Rapporto tra struttura e funzione della cistatina B e dei suoi mutanti in relazione all'epilessia mioclonica progressiva di tipo 1 (EPM1)

This work shows for the first time that native CSTB polymerizes on addition of Cu2+ and DnaK (Hsp70). Cysteines are involved in the polymerization process and in particular at least one cysteine is necessary. We propose that Cu2+ interacts with the thiol group of cysteine and oxidize it. The oxidized cysteine modifies the CSTB structure allowing interaction with DnaK/Hsp70 to occur. Thus, Cu2+ binding to CSTB exposes a site for DnaK and such interaction allows the polymerization of CSTB. The polymers generated from native CSTB monomers, are DTT sensitive and they may represent physiological polymers. Denatured CSTB does not require Cu2+ and polymerizes simply on addition of DnaK. The polymers generated from denatured CSTB do not respond to DTT. They have characteristics similar to those of the CSTB toxic aggregates described in vivo in eukaryotic cells following CSTB over-expression. Interaction between CSTB and Hsp70 is shown by IP experiments. The interaction occurs with WT CSTB and not with the cys mutant. This suggests that disulphur bonds are involved. Methal-cathalyzed oxidation of proteins involves reduction of the metal ion(s) bound to the protein itself and oxidation of neighboring ammino acid residues resulting in structural modification and de-stabilization of the molecule. In this work we propose that the cysteine thyol residue of CSTB in the presence of Cu2+ is oxidized, and cathalyzes the formation of disulphide bonds with Hsp70, that, once bound to CSTB, mediates its polymerization. In vivo this molecular mechanism of CSTB polymerization could be regulated by redox environment through the cysteine residue. This may imply that CSTB physiological polymers have a specific cellular function, different from that of the protease inhibitor known for the CSTB monomer. This hypothesis is interesting in relation to Progressive Myoclonus Epilepsy of type 1 (EPM1). This pathology is usually caused by mutations in the CSTB gene. CSTB is a ubiquitous protein, but EPM1 patients have problems only in the central nervous system. Maybe physiological CSTB polymers have a specific function altered in people affected by EPM1.

Sleep-related changes in blood pressure in hypocretin-deficient narcoleptic mice

Objectives. Blood pressure (BP) physiologically has higher and lower values during the active and rest period, respectively. Subjects failing to show the appropriate BP decrease (10-20%) on passing form diurnal activity to nocturnal rest and sleep have increased risk of target organ damage at the cardiac, vascular and cerebrovascular levels. Hypocretin (HCRT) releasing neurons, mainly located in the lateral hypothalamus, project widely to the central nervous system. Thus HCRT neurons are involved in several autonomic functions, including BP regulation. HCRT neurons also play a key role in wake-sleep cycle regulation, the lack of which becomes evident in HCRT-deficient narcoleptic patients. I investigated whether chronic lack of HCRT signaling alters BP during sleep in mouse models of narcolepsy. Methods. The main study was performed on HCRT-ataxin3 transgenic mice (TG) with selective post-natal ablation of HCRT neurons, HCRT gene knockout mice (KO) with preserved HCRT neurons, and Wild-Type control mice (WT) with identical genetic background. Experiments where replicated on TG and WT mice with hybrid genetic background (hTG and hWT, respectively). Mice were implanted with a telemetric pressure transducer (TA11PA-C10, DSI) and electrodes for discriminating wakefulness (W), rapid-eye-movement sleep (REMS) and non-REMS (NREMS). Signals were recorded for 3 days. Mean BP values were computed in each wake-sleep state and analyzed by ANOVA and t-test with significance at p<0.05. Results. The decrease in BP between either NREMS or REMS and W was significantly blunted in TG and KO with respect to WT as well as in hTG with respect to hWT. Conclusions. Independently from the genetic background, chronic HCRT deficiency leads to a decreased BP difference between W and sleep potentially adverse in narcoleptic subjects. These data suggest that HCRT play an important role in the sleep-dependent cardiovascular control.

Profilo psicopatologico e qualità della vita in bambini e adolescenti narcolettici: studio caso-controllo

Scopo del nostro lavoro è stato descrivere ed inquadrare gli aspetti psico-comportamentali e la qualità di vita della narcolessia in età evolutiva. Metodi: Abbiamo pertanto disegnato uno studio caso-controllo comprendente 30 pazienti narcolettici, 39 epilettici, e 39 controlli sani, appaiati per sesso e età. Risultati: La nostra popolazione di bambini e adolescenti affetti da narcolessia mostra un aumento delle problematiche internalizzanti. I due gruppi patologici hanno in comune punteggi più elevati rispetto ai controlli per i disturbi d’ansia, le difficoltà attentive e di socializzazione, i disturbi oppositivo-provocatori. Ciò che distingue, invece, i pazienti narcolettici, sono gli aspetti di ritiro e depressione, la tendenza alla somatizzazione, i problemi del pensiero ed i disturbi affettivi. Fattori di rischio psicopatologici per i giovani narcolettici sono risultati essere l’esordio precoce, il ritardo diagnostico, il sonno notturno disturbato, la minor latenza di sonno all’addormentamento, un maggior numero di SOREMP all’MSLT. Dall’altro lato la terapia farmacologica, un maggior numero di sonnellini spontanei e la durata di malattia, sembrano influenzare positivamente l’evoluzione comportamentale. La salute psicosociale dei giovani narcolettici, inoltre, risulta essere peggiore rispetto ai controlli sani, mentre la salute fisica non mostra differenze. I problemi internalizzanti influenzano negativamente tutti gli ambiti della salute di questi ragazzi, mentre la durata di malattia sembra migliorare il funzionamento scolastico. Conclusioni: Il nostro lavoro conferma che i giovani narcolettici presentano un maggior rischio psicopatologico sia rispetto ai controlli sani sia rispetto un’altra patologia neurologica cronica. Se da un lato alcuni aspetti comportamentali possono essere giustificati come una reazione adattativa verso una patologia neurologica invalidante, dall’altro un quadro distimico caratterizzato da ritiro e lamentele somatiche, sembra essere tipico dei bambini ed adolescenti narcolettici.

Measurement of volatile organic compounds and total OH reactivity in the atmosphere

Volatile organic compounds play a critical role in ozone formation and drive the chemistry of the atmosphere, together with OH radicals. The simplest volatile organic compound methane is a climatologically important greenhouse gas, and plays a key role in regulating water vapour in the stratosphere and hydroxyl radicals in the troposphere. The OH radical is the most important atmospheric oxidant and knowledge of the atmospheric OH sink, together with the OH source and ambient OH concentrations is essential for understanding the oxidative capacity of the atmosphere. Oceanic emission and / or uptake of methanol, acetone, acetaldehyde, isoprene and dimethyl sulphide (DMS) was characterized as a function of photosynthetically active radiation (PAR) and a suite of biological parameters, in a mesocosm experiment conducted in the Norwegian fjord. High frequency (ca. 1 minute-1) methane measurements were performed using a gas chromatograph - flame ionization detector (GC-FID) in the boreal forests of Finland and the tropical forests of Suriname. A new on-line method (Comparative Reactivity Method - CRM) was developed to directly measure the total OH reactivity (sink) of ambient air. It was observed that under conditions of high biological activity and a PAR of ~ 450 μmol photons m-2 s-1, the ocean acted as a net source of acetone. However, if either of these criteria was not fulfilled then the ocean acted as a net sink of acetone. This new insight into the biogeochemical cycling of acetone at the ocean-air interface has helped to resolve discrepancies from earlier works such as Jacob et al. (2002) who reported the ocean to be a net acetone source (27 Tg yr-1) and Marandino et al. (2005) who reported the ocean to be a net sink of acetone (- 48 Tg yr-1). The ocean acted as net source of isoprene, DMS and acetaldehyde but net sink of methanol. Based on these findings, it is recommended that compound specific PAR and biological dependency be used for estimating the influence of the global ocean on atmospheric VOC budgets. Methane was observed to accumulate within the nocturnal boundary layer, clearly indicating emissions from the forest ecosystems. There was a remarkable similarity in the time series of the boreal and tropical forest ecosystem. The average of the median mixing ratios during a typical diel cycle were 1.83 μmol mol-1 and 1.74 μmol mol-1 for the boreal forest ecosystem and tropical forest ecosystem respectively. A flux value of (3.62 ± 0.87) x 1011 molecules cm-2 s-1 (or 45.5 ± 11 Tg CH4 yr-1 for global boreal forest area) was derived, which highlights the importance of the boreal forest ecosystem for the global budget of methane (~ 600 Tg yr-1). The newly developed CRM technique has a dynamic range of ~ 4 s-1 to 300 s-1 and accuracy of ± 25 %. The system has been tested and calibrated with several single and mixed hydrocarbon standards showing excellent linearity and accountability with the reactivity of the standards. Field tests at an urban and forest site illustrate the promise of the new method. The results from this study have improved current understanding about VOC emissions and uptake from ocean and forest ecosystems. Moreover, a new technique for directly measuring the total OH reactivity of ambient air has been developed and validated, which will be a valuable addition to the existing suite of atmospheric measurement techniques.

Resa diagnostica di anamnesi, EEG intercritico ed home-made video negli episodi parossistici notturni: confronto con la VEPSG

Poiché la diagnosi differenziale degli episodi parossistici notturni è affidata alla VEPSG, tenendo conto dei limiti di tale metodica, il progetto attuale ha lo scopo di definire la resa diagnostica di strumenti alternativi alla VEPSG: anamnesi, home-made video ed EEG intercritico. Sono stati reclutati consecutivamente 13 pazienti, afferiti al nostro Dipartimento per episodi parossistici notturni. Ciascun paziente è stato sottoposto ad un protocollo diagnostico standardizzato. A 5 Medici Esperti in Epilessia e Medicina del Sonno è stato chiesto di formulare un orientamento diagnostico sulla base di anamnesi, EEG intercritico, home-made video e VEPSG. Attraverso l’elaborazione degli orientamenti diagnostici è stata calcolata la resa diagnostica delle procedure esaminate, a confronto con la VEPSG, attraverso il concetto di “accuratezza diagnostica”. Per 6 pazienti è stato possibile porre una diagnosi di Epilessia Frontale Notturna, per 2 di parasonnia, in 5 la diagnosi è rimasta dubbia. L’accuratezza diagnostica di ciascuna procedura è risultata moderata, con lievi differenze tra le diverse procedure (61.5% anamnesi; 66% home-made video; 69,2 % EEG intercritico). E’ essenziale migliorare ulteriormente l’accuratezza diagnostica di anamnesi, EEG intercritico ed home-made video, che possono risultare cruciali nei casi in cui la diagnosi non è certa o quando la VEPSG non è disponibile.

Studio neuroradiologico (morfologico e funzionale) nei pazienti con Epilessia Frontale Notturna

L'epilessia frontale notturna (EFN) è caratterizzata da crisi motorie che insorgono durante il sonno. Scopo del progetto è studiare le cause fisiopatologiche e morfo-funzionali che sottendono ai fenomeni motori nei pazienti con EFN e identificare alterazioni strutturali e/o metaboliche mediante tecniche avanzate di Risonanza Magnetica (RM). Abbiamo raccolto una casistica di pazienti con EFN afferenti al Centro Epilessia e dei Disturbi del Sonno del Dipartimento di Scienze Neurologiche, Università di Bologna. Ad ogni paziente è stato associato un controllo sano di età (± 5 anni) e sesso corrispondente. Tutti sono stati studiati mediante tecniche avanzate di RM comprendenti Spettroscopia del protone (1H-MRS), Tensore di diffusione ed imaging 3D ad alta risoluzione per analisi morfometriche. In particolare, la 1H-MRS è stata effettuata su due volumi di interesse localizzati nei talami e nel giro del cingolo anteriore. Sono stati inclusi nell’analisi finale 19 pazienti (7 M), età media 34 anni (range 19-50) e 14 controlli (6 M) età media 30 anni (range 19-40). A livello del cingolo anteriore il rapporto della concentrazione di N-Acetil-Aspartato rispetto alla Creatina (NAA/Cr) è risultato significativamente ridotto nei pazienti rispetto ai controlli (p=0,021). Relativamente all’analisi di correlazione, l'analisi tramite modelli di regressione multipla ha evidenziato che il rapporto NAA/Cr nel cingolo anteriore nei pazienti correlava con la frequenza delle crisi (p=0,048), essendo minore nei pazienti con crisi plurisettimanali/plurigiornaliere. Per interpretare il dato ottenuto è possibile solo fare delle ipotesi. L’NAA è un marker di integrità, densità e funzionalità neuronale. E’ possibile che alla base della EFN ci siano alterazioni metaboliche tessutali in precise strutture come il giro del cingolo anteriore. Questo apre nuove possibilità sull’utilizzo di strumenti di indagine basati sull’analisi di biosegnali, per caratterizzare aree coinvolte nella genesi della EFN ancora largamente sconosciute e chiarire ulteriormente l’eziologia di questo tipo di epilessia.

Rhythms of anorexigenic neuropeptides in senegalese sole: Effects of light, feeding regimes and developmental stage.

Increasing knowledge on the endocrine mechanisms that regulate feeding and growth in cultured fish can contribute to make improvement in fish holding conditions and feeding strategies, supporting the development of new techniques that could ameliorate feeding, food conversion efficiency and growth in aquaculture practice. The main objective of this study was to investigate how daily mRNA expression of three specific anorexigenic hormones, i.e. the corticotropin-releasing hormone (CRH) and the paralogues α- and β- proopiomelanocortin (POMC), is modulated by different photoperiods, light spectra and feeding regimes, in both adult and larvae of Solea senegalensis. In addition, as Senegalese sole exhibits a shift from diurnal to nocturnal in locomotor activity and feeding habits during metamorphic process, we tried to elucidate if this shift is accompanied by relevant daily variations in the expression of these anorexigenic hormones before, during and after the completion of metamorphosis. In order to reach this main objective, three main experiments were developed. In a first experiment, adults were reared under LD (12 h light: 12h dark) cycle and fed at mid-light (ML), mid-dark (MD) and at random (RND). In a second experiment, adult specimens were reared in constant darkness (DD) and fed at subjective mid-light (sML) or at RND. Larvae of Senegalese sole were reared under LD cycle with white, blue or red light for 40 days. Our results show an independence of crh mRNA expression from the feeding time and suggest an endogenous control of crh expression in sole. Both pomc paralogues showed significant daily rhythms under LD conditions. The rhythms were maintained or were even more robust under DD conditions for pomc_a, but were completely abolished for pomc_b. Our results indicate an endogenous control of pomc_a expression by the molecular clock in telencephalon and diencephalon, but not in the pituitary gland. Our findings confirm for the first time the significant influence that ambient lighting has on larval growth and development in Senegalese sole, revealing an important effect of light spectra upon functional elements of this species. Our results also emphasize the importance of maintaining cycling light-dark conditions of the adequate wavelengths in aquaculture practices during early development of sole.

Effects of photoperiod and feeding regimes on Neuropeptide Y (NPY) and Agouti-related Protein (AgRP) expression in central areas of senegalese sole, Solea senegalensis

In the present study, we have tried to expand our knowledge about the endocrine mechanisms that regulate feeding and growth in cultured fish, which could be relevant for the improvement of fish farming conditions and feeding strategies. In order to reach this goal, we have investigated some orexigenic hormones, Neuropeptide Y (NPY) and the paralogues of Agouti-related protein, (AgRP1, AgRP2) in Solea senegalensis, an important species for Mediterranean aquaculture. We focused on hormones synchronization to different feeding regimes (diurnal vs nocturnal and random feeding) and photoperiod (light-dark cycle vs constant darkness). Therefore, the achieved results could also be relevant from a chronobiological perspective. Solea senegalensis specimen were reared in two different photoperiods, i.e.LD Light-Dark conditions as well as in DD conditions (constant darkness) along with different feeding regimes (fed at ML, Med and RND times), so to determine if mRNA expression of orexigenic hormones (NPY, AgRP1 and AgRP2) are entrained by feeding time and/or photoperiod. Our results show an independence of npy mRNA expression from the feeding time and suggest an endogenous control of npy expression in telencephalon of sole, while in optice tectum, npy expression could be entrained by the light-dark cycle. Our results on Senegalese sole AgRP1 and AgRP2 showed the same pattern of expression, indicating that expression of AgRPs is related to photoperiod in optic tectum, instead to feeding time. However the involvement of AgRP1 and AgRP2 in feeding behaviour should not be discarded in sole, as further research will be carried out with specimens maintained under different fasting conditions. our results reinforce the role of the telencephalon as the main neural area involved in the neuroendocrine control of food intake in fish, where endogenous NPY rhythms have been found, while diencephalon statistical variations weren’t observed suggesting that this brain area could be less involved in the neuroendocrine control of food intake in fish than previously thought.

Analysis of evening and morning transitions in complex terrain: MATERHORN campaign 2013

A field study of thermal circulation over very gentle slope is described for a specific day characterised by weak synoptic conditions. The emphasis was on morning and evening transitions, but measurements cover the entire day; therefore a brief analysis is performed to represent the general thermal circulation pattern. Both transition periods are characterised by complex dynamic behaviours. During evening transition, the upslope flow has got through a stagnation condition characterised by wind velocity U<0:5 m=s. Only when the stagnating air has become negative buoyant, the flow is allowed to pour downslope like a slab. Some features of front formation has been found during the transition development, such as delay time of downslope flow start up along the slope, and the presence of positive turbulent kinetic energy at the onset of the motion. Eventually the observed evening transition has followed a mixed mechanisms, with features from different models. Therefore the Rayleigh number seems not to be a good criterion by which parametrise evening transition itself. Morning transition is characterised by destruction of nocturnal temperature inversion and the onset of upslope flow. Inversion destruction can be described in terms of CBL growth at surface and inversion decent from the top of the layer. KH has found to be a good indicator of inversion breakup, if used as parameter to study the inversion breakup in terms of temperature reversal. After the inversion breakup, buoyancy and mechanical productions supply the flow with the necessary energy to start the upslope wind. More quantitative analysis are provided by the study of stability parameters and turbulent kinetic energy budgets. Gradient Richardson number has been used in this terms, finding that a mixed SBL-CBL behaviour dominates the most of the observed layers. Tke budget has shown high turbulent behaviour during morning transition while the evening transition has developed entirely in laminar condition, apart from short intermittent turbulent events.

Hepatic-portal vein infusions of glucagon-like peptide-1 reduce meal size and increase c-Fos expression in the nucleus tractus solitarii, area postrema and central nucleus of the amygdala in rats

We recently reported that brief, remotely controlled intrameal hepatic-portal vein infusions of glucagon-like peptide-1 (GLP-1) reduced spontaneous meal size in rats. To investigate the neurobehavioural correlates of this effect, we equipped male Sprague-Dawley rats with hepatic-portal vein catheters and assessed (i) the effect on eating of remotely triggered infusions of GLP-1 (1 nmol/kg, 5 min) or vehicle during the first nocturnal meal after 3 h of food deprivation and (ii) the effect of identical infusions performed at dark onset on c-Fos expression in several brain areas involved in the control of eating. GLP-1 reduced (P < 0.05) the size of the first nocturnal meal and increased its satiety ratio. Also, GLP-1 increased (P < 0.05) the number of c-Fos-expressing cells in the nucleus tractus solitarii, the area postrema and the central nucleus of the amygdala, but not in the arcuate or paraventricular hypothalamic nuclei. These data suggest that the nucleus tractus solitarii, the area postrema and the central nucleus of the amygdala play a role in the eating-inhibitory actions of GLP-1 infused into the hepatic-portal vein; it remains to be established whether activation of these brain nuclei reflect satiation, aversion, or both.

Mitochondrial tRNA(Leu(UUR)) mutation m.3302A > G presenting as childhood-onset severe myopathy: threshold determination through segregation study

Mitochondrial tRNA(Leu(UUR)) mutation m.3302A > G is associated with respiratory chain complex I deficiency and has been described as a rare cause of mostly adult-onset slowly progressive myopathy. Five families with 11 patients have been described so far; 5 of them died young due to cardiorespiratory failure. Here, we report on a segregation study in a family with an index patient who already presented at the age of 18 months with proximal muscular hypotonia, abnormal fatigability, and lactic acidosis. This early-onset myopathy was rapidly progressive. At 8 years, the patient is wheel-chair bound, requires nocturnal assisted ventilation, and suffers from recurrent respiratory infections. Severe complex I deficiency and nearly homoplasmy for m.3302A > G were found in muscle. We collected blood, hair, buccal swabs and muscle biopsies from asymptomatic adults in this pedigree and determined heteroplasmy levels in these tissues as well as OXPHOS activities in muscle. All participating asymptomatic adults had normal OXPHOS activities. In contrast to earlier reports, we found surprisingly little variation of heteroplasmy levels in different tissues of the same individual. Up to 45% mutation load in muscle and up to 38% mutation load in other tissues were found in non-affected adults. The phenotypic spectrum of tRNA(Leu(UUR)) m.3302A > G mutation seems to be wider than previously described. A threshold of more than 45% heteroplasmy in muscle seems to be necessary to alter complex I activity leading to clinical manifestation. The presented data may be helpful for prognostic considerations and counseling in affected families.

The ocular motor features of adult-onset alexander disease: a case and review of the literature

A 51-year-old Chinese man presented with gaze-evoked nystagmus, impaired smooth pursuit and vestibular ocular reflex cancellation, and saccadic dysmetria, along with a family history suggestive of late-onset autosomal dominant parkinsonism. MRI revealed abnormalities of the medulla and cervical spinal cord typical of adult-onset Alexander disease, and genetic testing showed homozygosity for the p.D295N polymorphic allele in the gene encoding the glial fibrillary acidic protein. A review of the literature shows that ocular signs are frequent in adult-onset Alexander disease, most commonly gaze-evoked nystagmus, pendular nystagmus, and/or oculopalatal myoclonus, and less commonly ptosis, miosis, and saccadic dysmetria. These signs are consistent with the propensity of adult-onset Alexander disease to cause medullary abnormalities on neuroimaging.

Progressive dopamine and hypocretin deficiencies in Parkinson's disease: is there an impact on sleep and wakefulness?

Sleep-wake disturbances are frequent in patients with Parkinson's disease, but prospective controlled electrophysiological studies of sleep in those patients are surprisingly sparse, and the pathophysiology of sleep-wake disturbances in Parkinson's disease remains largely elusive. In particular, the impact of impaired dopaminergic and hypocretin (orexin) signalling on sleep and wakefulness in Parkinson's disease is still unknown. We performed a prospective, controlled electrophysiological study in patients with early and advanced Parkinson's disease, e.g. in subjects with presumably different levels of dopamine and hypocretin cell loss. We compared sleep laboratory tests and cerebrospinal fluid levels with hypocretin-deficient patients with narcolepsy with cataplexy, and with matched controls. Nocturnal sleep efficiency was most decreased in advanced Parkinson patients, and still lower in early Parkinson patients than in narcolepsy subjects. Excessive daytime sleepiness was most severe in narcolepsy patients. In Parkinson patients, objective sleepiness correlated with decrease of cerebrospinal fluid hypocretin levels, and repeated hypocretin measurements in two Parkinson patients revealed a decrease of levels over years. This suggests that dopamine and hypocretin deficiency differentially affect sleep and wakefulness in Parkinson's disease. Poorer sleep quality is linked to dopamine deficiency and other disease-related factors. Despite hypocretin cell loss in Parkinson's disease being only partial, disturbed hypocretin signalling is likely to contribute to excessive daytime sleepiness in Parkinson patients.

Stimulus-induced, sleep-bound, focal seizures: a case report

In nocturnal frontal lobe epilepsy (NFLE), seizures occur almost exclusively during NREM sleep. Why precisely these seizures are sleep-bound remains unknown. Studies of patients with nonlesional familial forms of NFLE have suggested the arousal system may play a major role in their pathogenesis. We report the case of a patient with pharmaco-resistant, probably cryptogenic form of non-familial NFLE and strictly sleep-bound seizures that could be elicited by alerting stimuli and were associated with ictal bilateral thalamic and right orbital-insular hyperperfusion on SPECT imaging.

Endothelial dysfunction and arterial stiffness in ischemic stroke: the role of sleep-disordered breathing

Sleep-disordered breathing (SDB) represents a risk factor for cardiovascular morbidity after a cerebral ischemic event (acute ischemic event, ischemic stroke, or transient ischemic attack). In the present study, endothelial function and arterial stiffness were analyzed in patients who experienced a postacute ischemic event with relation to SDB, sleep disruption, and nocturnal oxygenation parameters.