To the Editor: To thoroughly investigate avian influenza outbreaks, identifying highly pathogenic avian influenza (HPAI) and low pathogenic avian influenza (LPAI) is essential. Currently, determination of inserted basic amino acids within the hemagglutinin cleavage site of HPAI relies on nucleotide sequencing , then incubation at 95°C for 10 min (predenaturation), followed by 40 cycles of amplification alternating between 94°C for 15 s (denaturation) and 68°C for 40 s . The SYBR Green I fluorescent signal was obtained once per cycle at the end of the extension step. After amplification, melting curve analysis was performed by heating the sample to 95°C for 15 s, then cooling it to 70°C for 1 min, followed by a linear temperature increase to 95°C at a rate of 0.5°C/s, while continuously monitoring the fluorescent signal. Data were analyzed by the 7500 System SDS Software version 1.2 (Applied Biosystems).One-step, real-time RT-PCR with melting curve analysis was performed in an ABI 7500 system . In each reaction, 3.0 μL of RNA sample was combined with a reaction mixture containing 10 μL 2× SYBR Green PCR Master Mix (Applied Biosystems), 0.5 μL 40× MultiScribe (Applied Biosystems) and RNase inhibitor, each primer , 1.5 mmol/L MgCl2 copies/μL. To assess the specificity, viral RNA extracted from other subtypes of influenza A viruses (H1–H4 and H6–H15) was tested. The assay was specific for the H5 subtype, since no amplification was detected from other subtypes.To develop the assay, samples previously identified as influenza A/chicken/Nakorn-Patom/Thailand/CU-K2/04 (H5N1) served as a control for HPAI, and A/duck/Hong Kong/308/78 (H5N3) served as a control for LPAI. The H5 genes (nucleotides 914–1728) of each strain were inserted into pGEM-T Easy Vector and then transcribed in vitro by using RiboMAX Large Scale RNA Production System-T7 . Serial 10-fold dilutions of the standard H5 RNA were subjected to a sensitivity test (m) between runs were experimentally determined. The mean (standard deviation) of Tm values for HPAI and LPAI were 77.43°C (0.21°C) and 79.57°C (0.23°C), respectively. This assay provided high reliability and reproducibility, since the coefficients of variation were <0.30.Three preliminary melting curve analyses showed that this assay was effective in discriminating between the melting peaks of HPAI and LPAI . The varm varied from 77.2°C to 78.1°C for HPAI and 78.75°C to 79.5°C for LPAI. The melting curve analysis results were completely concordant with the results of direct sequence analysis of the H5 gene for all samples tested.Seventy-eight specimens of influenza A virus were used to validate the assay. The 75 HPAI samples were isolated during the 2004 outbreak in Thailand; 3 LPAI samples, including A/avian/NY/01 (H5N2), A/Chicken/Mexico/31381-3/94 (H5N2), and A/shoveler/Egypt/03 (H5N2), were provided by the Centers for Disease Control and Prevention. The viruses were isolated in embryonated chicken eggs as described previously (The melting curve analysis described here provides a rapid, accurate, and high-throughput approach to discriminate between HPAI and LPAI. This assay is particularly attractive for large-scale screening of suspected subtype H5 influenza A virus during outbreaks to identify candidate LPAI that could be used as vaccine strains.


Flap endonuclease 1 (Fen1) is a highly conserved structure-specific nuclease that catalyses a specific incision to remove 5′ flaps in double-stranded DNA substrates. Fen1 plays an essential role in key cellular processes, such as DNA replication and repair, and mutations that compromise Fen1 expression levels or activity have severe health implications in humans. The nuclease activity of Fen1 and other FEN family members can be stimulated by processivity clamps such as proliferating cell nuclear antigen (PCNA); however, the exact mechanism of PCNA activation is currently unknown. Here, we have used a combination of ensemble and single-molecule Förster resonance energy transfer together with protein-induced fluorescence enhancement to uncouple and investigate the substrate recognition and catalytic steps of Fen1 and Fen1/PCNA complexes. We propose a model in which upon Fen1 binding, a highly dynamic substrate is bent and locked into an open flap conformation where specific Fen1/DNA interactions can be established. PCNA enhances Fen1 recognition of the DNA substrate by further promoting the open flap conformation in a step that may involve facilitated threading of the 5′ ssDNA flap. Merging our data with existing crystallographic and molecular dynamics simulations we provide a solution-based model for the Fen1/PCNA/DNA ternary complex. The activity of Flap Endonuclease 1 (Fen1) as a divalent metal ion-dependent phosphodiesterase is essential to maintain genome integrity in all domains of life . As a cein vitro by up to 50-fold, depending on the experimental conditions (Sulfolobus solfataricus Xeroderma Pigmentosum Group F endonuclease (XPF)  ,20. Remase (XPF) , there iethality ,22. In fethality ,22. In gethality . The triethality ,22,23. Iase Lig1 . Althoug its own  are avaiS. solfataricus Fen1 by the sliding clamp PCNA and propose a nuclease-reaction profile. Our data suggest a model in which in the absence of Fen1, the double-flap DNA exhibits a Mg2+-dependent fluctuation between a Y-shape structure (in the absence of Mg2+) and a more extended duplex conformation. Fen1 binding significantly distorts the overall structure of the DNA flap, inducing a pronounced opening of the flap structure at the branch point, as observed in the x-ray crystal structure. PCNA association increases Fen1 affinity for the flap structure without altering the catalytic step and additionally cooperates with Fen1 to promote the flap opening/threading step. Finally, we use our solution-based FRET and PIFE data to refine current models of the Fen1/PCNA/DNA ternary complex based on existing crystal structures and molecular dynamics (MD) simulations.In this study, we have used a combination of Förster resonance energy transfer (FRET) and protein-induced fluorescence enhancement (PIFE) at ensemble and single-molecule level to investigate the activation mechanism of Supplementary Materials and Methods section and Supplementary Table S1) and mixed with hybridization buffer . Samples were then heated at 93°C for 2 min followed by slow overnight cooling to 4°C.Oligonucleotides were purchased from Integrated DNA Technologies either unlabelled, or labelled with Cy3/Fluorescein and/or an internal amino modifier C6-dT. Succinimidyl ester derivatives of the fluorophores Cy3, Cy5  and Alexa 488 (Invitrogen) were used according to the manufacturer’s protocol for the specific labelling of DNA oligonucleotides. The various double-flap substrates were assembled using 0.1 OD of each of the relevant strands . Sulfolobus solfataricus PCNA heterotrimer subunits were expressed and purified as described previously  was introduced to the PCNA1 expression plasmid by QuickChange (Agilent Technologies), and the mutant protein was expressed and purified as per the wild type. Pure protein was conjugated to Cy5-maleimide  according to the manufacturer instructions. Briefly, the protein was incubated in 50 mM Tris–HCl buffer, pH 7.2, 500 mM NaCl, 10-fold molar excess of Cy5 maleimide  for 1 h at room temperature. Reaction was stopped with 10 mM dithiothreitol (DTT). Cy5 labelled enzyme was separated from the free dye using two successive PD-10 columns. Labelling yield (∼80%) was assessed by UV-VIS absorbance spectra, correcting for the dye absorbance at 280 nm.The point mutation N131C , equipped with a Peltier temperature controller set to 20°C as described in ‘Supplementary Methods section.Single-molecule FRET (sm-FRET) trajectories were acquired from immobilized single-molecules using a prism-type total-internal reflection setup based on an inverted microscope as described in the Supplementary ‘Materials and Methods’ section).A flow cell was constructed by affixing plastic tubing to a drilled polyethylenglycol (PEG) passivated quartz slide . The una2+ ions. Three FRET constructs were engineered carrying the donor and the acceptor dye at different positions . The double-flap substrate contains two duplex regions that we have termed the 5′-flap-duplex (5F-duplex which refers to the duplex containing the 5′-flap strand) and the 3′-flap-duplex , indicative of a global reorganization of the flap structure (2+ ion concentration and were well fitted by a two-state model (Supplementary Equation S1). Indeed, a global fit of the three flap vectors yielded a good fit of all the experimental data with a value of n = 0.96 for the Hill coefficient and a half-point of [Mg2+]1/2 = 1.14 mM (R = 0.998) (Supplementary Table S2).Despite the fact that crucial cellular process including DNA replication and lpBER generate 5′-flap DNA substrates as intermediate products ,11,12,29tructure a–c. All 2+ ions . For Flap-12 and Flap-23, the FRET efficiency increased from 0.51 ± 0.04 and 0.50 ± 0.01, in the absence of Mg2+, to values of 0.56 ± 0.02 and 0.84 ± 0.01 at 20 mM concentration of Mg2+ ions, respectively. This implies a moderate 2 Å decrease in distance for Flap-12 and ∼13 Å for Flap-23 (Supplementary Table S3). In contrast, Flap-13 exhibited a decrease in FRET efficiency from a value of 0.13 ± 0.01 with no Mg2+ ions to a value of 0.034 ± 0.004 in the presence of 30 mM Mg2+ ions concentration, which represents a ∼18 Å decrease in the inter-dye distance (Supplementary Table S2). A significant shortening of the end-to-end distance associated with to Flap-13 can be explained by a change in the kink angle between the upstream and downstream complexes centred at the phosphate opposite the ss/dsDNA junction. Using the calculated inter-dye distance for Flap-13 and taking into account the length of the fluorescein linker and that Cy3 stacks at 5 Å distance from the terminal base pair of duplex DNA  histograms were obtained for different Mg2+ ion concentrations . In the absence of Mg2+ ions, the flap substrate remained in a low-FRET state (Eapp ∼ 0.37) until photobleaching occurred  to that obtained in the absence of Mg2+. At these conditions, the single-molecule trajectories exhibited fast, but clear transitions between these two FRET states. Because the observed fluctuations were too fast to be clearly resolved using Hidden Markov modelling methods, we performed cross-correlation analysis to determine their rates  (2+ ions known to inhibit Fen1 function (>30 mM), the flap substrate remained locked in the extended conformation. The fast inter-conversion observed here for the flap substrate using sm-FRET is in good agreement with recent studies on the structure of nicked, gapped and bulged structures, which have shown that these intermediates are highly dynamic and able to adopt a broad range of conformations . T. T2+ ion(∼10 mM) , we obsermations .Figure KD = 8.5 µM) and 5F-duplex (KD = 6.95 µM) regions with equal probability and with a 1:1 stoichiometry  (KD of 360 ± 30 nM for the PCNA12/Fen1 interaction. A moderate decrease (∼2-fold) in the affinity of PCNA12 when compared to the heterotrimeric PCNA provides strong experimental evidence for selective recruitment of Fen1 to PCNA12 but not PCNA3 as observed from the crystal structure of the PCNA12/Fen1 complex  . These d∼210 nM)  and rece∼210 nM) . For com complex . Also, tS. solfataricus PCNA . Titration of acceptor-labelled PCNA (PCNA-Cy5) with upstream or downstream donor-labelled DNA substrates, identical to those used for the PIFE experiments, and pre-equilibrated with 5 µM Fen1, revealed a significantly higher intermolecular FRET efficiency (Supplementary Equation S2) for the 3F-duplex labelled substrate (Eapp ∼ 0.48) corresponding to a distance of 63 ± 5 Å, compared to the 5F-duplex labelled complex  (KD of 117 ± 34 nM) also matches that obtained by fluorescence enhancement using non-labelled PCNA, indicating that the presence of Cy5 acceptor on the PCNA does not affect the formation of the ternary complex.FRET studies to investigate the structural organization of the eukaryotic Fen1/PCNA/DNA complex in solution are challenging due to the homotrimeric nature of the sliding clamp, which facilitates binding of up to three copies of the same client protein, as observed in the crystal structure of Fen1 with PCNA . Here, wcus PCNA ,25 to ci2 ± 7 Å) b. A shor2 ± 7 Å) a and conRecently, a structural model of the human Fen1/PCNA/DNA ternary complex was reported . This wa2+ ions to prevent cleavage (Supplementary Table S4). Global fitting of the three binding curves to Supplementary Equation S3 yielded a KD of 14 ± 3 nM that fitted accurately (R = 0.998) all Fen1 binding isotherms (KD ∼ 5–10 nM), suggesting that the presence of the fluorophores has no significant effect on complex formation (KD ∼ 3 ± 1 nM). This result is in good agreement with the 4-fold reduction in KM observed at room temperature under steady-state conditions using a similar flap substrate  in the presence of 2 µM PCNA .Once the structures and dynamics of the flap DNA substrate in the unbound state and the organization of the ternary complex were determined, we investigated Fen1 association to the 9-nt ssDNA 5′ flap in the absence and presence of PCNA (2 µM). FRET-binding curves were obtained at room temperature by titrating each vector with increasing concentrations of Fen1 in a background of 10 mM Casotherms a–c. Thisormation ,39. FRETormation a–c, reveubstrate . For comArchaeoglobus fulgidus Fen1 promotes a bent conformation between the 5F-duplex and 3F-duplex regions (Supplementary Table S5). At saturating concentrations of Fen1 (>1 µM), the inter-dye distance associated with Flap-12 increased only by ∼3 Å, whereas the equivalent duplex DNA to single-strand distance reported by Flap-23 exhibited a 10 Å increase in the presence of Fen1 (Supplementary Equation S4). For Flap-12 and Flap-23, addition of PCNA promoted an additional increase in the inter-dye distance of ∼2 Å  upon Fen1 and Fen1/PCNA binding allowed us to use sm-FRET to test this flap opening model in the presence and absence of the sliding clamp. A double-flap substrate (Flap-23) carrying a 9-nt 5′-ssDNA flap, similar to that used for ensemble-FRET measurements, was modified for sm-FRET using the donor–acceptor FRET pair Cy3-Cy5 (Ro ∼ 53 Å)  . A bioti ∼ 53 Å) .Eapp ∼ 0.62 ± 0.1 (76%) and a minor contribution at Eapp ∼ 0.38 ± 0.2  and a concomitant increase in the contribution of the low-FRET state (Eapp ∼ 0.44 ± 0.2), reaching a value of 82% at 100 nM concentration of Fen1 (Eapp ∼ 0.44 ± 0.2) very close to that observed in the absence of Mg2+ ions (Eapp ∼ 0.37 ± 0.1) (Eapp ∼ 0.44 ± 0.2) as arising from Fen1–DNA complexes in which Fen1 binding to the flap substrate increases the inter-dye distance as previously found using ensemble FRET (The variation in FRET efficiency of surface-immobilized flap substrates (Flap-23) was monitored as a function of Fen1 concentration a. In the38 ± 0.2 , these t7 ± 0.1) . Howeverble FRET b and d.Supplementary ‘Materials and Methods’ section)  (Eapp ∼ 0.32 ± 0.08). This low-FRET population became the predominant contribution at concentrations of Fen1 higher than ∼3 nM (∼65%) and therefore can be assigned to the formation of the PCNA:Fen1:DNA ternary complex. From these data it is clear that the presence of PCNA had two major effects in the mechanism of Fen1 recognition of the flap substrate. First, it increased Fen1’s affinity for the flap substrate. Indeed, average FRET values extracted at each Fen1 concentration from the sm-FRET histograms and fitted to the model described by Supplementary Equation S3 provided a KD-value of 1.6 ± 0.5 nM (Eapp ∼ 0.32 ± 0.08) in the ternary complex, compared to Fen1 alone (Eapp ∼ 0.44 ± 0.2), confirmed the moderate increase in the inter-dye distance observed in ensemble data  d, theref± 0.5 nM b which ible data b and d.2+ to avoid cleavage. Representative time-intensity traces and corresponding FRET trajectories in the absence and presence of Fen1 are shown in Eapp ∼ 0.62 ± 0.2) with occasional short-lived fluctuations (<1s) to the bent conformation (Eapp ∼ 0.37 ± 0.2) (Eapp ∼ 0.44 ± 0.2) for long periods of time (∼5–8 min) before photobleaching . Because the probability of these long-lived low-FRET trajectories increased with the concentration of pre-incubated Fen1, we assigned them as representing Fen1/DNA bound complexes exhibiting a very low dissociation dynamics. At each Fen1 concentration analysed, a small subset of these low-FRET trajectories (<15–20%) displayed very occasional transitions between both FRET states that we interpreted as Fen1 binding/unbinding events . The statistical frequency of these fluctuations remained low even when the observation time was significantly increased (>15 min). We additionally confirmed that the slow dissociation kinetics did not result from surface-immobilization artefacts using an ensemble-FRET competition assay . To get additional insights into the effect of PCNA, we next investigated the single-molecule equilibrium dynamics of flap substrates incubated with Fen1 in background of 2 µM PCNA and 10 mM Ca2+. Although the lack of a significant PCNA-induced effect on the predominant structure of the flap substrate was already confirmed (Eapp ∼ 0.37) observed for the flap alone  a. In cononfirmed d, and beonfirmed d. We obsap alone a. We proEapp ∼ 0.6), corresponding to Fen1/PCNA dissociation events, were detected during the time scale of our observation window (∼5 min) . Surprisingly, a high percentage (>70%) of the single-molecule trajectories showed a non-anticorrelated behaviour where the donor emission fluctuated between two intensity levels, differing by ∼2-fold; while the acceptor intensity remained practically unchanged . As a result, the corresponding FRET traces displayed transitions between efficiency values of Eapp ∼ 0.3 and Eapp ∼ 0.44. Taking into account that the two-state dynamics were only detected for Fen1/DNA complexes in the presence of PCNA and that a 2-fold fluorescence enhancement (PIFE) of the Cy3 donor located in the 3F-duplex was already confirmed by ensemble measurements (KD ∼ 1 µM) to PCNA12 permanently bound to the Fen1/DNA complex, was ruled out because a control experiment using only PCNA12 exhibited a similar non-anticorrelated dynamics . Overall, the invariance of the Cy5 acceptor signal can be explained by the formation of the Fen1/PCNA/DNA complex leading to two competing processes that counteract one another. PCNA binding promotes an increase in quantum yield of the Cy3 donor in the 3F-duplex, which would normally lead to a concomitant increase in acceptor signal; however, this effect is almost entirely compensated for by a simultaneous PCNA-induced opening of the Flap-23 distance. Such an increase in inter-dye distance leads to a less efficient energy transfer process to the acceptor dye and therefore lower Cy5 fluorescence. The fact that this increase in distance in the ternary complex compared to Fen1/DNA has been observed in both, ensemble  or Fen1/DNA/PCNA (Eapp ∼ 0.32) complexes can be easily distinguished by their distinctive FRET levels as shown in the previous sections. Representative real-time single-molecule trajectories and corresponding FRET traces obtained at each condition are shown in Supplementary Figures S8 and S9.We used real-time injection experiments to monitor Fen1 nuclease activity at single-molecule level. Because the acceptor dye is located at the 5′ end of the single-strand flap, cleavage of this single-stranded region in the Flap-23 substrate should lead to a complete loss of the FRET signal, while the formation of Fen1/DNA  for a variable period of time (25–500 s) before the FRET signal abruptly decreased to Eapp ∼ 0.4–0.45. This change in FRET corresponds to the formation of the Fen1/DNA complex .After injection of Fen1 and 10 mM MgEapp ∼ 0.6 (unbound state) to Eapp ∼ 0.3, confirming the assembly of the ternary complex (Eapp ∼ 0.4), followed by PCNA assembly (Eapp ∼ 0.3). Interestingly, the majority of reaction profiles analysed for the Fen1/PCNA/DNA ternary complex (∼90%) showed a quenching in Cy3 emission simultaneous to the cleavage event  and without PCNA, respectively. These values obtained at 20°C are very similar to those previously reported by us (0.022 s−1) at 25°C using an ensemble fluorimetric assay to monitor cleavage  between two conformations; a Y-shape structure and an extended form with a 168° angle between the 5F-duplex and the 3F-duplex  but unable to cleave , inducing a pronounced kink (90–100°) at the flap junction between both DNA duplex regions. Based exclusively on these ensemble data, it would be reasonable to conclude that an induced-fit binding model was in operation  and sm-FRET experiments , which agrees with previous studies reporting dissociation half-times >10 min for 5-nt ssDNA flaps (kobs = 0.006 ± 0.002 s−1). It is important to note that in these experiments Fen1 association is reported by flap opening using an intra-molecular FRET assay; thus the measured rate actually represents a combination of the initial binding event, most likely involving interaction of the H2TH domain with the 5F-duplex, plus any subsequent conformational rearrangements taking place on the flap   b and c. p ∼ 0.3) c and a rp ∼ 0.3)  and furtp ∼ 0.3) c. Altern2+ > 2 mM) or presumably a conformational selection model at lower Mg2+ ion concentrations. Our work confirms that Fen1 binding to the DNA opens the substrate at the flap base, as seen in the crystal structure, in addition to promoting a kink between the 5F- and 3F-duplex regions of the extended conformer (In summary, we demonstrate that unbound flap DNA substrates fluctuate rapidly between a Y-shaped conformer, with a structure relatively close to that observed in complex with Fen1, and an extended conformation . Fen1 bionformer . Fen1’s Supplementary Data are available at NAR Online, including [50–57].


Innate immune responses play a central role in neuroprotection and neurotoxicity during inflammatory processes that are triggered by pathogen-associated molecular pattern-exhibiting agents such as bacterial lipopolysaccharide (LPS) and that are modulated by inflammatory cytokines such as interferon γ (IFNγ). Recent findings describing the unexpected complexity of mammalian genomes and transcriptomes have stimulated further identification of novel transcripts involved in specific physiological and pathological processes, such as the neural innate immune response that alters the expression of many genes. We developed a system for efficient subtractive cloning that employs both sense and antisense cRNA drivers, and coupled it with in-house cDNA microarray analysis. This system enabled effective direct cloning of differentially expressed transcripts, from a small amount (0.5 µg) of total RNA. We applied this system to isolation of genes activated by LPS and IFNγ in primary-cultured cortical cells that were derived from newborn mice, to investigate the mechanisms involved in neuroprotection and neurotoxicity in maternal/perinatal infections that cause various brain injuries including periventricular leukomalacia. A number of genes involved in the immune and inflammatory response were identified, showing that neonatal neuronal/glial cells are highly responsive to LPS and IFNγ. Subsequent RNA blot analysis revealed that the identified genes were activated by LPS and IFNγ in a cooperative or distinctive manner, thereby supporting the notion that these bacterial and cellular inflammatory mediators can affect the brain through direct but complicated pathways. We also identified several novel clones of apparently non-coding RNAs that potentially harbor various regulatory functions. Characterization of the presently identified genes will give insights into mechanisms and interventions not only for perinatal infection-induced brain damage, but also for many other innate immunity-related brain disorders. Innate immune responses are pivotal in neuroprotection and neurotoxicity during various inflammatory processes, which are triggered by agents such as bacterial lipopolysaccharide (LPS) and modulated by inflammatory cytokines including interferon γ (IFNγ) produced mainly by T cells and natural killer cells Microglia and astrocytes are highly responsive to IFNγ, and microglia also to LPS The recent characterization of mammalian genomes and transcriptomes harboring unexpected complexity has prompted us to further isolate novel transcripts involved in specific physiological and pathological processes such as neural innate immunity. Besides microarray analysis Pregnant C57BL/6 mice were purchased from Nippon Clea Co. , and housed at 24±1°C on a 12-h light/12-h dark cycle with free access to food and water in the Laboratory Animal Center of Chiba University School of Medicine. All experimental procedures in this study were approved by the Animal Experiment Committee of Chiba University (Permit Numbers: 980005 and 20000008), and were conducted in accordance with the Guidelines for Proper Conduct of Animal Experiments of the Science Council of Japan.l-glutamine , 0.2% NaHCO3, 10% fetal bovine serum (Invitrogen), 10% horse serum (Invitrogen), 2% Nu serum , and 12 ng/mL of nerve growth factor . Cells were mechanically dissociated by trituration using fire-polished Pasteur pipettes and seeded in 100-mm dishes coated with 0.2% polyethyleneimine (Sigma). The cultured cells were maintained in the above mixture without the fetal bovine serum at 37°C in a humidified incubator with 5% CO2 atmosphere for 1 week. The cells were stimulated by using 10 µg/mL LPS  and 500 units/mL IFNγ  twice at 6 and 24 h before harvesting for the subtraction experiments.The neonatal mouse neuronal/glial mixed culture was essentially performed as previously described + RNA derived from 0.5 µg of total RNA was absorbed onto 50 µg of oligo(dT) magnetic beads Dynabeads Oligo(dT)25 , and was subjected to first-strand and second-strand cDNA synthesis ] in 20 µL of the buffer solution consisting of 20 mM Tris-HCl (pH 7.9), 10 mM MgCl2, 50 mM KCl, and 1 mM DTT at 37°C for 1 h. The reaction was stopped by adding 0.8 µL of 0.5 M EDTA-Na (pH 8.0), and the enzymes were inactivated by heating the mixture at 65°C for 20 min. The three 3′-trimmed cDNA-bead suspensions were combined, and the beads were washed three times with 50 µL of 10 mM Tris-HCl (pH 8.0)/1 mM EDTA . The bead-fixed 3′-trimmed cDNA was blunt-ended with 0.5 units of T4 DNA polymerase  in 20 µL of a mixture containing 50 mM Tris·HCl (pH 8.8), 15 mM (NH4)2SO4, 7 mM MgCl2, 0.1 mM EDTA, 10 mM 2-mercaptoethanol, 0.02 mg/mL bovine serum albumin, and 0.1 mM each of dATP, dCTP, dGTP, and dTTP at 16°C for 10 min. The reaction was stopped by adding 0.8 µL of 0.5 M EDTA-Na (pH 8.0), and the beads were washed three times with 50 µL of TE. The subsequent reactions were performed essentially as previously described Total RNA was prepared from the stimulated (LPS and IFNγ) and non-stimulated primary-cultured neuronal/glial cells using the acid-guanidine-phenol-chloroform method ynthesis , Step 1.2, 10 mM DTT, 2 mM spermidine, 1 mM ATP, 1 mM CTP, 1 mM GTP, 0.9 mM UTP, 0.1 mM biotin-16-UTP , 20 units of human placental RNase inhibitor , 0.3 µg of the amplified total cDNA, and 40 units of T7 or SP6 RNA polymerase (Roche Diagnostics) at 37°C for 2 h. Then, 20 units of DNase I (Roche Diagnostics) were added to digest the template cDNA, and the mixture was further incubated for 15 min. The synthesized cRNA was recovered by ethanol precipitation, washed with 70% ethanol, and dissolved in 20 µL of H2O.Driver cRNAs were prepared using the amplified total cDNA derived from the non-stimulated control cells as a template , Step 7a2O. To avoid hybridization at the linker sequences of both ends, 2 µg of the upper strand of the 5′-T7 linker and 2 µg of the 3′-SP6 oligo(dT) primer were added. The samples were made up to 0.3 M sodium acetate, precipitated with two volumes of ethanol, washed with 70% ethanol, and dissolved in 5 µL of 50 mM Hepes-Na (pH 8.3), 0.5 M NaCl, 0.02 mM EDTA, and 30% formamide. After heat denaturation at 98°C for 90 s to dissociate the tester double-strand cDNA (Step 7b), hybridization was performed at 68°C for 21 h (Step 8).Tester cDNA (0.5 µg) derived from the LPS/IFNγ-stimulated cells was mixed with 5 µg of the driver biotinylated cRNAs (2.5 µg each of sense and antisense strands) and dissolved in 30 µL of H2O plus 50 µL of the buffer solution containing 10 mM Tris-HCl (pH 8.0), 1 mM EDTA, and 2 M NaCl was added to the hybridized mixtures. The binding reaction of biotin and avidin was performed at room temperature for 10 min. After incubation at 55°C for 3 min to release the non-specific binding, the streptavidin beads harboring biotinylated cRNA-containing mixtures were absorbed using magnets, and the subtracted cDNA in the solution was recovered. After the addition of 20 µg of glycogen as a carrier, the subtracted cDNA was ethanol-precipitated, washed with 70% ethanol, and dissolved in 20 µL of TE.To remove the biotinylated cRNA-containing mixtures (Step 9), 1 mg of streptavidin-magnetic-beads Dynabeads M-280 Streptavidin  in 45 µL of H4)2SO4, 2 mM Mg SO4, 0.1% Triton X-100, 0.2 mM each of dATP, dCTP, dGTP, and dTTP, 0.1 µg/µL bovine serum albumin, 1 µL of the subtracted cDNA solution, 50 pmol each of the 5′ and 3′ PCR primers, and 1.8 units of PfuTurbo DNA polymerase . After heat-treatment at 94°C for 3 min, amplification was performed using 13 cycles of 1 min at 94°C, 2 min at 64°C, and 4 min at 72°C. The eight reactions were combined and supplemented with 20 µg of glycogen as a carrier. After extraction with phenol twice, phenol/chloroform twice, and chloroform twice, the PCR products were recovered by ethanol precipitation, washed with 70% ethanol, and dissolved in 20 µL of TE. Typically, 2 µg of PCR products from the subtracted cDNA were obtained. Using 0.5 µg of the subtracted cDNA as a tester, subtractive hybridization and PCR amplification were repeated.To amplify the subtracted cDNA (Step 10), PCR was performed in eight tubes each containing 50 µL of a mixture consisting of 20 mM Tris-HCl (pH 8.8), 10 mM KCl, 10 mM  was then transformed using the ligation products.Restriction ends for 2, 10 mM DTT, 0.5 mM each of dATP, dCTP, and dGTP, 0.2 mM 5--dUTP , and 400 units of reverse transcriptase SuperScript II (Invitrogen). The reaction was allowed to proceed at 42°C for 60 min. After alkaline degradation of the template RNA, the aminoallyl-modified cDNA was purified using MinElute . Coupling of the aminoallyl-modified cDNA (0.5 µg) with Cy3 or Cy5 CyDye  was performed according to the manufacturer’s protocol. The Cy3- and Cy5-labeled cDNAs were mixed, and made up to 40 µL of a solution containing 1.25 µg/µL yeast RNA, 1.25 µg/µL poly(A), 3.4×SSC (1×SSC: 0.15 M NaCl/15 mM sodium citrate), and 0.3% SDS. Hybridization with the microarray was performed at 65°C overnight under humidified conditions. After the hybridization, the array was washed twice for 5 min with 2×SSC/0.1% SDS at room temperature, twice for 5 min with 0.2×SSC/0.1% SDS at 40°C, and finally rinsed with 0.2×SSC. The array was centrifuged at 1,000 rpm for 1 min, and then scanned using the fluorescence laser-scanning device ScanArray4000 .A microarray chip was prepared as previously described Amplified total cDNA mixtures (0.2 µg per lane) were separated by 1% agarose gel electrophoresis. DNAs were visualized by ethidium bromide staining, alkaline-denatured, neutralized, and transferred to nylon membranes. Total RNAs (0.5 µg per lane) were electrophoresed in denaturing formaldehyde-agarose (1%) gels. RNAs were visualized by ethidium bromide staining, and blotted onto nylon membranes. For preparation of antisense strand-specific probes, plasmids harboring cloned cDNAs were linearized with an appropriate restriction enzyme, and then subjected to the synthesis of digoxigenin-labeled cRNA probes with SP6 polymerase, using a transcription kit (Roche Diagnostics). Hybridization, washing, and chemiluminescent detection on X-ray films were done as recommended by Roche Diagnostics. Densitometry was performed using ImageJ 1.46r .The statistical significance of the enrichment of LPS/IFNγ-induced clones by the subtraction was assessed using a Fisher’s exact test.in vivoTo isolate candidate genes involved in brain injuries such as PVL that is caused by perinatal infections, we used a primary mixed neuronal/glial culture derived from the cerebral cortex of postnatal day 1 mice. At this time point, the culture is expected to resemble the perinatal state of the brain. In addition, the mixed neuronal/glial culture more closely simulates physiological brain conditions than cell-type-specific cultures, and thus provides an excellent starting point for systematic investigations of the neural responses to LPS and IFNγ. The neuronal/glial cells were stimulated with 10 µg/mL LPS and 500 units/mL IFNγ. The LPS dose was chosen based on our previous studies, in which we examined NOS mRNA induction in the rat astroglioma C6 cell line + RNA derived from 0.5 µg of total RNA of the stimulated (LPS and IFNγ) or non-stimulated cells was absorbed onto oligo(dT) magnetic beads, and was used as a template for cDNA synthesis  prior to PCR. The restriction sites for each of these enzymes appear once in an average of 1,024 bp in random DNA sequences, and the combination of separate digestion with the three enzymes allows 99.7% of the cDNA population to be trimmed from the 3′ termini into less than 2,000 bp lengths. The restriction ends were blunted and ligated with a linker harboring the T7 promoter sequence (Step 3). The sense-strand cDNA liberated by heat denaturation (Step 4) was subjected to antisense-strand cDNA synthesis-coupled construction of the SP6 promoter sequence onto the 3′ terminus (Step 5). The total cDNA was amplified by PCR, using the linker sequences of both ends as primers (Step 6).To enrich the genes activated by LPS and IFNγ in the neuronal/glial cultured cells, we employed the subtractive cloning procedure described in the Materials and Methods and illustrated in ynthesis , Step 1.In the subtractive hybridization , we usedAs shown in P<0.001, Fisher’s exact test) higher than that of the clones derived from the LPS/IFNγ-stimulated cells, thereby confirming the successful enrichment of the LPS/IFNγ-induced clones by subtraction.Our system enabled the construction of unique restriction ends on 5′ and 3′ termini of amplified cDNA fragments Ccl2; No. 8), Ccl5 (No. 1), Ccl7 (No. 5), Ccl19 (No. 45), chemokine (C-X-C motif) ligand 1 , Cxcl9 (No. 4), Cxcl10 (No. 11), and Cxcl11 (No. 14). The second contains genes for GTPases involved in protection against intracellular pathogens Gbp2; No. 10), Gbp3 (No. 40), Gbp4 (No. 20), Gbp6 (No. 7), Ifi47 (No. 16), immunity-related GTPase family M member 2 , and T-cell specific GTPase 2 . The third category pertains to genes involved in antigen presentation: histocompatibility 2 (H2), class II antigen A, β1 , H2-DMa (No. 25), H2-K1 (No. 22), H2-Q10 (No. 58), β2 microglobulin , proteasome subunit β8 , Psmb9 (No. 18), Psmb10 (No. 32), transporter associated with antigen processing 1 ; and possibly proteasome assembly chaperone 4 , ubiquitin D , and ubiquitin-conjugating enzyme E2M .The LPS/IFNγ-activated genes with at least 1.5-fold change in the array analysis are listed in For several selected genes, the time-dependent changes in the mRNA levels in response to LPS and/or IFNγ were examined by Northern analysis . In the Cxcl10 and Cxcl11 loci, which in turn are located in a large (∼55 kb) intron of Art3. As previously noted Cxcl10 and Cxcl11 are activated by LPS and IFNγ  gene. This location is reminiscent of roles of antisense RNAs complementary to exon-intron boundaries of mRNA precursors in the regulation of alternative splicing, as has been demonstrated for a number of genes Clone No. 61  containeRiok2) gene. Riok2 has two major transcription start sites, and the clone No. 74 sequence is complementary to intron 3 or exon 1 of each alternative transcript. It is tempting to speculate that this antisense RNA differentially regulates alternative Riok2 transcripts in mRNA precursor processing and/or mRNA activity.Clone No. 74  containeIdentification of genes with altered expression levels in physiological and pathological processes is a promising strategy to obtain insights into underlying genetic mechanisms. We have developed a system of subtractive cloning coupled with in-house cDNA microarray analysis for the efficient isolation of differentially expressed genes. It enabled the identification of transcripts induced by LPS and IFNγ in primary-cultured mixed neuronal/glial cells derived from neonatal mice. Newly identified genes included those for novel putative non-coding RNAs potentially possessing various regulatory functions, suggesting the feasibility of the present system for direct isolation of the transcripts that represent emerging complexity of mammalian genomes and transcriptomes.H2-Kb and H2-Db genes, have been recently identified for their roles in the regulation of developmental ocular dominance As for protein-coding genes, the list  includedAn example of innate immunity-related disorders resulting in severe brain damage is PVL, which is a predominant form of cerebral palsy with white matter injury in preterm infants Many of the regulatory mechanisms underlying the cooperative or distinctive gene activation by LPS and IFNγ observed in Besides PVL, TLR4 stimulation is implicated in a number of brain injuries, as well as pathogen elimination


The DNA G+C content was 33.6 mol%. As determined in 16S rRNA gene sequence phylogeny analysis, the isolate belongs to the genus Sulfurimonas, within the class Epsilonproteobacteria, with 93.7 to 94.2 % similarity to the other species of the genus Sulfurimonas, Sulfurimonas autotrophica, Sulfurimonas paralvinellae and Sulfurimonas denitrificans. However, the distinct physiological and genotypic differences from these previously described taxa support the description of a novel species, Sulfurimonas gotlandica sp. nov. The type strain is GD1T ( = DSM 19862T = JCM 16533T). Our results also justify an emended description of the genus Sulfurimonas.A psychro- and aerotolerant bacterium was isolated from the sulfidic water of a pelagic redox zone of the central Baltic Sea. The slightly curved rod- or spiral-shaped cells were motile by one polar flagellum or two bipolar flagella. Growth was chemolithoautotrophic, with nitrate or nitrite as electron acceptor and either a variety of sulfur species of different oxidation states or hydrogen as electron donor. Although the bacterium was able to utilize organic substances such as acetate, pyruvate, peptone and yeast extract for growth, these compounds yielded considerably lower cell numbers than obtained with reduced sulfur or hydrogen; in addition, bicarbonate supplementation was necessary. The cells also had an absolute requirement for NaCl. Optimal growth occurred at 15 °C and at pH 6.6–8.0. The predominant fatty acid of this organism was 16 : 1ω7 Epsilonproteobacteria. Moreover, deep-sea vent chemolithoautotrophs are thought to be representatives of the earliest biological communities on Earth , a close phylogenetic relative (16S rRNA similarity of 95.7 %) and thus representative of the Baltic Sulfurimonas ‘Uncultured Helicobacteraceae G138eps1/GD17’ subgroup. Selected genomic and physiological data suggested an ecological role for GD1T, especially with respect to its sulfide detoxification ability . Water was collected in a free-flow bottle attached to a CTD-rosette from a depth of 215 m. The in situ temperature was 6 °C, the salinity 13 practical salinity units (PSU), and the sulfide concentration 11 µM. Directly on board, 100 µM KNO3 and 100 µM Na2S2O3 were added to the water samples, which were then incubated in the dark at 10 °C under anoxic conditions. For further isolation and cultivation in the laboratory, a modified version of artificial brackish water medium (ABW) . For anaerobic cultivation, the medium was boiled, bubbled with N2 for 30 min, and then autoclaved under anoxic conditions. Subsequently, anoxic and sterile-filtered 0.1 % (v/v) of the trace element solution SL10 (3 were added. The standard medium ABW+nitrate+thiosulfate (ABW+NS) was prepared by the variable addition of 10 mM KNO3 and 10 mM Na2S2O3, with the final concentration depending on the experiment. A pure culture was acquired by the dilution to extinction method and was cryopreserved at −80 °C in glycerol for long-term storage.Strain GD1ion SL10 , and 2–5T was cultivated in triplicate for 7–10 days at 15 °C in the dark. Growth was usually measured by counting 4′,6′-diamidino-2-phenylindol (DAPI) stained cells, observed using epifluorescence microscopy, or by flow cytometric determinations of SYBR-Green I (Molecular Probes) stained cells  whereas cell length, i.e. from pole to pole, was variable . The cells had a positive chemotactic response to nitrate /MgCl2 . 6H2O (g l−1)]: 0/0, 0/0.50, 2.50/0.38, 5.00/0.75, 7.50/1.13, 10.00/1.50, 12.50/1.88, 15.00/2.25, 17.50/2.63 and 20.00/3.00. The isolate had an absolute requirement for NaCl and grew best with between 10 and 20 g NaCl l−1; the upper limit for growth was not further determined. No growth was observed in media without added NaCl, in contrast to Sulfurimonas denitrificans DSM 1251T, which grew equally well without NaCl and at all NaCl concentrations tested  throughout the experiment whereas below and above this range it decreased by about 0.18–0.25 pH units. Optimal growth occurred over a wide pH range (6.7–8.0) but no growth occured at pH 6.0 and 8.4. The NaCl requirement was determined by cultivation in ABW+NS containing the following salt concentrations pyruvate (specific activity 0.6 GBq mmol−1) to cultures grown solely on pyruvate or on thiosulfate/pyruvate. After 24 h or 72 h of incubation, CO2 was degassed by the acidification of cell-free medium and trapped in ethanolamine. In nitrate/pyruvate medium, the growth of strain GD1T was accompanied by elevated CO2 production  on d-lactate  and rificans . This maT was determined to be 33.6 mol%, as calculated by analysis of the whole genome (The DNA guanine-plus-cytosine (G+C) content of strain GD1T based on 16S rRNA sequencing, preliminary searches in the EMBL Data Library were performed with the program fasta (arb (Proteobacteria (data not shown). In a pairwise analysis, it displayed highest (93.7–94.2 %) 16S rRNA gene sequence similarity to species of the genus Sulfurimonas and to the Baltic ‘Uncultured Helicobacteraceae G138eps1/GD17’ subgroup (95.7 %). Lower levels of relatedness (≤91 % sequence similarity) were determined for the other examined species belonging to the epsilon class of the phylum Proteobacteria.To establish the closest relatives of strain GD1am fasta . CloselyT, amongst the members of the class Epsilonproteobacteria .2, HS−, S0 and thiosulfate. Supplementation of bicarbonate can enable growth on formate, acetate, yeast extract, pyruvate or amino acid mix, but yields much lower cell numbers compared with growth on reduced sulfur or hydrogen. Sulfide concentrations of more than 20 µM inhibit, but up to 10 % of oxygen in the medium does not influence growth. Dominant cellular fatty acid is 16 : 1ω7c, with 14 : 0, 16 : 0, 16 : 1ω5c+t, and 18 : 1ω7c present in smaller amounts.Gram-negative, slightly curved or spirilla-shaped cells. Motile by one polar flagellum or two flagella at opposite poles. Cells exhibit a positive chemotactic response to nitrate. Cell sizes are 0.66±0.083×2.1±0.54 µm. Cells have a tendency to aggregate at older stages. Psychro- and aerotolerant. The temperature range for growth is 4–20 °C. Optimal growth occurs at 15 °C and pH 6.7–8.0. The cells have an absolute requirement for NaCl. Chemolithoautotrophic growth occurs with HT ( = DSM 19862T = JCM 16533T), isolated from water of a pelagic redox zone of the central Baltic Sea. The G+C content of the type strain is 33.6 mol%.The type strain is GD1


After 10 seconds of optogenetic stimulation, the evoked intracellular Ca2+ level and dopamine current of channelrhodopsin-2-transfected PC12 cells were 1.6- and 3.5-fold higher than those of the control cells. The optogenetic stimulation effects on Ca2+ influx and dopamine release were 81% and 63% inhibition by using a Ca2+ channel antagonist Nifedipine. The results indicate that optogenetic stimulation can evoke voltage-gated Ca2+ channel-dependent dopamine exocytosis from PC12 cells in a cell specific, temporally precise and dose-dependent manner. This proposed dopamine recording system can be developed to be a good cell model for dopamine regulation and drug screening in vitro, or dopaminergic cell implantation therapy in vivo using optogenetic stimulation in a precise and convenient way.Dopaminergic PC12 cells can synthesize and release dopamine, providing a good cellular model for investigating dopamine regulation. Optogenetic stimulation of channelrhodopsin-2 provides high spatial and temporal precision for selective stimulation as a powerful neuromodulation tool for neuroscience studies. The aim of this study is to measure dopamine release from dopaminergic PC12 cells under optogenetic stimulation using electrochemical recording of self-assembled monolayers modified microelectrode with amperometric measurement in real time. The activation of PC12 cells under various optogenetic stimulation schemes are characterized by measuring single-cell Ca Dopamine is an important catecholamine neurotransmitter implicated in physiological functions. Abnormal neurotransmission of dopamine can lead to numerous neurological disorders, such as Parkinson’s disease in vivo dopaminergic neuron stimulation successfully induced dopamine release in brain and conditioned animal behaviors Optogenetic stimulation approach that uses channelrhodopsin-2 (ChR2) has been developed for providing more precise and targeted stimulation effect on cells. ChR2-transfected neurons can be depolarized by exposure to ∼473 nm blue light in a very short duration (within 1 to 3 milliseconds), which immediately stops after the light is turned off. Because ChR2 expression can be targeted in specific types of cells, optogenetic stimulation allows potential therapeutic strategies to be investigated for neuroscience research and applications Various techniques, including neuroimaging 2+ is known to be essential for dopamine release from PC12 cells 2+ cascade effect in PC12 cells under optogenetic stimulation could provide in-depth information of the underlying mechanism. The results from Ca2+ signaling and real-time electrochemical monitoring of dopamine according to molecular imaging, we can delineate the Ca2+ dependency of dopamine release using blue light stimulation under different various Ca2+ buffers and voltage-gated Ca2+ channel (VGCC) antagonist. Combination of the ChR2-transfected PC12 cells, optogenetic stimulation and electrochemical recording could be a powerful model for dopamine research in vitro, which also has a high potential application to therapeutic treatment in animals.To better understand the optogenetic regulation of dopamine release from PC12 cells, the present study evaluates dopamine release from ChR2-tranfected PC12 cells using a surface-modified dopamine sensing electrode under various optogenetic stimulation schemes. Since intracellular CaChlamydomonas reinhardtii (UTEX 2247) was purchased from the University of Texas Algal Collection . Total RNA extraction and reverse transcription by Trizol® Reagent  and SuperScript® III  were performed according to the manufacturer’s instructions. The cDNA encoding the first 315 amino acids of ChR2 (GenBank accession no. AF461397) was gendered by polymerase chain reaction amplified with the primers 5′-AACTGCAGATGGATTATGGAGGCGCCCT and 3′-GGATCCCGCTTGCCGGTGCCCTTGTTGA. The cDNA fragment of ChR2 was then inserted into cloning vector pEGFP-N1  via the restriction sites of PstI and BamHI. The ChR2-EGFP plasmid was verified by DNA sequencing.The green algae PC12 cells , a cell line derived from a pheochromocytoma of the rat adrenal medulla, were cultured at 37°C in RPMI 1640 medium supplemented with 15% horse serum, 5% fetal bovine serum, 1 mM sodium pyruvate, 25 units/mL penicillin, and 25 units/mL streptomycin. For plasmid DNA transfection, the ChR2-EGFP plasmid was transfected using Lipofetamine® 2000 . The ChR2-EGFP or the controlled EGFP stable expressing PC12 cells were selected by 600 mg/mL G418 over one month.Φ = 75 µm, A–M Systems, USA) and inserted into a glass capillary which was drawn with a microelectrode puller. The end of the glass capillary was sealed with silicone rubber  and cured at 90°C for 12 hours. The tip of platinum microelectrode was cut by a scalpel, and the length of platinum micro-wire is extended ∼0.5 mm from the glass seal. For cleaning, the platinum plate (1.0 mm×1.2 mm) or microelectrode was cleaned for 30 minutes in each of ethanol and deionized water (18 MΩ·cm resistivity) and then immersed in 0.5 M H2SO4. Cyclic voltammetry (CV) was performed using an electrochemical analyzer  from −0.2 V to 1.5 V (vs. Ag/AgCl electrode) until the CV curves were stable. In order to coat Au-NP, the platinum plate or microelectrode was immersed in 0.1 M HClO4 solution  containing 10 mM hydrogen tetrachloroaurate . A constant voltage of −0.5 V relative to the Ag/AgCl was applied. Au-NP were uniformly coated onto the surfaces of platinum plates and microelectrodes via electrodeposition. The surface morphologies of native platinum and Au-NP deposited platinum plates were evaluated by scanning electron microscopy . To modify the self-assembled Au-NP monolayer, Au-NP deposited microelectrode was immersed in solutions of 5 mM mercaptopropionic acid  dissolved in 95% ethanol for 15 minutes. The microelectrodes were finally washed copiously with ethanol to remove any non-binding thiols. Cells were stimulated by blue laser and recorded extracellular dopamine by electrode simultaneously at the sampling rate of 10 Hz.For dopamine release recording, SAM-modified microelectrodes with high sensitivity and selectivity to dopamine were designed and fabricated. The working electrode was prepared with platinum micro-wire . The tip of the 400-µm fiber was fixed on a micromanipulator for guiding the blue light laser to the targeted cells. 1×106 cells/mL of ChR2-EGFP or EGFP stable expressing PC12 cells were seeded on a 25 µg/mL poly-D-lysine (PDL)-coated 6-well plate  with complete growth medium before optogenetic stimulation experiments. After culture for 24 hours at 37°C, the medium was replaced by 1.8 mM Ca2+-contained phosphate-buffered saline (PBS), Ca2+-free PBS, or 10 µM Nifedipine  in Ca2+-containing PBS. For optogenetic stimulation and dopamine sensing, the optical fiber and electrodes were fixed on the micromanipulator and aimed at the targeted cell as closely as possible, as shown in For all optogenetic stimulation experiments, a 473-nm fiber-coupled diode-pumped solid-state (DPSS) laser  with TTL modulation was used. The output power of the laser was adjusted to 10 mW/mm2+ levels were measured using the ratiomatric fluorescent dye fura-2/AM  under a single-cell fluorimeter . 1×106 PC12 cells/mL were seeded on a 25 µg/mL PDL-coated glass bottom dish and cultured in complete growth medium at 37°C for 24 hours. Cells were loaded with 5 µM fura-2/AM in PBS at 26°C for 1 hour. After loading, cells were washed three times with PBS and recovered for 30 minutes. Dishes were then placed on the stage of the single-cell fluorimeter for Ca2+ measurement. The ratiometric Ca2+ indicator, fluorophore fura-2/AM, was excited by fast switched 340 nm and 380 nm wavelengths for 50 ms, and emissions were the same at 510 nm. The duration between fast switched 340 nm and 380 nm wavelengths is 2 ms, and the illumination intensity is 10 mW. The relative Ca2+ level was calculated from the ratio of the emission intensities with excitations of 340 nm and 380 nm (ratio 340/380). Intracellular fura-2 fluorescence was sequentially imaged and the mean fluorescence intensity of individual PC12 cells was quantitatively analyzed by TILLvisION software of the TILL Photonics system . The Ca2+ imaging techniques were applied to ChR2-transfected PC12 cells with various optogenentic stimulation schemes.Intracellular Cat-test and ANOVAs were used for statistical analyses. A p value < 0.05 is considered as significantly different.All data were represented as mean ± SEM (standard error of the mean) in each experiment. Student’s −1. A pair of well-defined peaks for dopamine redox can be observed at Au-NP deposited microelectrode, with an oxidative peak potential at 0.25 V (vs. Ag/AgCl) and corresponding reductive peak potential at around 0.16 V. In contrast, these peaks of dopamine redox were not obvious on the CV curves of native platinum microelectrodes. In R2) of 0.999 and a detection limit of 7 nM at a signal-to-noise ratio of 3.−9 C can be observed in the controlled EGFP-expressing cells. In contrast, optogenetic stimulation evoked immediate elevations of dopamine signals about 1.36±0.08×10−9 C in ChR2-EGFP-expressing cells, which is significantly higher than that of the control (t(8) = −11.91, p<0.001; Student’s t-test). After termination of the blue light, the dopamine signals slowly decreased and returned towards the base values. Then, four durations  of blue light illumination were used to investigate the influence of stimulation duration on dopamine release from ChR2-expressing PC12 cells. As shown in R2 = 0.996) increased from 0.33±0.02, 0.99±0.03, 1.55±0.07 to 2.06±0.10×10−9 C for 5, 10, 15, and 20 seconds of stimulation duration, respectively. The photoelectrical artifacts were removed by direct subtraction.The controlled EGFP- or ChR2-EGFP-expressing PC12 cells were cultured in dish for dopamine release monitoring. After the medium was replaced with PBS, the sensing electrode recorded the extracellular concentration of dopamine change upon 10 seconds of blue light illumination. As shown in 2+ cascade is not clear. To investigate the optogenetic effect on the Ca2+ cascade, optogenetic stimulation were applied to PC12 cells in Ca2+-free buffer, 1.8 mM Ca2+ buffer and L-type VGCC antagonist Nifedipine-containing buffer (10 µM Nifedipine in 1.8 mM Ca2+ buffer). Relative intracellular Ca2+ levels are presented as the ratiometric fura-2 fluorescence intensity (ratio 340/380) by single-cell fluorimeter (two-way ANOVA of ChR2-EGFP/Ca2+ vs. EGFP/Ca2+ (p<0.001), ChR2-EGFP/Ca2+ vs. ChR2-EGFP/Ca2+-free (p<0.001), ChR2-EGFP/Ca2+ vs. ChR2-EGFP/Ca2+/Nifedipine (p<0.01); ChR2-EGFP/Ca2+/Nifedipine vs. ChR2-EGFP/Ca2+-free (p<0.001), ChR2-EGFP/Ca2+/Nifedipine vs. EGFP/Ca2+ (p<0.001) with Bonferroni’s post test; 2+ change of ChR2-EGFP-expressing PC12 cells (Δ ratio 340/380 = 0.121±0.007) was significantly higher than that of the controlled EGFP-expressing PC12 cells (Δ ratio 340/380 = −0.002±0.001) after 10 seconds of blue light illumination. In contrast, the intracellular Ca2+ change was inhibited (Δ ratio 340/380 = 0.009±0.004) when ChR2-EGFP-expressing PC12 cells were immersed in Ca2+-free buffer. Similarly, Nifedipine significantly inhibited the change of Ca2+ level (Δ ratio 340/380 = 0.045±0.003) upon optogenetic stimulation  = 146.8, p<0.001; one-way ANOVA with Tukey’s multiple comparisons test).It is well known that L-type VGCC plays an important role on dopamine release in PC12 cells. However, the optogenetic effect on the Ca2+ influx during optogenetic stimulation from 2+-free buffer (0.17±0.02×10−9 C) and Nifedipine/1.8 mM Ca2+ (0.09±0.01×10−9 C) (two-way ANOVA of Ca2+ vs. Ca2+-free (p<0.001), Ca2+ vs. Ca2+/Nifedipine (p<0.001), Ca2+-free vs. Ca2+/Nifedipine  with Bonferroni’s post test). The dopamine currents were significantly inhibited in both Ca2+-free buffer (0.17±0.02×10−9 C) and Nifedipine/1.8 mM Ca2+ (0.19±0.01×10−9 C) conditions in comparison to that for the 1.8 mM Ca2+ buffer (1.00±0.13×10−9 C) condition with the artifacts removed  = 33.52, p<0.001; one-way ANOVA with Tukey’s multiple comparisons test; It is known that that the L-type VGCC antagonist Nifedipine could block Ca2+ from ChR2-EGFP-expressing PC12 cells. For comparison, continuous illumination (CI) and stimulatory pulses of blue light with frequencies of 10, 20, and 30 Hz and durations of 10, 20, and 30 ms were applied to cells for 10 seconds. 2+ levels of PC12 cells increased with the increase of stimulatory frequency from 10 Hz to 30 Hz as well as with the increase of stimulation pulse duration. The highest elevation of Ca2+ (Δ ratio 340/380 = 0.130±0.020) occurred at stimulation pulses of 30 Hz and 30 ms which is about 3-fold of 10 Hz/10 ms stimulation pulse (Δ ratio 340/380 = 0.043±0.006). CI led to lower Ca2+ elevation (Δ ratio 340/380 = 0.095±0.004) compared to the 30 Hz/30 ms illumination  = 3.135, p<0.035; 20 Hz/30 ms vs. 20 Hz/10 ms, t(4) = 4.855, p<0.0083; 20 Hz/20 ms vs. 20 Hz/10 ms, t(4) = 2.854, p<0.0462; 30 Hz/30 ms vs. 30 Hz/10 ms, t(4) = 5.048, p<0.0072; 30 Hz/10 ms vs. 10 Hz/10 ms, t(4) = 3.264, p<0.031; 30 Hz/20 ms vs. 10 Hz/20 ms, t(4) = 2.835, p<0.0471; 30 Hz/30 ms vs. 10 Hz/30 ms, t(4) = 4.467, p<0.0111; 30 Hz/10 ms vs. 20 Hz/10 ms, t(4) = 2.858, p<0.046; 30 Hz/30 ms vs. 20 Hz/30 ms, t(4) = 3.704, p<0.0.0208 with Student’s t-test).To examine the effect of different stimulation schemes including stimulation frequency and pulse duration on the dopamine release and influx of Ca−9 C) and the 30 Hz/30 ms pulse (0.314±0.034×10−9 C) of blue light illumination evoked higher background dopamine currents compared to the 10 Hz/10 ms pulse (0.007±0.011×10−9 C). −9 C) was far lower than those of the 30 Hz/30 ms stimulation (1.215±0.135×10−9 C) and CI (1.006±0.128×10−9 C) with artifacts removed.The dopamine release of controlled EGFP- and ChR2-EGFP-expressing cells under various stimulation schemes was also observed. The photoelectrical artifacts of detected currents of EGFP-expressing PC12 cells under various stimulation schemes of blue light illumination were examined. The results are shown in 4 PC12 cells in 100 µL of medium under long period (5 hours) of high K+ stimulation + buffer (60 mM) for PC12 cell membrane depolarization. Our approach overcomes the disadvantages of a long diffusion time and the continuous replacement of K+ buffer, which is infeasible for studies in vivo. The present study validated that the evoked dopamine signals in ChR2-EGFP-expressing cells are significantly higher than those of EGFP-expressing cells . ChR2-mediated Ca2+ influx can active depolarized dopaminergic PC12 cells. An immediate dopamine release signal of around ∼30 pA (artifact-removed) can be recorded within 100 ms. On the other hand, the evoked dopamine signals can reach to 155 pA after 10 seconds of optogenetic stimulation. The signal stops rising immediately and slowly returns to the basal line in around 3 minutes upon dopamine reuptake and diffusion after turn off blue light.It is important to develop a delicate tool for effective control of dynamic process of dopamine, release or reuptake, from cells and real-time monitoring of extracellular dopamine levels for neuroscience investigation or clinical application. The developed microelectrodes at a diameter of 0.1 µm was enhanced with Au-NP sensing surface area for high sensitivity of detection limit of 7 nM for monitoring dopamine release from cells. Previous studies recorded the elevation of environmental dopamine levels at 10 nM/min/5×10+ stimulation passive depolarized cells only elevated 1.8-fold of dopamine level in 20 minutes from 5×105 cells/well of PC12 cells Moreover, the dopamine signal was elevated about 3.5-fold higher than those of cells without optogenetic stimulation, whereas the conventional high Ketal. measured Ca2+ dynamics in PC12 cells upon optogenetic stimulation by fluorescent indicators 2+ influx and the relationship to dopamine release. Single-cell Ca2+ measurement of fura-2/AM was used to characterize the optogenetic stimulation effect on the Ca2+ cascade in PC12 cells. In our findings, blue light did not affect the intracellular Ca2+ level in PC12 cells without ChR2 expression. In contrast, the Ca2+ level was significantly elevated by activation of ChR2 upon blue light illumination. The 340/380 ratio was elevated about 1.6-fold of control upon 10 seconds of optogenetic stimulation. However, the elevation immediately stopped and slowly returned to the basal level after the blue light was turned off. These results show the specific effect and temporal precision of optogenetic stimulation. Moreover, our findings indicate that PC12 cells with ChR2-EGFP expression had a higher basal intracellular Ca2+ level than that of EGFP-expressing cells, which might be caused by the self-leakage of ChR2 itself and/or activation of ChR2 by the ultraviolet light (340 nm and 380 nm) used for Ca2+ recording. Ca2+-free buffer and the L-type VGCC antagonist, Nifedipine, were used to explore the effect of optogenetic stimulation on extracellular Ca2+ influx. Nifedipine is a kind of L-type VGCC inhibitor, which can be a blocker in Ca2+-dependent dopamine release. Abu-Raya etal. showed that Nifedipine can block about 25% of intracellular Ca2+ elevation upon high K+ stimulation 2+ elevation induced by optogenetic stimulation. Additionally, the intracellular Ca2+ level barely changed when cells were immersed in Ca2+-free buffer. These results indicate that extracellular Ca2+ influx is the major source for cytosolic Ca2+ elevation during optogenetic stimulation. Then, the L-type VGCC plays an essential role in optogenetic stimulation-evoked Ca2+ influx. This phenomenon can be explained as ChR2-mediated extracellular Ca2+ influx slightly depolarized PC12 cells, which turns to activate L-type VGCC then following large Ca2+ influx. Compared to those using high K+ buffer, the optogenetic approach is more controllable and can be easily manipulated using Ca2+-free buffer or L-type VGCC blocker.Previous studies have demonstrated that optogenetic stimulation can induce cell membrane depolarization of PC12 cells. The relation between blue light intensity and evoked potential has been examined 2+-free buffer and Nifedipine on the modulation of dopamine release were examined after the cascade of Ca2+ influx was characterized. Our results in 2+-free PBS) and 81% (Nifedipine in 1.8 mM Ca2+ PBS) inhibition of dopamine signals were found in comparison to that of the controlled group (1.8 mM Ca2+-containing PBS). The high correlation between Ca2+ influx and dopamine release indicates that L-type VGCC might play a major role in dopamine release from PC12 during optogenetic stimulation. As shown in 2+ elevation by blue light is in a frequency- and time-dependent manner. The effects of illumination parameters were similar in Ca2+ influx and dopamine release experiments. However, a higher power-level of blue light caused stronger artifacts in the dopamine signal as The effects of Ca2+ imaging (340 nm and 380 nm) and for activation of ChR2 (325–550 nm). It may cause a large portion of ChR2 has already activated by 340 nm and 380 nm before a start of 473 nm pulses. Also, 380 nm is more efficient in activating ChR2 than 340 nm, causing a biased readout on the 340/380 ratio. In order to obtain continuous Ca2+ measurement during 473 nm illumination for ChR2 excitation, the potential solutions to overcome this problem by using red Ca2+ indicator with ChR2, green Ca2+ indicator with red-shifted ChR2, or using the bioluminescent Ca2+ indicator Nano-lantern 2+ indicators are available with the apply on ChR2 stimulation, including synthetic dyes  and protein-based red Ca2+ sensors  The problem and limitation on this study is the overlap between excitation wavelengths for Ca2+ influx and dopamine release under optogenetic stimulation. Furthermore, varied stimulation schemes to modulate ChR2 activation of PC12 cells have been examined. The 30 Hz with 30 ms pulse duration evoked more dopamine release than CI stimulation. The cationic channel ChR2 with sequenced activation and inactivation cycles based on its channel nature. So, the CI stimulation didn’t evoke more dopamine release compared to the 30 Hz with 30 ms pulse duration might due to it unreached the maximal activation of ChR2. The traditional stimulation approaches for dopamine release always limited the therapeutic potential of their studies by used of pharmaceutical or electrical stimulations. Based on our result, novel optogenetic stimulation approach would open powerful modulation scheme for further clinical application and investigation of dopamine regulation.Our results demonstrated that optogenetic stimulation could evoke and precisely control dopamine signals from PC12 cells. The extracellular dopamine signals can be evoked immediately within 100 ms when the ChR2-expressing cells were illuminated by blue light and slowly returned to the baseline after the stimulation was turned off. Optogenetic stimulation could be a powerful tool for activation of PC12 cells and controlling of dopamine release at high temporal resolution, easy manipulation and less side effects. In addition, the L-type VGCC on membrane might play a key role in the evoked Ca


Escherichia coli uropathogenic-specific protein (Usp) is a bacteriocin-like genotoxin, active against mammalian cells and associated with E. coli strains that provoke pyelonephritis, prostatitis and bacteraemia. Usp is encoded by a small pathogenicity island with three downstream small open reading frames (Imu1-3) that are believed to provide immunity to the producer. To prevent host suicide, colicins, bacteriocins of E. coli, form tight complexes with their cognate immunity proteins. Colicin – immunity protein complexes are among the strongest protein complexes known. Here, the Usp associated immunity protein 3 (Imu3) was partially characterized to gain insight into its role and mechanism of activity.The Isolation and partial characterisation of the Usp-associated immunity protein-3 (Imu3) revealed that, while Usp and Imu3 do not form a high affinity complex, Imu3 exhibits DNA and RNA binding activity. Imu3 was also shown to protect DNA against degradation by colicin E7.E. coli producing the genotoxin Usp.Our data infer that nonspecific DNA binding of the Imu3 immunity protein, prevents suicide of  Escherichia coli uropathogenic-specific protein (Usp) has been shown to be associated with E. coli strains that provoke pyelonephritis, prostatitis and bacteraemia, and with increased virulence and fitness of pathogenic strains of E. coli[E. coli bacteriocin colicin E7, which has nuclease activity, while the Usp N-terminal region is similar to the Type VI protein secretion system component (Hcp like)[E. coli strains and that it also enhances infectivity in the urinary tract. Recently, we demonstrated the genotoxic activity of Usp against mammalian cells[usp gene, there are three short open reading frames designated orfU1-3 , that are believed to be involved in the protection of the Usp-producing cell from its own nuclease activity[usp gene operon have a characteristic two-histidine region which appears to enable the inactivation of the Usp DNase activity[orfU immunity protein genes have been described. All three immunity proteins are thus not essential for the protection of the Usp producers, although Usp is lethal when it is expressed alone in E. coli. It has been postulated that none of the three proteins is exclusively required for Usp protein synthesis[E. coli Usp-associated immunity protein Imu3, previously designated OrfU3. Our study indicates that Imu3 has protective non specific DNA-binding abilities that could have possible biotechnological potential.The f E. coli-4. Nuclecp like)-7. It haan cells,8. To pran cells. Colicinan cells,11. In cactivity. The immactivity. Howeverynthesis. As protusp gene (data not shown) which were previously proposed to protect the Usp-producing cell against its endonucleolytic activity[Isolation of Immunity protein 3 (Imu3) with Ni-NTA affinity chromatography provided protein fractions with appropriate purity; Figure A. DNA biactivity. Neverthusp gene (without the immunity protein genes) was either lethal (liquid media) or resulted in severely diminished growth (plates). Of the three potential immunity proteins, when cloned separately downstream of the usp gene, Imu3 showed the greatest degree of protection as the number of transformants obtained was repeatedly higher, with larger colonies than for the other two , it repeatedly precipitated the DNA, which resulted in total retardation/precipitation of DNA in the electrophoresis , the effects of DNA retardation were decreased .Our data thus indicate that the Usp-producing cell is protected from the DNase activity of its own Usp by a mechanism that is distinct from that of colicin-producing cells. Surprisingly, EMSA showed that Imu3 binds linear and circular Figure B DNA as  Figure A. When I Figure A and C.  Figure B. The EM260) showed a stabilising effect of Imu3 on the linear double-stranded DNA molecule. The melting temperature  of DNA alone was 73°C, which increased by 3°C (Tm = 76°C) when an aliquot of 0.3 μg Imu3 was added in the EMSA studies. The DNA melting temperature was further raised by an additional 13°C (Tm = 89°C) when a 1 μg aliquot of Imu3 was added. This concentration of Imu3 saturated the DNA, and the melting curve revealed a two-phase thermal transition. One transition showed a stabilisation effect (89°C), whereas the other transition (at 63°C) was shown to be destabilising , most probably due to partial DNA precipitation . These EMSA tests showed that free Imu3 starts to bind to oligonucleotides longer than 11 base pairs, observed as the reappearance of unbound indicator DNA (absence of precipitation). These results indicate that 11 base pairs is the minimal DNA length required for Imu3 binding  were also performed however, the results were inconclusive since we repeatedly observed the recurring effect of unbound Imu3 that re-/dis-appeared every 3-5 nucleotides of the oligonucleotide length; however, the underlying basis of this phenomena is unclear.Separation of the DNA-Imu3 complex, was examined under different conditions. Exposure of the DNA–Imu3 complex to pH values between pH 3 and pH 13 showed separation at values between pH 11 and pH 12. However, at pH values higher than pH 12.5, DNA degradation was also observed. When the DNA–Imu3 complex was heated to 100°C for 5 min in the presence of different NaCl concentrations, separation of Imu3 from DNA was observed at 0.5 M NaCl or higher . All procedures were also performed with DNA that had not been previously complexed as a control, and no apparent losses in quantity or quality of DNA were observed (with exception of losses originating from the DNA purification procedure)  diluted solutions, where 1 μL (100 ng) of linear plasmid DNA was diluted in 15 mL. This procedure yielded less DNA as the control but could without doubt be optimised with appropriate protocol modification . Although all three immunity proteins of the Usp cluster share a high degree of sequence similarity, the present study shows that Imu3 has a distinct RNA- and DNA-binding ability. Nevertheless, additional as of yet unresolved mechanisms could be involved in protection of Usp producing cells by its cognate immunity proteins. Interestingly, protein-mediated DNA precipitation has been reported in studies describing eukaryotic histones and the E. coli global regulator, protein HU, a known DNA-binding protein[imu3 (IPTG induced for protein isolation) are toxic for producing cells. DNA-binding (basic) proteins usually have an overall positive charge that facilitates their binding to DNA. The Imu3 protein, has a theoretical isoelectric point of ca. 4.4, which implies that the DNA-binding region must be localised only on part of the tertiary structure of the molecule. Different online DNA-binding motif search tools were used to identify a potential Imu3 DNA binding motif[To the best of our knowledge, no known functions have been described yet for the protein products of  protein,14. Oper protein-17. We cng motif,19. The In conclusion, our study shows that Imu3 like the colicin E7 immunity protein Cei, does not form dimers and in addition, does not form a tight complex with the Usp protein. However, in contrast to the two other small proteins of the Usp pathogenicity island, Imu1 and 2, Imu3 does bind DNA and RNA. We propose that Usp producing cells are protected from genome fragmentation by Imu3 DNA masking. Further, as Imu3 precipitates but does not damage DNA we believe that could have biotechnological potential.E. coli) were amplified from the genomic DNA of the uropathogenic E. coli strain TA211 using standard PCR reactions. The Imu3n-F 5′-TTTCTCGAGCTATAATTTTAAAGATGAAATAG-3′ and Imu3n.R 5′-TTTACGCGTTATTTAGAGTCTTTAAACAAG-3′ primers were used, with XhoI and MluI restriction sites, respectively . The PCR product was cloned into the blunted pJET1.2 plasmid (Fermentas), and Usp-coding sequences were subsequently excised and re-cloned into the XhoI and MluI sites of the pET8c expression plasmid, with an N-terminal His tag (Novagen). Subsequently, Imu3 was expressed in the E. coli strain BL21(DE3) pLysE as described by the manufacturer (QIAgen). Briefly, an overnight culture of E. coli BL21(DE3) pLysE was diluted in liquid Luria Bertani medium supplemented with 120 mg/L ampicillin (LBAp medium) to an OD600 of 0.05 at 37°C, and grown to an OD600 of 0.6. Imu3 production was induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) to 0.8 mM final concentration. The culture was grown for an additional 4 h, and then the biomass was collected by 10 min centrifugation at 4,000× g.The nucleotide sequences that encode Imu3 , and complete EDTA-free protease inhibitor cocktail (Roche) was added. The cells were ruptured with 12 consecutive ultrasonication bursts  at the 55 setting (Sonics Vibra Cell). The cell lysates were cleared by three 20 min centrifugations at 20,000× g. All of the other protein isolation steps were carried out. When needed, Imu3 was further purified with size-exclusion FPLC chromatography  equilibrated with 50 mM Tris-HCl, pH 7.5, containing 0.15 M NaCl. Buffer exchanges were carried out using Amicon MWCO 3 kDa microconcentrators (Millipore). The his-tag was removed with the Thrombin Cleavage Capture Kit (Novagen) as described by the manufacturer. Actual mass of Imu3 protein was determined via mass spectrometry ESI + and Q-Tof .E. coli BL21(DE3) pLysE cells that were transformed with the plasmid pET8c carrying the combination of Usp and either Imu1, Imu2 or Imu3. The transformants were isolated on LB Ap plates with IPTG  after being grown overnight at 37°C.The degree of Usp-producing cell protection provided by each of the three individual immunity proteins (Imu1-3) was examined in Formation of a Imu3 dimer was checked using the cross-linking glutaraldehyde assay as previously described, native EcoRI) were initially incubated at 37°C for 30 min, to allow for potential multimerization. Samples were then subjected to either native PAGE resolution or to the glutaraldehyde cross-linking procedure and SDS-PAGE resolution, with the LexA protein as a dimerisation-positive control. Aditionally, Imu3 was checked for dimerisation with size exclusion chromatography , self-cleaved LexA protein was used as a standard .Imu3 samples (2 mg/mL) with or without the addition of 2.7 kbp double-stranded linear DNA  and the Imu3 protein were used to establish the nucleic-acid-binding ability of Imu3. The Imu3 was incubated with either the DNA or RNA in TE buffer  at 37°C for 30 min, prior to the electromobility shift assays (EMSAs) with 0.8% agarose gels. The effects of temperature , pH , NaCl (0-500 mM) and Mg2+ ions concentrations (0-50 mM) on the DNA binding ability of Imu3 were studied.Various concentrations of either -1 and an equilibration time of 1 min. The melting curves of buffer and of the Imu3 protein alone were subtracted from the melting curves of the DNA–Imu3 protein complex, providing curves that show only the changes in the thermal stability of the DNA.Thermal denaturation curves of linearised pUC19 DNA and the Imu3 protein were carried out in 5 mM cacodylic buffer (pH 6.5) using a UV-vis spectrophotometer  equipped with a thermoelectrically controlled cell holder. UV absorption was measured as a function of temperature (UV melting curves) for different ratios of linear DNA and Imu3 , at 260 nm. The UV melting temperature ranged from 25°C to 99°C, with a heating rate of 1°C•min2, 0.7% SDS, 1-3 M (NH4)2SO4 and 2.3 M guanidinium thiocyanate. The effects of temperature were studied 80°C and 95°C, with a 10 min incubation of the complex, and at 100°C, with a 5 min incubation.Further, the influences of pH, temperature and ionic strength on the separation of the DNA–Imu3 complex were examined. The effects of pH, were examined in the range from pH 3 to pH 13. Buffers used for these pH values were the following: pH 3-5, citric buffer; pH 6, MES buffer; pH 7-9, TRIS buffer; pH 10-12, glycine/NaOH buffer; pH 13, NaOH. The impacts of various ions on the separation of the DNA–Imu3 complex were studied as 0-1 M NaCl, 350 mM KCl, 350 mM NaSCN, 70 mM MgClE. coli DH5α, while the other half was subjected to a second restriction (EcoRI).To examine whether Imu3 binding to DNA triggers any DNA damage, religation experiments were performed. Initially, the linear plasmid DNA (pUC19) was incubated with the Imu3 protein at 37°C for 30 min, to allow for the DNA–Imu3 complex to form. The samples were subsequently purified using the QIAprep Spin Miniprep kits (QIAgen). To check DNA integrity, the linearised DNA was used for a (self) ligation reaction (Fermentas); half of the ligation mixture was transformed into PstI, with a single restriction site within the ampicillin resistance gene to yield one linear DNA fragment. Following gel electrophoresis the linear plasmid DNA was precipitated with Imu3 and centrifuged for 10 minutes at 4°C, followed by washing with 0.5 ml of TE buffer. The pellet was subsequently treated with the PCR Cleaning Kit (Thermo Scientific) and several μl of the isolate were employed for re-ligation. In control experiments, ligase was omitted. Transformants were isolated upon selection for tetracycline resistance. Five hundred transformants were further screened and all found to express ampicillin resistance.The structural integrity of the Imu3 precipitated plasmid DNA was also investigated on the basis of detection of potential mutations within a non-selected marker, the ampicillin resistance gene. For this purpose, plasmid pBR322 carrying both tetracycline and ampicillin resistance genes was employed. Plasmid DNA was digested with EcoRI). Oligonucleotides too short to form a complex with Imu3, bind to the indicator DNA provoking an electro-mobility shift. Samples were incubated for another 30 min at 37°C, to allow potential binding of Imu3 to the indicator DNA. The samples were later resolved on 0.8% agarose Tris-borate gels. The short DNA fragments were all synthesised as single-stranded oligonucleotides, the sequences of which are given in Table The minimum length of single-stranded DNA required for Imu3 binding was studied by initially saturating Imu3 with short DNA fragments of known lengths. The reaction mixtures of Imu3 (10 μg) and an at least 5-fold excess of oligonucleotides, were incubated for 30 min at 37°C  prior to the addition of the indicator DNA ; 4. 100 ng of pUC19/EcoRI complexed with Imu3 with His-tag removed (AE).Effect of His-tag presence on Imu3 DNA-binding ability. M: PageRuler Prestained Protein Ladder (Fermentas); 1. Imu3 with His-tag (SDS-PAGE); 2. Imu3 with His-tag removed (SDS-PAGE); 3. 100 ng of pUC19/Click here for fileDimerisation of Imu3 and USP proteins, 10% SDS PAGE gel. M: PageRuler Prestained Protein Ladder (Fermentas); 1. Imu3 protein (11.5 kDa); 2. USP protein (67 kDa). Samples following cross-linking with glutaraldehyde (3-7); 3. Imu3 protein, 4. USP protein, 5. Imu3 and USP, 6. Imu3 and USP with addition of DNA, 7. LexA protein mono/dimer (24/48 kDa).Click here for fileEcoRI DNA (100 ng); lane 2: Imu3-pUC19/EcoRI untreated complex; lanes 3-7: Imu3-pUC19/EcoRI complex treated with pH values 10, 11, 12, 12.5, 13; lanes 8-11: Imu3-pUC19/EcoRI complex incubated 5 min at 100°C in the presence 0, 0.2, 0.5 and 1 M NaCl; lanes 12-14: complex treated with 0, 0.5 and 1 M NaCl at pH 12.Representative electromobility shift assays on 0.8% agarose gels. Effects of pH, NaCl and temperature on DNA–Imu3 complex relaxation. Lane 1: pUC19/Click here for fileEcoRI (100 ng); 2. pUC19/EcoRI purified with GeneJet PCR purification Kit (Fermentas); 3. 100 ng of pUC19/EcoRI diluted 1.5 × 10-4 in 15 mL buffer and salvaged with Imu3 precipitation and subsequent GeneJet PCR purification Kit Imu3 removal.DNA precipitation from diluted systems. On agarose gel: 1. pUC19/Click here for file


Less predictable were the effects of the R107G substitution in MjHSP16.5 because of the differences in the oligomerization of metazoan and non-metazoan sHSPs. We present the crystal structure for MjHSP16.5 R107G and compare this with an improved (2.5 Å) crystal structure for wild-type (WT) MjHSP16.5. Although no significant structural differences were found in the crystal, using cryo-electron microscopy, we identified two 24mer species with octahedral symmetry for the WT MjHSP16.5 both at room temperature and at 60°C, all showing two major species with the same diameter of 12.4 nm. Similarly, at room temperature, there are also two kinds of 12.4 nm oligomers for R107G MjHSP16.5, but in the 60°C sample, a larger 24mer species with a diameter of 13.6 nm was observed with significant changes in the fourfold symmetry axis and dimer–dimer interface. This highly conserved arginine, therefore, contributes to the quaternary organization of non-metazoan sHSP oligomers. Potentially, the R107G substitution has functional consequences as R107G MjHSP16.5 was far superior to the WT protein in protecting βL-crystallin against heat-induced aggregation.The archael small heat-shock protein (sHSP), MjHSP16.5, forms a 24-subunit oligomer with octahedral symmetry. Here, we demonstrate that the IXI motif present in the C-terminal domain is necessary for the oligomerization of MjHSP16.5. Removal increased the  How such properties relate to the structure of sHSP oligomers is a key question.Methanocaldococcus jannaschii (MjHSP16.5) was the first sHSP to be structurally characterized by crystallography ).Escherichia coli. Recombinant protein expression was induced using 0.5 mM IPTG for 4 h, once the bacterial culture had reached an OD600 of 0.6. Harvested pellets were resuspended in 50 mM Tris–HCl pH 8, 1 mM EDTA, 300 mM NaCl, 1 mM DTT, 0.2 mM PMSF containing complete protease inhibitor cocktail (Roche) and lysed by several freeze/thaw cycles. A supernatant fraction containing the soluble MjHSP16.5 was prepared by centrifugation of the lysate at 15 000 rpm in a JA-20 rotor (Beckman) for 30 min at 4°C after incubation for 30 min with 10 U ml−1 of benzonase nuclease (Merck Bioscience) at 4°C followed by 0.005% (w/v) polyethyleneimine for 10 min on ice.The expression constructs of both WT and engineered  MjHSP16.5 were transformed into BL21(DE3) pLysS (b)Methanocaldococcus jannaschii is a hyperthermophile and lives in hyperthermal oceanic vents, which experience temperatures of approximately 50–95°C and NaCl concentrations of approximately 0.2–1.0 M [g for 30 min. By using this approach, almost all contaminating proteins are denatured and pelleted, and the resulting soluble fraction was 90–95% MjHSP16.5. The protein was then further purified by anion exchange chromatography and size exclusion chromatography (SEC). Purification by anion exchange chromatography used a Fractogel EMD-650S TMAE column. Peak fractions were pooled and then dialysed into 20 mM Tris–HCl pH 7.4, 100 mM NaCl in preparation for SEC..2–1.0 M . These o.2–1.0 M . With thThe R107G and G143X MjHSP16.5 proteins were purified using the same purification methods as WT MjHSP16.5, by heating and centrifugation, anion exchange and SEC. Purity was checked by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometry as described . For G14(c)hromeleon 6.30 software .To estimate the molecular size of the recombinant sHSP complexes, approximately 5 mg of purified proteins were subjected to SEC. Purified MjHSP16.5 proteins were analysed using a Superose 6 column (290 × 10 mm) at a flow rate of 0.2 ml per minute at room temperature using a Merck–Hitachi biochromatography system. The column was calibrated with the following molecular mass standards: thyroglobulin (669 kDa), apoferritin (440 kDa), β-amylase (200 kDa), bovine serum albumin (67 kDa) and carbonic anhydrase (29 kDa). The void volume of the column was determined by dextran blue (2000 kDa). The proteins were separated on the column in buffer containing either 20 mM Tris–HCl, pH 7.4 or 10 mM HEPES, pH 7.4 and 100 mM NaCl at room temperature, and the elution volume of each sample was used to estimate the molecular weight. The data were analysed using C(d)−1. The assay mixture contained citrate synthase and MjHSP16.5 at a substrate : chaperone (4 : 1) ratio in 220 μl at a temperature of 42°C. The effectiveness of MjHSP16.5 as a chaperone at 60°C was measured using βL-crystallin as the client protein [L-crystallin from bovine lens (Sigma-Aldrich) were dissolved in 50 mM sodium phosphate (pH 7.4), 100 mM NaCl. A chaperone-to-client ratio (1 : 1) by weight was used. Time-dependent light scattering was measured at 360 nm every 15 s over 30 min, using a Beckman DU640 spectrophotometer for both chaperone assays.Citrate synthase from porcine heart  was dialysed for 16 h at 4°C against 50 mM Tris–HCl pH 8.0, 2 mM EDTA and diluted to 0.49 mg ml protein  and α-cr(e)2 and 35 per cent MPD as precipitating agents. Crystals grew over a period of weeks to maximum dimensions of 1.0 × 0.5 × 0.5 mm.Initial crystallization experiments were performed using an Innovadyne screenmaker and standard sitting drop trays with drop sizes of 100 and 200 nl protein solution, respectively, mixed with 100 nl reservoir solution. Small crystals were observed in a number of conditions containing 2-methyl-2,4-pentanediol (MPD) and low molecular weight polyethylene glycol. The largest, best diffracting crystals were obtained from follow-up trays with hanging drop geometry using a reservoir solution of 0.02 M sodium acetate pH 5.5, 0.02 M CalCl(f)Crystals were typically tested using the in-house Bruker MicroStar rotating anode equipped with a platinum 135 CCD detector . All fin(g)−1. The sample (3.5–4.5 µl) was applied to the GIG holey grids  after the girds were treated with a glow-discharge machine (Master Plasmer). Then, the samples were blotted for 3 s with blot force three at 100 per cent humidity using the VitrobotMark IV . The temperature of the Vitrobot chamber was set to room temperature and 60°C, respectively, for both the WT and the R107G MjHSP16.5 proteins. The grids were fast frozen into liquid-nitrogen-cooled liquid ethane.The final concentrations of both R107G and WT MjHSP16.5 used for cryo-electron microscopy (cryoEM) study were 0.8 mg ml− Å−2. For the R107G MjHSP16.5 at room temperature, 126 raw CCD electron micrographs were collected manually. For R107G MjHSP16.5 at 60°C and the WT MjHSP16.5 at both room temperature and at 60°C, electron micrograph exposures were made with the automatic collection package Leginon [a); EMD–2289 for the second model of WT MjHSP16.5 at room temperature (b); EMD–2290 for the first model of WT MjHSP16.5 at 60°C (c); EMD–2291 for the second model of WT MjHSP16.5 at 60°C (d); EMD–2292 for the first model of R107G MjHSP16.5 at room temperature (e); EMD–2293 for the second model of R107G MjHSP16.5 at room temperature (f); EMD–2294 for the first model of R107G MjHSP16.5 at 60°C (g); EMD–2295 for the second model of R107G MjHSP16.5 at 60°C (h)).The frozen samples were imaged with a FEI 300 kV Titan Krios cryo-electron microscope equipped with a Gatan UltraScan4000 (model 895) 16-megapixel CCD. A nominal magnification of 96 000× corresponding to the pixel size 0.933 Å and a defocus range between −2 and −3 µm were used during data collection for the WT MjHSP16.5 both at room temperature and at 60°C and for R107G MjHSP16.5 at 60°C. For the mutant R107G at room temperature, a nominal magnification of 75 000× was used, which corresponds to the pixel size 1.196 Å. The electron dose per image was kept around 20 eperature b; EMD–22 at 60°C c; EMD–22 at 60°C d; EMD–22perature e; EMD–22perature f; EMD–22 at 60°C g; EMD–22 at 60°C h).(h)automatch, developed in Fei Sun's laboratory (to be published). The initial models for the earlier-mentioned four different samples were obtained by using ‘startoct’ in EMAN1 [actor [The defocus and astigmatism of each micrograph were estimated with CTFFIND3  and corrin EMAN1 . Two difin EMAN1 . During 1 [actor . CryoEM 1 [actor . The cry1 [actor .y-direction to obtain the respective one-dimensional profiles. The distance between the two minimal values of the one-dimensional profile was measured as the diameter of the reconstructed particle.The diameters of the reconstructed HSP16.5 particles were measured using the rotation-averaged projection method. First, the reconstructed volumes were projected by the same azimuthal angle to get the corresponding projections. Second, the projections were rotation-averaged using ‘proc2d’ in EMAN1 . Finally3.(a)E. coli (a). The WT and R107G MjHSP16.5 were both purified using the modified protocol, which included a heating step (80°C for 30 min in the presence of 1M NaCl) to remove as many E. coli proteins as possible. Purification was completed by a final SEC step. Although it was previously found that at 60°C the protein tended to aggregate [b). WT and R107G MjHSP16.5 were very similar in their elution profile and consistent with a calculated oligomer of 24 subunits. Interestingly, the G143X MjHSP16.5 SEC characteristics were consistent with a dimer being the most prevalent oligomer in solution (b). WT MjHSP16.5 prepared in this way did not aggregate when heated to 80°C, the protein remaining in the supernatant fraction after sedimentation (c). SEC was also used to prepare the highly pure material for crystallization experiments (d).The in vitro chaperone activity of WT, G143X and R107G MjHSP16.5 proteins was determined using an assay based on the heat denaturation of citrate synthase (e). Results show that both WT and R107G MjHSP16.5 can protect against the heat-induced aggregation of citrate synthase, providing comparable protection (e). Interestingly, the G143X MjHSP16.5 protein provided significantly increased (approx. two times) protection (e).Wild-type MjHSP16.5 and the two engineered proteins R107G (the G107 substitution) and G143X MjHSP16.5 (the IXI motif deletion construct) were produced recombinantly in E. coli a. The WTggregate , this wasolution b. WT MjHentation c. SEC waeriments d.The in synthase e. Resultotection e. Intereotection e.Figure(b)a). Clearly, the mutation does not cause any significant change in oligomeric state and overall structure (b).The R107G MjHSP16.5 crystallizes in the same rhombohedral space group with eight independent molecules per asymmetric unit as the WT protein [tructure b.Figureb). The root-mean-square deviation of 6299 equivalent main chain atoms is only 0.45 Å—clearly within the accuracy of the crystal structure determination. Hence, even locally, there are no significant structural changes detectable. However, it is noteworthy that the R107 in WT MjHSP16.5 forms hydrogen bonds to the main chain carbonyl oxygen atoms of G41 and M43, and forms electrostatic interactions with the residue E98 of the partner chain (c). Although the R107G substitution does not affect the overall crystal structure of the protein in the crystal environment, the loss of these two H-bonds and the inter-molecular electrostatic interaction in the R107G MjHSP16.5 variant may very well change the dynamic behaviour in solution.Closer comparison between the higher resolution WT structure presented here and the R107G MjHSP16.5 shows that there are no significant changes in the main chain positions b. The roer chain c. Althou(c)a), whereas the other is filled with very obvious central density (figure 4b). For the WT MjHSP16.5 at 60°C, there are also major octahedral structures obtained with the same diameter of 12.4 nm , similar to the room temperature particles. The final reconstructions of R107G MjHSP16.5 at room temperature were also converged to two structures with the same diameters 12.4 nm; one has an open structure around the fourfold axes  and the other has a closed structure at the same region . The chamber of both structures for R107G MjHSP16.5 at room temperature contains electron density.The WT and R107G MjHSP16.5 incubated at room temperature and at 60°C were imaged and analysed by cryo-electron microscopic single particle reconstruction see . Althougfigure 4g,h and electronic supplementary material, figure S1c,d). The two different diameters of R107G MjHSP16.5 particles at 60°C were obvious after two-dimensional reference free classification . The smaller of the two particles is similar in size (diameter 12.4 nm) to the R107G and WT MjHSP16.5 particles at room temperature. Interestingly, the central chamber of this smaller (12.4 nm) particle for R107G MjHSP16.5 is empty in contrast to similar-sized particles at room temperature. The larger of the two, however, has a significantly increased diameter of 13.6 nm and was larger than any other structure derived in this study. These two reconstructions of the particles revealed different oligomer arrangements in the fourfold symmetry axis and the dimer–dimer interface for R107G MjHSP16.5 oligomers . However, the distance between the dimers in the 13.6 nm particle is increased to reveal a large space at the dimer–dimer interface .The final reconstructions of R107G MjHSP16.5 at 60°C, however, revealed two significantly different structures with distinct diameters in vitro chaperone activity of MjHSP16.5. For this purpose, we used one of the earliest assays used to demonstrate the chaperone activity of sHSPs [L-crystallin (a) when present in a 1 : 1 ratio (b). Similarly, R107G MjHSP16.5 was equally effective in suppressing the heat-induced aggregation of this artificial client protein. This is in stark contrast to the WT MjHSP16.5 (a), which appeared ineffective. Under the conditions of this assay and in the absence of an effective chaperone, βL-crystallin formed aggregates that over the course of the assay were large enough to settle under gravity, which is the reason for the variable signal towards the end of the assay. Clearly, the effect of the addition of MjHSP16.5 was quite distinct from that of either α-crystallin or R107G MjHSP16.5.The cryo-electron microscopy data suggest that the R107G substitution allows for some oligomer flexibility and therefore we considered whether this could influence the of sHSPs  because of sHSPs , α-crystystallin a when pr 1 ratio b. SimilajHSP16.5 a, which 4.(a)e). Our data are consistent with previous reports and with a key role for this domain in the oligomerization of MjHSP16.5 [in vitro chaperone assays [Here, we have shown that removal of the C-terminal 5 residues from MjHSP16.5 is sufficient to significantly alter its oligomerization and also its chaperone activity towards citrate synthase e. Our dae assays . SimilarAltering the N-terminal sequences in MjHSP16.5 can also increase the particle size with subunit number increased from 24 to 48 and also the client-protein-binding properties of the protein complex . Swappin(b)Recent data suggest that the origin of metazoan sHSP particle size heterogeneity resides in the protein dimer and specifically the ACD . The CRYAs shown in this study, the introduction of glycine instead of arginine at position 107 had several interesting structural consequences for MjHSP16.5. In the first instance, the crystal structures revealed no significant difference in the electron density maps for the crystals of WT or R107G MjHSP16.5. This is likely due to the fact that in non-metazoans, the β6-strand exchanges between partner chains, which is absent in metazoan sHSPs ,15,45, tThe dimer–dimer interface within the 12.4 nm diameter particle is equivalent to the arrangement in the crystal structures of WT  and R107G MjHSP16.5 . The dis(c)L-crystallin at 60°C (The R107G substitution in MjHSP16.5 also has functional consequences as evidenced by the increased chaperone activity towards β at 60°C . The R12in vitro chaperone activity, which parallels data obtained for CRYAB [L-crystallin at 60°C as a result of the R107G substitution in MjHSP16.5 illustrates how such substitutions within the ACD can also contribute to structural and functional diversity of MjHSP16.5.Thermal-induced plasticity in chaperone activity combined with oligomerization has been observed for pea HSP18.1 . HSP18.1or CRYAB . While oor CRYAB ,52, the et al. [During the review of this manuscript, Shi et al.  reported