Coturnix c. coturnix) may share with Japanese quail (Coturnix c. japonica) its potential as an intermediate host and reservoir of avian influenza viruses (AIV). To elucidate this question, European quail were experimentally challenged with two highly pathogenic AIV (HPAIV) (H7N1/HP and H5N1/HP) and one low pathogenic AIV (LPAIV) (H7N2/LP). Contact animals were also used to assess the viral transmission among birds. Severe neurological signs and mortality rates of 67% (H7N1/HP) and 92% (H5N1/HP) were observed. Although histopathological findings were present in both HPAIV-infected groups, H5N1/HP-quail displayed a broader viral antigen distribution and extent of microscopic lesions. Neither clinical nor pathological involvement was observed in LPAIV-infected quail. Consistent long-term viral shedding and effective transmission to naive quail was demonstrated for the three studied AIV. Drinking water arose as a possible transmission route and feathers as a potential origin of HPAIV dissemination. The present study demonstrates that European quail may play a major role in AI epidemiology, highlighting the need to further understand its putative role as an intermediate host for avian/mammalian reassortant viruses.European quail ( Avian influenza (AI) represents a major disease problem, not only for poultry but also for other avian species, mammals, and human beings . The conCoturnix c. japonica) in Italy (1966–1968)  was obtained from the ongoing surveillance program carried out in Catalonia (Northeast Spain). The deduced amino acid sequence of the region coding for the cleavage site of the precursor of the HA molecule were PEIPKGSRVRR*GLF for the H7N1/HP and PEIPKGR*GLF for the H7N2/LP, being typical of HPAIV and LPAIV, respectively  was isolated during the 1999–2000 Italian epidemic . The H5 ectively .50) and the mean embryo infectious dose (EID50) for the HPAIV and LPAIV isolates, respectively, were determined [Virus stocks were produced in specific pathogen free (SPF) chicken eggs. The allantoic fluids were harvested at 48 hours post-inoculation (hpi) (H7N1/HP and H5N1/HP) and 72 hpi (H7N2/LP). Viruses were tenfold diluted in phosphate buffer saline (PBS) for titration in 9-day-old embryonating SPF chicken eggs. The mean embryo lethal dose . Quail were kept one week for acclimation, and feed and water were provided ad libitum throughout the experiment. All procedures were performed according to the requirements of the Ethical Commission of Animal Experimentation of the Autonomous Government of Catalonia.European quail  of approximately two months of age were used in this study. Male and female birds were included in almost equal numbers. Before the infection, serum samples of all individuals were confirmed to be seronegative for AIV by a competition ELISA test (C-ELISA) . Furthermore, oropharyngeal (OS) and cloacal (CS) swabs were ensured to be negative for AIV by real time RT-PCR (RRT-PCR). Each experimental group was housed in a different negative pressured isolator with HEPA-filtered air in the animal biosafety level 3 (ABSL-3) facilities of n = 12/group). Groups 1A, 2A, and 3A were used to evaluate morbidity, mortality, transmissibility, and viral shedding pattern. Groups 1B, 2B, and 3B were used for the pathological studies. All animals were inoculated intranasally with 106 EID50 (for the LPAIV) or 106 ELD50 (for the HPAIV) of the corresponding challenge virus in a volume of 0.5 mL, except four birds of each A group which were used as contact animals. Contact birds were placed into the isolators four hours after inoculating the other birds and after changing drinking water. A seventh group (group C) (n = 8) was used as negative controls; these quail were inoculated intranasally with PBS solution. Amounts of virus were verified by performing a RRT-PCR of both the original non-diluted viruses and the inocula.Eighty birds were randomly separated into seven groups: six challenged groups with 12 birds/group and one control group with 8 birds , at 12 dpi, and 15 dpi, OS, CS and feather pulp (FP) samples were obtained from quail from the A groups to measure viral shedding by RRT-PCR. Drinking water was collected with a 1 mL syringe at the same time points, and it was changed on a daily basis. The same samples were collected from group C. Mortality and mean death times (MDT) were calculated from the A groups. At 3, 5, 8, and 15 dpi, three animals from groups B and two animals from group C were euthanized using intravenous sodium pentobarbital . Surviving birds were euthanized at the end of the experiment (15 dpi). Blood samples were collected before euthanasia to detect AI antibodies by C-ELISA testing. As it was terminal, bleeding was done from the heart after previous anesthesia with intramuscular injection of ketamine/xylazine . All euthanized and naturally dead quail from the B groups were necropsied to evaluate gross lesions and obtain samples for histopathological studies. Swabs and FP samples were placed in 0.5 mL of Dulbecco’s Modified Eagle’s Medium (DMEM)  with 600 μg/mL penicillin and streptomycin. These samples, together with drinking water samples and serum samples, were stored at −80 °C until further use.Necropsies and tissue sampling were performed according to standard protocols . After fM) gene in Fast7500 equipment  using the primers and probe previously described [p < 0.05) using the Statistical Package for the Social Sciences for Windows Version 20.0.Viral RNA from OS, CS, FP, and drinking water samples was extracted with NucleoSpin® RNA virus kit  following the manufacturer’s instructions. The resulting viral RNA extracts were tested by one-step RRT-PCR for the detection of a highly conserved region of the matrix (escribed  and the escribed . Samples2); GMT of 3 log2 or greater were considered positive. Previously known positive and negative sera were used as controls.A C-ELISA test was carried out to detect antibodies against the NP of AIV using the commercially available kit ID Screen® Influenza A Antibody Competition , according to the manufacturer’s instructions. In addition, a hemagglutination inhibition (HI) test was performed to titrate antibodies against specific H5- (in H5N1/HP serum samples) and H7- (in H7N2/LP and H7N1/HP serum samples) subtypes. The HI assays were performed according to standard procedures  with chiClinical signs and mortality were only observed in HPAIV-infected groups groups 2 and 3) and were similar between inoculated and contact birds. Some of the quail  displayed nonspecific clinical signs, consisting of lethargy, anorexia, and ruffled feathers, that progressed to death or severe neurological signs  within 24 h. The onset times of these nonspecific signs were 6 dpi for H7N1/HP-group and 4 dpi for H5N1/HP-group. Two H7N1/HP-challenged quail (17%) and three H5N1/HP-challenged quail (25%) presented an acute fatal progression of the infection, displaying neurological signs without previous nonspecific signs at 7 dpi and 5 dpi, respectively. However, in other cases (33% in H7N1/HP-group and 8% in H5N1/HP-group) quail were found dead without previous clinical signs. Only one bird, belonging to the H5N1/HP-group, recovered after showing nonspecific clinical signs at 6–7 dpi. All animals with neurological signs, recumbent or both were euthanized for ethical reasons. The survival rates and the MDT of the HPAIV-infected groups (groups 2 and 3) throughout the experiment are summarized in Figure  and 3 anConsistent gross lesions were only observed in HPAIV-infected groups (groups 2 and 3) and were similar between inoculated and contact birds. At 3 dpi, one H7N1/HP-quail (group 2) presented multifocal petechia on the proventriculus-gizzard junction mucosa. However, the foremost lesions in the H7N1/HP-group were observed at 5 dpi, which consisted of moderate splenomegaly with pallor or parenchymal mottling and pancreatic lesions characterized by multifocal necrotic areas of 1 mm-diameter. Lesions in H5N1/HP-quail (group 3) were most pronounced and were detected throughout the experiment in all necropsied birds. At 3 dpi, liver pallor in one bird was observed. The quail found dead at 4 dpi presented spleen pallor and multifocal areas in the pancreas. Such pancreatic lesion, as well as thymus atrophy, was observed until the end of the experiment in all necropsied birds. At 5 dpi, spleen pallor was observed in one bird. No gross lesions were observed in H7N2/LP-infected birds (group 1) or in birds from the control group (group C).Histological lesions and influenza A viral NP were only observed in HPAIV-infected quail groups 2 and 3) Tables . In H7N1 and 3 Tap < 0.05), especially on 1, 2, and 4 dpi. Moreover, FP from H5N1/HP-challenged quail contained significantly more viral RNA than FP from H7N1/HP-infected quail (p < 0.05).Real time RT-PCR was performed on OS, CS, FP, and drinking water samples of the A groups. Oropharyngeal swabs of H7N2/LP-challenged birds (group 1) tested positive until 9 dpi for inoculated birds peaking at 3 dpi, and until 12 dpi for contact birds peaking at 7 dpi . H7N1/HP viral RNA was present in water during almost all the experiment (3–15 dpi), being quite stable in time. Existence of H5N1/HP viral RNA in drinking water was manifested at 2 dpi and at 6 dpi, at levels akin to those of H7N1/HP.Before infection, all serum samples tested negative for antibodies against influenza A NP. Almost all the H7N2/LP-inoculated quail (7/8) and all the H7N2/LP-contact quail (4/4) were antibody-positive at 15 dpi, with GMT of 7.9 and 7.3, respectively (Table Coturnix c. coturnix) can be infected with both HPAIV and LPAIV. This quail subspecies can be found not only in the wild all over the Palearctic region, but also in many parts of Europe as a game bird species raised in outdoor operations. Despite the relevance of this game bird species, no studies had previously investigated its AIV infection dynamics. The aim of the present study was to elucidate the putative role of European quail in the ecology of influenza A viruses by assessing the pathogenesis, transmissibility, and viral shedding of quail experimentally infected with two different HPAIV subtypes (H7N1 and H5N1) and one LPAIV (H7N2).This is the first experiment which demonstrates that European quail . Therefore, spreading disease into the wild by releasing apparently healthy farm-reared quail for hunting purposes could represent a substantial threat, even higher if assuming that this species could act as a mixing vessel like already stated for the Japanese quail. Furthermore, European quail may be considered sentinels (both for HPAIV and LPAIV) and reservoirs (for LPAIV), which is of special interest as most wild individuals are migratory . The appCurrent active AI surveillance activities include sampling of both OS and CS, as well as blood ,56. PassThe authors declare that they have no competing interests.KB, NB, and FXA prepared the viruses used in this study. KB, RD, VG, JVA, AJC, and NM participated in the daily monitoring of the clinical signs and the sampling of the animals. KB, RD, VG, JVA, AJC, AR, and NM performed the necropsies and the tissue sampling. KB and NM carried out the histopathological examinations. KB carried out the RRT-PCR and the serology assays, together with FXA in the case of H5N1/HP samples. RD, NB, FXA, UH, and NM conceived the study and participated in its design and coordination. All authors read and approved the final manuscript.


NLRP3 gene are associated with the dominantly inherited cryopyrin-associated periodic syndrome (CAPS) characterized by episodes of fever, urticarial rash, arthralgia, myalgia, eye inflammation, and, in its more severe forms, bony abnormalities and CNS inflammation. Of the 145 sequence variants in NLRP3 reported to date, 30 are either nonpathogenic or of undetermined significance, the commonest of which, Q703K, has been reported in 5 to10% of general population.Mutations in the To characterize the clinical phenotype in individuals with Q703K in a single UK center.NLRP3 gene, individuals in whom genetic variants were not identified or those with low penetrance mutations underwent additional screening of MEFV; TNFRSF1A; MVK and NOD2.1017 subjects with hereditary periodic fever syndromes (HPFS) were screened for mutations in the NLRP3 Q703K was identified in 69 subjects (7% of screened), clinical information was available on 56. 4 cases had another mutation in NLRP3: 1 had A439V and 3 siblings had R260W. 18 subjects (32%) had aberration in another HPFS gene: 4 in TNFRSF1A: R92Q, C29F, H22Q and S57_E64del; 1 in MVK: V377I; 2 in NOD2: P268S and 12 in MEFV: 2 were compound heterozygotes, M680I/V726A and M694V/V726A, 2 were homozygous and 1 was heterozygous for M694V, 1 was heterozygous for V726A, 2 for S208C, 1 for S154P and 3 for E148Q.NLRP3 variants other than Q703K; 4 subjects with TNFRSF1A mutations; a subject with MVK variant and 5 of the 12 cases with MEFV substitutions respectively. One subject with Q703K and E148Q was an asymptomatic carrier and in 4 cases a diagnosis of disease other than HPFS was made. 14 cases (25%) were diagnosed with AA amyloidosis  the nature of the underlying inflammatory disease in 12 remains uncertain. In total we were unable to make a clinical diagnosis in 25 subjects (44%): in this group the median age at disease onset was 5 years (birth-59 years); fever, arthralgia and myalgia were the most prominent features - identified in over 50% of cases; 11 subjects (44%) had rash during febrile attacks ; 7 (28%) had symptoms triggered or worsened by cold exposure; 5 (20%) suffered from headache, GI symptoms or lymphadenopathy; 4 (16%) had hearing impairment; a delayed puberty was identified in 4 (16%) and one had growth deficit. Episodes occurred irregularly and lasted from 1 day to 2 weeks.The inflammatory syndromes were thought to be fully consistent with CAPS, TRAPS, MKD and FMF in the 4 cases with In 10 the inflammatory markers, serum amyloid A protein (SAA) and C-reactive protein (CRP), were measured during disease flare and were elevated to median values of 106.5 mg/L (range 40-438) and 68 mg/L (range 34 – 220) respectively.We have identified Q703K in subjects displaying FCAS-like symptoms, in individuals with HPFS other than CAPS, in cases with uncharacterised autoinflammatory diseases, in AA amyloidosis, and in asymptomatic individuals. Given the high frequency of healthy carriers, the interpretation of Q703K presents a diagnostic challenge and the genetic and/or environmental factors that may influence pathogenic consequences of this variant remain unknown.None declared


Cadherins form inter-junctional lattices by the formation of both cis and trans dimers. Here, we directly visualize and quantify the spatiotemporal dynamics of wild-type and dimer mutant N-cadherin interactions using time-lapse imaging of junction assembly, disassembly and a FRET reporter to assess Ca2+-dependent interactions. A trans dimer mutant (W2A) and a cis mutant (V81D/V174D) exhibited an increased Ca2+-sensitivity for the disassembly of trans dimers compared to the WT, while another mutant (R14E) was insensitive to Ca2+-chelation. Time-lapse imaging of junction assembly and disassembly, monitored in 2D and 3D (using cellular spheroids), revealed kinetic differences in the different mutants as well as different behaviors in the 2D and 3D environment. Taken together, these data provide new insights into the role that the cis and trans dimers play in the dynamic interactions of cadherins. Cadherins, Ca For W2A, the roundness of the spheroid did not change regardless of the [Ca2+]ext, indicating that with long-term imaging in a 3D environment, the W2A junctions cannot be rescued by elevated extracellular Ca2+-levels . For both the R14E and V81D/V174D mutants, an extracellular Ca2+-concentration of 0.8 mM (vs. 0.4 mM) was required to see a significant change in spheroid roundness relative to WT-N-cadherin . This emphasizes that in 3D, loss of either the X-dimer or the cis interface leads to a decreased Ca2+-sensitivity in junction assembly . Given that cadherins are CaOnce spheroid formation was completed, we imaged each spheroid with high spatial resolution in order to determine whether there were any morphological differences apparent at the cellular or global levels. As cellular spheroids are thick specimens (hundreds of microns in diameter) and prone to light scattering we used a monolith digital scanned laser light sheet-based fluorescence microscope (mDSLM) . This entrans interactions, the X-dimer and the strand-swapped dimer, and the third is involved in cis interactions. Here, we analysed the functional role of these interfaces in living cells examining their contribution to the Ca2+-dependent interactions of cadherins as well as the assembly and disassembly of cell-cell junctions. Three binding interfaces for cadherin-cadherin interactions have been reported thus far: two of them are involved in cis junction to be more volatile and transient than the WT junctions. This was also true for the X-dimer W2A, which showed a reduced lifetime of de novo assembled junctions. The reported reduced binding affinity of the X-dimer compared to the strand-swapped dimer ext. The role of extracellular Ca2+-signaling has been shadowed by many years of extensive work done on understanding the signaling by intracellular Ca2+. There is, however, accumulating evidence that extracellular Ca2+-concentration is dynamic and fluctuations have been shown to occur in cardiac muscle [2+-concentration, it is of particular interest as a sensor of physiological fluctuations in extracellular Ca2+-concentrations. Our results shed new light on the role the of the various N-cadherin binding interfaces in cell-cell junctions assembled in two- and three-dimensional contexts in living cells. In addition, we elucidate the interaction of these interfaces with the dynamics of [Cac muscle , islets c muscle  and neurc muscle . Due to Mutations or altered expression levels of cadherins are associated with several cancers -37. For 12bit-images with a size of 512 x 512 pixels (106.07x106.07 µm) were acquired with a confocal laser scanning microscope LSM780  using the spectral imaging “lambda mode”. A Plan-Apochromat 40x/1.4 oil objective  and the 458nm line of an argon laser were used to obtain an optimal excitation of both fluorophores. Spectral detection occurred from 473nm to 569nm with a bandwidth of 8.7nm. Individual spectra were obtained from adherens junctions of cells expressing the same N-cadherin fluorescent fusion protein. Spectra were saved to the databank and used to unmix images taken from cells that express the two different fluorescent fusion proteins using the “linear unmixing”-function of the Zeiss software ZEN. After the acquisition of a cell pair image expressing different fluorescent fusion proteins the region of the adherens junction was marked by a rectangular region of interest (ROI) and the Venus signal within this ROI was bleached with high laser power . This bleaching was followed by the acquisition of a second image and the subsequent unmixing of both images. 2+-dependence of the WT and the mutant junctions were addressed by ratiometric FRET measurements. Time-lapse images of a ROI containing the adhesion junction formed by one N-cadherin-Venus- and one N-cadherin-Cerulean-expressing cell were obtained in the lambda mode  with a time interval of 100 ms for 35 sec. The Ca2+-chelator BAPTA was added to the dish up to a final concentration of 20 mM after a baseline recording (10-15 sec). Images were unmixed using spectra acquired from single-colored junctions. Kinetic measurements and the Ca6 cells, resuspended in Hibernate A low fluorescence buffer , were added to a 35 mm MatTek dish . The dish was immediately placed in the incubator that was set to 37°C and 5% CO2 on the spinning disc microscope BD Pathway 855 (BD Biosciences) and allowed to acclimatize for 30 min. A z-stack (16-bit 672 x 512) of 6 images was captured every 2 min for 2 hours using 40x 0.90 N.A. air Apochromat objective lens (Olympus), 514 nm excitation and an Orca ER-1394 . N-cadherin-L cells  so that all mutants had similar cell densities on the day of imaging. Cells were allowed to form junctions over a period of 24 - 48 hours. Prior to imaging, cell nuclei were stained with Hoechst 33342 (Sigma Aldrich) for a minute. Cells were imaged in Hibernate A low fluorescence buffer . The cells were allowed to acclimatize inside a custom-built chamber heated to 30°C mounted on a LSM 780  microscope for 30 min before imaging. A 40x 1.4 N.A. oil immersion plan-Apochromat objective lens  was used to acquire a z-stack (16-bit 1024 x 1024) consisting of 6 z-planes every 5 min using 514nm and 405nm argon ion laser excitation. After 15 min of baseline imaging, 5 mM BAPTA (Alfa Aesar) was pipetted into the dish and imaging was continued for 1-hour post treatment. A final concentration of 5 mM BAPTA was chosen for time lapse imaging because on a longer time scale 20 mM BAPTA led to cells detaching from the dish. 2+-assay, Ca2+-free DMEM  was used in order to remove extracellular Ca2+ and adjust the Ca2+-concentration to 0, 0.4, 0.8, 1.3 or 2.1 mM using CaCl2. At 0 mM Ca2+, Ca2+-free serum  was used to avoid effects that background Ca2+ may have on spheroid formation. The dish was centrifuged for 2 min at 300 g to bring all the cells to the center of the well and was placed in the Cell Observer  with the incubator set to 37°C and 5% CO2. After determining the position of each well, image acquisition commenced. Images were captured every 5 min for 48 hours using a 5x 0.16 N.A. plan-Apochromat objective lens , transmitted light and an AxioCam MRm . The position of the well was updated periodically if the spheroids moved out of the field of view. L cells were split and then counted. 2000 cells were added to a well in a coated HydroCell 96-U well dish (Nunc) (to avoid cells sticking to the bottom of the dish). Normal growth DMEM medium  was used for spheroid formation. For the Ca48 h post spheroid formation, the spheroids were fixed in freshly prepared 4% PFA in PBS at 4°C for 30 min. The cell nuclei were stained with Hoechst 33342 for 2 h at room temperature. The spheroids were then embedded in 1% low-melting agarose and drawn into a 20 µl Brand intra MARK micropipette according to the method described in Pampaloni et. al . The micFigure S1Examples of acceptor bleach experiments for mutants and analysis of control acceptor bleach experiments. (A) Examples for acceptor bleach experiments with COS7-cells expressing either N-cadherin-FP-W2A (strand-swap mutant), -R14E (X-dimer mutant) or -V81D/V174D (cis mutant). The upper two images show the Venus-channel before and after the bleaching of the Venus signal in the junction (boxed region). The Cerulean channel is shown in the lower two images. Bleaching of the Venus fluorescence leads to a dequenching of the FRET donor Cerulean, which can be observed in the lower two images. An enlargement of the junction (boxed region) is shown next to the images. Scale bars = 20 µm. (B) Quantitative comparison of the acceptor bleach experiments for N-cadherin-WT with the insertion of the fluorescent protein within EC2 (WT) or close to EC5 (EC5). The bars represent the mean ± SEM. The mean of the WT (n = 8) is significantly increased compared to EC5 . (TIF)Click here for additional data file.Figure S2Curve fitting for junction assembly and disassembly. (A) A linear function following a constant baseline was fit to the junction assembly data and the slope was used to determine the rate of junction assembly. WT has the fastest rate of junction formation, followed by R14E, W2A and V81D/V174D. (B) An exponential fit for temporal dynamics of disassembly was performed from the time point of BAPTA addition. The fit for R14E was started 10 min after BAPTA addition, the time point at which the decay started. (TIF)Click here for additional data file.Figure S3Curve fitting for spheroid formation and analysis of cryo-sections. (A) A sum of two exponentials – fast and slow – following a constant baseline were fit to the roundness data up to 20 h. The tau is displayed for the fast exponential. WT has the fastest rate of spheroid formation, followed by R14E, V81D/V174D and W2A. (B) 10 µm cryo-sections of the spheroids were imaged with 20x 0.8 N.A. Plan-Apochromat objective lens. Using a 99-pixel wide line, each section was straightened along 3 axes, distance binned, and relative fluorescence intensity profiles were plotted. Comparing the intensity profiles between bins 30-50 for all the mutants, only R14E was significantly different compared to WT. . Error bars indicate SEM. n(WT) = 18, n(W2A) = 20, n(R14E) = 25, n(V81D/V174D) = 18 straightened sections. (C) The cryo-sections were immuno-labeled using an anti-laminin antibody. Using automated detection of laminin on the periphery of the sections, the mean fluorescence intensity of laminin staining was measured. The mean fluorescence intensity of W2A was significantly lower than WT . Error bars indicate SEM. n(WT) = 7, n(W2A) = 5, n(R14E) = 6, n(V81D/V174D) = 7 spheroids. Scale bars (B) and (C) = 50 µm. (TIF)Click here for additional data file.Figure S4Analysis pipeline and 3D imaging illustration. (A) Identification of junction location across time series. (A1) Maximum intensity projection of two central images of z-stack  at time point -15 min. (A2) Threshold mask of Venus channel. (A3) Cell centers (yellow dots) calculated based on a watershed segmentation (blue lines). The watershed was calculated in 3D , so that the ‘identity’ of the cells was preserved across the whole time series. (A4) Two-closest neighbors’ segmentation: Each color patch represents the pixels that share the same pair of cells as their closest neighbors (based on the cells’ centers shown in A3). (A5) Masking segmentation based on distance from the closest cell center. (A6) Combination of the threshold mask (A2) with the two-closest neighbor segmentation (A5). Since cell identity is preserved across frames, the junction identity is too, making it possible to track junctions even when they are discontinuous in space and/or time. (B) Examples of segmentation across time. The top row (B1-B3) shows the fluorescence images (similar to A1) at time points 0, 15 and 30 min. The bottom row (B4-B6) shows the corresponding segmentation (similar to A6). Note that junctions preserve identity (color) across frames. Segmentations that do not represent individual (e.g. star in B5) or complete (e.g. arrow heads in B4) junctions were excluded from further analysis. (C) Identification of spheroid formation over time. Raw data shows the image as it was acquired. An Ilastik classifier was used to define two classes – spheroid (green) and background (red). The pixel segmentation based on the classifier followed by object detection based on the size of spheroid-classified pixels. (D) Imaging the spheroids with mDSLM. The spheroid (yellow) was illuminated (blue arrow) from the side and the detection was done at a 90° angle (green arrow). The spheroid was rotated 12 times in steps of 30° as illustrated. (TIF)Click here for additional data file.Methods S1Supporting materials and methods.(DOC)Click here for additional data file.Movie S1Junction assembly of N-cadherin-Venus-WT L cells and its mutants W2A, R14E and V81D/V174D. L cells stably expressing N-cadherin-Venus WT and its mutants were used to monitor junction assembly. Cells were imaged every 2 min for 2 hours using a BD Pathway 855 spinning disc microscope (BD Biosciences). (MOV)Click here for additional data file.Movie S2Junction disassembly of N-cadherin-Venus-WT L cells and its mutants W2A, R14E and V81D/V174D. Cells with pre-established junctions were used to monitor junction disassembly following addition of 5mM BAPTA. Baseline images were captured every 5 min for 15 min, followed by drug addition and then imaging was continued for 1 hour. Images were acquired using LSM 780 microscope with an incubator at 30°C .(MOV)Click here for additional data file.Movie S3Spheroid formation with N-cadherin-Venus-WT L cells and its mutants W2A, R14E and V81D/V174D. The cells were added to a 96-U well dish and spheroid formation was monitored using the Cell Observer  by acquiring images in transmitted light every 5 min for 48 hours. (MOV)Click here for additional data file.Movie S43D rendering of spheroids imaged using a light-sheet microscope. The N-cadherin-Venus-WT and the mutant spheroids were imaged using the mDSLM from 12 different angles, which were combined post-hoc in Amira . A volume rendering was used for visualization of the 3D spheroid.(MOV)Click here for additional data file.


Deltaproteobacteria in comparison to control biofilms formed on non-polarized electrodes, which were preferentially composed of Gammaproteobacteria and Firmicutes. Among Deltaproteobacteria, sequences affiliated with Pelobacter and Geobacter genera were identified. A bacterial consortium was cultivated, in which 25 isolates were identified as Geobacter bremensis. Pure cultures of 4 different G. bremensis isolates gave higher current densities  than the original multi-species biofilms (in average 300 mA/m2 on DSA) and the G. bremensis DSM type strain . FISH analysis confirmed that G. bremensis represented a minor fraction in the original EA biofilm, in which species related to Pelobacter genus were predominant. The Pelobacter type strain did not show EA capacity, which can explain the lower performance of the multi-species biofilms. These results stressed the great interest of extracting and culturing pure EA strains from wild EA biofilms to improve the current density provided by microbial anodes.Electrochemically active (EA) biofilms were formed on metallic dimensionally stable anode-type electrode (DSA), embedded in garden compost and polarized at +0.50 V/SCE. Analysis of 16S rRNA gene libraries revealed that biofilms were heavily enriched in  Since their discovery Ochrabactrum anthropi isolates have given 89 mW/m2, while the type strain provided only 45 mW/m2 in identical conditions 2). In contrast, Rhodopseudomonas palustris isolated from a MFC has produced 56% larger power (2720 mW/m2) than the original biofilm (1740 mW/m2) Most studies devoted to single EA species have been carried out with type strains that corresponded to predominant species identified in wild EA biofilms. Thus far, only a few strains of bacteria have been directly isolated from EA biofilms In this framework, a new procedure has recently been proposed in the literature to enlarge the possibilities of isolation of EA strains. It consisted of a two-chamber U-tube MFC coupled to successive dilution-to-extinction steps 2 of current without the addition of acetate to the anode chamber The purpose of this study was to identify the microbial community and then isolate and test in pure culture EA species that are responsible for harvesting electricity in biofilms formed from garden compost th day, when the current densities stabilized and averaged 300 mA/m2, the electrodes were removed to analyse the microbial communities attached on both polarized and control electrodes. Suspensions of microbial consortia obtained by scrapping were characterized by, total cell count of DAPI-stained cells, enumeration of cultivable heterotrophic bacteria and DNA gel quantification. The polarized electrodes (PE) were covered by a more important biofilm than the non polarized electrodes (NPE) as indicated by the higher amount of DNA extracted from biofilms formed on PE and also by a tenfold increase in the number of cultivable bacteria recovered from the latter biofilms.  electrodes were embedded in garden compost that contained acetate (10 mM) used as electron donor. Six electrodes were individually polarized at 0.50 V/SCE and 6 control electrodes were not polarized. After one day, the current gradually increased due to the formation of an electrochemically active biofilm on the electrode surface, as previously reported iofilms. .DGGE analysis showed different microbial compositions of the biofilms collected from the PE and from the NPE . Some baDeltaproteobacteria and Betaproteobacteria. The PE were heavily enriched in Deltaproteobacteria in comparison to NPE that were preferentially colonized by Gammaproteobacteria and Firmicutes. The polarized electrodes showed mainly Pelobacter, Geobacter, Azoarcus and Burkholderia, whereas control electrodes were mainly colonized by bacteria related to Pseudomonas, Bacillus, Sphingomonas, Acinetobacter and Acidovorax  fingerprinting  strains . As bothd on NPE .G. bremensis isolate ONC105 and isolate ONC106, two isolates with ERIC profile 2, G. bremensis isolate ONC102 and isolate ONC104, and the type strain DSM 12179 were cultured and used to inoculate (10% v/v) 5 different anaerobic reactors, which contained 10 mM ethanol and a DSA electrode polarized at 0.50 V vs. Ag/AgCl as the sole electron acceptor. In parallel, a control reactor was not inoculated. Current increase was observed in each reactor, except the control, showing after 3 days maximal values of 1100, 735, 485, 360 and 350 mA/m2 for isolates ONC106, ONC105, ONC102, ONC104 and the type strain, respectively  oxides G. bremensis strains were found to be able to transfer electrons to a solid electrode. Moreover, each isolate gave higher current density than the type strain and than the original multi-species biofilm. These results reinforced the few previous examples that indicated better performance of the isolates than the type strain Pelobacter venetianus strain DSM 2395, which was related to Pelobacter spp. detected in the EA biofilms and run for more than 15 days did not exhibit any current production. Bacteria related to Pelobacter genus have already been described several times as major EA-biofilm-forming bacteria Geobacter genus represented a minor fraction of the original EA biofilm, whereas Pelobacter was predominant. Neither Geobacter nor Pelobacter genus were evidenced on control electrodes. Consequently, the development of Pelobacter spp. on the electrode surface was favoured by polarization, while pure cultures put in doubt its capacity to use the electrode as electron acceptor. It can be concluded that the enrichment of Pelobacter spp., which did not contribute to current harvesting, is a cause of the lower performance observed here for the original biofilms compared to the Geobacter pure cultures. It may be thought that Pelobacter related bacteria are non-EA bacteria that are enriched in EA biofilms and impediment the colonization by Geobacter spp.Several similar chronoamperometries performed with pure cultures of G. bremensis isolate ONC105 and isolate ONC102, which showed the highest current density for each ERIC profile, and the G. bremensis type strain were inoculated in similar anaerobic conditions (10 mM ethanol) in different reactors with DSA or graphite electrodes polarized at 0.50 V/SCE. On DSA electrodes, current increased from day 1.5 reaching maximal values of 1400, 750 and 100 mA/m2 for isolate ONC105, isolate ONC102 and type strain, respectively , with circadian oscillation as commonly observed with EA biofilms Finally, a reactor containing a graphite electrode polarized at 0.50 V vs Ag/AgCl was inoculated with 35 mA/m2 . Since dGeobacter bremensis that were extracted from wild EA biofilms revealed thus able to produce current density three-fold higher (up to 1400 mA/m2) than the original biofilms (average 300 mA/m2) in identical conditions (DSA electrode) and up to eight-fold higher when the electrode was changed to graphite (up to 2490 mA/m2 and around 1730 mA/m2 stable for days). This work is a supplementary example, among the few that have already been reported, of the great interest of extracting and culturing pure EA strains to improve the current density provided by microbial anodes.The different strains of Twelve Dimensionally Stable Anode  electrodes (50×25×1 mm) were cleaned and embedded in a 10 L tank containing garden compost mixed with 10 mM acetate and 10 mM NaCl as previously described 4Cl (0.25), NaH2PO4 (0.6), KCl (0.1), NaHCO3 (2.5), AQDS (0.515), 1 mL trace elements solution SL-10 and 1 mL vitamin solution (medium DSMZ 320). The Fe-citrate–Freshwater basal medium was prepared according to the medium DSMZ 579 with slight modification and consisted in (in grams per liter) FeIII-citrate (13.7), NaHCO3 (2.5), NH4Cl (1.5), NaH2PO4 (0.6), KCl (0.1), 1 mL trace elements solution SL-10, and 1 mL vitamin solution. Acetate (10 mM) and lactate (10 mM) were provided as carbon and electron sources in these two media. The Fe(III)-nitrilotriacetic acid (NTA))-Freshwater basal medium was prepared according to the medium DSMZ 298 with slight modification and consisted in (in grams per litre of water) NaH2PO4 (0.2), NH4Cl (0.25), NaCl (1), MgCl2. 6 H2O (0.4), KCl (0.5), CaCl2.2H2O (0.15), NaHCO3 (2.5), FeIII-NTA (5 mM), 1 mL trace elements solution SL-10, and 1 mL selenite/tungstate solution (medium DSMZ 385). Fe(III)-NTA was obtained from a 100 mM solution prepared in dissolving 1.64 g NaHCO3, 2.56 g C6H6NO6Na3 (sodium nitrilotriacetic acid) and 2.7 g FeCl3.6H2O in distilled water for a total volume of 100 mL. Solutions of Fe(III)-NTA, ethanol, K2HPO4, NaHCO3 were sterilized by filtration at 0.2 µm and added after autoclaving. Na2S was autoclaved separately. The medium was then dispensed into 50-mL sealed bottles and bubbled with N2/CO2 (80∶20) for 15 min. pH was 6.6, incubation was done at 30°C. A modified version of this media was also used and consisted in (in grams per litre of water): NH4Cl (1), MgSO4.7 H2O (0.2), CaCl2.2H2O (0.1), K2HPO4 (0.05), NaHCO3 (0.43), Fe(III)-NTA (5 mM), 1 mL trace elements solution SL-10, and 1 mL selenite/tungstate solution. Ethanol (10 mM) was provided as carbon and electron sources in these two media. Cultures were performed at 28°C in the dark. Positive cultures were obtained by three tenfold-serial dilutions, and were further purified by successive transfer in fresh medium. Finally, single colonies were obtained in agar-solidified media. The isolates were identified by sequencing the 16S rRNA gene. The type strains Geobacter bremensis (DSM 12179) and Pelobacter venetianus (DSM 2395) and isolates were grown in medium containing 5 mM Fe(III)-NTA as the electron acceptor and 10 mM ethanol as the electron donor and (in grams per litre of water) NH4Cl (1); (0.2) MgSO4.7H2O (0.2); CaCl2.2H2O (0.1); K2HPO4 (0.05); NaHCO3 (0.43); 50 µL of 10 mg/L resazurin; 1 mL of Na2S 10% (w/v); 1 mL element trace solution SL10 ; and 1 mL of a selenite/tungstate solution .Microbial suspensions obtained from the polarized and control DSA electrodes were used to inoculate the anthraquinone-2,6- disulfonate (AQDS) Freshwater basal medium, which was slightly modified and consisted in (in grams per litre of water) NH2/CO2 (80∶20) was continuously bubbled in the medium through a 0.2-µm filter. All experiments were performed at 30°C. The reactors were filled with the medium used for bacterial growth that did not contain the electron acceptor Fe(III)-NTA, and Na2S . Reactors were inoculated with 10% of culture solution, 24 hours after Fe(III)-(NTA) had been completely reduced to Fe(II), as indicated by the disappearance of the brown colour. Control experiments performed with the culture medium showed no current production in the absence of Fe(III)-(NTA). Cyclic voltammetry at 10 mV/s started from 0.50 V towards positive values between −0.80 and 0.80 V vs. Ag/AgCl with graphite electrodes.DSA (50×25×1 mm) and graphite  electrodes were cleaned as described elsewhere 595 to estimate bacterial suspensions from biofilms. DNA was extracted from biofilms according to Erable et al.Biofilms were recovered from electrodes as previously described rrs) amplification was performed using bacterial primers  as described previously by Achouak et al.rrs fragments were screened by denaturing gradient gel electrophoresis (DGGE), and those representing different DGGE patterns were sequenced.DNA samples extracted from the 6 connected DSA were pooled. The entire 16S rDNA gene  and the use of epifluorescence microscopy. Fluorescence In Situ Hybridization (FISH) was performed as previously described Figure S1Impact of electrodes polarisation on biofilms formation. Nucleic acids contents and cultivable bacterial cells number from polarized DSA (PE) and non-polarized DSA (PE) electrodes.(TIF)Click here for additional data file.Figure S2Bacterial diversity of EAB. Bacterial diversity of rrs clones library from polarised and non-polarised DSA, evaluated by number of different DGGE profile.(TIF)Click here for additional data file.Figure S3Genotyping of Geobacter sp. isolates. Selected isolates of Geobacter sp. ONC1001-1006 (lines A–F) and G. bremensis (Lline G) type strain DSM 12179 were characterised by ERIC-PCR.(TIF)Click here for additional data file.


Reperfusion into severely ischemic myocardium can lead to myocardial hemorrhage (MH). Recent studies have employed T2- and T2*-weighted imaging to assess MH. However, a direct comparison of myocardial T2 and T2* changes in the setting of MH is not available.To examine the T2 and T2* changes associated with MH and thereby determine the effectiveness of each method for detecting MH.Canines (n = 9) were subjected to a 3-hour occlusion of the LAD followed by reperfusion. Serial CMR studies (1.5T Siemens Espree) were performed post-reperfusion on days 2, 5 and 7. Short-axis images of the entire LV (resolution = 1.1x1.1x8mm3) were obtained using T2-prepared SSFP , multi gradient-echo  and PSIR Late-Enhancement (LE-PSIR) imaging. T2 and T2* maps were computed from T2-prepared and gradient-echo acquisitions. Hemorrhagic infarctions (MH+) were determined by the presence of hypointense territories on T2* maps within the infarcted zones identified from LE-PSIR images. In the MH+ group, manually drawn ROIs on the T2* maps around the hemorrhagic cores and remote territories were copied to the T2 maps. In non-hemorrhagic infarctions (MH-), manually drawn ROIs on LE-PSIR images around the infarcted zones and remote territories were copied to T2 and T2* maps. T2 and T2* values from the MH+, MH- and remote territories were measured and compared (p<0.05).MH was observed in 6 dogs (MH+), but not in the remaining 3 dogs (MH-). Figure T2* of MH+ territories were significantly lower than those of MH- and remote territories. This was not the case amongst T2 of MH+, MH- and remote territories. The reduced conspicuity of MH on T2 maps is likely due to the refocusing effects of the 180o pulses and its intrinsic sensitivity to myocardial edema. We conclude that the potential insensitivity of T2*-weighted CMR to myocardial edema and strong sensitivity to hemorrhagic byproducts makes T2* mapping a more effective approach for detecting MH.


TOC Summary: Viruses belonging to these novel genotypes are indistinguishable phenotypically from endemic swine viruses. As a result of human-to-pig transmission, pandemic influenza A (H1N1) 2009 virus was detected in pigs soon after it emerged in humans. In the United States, this transmission was quickly followed by multiple reassortment between the pandemic virus and endemic swine viruses. Nine reassortant viruses representing 7 genotypes were detected in commercial pig farms in the United States. Field observations suggested that the newly described reassortant viruses did not differ substantially from pandemic (H1N1) 2009 or endemic strains in their ability to cause disease. Comparable growth properties of reassortant and endemic viruses in vitro supported these observations; similarly, a representative reassortant virus replicated in ferrets to the same extent as did pandemic (H1N1) 2009 and endemic swine virus. These novel reassortant viruses highlight the increasing complexity of influenza viruses within pig populations and the frequency at which viral diversification occurs in this ecologically important viral reservoir. Since its detection in humans in April 2009  dominated from 1918 through 1998. In ≈1998, influenza (H3N2) triple reassortant viruses appeared, accompanied by a transient increase in disease severity  strains in 2003–2005; and H1γ strains in 1999–2000 (We describe the isolation of 9 pandemic (H1N1) 2009/endemic swine reassortant influenza viruses representing 7 distinct genotypes in pigs in the United States. Our study highlights the effect of reverse zoonotic transmission of the pandemic virus on this population.Samples used in this study were nasal swabs or lungs collected from pigs with clinical signs of respiratory disease, with the exception of A/swine/Indiana/240218/2010, which was isolated from a healthy pig within the framework of an active swine influenza surveillance program. In this program, nasal swab specimens had been randomly collected on a monthly basis since June 2009 from commercial farms in Iowa, Indiana, Minnesota, North Carolina, and Illinois. A/swine/Indiana/240218/2010 was 1 of 176 viruses detected. Vaccination status of the pigs was unknown. The specimens were transported in virus transport media at 4°C to the laboratory  for influenza screening. Samples were either tested within 48 h or frozen at −80°C before being processed.2. The ABI Fast real-time PCR system 7500 thermocycler and corresponding software  were used with the following cycling conditions: 50°C for 30 min, 95°C for 15 min, followed by 40 cycles of 95°C for 10 sec, and 60°C for 30 sec. For the growth curves, viral titers were monitored by rRT-PCR by using the method described by Harmon et al.  on a Kingfisher Flex , or with QIAGEN viral RNA kit , following the manufacturers’ instructions. Real-time reverse transcription PCR (rRT-PCR) was performed to initially screen for all influenza A viruses (Swab samples were added to MDCK or swine testicle (ST) cells (American Type Culture Collection) as described . Polymerase gene primers were added to optimize the sequencing reaction . An RT-PCR was performed on RNA templates by using Uni-12 and Uni-13 primers to amplify all 8 segments in 1 reaction with Invitrogen SuperScript III One-Step Reverse Transcriptase and Platinum For sequence analyses, samples were de-multiplexed and each genome was assembled by using CLC Genomics Workbench software  by running a high stringency de novo assembly. Sequences were compared by using BioEdit (Mustela putorius furo) 3–4 months of age obtained from Triple F Farms . All ferrets were seronegative for seasonal influenza A (H1N1) and (H3N2) viruses, pandemic (H1N1) 2009, and influenza B viruses by hemagglutination-inhibition assay. Five ferrets were inoculated intranasally under light isoflurane anesthesia with 106 50% egg infectious dose (EID50) of the subtype H3N2 reassortant, A/swine/Minnesota/239105/2009, in 1 mL of sterile phosphate-buffered saline. Pandemic (H1N1) 2009 strain A/Tennessee/560–1/2009 (TN/560/09) and influenza (H3N2) strain A/swine/Texas/4199–2/98 (sw/TX/4199/98) were used as controls for comparison. Clinical signs of infection, relative inactivity index . All animal studies were approved by the St. Jude Children’s Research Hospital Animal Care and Use Committee and were conducted according to applicable laws and guidelines.During routine surveillance for influenza viruses in pigs, 9 reassortant viruses were detected during 2009 and 2010. These viruses came from asymptomatic animals or from animals showing classic influenza symptoms, including coughing, respiratory distress, fever, or nasal discharge. These viruses were detected in Minnesota, Indiana, and North Carolina. Complete genome sequences were obtained for 7 strains: A/swine/Indiana/240218/2010(H1N2) (sw/IN/240218/10), A/swine/Minnesota/239105/2009(H3N2) (sw/MN/239105/09), A/swine/Minnesota/239106/2010(H1N2) (sw/MN/239106/10), A/swine/North Carolina/239108/2010(H1N2) (sw/NC/239108/10), A/swine/North Carolina/226124/2010(H1N2) (sw/NC/226124/10), A/swine/North Carolina/226125/2010(H1N2) (sw/NC/226125/10), and A/swine/North Carolina/226126/2010(H1N2) (sw/NC/226126/10). Partial sequences were obtained from 2 additional samples, A/swine/Minnesota/340304/2010(H1N2) (sw/MN/340304/10), and A/swine/Minnesota/226128/2010(H1N1) (sw/MN/226128/10). Of the 9 viruses, 8 displayed HA and NA genes of endemic swine influenza viruses and pandemic (H1N1) 2009 M gene segments; the origin of the remaining gene segments differed depending on the virus . The phyEight of the 9 reassortant viruses were successfully isolated on either MDCK or ST cells. Sw/MN/340304/10 could not be isolated, most likely because of lack of initial material, because this specimen had the highest cycle threshold value by rRT-PCR targeting the M gene . Isolates were plaque-purified on MDCK or ST cells, and the genotypes were all confirmed. Taken together these data show that several novel genotypes of swine influenza viruses had been generated after the reverse zoonotic transmission of pandemic (H1N1) 2009 virus to pigs.To understand whether the increased genetic diversity created through the reassortment was associated with an increase in phenotypic diversity, select reassortants were assessed for growth in vitro and in ferrets. The growth characteristics of 6 endemic viruses from 2009–2010 and 3 pandemic reassortant viruses  were compared on ST cells. We found no difference in replication potential between any of these viruses, which suggests that no selective growth advantages had occurred through reassortment , panel A6 EID50 of sw/MN/239105/09 (H3N2). This reassortant virus caused only mild clinical signs (relative inactivity index ≈0.1) without marked weight changes or body temperature elevation . The virus replicated to similar titers, as did the pandemic (H1N1) 2009 virus, TN/560/09, and the triple reassortant swine subtype H3N2 virus, sw/TX/4199/98, with a peak titer of infection of 5–6 log10 of virus and with viral clearance occurring ≈1 week postinfection (Because of the recent zoonotic transmissions of triple reassortant swine influenza (H3N2) viruses in the United States during 2010  2009 origin, a novel gene segment introduced into this animal population after human-to-pig transmission of the pandemic strain. The TRIG cassette in the reassortant viruses, a cassette that had remained relatively unchanged since 1998, was disrupted to include not only the M gene segment but also variably the NS, NP, and PA genes of pandemic (H1N1) 2009 virus. Because the pandemic virus contains M and NA gene segments from Eurasian-lineage swine influenza viruses and PB2, PB1, PA, NP, and NS gene segments from TRIG viruses, it is not surprising that several pandemic (H1N1) 2009 genes could be introduced into endemic US swine influenza viruses without altering the viability of the progeny viruses.The inclusion of the pandemic (H1N1) 2009 M gene in the reassortants suggests a selective advantage to viruses containing it, although we were unable to measure any phenotypic differences in these viruses in our in vitro and in vivo assays. One phenotype that we did not measure was transmission, and it is tempting to speculate that the pandemic (H1N1) 2009 M gene segment could play a role here, both in terms of its selection in the reassortants described and in the human pandemic virus itself. Further studies are required to test this hypothesis.The effect of these reassortants on the US pig industry is somewhat difficult to predict, although on the basis of the data generated here it is not likely to be great in terms of animal health. Antigenically, the reassortant influenza viruses carried HA genes already within the population (either endemic or pandemic (H1N1) 2009 viruses), and we were unable to detect any replication differences or unusual clinical signs in the field. Thus, and because HA-specific immunity is the target of current vaccines, the generation of these viruses is not expected to have any adverse effect on vaccine efficacy levels or disease severity in the field unless further adaptive changes occur as a result of continued circulation of these viruses.Although the reassortants invariably contained the pandemic virus M gene and, with the exception of 1 reassortant, endemic virus HA and NA genes, the fact that we only saw viruses of the exact same genotype in limited spatial and temporal space suggests that there is no single dominant reassortant yet. Indeed, it is possible that these reassortants are generated but quickly displaced by other influenza viruses. Nevertheless, the data presented here once again highlight the dynamic nature of influenza viruses in pig populations and the continued monitoring of viruses in US pigs at the level of full genome sequencing is absolutely required.


Populus euphratica. Application of NaCl (200 mM) shock induced a transient elevation in [eATP]. We investigated the effects of eATP by blocking P2 receptors with suramin and PPADS and applying an ATP trap system of hexokinase-glucose. We found that eATP regulated a wide range of cellular processes required for salt adaptation, including vacuolar Na+ compartmentation, Na+/H+ exchange across the plasma membrane (PM), K+ homeostasis, reactive oxygen species regulation, and salt-responsive expression of genes related to K+/Na+ homeostasis and PM repair. Furthermore, we found that the eATP signaling was mediated by H2O2 and cytosolic Ca2+ released in response to high salt in P. euphratica cells. We concluded that salt-induced eATP was sensed by purinoceptors in the PM, and this led to the induction of downstream signals, like H2O2 and cytosolic Ca2+, which are required for the up-regulation of genes linked to K+/Na+ homeostasis and PM repair. Consequently, the viability of P. euphratica cells was maintained during a prolonged period of salt stress.Extracellular ATP (eATP) has been implicated in mediating plant growth and antioxidant defense; however, it is largely unknown whether eATP might mediate salinity tolerance. We used confocal microscopy, a non-invasive vibrating ion-selective microelectrode, and quantitative real time PCR analysis to evaluate the physiological significance of eATP in the salt resistance of cell cultures derived from a salt-tolerant woody species,  Recently, H2O2 has been implicated in the mediation of K+/Na+ homeostasis in salt-tolerant poplar cells 2O2 stabilized SOS1 mRNA 2+-permeable channels in Arabidopsis 2+, H2O2 was suggested to upregulate the activity of the PM H+-ATPase, which is fundamental to plant salt tolerance +-ATPase was shown to create an H+ gradient for Na+/H+ exchange at the PM; furthermore, a high H+-pumping activity inhibited K+ efflux through depolarization-activated K+ channels in the face of high salinity 2O2 production typical in response to a salt shock; as a result, K+/Na+ homeostasis was no longer retained during the following 24-h of salt stress Plant tolerance to salinity is mediated by a multi-trait, regulatory network. In recent years, plant regulation of ion homeostasis has received much attention. Ca2+ ([Ca2+]cyt), reactive oxygen species (ROS), and NO 2+]cyt and ROS production in Arabidopsis, and these ATP-mediated responses were blocked with antagonists of animal purinergic receptors (P2 receptors) In plant cells, extracellular ATP (eATP) has been postulated to serve as a signal in growth and stress responses Populus euphratica. Callus cells of P. euphratica have exhibited high efficiency in regulating K+/Na+ and ROS homeostasis under salt stress 2O2 and [Ca2+]cytIn this study, we attempted to clarify the contribution of eATP to salinity tolerance in higher order plants. We used an ideal model system: cell cultures of a salt-resistant woody species, Populus euphratica Oliver were prepared as described previously −1 benzyladenine (BA) and 0.50 mg L−1 α-naphthaleneacetic acid (NAA), and raised in the dark at 25°C. Callus cells were subcultured every 15 days, and all experiments were performed at 10 days after cells were transferred to fresh propagation medium. Prior to experimental treatments, cell cultures were suspended in liquid MS (LMS) medium without hormones for 1 h equilibration  2O2, and Ca2+ flux during 24 h experiment  and non-hydrolyzable ATP ; in the H-G trap, hexokinase phosphorylates glucose in a reaction that consuming one molecule of ATP a2+ flux , activita2+ flux , and expa2+ flux . Furtherreatment . Compare2O2 accumulation, membrane potential (MP), Na+ levels in the cytosol and vacuole, expression levels of salt-responsive genes, and steady-state fluxes of Na+, H+, and K+. Activities of antioxidant enzymes  were examined in untreated control and saline-stressed cells that had been pretreated with or without suramin, PPADS, or H-G.Suspended cells were pretreated without or with suramin (300 µM for 2 h), PPADS (300 μΜ for 2 h), or an H-G system (50 mM glucose and 100 units/mL hexokinase for 6 h), followed by the addition of NaCl (200 mM). After 24 h, we measured cell viability, H2O2, cytosolic Ca2+, Na+ compartmentation, and transient fluxes of H+, K+, and Ca2+ across the PM.Suspended cells were subjected to suramin or PPADS (300 µM) for 2 h or the H-G solution (50 mM glucose and 100 units/mL hexokinase) for 6 h prior to the addition of NaCl (200 mM). Immediately after NaCl addition, during the following observation period (30–60 min), we recorded HP. euphratica cells. The pharmacological experiments were designed as described in Series 1 and 2, except that different concentrations of ATP  were added to the 200 mM NaCl solution. The addition of 200 µM ATP exhibited a pronounced rescue from the H-G inhibition; both early H2O2 production (30 min) and late Na+ extrusion (24 h) were rescued in the presence of high salinity  treatment of Series 1, we measured cell viability, H2O2 accumulation, MP, Na+ levels in the cytosol and vacuole, expression of salt-responsive genes, and steady-state fluxes of Na+, H+, and K+.In this series, ATP was introduced to inhibitor-pretreated cells to confirm that eATP mediated the salt response in salinity . TherefoP. euphratica cells (0.1 g) were suspended in 0.5 mL LMS that contained a P2 receptor antagonist  or the H-G solution (LMS supplemented with 50 mM glucose and 100 units/mL hexokinase). After incubation at room temperature for 2 h or 6 h, respectively, the medium was exchanged with a solution of high NaCl (200 mM) prepared in LMS with the corresponding inhibitors. Control cells were not exposed to NaCl or pharmacological agents. Cell-free supernatants were collected at the indicated time points and immediately frozen in liquid nitrogen for later analyses. ATP was determined in an assay with luciferin-luciferase. All samples were assessed with a Turner Designs Modulus™ Microplate Multimode Reader . Two individual 10 µL samples were assayed from each replicate to ensure internal consistency of the sample. The concentration of eATP was calculated from a standard curve; the [eATP] varied over a linear range of 0.01 to 100 nM.ATP levels in the ECM were measured with the Enlighten ATP assay system bioluminescence kit . In brief, −1 FDA (Sigma-Aldrich) and then incubated in the dark for 10 min at room temperature. Samples were observed under a Leica inverted fluorescence microscope  at an excitation wavelength of 480 nm. Cell viability was calculated by counting 8–10 randomly selected fields, each, with at least 300 cells.Cell viability was measured with a fluorescein diacetate stain , as described previously 2O2 was detected with dichlorodihydrofluorescein diacetate  2DCF-DA (prepared in LMS) for 5 min at room temperature in the dark. Then the H2DCF-DA-loaded cells were washed 3–4 times with LMS and analyzed with a Leica SP5 confocal microscope . The confocal settings were as follows: excitation 488 nm, emission 510–530 nm, frame 512×512. Three-dimensional (3D) scanning was performed with a 3-µm Z-series project step, and 3D reconstructed images of cells were used to calculate relative fluorescence. Image processing software  was used to determine the fluorescent intensity of all the individual cells, and each measurement was expressed as the number of pixels on a scale of 0 to 255.The specific fluorescence of H2O2 to NaCl. Control or inhibitor-pretreated cells from Series 2 and 3 were loaded with 50 µM H2DCFDA for 5 min prior to the addition of NaCl, supplemented with or without ATP. DCF-dependent fluorescence was measured every 5 min with a confocal microscope.We also recorded the transient response of H4(3); Molecular Probe, Eugene, OR, USA) 4(3) (200 µM in DMSO) was added to suspended cells that had been treated with NaCl, inhibitors, and ATP (Series 1 and Series 3); the final concentration of DiBAC4(3) was 2 µM (10 min). A total of 200 µL cells were placed in the centers of poly-L-lysine-pretreated cover slips (2×5 cm), and DiBAC4(3)-dependent fluorescence was measured with a confocal microscope. The confocal settings were the same as those described above for H2O2 detection The MP was detected with a fluorescent probe, Bis-trimethine oxonol . In addition to the effects of long-term salt stress (24 h), we also examined the effects of suramin, PPADS, and H-G on Na+ compartmentation after a short-term treatment .To evaluate the effects of eATP on the pattern of intracellular Na+, H+, K+, and Ca2+ were measured non-invasively with the Scanning Ion-selective Electrode Technique . Recordings of transient H+, Ca2+, K+, and steady-state Na+, H+, K+ fluxes were performed as described previously +, K+, and Ca2+ recordings, control or inhibitor-pretreated cells (Series 2 and Series 3) were settled on the bottom of a poly-L-lysine-pretreated petri dish in 4 mL measuring solution , with added H-G, PPADS, and suramin. First, the steady-state H+, K+, and Ca2+ fluxes were recorded (5–6 min) prior to the NaCl and ATP treatment. Stock solutions of NaCl (400 mM) and ATP (400 µM) were slowly added to the measuring solution until the final concentration in the solution reached 200 mM NaCl, with or without 200 µM ATP. The flux recording was restarted and continued for 30–35 min. The data measured during the first 30 s were discarded, due to the diffusion effects of the stock addition . We compared the kinetics of Ca2+ transients elicited by 100 and 200 mM NaCl.Net fluxes of Na+, H+, and K+ flux measurements, cells pretreated with NaCl and inhibitors (Series 1 and Series 3) were placed in the centers of poly-L-lysine-pretreated coverslips in 4 mL measuring solution . The steady-state flux measurements were, as a rule, continuously recorded for 8–10 min . Three-dimensional ionic flux signals were plotted with MageFlux software, developed by Yue Xu (http://xuyue.net/mageflux).For steady-state Na2+ was visualized with a Ca2+-sensitive fluorescent dye, Rhod-2/AM (Biotium) Cytosolic CaP. euphratica cells were treated with 200 mM NaCl for 24 h in the absence or presence of corresponding agents . Control cells were incubated in LMS without addition of NaCl, inhibitors, or ATP  containing 1 mM EDTA and 1% polyvinylpyrrolidone (PVP) g for 20 min at 4°C, and the supernatant was examined for total CAT and GR activities, as described previously We also examined activities of antioxidant enzymes, CAT, APX, and GR, under salt and ATP treatments. NHX1, SOS1, and +-ATPase-subunit avacuolar H (VHA-a) were based on expressed sequence tags (ESTs) from P. euphratica; and primers designed to target the +-ATPasePM H (AHA), +-pyrophosphatasevacuolar H (AVP), VHA-b, VHA-c, mitogen-activated protein kinase (MPK), and synaptotagmin (SYT) were based on P. trichocarpa homologs (http://www.ncbi.nlm.nih.gov/guide/). The resulting amplicons were between 150 and 300 bp. The total 25 µL qRT-PCR reaction volume contained 12.5 µL SYBR Green PCR Master Mix (Applied Biosystems), 1 µL of 1∶1 (v/v) diluted cDNA, and 0.12 µM gene-specific primers. The PCR was performed on an Applied Biosystems 7500 Fast Real-Time PCR System . The melting-curves were analyzed immediately to confirm the specificity of the products; the mean Ct value for each gene was obtained from three independent PCR experiments. The relative expression level for each target gene was normalized to the Populus housekeeping gene, Actin (GeneBank number XM_002322628), with the forward primer 5′-CCCTCTATGCCAGTGGTCGTA-3′ and the reverse primer 5′-ACGCTCTGCTGTGGTTGTGAA-3′. Relative expression levels were calculated with the 2−ΔΔC method. In addition to the gene expression elicited by high salt exposure, we examined the effects of suramin, PPADS, H-G, ATP (100 and 200 µM), and ATPλS (200 µM) on the expression of salt-responsive genes in control cells that were not exposed to high salt conditions , followed by an on-column DNase treatment. Then, 2 µg of total RNA was reverse transcribed with SuperScript III (Invitrogen) and oligo (dT) primers (Invitrogen). Next, 1 µL of synthesized cDNA was used as template for real-time PCR (RT-PCR) amplification. The PCR products were sequenced and validated for quantitative RT-PCR (qRT-PCR). Primers designed to target homologs . All thenditions .p<0.05.All mean data were subjected to an analysis of variance. Significant differences between means were determined with Duncan’s multiple range test. Unless otherwise stated, differences were considered statistically significant when P. euphratica by adding suramin or PPADS to block eATP signaling P. euphratica cells. It was previously shown in Arabidopsis seedlings that plants responded to increased hypertonic stress with a transient increase in [eATP] P. euphratica cells exposed to high salt. First, we found that a 24-h exposure to NaCl (200 mM) did not suppress cell viability , the pattern of Na, or H-G . This da+ flux depends on MP in salt-stressed P. euphratica cells + homeostasis of P. euphratica cells exposed to long- and short-term salt stress. After 24 h of high NaCl treatment, the PM was depolarized and K+ efflux was increased in P. euphratica cells  reduced the enhancement in K+ efflux and PM depolarization mediated by H-G in salinized cells, but not that mediated by suramin or PPADS treatment  was not affected by suramin, PPADS, H-G, or exogenously applied ATP  compared to short-term NaCl stress , the PM Na+/H+ antiporter (SOS1), synaptotagmin (SYT), mitogen-activated protein kinase (MPK), vacuolar Na+/H+ antiporter (NHX1), vacuolar H+-pyrophosphatase (AVP), and vacuolar H+-ATPase subunits a–c . We used suramin, PPADS, and the H-G system to study eATP regulation of the transcription of these salt-responsive genes. We found that, in salinized cells, application of suramin, PPADS, and H-G markedly inhibited the abundance of the mRNA levels of these selected genes  or ATPλS (200 µM) under control conditions  rescued the salt-induced H2O2 production in H-G treated cells, and this effect was dose-dependent over the tested ATP concentrations  rescued cells from H-G inhibition of salt-induced [Ca2+]cyt, but not from the effects of suramin or PPADS , but the flux rate fluctuated over the recording period  during SIET recording period  was able to rescue the Ca2+ influx elicited by NaCl in H–G treated cells, but not in suramin or PPADS-treated cells , P. euphratica cells were unable to perform processes of acclimation to the salt medium, including cytosolic Na+ exclusion, vacuolar salt compartmentation, K+ homeostasis, ROS control, antioxidant defense, and induction of salt-resistant gene expression , who found that salinized P. euphratica suspension cultures displayed high tonoplast Na+/H+ exchange activity + exclusion and vacuolar ion compartmentation were both diminished by H-G, PPADS, or suramin in salinized cells  and PM H+-ATPase (AHA) in P. euphratica cells  could not generate H+ gradients across the vacuolar membrane, and this led to insufficient Na+ compartmentation in the vacuole  channels + permeable and thus directly contribute to Na+ sequestration in vacuoles (by preventing its back leak into cytosol), further investigations are necessary to elucidate how salt-induced signaling molecules mediate SV channels and Na+ compartmentation. Our previous studies showed that increases in eATP caused increases in the intracellular ATP level +-coupled transporters (H+-ATPase) or regulated other signaling pathways in these cells. However, our experiments did not differentiate between effects due to intracellular ATP and those due to eATP.Our results showed that salt-induced increase in eATP contributed to regulating Nareatment . This ret stress . This aged cells . Additiomentaion . These rca cells . We conc vacuole . In addi+ efflux in P. euphratica cells  during NaCl stress , was shown to be involved in Na+ detoxification in Arabidopsis. NaCl stress stimulated PA production and MPK6 activity, which phosphorylated the C-terminal of SOS1 Solanum lycopersicum) and Arabidopsis suspensions + homeostasis in NaCl-stressed P. euphratica cells.In Arabidopsis, rice, and poplar, high salt treatment stimulated a SOS pathway that caused an increase in NaP. euphratica cells ; this led to uncontrolled oxidation and cell death in salt stressed P. euphratica cells , which triggered H+ influx across the PM, and this led to rises in H2O2 and [Ca2+]cyt in P. euphratica cells + flux in NaCl-treated cells  and the PM H+-coupled ion transporters  in the salt-resistant species P. euphratica  and 0.50 mg L−1 α-naphthaleneacetic acid (NAA) for 24 h, then cell viability, H2O2, and Ca2+ flux were measured. Bars represent the means from four independent experiments and whiskers represent the error of the mean. The same letter denotes no significant difference between treatments.(DOC)Click here for additional data file.Figure S22O2, and Ca2+ flux in Effects of H-G on cell viability, HP. euphratica cells.P. euphratica cells were incubated in LMS containing an ATP trap  for 6 h, then cell viability, H2O2, and Ca2+ flux were measured. Bars represent the means from four independent experiments and whiskers represent the error of the mean. The same letter denotes no significant difference between treatments.(DOC)Click here for additional data file.Figure S3Effects of pharmacological agents and ATP on antioxidant enzyme activity in control and NaCl-stressed cells of P. euphratica.P. euphratica cells were treated with 200 mM NaCl for 24 h in the absence and presence of suramin (300 µM), PPADS (300 µM), or H-G (50 mM glucose and 100 units/mL hexokinase). Control cells were incubated in LMS supplemented with or without ATP (200 and 500 µM) for 24 h. Then, the activities of antioxidant enzymes, ascorbic peroxidase (APX), catalase (CAT), and glutathione reductase (GR) were measured; activities are expressed as the amount of ascorbate (ASA), H2O2, and NADPH consumed, respectively. Each bar represents the mean of four independent experiments, and whiskers represent the standard error of the mean. Different letters  indicate significant differences between treatments (P<0.05).(DOC)Click here for additional data file.Figure S4Effects of pharmacological agents and ATP on the expression of salt-responsive genes in no-salt control cells of P. euphratica.P. euphratica cells were treated without (control) or with suramin (300 µM), PPADS (300 µM), H-G (50 mM glucose and 100 units/mL hexokinase), ATP (100 or 200 µM), or ATPλS (200 µM) for 24 h; then, total RNA was isolated for quantitative Real-Time PCR analysis. Each bar represents the mean of four replicates and whiskers represent the standard error of the mean. Different letters  indicate significant differences between treatments (P<0.05).(DOC)Click here for additional data file.Figure S52O2 accumulation in Concentration tests for effects of suramin or PPADS on cell viability and HP. euphratica cells. Suspended cells were untreated (control) or treated with suramin or PPADS  for 24 h, then cell viability and H2O2 levels were measured under a fluorescence microscope. (A) Cell viability. Bars represent the mean of three independent experiments in which at least 300 cells were counted. (B) H2O2 accumulation. Bars represent the mean H2O2 levels quantified from 45 to 50 individual cells in three independent experiments. Whiskers represent the standard error of the mean. N.S. = no significant difference.(DOC)Click here for additional data file.Figure S62O2 burst in Effects of P2 receptor antagonists (suramin and PPADS) on the early HP. euphraticaλS). cells elicited by NaCl or non-hydrolysable ATP  for 2 h, then subjected to (A) 200 mM NaCl or (B) 200 µM ATPλS for 30 min. H2O2 levels were measured under a fluorescence microscope. Bars represent the mean H2O2 levels quantified from 45 to 50 individual cells in three independent experiments. Whiskers represent the standard error of the mean. Different letters  indicate significant differences between treatments (P<0.05).(DOC)Click here for additional data file.Figure S7+ flux and early H2O2 burst in NaCl-stressed Effects of exogenous ATP on NaP. euphratica cells in the presence and absence of pharmacological agents. Suspended cells were untreated (control) or pretreated with suramin (300 µM for 2 h), PPADS (300 µM for 2 h), or the H-G system (50 mM glucose and 100 units/mL hexokinase for 6 h). This was followed by exposure to 200 mM NaCl supplemented without or with ATP . (A) Steady Na+ fluxes after 24 h or (B) early H2O2 production after 30 min of NaCl stress. Bars represent the mean of 14–16 (Na+ fluxes) and 40–50 (H2O2) individual cells; whiskers represent the standard error of the mean. Different letters  indicate significant differences between treatments (P<0.05).(DOC)Click here for additional data file.Figure S8+ compartmentation in NaCl-stressed Effects of pharmacological agents on NaP. euphratica cells.P. euphratica cells were treated with 200 mM NaCl for 1 h in the absence (control) or presence of suramin (300 µM), PPADS (300 µM), or the H-G system (50 mM glucose and 100 units/mL hexokinase). The Na+-specific fluorescent probe, CoroNa-Green/AM, was added to detect Na+ levels in the cytoplasm and vacuole. Each measurement was based on at least 100 individual cells. Bars are the mean of three independent experiments. Whiskers represent the standard error of the mean. Different letters  indicate significant differences between treatments (P<0.05).(DOC)Click here for additional data file.Figure S92+ flux in response to NaCl shock in Transient CaP. euphratica cells. (A) P. euphratica cells were subjected to 100 and 200 mM NaCl shock, respectively. Each point represents the mean value for six individual cells. (B) Mean flux rates of Ca2+ before (-) and after (+) the addition of NaCl. Each bar represents the mean of six individual cells and whiskers represent the standard error of the mean. Different letters  indicate significant differences (P<0.05).(DOC)Click here for additional data file.Table S1Sequences of gene-specific primers used in quantitative Real time PCR analysis.(DOC)Click here for additional data file.


The experimental exposure groups were incubator control, sham-exposed, combined RF radiation-exposed with or without either H2O2 or menadione groups. The intracellular ROS level was measured by flow cytometry using the fluorescent probe dichlorofluorescein diacetate. Intracellular ROS levels were not consistently affected by combined RF radiation exposure alone in a time-dependent manner in U87, PC12 or SH-SY5Y cells. In neuronal cells exposed to combined RF radiation with either H2O2 or menadione, intracellular ROS levels showed no statically significant alteration compared with exposure to menadione or H2O2 alone. These findings indicate that neither combined RF radiation alone nor combined RF radiation with menadione or H2O2 influences the intracellular ROS level in neuronal cells such as U87, PC12 or SH-SY5Y.The objective of this study was to investigate the effects of the combined RF radiation (837 MHz CDMA plus 1950 MHz WCDMA) signal on levels of intracellular reactive oxygen species (ROS) in neuronal cells. Exposure of the combined RF signal was conducted at specific absorption rate values of 2 W/kg of CDMA plus 2 W/kg of WCDMA for 2 h. Co-exposure to combined RF radiation with either H As a large number of individuals have been exposed to the radiofrequency (RF) signals from cellular phones, social concerns regarding the possible biological effects of RF radiation emitted from cellular phones on human health have been increasing. While a large part of the world, including Europe, Australia and East Asia, use wideband code division multiple access (WCDMA), several countries use the code division multiple access (CDMA), and some countries, such as Korea, use both . HoweverIncomplete one-electron reduction of oxygen, such as singlet oxygen, superoxides, peroxides, hydroxyl radical and hypochlorous acid, form the reactive oxygen species (ROS). ROS causes oxidative modification of DNA, proteins, lipids and small intracellular molecules, which are associated with cell or tissue damage, and are the contributing factors for cellular events including gene expression, cell proliferation, apoptosis, differentiation and senescence . Furtheret al. (2008) [et al. (2009) [A number of studies have been made into possible adverse biological effects of RF fields on oxidative stress. While most studies have shown no effects of RF radiation on ROS formation –11, some. 2008)  also rep. (2008) . Xu et a08  also Information related to the biological effects of multiple RF radiation is still very minimal, and studies related to the RF radiation effect on oxidative stress are still controversial. In order to further investigate this issue, we examined the possible effects of combined (837 MHz and 1950 MHz) RF radiation on the intracellular ROS level in neuronal cell culture systems. The present study focused on co-exposures, i.e. combining RF radiation with chemicals that induce oxidative stress, in U87, PC12 and SH-SY5Y cells as neuronal cell models.in vitro multi-frequency radiation exposure system for this study. The details about this exposure system have been described previously [The Radial Transmission Line (RTL) exposure system was used as an eviously . A typic6) seeded per dish was selected from preliminary experiments to obtain a subconfluent culture at the end of each exposure. The exposure system was then warmed up for 2 h to equilibrate it prior to RF exposure. The petri dishes (8 dishes/exposure) were placed within the exposure chamber. Exposure of RF radiation for combined (CDMA at 2 W/kg plus WCDMA at 2 W/kg) signals was performed for 2 h. During the exposure period, the temperature in the chamber was maintained within a range of 37 ± 0.3°C by circulating water within the cavity. The temperature of the culture media was monitored twice per second throughout the exposure period in the sham- and the RF radiation-exposed groups. A temperature-controlled mixture of air and CO2 (5% CO2 inside the chambers) was provided by ventilation from a modified cell culture incubator. For the sham exposure, the cells were kept in the RF radiation exposure device, but were not exposed to RF radiation. The cells were exposed in six groups: (i) incubator control group, (ii) sham exposure group, (iii) RF radiation exposure group, (iv) ROS inducers (in the form of hydrogen peroxide (H2O2)  or menadione ) exposure group, (v) sham + ROS inducers co-exposure group, (vi) RF radiation + ROS inducers co-exposure group. After 2 h RF radiation exposure, the cells were immediately transferred to a cell culture incubator and further analyzed at 1, 3, 6 and 12 h after H2O2 treatment, or at 0.5, 1 and 3 h after menadione treatment.For these experiments, cells were plated in plastic 65-mm cell culture dishes ∼ 16 h prior to each exposure. The cell count  . NIH3T3 cells were maintained in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% fetal bovine serum  and 25 U/ml penicillin/streptomycin. U87 cells were cultured in DMEM supplemented with 10% FBS  and 25 U/ml penicillin/streptomycin. PC12 cells were maintained in DMEM medium supplemented with 10% horse serum , 5% FBS, and 25 U/ml penicillin/streptomycin. SH-SY5Y cells were cultured in a 1:1 mixture of ATCC-formulated Eagle's Minimum Essential Medium  and Ham's-F12 medium  supplemented with 15% FBS and 25 U/ml penicillin/streptomycin. Cells were kept at 37°C in a cell culture incubator with a humidified atmosphere of 5% COTo quantify intracellular ROS production the fluorescent probe 2′7′-dichlorofluorescein-diacetate (DCFH-DA) was employed. It is a non-polar compound that easily penetrates the cell membrane and is hydrolyzed by intracellular esterases to its nonfluorescent polar derivate DCFH. In the presence of ROS, DCFH is oxidized to the fluorescent dichlorofluorescein (DCF) . After epost hoc test for the multiple comparisons  in the incubator control, sham-exposed and the RF-exposed groups with or without ROS inducers. Values of P < 0.05 were considered as significantly different from either sham-exposed or incubator control, respectively.All values were expressed as the mean value of duplicates of at least six independent experiments ± SD. The results were subjected to an analysis of the variance (ANOVA) using the Tukey post hoc test for the multiple comparisons. Our results indicated that the ROS level was neither consistently nor sustainably affected by the combined RF radiation exposure, with statistical significance in four different types of cell lines.To examine whether combined RF radiation (CDMA at specific absorption rate (SAR) of 2 W/kg plus WCDMA at SAR of 2 W/kg for 2 h) affects the intracellular ROS levels in neuronal cells, we measured ROS levels by DCF-DA staining in three different neuronal cell lines  at various time-points  after combined RF exposure , cell viability was also measured by MTT assay in U87, PC12, SH-SY5Y and NIH3T3 cells 12 and 24 h post exposure . Therefore, we selected 100 µM H2O2 concentration for the co-exposure of H2O2 and combined RF radiation.To investigate the effect of co-exposure with combined RF radiation and the ROS inducer H2O2 on ROS levels in three different neuronal cell lines  and additionally in NIH3T3 mouse fibroblast cells. Cells were exposed to combined RF radiation (CDMA at SAR of 2 W/kg plus WCDMA at SAR of 2 W/kg) for 2 h prior to 100 µM H2O2 treatment, and then incubated for the indicated periods for the ROS measurements. ROS levels in cells exposed to combined RF radiation with H2O2 showed no statistically significant changes compared with incubator control or sham-exposed groups of 100 µM H2O2-treated cells  after menadione treatment. Since U87 and NIH3T3 cells did not show detectable changes in ROS levels up to 12 h after menadione treatment up to a concentration of 400 µM (data not shown), we measured the ROS level and cell viability only in PC12 and SH-SY5Y cells. In PC12 cells, from our statistical analyses using ANOVA, a statistically significant increase in the ROS level (>1.5-fold) was detected at 0.5 h and 1 h, but cell viability did not show a statistically significant change (still above 80%) at 24 h after 100 µM of menadione treatment  or degrade into H2O and O2 via the enzymatic reaction of catalase and glutathione peroxidase [2−) and H2O2. Second, menadione is capable of reacting with the thiol groups of proteins and glutathione (GSH) [2O2 and menadione as well as to combined RF produces additive or synergistic effects on ROS production. We observed that none of the responses to either combined RF alone or combined RF with ROS inducers showed additive or synergistic effects on ROS production. In our experiments, only a few conditions presented a slight increase in ROS in comparison with sham exposure, and those slight increases were not consistent or sustained. Living organisms possess natural defense systems of complex antioxidant mechanisms to detoxify intracellular ROS [et al. [In this study, we present the results of a study aimed at assessing the effects of combined RF radiation (CDMA at 2 W/kg plus WCDMA at 2 W/kg for 2 h) on the intracellular ROS level in neuronal cell models: U87 human glioma cells, PC12 rat pheochromcytoma cells, and SH-SY5Y human neuroblastoma cells. Three experimental systems were used to test the hypothesis that multiple RF signals might act as promoters of ROS formation: combined (837 MHz and 1950 MHz) RF radiation alone, and combined RF radiation in combination with either Hmembrane . H2O2 caroxidase , 41. Thune (GSH) –44. In tular ROS . ROS is ular ROS , 47. Nonular ROS . Since v [et al. .In conclusion, we found a transient small increase in the ROS level after exposure of neuronal cells to multiple RF signals Fig. . HoweverThis study was supported by the Korea Communications Commission (2012) grant funded by the Korean government.


To establish the allele distribution and statistical parameters of forensic interest for the D10S1248, D22S1045, D2S441, D1S1656, D12S391, and SE33 loci in Slovenian population and to compare allele frequencies with those from other populations.We analyzed blood and buccal swab samples from 333 unrelated, healthy Slovenian individuals. All samples were genotyped using the AmpFlSTR NGM Kit to obtain the allele frequency data for the loci D10S1248, D22S1045, D2S441, D1S1656, and D12S391. Samples from 113 individuals were also analyzed using the PowerPlex ESX 17 system to obtain the allele frequency data for the SE33 locus. Allele frequencies and statistical parameters of forensic interest were determined and frequency profiles compared between Slovenian and other European Caucasian populations using the Arlequin software, version 3.5.1.3.The investigated short tandem repeat (STR) loci in Slovenian population had a great discriminating potential with a combined discrimination power of 0.99999998. The highest discrimination power and polymorphism information content were observed for the SE33 locus, followed by loci D1S1656, D12S391, D10S1248, D2S441, and D22S1045. When Slovenian allele frequency distribution was compared with other European populations, deviations were found only for Spanish and Italian population for D2S441 and D12S391.Slovenian population does not differ significantly from other European populations in terms of allele frequency distributions for the six analyzed STR loci. Based on forensic efficiency values, SE33 may be considered the most informative locus, which makes it especially useful in forensic investigations. TM PCR Amplification Kit  and the PowerPlex ESX 17 System , which we used for our population study. Allele frequencies for autosomal STRs  DNA typing in forensic casework, the European Network of Forensic Institutes (ENFSI) and the European DNA Profiling (EDNAP) Group proposed the use of additional five STRs, three mini-STRs , and two highly polymorphic STRs (D1S1656 and D12S391). The increased number of the European Standard Set (ESS) loci resulted in improved discrimination power, sensitivity, and reproducibility for the analysis of minute amounts of DNA . The DNA was extracted and purified in a Biorobot EZ1 (Qiagen) device using the EZ1 DNA Investigator Card and EZ1 DNA Investigator Kit (Qiagen). Following the manufacturer’s instructions (TM Human DNA Quantification Kit (Applied Biosystems) and the quantity of DNA was determined. The reactions were carried out in a 7500 Real Time PCR System (Applied Biosystems), using the HID Real-Time PCR Analysis Software, version 1.1 (Applied Biosystems) according to the manufacturer’s instructions . Additionally, 113 of 333 samples underwent duplicate analysis using the PowerPlex ESX 17 System (Promega) to obtain the data for SE33 locus. We used both kits to increase the posterior probability in complex kinship analyses in cases when the probability for inclusion was too low after typing autosomal STRs using NGM kit. With typing of additional highly polymorphic SE33 locus using PowerPlex ESX 17 it was possible to achieve higher evidential value of autosomal STR typing. We performed a population analysis of 5 new ESS loci including 333 individuals and of the SE33 locus including 113 individuals. The NGM and ESX 17 System contain the same 15 core STR loci, including five new ESS loci, and amelogenin, whereas the ESX 17 System also contains the SE33 locus. The polymerase chain reaction (PCR) amplifications were performed according to the manufacturer’s instructions  using the 3130 Performance Optimized Polymer 4 (Applied Biosystems) and the GeneScan-500 LIZ (Applied Biosystems) internal size standard with the NGM kit, and CC5 Internal Lane Standard 500 (Promega) with the ESX 17. The genetic profiles were determined using the Data Collection v. 3.0 and GeneMapper ID v. 3.2 software (Applied Biosystems). Samples with questionable allele calls and with peak imbalance were reanalyzed. For quality control, our laboratory regularly participates in the quality control proficiency testing provided by the GEDNAP group (German DNA profiling group).DNA typing of autosomal STR loci was performed for all samples using the AmpFlSTR NGMructions ,23. BothWe used the Arlequin software, version 3.5.1.3 to determine allele frequencies, observed heterozygosity, expected heterozygosity, probability value of the Hardy-Weinberg equilibrium (HWE) exact test, and standard error . The samP = 0.0497) and D12S391 (P = 0.0217)  for the six STR loci showed no deviations from the HWE, except for the D22S1045 locus (0.0475), which was rejected after Bonferroni correction  (Supplem P = 0.02 (17). Th 0.0217)  , followed by D1S1656, D12S391, D10S1248, D2S441, and D22S1045. The highest typical paternity index and power of exclusion were observed for the SE33 locus  followed by D12S391, D1S1656, D10S1248, D2S441, and D22S1045. The lowest matching probability was calculated for the SE33 locus (0.014), followed by D12S391, D1S1656, D10S1248, D2S441, and D22S1045 (Supplementary Table 1).The analyzed STR loci in Slovenian population were highly polymorphic, as it was shown previously in other European populations -19. SE33Allele frequencies for five new ESS loci and SE33 in Slovenian population did not significantly differ from those in Croatia , Czech RThe obtained data demonstrate that analyzed loci are very useful for forensic purposes due to their shortness and high polymorphism. We recommend that 5 new ESS STR loci are adopted for the analysis of degraded challenging DNA samples to improve the discrimination power of national databases and to facilitate standardization within Europe ,2. Becau


Thermus sp. enzyme family that we propounded previously within Type IIS restriction endonucleases, containing related thermophilic bifunctional endonucleases-methyltransferases from various Thermus sp.: TaqII, Tth111II, TthHB27I, TspGWI, TspDTI and TsoI. These enzymes show significant nucleotide and amino acid sequence similarities, a rare phenomenon among restriction endonucleases, along with similarities in biochemical properties, molecular size, DNA recognition sequences and cleavage sites. They also feature some characteristics of Types I and III.The TaqII enzyme is a member of the Barker et al. reported the Type IIS/IIC restriction endonuclease TaqII as recognizing two distinct cognate site variants (5'-GACCGA-3' and 5'-CACCCA-3') while cleaving 11/9 nucleotides downstream. We used four independent methods, namely, shotgun cloning and sequencing, restriction pattern analysis, digestion of particular custom substrates and GeneScan analysis, to demonstrate that the recombinant enzyme recognizes only 5'-GACCGA-3' sites and cleaves 11/9 nucleotides downstream. We did not observe any 5'-CACCCA-3' cleavage under a variety of conditions and site arrangements tested. We also characterized the enzyme biochemically and established new digestion conditions optimal for practical enzyme applications. Finally, we developed and propose a new version of the Fidelity Index - the Fidelity Index for Partial Cleavage (FI-PC).Thermus aquaticus has been redefined as recognizing only 5'-GACCGA-3' cognate sites. The reaction conditions  were designed either to minimize (pH = 8.0 and 10 mM ammonium sulphate) or to enhance star activity . Redefinition of the recognition site and reaction conditions makes this prototype endonuclease a useful tool for DNA manipulation; as yet, this enzyme has no practical applications. The extension of the Fidelity Index will be helpful for DNA manipulation with enzymes only partially cleaving DNA.The DNA recognition sequence of the bifunctional prototype TaqII endonuclease-methyltransferase from  Restriction endonucleases (REases) are indispensable as molecular scissors in the analysis, rearrangement, cloning and sequencing of DNA -3. Restrin vitro  Δ(lac-proAB) Δ(recA1398) deoR, rpsL, srl-thi, supE/F' proAB+ lacIQZΔM15}  was used for the transformation of ligation mixtures and DNA propagation.The The Plasmid Mini, Gel-Out and Clean-Up DNA purification kits were from A&A Biotechnology ; SmaI restriction endonuclease and T4 DNA polymerase from Eurx Molecular Biology Products ; the PCR 20-bp Low Ladder from Sigma-Aldrich Poland; Taq DNA Polymerase, λ DNA and plasmids pBR322 and pUC19 from Vivantis Technologies . The DNA sequencing, GeneScan and PCR primer synthesis were performed at Genomed . All other reagents were purchased from Sigma-Aldrich .Since TaqII does not yield complete digestion, regardless of the amount of the enzyme used, one unit of TaqII enzymatic activity is defined as the minimum amount of the enzyme needed to yield a stable digestion pattern during 1 h at optimum temperature in optimum buffer, using 1 μg of pUC19 plasmid DNA.Taq DNA polymerase and a pair of primers: RTaqII 5'-GGTGCAGGGCGCTGACTTCC-3' and 1FTaqII 5'-TCGACCTGAATGGAAGCCGGCGGCACCTCGCTgACcGATTCACCACT-3' or 2FTaqII 5'-TCGACCTGAATGGAAGCCGGCGGCACCTCGCTcACccATTCACCACT-3', respectively. The cleavage pattern of the DNA substrates used, expected from published data [CAccCAAACCGAAGA-3'  within the PCR fragment amplified from pBR322 plasmid DNA. Two alternative PCR fragments (390 bp): PCR(GACCGA) and PCR(CACCCA) containing single (→) originally published canonical sites for TaqII , either hed data , should ' Figure . TaqII c gelatin . The proet al. [Cleavage was carried out in the reaction buffer proposed by Barker et al. . The reaThe investigations into the influence of pH on recombinant TaqII/TaqII* REase activity were carried out for the range from 5.0 to 9.5. The pH of all the reaction buffers was determined at the reaction temperature (65°C). The following 40 mM biological buffers were used: sodium acetate buffer for pH from 5.0 to 5.5, HEPES buffer for pH from 6.0 to 7.0, and Tris-HCl buffer for pH from 7.5-9.5.2, 1 mM DTT, BSA 100 μg/mL. All the reaction mixtures contained 1.2 pmol recognition site and 0.75 pmol recombinant TaqII protein. The molar ratio of protein to DNA recognition sites was approximately 0.625:1. The reaction volume was 50 μL. After 30 min, the reactions were quenched with phenol/chloroform, and DNA was ethanol-precipitated from the aqueous phase. The DNA precipitate was collected by centrifugation and dissolved in 10 mM Tris-HCl, pH 8.0, at 25°C. The products were analysed by electrophoresis in either 2% agarose or 15% polyacrylamide gels.The TaqII cleavage reactions of the PCR (GACCGA) DNA substrate were performed at 65°C in the reaction buffers: 40 mM of the appropriate buffering agent, 10 mM MgClThe intensity of the band corresponding to the 48 bp TaqII restriction fragment was measured using UnScan-It software and the relative enzymatic activity was calculated as a percentage. The optimal reaction buffer was chosen and the influence of the increasing ionic strength on the reduction of TaqII* star activity was determined. Ammonium sulphate in the concentration range from 0 to 70 mM was added to the reaction buffer in order to obtain maximum TaqII activity with simultaneous elimination of TaqII* activity.CAccCAAACCGAAGA-3'  substrate, an additional 5'-CACCCA-3' site was introduced to the PCR(GACCGA) DNA fragment using the analogous reverse primer 2RTaqII 5'-GGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAA' Figure . The dis' Figure .et al.: 6 mM Tris-HCl, pH 7.5, 6 mM MgCl2, 6 mM β-mercaptoethanol and 100 μg/ml gelatin [® Terminator v3.1 Applied Biosystems (Life Technologies).The TaqII cleavage of the PCR (sequencing) DNA substrate obtained was carried out in the appropriate reaction buffer at 65°C. The TaqII cleavage reaction was performed in the reaction buffer proposed by Barker  gelatin . The reaSmaI site of pUC19 vector, transformed into E. coli DH11S, and plated onto X-gal/IPTG plates [The TaqII recognition site and cleavage positions were established by shotgun cloning and sequencing of the digestion products of bacteriophage λ DNA. The TaqII-generated restriction fragment ends were blunted with T4 DNA polymerase in the presence of dNTPs, cloned into the G plates . PlasmidFor the PCR(GeneScan) substrate, an additional 5'-CACCCA-3' site was introduced to the PCR(GACCGA) DNA fragment . In the second round of the PCR reaction, the fluorescence dyes 6-FAM and Hex were added to the 5'- and 3'-ends of DNA fragment using a pair of primers, complementary to the PCR (GACCGA/CACCCA) substrate. The DNA sequences of the primers used were F6FAM-390: 5'-ggaggCTCGACCTGAATGGAA-3' and RHex-390 5'-aaagagaGGTGCAGGGCG-3'. The PCR (GeneScan) fragment obtained was 402 bp in length Figure .2, 6 mM β-mercaptoethanol and 100 μg/ml gelatin 38 pM PCR (GeneScan) DNA substrate and 380 pM of TaqII protein were incubated at 65°C for 16 hours. The products were analysed by electrophoresis in either 2% agarose or 15% polyacrylamide gels. The 345 bp restriction fragment was isolated from agarose and subjected to GeneScan analysis using an ABI 3730xl DNA Analyzer and internal size standard: GeneScan ROX-500 (PE Applied Biosystems). The data obtained were analysed using PeakScanner v 1.0 software (Applied Biosystems).The cleavage reaction mixture (500 μl) containing 6 mM Tris-HCl, pH 7.5 at 65°C, 6 mM MgClPolyacrylamide gels (15%) were prepared in 1× Tris-Borate-EDTA (TBE) buffer . The gelThermus sp. enzyme family and FI-PC, participated in the design and interpretation of the experiments, coordinated the execution of the project and drafted the manuscript. All the authors read and approved the final manuscript.AŻS conceived the project, coordinated its execution, participated in the design and interpretation of the experimental analyses, performed some of the experiments and drafted the manuscript. OŻ performed most of the experiments. KŚ purified TaqII REase. JJF performed some of the experiments and participated in the preparation of the figures. PMS was the author of the concept of the


Please see the correct Figure 3 here: In addition, there was an error in the legend for Figure 3B. The legend should read: B. Introduction of CaM binding perturbing mutations in helix A or helix B of Kv7.3 lead to non-functional channels. Left, schematic representation of the mutants analyzed. Middle column, representative current traces of whole-cell patch-clamp recordings from HEK293T expressing homomeric Kv7.3T WT, I379D or A518D. The pore A315T mutation was introduced to boost expression [19]-[21].