A Novel MOS Nanowire Gas Sensor Device (S3) and GC-MS-Based Approach for the Characterization of Grated Parmigiano Reggiano Cheese.

To determine the originality of a typical Italian Parmigiano Reggiano cheese, it is crucial to define and characterize its quality, ripening period, and geographical origin. Different analytical techniques have been applied aimed at studying the organoleptic and characteristic volatile organic compounds (VOCs) profile of this cheese. However, most of the classical methods are time consuming and costly. The aim of this work was to illustrate a new simple, portable, fast, reliable, non-destructive, and economic sensor device S3 based on an array of six metal oxide semiconductor nanowire gas sensors to assess and discriminate the quality ranking of grated Parmigiano Reggiano cheese samples and to identify the VOC biomarkers using a headspace SPME-GC-MS. The device could clearly differentiate cheese samples varying in quality and ripening time when the results were analyzed by multivariate statistical analysis involving principal component analysis (PCA). Similarly, the volatile constituents of Parmigiano Reggiano identified were consistent with the compounds intimated in the literature. The obtained results show the applicability of an S3 device combined with SPME-GC-MS and sensory evaluation for a fast and high-sensitivity analysis of VOCs in Parmigiano Reggiano cheese and for the quality control of this class of cheese.

Interactions between permeation and gating in the TMEM16B/anoctamin2 calcium-activated chloride channel.

At least two members of the TMEM16/anoctamin family, TMEM16A (also known as anoctamin1) and TMEM16B (also known as anoctamin2), encode Ca(2+)-activated Cl(-) channels (CaCCs), which are found in various cell types and mediate numerous physiological functions. Here, we used whole-cell and excised inside-out patch-clamp to investigate the relationship between anion permeation and gating, two processes typically viewed as independent, in TMEM16B expressed in HEK 293T cells. The permeability ratio sequence determined by substituting Cl(-) with other anions (PX/PCl) was SCN(-) > I(-) > NO3 (-) > Br(-) > Cl(-) > F(-) > gluconate. When external Cl(-) was substituted with other anions, TMEM16B activation and deactivation kinetics at 0.5 µM Ca(2+) were modified according to the sequence of permeability ratios, with anions more permeant than Cl(-) slowing both activation and deactivation and anions less permeant than Cl(-) accelerating them. Moreover, replacement of external Cl(-) with gluconate, or sucrose, shifted the voltage dependence of steady-state activation (G-V relation) to more positive potentials, whereas substitution of extracellular or intracellular Cl(-) with SCN(-) shifted G-V to more negative potentials. Dose-response relationships for Ca(2+) in the presence of different extracellular anions indicated that the apparent affinity for Ca(2+) at +100 mV increased with increasing permeability ratio. The apparent affinity for Ca(2+) in the presence of intracellular SCN(-) also increased compared with that in Cl(-). Our results provide the first evidence that TMEM16B gating is modulated by permeant anions and provide the basis for future studies aimed at identifying the molecular determinants of TMEM16B ion selectivity and gating.

The voltage dependence of the TMEM16B/anoctamin2 calcium-activated chloride channel is modified by mutations in the first putative intracellular loop.

Ca(2+)-activated Cl(-) channels (CaCCs) are involved in several physiological processes. Recently, TMEM16A/anoctamin1 and TMEM16B/anoctamin2 have been shown to function as CaCCs, but very little information is available on the structure-function relations of these channels. TMEM16B is expressed in the cilia of olfactory sensory neurons, in microvilli of vomeronasal sensory neurons, and in the synaptic terminals of retinal photoreceptors. Here, we have performed the first site-directed mutagenesis study on TMEM16B to understand the molecular mechanisms of voltage and Ca(2+) dependence. We have mutated amino acids in the first putative intracellular loop and measured the properties of the wild-type and mutant TMEM16B channels expressed in HEK 293T cells using the whole cell voltage-clamp technique in the presence of various intracellular Ca(2+) concentrations. We mutated E367 into glutamine or deleted the five consecutive glutamates (386)EEEEE(390) and (399)EYE(401). The EYE deletion did not significantly modify the apparent Ca(2+) dependence nor the voltage dependence of channel activation. E367Q and deletion of the five glutamates did not greatly affect the apparent Ca(2+) affinity but modified the voltage dependence, shifting the conductance-voltage relations toward more positive voltages. These findings indicate that glutamates E367 and (386)EEEEE(390) in the first intracellular putative loop play an important role in the voltage dependence of TMEM16B, thus providing an initial structure-function study for this channel.

Blood-brain barrier damage and brain penetration of antiepileptic drugs: role of serum proteins and brain edema.

PURPOSE: Increased blood-brain barrier (BBB) permeability is radiologically detectable in regions affected by drug-resistant epileptogenic lesions. Brain penetration of antiepileptic drugs (AEDs) may be affected by BBB damage. We studied the effects of BBB damage on brain distribution of hydrophilic [deoxy-glucose (DOG) and sucrose] and lipophilic (phenytoin and diazepam) molecules. We tested the hypothesis that lipophilic and hydrophilic drug distribution is differentially affected by BBB damage. METHODS: In vivo BBB disruption (BBBD) was performed in rats by intracarotid injection of hyperosmotic mannitol. Drugs (H3-sucrose, 3H-deoxy-glucose, 14C-phenytoin, and C14-diazepam) or unlabeled phenytoin was measured and correlated to brain water content and protein extravasation. In vitro hippocampal slices were exposed to different osmolarities; drug penetration and water content were assessed by analytic and densitometric methods, respectively. RESULTS: BBBD resulted in extravasation of serum protein and radiolabeled drugs, but was associated with no significant change in brain water. Large shifts in water content in brain slices in vitro caused a small effect on drug penetration. In both cases, total drug permeability increase was greater for lipophilic than hydrophilic compounds. BBBD reduced the amount of free phenytoin in the brain. DISCUSSION: After BBBD, drug binding to protein is the main controller of total brain drug accumulation. Osmotic BBBD increased serum protein extravasation and reduced free phenytoin brain levels. These results underlie the importance of brain environment and BBB integrity in determining drug distribution to the brain. If confirmed in drug-resistant models, these mechanisms could contribute to drug brain distribution in refractory epilepsies.

Antagonism of peripheral inflammation reduces the severity of status epilepticus.

Status epilepticus (SE) is one of the most serious manifestations of epilepsy. Systemic inflammation and damage of blood-brain barrier (BBB) are etiologic cofactors in the pathogenesis of pilocarpine SE while acute osmotic disruption of the BBB is sufficient to elicit seizures. Whether an inflammatory-vascular-BBB mechanism could apply to the lithium-pilocarpine model is unknown. LiCl facilitated seizures induced by low-dose pilocarpine by activation of circulating T-lymphocytes and mononuclear cells. Serum IL-1beta levels increased and BBB damage occurred concurrently to increased theta EEG activity. These events occurred prior to SE induced by cholinergic exposure. SE was elicited by lithium and pilocarpine irrespective of their sequence of administration supporting a common pathogenetic mechanism. Since IL-1beta is an etiologic trigger for BBB breakdown and its serum elevation occurs before onset of SE early after LiCl and pilocarpine injections, we tested the hypothesis that intravenous administration of IL-1 receptor antagonists (IL-1ra) may prevent pilocarpine-induced seizures. Animals pre-treated with IL-1ra exhibited significant reduction of SE onset and of BBB damage. Our data support the concept of targeting systemic inflammation and BBB for the prevention of status epilepticus.