A quorum sensing small volatile molecule promotes antibiotic tolerance in bacteria.

Bacteria can be refractory to antibiotics due to a sub-population of dormant cells, called persisters that are highly tolerant to antibiotic exposure. The low frequency and transience of the antibiotic tolerant "persister" trait has complicated elucidation of the mechanism that controls antibiotic tolerance. In this study, we show that 2' Amino-acetophenone (2-AA), a poorly studied but diagnostically important small, volatile molecule produced by the recalcitrant gram-negative human pathogen Pseudomonas aeruginosa, promotes antibiotic tolerance in response to quorum-sensing (QS) signaling. Our results show that 2-AA mediated persister cell accumulation occurs via alteration of the expression of genes involved in the translational capacity of the cell, including almost all ribosomal protein genes and other translation-related factors. That 2-AA promotes persisters formation also in other emerging multi-drug resistant pathogens, including the non 2-AA producer Acinetobacter baumannii implies that 2-AA may play an important role in the ability of gram-negative bacteria to tolerate antibiotic treatments in polymicrobial infections. Given that the synthesis, excretion and uptake of QS small molecules is a common hallmark of prokaryotes, together with the fact that the translational machinery is highly conserved, we posit that modulation of the translational capacity of the cell via QS molecules, may be a general, widely distributed mechanism that promotes antibiotic tolerance among prokaryotes.

Oxygen-sensing reporter strain of Pseudomonas fluorescens for monitoring the distribution of low-oxygen habitats in soil.

The root-colonizing bacterium Pseudomonas fluorescens CHA0 was used to construct an oxygen-responsive biosensor. An anaerobically inducible promoter of Pseudomonas aeruginosa, which depends on the FNR (fumarate and nitrate reductase regulation)-like transcriptional regulator ANR (anaerobic regulation of arginine deiminase and nitrate reductase pathways), was fused to the structural lacZ gene of Escherichia coli. By inserting the reporter fusion into the chromosomal attTn7 site of P. fluorescens CHA0 by using a mini-Tn7 transposon, the reporter strain, CHA900, was obtained. Grown in glutamate-yeast extract medium in an oxystat at defined oxygen levels, the biosensor CHA900 responded to a decrease in oxygen concentration from 210 x 10(2) Pa to 2 x 10(2) Pa of O(2) by a nearly 100-fold increase in beta-galactosidase activity. Half-maximal induction of the reporter occurred at about 5 x 10(2) Pa. This dose response closely resembles that found for E. coli promoters which are activated by the FNR protein. In a carbon-free buffer or in bulk soil, the biosensor CHA900 still responded to a decrease in oxygen concentration, although here induction was about 10 times lower and the low oxygen response was gradually lost within 3 days. Introduced into a barley-soil microcosm, the biosensor could report decreasing oxygen concentrations in the rhizosphere for a 6-day period. When the water content in the microcosm was raised from 60% to 85% of field capacity, expression of the reporter gene was elevated about twofold above a basal level after 2 days of incubation, suggesting that a water content of 85% caused mild anoxia. Increased compaction of the soil was shown to have a faster and more dramatic effect on the expression of the oxygen reporter than soil water content alone, indicating that factors other than the water-filled pore space influenced the oxygen status of the soil. These experiments illustrate the utility of the biosensor for detecting low oxygen concentrations in the rhizosphere and other soil habitats.

Aldosterone paradox: differential regulation of ion transport in distal nephron.

The mechanisms through which aldosterone promotes apparently opposite effects like salt reabsorption and K(+) secretion remain poorly understood. The identification, localization, and physiological analysis of ion transport systems in distal nephron have revealed an intricate network of interactions between several players, revealing the complex mechanism behind the aldosterone paradox. We review the mechanisms involved in differential regulation of ion transport that allow the fine tuning of salt and K(+) balance.

Messenger RNA expression of transporter and ion channel genes in undifferentiated and differentiated Caco-2 cells compared to human intestines.

PURPOSE: The purpose of this work was to study the influence of cell differentiation on the mRNA expression of transporters and channels in Caco-2 cells and to assess Caco-2 cells as a model for carrier-mediated drug transport in the intestines. METHOD: Gene mRNA expression was measured using a custom-designed microarray chip with 750 deoxyoligonucleotide probes (70mers). Each oligomer was printed four times on poly-lysine-coated glass slides. Expression profiles were expressed as ratio values between fluorescence intensities of Cy3 and Cy5 dye-labeled cDNA derived from poly(A) + RNA samples of Caco-2 cells and total RNA of human intestines. RESULTS: Significant differences in the mRNA expression profile of transporters and channels were observed upon differentiation of Caco-2 cells from 5 days to 2 weeks in culture, including changes for MAT8, S-protein, and Nramp2. Comparing Caco-2 cells of different passage number revealed few changes in mRNAs except for GLUT3, which was down-regulated 2.4-fold within 13 passage numbers. Caco-2 cells had a similar expression profile when either cultured in flasks or on filters but differed more strongly from human small and large intestine, regardless of the differentiation state of Caco-2 cells. Expression of several genes highly transcribed in small or large intestines differed fourfold or more in Caco-2 cells. CONCLUSIONS: Although Caco-2 cells have proven a suitable model for studying carrier-mediated transport in human intestines, the expression of specific transporter and ion channel genes may differ substantially.

Glutamatergic regulation of mitochondrial ion dynamics in astrocytes

Résumé grand public :Le cerveau se compose de cellules nerveuses appelées neurones et de cellules gliales dont font partie les astrocytes. Les neurones communiquent entre eux par signaux électriques et en libérant des molécules de signalisation comme le glutamate. Les astrocytes ont eux pour charge de capter le glucose depuis le sang circulant dans les vaisseaux sanguins, de le transformer et de le transmettre aux neurones pour qu'ils puissent l'utiliser comme source d'énergie. L'astrocyte peut ensuite utiliser ce glucose de deux façons différentes pour produire de l'énergie : la première s'opère dans des structures appelées mitochondries qui sont capables de produire plus de trente molécules riches en énergie (ATP) à partir d'une seule molécule de glucose ; la seconde possibilité appelée glycolyse peut produire deux molécules d'ATP et un dérivé du glucose appelé lactate. Une théorie couramment débattue propose que lorsque les astrocytes capturent le glutamate libéré par les neurones, ils libèrent en réponse du lactate qui servirait de base énergétique aux neurones. Cependant, ce mécanisme n'envisage pas une augmentation de l'activité des mitochondries des astrocytes, ce qui serait pourtant bien plus efficace pour produire de l'énergie.En utilisant la microscopie par fluorescence, nous avons pu mesurer les changements de concentrations ioniques dans les mitochondries d'astrocytes soumis à une stimulation glutamatergique. Nous avons démontré que les mitochondries des astrocytes manifestent des augmentations spontanées et transitoires de leur concentrations ioniques, dont la fréquence était diminuée au cours d'une stimulation avec du glutamate. Nous avons ensuite montré que la capture de glutamate augmentait la concentration en sodium et acidifiait les mitochondries des astrocytes. En approfondissant ces mécanismes, plusieurs éléments ont suggéré que l'acidification induite diminuerait le potentiel de synthèse d'énergie d'origine mitochondriale et la consommation d'oxygène dans les astrocytes. En résumé, l'ensemble de ces travaux suggère que la signalisation neuronale impliquant le glutamate dicte aux astrocytes de sacrifier temporairement l'efficacité de leur métabolisme énergétique, en diminuant l'activité de leurs mitochondries, afin d'augmenter la disponibilité des ressources énergétiques utiles aux neurones.Résumé :La remarquable efficacité du cerveau à compiler et propager des informations coûte au corps humain 20% de son budget énergétique total. Par conséquent, les mécanismes cellulaires responsables du métabolisme énergétique cérébral se sont adéquatement développés pour répondre aux besoins énergétiques du cerveau. Les dernières découvertes en neuroénergétique tendent à démontrer que le site principal de consommation d'énergie dans le cerveau est situé dans les processus astrocytaires qui entourent les synapses excitatrices. Un nombre croissant de preuves scientifiques a maintenant montré que le transport astrocytaire de glutamate est responsable d'un coût métabolique important qui est majoritairement pris en charge par une augmentation de l'activité glycolytique. Cependant, les astrocytes possèdent également un important métabolisme énergétique de type mitochondrial. Par conséquent, la localisation spatiale des mitochondries à proximité des transporteurs de glutamate suggère l'existence d'un mécanisme régulant le métabolisme énergétique astrocytaire, en particulier le métabolisme mitochondrial.Afin de fournir une explication à ce paradoxe énergétique, nous avons utilisé des techniques d'imagerie par fluorescence pour mesurer les modifications de concentrations ioniques spontanées et évoquées par une stimulation glutamatergique dans des astrocytes corticaux de souris. Nous avons montré que les mitochondries d'astrocytes au repos manifestaient des changements individuels, spontanés et sélectifs de leur potentiel électrique, de leur pH et de leur concentration en sodium. Nous avons trouvé que le glutamate diminuait la fréquence des augmentations spontanées de sodium en diminuant le niveau cellulaire d'ATP. Nous avons ensuite étudié la possibilité d'une régulation du métabolisme mitochondrial astrocytaire par le glutamate. Nous avons montré que le glutamate initie dans la population mitochondriale une augmentation rapide de la concentration en sodium due à l'augmentation cytosolique de sodium. Nous avons également montré que le relâchement neuronal de glutamate induit une acidification mitochondriale dans les astrocytes. Nos résultats ont indiqué que l'acidification induite par le glutamate induit une diminution de la production de radicaux libres et de la consommation d'oxygène par les astrocytes. Ces études ont montré que les mitochondries des astrocytes sont régulées individuellement et adaptent leur activité selon l'environnement intracellulaire. L'adaptation dynamique du métabolisme énergétique mitochondrial opéré par le glutamate permet d'augmenter la quantité d'oxygène disponible et amène au relâchement de lactate, tous deux bénéfiques pour les neurones.Abstract :The remarkable efficiency of the brain to compute and communicate information costs the body 20% of its total energy budget. Therefore, the cellular mechanisms responsible for brain energy metabolism developed adequately to face the energy needs. Recent advances in neuroenergetics tend to indicate that the main site of energy consumption in the brain is the astroglial process ensheating activated excitatory synapses. A large body of evidence has now shown that glutamate uptake by astrocytes surrounding synapses is responsible for a significant metabolic cost, whose metabolic response is apparently mainly glycolytic. However, astrocytes have also a significant mitochondrial oxidative metabolism. Therefore, the location of mitochondria close to glutamate transporters raises the question of the existence of mechanisms for tuning their energy metabolism, in particular their mitochondrial metabolism.To tackle these issues, we used real time imaging techniques to study mitochondrial ionic alterations occurring at resting state and during glutamatergic stimulation of mouse cortical astrocytes. We showed that mitochondria of intact resting astrocytes exhibited individual spontaneous and selective alterations of their electrical potential, pH and Na+ concentration. We found that glutamate decreased the frequency of mitochondrial Na+ transient activity by decreasing the cellular level of ATP. We then investigated a possible link between glutamatergic transmission and mitochondrial metabolism in astrocytes. We showed that glutamate triggered a rapid Na+ concentration increase in the mitochondrial population as a result of plasma-membrane Na+-dependent uptake. We then demonstrated that neuronally released glutamate also induced a mitochondrial acidification in astrocytes. Glutamate induced a pH-mediated and cytoprotective decrease of mitochondrial metabolism that diminished oxygen consumption. Taken together, these studies showed that astrocytes contain mitochondria that are individually regulated and sense the intracellular environment to modulate their own activity. The dynamic regulation of astrocyte mitochondrial energy output operated by glutamate allows increasing oxygen availability and lactate production both being beneficial for neurons.

Compressed Sensing Single-Breath-Hold CMR for Fast Quantification of LV Function, Volumes, and Mass.

OBJECTIVES: The purpose of this study was to compare a novel compressed sensing (CS)-based single-breath-hold multislice magnetic resonance cine technique with the standard multi-breath-hold technique for the assessment of left ventricular (LV) volumes and function. BACKGROUND: Cardiac magnetic resonance is generally accepted as the gold standard for LV volume and function assessment. LV function is 1 of the most important cardiac parameters for diagnosis and the monitoring of treatment effects. Recently, CS techniques have emerged as a means to accelerate data acquisition. METHODS: The prototype CS cine sequence acquires 3 long-axis and 4 short-axis cine loops in 1 single breath-hold (temporal/spatial resolution: 30 ms/1.5 × 1.5 mm(2); acceleration factor 11.0) to measure left ventricular ejection fraction (LVEFCS) as well as LV volumes and LV mass using LV model-based 4D software. For comparison, a conventional stack of multi-breath-hold cine images was acquired (temporal/spatial resolution 40 ms/1.2 × 1.6 mm(2)). As a reference for the left ventricular stroke volume (LVSV), aortic flow was measured by phase-contrast acquisition. RESULTS: In 94% of the 33 participants (12 volunteers: mean age 33 ± 7 years; 21 patients: mean age 63 ± 13 years with different LV pathologies), the image quality of the CS acquisitions was excellent. LVEFCS and LVEFstandard were similar (48.5 ± 15.9% vs. 49.8 ± 15.8%; p = 0.11; r = 0.96; slope 0.97; p < 0.00001). Agreement of LVSVCS with aortic flow was superior to that of LVSVstandard (overestimation vs. aortic flow: 5.6 ± 6.5 ml vs. 16.2 ± 11.7 ml, respectively; p = 0.012) with less variability (r = 0.91; p < 0.00001 for the CS technique vs. r = 0.71; p < 0.01 for the standard technique). The intraobserver and interobserver agreement for all CS parameters was good (slopes 0.93 to 1.06; r = 0.90 to 0.99). CONCLUSIONS: The results demonstrated the feasibility of applying the CS strategy to evaluate LV function and volumes with high accuracy in patients. The single-breath-hold CS strategy has the potential to replace the multi-breath-hold standard cardiac magnetic resonance technique.

GLUT2, glucose sensing and glucose homeostasis.

The glucose transporter isoform GLUT2 is expressed in liver, intestine, kidney and pancreatic islet beta cells, as well as in the central nervous system, in neurons, astrocytes and tanycytes. Physiological studies of genetically modified mice have revealed a role for GLUT2 in several regulatory mechanisms. In pancreatic beta cells, GLUT2 is required for glucose-stimulated insulin secretion. In hepatocytes, suppression of GLUT2 expression revealed the existence of an unsuspected glucose output pathway that may depend on a membrane traffic-dependent mechanism. GLUT2 expression is nevertheless required for the physiological control of glucose-sensitive genes, and its inactivation in the liver leads to impaired glucose-stimulated insulin secretion, revealing a liver-beta cell axis, which is likely to be dependent on bile acids controlling beta cell secretion capacity. In the nervous system, GLUT2-dependent glucose sensing controls feeding, thermoregulation and pancreatic islet cell mass and function, as well as sympathetic and parasympathetic activities. Electrophysiological and optogenetic techniques established that Glut2 (also known as Slc2a2)-expressing neurons of the nucleus tractus solitarius can be activated by hypoglycaemia to stimulate glucagon secretion. In humans, inactivating mutations in GLUT2 cause Fanconi-Bickel syndrome, which is characterised by hepatomegaly and kidney disease; defects in insulin secretion are rare in adult patients, but GLUT2 mutations cause transient neonatal diabetes. Genome-wide association studies have reported that GLUT2 variants increase the risks of fasting hyperglycaemia, transition to type 2 diabetes, hypercholesterolaemia and cardiovascular diseases. Individuals with a missense mutation in GLUT2 show preference for sugar-containing foods. We will discuss how studies in mice help interpret the role of GLUT2 in human physiology.

Sensing of glucose in the brain.

The brain, and in particular the hypothalamus and brainstem, have been recognized for decades as important centers for the homeostatic control of feeding, energy expenditure, and glucose homeostasis. These structures contain neurons and neuronal circuits that may be directly or indirectly activated or inhibited by glucose, lipids, or amino acids. The detection by neurons of these nutrient cues may become deregulated, and possibly cause metabolic diseases such as obesity and diabetes. Thus, there is a major interest in identifying these neurons, how they respond to nutrients, the neuronal circuits they form, and the physiological function they control. Here I will review some aspects of glucose sensing by the brain. The brain is responsive to both hyperglycemia and hypoglycemia, and the glucose sensing cells involved are distributed in several anatomical sites that are connected to each other. These eventually control the activity of the sympathetic or parasympathetic nervous system, which regulates the function of peripheral organs such as liver, white and brown fat, muscle, and pancreatic islets alpha and beta cells. There is now evidence for an extreme diversity in the sensing mechanisms used, and these will be reviewed.

Effects of tetrodotoxin and ion replacements on the short-circuit current induced by Escherichiacoli heat stable enterotoxin across small intestine of the gerbil (Gerbillus cheesmani)

The effects of mucosally added Escherichia coli heat stable enterotoxin (STa 30 ng ml-1) on the basal short-circuit current (Isc in µA cm-2) across stripped and unstripped sheets of jejuna and ilea taken from fed, starved (4 days, water ad lib) and undernourished (50% control food intake for 21 days) gerbil (Gerbillus cheesmani) were investigated. The effect of neurotoxin tetrodotoxin (TTX 10 µM) and the effects of replacing chloride by gluconate or the effects of removing bicarbonate from bathing buffers on the maximum increase in Isc induced by STa were also investigated. The maximum increase in Isc which resulted from the addition of STa were significantly higher in jejuna and ilea taken from starved and undernourished gerbils when compared with the fed control both using stripped and unstripped sheets. In the two regions of the small intestine taken from fed and starved animals TTX reduced the maximum increase in Isc induced by STa across unstripped sheets only. Moreover in jejuna and ilea taken from undernourished gerbils TTX reduced significantly the maximum increase in Isc induced by STa across stripped and unstripped sheets. Replacing chloride by gluconate decreased the maximum increase in Isc induced by STa across jejuna and ilea taken from undernourished gerbils only. Removing bicarbonates from bathing buffer decreased the maximum increase in Isc across the jejuna and ilea taken from starved and undernourished gerbils.

The Quorum-Sensing Molecules Farnesol/Homoserine Lactone and Dodecanol Operate via Distinct Modes of Action in Candida albicans.

Living as a commensal, Candida albicans must adapt and respond to environmental cues generated by the mammalian host and by microbes comprising the natural flora. These signals have opposing effects on C. albicans, with host cues promoting the yeast-to-hyphal transition and bacteria-derived quorum-sensing molecules inhibiting hyphal development. Hyphal development is regulated through modulation of the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, and it has been postulated that quorum-sensing molecules can affect filamentation by inhibiting the cAMP pathway. Here, we show that both farnesol and 3-oxo-C(12)-homoserine lactone, a quorum-sensing molecule secreted by Pseudomonas aeruginosa, block hyphal development by affecting cAMP signaling; they both directly inhibited the activity of the Candida adenylyl cyclase, Cyr1p. In contrast, the 12-carbon alcohol dodecanol appeared to modulate hyphal development and the cAMP signaling pathway without directly affecting the activity of Cyr1p. Instead, we show that dodecanol exerted its effects through a mechanism involving the C. albicans hyphal repressor, Sfl1p. Deletion of SFL1 did not affect the response to farnesol but did interfere with the response to dodecanol. Therefore, quorum sensing in C. albicans is mediated via multiple mechanisms of action. Interestingly, our experiments raise the possibility that the Burkholderia cenocepacia diffusible signal factor, BDSF, also mediates its effects via Sfl1p, suggesting that dodecanol's mode of action, but not farnesol or 3-oxo-C(12)-homoserine lactone, may be used by other quorum-sensing molecules.

Distinct ASIC currents are expressed in rat putative nociceptors and are modulated by nerve injury.

The H(+)-gated acid-sensing ion channels (ASICs) are expressed in dorsal root ganglion (DRG) neurones. Studies with ASIC knockout mice indicated either a pro-nociceptive or a modulatory role of ASICs in pain sensation. We have investigated in freshly isolated rat DRG neurones whether neurones with different ASIC current properties exist, which may explain distinct cellular roles, and we have investigated ASIC regulation in an experimental model of neuropathic pain. Small-diameter DRG neurones expressed three different ASIC current types which were all preferentially expressed in putative nociceptors. Type 1 currents were mediated by ASIC1a homomultimers and characterized by steep pH dependence of current activation in the pH range 6.8-6.0. Type 3 currents were activated in a similar pH range as type 1, while type 2 currents were activated at pH < 6. When activated by acidification to pH 6.8 or 6.5, the probability of inducing action potentials correlated with the ASIC current density. Nerve injury induced differential regulation of ASIC subunit expression and selective changes in ASIC function in DRG neurones, suggesting a complex reorganization of ASICs during the development of neuropathic pain. In summary, we describe a basis for distinct cellular functions of different ASIC types in small-diameter DRG neurones.

Plasma membrane cyclic nucleotide gated calcium channels control land plant thermal sensing and acquired thermotolerance.

Typically at dawn on a hot summer day, land plants need precise molecular thermometers to sense harmless increments in the ambient temperature to induce a timely heat shock response (HSR) and accumulate protective heat shock proteins in anticipation of harmful temperatures at mid-day. Here, we found that the cyclic nucleotide gated calcium channel (CNGC) CNGCb gene from Physcomitrella patens and its Arabidopsis thaliana ortholog CNGC2, encode a component of cyclic nucleotide gated Ca(2+) channels that act as the primary thermosensors of land plant cells. Disruption of CNGCb or CNGC2 produced a hyper-thermosensitive phenotype, giving rise to an HSR and acquired thermotolerance at significantly milder heat-priming treatments than in wild-type plants. In an aequorin-expressing moss, CNGCb loss-of-function caused a hyper-thermoresponsive Ca(2+) influx and altered Ca(2+) signaling. Patch clamp recordings on moss protoplasts showed the presence of three distinct thermoresponsive Ca(2+) channels in wild-type cells. Deletion of CNGCb led to a total absence of one and increased the open probability of the remaining two thermoresponsive Ca(2+) channels. Thus, CNGC2 and CNGCb are expected to form heteromeric Ca(2+) channels with other related CNGCs. These channels in the plasma membrane respond to increments in the ambient temperature by triggering an optimal HSR, leading to the onset of plant acquired thermotolerance.