After treatment with different concentrations of EDT, the decreased hemodynamic parameters induced by myocardial I/R injury were significantly attenuated by EDT. The elevated levels of IL-6, CRP, IL-8, and TNF-α were effectively reduced by EDT treatment. EDT also remarkably suppressed the levels of Bax and cleaved Caspase-3, and up-regulated the level of Bcl-2 in cardiac tissues from EDT-treated groups. Further studies showed that EDT could increase the levels of p-JAK2 and p-STAT3 in cardiac tissues. Meanwhile, treatment of AG490, a specific inhibitor of JAK2, abolished the protective effect of EDT on hemodynamic parameters, myocardial inflammation and myocardial cell apoptosis induced by I/R injury. These results demonstrated that EDT could protect against myocardial I/R injury through the activation of JAK2, providing a potential treatment with EDT during myocardial I/R injury.Myocardial ischemia-reperfusion (I/R) injury remains the leading risk factor of disability and mortality worldwide. In this study, the myocardial protective effect of eriodictyol (EDT) and the underlying mechanism in an  Myocardial ischemia, caused by a decrease of blood supply to cardiac tissue, remains the leading risk factor of disability and mortality worldwide . AlthougThe Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling, transducing cellular signals from the plasma membrane to the nucleus, has been proven to mediate cardio-protection against myocardial I/R injury. Until now, four JAKs molecules  and seven STATs molecules  have been discovered . EspeciaEriodictyol , a bitter- masking flavanone, is commonly present in fruits and vegetables, especially citrus such as lemon . Recent ex vivo model of global myocardial I/R. Next, the apoptosis-related proteins and the activation of JAK2 and STAT3 in the cardiac tissues were examined by Western blot. Based on these results, we employed the special inhibitor of JAK2 in this I/R model, and then re-evaluated the heart Hemodynamic parameters, myocardial infarct size, proinflammatory cytokines and myocardial cell apoptosis to investigate the role of JAK2 activation in the myocardial protection of EDT.In this study, we first evaluated the effects of EDT on the heart hemodynamic parameters, myocardial infarct size, proinflammatory cytokines and myocardial cell apoptosis in TM ELISA kit (Cat No. 1030026) were purchased from Beijing Solarbio Science & Technology Co., Ltd., Rat IL-8 ELISA Kit (Cat No. BP-E30583) was purchased from Shanghai Langton Biotechnology Co., Ltd., Anti-Bax antibody (Cat No. sc-7480), Anti-Bcl-2 antibody (Cat No. sc-7382), Anti-Caspase-3 antibody (Cat No. sc-56053), Anti-JAK2 antibody (Cat No. sc-390539), Anti-STAT3 antibody (Cat No. sc-8019), Anti-p-STAT3 antibody (Cat No. sc-8059) and anti-β-actin antibody (Cat No. sc-47778) were all purchased from Santa Cruz Biotechnology. Anti-p-JAK2 antibody (Cat No. ab32101) was purchased from Abcam .Eriodictyol (EDT) , AG 490  and 2,3,5-Triphenyltetrazolium chloride , were purchased from Sigma-Aldrich . Lactate dehydrogenase (LDH) Cytotoxicity Assay Kit (Cat No. C0017), Rat IL-6 Enzyme-Linked ImmunoSorbent Assay (ELISA) Kit (Cat No. PI328), Rat TNF-α ELISA Kit (Cat No. PT516) and Colorimetric TUNEL Apoptosis Assay Kit (Cat No. C1098) were purchased from Beyotime Biotechnology . Creatine Kinase Activity Assay Kit (Cat No. BC1140) Rat CRP EasyTestad libitum. The room was maintained at a constant temperature of 25°C relative humidity of 70%, and a 12 h light/12 h dark cycle. Then, the ex vivo model of global myocardial I/R was established according our previous study  buffer , equilibrated with a gas mixture comprised of 5% CO2 and 95% O2 at 37°C, and then incubated in a water-jacketed organ chamber at 37°C. After 15 min of perfusion to stabilize the hearts, the hearts were subjected to 15 min of perfusion, 15 min of zero-flow global ischemia and subsequently 45 min of reperfusion. All the experimental protocols were approved by the Committees of Animal Ethics and Experimental Safety of Shihezi University are conform with the NIH guidelines for the care and use of laboratory animals.Ninety male Sprague Dawley (SD) rats, 250–300 g, were obtained from Xinjiang Medicine University Medical Laboratory Animal Center [License No. SCXK (xin) 2016-0016]. The rats were kept in the animal room at Shihezi University in accord with a commercial standard rat diet and water us study . Briefly-1⋅d-1, i.p., 3 days before the operation), then the hearts were excised from the rats and were administrated with a K–H buffer containing EDT (5 mg/L) for 15 min, 15 min of zero-flow global ischemia and subsequently 45 min of reperfusion after 15 min of stabilization, (4) I/R+EDT-10 treatment group (I/R+EDT-10): the rats were treated with EDT , then the hearts were excised from the rats and were administrated with a K–H buffer containing EDT (10 mg/L) for 15 min, 15 min of zero-flow global ischemia and subsequently 45 min of reperfusion after 15 min of stabilization, (5) I/R+EDT-20 treatment group (I/R+EDT-20): the rats were treated with EDT , then the hearts were excised from the rats and were administrated with a K–H buffer containing EDT (20 mg/L) for 15 min, 15 min of zero-flow global ischemia and subsequently 45 min of reperfusion after 15 min of stabilization.To evaluate the protective effects of EDT on myocardial I/R injury and to confirm the role of JAK2 activation during the myocardial protection of EDT, two experimental studies were designed in the present study. For study 1: Forty rats were randomly subdivided into five groups, including (1) Control group (Control): the hearts were excised from normal rats and were stabilized for 15 min and subsequently perfused for 75 min, (2) myocardial I/R group (I/R): the hearts were excised from normal rats and were subjected to 15 min of perfusion, 15 min of zero-flow global ischemia and subsequently 45 min of reperfusion after 15 min of stabilization, (3) I/R+EDT-5 treatment group (I/R+EDT-5): the rats were treated with EDT , then the hearts were excised from the rats and were administrated with a K–H buffer containing EDT (20 mg/L) for 15 min, 15 min of zero-flow global ischemia and subsequently 45 min of reperfusion after 15 min of stabilization, (3) I/R+EDT+AG490 treatment group (I/R+EDT+AG490): the rats were treated with EDT  and AG490 , then the hearts were excised from the rats and were simultaneously administrated with a K–H buffer containing EDT (20 mg/L) and AG490 (5 mg/L) for 15 min, 15 min of zero-flow global ischemia and subsequently 45 min of reperfusion after 15 min of stabilization, (4) I/R+AG490 treatment group (I/R +AG490): the rats were treated with AG490 , then the hearts were excised from the rats and were administrated with a K–H buffer containing AG490 (5 mg/L) for 15 min, 15 min of zero-flow global ischemia and subsequently 45 min of reperfusion after 15 min of stabilization.For study 2: Thirty-two rats were randomly subdivided into 4 groups, including (1) myocardial I/R group (I/R): the hearts were excised from normal rats and were subjected to 15 min of perfusion, 15 min of zero-flow global ischemia and subsequently 45 min of reperfusion after 15 min of stabilization, (2) I/R+EDT treatment group (I/R+EDT): the rats were treated with EDT (20 mg⋅kgmax). Meanwhile, an ultrasonic flowmeter (model T106) was used to determine the coronary flow (CF). The recorded data were analyzed by a 4S AD Instruments biology polygraph .After the perfusion of the isolated rat hearts in Langendorff’s apparatus, a water-filled latex balloon coupled to a pressure transducer (Statham) was inserted into the left ventricular cavity via the left auricle for pressure recording. The PowerLab data acquisition (DAQ) device was employed to continuously monitor the hemodynamic parameters, including the left ventricular end-diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), left ventricular developed pressure  and maximum rise/down velocity of the left intraventricular pressure . Then the hearts were stored and frozen at -80°C for 5 min. The frozen hearts sectioned into five pieces perpendicularly along the long axis from apex to base. Subsequently, the heart slices were incubated with 1% TTC buffer (pH 7.4) for 15 min at 37°C. After the incubation, the heart slices were fixed using a 4% formaldehyde solution. Finally, the fixed heart slices were photographed by a digital camera. The areas of white-unstained necrotic tissues and red-stained viable tissues were measured by Image-Pro Plus 7.0  and used to calculate the infarct size percentage of myocardium. The concrete formula is as follows: infarct volume percentage =  × 100.TM BCA Protein Assay Kit . Then, the content of IL-6, CRP, IL-8, and TNF-α were examined using the commercial ELISA kits. All the experimental procedures were performed according to the manufacturer’s instructions.After the reperfusion, cardiac tissues were harvested and homogenized (100 mg/mL) in ice-cold physiological saline. The homogenates were centrifuged at 3000 g and 4°C for 15 min. The supernatant was isolated and the protein concentration of the supernatant was measured by PierceThe hearts were fixed by 4% paraformaldehyde in PBS solution (pH 7.4) at room temperature for 24 h. The fixed cardiac tissues were then embedded in paraffin and sliced into 4 μm sections. The slices were deparaffinized, rehydrated, and treated with protease K (10 mmol/L) for 15 min. After treatment, the slides were employed for TUNEL staining according to the manufacturer’s instructions as described in our previous study .TM BCA Protein Assay Kit. Then the proteins were boiled for 5 min and subsequently separated by electrophoresis on a 12% SDS polyacrylamide gel. After separating, the proteins were transferred to PVDF membranes. The membranes were incubated with the primary antibodies against Bax, Bcl-2, Caspase-3, JAK2, p-JAK2, STAT3, p-STAT3, and β-actin overnight at 4°C. Then, the membranes were washed with TBST buffer and incubated with horseradish peroxidase-conjugated secondary antibody in TBST buffer at 37°C for 2 h. Finally, the membranes were washed and visualized using PierceTM ECL Plus Western Blotting Substrate , and the bands were scanned and quantified using Bio-Rad Gel Doc 2000 imaging system.The RIPA buffer  containing a cocktail of protease inhibitors  was used to extract proteins from rat hearts. The concentrations of the extracted proteins were quantified by Piercet-test for unpaired examinations or a one-way ANOVA followed by the Bonferroni post hoc test for multiple comparisons was performed for statistical analyses. P < 0.05 was considered statistically significant.The data are presented as the mean ± standard deviation (SD). A two-tailed Student max, -dP/dtmax, and CF) were remarkably decreased in I/R group . After pretreatment and treatment with EDT, we found that the decreased Hemodynamic parameters induced by myocardial I/R were significantly attenuated in M-EDT and H-EDT treatment groups . Moreover, the LDH and CK levels in K–H buffer were increased in I/R group when compared with the control group, and EDT treatment effectively improved the changes induced by I/R injury . Furthermore, we evaluated the infracted myocardium using TTC staining. The myocardial infarct size was markedly decreased by treatment with EDT when compared with the I/R group .In order to confirm the myocardial protective effect of EDT, the hemodynamic parameters were examined in heart after pretreatment and treatment with different concentrations of EDT. Compared with the control group, the Hemodynamic parameters .To determine whether EDT treatment can interfere with inflammatory response induced by I/R in the myocardium, the cardiac tissue obtained from each group were examined for the levels of IL-6, CRP, IL-8, and TNF-α. The levels of IL-6, CRP, IL-8, and TNF-α in cardiac tissue were significantly upregulated in I/R-injured myocardium when compared to those in control group. Notably, the elevated levels of IL-6, CRP, IL-8, and TNF-α were effectively reduced by EDT treatment in a dose-dependent manner . After treatment with EDT, the levels of Bax and Caspase-3 were significantly decreased and the Bcl-2 level was increased in the cardiac tissue when compared to those in the I/R group .Considering myocardium cell apoptosis is one important process in the pathogenesis of myocardial I/R injury, we next evaluated the effect of EDT on cell apoptosis induced by I/R in the myocardium. Compared with the control group, there was a significant higher myocardium apoptotic cell rate in I/R group. However, the EDT treatment significantly decreased the elevated cell apoptotic rate induced by myocardial I/R in the myocardium . Meanwhile, the ratios of p-JAK2/JAK2 and p-STAT3/STAT3 were both increased in I/R+EDT groups when compared with I/R group .To further explore the molecular mechanism of EDT’s cardio-protective action, we examined the protein levels and the protein phosphorylation levels of JAK2 and STAT3 in cardiac tissue. Western blot analysis showed that no obvious change of the levels of JAK2 and STAT3 was found between control group and I/R group or between I/R group and I/R+EDT groups. But the levels of p-JAK2 and p-STAT3 in EDT-treated groups were both significantly elevated in cardiac tissue when compared to those with the I/R group  induced by EDT were blocked by AG490 co-treatment . AG490 alone (I/R+AG490 group) didn’t have a significant effect on those Hemodynamic parameters when compared with the I/R group . Meanwhile, compared with I/R group, the decreased LDH and CK levels in I/R+EDT group were also obviously blocked by AG490 co-treatment . Subsequently, the protective effect of EDT on myocardial infarct size was suppressed by AG490 co-treatment .To further confirm whether JAK2 activation can participate in the cardio-protection of EDT in I/R injury, we employed AG490 to inhibit the JAK2 activation and subsequently evaluated the influence of AG490 on the cardio-protection of EDT in rat heart I/R model. We found that the elevated Hemodynamic parameters . Moreover, the inhibition effect of EDT on myocardium cell apoptosis rate was attenuated by AG490 co-treatment, whereas no significant differences in the myocardium cell apoptosis rate were found between I/R group and I/R+AG490 group .Compared with I/R group, the reductions of IL-6, CRP, IL-8, and TNF-α in I/R+EDT group were suppressed by AG490 co-treatment, but AG490 alone (I/R+AG490 group) didn’t have an obvious effect on those inflammatory factors when compared with the I/R group . In addition, the increased ratios of p-JAK2/JAK2 and p-STAT3/STAT3 induced by EDT were also suppressed by AG490 co-treatment in I/R+EDT+AG490 group when compared with those in I/R+EDT group, while no change was found in AG490-treated group when compared with the I/R group .We further examined the influence of AG490 on the expression of apoptosis-related proteins and the activation of JAK2 in EDT-treated rat hearts. Compared with I/R group, the decreases of pro-apoptotic proteins (Bax and Caspase-3) and the increase of Bcl-2 induced by EDT were inhibited by AG490 co-treatment in I/R+EDT+AG490 group, but AG490 alone didn’t have a significant effect on these protein levels in AG490-treated group , the increased myocardial infarct size, proinflammatory cytokine levels and myocardial cell apoptosis rate induced by myocardial I/R. Importantly, the JAK2 inhibitor AG490 attenuated the protective effect of EDT on myocardial I/R injury, indicating JAK2 activation could play a regulatory role in this process. This study could help us to understand the pharmacology and mechanism of EDT in the treatment of myocardial ischemia disease.Until now, EDT has attracted more interesting as a therapeutic agent in many diseases, such as hypercholesterolemia, cerebral ischemia, cardiovascular diseases and neurodegenerative diseases . In the Inflammatory response is an important process during the pathogenesis of myocardial I/R injury. Many pro-inflammatory factors, e.g., IL-1β, IL-6, IL-8, IL-10, and TNF-α, are up-regulated and contribute to myocardial I/R injury. In vitro myocardial I/R model indicated that myocardial cells appeared apoptosis at 10 min after ischemia and reached peak at 30 min  and decreased LDH and CK levels in EDT-treated group were also suppressed by AG490, indicating that the activation of JAK2 was involved in the cardio-protection of EDT.Recent studies have confirmed that the JAK2/STAT3 signaling plays a pivotal role in many physiological processes, e.g., inflammation and apoptosis. It is reported that cilostazol decreases the inflammatory cytokines  by the activation of JAK2 and STAT3 during myocardial I/R injury. On the other hand, ex vivo model of global myocardial I/R, but the protective effect or the long-term consequence of EDT on a in vivo myocardial I/R injury was not characterized. Second, we utilized only one JAK2 inhibitor (AG490) in this study. It is reported that AG490 and WP1066 are the two commonly used JAK2 inhibitors (There are two limitations in the study should be acknowledged. First, we evaluated the protective effect of EDT on an hibitors . Howeverhibitors . Thus, wTaken all the above findings, we speculate that EDT could suppress pro-inflammatory response and myocardial cell apoptosis through the activation of JAK2. These results may provide a potential treatment with EDT during myocardial I/R injury.DL and QZ supervised the whole project. DL and NL performed the major research and wrote the manuscript in equal contribution. JH, XC, WH, WX, and XL provided the technical support. LY provided her professional expertise.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Mycoplasma synoviae is an economically significant pathogen in the poultry industry, inducing respiratory disease and infectious synovitis in chickens and turkeys, and eggshell apex abnormality in chickens. Eradication, medication and vaccination are the options for controlling M. synoviae infection. Currently there are two commercial, live, attenuated vaccines available against M. synoviae: the temperature sensitive MS-H vaccine strain and the NAD independent MS1 vaccine strain. Differentiation of vaccine strains from field isolates is essential during vaccination and eradication programs. The present study provides melt-curve and agarose gel based mismatch amplification mutation assays (MAMA) to discriminate the MS1 vaccine strain from the MS-H vaccine strain and wild-type M. synoviae isolates. The assays are based on the A/C single nucleotide polymorphism at nt11 of a HIT family protein coding gene. The melt- and agarose-MAMAs reliably distinguish the MS1 vaccine strain genotype from the MS-H vaccine strain and wild-type M. synoviae isolate genotype from 102 template number/DNA sample. No cross-reactions with other avian Mycoplasma species were observed. The assays can be performed directly on clinical samples and they can be run simultaneously with the previously described MAMAs designed for the discrimination of the MS-H vaccine strain. The developed assays are applicable in laboratories with limited facilities and promote the rapid, simple and cost effective differentiation of the MS1 vaccine strain. Mycoplasma synoviae occurs worldwide leading to respiratory diseases and synovitis in chickens and turkeys, and eggshell apex abnormality in chickens, and as a consequence to high economic losses in the poultry industry [Escherichia coli or infectious bronchitis virus) [M. synoviae can be transmitted horizontally, colonizing first the respiratory tract or vertically through the eggs [M. synoviae into a flock effective biosecurity programs should be applied and mycoplasma-free sources should be used for restocking. Medication can reduce the economic losses in the infected flocks and inhibit the transmission through the eggs [M. synoviae: the temperature sensitive (ts+) MS-H vaccine strain  which was developed by the chemical mutagenesis of an Australian field strain (86079/7NS); and the NAD independent MS1 vaccine strain  which was obtained spontaneously during in vitro passages of the type strain  [+ MS-H strain, its ts- re-isolates and field isolates, such as the sequence analysis of the vlhA gene or identification of specific point mutations in the obg gene by high resolution melting-curve (HRM) analysis or mismatch amplification mutation assays (MAMAs) [+ MS-H vaccine, ts- MS-H vaccine re-isolate and field strains in one simple and cost-efficient step.Infection by industry –3. The ss virus) –2,4. M. the eggs . To prevthe eggs . Vaccinathe eggs –7. At prd strain 6079/7NS;+ MS-H (Vaxsafe® MS-H) used during the examinations originated from their commercial distributors. M. synoviae  type strain was used as control in the assays. M. anatis (ATCC 25524), M. anseris (ATCC 49234), M. cloacale (ATCC 35276), M. columbinasale (ATCC 33549), M. columbinum (ATCC 29257), M. columborale (ATCC 29258), M. gallinaceum (ATCC 33550), M. gallinarum (ATCC 19708), M. gallisepticum (ATCC 19610), M. gallopavonis (ATCC 33551), M. iners (ATCC 19705), M. iowae (ATCC 33552) and M. meleagridis (NCTC 10153) type strains and a M. sp. 1220 strain isolated by the authors were used for the examinations of cross-reactions. The M. sp. 1220 strain originated from a cloaca swab of a goose sampled during routine diagnostic examinations in 2016 in Csongrád county, Hungary.The vaccine strains MS1 (Nobilis® MS Live) and the ts- MS-H vaccine re-isolate and 26 M. synoviae wild-type strains) were collected and isolated by the authors during routine diagnostic examinations of live animals  and nt629 (C) in the obg gene [DNA was extracted from the strains, trachea swabs , tissue samples and FTA cards with the Qiamp DNA Mini kit . The tsobg gene .5’–ACT GTA AAT GAC GCC TTT TCT AC– 3’) and MS-HL2 (5’–ACC GCT TAT GCA AGT AAA TTA TT– 3’), designed in the present study. The reaction mixture contained 5 μl 5X Green GoTaq Flexi Buffer , 2.5 μl MgCl2 , 0.5 μl dNTP , 1 μl of each primer (10 pmol/μl), 0.25 μl GoTaq DNA polymerase  and 2 μl of target DNA in a total volume of 25 μl. Thermocycling parameters consisted of 95°C for 2 min, then 40 cycles of 95°C for 30sec, 55°C for 30 sec and 72°C for 1 min and a final elongation step at 72°C for 5 min. The amplicons were visualised in 1% agarose gel  under UV light. The amplicons were extracted from the gel using the QIAquick Gel Extraction kit (Qiagen) and submitted for Sanger sequencing on an ABI 3700 DNA Analyzer . Sequence of the amplicon of the MS1 strain (660 bp) contains the complete sequence of the HIT family protein coding gene (321 bp) and fragments of the flanking regions (GenBank accession number: KY712765). Alignment of the sequences of the HIT family protein coding genes of the M. synoviae type strain : 195625–195945) and the MS1 strain (KY712765) revealed a point mutation at nucleotide (nt) 11 in accordance with the previous publication [The HIT family protein coding gene of the MS1 strain was amplified by conventional PCR using the primers MS-HL1 (m) and the size of the resulting PCR product. The difference between the Tm and the product size is detectable by fluorescent dye on a real-time PCR platform (melt-MAMA) or by 3% agarose gel electrophoresis (agarose-MAMA), respectively.MAMA is based on allele-specific competing primers and is used widely for SNP detection . The allM. synoviae : 195625–195945). Genome location, primer sequences, annealing and melting temperatures for the assays and PCR product sizes can be found in In the current study melt- and agarose-MAMAs were developed to identify the point mutation at nt11 in the HIT family protein coding gene of 2 (25mM), 0.3 μl dNTP , 0.5 μl EvaGreen , primers (10 pmol/μl) according to TM v2.2.2 and on Qiagen Rotor-Gene Q real-time PCR system, software version 2.3.1. Thermocycling parameters were 95°C for 10 min, followed by 39 cycles of 95°C for 15 sec and 58°C for 1 min and a dissociation protocol comprising 95°C for 15 sec, followed by incremental temperature ramping (0.2°C) from 58°C to 95°C. EvaGreen fluorescent intensity was measured at 525 nm at each ramp interval and plotted against temperature.Melt-MAMA PCR reactions were performed in 10 μl total volume, containing 1μl target DNA diluted in 2 μl 5X Color-less GoTaq Flexi Buffer (Promega), 1 μl MgCl2 (25mM), 0.5 μl dNTP , primers (10 pmol/μl) according to Agarose-MAMA PCR reaction mixtures contained 2 μl target DNA diluted in 5 μl 5X Green GoTaq Flexi Buffer (Promega), 2.5 μl MgClIn order to test the intra-run repeatability at least four replicates of the two genotypes (wild-type and MS1 vaccine-type) were examined within the same run. For inter-run reproducibility test, the duplicate of at least ten samples from each genotype was examined in separate tests.M. synoviae type strain  were used  is higher by approximately 5°C than for the wild-type or MS-H vaccine genotype . For the same reason, the PCR product size in the agarose-MAMA for the MS1 vaccine genotype (83 bp) is longer by 16 bp than for the wild-type or MS-H vaccine genotype (67 bp)  , Fig 2.2 template number displaying a Ct range of 31–35. Negative controls or templates of other avian Mycoplasma species either were not amplified or generated non-specific products (with Ct above 37) with melt-profiles differing from the profiles of the expected two allelic states. Similarly, agarose-MAMAs worked reliable on samples containing 102 template number and neither of the tested other avian Mycoplasma species caused cross-reaction (no amplicons observed). The M. synoviae type strain , the ts+ MS-H vaccine strain (Vaxsafe® MS-H), the ts- MS-H re-isolate, the 26 clinical M. synoviae isolates, the two tissue samples, the two FTA cards and the 11 DNA pools from the trachea swabs taken from clinically infected chickens and turkeys showed the wild-type strain profile while the MS1 vaccine strain (Nobilis® MS Live) and the 32 DNA pools from the trachea swabs taken from vaccinated chickens showed the MS1 vaccine strain profile in the melt- and agarose-MAMAs  is crucial in the vaccination programs, animal trading and eradication programs.M. synoviae are the ts+ MS-H vaccine (Vaxsafe® MS-H) and the MS1 vaccine (Nobilis® MS Live) strains. The MS-H vaccine strain was developed by chemical mutagenesis and the discriminatory phenotypic characteristic of this strain is its temperature sensitivity [in vitro passages and it does not require NAD for its growth [vlhA gene is used worldwide for the molecular differentiation of M. synoviae strains, but this method was proved to be unreliable in the discrimination of the MS-H vaccine strain [obg gene in order to distinguish the ts+ MS-H vaccine strain from its ts- re-isolates and field strains [obg gene to enable rapid, simple and cost efficient detection of the MS-H vaccine strain [M. synoviae infections in one rapid, convenient and cost efficient step.The live, attenuated vaccine strains currently available against sitivity . The MS1s growth . The re-s growth . Genotype strain ,18–19. H strains . In our e strain . The pur


Here, we use miR-122a that exhibits liver-specific expression for developing a post-transcriptional regulative system mediated by microRNAs.Gene therapy with adenovirus (Ad) vectors that express herpes simplex virus thymidine kinase (HSVtk) and ganciclovir (GCV) have been suggested as a therapeutic strategy to cancer. However, Ad vectors injected into tumors are dispersed into the systemic circulation and transduce liver cells, resulting in severe hepatotoxicity. To be effective, the delivery and expression of suicide genes to cancer treatment ought to be specific to tumor cells, and avoid death of healthy cells. Researchers have demonstrated that expression of transgene could be suppressed in healthy cells with use of vectors that are reactive to microRNA regulation. We constructed an Ad vector carrying four tandem copies of target sequences of miR-122a that were incorporated into 3'-UTR of HSVtk gene. The expression level of miR-122a was quantified in HepG2 and Huh7 cell lines. Quantitative RT- PCR analysis demonstrated that Huh7 cells express large amounts of miR-122a compared to HepG2 cells. The viability of Huh7 cells and HepG2 cells after infection by Ad-tk-122aT vector was 83% and 23.5%, respectively. The viability of Huh7 cells was not reduced in the presence of GCV after infection by Ad-tk-122a vector. In contrast, cytotoxicity of HSV-tk/GCV was similar in Huh7 cells and HepG2 cells by Ad-tk vector, with 35.3% and 27% viability, respectively. Inclusion of the miR-122a target sequences in the HSVtk expression cassette yielded a feasible strategy for reducing cytotoxicity of suicide gene in a liver cell line with high miR-122a expression  Of various approaches to cancer gene therapy, suicide cancer gene treatments using the herpes simplex virus thymidine kinase (HSVtk) gene and ganciclovir (GCV) have been demonstrated in clinical and preclinical researches , 2. The Regulation of virus host range is of particular importance. For best therapeutic effects, gene therapy vehicles should target cancer cells while avoiding other organs and normal cells , 6. Suic Insertion of miRNA target sequences into the 3′-UTR of a gene decreases the expression level of the gene, with the amount of reduction dependent on the cellular expression levels of the miRNA , 10, 11.To test this hypothesis, we constructed an Ad vector containing four tandem copies of miRNA-binding sites for miR- 122a in 3′-UTR of HSVtk gene and used the hepatoma cell lines, Huh7 and HepG2, as models for suicide gene therapy with this Ad vector. We showed that endogenous miR-122a regulated HSVtk expression in Huh7 cell line with high level of endogenous miR-122a in comparison to HepG2 cell line with low level of endogenous miR-122a. Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Gibco. The human hepatomata cell lines HepG2 and Huh7, and human embryonic kidney cell line AD-293 were obtained from the Groningen research institute of pharmacy . The replication-defective recombinant adenovirus vector, AdEasy™ Adenoviral Vector System, was obtained from Agilent Technologies.TriPure Isolation Reagent was purchased from Roche. Universal cDNA Synthesis kit, LNA™-enhanced microRNA qPCR primer sets and SYBR® Green Master Mix kits were purchased from Exiqon. MTT Assay Kit was purchased from Promega. All experiments were performed at least three times and their average values are reported here. Cell cultureThe human hepatomata cell lines HepG2 and Huh7, and human embryonic kidney cell line AD-293 were grown in high-glucose Dulbecco's modified Eagle's medium supplemented with 2 mM l-glutamine, 100 U of penicillin/mL, 100 μg of streptomycin/mL, and 10% fetal bovine serum. Cells were grown in 25 cm2 polystyrene tissue culture flasks in a humidified atmosphere of 5% CO2 at 37 ◦C.Plasmids and Ad vectorsRecombinant adenovirus vector was generated from adenovirus type 5, and the E1 and E3 regions were deleted to prevent virus replication. The pSELECT-zeo-HSV1tk was purchased from Invivogen Company. We amplified tk gene with forward (F XhoI: 5- AATCTCGAGATGGCCTCGTACCCCGGCCA -3) and reverse (R EcoRI: 5-CCGGAATTCTTACATCTCACGGGCAAACG-3) primers containing Xho1 and EcoR1 site, respectively, in a PCR from pSELECT-zeo-HSV1tk. Briefly, a 1.7-kb XhoI/EcoRI fragment was purified and subcloned into pAd track shuttle, to form the pAd track-tk shuttle. Ad vectors containing miR-122a target sequences were established by in vitro ligation. A pAd track-tk/122a shuttle plasmid with four tandem copies of perfectly complementary sequences to miR-122a in the 3′-UTR of the HSVtk expression cassette, was established as follows: A EcoRI/BamHI fragment of miR-122a target sequences (5′- AATTCACAAACACCATTGTCACACTCCACAGCACAAACACCATTGTCACACTCCATTCGAAACAAACACCATTGTCACACTCCAGGACACAAACACCATTGTCACACTCCAA -3) (perfect complementary sequences of miR-122a are underlined) was ligated to pAd track-tk, resulting in pAd track-tk/122a. For generating Ad vectors , the resultant plasmids were linearized by digesting with restriction endonuclease PmeI, and were subsequently co-transformed into E. coli (BJ5183 cells) with the pAdEasy-1 adenoviral backbone plasmid. Recombinants were selected for kanamycin resistance, and recombination was confirmed by restriction endonuclease analyses. Finally, the linearized recombinant plasmids were transfected into AD-293 cells with FuGENE® HD Transfection Reagent , in accordance with the manufacturer’s instructions. Biological titers were measured using the Adeno-X rapid titer kit  according to the manufacturer’s instructions.miRNA expression analysisTotal RNA was extracted from HepG2 and Huh7 cell lines using TriPure Isolation Reagent (Roche) following the manufacturer's protocol. Residual DNA was removed with use of RNase-free DNase I (Fermentase) following the manufacturer's protocol. To quantify miR-122a expression, real-time PCR was performed using the TaqMan miRNA assay kit for miR-122a by the Applied Biosystems 7500 Sequence Detection System (Applied Biosystems). The expression of miRNA was defined from the threshold cycle (Ct), and relative expression levels were calculated after normalizing with reference to expression of U6 small nuclear RNA.In vitro gene expression analysisHepG2 and Huh7 cells were seeded at 1000 cells/well on 96-well plates. On the following day, the cells were infected with Ad, Ad-tk and Ad-tk-122aT for 1.5 hours at 400 VP/cell. Following 24-hour incubation, 20µL of GCV was added to cells. After 3 days, the viability of cells was measured using a MTT Assay Kit (Promega). Briefly, MTT 10.0 µL (5.0 mg/mL) was added to each well and the cells were incubated at 37 ◦C for 4 hours. Then, the MTT-containing medium was removed and the formazan crystals formed by living cells were dissolved in 200.0 µL DMSO. Absorbance at 570 nm was determined by a microplate reader  at test and reference wavelengths of 570 nm .Statistical analysisFor calculation of miR-122a expression fold change, the expression level of miR-122a in each cell line was normalized to that of U6 snRNA, as the internal control. Then, miR-122a expression in Huh7 cell line was compared to the HepG2 cell line (2^-ΔΔCT). We used samples t-test to calculate statistical significance and data were considered statistically significant if P-values < 0.05. Establishment of an Adeno vector that carry tandem copies of miR-122a-target sequencesTo test the effect of endogeneous miR-122a on transgene expression in hepatic cells, Ad vector was constructed containing miRNA-binding sites for miR- 122a . HSVtk gEndogenous level of miR-122a in Huh7 and HepG2 cell linesmiR-122a is highly expressed in the liver. Expression level of miR-122a in two human hepatoma cell lines, Huh7 and HepG2, were evaluated by quantitative RT- PCR. Quantitative RT- PCR analysis demonstrated that Huh7 cells express higher expression level of miR-122a compared to HepG2 cells (P = 0.000; Regulation of HSVtk gene in liver cell lines by miR-122aWe evaluated the Ad vector containing the miR- 122a target sequences for HSVtk/GCV suicide gene therapy in treating cancer. Ad-tk-122aT vector was constructed , and theThe potential of HSVtk/GCV suicide gene therapy by Ad vectors has been widely reported . HoweverHuman hepatoma cell lines, Huh7 and HepG2, expressing different levels of miR-122a  were cho


Idh1R132H in the subventricular zone (SVZ) of the adult mouse brain. The mice developed hydrocephalus and grossly dilated lateral ventricles, with accumulation of 2-hydroxyglutarate and reduced α-ketoglutarate. Stem and transit amplifying/progenitor cell populations were expanded, and proliferation increased. Cells expressing SVZ markers infiltrated surrounding brain regions. SVZ cells also gave rise to proliferative subventricular nodules. DNA methylation was globally increased, while hydroxymethylation was decreased. Mutant SVZ cells overexpressed Wnt, cell-cycle and stem cell genes, and shared an expression signature with human gliomas. Idh1R132H mutation in the major adult neurogenic stem cell niche causes a phenotype resembling gliomagenesis.Isocitrate dehydrogenase 1 mutations drive human gliomagenesis, probably through neomorphic enzyme activity that produces D-2-hydroxyglutarate. To model this disease, we conditionally expressed   •Idh1R132H knockin in the mouse brain SVZ recapitulates features of gliomagenesis•Self-renewal and proliferation of neural stem cells and progenitors increase•SVZ cells proliferate ectopically, infiltrate the brain parenchyma, and form nodules•Increases occur in 2HG levels, Wnt and telomere pathway activity, and DNA methylation IDH1R132H in the subventricular zone of the adult mouse brain causes features of gliomagenesis, including increased numbers of neural stem cells, cellular infiltration into surrounding brain regions, and a gene expression profile overlapping that of human gliomas.Bardella et al. report that expression of  IDH1Few curative treatments are available for human brain tumors. R132HIdh1is a driver mutation in gliomas and other malignancies, probably causing tumorigenesis through D-2-hydroxyglutarate accumulation, although the downstream mechanisms remain unclear. We found that adult mice expressing R132HIdh1in the brain subventricular zone (SVZ) develop features of gliomagenesis, including increased numbers of neural stem cells and their progeny. Other abnormalities included cellular infiltration into surrounding brain regions, reminiscent of tumor invasion. The gene expression profile of the R132HIdh1SVZ closely overlaps those of human gliomas. Likely, non-exclusive tumorigenic mechanisms included promotion of a neural stem cell phenotype, Wnt pathway activation, maintenance of telomeres, and DNA hypermethylation. Our R132Hmouse provides a system for assessing brain tumor therapies in vivo.IDH1 or IDH2  , the most common and most aggressive glioma, may develop rapidly without evidence of a less malignant precursor lesion (primary), or less often by progression of a lower grade tumor (secondary). Most grade II/III gliomas and secondary GBMs carry mutations in one of isocitrate dehydrogenase genes  or IDH2 . IDH1 ormutation . IDH driia (AML) , cholangia (AML) , enchondia (AML) , chondroia (AML) , and occIDH1 and IDH2 convert isocitrate to α-ketoglutarate (αKG) by oxidative decarboxylation, also generating NADPH. These enzymes share sequence and functional homology. IDH1 is found in the cytoplasm and peroxisome where it acts in lipid and glucose metabolism and protects against reactive oxygen species (ROS). IDH2 localizes to the mitochondria, where it regulates the tricarboxylic acid (TCA) cycle .IDH1R132H is the most common mutation  (L2HGDH (L-2-hydroxyglutarate dehydrogenase) deficiency, in whom L2HG accumulates because it cannot be metabolized to αKG  . D2HG wad to αKG . Furtherutations . The tumutations , and proutations . EvidencIdh1 or Idh2 mutations have been analyzed. Idh1R132H in hematopoietic lineages, leading to raised progenitor cell numbers and extra-medullary hematopoiesis. Mutant cells showed increased histone and DNA methylation, consistent with findings in human AML  in 5-week-old mice, resulting in cardiomyopathy and white matter abnormalities throughout the CNS. None of these Idh-mutant mice developed phenotypes clearly related to brain tumorigenesis.Several mice carrying pathogenic uman AML . Knockinpoptosis . No stemKras, Pdgfb, Trp53, Ptch1, and Pten in the SVZ of mice can elicit tumors , the largest NSC niche in the adult mammalian brain . In the t tumors . MultiplIdh1R132H mice had not developed features of brain tumors because the mutant protein was deleterious in embryos or young animals. Consistent with this notion, we noted that germline IDH mutations had not been reported in patients with gliomas or AML, and that the few patients with constitutional IDH mutations were mosaics /+ knockin mice, we designed a replacement targeting construct to conditionally express the Idh1R132H mutation under the control of the endogenous promoter /+ animals with nestin-Cre mice, thus inducing efficient recombination throughout the CNS from E10.5 (Idh1fl(R132H)/+ animals with mice carrying a tamoxifen-inducible nestin-CreER(T2), which in adult mice targets Cre to the SVZ and the other major neurogenic niche, the subgranular zone (SGZ) of the hippocampal dentate gyrus (Nes-CreER(T2); Idh1fl(R132H)/+ mice at 5–6 weeks of age for 5 consecutive days (Tam-Idh1-KI mice)  C. We shoKI mice) D.Idh1-wild-type controls  and mice with no abnormal physical signs or gross brain enlargement (details not shown). There was also enlargement of the SVZ, with irregular lateral borders, and an expansion of the rostral migratory stream (RMS) A. FurtheWe next examined Tam-Idh1-KI mice using immunofluorescence (IF) for cellular phenotypes related to tumorigenesis, including proliferation, differentiation, and migration. Owing to the abnormalities found in H&E sections, we focused on the SVZ. IF-based quantitation confirmed that, compared with controls, Tam-Idh1-KI mice showed a larger SVZ volume , similar cell density (p = 0.20), and a larger number of cells (p = 0.047).To better evaluate the proliferative dynamics of the SVZ and to obtain information regarding which cell populations A were ex+ (rapidly proliferating or re-entering cell cycle), GFAP+ (mainly NSCs and niche astrocytes), and EdU+GFAP+ cells (proliferating NSC or niche astrocytes) were increased in Tam-Idh1-KIs (+ (cycling) and BrdU+ (quiescent or slowly cycling) cells also increased in the mutant SVZ . The increased cell populations included quiescent and activated NSCs and, to a smaller degree, TAPs .We also assessed the oligodendrocyte progenitor marker Pdgfrα and Olig2, a marker of the entire oligodendroglial lineage that is universally expressed in diffuse gliomas  FiguresF and 3G.We assessed Ki67, GFAP and Olig2 expression in younger mice that had received tamoxifen at 5–6 weeks of age and were analyzed 3–8 weeks later. Compared with controls, the younger Tam-Idh1-KI mice showed similar GFAP and Olig2 changes to those observed previously in older animals, although the Ki67 increase was smaller (details not shown). The younger mice also had an expanded SVZ, a diffuse RMS and ectopic cells. These data suggest that the Tam-Idh1-KI phenotype can develop quite rapidly but may also progress over time.Idh1R132H increased the proportions and numbers of quiescent NSCs and their proliferative progeny  in both young and older mice, thereby enlarging the SVZ  were present in the striatum of Tam-Idh1-KI brains but absent in control mice , with few emigrating from the SVZ or RMS to the adjacent parenchyma . The Tamype mice A and S3.+ cells and Olig2+ cells in the striatum and corpus callosum  and the OB as a whole  was similar in controls and mutants, suggesting that the cells retained much of their normal differentiation capacity (data not shown).Migration in the RMS is highly regulated, similar to the SVZ. All seven Tam-Idh1-KI mice analyzed (five old and two young) had a more diffuse RMS A, with d from it A. This w from it . Additio a whole A, indica+ cells that were negative for neuronal markers . This suggests that the emigration/infiltration of the neuroblast progenitors, as well as the glial progenitors, into the regions adjacent to RMS and SVZ is probably cell autonomous.Disruption of the glial tubes composed of B1 astrocytes and B2 niche astrocytes is often characteristically associated with abnormal migration of neuroblasts . To addrIdh1R132H in adult mouse stem/progenitor cells robustly induced expansion of the SVZ and RMS and infiltration into adjacent regions, similar to observations of glioma cell invasion. The SGZ showed similar features  nodules originating from the SVZ and protruding into the LVs A. The lerker Dcx C and 6D.rker Dcx E. Microsrker Dcx A. Some or lining F. The epr lining G.Idh1fl(R132H)/+ mice without the Nes-Cre transgene and 8% (5/62) of non-induced Nes-CreER(T2);Idh1fl(R132H)/+ animals developed rounded and enlarged skulls at 3–6 weeks of age. This phenotype, reminiscent of human hydrocephalus prior to fusion of the skull sutures, necessitated immediate culling. Ventricular nodules, similar to those in Tam-Idh1-KI mice, were found in these animals (Idh1fl(R132H)/+ animals (after tamoxifen injections) for 1–2 years. Although none showed symptoms or signs of disease while alive, upon postmortem investigation, 8/34 animals (24%) had ventricular enlargement. Of those eight mice, histological examination showed one to have a clearly enlarged, diffuse SVZ (Nes-CreER(T2);Idh1fl(R132H)/+ animals that had not received tamoxifen, and all showed a similar phenotype to the eight Idh1fl(R132H)/+ mice without Nes-Cre. Further investigation strongly suggested that the phenotype resulted from expression of an Idh1 mRNA that lacked exons 1 and 2 and was derived from the mouse construct  of  animals A. We agefuse SVZ B and anofuse SVZ C. The bronstruct D, and 7Ee intact F. We conIdh1-mutant neurospheres from the SVZ of Tam-Idh1-KI mice were unsuccessful. We therefore stably expressed IDH1R132H both in human neuronal stem/progenitor cells (ReNcell CX) and in primary SVZ cells dissected from wild-type BL6 mouse pups. In each case, IDH1R132H increased the size and number of neurospheres cultured from human NSC/NPCs  conditions. IDH1-mutant cells were significantly more motile than control cells . These data were in accordance with the infiltrative behavior of Idh1-mutant NSC/NPCs in vivo.We also measured the capability of ReNcell CX expressing IDH1Nes-Cre; Idh1fl(R132H)/+ embryos using IC-MS (Using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS), we performed a comprehensive analysis of metabolites in Tam-Idh1-KI and control mice. In Tam-Idh1-KI samples, we detected ∼2-fold increase in total 2HG and ∼30% decreased αKG compared with controls C. The leng IC-MS A. No sigIdh1R132H mutation led to a global decrease of 5hmC and increase of 5mC, consistent with tumor-promoting effects.We found no clear evidence that collagen-PHDs, ROS, HIF-PHDs, or JHDMs were altered in the Tam-Idh1-KI mice . In contIDH1  revealed several pathways and processes of note that were more active in Tam-Idh1-KIs than controls at a false discovery rate <0.05 . NotablyIDH1   increased numbers of label-retaining stem cells as well as acutely proliferating cells in and around the SVZ and RMS; (2) significant infiltration of neuronal and glial progenitors from the SVZ and RMS into surrounding regions; and (3) subventricular nodules containing proliferating stem/progenitor cells that protruded into the LV from the SVZ. The mutant animals developed dilation of the LVs, with accompanying distortion of the cerebrum and clinically apparent hydrocephalus. However, the brains showed little, if any, evidence of neural damage or degeneration and appeared largely normal histologically in regions away from the niche.We have shown that conditional, inducible expression of the Idh1/2 models probably resulted from our targeting of Idh1R132H to the neurogenic niches of the adult brain. Nestin-Cre causes recombination early in the SVZ lineage and can affect large numbers of NSCs /+ allele may also have contributed to the Tam-Idh1-KI phenotype. Evidence strongly suggests that the phenotype caused by the leaky allele also originates in the SVZ. First, although the leaky allele causes 2HG accumulation in all tissues examined, no phenotype outside the brain was detected. Second, the phenotypes of Tam-Idh1-KI and leaky mice are very similar, notwithstanding much reduced penetrance and severity in the latter. Third, nestin expression is not induced by 2HG outside the SVZ and SGZ in the leaky mice; thus, while Tam-Idh1-KI mice are produced from an allele that is already leaky, when tamoxifen is given, full R132H knockin occurs in the SVZ and SGZ. Although leaky R132H expression in cells outside the SVZ could in theory explain the more numerous and misplaced cells in the striatum, corpus callosum, RMS, and OB in Idh1 mutants, lineage tracing in Tam-Idh1-KIs showed that the cells undergoing recombination in the SVZ after tamoxifen administration could account for the great majority of these cells.The phenotypic dissimilarities between our Tam-Idh1-KI and other  of NSCs . However of NSCs , and allNes-CreER(T2); Tsc1loxP/loxP) mice also develop hydrocephalus, periventricular nodules, and tumor-like structures near the inter-ventricular foramen  5mC and 5hmC (HPLC and next-generation sequencing), (2) TCA cycle and other metabolites (mass spectrometry); and (3) mRNA expression profiling (Illumina Mouse WG6-v2 microarrays). Western blotting was performed to assess the expression of selected proteins , supplemented by qRT-PCR for mRA where appropriate.Idh1+/+, with or without Nes-Cre transgene). Cell lineage and proliferation analysis was performed using immunofluorescence for DNA labeling agents , proliferation marker (Ki67), and markers of differentiation . Assessment of tissue morphology was performed using H&E-stained sections, supplemented by IHC for selected markers.For all phenotypic assessments, including molecular studies, tamoxifen-treated, Tam-Idh1-KI animals were compared with paired, tamoxifen-treated, littermate controls (IDH1R132H were performed by expressing the mutant protein in ReNcell CX from human fetal cerebral cortex or neural stem/progenitor cells from P4 wild-type mice. Assays performed on these cells included primary and second neurosphere formation and transwell motility.Assays to assess the effect of All data are presented as means ± SDs, with nominal p values from unpaired t tests, unless specified otherwise in the figure legend.Planned experiments: C.B., O.A.D., J.A., P.J.P., J.S.M., C.J.S., S.K., P.J.R., F.G.S., I.T. Performed experiments and analyses: C.B., O.A.D., O.A., D.K., J.A., P.J.P., K.A., I.P., L.F.M., J.S.M., H.L., S.S., N.S., R.G., T.S., O.A., P.B., M.T., F.G.S., and I.T. Wrote the manuscript: C.B., O.A.D., P.J.R., F.G.S., and I.T. Oversaw the study: C.J.S., P.M., O.A., B.S.-B., S.K., P.J.R., F.G.S., and I.T.


Escherichia coli O104:H4, a Shiga toxin-producing hybrid pathotype that was implicated in a major foodborne outbreak in Germany in 2011, has not been detected in cattle. However, serotypes of O104, other than O104:H4, have been isolated from cattle feces, with O104:H7 being the most predominant. In this study, we investigated, based on whole genome sequence analyses, the virulence potential of E. coli O104 strains isolated from cattle feces, since cattle are asymptomatic carriers of E. coli O104. The genomes of ten bovine E. coli O104 strains  and five O104:H7 isolated from human clinical cases were sequenced. Of all the bovine O104 serotypes  that were included in the study, only E. coli O104:H7 serotype possessed Shiga toxins. Four of the six bovine O104:H7 strains and one of the five human strains carried stx1c. Three human O104 strains carried stx2, two were of subtype 2a, and one was 2d. Genomes of stx carrying bovine O104:H7 strains were larger than the stx-negative strains of O104:H7 or other serotypes. The genome sizes were proportional to the number of genes carried on the mobile genetic elements . Both bovine and human strains were negative for intimin and other genes associated with the type III secretory system and non-LEE encoded effectors. Plasmid-encoded virulence genes  were also present in bovine and human strains. All O104 strains were negative for antimicrobial resistance genes, except one human strain. Phylogenetic analysis indicated that bovine E. coli O104 strains carrying the same flagellar antigen clustered together and STEC strains clustered separately from non-STEC strains. One of the human O104:H7 strains was phylogenetically closely related to and belonged to the same sequence type (ST-1817) as the bovine O104:H7 STEC strains. This suggests that the bovine feces could be a source of human illness caused by E. coli O104:H7 serotype. Because bovine O104:H7 strains carried virulence genes similar to human clinical strains and one of the human clinical strains was phylogenetically related to bovine strains, the serotype has the potential to be a diarrheagenic pathogen in humans. Escherichia coli (STEC) O157 and six serogroups of non-O157 STEC, O26, O45, O103, O111, O121, and O145, are responsible for foodborne illnesses that range from mild to bloody diarrhea to serious complications of hemolytic uremic syndrome (HUS) in humans  and STEC  , a subset of STEC, and enteropathogenic E. coli (EPEC), is responsible for the intimate attachment of bacteria to the host epithelial cells leading to attachment and effacement lesion  are major virulence factors of STEC and are responsible for life-threatening complications, such as HUS, associated with STEC infections  were included in the study. Details on the study population, sampling and isolation procedures of cattle E. coli O104 strains have been described . A single colony of each strain was inoculated into Luria Bertani (LB) broth and incubated on a shaker at 37°C. Genomic DNA was isolated from overnight culture using the Qiagen DNeasy blood and tissue kit . The purity of the DNA was assessed spectrophotometrically using the Nanodrop . Genomic libraries of the strains were constructed using Nextera XT DNA Library Preparation kit and whole genome sequencing was performed on an Illumina MiSeq benchtop sequencer  using the MiSeq version 2 reagent kit with 2 × 250 cycles. De novo assembly of quality-controlled trimmed sequenced reads was performed using the SPAdes genome assembler version 3.8.2 (http://cab.spbu.ru/software/spades/)  server . Different types of plasmid sequences were identified by PlasmidFinder 1.3 (https://cge.cbs.dtu.dk/services/PlasmidFinder/) tool . The tool identifies intact, questionable, and incomplete prophage sequences by scores of >90, 70–90, < 70, respectively . The tool provides class, type, and subtype of CRISPR-Cas system and number, length and nucleotide sequences of repeats and spacers   was used to align the core genomes of human and bovine E. coli O104 strains, followed by the construction of maximum likelihood tree. Escherichia coli O104:H21, a human outbreak strain  was also included in the phylogenetic analysis to determine the genetic relatedness to the bovine O104 strains. The phylogenetic tree was subsequently imported to FigTree 1.4.3 software (http://tree.bio.ed.ac.uk/software/figtree/) , then Tukey adjustment for multiple comparisons was performed using PROC GLIMMIX procedure of SAS version 9.4 , to test each pairwise comparison.A single factor analysis of variance (ANOVA) test was performed to determine whether genome size and number of genes associated with different functional categories were significantly different between bovine STEC, non-STEC and human strains. If the means were significantly different (n = 6) and human O104:H7 (n = 5) strains were confirmed by SerotypeFinder 1.1 and the other four unknown serotypes from cattle were identified as O104:H8, O104:H12, and 104:H23 (n = 2).Serotypes of bovine (E. coli O104 strains ranged from 4.9 to 5.4 Mb. The average genome size of bovine STEC strains (5.3 Mb) were significantly (P < 0.01) larger than bovine non-STEC strains (4.9 Mb). The functional categorization of genes revealed that the number of genes associated with virulence, disease, and defense ranged from 109 to 116 in bovine strains and 110 to122 in human O104 strains. The number of genes carried on mobile elements such as phages, prophages, transposable elements and plasmids ranged from 60 to 201 in bovine O104 strains, and 80 to 201 in human O104 strains. The average number of genes associated with mobile genetic elements was significantly (P < 0.01) higher in bovine STEC strains (190) compared to non-STEC strains (71). The human strains had lower (P < 0.01) number of genes associated with mobile genetic elements than bovine O104:H7 STEC strains. There was no significant difference in the average number of genes associated with other subsystem categories  between bovine and human strains . The gene subA (subtilase toxin subunit) was present in 4 of 5 human strains of O104:H7, but absent in all bovine strains. Both bovine and human O104 strains were negative for intimin (eae) and other genes associated with LEE Pathogenicity Island, and non-LEE encoded effectors. However, both bovine and human E. coli O104 strains carried other adhesins, such as irgA homolog adhesin (iha) and long polar fimbriae (lpfA).Four of the six bovine and one of the five human O104:H7 strains carried ehxA, epeA, espP, katP) were present in bovine and human strains. Four of the bovine strains, and three of the human strains carried ehxA (enterohemolysin). Enterohemorrhagic E. coli plasmid-encoded autotransporter (epeA) was present in one of the bovine strains , and one human strain . Extracellular serine protease  and catalase peroxidase  were present only in human strains.Plasmid-encoded virulence genes (gad (Glutamate decarboxylase). Four bovine O104:H7 strains  and one human O104:H7 strain (2011C-3665) was positive for aaiC (Type VI secretory system protein). The gene encoding increased serum survival (iss) was present in two bovine (2013-6-685A and 2013-6-210A) and four human O104:H7 strains . One of the bovine strains  carried pic . Different types of genes that code for colicin, a bacteriocin, were also present in bovine and human E. coli O104 strains. The colicin B gene (cba) was present in one bovine strain (2013-6-148B) and two of the human strains (08-4061 and 2011C-3665), celb (endonuclease colicin E2) was present in three human strains , cma (Colicin M) was present only in one human strain (2011C-3665). Virulence gene profiles of bovine and human strains are provided in Table All bovine and human strains carried tet (tetracycline resistance), aadA1 (aminoglycoside resistance), and sul1 (sulfonamide resistance), were present only in one of the human strains (2011C-3665). All the bovine and other human strains were negative for antimicrobial resistance genes  of the non-STEC O104:H7 strains carried IncFIB and IncFII plasmid sequences  carried four intact, six incomplete and zero questionable prophage sequences (Table E. coli O104 strains was I-A and I-E. Other subtypes identified were I-D (2011C-3665), I-F , III-B (2013-6-148B), and VI-C (2013-6-148B). All the four bovine O104:H7 STEC strains and one the human O104:H7 STEC strain (2011C-3665) carried a single CRISPR locus, four human O104:H7 STEC strains and three stx-negative bovine strains carried two loci, and two stx-negative bovine strains carried four loci. One of the bovine O104:H12 strain (2013-6-210A) carried CRISPR region lacking cas genes. Total number, nucleotide sequence and average length of repeats and spacers were highly conserved across all stx-positive bovine O104:H7 STEC strains. Average length and nucleotide sequence of repeats and spacers of one of the human O104:H7 STEC strain (2011C-3665) was similar to bovine O104:H7 STEC strains, however, it carried fewer repeats and spacers compared to bovine O104:H7 STEC strains. Similarly, total number, nucleotide sequence and average length of repeats and spacers were highly conserved across all human O104:H7 STEC strains. Average length and nucleotide sequences of spacers and repeats were similar among bovine stx-negative O104:H7 strains, but they differed in number of repeats and spacer units. However, repeats and spacer units were not conserved among O104:H23 strains  was distantly related to other O104 serotypes. All human O104:H7 strains, except one, clustered together. The human O104:H7 strain, 2011C-3665, which carried stx1c gene similar to bovine STEC O104:H7 strains clustered with the bovine O104:H7 STEC strains. The human Montana outbreak strain of O104:H21 (Montana outbreak) clustered more closely with one of the human (2011C-3665) and bovine O104:H7 STEC strains , which clustered with the bovine strains, belonged to ST-1817. The other 4 human O104:H7 strains that clustered separately belonged to ST-2773. One non-STEC bovine O104:H7 strain (2013-6-289D) belonged to ST-5014. The remaining bovine non-STEC O104 strains belonged to ST-678, ST-10, ST-6494, ST-129, and ST-939  and an O104:H7 STEC strain isolated from cattle and compared them to E. coli O104:H4  strains. They reported that the O104:H7 STEC strain was more similar to the Montana strain (O104:H21) than to the German strain (O104:H4). To our knowledge, this is the first study to analyze the whole genome sequences of human O104:H7 strains and to compare the virulence gene profiles of bovine O104:H7 strains.Shiga toxin-producing Hussein, . Only onHussein, . SeveralHussein, . Howeverstx carrying bovine O104:H7 strains were larger than the stx-negative strains of O104:H7 or other serotypes. The genome sizes were proportional to the number of genes carried on the mobile genetic elements . Yan et al.  that were included in the study, only h et al.  reportedeae has been reported to be a feature of O104 human outbreak strains (O104:H4 German outbreak and O104:H21 Montana outbreak strains)  and iha that encode for IrgA homologous adhesins were the most prevalent adhesins identified in LEE-negative E. coli strains isolated from cattle and human sources  were also carried by E. coli O104:H4 German outbreak strains and E. coli O104:H21 Montana outbreak strain , which is not surprising because previous studies have suggested a strong association between eae and efa1 genes , and epeA (encoded by pO113). Enterohemolysin, encoded by ehxA, has been reported to be involved in increased production of IL-1β and cytotoxicity  is responsible for mucosal hemorrhage observed in patients suffering from hemorrhagic colitis, due to cleavage of pepsin A and human coagulation factor V , which encodes for serine protease autotransporter. The protease, also secreted by Shigella flexneri and enteroaggregative E. coli, is involved in intestinal colonization  , agg3A (pilin subunit of aggregative adherence fimbriae III), aggR , and serine protease autotransporter toxins  were present in E. coli O104:H4 German outbreak strains, but absent in E. coli O104:H21 Montana outbreak strain . Antimicrobial resistance genes, such as blaTEM−1, blaOXA−1, and aac(6′)-Ib-cr, are carried on IncF plasmids  was negative for IncF1, but positive for IncFII plasmids. Tetracycline resistance was the most common resistance in E. coli O157 strains isolated from bovine and human sources  were present in all bovine O104 STEC strains, except one  were all positive for the stx1c gene and clustered together phylogenetically. In contrast, four human O104:H7 strains of ST-2773, positive for either stx2a, stx2d or no Shiga toxin genes, clustered together, but were apart from the bovine ST-1817 strains. Similarly, CRISPR loci of four of the bovine O104:H7 STEC strains and one of the human O104:H7 STEC strain (2011C-3665) were conserved. These findings suggests that bovine feces could be a source of human O104:H7 STEC strains. The WGS-based analysis of virulence genes suggests that bovine O104:H7 STEC has the potential to be a diarrheagenic pathogen in humans.Whole genome analyses of NEKE00000000 to NEKS00000000.The whole genome shotgun sequences have been deposited at DDBJ/ENA/GenBank under the accession numbers TN, JB, CE, and NS conceived and designed the experiments. PS, IP, JG, LN, and XS performed the experiments. PS, IP, and JG analyzed the data. PS, TN, JB, and IP wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We show that activation of protein kinase C (PKC) caused phosphorylation of S313, S319 and S1169. Activation of growth factor receptors induced phosphorylation of S1169 through a mechanism that was independent of PKC, indicating that distinct kinases and mechanisms control the phosphorylation of P-Rex1 at different regulatory serines. Genetic and biochemical studies confirmed that the PKC isoform PKCδ was able to directly phosphorylate P-Rex1 at S313. Functional studies using cells with very low endogenous P-Rex1 expression, transfected with wild type P-Rex1 or a mutant form in which S313 was substituted by alanine, indicated that phosphorylation at that residue negatively regulated P-Rex1 exchange activity. We suggest that control of P-Rex1 activity depends on a highly dynamic interplay among distinct signalling routes and its multisite phosphorylation is controlled by the action of different kinases.P-Rex proteins are guanine nucleotide exchange factors (GEFs) that act on the Rho/Rac family of GTP binding proteins. The activity of P-Rex proteins is regulated by several extracellular stimuli. In fact, activation of growth factor receptors has been reported to activate a phosphorylation/dephosphorylation cycle of P-Rex1. Such cycle includes dephosphorylation of serines 313 and 319 which negatively regulate the GEF activity of P-Rex1, together with phosphorylation of serines 605 and 1169 which favour P-Rex1 GEF activity. However, the kinases that regulate phosphorylation at these different regulatory sites are largely unknown. Here we have investigated the potential regulatory action of several kinases on the phosphorylation of P-Rex1 at S PREX1 and PREX2 have been described, each of them coding for three different isoforms [−/−P-Rex1 mice are resistant to metastatic spreading of the melanoma cells [PREX2 have also been reported [P-Rex proteins belong to the family of guanine nucleotide exchange factors (GEFs) which act on the Rho/Rac family of GTP binding proteins , 2. In aisoforms . While kisoforms –8, severisoforms , 4, 9–17isoforms . In melaisoforms . Furtherma cells . In pancreported .Several structural domains have been identified in P-Rex proteins , 20 Fig. The N-t313 and Ser319) accompanied by phosphorylation of other residues (Ser605 and Ser1169). Mutagenesis as well as functional studies showed that under resting conditions P-Rex1 is phosphorylated at Ser313 and Ser319 and such phosphorylations inhibit P-Rex1 GEF activity [313 and Ser319 are included in the PH domain of P-Rex1, a region whose deletion augments the GEF activity of P-Rex1 in vitro [605 and particularly Ser1169 increases the GEF activity of P-Rex1 [1169 increases, and this facilitates the GEF activity of P-Rex1 towards Rac [Phosphorylation of P-Rex represents an important regulatory mechanism. Various reports have demonstrated that P-Rex1 is phosphorylated at multiple sites , 22, 23,activity . Of notein vitro . Therefof P-Rex1 . Under rards Rac . TherefoAdditional evidence that phosphorylation of P-Rex1 may control its GEF activity has been reported upon activation of β-adrenergic receptors . Agonist313, S319, S605 and S1169 were included among those sites and we centered our attention on those residues because of their reported regulatory role on P-Rex1 GEF activity [313 (score: 0.92) and S605 (score: 0.64) of P-Rex1  and S1169 (score: 0.58). The latter residue was also expected to be a target for casein kinase II (CKII) (score: 0.55).In an attempt to identify P-Rex1 phosphorylating kinases, we performed bioinformatic analyses using the NetPhosK 1.0 online tool, which allows predictions of kinase specific eukaryotic protein phosphorylation sites . Upon anactivity . These s1 Figure . In addi313 was identified by the program as a target for kinases of the PKC family  as compared to other predicted phosphorylation sites whose surface availability was below 3.To further explore P-Rex1 phosphorylating kinases in silico, we also used the Scansite 3 beta online tool. Scansite searches for specific motifs within proteins that may be phosphorylated by mammalian protein kinases or bind to domains such as SH2 domains, 14-3-3 domains or PDZ domains . A totalC family . In cont313, S319, S605 and S1169 sites. This, together with previously published data indicating that PKA activity may phosphorylate P-Rex1 [313, S319, S605 and S1169. To this end, MCF7 breast cancer cells were treated with the PKC agonist Phorbol 12-myristate,13-acetate (PMA), or with forskolin, which is known to stimulate PKA activity by augmenting cAMP levels. These cells, which mainly express isoform 1 of P-Rex1, were treated with these drugs for 15 minutes and the phosphorylation of S313, S319, S605 or S1169 was analyzed by Western blotting using antibodies which specifically recognize those residues when phosphorylated [313 -regulated GEF . Becausenhibitor  also fai9 Figure . As expe3 Figure  and S6 , and sustained for several hours  showed a substantial reduction in PKCδ levels  as well as cell lines of the HER2+ subtype  expressed P-Rex1, while HCC1954 which also belongs to the HER2+ subtype did not express P-Rex1  involved in the regulation of such phosphorylation upon activation of growth factor receptors. Since the inhibitor studies failed to define the S1169 kinase, the use of alternative approaches, e. g. kinome targeting with siRNA libraries, may help in the identification of such kinase. These results were taken to indicate the highly regulated nature of P-Rex1 phosphorylation, and the fact that distinct kinases/signaling routes control multisite phosphorylation of P-Rex1.Phosphorylation of P-Rex1 at S313. Moreover, in vitro kinase experiments confirmed that this kinase could directly phosphorylate P-Rex1 at S313. However, our studies cannot exclude that kinases other than PKCδ may phosphorylate P-Rex1 at S313. While in vivo activation of PKC resulted in phosphorylation of P-Rex1 at S1169, the in vitro studies failed to demonstrate direct PKCδ phosphorylation of that residue. It is therefore possible that such failure may be caused by the requirement of another component absent in the in vitro kinase assay. Another possibility is that a PKC isoform other than PKCδ may be involved in the phosphorylation of S1169.Genetic studies, which included gene expression profiling as well as knockdown experiments identified PKCδ as an important kinase in the regulation of P-Rex1 phosphorylation at S313 and S319 of P-Rex1 are located within the β3-β4 loop of the PH domain of P-Rex1, a region that is not expected to directly interact with Rac. However, those studies have shown that the β3-β4 loop is endowed with a high degree of mobility and surface availability. This property may facilitate accessibility for interaction with other regulatory regions of P-Rex1 or with other proteins, such as kinases or phosphatases that may regulate the GEF action of P-Rex1. The high surface availability of S313 could favor its phosphorylation, causing a conformational change that could facilitate phosphorylation of other P-Rex sites. In fact, the delayed time course of phosphorylation of other residues with respect to S313 could be due to such phenomenon, or could be indirect. Phosphorylation of P-Rex1 at S313 has been formerly reported to negatively regulate the GEF function of P-Rex1 against Rac [313 was substituted by non-phosphorylatable alanine. While informative, those studies had limitations, since the knockdown of P-Rex1 was not complete, and some endogenous protein remained. To better assess the relevance of the phosphorylation of P-Rex1 at S313 we analyzed several breast cancer cell lines which belong to the three major clinicopathological subtypes. These studies informed that expression of P-Rex1 was very low in the cell lines representative of the triple negative subgroup. We took advantage of the lack of substantial expression of P-Rex1 in these cell lines to explore the relevance of P-Rex1 phosphorylation at S313 on Rac activity. When expressed at similar levels, the mutated form of P-Rex1 which contained a non phosphorylatable residue at position 313 had a stronger effect on Rac activity than wild type P-Rex1. This is consistent with the concept that phosphorylation of P-Rex1 at S313 restricts P-Rex1 GEF activity towards Rac. We observed that expression of wild type P-Rex1 form in the three cell lines tested substantially augmented resting Rac activity, indicating that overexpression of P-Rex by itself is sufficient to raise Rac activity. This conclusion is relevant, since elevated Rac activity may contribute to progression of diseases such as cancer. Moreover, the expression of elevated levels of P-Rex1 has been linked to a worse patient outcome in breast cancer, particularly in the luminal type [Crystallographic analyses indicated that Rac interacts with the DH domain of P-Rex . Residuenal type  suggestenal type  indicateCell culture media, puromycin, PMA , dbcAMP 473) antibody was from BD Biosciences. The antibodies against P-Rex1 (anti-pan-P-Rex) and phospho P-Rex1  have been described [The antibody against GAPDH was purchased from Santa Cruz Biotechnology. The anti-PKC antibodies were from BD Transduction Laboratories, Inc . The anti-pAKT S73 antibo2 and 95% air. Cells were grown in DMEM or in RPMI medium containing high glucose concentration  and antibiotics  and supplemented with 10% FBS. Cell lines were provided by Drs. J. Losada and A. Balmain  [All cell lines were cultured at 37°C in a humidified atmosphere in the presence of 5% COsources) . Cell idKnockdown of PKCδ in MCF7 cells was performed by infection with lentiviral particles. The lentiviral vectors containing short hairpin RNA (shRNA) for PKCδ were obtained from Thermo Scientific . A minimum of 5 different shRNA sequences were tested and the two that produced higher knockdown levels were used. Preparation of lentiviral vectors was performed as described previously .313A, cells were transfected with pCMV3-myc-P-Rex1-WT and pCMV3-myc-P-Rex1-S313A using JetPEI. Transfections were performed according to the instructions provided by the manufacturer. The expression of exogenous P-Rex1 was analyzed by Western blotting with anti-P-Rex antibodies.To express exogenous P-Rex1-WT and P-Rex1-S2HPO4, 8 mM; KH2PO4, 1.5 mM) and lysed in ice-cold lysis buffer . Lysates were centrifuged at 10,000 xg at 4°C for 10 minutes and supernatants were transferred to new tubes with the corresponding antibody and protein A-Sepharose. Immunoprecipitations were performed at 4°C for at least 2 hours. Immune complexes were recovered by a short centrifugation at 10,000 xg for 15 seconds, followed by three washes with 1 mL ice-cold lysis buffer. Samples were then boiled in electrophoresis sample buffer and placed on SDS-PAGE gels at varying acrylamide concentrations, depending on the molecular weight of the proteins to be analyzed. After electrophoresis, the separated proteins in the gel were transferred to polyvinylidene difluoride membranes (PVDF) . Membranes were blocked in TBST  containing 1% BSA or 5% skimmed milk for 1-3 hours and then incubated with the corresponding antibody for 2-16 hours. After washing three times with TBST during 10 minutes, membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies for 30 minutes. After the secondary antibody, the membranes were washed three times with TBST and the bands were visualized by using enhanced chemiluminescence [Cultured cells were washed with phosphate-buffered saline (PBS)  following the manufacturer's instructions. Data are plotted as mean±s.d of duplicates from an experiment which was repeated at least twice.313A) was generated as described [The pCMV3 plasmid containing wild type human P-Rex1 tagged with myc was provided by Dr. Heidi C. E. Welch . The P-Rex1 phosphorylation mutant ; distilled water until 60 μl.The PKCδ kinase assay was performed as described in the assay procedure in Sigma, Product Code P 6862, with slight modifications. The reaction mix was as follows: HEPES (pH 7.4), 20 mM; MgClAs substrate of the kinase (PKCδ), P-Rex1 immunoprecipitated from MCF7 cells was used. MCF7 cells were lysed and 1 mg of protein was immunoprecipitated with the anti-pan-P-Rex1 antibody. The immunocomplex were washed three times with lysis buffer and twice with water. 60 μl of the reaction mix was added into each immunocomplex and these were placed at 30°C. Then it was added 2 μl of diluted enzyme  to each assay tube and the reaction was continued up to 10 minutes. The reaction was stopped by adding loading buffer 4X containing β-mercaptoethanol (20 μl). The reaction of kinase assay was evaluated in Western blot using the anti-phospho-P-Rex1 antibodies.Gene expression studies were performed as described . Brieflyhttp://www.cbs.dtu.dk/services/NetPhosK/). The NetPhosK program produces neural network predictions of kinase specific eukaryotic protein phosphorylation sites. Currently NetPhosK covers the following kinases: PKA, PKC, PKG, CKII, Cdc2, CaM-II, ATM, DNA PK, Cdk5, p38 MAPK, GSK3, CKI, PKB, RSK, INSR, EGFR and Src [http://scansite3.mit.edu/#home) [The sequence in FASTA format of P-Rex1 was introduced in the NetPhos web page ( and Src . Similaru/#home) .t test. Differences were considered to be statistically significant when p-values were less than 0.05. Statistical data are presented as the mean±s.d. All data were analyzed using the statistical software SPSS 21.0 .Comparisons of continuous variables between two groups were performed using a two-sided Student's


Mus musculus) basal GC receptor  expression in dorsal root ganglion (DRG) sensory neurons is 15-fold higher than in neurons in canonical stress-responsive brain regions (M. musculus). In response to stress or GCs, adult DRG neurite growth increases through mechanisms involving GR-dependent gene transcription. In vivo, prior exposure to an acute systemic stress increases peripheral nerve regeneration. These data have broad clinical implications and highlight the importance of stress and GCs as novel behavioral and circulating modifiers of neuronal plasticity.Stress and glucocorticoid (GC) release are common behavioral and hormonal responses to injury or disease. In the brain, stress/GCs can alter neuron structure and function leading to cognitive impairment. Stress and GCs also exacerbate pain, but whether a corresponding change occurs in structural plasticity of sensory neurons is unknown. Here, we show that in female mice ( Nerve injury-induced pain affects millions and is a comorbidity factor for individuals living with traumatic spinal cord or peripheral nerve injuries. Pain is associated with aberrant plasticity and sprouting in the injured peripheral and central nervous systems. However, the mechanisms underlying these structural changes are not understood. Here, new data implicate stress hormones (steroids) and GR activation as a novel mechanism underlying enhanced sensory neuron plasticity in injured peripheral nerves. These data also have important implications for the development and care of nerve-injury induced pain, including the use of steroids as a treatment for inflammatory pain.Stress, a ubiquitous and recurrent part of daily life, activates complex regulatory mechanisms in the body that cause glucocorticoids (GCs) to increase in the circulation. GCs are steroid hormones that dramatically affect cell function. They do so mainly by binding to GC receptors (GRs), which act in the nucleus to regulate gene transcription. In the brain, neurons in the cerebral cortex and subcortical structures  express high levels of GRs  and GCs/Peripheral nerve injury  activates transcriptional programs that enhance DRG neuron regenerative growth and sprouting . Periphein vitro, they grow neurites that are both longer and more complex than those that grow from control DRG neurons. These growth-promoting effects of stress can be blocked using GR antagonists, and recapitulated in vivo by injecting synthetic GCs into naïve mice before DRG neuron harvest. Stress increases GR nuclear localization in DRG neurons but without markedly increasing the expression of regeneration-associated genes (RAGs). In fact, unlike the ability of nerve injury  to elicit RAG expression  while other potentially novel GR-dependent RAGs  are increased by stress. Finally, we show that the axon growth-promoting effects of stress/GCs can also be identified in vivo. These data highlight the importance of stress and GCs as novel behavioral and circulating modifiers of sensory neuron plasticity, and bring into question whether the inflammation associated with pain should be treated with steroids.Our new data indicate that GCs act as circulating modifiers of sensory neuron structure and function and that DRG neurons express surprisingly high levels of GRs, even compared to hippocampal neurons, which have been considered the most GR-rich and stress-responsive neurons in the nervous system . When DRG neurons are isolated from stressed mice and grown pression , stress Thy1-GFP-M mice (ad libitum access to food and water. Mice were maintained in a vivarium with controlled temperature (∼20°C) on a 12/12 h light/dark cycle and assigned randomly to experimental groups after a one week habituation period. All procedures were conducted by protocols approved by The Ohio State University and the University of Miami Institutional Laboratory Animal Care and Use Committee and with the guidelines of the Committee for Research and Ethical Issues of International Association for the Study of Pain.Adult female C57BL/6 mice (Taconic) or P-M mice  were grog for 5 min), protein concentration was determined using a BCA protein assay kit . Samples (10 μg) were separated on 10% Bis-Tris gels and transferred to a nitrocellulose membrane in a wet-transfer apparatus (Invitrogen). After protein transfer, membranes were incubated with 5% BSA for 1 h at room temperature (RT), then with one of the following primary antibodies (1:200-1:2000): GR #PA1-511A from Thermo Scientific, RRID: AB_2236340; Actin #A1978 from Sigma-Aldrich, RRID: AB_476692, in 5% BSA at 4°C for at least 12 h, and finally with HRP-conjugated anti-rabbit IgG antibody  in 5% BSA for 1 h at RT. Between incubations, the membrane was washed three times with PBS + 5% Tween for 10 min each. HRP activity was visualized using a chemiluminescent substrate (Thermo Scientific) and signal density quantified with a Kodak Image Station 4000MM Pro . A ratio of signal density of phosphorylated protein to total protein was calculated. For N = 4 per condition for each experiment.Mice were sacrificied in accordance to Institutional Animal Care and Use Committee guildelines and DRG cell lysates were prepared by homogenization in 250-μl T-PER Tissue Protein Extraction Reagent  supplemented with protease and phosphatase inhibitors  immediately after harvest. Following centrifugation  for 1 h elicits a transient, but significant increase in circulating corticosterone cort; . This mein vivo experiments, mifepristone , cort , and dexamethasone  were prepared in a sterile peanut oil vehicle (veh) and then injected in a 0.1-ml volume 1 h before cell harvest. Cells were plated and kept in Neurobasal A media for 72 h. The dose of injected cort was previously determined to reproduce stress-induced plasma cort concentrations (For trations . All dru2 humidified incubator. Different culture durations were used to interrogate different mechanisms of GC-GR-dependent growth (see Results). All DRG neurons were fixed with 2% paraformaldehyde (PFA) in PBS for subsequent immunohistochemistry. For each experiment, DRG neurons were prepared from three animals, and at least three coverslips or wells were used to quantify neurites using either Scholl Analysis or high-density neurite growth assays (below).Mouse DRG were harvested after 1 h of restraint (stress) or without , as described previously . Briefly, we dissected cervical, thoracic and lumbar DRG neurons from terminally anesthetized adult mice and incubated them in dispase 2  and collagenase type 2  for 45-60 min at 37°C in HBSS (Mediatech), followed by DNase 1 type 2  treatment for 5 min. Next, DRG were triturated in 0.5 ml of HBSS media through fire-polished Pasteur pipettes and spun at 3000 rpm for 3 min. The neuron-enriched pellet was resuspended in 0.1 ml of Neurobasal A media supplemented with 2% B27, 1% Glutamax, and 1% penicillin-streptomycin. Neurons were plated onto coverslips at 400 cells/coverslip, or for high-density growth analysis at 10,000 cells/coverslip. Cells assessed using automated microscopy were plated into 12-well tissue culture plates. All coverslips and tissue culture plates were pre-coated with 0.1 mg/ml of poly-L-lysine (Sigma) and 10 µg/ml of laminin (Invitrogen). DRG neurons grew for 15, 24, or 72 h at 37°C in a 5% COAB_262133) or a combination of chicken anti-neurofilament (NF) 200 and 68-kDa antibodies  were used to visualize DRG neurons and processes. Alexa Fluor 546-conjugated goat anti-rabbit or anti-chicken IgG secondary antibodies were paired with primary antibodies to detect DRG neurons . For high-density assays at least 45% of the coverslip was randomly sampled and digitized to quantify neurites . Using automatic densitometric threshold detection, neurons that were positively stained for β-tubulin III or NF were quantified then neurite density was normalized to total soma number in each field. Individual neuron neurite length in low-density cultures was quantified randomly on isolated DRG neurons after digitizing them on a light microscope followed by unbiased automated Sholl analysis  followed by 100 ml of 4% PFA in PBS. DRGs were embedded in optimal cutting temperature compound  and frozen at -80°C; 10- to 20-μm sections were cut using a cryostat and thaw-mounted on SuperFrost Plus slides (Fisher Scientific), then stored at -20°C until use. After drying at RT, slides were rinsed with 0.1 M PBS and overlaid with blocking solution for 1 h. Sections were subsequently washed and incubated overnight with the primary antibody and appropriate secondary antibodies. We used the following primary and secondary antibodies: GR: 1:200, #PA1-511A, Thermo Fisher, RRID: A 30-s sciatic nerve crush was performed as described below. In stress + injury groups, 1 h of restraint stress (see above) was performed before injury. L3-L5 DRGs ipsilateral to the injury were removed, trimmed of their axons and then placed into Trizol for RNA extraction. A total of 500 ng of RNA was used to prepare cDNA with the RT-for-PCR cDNA kit (Clontech), and qPCR was performed as described below.Ankrd1: CAATGGGGCCGCAGGGGAAT, GCTTCACGCTGTTGGCCGGA; Atf3: AAGGGGTGATGCAACGCGCT, CGCGGGTTAGCCGATTGGCT; Jun: GAGTGGGAAGGACGTGGCGC, TCCATCGTTCTGGTCGCGCG; Gap43: GCCCCCTCCGAGGAAAAGGC, TGGCTGGGCCATCTTCAGCC; Il6: GCCTTCTTGGGACTGATGCT,AGTCTCCTCTCCGGACTTGTG; Sprr1a: CCATTGCCTTGTGCTACCAA,TCAGGAGCCCTTGAAGATGAG; Tp53: AGCAGGGCTCACTCCAGCTACC,GGCTGGTGATGGGGACGGGAT; Stat3: CGGCAGCCTGTCTGCAGAGTT,ACCAGGCAATCACAATTGGCACG; Tsc22d3: bhCTCGAGGGGTCGACGGAGCC,AGGTGAGCGGCACTCGGTCT; Sgk: GCCGGAGCGCACTGTTGTCTT,CCGATGCCCGGAAAGAGCCC; Cebpa: GGTACGGCGGGAACGCAACA, GAAGATGCCCCGCAGCGTGT; Hspb2: GGGCTCCAGTCCGGCACTTC,GCGGCGCTCGGTCATGTTCT; Cebpb: CGCGTTCATGCACCGCCTGC, CCAGGCAGTCGGGCTCGTAGTAG; and Hif1a: TCTCGGCGAAGCAAAGAGTCTG, CCCACACTGAGGTTGGTTACTGTTG. Primer sequence specificity was confirmed by performing BLAST analysis for similar sequences against known sequence databases. PCRs were conducted in triplicate using 0.001 g of cDNA/reaction and Power SYBR Green master mix (Applied Biosystems) in 0.010-ml reactions. PCR product was measured using SYBR Green fluorescence on an Applied Biosystems 7900HT system. Standard curve and melting point analyses were performed to confirm amplification efficiency and single amplified products, respectively. The ΔΔCt analysis was used to normalize gene data to 18s ribosomal RNA reference gene expression. In all cases, expression was normalized to cDNA derived from L3-L5 DRGs from ns and noninjured mice; N = 4 mice per group.Gene-specific primers were used for qRT-PCR to compare expression between ns and stress groups. All primers are listed 5’-3’: Cebpa, Cebpb, Cebpd, Stat3, Hif1a, Il6, and Tp53 were amplified with promoter-specific primers which also contained restriction sites so that the promoter fragments (-1000 to +300 bp from the transcription start site) could be cloned into the pLightSwitch_Prom plasmid (SwitchGear Genomics) using the In-Fusion HD Cloning Kit (Clontech). The primers are show 5’-3’: Sgk1: CTAGCCCGGGCTCGAGTGAACGCTTACTGGTTTTGG, TTACTTAGTTAAGCTTTGCGGCAGCGACTGCAGTAA; Hspb2: CTAGCCCGGGCTCGAGTGTTGATAAATTTGCATGTG, TACTTAGTTAAGCTTTTTGGACACGGAAGTCAATG; Cebpa: CTAGCCCGGGCTCGAGTCCCCGATTCAAGTTCACTC, TACTTAGTTAAGCTTCGTGCTCGCAGATGCCGCCC; Cebpb: CTAGCCCGGGCTCGAGGTCAATGGGTCGGGGGTCAG, TACTTAGTTAAGCTTGGGCTGAAGTCGATGGCGCG; Cebpd: CTAGCCCGGGCTCGAGGGAGCTAGGCTGCTCTGTG, TTACTTAGTTAAGCTTTGTTGAAGAGGTCGGCGAAG; Stat3: CTAGCCCGGGCTCGAGGTTTTTCTGCACAAGGTGTG, TACTTAGTTAAGCTTCCTGCACCCCCTTCACCTGT; Hif1a: CTAGCCCGGGCTCGAGCACCAGCGGACCACAGGGCT, TACTTAGTTAAGCTTCGACTTTCTCAGGCAGAAAA; Il6: CTAGCCCGGGCTCGAGGAGGTCCTTCTTCGATATCT, TTACTTAGTTAAGCTTGAAGTCTCCTCTCCGGACTT; and Tp53: CTAGCCCGGGCTCGAGACACCTCAAGCGCTGGAGAA, TACTTAGTTAAGCTTGCCAAGCTTCCATTCCGCCC. All plasmids were validated by restriction digest and sequencing. The promoter containing plasmids were individually cotransfected with wtGR, constitutively active GR (caGR)  or EGFP into HEK293 cells grown in a 96-well format. All transfections were conducted according to the manufacturer’s recommendations . HEK293 cells were cultured for 2 d in vitro, fluorescence was checked visually to confirm a successful transfection, and then the luciferase assay was performed following the manufacturer’s recommendations (SwitchGear Genomics). For each experiment 8 replicate wells were transfected for each condition  and the values attained from the luminometer were normalized to the average of the promoter only wells to generate a fold change value; N = 8 replicates per plate, N = 3 independent experiments.The promoters of GR was visualized in DRG sections using an Axioplan 2 imaging microscope (Zeiss). All images were captured using identical exposure times, which were optimized for detecting GR in DRG from ns mice. Ganglia from L3-L5 were collected and imaged from three separate mice for each condition, and at least two sections were imaged from each ganglion. GR intensity was quantified in at least 50 randomly selected neurons using the Measure tool in ImageJ . Neuron N = 4 nerves/mouse; N = 3 mice/group.Eight-week-old female C57BL/6 mice (Taconic) were anesthetized  then, following shaving and aseptic preparation of the right hind leg, a Dumont #5 forceps was used to crush (30-s duration) the sciatic nerve. Stressed mice underwent 1 h of restraint just before injury. The site of nerve injury was marked by charcoal. This crush duration did not produce gross motor impairment  postoperatively. The muscle/fascia layer was pulled together and the skin was sealed with a single wound clip (Stoelting). Three days after crush injury, mice were perfused, and sciatic nerves were harvested then prepared for histology as described above. Slides were incubated overnight at 4°C with the anti-SCG10 antibody. The next day, sections were washed then incubated for 1-2 h with Alexa Fluor 555- or 488-conjugated goat anti-rabbit IgG antibodies. Nerves were imaged with identical exposure times . A photo montage was created in Photoshop then SCG10 intensity was quantified using ImageJ as described previously . BrieflyN = 3/group) then compared between groups (stress vs no-stress).A separate cohort of mice was used for 3-dimensional (3D) imaging and axon quantification. The same 30-s unilateral sciatic nerve crush injury was performed on eight-week-old female Thy1-GFP-M  mice . These mIn vitro and in vivo neurite/axon growth was analyzed using one- or two-way repeated measures analysis of variance with Bonferroni or Fisher post hoc analyses. Student’s t test was used to analyze data from histologic experiments comprising only two groups. A ratio t test for paired data were applied to Western blot analyses to compare fold change between groups. An α-level of p < 0.05 was used to indicate statistical significance.Stress activates the hypothalamic-pituitary-adrenal axis leading to an increase in the synthesis and release of GCs into the circulation. High levels of circulating GCs directly regulate gene transcription by binding to GRs. Hippocampal neurons express high levels of GRs, and GC-GR interactions profoundly affect hippocampal neuron structure/function . In sensin vitro  phase of neurite growth. In the second phase (≥24 h), most DRG neurons develop the capacity for rapid and sustained axonal elongation. This later and distinct phase of axon growth requires transcription of new genes including various canonical RAGs  or a synthetic GC  1 h before DRG harvest. Neurons were evaluated at 24 h, well before the marked increases in RAG expression that occurs in cultured DRG neurons . Structuin vivo, mice were injected with the GR-antagonist RU486 (50 mg/kg) before stress. High-density DRG neuron cultures were used since they require less effort to prepare and analyze, and because stress and cort were found to increase the density of neurites in high-density cultures (data not shown). As shown in To determine if the stress/GC effects were mediated via binding to GR Combined, these data indicate that stress enhances axon outgrowth from sensory neurons in part via GC-GR-dependent interactions. These changes occur early (<24 h), before axotomy induced transcriptional programs, and thus might work through GC-GR-dependent transcription pathways.Atf3, Ankrd1, Sprr1a, Il-6, Gap43, and Jun) also are upregulated by stress, mRNA was prepared from DRG neurons that were isolated from mice immediately following stress (0 d), or 1 or 3 d after exposing them to 1 h of restraint stress , a finding consistent with GR-dependent regulation of Jun  before nerve injury caused a partial or complete block of the injury-induced increase in RAG expression .To test this hypothesis, acute stress was applied for 1 h before a sciatic nerve crush. One or three days later, DRGs were dissected and gene expression was analyzed using qPCR. Using this approach, gene expression changes caused by stress and injury will occur together and interact in DRG neurons pression F. When cAtf3, with near complete inhibition of Atf3 upregulation at 1 dpi . GREs were identified in the promoters of several TFs and RAGs expressed in DRG neurons  or stressed mice. Expression of Tsc22d3 , a stress-induced gene with GREs in its promoter region, was analyzed as a positive control gene , Il-6, Hif1a, Tp53 , and Stat3, were also affected by stress, although gene expression changes were highly variable and were not significantly different from controls . In contrast, transfection with caGR consistently increased luciferase activity from the promoters of Cebpa, Cebpb, and Cebpd  from stressed or ns mice were labeled with SCG10, a protein that is preferentially expressed in regenerating sensory axons , and white dots show the results of the algorithm, indicating counted axons.10.1523/ENEURO.0246-17.2017.video.2Chromeless Video PlayerV4Issue4-0246-Movie2Movie 2.Thy1-GFPM sciatic nerve in a stressed mouse 14 d after injury crush injury. Green fibers are axons distal to the crush site. The box through the image shows an area quantified using the spots algorithm (Imaris) and white dots show the results of the algorithm, indicating counted axons.in vitro and in vivo. Using in silico modeling strategies, we identified several TFs and RAGs with GC-response elements and show that some of these genes are regulated by stress via GR-dependent mechanisms.Novel data in this report show that stress and GCs enhance the sprouting and regenerative growth of adult sensory axons, In the brain, stress and GCs enhance structural plasticity , neurogenesis, memory, and neuronal excitability . Becausein vitro and in vivo. Instead, we found that although stress enhances axon outgrowth through GC-GR-dependent mechanisms, these effects likely occur rapidly (immediately poststress) and do not involve transcriptional regulation of traditional RAGs. Indeed, stress only modestly increased expression of a subset of RAGs and such changes typically took 1-3 d. Other traditional RAGs  were repressed. GR repression of Il6 and Atf3 is consistent with previous studies of Il6 in COS cells and Atf3 in macrophages  are at least one gene family that is positively regulated by GC-GR binding.The axon growth promoting effects of stress and GCs are reminiscent of the effects of injuring the peripheral branch of DRG sensory neurons . These “rophages . Moreovete genes -3 and deCebps are transcriptional regulators of cellular differentiation, terminal function and responses to inflammation and tissue injury (Cebpb expression increases in regenerating facial motor neurons and may control the expression of other genes necessary for axon growth (Cebpd function in mice delays regeneration of injured peripheral nerve (Cebps are responsible for the axon growth promoting effects of stress, our data do show that Cebpa and Cebpb expression are induced by acute stress and that GC-GR interactions drive expression of Cebp-a, -b, and -d. Although these data implicate Cebps as regulators of stress-induced neurite growth, additional studies are needed to unequivocally prove a causal role for these TFs in regulating stress-induced axon plasticity.e injury . In the n growth . Loss ofal nerve . AlthougThe current experiments focused on the effects of GC-GR interactions in adult sensory neurons. Other neurons and glia also express GRs, and in response to injury or stress, GCs undoubtedly affect structure and function in these cells. As an example, after spinal cord injury basal GR expression increases in spinal neurons, but it is not known how GR activation influences neural plasticity . GR actiThe current data may have broad clinical implications, since stress is ubiquitous and synthetic corticosteroids are widely used as anti-inflammatory agents to treat various diseases in animals and humans. Perhaps most notable are the consequences that the present data could have for the diagnosis and treatment of neuropathic pain. Consider that in animal models of nerve injury or inflammatory pain , stress