In the rat SCN neurons, the clearance of somatic Ca2+ following depolarization-induced Ca2+ transients involves Ca2+ extrusion via Na+/Ca2+ exchanger (NCX) and mitochondrial Ca2+ buffering. Here we show an important role of intracellular Na+ in the regulation of [Ca2+]i in these neurons. The effect of Na+ loading on [Ca2+]i was determined with the Na+ ionophore monensin and the cardiac glycoside ouabain to block Na+/K+-ATPase (NKA). Ratiometric Na+ and Ca2+ imaging was used to measure the change in [Na+]i and [Ca2+]i, and cell-attached recordings to investigate the effects of monensin and ouabain on spontaneous firing. Our results show that in spite of opposite effects on spontaneous firing and basal [Ca2+], both monensin and ouabain induced Na+ loading, and increased the peak amplitude, slowed the fast decay rate, and enhanced the slow decay phase of 20 mM K+-evoked Ca2+ transients. Furthermore, both ouabain and monensin preferentially enhanced nimodipine-insensitive Ca2+ transients. Together, our results indicate that in the SCN neurons the NKA plays an important role in regulating [Ca2+]i, in particular, associated with nimodipine-insensitive Ca2+ channels.Transmembrane Ca Intr2+ flux in regulating clock genes expression, glutamate-induced phase shifts, and neuropeptide release  K+-induced Ca2+ transients involves NCX and mitochondria, with NCX mediating fast Ca2+ decay and mitochondria regulating slow Ca2+ decay and basal [Ca2+]i. In contrast, blockade of plasmalemmal and sarco(endo)plasmic reticulum Ca2+-ATPase, ryanodine receptors, or IP3 receptors had little effect on the decay kinetics i, we first determined the effects of the Na+ ionophore monensin on [Ca2+]i in the SCN neurons. In our previous studies, monensin at a concentration of 10 µM increases [Na+]i and activates Na+/K+-ATPase (NKA) to hyperpolarize the resting membrane potential [+]i to allow for Ca2+ uptake via Na+/Ca2+ exchanger (NCX) in response to the removal of external Na+ [+]i, spontaneous firing, and [Ca2+]i. The addition of 10 µM monensin increased [Na+]i , during (open circle trace), and after (grey trace) the application of 10 µM monensin indicates that 10 µM monensin reversibly increased the amplitude of Ca2+ transient and slowed the rate of Ca2+ clearance following the termination of 20 mM K+ stimulation  and presence (open circles) of monensin  in control and 20.8 ± 1.7 s  in 10 µM monensin, respectively.The monensin-induced slowing of the Caactivity . For thi2+ decay, as can be seen by superimposing the Ca2+ transient in the absence (filled dark circles) and presence (open circles) of monensin , which represents the monensin-enhanced Ca2+ transient, has a slow rate of Ca2+ rise and decay. Notably, there is a delayed onset of Ca2+ clearance, when [Ca2+]i appears to increase (marked by arrow), after the termination of 20 mM K+ stimulation (marked by dotted line). Together, the results indicate that in addition to slowing the fast Ca2+ decay, monensin-induced Na+ loading also enhances the slow decay phase, which is thought to be associated with mitochondrial Ca2+ buffering i in the SCN neurons i, could play an important role in the regulation of NCX activity. For comparison with the effects of monensin, ouabain at a concentration of 10 µM was also applied to determine its effects on [Na+]i, spontaneous firing, and [Ca2+]i , and thereafter 20 mM K+ solution elicited Ca2+ responses with progressively larger amplitude and slower decay on repetitive application to reach steady state generally after ~15 min (traces d–h) (2+ transients in control (trace a) and 5 min into ouabain (trace b) indicates a smaller amplitude and a slightly slower kinetics in ouabain (2+ transients (inset). On the other hand, superimposition of the Ca2+ transients in ouabain indicates an increase in the amplitude and a slowing in the fast decay rate from 5 (trace b), 10 (trace c), to 15 (trace d) min and then leveling off thereafter to 35 (trace h) min into the application of ouabain   to 15.0 ± 1.0 s .As 10 µM ouabain evoked a biphasic, or even triphasic see B, effect ouabain . The resces d–h) A. Superi ouabain B, as can ouabain C. This ices d–h) D. Note t trace h E. On ave2+ decay, as evidenced by superimposing the Ca2+ transient in the absence (filled dark circles) and presence (open circles) of ouabain, as well as the digitally subtracted Ca2+ transient enhanced by ouabain   after the termination of 20 mM K+ stimulation. Comparison of normalized Ca2+ in ouabain and monensin  F. The ousin from  indicatemonensin . Taken t2+ transients evoked after ~15 min into 10 µM ouabain have virtually identical fast decay but widely varied slow decay phase, suggesting that the slow decay phase as observed in ouabain is not associated to the fast decay mediated by Ca2+ extrusion via NCX, the activity of which depends on both extracellular i when Ca2+ transient was elicited by 20 mM K+. This is evident by comparing trace d with trace f  to 20 mM K+ in the absence and presence of 10 µM ouabain to demonstrate the appearance of prominent slow decay phase in parallel to the decrease of basal level of [Ca2+]i back to or even below the resting level, following an initial increase by ouabain. 2+ response in ouabain from three representative cells. For the first cell (top), after an initial increase in ouabain the basal level of [Ca2+]i returned to near its resting level for as long as 30 min along with the appearance of prominent slow decay phase (marked by arrowheads) for the 20 K+-evoked Ca2+ transients. For the second cell (middle), after an initial increase in ouabain the basal [Ca2+]i return to near its resting level for about 10 min only to increase back to a level similar to or higher than the initial increase by ouabain. Notably, the slow decay phase for the Ca2+ transient appeared (marked by arrowheads) when the basal [Ca2+]i decreased back to close to the resting level and then disappeared (marked by arrows) altogether when the basal [Ca2+]i increased again to the higher levels. For the third cell (bottom), there is only a small decrease in basal [Ca2+]i following ouabain-induced initial increase and so is only a small increase in the slow decay phase.2+]i and the appearance of plateau-like slow decay phase in ouabain. Nevertheless, it is worthy of mentioning that high K+-evoked Ca2+ transients with pronounced plateau slow decay are known to be present in peripheral neurons, with the plateau being attributed to mitochondrial Ca2+ uptake and release i may increase the electrochemical potential for Ca2+ influx to enhance Ca2+ response to 20 K+-evoked depolarization. In particular, monensin slows the fast Ca2+ decay, suggesting that monensin-induced Na+ loading inhibits forward NCX activity [We show that 10 µM monensin increases [Na [Ca2+]i . The resotential . Unlike 2+ transient has a slow rate of Ca2+ rise and decay, with the latter characterized by a delay of continuing increase in [Ca2+]i before the slow Ca2+ decay .2+ transient, 1 μM monensin had minimal effect on the amplitude and the fast decay phase of Ca2+ responses to 20 mM K+ solution in this study. The unexpected result is contrary to our previous finding that 1 μM monensin increases intracellular [Na+]i to promote Ca2+ uptake via reverse NCX activity in response to the removal of extracellular Na+ [+ concentration around the NCX might not be high enough to significantly inhibit forward NCX activity.In contrast to the marked effects of 10 μM monensin on the Ca2+]i in view of our recent finding showing that intracellular pH also plays a role in the regulation of [Ca2+]i in rat SCN neurons i .Contrary to the suppressive effect of monensin, inhibition of NKA with 10 µM ouabain increases both spontaneous firing and basal [Ca2+]i . The oua neurons . Howeveron block . In cont+-evoked Ca2+ transient, i.e., increasing the amplitude and slowing the kinetics of Ca2+ transients, except for its initial inhibition of the nimodipine-sensitive Ca2+ transient. Our results indicate that the inhibition of NKA with 10 µM ouabain increases basal [Ca2+]i and inhibits 20 K+-induced Ca2+ transients at 5 min into ouabain, and then gradually increases the amplitude and slows the kinetics of Ca2+ transients, on top of delayed decrease in basal [Ca2+]i, to reach steady state after ~15 min. Notably, the time course of ouabain-induced changes in the Ca2+ transients is in parallel to that of ouabain-induced Na+ loading, suggesting that the gradual increase in cytosolic Na+ and decrease in the transmembrane Na+ gradient bring about these changes.Nevertheless, ouabain is similar to monensin in its effects on the 20 K+ loading inhibits forward NCX activity. Second, similar to the monensin-enhanced Ca2+ transient, the slow decay of the digitally subtracted ouabain-enhanced Ca2+ transient is also characterized by a delay of slightly elevated [Ca2+]i before the slow Ca2+ decay , in spite of its ability to increase [Na+]i in the SCN neurons as shown in our previous study i has began to decrease back to or even below the resting level, as if ouabain-induced increase in basal [Ca2+]i actually promoted mitochondrial Ca2+ uptake to bring it down. If this is the case, then a functional linkage between NKA and mitochondria should play an important role in regulating Ca2+ entering the nimodipine-insensitive Ca2+ channels. Furthermore, as Ca2+ entering mitochondria is known to activate dehydrogenase to increase oxidative phosphorylation i as presented in this study suggests a mechanism how metabolic stress such as glucose shortage could alter the circadian functioning of the SCN . Met neurons  and the  neurons . This is the SCN . Further2–5% CO2. The ACSF contained (in mM): 125 NaCl, 3.5 KCl, 2 CaCl2, 1.5 MgCl2, 26 NaHCO3, 1.2 NaH2PO4, 10 glucose. A coronal slice (200–300 µm) containing the SCN and the optic chiasm was cut with a DSK microslicer DTK-1000 , and was then incubated at room temperature (22–25 °C) in the incubation solution, which contained (in mM): 140 NaCl, 3.5 KCl, 2 CaCl2, 1.5 MgCl2, 10 glucose, 10 HEPES, pH 7.4, bubbled with 100% O2.All experiments were carried out according to procedures approved by the Institutional Animal Care and Use Committee of Chang Gung University . Sprague–Dawley rats (18–24 days old) were kept in a temperature-controlled room under a 12:12 light:dark cycle (light on 0700–1900 hr). Lights-on was designated Zeitgeber time (ZT) 0. For daytime (ZT 4–11) and nighttime (ZT 13–20) recordings, the animal was killed at ZT 2 and ZT 10, respectively. Hypothalamic brain slices and reduced SCN preparations were made as described previously . An anim2+ and Na+ imaging, a reduced SCN preparation was obtained by excising a small piece of tissue (circa one-ninth the size of SCN) from the medial SCN using a fine needle , followed by further trimming down to 4–10 smaller pieces with a short strip of razor blade. The reduced preparation was then transferred to a coverslip precoated with poly-D-lysine  in a recording chamber for recording. The SCN cells of the reduced preparation could be identified visually with an inverted microscope . The preparation thus obtained allows rapid application of drugs [+ and Ca2+ fluorescent imaging [For electrical recordings and fluorescent Caof drugs  and has  imaging  and to d imaging .2, 1.5 MgCl2, 10 glucose, 10 HEPES, pH adjusted to 7.4 with NaOH. All recordings were made with Axopatch 200B amplifier  at room temperature (22–25 °C). The spontaneous firing rate was recorded in the cell-attached configuration. The patch electrode was filled with the bath solution or with the patch solution containing (in mM): 20 NaCl, 1 CaCl2, 2 MgCl2, 110 K-gluconate, 11 EGTA, 10 HEPES, 3 Na-ATP, 0.3 Na-GTP, pH adjusted to 7.3 with KOH. The spike counts, in 6-s epochs, always began only after stable recordings were made. At least one or two minutes of spontaneous firing rate were counted before the application of drugs. The signal was low-pass filtered at 1–5 KHz and digitized on-line at 2–10 KHz via a 12-bit A/D digitizing board  with a custom-made program written in the C Language.Patch clamp recording was carried out as described previously . The red2+ and Na+ imaging was performed, respectively, by pre-loading the SCN cells with the Ca2+-sensitive fluorescent indicator Fura2-acetoxymethyl ester (Fura2-AM) [+-sensitive fluorescent indicator sodium-binding benzofuran isophthalate (SBFI-AM) [+ and 0.5 Hz for Ca2+. Ca2+ and Na+ levels in regions of interest (ROI) over the soma were spatially averaged and presented by fluorescence ratios (F340/F380) after background subtraction. Data were analyzed and plotted with custom-made programs written in Visual Basic 6.0 and the commercial software GraphPad PRISM . Data were given as means ± SEM and analyzed with paired t-test.Ratiometric fluorescence imaging was carried out as described previously ,14. Fluoura2-AM)  and the SBFI-AM) . The red+ solution was prepared with equal molar substitution of K+ for Na+. All solutions were adjusted to pH 7.4 before use.Stock solutions of nimodipine (20 mM in DMSO) and monensin (10 mM in 100% ethanol) were stored at –20 °C and were diluted at least 1000 times to reach desired final concentrations. Nimodipine was purchased from Tocris Cookson , and ouabain and monensin from Sigma-Aldrich . 20 mM K


Mitochondrial dysfunction and specifically mitochondrial Ca2+ handling has been linked to the pathogenesis of PD. Here we describe for the second time a mitochondrial Ca2+ efflux deficiency in a model displaying alterations in a PD-associated risk protein. LRRK2 deletion, inhibition and mutations led to an impaired mitochondrial Ca2+ extrusion via Na+/Ca2+/Li+ exchanger (NCLX) which in turn lowered mitochondrial permeability transition pore (PTP) opening threshold and increased cell death. The mitochondrial membrane potential was found not to be the underlying cause for the Ca2+ extrusion deficiency. NCLX activity was rescued by a direct (phosphomimetic NCLX mutant) and indirect (protein kinase A) activation which in turn elevated the PTP opening threshold. Therefore, at least two PD-associated risk protein pathways appear to converge on NCLX controlling mitochondrial Ca2+ extrusion and therefore mitochondrial health. Since mitochondrial Ca2+ overload has been described in many neurological disorders this study warrants further studies into NCLX as a potential therapeutic target.Variants of leucine-rich repeat kinase 2 ( One of the most extensively studied genes containing these mutations is the leucine-rich repeat kinase 2. In particular, the missense mutation G2019S has been reported to be the underlying cause for PD in 1–2% in the UK/USA rising to 10% in Ashkenazi Jews and 40% in North African Arab Berbers3. LRRK2 possesses a kinase and a ROC-GTPase domain whose exact physiological function is yet to be fully unravelled but studies provided evidence for a role of LRRK2 in vesicle trafficking, inflammatory responses, autophagy and neurite outgrowth7; with few phosphorylation substrates identified9. The multitude of pathways regulated by LRRK2 demonstrate the complexity of LRRK2 mutations and their potential effects on cellular signalling pathways. In PD, it has long been known that LRRK2 mutations (in particular G2019S) lead to an increased kinase activity and this upregulation is currently being targeted by LRRK2 kinase inhibitors as a therapeutic agent in PD12.Parkinson’s disease is a common, disabling, incurable neurodegenerative condition affecting 1% of the population over the age of 60. The past 10 years have seen a shift in the aetiological understanding of PD, moving from a nearly exclusively environmentally mediated disease towards a complex disorder with important genetic contributions2+ homoeostasis suggesting a potential role for the recently described sodium Ca2+ exchanger (NCLX) in PD pathology15. While mitochondrial Ca2+ shuttling couples between Ca2+ signalling and metabolic activity, its impairment can lead to mitochondrial Ca2+ overload that can be a trigger for neuronal cell death. We have shown that the mitochondrial Ca2+ overload phenotype in PINK1 deficient cells can be rescued by a PKA/NCLX-mediated pathway16. Furthermore, α-synuclein has been linked to Ca2+-induced cell death18 suggesting that inhibition of mitochondrial Ca2+ efflux may be a common feature in the mechanism of neurodegeneration observed in PD. To test this hypothesis, we used LRRK2 deficient cells and assessed mitochondrial Ca2+ homoeostasis.Mitochondrial dysfunction is strongly implicated in PD as toxic models of PD which employ mitochondrial toxins as well as the majority of proteins associated with familial forms of PD affect mitochondrial function. Recently, we have shown how another PD-associated protein, PINK1, is involved in mitochondrial Ca2+ homoeostasis is a common phenotype in PD and not specific to PINK1 deficiency only.This current study established that mitochondrial CaWild-type and LRRK2 KO mice were obtained from breeding colonies generated by Jackson Laboratories and Sprague Dawley rat pups (1–3 days postpartum) were acquired from the University College London breeding colony. Wild-type rat of either sex were used for neuronal co-cultures and experimental procedures were performed in full compliance with the United Kingdom Animal (Scientific Procedures) Act of 1986.2+/Mg2+ free PBS . The tissue was minced and trypsinised , triturated and plated on poly-D-lysine-coated coverslips. The tissue was cultured in Neurobasal A medium  supplemented with B-27  and 2 mM GlutaMAX 19. Cultures were maintained at 37 °C in a humidified atmosphere of 5% CO2 and 95% air; media was changed twice weekly and maintained for a minimum of 12 days before experimental use to ensure expression of glutamatergic and other receptors. Only neurons were used for analysis. Neurons were distinguishable from glia: they appeared phase bright, had smooth rounded somata and distinct processes, and lay just above the focal plane of the glial layer. Cells were used at 12–15 days in vitro. Experimental procedures were performed in full compliance with the United Kingdom Animal (Scientific Procedures) Act of 1986.Mixed cultures of cortical and midbrain neurons and glial cells were prepared from postnatal pups . Midbrain were removed and placed into ice-cold Ca20. Biopsies were dissected into ~1mm pieces and cultured in DMEM, 10% FBS, 1% GlutaMAX until fibroblasts were seen to grow out from the explants. When fibroblasts reached confluency, they were detached from culture dishes using TrypleE (Invitrogen) and transferred to larger culture vessels for further expansion and cryopreservation.Fibroblasts were generated from a skin punch biopsy taken under local anaesthetic following local ethical approval and full informed consent21. Plasmids were transfected using Effectene (Qiagen) according to manufacturers protocol. NCLX plasmids were co-transfected with an empty GFP vector (pcDNA3 backbone) to allow for selection of transfected cells for experimentation and analysis.Preparation of human WT NCLX plasmids and site-directed mutagenesis was carried out as previously described2+ imaging, cells were loaded for 30 min with 0.005% Pluronic and the Ca2+ indicator Fluo-4 AM . Mitochondrial Ca2+ was visualised by pre-loading cells with 0.005% Pluronic and either Rhod5N AM  or Rhod-2 AM  for 20 min. Whole-cell imaging was carried out in a HEPES-buffered salt solution (HBSS). Cells were loaded for 30 min with 0.005% Pluronic and CoroNa green AM  to visualise Na+.For cytosolic CaFor TMRM experiments, cells were placed in a HBSS containing 25 nM TMRM for 40 min at room temperature. TMRM was excited using the 560 nm laser line and fluorescence was measured > 580 nm using a Zeiss 710 VIS CLSM equipped with a META detection system and a ×40 oil-immersion objective. Z-stack images were obtained by confocal microscopy and the basal Δψm was measured using Zen software (Zeiss).2+ efflux was assessed in permeabilized cells. To permeabilize, a buffer  containing low concentration of digitonin (20 μM) was added to the cells. Upon permeabilization, the buffer and digitonin were replaced by fresh buffer containing mitochondrial substrates . Cells were allowed to rest for at least 5 min before measurements were taken. 20 μM CaCl2 was applied to assess mitochondrial Ca2+ handling. Confocal images were obtained using a Zeiss 710 equipped with a META detection system and a ×40 oil-immersion objective. Mitochondrial Ca2+ measurements were undertaken using the 543 nm laser line and 560 nm longpass filter. A 488 nm Argon laser line was used to excite Fluo-4 which was measured between 510 and 550 nm. The pinhole set to give an optical slice of ~ 2 μm and illumination intensity was kept to a minimum (at 0.1–0.2% of laser output) to avoid phototoxicity.Mitochondrial Ca2+ ionophore ferutinin in stepwise fashion22. Briefly, ferutinin induces a rise in mitochondrial calcium and the concentration of ferutinin required to induce PTP opening  was determined.PTP opening threshold was determined with increasing concentrations of the electrogenic CaCells were incubated with propidium iodide  and Hoechst 33342 . Viable cells exclude the red fluorescent PI whereas Hoechst stains chromatin blue in all cells thus allowing dead cells to be quantified.p < 0.05.Statistical analysis and exponential curve fitting were performed using Origin 9 software. Experimental data are shown as means ± SEM. Statistical analysis between samples was performed using a one-way ANOVA with Bonferroni correction. Differences were considered to be significantly different if 2+ homeostasis. To do so, we used primary neuron/glia co-cultures from the cerebral cortex/midbrain of wild-type (WT) and LRRK2 knockout (KO) mice. Both cytosolic and mitochondrial Ca2+ was assessed upon adenosine triphosphate (ATP) application. Intracellular Ca2+ levels are tightly controlled by the ER which is the main Ca2+ store and mitochondria which “fine-tunes” Ca2+ transients. Exogenous applied ATP triggers an intracellular Ca2+ release from the endoplasmic reticulum (ER) via P2Y receptors followed by activation of the phospholipase C and IP3 pathway in astrocytes23. The Ca2+ release from the ER into the cytosol leads to a simultaneous increase in mitochondrial Ca2+. Application of ATP led to increased cytosolic and mitochondrial Ca2+ in WT astrocytes and Ca2+ levels recovered back to basal levels typically within 1 min  in KO mitochondria when compared with WT  and, to a lesser extent, by other efflux pathways. NCLX translocates Na+ and Ca2+ ions across the inner mitochondrial membrane which can be visualised by loading cells with CoroNA (Na2+ indicator) as well as xRhod1 . In permeabilized WT neurons, application of CaCl2 resulted in an increase in mitochondrial Ca2+ and decrease in mitochondrial Na+, before Ca2+ is extruded and Na+ is taken up by the mitochondria , CZC-25146 (5 µM) or DMSO vehicle control and mitochondrial Ca2+ homoeostasis was assessed in neurons. Inhibition of LRRK2 kinase mirrored the significant inhibited mitochondrial Ca2+ efflux observed in KO mitochondria . As a positive control, the NCLX inhibitor CGP 37157 (10 µM) was employed which mirrored the significantly inhibited mitochondrial Ca2+ efflux similar to that observed in LRRK2 deficient models . Figure 2+ uptake was not significantly different when compared with control. Further, non-normalised data mirror the normalised efflux data.Inhibition of kinase activity (loss of function) was employed to confirm the mitochondrial Ca2+ buffering was a common phenotype in PD as well as LRRK2 deficiencies. Indeed, the mitochondrial Ca2+ efflux was significantly inhibited in G2019S , R1441G  and Y1699C  when compared with the three matched control cell lines , R1441G (ROC domain) or Y1699C (COR domain) mutation and unaffected controls were employed to assess whether mitochondrial Capes Fig. 2+ efflux deficiency is likely to lower the mitochondrial Ca2+ buffering capacity. To test this, we employed a natural electrogenic Ca2+ ionophore ferutinin to promote mitochondrial Ca2+ overload independently of MCU and trigger Ca2+-dependent PTP opening25. PTP opening can be visualised by a significant drop in ΔΨm  where an altered potential could lead to the observed inhibited mitochondrial Ca2+ efflux in LRRK2 deficiency. Therefore, we assessed ΔΨm in LRRK2 KO, LRRK2 inhibitor treated neuronal co-cultures and LRRK2 mutation bearing fibroblasts  and astrocytes  when compared with WT  and astrocytes  when compared with cultures treated with DMSO vehicle control  prevents mitochondrial Ca2+ overload in a PINK1 deficient model16. Hence, this study expressed NCLXS258D in LRRK2 KO primary cultures and mitochondrial Ca2+ was recorded in neurons. Expression of NCLXS258D restored the efflux back to levels similar to those observed in WT neurons  and CZC-25146  inhibits mitochondrial Ca2+ efflux when compared with DMSO vehicle control  or CZC-25146 .An indirect upregulation of NCLX can be achieved through pre-exposure to the PKA activator forskolin. Forskolin activates adenylyl cyclase which in turn raises cyclic AMP (cAMP) levels. cAMP is required for PKA activation which in turn enables NCLX phosphorylation and therefore NCLX activation. WT neuronal co-cultures were pre-exposed to both LRRK2 inhibitors and forskolin before mitochondrial Caons Fig. . As prev2+ efflux was assessed. Forskolin restored the efflux rate in G2019S (0.29 ± 0.03), R1441G (0.29 ± 0.02) and Y1669C (0.31 ± 0.04) back to levels observed in unaffected control cell line , 58% of Y1699C cells  and 60% of R1441G cells  opened PTP at this forskolin concentration. Pre-incubation of control fibroblasts with forskolin also significantly increased the Ca2+ handling capacity in control 1 and control 3 cells  and CZC-25146 to 44.6% ( ± 2.6%) when compared with basal cell death of 15.7% .Since inhibition of NCLX leads to a slower mitochondrial Ca2+ homoeostasis28. Mitochondrial Ca2+ overload has a direct effect on mitochondrial bioenergetics and lowers the threshold for PTP opening leading to premature cell death30 and has previously been demonstrated in loss-of-function, recessively inherited mutations of PINK131.Mitochondrial dysfunction has been linked to both familial and sporadic forms of PD. Several PD risk genes, such as PINK-1, Parkin and DJ-1 have been associated with mitochondrial pathogenesis observed in PD with a key role in mitochondrial Ca2+ efflux; therefore linking another PD risk gene to NCLX leading to decreased PTP opening threshold and an increased dopamine-induced neuronal death , inhibition (de-phosphorylation) or mutagenesis of LRRK2 alters the mitochondrial Cath Figs. , 2, 5. Lm can result in reduced NCLX Ca2+ efflux17. However, ΔΨm in all LRRK2 models employed in this current study is either not changed (fibroblasts) or elevated (inhibitor/KO) when compared with control conditions suggesting that the ΔΨm is not the underlying reason for the mitochondrial Ca2+ efflux deficiency  and could not find any significant changes in the ΔΨm.We and other have previously described that a lower ΔΨncy Fig. . Papkovs2+ dysregulation can lead to mitochondrial Ca2+ dysregulation, For example, Bedford et al.34 have provided evidence that LRRK2 stimulates voltage-gated Ca2+ channels, while the PD mutation G2019S leads to an increased channel stimulation when compared with wild-type LRRK2. Furthermore, LRRK2 has been proposed to stimulate plasma membrane sodium/calcium exchanger activity35 and nicotinic acid adenine dinucleotide phosphate receptors36. These reports provide evidence for a global Ca2+ regulation through LRRK2 where mutations can lead to a Ca2+ dysregulation affecting organelles and cellular functions. Indeed, Verma et al.37 has shown that mitochondrial Ca2+ contributes to dendrite injury in a LRRK2 PD model. The authors have also reported transcriptional upregulation the mitochondrial calcium uniporter and the mitochondrial calcium uptake 1 protein leading to increased mitochondrial Ca2+ influx. Interestingly, we did not observe an altered mitochondrial Ca2+ uptake in any of the models employed in this current study indicating other driving factor(s) for the mitochondrial Ca2+ efflux pathology 2+ efflux in PINK1 deficient models16. In this current study expression of the constitutively active phosphomimetic mutant of NCLX (NCLXS258D) also rescued the mitochondrial Ca2+ efflux in LRRK2 mutation bearing fibroblast and LRRK2 inhibitor treated cells in a similar manner previously described in a PINK PD model45  restored the Ca2+ buffering capacity back to control levels in LRRK2 mutation bearing fibroblasts  which can have particular detrimental effects in cells with a high-energy demand such as dopaminergic neurons which are predominantly affected in PD47. Here, we have shown that LRRK2 inhibitors make neurons more prone to dopamine-induced cell death (Ca2+-dependent). By upregulation of NCLX and therefore enhancing mitochondrial Ca2+ efflux, we were able to rescue this dopamine-induced neuronal cell death . This phenotype can be rescued by an upregulation of NCLX via a direct or indirect cAMP/PKA-dependent approach which may offer a novel therapeutic strategy in PD. The observed phenotype associated with LRRK2 modulation may be of particular importance when employing LRRK2 inhibitors as mitochondrial bioenergetics are likely to be negatively affected. Prolonged mitochondrial Ca2+ dysregulation has detrimental bioenergetic consequences and is of particular importance to high-energy-demanding cells such as neurons. Since mitochondrial Ca2+ dysregulation is a common phenotype in many neurodegenerative disease models NCLX and its regulators may serve as a novel therapeutic strategy.This study provides evidence that mitochondrial Ca


Alterations in the expression, activity and regulation of Ca2+-transport systems both at the plasma membrane and at organelles like the endoplasmic reticulum (ER) and mitochondria have been implicated in oncogenesis and neoplasia 12+-signaling events that could favor resistance to cell death, migration or senescence escape Intracellular Ca2+ exchanges between the ER, the main intracellular Ca2+-storage organelle, and the mitochondria are pivotal for cell-fate decisions 3672+-flux systems: the IP3 receptors (IP3Rs) at the ER side and the voltage-dependent anion channels (VDACs) at the mitochondrial outer membrane side 10112+ fluxes between ER and mitochondria sustain anabolic pathways for mitochondrial metabolism, ensuring proper cell cycle progression 2+ transfers result in loss of mitochondrial membrane integrity and release of apoptogenic factors 2+ transfer is therefore key to cells’ response to pro-apoptotic stimuli: the failure of which results in cell death resistance, as often observed in cancer cells 152+ exchanges 3R isoform (IP3R3) 3R isoforms can contribute to the initiation of cell death programs 19Over the last decade, we learnt that tight contacts and functional connections involving Ca3R3 in pro-apoptotic Ca2+ transfers from ER to mitochondria might relate to its ability to preferentially partner with the VDAC1 complex 2+ homeostasis and dynamics 102+ fluxes, whereas oncogenes suppress ER-mitochondrial Ca2+ fluxes. The actions of both tumor suppressors and oncogenes at the ER can involve changes of the steady-state ER Ca2+-filling state through modulation of sarco/endoplasmic reticulum Ca2+ ATPases (SERCA) and/or ER Ca2+-leak channels. For instance, during stress, the tumor suppressor p53 accumulates at the ER and enhances the Ca2+-pump activity SERCA, causing ER Ca2+ overload and the likelihood for pro-apoptotic ER-mitochondrial Ca2+ fluxes 22233R phosphorylation and its sensitivity to IP3, enhancing the passive Ca2+ leak from the ER, lowering ER Ca2+ levels and so pro-apoptotic ER-mitochondrial Ca2+ fluxes 3R, whereby tumor suppressors  and oncogenes  that respectively promote and suppress the activity of IP3R channels by impacting their gating and consequently their open probability 103R gating, it is clear that total IP3R-protein levels impact Ca2+ flux from the ER to the mitochondria and in turn cellular sensitivity to death as well. Insights are now available on IP3R degradation by ER-assisted and 26S proteasomal turnover after cell stimulation and IP3R activation 3Rs become ubiquitinated due to recruitment of the erlin1/2 complex and RNF170, an E3 ubiquitin ligase 2728In some cells, the role played by the IP3R turn-over and controlling their steady-state in stressed cells; equally, whether dysregulation of these mechanisms was involved in oncogenesis and/or tumor progression. Nevertheless, it is clear that IP3R levels do impact apoptotic sensitivity 2030313R levels 33Hitherto, not much was known about the molecular mechanisms impacting basal IPet al. revealed an unexpected role for the tumor suppressor lipid/protein phosphatase PTEN, an allele frequently lost in cancer 3R channels by protecting them from proteasomal degradation Fig. 1). This novel function adds to its recently discovered presence at the mitochondria-associated membranes (MAMs), where it contributes to cell death sensitivity by suppressing IP3R3-mediated Ca2+ fluxes 383R3 channels, in particular to a region in the ligand-binding domain. Normal cells that express PTEN will have a low level of the IP3R3/FBXL2-complex formation, preventing the ubiquitination of IP3R3 channels and subsequent targeting to the proteasome. Consistently with previous observations 273R3-complex formation and subsequent IP3R3 post-transcriptional regulation via ubiquitination similarly to RNF170-mediated ubiquitination of IP3Rs 2+ dependent, but antagonized by calmodulin 3Rs may not only make these channels more susceptible for degradation by increased interaction with FBXL2 but release of Ca2+ itself through IP3Rs may trigger local activation of FBXL2 associated with IP3R3 leading to IP3R3 degradation. Notably, FBXL2 binding to its substrates (like cyclin D3) was found to occur via canonical calmodulin-binding motif thereby preventing ubiquitination. It is nonetheless likely that FBXL2 binding to IP3Rs does not occur by targeting its calmodulin-binding motif even though a role for calmodulin in regulating IP3R3/FBXL2-complex formation may not be ruled out. In any case, access to the degron region in the ligand-binding core of IP3R3 for FBXL2 is facilitated by deletion of the suppressor domain and loss of PTEN is associated with increased FBXL2 binding to IP3R3 and degradation of IP3R3, contributing to the apoptotic resistance of cells 3R3 levels and apoptotic sensitivity.Recent work from Kuchay 3R3 the FBXL2 binding and IP3R3 degradation were both prevented. Using a knockin approach in which wild-type IP3R3 was altered into a non-degradable IP3R3 mutant version (IP3R3Q550A/F553A/R554A), the authors could restore the rise of Ca2+ induced by photodynamic therapy and apoptosis in PTEN-negative cancer cells. This correlation was eloquently observed in human tumor samples beyond the convincing in vivo xenograft models, in which tumor cells expressing a non-degradable IP3R3 version or treated with geranylgeranyltransferase inhibitor  Fig. 1). BAP1 is a potent tumor suppressor, which protects against environmental stress and damage 3R3-interacting protein that impacts its post-translational modification. BAP1 causes IP3R3 stabilization and prevents the channel to be degraded by the proteasome. Loss of only 1 allele in BAP1 is sufficient to protect cells from undergoing apoptotic cell death via suppressed Ca2+ release triggered by apoptotic stimuli like H2O2. An effect operated via the IP3R3 degradation due to a decreased BAP1-mediated deubiquitylation of IP3R3. As a consequence, exposure of cells being BAP1+/- to DNA-damaging conditions will result in a higher percentage of cells surviving despite having damaged DNA. Such cells bearing genomic aberrations are at high risk for neoplastic behavior and oncogenesis, resulting in malignant cell growth and tumor formation. However, restoring IP3R3 in these cells could be an attractive strategy to reinstate cell death sensitivity of BAP1+/- cells.In a separate study, Bononi 3R3 ubiquitination observed when BAP1 levels are declined is mediated through FBXL2 and whether lack of BAP1 results in increased FBXL2 association with IP3R3 channels. In any case, by inhibiting FBXL2’s action on IP3R3 without inhibiting its action on cell cycle targets could be an attractive avenue to restore IP3R3 levels in these cancers. Another strategy can be boosting ER Ca2+-store loading in tumor cells by activating the SERCA pumps, thereby increasing the likelihood for ER-mitochondrial Ca2+ fluxes and restoring cellular sensitivity to apoptosis 222223Based on these recent developments, it will be therefore important to examine the interplay between BAP1 and FBXL2. It is indeed not clear whether the increased IP3R3 ubiquitination homeostasis not only impacts death and survival of cells but also contributes to the oncogenic behavior of cells with dysfunctional tumor suppressors by either (i) lacking PTEN 2+ signaling at the MAM level as a key event in apoptosis resistance that contributes early events associated with oncogenesis and tumor formation. Challenging will be the translation of these insights into anti-cancer therapies for which not only tumor-selective applications will be required but also further understanding in the selective targeting at the level of the IP3R3.These recent papers highlight that deregulation of IP


The efficiency of this process is dependent on the density and macroscale arrangement of dyads, but also on the nanoscale organization of LTCCs and RyRs within them. We presently review accumulating data demonstrating the remarkable plasticity of these structures. Dyads are known to form gradually during development, with progressive assembly of both t-tubules and junctional SR terminals, and precise trafficking of LTCCs and RyRs. While dyads can exhibit compensatory remodeling when required, dyadic degradation is believed to promote impaired contractility and arrythmogenesis in cardiac disease. Recent data indicate that this plasticity of dyadic structure/function is dependent on the regulatory proteins junctophilin-2, amphiphysin-2 (BIN1), and caveolin-3, which critically arrange dyadic membranes while stabilizing the position and activity of LTCCs and RyRs. Indeed, emerging evidence indicates that clustering of both channels enables “coupled gating”, implying that nanoscale localization and function are intimately linked, and may allow fine-tuning of LTCC-RyR crosstalk. We anticipate that improved understanding of dyadic plasticity will provide greater insight into the processes of cardiac compensation and decompensation, and new opportunities to target the basic mechanisms underlying heart disease.Contraction of cardiomyocytes is dependent on sub-cellular structures called dyads, where invaginations of the surface membrane (t-tubules) form functional junctions with the sarcoplasmic reticulum (SR). Within each dyad, Ca Ca2+ influx through LTCCs triggers release of Ca2+ from ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR), and contraction as this released Ca2+ binds to the myofilaments. Tight control of contractility thus requires efficient crosstalk between LTCCs and RyRs, which is afforded by close apposition of the sarcolemmal and SR membranes at junctions called dyads , where they are oriented along the long axis of the cell  and rapid contraction. Of note, findings from a range of species indicate that RyR organization has greater regularity than the t-tubule network, resulting in the presence of “orphaned” or non-junctional RyRs along z-lines which do not have colocalized t-tubules  including hyperosmotic shock  and super-resolution imaging have indicated that dyads are not completely filled with RyRs, but often contain multiple, smaller RyR clusters  which is capable of initiating t-tubule growth even in non-muscle cells , which is known to play an important role in the formation of caveolae and t-tubules , interac surface , likely  surface . The Gor surface . They fu surface . These f2+ influx is rapid and large enough to drive efficient RyR opening during Ca2+-induced Ca2+ release. In light of findings described above, it seems plausible that loss of t-tubular L-type current during heart failure   , and mays (CRUs) . EM datas (CRUs) . This prs (CRUs) , 6. We hctivated . Slowingransient . We obseillation . An assoWhat is the timescale of RyR cluster dynamics? Some insight has been provided by a recent study employing direct visualization of RyR clusters using GFP-labeled RyR in live cells . The aut2+ were found to promote movement whereas conditions known to suppress RyR function such as low Ca2+ and tetracaine retarded their movement . This raises the possibility that hyperactivity of the RyR, a hallmark of diseased cardiomyocytes  but with many channels present in side-by-side or disordered arrangements  and checkerboard (further classified as oblique and center-to-corner). At low Ca2+ concentrations the channels were found to be relatively evenly distributed between the two conformations but, consistent with Asghari et al., stimulatory conditions (high Ca2+) favored an oblique and center-to-corner conformation, the basis of the crystalline array . The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We quantify neuronal Ca2+ activity using intracellular Ca2+ imaging in cultured hippocampal neurons and confirm that FIV envelope glycoprotein gp95 also elevates neuronal Ca2+ activity. In addition, we reveal that gp95 interacts with the chemokine receptor, CXCR4, and facilitates the release of intracellular Ca2+ by the activation of the endoplasmic reticulum (ER)-associated Ca2+ channels, inositol triphosphate receptors (IP3Rs), and synaptic NMDA receptors (NMDARs), similar to HIV gp120. This suggests that HIV gp120 and FIV gp95 share a core pathological process in neurons. Significantly, gp95’s stimulation of NMDARs activates cGMP-dependent protein kinase II (cGKII) through the activation of the neuronal nitric oxide synthase (nNOS)-cGMP pathway, which increases Ca2+ release from the ER and promotes surface expression of AMPA receptors, leading to an increase in synaptic activity. Moreover, we culture feline hippocampal neurons and confirm that gp95-induced neuronal Ca2+ overactivation is mediated by CXCR4 and cGKII. Finally, cGKII activation is also required for HIV gp120-induced Ca2+ hyperactivation. These results thus provide a novel neurobiological mechanism of cGKII-mediated synaptic hyperexcitation in HAND.Over half of individuals infected with human immunodeficiency virus (HIV) suffer from HIV-associated neurocognitive disorders (HANDs), yet the molecular mechanisms leading to neuronal dysfunction are poorly understood. Feline immunodeficiency virus (FIV) naturally infects cats and shares its structure, cell tropism, and pathology with HIV, including wide-ranging neurological deficits. We employ FIV as a model to elucidate the molecular pathways underlying HIV-induced neuronal dysfunction, in particular, synaptic alteration. Among HIV-induced neuron-damaging products, HIV envelope glycoprotein gp120 triggers elevation of intracellular Ca Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HANDs) occur in as many as 50% of individuals infected with HIV, including patients receiving combination antiretroviral therapy (cART). Notably, while neuronal death is mitigated with cART, neuronal dysfunction persists. This study investigates HAND-associated alteration of neuronal function, in particular, synaptic activity. The development of therapies designed to prevent HAND requires a detailed understanding of pathogenic mechanisms—processes difficult to study in humans. Here, we develop a feline immunodeficiency virus (FIV) model to study this question. FIV is genetically and functionally similar to HIV and produces a naturally occurring AIDS that is frequently associated with the development of neurological disease. We demonstrate that FIV and HIV share the core cellular pathway that alters neuronal activity via aberrant neuronal activation of cGMP-dependent protein kinase II. Thus, FIV infection of cats can be a valuable model to investigate the neurobiological mechanisms of HAND-associated neuropathogenesis. Human immunodeficiency virus (HIV)-associated neurocognitive disorders (HANDs) occur in as many as 50% of individuals infected with HIV, including patients receiving combination antiretroviral therapy (cART) . HANDs rOne of the major limitations in searching for HAND cures has been the lack of an animal model that recapitulates all of the features of HIV infection in humans . Thus, n2+-regulating systems through NMDA receptors (NMDARs) in the synaptic membrane and inositol trisphosphate receptors (IP3Rs) in the endoplasmic reticulum (ER), resulting in apoptosis [2+ fluxes through NMDARs promoting the production of nitric oxide (NO) by neuronal nitric oxide synthase (nNOS), which is tethered by the scaffolding protein postsynaptic density 95 (PSD95) [Both HIV and FIV are tropic for lymphocytes and monocytes, utilizing CD4 (HIV) and CD134 (FIV) primary receptors together with the alpha chemokine receptor CXCR4 as a co-receptor to infect cells –19. Evenpoptosis –34. In a (PSD95) –39. NO s (PSD95) . Notably (PSD95) ,42. Howe2+ release [2+ activity and synaptic activity in neurons. Thus, our results indicate that FIV serves as a model for HAND-associated synaptic hyperexcitation. Most notably, our study reveals the inclusion of cGKII in both FIV gp95 and HIV gp120-induced Ca2+ hyperactivity, suggesting that cGKII inhibition may be a novel therapeutic target for HAND.We have shown previously that cGKII can phosphorylate serine 1756 in neuronal IP3Rs and increase ER Ca release . cGKII a release ,44. This2+ is the second messenger responsible for transmitting depolarization status and synaptic activity [2+ regulation a critical process in neurons, and thus altered Ca2+ activity in neurons is one of the major contributors to many neurological disorders, including HAND [2+ dynamics in neurons, contributing to neuronal dysfunction [2+ activity in neurons, as seen with HIV gp120. To test this idea, we measured Ca2+ activity in cultured mouse hippocampal neurons transfected with GCaMP5, a genetically encoded Ca2+ indicator, as described previously (2+ activity immediately after gp95 treatment (2+ transients in control cells (CTRL) and neurons treated with gp95 , confirming that 1-nM gp95 was sufficient to increase neuronal Ca2+ activity, while 10-pM (1.34 ± 0.18 ΔF/Fmin) and 100-pM gp95 (1.35 ± 0.23 ΔF/Fmin) slightly elevated Ca2+ activity but were not significantly different from control cells (p = 0.001) and amplitude  were significantly elevated in gp95-treated neurons   (2+ activity (1.04 ± 0.16 ΔF/Fmin)  (min)  (2+ activity (0.92 ± 0.11 ΔF/Fmin)  (2+ activity (0.77 ± 0.13 ΔF/Fmin)   . We used 0.0001) , while 2ΔF/Fmin) . This sus, IP3Rs –53. To c 0.0001) . However1) (min) . Ryanodi channel . To test 0.0047) . TreatmeΔF/Fmin) . This su 0.0001) . HoweverΔF/Fmin) . We thus 0.0001) . This su2+ flux through the NMDAR-nNOS pathway activates cGKII by the production of cGMP [2+ dynamics. Indeed, we found that inhibition of nNOS blocked gp95 effects on Ca2+ activity by treating neurons with 2-μM Nω-Propyl-L-arginine hydrochloride (NPA), a nNOS inhibitor  (2+ activity when cGKII activity was blocked by treating neurons with 1-μM Rp8-Br-PET-cGMPS (RP), a cGKII inhibitor    , increasing Ca2+ currents in neuronal tissues [2+ activity (cGKII gene abolished the gp95-mediated elevation of pIP3Rs (2+ release.Ca of cGMP . We thus<0.0001) . Next, w 0.0001) . NotablyΔF/Fmin) . We addi = 0.39) . This su tissues ,55. We tactivity . Howeverf pIP3Rs . In summp = 0.04) and frequency  (cGKII mediates phosphorylation of serine 845 of GluA1 (pGluA1), important for activity-dependent trafficking of GluA1-containing AMPARs, and increases the level of extrasynaptic receptors ,44. More 0.0001) . This su2+ hyperactivity. We carried out Ca2+ imaging in the presence of an AMPAR inhibitor, 6-Cyano-7-nitroquinoxaline-2,3-dione (CNQX) (2+ activity (0.9 ± 0.12 ΔF/Fmin)    are generally sensitive to polyamine block [2+ hyperactivity. We found that 20-μM NASPM treatment was sufficient to block the gp95 effects on Ca2+ activity  (2+ dynamics (0.96 ± 0.13 ΔF/Fmin)  . Treatmee (CNQX) . HoweverΔF/Fmin) ; thus, w 0.0001)  was comp= 0.009) , suggesteceptors . Ca2+-pene block . As we f = 0.02) , while NΔF/Fmin) . To furtΔF/Fmin) . Taking 2+ overactivation in cultured feline neurons. We first treated 14–16 DIV cultured feline hippocampal neurons with 1-nM gp95 and determined Ca2+ activity. Consistent with the findings in cultured mouse neurons       , indicat 0.0001) . We also 0.0001) . This co 0.0001) . NotablyΔF/Fmin) . This su2+ hyperactivity. We treated 12–14 DIV cultured mouse hippocampal neurons with 1-nM CXCR4-tropic gp120 (IIIB) and determined Ca2+ activity , 2.20 ± 0.51 ΔF/Fmin, p = 0.0075)   , 1.87 ± 0.15 ΔF/Fmin, p < 0.0001)  (2+ hyperactivity. We further examined whether CXCR4 inhibition affected the CCR5-tropic gp120 (JRFL) effects on Ca2+ activity by treating neurons with 1-nM gp120 (JRFL) and 200-nM AMD3100 together. We found that AMD3100 treatment was also able to block gp120 (JRFL)-induced Ca2+ hyperexcitation     , consist 0.0025) , confirm 0.0141) , suggestreceptor . We next 0.0001) , which w 0.0014) . This su 0.0001) . This su 0.0002) , which w 0.0028) , confirm2+ and induce cell death in neurons [2+ through mobilizing ER Ca2+ via the stimulation of IP3Rs, as well as NMDARs, the same pathway of HIV gp120-induced Ca2+ overactivation [2+ hyperactivity caused by HIV gp120 .2+ imaging. Mouse hippocampal neurons were also plated on 12-mm coverslips for electrophysiology . Cells were grown in Neurobasal Medium with B27 and 0.5 mM Glutamax and penicillin/streptomycin (Life Technologies).Mouse hippocampal and cortical neuron cultures were prepared as described previously ,46,47. NStaphylococcus Protein A-Sepharose [Escherichia coli and purified using a GST-tag [Expression, amplification, and purification of FIV envelope glycoprotein gp95 and capsid p26 recombinant proteins were performed using previously described methods ,101. Briepharose ,103, or  GST-tag . The fol2+ imaging was carried out by a modification of the previously reported method [2 using a Tokai Hit heating stage and digital temperature and humidity controller. The imaging was captured for periods of 50 milliseconds using a 60× oil-immersion objective. A total of 100 images was obtained with 1-second interval, and Ca2+ activity in the cell body (excluding dendrites) was analyzed using the Olympus CellSens software. Fmin was determined as the minimum fluorescence value during the imaging. Total Ca2+ activity was obtained by combining 100 values of ΔF/Fmin = (Ft − Fmin)/Fmin in each image, and values of ΔF/Fmin < 0.2 were rejected due to bleaching. Twenty to thirty neurons were used for imaging in one experiment, and one individual neuron was assayed in one imaging.GCaMP5 Cad method ,46,47. Dd method  by usingSynaptosomal fractions from DIV14 primary cortical neurons were prepared as described previously ,46,47. S2, 1.5 MgCl2, 30 glucose, and 20 HEPES and was kept at a constant temperature of 31.0 °C. Patch pipettes were filled with (in mM) 120 KGlu, 20 KCl, 2 MgCl2, 10 HEPES, 2 MgATP, 200-μM GTP, and 12.5 mg sucrose and were pH’d with KOH to 7.4. Cells were voltage clamped at −70 mV and input resistance and series resistance were monitored throughout experiments. mEPSCs were amplified and recorded using pClamp10.3. Mini Analysis Program Demo  was used to measure the peak mEPSC amplitude and decay time. CellSens software (Olympus) was used to visualize cells. Patching pipettes were pulled from borosilicate capillary tubing  and the electrode resistance was typically 4–6 mOhms.To record mEPSCs, aCSF contained bicuculline (20 μM), TTX (1 μM), and glycine (1 μM). The recording chamber contained aCSF with a composition of (in mM) 119 NaCl, 5 KCl, 2.5 CaClt tests were used in single comparisons. For multiple comparisons, we used one-way ANOVA followed by Fisher’s Least Significant Difference (LSD) test to determine statistical significance. Results were represented as mean ± SEM, and p < 0.05 was considered statistically significant.Statistical comparisons were analyzed with the GraphPad Prism6 software. Unpaired two-tailed Student S1 FigAn example of time-lapse images and their responses. A scale bar indicates 10 μm.(TIF)Click here for additional data file.S2 Fig2+ activity in each condition showing that a higher dose of DL-APV completely blocks Ca2+ activity in both control and gp95-treated neurons, while a lower dose of DL-APV selectively inhibits the gp95 effects  = 9.238). A scale bar indicates 20 seconds. LSD, Least Significant Difference; NMDAR, NMDA receptor.Representative traces of GCaMP5 fluorescence intensity and a summary graph of the normalized average of total Ca(TIF)Click here for additional data file.S3 FigThe quality of synaptosomes used in (TIF)Click here for additional data file.S4 Fig(A) GluA2 (n = 12 experiments) and (B) NR1 . Actin is used as an intracellular negative control and absent in the biotinylated samples.Representative immunoblots and quantitative analysis of surface biotinylation in each condition showing that gp95 has no effect on surface expression of (TIF)Click here for additional data file.S5 Fig2+ activity in each condition showing that a higher dose of CNQX completely blocks Ca2+ activity in both control and gp95-treated neurons, while a lower dose of CNQX selectively inhibits the gp95 effects. Furthermore, lower doses of DL-APV and CNQX in combination completely inhibited Ca2+ activity in both control and gp95-treated neurons, suggesting that inhibition of both receptors induces additive effects on Ca2+ activity  = 5.933). A scale bar indicates 20 seconds. AMPAR, AMPA receptor; CNQX, 6-Cyano-7-nitroquinoxaline-2,3-dione; LSD, Least Significant Difference.Representative traces of GCaMP5 fluorescence intensity and a summary graph of the normalized average of total Ca(TIF)Click here for additional data file.S6 Fig2+ activity in each condition showing that inhibition of (A) neuronal activity by TTX and (B) L-type Ca2+ channels abolish GCaMP5 activity in both control and gp95-treated neurons  F  = 25.61 and (B) F  = 17.17). A scale bar indicates 20 seconds. LSD, Least Significant Difference; TTX, tetrodotoxin.Representative traces of GCaMP5 fluorescence intensity and a summary graph of the normalized average of total Ca(TIF)Click here for additional data file.S1 Data(XLSX)Click here for additional data file.


Therefore, we evaluated mepivacaine’s effects on Ca2+ indicator Fluo-4-AM to record intracellular Ca2+ transients upon electrical stimulation. The mepivacaine effects at half-maximal inhibitory concentration (IC50) was determined on calibrated cardiomyocytes’ Ca2+ transients by non-parametric statistical analyses on biophysical parameters. Combination of mepivacaine with NCX blockers ORM-10103 or NiCl2 were used to test a possible mechanism to explain mepivacaine-induced Ca2+ transients’ reduction.Single ventricular myocytes were enzymatically isolated from wild-type C57Bl/6 mice and loaded with 10 μM fluorescent Ca50 (50 μM) on Ca2+ transients was measured in biophysical parameters such as peak , peak area , slope , time to peak  and D50 . Combination of mepivacaine with NCX blockers ORM-10103 or NiCl2 showed a significant increase in the baseline of [Ca2+] and arrhythmic activity upon electrical stimulation.A significant inhibition at mepivacaine’s IC+ channels, enhancing the reverse mode activity of NCX, leading to a significant reduction of Ca2+ transients. These results suggest a new mechanism for the mepivacaine-reduction contractility effect.At cellular level, mepivacaine blocks Na The final result supports this hypothesis showing that the combination of mepivacaine and an specific NCX blocker ORM10103 or NiCl2 alters the Ca2+ homeostasis.Hemodynamic changes caused by local anesthetics may be of major clinical concern. Among others, these hemodynamic effects associated with local anesthetics include a decrease in ventricular conduction velocity leading to arrhythmias and a decrease in myocardial contractility –3. Mepiv (APD20) . As consreported , 9, withboratory –12. We oAll animal experiments were performed in accordance with the governmental guidelines of the state Baden Württemberg, including the German law on animal experimentation, and were approved by the ethics committee of Heidelberg University Interfaculty Biomedical Research Facility (T84/14) and followed relevant aspects of ARRIVE guidelines. Isolated cardiomyocytes were obtained from five 6-month male C57 BL/6 NCrl mice purchased from Charles River, Europe and kept in the Interfaculty Biomedical Research Facility until use under 12 h light/ dark cycle with food and water ad libitum.2+ free perfusion solution for 5 min containing the following (in mM): NaCl: 135; KCL: 4; MgCl2*6H2O: 1; HEPES: 10; NaH2PO4: 0.33; Glucose: 10; BDM: 20; Taurine: 5 with a pH value of 7.2 at room temperature. Following the perfusion solution, a Ca2+ free digestion solution was applied which was consisting of Collagenase D 0,36 mg/g (Roche cat no. 11088858001), Collagenase B 0,48 mg/g (Roche cat no. 11088807001) and protease XIV: 0,06 mg/g (Sigma-Aldrich cat no. P5147) dissolved in 30 ml perfusion solution. After 20 min appropriate digestion, the heart was once more perfused with perfusion solution for 5 min to wash out the enzymes. The ventricles were then mechanically dissociated in Transfer Buffer-A (TB-A) solution containing (in mM): NaCl: 135; KCL: 4; MgCl2*6H2O: 1; HEPES: 10; NaH2PO4: 0.33; Glucose: 5.5; BDM: 15; BSA: 5 mg/ml; pH: 7.2 at room temperature. For plating, the cardiomyocytes were placed on 35 mm petri dishes  previously coated with extracellular matrix . Extracellular Ca2+ content was increased in steps  by mixing TB-A with a phosphate-free HEPES buffered Transfer Buffer-B (TB-B) solution containing the following (in mM): NaCl: 137; KCL: 5.4; MgCl2*6H2O: 1; CaCl2*2H2O: 1.8; HEPES: 10; Glucose: 5.5; pH: 7.4 at room temperature. Isolated myocytes were then incubated in TB-B containing 10 μM Fluo-4-acetoxymethyl ester (Fluo-4 AM), at 37 °C for 30 min until introduced into the experimental chamber.Isolation of single cardiac cells was carried out in a modified Langendorff system by the method of Liao and Jain as described previously  and adap2+ transients were examined with a fluorescence light system (Olympus) through a 40X objective (Olympus) using the 488-nm filter for a 75 W Xenon light source. Fluorescence emission was monitored at 510 nm. During recordings the cells were flanked with two platinum electrodes and field electrically stimulated  using SI-Heidelberg Stimulator . The signals were digitally recorded using PowerLab 4/35 (AD Instruments) and analyzed offline after calibration utilizing LabChart 8 software. For each cardiomyocyte ten consecutive stimulations were recorded, but only the last seven Ca2+ transients were averaged and analyzed in order to avoid differences in the sarcoplasmic reticulum’s Ca2+ loading due to inactivation between two recordings i = Kd [(F - Fmin)/ (Fmax - F)]  . Kd is t2+ handling properties of the cells, biophysical parameters were analyzed in Peak Analysis extension from LabChart 8 software as previously mentioned i i activates NCX forward mode in order to avoid arrhythmias, resulting in a reduced sarcoplasmic reticulum Ca2+ after few Ca2+ transients. In order to test this hypothesis, we combined the use of mepivacaine with either two NCX blockers: NiCl2 and ORM-10103 both reduce the NCX forward and reverse currents i –34. It iing Ca2+ –38. Unde+ levels . This wocurrents . The alohed data , 40. The2+ release from the sarcoplasmic reticulum. David and colleagues [2+ in the sarcoplasmic reticulum. However, the fact that the post-rest potentiation was not significantly modified by mepivacaine or by lidocaine, indicating that Ca2+ efflux from the sarcoplasmic reticulum was not significantly influenced. Regarding the effects of mepivacaine on Ca2+ release, the results of David and colleagues [2+ release that was not confirmed by David and colleagues. Since the potential mechanism of mepivacaine’s myocardial depressant effect was not completely cleared, we investigated the consequences of mepivacaine influence in the general aspects of the Ca2+ transients, the parameters peak area, duration and D50 were analyzed. As shown in Fig. 50 significantly. The present results suggest that the reduced Ca2+ transients could be caused by the activity of Na+-Ca2+ exchanger, inwarding Na+ and extruding Ca2+, which would lead to reduced sarcoplasmic reticulum Ca2+ concentration; alternatively, mepivacaine would also altered kinetics of Ca2+ release and/or Ca2+ uptake in the sarcoplasmic reticulum by an unknown mechanism. The biophysical parameters analyzed related to Ca2+ release and Ca2+ uptake showed that mepivacaine significantly reduced all three parameters which evaluate the Ca2+ release: peak, slope and time to peak. All together, these parameters suggest that mepivacaine would reduce the amount of Ca2+ released into the cytosol and its rate of release and thus, the time to reach its maximum. Finally, we observed no effect of mepivacaine on cardiomyocytes’ Ca2+ uptake by analyzing the parameters tau and time to uptake. However, this no effect cannot completely be excluded due to mepivacaine’s major effect on the Ca2+ release without challenging cardiomyocytes’ Ca2+ uptake mechanisms.The negative inotropic effects of mepivacaine in ventricular papillary muscles have been previously reported , 9. Parklleagues  comparedlleagues  were in 2+ transients in isolated, adult mouse ventricular myocytes. Our findings in isolated cardiomyocyte preparations provide evidences to explain that the negative inotropic effect of mepivacaine found previously in cardiac muscle is due to its modulating effects on Ca2+ regulation at cellular level, specifically on Ca2+ release.This is the first study that evaluated the direct effects of mepivacaine on Ca


The detection was based on a colorimetric reaction as a result of the aggregation of modified AuNPs induced by Ca2+ due to the ability to form strong electrostatic interactions between positively charged Ca2+ and negatively charged modified AuNPs. Probe solution changes color from red to blue in the presence of Ca2+ and can be observed by the naked eyes. To verify the complete self-assembly of the AHMP onto the AuNP surface, the modified AuNPs were characterized using ultraviolet–visible spectroscopy and zeta potential measurements. Under optimal conditions, a quantitative linearity was 10 to 100 ppm (R2 = 0.9877) with a detection limit of 3.05 ppm. The results obtained by the developed method were in good agreement with standard atomic absorption spectrometry (AAS) results and demonstrated that this method could reliably measure Ca2+. Overall, this novel alternative approach presents a low-cost, simple, sensitive, rapid, and promising device for the detection of Ca2+. A simple and novel transparency sheet-based colorimetric detection device using gold nanoparticles (AuNPs) modified by 4-Amino-6-hydroxy-2-mercaptopyrimidine monohydrate (AHMP) was fabricated and developed for the determination of calcium ions (Ca Ca2+ plays a role in the growth and maintenance of bones, teeth, and nails. In the ionized form, Ca2+ is significant in many vital processes, such as hormone secretion, nerve conduction, and blood coagulation. The World Health Organization (WHO) and Food and Agriculture Organization (FAO) recommend a minimum daily intake of 1200 mg of Ca2+ for adults [2+ concentration, which usually is relatively stable in body fluids (range in urine: 100 to 300 mg/day) [2+ concentration from body fluids should be determined for the evaluation of and protection against any disease that would be manifested by an abnormal calcium concentration. This emphasizes the need to develop highly sensitive devices for the portable, simple, and rapid deficiency screening in patient care. Numerous methods have been proposed for the measurement of Ca2+ in biological materials. The classical method involves titration with a standardized solution of ethylenediaminetetraacetic acid (EDTA) [2+ in body fluids is atomic absorption spectrometry (AAS) [2+ determination. Currently, the determination of Ca2+ developed with colored complexes using metallochromic reagents, such as Eriochrome Black T (EBT), has been reported [2+ determination. Given their precise and selective performance, this electrochemical measurement technique has been widely developed for the detection of Ca2+ in biological fluids [2+ detection, including solid-contact ion selective electrodes [2+ in urine samples using colorimetric gold nanoparticles. Recently, paper-based analytical devices (PADs) are one of the most common devices used as a miniaturized analytical platform for economical and portable sensing applications. Paper-based platforms can be beneficial for applications in diagnostics, food safety, and environmental monitoring due to the advantages of natural abundance, simplicity, disposability, low sample-reagent usage, and suitability for routine measurement. Unfortunately, the use of paper as substrate for colorimetric detection is usually suffer from the adsorption of color by porosities of paper lead to difficulty in the measurement of the color change at low concentration of the analyte. Therefore, the sensitivity in detection is low in comparison to those obtained from solution. To overcome this limitation, the search for new materials to be used as a substrate to fabricate the analytical platform is a challenge. In this research, we propose to use transparency sheets as the substrate for creating a new colorimetric device platform. Calcium Ca+ is an e mg/day) . Thus, Cd (EDTA) ,6. Howevry (AAS) ,8,9,10. ry (AAS) , spectrory (AAS) , inductiry (AAS) , and flury (AAS) ,16,17 hareported ,19. Evenl fluids ,22. Therectrodes  and polyectrodes . Unfortu2+ detection. The electrostatic force between the negatively charged AHMP-AuNPs and positively charged Ca2+ may induce aggregation. The color change of AHMP-AuNPs has become a useful tool in the development of colorimetric sensing, transparency sheet-based analytical devices. These devices can determine the Ca2+ concentration in urine samples by assessment with the naked eye. The proposed mechanism for Ca2+ detection, the induction of the aggregation of AHMP-AuNPs, is shown in 2+ can induce the aggregation of AHMP-modified AuNPs via electrostatic force. The red color of the AHMP-AuNPs changes to blue in accordance with the Ca2+ level added. Moreover, the proposed approach has been successfully applied to detect Ca2+ levels in urine samples. The verified results were in good agreement with atomic absorption spectroscopy (AAS). The simplicity, speed, suitability for real-time on-site detection, and lack of required complex and expensive instruments could make this method a promising tool for point-of-care (POC) diagnosis. Gold nanoparticles (AuNPs) have been widely used as colorimetric probes for metal ions, anions, small molecules, proteins, nucleic acids, and other analytes because of their unique properties, such as optical properties dependent on their spacing or surface plasmon resonances. The extinction spectrum and the wavelength at which AuNPs absorb and scatter light depends on the distance between particles ,26. For To investigate the incorporation of AHMP onto the AuNP surface, UV–Vis spectroscopy and zeta potentials were determined. 2+. The color and absorption spectra of the AHMP-AuNPs, with and without Ca2+, are shown in 2+, the zeta potential dramatically decreased from −58.2 mv, intensity 35 a.u., to −50.3 mv, intensity 26 a.u., while the average size of the AHMP-AuNPs increased from 29.1 to 65.4 nm. These results indicate that the AHMP-AuNPs aggregation was induced by the electrostatic force between the positive charge of Ca2+ and the negative charge of AHMP-AuNPs. Moreover, the results obtained corresponded to the decrease of the absorption bands at 273 and 523 nm along with the formation of a new absorption band at 680 nm (redshift), as illustrated in 2+ could quickly bind to their functional groups (–NH2 and –OH) onto modified AuNPs [2+, the development of a novel colorimetric AHMP-AuNPs device for Ca2+ determination was successfully accomplished. Following the AuNP modification, a colorimetric analysis using AHMP-AuNPs was performed for the detection of Caed AuNPs ,32. Thes2+ was performed at room temperature by the sequential addition of standard Ca2+ and AHMP-AuNPs on the detection zone of the transparency sheet. The red/blue intensity was measured and calculated by mobile phone and Image J software, respectively. The result of the aggregation capability between AHMP-AuNPs and Ca2+ in each condition was observed by the red/blue intensity from each transparency sheet image. First, the volume of 4 mM AHMP modifier was varied at 0.1, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, and 1.5 mL with the same volume of concentrated AuNPs (1.5 µL). Then, 15 µL of the varied AHMP-AuNPs were investigated with 15 µL of Ca2+ at 0, 10, 50, and 100 ppm concentration. In 2+. Moreover, this change could be easily detected with the naked eye. Therefore, 0.1 mL AHMP was selected as an optimum modifier volume for further studies.In this section, the modifier volume, pH, incubation time, and AHMP-AuNP: sample volume ratios were examined for optimization in order to accelerate the performance of the transparency sheet-based colorimetric device. The colorimetric detection of Ca2+ at a 1:1 volume ratio. The results demonstrated that pH 6 provided the maximum color change. At this pH, Ca2+ has a high potential to induce the aggregation of AHMP-AuNPs. Therefore, phosphate buffer pH 6 was chosen for further studies.Then, the effect of pH (range of 3–12) on the aggregation process was studied. As shown in 2+ was investigated at various incubation times (1–30 min). The aggregation of the AHMP-AuNPs occurred immediately after the addition of Ca2+ and was completed in one minute. In other words, the red/blue intensity from the aggregation of AHMP-AuNPs induced by Ca2+ can be measured at one minute. When AHMP-AuNPs were left on the device for over five minutes, the red/blue intensity of the blank was increased from the initial . The red/blue intensity of AHMP-AuNPs after mixing with Ca2+ was measured at various reagent volume ratios with 30 µL maximum volume. As shown in 2+ at 25:5 provided the highest red/blue intensity. This indicated that a 25:5 ratio of AHMP-AuNPs:Ca2+ was sufficient and appropriate for the determination of Ca2+.Next, the aggregation of AHMP-AuNPs induced by Ca initial  because 2+, K+, Na+, Cl−, CO3−, PO42−, and SO42−), since they may also affect the aggregation of AHMP-AuNPs and might interfere with the method accuracy. An interference was defined to happen when the Δred/blue intensity in the mixture varied by more than ±5% of the Ca2+ intensity. As emphasized previously, the incubation time directly affects the selectivity. Therefore, the change of color was measured at one minute after mixing to neglect the interference from Mg2+. As shown in 2+ did not significantly affect the signal of Ca2+ and the tolerance limits were shown in 2+, Cu2+, Cd2+, and Zn2+. According to the results, it can be seen that the interferences from almost all metal ions studied did not affect Ca2+ detection. Therefore, this probe provides high selectivity for only Ca2+ detection at an incubation time of one minute.After getting the optimal conditions, selectivity was evaluated by the Δred/blue intensity (red/blue intensity of blank—red/blue intensity of sample) of AHMP-AuNPs mixed with other substances and metal ions possibly found in urine  was found to be 3.05 ppm. The proposed method provided a good recovery in the range of 91.7%–106.7%. These recoveries were within the acceptable recovery percentages, 90%–107%, for a concentration of 100 ppm (mg/kg) or at a Ca2+ concentration lower than 100 ppm, as recommended by the Association of Official Analytical Chemists (AOAC). The repeatability of this probe was investigated using 40 and 80 ppm standard solutions of Ca2+ (n = 5 measurements with different probes). The relative standard deviations were 2.54% and 5.16%, which indicated that there was good probe-to-probe reproducibility.Under the optimal conditions, the analytical performance for determination of Ca2+ and proposed devices were summarized in 2+, even though this proposed method has a detection limit higher than previous works, as shown in 2+ in urine samples. Moreover, this proposed platform offers the benefit in terms of simplicity, speed, suitability for real-time on-site detection, and lack of required complex and expensive instruments. Therefore, it can provide a promising tool for Ca2+ diagnosis and could be an alternative choice for point-of-care analysis in the future. The comparisons between some previous report for the determination of Ca2+ in artificial urine samples, and the results were compared to the AAS method as shown in 2+ at 80, 160, and 320 ppm. Then, the samples were further diluted to produce a final appropriate dilution factor before analysis. All artificial urine samples were analyzed by the AAS standard method and the proposed method. The obtained concentrations from the two methods were statistically compared using the t-test at the 95% confidence level. A t-test result was obtained with a value of 1.75, which is lower than the tcritical of 4.30. Thus, there was no significant difference between the concentrations obtained from these two methods. As a result, this proposed colorimetric method can be applied for the determination of Ca2+ in urine samples with satisfactory results, which suggests that the transparency sheet-based colorimetric device is accurate and reliable for practical applications in the clinical field.To evaluate the efficiency of the proposed method, the transparency sheet-based devices were used to detect Caw/v (100 ppm), diameter 20 nm) were purchased from Kestrel Bio Sciences . Calcium carbonate (CaCO3), sodium chloride (NaCl), magnesium chloride (MgCl2), disodium hydrogen orthophosphate (Na2HPO4·2H2O), sodium dihydrogen orthophosphate (NaH2PO4·2H2O),) and hydrochloric acid were purchased from Ajax Finechem Pty Ltd . 4-Amino-6-hydroxy-2-mercaptopyrimidine monohydrate (AHMP), glucose, uric acid, albumin, and ascorbic acid (vitamin C) were purchased from Merck . Artificial urine was purchased from Carolina Biological Supply Company . Analytical grade reagents and 18 MΩ cm−1 resistance deionized (DI) water (obtained from a Millipore Milli-Q purification system) were used throughout. Gold nanoparticles  was prepared from 0.1 M of NaH2PO4·2H2O and 0.1 M Na2HPO4·2H2O and the pH was adjusted with 0.1 M sodium hydroxide (Merck) solution.Phosphate buffers (pH 5–8) were prepared from 0.1 M of NaHA wax printer  was used to print wax onto transparency sheets to obtain the detection devices. Absorption spectra were measured by using a UV-2450 UV–visible spectrophotometer . The zeta potential measurements were performed on Zeta sizer Nano S90 . An atomic absorption spectrometer (AAS) with a hollow cathode lamp and standard air/acetylene flame was also used .First, the purchased gold nanoparticles were preconcentrated by pipetting the commercial gold nanoparticles into 1 mL micro centrifuge tubes. The tubes were centrifuged at 12,000 rpm (4 °C) for 30 min. Then, 0.8 mL of supernatant was removed, and the remaining 0.2 mL of solution was stirred for 10 s.AHMP-AuNPs were prepared by mixing 0.10 mL of 4 mM AHMP and 1.50 mL of the preconcentrated AuNP solution. After that, the mixture was stirred for 15 min to ensure the complete self-assembly of AHMP onto the surface of the AuNPs.Transparency sheet-based devices  were fabricated using a wax printing method. The pattern of the device was designed as an array containing a circular area with a detection zone 5 mm in diameter using the graphic software Adobe Illustrator CS4. The fabrication process has only two steps. First, the wax pattern was printed on the transparency sheet using the wax printer . The maximum liquid volume in the detection zone was 30 μL. The wax-covered area was hydrophobic, while the detection area without wax was hydrophilic. Second, white paper was attached to the back side of the transparency sheet with tape to enhance the visibility of the color change. Using this process, the transparency sheet-based devices could be fabricated within 2 min. 2+ was made by appropriate dilution of the stock standard solution on a daily basis as required. A colorimetric analysis of Ca2+ was performed as follows. First, 5 µL of standard Ca2+ solution or samples (prepared in pH 6.0 PBS) were added into the detection zone of a transparency sheet-based device, followed by the addition of 25 µL of AHMP-AuNPs. Then, the red/blue intensity of AHMP-AuNPs mixed with Ca2+ standard solution was captured by mobile phone  in a light control box and measured via Image J software (https://imagej.nih.gov). Under optimal conditions, the relationship between the red/blue intensity of triplicate measurements and concentrations of the Ca2+ solution between 10–100 ppm was plotted to obtain the linear range.The working standard solution for the Ca2+ in artificial urine samples. The artificial urine was purchased from Carolina Biological Supply Company (controlled level). The proposed method was validated against atomic absorption spectrophotometry (AAS). An appropriate volume of 1000 ppm Ca2+ was added to 25 mL of artificial urine for preparation of the 500 ppm Ca2+ stock solution. Then, the stock solution was diluted with artificial urine to 320, 160, or 80 ppm Ca2+ concentration. The samples were adjusted to pH 6 by the addition of 3 M sodium hydroxide and additional urine was added to reach a final volume. Prior to analysis with the proposed method, the 320 and 160 ppm Ca2+ samples were diluted with 50 mM phosphate buffer pH 6, 4-fold and 2-fold, respectively. The AAS method can detect Ca2+ within the range of 0.5–5 ppm. Therefore, the 320, 160, and 80 ppm Ca2+ samples were diluted with 50 mM phosphate buffer pH 6, 150-fold, 45-fold, and 80-fold, respectively. Under optimal conditions, the proposed method was used for the determination of Ca2+ in urine samples. This method was based on the aggregation of AHMP-AuNPs on the device by the electrostatic force between the negatively charged AHMP-AuNPs and the positively charge Ca2+. Under optimal conditions, the color change of the AHMP-AuNPs can be clearly observed by the naked eye. Furthermore, the proposed method was successfully applied to the quantitative analysis of Ca2+ in artificial urine samples. This colorimetric device provided a result within one minute, much faster than other mentioned methods. This simple device is highly suitable for Ca2+ quantitative analysis and can act as an alternative method for the rapid screening of Ca2+ deficiency without the need for any special instrumentation. The advantages of the transparency sheet-based colorimetric device developed from this work include its high sensitivity and selectivity, good precision, simplicity, and reproducibility. These findings offer a high potential for applications in clinical diagnosis.This paper demonstrated, for the first time, the use of AHMP-AuNPs for a transparency sheet-based colorimetric method to provide simple, rapid, inexpensive, reliable, and portable devices for the determination of Ca


Rabbits (Oryctolagus cuniculus) are one of the most widely used animal models for the study of human lipid metabolism and atherosclerosis because they are more sensitive to a cholesterol diet than other experimental animals such as rodents. Currently, two hypercholesterolemic rabbit models are frequently used for atherosclerosis studies. One is a cholesterol-fed wild-type rabbit and the other is the Watanabe heritable hyperlipidemic (WHHL) rabbit, which is genetically deficient in low density lipoprotein (LDL) receptor function. Wild-type rabbits can be easily induced to develop severe hypercholesterolemia with a cholesterol-rich diet due to the marked increase in hepatically and intestinally derived remnant lipoproteins, called β-very low density lipoproteins (VLDL), which are rich in cholesteryl esters. WHHL rabbits are characterized by elevated plasma LDL levels on a standard chow diet, which resembles human familial hypercholesterolemia. Therefore, both rabbit models develop aortic and coronary atherosclerosis, but the elevated plasma cholesterol levels are caused by completely different mechanisms. In addition, cholesterol-fed rabbits but not WHHL rabbits exhibit different degrees of hepatosteatosis. Recently, we along with others have shown that there are many differentially expressed genes in the atherosclerotic lesions and livers of cholesterol-fed rabbits that are either significantly up- or down-regulated, compared with those in normal rabbits, including genes involved in the regulation of inflammation and lipid metabolism. Therefore, dietary cholesterol plays an important role not only in hypercholesterolemia and atherosclerosis but also in hepatosteatosis. In this review, we make an overview of the recent progress in genomic and transcriptomic analyses of hypercholesterolemic rabbits. These transcriptomic profiling data should provide novel insight into the relationship between hypercholesterolemia and atherosclerosis or hepatic dysfunction caused by dietary cholesterol. Hypercholesterolemia, atherosclerosis and hepatosteatosis, such as non-alcohol steatosis hepatitis and non-alcohol fatty liver disease, are life-style-related metabolic disorders, and are major health problems in modern society ,2. Althohttps://www.genome.gov/11006943/human-genome-project-completion-frequently-asked-questions/). After that, mouse [http://www.picb.ac.cn/RabGTD/.The Human Genome Project was completed by an international team on 14 April 2003 , it is found that the transcriptional response patterns in the aortas of cholesterol-fed and WHHL rabbits are essentially similar  rabbits and hypercholesterolemic (cholesterol-fed and WHHL) rabbits, and then performed RNA expression analyses for cholesterol-fed NZW versus chow-fed NZW rabbits, chow-fed WHHL versus chow-fed JW rabbits and cholesterol-fed NZW rabbits versus chow-fed WHHL rabbits. In atherosclerosis research using rabbits, aortas are mostly used for pathological observation and quantitation of atherosclerosis because aortas are susceptible to atherosclerosis and easy to quantify gross and microscopic lesions . Represe similar B. Among  similar C which i similar . The mosAs the liver is an important organ for mediating lipid metabolism, we further investigated transcriptional changes in the livers of these two hypercholesterolemic rabbits by RNA sequencing analysis. As WHHL rabbits exhibit no histological changes in the liver, there were no significant changes in the DEGs compared with control rabbits. On the contrary, cholesterol-fed rabbits often have varying degrees of fatty liver changes  Figure . RNA seqN-glycan profiling of hypercholesterolemic rabbits along with hypercholesterolemic patients by electrospray ionization mass spectrometry and showed that there are increased plasma levels of high-mannose and high complex/hybrid N-glycan levels [Genomic and transcriptomic sequencing analyses have provided a novel platform to disclose a number of molecular pathways that may be responsible for or associated with the metabolic diseases such as atherosclerosis , hepatosn levels . Therefo