Functionally discrete fine roots differ in microbial assembly, microbial functional potential, and produced metabolites.

Traditionally, fine roots were grouped using arbitrary size categories, rarely capturing the heterogeneity in physiology, morphology and functionality among different fine root orders. Fine roots with different functional roles are rarely separated in microbiome-focused studies and may result in confounding microbial signals and host-filtering across different root microbiome compartments. Using a 26-year-old common garden, we sampled fine roots from four temperate tree species that varied in root morphology and sorted them into absorptive and transportive fine roots. The rhizoplane and rhizosphere were characterized using 16S rRNA gene and internal transcribed spacer region amplicon sequencing and shotgun metagenomics for the rhizoplane to identify potential microbial functions. Fine roots were subject to metabolomics to spatially characterize resource availability. Both fungi and bacteria differed according to root functional type. We observed additional differences between the bacterial rhizoplane and rhizosphere compartments for absorptive but not transportive fine roots. Rhizoplane bacteria, as well as the root metabolome and potential microbial functions, differed between absorptive and transportive fine roots, but not the rhizosphere bacteria. Functional differences were driven by sugar transport, peptidases and urea transport. Our data highlights the importance of root function when examining root-microbial relationships, emphasizing different host selective pressures imparted on different root microbiome compartments.

Resting cardiac sympathetic firing frequencies suppress terminal norepinephrine transporter uptake.

The prejunctional norepinephrine transporter (NET) is responsible for the clearance of released norepinephrine (NE) back into the sympathetic nerve terminal. NET regulation must be tightly controlled as variations could have important implications for neurotransmission. Thus far, the effects of sympathetic neuronal activity on NET function have been unclear. Here, we optically monitor single-terminal cardiac NET activity ex vivo in response to a broad range of sympathetic postganglionic action potential (AP) firing frequencies. Isolated murine left atrial appendages were loaded with a fluorescent NET substrate [Neurotransmitter Transporter Uptake Assay (NTUA)] and imaged with confocal microscopy. Sympathetic APs were induced with electrical field stimulation at 0.2-10 Hz (0.1-0.2 ms pulse width). Exogenous NE was applied during the NTUA uptake- and washout phases to investigate substrate competition and displacement, respectively, on transport. Single-terminal NET reuptake rate was rapidly suppressed in a frequency-dependent manner with an inhibitory EF50 of 0.9 Hz. At 2 Hz, the effect was reversed by the α2-adrenoceptor antagonist yohimbine (1 µM) (p < 0.01) with no further effect imposed by the muscarinic receptor antagonist atropine (1 µM). Additionally, high exogenous NE concentrations abolished NET reuptake (1 µM NE; p < 0.0001) and displaced terminal specific NTUA during washout (1-100 µM NE; p < 0.0001). We have also identified α2-adrenoceptor-induced suppression of NET reuptake rate during resting stimulation frequencies, which could oppose the effect of autoinhibition-mediated suppression of exocytosis and thus amplify the effects of sympathetic drive on cardiac function.

ß-Adrenoceptor blockade prevents carotid body hyperactivity and elevated vascular sympathetic nerve density induced by chronic intermittent hypoxia.

Carotid body (CB) hyperactivity promotes hypertension in response to chronic intermittent hypoxia (CIH). The plasma concentration of adrenaline is reported to be elevated in CIH and our previous work suggests that adrenaline directly activates the CB. However, a role for chronic adrenergic stimulation in mediating CB hyperactivity is currently unknown. This study evaluated whether beta-blocker treatment with propranolol (Prop) prevented the development of CB hyperactivity, vascular sympathetic nerve growth and hypertension caused by CIH. Adult male Wistar rats were assigned into 1 of 4 groups: Control (N), N + Prop, CIH and CIH + Prop. The CIH paradigm consisted of 8 cycles h-1, 8 h day-1, for 3 weeks. Propranolol was administered via drinking water to achieve a dose of 40 mg kg-1 day-1. Immunohistochemistry revealed the presence of both ß1 and ß2-adrenoceptor subtypes on the CB type I cell. CIH caused a 2-3-fold elevation in basal CB single-fibre chemoafferent activity and this was prevented by chronic propranolol treatment. Chemoafferent responses to hypoxia and mitochondrial inhibitors were attenuated by propranolol, an effect that was greater in CIH animals. Propranolol decreased respiratory frequency in normoxia and hypoxia in N and CIH. Propranolol also abolished the CIH mediated increase in vascular sympathetic nerve density. Arterial blood pressure was reduced in propranolol groups during hypoxia. Propranolol exaggerated the fall in blood pressure in most (6/7) CIH animals during hypoxia, suggestive of reduced sympathetic tone. These findings therefore identify new roles for ß-adrenergic stimulation in evoking CB hyperactivity, sympathetic vascular hyperinnervation and altered blood pressure control in response to CIH.

Dynamic monitoring of single-terminal norepinephrine transporter rate in the rodent cardiovascular system: A novel fluorescence imaging method.

Here, we validate the use of a novel fluorescent norepinephrine transporter (NET) substrate for dynamic measurements of transporter function in rodent cardiovascular tissue; this technique avoids the use of radiotracers and provides single-terminal resolution. Rodent (Wistar rats and C57BL/6 mice) hearts and mesenteric arteries (MA) were isolated, loaded with NET substrate Neurotransmitter Transporter Uptake Assay (NTUA) ex vivo and imaged with confocal microscopy. NTUA labelled noradrenergic nerve terminals in all four chambers of the heart and on the surface of MA. In all tissues, a temperature-dependent, stable linear increase in intra-terminal fluorescence upon NTUA exposure was observed; this was abolished by NET inhibitor desipramine (1 µM) and reversed by indirectly-acting sympathomimetic amine tyramine (10 µM). NET reuptake rates were similar across the mouse cardiac chambers. In both species, cardiac NET activity was significantly greater than in MA (by 62 ± 29% (mouse) and 21 ± 16% (rat)). We also show that mouse NET reuptake rate was twice as fast as that in the rat (for example, in the heart, by 94 ± 30%). Finally, NET reuptake rate in the mouse heart was attenuated with muscarinic agonist carbachol (10 µM) thus demonstrating the potential for parasympathetic regulation of norepinephrine clearance. Our data provide the first demonstration of monitoring intra-terminal NET function in rodent cardiovascular tissue. This straightforward method allows dynamic measurements of transporter rate in response to varying physiological conditions and drug treatments; this offers the potential to study new mechanisms of sympathetic dysfunction associated with cardiovascular disease.

Use of Participatory Action Research in the Development of a Survey of Physiotherapy Services for People with Multiple Sclerosis in Canada.

Purpose: Currently, there is a paucity of research describing physiotherapy services for individuals with multiple sclerosis (MS) in Canada. Using qualitative methods, we aimed to develop a survey to examine physiotherapy practice patterns for people with MS receiving services in Canada. Method: We began by conducting a review of the current literature and combining participatory action research methods with the expertise of registered physiotherapists and individuals with MS. Semi-structured interviews were conducted with 10 participants to obtain their input into survey development. The interviews were then transcribed verbatim and analyzed thematically. Results: Five key themes emerged from the thematic analysis: (1) provide additional answer options, (2) reformat or clarify questions, (3) ensure that questions or options are appropriate, (4) ensure good readability and flow, and (5) determine the appropriate length of the survey. After a final revision, the survey consisted of 24 items in the following domains: demographics, MS programme and patient population, interdisciplinary care, and programme and service barriers. Conclusions: This survey is the first of its kind in Canada and is the first step toward improving the quality of health of people living with MS and the effectiveness of current physiotherapy practices for them.

Objectif : très peu d'études portent sur les services de physiothérapie pour les personnes atteintes de sclérose en plaques (SP) au Canada. À l'aide de méthodes qualitatives, la présente étude visait à préparer un sondage sur les modes d'exercice de la physiothérapie pour la SP au Canada. Méthodologie : analyse des publications à jour et combinaison de méthodes de recherche-action participatives avec les compétences de physiothérapeutes diplômés et de personnes atteintes de SP. Les chercheurs ont réalisé des entrevues semi-structurées avec dix participants pour obtenir leur avis sur l'élaboration du sondage. Les entrevues ont ensuite été transcrites textuellement, puis analysées par thèmes. Résultats : cinq thèmes principaux ont émergé de l'analyse thématique : 1) fournir d'autres possibilités de réponses, 2) reformuler ou clarifier les questions, 3) s'assurer que les questions ou les options sont appropriées, 4) s'assurer d'une bonne lisibilité et d'un bon enchaînement et 5) déterminer la bonne longueur du sondage. Après la dernière révision, le sondage se composait de 24 points dans les domaines suivants : démographie, programme pour la SP et population de patients, soins interdisciplinaires et obstacles aux programmes et aux services. Conclusion : le sondage est le premier du genre au Canada et représente la première étape vers l'amélioration de la qualité de vie des personnes ayant la SP ainsi que de l'efficacité des pratiques actuelles de physiothérapie auprès d'elles.

Enhanced GABAergic actions resulting from the coapplication of the steroid 3α-hydroxy-5α-pregnane-11,20-dione (alfaxalone) with propofol or diazepam.

Many GABAergic drugs are in clinical use as anesthetics, sedatives, or anxiolytics. We have investigated the actions of the combinations of the neuroactive steroid 3α-hydroxy-5α-pregnane-11,20-dione (alfaxalone) with the intravenous anesthetic propofol or the benzodiazepine diazepam. The goal of the study was to determine whether coapplication of alfaxalone reduces the effective doses and concentrations of propofol and diazepam. Behavioral effects of alfaxalone, propofol, diazepam, and the combinations of the drugs were evaluated during a 30-min activity test in mice. Functional effects of the individual drugs and drug combinations were tested by measuring the decay times of spontaneous inhibitory postsynaptic currents in rat hippocampal neurons, and peak current responses from heterologously expressed concatemeric α1ß2γ2L GABAA receptors. Co-administration of alfaxalone increased the sedative actions of propofol and diazepam in mice. The combination of alfaxalone with propofol or diazepam increased the decay times of sIPSCs and shifted the concentration-response relationships for GABA-activated receptors to lower transmitter concentrations. We infer that alfaxalone acts as a co-agonist to enhance the GABAergic effects of propofol and diazepam. We propose that co-administration of alfaxalone, and possibly other neuroactive steroids, can be employed to reduce dosage requirements for propofol and diazepam.

Multiple Non-Equivalent Interfaces Mediate Direct Activation of GABAA Receptors by Propofol.

BACKGROUND: Propofol is a sedative agent that at clinical concentrations acts by allosterically activating or potentiating the γ-aminobutyric acid type A (GABAA) receptor. Mutational, modeling, and photolabeling studies with propofol and its analogues have identified potential interaction sites in the transmembrane domain of the receptor. At the &quot;+&quot; of the ß subunit, in the ß-α interface, meta-azipropofol labels the M286 residue in the third transmembrane domain. Substitution of this residue with tryptophan results in loss of potentiation by propofol. At the &quot;-&quot; side of the ß subunit, in the α-ß interface (or ß-ß interface, in the case of homomeric ß receptors), ortho-propofol diazirine labels the H267 residue in the second transmembrane domain. Structural modeling indicates that the ß(H267) residue lines a cavity that docks propofol with favorable interaction energy. METHOD: We used two-electrode voltage clamp to determine the functional effects of mutations to the "+" and "-" sides of the ß subunit on activation of the α1ß3 GABAA receptor by propofol. RESULTS: We found that while the individual mutations had a small effect, the combination of the M286W mutation with tryptophan mutations of selected residues at the α-ß interface leads to strong reduction in gating efficacy for propofol. CONCLUSION: We conclude that α1ß3 GABAA receptors can be activated by propofol interactions with the ß-ß, α-ß, and ß-α interfaces, where distinct, non-equivalent regions control channel gating. Any interface can mediate activation, hence substitutions at all interfaces are required for loss of activation by propofol.

Mutational Analysis of the Putative High-Affinity Propofol Binding Site in Human ß3 Homomeric GABAA Receptors.

Propofol is a sedative and anesthetic agent that can both activate GABA(A) receptors and potentiate receptor activation elicited by submaximal concentrations of the transmitter. A recent modeling study of the ß3 homomeric GABA(A) receptor postulated a high-affinity propofol binding site in a hydrophobic pocket in the middle of a triangular cleft lined by the M1 and M2 membrane-spanning domains of one subunit and the M2 domain of the neighboring subunit. The goal of the present study was to gain functional evidence for the involvement of this pocket in the actions of propofol. Human ß3 and α1ß3 receptors were expressed in Xenopus oocytes, and the effects of substitutions of selected residues were probed on channel activation by propofol and pentobarbital. The data demonstrate the vital role of the ß3(Y143), ß3(F221), ß3(Q224), and ß3(T266) residues in the actions of propofol but not pentobarbital in ß3 receptors. The effects of ß3(Y143W) and ß3(Q224W) on activation by propofol are likely steric because propofol analogs with less bulky ortho substituents activated both wild-type and mutant receptors. The T266W mutation removed activation by propofol in ß3 homomeric receptors; however, this mutation alone or in combination with a homologous mutation (I271W) in the α1 subunit had almost no effect on activation properties in α1ß3 heteromeric receptors. We hypothesize that heteromeric α1ß3 receptors can be activated by propofol interactions with ß3-ß3, α1-ß3, and ß3-α1 interfaces, but the exact locations of the binding site and/or nature of interactions vary in different classes of interfaces.

Protein modulator of multidrug efflux gene expression in Pseudomonas aeruginosa.

nalC multidrug-resistant mutants of Pseudomonas aeruginosa show enhanced expression of the mexAB-oprM multidrug efflux system as a direct result of the production of a ca. 6,100-Da protein, PA3719, in these mutants. Using a bacterial two-hybrid system, PA3719 was shown to interact in vivo with MexR, a repressor of mexAB-oprM expression. Isothermal titration calorimetry (ITC) studies confirmed a high-affinity interaction (equilibrium dissociation constant [K(D)], 158.0 +/- 18.1 nM) of PA3719 with MexR in vitro. PA3719 binding to and formation of a complex with MexR obviated repressor binding to its operator, which overlaps the efflux operon promoter, suggesting that mexAB-oprM hyperexpression in nalC mutants results from PA3719 modulation of MexR repressor activity. Consistent with this, MexR repression of mexA transcription in an in vitro transcription assay was alleviated by PA3719. Mutations in MexR compromising its interaction with PA3719 in vivo were isolated and shown to be located internally and distributed throughout the protein, suggesting that they impacted PA3719 binding by altering MexR structure or conformation rather than by having residues interacting specifically with PA3719. Four of six mutant MexR proteins studied retained repressor activity even in a nalC strain producing PA3719. Again, this is consistent with a PA3719 interaction with MexR being necessary to obviate MexR repressor activity. The gene encoding PA3719 has thus been renamed armR (antirepressor for MexR). A representative "noninteracting" mutant MexR protein, MexR(I104F), was purified, and ITC confirmed that it bound PA3719 with reduced affinity (5.4-fold reduced; K(D), 853.2 +/- 151.1 nM). Consistent with this, MexR(I104F) repressor activity, as assessed using the in vitro transcription assay, was only weakly compromised by PA3719. Finally, two mutations (L36P and W45A) in ArmR compromising its interaction with MexR have been isolated and mapped to a putative C-terminal alpha-helix of the protein that alone is sufficient for interaction with MexR.

nalD encodes a second repressor of the mexAB-oprM multidrug efflux operon of Pseudomonas aeruginosa.

The Pseudomonas aeruginosa nalD gene encodes a TetR family repressor with homology to the SmeT and TtgR repressors of the smeDEF and ttgABC multidrug efflux systems of Stenotrophomonas maltophilia and Pseudomonas putida, respectively. A sequence upstream of mexAB-oprM and overlapping a second promoter for this efflux system was very similar to the SmeT and TtgR operator sequences, and NalD binding to this region was, in fact, demonstrated. Moreover, increased expression from this promoter was seen in a nalD mutant, consistent with NalD directly controlling mexAB-oprM expression from a second promoter.

Mutations in PA3574 (nalD) lead to increased MexAB-OprM expression and multidrug resistance in laboratory and clinical isolates of Pseudomonas aeruginosa.

Mutations in genes mexR and nalC have previously been shown to drive overexpression of the MexAB-OprM multidrug efflux system in Pseudomonas aeruginosa. A transposon insertion multidrug-resistant mutant of P. aeruginosa overproducing MexAB-OprM was disrupted in yet a third gene, PA3574, encoding a probable repressor of the TetR/AcrR family that we have dubbed NalD. Clinical strains overexpressing MexAB-OprM but lacking mutations in mexR or nalC were also shown to carry mutations in nalD. Moreover, the cloned nalD gene reduced the multidrug resistance and MexAB-OprM expression of the transposon mutant and clinical isolates, highlighting the significance of the nalD mutations vis-a-vis MexAB-OprM overexpression in these isolates.

MexAB-OprM hyperexpression in NalC-type multidrug-resistant Pseudomonas aeruginosa: identification and characterization of the nalC gene encoding a repressor of PA3720-PA3719.

MexAB-OprM is a multidrug efflux system that contributes to intrinsic and acquired multidrug resistance in Pseudomonas aeruginosa, the latter as a result of mutational hyperexpression of the mexAB-oprM operon. While efflux gene hyperexpression typically results from mutations in the linked mexR repressor gene, it also occurs independently of mexR mutations in so-called nalC mutants that demonstrate more modest mexAB-oprM expression and, thus, more modest multidrug resistance than do mexR strains. Using a transposon insertion mutagenesis approach, nalC mutant strains were selected and the disrupted gene, PA3721, identified. Amplification and sequencing of this gene from previously isolated spontaneous nalC mutants revealed the presence of mutations in all instances and as such, PA3721 has been renamed nalC. PA3721 (nalC) encodes a probable repressor of the TetR/AcrR family and occurs upstream of an apparent two-gene operon, PA3720-PA3719, whose expression was negatively regulated by PA3721. Thus, PA3720-PA3719 was hyperexpressed in transposon insertion and spontaneous nalC mutants. The loss of PA3719 but not of PA3720 expression in a spontaneous nalC mutant reduced MexAB-OprM expression to wild-type levels and compromised multidrug resistance, an indication that hyperexpression of PA3719 only was necessary for the nalC phenotype. Introduction of PA3719 into wild-type P. aeruginosa on a multicopy plasmid was, in fact, sufficient to promote elevated MexAB-OprM expression and multidrug resistance characteristic of a nalC strain. Thus, the nalC (PA3721) mutation serves only to enhance PA3720-PA3719 expression, with expression of PA3719 (encodes a 53 amino acid protein of predicted pI 10.4) directly or indirectly impacting MexAB-OprM expression. Intriguingly, nalC strains produce markedly elevated levels of stable MexR protein suggesting that PA3720-PA3719 hyperexpression somehow modulates MexR repressor activity. The deduced products of PA3720-PA3719 show no homology to sequences presently in the GenBank databases, however, and as such provide no clues as to how this might occur.

Rapid uptake of oxidized ascorbate induces loss of cellular glutathione and oxidative stress in liver slices.

The observation that ascorbate known to retain pro-oxidant properties induces cell death in a number of immortal cell lines, led us to examine its mechanism and whether it is involved in oxidative stress injury in such asocorbate-enriched tissue cells as hepatocytes. In rat liver homogenates, higher concentrations (1 and 3 mM) of ascorbate suppressed lipid peroxide productions but lower concentrations (0.1 and 0.3 mM) did not. In contrast to the homogenate, ascorbate increased lipid peroxide production in liver slices in a concentration dependant manner. Iso-ascorbate, the epimer of ascorbate did not cause an increase the oxidative stress in liver slices. This differential effect between homogenates and liver slices implies that cellular integrity is required for ascorbate to induce oxidative stress. Wortmannin, an inhibitor of the GLUT (glucose transporter) thought to transport dehydroascorbate into cells, inhibited [(14)C]-ascorbate uptake and suppressed oxidative stress in liver slices. Wortmannin suppressed that [(14)C]-ascorbate uptake by GLUT following oxidation to [(14)C]dehydroascorbate. Taken together, these observations support our hypothesis that ascorbate is oxidized to dehydroascorbate by molecular oxygen in solution (i.e., plasma and culture medium) which is then carried into hepatocytes (via a GLUT) where it is reduced back to ascorbate causing oxidative stress.