The most common cause of neonatal seizures is hypoxic encephalopathy, and here we show in an established model of neonatal hypoxia-induced seizures that the NKCC1 inhibitor, bumetanide, in combination with phenobarbital is significantly more effective than phenobarbital alone. A sensitive mass spectrometry assay revealed that bumetanide concentrations in serum and brain were dose-dependent, and the expression of NKCC1 protein transiently increased in cortex and hippocampus after hypoxic seizures. Importantly, the low doses of phenobarbital and bumetanide used in the study did not increase constitutive apoptosis, alone or in combination. Perforated patch clamp recordings from ex vivo hippocampal slices removed following seizures revealed that phenobarbital and bumetanide largely reversed seizure-induced changes in EGABA. Taken together, these data provide preclinical support for clinical trials of bumetanide in human neonates at risk for hypoxic encephalopathy and seizures.Neonatal seizures can be refractory to conventional anticonvulsants, and this may in part be due to a developmental increase in expression of the neuronal Na Neonatal seizures occur most commonly in the setting of perinatal asphyxia and hypoxic-ischemic encephalopathy (HIE), and can be resistant to conventional antiepileptic therapies. Refractory neonatal seizures increase risk of subsequent epilepsy and neurocognitive morbidity. A receptors results in membrane hyperpolarization due to Cl− influx through its ion channel, and hence are inhibitory. − through the GABA receptor ion channel, resulting in neuronal excitation. − homeostasis in neurons: the Na+-K+-2 Cl− cotransporter isoform 1 (NKCC1) that transports Cl− into the cell, and the K+-Cl− cotransporter isoform 2 (KCC2) that moves Cl− out of the cell. A receptor polarity appears much later in male than in female rats, − concentrations are higher than in the adult due to a high NKCC1 expression coincident with a low KCC2 expression, relative to normal adult expression patterns. A receptors, also occurs around P14. The neonatal period is one of heightened synaptic plasticity and synaptogenesis during brain development. Excitatory ionotropic glutamate receptors are expressed at higher levels than in later life, while the expression of classical inhibitory γ-amino-butyric acid (GABA) receptors is significantly lower than adult. A receptor agonists in newborns. A receptor function in ex vivo hippocampal slices following hypoxia-induced seizures (HS) in immature rats. Finally, as some AEDs induce apoptosis in developing cortex, NKCC1 potentially represents an age-specific therapeutic target and is postulated to contribute to the relative lack of efficacy of GABAGuide for the Care and Use of Laboratory Animals. The protocol was approved by the Animal Care and Use Committee at Children’s Hospital Boston (protocol number: 11-05-1952R). We made all efforts to minimize animal suffering and the number of animals used.All procedures were in accordance with the guidelines of the National Institutes of Health’s Male Long Evans rat pups  were maintained in a temperature-controlled animal care facility with a 12 hr light-dark cycle.P10 rat pups were exposed to nonlethal graded global hypoxia for 15 min in an airtight chamber as previously described. Prior to seizure induction, phenobarbital (15 mg/kg) and/or bumetanide 0.15 mg/kg (low dose) or 0.3 mg/kg (high dose) or vehicle were administered to P10 rat pups (18–22 g) by intraperitoneal (i.p.) injection. Phenobarbital was diluted in 0.9% saline, and bumetanide  was dissolved in DMSO, then diluted in 0.9% saline. Phenobarbital and bumetanide were administered as separate injections at intervals 30 and 15 min prior to hypoxia, respectively. Five treatment groups and one vehicle group were tested: phenobarbital alone, bumetanide alone (low or high dose), and phenobarbital in combination with either of the two bumetanide doses. Rats in the phenobarbital only and bumetanide only groups received an additional vehicle i.p. injection (DMSO in 0.9% NaCl or 0.9% NaCl) of the same volume they would have received if they were in a combined treatment group. Vehicle-treated rats received two vehicle i.p. injections (DMSO in 9% NaCl and 0.9% NaCl) of the same volume as those in the combined treatment group.Seizures during hypoxia were videotaped and reviewed by an investigator blind to treatment, and scored for the number and cumulative duration of tonic-clonic seizures during hypoxia. A subgroup of rats was monitored by video-electroencephalogram (video-EEG) before, during, and after hypoxia. Continuous video-EEG was acquired using three thin silver/silver-chloride Teflon coated subcutaneous wire electrodes (SWE) for a total of 90 min for each rat . [15], [Analysis of EEG was performed on amplified  signals digitized at 200 Hz, filtered using a digital 32-channel EEG-recording apparatus from Stellate Systems Inc. , and displayed in a referential montage for post hoc analysis by visual inspection. An investigator blind to treatment conditions reviewed EEG recordings to assess seizure activity. Seizures were defined as electrographic seizures recorded from both hemispheres, only when they consisted of paroxysmal rhythmic spikes of high amplitude, diffuse frequency of ≥ 8 Hz lasting ≥ 3 s , and werControl rats were treated with phenobarbital and bumetanide (0.15 or 0.3 mg/kg), anesthetized , and perfused transcardially with 0.9% saline at 10, 30, 60, and 120 min following bumetanide injection. An additional group of rats was also exposed to global hypoxia prior to sacrifice, using the treatment paradigm described earlier. The 10 min group could only be collected for control rats due to the treatment paradigm. Blood was collected by cardiac puncture before perfusion, then clotted to separate out serum. Brains were collected following perfusion, halved along the sagittal midline, then frozen. All brains were visually inspected for signs of blood during collection. In the occasional cases where brain perfusion was incomplete, serum and brain tissue from those animals was excluded from all further analysis.5-bumetanide in acetonitrile), then vortexed for 1 min to precipitate protein, and centrifuged for 5 min at 16,200 relative centrifugal force. For brain samples, tissue from one hemisphere (∼0.5 g) was homogenized in 270 µl of internal standard and additional acetonitrile, then centrifuged for 10 min at 16,200 relative centrifugal force. Supernatant was analyzed using a Waters Quattro Premier mass spectrometer equipped with an electrospray ionization source, and a Waters Acquity UPLC system  with an Atlantis T3 column . Selected reaction monitoring (SRM) transitions were monitored for both bumetanide and D5-bumetanide. Additionally, the ratio of bumetanide concentration in brain to that in serum was calculated for each animal as a relative measure of drug elimination rates from the brain and serum. Of the 51 rats tested, three  had brain levels below the assay’s lower limit of quantitation (<0.01 ng/g) Levels of bumetanide in serum and brain were analyzed by LC-MS/MS, using a previously described method. Many commonly used antiepileptic drugs, including high doses of phenobarbital, acutely increase constitutive apoptosis in the developing rodent brain following a single dose, 2 for caudate putamen to over 4 mm2 for thalamus. Brain regions were analyzed separately and as a combined area (15 mm2) for the number of TUNEL-positive cells. TUNEL positive cells were evident as darkly stained pyknotic nuclei within a background of healthy appearing cells counterstained with methyl green. Parietal cortex from a rat treated with MK-801 was used as a positive control.Coronal sections in the region of the mid-dorsal hippocampus  Cortex and hippocampal tissue from rats sacrificed at 1, 12, 24, 48, and 168 hr following HS was dissected over cold PBS and membrane protein fractions were obtained, as previously described. 2–5% CO2), ice-cold (2–5°C) cutting solution containing 210 mM sucrose, 2.5 mM KCl, 1.02 mM NaH2PO4, 0.5 mM CaCl2, 10 mM MgSO4, 26.19 mM NaHCO3, and 10 mM D-glucose (pH 7.4). Coronal hippocampal slices were sectioned at 300 µm from the middle third of the hippocampus using a vibratome . For electrophysiological recording, slices were transferred to a chamber containing oxygenated artificial cerebral spinal fluid (ACSF) containing 124 mM NaCl, 5 mM KCl, 1.25 mM NaH2PO4, 2 mM CaCl2, 1.2 mM MgSO4, 26 mM NaHCO3, and 10 mM glucose (pH 7.4 ) at 35°C for 30 min and thereafter at room temperature for at least 1 hr before use.Hippocampal slices were prepared at 24 hr after HS as previously described. − concentration, as per our previously published protocol. Gramicidin perforated patch clamp recordings were used in order to maintain the endogenous intracellular ClA receptor–mediated currents were pharmacologically isolated by application of 10 µM NBQX, and 100 µM DL-AP5 to block AMPA and NMDA receptors. The reversal potentials for the GABA-induced currents (EGABA) were determined by varying the holding potential of the recorded neuron in 10 mV increments, and then measuring peak amplitude of the GABA-activated currents. To parallel the in vivo treatment, phenobarbital (100 µM) and bumetanide (10 µM) were applied alone and in combination to test their effects on EGABA. Data were collected 10 min after drugs were applied, and each drug was applied for a total of 15 to 20 min. Recordings for each condition were compared to control recordings obtained prior to drug application. Current-voltage curves and data analysis were performed using Clampfit 9 (Molecular Devices).GABA (100 µM) was briefly (100–200 ms) puffed onto the dendrites of the recorded neurons via a patch-type electrode using a valve-controlled pressure application system . We allowed at least 25 s between GABA applications. GABAt-test and one-way analysis of variance (ANOVA) for multiple group comparisons.Group measures are expressed as mean ± standard error of the mean (SEM); error bars also indicate SEM. Statistical analysis for differences between group means was performed using Consistent with previous studies, To confirm that the observed behavioral events were in fact ictal EEG activity, video-EEG and FFT analysis was performed in a subgroup of rats during hypoxia. Of the 201 seizures recorded from all treatment groups, 52.7% were between 3 and 15 s in duration, and 24.9% were 15–30 s in duration. Seizure duration ranged from 3–113 s, with an average duration of 21.8±1.48 s, and all seizures were accompanied by the behavioral automatisms described earlier. FFT analysis was performed on frequency bands 4–12 Hz and 12–32 Hz, however, significant changes were only seen in the 4–12 Hz band (12–32 Hz data not shown).p<0.001), and cumulative seizure duration was reduced from 229±16.3 s to 122±20.8 s (p<0.001). Low dose bumetanide (0.15 mg/kg) alone had very little effect on seizures in our model , and monotherapy with the higher dose  only moderately attenuated seizure activity . In contrast, the combination of phenobarbital with low dose bumetanide (n = 35) reduced seizure incidence by approximately 75% compared to vehicles , while combination with the high dose further decreased seizure activity by 85% from vehicle-treatment , and ∼75% from phenobarbital alone (p<0.01). The low and high dose combined treatments completely suppressed seizures in 46% and 64% of the rats treated, respectively.Treatment with phenobarbital alone (n = 33) prevented seizures in only 23% during hypoxia . Comparep<0.01). When administered alone, no significant effect on EEG power was seen with phenobarbital , low dose bumetanide , or high dose bumetanide , as compared to vehicle. While combination of phenobarbital with low dose bumetanide appeared to have only a modest effect , its combination with high dose bumetanide significantly reduced summed power compared to vehicle , approximating levels seen in baseline EEG recordings.FFT analysis was used to quantitate differences in ictal activity between treatment groups. Vehicle-treated rats (n = 4) showed increased spectral power between 4–12 Hz (summed power 43.3±3.7 µV) during seizures  compared2 in 5 different anatomical regions: parietal cortex, amygdala, caudate putamen, hippocampus, and thalamus. p = 0.183), amygdala (p = 0.727), caudate putamen (p = 0.317), hippocampus (p = 0.724), or thalamus (p = 0.096) (p = 0.129).In vehicle-treated rats (n = 10) the distribution of TUNEL positive cells was sparse, consistent with prior reports, ranging between 1.4±0.14 to 1.9±0.21 cells/mm= 0.096) . CompariBumetanide serum and brain levels were determined in 51 rat pups , p = 0.003), followed by a nearly two-fold increase between 12 and 24 hr post-hypoxia  before stabilizing to levels similar to controls (p = 0.016) before returning to normal levels.The effects of hypoxic seizures on the relative expression of NKCC1 and KCC2 were assessed by western blotting in cortical and hippocampal tissue removed at 1, 12, 24, 48, and 168 hr post-HS. In the cortex, HS caused an initial decrease in the expression of NKCC1 relative to KCC2 , with no changes in resting membrane potential between the two groups (p>0.05). We next compared the effects of phenobarbital (100 µM) alone or in combination with bumetanide (10 µM) to control conditions . In contrast, the combination of phenobarbital with bumetanide significantly decreased reversal potential  to baseline values seen in naïve age-matched littermates  are expressed as mean ± SEM.(DOCX)Click here for additional data file.Table S2Serum and brain bumetanide levels, and brain:serum ratio after hypoxic seizures. Dose = bumetanide dosage. Time = min after bumetanide injection. n = number of rats. Concentrations (Conc.) are expressed as mean ± SEM.(DOCX)Click here for additional data file.Methods S1.(DOCX)Click here for additional data file.


Lactobacillus fabifermentans strain T30PCM01 isolated from grape marc. Its genome is the largest (3.58 Mbp) among Lactobacillus species and reveals an enormous potential for carbohydrate utilization and transcriptional regulation.Here, we report the draft genome assembly of  Lactobacillus is the most diverse genus among lactic acid bacteria (LAB), a group of Gram-positive bacteria that produce lactic acid as their major fermented end product and that are often involved in food fermentation (Lactobacillus fabifermentans was proposed as a new species and was associated with spontaneous cocoa bean fermentation (L. fabifermentans T30PCM01) were isolated in northeast Italy from grape marc collected after a prolonged storage period to allow spontaneous alcoholic fermentation  obtaining 94 scaffolds >1 kb, for a total of 3,580,413 bp, with a mean G+C content of 44.9%. A second assembly was performed with Velvet 1.2.10  obtaining 149 scaffolds >1 kbp. The two assemblies were aligned using Mauve .Sequencing was performed by the Ramaciotti Centre, Sydney, Australia, using a MiSeq sequencer (Illumina) that produced 1,413,271 paired-end reads of 250 + 250 bases. Libraries were produced with the Nextera XT kit (Illumina) that gave an insert size between ~350 bp and 1.5 kbp. Short reads were assembled with ABySS 1.3.6  . The regulation and cell signaling category comprises 42 functional roles, a large number in comparison to other Lactobacillus species, suggesting a high regulatory potential.Gene prediction and annotation were performed with RAST , obtainilantarum , and witlantarum , obtainilantarum . Analysilantarum  revealedL. fabifermentans T30PCM01 has been deposited at DDBJ/EMBL/GenBank under the accession no. AWWK00000000.The whole-genome shotgun project of


This carbohydrate specificity is mediated by the type 1 fimbrial lectin FimH. In this account, we have employed four different set-ups to assay type 1 fimbriae-mediated bacterial adhesion, including tailor-made glycoarrays. The focus of our study was on testing FimH specificity with regard to the glycone part of a glycosidic ligand by testing a series of synthetic α-mannosides, as well as α-glucosides and α-galactosides. Unexpectedly, it was found that in solution all tested aminothiahexyl glycosides inhibit bacterial adhesion but that this effect is unspecific. Instead it is due to cytotoxicity of the respective glycosides at high mm concentrations.Adhesion of bacteria to the glycosylated surface of their target cells is typically mediated by fimbrial lectins, exposed on the bacterial surface. Among the best-investigated and most important fimbriae are type 1 fimbriae, for which α- Escherichia coli is a widely distributed bacterial species that is responsible for many serious infections. Urogenital infections, for example, are caused by uropathogenic E. coli (UPEC). In order to infect their target cells, UPEC first have to accomplish adhesion to the glycosylated cell surface and establish colonization of the cell surface [E. coli and constitute important virulence factors of uropathogenic strains. They are used to mediate attachment to specific niches in the urinary tract [ surface ,2. The b surface ,4. Type ry tract . Thus, try tract ,7.N-linked high-mannose type oligosaccharides serve as receptor molecules for type 1 fimbriae. Early studies by Sharon and Ofek et al. have revealed the affinities of various oligosaccharides of different complexity [d-mannosyl moieties, which varies in different oligosaccharides, is important for binding to type 1-fimbriated bacteria. This assumption is also supported by recent literature on carbohydrate binding of selectins [It has been shown that glycoproteins carrying one or more mplexity ,10,11,12electins . Additioelectins ,22,23,24electins ,28,29,30electins ,32.P anchoring the lectin at the tip of the type 1 fimbrial shaft and its lectin domain, FimHL, harboring the carbohydrate-binding site. X-ray analysis of FimH has shown that [d-mannosyl residue can be accommodated within the carbohydrate-binding pocket (β-glycosides do not fit into the binding site). The aglycone moiety of a natural oligosaccharide exerts additional interactions at the periphery of the carbohydrate-binding site [d-mannoside according to computer docking and biological testing [d-mannosides more strongly [Apparently, carbohydrate binding of type 1 fimbriae is mediated by the lectin FimH, which is located at the fimbrial tips . FimH isown that ,35,36,37 testing ,38. This testing ,40,41,42 testing . Tensilestrongly .manno-, gluco-, and galacto-series, 1–6  A binding assay with GFP-tagged E. coli to microtiter plate-based glycoarrays testing varied bacterial concentration and (ii) varied glycoarray density; (iii) an adhesion-inhibition assay to test the prepared series of synthetic α-glycosides as inhibitors of bacterial adhesion to a mannan-coated surface in solution, and (iv) a preincubation-inhibition-adhesion assay in which the bacteria are allowed to interact with the glycosides in solution before they are transferred to the microplates. This approach should allow us to test if in any set-up an altered carbohydrate specificity of type 1 fimbriae-mediated bacterial adhesion can be seen.Carbohydrate specificity of lectins is typically tested in solution or on polystyrene microtiter plates, which can be carbohydrate-decorated to allow type 1 fimbriae-mediated adhesion of E. coli . The pot1–6) were synthesized according to known procedures. The aminophenyl glycosides, 1, 3, and 5, were obtained by catalytic reduction from the corresponding nitrophenyl glycosides, which are commercially available. The 6-amino-4-thiahexyl glycosides, 2, 4, and 6, where synthesized starting with the respective allyl glycosides by radical addition of cysteamine [All glycosides used for the bacterial adhesion assays , 1–6 wersteamine . All pre®20 (0.05% v/v). Lysogeny broth (LB) medium: tryptone (10.0 g), sodium chloride (10.0 g), and yeast extract (5.00 g) were dissolved in distilled deionized water (1.00 L) and autoclaved, and ampicillin (100 mg) and chloramphenicol (50 mg) were added. Agar plates: tryptone (10.0 g), sodium chloride (10.0 g), yeast extract (5.00 g), and agar (15.0 g) were dissolved in distilled deionized water (1.00 L) and autoclaved, and ampicillin (100 mg) and chloramphenicol (50 mg) were added. All pH values were adjusted with HCl (1 m) and NaOH (1 m).Carbonate buffer solution (pH 9.2): sodium carbonate (1.59 g) and sodium hydrogen carbonate (2.52 g) were dissolved in distilled deionized water. PBS buffer solution (pH 7.2): sodium chloride (8.00 g), potassium chloride (200 mg), sodium hydrogen phosphate dihydrate (1.44 g), and potassium dihydrogen phosphate (200 mg) were dissolved in distilled deionized water (1.00 L). PBST buffer solution: PBS buffer + TweenE. coli strain pPKL1162 was used [E. coli mutant exclusively expresses type 1 fimbriae as well as GFP (green fluorescent protein) allowing for quantification of bacterial adhesion by fluorescence read-out [E. coli strain pPKL1162 was constructed by inserting the pPKL1174 plasmid into strain SAR18. The pPKL1174 plasmid contains the fim gene cluster, which is responsible for the expression of type 1 fimbriae. SAR18 includes the GFP gene on its genome, controlled by a constitutive promoter [600 = 0.4 (≙ 2 mg/mL) with PBS.For testing the GFP-expressing was used . This E.read-out . The E. promoter . The pPKSaccharomyces cerevisiae  and dried overnight at 37 °C. After washing with PBST (3 × 150 µL/well) the wells were blocked with BSA  and incubated at 37 °C for 2 h. Following this, the microtiter plates were washed with PBST (2 × 150 µL/well) and PBS (150 µL/well) [Black 96-well microtiter plates  were incubated with mannan from µL/well) .m in carbonate buffer, 120 µL/well) and incubated at ambient temperature and 100 rpm for 2 h. Following this, the microtiter plates were washed with PBST (2 × 150 µL/well) and PBS (150 µL/well) [For glycoarray preparation, black Immobilizer Amino™ F96 MicroWell™ plates (Nunc) were incubated with solutions of the respective glycosides in carbonate buffer  at ambient temperature and at 100 rpm overnight. Following washing with PBST (3 × 150 µL/well), the wells were blocked with ethanolamine (10 mµL/well) .a) Variation of bacteria concentration: Glycoarrays were prepared with 10 mm of the respective glycoside in carbonate buffer and to these glycoside-functionalized microtiter plates PBS buffer was added (100 µL/well). Then, the prepared bacterial solution (OD600 0.4) was added to well 1 and 2 and serially diluted (starting with well 2). The plate was incubated for 1 h at 37 °C, at 100 rpm, washed with PBS (3 × 150 µL/well), and then PBS was added (100 µL/well) and the fluorescence intensity (485 nm/535 nm) determined [(termined .b) Variation of glycoside concentration: Glycoarrays were prepared using serial dilutions of the respective glycoside (starting with 200 mm glycoside solution in carbonate buffer). Following this, the prepared bacterial solution  was added and the plate incubated for 1 h at 37 °C at 100 rpm. The plate was washed with PBS (3 × 150 µL/well), and then PBS was added (100 µL/well) and the fluorescence intensity (485 nm/535 nm) determined [(termined .m carbonate buffer except for pAPMan: 10 mм) and serial dilutions of the inhibitor solution added to the mannan-coated microtiter plate wells. The prepared bacterial solution  was added and the plate incubated for 1 h at 37 °C and 100 rpm. The plates were washed with PBS buffer (3 × 150 µL/well), and then the wells were filled with PBS (100 µL/well) and the fluorescence intensity (485 nm/535 nm) was determined [Solutions of the respective inhibitory glycosides were prepared (200 mtermined .pAPMan: 10 mм) and mixed with the prepared bacterial solution . /well) for preincubation. These mixtures were incubated for 1 h at 37 °C and 100 rpm and were then transferred to mannan-coated microtiter plates followed by incubation for 1 h at 37 °C and 100 rpm. The plates were washed with PBS buffer (3 × 150 µL/well), the wells filled with PBS (100 µL/well) and the fluorescence intensity (485 nm/535 nm) was determined [Serial dilutions of the respective inhibitory glycoside solution were prepared (starting concentration 200 mм in carbonate buffer except for termined .m in carbonate buffer) and mixed with a bacterial solution . The resulting mixtures were incubated for 1 h at 37 °C and 100 rpm. Following this, 50 µL of each mixture were transferred to an agar plate, which were incubated overnight at 37 °C. Bacterial growth was determined by visual inspection of the agar plates.Serial dilutions of the respective inhibitory glycoside solutions were prepared . On the other hand, a constant number of fluorescent E. coli cells were used in the adhesion experiments. Fluorescence read-out showed a plateau of maximal adhesion efficiency at “densely” functionalized plate surfaces and diminishing bacterial adhesion starting at lower glycoside concentrations when mannosides 1 or 2 were used for the functionalization of the microtiter plates (3 or 4) or galactosides (5 or 6), respectively, did not lead to an adhesive surface.Thus, glycoside solutions used for glycoarray fabrication were serially diluted starting with a comparatively high concentration , were now used as soluble inhibitors of type 1 fimbriae-mediated bacterial adhesion in solution. It was important to test, if the effect of the putative FimH ligands in solution was different from their effect as immobilized ligands. Hence, serial dilutions of inhibitor solutions were used to interfere with bacterial adhesion to mannan-coated microtiter plates. On each individual plate, we tested for methyl α-d-mannopyranoside (MeMan) to provide a universal reference inhibitor of type 1 fimbriae-mediated bacterial adhesion. Inhibition curves were determined for each tested inhibitor and IC50 values were deduced where possible. The IC50 value represents the inhibitor concentration at which 50% of bacterial adhesion is prevented. As MeMan was always simultaneously tested, relative inhibitory potencies  can be specified for all tested glycosides with respect to the IC50 obtained for MeMan, which was defined as reference inhibitor potency with IP≡1.In the next step of our study, all glycosides, which had been used before for glycoarray preparation  a soluble inhibitor has to compete against a highly mannosylated (mannan-coated) surface for binding to the type 1 fimbrial lectin. In contrast to this, in the preincubation-inhibition-adhesion assay [E. coli were first incubated with the respective inhibitor under physiological conditions for 1 h. Only then, this suspension was transferred to the mannan-coated microtiter plate surface. In the adhesion-inhibition assay both ligands, mannan and the tested inhibitor, are presented to the bacteria at the same time. In contrast, preincubation of the bacteria with inhibitor solution can lead to saturation of the lectin’s carbohydrate binding sites prior to the adhesion experiment on the mannan-coated surface. Thus, in this assay bacterial binding to mannan is only possible if the fimbrial binding site is not effectively saturated with the inhibitor after preincubation.In the adhesion-inhibition assay  are cytotoxic at high concentrations. However, all aminothiahexyl glycosides  were cytotoxic at high concentrations, the glucoside 4 at concentrations ≥100 mm, galactoside 6 at concentrations ≥50 mm, and, surprisingly, mannoside 2 inhibits bacterial growth even at a concentration of 25 mm.As a next step, it was important to analyze the effect of galactoside manno-, but also the gluco- and galacto-series (1–6) were investigated with type 1 fimbriae-mediated bacteria. Two different aglycone moieties were used, namely an aromatic p-aminophenyl unit and the 6-aminothiahexyl spacer (3 and 4) and galactosides (5 and 6) were tested in solution as inhibitors of bacterial adhesion to a mannan surface, rather unexpectedly, an inhibitory effect was observed  inhibit bacterial growth at high concentrations. Thus, the measured inhibitory effect could be shown to stem from toxicity rather than from carbohydrate-specific inhibition of adhesion. The question of why mannoside 2 shows the highest toxicity in this series cannot be answered at this stage, as E. coli bacteria have transporters for all three types of sugars, glucose, galactose, as well as mannose [To analyze the specificity of these effects, bacterial growth was tested after incubation with the employed glycosides at high concentrations . In this mannose . Recentl mannose . Interes mannose ,58,59,602. On the other hand, when glycosides such as 2, 4, or 6 are used to decorate surfaces, the resulting glycoarrays exert no cytotoxicity, suggesting that cellular uptake is a prerequisite for the effect (cf. Our study has important implications for further utilization of aminothiahexyl glycosides, regardless whether they are used as specific inhibitors or as control compounds in solution. As the aminothiahexyl aglycone is especially easily accessed in synthetic organic chemistry using the “thiol-ene” reaction of cysteamine to allyl glycosides, respective compounds are quite popular . It has fect cf. .E. coli with the highly mannosylated polysaccharide mannan. In contrast to this, in the preincubation assay the bacteria are initially exclusively presented to the glycoside ligand, and no competing compound is present in the first step of the assay. In accordance with these considerations, the IC50 of mannoside 1 is ~0.6 mm in the adhesion-inhibition assay, but 0.035 mm in the preincubation-inhibition-adhesion assay. An analogous observation was made with mannoside 2. In addition, the IC50 of the glucoside 3 is ~120 mm in the first assay and ~50 in the second.The effect of the assay set-up can be seen when data, which were obtained in the adhesion-inhibition assay are compared with those measured in the preincubation-inhibition-adhesion assay . In the At this end it remains unclear, whether conformational rearrangement of FimH can result in altered carbohydrate specificity. In the herein performed tests, a “relaxed” conformation of the FimH carbohydrate-binding site must be assumed. As a next step, we will perform adhesion studies under tensile force, based on the herein reported results. After allosteric activation of FimH according to the catch bond mechanism, carbohydrate specificity of FimH will be tested accordingly.


Here we report a cross-sectional study investigating the influence of instrumental music practice on the ability to monitor for and respond to processing conflicts and performance errors. Behavioural and electrophysiological indicators of response monitoring in amateur musicians with various skill levels were collected using simple conflict tasks. The results show that instrumental musicians are better able than non-musicians to detect conflicts and errors as indicated by systematic increases in the amplitude of the error-related negativity and the N200 with increasing levels of instrumental practice. Also, high levels of musical training were associated with more efficient and less reactive responses after experience of conflicts and errors as indicated by reduced post-error interference and post-conflict processing adjustments. Together, the present findings suggest that playing a musical instrument might improve the ability to monitor our behavior and adjust our responses effectively when needed. As these processes are amongst the first to be affected by cognitive aging, our evidence could promote musical activity as a realistic intervention to slow or even prevent age-related decline in frontal cortex mediated executive functioning. •We evaluated the effects of instrumental practice on cognitive control functions.•We show that Instrumental practice affects the ability to detect errors and conflicts.•We found an enlarged ERN and N200 in musicians compared to non-musicians.•Musical training predicted less reactive control adjustments after conflicts/errors. Nevertheless, in times of economic hardship, funds for music education are often amongst the first to be cut e.g., . This isBoth professional as well as advanced amateur musicians spend a substantial amount of time on instrumental practice, with the amount of practice time being one of the best predictors for musical skill achievement . For exaIt is not surprising that such extensive motor skill practice does result in substantial structural as well as functional brain adaptations. Both cross-sectional as well as longitudinal studies have shown increased primary motor area representations, depending on the specific type of skill practice e.g., . These aAn influential theory about how frontal cortex mediated response monitoring is exercised is the conflict monitoring theory, recently put forward by Although the studies mentioned above have suggested an enhanced ability in musicians to inhibit task-irrelevant information, it remains unclear how cognitive control functions are generally altered in musicians. Furthermore, it remains unclear how these behavioral findings relate to underlying changes in brain response. Limited evidence suggests that instrumental musicians might have better response monitoring skills, as indicated by faster error detection and correction speed in intermediate and advanced piano players e.g., . FurtherThe present study therefore aims to evaluate differences in cognitive control functions between non-musicians and amateur musicians, more specifically, the ability to monitor behavior and to implement necessary control adjustments. Implementation of control adjustments are quantified here as (A) the size of the Gratton effect, that is, the reduction in interference effects after the experience of conflict in the previous trial e.g., ; (B) as We predict that intensive musical practice will not only be associated with an increased ability to detect processing conflicts and errors, but also an improved effectiveness of control adjustments after error and conflict experience.22.1Thirty-six young adults were divided into four groups (8–10 participants per group) according to the number of accumulated hours of practicing a musical instrument over their lifetime: ‘High’ (>5000 h), ‘Intermediate’ (2000 to 5000 h), ‘Low’ (200 to 2000 h) and ‘No’ (<200 h), see also 2.2Participants completed the trait version of the PANAS Mood Questionnaire  and a deIn the Stroop task, participants were presented with the words “blue” and “red” printed in either blue or red ink color and were asked to respond to the ink color (blue or red). In the Simon task, one of two stimulus shapes (square or diamond) was presented in black either to the left or the right  of the fixation point, and participants were asked to respond to the stimulus identity. Assignment of stimulus color  or stimulus identity  to response side was counterbalanced across participants. Compatible (C) and incompatible (IC) trials were presented with equal probability. The stimulus size (shapes and letters) was approximately 7×7 mm. Stimuli were presented on a 17-inch CRT monitor at a viewing distance of about 80 cm. Responses were recorded with two response keys mounted 15 cm apart in horizontal direction. Participants used the index finger of each hand to indicate their response on an ERTS keypad.EEG activity was recorded using a BIOSEMI Active-Two amplifier with 72 Ag/AgCl electrodes. Four of these electrodes were placed at the outer canthi of and below each eye to record electro-occulographic activity. The usual ground electrode was replaced by a Common Mode Sense (CMS) active electrode and driven right leg (DRL) passive electrode feedback loop. The CMS also served as the recording reference. Data were recorded at a sampling rate of 256 Hz.2.3Participants performed both the Stroop and the Simon task, with task order balanced across participants. For each of the two tasks, participants completed one practice block  followed by six blocks . Blocks were separated by short breaks where feedback about mean reaction time (RT) and error rate was provided. Stimuli were presented until response or for a maximum of 2000 ms, followed by a 1200 ms blank interval before the start of the next trials. Stimuli were presented randomly and with equal probability. At the end of the experiment, all participants completed both the PANAS and the demographic music questionnaire.2.4N-1 and/or trial N were removed from the analysis (1.8%). Post-error choice error rates were determined by dividing the number of errors following an error by the total number of posterror trials, multiplied by one hundred. Post-correct choice error rates were determined by dividing the number of errors following correct responses by the total number of postcorrect trials, multiplied by one hundred.Trials with RTs smaller than 100 ms and larger than 1500 ms in trials To investigate group differences in behavioral conflict adjustments, a mixed ANOVA including between-subjects factor musical practice level  and within-subjects factors current compatibility , previous compatibility  and task  was conducted on mean RTs and arcsine-transformed (post-correct) choice error rates. For RT analysis only correct trials preceded by a correct trial were considered.N<5).To investigate group differences in posterror behavior, a mixed ANOVA including between-subjects factor musical practice level  and within-subjects factors trial type (post-error vs. post-correct) and task  was conducted on mean RTs and arcsine-transformed choice error rates. Three participants had to be excluded from this analysis  as they had too few trials per analysis cell . Blinks and horizontal eye movements were corrected using the adaptive artifact correction method of Brain Electromagnetic Source Analysis  software. Epochs were re-referenced off-line to average reference. A 0.1 Hz low cutoff and a 20 Hz high cutoff filter were applied. One participant (from the high level practice group) had to be excluded from EEG analyses due to a recording fault.The mean amplitude of the N200 component in a 50 ms time window around the peak latency of the grand mean (260–310 ms) was analyzed in difference waves  at electrodes FCz and Cz in stimulus-locked data relative to a 100 ms baseline starting 100 ms before stimulus onset. A mixed ANOVA including between-subjects factor musical practice level and within-subjects factors electrode  and task  was used to analyse N200 amplitudes. Only trials with correct responses were included in this analysis.N=62 . A mixed ANOVA including between-subjects factor musical practice level and within-subjects factor electrode  was conducted on ERN peak amplitudes.ERN peak amplitudes were measured in difference waves (errors minus correct response) at electrodes FCz and Cz in response-locked data, using a 100 ms baseline starting 150 ms before response onset, with automatic peak detection carried out in a search window 0 to 100 ms relative to response onset. For this analysis we averaged across the two tasks in order to achieve sufficiently high trial numbers. For ERN analysis the mean number of artifact-free trials per analysis cell was Group effects in the different dependent measures were assessed by testing for a linear trend across the four levels of group using the polynomial contrasts function in SPSS, as our a priori prediction was that observed effects should systematically relate to the amount of musical practice.33.13.1.13.1.1.1F=6.39, p=.017. There was a main effect of task, F=27.96, p<.001, ηp2=.47, due to overall faster RTs in the Stroop task (352 ms) than the Simon Task (385 ms). Participants were faster on compatible (359 ms) than incompatible trials (378 ms), F=84.42, p<.001, ηp2=.73. Task interacted with previous compatibility, F=5.36, p=.027, ηp2=.14. Task also interacted with current compatibility, F=5.91, p=.021, ηp2=.16, due to the compatibility effect (IC minus C) being larger in the Simon task (25 ms) than in the Stroop task (13 ms). Indicating the presence of the Gratton effect, there was a significant interaction between previous and current compatibility, F=113.49, p<.001, ηp2=.78, due to larger compatibility effects when the previous trial was compatible (38 ms) than when the previous trial was incompatible (0 ms). Importantly, this effect was modulated by musical practice levelF=6.65, p=.015, see also F=86.87, p<.001, ηp2=.73. The Gratton effect ([C–IC minus C–C] minus [IC–IC minus IC–C]) was larger in the Simon task (50 ms) than in the Stroop task (11 ms).RTs decreased with increasing levels of instrumental music practice , Flinear: ,32=6.39,3.1.1.2F=12.71, p=.001, ηp2=.28. There was a main effect of previous compatibility, F=36.15, p<.001, ηp2=.53. There was also a main effect of current compatibility, F=56.55, p<.001, ηp2=.64, due to participants making fewer errors in compatible (4.9%) than incompatible trials (8.1%). There was an interaction between task and previous compatibility, F=4.23, p=.048, ηp2=.12, as well as an interaction between task and current compatibility, F=13.37, p=.001, ηp2=.30, due to a larger compatibility effect (IC minus C) in the Simon task (5.5%) than in the Stroop task (1.0%). There was a significant interaction between previous and current compatibility, F=202.70, p<.001, ηp2=.86, due to larger compatibility effects when the previous trial was compatible (8.0%) compared to when the previous trial was incompatible (−1.5%), indicating the presence of a Gratton effect. Importantly, this effect was modulated by musical practice level, indicating a decrease in the Gratton effect with increasing levels of musical skills, F=6.70, p=.014. Finally, there was a three-way interaction between task, previous and current compatibility, F=42.80, p<.001, ηp2=.57, that is, the Gratton effect ([C–IC minus C–C] minus [IC–IC minus IC–C]) was larger in the Simon task (15.7%) than in the Stroop task (3.2%).Participants made fewer errors in the Stroop task (5.1%) than the Simon task (7.9%), 3.1.23.1.2.1F=71.06, p<.0001, ηp2=.71. There was an interaction between trial type and task, F=22.09, p<.001, ηp2=.43, due to the post-error slowing effect (posterror minus postcorrect) being larger in the Stroop task (78 ms) than the Simon task (32 ms). Importantly this effect was further modulated by musical practice level, F=5.42, p=.027; post-error slowing decreased with increasing levels of musical skill in the Stroop task, F=5.65, p=.024, but not in the Simon task, F=0.49, n.s.Post-error trials were significantly slower (414 ms) than post-correct trials (358 ms), 3.1.2.2F=10.64, p=.003, ηp2=.27. Participants made more errors on post-error (9.0%) than post-correct trials (5.4%) in the Stroop task (p=.036), but less errors on post-error (7.4%) than post-correct trials (8.6%) in the Simon task (p=.027). Important for present purpose, the effect of trialtype was modulated by musical practice level, F=4.80, p=.037, indicating increasingly less compromised posterror accuracy with increasing levels of musical skill =10.60, p=.003, ηp2=.26. More important for present purpose, the N200 amplitude increased (became more negative) with increasing levels of musical skills =5.05, p=.032.The N200 amplitude was larger in the Simon (−0.80 μV) than in the Stroop task (−0.08 μV), ills see , F=4.67, p=.039, see The amplitude of the ERN increased (became more negative) with increasing levels of musical skill, 4The aim of the present study was to investigate the effects of musical training on behavioural and neural mechanisms involved in conflict and post-error adjustment. The amount of musical practice was positively associated with response speed. This result suggest that higher levels of musical training might result in more efficient information processing in general , and confirms earlier reports indicating a positive link between mental speed and musical ability e.g., . More imIf active engagement in musical activity, including extensive instrumental practice, can potentially increase frontal brain mediated error and conflict monitoring functions, such activity could be used as a powerful intervention that may remediate or slow age related cognitive decline in frontal brain functions e.g.,  and it hcause differences in brain activity and cognitive functioning (e.g., However, it is important to be cautious in interpreting our findings in support of a causal link between musical activity and effectiveness of frontal brain functions, as unspecific group selection confounds might explain possible differences between participant groups. Some researchers argue that, although links between music training and cognitive functioning are relatively well established, the direction of causation remains unclear e.g., . As Scheng e.g., . Our stuThe present results have also theoretical implications for models of error/conflict detection. Traditional models of conflict/error detection assume a direct link between error detection and subsequent control adjustments, more specifically, that increased error/conflict signals result in stronger posterror and post-conflict adjustments e.g., . SeveralAn important aspect of this study is the choice of amateur rather than professional musicians. Most previous studies reporting cognitive and structural brain changes related to musical activity used professional musicians with extensive levels of accumulated practice time. Here we show that already moderate levels of musical activity are associated with improved executive functioning when performing basic non-musical cognitive task. This is particularly important if musical activity were to be suggested as a realistic intervention method to slow or even prevent age-related decline in frontal brain functioning. Furthermore, people who have never played an instrument or felt too old to start engaging in such an activity might be encouraged to take up music, and the associated benefits for physical and mental functioning could be much more far-reaching than proposed in this study.


Rhizobium leguminosarum bv. trifolii (Rlt) are soil bacteria inducing nodules on clover, where they fix nitrogen. Genome organization analyses of 22 Rlt clover nodule isolates showed that they contained 3–6 plasmids and majority of them possessed large (>1 Mb), chromid-like replicon with exception of four Rlt strains. The Biolog phenotypic profiling comprising utilization of C, N, P, and S sources and tolerance to osmolytes and pH revealed metabolic versatility of the Rlt strains. Statistical analyses of our results showed a clear bias toward specific metabolic preferences, tolerance to unfavorable osmotic conditions, and increased nodulation activity of the strains having smaller amount of extrachromosomal DNA. The K5.4 and K4.15 lacking a large megaplasmid possessed substantially diverse metabolism and belonged to effective clover inoculants. In conclusion, besides overall metabolic versatility, some metabolic specialization may enable rhizobia to persist in variable environments and to compete successfully with other bacteria.The online version of this article (doi:10.1007/s00203-013-0874-x) contains supplementary material, which is available to authorized users. The rhizosphere is the microbe-rich zone surrounding plant roots. It is a dynamic environment, where resource distribution varies spatially and temporally, with plants providing a plethora of carbon and energy sources that significantly affect the populations of microorganisms in a manner specific to the host  have complex and large, >6 Mb, genomes that reflect their diverse metabolic capabilities (Konstantinidis and Tiedje Rhizobium leguminosarum bv. trifolii (Rlt) is a microsymbiont of clover and is able to fix atmospheric nitrogen in root nodules of this plant. Our previous studies of Rlt isolates from root nodules of clover plants growing at the same site showed a substantial divergence of their genome organization, especially as regards the plasmid DNA content  kb to 3,250 kb . Furthermore, most of the strains had large (>1 Mb), chromid-like replicon with the exception of four Rlt strains K3.6, K3.16, K4.15, and K5.4, in which this type of replicon was substantially smaller  technology, to test the utilization of numerous sources of carbon, nitrogen, phosphorus, and sulfur, as well as tolerance to osmolytes and different pH conditions in the clover nodule isolates, which was followed by plant tests to estimate their symbiotic activity. The potential interrelation between observed phenotypic traits and amount of extrachromosomal DNA in the sampled Rlt strains was assessed by comprehensive statistical analyses.The purpose of this study was to extend and deepen the analyses of metabolic capacities of the previously genetically characterized 22 Rlt isolates were obtained from nodules of red clover (Trifolium pratense L. cv. Dajana) growing in sandy loam (N:P:K 0.157:0.014:0.013 %), as described previously  complex media at 28 °C. Vincent . Pure cuRlt isolates and tolerance to different osmotic and pH conditions were performed using Biolog GN2, PM2A, PM3B, PM4A, PM9, and PM10 microplates, Biolog Inc. . GN2 and PM2A plates were used to study C sources metabolism, PM3B plates to assess N metabolism, and PM4A for P and S sources, respectively. In addition, PM9 and PM10 plates were used to test the growth under various stress conditions and different pH , nitrogen (N), phosphorus (P), and sulfur (S) sources by 550) of 0.1, and a 100 μl suspension of rhizobia was inoculated into each well of the GN2 microplate. For the remaining plates , the washed bacteria were suspended in 10 ml of inoculating fluid (IF-0a) and OD550 was adjusted to 0.5. The bacterial suspensions were further diluted into 12 ml (per plate) of a relevant inoculating fluid. IF-0a inoculating fluid supplemented with Dilworth’s vitamins was used for PM2A plate; IF-0a with 20 mM sodium pyruvate and vitamins was used for plates PM3B and PM4A; IF-10 Base was used for PM9 and PM10 plates, respectively. The prepared bacterial suspensions were inoculated into wells of the appropriate microplates. The cells were incubated for 72 h at 28 °C, and color development (absorbance at 590 and 750 nm) in the wells was monitored using Benchmark Plus™ microplate reader . The conversion of colorless tetrazolium violet to a purple-colored compound meant normal process of respiration (positive phenotype), whereas when the phenotype was negative, the wells remained colorless. The optical density (OD) values of Biolog microplate wells were corrected using background color developed in the control well A1.Bacteria growing overnight at 28 °C in TY medium were pelleted and washed twice with sterile water. After that, for GN2 plates, the pellet was diluted in water to an initial optical density at 550 nm (ODTrifolium pratense L. cv. Dajana) were surface-sterilized and germinated on nitrogen-free Fåhraeus medium. Clover seedlings were inoculated with 0.2 ml of Rlt cell suspension at an approximate density of 1.0 × 109 cells/ml and were grown (one per tube) in a greenhouse under natural light supplemented with artificial light (14/10-h light/dark) regime and at 24/19 °C day/night temperature. After 4 weeks, the plants were harvested, the nodules were counted, and fresh masses of shoots and roots were estimated. For each strain, 20 clover plants were used.Red clover seeds , the results of Biolog test were coded in the binary system, and PCA with varimax rotation Morrison  was usedRlt genome and the number of utilized substrates classified into different PCs, as well as (b) the organization of Rlt genome and the symbiotic properties of strains, were studied using one-way analysis of variance (ANOVA), which was performed using Statistica software.The relationships between (a) the organization of Rlt strains involved in this study have been previously analyzed with respect to genome organization and genetic diversity , nitrogen (N), sulfur (S), and phosphorus (P) (Table S1). To gain general overview of metabolic divergence of Rlt strains and to simplify initial examination, the substrates contained in GN2, PM2A, PM3B, PM4A microplates were arbitrarily divided into 19 groups: monosaccharides, oligosaccharides, polysaccharides, sugar alcohols, modified sugars, phospho-sugars, d-amino acids, l-amino acids, modified amino acids, oligopeptides (dipeptides mainly), amines, glycosides, carboxylic acids, sugar acids, modified carboxylic acids, nitrogen bases, nucleosides and nucleotides, inorganic compounds and miscellaneous, that is, other organic compounds not classified into the aforementioned groups (herein referred to as “other organic”) (Table S2). The number of the various substrates taken into account in this analysis was 301 and was less than the number of all the possible compounds assayed in the four types of microplates (379 compounds) and resulted from exclusion of some repeated substrates on different plates.The Rlt strains. The individual Rlt isolates metabolized from 113 (37.5 %) to 157 (52.2 %) substrates  and two strains used none of them  used approximately half of the tested substrates (150–157). The lowest number of substrates (113) was utilized by K3.16 strain . Of all the compounds, 71 (23.6 %) were metabolized by all the tested Rlt strains were extremely sensitive to NaCl and Na2SO4, with the exception of K4.15, K5.4, and K9.8 strains, which tolerated the presence of 1 % NaCl and 2 % Na2SO4, respectively. Majority of the strains showed no metabolic activity in the presence of sodium formate and sodium lactate . Majority of Rlt strains tolerated pH from slightly acidic (pH 5.0–5.5) to neutral (pH 7), only few of them showed metabolic activity also at slightly alkaline pH (pH 8.0–8.5). Strain K5.4 was the most resistant to pH changes and was metabolically active at pH 5.0–8.5  were classified into principal components (PCs). This PCA will be referred as general (PCAG) throughout the manuscript. The main criterion for the classification of every substrate group to the individual principal component was high correlation coefficient (positive or negative) of the substrate group with a given principal component and low rates for the remaining principal components. PC1G to PC7G were responsible for 81.12 % of the total variance of metabolic capabilities of Rlt strains (Table S3A). The PC1G included monosaccharides, oligosaccharides, sugar alcohols, and carboxylic acids; PC2G constituents were modified sugars, d-amino acids, and amines; inorganic compounds and “other organic” were classified into PC3G; PC4G comprised sugar acids, modified carboxylic acids, and nitrogen bases; PC5G—nucleosides and nucleotides; PC6G—glycosides; PC7G contained oligopeptides (Table S3A).To qualitatively analyze metabolic differentiation between the sampled G–PC4G) were composed of more than one group of substrates, in total encompassing twelve groups of compounds, and contributed to 60.37 % of variance of the tested strains. PC1G containing 61 compounds . Rlt strains metabolized from 22 to 41 substrates classified into PC1G. K4.11 and K4.16 strains utilized the most compounds included into the PC1G, while the K3.16, K4.15, and K5.4 used few of these substrates  and contributed to 17.97 % of variance (Table S3A). The Rlt isolates metabolized from 6 to 15 substrates from PC2G (on average 31.4 % of substrates) , respectively (Table S3A), and these principal components did not contribute substantially to the metabolic diversity of the tested strains.The PC5G, the individual PCs encompassed groups of substrates, which may be both sources of carbon, nitrogen, sulfur, and phosphorus. To find out whether the specific metabolism of carbon, nitrogen, sulfur, or phosphorus is responsible for the Rlt strains metabolic variance, another PCA was done in which utilization of (a) carbon sources, (b) nitrogen sources, and (c) phosphorus and sulfur sources were studied separately . Classification of individual substrates remained almost the same as in previous PCAG, with minor exceptions. Substrates previously included in the group of nucleosides and nucleotides were now classified as nucleosides group (when carbon or nitrogen sources were studied) or as nucleotides group . Moreover, in PCAP/S thio-β-d-glucose, previously belonging to the group of modified sugars was included into other organic compounds. In the PCA concerning utilization of carbon, nitrogen, phosphorous, and sulfur sources, 15, 11, and 8 groups of substrates were analyzed, respectively. Furthermore, this kind of analysis allowed us to consider utilization of all possible 379 compounds without excluding of any repeating sources.In the performed PCAC (comprising 15 groups of substrates)—six principal components were identified and the most important PC1C, PC2C, and PC3C together contributed to 59.5 % of variance of tested strains (Table S3B). PC1C was mainly composed of monosaccharides, oligosaccharides, carboxylic acids, nucleosides, as well as sugar alcohols (with lower correlation coefficient). PC2C contained l-amino acids and oligopeptides, whereas PC3C comprised sugar acids and modified carboxylic acids.In PCA focusing on carbon sources—PCARlt strains in the PC1C and PC2C, as well as in PC1C and PC3C coordinate system , only five principal components were identified, with the most important PC1 N, PC2 N, and PC3 N, which contributed together to 66.8 % of metabolic variance of studied strains (Table S3C). PC1 N was mainly composed of modified sugars, l-amino acids, amines, and nitrogen bases (with lower correlation coefficient). PC2 N contained nucleosides, other organic compounds, and inorganic nitrogen sources (with lower correlation coefficient). PC3 N was highly correlated only with oligopeptides (dipepetides mainly) (Table S3C). Strains, which differed substantially from others in carbon metabolism revealed comparable nitrogen metabolic properties to other strains from studied group  were submitted for PCA , in which four principal components were identified (Table S3D). PC1P/S was mainly composed of inorganic P and S compounds as well as phospho-sugars and other organic compounds (with lower correlation coefficients) and contributed to 33.7 % of variance. PC2P/S contained l-amino acids and modified amino acids, resulting in further 21.8 % of metabolic variance (Table S3D). PC3P/S comprised mainly d-amino acids and oligopeptides, PC4P/S comprised only nucleotides contributing, respectively, to 12.6 % and 11.9 % of metabolic variance of studied strains. It was observed that some of the strains, namely K4.15 and K5.4, differed mostly in the utilization of compounds classified to PC1P/S and PC3P/S  . Furthermore, in the cases of K4.15 and K5.4 differences in the utilization of inorganic compounds, phospho-sugars, other organic compounds, d-amino acids, and oligopeptides as phosphorus and sulfur sources  were observed. Interestingly, the metabolically distinct K3.16, K4.15, and K5.4 represent the subgroup of the Rlt strains lacking a very large plasmid (>1 Mb).According to the data obtained with Biolog microplates, majority of the nds Fig. a. FurtheRlt strains. Significant difference (p < 0.05) was observed in the number of utilized substrates classified into general PC1G and PC4G for Rlt isolates, which possessed a very large plasmid (>1 Mb) versus those which did not have such replicon. In general, the strains lacking the very large replicons utilized significantly lower number of PC1G classified compounds, especially monosaccharides and oligosaccharides, in comparison with strains having such plasmids (an average 26.5 versus 33.7). Simultaneously, the same strains demonstrated higher metabolic versatility in the utilization of various sugar acids, modified carboxylic acids, and nitrogen bases of PC4G-classified compounds (an average 11.8 versus 9.5). In the subgroup of Rlt strains without the very large plasmids, K4.15 and K5.4 were of special interest. These strains not only metabolized less substrates of PC1G in comparison with other strains but, concomitantly, utilized more inorganic compounds and other organic compounds of PC3G (an average 24.5 versus 18.2), as well as sugar acids, modified carboxylic acids, and nitrogen bases of PC4G (13 versus 9.6). In the PCAs taking into account utilization C, N, P, and S sources, significant difference (p < 0.05) was also observed in the number of utilized carbon sources classified into PC1C for Rlt isolates differing in the possession of >1 Mb plasmid. The strains lacking the very large replicons utilized significantly lower number of PC1C classified compounds, that is, monosaccharides, oligosaccharides, carboxylic acids, nucleosides, as well as sugar alcohols and modified sugars in comparison with strains having such plasmids (an average 22.3 vs. 34.9). Concomitantly, the strains lacking >1 Mb plasmid utilized more inorganic compounds, phospho-sugars, other organic compounds, d-amino acids, and oligopeptides included into PC1P/S and PC3P/S .We subsequently focused on the potential relationship between the genome complexity and metabolic capabilities of the sampled K4.15 and K5.4 strains were also less sensitive to unfavorable osmotic conditions in comparison with the others; K5.4 was found to be the most tolerant strain within the sampled group. However, we did not find any significant correlation between the tolerance to osmotic stresses, or a wide range of pH, and the size of the extrachromosomal genome or the presence of the very large plasmids.Rlt strains was examined in a laboratory plant test, in which red clover plants were inoculated with the relevant bacteria. Average shoot fresh mass ranged from 22 mg (K3.12 and K9.2) to more than 40 mg/plant   to 10–20 nodules/plant . An increased nodule number was not correlated with an increase in shoots mass of plants. Interestingly, significant differences were noted for the wet masses of clover shoots. It was observed that plants inoculated with strains without the very large plasmids had a significantly higher fresh shoot mass than plants infected with strains harboring such plasmid . Thus, the most metabolically distinct K5.4 and K4.15 strains belonged to the group of symbiotically effective inoculants  Fig. . Clover Rlt strains included in this study could be considered champions in clover nodulation competition, thus might provide valuable information about bacteria metabolic approaches helpful for root colonization. The strains have been previously characterized on a genetic level and the diversity of their genomic organization had been described  was lower by about 551 kb than in the remaining strains . The question was whether these differences in amount of extrachromosomal DNA and overall genomic heterogeneity of the strains reflect the metabolic potential and symbiotic activity of the sampled rhizobia. Therefore, we compared substrate utilization data gathered from high-throughput phenotyping microarrays for a set of environmental isolates, which differed in genomic architecture and plasmid patterns.The metabolic properties of an organism may contribute toward a particular niche adaptation. Metabolic differences that have enabled rhizobia to adapt to specific environments, such as the soil and plant tissues, are poorly understood but may be key in understanding how bacteria survive and compete for host’s nodulation. The Rlt isolates was performed using Biolog microplates, comprising analyses of the utilization of nutritional compounds , as well as tolerance to osmolytes and different pH conditions. The obtained patterns of phenotypes suggested that Rlt strains are capable of utilizing a variety of metabolic substrates (68.4 % of the tested sources), with significant preferences for sugar alcohols, glycosides, nucleosides, nucleotides, and oligopeptides (comprising mainly dipeptides), with an average utilization of ~73 % of the compounds. Monosaccharides were also quite well utilized with an average of 67.6 % and specific sugars, such as d-fructose, l-fucose, d-glucose, d-psicose, and l-, d-arabinose, were utilized by all 22 strains. All the tested strains also utilized sucrose and d-trehalose, and up to 50 % of oligopeptides (9 of 18) or glycosides (4 of 8), respectively. About 30 % of the tested nucleotides and nucleosides were metabolized by all strains. In general, the phenotypic profiling in conjunction with statistical analyses revealed versatility of the sampled Rlt strains. Only a few strains were metabolically distinct in relation to others, with two, namely K5.4 and K4.15, remarkably dissimilar. In comparison with the remaining isolates, these strains metabolized less monosaccharides and oligosaccharides, and more inorganic and other organic compounds, as well as sugar acids, modified carboxylic acids, and nitrogen bases, respectively. With respect to the specific metabolism, K5.4 and K4.15 utilized less monosaccharides, oligosaccharides, carboxylic acids, nucleosides, as well as sugar alcohols and modified sugars as carbon sources and more inorganic compounds, phospho-sugars, other organic compounds, d-amino acids and oligopeptides (dipeptides) as P and S sources, but they did not differ substantially in nitrogen sources utilization.Metabolic profiling of 22 R. leguminosarum adaptation to rhizospheres of legumes Supplementary material 2 (DOC 53 kb)Supplementary material 3 (DOCX 25 kb)Below is the link to the electronic supplementary material.


Neuregulin switches oligodendrocytes between two modes of myelination: from a neuronal activity–independent mode to a myelin-increasing, neuronal activity–dependent, mechanism that involves glutamate release and NMDA receptor activation. In vivo, we demonstrate that remyelination after white matter damage is NMDA receptor-dependent. These data resolve controversies over the signalling regulating myelination and suggest novel roles for neuregulin in schizophrenia and in remyelination after white matter damage.Myelination is essential for rapid impulse conduction in the CNS, but what determines whether an individual axon becomes myelinated remains unknown. Here we show, using a myelinating coculture system, that there are two distinct modes of myelination, one that is independent of neuronal activity and glutamate release and another that depends on neuronal action potentials releasing glutamate to activate NMDA receptors on oligodendrocyte lineage cells. Neuregulin switches oligodendrocytes from the activity-independent to the activity-dependent mode of myelination by increasing NMDA receptor currents in oligodendrocyte lineage cells 6-fold. With neuregulin present myelination is accelerated and increased, and NMDA receptor block reduces myelination to far below its level without neuregulin. Thus, a neuregulin-controlled switch enhances the myelination of active axons.  Myelination acts as an insulator for neurons and as such is essential for normal brain function, ensuring fast neuronal communication. Oligodendrocytes are the cells that wrap their membrane around nerve cell axons to form the myelin sheath that enables fast action potential propagation. However, what determines whether an individual axon becomes myelinated remains unknown. We show that there are two distinct modes of myelination: one that is independent of neuronal activity and the release of the neurotransmitter glutamate and another that depends on nerve cell action potentials releasing glutamate, which then activates a class of glutamate receptor (NMDA receptors) on oligodendrocyte lineage cells. We find that the protein neuregulin switches oligodendrocytes between these two modes of myelination; neuregulin increases oligodendrocyte lineage cells' sensitivity to glutamate by increasing the current flowing through their glutamate receptors. With neuregulin present, myelination is accelerated and increased. Blocking NMDA receptors reduces the amount of myelination to far below its level without neuregulin. Thus, a neuregulin-controlled switch enhances the myelination of active axons. We also demonstrate that remyelination after white matter damage  is NMDA receptor-dependent. These data help us understand the signalling that regulates myelination and suggest the possible involvement of neuregulin in schizophrenia and in remyelination after white matter damage. Myelination is essential for normal brain function as myelin sheaths provide trophic support for axons and increase the conduction speed of action potentials NRG on axons can regulate myelination by signalling to ErbB receptors on ensheathing Schwann cells or oligodendrocytes, but there is controversy over its actions. In the peripheral nervous system, a decrease of NRG signalling leads to less myelination Further uncertainty relates to how neuronal activity regulates CNS myelination. Neuronal activity can promote myelination in vivo is NMDA receptor dependent, suggesting enhancement of NRG- and NMDAR-dependent remyelination as a therapeutic approach for promoting recovery after demyelination.Here we show for the first time that there are two distinct modes of myelination by oligodendrocytes, one independent of neuronal activity and the other dependent on neuronal action potentials. Furthermore, we demonstrate that NRG switches oligodendrocytes between these two myelination programmes by increasing NMDA receptor-mediated currents in oligodendrocyte lineage cells, making these cells more sensitive to glutamate released from active axons. As a result, this interaction of NRG and glutamatergic signalling accelerates and increases myelination, but most importantly provides a mechanism by which myelination is focussed on the axons of active neurons. Investigations of white matter pathology based on this finding revealed that remyelination of axons in a demyelinated lesion n = 6) and a g-ratio of 0.74±0.02 (n = 6), as expected for CNS myelination To assess whether NRG-ErbB signalling and activation of NMDA receptors in oligodendrocytes interact to control myelination, we studied myelination of cultured dorsal root ganglion (DRG) neurons by forebrain oligodendrocytes p = 7×10−4; p = 0.03; ErbB signalling was activated by applying the extracellular domain of NRG , which includes the EGF domain that binds to ErbB receptors expressed on oligodendrocytes p = 0 .024), and its onset  occurred 5 d earlier (p = 0.0037), according to the fitted curves in Varying the time at which myelination was assessed after plating the OPCs onto the neurons showed that NRG both accelerated myelination and increased its steady state level . The myeTo assess whether changes in cell proliferation, growth, or survival were responsible for the increased myelination in NRG, we counted cells of different types identified with antibodies. In the presence of NRG, there was no significant change in the density of axons , the totp = 0.65), but reduced myelination by 50% in the presence of NRG  to block action potentials. TTX had no effect on myelination in the absence of added NRG  but reduced myelination by 82% when NRG was present (p = 3×10−15). In NRG+MK-801, myelination was reduced to only 25% of the level seen in control conditions in the absence of NRG (p = 4×10−9). The same results were obtained (without and with NRG) when the AMPA/kainate receptor blocker NBQX (25 µM) was added with the MK-801 (p = 0.66) but reduced myelination in the presence of NRG by 80% (p = 4×10−6), that is, to only 28% of the control level with no NRG  in the absence of NRG, but reduced myelination by 46% in NRG  had no effect on myelination in the absence of added NRG , but the NRG effect became much smaller when the control level of myelination was large . Strikinp = 0.92; differentiated oligodendrocytes, control 130±25 MΩ versus NRG 208±65 MΩ, p = 0.20). However, with added NRG the NMDA-evoked currents at −64 mV in oligodendrocyte precursor cells and in differentiated oligodendrocytes were ∼6-fold larger than in cells not exposed to added NRG  or differentiated oligodendrocytes , using postrecording antibody labelling  and membp = 0.02; p = 0.85; p = 0.02; NMDA receptors in oligodendrocyte lineage cells have been suggested to mainly comprise NR1, NR2C, and NR3A subunits 1 integrin (1 µg/ml) to block its function p = 0.27), but in the presence of NRG it reduced myelination by 82% . Thus, unlike the activity-independent mode of myelination, the activity and NMDA receptor-dependent myelination induced by NRG is dependent on integrin function.Myelination depends on integrins p = 0.047; p = 0.79; p = 0.01) with no effect on pErk (p = 0.26), and there was no significant difference between the levels of pAkt in NRG, with or without MK-801 (p = 0.50). Thus, activation of Akt does not occur downstream of NMDA receptor activation . NRG also increased by 65% (p = 0.02) the proportion of Nkx2.2-expressing cells (late OPCs/immature oligodendrocytes) that expressed phosphorylated CREB (pCREB), an Akt target p = 0.35; NRG can activate the PI3K-Akt or MAPK/Erk pathways in oligodendrocytes Egr-1  and had no effect on the level of c-Jun , to only 47% of its value without BDNF (p = 0.019). Like NRG, BDNF induced phosphorylation of Akt but not Erk , and downregulated expression of the NR3A subunit but not the NR1 subunit of NMDA receptors . Thus, by acting through similar signalling pathways, BDNF can also switch oligodendrocyte myelination to a pathway that depends on glutamate release activating NMDA receptors. Consequently, when the NRG/ErbB pathway is knocked out, myelination may still occur The role of NRG/ErbB signalling in CNS myelination is controversial, as decreasing NRG or ErbB function reduced myelination Our demonstration of NRG- and NMDAR-dependent myelination of axons by oligodendrocytes raises the question of whether this same mechanism operates during remyelination after white matter damage. A lack of NRG signalling has been suggested to result in poor remyelination in multiple sclerosis 2; MK-801, 0.22±0.04 mm2, p = 0.28) nor axon density  between the conditions, but a blinded analysis ranking remyelination revealed that blocking NMDA receptors with MK-801 significantly inhibited remyelination  from the 3rd day postlesion , for 3 wk, with medium changes twice per week thereafter.in vitro.These were as described previously fraction of oligodendrocytes myelinating (F) on axon density (D),K is a free constant and the slope A is the measure of myelination plotted in K that were not significantly different . This quantification of myelination gave results similar to those obtained using other assumed dependencies of fraction of oligodendrocytes myelinating (F) on axon density (D) as follows:For each coverslip, 30 randomly located images were taken of MBP and NF staining . Within each image, myelination was quantified by counting the number of myelinating MBP-positive oligodendrocytes  as a perthresholdD, a free parameter gave thresholds that were not significantly different from thresholdD = 17% , so thresholdD was set to 17%; we refitted the data, and used the value of A as a measure of myelination , so we fixed the power at 2, re-fitted the data, and then used the amplitude (A) of the best fit curve as the measure of myelination , or 130 Cs-gluconate, 4 NaCl, 0.5 CaCl2, 10 HEPES, 10 BAPTA, 4 MgATP, 0.5 Na2GTP, 2 K-Lucifer yellow, pH set to 7.3 with CsOH (ECl = −88 mV). Series resistance was 5–20 MΩ, and electrode junction potentials were compensated. Cultures were superfused at 24±1°C with HEPES-buffered solution containing (mM) 144 NaCl, 2.5 KCl, 10 HEPES, 1 NaH2PO4, 2.5 CaCl2, 10 glucose, 0.1 glycine (to coactivate NMDA receptors), 0.005 strychnine (to block glycine receptors), pH set to 7.4 with NaOH, bubbled with 100% O2. Cells were identified .Cultures were fixed at 21°C for 20 min in 4% PFA and incubated for 1 h in 0.1% Triton X-100, 10% goat serum in phosphate-buffered saline at 21°C, then with primary antibody at 21°C for 2 h or for overnight at 4°C, and then for 1 h at 21°C with secondary antibody. Primary antibodies were guinea pig NG2 , rabbit NG2 , rabbit Olig2 , rabbit Caspr , rabbit SCIP , rabbit GalC , mouse NF 160/200 , rat MBP , mouse MOG , mouse CNPase , rabbit GAD 65/67 , rabbit GFAP , chicken P0 , mouse Nkx2.2 , CD11b , and rabbit pCREB . Alexa 488–conjugated isolectin B4  was used to label microglia. DAPI  was used to label nuclei. Secondary antibodies (goat) were for rabbit , rat IgG , mouse IgG , chicken , and guinea pig .Ser473 , rabbit anti-phosphorylated-ERK1/2Thr202/Tyr214 , mouse anti-ERK1/2 , rabbit anti-MBP , mouse anti-NR1 , rabbit anti-NR2A , rabbit anti-NR2B , rabbit anti-phosphorylated NR2BTyr1472 , rabbit anti-NR2C , rabbit anti-phosphorylated NR2CS1096 , rabbit anti-NR3A , rabbit anti-NR3B , rabbit anti-NR2D , or mouse anti-β-actin  in 3% BSA in PBS-T. This was followed by incubation with the secondary horseradish peroxidise-linked anti-rabbit, anti-mouse, or anti-goat antibodies  for 1 h at room temperature in 3% BSA in PBS-T. Immunoreactive proteins were visualised with enhanced chemiluminescence . When phosphorylated and total amounts of the same protein were measured, this was done by first probing with the antibody to the phosphorylated protein, then stripping the membranes to remove the antibody, and reprobing with antibody recognizing both phosphorylated and unphosphorylated forms. Stripping membranes was performed with 50 mM dithiothreitol, 50 mM Tris (pH 6.8), and 2% SDS at 70°C for 30 min and washing three times in PBS-T before blocking and incubating with the primary antibody. Densitometric analysis was conducted using ImageJ gel analysis software (version 1.43u). For each sample, pAkt, pERK, pNR2B, and pNR2C signals were normalised to the total levels of their respective proteins present. The levels of nonphosphorylated proteins were normalized against β-actin.For protein analysis, cultures were scraped from 22 mm coverslips, 6 well plates, or 10 cm dishes and lysed mechanically in solution containing 0.1 M phosphate buffered saline (PBS), 10% or 20% (w/v) sucrose and Halt protease, and phosphatase inhibitor cocktail (Thermo Scientific). Protein content was determined by Bradford assay and quantified using a standard curve obtained from Quick Start BSA protein standards (Bio-Rad). Equal amounts of protein (8 or 10 µg) from samples were resolved on 4–%12% NuPage Novex Bis-Tris mini gels (Invitrogen), with prestained molecular weight protein standards (Bio-Rad). Proteins were transferred to a nitrocellulose membrane  using a wet transfer system. Nitrocellulose membranes were blocked for 1 h at room temperature with 3% BSA in PBS with 0.1% Tween-20 (PBS-T). Immunoblots were then incubated overnight at 4°C with goat anti-Akt , rabbit anti-phosphorylated-AktRNA was extracted from DRG-OPC cocultures 21 d after addition of OPCs using an RNeasy mini kit (QIAGEN). RNA was converted to cDNA using a SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). qPCR was performed with a QuantiFast SYBR Green PCR Kit (QIAGEN) using a LightCycler 480 II. Samples were normalised to GAPDH levels using commercially available QuantiTect Primers (QIAGEN) for analysis.Female Sprague-Dawley rats aged 9–12 wk of age (200–225 g) were used for remyelination studies, which were performed in compliance with UK Home Office regulations. Focal demyelination was induced unilaterally by stereotaxically injecting 4.0 µl of 0.01% ethidium bromide (w/v) in saline into the caudal cerebellar peduncle (CCP)  in phosphate buffer containing 0.72 mM CaCl2. All samples were then left to fix in 2% osmium tetroxide (Oxkem Ltd) in phosphate buffer at 4°C overnight. This was followed by dehydration in 70% ethanol for 15 min, 95% ethanol for 15 min, and 100% ethanol for 3×10 min, then the tissue was placed in propylene oxide for 2×15 min, and then left for at least 3 h in a 50%/50% mixture of propylene oxide and resin mix . They were then transferred to 100% resin mix overnight. New resin was made and the samples left in it for at least 6 h before being placed in embedding capsules and further incubated at 60°C for 15–24 h until the resin was solid. Embedded samples were cut in 90 nm sections on an ultramicrotome (Reichert Ultracut E) with a diamond knife (Diatome) and visualised using a Transmission Electron Microscope (Hitachi H600). Images were developed on electron microscope film 4489 (Kodak), then scanned at high resolution , and analyzed with ImageJ software, version 1.34s.Co-cultures were fixed at room temperature for 1 h in PBS containing 2.5% glutaraldehyde (Agar Scientific) and 0.72 mM CaCl2), and the brains were immersion fixed in 4% glutaraldehyde for 7 d. Tissue blocks encompassing the caudal cerebellar peduncle were cut as detailed previously Animals were perfused with 4% glutaraldehyde  or Dunnett's post hoc test. Normality of data was assessed using Shapiro–Wilk tests, and nonparametric Kruskal–Wallis and Mann–Whitney tests, which do not assume data follow a normal distribution, gave the same conclusions for significant and nonsignificant differences in all cases. For p value (>0.05), we used the more conservative Student's two-tailed t test without correction for multiple comparisons to give a lower limit to the p value for post hoc comparisons .Data are mean ± s.e.m. The Figure S1Characterization of myelination in the co-cultures. (A) Electron microscopic image of myelination in the cocultures showing formation of compact myelin. (B) Labelling of coculture for Caspr shows formation of axon-oligodendrocyte junctions at the end of internodes. Note that some axons have single internodes so that only one Caspr-labelled region is visible at a heminode (open arrows), while some have multiple adjacent internodal segments, so that two Caspr-labelled regions are visible at nodes (filled arrows). (C) Enlarged view of a node of Ranvier labelled for Caspr. (D) DRG cells with no added OPCs show no myelination, ruling out the possibility of myelination by Schwann cells or precursors added with the DRG cells. Blue is DAPI to label nuclei, red is NF 160/200, and green is MBP (which is absent). (E) Myelination is by MBP-expressing OPCs and not P0-expressing Schwann cells. Mauve is NF, green is MBP, and red is P0 protein . (F) The processes of DRG cells but not of interneurons become myelinated. Red is NF, green is MBP, and white is the GABA synthesizing enzyme glutamate decarboxylase (GAD 65/67). (G) Western blots of control (Con), NRG-treated and BDNF-treated cocultures for MBP (β-actin acts as a loading control), and densitometric quantification of subunit protein levels . (H) Change in myelin gene mRNA level (by qPCR) in NRG-treated cocultures normalized to control levels. All p values are from one-sample Student's t tests. Numbers of cultures are shown on bars.(EPS)Click here for additional data file.Figure S2Quantification of myelination. Each set of graphs shows the same data (from 2.(EPS)Click here for additional data file.Figure S3Myelination in various conditions quantified in different ways. Graphs show the myelination parameter for the data analysed for p = 0.72); for with NRG, ANOVA showed significant differences across bars (p<0.0001). (B) Threshold-linear relation with threshold of 17%. ANOVA gave p = 0.77 for no NRG and p<0.0001 for with NRG. (C) Power 2 relation. ANOVA gave p = 0.76 for no NRG and p<0.0001 for with NRG. (D) Fraction of oligodendrocytes myelinating, averaged across all images. ANOVA gave p = 0.096 for no NRG and p<0.0001 for with NRG. For all panels the p values, comparing data in no NRG with control, and comparing data in with NRG with NRG alone, were from Holm–Bonferroni tests.(EPS)Click here for additional data file.Figure S4Electrophysiological characteristics of the cells in the cocultures. Specimen responses to voltage steps in 20 mV increments from a holding potential of −64 mV  an OPC with voltage-gated Na+ current (NaV), (B) an OPC without NaV, (C) the OPC in (A) on a faster time scale, (D) the OPC in (B) on a faster time scale, (E) a mature oligodendrocyte, (F) an interneuron, (G) a DRG neuron, (H) a satellite cell, and (I) an astrocyte. In (F and G) transient currents generated by unclamped action potentials are visible.(EPS)Click here for additional data file.Figure S5Details of the signalling pathways. (A) Mean myelination parameter of cocultures treated with NRG with TTX either added at the same time as NRG (TTX) or 3 d later (TTX 3 d). ANOVA showed significant differences across all bars (p = 0.025); the p values above bars are for comparison with NRG alone. (B–C) Western blot of control (Con) and NRG-treated pure DRG cultures (cultured as for myelinating OPC-DRG cocultures), for phosphorylated  and total Akt and ERK, with the level of phosphorylated enzyme normalized to total enzyme in the bar charts below. The p values are from Student's t tests; numbers of experiments are shown on bars.(EPS)Click here for additional data file.