Advantages and challenges of the implementation of a low-cost particulate matter monitoring system as a decision-making tool.

The integration of monitoring technologies in the last decades has been a key factor in the development of new ways to track air pollutants and supplementing the network of traditional monitoring systems. In this regard, the appearance of affordable and accurate sensor devices to monitor air quality has made possible to obtain relevant data about the state of the air, and moreover, eminent institutions are interested in promoting the use of novel and more affordable tools for air pollution, such as the United States Environmental Protection Agency and European institutions, within a new approach to environmental surveillance, known as Next Generation Compliance and Enforcement technologies. On other hand, in order to get more reliable measurements, the use of machine learning to support adjustment or calibration process has been used in some studies to improve the performance of monitoring devices. On this paper, led by a group of specialists of the Chilean Superintendence of Environment (henceforth, SMA from its Spanish initials), a first approach case study related to the convenience of the usage of low-cost devices in environmental enforcement will be presented. The study was made in the Metropolitan Region of Santiago and considers the spatial distribution of different particulate matter sensors in the region. Some aspects regarding communication and technical issues are presented as well as the main findings about their performance. Results illustrate that low-cost sensors, aided by machine learning algorithms, could provide a reliable enough general screening of particulate matter within a large city, constituting a valuable decision-making tool for environmental oversight, as well as a powerful preventive and deterrent approach for compliance.

TMBIM3/GRINA is a novel unfolded protein response (UPR) target gene that controls apoptosis through the modulation of ER calcium homeostasis.

Transmembrane BAX inhibitor motif-containing (TMBIM)-6, also known as BAX-inhibitor 1 (BI-1), is an anti-apoptotic protein that belongs to a putative family of highly conserved and poorly characterized genes. Here we report the function of TMBIM3/GRINA in the control of cell death by endoplasmic reticulum (ER) stress. Tmbim3 mRNA levels are strongly upregulated in cellular and animal models of ER stress, controlled by the PERK signaling branch of the unfolded protein response. TMBIM3/GRINA synergies with TMBIM6/BI-1 in the modulation of ER calcium homeostasis and apoptosis, associated with physical interactions with inositol trisphosphate receptors. Loss-of-function studies in D. melanogaster demonstrated that TMBIM3/GRINA and TMBIM6/BI-1 have synergistic activities against ER stress in vivo. Similarly, manipulation of TMBIM3/GRINA levels in zebrafish embryos revealed an essential role in the control of apoptosis during neuronal development and in experimental models of ER stress. These findings suggest the existence of a conserved group of functionally related cell death regulators across species beyond the BCL-2 family of proteins operating at the ER membrane.

A functional assay for paralytic shellfish toxins that uses recombinant sodium channels.

Saxitoxin (STX) and its derivatives are highly toxic natural compounds produced by dinoflagellates commonly present in marine phytoplankton. During algal blooms ("red tides"), shellfish accumulate saxitoxins leading to paralytic shellfish poisoning (PSP) in human consumers. PSP is a consequence of the high-affinity block of voltage-dependent Na channels in neuronal and muscle cells. PSP poses a significant public health threat and an enormous economic challenge to the shellfish industry worldwide. The standard screening method for marine toxins is the mouse mortality bioassay that is ethically problematic, costly and time-consuming. We report here an alternative, functional assay based on electrical recordings in cultured cells stably expressing a PSP target molecule, the STX-sensitive skeletal muscle Na channel. STX-equivalent concentration in the extracts was calibrated by comparison with purified STX, yielding a highly significant correlation (R=0.95; N=30) between electrophysiological determinations and the values obtained by conventional methods. This simple, economical, and reproducible assay obviates the need to sacrifice millions of animals in mandatory paralytic shellfish toxin screening programs.

Specificity in signaling pathways: assembly into multimolecular signaling complexes.

A critical issue in the field of signal transduction is how signaling molecules are organized into different pathways within the same cell. The importance of assembling signaling molecules into architecturally defined complexes is emerging as an essential cellular strategy to ensure specificity and selectivity of signaling. Scaffold proteins function as the pillars of these transduction complexes, bringing together a diversity of signaling components into defined ultramicrodomains of signaling.

A multivalent PDZ-domain protein assembles signalling complexes in a G-protein-coupled cascade.

How are signalling molecules organized into different pathways within the same cell? In Drosophila, the inaD gene encodes a protein consisting of five PDZ domains which serves as a scaffold to assemble different components of the phototransduction cascade, including the principal light-activated ion channels, the effector phospholipase C-beta and protein kinase C. Null inaD mutants have a dramatically reorganized subcellular distribution of signalling molecules, and a total loss of transduction complexes. Also, mutants defective in a single PDZ domain produce signalling complexes that lack the target protein and display corresponding defects in their physiology. A picture emerges of a highly organized unit of signalling, a 'transduclisome', with PDZ domains functioning as key elements in the organization of transduction complexes in vivo.

Modal gating in neuronal and skeletal muscle ryanodine-sensitive Ca2+ release channels.

The bursting behavior of ryanodine-sensitive single Ca2+ release channels present in chicken cerebellum endoplasmic reticulum (ER), rat hippocampus ER, and frog and rabbit skeletal muscle sarcoplasmic reticulum was established. Unconditional dwell time distributions fitted by the maximum likelihood method reveal at least three open and closed exponential components. Trains of low open probability (P(o)) bursts were interspersed with trains of high P(o) bursts (> or = 0.8) in all the ryanodine receptor isotypes tested. The gating kinetics of the Ca2+ release channels were defined in long recordings by analyzing burst sequences and gamma distributions of average intraburst open (T(o)) and closed times (Tc). The gamma distributions of T(o) had two gamma components, suggesting the existence of two distinct burst types. In contrast, the gamma distributions of Tc had only one component. The correlation between consecutive burst pairs was defined in terms of T(o) and then statistically tested by 2 x 2 matrix contingency analysis. The probability that the ubiquitous sequential burst pattern was generated by random occurrence was < 0.01 (two-tailed Fisher's exact test). Temporal correlations were observed in all ryanodine receptor isotypes under a variety of experimental conditions. These data strongly suggest that single Ca2+ release channels switch slowly between modes of gating. We propose that the effects of agonists of Ca2+ release channels such as Ca2+ itself can be explained as concentration-dependent changes in the availability of each mode.

Functionally heterogenous ryanodine receptors in avian cerebellum.

The functional heterogeneity of the ryanodine receptor (RyR) channels in avian cerebellum was defined. Heavy endoplasmic reticulum microsomes had significant levels of ryanodine and inositol 1,4,5-trisphosphate binding. Scatchard analysis and kinetic studies indicated the existence of at least two distinct ryanodine binding sites. Ryanodine binding was calcium-dependent but was not significantly enhanced by caffeine. Incorporation of microsomes into planar lipid bilayers revealed ion channels with pharmacological features (calcium, magnesium, ATP, and caffeine sensitivity) similar to the RyR channels found in mammalian striated muscle. Despite a wide range of unitary conductances (220-500 picosiemens, symmetrical cesium methanesulfonate), ryanodine locked both channels into a characteristic slow gating subconductance state, positively identifying them as RyR channels. Two populations of avian RyR channels were functionally distinguished by single channel calcium sensitivity. One population was defined by a bell-shaped calcium sensitivity analogous to the skeletal muscle RyR isoform (type I). The calcium sensitivity of the second RyR population was sigmoidal and analogous to the cardiac muscle RyR isoform (type II). These data show that there are at least two functionally distinct RyR channel populations in avian cerebellum. This leads to the possibility that these functionally distinct RyR channels are involved in different intracellular calcium signaling pathways.

Properties of the ryanodine-sensitive release channels that underlie caffeine-induced Ca2+ mobilization from intracellular stores in mammalian sympathetic neurons.

The most compelling evidence for a functional role of caffeine-sensitive intracellular Ca2+ reservoirs in nerve cells derives from experiments on peripheral neurons. However, the properties of their ryanodine receptor calcium release channels have not been studied. This work combines single-cell fura-2 microfluorometry, [3H]ryanodine binding and recording of Ca2+ release channels to examine calcium release from these intracellular stores in rat sympathetic neurons from the superior cervical ganglion. Intracellular Ca2+ measurements showed that these cells possess caffeine-sensitive intracellular Ca2+ stores capable of releasing the equivalent of 40% of the calcium that enters through voltage-gated calcium channels. The efficiency of caffeine in releasing Ca2+ showed a complex dependence on [Ca2+]i. Transient elevations of [Ca2+]i by 50-500 nM were facilitatory, but they became less facilitatory or depressing when [Ca2+]i reached higher levels. The caffeine-induced Ca2+ release and its dependence on [Ca2+]i was further examined by [3H]ryanodine binding to ganglionic microsomal membranes. These membranes showed a high-affinity binding site for ryanodine with a dissociation constant (KD = 10 nM) similar to that previously reported for brain microsomes. However, the density of [3H]ryanodine binding sites (Bmax = 2.06 pmol/mg protein) was at least three-fold larger than the highest reported for brain tissue. [3H]Ryanodine binding showed a sigmoidal dependence on [Ca2+] in the range 0.1-10 microM that was further increased by caffeine. Caffeine-dependent enhancement of [3H]ryanodine binding increased and then decreased as [Ca2+] rose, with an optimum at [Ca2+] between 100 and 500 nM and a 50% decrease between 1 and 10 microM. At 100 microM [Ca2+], caffeine and ATP enhanced [3H]ryanodine binding by 35 and 170% respectively, while binding was reduced by > 90% with ruthenium red and MgCl2. High-conductance (240 pS) Ca2+ release channels present in ganglionic microsomal membranes were incorporated into planar phospholipid bilayers. These channels were activated by caffeine and by micromolar concentrations of Ca2+ from the cytosolic side, and were blocked by Mg2+ and ruthenium red. Ryanodine (2 microM) slowed channel gating and elicited a long-lasting subconductance state while 10 mM ryanodine closed the channel with infrequent opening to the subconductance level. These results show that the properties of the ryanodine receptor/Ca2+ release channels present in mammalian peripheral neurons can account for the properties of caffeine-induced Ca2+ release. Our data also suggest that the release of Ca2+ by caffeine has a bell-shaped dependence on Ca2+ in the physiological range of cytoplasmic [Ca2+].

Characterization and subcellular distribution of G-proteins in highly purified skeletal muscle fractions from rabbit and frog.

The presence of G-proteins in highly purified fractions from frog and rabbit skeletal muscles was analyzed by Bordetella pertussis toxin-catalyzed ADP-ribosylation and by immunoblots. Two B. pertussis toxin substrates were present in transverse tubules and sarcoplasmic reticulum from frog and rabbit skeletal muscle. Immunoblot analysis suggested that the two B. pertussis toxin substrates present in rabbit sarcoplasmic reticulum are possibly alpha i2 and alpha i3, but the corresponding substrates in sarcoplasmic reticulum from frog are still unidentified. Transverse tubules isolated from both rabbit and frog also contained alpha i2 and alpha i3; additionally, beta 35, beta 36, alpha o (weak immunoreactivity), alpha i1 (and/or alpha i3 in frog), alpha s (short form only in frog), and alpha q were found. Sarcoplasmic reticulum from rabbit also contained alpha s (predominantly alpha s short), beta 36, and alpha q. The presence of alpha q in sarcoplasmic reticulum, in addition to transverse tubules, is potentially a very important finding since this G-protein has been described only in plasma membranes so far. Also, this study describes the candidate G-proteins for the control of excitation-contraction coupling in skeletal muscle.

Phospholipase C activity in membranes and a soluble fraction isolated from frog skeletal muscle.

Highly purified triads and transverse tubules, as well as soluble fraction isolated from frog skeletal muscle, hydrolyze exogenous phosphatidylinositol 4,5-bisphosphate forming inositol 1,4,5-trisphosphate with maximal rates in the range 0.5-1 nmol/mg per min at pCa 3. Sarcoplasmic reticulum membranes present a minor activity. The hydrolysis rates in triads were 0.072 +/- 0.015 nmol/mg per min at pCa 7, increasing to 0.263 +/- 0.026 nmol/mg per min at pCa 5 with 1.0 mM Mg and 0.1 mM substrate. The phospholipase C activity of isolated transverse tubules at pCa 3 was 0.570 +/- 0.032 nmol/mg per min. Since triads contain 10% transverse tubules, and correcting for the small contribution of sarcoplasmic reticulum, the calculated phospholipase C activity of transverse tubules at pCa 3 is about 10-times higher than the observed values, suggesting loss of activity during isolation. The activation by calcium was also observed in a soluble fraction and was neither replaced nor inhibited by magnesium. No effect of GTP analogs on phospholipase C activity was detected.

Effect of dopamine depletion upon the K(+)-evoked release of CCK from superfused striatal slices.

The effect of a lack of dopamine (DA) in the striatum upon the K(+)-evoked release of cholecystokinin (CCK) from superfused rat striatal slices has been studied. Two pharmacological tools were used to deplete the nigrostriatal DA system: administration of alpha-methyl-p-tyrosine which competitively inhibits DA synthesis and lesions with 6-hydroxydopamine of the medial forebrain bundle. In both cases there was a significant inhibition of the K(+)-evoked release of CCK. The observed effects might be relevant on pathological conditions implying depletion of the DA system.