Echocardiographic heart ageing patterns predict cardiovascular and non-cardiovascular events and reflect biological age: the SardiNIA study.

AIMS: Age is a crucial risk factor for cardiovascular (CV) and non-CV diseases. As people age at different rates, the concept of biological age has been introduced as a personalized measure of functional deterioration. Associations of age with echocardiographic quantitative traits were analysed to assess different heart ageing rates and their ability to predict outcomes and reflect biological age. METHODS AND RESULTS: Associations of age with left ventricular mass, geometry, diastolic function, left atrial volume, and aortic root size were measured in 2614 healthy subjects. Based on the 95% two-sided tolerance intervals of each correlation, three discrete ageing trajectories were identified and categorized as 'slow', 'normal', and 'accelerated' heart ageing patterns. The primary endpoint included fatal and non-fatal CV events, and the secondary endpoint was a composite of CV and non-CV events and all-cause death. The phenotypic age of the heart (HeartPhAge) was estimated as a proxy of biological age. The slow ageing pattern was found in 8.7% of healthy participants, the normal pattern in 76.9%, and the accelerated pattern in 14.4%. Kaplan-Meier curves of the heart ageing patterns diverged significantly (P = 0.0001) for both primary and secondary endpoints, with the event rate being lowest in the slow, intermediate in the normal, and highest in the accelerated pattern. In the Cox proportional hazards model, heart ageing patterns predicted both primary (P = 0.01) and secondary (P = 0.03 to <0.0001) endpoints, independent of chronological age and risk factors. Compared with chronological age, HeartPhAge was 9 years younger in slow, 4 years older in accelerated (both P < 0.0001), and overlapping in normal ageing patterns. CONCLUSION: Standard Doppler echocardiography detects slow, normal, and accelerated heart ageing patterns. They predict CV and non-CV events, reflect biological age, and provide a new tool to calibrate prevention timing and intensity.

Age is the main risk factor for cardiovascular (CV) disease. Since people age and develop diseases at very different rates, biological age has been proposed as a more accurate measure of the body's functional decline. This study aimed to investigate the ageing rates of the heart and to assess their impact on CV events. The phenotypic age of the heart was also estimated as a proxy for biological age. Associations of age with Doppler echocardiographic parameters were analysed in a subgroup of 2614 clinically healthy subjects, part of a larger cohort of 3817 adults of both sexes.Three patterns of slow, normal, and accelerated ageing rates of the heart were detected. They predicted both CV and non-CV events, with different and progressively increasing event rates from the slow to the accelerated pattern. Compared with chronological age, the phenotypic (biological) age of the heart was 9 years younger in the slow pattern, 4 years older in the accelerated pattern, and comparable in the normal pattern.A standard Doppler echocardiogram is therefore able to detect three distinct heart ageing patterns, which reflect different biological susceptibilities to age-dependent diseases and provide a new tool for personalizing timeliness and intensity of prevention.

Glucose-6-phosphate dehydrogenase deficiency accelerates arterial aging in diabetes.

AIMS: High glucose levels and Glucose-6-Phosphate Dehydrogenase deficiency (G6PDd) have both tissue inflammatory effects. Here we determined whether G6PDd accelerates arterial aging (information linked stiffening) in diabetes. METHODS: Plasma glucose, interleukin 6 (IL6), and arterial stiffness (indexed as carotid-femoral Pulse Wave Velocity, PWV) and red blood cell G6PD activity were assessed in a large (4448) Sardinian population. RESULTS: Although high plasma glucose in diabetics, did not differ by G6DP status (178.2 ± 55.1 vs 169.0 ± 50.1 mg/dl) in G6DPd versus non-G6PDd subjects, respectively, IL6, and PWV (adjusted for age and glucose) were significantly increased in G6PDd vs non-G6PDd subjects (PWV, 8.0 ± 0.4 vs 7.2 ± 0.2 m/sec) and (IL6, 6.9 ± 5.0 vs 4.2 ± 3.0 pg/ml). In non-diabetics, neither fasting plasma glucose, nor IL6, nor PWV were impacted by G6PDd. CONCLUSION: G6PDd in diabetics is associated with increased inflammatory markers and accelerated arterial aging.

Metabolic and immune dysfunctions in post-traumatic stress disorder: what can we learn from animal models?

Highly stressful experiences such as terrorist attacks, domestic and sexual violence may lead to persistent pathological symptoms such as those seen in posttraumatic stress disorder (PTSD). There is growing evidence of multiple metabolic and immune disorders underlying the etiology and maintenance of PTSD. However, changes in the functioning of various systems and organs associated with PTSD are not well understood. Studies of reliable animal models is one of the effective scientific tools that can be used to gain insight into the role of metabolism and immunity in the comorbidity associated with PTSD. Since much progress has been made using animal models to understand mechanisms of PTSD, we summarized metabolic and immune dysfunction in mice and humans to compare certain outcomes associated with PTSD. The systemic effects of PTSD include chronic activation of the sympathetic nervous system (psycho-emotional stress), that leads to impairment of the function of the immune system, increased release of stress hormones, and metabolic changes. We discuss PTSD as a multisystem disease with its neurological, immunological, and metabolic components.

Structural accreditation of healthcare facilities: comparison of the requirements by Italian Presidential Decree 14/01/1997 and regional regulations. A proposal for updating the minimum environmental units at national level.

BACKGROUND AND AIM: The Decree of the President of the Italian Republic 14/01/1997 is the reference norm related to the accreditation of public and private healthcare structures. This guideline establishes the minimum structural, technological and organizational requirements that each structure operating in the Italian territory must comply with. METHODS: In occasion of the project work for the postgraduate training course in healthcare management by ALTEMS School, a team of researchers conducted a survey on the state of updating of the minimum structural requirements indicated in the norm-in particular those relating to hospital facilities- with those adopted by the individual regions through the analysis of the most up-to-date regional regulations. RESULTS: Precisely starting from the comparison of regional references and from the regulations on the subject of structural accreditation which suggest strategic environmental units and which address some key-aspects relating to the contemporary design of healing environments (i.e. semi-intensive care units, hybrid operating theatres, etc.), the outcome of the project work is to define a proposal to update the national reference document, also in the light of the currently changing needs in terms of hospital design. CONCLUSIONS: The research aims to become a starting milestone for future investigations. The team investigated - in this first phase - the functional areas listed in the norm, and the next step aims to extend the analysis also to the innovative functions (i.e. buffer spaces, hybrid operating theatres, sub-intensive care units, etc.) and/or introduced only the last years which have only been regulated in some regions.

Computer-aided detection of arrhythmogenic sites in post-ischemic ventricular tachycardia.

Nowadays, catheter-based ablation in patients with post-ischemic ventricular tachycardia (VT) is performed in arrhythmogenic sites identified by electrophysiologists by visual inspection during electroanatomic mapping. This work aims to present the development of machine learning tools aiming at supporting clinicians in the identification of arrhythmogenic sites by exploiting innovative features that belong to different domains. This study included 1584 bipolar electrograms from nine patients affected by post-ischemic VT. Different features were extracted in the time, time scale, frequency, and spatial domains and used to train different supervised classifiers. Classification results showed high performance, revealing robustness across the different classifiers in terms of accuracy, true positive, and false positive rates. The combination of multi-domain features with the ensemble tree is the most effective solution, exhibiting accuracies above 93% in the 10-time 10-fold cross-validation and 84% in the leave-one-subject-out validation. Results confirmed the effectiveness of the proposed features and their potential use in a computer-aided system for the detection of arrhythmogenic sites. This work demonstrates for the first time the usefulness of supervised machine learning for the detection of arrhythmogenic sites in post-ischemic VT patients, thus enabling the development of computer-aided systems to reduce operator dependence and errors, thereby possibly improving clinical outcomes.

Spectral characterisation of ventricular intracardiac potentials in human post-ischaemic bipolar electrograms.

Abnormal ventricular potentials (AVPs) are frequently referred to as high-frequency deflections in intracardiac electrograms (EGMs). However, no scientific study performed a deep spectral characterisation of AVPs and physiological potentials in real bipolar intracardiac recordings across the entire frequency range imposed by their sampling frequency. In this work, the power contributions of post-ischaemic physiological potentials and AVPs, along with some spectral features, were evaluated in the frequency domain and then statistically compared to highlight specific spectral signatures for these signals. To this end, 450 bipolar EGMs from seven patients affected by post-ischaemic ventricular tachycardia were retrospectively annotated by an experienced cardiologist. Given the high variability of the morphologies observed, three different sub-classes of AVPs and two sub-categories of post-ischaemic physiological potentials were considered. All signals were acquired by the CARTO® 3 system during substrate-guided catheter ablation procedures. Our findings indicated that the main frequency contributions of physiological and pathological post-ischaemic EGMs are found below 320 Hz. Statistical analyses showed that, when biases due to the signal amplitude influence are eliminated, not only physiological potentials show greater contributions below 20 Hz whereas AVPs demonstrate higher spectral contributions above ~ 40 Hz, but several finer differences may be observed between the different AVP types.

The adverse effects of pramipexole on probability discounting are not reversed by acute D2 or D3 receptor antagonism.

Pramipexole (PPX) is a D2 and D3 dopamine receptor agonist approved for clinical use, which is associated with a higher risk of impulse-control disorders. Using a rat model, we recently found that low doses of the monoamine-depleting agent reserpine (RES; 1 mg/kg/day, SC) dramatically increased the untoward effects of PPX (0.3 mg/kg/day, SC) on probability discounting, a key impulsivity function. To further understand the neurobehavioral mechanisms underlying these effects, we first tested whether the combination of PPX and RES may lead to a generalized enhancement in risk taking, as tested in the suspended wire-beam paradigm. The association of RES and PPX did not augment the proclivity of rats to cross the bridge in order to obtain a reward, suggesting that the effects of RES and PPX on probability discounting do not reflect a generalized increase in impulsivity. We then studied what receptors mediate the effects of PPX in RES-treated rats. The combination of RES and PPX increased membrane expression and binding of D3, but not D2 dopamine receptors, in the nucleus accumbens. However, the behavioral effects of PPX and RES were not reduced by acute treatments with the D2/D3 receptor antagonist raclopride (0.01-0.05 mg/kg, SC), the highly selective D2 receptor antagonist L-741,626 (0.1-1 mg/kg, SC) or the D3 receptor antagonists GR 103691 (0.1-0.3 mg/kg, SC) and SB 277011A (1-10 mg/kg, SC). These findings collectively suggest that the effects of PPX in probability discounting do not reflect generalized enhancements in impulsivity or acute dopamine D2 or D3 receptor activation.

Arterial stiffness and multiple organ damage: a longitudinal study in population.

AIMS: Previous cross-sectional observation identified arterial aging, indexed as pulse-wave velocity (PWV), as a key determinant of the simultaneous multiple organ damage (heart, carotid artery, and kidney). The aim of the present cohort study is to investigate trajectories of repeated measures of PWV and traditional CV risk factors in subjects who eventually presented clinical evidence of multiple organ damage in the SardiNIA study. METHODS AND RESULTS: Organ damage was measured in the heart (left ventricular hypertrophy, LVH), the common carotid artery (intima-media thickness > 0.9 mm and/or plaque), and the kidney (eGFR < 60 ml/min/1.73 m2) of 2130 men and women of a broad age range participating the SardiNIA study. SHATS was defined as the simultaneous occurrence of all the three-organ damages. Trajectory in traditional CV risk factors and PWV was analyzed retrospectively (four observations over 9 years) according to the number of organ damage (from 0 to 3). Compared to subjects with no organ damage, after controlling for traditional CV risk factors, each 1 m/s increase in baseline PWV was accompanied by a 93% higher odds of developing SHATS; and each 1 cm/s (0.01 m/s) annual increase in PWV by a 31% greater odds of developing SHATS. CONCLUSIONS: Arterial stiffness, a proxy of arterial aging that can be measured clinically as PWV, is an integrated predictive marker of multiple age-associated organ damage recognized as clinical diseases.

Impact of Stiffer Arteries on the Response to Antihypertensive Treatment: A Longitudinal Study of the SardiNIA Cohort.

OBJECTIVES: Carotid-femoral pulse wave velocity (PWV), an index of arterial stiffness and a proxy of arterial aging, has been reported to be an independent determinant of cardiovascular health. Whether the effects of antihypertensive treatment vary in the presence of accelerated arterial aging (stiffer artery, ie, PWV >10 m/s) has not been established. We tested this hypothesis in a longitudinal study in a large community-dwelling population. DESIGN: Longitudinal population study with repeated measures. SETTING AND PARTICIPANTS: Study population consisted of a cohort of 6011 volunteers (2546 men and 3465 women, age range 14-101 years; 15,011 observations over a median follow-up of 6.8 years) participating in the SardiNIA Study. MEASURES: Repeated measures of PWV, blood pressure (BP), and metabolic risk factors and the antihypertensive medication trajectories of BP and PWV over time were assessed via mixed effects models. RESULTS: Antihypertensive treatment significantly affected the trajectory of BP in both participants with (-0.47 ± 0.20 mmHg/y, P = .02) and participants without stiffer arteries (-0.47 ± 0.07 mmHg/y, P = .001). They also affected the trajectory of PWV in participants with stiffer artery, independent of the BP values. CONCLUSIONS AND IMPLICATIONS: Antihypertensive treatment is effective in reducing both BP and PWV in older individuals with stiffer arteries.

Equilibrative nucleoside transporter ENT1 as a biomarker of Huntington disease.

The initial goal of this study was to investigate alterations in adenosine A2A receptor (A2AR) density or function in a rat model of Huntington disease (HD) with reported insensitivity to an A2AR antagonist. Unsuspected negative results led to the hypothesis of a low striatal adenosine tone and to the search for the mechanisms involved. Extracellular striatal concentrations of adenosine were measured with in vivo microdialysis in two rodent models of early neuropathological stages of HD disease, the Tg51 rat and the zQ175 knock-in mouse. In view of the crucial role of the equilibrative nucleoside transporter (ENT1) in determining extracellular content of adenosine, the binding properties of the ENT1 inhibitor [3H]-S-(4-Nitrobenzyl)-6-thioinosine were evaluated in zQ175 mice and the differential expression and differential coexpression patterns of the ENT1 gene (SLC29A1) were analyzed in a large human cohort of HD disease and controls. Extracellular striatal levels of adenosine were significantly lower in both animal models as compared with control littermates and striatal ENT1 binding sites were significantly upregulated in zQ175 mice. ENT1 transcript was significantly upregulated in HD disease patients at an early neuropathological severity stage, but not those with a higher severity stage, relative to non-demented controls. ENT1 transcript was differentially coexpressed (gained correlations) with several other genes in HD disease subjects compared to the control group. The present study demonstrates that ENT1 and adenosine constitute biomarkers of the initial stages of neurodegeneration in HD disease and also predicts that ENT1 could constitute a new therapeutic target to delay the progression of the disease.

Local Control of Extracellular Dopamine Levels in the Medial Nucleus Accumbens by a Glutamatergic Projection from the Infralimbic Cortex.

It is generally assumed that infralimbic cortex (ILC) and prelimbic cortex, two adjacent areas of the medial prefrontal cortex (mPFC) in rodents, provide selective excitatory glutamatergic inputs to the nucleus accumbens (NAc) shell and core, respectively. It is also generally believed that mPFC influences the extracellular levels of dopamine in the NAc primarily by an excitatory collateral to the ventral tegmental area (VTA). In the present study, we first established the existence of a selective functional connection between ILC and the posteromedial portions of the VTA (pmVTA) and the mNAc shell (pmNAc shell), by measuring striatal neuronal activation (immunohistochemical analysis of ERK1/2 phosphorylation) and glutamate release (in vivo microdialysis) upon ILC electrical stimulation. A novel optogenetic-microdialysis approach allowed the measurement of extracellular concentrations of glutamate and dopamine in the pmNAc shell upon local light-induced stimulation of glutamatergic terminals from ILC. Cortical electrical and local optogenetic stimulation produced significant increases in the extracellular concentrations of glutamate and dopamine in the pmNAc shell. Local blockade of glutamate release by perfusion of an adenosine A2A receptor antagonist in the pmNAc shell blocked the dopamine release induced by local optogenetic stimulation but only partially antagonized dopamine release induced by cortical electrical stimulation. The results demonstrate that ILC excitatory afferents directly modulate the extracellular concentration of dopamine in the pmNAc shell, but also support the involvement of an indirect mechanism of dopamine control, through a concomitant ILC-mediated activation of the pmVTA. Significance statement: We established the existence of a functional connection between the infralimbic cortex (ILC) and the posteromedial portions of the ventral tegmental area (pmVTA) and the medial nucleus acumbens shell (pmNAc shell). A novel optogenetic-microdialysis approach allowed us to demonstrate that local glutamate release from glutamatergic terminals from the ILC exert a significant modulation of extracellular concentration of dopamine in the pmNAc shell. This mechanism provides the frame for a selective cortical-mediated tonic dopaminergic modulation of specific striatal compartments.

Serum free thyroxine levels are positively associated with arterial stiffness in the SardiNIA study.

OBJECTIVE: Thyroid dysfunction may accelerate atherosclerosis. Aortic pulse wave velocity (PWV) is an early index of arterial stiffness and an important risk factor for cardiovascular disease and might therefore be linked to changes in thyroid activity. We investigated the relationship between thyroid function and carotid-femoral PWV, as an index of arterial stiffness. DESIGN: Cross-sectional cohort study. PATIENTS: Participants from the SardiNIA study. Those being treated for thyroid diseases were excluded, yielding a sample of 5875 aged 14-102. MEASUREMENTS: Clinical parameters, blood tests including serum TSH and serum FT4, and carotid-femoral PWV were measured. RESULTS: After adjusting for confounders, a direct and linear association between FT4 and PWV was shown (multiple regression analysis). The model containing age, mean blood pressure, body mass index, heart rate, FT4, hypertension, diabetes and dyslipidaemia accounted for 55% of the variation in PWV. CONCLUSIONS: Like several other known risk factors, serum FT4 levels are associated with carotid-femoral PWV, suggesting that high FT4 levels have a detrimental effect on aortic stiffness and may contribute to ageing process of the vascular system. This finding may help to understand the pathogenesis of cardiovascular disease and contribute to improve prevention therapy.

Are personality traits associated with white-coat and masked hypertension?

OBJECTIVES: Anxiety and other psychological dispositions are thought to be associated with blood pressure. This study tests whether personality traits have long-term associations with masked and white-coat effects. METHODS: A community-based sample of 2838 adults from Sardinia (Italy) completed the Revised NEO Personality Inventory, and 7 years later, blood pressure was assessed in the clinic and with ambulatory monitoring. Logistic regressions were used to test whether anxiety, neuroticism, extraversion, openness, agreeableness, and conscientiousness predicted the white-coat and masked hypertension phenomena. Age, sex, and antihypertensive medication use were tested as moderators. RESULTS: Significant interactions were found between personality traits and antihypertensive medications in predicting masked and white-coat effects. Only among those taking antihypertensive medication, higher anxiety was associated with a higher risk of pseudo-resistant hypertension due to white-coat effect (odds ratio 1.39, 95% confidence interval 1.01-1.91) and higher conscientiousness was associated with a lower risk of masked uncontrolled hypertension (odds ratio 0.70, 95% confidence interval 0.49-0.99). There were no significant interactions with age or sex. CONCLUSIONS: Among those on antihypertensive medications, anxious individuals were more likely to have pseudo-resistant hypertension due to white-coat effect and less conscientious individuals were at increased risk of masked uncontrolled hypertension. Particularly among anxious and less conscientious individuals, ambulatory monitoring may improve the tailoring of pharmacological treatments.

Longitudinal perspective on the conundrum of central arterial stiffness, blood pressure, and aging.

The age-associated increase in arterial stiffness has long been considered to parallel or to cause the age-associated increase in blood pressure (BP). Yet, the rates at which pulse wave velocity (PWV), a measure of arterial stiffness, and BP trajectories change over time within individuals who differ by age and sex have not been assessed and compared. This study determined the evolution of BP and aortic PWV trajectories during a 9.4-year follow-up in >4000 community-dwelling men and women of 20 to 100 years of age at entry into the SardiNIA Study. Linear mixed effects model analyses revealed that PWV accelerates with time during the observation period, at about the same rate over the entire age range in both men and women. In men, the longitudinal rate at which BP changed over time, however, did not generally parallel that of PWV acceleration: at ages>40 years the rates of change in systolic BP (SBP) and pulse pressure (PP) increase plateaued and then declined so that SBP, itself, also declined at older ages, whereas PP plateaued. In women, SBP, diastolic BP, and mean BP increased at constant rates across all ages, producing an increasing rate of increase in PP. Therefore, increased aortic stiffness is implicated in the age-associated increase in SBP and PP. These findings indicate that PWV is not a surrogate for BP and that arterial properties other than arterial wall stiffness that vary by age and sex also modulate the BP trajectories during aging and lead to the dissociation of PWV, PP, and SBP trajectories in men.

Personality traits and circadian blood pressure patterns: a 7-year prospective study.

OBJECTIVE: A nighttime dip in blood pressure is associated with decreased risk of cardiovascular morbidity and mortality. We examined whether personality traits predict nighttime dipping blood pressure. METHODS: A community-based sample of 2848 adults from Sardinia (Italy) completed the Revised NEO Personality Inventory and 7 years later were examined with 24-hour ambulatory blood pressure monitoring. The primary analyses examined the associations of personality traits with continuous and categorical measures of mean arterial, systolic, and diastolic blood pressure nighttime dipping. RESULTS: Agreeableness and conscientiousness were associated with more nocturnal blood pressure dipping (ß = .05 [p = .025] and ß = .07 [p < .001], respectively) and lower systolic blood pressure at night (ß = -.05 [p = .018] and ß = -.03 [p = .072], respectively). Nondippers were particularly more impulsive (p = .009), less trusting (p = .004), and less self-disciplined (p = .001), but there was no significant association between nocturnal dipping blood pressure and trait anxiety (p = .78) or depression (p = .59). The associations were stronger when comparing extreme dippers (nighttime drop ≥ 20%) to reverse dippers (nighttime increase in blood pressure). Indeed, scoring 1 standard deviation higher on conscientiousness was associated with approximately 40% reduced risk of reverse dipping (odds ratio = 1.43, confidence interval = 1.08-1.91). CONCLUSIONS: We found evidence that reduced nighttime blood pressure dipping is associated with antagonism and impulsivity-related traits but not with measures of emotional vulnerability. The strongest associations were found with conscientiousness, a trait that may have a broad impact on cardiovascular health.

Arterial stiffness and influences of the metabolic syndrome: a cross-countries study.

Specific clusters of metabolic syndrome (MetS) components impact differentially on arterial stiffness, indexed as pulse wave velocity (PWV). Of note, in several population-based studies participating in the MARE (Metabolic syndrome and Arteries REsearch) Consortium the occurrence of specific clusters of MetS differed markedly across Europe and the US. The aim of the present study was to investigate whether specific clusters of MetS are consistently associated with stiffer arteries in different populations. We studied 20,570 subjects from 9 cohorts representing 8 different European countries and the US participating in the MARE Consortium. MetS was defined in accordance with NCEP ATPIII criteria as the simultaneous alteration in ≥3 of the 5 components: abdominal obesity (W), high triglycerides (T), low HDL cholesterol (H), elevated blood pressure (B), and elevated fasting glucose (G). PWV measured in each cohort was "normalized" to account for different acquisition methods. MetS had an overall prevalence of 24.2% (4985 subjects). MetS accelerated the age-associated increase in PWV levels at any age, and similarly in men and women. MetS clusters TBW, GBW, and GTBW are consistently associated with significantly stiffer arteries to an extent similar or greater than observed in subjects with alteration in all the five MetS components--even after controlling for age, sex, smoking, cholesterol levels, and diabetes mellitus--in all the MARE cohorts. In conclusion, different component clusters of MetS showed varying associations with arterial stiffness (PWV).

Paraxanthine: Connecting Caffeine to Nitric Oxide Neurotransmission.

Recent results obtained in our laboratory indicate that paraxanthine, the main metabolite of caffeine in humans, produces a significantly stronger locomotor activation in rats than caffeine. Furthermore, paraxanthine also produced a very significant increase in striatal extracellular concentrations of dopamine. Searching for an additional mechanism other than adenosine antagonism responsible for these psychostimulant-like effects, it was found that paraxanthine, but not caffeine, inhibited cGMP-preferring phosphodiesterases. Furthermore, interrupting nitric oxide neurotransmision (inhibiting nitric oxide synthase) significantly decreased both the locomotor-activating and the dopamine-releasing effects of paraxanthine. These results open up some obvious questions about the role of paraxanthine in the pharmacological effects of caffeine.

Psychostimulant pharmacological profile of paraxanthine, the main metabolite of caffeine in humans.

Caffeine induces locomotor activation by its ability to block adenosine receptors. Caffeine is metabolized to several methylxanthines, with paraxanthine being the main metabolite in humans. In this study we show that in rats paraxanthine has a stronger locomotor activating effect than caffeine or the two other main metabolites of caffeine, theophylline and theobromine. As previously described for caffeine, the locomotor activating doses of paraxanthine more efficiently counteract the locomotor depressant effects of an adenosine A(1) than an adenosine A(2A) receptor agonist. In drug discrimination experiments in rats trained to discriminate a maximal locomotor activating dose of caffeine, paraxanthine, unlike theophylline, generalized poorly to caffeine suggesting the existence of additional mechanisms other than adenosine antagonism in the behavioral effects of paraxanthine. Pretreatment with the nitric oxide inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME) reduced the locomotor activating effects of paraxanthine, but not caffeine. On the other hand, pretreatment with the selective cGMP-preferring phosphodiesterase PDE9 inhibitor BAY 73-6691, increased locomotor activity induced by caffeine, but not paraxanthine. Ex vivo experiments demonstrated that paraxanthine, but not caffeine, can induce cGMP accumulation in the rat striatum. Finally, in vivo microdialysis experiments showed that paraxanthine, but not caffeine, significantly increases extracellular levels of dopamine in the dorsolateral striatum, which was blocked by l-NAME. These findings indicate that inhibition of cGMP-preferring PDE is involved in the locomotor activating effects of the acute administration of paraxanthine. The present results demonstrate a unique psychostimulant profile of paraxanthine, which might contribute to the reinforcing effects of caffeine in humans.

Designing of sequencing assay assisted by capillary electrophoresis based on DNA folding analysis: an application to the VCAM1 gene.

In this work, we describe a fast standardized molecular method for DNA sequencing assisted by capillary electrophoresis with a particular emphasis on bioinformatic approaches to avoid sequencing errors due to complex DNA regions. In this case, the method was applied on the human vascular adhesion molecule 1 (VCAM1) gene. VCAM1 sequence, in fact, shows many thermodynamically critical parameters such as very low GC content (30-40%), many nucleotide stack areas, i.e. hairpins, self-complementary regions. With a traditional primer design approach it was difficult to design correct PCR oligonucleotides, thus sometimes, the chromatogram showed an illegible profile. By a strategy involving various bioinformatic tools (Mfold, Oligo, Highter), we investigated the role of the DNA-folding analysis in the assistance of primer design for the DNA sequencing of fragments with high -ΔG stem-loop regions. This new approach allowed us to sequence nine different VCAM1 regions each containing the respective exon. Our results, based on different DNA samples recruited from oral brushes taken from ten different subjects, identified four different SNPs (c.662-7C/T, c.1793-79A>G, c.2079C/T, c.2208A>G) with high reproducibility.