In response to brief glutamate exposure, NMDA receptors produce excitatory currents that have sub-maximal amplitudes and characteristically slow kinetics. The activation sequence starts when glutamate binds to residues located on the upper lobe of extracellularly located ligand-binding domains (LBDs) and then contacts lower lobe residues to bridge the cleft between the two hinged lobes. This event stabilizes a narrow-cleft LBD conformation and may facilitate subsequent inter-lobe contacts that further stabilize the closed cleft. Agonist efficacy has been traced to the degree of agonist-induced cleft-closure and may also depend on the stability of the closed-cleft conformation. To investigate how cross-cleft contacts contribute to the amplitude and kinetics of NMDA receptor response, we examined the activation reaction of GluN1/GluN2A receptors that had single-residue substitutions at the interface between LBD lobes. We found that side-chain truncations at residues of putative contact between lobes increased glutamate efficacy through independent additive mechanisms in GluN1 and GluN2A subunits. In contrast, removing side-chain charge with isosteric substitutions at the same sites decreased glutamate efficacy. These results support the view that in GluN1/GluN2A receptors’ natural interactions between residues on opposing sides of the ligand-binding cleft encode the stability of the glutamate-bound closed-cleft conformations and limit the degree of cleft closure, thus contributing to the sub-maximal response and emblematically slow NMDA receptor deactivation after brief stimulation.  Glutamate-activated channels sensitive to the synthetic agonists AMPA, kainate, or NMDA mediate almost all fast excitatory transmission between central neurons. Among these, NMDA receptors have several characteristic features that make them uniquely suited to the functions they serve in synaptic physiology: high calcium permeability, voltage-dependent block by magnesium ions, and distinctively slow deactivation kinetics  with 10% Fetal Bovine Serum at 37°C in 5% COPlasmids encoding rat GluN1 (UO8261) and GluN2A (M91561) were subcloned into pcDNA3.1. Residues were selected for substitution based on predictions from the glycine-bound (PDB: 1PB7) and glutamate-bound (PDB: 2A5S) GluN1-LBD crystal structures. The following single-residue substitutions were introduced: K483A and K483M in GluN1, and K487A and N687L in GluN2A, where numbering included the signal peptide. 2+ to prevent glutamate toxicity. Cells were used for electrophysiological recordings 24 - 48 hours post transfection.Plasmids were transfected using the calcium phosphate method at a ratio N1:N2A:GFP = 1:1:1. After 2 hours, the transfection medium was washed and cells were changed into DMEM supplemented with 2 mM Mgwww.qub.buffalo.edu); were amplified, low pass filtered at 10 kHz, at an applied pipette potential of +100 mV with Axopatch 200B (Molecular Devices); and were sampled at 40 kHz (NIDAQ board) into digital files. Single-channel currents were recorded using the cell-attached patch-clamp technique as described in detail previously . Fire po2, 1 CaCl2, 10 HEPES, and 11 EGTA at pH 7.4 (CsOH). Extracellular solutions, which contained (in mM): 1 glutamate, 0.1 glycine, 150 NaCl, 2.5 KCl, 0.01 EDTA, 10 HEPBS, 0.5 CaCl2 at pH 8.0 (NaOH), were superfused onto the recorded cell to elicit currents while clamping the cell at -70 mV. Currents were amplified, filtered at 2 kHz (Axopatch 200B), sampled at 5 kHz (Digidata 1440A) and acquired into digital files using pClamp suite software (Axon Instruments). Macroscopic currents were recorded with the whole-cell patch-clamp technique. Fire polished pipettes (4 - 6 MΩ) were filled with an intracellular solution containing (in mM): 135 CsF, 33 CsOH, 2 MgClResponses from excised patches were recorded from outside-out membrane patches using the same solutions as described above for whole-cell experiments. Two extracellular solutions, without (wash) or with 1 mM glutamate, were applied simultaneously through at 2.0 mm diameter glass theta tube (Harvard Apparatus) mounted on a piezoelectric translator (Burleigh LSS-3100/3200) to create a fast exchange between the two solutions onto the patch pipette. Open-tip potentials were measured at the end of each experiment to confirm that the 10-90% exchange occurred faster than 0.25 ms. Recorded currents were low-pass filtered at 5 kHz (Axopatch 200B), sampled at 50 kHz (Digidata 1440A) and stored as digital files with pClamp software.Single-channel currents were converted from an analog signal to a digital file using a National Instruments A/D card and QuB acquisition software. Channel openings are denoted by downward deflections of 7 - 10 pA, whereas channel closures are designated by the absence of recorded current (0 pA). Segmental k-means (SKM) algorithm based on hidden Markov model was used to idealize the single channel data . Each dapk) and steady state (Iss) current levels, as well as decay time constants for excised patches were determined using pClamp software based on the averages of (at least 5) traces obtained for each cell. Results were expressed as means ± SEM.For macroscopic currents, peak  occupied the resting state and during the 1 mM glutamate pulse they bound glutamate with the rate constants determined previously for N1/N2A receptors . Traces Crystal structures solved for the agonist-bound LBDs of N1 (PDB: 1PB7) and N2A (PBD: 2A5S) subunits illustrate clefts of similar but not identical geometries. In both structures, the ligands straddle the cleft and contact directly residues on both D1 and D2 lobes. In addition, the side chains of K483 and E712 in N1 and K487 and N687 in N2A extend across the cleft within 2.9 Å of one other, a distance consistent with non-covalent interactions  Figure Figure 1K/A and N2AK/A, respectively  and N2A(K487A), herein referred to as N1ectively 1. TheseK/A/N2A (n = 6), N1/N2AK/A (n = 6), and N1K/A/N2AK/A (n = 6) receptors expressed in HEK293 cells, and we compared these with traces recorded from wild-type  receptors (n = 13)  from N1(n = 13) 2. Consihigher open probabilities (Po) relative to WT receptors  were excluded from analyses, the remaining bursts revealed more clearly slightly but significantly higher activities: by 8% for N1K/A/N2A and N1/N2AK/A, and by 14% for N1K/A/N2AK/A .We were surprised to find that receptors carrying alanine substitutions, although presumably lacking stabilizing cross-cleft interactions, produced currents with eceptors 1. When K/A/N2A receptors solely to a 37% increase in mean open time , whereas that of N1/N2AK/A receptors originated solely from a 41% decrease in intra-burst mean closed time . These strikingly specific subunit-dependent effects carried over in the double mutant, which produced activity with both longer MOT (9.4 ± 0.8 ms) and shorter intra-burst MCT (1.6 ± 0.1 ms). These values were indistinguishable from the N1K/A/N2A and N1/N2AK/A, respectively (p > 0.16), but were significantly different from WT-values (p < 0.02). Inspecting the structures of open and closed interval distributions, we observed that although of similar magnitude, the K to A substitutions produced distinct and additive effects on NMDA receptor activity when introduced in N1 and/or N2A subunits. We traced the increased activity of N1Previously, we examined receptors with lobes that were covalently immobilized with disulfide bridges at cross-cleft distances comparable to the bonds illustrated by the LBD crystal structures and found that fixing the N1 lobes had no effect on channel kinetics whereas fixing the N2A lobes increased gating . Based oExisting views of glutamate receptor activation mechanism correlate an increase in agonist-elicited activity with an increase in the stability of closed-cleft conformations and/or to narrower cleft between D1 and D2 residues. The K-to-A substitutions we examined presumably eliminated the putative K-to-N interaction within the N2 pocket and simultaneously, according to the static crystal structure, created more cross-cleft space between these residues. Thus, the increased activity we observed may reflect not only the absence of interactions, but also the ability of the engineered cleft to become narrower than in wild-type receptors due to a reduction of steric hindrance. To investigate this possibility we designed and produced NMDA receptor subunits in which one of the residues within the native pair, K483/E712 in N1 and K487/N687 in N2A, were replaced with an isosteric residue that lacked charge.K/M, and in N2A we replaced N687 on D2 with leucine to produce N2AN/L  and N1/N2AN/L  (We expressed these mutated receptors in HEK293 cells and recorded currents from single receptors within a cell-attached membrane patch: N1 events) 1. For ao of N1K/M/N2A receptors, either across entire records or within bursts   1. Basedo for N1/N2AN/L receptors. No change occurred within the open durations (data not shown) and the lower Po resulted entirely from changes in the closed components similar to N1/N2K/A receptors  closed states and one aggregated open state 4 33]. . 33]. K/A/N2A receptors, the only rate constant affected was that for the closing transition C1O, which was significantly slower (160 ± 20 s-1) compared to WT   4, refleK/M/N2A receptors, we did not expect dramatic changes in rate constants. Several rates were significantly changed relative to WT, but these were in opposing directions, and, thus, had a nearly negligible effect on the overall gating characteristics of these mutants. The slower activity of N1/N2AN/L receptors was traced to the slowing of a single transition, C1 O, from 3,000 ± 300 s-1 to 1,900 ± 200 s-1 , whereas a less stable closed cleft, as in the N1/N2AN/L mutant, slows activation (slower kC1O).Since there were no gross kinetic changes in the gating reaction of N1 200 s-1 4. Inter closure 1 30]. . K/M/N2AK/A/N2A and N1/N2AK/A, and a small increase in macroscopic desensitization for the N1K/M/N2A and N1/N2AN/L receptors (vs. whole-cell for experiments) we observed a satisfactory concordance between model predictions and experimental measurement. If, as our single-channel observations indicate, the LBD cross-cleft interactions contribute to gating, it is important to determine how these structural features influence the NMDA receptor macroscopic response. A direct evaluation of how the mutations affect the amplitude of the ensemble current is not feasible experimentally because when measuring whole-cell currents the total number of receptors varies from one cell to the next and is unknown. Instead, macroscopic measurements inform about possible changes in the time course of the ensemble response. In an attempt to test our models, we simulated macroscopic currents with long (5 s) pulses of glutamate (1 mM), and we found largely WT-like kinetics for the K/A mutants, N1eceptors 5A. To tK/M/N2A and N1/N2AN/L, produced currents with similar or lower peak levels, respectively. More significantly, the deactivation rates were slower for both K/A substituted receptors, whereas the deactivation rate was increased for N1/N2AN/L. We should note that for these simulations we assumed that the microscopic association and dissociation rates for glutamate were not affected by the mutations, and, thus, the results only reflect the changes in gating we estimated in this study. To compare these simulated traces with macroscopically recorded responses, we measured currents from N1/N2AN/L receptors (n=4) residing in outside-out patches. Small membrane patches were pulled and exposed to 10 ms pulses of 1mM glutamate in the presence of continuous 0.1 mM glycine (N/L had faster deactivation time-constants relative WT receptors observed in the same conditions (280 ± 40 ms vs. 120 ± 13 ms), respectively [Next, we used the kinetic models we developed to evaluate how the LBD cross-cleft interactions may impact responses to brief (10 ms) synaptic-like exposures to glutamate 5B. Rela glycine 5C. Consectively . Based oK/M or N2AN/L, gating efficiency was unaffected if the mutation was in N1 and decreased, when the mutation was in N2A, the latter resulting in macroscopic currents with smaller peaks and faster deactivation. These results reinforce the distinct roles in NMDA receptor gating played by N1 or N2A cleft residues.In this study, we investigated the role of putative direct D1-D2 interactions within the LBDs of NMDA receptors and determined that the side chains examined contributed substantially to setting the time course of receptor deactivation. When a single lysine residue was replaced with alanine in either N1 (K483A) or N2A (K487A), presumably abolishing a native cross-cleft interaction and reducing bulk at the cross-cleft interface, gating efficiency was enhanced resulting in macroscopic currents with larger peaks and slower deactivation. In contrast, when the interaction was disrupted by eliminating the side chain charge on in N1CC/N2, were constitutively active and had WT-like kinetics (o: 0.57 ± 0.05 vs. 0.51 ± 0.03) [Previous studies have suggested that the stability of a narrow LBD cleft determines, in part, agonist efficacy . Thus, w ± 0.03) , an indiCC, produced significantly increased gating (o: 0.69 ± 0.04 vs. 0.51± 0.03). Further, the side chain truncation at K487 and the N1/N2CC increased gating efficiency with the same mechanism: a significant decrease in MCT (vs. 6.5 ± 0.8 ms). In addition, exchanging N687 for aspartate, which contributes a negative charge and promotes electrostatic contact, gating was also enhanced [In contrast, our results are consistent with the view that the D1-D2 interaction illustrated in the N2A LBD crystal structure between K487 and N687 contributes substantially to the shape of the glutamate elicited response of NMDA receptors. Exchanging N687 for leucine, which eliminates a hydrogen bond and adds bulk, significantly impaired NMDA receptor gating. However, a covalent bond engineered to fix these residues at the distance illustrated by the N2A LBD crystal structure, referred to as N1/N2enhanced ,27,30. TAn important observation in this study was that cross-cleft interactions we studied did not alter NMDA receptor modal behavior, as measured by the relative abundance of the three types of openings. In AMPA receptors modal behavior varies with agonist concentrations and is affected by mutations within the LBD cleft . Our resLastly, a consistent finding across several studies is that similar perturbations in N1 and N2A subunits make distinct contributions to gating. In the current study, we found the alterations in N1 and N2 to have additive effects suggesting independent contributions to gating by the N1 and N2 LBDs. Although agonist-binding to both subunits results in cleft closure each subunit implements its closed-cleft conformation via distinct interactions, these have separate contributions to the gating reaction, with N1 interactions in affecting the stability of the open states and N2 affecting mostly closed durations.In summary, our results demonstrate that changing the size or charge of residues facing the LBD clefts of NMDA receptors that may affect closed-cleft dimensions and/or stability have distinct and measurable effects on receptor gating. Thus, the interactions between mobile lobes are important determinants of gating efficacy, and they contribute to the characteristically slow deactivation kinetics of NMDA receptors.


The domestic chicken is an attractive model system to explore the development and function of brain circuits. Electroporation-mediated and retrovirus (including lentivirus) vector-mediated gene transfer techniques have been widely used to introduce genetic material into chicken cells. However, it is still challenging to efficiently transduce chicken postmitotic neurons without harming the cells. To overcome this problem, we searched for a virus vector suitable for gene transfer into chicken neurons, and report here a novel recombinant virus vector derived from avian adeno-associated virus (A3V). A3V vector efficiently transduces neuronal cells, but not non-neuronal cells in the brain. A single A3V injection into a postembryonic chick brain allows gene expression selectively in neuronal cells within 24 hrs. Such rapid and neuron-specific gene transduction raises the possibility that A3V vector can be utilized for studies of memory formation in filial imprinting, which occurs during the early postnatal days. A3V injection into the neural tube near the ear vesicle at early embryonic stage resulted in persistent and robust gene expression until E20.5 in the auditory brainstem. We further devised an A3V-mediated tetracycline (Tet) dependent gene expression system as a tool for studying the auditory circuit, consisting of the nucleus magnocellularis (NM) and nucleus laminaris (NL), that primarily computes interaural time differences (ITDs). Using this Tet system, we can transduce NM neurons without affecting NL neurons. Thus, the A3V technology complements current gene transfer techniques in chicken studies and will contribute to better understanding of the functional organization of neural circuits. Gallus gallus domesticus) has provided an attractive model system to understand the development and function of brain circuits. The chicks are born with their eyes open, and can actively explore from the moment they hatch The domestic chicken  vector is broadly applied to mammalian studies In this study, we demonstrate that the recombinant A3V vector efficiently transduces neuronal cells, but not non-neuronal cells in the chicken brain. We show that a single A3V injection into the postembryonic chick brain allows transgene expression selectively in neuronal cells within 24 hrs. Such rapid induction of gene expression raises the possibility that A3V vector can be utilized for studies of filial imprinting, which occurs during the early postnatal days Previous studies have shown that recombinant A3V vector transfers genes into various types of chicken cell lines and primary cultured cells 3 genome copies (GC) per cell. Then, 3 days after infection, cells were fixed and counterstained with DAPI (upper panels in +/DAPI+)   revealed+/DAPI+)  and S2. +/DAPI+) . On the +/DAPI+) . BecauseIn contrast to the species-specific transduction by these AAV vectors, the LV vector transduced not only HEK293T cells but also an appreciable number of chicken neural cells . Howeverinvivo and in ovo.As shown in 7, 5×108, and 5×109 GC) into the unilateral striatum at post-hatch day 5 or 6 (PHD5 or 6), conducted immunofluorescence labeling of EGFP, and analyzed the effects of the transductions 7 days after injection. As shown in 7 GC . This indicates that the A3V vector can efficiently and selectively transduce chicken neurons not only invitro but also invivo.Because young chicks can actively explore and rapidly learn from the moment they hatch, these precocial chicks have been considered as an attractive model to study neurobiological processes underlying memory formation  in 7 GC , but was5×109 GC . To exam2, n = 4) within 24 hrs. To examine the transduction selectivity of A3V for neuronal cells in the IMM, we also performed immunofluorescence labeling for NeuN. All the EGFP-positive cells in the IMM expressed NeuN , indicating that A3V selectively transduces neurons in the IMM. These results demonstrate that A3V technology can become a powerful tool to study memory formation in filial imprinting that occurs during the early posthatch period.Establishment of memory underlying filial imprinting is limited within the early postnatal days Animals utilize binaural interaural time differences (ITDs) in the microsecond range as a cue for localizing the sound source + NeuN+/NeuN+) in the following auditory brainstem nuclei: NM, NL, nucleus angularis (NA), and superior olivary nucleus (SON). As summarized in + EGFP+/EGFP+) were almost 100% in these four nuclei , indicating that the A3V vector can transfer genetic material efficiently to neurons in the embryonic auditory brainstem.Because the embryonic central nervous system contains various types of neural cells in different differentiated states, transduction efficiency and neuronal selectivity of A3V may depend on the embryonic stage. We therefore examined how the transgene expression pattern in the embryonic auditory brainstem varies, dependent on the injection timing, and addressed whether A3V selectively transduces neurons in the developing auditory brainstem. A3V-RSV-EGFP was injected into the lumen of the neural tube near the ear vesicle at E1.5, E2.5, E3.0, E3.5, and E4.5, and EGFP expression in the auditory brainstem was assessed at E17. A3V injection at E1.5 scarcely induced EGFP expression in these auditory nuclei (data not shown), whereas injection at E2.5 to E4.5 resulted in persistent EGFP expression in the auditory brainstem nuclei until E17 . To quanBecause NM axons serve as neural delay lines for ITD detection  [37], thFirst, to examine the sustained effect of a single injection of the Tet analogue doxycyclin (Dox), we performed A3V-TRE-EGFP and A3V-RSV-rtTAV16 injection into the neural tube at E3.0, applied Dox in the yolk at E6.0, and performed immunofluorescence labeling at E20 (n = 2 embryos) and E20.5 (n = 2 embryos) . As showTo more selectively transduce NM neurons in the NM-NL circuit, we next performed A3V-TRE-EGFP injection into the neural tube at E2.5, followed by A3V-RSV-rtTAV16 injection at E3.5, and then applied Dox in the yolk . As showDuring the embryonic stage, neurons in the NM and NL form tonotopically organized connections and show remarkable diversity in morphological properties depending on the characteristic frequency of sound. For morphological analysis of neural circuits, sparse and robust labeling of cells, such as Golgi staining, is useful. When diluted A3V-RSV-EGFP was injected, the EGFP signal in the auditory brainstem became sparse but weak (data not shown). To allow sparse and strong transgene expression in the NM and NL neurons, we employed the A3V-mediated Tet-inducible expression system. We injected A3V-TRE-EGFP and A3V-RSV-rtTAV16 at E3.5, followed by Dox administration at E6.5, and then EGFP expression was assessed at E9 and E17 . As showIn this study, we characterized recombinant A3V vector-mediated gene transduction in chicken brain cells. In comparison with AAV2 and LV vectors, A3V showed highly efficient, selective transduction of differentiated neurons. A single injection of A3V vector into postembryonic chick brain induced gene expression selectively in neuronal cells within 24 hrs, suggesting that A3V can be applied as a gene delivery tool to study imprinting memory, which becomes established in a relatively short period of time after hatching. We also applied A3V technology to the primary neural circuit that computes binaural ITDs, and engineered a method to transduce NM neurons without affecting NL neurons during the formation of the ITD detection circuit.Dependovirus) that is a naturally replication-defective DNA virus that can replicate only in the presence of a helper virus such as adenovirus or herpesvirus 14 GC/ml) based upon the standard protocols for mammalian AAV vectors . Future advanced technologies devised for other AAV vectors could also easily be introduced to the design and production of the A3V vectors.A3V belongs to an AAV group  supplemented with 10% fetal bovine serum , 100 units/ml penicillin, and 100 µg/ml streptomycin.Human embryonic kidney 293T cells  were maintained at 37°C with 5% CO5 cells/well). The medium was refreshed 1 hr after plating to remove excess debris. Half of the medium was changed after 3 days in vitro (DIV3).Primary cultures of chicken and zebra finch brains were prepared essentially by the methods described earlier S-M2 gene of pTet-On advanced vector (Clontech) so as to increase the sensitivity for doxycycline (Dox), as previously reported AAV transfer vector plasmids  were constructed by replacing the β-galactosidase gene of pA3V-RSV-β-Gal 6 cells per 15 cm dish the day before transfection. The medium was replaced with fresh medium 1–2 hrs before transfection. 293T cells were co-transfected with transfer vector plasmid, rep/cap-expressing plasmid and helper plasmid  by calcium phosphate precipitation (100 µg each for 12 dishes). The medium was replaced with DMEM containing 2% FBS 16–20 hrs after transfection. Cells were harvested 48 hrs after transfection, and pelleted by centrifugation at 1,100×g for 5 min. The pellets were lysed in 2.5 ml of 150 mM NaCl, 100 mM Tris-HCl pH 8.0 by a triple freeze-and-thaw procedure. The cell lysate was treated with 250 U/ml of benzonase  for 30 min at 37°C and centrifuged at 7,900×g for 60 min. The virus-containing supernatant was further purified by iodixanol  step gradient ultracentrifugation 5′-CCGTTGTCAGGCAACGTG-3′; reverse primer, 5′-AGCTGACAGGTGGTGGCAAT-3′; probe, 5′-FAM-TGCTGACGCAACCCCCACTGGT-TAMRA-3′ 13–1014 genome copies (GC)/ml.For the production of AAV2 and A3V, 293T cells were plated at 6×1012–1013 GC/ml.VSV-G-pseudotyped lentivirus particles were produced basically as previously described 8 genome copies (GC) of each virus vector was added to cultured neuronal cells at DIV3, or 293T cells soon after plating on 24-well plates at a density of 105 cells/well. For the quantitative analysis of virus transduction, cells 3 days after viral infection were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 30 min. To measure average EGFP intensity, fluorescent images of 3 random fields per well were taken with the same exposure using a 10×objective and a BZ-9000 fluorescence microscope . The background fluorescence was subtracted from each image using NIH ImageJ software  A single dose of 10Fertilized eggs of Barred Plymouth Rock chicken (n = 55) were obtained from a local supplier  and incubated in a humidified incubator at 37.5°C to desired stage For the striatum infection studies, we used White Leghorn chicks  at PHD5–6 (n = 12). For the IMM studies, we used Barred Plymouth Rock chicken (n = 3). Newly hatched chicks were used within several hrs after hatch. The chicks were deeply anesthetized with ketamine  and xylazine , and fixed on stereotaxic apparatus. Stereotaxic coordinates for the striatal injection were as follows: 6 mm anterior from the bregma; 1.8 mm lateral from the midline; 5 mm ventral from the pial surface After 24 hrs or 7 days from viral infection, chicks were perfused intraventricularly with 4% paraformaldehyde in 0.1 M phosphate buffer under deep anesthesia. Whole brains were dissected and post-fixed overnight. The fixed brains were cryoprotected with 30% sucrose in PBS overnight and embedded in O.T.C. compound . Frozen brains were cut into 40-µm-thick sections on a freezing microtome  and processed as free-floating for immunofluorescence labeling as follows. The sections were permeabilized and blocked with PBS containing 10% normal goat serum and 0.3% Triton X-100 for 60 min at room temperature, and incubated with rabbit anti-EGFP  and mouse anti-NeuN  or rat anti-EGFP  and rabbit anti-DsRed to visualize mCherry  in PBS containing 10% normal goat serum and 0.3% Triton X-100 overnight at 4°C. Following three washes with PBS, sections were incubated with Alexa Fluor 488-conjugated anti-rabbit IgG and 555-conjugated anti-mouse IgG or 488-conjugated anti-rat IgG and 555-conjugated anti-rabbit IgG  in PBS containing 0.5% normal goat serum and 0.1% Triton X-100 for 1 hr at room temperature. After three washes with PBS, sections were counterstained with DAPI, and mounted onto glass slides with Fluor Save mounting media . Images were obtained using 10×and 40×objectives with a BZ-9000 fluorescence microscope, and a 63×objective with a TCS-SP5 confocal microscope system. To quantify the EGFP-positive area per section, images through the section containing the injection site were tiled together using a BZ-II Analyzer. The tiled images were saved as 8-bit TIFF files (fluorescence intensity range 0–255) and the pixels (fluorescence intensity >99) were summed over the EGFP-positive area using NIH ImageJ software. The immunoreactive cells were counted manually.Results were presented as mean ± SD. Statistical comparisons were performed with a two-tailed Student’s t-test. Results were considered to be statistically significant when p<0.05.Table S1Raw data of . Quantification of overall gene expression.(DOC)Click here for additional data file.Table S2Raw data of . Quantification of overall gene transduction rate.(DOC)Click here for additional data file.Table S3Raw data of . The neuronal transduction rates of A3V and LV are represented as the percentage of MAP2 and EGFP double-positive cells within EGFP-positive cells.(DOC)Click here for additional data file.Table S4Raw data of . Gene transduction after LV or A3V injection was quantified by measurements of EGFP-expressing area in the parasagittal sections containing injection sites.(DOC)Click here for additional data file.Table S5Raw data of . A3V transduction rates in the embryonic auditory nuclei were quantified as the percentage of EGFP-expressing cells within total NeuN-positive cells in each nucleus.(DOC)Click here for additional data file.


Recently, it was shown that K2P5.1 is upregulated upon (autoimmune) T cell stimulation. The aim of this study was to correlate expression levels of K2P5.1 on T cells from patients with rheumatoid arthritis (RA) to disease activity in these patients.CD42P5.1 were measured by RT-PCR in the peripheral blood of 58 patients with RA and correlated with disease activity parameters  scores). Twenty patients undergoing therapy change were followed-up for six months. Additionally, synovial fluid and synovial biopsies were investigated for T lymphocytes expressing K2P5.1.Expression levels of K2P5.1 expression levels in CD4+ T cells show a strong correlation to DAS28 scores in RA patients. Similar correlations were found for serological inflammatory parameters . In addition, K2P5.1 expression levels of synovial fluid-derived T cells are higher compared to peripheral blood T cells. Prospective data in individual patients show a parallel behaviour of K2P5.1 expression to disease activity parameters during a longitudinal follow-up for six months.K2P5.1 expression levels in CD4+ T lymphocytes in the peripheral blood in cross-sectional as well as in longitudinal observations. Further studies are needed to investigate the exact pathophysiological mechanisms and to evaluate the possible use of K2P5.1 as a potential biomarker for disease activity and differential diagnosis.Disease activity in RA patients correlates strongly with K Rheumatoid arthritis (RA) is a chronic inflammatory disease which is characterized by pain, swelling and progressive destruction of multiple joints. The systemic nature of RA causes, next to loss of joint function, substantially decreased quality of life and increased mortality of patients. Current treatments are mainly based on immunosuppressive disease-modifying antirheumatic drugs, among them the rapidly expanding family of biologic agents . Close m2P5.1 (TWIK-related acid-sensitive potassium channel 2 (TASK2); KCNK5) belongs to the family of two-pore domain potassium channels (K2P channels) which has recently been shown to be expressed on T lymphocytes [2P5.1 is important for T cell functions such as proliferation or cytokine production [2+ levels during T cell stimulation [2P5.1 channel expression whereas pharmacological blockade or siRNA-induced gene silencing of K2P5.1 results in a reduction of T cell effector functions. It has additionally been shown that expression levels of this channel are strongly increased on T lymphocytes from the peripheral blood from clinically active relapsing-remitting multiple sclerosis (MS) patients. Interestingly, expression and MS-specific upregulation were found predominantly on CD8+ T cells rather than on CD4+ T cells which may be due to a disease-specific pathogenic role of cytotoxic T lymphocytes. Expression of K2P5.1 was even higher on cerebrospinal fluid (CSF)-derived T lymphocytes than in the peripheral blood and K2P5.1-positive T lymphocytes can be found within inflammatory lesions from MS patients. So far, it was not known whether these findings are MS-specific or can be similarly found in other autoimmune disorders. CD4+ T helper cells play an important role in the pathogenesis of RA. This is suggested by its association with certain MHC II loci, especially HLA-DRB1, and PTPN22, which is relevant for T cell function [The potassium channel Kphocytes . K2P5.1 oduction  as it ismulation . Moreovefunction .2P5.1 expression levels on T lymphocytes from RA with different disease activity parameters. The influence of different therapies was taken into account as they might potentially influence K2P5.1 expression. Finally, a longitudinal study was conducted in a subset of patients who underwent therapy change due to disease exacerbation and these patients were followed up for six months.Therefore, we investigated the correlation of KT lymphocytes were isolated from the peripheral blood of 73 RA patients  and 10 age- and sex-matched healthy donors. The patients were seen between April 2009 and August 2010 in the outpatient RA center at the University of Wuerzburg. We differentiated the patients according to EULAR criteria in patients in remission (DAS28 ≤2.6), patients with low disease activity (2.6 <DAS28 ≤3.2), with moderate disease activity (3.2 <DAS28 ≤5.1) and high disease activity (DAS28 >5.1). The DAS28 specifies the disease activity measuring the number of tender and swollen joints, the ESR and VAS of general health. Conventional therapy with disease modifying anti-rheumatic drugs (DMARDs) included leflunomide, hydroxychloroquine, sulfasalazine, methotrexate and glucocorticoids. Treatment with biologic agents included tumor necrosis factor alpha (TNFα) blockers  and anti-CD20 treatment (rituximab). Patients receiving interleukin (IL)-6 receptor blocking antibodies (tocilizumab) were excluded from the primary analysis because of its direct influence on CRP and ESR levels. However, they were included into the longitudinal study due to their influence on CRP and ESR values. In additional sets of experiments, the synovial fluid of patients who underwent joint punctures for diagnostic or therapeutic purposes was investigated. See Table + and CD8+ T cells were separated by magnetic cell sorting (MACS) according to the manufacturer's instruction  and purity was >95%. Direct cell isolation had no effect on K2P5.1 expression when compared to indirect cell isolation (data not shown). Synovial fluid from patients undergoing joint puncture for therapeutic or diagnostic purposes was processed accordingly.Peripheral blood mononuclear cells (PBMCs) from RA patients and healthy donors were isolated out of fresh blood samples by density gradient centrifugation using a lymphocyte separation medium . CD42P5.1-mRNA expression, RNA was purified using Trizol reagent  and cDNA synthesis was performed using a standard protocol with random hexamer primers . This cDNA was used in a RT-PCR assay with specific primers for KCNK5  and endogen control primers for 18sRNA . Real time RT-PCR was performed according to the manufacturer's protocol.For analysis of K+ T cells from healthy donors were treated with methotrexate , etanercept , adalinumab , certolizumab , tocilizumab  or hydroxychloroquine  over 24 hours before PCR analysis.In one set of experiments cultured CD4+ T lymphocytes were analyzed as described previously [2P5.1 and HRP-donkey anti-rabbit . HRP was inactivated with 2% NaN3 and blots were stained with β-actin antibody for protein loading control. Quantification of Western blot results was performed using Image J.Whole cell lysates from unstimulated and CD3/CD28-bead stimulated MACS-isolated CD4eviously  using ra2P5.1  and goat anti-rabbit Cy2 , CD4-FITC (RPA-T4), CD69-PerCP , and CD25-PE (Miltenyi). Flow cytometry was done using a FACSCalibur system  and CellQuest Pro Software (BD Bioscience).The following antibodies were used: rabbit anti-KImmunfluorescence staining was performed on human synovial tissue sections (n = 5). For double labelling, slices were postfixated in 4% paraformaldehyde (PFA) and incubated in blocking solution. Slices were then incubated consecutively with anti-CD3  and TASK2 (Sigma). Secondary antibodies were Alexa goat anti-mouse Fluor 488 and goat anti-rabbit Cy3 .t-test [P-values < 0.05 were considered statistically significant.All results are presented as mean ± standard error of measure (SEM). Statistical analysis was performed using a modified Student's t-test  in case 2P5.1 has been previously shown to regulate T cell function in vitro and in vivo. In a first set of experiments, freshly isolated CD4+ T cells from healthy donors were either left unstimulated or stimulated with CD3/CD28 beads for two days. A clear upregulation of K2P5.1 could be shown on protein level  and RA (CD4+ T cells). Finally, TASK2 expressing T cells could be identified immunohistochemically within human synovial tissue sections. Exemplary costainings for the T cell marker CD3 and TASK2 can be found in Figure The potassium channel Kpatients . Based od Figure . In pati+ and CD8+ T lymphocytes critically depend on constitutively expressed K2P5.1 potassium channels. Chronic stimulation as it may occur under autoimmune conditions leads to an upregulation of these channels on RNA and protein level. In a first set of experiments, MACS-isolated T cells from the peripheral blood of 58 RA patients were analyzed for expression of K2P5.1 by RT-PCR. We compared the correlation of K2P5.1 channel expression on both CD4+ and CD8+ T lymphocytes with different disease activity parameters. We found a positive correlation between K2P5.1 expression levels measured as Δct values and DAS28 on CD4+ T lymphocytes  showed at Month 3 a reduction in the DAS28 score Figure  and ESR s Figure . It shou2P5.1 levels were clearly downregulated in four and strongly upregulated in two patients to 3.0 and 3.5, respectively  which are also part of composite scores like DAS28 or DAS28-CRP reflect rather the level of systemic inflammation than the specific activation status of pathogenic immune cells. Moreover, their use seems to be limited especially in the case of tocilizumab which inhibits the systemic acute phase reaction at least in part independently from its well-proven anti-rheumatic effects [et al. who evaluated neutrophil CD64 as a biomarker for otherwise masked infection under tocilizumab therapy [2P5.1 expression changes behaved differently in tocilizumab-treated patients. Two out of nine patients even showed an opposite upregulation of K2P5.1 at three months preceding a relapse about three months later. At the present, it seems too early and patient numbers are too low to state that K2P5.1 upregulation generally precedes clinical relapses in RA, whether it reveals otherwise masked clinical developments under tolizumab therapy or whether its validity is limited under these circumstances. Therefore, in light of the current results further patient studies need to assess the prognostic value, time course and clinical validity of monitoring K2P5.1 expression in RA patients. Furthermore, it has to be proven whether K2P5.1 can be used for differential diagnosis.However, a number of questions remain to be addressed which are beyond the focus of this initial study, for example: Can K effects . This pr therapy . We obse2P5.1 function and regulation in the pathophysiology of RA. Especially the use of animal models for RA may help to provide insight in this context.Additionally, research efforts are needed concerning the mechanisms underlying K2P5.1 expression levels in CD4+ T lymphocytes and disease activity in patients suffering from RA. Since other studies already showed a functional role of K2P5.1 for T cell effector function this member of the two-pore domain potassium channel family might represent an interesting molecular target for diagnostic and/or therapeutic applications. However, further studies from independent cohorts are warranted to confirm and extend the presented findings. Furthermore, the use of animal models for RA might help to shed more light on the functional role of K2P5.1 in RA pathogenesis.We show here for the first time a correlation of KCRP: C-reactive protein; CSF: cerebrospinal fluid; DAS28: disease activity score of 28 joints; DMARDs: disease modifying anti-rheumatic drugs; ESR: erythrocyte sedimentation rate; MACS: magnetic cell sorting; MS: Multiple Sclerosis; PBMCs: peripheral blood mononuclear cells; PFA: paraformaldehyde; RA: rheumatoid arthritis; TASK2: TWIK-related acid-sensitive potassium channel 2; VAS: visual analogy scale.2P5.1-biology in inflammatory conditions.The authors declare no conflict of interest. AJH, SB, NB, HW and SGM collaborate in a project on KSB, NB and AHM isolated cells and performed RT-PCR. SB performed flow cytometry and NB was responsible for Western blotting. MF recruited the RA patients and assessed clinical data. KG and AJH performed immunohistochemical stainings on synovial biopsies, which were provided by RWK and AJH. SGM, HW, HPT and TB conceived and supervised the project. They provided continuous conceptual input, designed the experiments and provided financial support. SB, NB and MF wrote the first draft of the manuscript, which was finalized by AJH, RWK, SGM, HW, HPT and TB.Supplementary Figure S1. Quantitative K2P5.1 expression on the protein level in cells from RA patients compared to healthy controls. Western blot analysis of five individual RA patients (one to five) compared to two healthy controls (HD). Respective DAS28 scores are indicated at the upper part of the figure.Click here for fileSupplementary Figure S2. Influence of therapeutic agents on K2P5.1 expression. A) Correlation between K2P5.1 expression levels  on CD4+ T lymphocytes and DAS28 scores is shown for patients with convential treatments (black diamonds), TNFα inhibitors (grey diamonds) and rituximab therapy (white diamonds). B) Patient subgroups are shown with conventional treatment (left side), TNFα inhibitors (middle side) and rituximab (right side). C) The left bar graph representation shows the correlation coefficients between DAS28 and K2P5.1 expression levels. The DAS28 score for the treatment subgroups is shown on the right side.Click here for file


PIK3CA mutations, were most responsive to IGF-I induction resulting in upregulated AKT and p70S6K phosphorylation via PDK1 activation. PF-5177624 downregulated AKT and p70S6K phosphorylation, blocked cell cycle progression, and decreased cell proliferation and transformation to block IGFR-I induced activation in breast cancer cells. These results may provide insight into clinical strategies for developing an IGFR-I inhibitor and/or a PDK1 inhibitor in luminal breast cancer patients.Binding of IGF to IGF-IR activates PI3K to generate PIP3 which in turn recruits and activates proteins that contain a pleckstrin homology (PH) domain, including AKT and PDK1. PDK1 is highly expressed in breast tumor samples and breast cancer cell lines. Here we demonstrate that targeting PDK1 with the potent and selective PDK1 inhibitor PF-5177624 in the IGF-PI3K pathway blocks breast cancer cell proliferation and transformation. Breast cancer cell lines MCF7 and T47D, representing the luminal ER positive subtype and harboring  The insulin-like growth factor (IGF) system is a complex set of interactions comprised of the ligands IGF-I and IGF-II, their corresponding receptors (IGFR-I and IGFR-II), IGF binding proteins 1–6 (IGFBPs), insulin receptor substrate (IRS), and related downstream pathways. The IGF signaling pathway plays a critical role in cellular proliferation and inhibition of apoptosis. Multiple studies using cultured breast cancer cells and xenograft or transgenic mouse models have demonstrated a critical role for IGF-IGFR signaling in breast cancer progression and metastasis Binding of IGF to IGF-I receptor (IGF-IR) stimulates conformational change of the receptor and receptor tyrosine kinase activation, recruits and phosphorylates intracellular adaptor proteins such as IRS family members and SHC, and results in the activation of the PI3K pathway PIK3CA mutant breast cancer cells suppresses anchorage-independent growth, indicating a functional dependence on PDK1 in these cells PIK3CA mutations in human breast tumor samples The oncogenic activity of aberrant PI3K pathway signaling through PDK1 has been extensively studied. Hypomorphic mutation of PDK1 in PTEN+/− mice markedly protects these animals from developing a wide range of tumors PF-5177624 was synthesized as previously described http://www.sanger.ac.uk/. Prior to stimulation, cells were cultured without serum for 24 hours. Cells were stimulated with EGF , TNF-α, , IGF-I , or insulin  as indicated.BT20, HCC1954, MCF7, T47D, and MCF-10-2A cell lines were purchased from the American Type Culture Collection and cultured according to ATCC instructions. The gene mutation status of all cell lines was obtained from the Sanger COSMIC database: 2, 50 mM HEPES, 10% glycerol, 1 mM EGTA, 1% Triton X-100, 0.5% NP-40) supplemented with 1 mM Na3VO4, 1 mM PMSF, 1 mM NaF, 1 mM β-glycerophosphate, protease inhibitor cocktail (Roche), and phosphatase inhibitor cocktail (Roche) prior to use. Protein concentration was determined using the BCA Protein Assay (Pierce/Thermo Fisher Scientific) per the manufacturer’s instructions. Protein (30–50 µg) was resolved by SDS-PAGE and transferred onto nitrocellulose membrane. Blots were probed with primary antibodies to detect proteins of interest. After incubation with secondary antibodies, membranes were visualized by chemiluminescence (Pierce/Thermo Fisher Scientific). All antibodies were from Cell Signaling Technology, Inc with the exception of GAPDH (Santa Cruz Biotechnology) and phospho-p70S6K T252 (custom order). All western experiments were repeated at least two times.Cells were lysed in lysis buffer  and allowed to adhere overnight prior to serum-starvation. After 24 hours of serum-starvation, compound was added for 2 hours and cells were then subjected to IGF-I stimulation for 6, 18, 24, 48, or 72 hours. At each time point, cells were collected, fixed, and permeablized using the Cell Cycle Phase Determination Kit  following the manufacturer’s protocol. Samples were stored at −20°C until staining DNA with propidium iodide. Samples were also stained by anti-phosphorylated Histone H3 and anti-Bromodeoxyuridine (BrdU) to determine the number of cells in the mitotic and S phase stages. Anti-pH3 Fluor488-conjugated, anti-BrdU FITC-conjugated antibodies and BrdU were obtained from BD Bioscience and Sigma-Aldrich, respectively. Cells were labeled with 33 µM BrdU for 30 minutes before subjected to BrdU staining. A minimum of 10000 events were collected per sample on a BD FACSCalibur (BD Biosciences). Data analysis was done with FCS Express (De Novo Software). All assays were run in duplicate, and have been repeated at least twice.Cells were plated at 50,000 cells/well in a 96-well microtiter plate and allowed to adhere overnight. Cells were serum starved for 24 hours prior to compound treatment; PF-5177624 was added to each well  for two hours, followed by stimulation with IGF-I  as noted. After compound treatment and stimulation, cells were lysed and lysate transferred to the ELISA plate. The pAKT T308 chemilumiscent ELISA was performed per the manufacturer’s directions . All assays were run in duplicate, and have been repeated at least twice.Cells were plated at 3,000 cells/well in a 96-well microtiter plate and allowed to adhere overnight. PF-5177624 was added to each well  and incubated for three days. At 72 hours post-compound addition, Resazurin  was added to each well. Plates were incubated at 37°C for six hours and then analyzed for florescence . All assays were run in duplicate, and have been repeated at least twice.MCF7  and T47D  cells were plated for anchorage independent growth with compound in 0.35% BD Difco Noble agar (BD Diagnostic Systems) over a bottom layer of 0.5% BD Difco Noble agar containing MEM or RPMI, respectively. Cultures were maintained for a minimum of four weeks by weekly addition of compound in fresh agar (0.35%) containing medium. Microscope images of colony morphology were taken prior to colony visualization by addition of iodonitrotetrazolium chloride  for 18 hours. Colonies were counted using the colony count function on the AlphaImager Gel Imaging System (Alpha Innotech/Protein Simple). All assays were run in duplicate, and have been repeated at least twice.PIK3CA mutations yet basal AKT phosphorylaton levels are not similar and induced AKT and p70S6K phosphorylation by RTKs are also different. The basal phosphorylation levels of both AKT and p70S6K are not significantly high in MCF7 and T47D cells, and are stimulated by EGF, IGF-I, and insulin but not by TNF-α. Among these four growth factors, IGF-I induced the highest activation of pAKT in MCF7 and T47D cells  and pP70S6K (both T389 and T252) were markedly reduced in MCF7 , and resNext, we looked at cell cycle progression to investigate whether the PDK1 inhibitor PF-5177624 was able to block IGF-I induced cell growth. MCF7 and T47D cells were serum starved for 24 hours to synchronize cells in the G0/G1 phase and then treated with PF-5177624 or DMSO two hours prior to IGF-I stimulation (200 ng/ml). Cells were harvested at the indicated time points post-stimulation and subjected to FACS analysis to determine cell cycle progression under IGF-I stimulation conditions. In MCF7 cells, IGF-I induced DNA synthesis with an increasing S phase starting at 18 hours and one cell cycle was completed at approximately 48 hours . Additio50 values determined from the pAKT (T308) ELISA and cell proliferation assays treated with PF-5177624 are relatively similar, indicating a good correlation between kinase activity and cell proliferation. Furthermore, an anchorage independent growth assay was performed to evaluate whether inhibition of PDK1 activity would block cell transformation of MCF7 and T47D cells. Both MCF7 and T47D cells form colonies when cultured in soft agar, and addition of PF-5177624 decreased both colony size and colony numbers in the soft agar assay , while MCF7 and T47D cells represent luminal breast cancer cells. Although all four of these cell lines harbor a PIK3CA mutation, phosphorylation of AKT and p70S6K in MCF7 and T47D cells is not constitutively high and can be induced by IGF-I and other growth factors. These results highlight the difference in PI3K-AKT signaling in different breast cancer subtypes, and imply that growth factor stimulation may play an important role in luminal breast cancer cells harboring a PIK3CA mutation. Phosphorylation of AKT at T308 and p70S6K at T252 are indicators of PDK1 activity. Our data clearly show that IGF-I induced high levels of phosphorylation of AKT and p70S6K and that this activity could be modulated by inhibition of PDK1 activity in MCF7 cells. These data are also in line with recent publications demonstrating a role for IGF-IR/PDK1 signaling in the mediation of breast cancer cell growth PDK1 is downstream of PI3K and elevated phosphorylation of PDK1 has been associated with Cross-talk between the ErbB/HER family and the IGFR-I receptor signaling pathway has been described and suggested as a possible mechanism of resistance in breast cancer patients treated with the anti-HER2/neu antibody trastuzumab Collectively, there are multiple pieces of evidence indicating that PDK1 plays an important role in mediating cell proliferation, transformation, migration and other oncogenic mechanisms as previously described. In this study, we further demonstrated that IGF-I induced cell proliferation is mediated by PDK1 activity in luminal breast cancer cells. Therefore, the driving mechanism of PDK1 mediated tumorigenesis may be cell type dependent or may be variable based on the genetic background of specific tumor cells. For example, a recent study used knockdown of PDK1 by an RNAi approach but did not observe a block in prostate or leukemia tumor development in PTEN-deficient transgenic mouse models in vivo studies.Several PDK1 inhibitors have been described In conclusion, we have demonstrated that IGF-I signaling through the activation of PDK1 plays a critical role in the induction of cell cycle progression in luminal breast cancer cells. Furthermore, using the PDK1 inhibitor PF-5177624, we confirmed that inhibition of AKT and p70S6K can block IGF-I induced cell proliferation and transformation in MCF7 and T47D cells. These results provide evidence that inhibition of PDK1 may have greater anti-cancer therapeutic benefits in breast cancer patients with higher IGF-I/IGFR-I levels, and that a combination therapy featuring a PDK1 inhibitor and an IGFR-I inhibitor may have synergistic effects in this patient population.Figure S1(A), TNFα (B), IGF-I (C), or insulin (D) were added to the culture media for 15 minutes after starvation. Cells were subsequently harvested and lysed, and lysates were subjected to SDS-PAGE. Western blot analysis was performed to examine the phosphorylation levels of AKT and p70S6K. An untransformed breast cell line, MCF-10-2A, was cultured in growth media with or without FBS and treated with IGF-I for 15 minutes after starvation. Cell lysates were evaluated by western blot to examine the phosphorylation levels of AKT and p70S6K (E). GAPDH was included in each western as protein loading control.Breast cancer cell lines HCC1954 and BT20 were cultured in normal growth media (with 10% FBS) or serum starved for 24 hours. Growth factors EGF (TIF)Click here for additional data file.Figure S2(A) and (B). Relative activity of pIGFR, pAKT, and pS6K was determined by normalization of the density of the phosphorylated protein bands to GAPDH and total IGFR, AKT, and S6K. Data are from a representative experiment in MCF7 (A) and T47D (B). Similarly, relative activity of pIGFR, pAKT, and pS6K after PF-5177624 treatments was calculated, and plotted by normalization to DMSO treated samples, and the graphs of 2 hour treatment were shown for MCF7 (C) and T47D (D). The t-test was performed to determine if there were any difference compared to the DMSO treatment group; *  =  p<0.05, **  =  p<0.001, ***  =  p<0.005.The digital density of protein band in each western blot analysis was determined by FluorChem Q software for the blots shown in (TIF)Click here for additional data file.Figure S3MCF7 and T47D cells cultured in normal growth media were treated with 0.2, 1 or 5 µM of PF-5177624 at various time points, and phosphorylation levels of IGFR-I, AKT and p70S6K were determined by western blot.(TIF)Click here for additional data file.Figure S4(A) and T47D (B).Cells were serum-starved for 24 hours in order to synchronize cells at stage G0/G1. Cells were pre-treated with DMSO or PF-5177624 for 2 hours prior to addition of IGF-I for 6, 18, 24, 48, or 72 hours. Cells were subsequently harvested, fixed, and stained with PI and cell cycle profiles were obtained by flow cytometry. DNA content is shown in MCF7 (TIF)Click here for additional data file.Table S1The detailed relative activity changes of pIGFR-I, pAKT and pS6K by PF-5177624 treatment in MCF7 cells at all time points are summarized.(TIF)Click here for additional data file.Table S2The detailed relative activity changes of pIGFR-I, pAKT and pS6K by PF-5177624 treatment in T47D cells at all time points are summarized.(TIF)Click here for additional data file.Table S3The detailed cell population at the various cell cycle stages at each time point are summarized in MCF7 and T47D cells.(TIF)Click here for additional data file.


The interferon-induced dynamin-like MxA GTPase restricts the replication of influenza A viruses. We identified adaptive mutations in the nucleoprotein (NP) of pandemic strains A/Brevig Mission/1/1918 (1918) and A/Hamburg/4/2009 (pH1N1) that confer MxA resistance. These resistance-associated amino acids in NP differ between the two strains but form a similar discrete surface-exposed cluster in the body domain of NP, indicating that MxA resistance evolved independently. The 1918 cluster was conserved in all descendent strains of seasonal influenza viruses. Introduction of this cluster into the NP of the MxA-sensitive influenza virus A/Thailand/1(KAN-1)/04 (H5N1) resulted in a gain of MxA resistance coupled with a decrease in viral replication fitness. Conversely, introduction of MxA-sensitive amino acids into pH1N1 NP enhanced viral growth in Mx-negative cells. We conclude that human MxA represents a barrier against zoonotic introduction of avian influenza viruses and that adaptive mutations in the viral NP should be carefully monitored. Influenza A viruses of avian or swine origin sporadically enter into the human population but do not transmit between individuals. In rare cases, however, they establish a new virus lineage in humans. The mechanisms by which invading viruses overcome the species barrier are not well understood, but multiple adaptations to the new host are required. Surprisingly little is known about adaptive mutations that overcome restriction factors of the intrinsic and innate host defense system. In this study, we have identified adaptive mutations in pandemic strains A/Brevig Mission/1/1918 and A/Hamburg/4/2009 that confer resistance to the interferon-induced antiviral factor MxA which is a dynamin-like large GTPase that recognizes the incoming viral nucleocapsids and blocks their function. The resistance-enhancing mutations changed several amino acids in the viral nucleoprotein which is the main nucleocapsid component. These mutations were sufficient to increase the pathogenicity of an avian influenza virus strain in a Mx-positive mouse model. Interestingly, the resistance-associated amino acids are counter-selected in circulating avian influenza strains, because they compromise general viral replication fitness. The present data indicate that the innate immunity factor MxA provides a barrier against zoonotic introduction of influenza A viruses and that adaptive mutations in the nucleoprotein must be carefully monitored. Avian influenza A viruses sporadically transmit from waterfowl, their natural reservoir, into the human population To overcome the species barrier, multiple adaptations to the new host are required The human interferon (IFN) system represents a major innate defense against zoonotic viruses. Among the many antiviral factors induced by IFNs, the MxA protein is one of the most potent characterized to date In accordance with this model, recent findings suggest that NP determines the relative sensitivity of influenza A viruses toward the antiviral action of MxA. Avian influenza viruses were found to be generally more sensitive to MxA than human strains Here, we identified the amino acids critical for MxA resistance in the two NP proteins introduced into the human population in 1918 (by the “Spanish” H1N1 influenza A virus) and in 2009 (by the pH1N1 strain). These residues clustered into two distinct but overlapping “patches” in the body domain of the protein. We have previously shown that the polymerase activity of A/Thailand/1(KAN-1)/04 (H5N1) is highly sensitive to inhibition by murine Mx1, a close homolog of human MxA, in a polymerase reconstitution assay, and that this sensitivity is determined by the NP gene To test whether the amino acids apparently responsible for Mx1 resistance of 1918 NP could also confer Mx1 resistance to an Mx1-sensitive NP, we introduced the mutations R100I, L283P, and F313Y into H5N1 NP. We also tested the exchange R100V in NP, since screening of the NCBI influenza database revealed that valine rather than isoleucine is commonly found at this position in seasonal strains Next, we evaluated the capacity of NP from the 2009 pandemic H1N1 influenza A virus (pH1N1) to confer Mx1 resistance in the context of the H5N1 polymerase. Next, we investigated whether the identified amino acid clusters in NP of the 1918 and the pH1N1 strains also confer resistance to human MxA. Consistent with the findings observed with Mx1, both 1918 and pH1N1 NP increased resistance in the H5N1 polymerase reconstitution assay , howeverThe atomic crystal structure of the H5N1 NP V100, P283, and V/Y313 mainly responsible for Mx1 or MxA resistance , we analSince pH1N1 NP is derived from an influenza A virus of the classical swine lineage Sus scrofa domestica) decreased the H5N1 polymerase activity to 50%  assays Influenza A viruses carrying a novel NP gene were introduced into the human population in 1918 and 2009 We observed that the acquisition of Mx resistance had a negative effect on viral growth in the absence of MxA. When MxA resistance-enhancing mutations were introduced into highly pathogenic avian H5N1 viruses, the recombinant viruses grew less well than the wild-type H5N1 virus in MxA-negative MDCKII cells , in BALBCorrespondingly, we observed an increase in viral growth of pH1N1 lacking the MxA resistance-enhancing mutations D53, V313 and M316 in MDCKII cells  which doThe mechanism by which MxA exerts its antiviral function during infection or in the polymerase reconstitution assays is presently not known. MxA may block the viral life cycle at several early steps by interfering with the processes of vRNP entry and intracellular transport In summary, we have found functional and evolutionary evidence that the human MxA GTPase provides an efficient barrier against zoonotic introduction of influenza A viruses into the human population. Thus, the human MxA is a significant driving force in influenza A virus nucleoprotein evolution. We therefore propose that amino acids known to contribute to MxA resistance should be monitored as a strong indicator for the pandemic potential of newly emerging influenza A viruses.www.felasa.eu/guidelines.php) and the national animal welfare body GV-SOLAS (www.gv-solas.de/index.html). The animal welfare committees of the university of Freiburg, as well as the local authorities (Regierungspräsidium Freiburg) approved all animal experiments.All animal experiments were performed in compliance with the German animal protection law (TierSchG). The mice were housed and handled in accordance with good animal practice as defined by FELASA .The pHW2000 rescue plasmids The recombinant viruses A/Hamburg/4/09 (pH1N1) and A/Thailand/1(KAN-1)/04 (H5N1), and the NP-mutant viruses were generated by the eight-plasmid reverse-genetics system as described previously Renilla luciferase under the control of the simian virus 40 promoter to normalize variations in transfection efficiency. To evaluate the antiviral potential of Mx1 and MxA, we cotransfected Mx1- or MxA- encoding plasmid. A simultaneous experiment with cotransfection of the antivirally inactive mutants Mx1-K49A or MxA-T103A, respectively, was used as a control. To achieve equal amounts of transfected DNA, an empty vector plasmid was added. Twenty-four hours post transfection, cells were lysed and firefly and Renilla luciferase activities were measured using the dual luciferase reporter assay (Promega) according to the manufacturer's protocol. Reconstitution of the viral polymerase complex in avian and porcine cells was performed as above with the exception that the minigenome RNA was expressed under the control of a chicken HEK 293T cells seeded in 12-well plates were transfected using the Nanofectin transfection reagent (PAA Laboratories) according to the manufacturer's protocol. 10 ng of pCAGGS plasmids encoding PB2, PB1, and PA and 100 ng of NP-encoding plasmid were cotransfected with 100 ng of the firefly luciferase- encoding viral minigenome construct pPolI-FFLuc-RT, which is flanked by the noncoding regions of segment 8 of influenza A virus. The transfection mixture also contained 30 ng of pRL-SV40, a plasmid constitutively expressing +/+ containing 0.2% BSA for 1 h at 37°C. The inoculum was removed and 3 ml infection medium (DMEM supplemented with 0.2% BSA), additionally containing 1 µg/ml TPCK-treated trypsin for pH1N1 viruses, was added. Virus titers in cell culture supernatants were determined at the indicated time points by plaque assay and are expressed as PFU per ml.MDCKII cells seeded in 6-well plates were incubated with virus at a multiplicity of infection (MOI) of 0.001 in PBSFor determination of viral transcript levels in virus-infected MDCKII cells, cells were seeded in 6-well plates and infection was carried out with infection media. After the indicated time point post infection, cells were harvested in Trizol™ and RNA was purified according to the manufacturer's protocol (Invitrogen). Primer extension analysis was performed as described Mx1 mice (designated BALB-Mx1) carrying the functional Mx1 allele 50 values were calculated based on the infectious dose (PFU). All animal work was conducted under BSL 3 conditions in accordance with the guidelines of the local animal care committee.BALB/c mice were obtained from Janvier (Straßburg) and congenic BALB.A2G-www.pymol.org) was used to assign the indicated positions in the structural model of the NP of A/HK/483/97(H5N1) (PDB code:2Q06). The program I-TASSER (zhanglab.ccmb.med.umich.edu/I-TASSER) was used to generate a full length NP model of A/Thailand/1(KAN-1)/04 (H5N1), including amino acids 1–20.The program PyMOL  Reporter activity of 1918 polymerase. HEK293T cells were transfected with expression plasmids coding for PB2, PB1 and PA of the pandemic 1918 strain, the indicated NP proteins, the firefly luciferase encoding minigenome, 200 ng Mx1-encoding plasmid and a Renilla-expressing plasmid to normalize variation in transfection efficiency. Polymerase activity in the presence of antivirally inactive Mx1-K49A was used to normalize the data obtained with Mx1. Activity in the presence of the 1918*-NP was set to 100%. Error bars indicate the standard error of the mean of three independent experiments. Student's t-test was performed to determine the P value. **P<0.01. (B) 1918 polymerase activity in the presence of the antivirally inactive mutant Mx1-K49A. HEK293T cells were transiently transfected with expression plasmids coding for the vRNP components as described in (A) including 200 ng of Mx1-K49A-encoding plasmid. Renilla activity was used to normalize variation in transfection efficiency. The polymerase activity in the presence of the 1918*-NP was set to 100%. Error bars indicate the standard error of the mean of three independent experiments. Student's t-test was performed to determine the P value. NS, not significant. (C) H5N1 polymerase activity in the presence of either Mx1, the antivirally inactive mutant Mx1-K49A or empty vector. HEK293T cells were transiently transfected with expression plasmids coding for the vRNP components of H5N1 including 200 ng of Mx1, Mx1-K49A-encoding plasmid or empty vector and the indicated NP mutants. Renilla activity was used to normalize variation in transfection efficiency. The polymerase activity in the presence of the 1918-NP was set to 100%. Error bars indicate the standard error of the mean of three independent experiments. Student's t-test was performed to determine the P value. *P<0.05, **P<0.01, ***P<0.001; NS, not significant.(PDF)Click here for additional data file.Figure S2Identification of amino acids in 1918 NP responsible for MxA resistance. Reporter activity of the H5N1 polymerase in HEK293T cells after co-transfection of the expression plasmids coding for MxA (200 ng) and the indicated NP proteins (100 ng). The activity in the presence of MxA was normalized to the activity observed with the antivirally inactive mutant MxA-T103A. The activity observed with the 1918*-NP was set to 100%.(PDF)Click here for additional data file.Figure S3Polymerase activities in the presence of the antivirally inactive mutant MxA-T103A. H5N1 polymerase reporter activity was determined after co-transfection of expression plasmids coding for the indicated NP mutants (100 ng) and the antivirally inactive mutant MxA-T103A (200 ng). The reporter activity observed with the 1918-NP was set to 100%. Error bars indicate the standard error of the mean of three independent experiments. Student's t-test was performed to determine the P value. *P<0.05, **P<0.01; NS, not significant.(PDF)Click here for additional data file.Figure S4Localization of the adaptive mutation 16D in NP. The model for the full-length structure of H5N1 NP (A/Thailand/1(KAN-1)/04) harboring the mutations G16D, R100I, L283P and F313Y was generated utilizing I-TASSER. The N-terminus that was not resolved in the crystal structure (aa 1–20) is highlighted in light orange, whereas 1918-specific amino acids that confer Mx resistance are shown in blue .(PDF)Click here for additional data file.Figure S5Phylogenetic analysis of representative NP sequences and the presence or loss of Mx-resistance enhancing mutations. The maximum likelihood tree of 147 aligned sequences shows four genotypes, i.e., (i) the human H1N1, H2N2 and H3N2 viruses, (ii) the classical swine H1N1 viruses and pandemic (2009) H1N1 viruses, (iii) the European lineages of swine influenza viruses, and (iv) the North American avian influenza viruses. Strain designations and GenBank acc. nos. are presented. Numbers at nodes indicate bootstrap values obtained after 1,000 replications. Only bootstrap values greater than 50% are presented. The bar indicates substitutions per site. Alterations of amino acid positions shown to influence Mx resistance (sistance  are high(PDF)Click here for additional data file.Figure S6Mx resistance of NP variants of swine origin. (A–B) H5N1 or (C) pH1N1 polymerase activity in the presence of human MxA (A and C) or porcine Mx1 (poMx1) (B). The activity in the presence of human MxA or poMx1 was normalized to the activity observed after co-expression of the antivirally inactive MxA mutant MxA-T103A. The activity observed with the pH1N1-NP was set to 100%, respectively. Error bars indicate the standard error of the mean of three independent experiments. Student's t-test was performed to determine the P value. **P<0.01, ***P<0.001; NS, not significant.(PDF)Click here for additional data file.Figure S7Inhibition of polymerase activities by porcine and canine Mx proteins. (A) Swine NPTr or NSK cells were transfected with expression plasmids coding for NP, PB2, PB1 and PA of H5N1, the porcine Pol1-driven firefly luciferase encoding minigenome, 200 ng of either porcine Mx1 (poMx1), MxA, or the GTPase-inactive MxA mutant MxA-T103A and a Renilla-expressing plasmid to normalize variation in transfection efficiency. The activity in the presence of MxA-T103A was set to 100%. Error bars indicate the standard error of the mean of three independent experiments. Student's t-test was performed to determine the P value. **P<0.01; ***P<0.001. (B) HEK293T cells were transfected with expression plasmids coding for NP, PB2, PB1 and PA of H5N1, the firefly luciferase encoding minigenome, 200 ng of either canine Mx1 (cMx1), canine Mx2 (cMx2), MxA or the GTPase-inactive MxA mutant MxA-T103A and a Renilla-expressing plasmid to normalize variation in transfection efficiency. The activity in the presence of MxA-T103A was set to 100%. Error bars indicate the standard error of the mean of three independent experiments. Student's t-test was performed to determine the P value. ***P<0.001; NS, not significant.(PDF)Click here for additional data file.Figure S8Mx resistance-enhancing mutations influence transcription and viral growth in avian cells. (A) Comparison of viral transcription in MDCKII or avian LMH cells infected with an MOI of 5 of either H5N1 (KAN-1) or H5N1-NP-R100I,F313Y after the indicated hours post infection (h.p.i.). mRNA, cRNA, and vRNA levels were determined using primer extension analysis with primers specific for segment 6. Levels of cellular 5sRNA served as internal control. (B) Avian LMH cells were infected with an MOI of 0.001 of either H5N1 or H5N1-NP-R100I,F313Y and incubated at 37°C. At the indicated time points post infection (p.i.), virus titers were determined by plaque assay. Error bars indicate the standard error of the mean of three independent experiments.(PDF)Click here for additional data file.Figure S9Mutation D101G in NP of pH1N1 strongly reduces resistance to MxA. H5N1 polymerase activity after co-transfection of MxA (200 ng) and the indicated NP (100 ng) expression plasmids. The activity in the presence of MxA was normalized to the activity observed with the antivirally inactive mutant MxA-T103A. The reporter activity observed with the pH1N1-NP in the presence of MxA was set to 100%. Error bars indicate the standard error of the mean of two-three independent experiments.(PDF)Click here for additional data file.


JAK2 V617F and MPL W515L/K mutations in Philadelphia chromosome- (Ph-) negative myeloproliferative neoplasms (MPNs). The multiplex system comprises two snapback versus limiting primer sets for JAK2 and MPL mutation enrichment and detection, respectively. Linear-After exponential (LATE) PCR strategy was employed for the primer design to maximize the amplification efficiency of the system. Low ionic strength buffer and rapid PCR protocol allowed for selective amplification of the mutant alleles. Amplification products were analyzed by melting curve analysis for mutation identification. The multiplex system archived 0.1% mutation load sensitivity and <5% coefficient of variation inter-/intra-assay reproducibility. 120 clinical samples were tested by the multiplex snapback primer assay, and verified with amplification refractory system (ARMS), quantitative PCR (qPCR) and Sanger sequencing method. The multiplex system, with a favored versatility, provided the molecular diagnosis of Ph-negative MPNs with a suitable implement and simplified the genetic test process.A multiplex snapback primer system was developed for the simultaneous detection of  JAK2 exon 14 (JAK2 V617F) is presented in almost all the PV and half of the ET and PMF patients totally modified our understanding of these Philadelphia chromosome- (Ph-) negative MPNs [MPL exon 10 (MPL W515L/MPL W515K) were also identified in around 10% of the JAK2 V617F mutation-negative ET and PMF patients [JAK2 and MPL mutations are also considered as the driver molecular alternation of the disease because of the resulting constitutive activation of JAK-STAT pathway. Consequently, these mutations are now incorporated into the diagnostic criteria of the Ph-negative MPNs [The classical myeloproliferative noeplasms (MPNs), according to the definition of World Health Organization (WHO) classification criteria revised in 2008, encompass chronic myelogenous leukemia (CML), polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) . In 200514 JAK2 V17F is prMPL mutations in ET and PMF. Unfortunately, Sanger sequencing, the current gold standard for mutation identification, is not able to detect these low-abundance mutations (mutation load < 20%) [The prevalent tools for the identification of these mutations are mostly based on the common techniques such as sequencing, allele specific PCR , and quantitative PCR (qPCR). However, there are also several major defects in these methods. For instance, sequencing and allele specific PCR are both labor-intensive and time consuming due to the manual post-PCR procedures, which lead to the unfavorable turnaround time and latent risk of DNA contamination. Although the allele specific qPCR is an efficient closed-tube procedure for mutation identification, this fluorescent based method still costs false positive results . Moreoved < 20%) . Besidesd < 20%) . Thus, tInspired by the scorpion probe, Zhou et al.  developeJAK2 V617F and MPL W515L/K mutations in the classical Ph-negative MPNs patients.In this study, we employed the snapback primer concept and constructed a multiplex snapback primer system for simultaneous detection of trace-amount JAK2 V617F homozygous mutant human erythroleukemia (HEL) cell line and JAK2/MPL homozygous wild-type multiple myeloma (RPMI 8226) cell line were purchased from the cell bank of type culture collection of Chinese Academy of Sciences. The HEL cell line was used as JAK2 V617F mutation-positive control, while the RPMI8226 cell line was used as JAK2 V617F and MPL W515L/K mutation-negative control. Genomic DNA from the cells was extracted with QiaAmp DNA Blood Mini Kit  according to the manufacturer's instruction to achieve the final concentration of 20 ng/μL.MPL W515L and MPL W515K were constructed with pMD19-T simple vector utilizing the overlap mutagenesis technology [MPL amplicon copy number as in the 20 ng/μL genomic DNA from RPMI8226 cells with deionized distilled water (ddH2O) (3 × 10E-4 ng/μL).Artificial plasmids with the chnology . The ins2O to a final concentration of 15–25 ng/μL. In compliance with Helsinki Declaration of 1975 as revised in 1996, this study was approved by the Institutional Review Board of Huashan Hospital.The peripheral blood samples were obtained from 50 PV, 50 ET, and 20 PMF patients in the Department of Hematology in Huashan Hospital of Fudan University. All these Ph-negative patients were diagnosed according to . WrittenTm) of the limiting primer was 4-5°C higher than its paired snapback primer. Therefore, both the excessive snapback primer and the limiting primer can achieve the same annealing activity during the asymmetric PCR procedure.The multiplex snapback primer system was developed on a Rotor-Gene Q real-time PCR platform (Qiagen). The primer sequences used for PCR are listed in μL of reaction volume. The master mix contained 1U TaKaRa Ex Taq HS, 2 μL 10 × Ex Taq buffer (Mg2+ free), 0.5 mM MgCl2, 0.25 μM JAK2 V617F snapback primer, 0.02 μM JAK2 V617F limiting primer, 0.25 μM MPL W515L/K snapback primer, 0.02 μM MPL W515L/K limiting primer, 1.5 μM SYTO-9 DNA dye , and 15–25 ng DNA template. The PCR was performed on a Rotor-Gene Q real-time platform. The PCR protocol included an initial denaturation step (95°C for 10 min) followed by 70 cycles of 95°C for 1 sec, 58°C for 1 sec, and 72°C for 1 sec. The products were then heated for denaturation at 98°C for 2 min, followed by cooling down to 40°C for 2 min to facilitate the hybridization of snapback probe, and then melted at a ramping rate of 0.5°C/sec from 50°C to 99°C. High resolution melting- (HRM-) curve analysis was performed with the Rotor-Gene Q 1.7 software. For each assay, the positive and negative controls for both JAK2 V617F and MPL W515L/K mutations were always included.We performed asymmetric PCR with the TaKaRa Ex Taq Hot Start Version Kit  in a 20 MPL W515L and MPL W515K mutation stands were also made up with serial dilution of the MPL artificial plasmids and RPMI 8226 cell line DNA. The analytical sensitivity for JAK2 V617F and MPL W515L/K mutation of this snapback system was then evaluated by testing these standards. The reproducibility of this system was confirmed with 20 intra-assay duplications and a 20-day interassay duplication by testing the standards with the minimum detectable mutation load.Serial dilution of homozygous HEL cell line DNA was performed with the wild-type RPMI 8226 cell line DNA to prepare the standards of 10%, 1%, 0.1%, and 0.01% HEL cell DNA load. Meanwhile the JAK2 V617F mutation [MPL W515L/K mutations [JAK2 V617F identification utilized a pair of outer primers as well as wild-type/mutant specific primers to amplify the wild-type or mutant fragment with an internal amplification control sequence in a single reaction. Detection of MPL W515L/K mutations was carried out in a single PCR reaction with 2 outer primers designed to amplify the internal amplification control flanking the mutation site. The 2 specific primers for either the wild-type or the mutant sequence were employed to distinguish the wild-type allele from the mutant one by fragment size. A similar assay was also developed to detect MPL W515K mutation with the other mutation specific primer. All the primers used in the ARMS method are listed in The ARMS PCR testing for mutation  and MPL utations  was perfJAK2 V617F [MPL W515L/K mutations [JAK2 V617F detection system comprises a single set of primers and two probes that differed only at the position of V617F mutant nucleotide substitution and fluorescent labelling, so as to discriminate the mutation from the wild-type sequence in a single qPCR reaction. The MPL W515L/K mutation detecting system consisted of the same amplification primers but the distinct TaqMan probe that is specifically designed for the wild-type W515L and W515K mutant template. The amplification and fluorescent signal acquiring were performed for 40 cycles with the Quantitect Probe qPCR kit (Qiagen) on a LightCycler-480 II qPCR platform  according to the manufacturer's instructions. The qPCR assay data were acquired and analyzed with the LC-480 software Version 1.5.Two qPCR system based on the TaqMan hydrolysis probe was performed to detection of utations  as descrJAK2 V617F-positive samples, all the 5 MPL W515L/K-positive samples (3 W515L and 2 W515K), and 10 mutant allele-free samples with the sequencing primers listed in JAK2 V617F and MPL W515L/K detection were aligned with the reference sequence of JAK2 (NG_009904.1) and MPL (NG_007525.1), respectively. In consideration of the limited rare-allele detecting sensitivity, all the mutation-negative amplicons identified by direct sequencing were subjected to T-A cloning in order to produce the fragments with monoclonal allele. These amplicons (377 bp for JAK2 and 184 bp for MPL) were separated with 1.5% agarose electrophoresis and purified using the Qiaquick gel purification kit (Qiagen). For each mutation, the purified extracts were mixed with the pMD19-T simple vector master mix (Takara BIO) according to the manufacturer's instruction and ligated at 16°C for 2 hours. The vector plasmids with cloned insert were transformed into DH5α competent E. coli cells by 42°C heat shock for 15 seconds and iced chilling. Transformed E. coli cells were multiplied at 37°C in the Luria-Bertani (LB) broth for 1 hour and then spread onto the IPTG/x-GAL (Invitrogen) coated ampicillin-LB agar dishes. After 37°C incubation for 16 hours, the white clones were picked up and again enriched in the ampicillin-LB broth at 37°C overnight. Plasmid DNA of each single clone was extracted with Plasmid Mini Kit (Qiagen) according to the manufacturer's instruction and subjected to sequencing with the correspondent sequencing primers. For each dish, a maximum of 100 clones were sequenced, till the isolate with mutant allele was identified. All the Sanger sequencing assays were performed on an Applied Biosystems PRISM 3130 genetic analyzer in the Invitrogen Laboratory of Technical Services (Shanghai).To verify the result of snapback primer assay, we amplified 10 2+ concentration and rapid PCR protocol , the mutant hairpin could be more easily denatured for further template amplification. After dozens of PCR cycles, the mutant allele was selectively amplified.For each snapback primer set in the multiplex system, the asymmetric PCR produced two kinds of amplicon. The snapback primer and the limiting primer first generated double-strand DNA fragment. After the depletion of limiting primer, the excess snapback primer produced single strand amplicon with snapback probe tail in a linear amplification manner. The snapback probe was complementary to the target sequence in the single-strand amplicon, which led to the formation of the stem-loop hairpin of the snapback primer's own extension product. Since the snapback probe was designed totally complement to the wild-type allele, the wild-type hairpin possessed a higher thermal stability than the mutant one. Under the circumstance of low MgJAK2 V617F, MPL W515K, and MPL W515L allele was observed at 59.3°C, 63.2°C, and 67.6°C, respectively. Meanwhile, the double strand amplicons of JAK2 and MPL gene were presented at 81.1°C and 91.2°C  JAK2 V617F and MPL W515L/K mutation can be discriminated from the wild-type allele after the melting curve analysis . Therefore, up to 0.1%  of melting peaks for each mutation allele. At a  JAK2 V617F mutation. This system also detected 2 MPL W515L (2.5%), 1 MPL W515K (1.3%) mutation in the ET patients and 1 MPL W515L (5%), 1 MPL W515K (5%) in the PMF patients. The melting curve plots relating to the MPNs patient were illustrated in MPL W515L or K mutation was found in the PV patients. No patient harbored both the MPL W515L and MPL W515K mutation. It is worth noting that one of the ET sample turned out to be JAK2 V617F and MPL W515K mutation-positive, which is a rare clinical phenomenon.The snapback system identified 47 out of 50 PV (94%), 23 out of 50 ET (46%), and 8 out of 20 PMF (40%) patients harboring theJAK2 V617F ARMS assay utilized a pair of outer primers to produce a 463 bp amplification control flanking the mutation region. The 229 bp product of wild-type specific primer and reverse outer primer indicate the presence of wild-type allele in the sample. The mutation-specific primer and forward outer primer will generate a 279 bp fragment when the JAK2 V617F mutant allele is present  and VIC (wild-type) fluorescence channel, while results of the MPL W515L/K mutation detecting system should be analyzed with the amplification curves obtained from three reaction wells . After the qPCR procedure, a sigmoidal shaped amplification curve in the fluorescent signal versus cycle number plot with a threshold cycle (Ct) < 35 indicated the explicit existence of the corresponding allele, while the sample with a Ct > 38 amplification curve was considered free from the particular allele  identified as MPL W515L positive by snapback assay showed a weak-positive curve with Ct = 38.623 in the W515L channel , and 1 for MPL W515K. After T-A cloning, the subclone of these 4 samples was again subjected to sequencing and turned out to harbor the mutant alleles, and supporting the snapback primer system enjoyed a higher sensitivity over the commonly used sequencing method.Results from the snapback primer system were further verified with Sanger sequencing. Ten JAK2 and MPL in the middle 2000s, these molecular abnormalities have now become one of the most important diagnostic markers for the Ph-negative MPNs [The last decade witnessed great strides in the development of molecular pathology of myeloproliferative malignancies. Soon after the discovery of mutations in ive MPNs –21. Moreive MPNs , 23. AllJAK2 V617F and common MPL mutations utilizing the minor grove binder (MGB) probe. However, the additional rare mutations in JAK2 exon 14 and MPL exon 10, such as JAK2 c.1860C>A (D620E) [MPL c.1543T>C (W515R) [MPL c.1543T>G/1544G>C (W515A) [At present, sequencing, ARMS, and fluorescent probe-based qPCR still remain the most available approaches for the detection of these genetic alternations. However, both the ARMS and sequencing technology are technically challenging and time/labor consuming. Besides, genetic analyzer is also rather expensive for the small-scale laboratories to afford. ARMS qPCR is a powerful tool for rare allele mutation detecting, because this system minimalized the time/labor cost of each test . However (D620E) , c.1848T (D620E) , c.1849G (D620E) , c.1849G (D620E) , MPL c.1 (W515R) , and MPL (W515A) , could lThe Wittwer Lab innovated snapback primer genotyping for single nucleotide polymorphism (SNP) in 2008  and thenJAK2 V617F and MPL W515L/K mutations simultaneously. This multiplex system comprises two pairs of primers, each designed for the amplification of stem-loop hairpin flanking the JAK2 and MPL mutation, respectively.In this study, we developed a robust multiplex snapback primer assay to detect MPL W515L/K, extremely high sensitivity is necessary, since this group of diseases is characterized with polyclonal events [JAK2 617F and MPL W515L/K mutations, with the analytical sensitivity around 1–5% mutation load [JAK2 V617F and MPL W515L/K mutations simultaneously down to the 0.1% level.For the detection of mutations in the Ph-negative MPNs, especially the l events . Previouion load , 33–35. MPL W515L mutation in a PMF patient, indicating a favorable detection sensitivity of the snapback primer assay.The mutation detecting performance of the snapback primer assay was validated with 120 peripheral blood DNA from MPNs patients, in comparison with the electrophoresis based ARMS method and TaqMan qPCR. The test results were further verified with T-A cloning sequencing. Results of snapback primer assay and ARMS method were totally identified, and the new method also detected one indeterminable weak-positive JAK2 V617F is the initial test of all the suspicious PV, ET, and PMF patients [JAK2 exon 14, further MPL W515L/K mutation identification should be performed if the patients are suspicious for ET/PMF. Here, we combined the JAK2 V617F and MPL W515L/K mutation detection with a multiplex system, which reduced the total processing time of molecular diagnosis of MPNs.Our multiplex system condenses the genetic test strategy for classical Ph-negative MPNs. According to the molecular diagnostic pipeline of disease, patients . For theThere are additional benefits of this system. Since the melting curve analysis strategy combines the fluorescent signal with the melting temperature of the PCR products, it provides our method with a favorable analytical specificity . BesidesJAK2 V617F and MPL W515K mutations in a clinical sample of ET patient. Although previous studies from Pardanani et al. [JAK2 V617F and MPL W515L in ET patients, we could not exclude the possibility of plasmid/sample contamination. Although we did not detect any other mutations in this study except the JAK2 V617F and MPL W515L/K, this snapback primer system might have the ability to identify other JAK2 exon 14 and MPL exon 10 genetic alternations within the snapback probe covered region theoretically.Of note, we observed the coexistence of i et al.  reportedHowever, our system still has room for improvement. Firstly, the entire method was developed and validated on a high-resolution melting platform. The practicability of this system on other classical real-time instruments still needs to be further tested, although our previous work has reported the application of oligonucleotide probe melting analysis on the conventional devices . FurtherJAK2 V617F and MPL W515L/K mutations by melting curve analysis. With a favorable sensitivity and reproducibility, this closed tube system might be applied to the molecular diagnosis of Ph-negative MPNs.In summary, we developed a novel labor-saving snapback primer system for simultaneous detection of