggest the possible involvement of Pb2+ in the pathogenesis of Alzheimer disease and provide important insights into the mechanism of lead toxicity.Tau binds to microtubules through repeat domain in their C-terminal part 3)2 were made in China and were of analytical grade.Heparin (average MW = 6 kDa), ThT, and 8-anilino-1-naphthalene -sulfonic acid (ANS) were obtained from Sigma-Aldrich . Dithiothreitol (DTT) was obtained from Ameresco . All other chemicals used including Pb(NO244–372 was amplified using the plasmid for human Tau40 (kindly provided by Dr. Michel Goedert) as a template. The PCR-amplified Tau244–372 was subcloned into pRK172 vector. Single histidine mutants H330A and H362A and double mutant H330A/H362A of Tau244–372 were generated using primers CACCTCCTGGTTTGGCATGGATGTT/AACATCCATGCCAAACCAGGAGGTG for H330A and GACAATATGCCCCACGTCCC/GGGACGTGGGGCATATTGTC for H362A. Triple mutant H268A/H299A/H329A and histidine-less mutant H268A/H299A/H329A/H330A/H362A were generated in a similar manner. Recombinant Tau244–372 and its mutants were expressed in Escherichia coli and purified to homogeneity by SP sepharose chromatography as described The cDNA encoding human Tau fragment Tau2+ at 37°C for up to 8 h in the presence of fibrillization inducer heparin used in a Tau ∶ heparin molar ratio of 4 ∶ 1. The solutions with a volume of 200 µl were placed into a well of a 96-well plate in SpectraMax M2 microplate reader  using excitation at 440 nm and emission at 480 nm with a wavelength cut-off at 475 nm A 2.5 mM ThT stock solution was freshly prepared in 10 mM HEPES buffer (pH 7.4) and passed through a 0.22-µm pore size filter before use to remove insoluble particles. Under standard conditions, 10 µM human Tau fragment was incubated in 10 mM HEPES buffer (pH 7.4) containing 100 mM NaCl, 1 mM DTT, and 20 µM ThT with or without Pbversus time to the empirical Hill equation F(∞) is the fluorescence intensity in the long time limit, t50 is the elapsed time at which F is equal to one-half of F(∞), and n is a cooperativity parameter.Kinetic parameters were determined by fitting ThT fluorescence intensity 2+ at 37°C for up to 1 h in the presence of heparin used in a Tau ∶ heparin molar ratio of 4 ∶ 1. The fluorescence of ANS was excited at 350 nm with a slit-width of 5.0 nm and the emission was 470 nm with a slit-width of 7.5 nm on an LS-55 luminescence spectrometer . Assays in the absence of the protein were performed to correct for unbound ANS emission fluorescence intensities.A 2.5 mM ANS stock solution was freshly prepared in 10 mM HEPES buffer (pH 7.4) and passed through a 0.22-µm pore size filter before use to remove insoluble particles. Under standard conditions, 10 µM human Tau fragment was incubated in 10 mM HEPES buffer (pH 7.4) containing 1 mM DTT and 20 µM ANS with or without Pb2PO4-Na2HPO4 buffer (pH 7.4) (or 50 mM sodium acetate buffer at pH 7.4) containing 1 mM DTT and 2.5 µM heparin with or without Pb2+ at 37°C for up to 1 h. Circular dichroism spectra were obtained by using a Jasco J-810 spectropolarimeter  with a thermostated cell holder. Quartz cell with a 1 mm light-path was used for measurements in the far-UV region. Spectra were recorded from 195 to 250 nm for far-UV CD. The scan number for one sample was 45, and the scan time for each scan was about 74 s. No time interval was set between the sequential two scans. The final concentration of Tau244–372 was kept at 10 µM. The spectra of all scans were corrected relative to the buffer blank. The mean residue molar ellipticity [θ] (deg⋅ cm2⋅dmol−1) was calculated using the formula [θ] = (θobs/10)(MRW/lc), where θobs is the observed ellipticity in deg, MRW the mean residue molecular weight , l the path length in cm, and c the protein concentration in g/ml.Under standard conditions, 10 µM human Tau fragment was incubated in 30 mM NaH2+ at 37°C for overnight. Then the samples were lyophilized (freeze drying) at −40°C for FTIR measurements. FTIR spectra were recorded in the range from 400 to 4000 cm−1 at 4 cm−1 resolution. The sample was scanned 128 times in each FTIR measurement, and the spectrum acquired is the average of all these scans. After FTIR assays, the spectra are analyzed by OMNIC 8 software to obtain FTIR second derivative spectra.FTIR spectra of human Tau fibril samples were recorded in KBr pellets using a Nicolet 5700 FTIR spectrophotometer . 200 µM human Tau fragment was incubated in 10 mM HEPES buffer (pH 7.4) containing 100 mM NaCl, 1 mM DTT, and 5 µM heparin with or without Pb2O twice, and then stained with 2% (w/v) uranyl acetate for another 1–2 min. The stained samples were examined using an H-8100 transmission electron microscope  operating at 100 kV.The formation of filaments by human Tau fragment was confirmed by electron microscopy of negatively stained samples. Sample aliquots of 10 µl were placed on copper grids, and left at room temperature for 1–2 min, rinsed with H2+ with Tau244–372 and its mutants were carried out at 25.0°C using a VP-ITC titration calorimetry . Freshly purified Tau proteins  were dialyzed against 50 mM Bis-Tris buffer (pH 7.4) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 100 mM NaCl, overnight at 4°C and then dialyzed against 50 mM Bis-Tris buffer (pH 7.4) containing 100 mM NaCl extensively to remove EDTA. A solution of 100 µM Tau protein was loaded into the sample cell (1.43 ml), and a solution of 1.5 mM Pb2+ was placed in the injection syringe (280 µl). The first injection (2 µl) was followed by 19–24 injections of 10 µl. Dilution heats of Pb2+ were measured by injecting Pb2+ solution into buffer alone and were subtracted from the experimental curves prior to data analysis. The stirring rate was 300 rpm. The resulting data were fitted to a single set of identical sites model using MicroCal ORIGIN software supplied with the instrument, and the standard molar enthalpy change for the binding, Kd, and the binding stoichiometry, n, were thus obtained. The standard molar free energy change, ITC experiments on the interaction of Pbin vivo, heparin has been often employed to induce Tau filament formation in vitroThe enhanced fluorescence emission of the dye ThT, a specific marker for the β-sheet conformation of fibril structures, has been widely used for monitoring the kinetics of amyloid fibril formation 244–372 was incubated with Pb2+ ranging from 5 to 40 µM. Fitting human Tau fragment aggregation kinetic data (t50 and F(∞) values which reflect the lag phase and the final quantity of Tau244–372 amyloid formation respectively. The corresponding kinetic parameters are summarized in t50 of Tau244–372 aggregation monitored by ThT binding assays were 143, 103, 142, 164, and 146 min in the presence of 5, 10, 20, 30, and 40 µM Pb2+ respectively, remarkably shorter than that in the absence of Pb2+ (255 min). The value of t50 reached the minimum at the molar ratio of Pb2+ to Tau of 1∶1, and then got longer at larger ratios of Pb2+/Tau (from 2∶1 to 4∶1). Therefore, as revealed by ThT binding assays  changed larger and faster than those in the absence of Pb2+ . As shown in 218 of Tau244–372 aggregation in the absence of Pb2+ increased gradually from −2900 to −6100 deg⋅cm2⋅dmol−1 when the incubation time increased gradually from 0 to 54.4 min. In the presence of 10 µM Pb2+, however, the value of [θ]218 of Tau244–372 aggregation increased from −3200 to about −9000 deg⋅cm2⋅dmol−1 in the same incubation time range, reaching the maximum at 37.1 min. Similar phenomena were observed in the presence of 20, 30, and 40 µM Pb2+ in phosphate buffer 2 is soluble. As shown in 218 of Tau244–372 aggregation in the absence of Pb2+ increased gradually from −4500 to −6300 deg⋅cm2⋅dmol−1 when the incubation time increased gradually from 0 to 36 min. In the presence of 10–40 µM Pb2+, however, the value of [θ]218 of Tau244–372 aggregation increased from −4300 to about −8000 deg⋅cm2⋅dmol−1 in the same incubation time range, reaching the maximum at 16 min. Therefore, as evidenced by CD spectroscopy .CD spectroscopy was used to detect the conformational conversion of human Tau fragment during fibril formation in the presence and absence of Pb of Pb2+ . Fig. 2Dtroscopy  and S2,  similar  and S2 n244–372 fibrils in the presence and absence of Pb2+. 244–372 fibrils and −1 is characteristic for β-sheet formed by amyloid fibrils 2+, an increase of the band at 1630 cm−1 was clearly observed for Tau244–372 fibrils in the presence of 20 µM Pb2+, further supporting the conclusion reached by CD spectroscopy that fibrils formed by wild-type Tau244–372 in the presence of Pb2+ contain more β-sheet structure than the same amount of fibrils formed by the protein in the absence of Pb2+.FTIR was used to confirm the change in β-sheet structure of human Tau2+. Our TEM studies confirmed the formation of fibrils by wild-type Tau244–372. As shown in 2+ had no significant effect on the morphology of Tau samples.TEM was used to study the morphology of human Tau samples incubated with 0–20 µM Pb2+ on Tau fibrillization, histidine mutants of Tau244–372 were employed. There are five histidine residues in Tau244–372: His-268, His-299, His-329, His-330, and His-362. In this study, Tau244–372 mutants containing single, double, triple, and quintuple histidine mutations were designed, and ThT binding assays and far-UV CD experiments using such mutants were performed in order to provide information about the binding sites of Pb2+ in Tau protein and the role of histidine residues in Tau assembly. 2+ on single mutants H330A and H362A and double mutant H330A/H362A of Tau244–372. Unlike wild-type Tau244–372, the presence of 5–40 µM Pb2+ had no obvious effects on fibrillization kinetics of single mutants H330A . The above results suggest that His-330 and His-362 are key residues in the interaction of Pb2+ with Tau protein. It should be pointed out that the significant intensity variations between different measurements in To determine the reason for the enhancing effect of Pbts H330A  and H362ts H330A  and doubts H330A  except ttructure . However2+ to Tau protein in the absence of DTT. ITC profiles for the binding of Pb2+ to wild-type Tau244–372 and its histidine mutants at 25.0°C are shown in 2+ into a solution of wild-type Tau244–372  molecule with a dissociation constant of 0.217 µM (or 0.286 µM). The binding affinity of Pb2+ to single histidine mutant H362A was significantly lower than that of wild-type Tau244–372, with a dissociation constant of 0.546 µM, and a weak binding reaction for Pb2+ with single histidine mutant H330A was observed. No binding reaction for Pb2+ with double histidine mutant H330A/H362A or histidine-less mutant was detected by ITC  and are carcinogenic to human being, pollution by heavy metals poses a great potential threat to the environment and human health 2+ used was 5–40 µM because of the following reasons. Firstly, it has been demonstrated that there is no significant cytotoxicity to SH-SY5Y cells for 1–50 µM of Pb2+ at either 48 or 72 h 2+ we used is one order of magnitude higher than that considered as lead poisoning by public health authorities in the United States and France 2+via interaction with His-330 and His-362, with sub-micromolar affinity. In other words, His-330 and His-362 are key residues in the interaction of Pb2+ with Tau protein. Moreover, fibrils formed by human Tau protein in the presence of 5–40 µM Pb2+ contained more β-sheet structure than the same amount of fibrils formed by the protein in the absence of Pb2+. In other words, exposure to 5–40 µM Pb2+ enhanced the conversion of random coil structure into β-sheet structure and thereby accelerated the fibrillization of human Tau protein. Our results suggest the possible involvement of Pb2+ in the pathogenesis of Alzheimer disease and provide critical insights into the mechanism of lead toxicity.In this paper the concentration of Pb244–372 was dialyzed overnight at 4°C in the absence of DTT. It has been reported that similar conditions  result in the oxidation of the two cysteines and lead to the formation of compact monomers and a minor population of dimers 244–372 monomers, but neither dimers nor compact monomers, in the ITC experimental conditions. Lane 2 serves as a standard where Tau244–372 was in the presence of DTT, resulting in a purely monomeric population  similar to Tau244–372, but with a binding stoichiometry (2.72±0.06) remarkably larger than Tau244–372  fibrils formed by human Tau protein in the presence of micromolar concentrations of Pb2+ contain more β-sheet structure than the same amount of fibrils formed by the protein in the absence of Pb2+; (iii) the fibrillization of human Tau protein is promoted by exposure to Pb2+via interaction with His-330 and His-362, with sub-micromolar affinity. Information obtained here can enhance our understanding of how low levels of inorganic lead interact with microtubule-associated protein Tau in pathological environments and thereby play a role in the pathology of Alzheimer disease.In conclusion we have shown that: (i) the addition of micromolar concentrations of PbFigure S12+ alone did not induce Tau filament formation.Pb 10 µM Tau244–372 was incubated with 0–10 µM Pb2+  in 10 mM HEPES buffer (pH 7.4) containing 100 mM NaCl, 1 mM DTT, 2.5 µM heparin, and 20 µM ThT, or incubated with 10 µM Pb2+ (red) in 10 mM HEPES buffer (pH 7.4) containing 100 mM NaCl, 1 mM DTT, and 20 µM ThT. ThT binding assays were carried out at 37°C, and all experiments were repeated at least twice.(DOC)Click here for additional data file.Figure S22+ on the relative change in the β-sheet content of Tau244–372 during fibril formation at 37°C, studied by monitoring the CD signal at 218 nm.Effect of Pb 10 µM Tau244–372 was incubated with 0–40 µM Pb2+  in 50 mM sodium acetate buffer (pH 7.4) containing 1 mM DTT and 2.5 µM heparin.(DOC)Click here for additional data file.Figure S32+ to Tau244–372mutants at 25.0°C.ITC profiles for the binding of Pb The top panels represent the raw data for sequential 10-µl injections of 1.5 mM Pb2+ into 150 µM single mutant H330A (A) and 150 µM double mutant H330A/H362A (B) in 50 mM Bis-Tris buffer (pH 7.4), respectively. The bottom panels (C and D) show the plots of the heat evolved  per mole of Pb2+ added, corrected for the heat of Pb2+ dilution, against the molar ratio of Pb2+ to Tau244–372. The data (solid squares) were best fitted to a single set of identical sites model and the solid lines represented the best fit.(DOC)Click here for additional data file.Figure S4244–372.Native gel electrophoresis of Tau Lane 1, Tau244–372 in the absence of DTT (oxidative conditions). Lane 2, Tau244–372 in the presence of 1 mM DTT (reducing conditions). Only one population of monomers, the extended Tau244–372 monomer (M), was visible in lanes 1 and 2. Freshly purified wild-type Tau244–372 was dialyzed against 50 mM Bis-Tris buffer (pH 7.4) containing 1 mM EDTA and 100 mM NaCl, overnight at 4°C and then dialyzed against 50 mM Bis-Tris buffer (pH 7.4) containing 100 mM NaCl extensively to remove EDTA. The samples were mixed with 2× loading buffer and separated by 15% native PAGE. Gel was stained by Coomassie Blue G250.(DOC)Click here for additional data file.Figure S52+ to full-length Tau protein at 25.0°C.ITC profiles for the binding of Pb The panel A represents typical calorimetric titration of full-length Tau (150 µM) with Pb2+ (1.0 mM) in 50 mM Bis-Tris buffer (pH 7.4). The first injection (5 µl) was followed by 19 injections of 10 µl. The panel B shows the plots of the heat evolved  per mole of Pb2+ added, corrected for the heat of Pb2+ dilution, against the molar ratio of Pb2+ to full-length Tau. The data (solid squares) were best fitted to a single set of identical sites model and the solid lines represented the best fit.(DOC)Click here for additional data file.


Cultures were assessed using whole-cell patch-clamp electrophysiology and immunofluorescence. We studied the functional maturation of developing neurons and the activity of the synaptic connections they formed. Neurons exhibited minor differences in the developmental patterns for their intrinsic parameters, such as resting membrane potential and excitability; with the MeCP2-/y cells showing a slightly accelerated development, with shorter action potential half-widths at early stages. There was no difference in the early phase of synapse development, but as the cultures matured, significant deficits became apparent, particularly for inhibitory synaptic activity. MeCP2-/y embryonic stem cell-derived neuronal cultures show clear developmental deficits that match phenotypes observed in slice preparations and thus provide a compelling tool to further investigate the mechanisms behind RTT pathophysiology.Neurodevelopmental diseases such as the Rett syndrome (RTT) have received renewed attention, since the mechanisms involved may underlie a broad range of neuropsychiatric disorders such as schizophrenia and autism. In vertebrates early stages in the functional development of neurons and neuronal networks are difficult to study. Embryonic stem cell-derived neurons provide an easily accessible tool to investigate neuronal differentiation and early network formation. We used  MECP2 gene on the X chromosome, coding for the methyl-CpG-protein 2 (MeCP2). More than 95% of individuals with classic RTT carry de novo mutations. MeCP2 is highly enriched in neurons in the central nervous system -derived neurons allow a straightforward functional analysis of neuron maturation from very early stages up to network formation.Here we investigate the differentiation of neural precursors derived from MeCP2-/y mice and from the corresponding E14Tg2a wt background. We compared intrinsic parameters, such as resting membrane potential (RMP), the function of voltage-gated sodium- and potassium channels as well as generation of action potentials (APs) from immature and mature neurons. As soon as the neurons formed synaptic networks, we studied spontaneous excitatory and inhibitory synaptic activity and its maturation over time.trans-retinoic acid  for additional 4 days. Embryoid bodies are dissociated and neuronal precursors were plated on poly-L-ornithine /laminin  – coated glass cover slips . At day in vitro (DIV) 0 and 1 neuronal precursors were cultured in neural medium containing DMEM/F12, N-2 Supplement (100X) and penicillin/streptomycin and 1 mM glutamine . From DIV 2 the medium was changed to the differentiation medium containing Neurobasal medium, B-27® Supplement (50X), N-2 Supplement (100X), 0.6 mM glutamine and penicillin/streptomycin .Embryonic stem cells derived from a E14Tg2a background with the MeCP2+/y (wt) and MeCP2-/y genotype were cultured and differentiated into neurons as described . Brieflyμm on a horizontal Puller . The intracellular solution was adapted to the medium the cells were cultivated in; for N2 medium it contained (mM): 110 K-D-gluconate, 5 KCl, 11 Tris-phosphocreatine, 1 EGTA, 4.5 MgATP, 10 HEPES, 0.3 Tris-GTP . The extracellular solution for cells coming from N2 medium used for DIV 0 and DIV 1 contained (in mM): 120 NaCl, 29 NaHCO3, 4 KCl, 1 CaCl2, 0.7 MgCl2, 18 glucose, pH 7.4 when bubbled continuously with 95% O2 and 5% CO2. Intracellular solution for cultures coming from complete medium contained (mM): 100 K-D-gluconate, 5 NaCl, 1 EGTA, 5 MgATP, 10 HEPES, and 0.5 Tris-GTP . The extracellular solution for complete medium contained (in mM): 125 NaCl, 26 NaHCO3, 1.25 NaH2PO4*H2O, 2.5 KCl, 1.0 MgSO4, 2.0 CaCl2 and 11 glucose, pH 7.4 when bubbled continuously with 95% O2 and 5% CO2. Voltage-gated sodium- and potassium channels were detected in voltage-clamp mode at a holding potential of -60 mV. The holding potential was changed in a stepwise fashion from -75 to +25 mV in 5 mV increments for 800 ms and the voltage-gated peak inward current and the sustained outward current (between 600–800 ms) were measured for each step. The inward currents were tetrodotoxin (TTX) sensitive, while the outward currents were blocked by tetraethyl-ammonium and 4-aminopyridine [TEA (3 mM) and 4-AP ]. For statistical comparisons the maximal evoked currents for a given cell were used. RMPs and APs were recorded in current-clamp mode. Somatic current injections were applied in 2.5 pA steps from -2.5 to +30 pA, in older cells up to +60 pA. Synaptic activity was measured in voltage-clamp mode using the same k-gluconate based internal in order to maximise cell-yield: to detect spontaneous excitatory synaptic currents (sEPSCs), cells were held at -60 mV, while for spontaneous inhibitory synaptic currents (sIPSCs), cells were held at -40 mV. This allowed us to unambiguously identify EPSCs and IPSCs in the same recording without pharmacological agents present, which might have affected network activity. Responses were filtered at 5 kHz and digitized at 20 kHz. The excitatory glutamate receptor blocker 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione  and antagonists of inhibitory GABAA receptors picrotoxin (100 μM) or bicuculline (20 μM) were added to the perfusate at the end of recordings to block the respective synaptic activity . All other chemicals were purchased from the same supplier . Recorded sEPSC and sIPSC were detected and analyzed using Mini Analysis 6 . All other data analysis was done with IGOR PRO 6.0  software. Two-way ANOVA were used for all statistical analysis (unless otherwise mentioned), with Tukey’s HSD tests where indicated.Cover slips with neurons were transferred to a bath chamber mounted to an inverted microscope . Experiments were performed on DIV 0–8 and DIV 11–23 neurons in culture using the whole-cell voltage-clamp technique. Data were collected using a Multiclamp 700A amplifier . We used electrodes with an open tip resistance of 4–5 MΩ obtained by pulling borosilicate pipettes  with 1.5 mm external diameter and 1.17 mm internal diameter without filament to a tip diameter of ~1 Cells cultured on glass coverslips were rinsed twice with phosphate buffered saline (PBS) pH 7.4 and fixed with 10% neutral buffered formalin  for 20 min at room temperature (RT). After rinsing with PBS, coverslips were permeabilized for 5 min in 0.2% TritonX-100/PBS, rinsed with PBS and incubated for 1 h at RT in a humidified chamber with the following primary antibodies and dilutions : doublecortin , microtubule-associated protein 2 , glutamic acid decarboxylase, 67 kDa isoform,  and synaptophysin . After several washes with PBS, coverslips were incubated for 1 h with corresponding secondary antibody: Cy5 , Alexa 488 , Alexa 488-phalloidin  and 4′,6-diamidino-2-phenylindole (DAPI) . After several washes in PBS, coverslips were mounted in Mowiol-1188 as previously described . ConfocaFigure 1A) showed already at DIV 0 a distinct, spindle shaped morphology , which transformed into a multipolar shape by DIV 1 . To characterize their morphological development, wt  and MeCP2-/y cells  were stained on DIV 3 with antibodies against doublecortin , a marker of immature neurons. In addition we used the actin cytoskeleton label phalloidin , the nuclear marker DAPI  and differential interference contrast (DIC) images . By DIV 3 more than 90% of the cells in culture were immature neurons with a multipolar shape. The DAPI staining indicated that the cell densities were similar in both genotypes .We differentiated MeCP2+/y (wt) and MeCP2-/y mES cells from the same E14Tg2a background into neurons using an established protocol . Progenin = 15) compared to -35 ± 3.0 mV for MeCP2-/y progenitors (n = 8). At DIV 2 RMP of wt cells (n = 21) depolarized to -41 ± 3.3 mV, while for MeCP2-/y (n = 16) we measured a RMP of -36 ± 2.1 mV. In the following days cells from both genotypes showed a gradual hyperpolarization to -57 ± 0.9 mV for wt and -57 ± 1.0 mV for MeCP2-/y, which stabilized around DIV 11 (n = 180 for wt and 151 for MeCP2-/y). Statistical analysis revealed a highly significant influence of developmental age (p < 0.01), but no significant effect of the genotype (p > 0.08) on the RMP.The wt progenitors exhibited a RMP of -56 ± 2.2 mV at DIV 0 (Na) with fast activation and inactivation kinetics, as well as slow, non-inactivating outward potassium currents (IK) during all stages of differentiation. Already 6 h after plating in 6 of 10 cells a small INa of 0.04 ± 0.02 nA in wt and 0.02 ± 0.01 nA in MeCP2-/y and a IK of 0.11 ± 0.03 nA in wt and 0.04 ± 0.01 nA in MeCP2-/y could be found. Both types of currents showed a substantial increase during development in culture . INaincreased continuously from 0.48 ± 0.04 nA in wt and 0.60 ± 0.04 nA in MeCP2-/y (DIV 3–5) to 3.06 ± 0.16 nA in wt and 3.14 ± 0.23 nA in MeCP2-/y . For IK an initial increase from 0.36 ± 0.025 nA in wt and 0.45 ± 0.03 nA in MeCP2-/y (DIV 3–5) to 1.24 ± 0.07 nA in wt and 1.78 ± 0.11 nA in MeCP2-/y (DIV 11–14) then leveled off at 1.52 ± 0.09 nA in wt and 1.31 ± 0.09 nA in MeCP2-/y . For INa the effect of genotype was not significant (p > 0.82), while the developmental age exerted a highly significant effect . Both genotype and developmental age significantly affected IK . Pairwise post hoc analysis did not reveal a significant difference between genotypes at any specific time point (p > 0.5).We observed voltage-dependent inward sodium currents  in both genotypes. Typical APs are shown for DIV 6 , DIV 12  and DIV 21 . A quantitative analysis of the APs was performed between the age ranges DIV 4–6, DIV 11–13, and DIV 20–23 in 61 wt and 44 MeCP2-/y cells  by measuring the size of the first AP in a train  and its half-width, as well as the initial frequency of APs in a train and their frequency adaptation. First AP size  increased from DIV 4 to DIV 23 in wt neurons from 59.5 ± 2.5 to 87.6 ± 3.7 mV and in MeCP2-/y from 60.3 ± 4.9 to 95.7 ± 2.8 mV. First AP half width  decreased over time in wt from 6.7 ± 0.5 to 2.2 ± 0.3 ms and in MeCP2-/y from 4.4 ± 0.7 to 1.6 ± 0.08 ms. Initial AP frequency within a train  showed no clear developmental pattern and varied between 10–20 Hz for both genotypes. Cells from both genotypes exhibited a weak frequency adaptation to between 60 and 90% of the initial frequency  throughout their development. Statistical analysis revealed a significant effect of age (p < 0.0.1), but not of genotype (p > 0.3) on AP size. Both age and genotype significantly affected the AP half-width (p < 0.01) with a significant interaction (p < 0.01). Initial AP frequency was significantly affected by both age (p < 0.01) and genotype (p < 0.05) without significant interaction (p > 0.2). AP frequency adaptation was significantly affected by age (p < 0.05), but not by genotype (p > 0.9).Action potentials were detected from DIV 3 . We measured sEPSCs as inward currents  at a holding potential of -60 mV, while sIPSCs were best visible and measured as outward currents at a holding potential of -40 mV . sEPSCs were blocked by the addition of the AMPA-receptor blocker NBQX (10 μM) to the bath medium, whereas sIPSCs were blocked by bath application of the GABAA receptor antagonists picrotoxin (100 μM) or bicuculline (20 μM) respectively, in all cultures tested .We detected the first spontaneous excitatory (sEPSCs) and inhibitory (sIPSCs) post-synaptic currents by DIV 11 in neurons from both wt and MeCP2-/y cultures (n = 186) and from 21.2 ± 2.6 to 43.8 ± 4.6 events per minute in MeCP2-/y . This represents a highly significant increase with age (p < 0.01) as well as a significant reduction at DIV 20–23 in excitatory activity in MeCP2-/y cultures compared to wt (p < 0.05).Between DIV 11–23 the frequency of sEPSCs increased from 26.2 ± 4.8 to 71.8 ± 7.4 events per minute in wt . Both developmental age (p < 0.01) and the cell’s genotypes (p < 0.01) exerted a highly significant influence in inhibitory synaptic activity.The frequency of sIPSCs rose from 23.5 ± 5.1 to 97.8 ± 13.3 events per minute between DIV 11–23 for wt neurons, while in neurons from MeCP2-/y cultures it went from 19.7 ± 3.4 to 58.8 ± 7.9 events per minute over the same time . We measured synapse density by counting the number of synaptophysin positive puncta along 6–12 MAP2-positive dendrites per age range and genotype. In wt cultures the densities were 61 ± 2.2, 89 ± 8.9, and 64 ± 8.1 puncta/100 μm at DIV 12, 18, and 21 respectively. In MeCP2-/y cultures the values were 46 ± 2.6, 76 ± 7.3, and 81 ± 7.1 puncta/100 μm for the same time points. While age had a significant influence on the density of synaptophysin puncta (p < 0.01), there was no significant effect of the genotype (p > 0.45) on synapse density. We also stained the cultures against GAD67, a specific marker for GABAergic neurons. A clear increase in staining could be observed from DIV 12 to 18 to 21, without an obvious difference in the amount of GAD67 immunoreactivity between the two genotypes . Between DIV 18–21 the percentage of GAD67- and MAP2-double-positive neurons among all MAP2-positive cells was 41+/-3 % for wt and 36+/-2 % for MeCP2 -/y cultures .To see whether the reduced synaptic activity was accompanied by a change in synaptic density we stained cultures at DIV 12, 18, and 21 with antibodies against the neuronal marker MAP2 and against the presynaptic marker synaptophysin  in MeCP2-/y and wt cultures; and while we could not statistically resolve it, the deficit in synaptic activity appears larger at later compared to early developmental stages. We did not detect a significant difference in the synapse density between wt and MeCP2-/y cultures. A deficit in initial synapse formation or later in the number of synapses can therefore not fully explain the reduced synaptic activity. While we did not observe substantial differences in cell densities between MeCP2-/y and wt cultures during development, we cannot rule out subtle differences in cell numbers, which may contribute to our findings. In particular, small changes in the number of interneurons can also not be ruled out as a reason for some of the deficit in GABAergic signaling, even though our GAD67 stains did not show overt differences between the two genotypes. Due to the technical difficulties in unambiguously differentiating between glutamatergic and GABAergic neurons we pooled the recordings from all recorded cells. Since glutamatergic cells are clearly dominant in our cultures, our findings certainly apply to this cell type. For future studies it would be interesting to look at synaptic activity in the different neuron types and to further investigate the mechanisms underlying the deficit in synaptic activity.We found small, but significant differences in voltage-gated potassium currents and in AP half-with and -frequency between genotypes. The biological significance of these small differences is not clear. Voltage-gated potassium currents are significantly stronger in young MeCP2-/y neurons compared to age-matched wt neurons; this is compatible with the shorter AP half-width that we observe in young MeCP2 deficient neurons. The AP frequency is significantly higher in MeCP2-/y cultures; however, the effect is not stable over time. Since MeCP2-/y neurons actually show a higher propensity to spike and since the effects on synaptic activity are most pronounced at later developmental stages the lower synaptic activity is clearly not a consequence of lower excitability in MeCP2-/y cultures.Na, but in IK between MeCP2-/y and wt cultures in our system. Several recent studies have looked at the impact of MeCP2 mutations on intrinsic excitability in different brain regions. In one study on locus coeruleus neurons, the authors found altered expression levels of several voltage-dependent conductances in MeCP2-/y mice compared to wt controls (Previous studies using ES and iPS cultures have found differences in the activity of voltage-sensitive sodium channels in cultures of MeCP2-/y neurons . Such dicontrols . APs hadcontrols . In othecontrols . MeCP2 eSeveral groups have studied synapse development and plasticiy in MeCP2 mutated mice . In all The molecular nature of the synaptic deficit has not yet been elucidated for any of the systems tested. The initially normal development and the different effects on either excitatory or inhibitory synaptic transmission in various brain regions argue against key elements of the synaptic release machinery to be affected. In some studies a reduced number of dendritic spines or of synapses was described . While wMurine embryonic stem cell cell-derived neuronal cultures can produce relatively homogeneous neuronal tissue with an essentially limitless supply. Transcriptome analysis of such tissue might yield candidate molecules responsible for the deficits we observe. Key elements of the known RTT pathology in early developing neural networks could be replicated in our system. This makes it a useful tool to further investigate functional deficits of MeCP2 deficient neurons and to study candidate interventions aimed at slowing or reversing the changes observed.Lydia Barth: conception and design, data collection, analysis and interpretation, manuscript preparation. Rosmarie Sütterlin: conception and design, data collection, analysis and interpretation. Markus Nenniger: data analysis and interpretation. Kaspar E. Vogt: conception and design, data analysis and interpretation, manuscript preparation.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


In contrast to our findings in sporadic AD, BIN1 was unchanged in the hippocampus from 6 cases of familial AD compared to 6 age-matched controls (p=0.488). BIN1 declined with age in a cohort of non-dementia control cases between 25 and 88 years but the correlation was not significant . Although BIN1 is known to have a role in endocytosis, and the processing of the amyloid precursor protein (APP) to form amyloid-β (Aβ) peptides is dependent on endocytosis, knockdown of BIN1 by targeted siRNA or the overexpression of BIN1 in a human neuroblastoma cell line (SH-SY5Y) had no effect on APP processing. These data suggest that the alteration in BIN1 is involved in the pathogenesis of sporadic, but not familial AD and is not a consequence of AD neurodegeneration or the ageing process, a finding in keeping with the numerous GWAS that implicate BIN1 in sporadic AD. However, the mechanism of its contribution remains to be established.Bridging integrator 1 (BIN1) has been implicated in sporadic Alzheimer’s disease (AD) by a number of genome wide association studies (GWAS) in a variety of populations. Here we measured BIN1 in frontal cortex samples from 24 sporadic AD and 24 age-matched non-dementia brains and correlated the expression of this protein with markers of AD. BIN1 was reduced by 87% (p=0.007) in sporadic AD compared to non-dementia controls, but BIN1 in sporadic AD did not correlate with soluble Aβ (r Genome-wide association studies (GWAS) have recently identified a number of genes that are related to sporadic AD -8. TheseDespite the identification of BIN1 gene variants as being associated with sporadic AD, little is known about BIN1 protein expression and its involvement in AD pathogenesis. In this study we measured BIN1 protein level in frontal neocortex from cases of sporadic AD and age-matched control brain samples, the hippocampus of familial AD cases compared to age-matched controls, and in a non-dementia cohort across a range of ages. We demonstrate that BIN1 protein level is significantly decreased in sporadic AD compared to age-matched controls, but not in familial AD. Although BIN1 level tended to decline with age, this trend was not significant. As BIN1 has a function in endocytosis, we investigated its potential role in amyloid precursor protein (APP) metabolism and amyloid-β (Aβ) generation in a cell culture system. Neither knocking down endogenous BIN1 expression with targeted siRNA nor overexpression of BIN1 in a human neuroblastoma cell line had an effect on APP processing.Quantitative immunoblotting was used to assess BIN1 level in the frontal cortex in sporadic AD and in age-matched non-dementia controls . BIN1 apTo determine whether the reduction in BIN1 expression is a primary disease process or secondary to other AD-associated changes, we measured the level of BIN1 in brain tissue in familial AD and age-matched controls . In hipps=-0.449, p = 0.081). We showed previously that there is no significant correlation between age and the expression of APP [s=0.322, p = 0.223).We also used quantitative immunoblotting to measure the level of BIN1 in a series of non-dementia brains covering a wide spectrum of ages . The levn of APP  and thers=-0.084, p=0.698 and rs=0.237, p = 0.269, respectively). In addition, Aβ plaque load, as measured immunohistochemically, did not correlate with BIN1 .As BIN1 is decreased in sporadic AD compared to age-matched controls  we invesAs BIN1 is known to play a role in endocytosis -16 and tTo confirm the results of the BIN1 overexpression on APP processing the converse experiment was performed in which endogenous BIN1 expression was knocked down by siRNA in the SH-SY5Y cells. BIN1 knockdown caused a 65% (p < 0.001) reduction in BIN1 protein when SH-SY5Y cells were incubated with BIN1 siRNA compared to a non-targeting siRNA control . FluoresIn this study we have shown that the level of BIN1 protein is lower in the brain in sporadic AD than in non-demented age-matched controls. There was a large variation of BIN1 protein levels across the whole cohort of sporadic AD and control samples, however, this is unsurprising as protein levels are heritable molecular phenotypes that exhibit considerable variation between individuals, populations and sexes . The obsThe levels of BIN1 protein were reduced in the frontal cortex of sporadic AD compared to age-matched non-dementia controls, but not in the hippocampus of familial AD. This implies that the alteration in BIN1 is involved in the pathogenesis of sporadic, but not familial AD and is not a consequence of AD neurodegeneration. The brain areas analysed did differ between the sporadic and familial AD cohorts, but both the frontal cortex and the hippocampus are areas known to be associated with AD -29. EachAmph, the BIN1 Drosophila ortholog, modulates Tau neurotoxicity in vivo in a Drosophila model system, the loss of Amph was able to suppress Tau-induced neurotoxicity [Our data show that BIN1 does not correlate with the amount of tau pathology in sporadic AD. This is in contrast to a recent publication that proposed that a 3 bp insertion allele 28 kb upstream of BIN1 (rs59335482) was associated with the tau load in AD brains . Over hatoxicity . In additoxicity . This maOne of the numerous roles of BIN1 is in endocytosis -16 . EndIn conclusion our data demonstrate that BIN1 is reduced in sporadic but not familial AD, showing that the alteration in BIN1 is probably a primary contributor to the disease and not a secondary consequence of AD. Our findings are consistent with the numerous GWAS that implicate variation in the gene encoding for BIN1 in sporadic AD. Finally, we did not find evidence that a decline in BIN1 is likely to contribute to the increasing risk of AD with age.Brain tissue was obtained from the HTA licensed South West Dementia Brain Bank, University of Bristol, UK (sporadic AD) and from the Medical Research Council London Neurodegenerative Diseases Brain Bank, Institute of Psychiatry, King’s College London, UK . The study was conducted with approval from The Cardiff Research Ethics Committee, North Somerset and South Bristol Research Ethics Committee and the Leeds Central Research Ethics Committee. All sporadic AD cases had been subjected to detailed neuropathological examination. Sporadic AD cases had been assessed according to the criteria of the Consortium to Establish a Registry for Alzheimer's Disease (CERAD) . The spog for 15 min at 4°C, and aliquots of the supernatant were stored at -80°C until used. Familial AD and age-spectrum control brain tissue samples from the hippocampus were homogenised in 9 volumes of phosphate buffered saline , 0.5% (v/v) Nonidet P40, 0.5% (w/v) sodium deoxycholate, pH 7.4 in the presence of a complete protease inhibitor cocktail  using an electrical homogeniser. Samples were centrifuged at 14,000 g for 10 min. For measurements of BIN1 protein and NSE in sporadic AD samples, approximately 200 mg of frontal neocortex (Brodmann area 6) was homogenised in 1 ml lysis buffer  sucrose containing aprotinin (1 µg/ml) and phenylmethane sulfonyl fluoride ) . Brain tissue was homogenised for 30 s in a Precellys 24 automated tissue homogeniser  with 2.3-mm silica beads  and total protein was measured using Total Protein kit . The homogenates were centrifuged at 20 817 g for 15 min at 4°C and the supernatant (soluble fraction) was stored at −80°C until used. The pellet was homogenised in 6.25 M guanidine HCl in 50 mM Tris/HCl, pH 8.0, incubated for 4 h at 25°C and centrifuged at 20 817 g for 20 min at 4°C. The resultant supernatant (guanidine-extractable fraction) was stored at −80°C until used.For measurements of Aβ, tissue (200 mg) was allowed to thaw to 4°C, homogenised in 5 volumes (w/v) of Tris-buffered saline (TBS) extraction buffer [140 mM NaCl, 3 mM KCl, 25 mM Tris/HCl, pH 7.4, containing 1% Nonidet P-40 (NP40), 5 mM EDTA, 2 mM 1,10-phenanthroline, 10 µM PMSF and 1 µg/ml aprotinin , as detailed in ,36. The The protein content of the brain homogenate was determined using bicinchoninic acid in a microtitre plate-based assay with bovine serum albumin (BSA) standards. Samples were mixed with an equal volume of SDS dissociation buffer  SDS, 20% (v/v) glycerol, 100 mM dithiothreitol, 0.002% (w/v) bromophenol blue), and boiled for 4 min. Proteins were resolved by SDS polyacrylamide gel electrophoresis using 7% (APP) and 10%  polyacrylamide gels. Resolved proteins were transferred to Immobilon P polyvinylidene difluoride membrane . The membrane was blocked by incubation for 1 h with PBS containing 0.1% (v/v) Tween-20 and 5% (w/v) dried milk powder. Antibody incubations were performed in PBS Tween containing 2% (v/v) bovine serum albumin. Antibody against BIN1  was used at 1:1000, anti-APP antibody 22C11  was used at 1:2500, anti-sAPPα antibody 6E10  was used at 1:4000 and 1A9 antibody  was raised against a neoepitope formed on wild type sAPPβ following β-cleavage of APP was used at 1:2500. Horseradish peroxidase-conjugated secondary antibody was used at 1:4000 in the same buffer. Bound antibody was detected using the enhanced chemiluminescence detection method . Densitometric analysis was performed using Image J 1.44p . Quantification was in relation to total protein. Total amount of protein was determined from amido black (0.1% (w/v) amido black, 1% (v/v) acetic acid) stained membranes using Image J 1.44p.The method of ELISA measurement of total soluble and insoluble Aβ was reported previously ,36. Solu2 with the help of Histometrix software  driving a Leica DM microscope with a motorised stage. Aβ-laden blood vessels were excluded from analysis.Parenchymal Aβ load had previously been measured in all cases . The fieThe precentage area of cerebral cortex immunopositive for phospho-tau had also been measured previously ,40, usin2. SH-SY5Y (human neuroblastoma) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM)  supplemented with 10% (v/v) fetal bovine serum (FBS) , in a humidified incubator at 37°C with 5% (v/v) CO695 in pIREShyg [cDNA encoding human BIN1 in expression vector pcDNA3.1(+)  or empty vector alone  was introduced by electroporation into SH-SY5Y cells stably expressing APPpIREShyg . Cells wUGGUUUACAUGUCGACUAA UGGUUUACAUGUUGUGUGA UGGUUUACAUGUUUUCUGA UGGUUUACAUGUUUUCCUA) were obtained as smartpools from Dharmacon . SH-SY5Y cells were seeded in routine culture medium and allowed to adhere overnight. Cells were transfected with 50 nM  BIN1 or non-targeting control siRNA as a complex with Dharmafect 3  according to the manufacturer’s instructions and incubated for 24 h at 37°C with 5% (v/v) CO2. siRNA specific for human BIN1 and a non-targeting sequence (2 (v/v). OptiMEM reduced sera medium was removed from the cells and centrifuged for 10 min at either 3913 g at 4°C. The supernatant was either stored at -20°C for Aβ ELISA or concentrated ~50 fold using Vivaspin 100,000 Da molecular weight cut off polyethersulfone membrane concentrator  by centrifugation at 3913 g at 4°C. Cells were incubated for 48 h in OptiMEM reduced sera medium  at 37°C with 5% CO2+ and Mg2+), and pelleted by centrifugation at 3193 g at 4°C. Cells were lysed in RIPA lysis buffer  Sodium deoxycholate, 1% (v/v) NP-40, pH 8.0) with EDTA complete protease inhibitor cocktail , incubated at 4°C for 30 min and centrifuged at 13,000 g for 10 min at room temperature. The subsequent supernatant was frozen at -20°C for protein analysis. Cells were washed and harvested in PBS (with CaSandwich ELISAs for the detection of human Aβ40 and Aβ42 were performed as described previously [~60% confluence on glass coverslips and siRNA treated (see above). Post-incubation, cells were fixed with 4% (v/v) paraformaldehyde for 10 min, permeabilised using 0.2% (v/v) Triton X-100 and blocked overnight at 4 °C in PBS containing Ca2+ and Mg2+ ions with 5% (v/v) fish skin gelatin. Coverslips were then incubated for 2 h in the same buffer containing BIN1 specific antibody , washed, and then incubated with the appropriate fluorescent probe-conjugated secondary antibody under the same conditions. Nuclei were counterstained by washing briefly in DAPI stain, and coverslips were mounted onto glass slides using Fluoromount-G (Southern Biotech). Cells were visualised using a DeltaVision Optical Restoration Microscopy System (Applied Precision). Fluorescence microscopy was carried out as described previously . BrieflyThe distribution of the AD cases compared to their age-matched controls was determined by the Kolmogorov-Smirnov test. Group data were compared using either an Independent T-test for parametric, or a Mann-Whitney U test for non-parametric data 2-tailed Spearman’s rank correlation coefficient was used on non-parametric data to assess the correlation between BIN1 and soluble and insoluble Aβ, Aβ load, tau load and age; p ≤ 0.05 was considered significant. The data were analysed using the Statistical Package for Social Sciences (SPSS 17.0) program  and GraphPad Prism (version 6) .


Myocardial oedema and salvaged area at risk (ARA) have been described as markers of prognosis and as surrogate markers for clinical trials. The time-course of oedema has been described in a canine infarct model but the in vivo time course following revascularised MI has not been described. CMR is able to characterise oedema in myocardial tissue using T2-weighted sequences. We therefore aimed to determine the time-course of post infarct myocardial oedema using serial CMR imaging.9 patients with acute ST-elevation MI who underwent primary PCI with stent implantation within 12 hours of symptom onset were recruited. Patients were scanned on days 1, 3, 10, 20 and 90 following their PPCI with a 1.5T Philips Achieva . Images were obtained as continuous short-axis stacks covering the left ventricle with a slice thickness of 8mm and gap of 2mm. Myocardial oedema was assessed at all time points using T2-weighted triple inversion turbo spin echo STIR imaging . Scans performed on days 3 and 90 included assessment of scar after administration of a bolus of Gadodoteric acid (0.15 mmol/kg) using a T1-weighted TFE sequence. Image analysis was performed using dedicated software, CMR42 . Scar and oedema volumes were calculated by manually drawing endocardial and epicardial contours followed by semi-automated selection of normal remote myocardium per slice. The scar and oedema were described as >5SD and >2SD in signal intensity from remote normal myocardium respectively. Values are expressed as a percentage of the LV mass (%LVM).Patient age was 56.6 ± 5.4 years . Myocardial oedema peaks at day 3 (31.1 ± 4.3%LVM) and is significantly greater than the area of scar (13.7 ± 4.5%LVM) p=<0.0001, with an average salvaged ARA of 17.3 ± 5.7%. There is no difference in oedema between days 1 and 3 or days 10 and 20. There was a reduction of 8% in oedema between days 3 and 10 . Oedema persists at 90 days and is the same size as the scar (10.6 ± 6% vs. 10 ± 5.1% p=ns). Figure Myocardial oedema post myocardial infarction peaks at day 3 and persists for 90 days. There is no significant change between days 10-20 which may provide the most appropriate time period to assess myocardial oedema. Myocardial oedema may also persist much longer than previously thought.


Corynebacterium falsenii is a member of the natural microflora of wild and domesticated birds and is rarely detected in human clinical specimens. The chromosomal sequence of the type strain C. falsenii DSM 44353 comprises 2,677,607 bp and provides detailed insights into the evolution of Corynebacterium species assigned to the highly diverse cluster 3. Corynebacterium falsenii was first described by Sjödén et al.  was obtained from the Leibniz Institute DSMZ (Braunschweig) and grown in brain heart infusion broth-yeast extract at 37°C (de novo assembler software (release 2.8) to yield 44 contigs in 12 scaffolds. The software r2cat (C. jeikeium K411 (in silico with the Consed software (version 24)  .C. falsenii DSM 44353 includes a circular chromosome of 2,677,607 bp (63.18% G+C content) and the circular corynephage ΦCFAL8171I genome of 42,009 bp (61.74% G+C content). An identical linear copy of ΦCFAL8171I is present in the chromosome as a prophage, suggesting that this corynephage had entered a lytic cycle in a subpopulation of the culture used to prepare the genomic DNA. The automatic annotation of the genome sequence with the NCBI Prokaryotic Genome Annotation Pipeline and the GeneMarkS+ software (version 2.3) revealed 2,248 protein-coding regions, 35 pseudogenes, 50 tRNA genes, 1 noncoding RNA (ncRNA) gene, and 3 rRNA operons in the chromosome of C. falsenii DSM 44353 and 58 protein-coding regions in the circular genome of ΦCFAL8171I.The genome sequence of CP007156 (chromosome) and CP007157 (ΦCFAL8171I).This genome project has been deposited in the GenBank database under accession no.