The endoplasmic reticulum: a sensor of cellular stress that modulates immune responses.

Many inflammatory and infectious diseases are characterized by the activation of signaling pathways steaming from the endoplasmic reticulum (ER). These pathways, primarily associated with loss of ER homeostasis, are emerging as key regulators of inflammation and infection. Recent advances shed light on the mechanisms linking ER-stress and immune responses.

Domain shuffling in a sensor protein contributed to the evolution of insect pathogenicity in plant-beneficial Pseudomonas protegens.

Pseudomonas protegens is a biocontrol rhizobacterium with a plant-beneficial and an insect pathogenic lifestyle, but it is not understood how the organism switches between the two states. Here, we focus on understanding the function and possible evolution of a molecular sensor that enables P. protegens to detect the insect environment and produce a potent insecticidal toxin specifically during insect infection but not on roots. By using quantitative single cell microscopy and mutant analysis, we provide evidence that the sensor histidine kinase FitF is a key regulator of insecticidal toxin production. Our experimental data and bioinformatic analyses indicate that FitF shares a sensing domain with DctB, a histidine kinase regulating carbon uptake in Proteobacteria. This suggested that FitF has acquired its specificity through domain shuffling from a common ancestor. We constructed a chimeric DctB-FitF protein and showed that it is indeed functional in regulating toxin expression in P. protegens. The shuffling event and subsequent adaptive modifications of the recruited sensor domain were critical for the microorganism to express its potent insect toxin in the observed host-specific manner. Inhibition of the FitF sensor during root colonization could explain the mechanism by which P. protegens differentiates between the plant and insect host. Our study establishes FitF of P. protegens as a prime model for molecular evolution of sensor proteins and bacterial pathogenicity.

A double layer model of the gas bubble/water interface

Zeta potential is a physico-chemical parameter of particular importance to describe sorption of contaminants at the surface of gas bubbles. Nevertheless, the interpretation of electrophoretic mobilities of gas bubbles is complex. This is due to the specific behavior of the gas at interface and to the excess of electrical charge at interface, which is responsible for surface conductivity. We developed a surface complexation model based on the presence of negative surface sites because the balance of accepting and donating hydrogen bonds is broken at interface. By considering protons adsorbed on these sites followed by a diffuse layer, the electrical potential at the head-end of the diffuse layer is computed and considered to be equal to the zeta potential. The predicted zeta potential values are in very good agreement with the experimental data of H-2 bubbles for a broad range of pH and NaCl concentrations. This implies that the shear plane is located at the head-end of the diffuse layer, contradicting the assumption of the presence of a stagnant diffuse layer at the gas/water interface. Our model also successfully predicts the surface tension of air bubbles in a KCl solution. (c) 2012 Elsevier Inc. All rights reserved.

Glucose sensing by the hepatoportal sensor is GLUT2-dependent: in vivo analysis in GLUT2-null mice.

In the preceding article, we demonstrated that activation of the hepatoportal glucose sensor led to a paradoxical development of hypoglycemia that was associated with increased glucose utilization by a subset of tissues. In this study, we tested whether GLUT2 plays a role in the portal glucose-sensing system that is similar to its involvement in pancreatic beta-cells. Awake RIPGLUT1 x GLUT2-/- and control mice were infused with glucose through the portal (Po-) or the femoral (Fe-) vein for 3 h at a rate equivalent to the endogenous glucose production rate. Blood glucose and plasma insulin concentrations were continuously monitored. Glucose turnover, glycolysis, and glycogen synthesis rates were determined by the 3H-glucose infusion technique. We showed that portal glucose infusion in RIPGLUT1 x GLUT24-/- mice did not induce the hypoglycemia observed in control mice but, in contrast, led to a transient hyperglycemic state followed by a return to normoglycemia; this glycemic pattern was similar to that observed in control Fe-mice and RIPGLUT1 x GLUT2-/- Fe-mice. Plasma insulin profiles during the infusion period were similar in control and RIPGLUT1 x GLUT2-/- Po- and Fe-mice. The lack of hypoglycemia development in RIPGLUT1 x GLUT2-/- mice was not due to the absence of GLUT2 in the liver. Indeed, reexpression by transgenesis of this transporter in hepatocytes did not restore the development of hypoglycemia after initiating portal vein glucose infusion. In the absence of GLUT2, glucose turnover increased in Po-mice to the same extent as that in RIPGLUT1 x GLUT2-/- or control Fe-mice. Finally, co-infusion of somatostatin with glucose prevented development of hypoglycemia in control Po-mice, but it did not affect the glycemia or insulinemia of RIPGLUT1 x GLUT2-/- Po-mice. Together, our data demonstrate that GLUT2 is required for the function of the hepatoportal glucose sensor and that somatostatin could inhibit the glucose signal by interfering with GLUT2-expressing sensing units.

Simultaneous determination of plasma levels of fluvoxamine and of the enantiomers of fluoxetine and norfluoxetine by gas chromatography-mass spectrometry.

A gas chromatographic-mass spectrometric method is presented which allows the simultaneous determination of the plasma concentrations of fluvoxamine and of the enantiomers of fluoxetine and norfluoxetine after derivatization with the chiral reagent, (S)-(-)-N-trifluoroacetylprolyl chloride. No interference was observed from endogenous compounds following the extraction of plasma samples from six different human subjects. The standard curves were linear over a working range of 10 to 750 ng/ml for racemic fluoxetine and norfluoxetine and of 50 to 500 ng/ml for fluvoxamine. Recoveries ranged from 50 to 66% for the three compounds. Intra- and inter-day coefficients of variation ranged from 4 to 10% for fluvoxamine and from 4 to 13% for fluoxetine and norfluoxetine. The limits of quantitation of the method were found to be 2 ng/ml for fluvoxamine and 1 ng/ml for the (R)- and (S)-enantiomers of fluoxetine and norfluoxetine, hence allowing its use for single dose pharmacokinetics. Finally, by using a steeper gradient of temperature, much shorter analysis times are obtained if one is interested in the concentrations of fluvoxamine alone.

The effect of vaccines based on ovalbumin coupled to gas-filled microbubbles for reducing infection by ovalbumin-expressing Listeria monocytogenes.

Gas-filled microbubbles (MB) are a very promising alternative to the currently evaluated lipid- or polymer-based particulate Ag delivery systems. We recently demonstrated the ability of MB to deliver associated Ag to DC, to activate them and thereby induce both humoral and cellular immune responses. We now extended the characterization of MB as antigen-delivery system by appraising the efficiency of MB-associated ovalbumin (OVA-MB) at protecting mice against pathogen infection. Ultrasound-mediated imaging demonstrated that the administration of OVA via MB generates a depot at the injection site that lasts for several hours. We found that OVA-MB injected subcutaneously is far more effective at inducing specific Ab and T cell immunity than immunization with free OVA. Moreover, a covalent link between MB and OVA causes a stronger bias towards a Th1-type of immune response than adsorption of the Ag or its covalent link to liposomes of the same lipid composition. Finally, vaccination of mice with OVA-MB partially protects against a systemic infection with OVA-expressing Listeria monocytogenes. The vaccine induces specific effector CD8 T cell responses capable of decreasing more than 100 fold the bacterial load. MB thus represent a potent Ag delivery system for vaccination against intracellular infectious agents.

Hyperpolarized lithium-6 as a sensor of nanomolar contrast agents.

Lithium is widely used in psychotherapy. The (6)Li isotope has a long intrinsic longitudinal relaxation time T(1) on the order of minutes, making it an ideal candidate for hyperpolarization experiments. In the present study we demonstrated that lithium-6 can be readily hyperpolarized within 30 min, while retaining a long polarization decay time on the order of a minute. We used the intrinsically long relaxation time for the detection of 500 nM contrast agent in vitro. Hyperpolarized lithium-6 was administered to the rat and its signal retained a decay time on the order of 70 sec in vivo. Localization experiments imply that the lithium signal originated from within the brain and that it was detectable up to 5 min after administration. We conclude that the detection of submicromolar contrast agents using hyperpolarized NMR nuclei such as (6)Li may provide a novel avenue for molecular imaging.

Indirect hydrogen monitoring by chemi-ionisation following an associative ionisation pathway after metastable helium atoms production by electron impact ionisation: Protonation and deuteration of carrier gas

Gas chromatography (GC) is an analytical tool very useful to investigate the composition of gaseous mixtures. However, hydrogen (H2) detection after a GC separation is only possible with a Thermal Conductivity Detector (TCD), a Helium Ionisation Detector (HID) or expensive Atomic Emission Detector (AED). Recently, indirect H2 detection by GC coupled to mass spectrometry (MS) was demonstrated but the mechanism of carrier gas protonation remained unclear. With electron impact as ionisation source of MS and helium (He) as GC carrier gas, H2 is not ionised according the expected Penning ionisation neither according to the Associative ionisation. Rearrangement ionisation (RI) was found to be the main channel for H2 and D2 ionisation under GC-MS conditions used in most of laboratories using GC-MS, leading to the formation of [He−H]+ and [He−D]+ ions.

Determination of plasma levels of citalopram and its demethylated and deaminated metabolites by gas chromatography and gas chromatography-mass spectrometry.

Sensitive and specific methods based on gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) for the determination of levels of citalopram, desmethylcitalopram and didesmethylcitalopram in the plasma of patients treated with citalopram are presented, as well as a GC-MS procedure for the assay of the citalopram propionic acid derivative. After addition of a separate internal standard for each drug, liquid-solvent extraction is used to separate the basic compounds from the acid compounds. The demethylated amines are derivatized with trifluoroacetic anhydride, and the acid metabolite with methyl iodide. GC-MS is performed in the electron impact mode, as mass spectrometry by the (positive-ion) chemical ionization mode (methane and ammonia) appeared to be unsuitable. The limits of quantification were 1 ng/ml for citalopram and desmethylcitalopram and 2 ng/ml for the other metabolites. The correlation coefficients for the calibration curves (range 10-500 ng/ml) were > or = 0.999 for all compounds, whether determined by GC or GC-MS.

A pilot assessment of alpha-stat vs pH-stat arterial blood gas analysis after cardiac arrest.

PURPOSE: Resuscitated cardiac arrest (CA) patients typically receive therapeutic hypothermia, but arterial blood gases (ABGs) are often assessed after adjustment to 37°C (alpha-stat) instead of actual body temperature (pH-stat). We sought to compare alpha-stat and pH-stat assessment of Pao2 and Paco2 in such patients. MATERIALS AND METHODS: Using ABG data obtained during the first 24 hours of intensive care unit admission, we determined the impact of measured alpha vs calculated pH-stat on Pao2 and Paco2 on patient classification and outcomes for CA patients. RESULTS: We assessed 1013 ABGs from 120 CA patients with a median age of patients 66 years (interquartile range, 50-76). Median alpha-stat Pao2 changed from 122 (95-156) to 107 (82-143) mm Hg with pH-stat and median Paco2 from 39 (34-46) to 35 (30-41) mm Hg (both P < .001). Using the categories of hyperoxemia, normoxemia, and hypoxemia, pH-stat estimation of Pao2 reclassified approximately 20% of patients. Using the categories of hypercapnia, normocapnia, and hypocapnia, pH stat estimation of Paco2 reclassified approximately 40% of patients. The mortality of patients in different Pao2 and Paco2 categories was similar for pH-stat and alpha-stat. CONCLUSIONS: Using the pH-stat method, fewer resuscitated CA patients admitted to intensive care unit were classified as hyperoxemic or hypercapnic compared with alpha-stat. These findings suggest an impact of ABG assessment methodology on Pao2, Paco2, and patient classification but not on associated outcomes.

Transcutaneous carbon dioxide and oxygen tension in newborn infants: reliability of a combined monitor of oxygen tension and carbon dioxide tension.

We evaluated a new combined sensor for monitoring transcutaneous carbon dioxide tension (PtcCO2) and oxygen tension (PtcO2) in 20 critically ill newborn infants. Arterial oxygen tension (PaO2) ranged from 16 to 126 torr and arterial carbon dioxide tension (PaCO2) from 14 to 72 torr. Linear correlation analysis (100 paired values) of PtcO2 versus PaO2 showed an r value of 0.75 with a regression equation of PtcO2 = 8.59 + 0.905 (PaO2), while PtcCO2 versus PaCO2 revealed a correlation coefficient of r = 0.89 with an equation of PtcCO2 = 2.53 + 1.06 (PaCO2). The bias between PaO2 and PtcO2 was -2.8 with a precision of +/- 16.0 torr (range, -87 to +48 torr). The bias between PaCO2 and PtcCO2 was -5.1 with a precision of +/- 7.3 torr (range, -34 to +8 torr). The transcutaneous sensor detected 83% of hypoxia (PaO2 less than 45 torr), 75% of hyperoxia (PaO2 greater than 90 torr), 45% of hypocapnia (PaCO2 less than 35 torr), and 96% of hypercapnia (PaCO2 greater than 45 torr). We conclude that the reliability of the combined transcutaneous PO2 and PCO2 monitor in sick neonates is good for detecting hypercapnia, fair for hypoxia and hyperoxia, but poor for hypocapnia. It is an improvement in that it spares available skin surface and requires less handling, but it appears to be slightly less accurate than the single electrodes.

Hydrogen sulfide measurement by headspace-gas chromatography-mass spectrometry (HS-GC-MS): application to gaseous samples and gas dissolved in muscle.

The aim of our study was to present a new headspace-gas chromatography-mass spectrometry (HS-GC-MS) method applicable to the routine determination of hydrogen sulfide (H(2)S) concentrations in biological and gaseous samples. The primary analytical drawback of the GC/MS methods for H(2)S measurement discussed in the literature was the absence of a specific H(2)S internal standard required to perform quantification. Although a deuterated hydrogen sulfide (D(2)S) standard is currently available, this standard is not often used because this standard is expensive and is only available in the gas phase. As an alternative approach, D(2)S can be generated in situ by reacting deuterated chloride with sodium sulfide; however, this technique can lead to low recovery yield and potential isotopic fractionation. Therefore, N(2)O was chosen for use as an internal standard. This method allows precise measurements of H(2)S concentrations in biological and gaseous samples. Therefore, a full validation using accuracy profile based on the β-expectation tolerance interval is presented. Finally, this method was applied to quantify H(2)S in an actual case of H(2)S fatal intoxication.