Relative protein quantification by isobaric SILAC with immonium ion splitting (ISIS)

Metabolic labeling techniques have recently become popular tools for the quantitative profiling of proteomes. Classical stable isotope labeling with amino acids in cell cultures (SILAC) uses pairs of heavy/light isotopic forms of amino acids to introduce predictable mass differences in protein samples to be compared. After proteolysis, pairs of cognate precursor peptides can be correlated, and their intensities can be used for mass spectrometry-based relative protein quantification. We present an alternative SILAC approach by which two cell cultures are grown in media containing isobaric forms of amino acids, labeled either with 13C on the carbonyl (C-1) carbon or 15N on backbone nitrogen. Labeled peptides from both samples have the same nominal mass and nearly identical MS/MS spectra but generate upon fragmentation distinct immonium ions separated by 1 amu. When labeled protein samples are mixed, the intensities of these immonium ions can be used for the relative quantification of the parent proteins. We validated the labeling of cellular proteins with valine, isoleucine, and leucine with coverage of 97% of all tryptic peptides. We improved the sensitivity for the detection of the quantification ions on a pulsing instrument by using a specific fast scan event. The analysis of a protein mixture with a known heavy/light ratio showed reliable quantification. Finally the application of the technique to the analysis of two melanoma cell lines yielded quantitative data consistent with those obtained by a classical two-dimensional DIGE analysis of the same samples. Our method combines the features of the SILAC technique with the advantages of isobaric labeling schemes like iTRAQ. We discuss advantages and disadvantages of isobaric SILAC with immonium ion splitting as well as possible ways to improve it

Precursor ion scans for the targeted detection of stable-isotope-labeled peptides.

Stable isotope labels are routinely introduced into proteomes for quantification purposes. Full labeling of cells in varying biological states, followed by sample mixing, fractionation and intensive data acquisition, is used to obtain accurate large-scale quantification of total protein levels. However, biological processes often affect only a small group of proteins for a short time, resulting in changes that are difficult to detect against the total proteome background. An alternative approach could be the targeted analysis of the proteins synthesized in response to a given biological stimulus. Such proteins can be pulse-labeled with a stable isotope by metabolic incorporation of 'heavy' amino acids. In this study we investigated the specific detection and identification of labeled proteins using acquisition methods based on Precursor Ion Scans (PIS) on a triple-quadrupole ion trap mass spectrometer. PIS-based methods were set to detect unique immonium ions originating from labeled peptides. Different labels and methods were tested in standard mixtures to optimize performance. We showed that, in comparison with an untargeted analysis on the same instrument, the approach allowed a several-fold increase in the specificity of detection of labeled proteins over unlabeled ones. The technique was applied to the identification of proteins secreted by human cells into growth media containing bovine serum proteins, allowing the preferential detection of labeled cellular proteins over unlabeled bovine ones. However, compared with untargeted acquisitions on two different instruments, the PIS-based strategy showed some limitations in sensitivity. We discuss possible perspectives of the technique.

Determination of radionuclide levels in rainwater using ion exchange resin and gamma-spectrometry.

The evaluation of radioactivity accidentally released into the atmosphere involves determining the radioactivity levels of rainwater samples. Rainwater scavenges atmospheric airborne radioactivity in such a way that surface contamination can be deduced from rainfall rate and rainwater radioactivity content. For this purpose, rainwater is usually collected in large surface collectors and then measured by gamma-spectrometry after such treatments as evaporation or iron hydroxide precipitation. We found that collectors can be adapted to accept large surface (diameter 47mm) cartridges containing a strongly acidic resin (Dowex AG 88) which is able to quantitatively extract radioactivity from rainwater, even during heavy rainfall. The resin can then be measured by gamma-spectrometry. The detection limit is 0.1Bq per sample of resin (80g) for (137)Cs. Natural (7)Be and (210)Pb can also be measured and the activity ratio of both radionuclides is comparable with those obtained through iron hydroxide precipitation and air filter measurements. Occasionally (22)Na has also been measured above the detection limit. A comparison between the evaporation method and the resin method demonstrated that 2/3 of (7)Be can be lost during the evaporation process. The resin method is simple and highly efficient at extracting radioactivity. Because of these great advantages, we anticipate it could replace former rainwater determination methods. Moreover, it does not necessitate the transportation of large rainwater volumes to the laboratory.

Fitness to drive and cannabis: Validation of two blood THCCOOH thresholds to distinguish occasional users from heavy smokers.

Many studies based on either an experimental or an epidemiological approach, have shown that the ability to drive is impaired when the driver is under the influence of cannabis. Baseline performances of heavy users remain impaired even after several weeks of abstinence. Symptoms of cannabis abuse and dependence are generally considered incompatible with safe driving. Recently, it has been shown that traffic safety can be increased by reporting the long-term unfit drivers to the driver licensing authorities and referring the cases for further medical assessment. Evaluation of the frequency of cannabis use is a prerequisite for a reliable medical assessment of the fitness to drive. In a previous paper we advocated the use of two thresholds based on 11-nor-9-carboxy-Δ9-tetrahydrocannabinol (THCCOOH) concentration in whole blood to help to distinguish occasional cannabis users (≤3μg/L) from heavy regular smokers (≥40μg/L). These criteria were established on the basis of results obtained in a controlled cannabis smoking study with placebo, carried out with two groups of young male volunteers; the first group was characterized by a heavy use (≥10 joints/month) while the second group was made up of occasional users smoking at most 1 joint/week. However, to date, these cutoffs have not been adequately assessed under real conditions. Their validity can now be evaluated and confirmed with 146 traffic offenders' real cases in which the whole blood cannabinoid concentrations and the frequency of cannabis use are known. The two thresholds were not challenged by the presence of ethanol (40% of cases) and of other therapeutic and illegal drugs (24%). Thus, we propose the following procedure that can be very useful in the Swiss context but also in other countries with similar traffic policies: if the whole blood THCCOOH concentration is higher than 40μg/L, traffic offenders must be directed first and foremost toward medical assessment of their fitness to drive. This evaluation is not recommended if the THCCOOH concentration is lower than 3μg/L and if the self-rated frequency of cannabis use is less than 1 time/week. A THCCOOH level between these two thresholds cannot be reliably interpreted. In such a case, further medical assessment and follow-up of the fitness to drive are also suggested, but with lower priority.

No healthy heart for heavy drinkers: a population-based study in Switzerland

Background: Low to moderate alcohol consumption has been associated with lower coronary heart disease (CHD) risk, an effect mainly mediated by an increase in HDL-cholesterol levels. However, data on the CHD risk associated with high alcohol consumption are conflicting. Methods: In a population-based study of 5,769 men and women, aged 35-75 years, without cardiovascular disease in Switzerland, last week alcohol consumption was categorized into 0, 1-6, 7-13, 14-20, 21-27, 28-34, 035 drinks/week and into nondrinkers (0 drink/week), moderate (1-13), high (14-34) and very high drinkers (035). Blood pressure, lipids and high sensitivity C-reactive protein (hs-CRP) were measured, and the 10-year CHD risk was calculated according to the Framingham risk score. Results: 73% (n = 4,214) of the participants consumed alcohol; 16% (n = 909) were considered as high drinkers and 2% (n = 119) as very high drinkers. In multivariate analysis, increasing alcohol consumption was associated with higher HDL-cholesterol (from 1.57 ± 0.01 [adjusted mean ± SE] in nondrinkers to 1.88 ± 0.03 mmol/L in very high drinkers); triglycerides (1.17 ± 1.01 to 1.32 ± 1.05 mmol/L), and systolic and diastolic blood pressure rose significantly (127.4 ± 0.4 to 132.2 ± 1.4 and 78.7 ± 0.3 to 81.7 ± 0.9 mm Hg, respectively, all p for trend <0.001). Predicted 10-year CHD risk increased from 4.31 ± 0.10 to 4.90 ± 0.37 (p = 0.03) with increasing alcohol use, with a J-shaped relationship. Conclusion: As measured by the 10-year CHD risk, the protective effect of alcohol consumption disappears in very high drinkers, namely because the beneficial increase in HDL-cholesterol may be blunt by a rise in blood pressure levels.

On the function of proton-gated ion channels ASICs and TRPV1 : a patch-clamp study

AbstractAcidosis is encountered during tissue inflammation and triggers pain in humans. H+-gated ion channels are expressed at high levels in sensory neurons of the peripheral nervous system. Ion channels from two different families present the required pH sensitivity to detect the acidosis associated with peripheral inflammation: Acid-Sensing Ion Channels (ASICs) and the Transient Receptor Potential Vanilloid-1 (TRPV1) channel.ASICs are members of the Degenerin/Epithelial Na+ Channel family of ion channels. Six ASIC subunits have been identified in mammals (ASICla, -lb, -2a, -2b, -3 and -4). ASICs form In-activated voltage-insensitive homo- or heterotrimeric Na+ channels. TRPV1 is a member of the TRP family of ion channels and forms non-selective cation channels that mediate a sustained current. TRPV1 is activated by H+, heat (T>43°C), lipids, capsaicin, voltage and other stimuli. A stimulus can increase TRPV1 response to a different stimulus. For example H+ can shift the capsaicin concentration dependence of TRPV1 to lower values. ASICs and TRPV1 have been shown to be involved in inflammatory pain. Using the patch-clamp technique, we studied different aspects of the function of ASICs and TRPV1 in the physiological context of pain.In the first part of this thesis, we characterize the effect of a temperature increase from 25 to 35°C on the function of ASICs and TRPV1 in transfected CHO cells and primary cultures of rat DRG sensory neurons. ASICs give rise to transient currents while TRPV1 mediates a sustained current. In addition, ASICs and TRPV1 respond to H+ with distinct pH dependences. We assess the relative contribution of ASICs and TRPV1 to H+-evoked electrical signaling in rat DRG neurons and we conclude that ASICs are the most important pH sensors in the pH range 7.4 to 6.0 at 35°C in sensory neurons.ASICs and TRPV1 are expressed in the epithelium lining the lumen of the bladder (urothelium). The Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) is a painful condition associated with a dysfunction of the urothelial barrier and with inflammation. In the second part of this thesis, we show that human urothelial cells -the cell line TEU2 and primary cultures of human bladder urothelium- express functional ASICs but no functional TRPV1 channels. In addition, we show that the levels of ASIC2 and ASIC3 mRNA are increased in the urothelium of patients suffering from BPS/IC. These data suggest that ASICs are involved in the pathology of BPS/IC.Finally, we demonstrate that APETx2 inhibits the sensory neuron specific voltage-dependent Na+ channel Nav1.8. APETx2 was previously shown to inhibit homo- or heterotrimeric ASIC3- containing channels with IC5o from 0.08 to 1 μΜ. We show that APETx2 also inhibits Nav1.8 with an ICsoof «2.6 μΜ. APETx2 reduces the maximal conductance and induces a depolarizing shift in the voltage dependence of activation of Nav1.8. In current-clamp experiments, APETx2 reduces the number of action potentials (APs) evoked by a current ramp. Nav1.8 mediates most of the current during the AP upstroke and has been shown to be an important mediator of inflammatory pain. The fact that APETx2 inhibits two ion channels involved in inflammatory pain suggests that APETx2 or derivatives may represent novel analgesic compounds.RésuméL'acidose tissulaire est observée durant l'inflammation et entraine la douleur chez l'humain. Des canaux ioniques activés par les protons (H+) sont fortement exprimés dans les neurones sensoriels du système nerveux périphérique. De ceux-ci, les Acid-Sensing Ion Channels [ASICs) et Transient Receptor Potential Vanilloid-1 (TRPV1) présentent une sensibilité adéquate à l'acidité pour servir de détecteurs d'acidose.Les ASICs sont membres de la famille Degenerin/Epithelial Na* Channel. Six sous-unités ASIC ont été identifiées chez les mammifères (ASICla, -lb, -2a, -2b, -3 et -4). Les ASICs forment des canaux sélectifs au Na\ insensibles au voltage et activés par les H+. Les canaux fonctionnels sont des homo- ou hétérotrimères de sous-unités ASIC. TRPV1 est un membre de la famille TRP de canaux ioniques. Les canaux TRPV1 sont activés par les H+, la chaleur (T>43°Ç), les lipides, la capsaicine, le voltage et d'autres stimulus. L'activation de TRPV1 entraine un courant soutenu non-sélectif. Un stimulus peut augmenter la réponse de TRPV1 à un autre stimulus. Les H+ peuvent, par exemple, induire un décalage vers des valeurs plus faibles de la courbe de dépendance à la concentration de TRPV1 pour la capsaicine. Il a été démontré que les ASICs et TRPV1 sont impliqués dans la douleur inflammatoire. En utilisant la technique du patch-clamp, nous avons étudié différents aspects de la fonction des ASICs et de TRPV1 dans des contextes associés à la douleur.Dans la première partie de cette thèse, nous caractérisons l'effet d'une augmentation de température de 25 à 35°C sur la fonction des canaux ASICs et TRPV1, dans des cellules CHO transfectées et dans des cultures primaires de neurones sensoriels (DRG) de rat. L'activation des ASICs entraine l'apparition d'un courant transitoire tandis que l'activation de TRPV1 entraine un courant soutenu. De plus, les ASICs et TRPV1 possèdent des dépendances au pH différentes. Nous évaluons la contribution relative des ASICs et de TRPV1 au signalement électrique induit par les H+ et nous concluons que les ASICs sont les senseurs d'acidité les plus importants dans les neurones sensoriels, dans le domaine de pH de 7.4 à 6.0, à température corporelle.Les ASICs et TRPV1 sont exprimés dans l'épithélium recouvrant l'intérieur de la vessie (l'urothélium). Le Bladder Pain Syndrome/Interstitial Cystitis (BPS/IC) est une condition médicale douloureuse associée à une dysfonction de la barrière urothéliale et à une inflammation. Dans la seconde partie de cette thèse, nous démontrons que des cellules urothéliales (de la lignée cellulaire TEU2) et des cellules provenant de cultures primaires d'épithéliums de vessies humaines expriment des canaux ASIC fonctionnels mais pas de TRPV1 fonctionnels. De plus, nous montrons que le niveau d'expression de ASIC2 et -3 est augmenté dans l'urothélium de la vessie de patients souffrant de BPS/IC. Ces données suggèrent que les ASICs sont impliqués dans la pathologie BPS/IC.Pour finir, nous démontrons que la toxine APETx2 inhibe le canal spécifique aux neurones sensoriels Nav1.8, un membre de la famille des canaux sodiques dépendants du potentiel. Il a été démontré précédemment que la toxine APETx2 inhibe les canaux contenant une ou plusieurs sous-unités ASIC3 avec un ICso entre 0.08 et 1 μΜ. Nous montrons que la toxine APETx2 inhibe Nav1.8 avec un IC50 de «2.6 μΜ. La toxine APETx2 réduit la conductance maximale et induit un décalage de la dépendance au potentiel de Nav1.8 vers des valeurs plus positives. Dans des expériences de courant imposé sur des neurones sensoriels, la toxine APETx2 réduit le nombre de potentiels d'action induits par une rampe de courant. Nav1.8 est responsable de la majeure partie du courant durant la phase ascendante du potentiel d'action et a été démontré comme étant un médiateur important de la douleur inflammatoire. L'inhibition de deux types de canaux, impliqués dans la douleurs inflammatoire, par la toxine APETx2, suggère que cette dernière ou ses dérivés représentent des composés analgésiques prometteurs.

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.

Influence of counselor characteristics and behaviors on the efficacy of a brief motivational intervention for heavy drinking in young men-a randomized controlled trial.

BACKGROUND: Brief motivational intervention (BMI) has shown promising results to reduce alcohol use in young adults. Knowledge on mechanisms that predict BMI efficacy could potentially improve treatment effect sizes through data that optimize clinical training and implementation. Particularly, little attention has been given to counselor influence on treatment mechanisms. METHODS: We investigated the influence of counselors on BMI efficacy in reducing alcohol use among non-treatment-seeking young men (age 20) screened as hazardous drinkers. Participants were randomly allocated to (i) a group receiving a single BMI from 1 of 18 counselors selected to maximize differences in several of their characteristics (gender, professional status, clinical experience, and motivational interviewing [MI] experience) or (ii) a control group receiving assessment only. Drinking at 3-month follow-up was first compared between the BMI and control groups to assess efficacy. Then, the influence of counselors' characteristics (i.e., gender, professional status, clinical experience, MI experience, BMI attitudes, and expectancies) and within-session behaviors (i.e., measured by the Motivational Interviewing Skill Code) on outcome was tested in regression analyses. RESULTS: There was a significant (p = 0.02) decrease in alcohol use among the BMI group compared to the control group. Counselors that were male, more experienced, that had more favorable BMI attitudes and expectancies, higher MI skills, but surprisingly less MI-consistent behaviors, had significantly better outcomes than the control group while their counterparts did not. CONCLUSIONS: The current study demonstrated BMI efficacy on alcohol use reduction within a sample of non-treatment-seeking young adult males. Moreover, BMI effect was related to interindividual differences among counselors, and results therefore provide recommendations for BMI training and implementation with similar populations.

Use of nicotine substitute prescribed at hourly plus ab libitum intake or ad libitum for heavy smokers willing to quit: a randomized controlled trial.

OBJECTIVE: To assess the impact of instructional guidance in the regular use of use nicotine nasal spray (NNS) on the true use of NNS during the first three weeks of smoking cessation for heavy smokers who are willing to quit. METHODS: This randomized, open, controlled trial included 50 patients who were heavy smokers, were willing to quit, and attending an academic outpatient clinic in Western Switzerland. Patients were randomised to instruction on NNS use as "ad libitum" (administration whenever cravings appear; control group) or to use NNS when craving appears and at least every hour when awake (intervention group). Intakes were monitored using an electronic device fixed in the spray unit (MDILog) during the first three weeks of use. Self reported abstinence from smoking at six months was confirmed by expired-air carbon monoxide. Using intention-to-treat analysis, random-effect GLS regression was used to calculate the mean difference of daily doses between groups controlling for lack of independence between measures from the same individual. RESULTS: One patient was lost to follow-up. At baseline randomization, the group receiving instruction to use NNS hourly included more women, patients with previous desires to quit, and patients with more psychiatric comorbidities and less somatic complaints compared to the group instructed to use NNS with cravings (group imbalance). Both groups self-administered more than the daily recommended dosage of 8 uses. Mean daily usage was 13.6 dose/day and 11.1 dose/day for the group instructed to use NNS hourly and with cravings, respectively. Adjusting for baseline imbalance, the increased daily doses in the intervention group (hourly use) remained nonsignificant compared to ad libitum use (-0.5 dose/day; CI 95% -6.2; 5.3, from day 1 to day 7; and 2.3 dose/day; CI 95% -5.4; 10.0, from day 8 to day 21). Instructing patients to use the NNS daily had no effect on smoking cessation at six months (RR = 0.69; CI 95% 0.34; 1.39). CONCLUSION: Heavy smokers willing to quit use NNS frequently, regardless of the instructions given. Recommending the use of NNS only when craving appears for heavy smokers willing to quit seems acceptable compared to prescribing hourly administration. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00861276.

Dynamic responses of oxygen uptake at the onset and end of moderate and heavy exercise in trained subjects.

Inconsistencies about dynamic asymmetry between the on- and off-transient responses in VO2 are found in the literature. Therefore the purpose of this study was to examine VO2 on- and off-transients during moderate- and heavy-intensity cycling exercise in trained subjects. Ten men underwent an initial incremental test for the estimation of ventilatory threshold (VT) and, on different days, two bouts of square-wave exercise at moderate (<VT) and heavy (>VT) intensities. VO2 kinetics in exercise and recovery were better described by a single exponential model (<VT), or by a double exponential with two time delays (>VT). For moderate exercise, we found a symmetry of VO2 kinetics between the on- and off-transients (i.e., fundamental component), consistent with a system manifesting linear control dynamics. For heavy exercise, a slow component superimposed on the fundamental phase was expressed in both the exercise and recovery, with similar parameter estimates. But the on-transient values of the time constant were appreciably faster than the associated off-transient, and independent of the work rate imposed (<VT and >VT). Our results do not support a dynamically linear system model of VO2 during cycling exercise in the heavy-intensity domain.

Genetic male infertility and mutation of CATSPER ion channels.

A clinically significant proportion of couples experience difficulty in conceiving a child. In about half of these cases male infertility is the cause and often genetic factors are involved. Despite advances in clinical diagnostics ∼50% of male infertility cases remain idiopathic. Based on this, further analysis of infertile males is required to identify new genetic factors involved in male infertility. This review focuses on cation channel of sperm (CATSPER)-related male infertility. It is based on PubMed literature searches using the keywords 'CATSPER', 'male infertility', 'male contraception', 'immunocontraception' and 'pharmacologic contraception' (publication dates from January 1979 to December 2009). Previously, contiguous gene deletions including the CATSPER2 gene implicated the sperm-specific CATSPER channel in syndromic male infertility (SMI). Recently, we identified insertion mutations of the CATSPER1 gene in families with recessively inherited nonsyndromic male infertility (NSMI). The CATSPER channel therefore represents a novel human male fertility factor. In this review we summarize the genetic and clinical data showing the role of CATSPER mutation in human forms of NSMI and SMI. In addition, we discuss clinical management and therapeutic options for these patients. Finally, we describe how the CATSPER channel could be used as a target for development of a male contraceptive.