Profiling the ionome of rice and its use in discriminating geographical origins at the regional scale, China

Element profile was investigated for their use to trace the geographical origin of rice (Oryza sativa L.) samples. The concentrations of 13 elements (calcium (Ca), potassium (K), magnesium (Mg), phosphorus (P), boron (B), manganese (Mn), iron (Fe), nickel (Ni), copper (Cu), arsenic (As), selenium (Se), molybdenum (Mo), and cadmium (Cd)) were determined in the rice samples by inductively coupled plasma optical emission and mass spectrometry. Most of the essential elements for human health in rice were within normal ranges except for Mo and Se. Mo concentrations were twice as high as those in rice from Vietnam and Spain. Meanwhile, Se concentrations were three times lower in the whole province compared to the Chinese average level of 0.088 mg/kg. About 12% of the rice samples failed the Chinese national food safety standard of 0.2 mg/kg for Cd. Combined with the multi-elemental profile in rice, the principal component analysis (PCA), discriminant function analysis (DFA) and Fibonacci index analysis (FIA) were applied to discriminate geographical origins of the samples. Results indicated that the FIA method could achieve a more effective geographical origin classification compared with PCA and DFA, due to its efficiency in making the grouping even when the elemental variability was so high that PCA and DFA showed little discriminatory power. Furthermore, some elements were identified as the most powerful indicators of geographical origin: Ca, Ni, Fe and Cd. This suggests that the newly established methodology of FIA based on the ionome profile can be applied to determine the geographical origin of rice.

Human dental pulp fibroblasts express the "cold-sensing" transient receptor potential channels TRPA1 and TRPM8.

Abstract
INTRODUCTION:
Transient receptor potential (TRP) channels comprise a group of nonselective calcium-permeable cationic channels, which are polymodal sensors of environmental stimuli such as thermal changes and chemicals. TRPM8 and TRPA1 are cold-sensing TRP channels activated by moderate cooling and noxious cold temperatures, respectively. Both receptors have been identified in trigeminal ganglion neurones, and their expression in nonneuronal cells is now the focus of much interest. The aim of this study was to investigate the molecular and functional expression of TRPA1 and TRPM8 in dental pulp fibroblasts.
METHODS:
Human dental pulp fibroblasts were derived from healthy molar teeth. Gene and protein expression was determined by polymerase chain reaction and Western blotting. Cellular localization was investigated by immunohistochemistry, and TRP functionality was determined by Ca(2+) microfluorimetry.
RESULTS:
Polymerase chain reaction and Western blotting showed gene and protein expression of both TRPA1 and TRPM8 in fibroblast cells in culture. Immunohistochemistry studies showed that TRPA1 and TRPM8 immunoreactivity co-localized with the human fibroblast surface protein. In Ca(2+) microfluorimetry studies designed to determine the functionality of TRPA1 and TRPM8 in pulp fibroblasts, we showed increased intracellular calcium ([Ca(2+)](i)) in response to the TRPM8 agonist menthol, the TRPA1 agonist cinnamaldehyde, and to cool and noxious cold stimuli, respectively. The responses to agonists and thermal stimuli were blocked in the presence of specific TRPA1 and TRPM8 antagonists.
CONCLUSIONS:
Human dental pulp fibroblasts express TRPA1 and TRPM8 at the molecular, protein, and functional levels, indicating a possible role for fibroblasts in mediating cold responses in human teeth.

Adaptor protein-2 sigma subunit mutations causing familial hypocalciuric hypercalcaemia type 3 (FHH3) demonstrate genotype-phenotype correlations, codon bias and dominant-negative effects

The adaptor protein-2 sigma subunit (AP2sigma;2) is pivotal for clathrin-mediated endocytosis of plasma membrane constituents such as the calcium-sensing receptor (CaSR). Mutations of the AP2sigma;2 Arg15 residue result in familial hypocalciuric hypercalcaemia type 3 (FHH3), a disorder of extracellular calcium (Ca<inf>o</inf>²⁺) homeostasis. To elucidate the role of AP2sigma;2 in Ca<inf>o</inf>²⁺ regulation, we investigated 65 FHH probands, without other FHH-associated mutations, for AP2sigma;2 mutations, characterized their functional consequences and investigated the genetic mechanisms leading to FHH3. AP2sigma;2 mutations were identified in 17 probands, comprising 5 Arg15Cys, 4 Arg15His and 8 Arg15Leu mutations. A genotype-phenotype correlation was observed with the Arg15Leu mutation leading to marked hypercalcaemia. FHH3 probands harboured additional phenotypes such as cognitive dysfunction. All three FHH3-causing AP2sigma;2 mutations impaired CaSR signal transduction in a dominant-negative manner. Mutational bias was observed at the AP2sigma;2 Arg15 residue as other predicted missense substitutions (Arg15Gly, Arg15Pro and Arg15Ser), which also caused CaSR loss-of-function, were not detected in FHH probands, and these mutations were found to reduce the numbers of CaSR-expressing cells. FHH3 probands had significantly greater serum calcium (sCa) and magnesium (sMg) concentrations with reduced urinary calcium to creatinine clearance ratios (CCCR) in comparison with FHH1 probands with CaSR mutations, and a calculated index of sCa × sMg/100 × CCCR, which was ≥ 5.0, had a diagnostic sensitivity and specificity of 83 and 86%, respectively, for FHH3. Thus, our studies demonstrate AP2sigma;2 mutations to result in a more severe FHH phenotype with genotype-phenotype correlations, and a dominant-negative mechanism of action with mutational bias at the Arg15 residue.

Identification of ML204, a novel potent antagonist that selectively modulates native TRPC4/C5 ion channels.

Transient receptor potential canonical (TRPC) channels are Ca(2+)-permeable nonselective cation channels implicated in diverse physiological functions, including smooth muscle contractility and synaptic transmission. However, lack of potent selective pharmacological inhibitors for TRPC channels has limited delineation of the roles of these channels in physiological systems. Here we report the identification and characterization of ML204 as a novel, potent, and selective TRPC4 channel inhibitor. A high throughput fluorescent screen of 305,000 compounds of the Molecular Libraries Small Molecule Repository was performed for inhibitors that blocked intracellular Ca(2+) rise in response to stimulation of mouse TRPC4ß by µ-opioid receptors. ML204 inhibited TRPC4ß-mediated intracellular Ca(2+) rise with an IC(50) value of 0.96 µm and exhibited 19-fold selectivity against muscarinic receptor-coupled TRPC6 channel activation. In whole-cell patch clamp recordings, ML204 blocked TRPC4ß currents activated through either µ-opioid receptor stimulation or intracellular dialysis of guanosine 5'-3-O-(thio)triphosphate (GTP?S), suggesting a direct interaction of ML204 with TRPC4 channels rather than any interference with the signal transduction pathways. Selectivity studies showed no appreciable block by 10-20 µm ML204 of TRPV1, TRPV3, TRPA1, and TRPM8, as well as KCNQ2 and native voltage-gated sodium, potassium, and calcium channels in mouse dorsal root ganglion neurons. In isolated guinea pig ileal myocytes, ML204 blocked muscarinic cation currents activated by bath application of carbachol or intracellular infusion of GTP?S, demonstrating its effectiveness on native TRPC4 currents. Therefore, ML204 represents an excellent novel tool for investigation of TRPC4 channel function and may facilitate the development of therapeutics targeted to TRPC4.

Feedback via Ca²⁺-activated ion channels modulates endothelin 1 signaling in retinal arteriolar smooth muscle

PURPOSE: To investigate the role of feedback by Ca²?-sensitive plasma-membrane ion channels in endothelin 1 (Et1) signaling in vitro and in vivo. Methods. Et1 responses were imaged from Fluo-4-loaded smooth muscle in isolated segments of rat retinal arteriole using two-dimensional (2-D) confocal laser microscopy. Vasoconstrictor responses to intravitreal injections of Et1 were recorded in the absence and presence of appropriate ion channel blockers using fluorescein angiograms imaged using a confocal scanning laser ophthalmoscope. Results. Et1 (10 nM) increased both basal [Ca²?](i) and the amplitude and frequency of Ca²?-waves in retinal arterioles. The Ca²?-activated Cl?-channel blockers DIDS and 9-anthracene carboxylic acid (9AC) blocked Et1-induced increases in wave frequency, and 9AC also inhibited the increase in amplitude. Iberiotoxin, an inhibitor of large conductance (BK) Ca²?-activated K?-channels, increased wave amplitude in the presence of Et1 but had no effect on frequency. None of these drugs affected basal [Ca²?](i). The voltage-operated Ca²?-channel inhibitor nimodipine inhibited wave frequency and amplitude and also lowered basal [Ca²?](i) in the presence of Et1. Intravitreal injection of Et1 caused retinal arteriolar vasoconstriction. This was inhibited by DIDS but not by iberiotoxin or penitrem A, another BK-channel inhibitor. Conclusions. Et1 evokes increases in the frequency of arteriolar Ca²?-waves in vitro, resulting in vasoconstriction in vivo. These responses, initiated by release of stored Ca²?, also require positive feedback via Ca²?-activated Cl?-channels and L-type Ca²?-channels.

A plasma membrane Ca2+-ATPase (PMCA) from the liver fluke, Fasciola hepatica

Fasciolosis is a parasitic infection by the liver fluke Fasciola hepatica, which costs the global agricultural community over US $2 billion per year. Its prevalence is rising due to factors such as climate change and drug resistance. ATP-dependent membrane transporters are considered good potential drug targets as they are essential for cellular processes and are in an exposed, accessible position in the cell. Immunolocalisation studies demonstrated that a plasma membrane calcium ATPase (PMCA) was localised to the parenchymal tissue in F. hepatica. The coding sequence for a F. hepatica PMCA (FhPMCA) has been obtained. This sequence encodes a 1,163 amino acid protein which contains motifs which are commonly conserved in PMCAs. Molecular modelling predicted that the protein has 10 transmembrane segments which include a potential calcium ion binding site and phosphorylation motif. FhPMCA interacts with the calmodulin-like protein FhCaM1, but not the related proteins FhCaM2 or FhCaM3, in a calcium-ion dependent manner. This interaction occurs through a region in the C-terminal region of FhPMCA which most likely adopts an a-helical conformation. When FhPMCA was heterologously expressed in a budding yeast strain deleted for its PMCA (Pmc1p), it restored viability. Microsomes prepared from these yeast cells had calcium ion stimulated ATPase activity which was inhibited by the known PMCA inhibitors, bisphenol and eosin. The potential of FhPMCA as a new drug target is discussed.

Modulation of intracellular calcium homeostasis blocks autophagosome formation

Cellular stress responses often involve elevation of cytosolic calcium levels, and this has been suggested to stimulate autophagy. Here, however, we demonstrated that agents that alter intracellular calcium ion homeostasis and induce ER stress-the calcium ionophore A23187 and the sarco/endoplasmic reticulum Ca (2+)-ATPase inhibitor thapsigargin (TG)-potently inhibit autophagy. This anti-autophagic effect occurred under both nutrient-rich and amino acid starvation conditions, and was reflected by a strong reduction in autophagic degradation of long-lived proteins. Furthermore, we found that the calcium-modulating agents inhibited autophagosome biogenesis at a step after the acquisition of WIPI1, but prior to the closure of the autophagosome. The latter was evident from the virtually complete inability of A23187- or TG-treated cells to sequester cytosolic lactate dehydrogenase. Moreover, we observed a decrease in both the number and size of starvation-induced EGFP-LC3 puncta as well as reduced numbers of mRFP-LC3 puncta in a tandem fluorescent mRFP-EGFP-LC3 cell line. The anti-autophagic effect of A23187 and TG was independent of ER stress, as chemical or siRNA-mediated inhibition of the unfolded protein response did not alter the ability of the calcium modulators to block autophagy. Finally, and remarkably, we found that the anti-autophagic activity of the calcium modulators did not require sustained or bulk changes in cytosolic calcium levels. In conclusion, we propose that local perturbations in intracellular calcium levels can exert inhibitory effects on autophagy at the stage of autophagosome expansion and closure.

Simultaneously multiply-configurable or superposed molecular logic systems composed of ICT (internal charge transfer) chromophores and fluorophores integrated with one- or two-ion receptors

Integrated "ICT chromophore-receptor" systems show ion-induced shifts in their electronic absorption spectra. The wavelength of observation can be used to reversibly configure the system to any of the four logic operations permissible with a single input (YES, NOT, PASS 1, PASS 0), under conditions of ion input and transmittance output. We demonstrate these with dyes integrated into Tsien's calcium receptor, 1-2. Applying multiple ion inputs to 1-2 also allows us to perform two- or three-input OR or NOR operations. The weak fluorescence output of 1 also shows YES or NOT logic depending on how it is configured by excitation and emission wavelengths. Integrated "receptor(1)-ICT chromophore-receptor(2)" systems 3-5 selectively target two ions into the receptor terminals. The ion-induced transmittance output of 3-5 can also be configured via wavelength to illustrate several logic types including, most importantly, XOR. The opposite effects of the two ions on the energy of the chromophore excited state is responsible for this behaviour. INHIBIT and REVERSE IMPLICATION are two of the other logic types seen here. Integration of XOR logic with a preceding OR operation can be arranged by using three ion inputs. The fluorescence output of these systems can be configured via wavelength to display INHIBIT or NOR logic under two-input conditions. The superposition or multiplicity of logic gate configurations is an unusual consequence of the ability to simultaneously observe multiple wavelengths.

Prograded Holocene beach-ridges with superimposed dunes in north-east Ireland: mechanisms and timescales of fine and coarse beach sediment decoupling and deposition

Two depositional models to account for Holocene gravel-dominated beach ridges covered by dunes, occurring on the northern coast of Ireland, are considered in the light of infrared-stimulated luminescence ages of sand units within beach ridges, and 14C ages from organic horizons in dunes. A new chronostratigraphy obtained from prograded beach ridges with covering dunes at Murlough, north-east Ireland, supports a model of mesoscale alternating sediment decoupling (ASD) on the upper beach, rather than macroscale sequential sediment sourcing to account for prograded beach ridges and covering dunes. The ASD model specifies storm or fair-weather sand beach ridges forming at high-tide positions (on an annual basis at minimum), which acted as deflationary sources for landward foredune development. Only a limited number of such late-Holocene beach ridges survive in the observed prograded series. Beach ridges only survive when capped by storm-generated gravel beaches that are deposited on a mesoscale time spacing of 50–130 years. The morphodynamic shift from a dissipative beach face for dune formation to a reflective beach face for gravel capping appears to be controlled by the beach sand volume falling to a level where reflective conditions can prevail. Sediment volume entering the beach is thought to have fluctuated as a function of a forced regression associated with the falling sea level from the mid-Holocene highstand (ca. 6000 cal. yr BP) identified in north-east Ireland. The prograded beach ridges dated at ca. 3000 to 2000 cal. yr BP indicate that the Holocene highstand’s regressive phase may have lasted longer than previously specified.

Electron impact excitation of carbon-like calcium

Collision strengths for transitions among the energetically lowest 46 fine-structure levels belonging to the (1s(2)) 2s(2)2p(2), 2s2p(3). 2p(4), 2s(2)2p3s, 2s(2)2p3p and 2s(2)2p3d configurations of Ca XV are computed. over an electron energy range of 50 less than or equal to E less than or equal to 300 Ryd. using the recent Dirac Atomic R-matrix Code (DARC) of Norrington and Grant. All partial waves with J less than or equal to 40.5 have been included, and the contribution of higher partial waves has been added to ensure the convergence of collision strengths for all transitions and at all energies. The results are compared with those available in the literature, and the accuracy of the data is assessed.