Ryanodine receptors (RyRs) are a family of intracellular Ca2+ release channels that control the flux of Ca2+ from internal stores into the cytosol. RyRs are primarily known for their role in excitation-contraction coupling in adult striated muscle and ryr gene mutations are implicated in several human diseases. Current evidence suggests that RyRs do not have a major role to play prior to organogenesis but regulate tissue differentiation.Calcium signals , fast (ryr3) and both types (ryr1b) of developing skeletal muscle. There are two ryr2 genes (ryr2a and ryr2b) which are expressed exclusively in developing CNS and cardiac tissue, respectively. In addition, ryr3 and ryr2a mRNA is detectable in the initial stages of development, prior to embryonic axis formation.The sequences of the five zebrafish ryr genes are differentially expressed throughout the developing embryo from cleavage onwards. The data suggests that RyR-regulated Ca2+ signals are associated with several aspects of embryonic development, from organogenesis through to the differentiation of the musculoskeletal, cardiovascular and nervous system. These studies will facilitate further work to explore the developmental function of RyRs in each of these tissue types.Our work reveals that zebrafish  Detailed imaging studies have revealed that the spatial and temporal organisation of Ca2+ signals during embryogenesis are associated with many of the major phases of development, from early cell division to the differentiation of tissues. Despite their importance little is known about the generation and regulation of embryonic Ca2+ signals. A comprehensive understanding of the pathways that regulate [Ca2+]i during development is essential to understand the functional relevance of these signals in the embryo.Transient changes in the concentration of intracellular calcium ions (i during excitation-contraction coupling (E-C coupling) in both skeletal and cardiac muscle. In humans, mutations in the ryr1 and ryr2 genes cause skeletal myopathies and cardiac disease respectively [ryr1(−/−) (skrrm1) and (−/−) ryr2knockout lines display gross morphological defects in either the skeletal muscle (ryr1) or heart tube (ryr2) and die at the perinatal or embryonic day 10 (E10) stages respectively [(−/−) ryr3knockout mice appear to have no gross developmental defects and evidence suggests that RyR3 act to augment the [Ca2+]i response of the other RyR isoforms in striated muscle i [2+]i continue to occur throughout gastrulation (up to 10 hpf)  that give rise to the embryonic body plan. Release of Ca2+ from intracellular stores via the PI signalling pathway is again implicated at this stage; however, the contribution to gastrulation from other Ca2+ signalling pathways remains undefined. Finally, the segmentation period (from 10 up to 24 hpf) is characterised by organogenesis and the emergence of the body systems. The Ca2+ signals that occur during segmentation are again more localised and typically associated with developing tissues. Transient changes in [Ca2+]i have been recorded within the nervous system, somites and cardiac tissue [The zebrafish has been used extensively as a model for vertebrate development. The rapid development iewed in ). Eviden [Ca2+]i . RyRs haiewed in ). Initiaon up to  hpf  during the cleavage and blastula periods suggestive of a novel role in early development. At 24 hours post fertilisation (hpf) ryr1a, ryr1b and ryr3 are expressed in skeletal muscle, whereas ryr2a is localized to the central nervous system (CNS) and ryr2b is found exclusively in the cardiac muscle. Our study suggests that RyR channels have a role in early development prior to organogenesis as well as in the differentiation of different cell types.This study set out to acquire a more comprehensive understanding of ryanodine receptor expression in early vertebrate development, using the zebrafish as an Tubingen and Tupfel long fin) were bred and raised in-house at the zebrafish facility of Queen Mary College, University of London, UK, as described previously , was reverse complemented using REQSEV program http://bioweb.pasteur.fr/seqanal/interfaces/revseq.html before performing a pairwise BLAST alignment with Gasterosteus aculeatus (stickleback) RyR2 peptide sequence on NCBI (http://www.ncbi.nlm.nih.gov/blast/bl2seq/wblast2.cgi) to confirm its sequence identity. Based on the alignment result, the full length zebrafish RyR2b peptide was first constructed with reference to Takifugu rubripes (pufferfish) and stickleback RyR2 peptide sequences using the Genewise program (http://www.ebi.ac.uk/Wise2/). The complete RyR2b peptide was finalised by the alignment of the translated genomic region using SHOWORF program http://bioweb.pasteur.fr/seqanal/interfaces/showorf.html. The sequence for the ryr2b gene was deposited in EMBL Nucleotide Sequence Database (Accession no. FR822741). For ryr1b, sequence was obtained via the NCBI CoreNucleotide search website [NCBI - AB247454] and isoform-specific PCR primers (set 3) were used, as described previously ) and RIH (RyR and IP3R Homology [pfam01365]) domains were identified within the N-terminal of all five zebrafish RyRs. The MIR and RIH domains are common to all the members of the intracellular Ca2+-release channel super family ) domain, which currently has no known function. EF-hand motifs may have a functional significance in activation of the channel by Ca2+ themselves. Putative Ca2+ binding sequences, EF1 and EF2 i, with the ryr3 gene product acting to amplify the signal. Our data in the embryonic fast muscle showed that ryr1b expression occurs prior to ryr3 and suggests that the RyR-generated [Ca2+]i increase occurs initially via ryr1b with the proposed amplification step via ryr3 developing subsequently. In zebrafish the role of the RyR in E-C coupling within the developing slow muscle appears more complex. Our data reveals that both ryr1a and ryr1b are expressed in the developing slow muscle, but ryr3 is not. This presents the possibility that zf-α1s-a could couple to both ryr1a and ryr1b and raises the issue of whether amplification of the Ca2+ signals occurs in this tissue and, if so, how is this achieved. Clearly there is still much to understand about the maturation of depolarization-induced Ca2+ signaling and its role during skeletal muscle differentiation in vivo.Several studies have implicated RyR function in organogenesis, particularly in striated muscle development (as reviewed in ). Our st the RyR . The cen+ signal . Thereforyr2 genes, ryr2a and ryr2b, which are exclusively expressed in either the developing nervous system or cardiac tissue, respectively. Studies in mammals revealed that RyR are expressed in the developing brain and that RyR-mediated Ca2+ signals may have a role in neuronal differentiation and neurite outgrowth i homeostasis in maturing neurons. In zebrafish ryr2a expression is localized to specific regions of the developing brains. In these regions ryr2a is likely to regulate neuronal Ca2+ signaling and therefore play a role in CNS development.Our data has revealed that there are two utgrowth -44. All  isoform . The pos2+ signals are generated by CICR via the activation of VOCs and the cardiac RyR (RyR2). Knockout mice which do not express ryr2 initially display spontaneous rhythmic contractions of the heart at embryonic day 9 (E9) but no heart beat by day 10 (E10) [2+ release does not play a significant role in the [Ca2+]i changes observed within the heart of new born rats [in vivo. The zebrafish cardiac ryr gene (ryr2b) is expressed exclusively in the developing heart tissue (precardiac mesoderm) from 14 hpf, 8 hours prior to the onset of cardiac contraction at 22 hpf, and may well contribute to early cardiac development. Investigation of ryr2 function during mammalian development is complicated by the fact that a single ryr2 gene is expressed in several tissues [ryr2a and ryr2b genes in the zebrafish embryo provides an excellent system to study individual receptor function in neuronal and cardiac tissues during vertebrate development.In mature cardiac muscle, Ca10 (E10) . Furtherorn rats . Thus inryr gene family in developing zebrafish embryos. This family of Ca2+-release channels are expressed predominantly in developing skeletal, cardiac and neuronal tissue, supportive of the view that RyRs function is relevant to later development events, such as tissue differentiation. In addition, the study has also revealed that maternal ryr mRNA is present in the very early embryo, suggestive of a function for this receptor prior to organogenesis. Ryanodine receptors have been implicated in human disease and the zebrafish is an important vertebrate developmental model which will facilitate work in this area. Future work will explore the function of RyR-regulated Ca2+ signal pathways during zebrafish embryogenesis.This study has provided a comprehensive overview of the spatial and temporal expression of the The authors declare that they have no competing interests.H.H.T.W performed all the experimental work and contributed to the writing of the manuscript. C.B. supervised and provided input on the design of the experiments. R.A. designed and supervised the experiments and wrote the manuscript. All authors read and approved the final manuscript.Figure S1. ryr1a, ryr1b and ryr3 mRNA is differentially expressed in the developing skeletal muscle. Double labelling in wholemount embryos at 24 hpf was performed using probes to myhz1, ryr1a, ryr1b and ryr3 and Fast Red as a substrate (red) followed by immunostaining using the F59 antibody and a fluorescent secondary (green). Images show Z-stacks of whole-mount double-labelled embryos (top row) or sections (bottom row) from dissected embryos. Cross-sectional images revealed that ryr1a co-localised exclusively with slow muscle staining, whereas myhz1, a marker of fast muscle, was not expressed in the slow muscle. Furthermore ryr1b expression could be observed throughout both muscle types whereas ryr3 did not co-localise with the slow muscle staining and appeared to be expressed exclusively in the fast muscle. Scale bars = 20 μm, unless otherwise indicated.Click here for fileryr3 mRNA expression is confined to the fast muscle fibres throughout the myotome at 24 hpf. Double fluorescent cross-sections (1-3) were prepared by labelling a 24 hpf zebrafish embryo with a fluorescence substrate  for ryr3 in situ hybridisation and the F59 antibody (green) for immunostaining. The position of cross-sections 1-3 are illustrated in the 24 hpf ryr3 WISH labelled embryo (top left) which has been stained with BM purple and laterally orientated with anterior to the left. Scale bars = 20 μm, unless otherwise indicated.Figure S2. Click here for file


The release kinetics of minocycline can be controlled by varying initial loading, Ca2+ concentration, and Ca2+ incorporation into different layers, enabling facile development of implant coatings with versatile release kinetics. This drug delivery platform can potentially be used for releasing any drug that has high Ca2+ binding affinity, enabling its use in a variety of biomedical applications.Infection and inflammation are common complications that seriously affect the functionality and longevity of implanted medical implants. Systemic administration of antibiotics and anti-inflammatory drugs often cannot achieve sufficient local concentration to be effective, and elicits serious side effects. Local delivery of therapeutics from drug-eluting coatings presents a promising solution. However, hydrophobic and thick coatings are commonly used to ensure sufficient drug loading and sustained release, which may limit tissue integration and tissue device communications. A calcium-mediated drug delivery mechanism was developed and characterized in this study. This novel mechanism allows controlled, sustained release of minocycline, an effective antibiotic and anti-inflammatory drug, from nanoscale thin hydrophilic polyelectrolyte multilayers for over 35 days at physiologically relevant concentrations. pH-responsive minocycline release was observed as the chelation between minocycline and Ca Infection and inflammation are common coexisting issues that seriously affect the functionality and longevity of a variety of medical implants including biosensors Minocycline Hydrochloride (MH), a tetracycline derivative, is a broad-spectrum antibiotic and effective anti-inflammatory drug that is widely used clinically to treat infection and inflammation 3 from 8 trilayers of LbL film. In contrast, the highest drug density reported so far is 220 µg/mm3 from 60 tetralayers of LbL film 2+ binding-mediated interactions. MH as a tetracycline derivative can chelate Ca2+ ions, without affecting its biological activity 2+ due to its numerous negatively charged sulfate groups 2+ as the linker to attach MH to DS. We further found that DS can form electrostatic layer by layer (LbL) assembly with gelatin type A (GA), a positively charged biodegradable natural polymer derived from collagen. Multilayers of DS-Ca2+-MH conjugate/GA were successfully constructed to form hydrophilic nanoscale thin coatings. We hypothesize that MH loading and release in the LbL assembly is mediated by calcium binding. Sustained MH release over 35 days was obtained from only 8 trilayers of LbL films with a thickness of 402 nm. We found that Ca2+-mediated MH release was pH-responsive, probably because the binding affinity of tetracycline for Ca2+ decreases with pH Acinetobacter baumannii (A. baumannii), and inhibited the activation of inflammatory macrophages. The combination of hydrophilic coatings that minimize bacterial and inflammatory cell adhesion with sustained release of anti-bacterial and anti-inflammatory MH holds great potential to combat implant-associated infection and inflammation.Layer-by-layer (LbL) self-assembly technique is a versatile method to construct thin film coatings via alternative adsorption of building blocks driven by weak molecular interactions such as electrostatic force and hydrogen bonding Escherichia coli (ATCC 25922), Escherichia coli O157∶H7, Acinetobacter baumannii (ATCC 19606), Staphylococcus aureus (ATCC 25923), methicillin-resistant Staphylococcus aureus USA300 (BAA 1860) and Staphylococcus epidermidis (ATCC 12228) strains were purchased from American Type Culture Collection . A multi-drug resistance Acinetobacter baumannii clinical isolate (referred as A. baumannii #22) was locally isolated from a hospitalized patient having invasive infection following a protocol approved by the Institutional Review Board of Drexel University. The study involves retrospective collection of isolates from clinical laboratory of the hospital, and the samples were coded so that the patient's details were not revealed and identity was not determined. Investigator is granted waiver for this protocol and no consent is required.All the chemicals were obtained from Sigma-Aldrich and used without further purification. Fluorescein isothiocyanate (FITC)-labeled GA was prepared as reported 2 solution or deionized (DI) water. The concentration of CaCl2 solution was 7.2 mM unless specified otherwise. The substrates were first coated with polyethyleneimine (PEI) as an initiating positively charged base layer, followed by alternating immersion in solutions of DS, MH, and GA with or without Ca2+ for 10 min. The excess molecules were removed by rinsing the substrates with DI water for 1 min between each step.LbL self-assembled films were deposited on UV-transparent 96 well plates and black 96 well plates for characterization of MH incorporation and film growth by UV absorption and fluorescence measurement, or on silicon substrates (University Wafer) for thickness measurement. 1 mg/ml solutions of dextran sulfate , MH, and GA were prepared in either CaClLbL film growth was monitored by UV-vis spectroscopy at 245 nm to detect MH incorporation (GA and DS have negligible absorbance at this wavelength), and fluorescence spectroscopy at an excitation wavelength of 485 nm and an emission wavelength of 535 nm to characterize FITC-labeled GA incorporation, using a Tecan M200 microplate reader . Thickness measurements were performed on films deposited on silicon wafers using a Zygo Newview 6000 optical profilometer .LbL films were incubated in Hank's Balanced Salt Solution (HBSS) at 37°C for quantification of MH release until no detectable MH was released. Every 24 h, HBSS was removed and replaced with fresh HBSS. The amount of MH released at each time point was determined by UV absorbance at 245 nm.4 cells per well. The wells in the plates were either pre-coated with 8 bilayers of (DS+Ca2+/GA+Ca2+) or 8 trilayers of (DS+Ca2+/MH+Ca2+/GA+Ca2+). Uncoated wells (polystyrene) were used as control. The biofilms were allowed to form by incubating at 37°C in a stationary incubator for 24 h Bacterial suspensions were inoculated in 96-well plates at a density of 2×10RAW264.7 murine macrophages  were treated with lipopolysaccharide (LPS), fresh MH (1 µg/ml), and MH released during a 24 h period on day 32 (diluted to 1 µg/ml). After 48 h, the accumulated levels of nitrite in the cell culture medium, as an indication of NO, were measured with Griess reagent (Promega).P<0.05 was considered statistically significant. Data are presented as mean ± standard deviations.Pairwise comparisons were conducted using a general linear ANOVA model and Tukey test (two-sided), and 2+ chelates were alternately adsorbed onto the wells of a UV-transparent 96 well plate, showed no increase in UV absorbance at 245 nm for MH as the number of bilayers increased  films, indicating an inefficient LbL assembly. To increase the efficiency of MH loading, we combined calcium binding-mediated MH loading mechanism with an efficient LbL assembly design 8 film before and after drug release was 402±19 and 294±65 nm respectively, indicating that most coating materials still remained after MH was depleted and MH release from the LbL coating was not mediated by degradation of the LbL films.We further studied the effect of adding Cayer film . The thi2+ ions in the coating solutions affect MH loading and release  of Ca2+ ions in the coating solution can overcome the attraction of MH by Ca2+ ions in the LbL film and causing detachment of MH from the coating. Thus, 7.2 mM Ca2+ was used in the following studies.We found that the concentration of Ca release . Increas2+ binding and gelatin also has Ca2+ binding affinity 2+ in MH and GA layers may affect the release kinetics. As shown in 2+/MH/GA)8 multilayer films there was an initial burst release during the first two days, and a second burst release between 6–8 days, followed by a slow, stable release until 24 days. Adding Ca2+ in DS and MH layers, but not GA layers resulted in a high initial burst during the first 5 days, with accelerating release of MH during this period, followed by a slow, stable release up to 21 days. Both release profiles indicate a destabilization of MH binding in the LbL films during release. These release profiles may be beneficial for the treatment of acute or recurrent pathological conditions where high initial burst or pulsed release is desired. However, stable drug release is required for the treatment of chronic conditions. We found that addition of Ca2+ in GA layers eliminated the destabilization effect. MH release from (DS+Ca2+/MH+Ca2+/GA+Ca2+)8 films showed a low initial burst release, followed by slow, stable release for over 35 days, suggesting a more stable MH binding. Incorporation of Ca2+ in the GA layers may increase the amount of Ca2+ ions in the LbL film and thereby increase the capacity of the film to attract MH. Control over release behavior using Ca2+ binding may allow its application to various pathological situations.Since MH loading and release are mediated by Ca2+/MH+Ca2+/GA+Ca2+) LbL films from 6 to 8 significantly increased the amount of MH released every day except the first day, and prolonged the release time from 21 to 35 days . Increasing the number of trilayers of (DS+Ca 35 days .2+/MH+Ca2+/GA+Ca2+)8 multilayer films only lasted 13 days at pH 6.0, with a high initial burst release, versus 35 days of sustained release and a small burst release at physiological pH , a potent inflammatory mediator and cytotoxic molecule The cytotoxicity of the coatings was evaluated using NIH 3T3 fibroblasts . Cells t2+ binding-mediated drug delivery mechanism was developed in this study. This mechanism allows for efficient loading and sustained release of MH, an effective antibiotic and anti-inflammatory drug, from nanoscale thin hydrophilic LbL films. The release kinetics of MH is mediated by calcium binding and can be controlled by varying initial loading, Ca2+ concentration, and adding Ca2+ into different layers, facilitating easy customization of versatile coatings with tailored release kinetics. This drug delivery system also allows pH-responsive MH release, enabling ‘smart’ drug release in response to the severity of infection and inflammation. This versatile drug delivery system will be especially useful for infection/inflammation-susceptible medical devices such as transcutaneous catheters, neural electrodes, and biosensors. In addition, the Ca2+ binding-mediated drug delivery mechanism can potentially be applied to other drugs and/or polymers with high Ca2+ binding affinity, enabling its use in a variety of biomedical applications.In conclusion, a CaFigure S1Cytotoxicity assay. (A) Cell viability under different treatments. Data shown are average ±STD (n = 3). Phase contrast images show the morphology of 3T3 fibroblast cells (stained with cresyl violet) following 24 h treatment with (A) no treatment control, (B) release medium from LbL films containing MH, and (C) release medium from LbL films without MH. Scale bar  = 100 µm.(TIF)Click here for additional data file.


The t translocation is a recurrent chromosomal aberration observed in myeloid malignancies. The translocation results in the generation of the ETV6/myelodysplastic syndrome 1 (MDS1)/ectopic viral integration site 1 (EVI1) fusion gene. However, the present case report is the first to present this rearrangement in acute myelogeneous leukemia (AML)-M4. Notably, this case is the first report of AML-M4 with an initial trisomy 8 and secondary acquired t. Cells harboring the t translocation were found to exhibit a higher proliferative capacity than cells with pure trisomy 8, which is consistent with the role of the ETV6/MDS1/EVI1 fusion transcript in the development and progression of malignancy. Chromosomal abnormalities are extremely common in malignancy, including leukemia. Acute myelogeneous leukemia (AML) has a wide variety of chromosomal rearrangements that involve specific regions –3. The 3The ETV6 gene, located at 12p13, encodes a transcription factor containing the 5′ helix-loop-helix dimerization motif and the 3′ ETS DNA-binding domain . Finallyde novo AML-M4 case with t and trisomy 8. Written informed consent was obtained from the patient.At present, mutual translocation of MDS1/EVI1 and ETV6 has been observed in only two AML-M4 cases; secondary to myelodysplastic syndrome (MDS) and in CML in blast crisis ,10. In t9 cells/l; composed of 32.4% neutrophils, 24.4% lymphocytes, 38.5% monocytes, 0.61% eosinophils and 4.1% basophils. The platelet count was 1.32×109 cells/l and hemoglobin levels were 9.61 g/dl. Serum LDH levels were 1,121 U/l . No treatment had been administered prior to the test. In December 2011, the patient succumbed to unknown causes whilst under treatment with 100 mg Cytosar.A 63-year old female was diagnosed with AML-M4 in October 2011 due to loss of weight and fever. Hematological parameters were as follows: White blood cell count, 5.43×10Chromosome analysis using GTG-banding was performed according to standard procedures prior to chemotherapeutic treatment . A totalin situ hybridization (FISH), using the LSI BCR/ABL dual color dual fusion translocation and chromosome enumeration probes (CEPs) for chromosomes 3 and 12 , were applied according to the manufacturer’s instructions, together with the TEL/AML1 translocation dual fusion probe   . A totalRT-PCR was performed to investigate the expression of human ETV6/MDS1/EVI1 fusion transcripts. Total RNA was extracted from the diagnostic peripheral blood sample using the InviTrap RNA kit  according to the manufacturer’s instructions. cDNA was prepared from 5 μg total RNA with the Genequality BCR-ABL kit  according to the manufacturer’s instructions. The primers used for ETV6/MDS1/EVI1 were as previously reported .Immunophenotyping of leukemic blasts was performed as described previously . BrieflyKaryotyping was performed prior to chemotherapy and the result was 47,XX,+8,t[18]/47,XX,+8[2] . This obThe t translocation was further studied by RT-PCR and the analysis revealed a typical fusion transcript of 144 bp in length, which confirmed the presence of an ETV6/MDS1/EVI1 fusion transcript .Immunophenotyping analysis of the peripheral blood showed that the abnormal cell population was CD45 (96%), CD33 (78%), CD13 (78%), CD18 (91%) and CD11c (55%), and that CD34 (32%), HLA-DR (58%), CD117 (32%), CD32 (26%), CD16 (54%), CD15 (20%) and CD235a (29%) were expressed heterogeneously. This immunophenotype is consistent with AML-M4 according to FAB classification.The tq26;p13) translocation is a rare cytogenetic abnormality and has been previously reported in 45 cases of myeloid malignancies, including MDS, AML and CML , monosomy 7 or del(7q), trisomy 8, t and rearrangements involving the MLL gene mapped to 11q23 [del(q22), inThe prognosis of AML with the tq26;p13) translocation has been reported to be poor, with a survival of only a few months. This is associated with treatment refractoriness and may reflect the fact that this AML is secondary to MDS with the presence of chromosome 7 abnormalities . This ha;p13 tranEVI1 is a transcription factor with two zinc finger motifs and its acquisition is known to be a poor prognostic factor for AML . It is lThe MDS1/EVI1 fusion generates a protein domain with homology to the positive regulatory domain of PRDI-BF1/Blimp-l, a transcriptional repressor of the interferon β gene and an inducer of genes that play a role in B-lymphocyte maturation .In conclusion, the current case report presents a novel cytogenetic case of AML-M4 with initial trisomy 8 and a secondary t, the latter having more proliferative capacity than cells with pure trisomy 8. The patient succumbed to unknown causes whilst under treatment. Therefore, we conclude that trisomy 8 with t has a negative prognosis.


The main goal of this paper is to explore the basis of this pro-arrhythmic capability of CO by incorporating changes in CO-induced ion channel activity with intracellular signaling pathways in the mammalian heart. To do this, a quite well-documented mathematical model of the action potential and intracellular calcium transient in the human ventricular myocyte has been employed. In silico iterations based on this model provide a useful first step in illustrating the cellular electrophysiological consequences of CO that have been reported from mammalian heart experiments. Specifically, when the Grandi et al. model of the human ventricular action potential is utilized, and after the Na+ and Ca2+ currents in a single myocyte are modified based on the experimental literature, early after-depolarization (EAD) rhythm disturbances appear, and important elements of the underlying causes of these EADs are revealed/illustrated. Our modified mathematical model of the human ventricular action potential also provides a convenient digital platform for designing future experimental work and relating these changes in cellular cardiac electrophysiology to emerging clinical and epidemiological data on CO toxicity.Carbon monoxide (CO) that is produced in a number of different mammalian tissues is now known to have significant effects on the cardiovascular system. These include: (i) vasodilation, (ii) changes in heart rate and strength of contractions, and (iii) modulation of autonomic nervous system input to both the primary pacemaker and the working myocardium. Excessive CO in the environment is toxic and can initiate or mediate life threatening cardiac rhythm disturbances. Recent reports link these ventricular arrhythmias to an increase in the slowly inactivating, or “late” component of the Na For this purpose, CO-induced changes in INa-L that are similar to those reported in the primary experimental data  increase in I2+ transient and Ca2+ homeostasis in the human ventricle. For the purpose of simulating some aspects of heart failure we have previously modified and utilized the original Grandi et al. model  modifying the Na+ current as in  both.To simulate the electrical activity of human ventricular myocytes, two relatively recent AP models were evaluated. The Grandi et al. model . In addition, INa-L was increased 2-fold compared to its values in the original Grandi et al. model  heart, as documented in the detailed studies of Campbell et al. (Ca-L (30–50%) under conditions of NO induced S-nitrosylation of this Ca2+ channel α-subunit. In addition, this paper also reported a nitrosylation induced change in ICa-L gating—specifically a small but significant change in the voltage dependent activation relationship—an approximately 6.5 mV shift in the hyperpolarizing direction. To reproduce these effects using the Grandi et al. model . The changes applied were slightly smaller than the ones reported experimentally, but were sufficient to trigger EADs, as shown in Figure CO is known to elevate NO levels  shifting the inactivation curve for the large transient component of INa in hyperpolarizing direction, and (iii) significantly increasing the slowly inactivating component of INa (which is denoted INa-L).The initial set of computations was done for the purpose of illustrating the consequences of the CO induced changes in mammalian heart NaNa and INa-L described above. For these computations, the steady-state inactivation curve was shifted in the hyperpolarizing direction by 5 mV and INa-L was increased 2-fold. Figure Na-L; Figure Ca-L; that is, the AP waveform lengthened, and this changed the ICa-L; and Figure 2+ transient, [Ca2+]i.The two superimposed human ventricular APs in Figure Na and INa-L, when combined, result in predictable decreases in AP rate-of-rise (not shown) and small increase in AP duration. However, these changes fail to elicit spontaneous firing (EADs on DADs) or produce any correlates of arrythmogenesis. Although this pattern of results was somewhat unexpected, it is important to recall that the primary experimental data on CO were obtained in rat hearts  small hyperpolarizing shift in the steady-state inactivation curve for INa, (iii) a marked enhancement (approximately 2-fold) in INa-L and (iv) an essential NO-induced S-nitrosylation of a defined residue on Nav1.5. These changes, when introduced into the Grandi model, also failed to result in increased arrhythmogenesis (results not shown).In fact, studies of NO-mediated effects in heterologous expression systems utilizing the alpha subunit of the human heart NaCa-L, in the mammalian (ferret) heart. These investigators reported a significant increase in ICa-L (30–50%) under conditions of NO induced S-nitrosylation of this Ca2+ channel α-subunit. Importantly, this paper also reported a nitrosylation-induced change in ICa-L gating, specifically, a small but significant shift in the voltage dependent activation relationship—approximately 6 mV in the hyperpolarizing direction. The superimposed APs in Figure (A) shows the AP waveforms  . INa-L and ICa-L records are shown in Panels , respectively. The L-type Ca2+ current is shown in Panel (C). As shown in Panel (D), these changes in ICa-L also resulted in a large increase in [Ca2+]i, and the [Ca2+]i exhibited pronounced frequency dependence or alternans  that INa-L now shows pronounced changes in peak amplitude, even in response to the fixed stimulus rate at 1 Hz. Presumably, this arises from the markedly prolonged AP and intrinsic voltage-dependence of reactivation of INa-L (see Discussion).Detailed inspection of Figure Na and INa-L , INa-L (Panel B), ICa-L (Panel C), and [Ca2+]i (Panel D) are shown in Figure Na-L and ICa-L “held” the membrane potential of the plateau near 0 mV in contrast with the approximately −10 mV plateau level in Figure The third set of computations in this study combined the reported CO induced changes in IL Figure , with thL Figure . The res), and Ca+i (Panel), and Ca+i  involves the non-linear interactions of the small overlapping Ca2+ channel—and Na+/Ca2+—exchanger mediated currents in this range of membrane potentials  almost identical to those for Figure Na-L  every 3rd AP in this 1 Hz AP train. An additional effect is also apparent: reducing INa-L changes the (i) waveform and (ii) the most negative membrane potential in some repolarizing components within this train of AP's. This primary “effect” appears to be linked functionally to the approximately normal repolarization of the next AP. In summary, when ranolazine is introduced in silico, the resulting AP waveforms show complex alternating patterns. However, there is an increased number of (i) near normal AP waveforms and (ii) monotonic [Ca2+]i records. The incidence of EADs is also reduced, consistent with the previously described actions of this drug in the settings of free radical challenge, genetic abnormalities in Na+ channel biophysics, hypoxia, or global ischemia/heart failure  ion channel mediated currents at the plateau of the AP are: (i) INa-L, (ii) ICa-L, (iii) IK1, a time-independent or background inwardly rectifying current, and (iv) the delayed rectifier K+ current, IK−R  of Figure The simulations in Figure (A) shows two superimposed pairs of APs, each elicited at steady state (1 Hz) before (black) and after (red) introducing the changes shown in Figure  show the corresponding INa-L and ICa-L records. Although this information is redundant with that in Figure (D), [Ca2+]i records; Panel (E), INCX; and Panel (F), the Ca2+ content of the SR.Panel Ca-L are critical. With each stimulus, a large “extra” Ca2+ influx prolongs the plateau and results in enhanced Ca2+ release and a much larger increase in [Ca2+]i. This change in [Ca2+]i can stimulate the INCX. Perhaps more importantly, however, this time-dependent and cumulative change in myocyte Ca2+ homeostasis results in markedly increased Ca2+ in the SR. Eventually, this causes anomalous Ca2+ release from the SR, and the Na+/Ca2+ exchanger (denoted by arrows) responds by generating an inward current that gives rise to the EAD. Under these conditions, approximately every second AP is associated with spontaneous release at the steady-state stimulus rate of 1 Hz.Taken together, these results suggest that in this model the CO/NO-induced changes in INa-L could be anti-arrhythmic (see Discussion). In brief, the answer appears to be that ranolazine, by blocking INa-L, can shorten the AP. This reduction in APD results in reduced Ca2+ influx through ICa-L during each AP and thus, there is less Ca2+-loading of the SR. The end result is that SR Ca2+ content does not reach the abnormal levels required for anomalous and spontaneous Ca2+ release. Hence, there is no intracellular Ca2+ “trigger” for the electrogenic INCX, that has an important role in initiating the EAD in this model  by generating an aberrant cellular electrophysiological pattern of responses, denoted EADs.+ current following CO exposure were not sufficient to provide the required pro-arrhythmic substrate. Additional changes in INa-L parameters may have given rise to EAD waveforms. However, we evaluated the possibility that other ion channel targets were involved in EAD initiation. Accordingly, our analyses of CO-induced EADs include changes in INa-L and ICa-L. As shown in Figure Ca-L can produce EAD generation. Although this pattern of results has not been reported when CO is used as the “toxic” stimulus; it is known that NO is a primary mediator of CO-induced pro-arrhythmic events in the heart  responsible for EAD generation can be deduced/confirmed by careful inspection of the new data from our simulations. EADs are slow, small depolarizations that arise in the ventricle (and in Purkinje tissue) within a transmembrane voltage range corresponding to the plateau of the ventricular AP (approximately −10 to +10 mV). A net inward current is required, and the work from the Marban  pattern of SR Ca2+ release. Each of these increases in [Ca2+]i gives rise to a significant “new” inward current generated by the sarcolemmal Na+/Ca2+ exchanger mechanism.However, the non-inactivating component of I+ and Ca2+ currents. These changes, alone or in combination, would enhance ICa-L and perhaps INa-L, both of which would contribute a net inward current at the plateau level of the ventricular AP. The Koren Laboratory  levels in the ventricular myocardium. Specifically, they have reported enhanced EAD activity either (i) following augmentation of ICa-L with isoproterenol, or (ii) a reduction of repolarizing K+ current(s) during hypokalemic conditions. Both of these maneuvers can alter the net current at the plateau of the AP and thus, change the repolarization reserve during the early repolarization phase of the AP  or in any setting involving stress, the known effects of stimulation of the sympathetic nervous system would be expected to augment both Na+ currents that are expressed throughout the mammalian heart, and have important roles in setting the resting potential and modulating repolarization. The Tamargo Group  hyperpolarization of the resting potential  for maintenance and generation of the plateau of the AP, in a way that involves complex, often non-linear interactions of a number of different currents  are not possible since many of the known subcellular details of CICR release are not included in this computational platform. After this model is developed further and e.g., the existing electrophysiological components are integrated with known properties of E-C coupling and mechanical activation  mediated relaxation.It is apparent that simulation of CO-mediated electrophysiological effects requires detailed understanding of each major component of CaNa-NO interactions (Ueda et al., Detailed studies of CO-mediated alterations of mammalian cardiac electrophysiological phenomena are relatively recent. At present, there is incomplete information on clinically relevant plasma levels, interspecies variations, and specific intracellular signaling cascades. This information is essential for more extensive simulations that could address cellular or molecular mechanisms for CO-I2+-activated K+ channels that modulate CO-induced vasodilation (Jaggar et al., Na-L in the myocardium (Ju et al., + channels, NO synthase isoform and G proteins are co-localized in caveolae (Besse et al., Our work is not intended to detract from or replace well-established principles of CO generation or action Maines,  based onNa, INa-L, and ICa-L dependent changes in AP waveform have not been ruled out as plausible pro-arrhythmic elements. These, along with the S-nitosylation induced change in mammalian heart K+ currents (Gómez et al., Our approach to the pro-arrhythmic effects of CO challenge has placed an emphasis on EAD initiation. We acknowledge that alterations in micro- or macroscopic conduction patterns or velocities; and/or IWe are aware that as presented our analysis of CO effects on the human ventricle is incomplete. Primary reasons for this include:Doctors Trenor, Saiz and Giles shared responsibilities for study design. Computations were performed at Universidad Politécnica de Valencia by Doctors Cardona and Trenor, Dr. Giles wrote this manuscript. All authors contributed to final editing and the R-1 revisions.The 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.


Due to its varied and essential roles, free Ca2+ is tightly regulated by complex mechanisms. These mechanisms are therefore of interest as putative drug targets. One pathway in Ca2+ homeostatic control in apicomplexans uses a Ca2+/H+ exchanger . The P. falciparum CAX (PfCAX) has recently been characterised in asexual blood stage parasites. To determine the physiological importance of apicomplexan CAXs, tagging and knock-out strategies were undertaken in the genetically tractable T. gondii and P. berghei parasites. In addition, a yeast heterologous expression system was used to study the function of apicomplexan CAXs. Tagging of T. gondii and P. berghei CAXs (TgCAX and PbCAX) under control of their endogenous promoters could not demonstrate measureable expression of either CAX in tachyzoites and asexual blood stages, respectively. These results were consistent with the ability of parasites to tolerate knock-outs of the genes for TgCAX and PbCAX at these developmental stages. In contrast, PbCAX expression was detectable during sexual stages of development in female gametocytes/gametes, zygotes and ookinetes, where it was dispersed in membranous networks within the cytosol . Furthermore, genetically disrupted parasites failed to develop further from “round” form zygotes, suggesting that PbCAX is essential for ookinete development and differentiation. This impeded phenotype could be rescued by removal of extracellular Ca2+. Therefore, PbCAX provides a mechanism for free living parasites to multiply within the ionic microenvironment of the mosquito midgut. Ca2+ homeostasis mediated by PbCAX is critical and suggests plasmodial CAXs may be targeted in approaches designed to block parasite transmission.Ca Plasmodium  and Toxoplasma , regulate their calcium levels. Here, we have demonstrated that removing apicomplexan genes for a protein that exchanges calcium for protons across membranes (a Ca2+/H+ exchanger) and a member of the cation exchanger (CAX) family, does not affect the survival of parasites during those stages when they live within host cells. It is, however, lethal for the mouse malaria P. berghei when the parasite is free living within its mosquito vector. When we removed calcium from around the parasites at this stage they were able to develop normally, suggesting that the protein provides a mechanism for the parasite to tolerate environmental calcium. Learning how this calcium transport protein impacts on the development of apicomplexan parasites may lead to the development of novel anti-parasitic interventions.Calcium is vital to all living organisms. It is used within cells to regulate many essential processes and, because of this, its cellular concentration is tightly controlled. To change cellular calcium levels, cells use calcium transport proteins. These proteins can alter calcium concentration by moving calcium into or out of the cell or specialised calcium storage compartments within the cell. We know little about how single-celled apicomplexan parasites, including  Plasmodium and Toxoplasma), including secretion of adhesins, motility, cellular invasion and egress, and intracellular development 2+ are therefore important to identify as novel drug targets and can provide fundamental insights into the biology of parasites when they are functionally characterised Free Ca2+ homeostatic control in apicomplexan parasites is limited. To control cytosolic free Ca2+ concentrations, eukaryotic cells use a range of Ca2+ binding proteins, Ca2+ diffusive “leak” pathways and active Ca2+ transporters. For example, several apicomplexan P-type Ca2+ ATPases have been characterised, such as the sarco(endo)plasmic reticulum Ca2+ ATPases (SERCAs) of P. falciparum, PfATP6, and T. gondii, TgSERCA Plasmodium P-type ATPases are already being investigated as new or existing drug targets. More recently, an initial characterisation of the P. falciparum Ca2+/H+ exchanger  has been undertaken Our current understanding of Ca2+/cation antiporter (CaCA) superfamily and members have been identified across the biological Kingdoms including some lower vertebrates, although not in more complex metazoa including mammals Dictyostelium, and lower vertebrates and Type III CAXs are found in bacteria, while Type I CAXs include bacterial, fungal, plant and protozoan CAXs. Type I CAXs are divided into 8 subgroups (A to H) and protozoa are classified into the Type I-C phylogenetic group. The first member of this group to be characterised functionally was a Ca2+/H+ exchanger (CrCAX1) from the unicellular green alga Chlamydomonas reinhardtiiPfCAX and other apicomplexan orthologues belong to the Ca+ coupled exchangers all transport Ca2+, with some being highly specific for Ca2+, whilst others mediate the transport of a broad range of additional divalent cations or, in some cases, transport additional monovalent cations 2+ concentrations, by internal sequestration of Ca2+ into acidic organelles when cytosolic levels rise 2+/H+ exchange activity, an ability to exchange a limited range of other divalent cations, and a high transport capacity but low affinity  for Ca2+, when expressed in Xenopus oocytes In vivo studies characterising PfCAX are consistent with an atypical localisation to the inner mitochondrial membrane, and an atypical function, where the protein provides a pathway for removal of Ca2+ from this organelle back into the parasite cytosol.Functional characterisation of CAX proteins (mainly from plants and fungi) has shown that these HP. berghei and T. gondii parasites were used with tagging and knock-out strategies to define expression and essentiality of their respective CAXs. The data demonstrate that under the control of their respective endogenous promoters only the expression of the tagged P. berghei CAX, PbCAX, could be established, and this, only in sexual stages of development . Furthermore, genetic disruption of pbcax, while having little or no effect on blood stage growth, was lethal during ookinete development. However, mutant parasites could be rescued by the removal of extracellular Ca2+.The aim of this study was to determine the physiological importance of apicomplexan CAXs. In addition to developing a yeast heterologous expression system for the functional characterisation of apicomplexan CAXs, the genetically amenable P. falciparum, P. berghei and T. gondii CAX genes, pfcax (PFF0170w), pbcax (PBANKA_010230) and TgCAX (TGME49_007910), have 1326, 1323 and 1506 base pair open reading frames, respectively, with only the latter having (12) introns. They are located on chromosomes 6, 1 and 1b in their respective genomes and encode polypeptides of 441, 440 and 501 amino acids, with estimated sizes of 48, 49 and 53 kDa, respectively  and C. reinhardtii CAX sequences, respectively. The phylogenetic relationship between the apicomplexan putative CAX transporters is shown in cax genes in the genomes of the Piroplasmida, Babesia bovis and bigemina or Theileria annulata, even though they are closely related to Plasmodium parasites.The ectively  and S1. www.cbs.dtu.dk/services/TMHMM/; Cryptosporidium ssp. CAXs to 126 amino acids for TgCAX). All contain conserved residues  in the case of P. berghei ookinetes.As with CrCAX1 and higher plant CAXs, the apicomplexan CAXs identified here are predicted to have 11 membrane spanning regions  and a Ca2+/H+ exchanger (Vcx1). This yeast mutant is unable to sequester Ca2+ into the vacuole and is therefore hypersensitive to external Ca2+, which causes toxicity due to elevation of cytosolic Ca2+ concentrations To investigate whether apicomplexan CAXs can provide a tolerance mechanism against elevated Capfcax cDNA , in which translation was initiated from AUG at nucleotide position 181 (encoding Met-61), was therefore generated for comparison with PfCAX and sCrCAX1.A codon optimized cax cDNA  was exprpfcax and spfcax in yeast was detectable by RT-PCR . The Ca2+ tolerance of yeast expressing PfCAX and sPfCAX was demonstrated further by cell growth in liquid YPD media supplemented with various concentrations of CaCl2 , while the cell growth of PfCAX-expressing yeast was significantly greater than that of the empty vector control yeast at all CaCl2 concentrations, including at 150 mM CaCl2 . A truncated version of TgCAX (sTgCAX), in which translation was initiated by introducing an AUG prior to nucleotide position 295 (encoding Ala-99), was also generated and expressed in K665 yeast. The transformation was assessed to have worked by PCR . Ca2+/H+ exchange activity mediated by full-length PfCAX was significantly reduced compared with that of sPfCAX  but was detectable over basal (empty vector) levels .To confirm that growth of PfCAX-expressing yeast on Ca variant . Howeverpfcax, using a strong but non-authentic/non-sequential promoter, was used to localise PfCAX to the parasite mitochondrion Plasmodium CAX to be determined over the parasite's full life cycle, the rodent parasite, P. berghei, was used. A GFP-tagged P. berghei parasite line was generated by endogenous C-terminus fusion of the gfp sequence onto pbcax, leaving control of gene expression under the endogenous promoter. This was achieved with a single crossing over homologous integration approach -tagged version of approach . PCR on approach  and pulsapproach .in situ. As a positive control for blotting and antibody staining, a P. berghei line that constitutively expresses GFP in the cytosol throughout the life cycle was used Expression of PbCAX-GFP in activated gametocytes was confirmed by Western blotting in the particulate fraction (contain membranes), using anti-GFP polyclonal antibodies . A protepbcax-gfp transgenic parasites, with stronger parasite-associated GFP signal observed only on rare occasions (data not shown). Stronger GFP signal was observed in female but not male gametocytes and the same was true for gametes . Little signal colocalised with MitoTracker, used as a marker for parasite mitochondrion . Non-con2+/H+ exchange activity during the erythrocytic stages of P. berghei, a direct pbcax knock-out strain was generated by double crossing over homologous recombination (P. berghei ANKA and a second line that constitutively expresses cytosolic GFP cl9 and cl5 gfp) were both positive for integration of the knock-out construct (using primers INT N43 and ol248) and disruption (using primers N43 KO1 and N43 KO2) of pbcax  suggest that PbCAX function is not essential for blood stages of the P. berghei life cycle.To determine the physiological importance of Cabination . After tof pbcax . Furtherof pbcax  and pulsof pbcax . The latin vitro sexual development of mutant parasite clones was studied cl9 Δpbcax parasites, in which no statistical difference was determined between the number of exflagellation centres produced by wild-type parasites compared with the mutant line . Apparently normal exflagellation was also observed for cl5 gfp Δpbcax parasites (not tested quantitatively).Next, the cl9 Δpbcax parasites in which no ookinete conversion was observed, while wild-type control parasites converted to ookinetes on average by 74±3% . No ookinete conversion was also observed for cl5 gfp Δpbcax parasites (n = 4), while in two matched experiments, using wild-type control parasites that constitutively express GFP, estimated conversion rates were 87 and 71%. Note that very occasional ookinete-like forms were observed in these in vitro cultures.Secondly, ookinete conversion was determined by culturing parasites for 24 h in ookinete medium and measuring the number of the motile ookinete forms in relation to unconverted “round” forms  at the end of this period, using fluorescently-labelled P28 antibody to aid identification . Δpbcax parasites remained round. At 24 h post-activation, wild-type parasites were predominantly fully converted into ookinetes (elongated forms with the nucleus in the centre). At this time, Δpbcax parasites were still round in form, fewer in number and were often smaller in size and had degenerated membranes (as judged from discontinuous P28 staining). Furthermore, they often had diffuse nuclei, suggestive of possible necrosis or late stage apoptosis. In a single semi-quantitative experiment with three repeats, the number of “round” form PbCAX knock-out parasites per field of view was counted in ookinete development cultures at 2, 6 and 24 h after activation . This is due to the fertilisation of female Δpbcax cl9 parasites by functional Δnek2 or Δnek4 male gametes. However, crossing Δpbcax cl9 with Δmap2 resulted in recovered ookinete conversion of 42±2% . This is due to the fertilisation of functional Δmap2 female gametes by male Δpbcax parasites. These data suggest that PbCAX activity, while not specific, is predominantly important to female gametes.Cross-fertilization experiments were performed in an effort to establish if PbCAX activity is an important male and/or female trait . This wain vivo by feeding Anopheles stephensi mosquitoes on mice infected with Δpbcax cl9 or cl5 gfp parasite mutants and measuring oocyst formation in the mosquito midgut 14 days later. Δpbcax cl9 and the second two using Δpbcax cl5 gfp parasites . It demonstrates that while mosquito infection with wild-type parasites produced mean numbers of oocysts per mosquito midgut of 110, 144 and 57, respectively, and infection prevalences (number of mosquitoes with oocysts) of 80, 90 and 65%, infection with parasites lacking PbCAX produced none. At day 21 post-infection, the infected mosquitoes were used to re-infect mice but no mutant asexual blood-stage parasites could be identified after 15 days, while wild-type asexual blood-stage parasites were observed by day 5 on average.Parasite development was assessed in vitro culture medium used here contained 0.42 mM Ca2+) and that CAX activity often provides a mechanism to enable organisms to survive in the presence of extracellular Ca2+2+ chelator EGTA (ethylene glycol tetraacetic acid) to restore ookinete conversion of both Δpbcax cl9 and cl5 gfp parasites . In case the rescuing effect of EGTA was osmotic rather than due to its Ca2+ chelation properties, additional controls were performed in the presence of 20 mM NaCl added to the culture medium immediately prior to gametogenesis. This had no effect on the ability of wild-type parasites to develop into ookinetes  or the inability of mutant parasite to develop (data not shown).Given that transmission stages of parasite development are extracellular  and monovalent (e.g. Na+) cations, in the case of CrCAX1 Nearly all Xenopus oocytes reported Ca2+/H+ exchange and, using competition assays, data implicating transport of other divalent cations, including Mg2+ and transition metal ions e.g.2+ transporters increased tolerance of yeast to extracellular Ca2+. Transport measurements in yeast vacuolar membrane preparations confirmed that PfCAX is a Ca2+/H+ exchanger and is localised to the tonoplast when expressed in yeast. In a previous study +, as would be predicted because sequences that maybe associated with Na+ transport 2+ tolerance and Ca2+ transport rates. Truncation of the protein may also improve delivery to the vacuole and provide an alternative explanation for improved functionality.Previous characterisation of PfCAX by expression in vcx1) and containing a calcium biosensor (apoaequorin) was published 2+/H+ exchanger and lend support to the present findings.While this paper was under revision, a report describing PfCAX/CHA expression in yeast lacking the endogenous CAX gene , consistent with mitochondrial localisation 2+ transport pathways, including in apicomplexan parasites, play critical roles in providing Ca2+ tolerance, by refilling internal Ca2+ stores, and by shaping cytosolic free Ca2+ transients or oscillations that act as intracellular signals 2+ efflux pathways include the plasma membrane and internally localised P. falciparum Ca2+-ATPase, PfATP4 P. falciparum and T. gondii, PfATP6 and TgSERCA, respectively T. gondii Ca2+-ATPase, TgA1, that localises to acidocalcisomes in vivo function of each Ca2+ efflux transporter (with respect to the cytosol) is unclear, although it is likely that there would be some overlap in the functions of these pathways. Thus, loss of one active Ca2+ transport mechanism might be compensated for by others, particularly during asexual development stages when the parasites are mainly intracellular, and do not face large extracellular Ca2+ concentrations 2+/H+ exchangers play major roles in providing tolerance to excess cytosolic Ca2+, due to their ability to function as low affinity, high capacity Ca2+ transporters. For example, in yeast the Ca2+/H+ exchanger Vcx1 efficiently sequesters Ca2+ into the vacuole when cytosolic Ca2+ levels are high without requiring the vacuolar Ca2+-ATPase 2+/H+ exchange activity, such as in the Arabidopsis cax1 knock-out, leads to up-regulation of vacuolar Ca2+-ATPase activity, as a compensatory mechanism Neither TgCAX nor PbCAX are required for asexually reproducing parasites. Most active Ca2+, this suggests that PbCAX's primary role is to provide a tolerance mechanism to environmental Ca2+ at a time when the parasite is extracellular and, at least initially, exposed to plasma concentrations of free Ca2+ of approximately 1.2 mM . This is consistent with its primary role in plants and fungi Interestingly, PbCAX was essential during ookinete conversion, with knock-out lines failing to transform from zygotes and over an extended time period (in excess of 8 h) becoming necrotic or possibly apoptotic. Given that this phenotype could be reversed by the removal of extracellular Ca2+ exchangers play a role in directly regulating cytosolic Ca2+ elevations and in modulating cellular signaling associated with stress responses Plasmodium parasites, it is well known that Ca2+ and Ca2+ interacting proteins regulate important parasite functions such as gametocyte differentiation into gametes, ookinete development and motility Evidence is accumulating that Ca2+/H+ exchangers, via protein modification or protein interaction, is consistent with their role as Ca2+ modulators. The Arabidopsis Ca2+/H+ exchanger AtCAX1 can be regulated via an autoinhibitory domain that is present on its long hydrophilic N-terminal tail Chlamydomonas may also share this mechanism of regulation. This CAX protein has an extended hydrophilic N-terminal tail which when truncated leads to increased Ca2+/H+ exchange activity P. falciparum and other apicomplexan organisms likewise have a long N-terminal tail  2+/calmodulin and is inhibited by immunosuppressant drugs (FK506 and cyclosporine A) is present in P. falciparumP. falciparum are being elucidated 2+ homeostasis including Ca2+ transporter regulation are unknown, but suggest alternative mechanisms of regulation and are worthy of investigation. Dysregulation of CAXs may prove equally as detrimental to plasmodial parasite survival at other life cycle stages, as PbCAX deletion does during ookinete conversion, and to other apicomplexans.The 2+/H+ exchange activity of apicomplexan CAXs, shed light on possible CAX regulation, fail to support substantial mitochondrial localisation, demonstrate that CAXs are neither expressed to a high level nor required during asexual apicomplexan parasite development but PbCAX is expressed to a measureable level during sexual stages of development and is likely essential during ookinete conversion by protecting parasites against extracellular Ca2+.The data presented here confirm the functional Ca2+/Ca2+ exchangers) being the best known 2+, and provides the experimental tools necessary to aid development of this therapeutic strategy.In a new age of malaria eradication there is renewed interest in the development of transmission blocking therapies All animal work has passed an ethical review process and was approved by the United Kingdom Home Office. Work was carried out in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986 and in compliance with European Directive 86/609/EEC for the protection of animals used for experimental purposes. All procedure were performed under Home Office licence number 40/3344.pfcax cDNA was synthesised (Geneart) and codon optimised for expression in S. cerevisiae , encoding proteins lacking the first 60 and 98 amino acids, respectively, were generated by PCR amplification using synthetic pfcax or T. gondii cDNA as template and the primers sPfCAXF/sTgCAXF and PfCAXR/TgCAXR  pfcax in yeast was confirmed by RT-PCR following extraction of yeast total RNA, using the acid phenol method sTgCAX in yeast was confirmed by PCR following extraction of genomic DNA. PCR was performed using the primers PUGF and PUGR (found approximately 200 base pairs either side of the multicloning site in piUGpd) and yeast actin primers  the isolated tonoplast fractions were all demonstrated to have no measurable contamination from other membrane fractions, as only the V-ATPase inhibitor bafilomycin (a tonoplast marker) inhibited H+-ATPase activity in these vesicles, while inhibitors of other membrane-localised H+-ATPases did not inhibit H+-ATPase activity (data not shown). Ca2+/H+ exchange activity was determined by measuring pH gradient-dependent 45Ca2+ uptake into membrane vesicles, as described previously Vacuolar membrane vesicles were isolated from yeast cells expressing P. berghei ANKA strain 2.34 parasites, as previously described pbcax knock-out vectors were constructed for a double cross-over homologous recombination in the pBS-DHFR plasmid that contains a Toxoplasma gondii dhfr/ts cassette conferring resistance to pyrimethamine pbcax 5′ untranslated (UTR) region upstream and 497 bp of the pbcax 3′ UTR region downstream of the dhfr cassette  microscope fitted with an AxioCam ICc1 digital camera . Hoechst 33342 (Sigma) was used for nuclear staining of all stages and the P28 cy3-labelled antibody was used as a marker for female gametes/zygotes/ookinetes.High resolution live cell imaging was performed using an Olympus-based personal Delta Vision work station at ×100 . Subsequent off-line image preparation was carried out using Applied Precision software and finalised with Adobe Photoshop. Images presented are 2D projections of 0.1 µm stepped Z-stacks.P. berghei-infected mice on day 4 post-infection into heparinised syringes, mixed with ookinete culture medium  fetal bovine serum, 10 mM sodium bicarbonate, 50 µM xanthurenic acid, pH 7.6) and cultured at 19°C for a further 21–24 h before assessment of conversion. For direct immunolabelling (to aid identification), cultured cells were pelleted for 2 min at 800× g and then labelled for 10 min on ice in 50 µl of ookinete medium containing Hoechst 33342 and Cy3-conjugated mouse monoclonal antibody specific for P28 For ookinete conversion assays, blood was taken by cardiac puncture from For exflagellation assays, mice were infected as described above. On day 4 to 5 post-infection, 10 µl of infectious tail snip blood was added to 40 µl of ookinete medium and incubated at room temperature for 15 mins. Gametocytaemia was 5–8%. The data were generated from the 15 min time point from 1 spot of tail blood performed in triplicate (from 3 different infected mice). An aliquot of 10 µl of tail blood was also analysed between 7 and 10 min in triplicate to ensure no exflagellation events were missed prior to microgametogenesis and in each case none were observed. Exflagellation was counted by measuring the number of exflagellation centres in 8 fields under magnification ×40 on a Zeiss Primostar microscope.in vivo was assessed using mosquitoes fed directly on P. berghei-infected mice, as described previously A. stephensi (SD 500 strain) mosquitoes in groups of approximately 100 for 30 min. Unfed mosquitoes were removed the next day, and the remaining mosquitoes were maintained at 19°C and 80% relative humidity on a 12-h light/dark cycle, being fed on a 2% (w/v) D-glucose solution that was replenished every 2 to 3 days. At 14 day post-feeding, mosquito midguts were dissected and analysed by fluorescence microscopy.Parasite transmission to mosquitoes T. gondii tachyzoites (RHhxgprt- and ΔKU80hxgprt- strains v/v) fetal calf serum, 2 mM glutamine and 25 µg/ml gentamicin.TgCAX was amplified with primers pairs TgCAX-1/TgCAX-2, cloned into the pGEM-Teasy vector (Promega) and then sub-cloned into pTUB8MycGFPPfMyoAtailTy-HX EcoRI and NsiI sites to create the pTUB8TgCAX-Ty vector. For the knock-in vector, a fragment of genomic DNA corresponding to the C-terminal part of the gene was amplified using primers TgCAX-3/TgCAX-2 and cloned between KpnI and NsiI sites of the pTUB vector TgCAX amplified with primers TgCAX-4/TgCAX-5 and cloned between the BamHI and NotI sites of the knock-in vector.Genomic DNA was prepared from tachyzoites (RH strain), using the Wizard SV genomic DNA purification system (Promega). Total RNA was isolated from tachyzoites, using Trizol (Invitrogen), and then total cDNA was generated by RT-PCR, using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's instructions. All amplifications were performed with LaTaq or ExTaq (TaKaRa) and the primers used are listed in hxgprt) gene was used as a positive selectable marker in the presence of mycophenolic acid (25 mg/ml) and xanthine (50 mg/ml) as described before Parasite transfections were performed by electroporation as described previously v/v) paraformaldehyde/0.05% (v/v) glutaraldehyde (PFA/GA) in phosphate buffered saline (PBS) and processed as described previously 2-GAPSRQLHLGLLSEGW-COOH was used to immunise two guinea pigs (Eurogentec) according to their standard protocol.For immunofluorescence assays (IFA), parasite-infected HFF cells were fixed with 4%  45 antibodies (a surface parasite marker). The number of parasites per vacuole was determined by counting the parasites in 100 vacuoles in duplicate in three independent experiments.v/v) dimethyl suphoxide (DMSO) or 3 µM of the Ca2+ ionophore A23187 from Streptomyces chartreusensis  before fixation. IFAs were performed using α-GAP45 antibodies and the average number of egressed vacuoles was determined by counting 100 vacuoles for each condition in at least 2 independent experiments.For egress assays, host cells were inoculated with freshly released parasites and grown for 30 h. Parasite-infected host cells were then incubated for 5 min at 37°C with DMEM containing 0.06%  with Dunnett's post-hoc test were performed, as noted in the text, using the GraphPad Prism software program (version 5 for PC).Unpaired, two-tailed Student's Figure S1Sequence alignments. Amino acid sequence alignment of PfCAX with other apicomplexan CAX genes. Eight apicomplexan CAX sequences are shown. The Clustal W program was used to generate the alignment. The residues highlighted by a bold black line above correspond to transmembrane segment predictions determined with the TMHMM program (http://www.cbs.dtu.dk/services/TMHMM/). The residues highlighted by a bold green line below correspond to the conserved CAX regions, c-1 and c-2. Green shading denotes residues shown to be essential for Ca2+ transport in AtCAX1 and OsCAX1a http://ihg.gsf.de/ihg/mitoprot.html). Note that the MitoProt II probabilities for predicted mitochondrial targeting are 0.99, 0.98, 0.98, 0.93, 0.96, 0.98, 0.93 and 0.98 for PfCAX, PvCAX, PkCAX, PbCAX, TgCAX, NcCAX, CpCAX, EtCAX, respectively. Using TargetP 1.1 (http://www.cbs.dtu.dk/services/TargetP/) the probabilities are 0.74, 0.78, 0.81, 0.77, 0.89, 0.90, 0.90 and 0.92, respectively. Red shading denotes phospo-acceptor sites ; Pv, Plasmodium vivax (XP_001616060.1); Pk, Plasmodium knowlesi (XP_002261646.1); Pb, Plasmodium berghei (XP_678577.1); Tg, Toxoplasma gondii (XP_002369594.1); Nc, Neospora caninum (CBZ49795.1); Cp, Cryptosporidium parvum; Et, Eimeria tenalla. Red letters, identical or conserved residues in all sequences; green letters, conserved substitutions; blue letters, semi-conserved substitutions.(DOC)Click here for additional data file.Figure S2Phylogenetic analysis. Phylogenetic relationship of 14 CAXs between 12 members of the phylum Apicomplexa and 2 of the green algae Chlamydomonas reinhardtii at the amino acid level. CAX sequences are from (accession no.): Pf, Plasmodium falciparum (XP_966025.1); Pv, Plasmodium vivax (XP_001616060.1); Pk, Plasmodium knowlesi (XP_002261646.1); Pc, Plasmodium chabaudi; Py, Plasmodium yoelii (XP_725194.1); Pb, Plasmodium berghei (XP_678577.1); Tg, Toxoplasma gondii (XP_002369594.1); Nc, Neospora caninum (CBZ49795.1); Et, Eimeria tenalla; Cp, Cryptosporidium parvum; Ch, Cryptosporidium hominis; Cm, Cryptosporidium muris (XP_002142215.1); Cr; Chlamydomonas reinhardtii (CAR92574.1). The tree was generated using a ClustalW alignment of the full-length sequence using maximum likelihood under the WAG+F model of amino acid substitution, as implemented in RAxML v7.1 and using the fast bootstrap approach to determine tree confidence (TIFF)Click here for additional data file.Figure S3Optimsed DNA sequence. DNA sequence alignment of PfCAX cDNA compared with the yeast codon optimised synthetic PfCAX cDNA (synPfCAX).(DOC)Click here for additional data file.Figure S42+ tolerance of yeast mediated by TgCAX.Ca (A) PCR analysis of sTgCAX transformation into yeast compared with yeast transformed with the empty vector control. (B) Saturated liquid cultures of K665 (pmc1 vcx1) yeast transformed with N-terminally truncated sTgCAX in piUGpd and empty vector alone were serially diluted to the cell densities as indicated, then spotted onto selection medium lacking uracil (SD –Ura) and YPD medium containing 50 mM CaCl2. Yeast growth at 30°C is shown after 3 days. A representative experiment is shown.(TIF)Click here for additional data file.Figure S5pbcax gfp-tagging strategy and confirmation. (A) Schematic representation of the gene targeting strategy used for tagging of the endogenous locus with gfp via single homologous recombination. Primers 1+2 (INT N43tag+ol492) used for diagnostic PCR are indicated. Probe location used for detection by pulse-field gel electrophoresis is indicated. (B) Diagnostic PCR confirming successful integration of the tagging sequence. Positive template controls (+ve control) amplifying a 517 bp region were performed using Control1 and Control2 primers. (C) Pulse-field gel electrophoresis blot hybridised with a probe to hdhfr, which detects the endogenous homologous locus on chromosome 7 and the disrupted locus on chromosome 1. (D) Western blot analysis using an anti-GFP antibody against control wild-type-GFP (wt) and transgenic (tag) activated gametocyte soluble and particulate fractions, showing bands of expected sizes of 29 kDa for wild-type-GFP and 77 kDa for PbCAX-GFP.(TIF)Click here for additional data file.Figure S6PbCAX-GFP expression. (A) Expression of PbCAX-GFP in live parasites at specific P. berghei life cycle stages is shown. Where appropriate, parasites are immunostained for the female gamete/zygote/ookinete marker P28 (red) and counterstained with the nuclear marker Hoechst 33342 (blue). GFP intensity is observed predominantly in female gameotyctes/female gametes/zygotes/ookinetes/oocysts and is far less prevalent in asexual blood stages/male gametocytes/male gametes. Scale bar: 5 µm. (B) High resolution deconvolution microscopy images of a live female gamete/zygote and an ookinete 24 h post activation expressing PbCAX-GFP and immunostained with P28 and counterstained with Hoechst 33342.(TIF)Click here for additional data file.Figure S7pbcax disruption strategy and confirmation. (A) Schematic representation of the gene targeting strategy used for gene disruption via double homologous recombination. Primers 1–4  used for diagnostic PCR are indicated, as well as the EcoRI sites used for Southern blotting. Probe location used for detection by Southern blotting is indicated. (B) Diagnostic PCR confirming successful integration of the disruption sequence of pbcax in mutants N43 clone 9 (cl9) and N43-GFP clone 5 (cl5 gfp). Primers 1+2 (INT N43+ol248) were used to verify successful integration at the correct locus. Primers 3+4 (N43 KO1+N43 KO2) were used to confirm loss of the endogenous gene. (C) Southern blot analysis of EcoRI digested N43 clone 9 genomic DNA using the 5′ UTR of the targeting construct as a probe. Band sizes for N43 clone 9 (cl9) and wild-type (wt) are indicated. (D) Pulse-field gel electrophoresis blot hybridised with a probe to tgdhfr/ts, which detects the endogenous locus on chromosome 7 and the disrupted locus on chromosome 1.(TIFF)Click here for additional data file.Figure S8Stability of Δpbcax parasites. Bar graph illustrating the numbers of Δpbcax cl9 parasites remaining in (ookinete) culture over time. As these parasite fail to convert into ookinetes, “round” form parasites were counted and their numbers presented per field of view . Bars represent the mean ± SEM of 3 repeats derived from cultured blood from a single infection.(TIF)Click here for additional data file.Figure S9TgCAX Ty-tagging and disruption strategies and confirmation. (A) Schematic representation of the TgCAX Ty transient transfection construct. (B) Immunofluorescence images of Toxoplasma tachyzoites stably transfected with TgCAX Ty, expressed under the control of the tubulin promoter. Note TgCAX-Ty (green) failed to colocalise with VP1 (red). (C) Schematic representation of the gene targeting strategy used for gene tagging the endogenous locus with ty via single homologous recombination. Primer set 1+2 (TgCAX-6+TgCAX-7) and 1+3 (TgCAX-6+P30A) used for diagnostic PCR are indicated. (D) Diagnostic PCR on cDNA confirming successful integration of the tagging sequence, expected sizes: 1+2: 843 bp and 1+3: 1008 bp. (E) Schematic representation of the strategy used for gene disruption via double homologous recombination. Primer sets 4+5 (TgCAX-8+TgCAX-2) and 4+6 (TgCAX-8+TgCAX-9) used for diagnostic PCR are indicated. (F) Diagnostic PCR on cDNA confirming successful gene deletion, expected sizes: 4+5: 1162 bp and 4+6: 695 bp. To confirm the presence of cDNA, two control genes were amplified GAP45 and profilin (PRF) with primers TgGAP45-1/2 and TgPRF-1/2, respectively. The expected sizes are 750 bp for GAP45 and 500 bp for PRF.(TIF)Click here for additional data file.Protocol S1Genotype and Western blot analysis of P. berghei transfectants.(DOC)Click here for additional data file.Table S1Oligonucleotides used in this study.(DOC)Click here for additional data file.


After 10 seconds of optogenetic stimulation, the evoked intracellular Ca2+ level and dopamine current of channelrhodopsin-2-transfected PC12 cells were 1.6- and 3.5-fold higher than those of the control cells. The optogenetic stimulation effects on Ca2+ influx and dopamine release were 81% and 63% inhibition by using a Ca2+ channel antagonist Nifedipine. The results indicate that optogenetic stimulation can evoke voltage-gated Ca2+ channel-dependent dopamine exocytosis from PC12 cells in a cell specific, temporally precise and dose-dependent manner. This proposed dopamine recording system can be developed to be a good cell model for dopamine regulation and drug screening in vitro, or dopaminergic cell implantation therapy in vivo using optogenetic stimulation in a precise and convenient way.Dopaminergic PC12 cells can synthesize and release dopamine, providing a good cellular model for investigating dopamine regulation. Optogenetic stimulation of channelrhodopsin-2 provides high spatial and temporal precision for selective stimulation as a powerful neuromodulation tool for neuroscience studies. The aim of this study is to measure dopamine release from dopaminergic PC12 cells under optogenetic stimulation using electrochemical recording of self-assembled monolayers modified microelectrode with amperometric measurement in real time. The activation of PC12 cells under various optogenetic stimulation schemes are characterized by measuring single-cell Ca Dopamine is an important catecholamine neurotransmitter implicated in physiological functions. Abnormal neurotransmission of dopamine can lead to numerous neurological disorders, such as Parkinson’s disease in vivo dopaminergic neuron stimulation successfully induced dopamine release in brain and conditioned animal behaviors Optogenetic stimulation approach that uses channelrhodopsin-2 (ChR2) has been developed for providing more precise and targeted stimulation effect on cells. ChR2-transfected neurons can be depolarized by exposure to ∼473 nm blue light in a very short duration (within 1 to 3 milliseconds), which immediately stops after the light is turned off. Because ChR2 expression can be targeted in specific types of cells, optogenetic stimulation allows potential therapeutic strategies to be investigated for neuroscience research and applications Various techniques, including neuroimaging 2+ is known to be essential for dopamine release from PC12 cells 2+ cascade effect in PC12 cells under optogenetic stimulation could provide in-depth information of the underlying mechanism. The results from Ca2+ signaling and real-time electrochemical monitoring of dopamine according to molecular imaging, we can delineate the Ca2+ dependency of dopamine release using blue light stimulation under different various Ca2+ buffers and voltage-gated Ca2+ channel (VGCC) antagonist. Combination of the ChR2-transfected PC12 cells, optogenetic stimulation and electrochemical recording could be a powerful model for dopamine research in vitro, which also has a high potential application to therapeutic treatment in animals.To better understand the optogenetic regulation of dopamine release from PC12 cells, the present study evaluates dopamine release from ChR2-tranfected PC12 cells using a surface-modified dopamine sensing electrode under various optogenetic stimulation schemes. Since intracellular CaChlamydomonas reinhardtii (UTEX 2247) was purchased from the University of Texas Algal Collection . Total RNA extraction and reverse transcription by Trizol® Reagent  and SuperScript® III  were performed according to the manufacturer’s instructions. The cDNA encoding the first 315 amino acids of ChR2 (GenBank accession no. AF461397) was gendered by polymerase chain reaction amplified with the primers 5′-AACTGCAGATGGATTATGGAGGCGCCCT and 3′-GGATCCCGCTTGCCGGTGCCCTTGTTGA. The cDNA fragment of ChR2 was then inserted into cloning vector pEGFP-N1  via the restriction sites of PstI and BamHI. The ChR2-EGFP plasmid was verified by DNA sequencing.The green algae PC12 cells , a cell line derived from a pheochromocytoma of the rat adrenal medulla, were cultured at 37°C in RPMI 1640 medium supplemented with 15% horse serum, 5% fetal bovine serum, 1 mM sodium pyruvate, 25 units/mL penicillin, and 25 units/mL streptomycin. For plasmid DNA transfection, the ChR2-EGFP plasmid was transfected using Lipofetamine® 2000 . The ChR2-EGFP or the controlled EGFP stable expressing PC12 cells were selected by 600 mg/mL G418 over one month.Φ = 75 µm, A–M Systems, USA) and inserted into a glass capillary which was drawn with a microelectrode puller. The end of the glass capillary was sealed with silicone rubber  and cured at 90°C for 12 hours. The tip of platinum microelectrode was cut by a scalpel, and the length of platinum micro-wire is extended ∼0.5 mm from the glass seal. For cleaning, the platinum plate (1.0 mm×1.2 mm) or microelectrode was cleaned for 30 minutes in each of ethanol and deionized water (18 MΩ·cm resistivity) and then immersed in 0.5 M H2SO4. Cyclic voltammetry (CV) was performed using an electrochemical analyzer  from −0.2 V to 1.5 V (vs. Ag/AgCl electrode) until the CV curves were stable. In order to coat Au-NP, the platinum plate or microelectrode was immersed in 0.1 M HClO4 solution  containing 10 mM hydrogen tetrachloroaurate . A constant voltage of −0.5 V relative to the Ag/AgCl was applied. Au-NP were uniformly coated onto the surfaces of platinum plates and microelectrodes via electrodeposition. The surface morphologies of native platinum and Au-NP deposited platinum plates were evaluated by scanning electron microscopy . To modify the self-assembled Au-NP monolayer, Au-NP deposited microelectrode was immersed in solutions of 5 mM mercaptopropionic acid  dissolved in 95% ethanol for 15 minutes. The microelectrodes were finally washed copiously with ethanol to remove any non-binding thiols. Cells were stimulated by blue laser and recorded extracellular dopamine by electrode simultaneously at the sampling rate of 10 Hz.For dopamine release recording, SAM-modified microelectrodes with high sensitivity and selectivity to dopamine were designed and fabricated. The working electrode was prepared with platinum micro-wire . The tip of the 400-µm fiber was fixed on a micromanipulator for guiding the blue light laser to the targeted cells. 1×106 cells/mL of ChR2-EGFP or EGFP stable expressing PC12 cells were seeded on a 25 µg/mL poly-D-lysine (PDL)-coated 6-well plate  with complete growth medium before optogenetic stimulation experiments. After culture for 24 hours at 37°C, the medium was replaced by 1.8 mM Ca2+-contained phosphate-buffered saline (PBS), Ca2+-free PBS, or 10 µM Nifedipine  in Ca2+-containing PBS. For optogenetic stimulation and dopamine sensing, the optical fiber and electrodes were fixed on the micromanipulator and aimed at the targeted cell as closely as possible, as shown in For all optogenetic stimulation experiments, a 473-nm fiber-coupled diode-pumped solid-state (DPSS) laser  with TTL modulation was used. The output power of the laser was adjusted to 10 mW/mm2+ levels were measured using the ratiomatric fluorescent dye fura-2/AM  under a single-cell fluorimeter . 1×106 PC12 cells/mL were seeded on a 25 µg/mL PDL-coated glass bottom dish and cultured in complete growth medium at 37°C for 24 hours. Cells were loaded with 5 µM fura-2/AM in PBS at 26°C for 1 hour. After loading, cells were washed three times with PBS and recovered for 30 minutes. Dishes were then placed on the stage of the single-cell fluorimeter for Ca2+ measurement. The ratiometric Ca2+ indicator, fluorophore fura-2/AM, was excited by fast switched 340 nm and 380 nm wavelengths for 50 ms, and emissions were the same at 510 nm. The duration between fast switched 340 nm and 380 nm wavelengths is 2 ms, and the illumination intensity is 10 mW. The relative Ca2+ level was calculated from the ratio of the emission intensities with excitations of 340 nm and 380 nm (ratio 340/380). Intracellular fura-2 fluorescence was sequentially imaged and the mean fluorescence intensity of individual PC12 cells was quantitatively analyzed by TILLvisION software of the TILL Photonics system . The Ca2+ imaging techniques were applied to ChR2-transfected PC12 cells with various optogenentic stimulation schemes.Intracellular Cat-test and ANOVAs were used for statistical analyses. A p value < 0.05 is considered as significantly different.All data were represented as mean ± SEM (standard error of the mean) in each experiment. Student’s −1. A pair of well-defined peaks for dopamine redox can be observed at Au-NP deposited microelectrode, with an oxidative peak potential at 0.25 V (vs. Ag/AgCl) and corresponding reductive peak potential at around 0.16 V. In contrast, these peaks of dopamine redox were not obvious on the CV curves of native platinum microelectrodes. In R2) of 0.999 and a detection limit of 7 nM at a signal-to-noise ratio of 3.−9 C can be observed in the controlled EGFP-expressing cells. In contrast, optogenetic stimulation evoked immediate elevations of dopamine signals about 1.36±0.08×10−9 C in ChR2-EGFP-expressing cells, which is significantly higher than that of the control (t(8) = −11.91, p<0.001; Student’s t-test). After termination of the blue light, the dopamine signals slowly decreased and returned towards the base values. Then, four durations  of blue light illumination were used to investigate the influence of stimulation duration on dopamine release from ChR2-expressing PC12 cells. As shown in R2 = 0.996) increased from 0.33±0.02, 0.99±0.03, 1.55±0.07 to 2.06±0.10×10−9 C for 5, 10, 15, and 20 seconds of stimulation duration, respectively. The photoelectrical artifacts were removed by direct subtraction.The controlled EGFP- or ChR2-EGFP-expressing PC12 cells were cultured in dish for dopamine release monitoring. After the medium was replaced with PBS, the sensing electrode recorded the extracellular concentration of dopamine change upon 10 seconds of blue light illumination. As shown in 2+ cascade is not clear. To investigate the optogenetic effect on the Ca2+ cascade, optogenetic stimulation were applied to PC12 cells in Ca2+-free buffer, 1.8 mM Ca2+ buffer and L-type VGCC antagonist Nifedipine-containing buffer (10 µM Nifedipine in 1.8 mM Ca2+ buffer). Relative intracellular Ca2+ levels are presented as the ratiometric fura-2 fluorescence intensity (ratio 340/380) by single-cell fluorimeter (two-way ANOVA of ChR2-EGFP/Ca2+ vs. EGFP/Ca2+ (p<0.001), ChR2-EGFP/Ca2+ vs. ChR2-EGFP/Ca2+-free (p<0.001), ChR2-EGFP/Ca2+ vs. ChR2-EGFP/Ca2+/Nifedipine (p<0.01); ChR2-EGFP/Ca2+/Nifedipine vs. ChR2-EGFP/Ca2+-free (p<0.001), ChR2-EGFP/Ca2+/Nifedipine vs. EGFP/Ca2+ (p<0.001) with Bonferroni’s post test; 2+ change of ChR2-EGFP-expressing PC12 cells (Δ ratio 340/380 = 0.121±0.007) was significantly higher than that of the controlled EGFP-expressing PC12 cells (Δ ratio 340/380 = −0.002±0.001) after 10 seconds of blue light illumination. In contrast, the intracellular Ca2+ change was inhibited (Δ ratio 340/380 = 0.009±0.004) when ChR2-EGFP-expressing PC12 cells were immersed in Ca2+-free buffer. Similarly, Nifedipine significantly inhibited the change of Ca2+ level (Δ ratio 340/380 = 0.045±0.003) upon optogenetic stimulation  = 146.8, p<0.001; one-way ANOVA with Tukey’s multiple comparisons test).It is well known that L-type VGCC plays an important role on dopamine release in PC12 cells. However, the optogenetic effect on the Ca2+ influx during optogenetic stimulation from 2+-free buffer (0.17±0.02×10−9 C) and Nifedipine/1.8 mM Ca2+ (0.09±0.01×10−9 C) (two-way ANOVA of Ca2+ vs. Ca2+-free (p<0.001), Ca2+ vs. Ca2+/Nifedipine (p<0.001), Ca2+-free vs. Ca2+/Nifedipine  with Bonferroni’s post test). The dopamine currents were significantly inhibited in both Ca2+-free buffer (0.17±0.02×10−9 C) and Nifedipine/1.8 mM Ca2+ (0.19±0.01×10−9 C) conditions in comparison to that for the 1.8 mM Ca2+ buffer (1.00±0.13×10−9 C) condition with the artifacts removed  = 33.52, p<0.001; one-way ANOVA with Tukey’s multiple comparisons test; It is known that that the L-type VGCC antagonist Nifedipine could block Ca2+ from ChR2-EGFP-expressing PC12 cells. For comparison, continuous illumination (CI) and stimulatory pulses of blue light with frequencies of 10, 20, and 30 Hz and durations of 10, 20, and 30 ms were applied to cells for 10 seconds. 2+ levels of PC12 cells increased with the increase of stimulatory frequency from 10 Hz to 30 Hz as well as with the increase of stimulation pulse duration. The highest elevation of Ca2+ (Δ ratio 340/380 = 0.130±0.020) occurred at stimulation pulses of 30 Hz and 30 ms which is about 3-fold of 10 Hz/10 ms stimulation pulse (Δ ratio 340/380 = 0.043±0.006). CI led to lower Ca2+ elevation (Δ ratio 340/380 = 0.095±0.004) compared to the 30 Hz/30 ms illumination  = 3.135, p<0.035; 20 Hz/30 ms vs. 20 Hz/10 ms, t(4) = 4.855, p<0.0083; 20 Hz/20 ms vs. 20 Hz/10 ms, t(4) = 2.854, p<0.0462; 30 Hz/30 ms vs. 30 Hz/10 ms, t(4) = 5.048, p<0.0072; 30 Hz/10 ms vs. 10 Hz/10 ms, t(4) = 3.264, p<0.031; 30 Hz/20 ms vs. 10 Hz/20 ms, t(4) = 2.835, p<0.0471; 30 Hz/30 ms vs. 10 Hz/30 ms, t(4) = 4.467, p<0.0111; 30 Hz/10 ms vs. 20 Hz/10 ms, t(4) = 2.858, p<0.046; 30 Hz/30 ms vs. 20 Hz/30 ms, t(4) = 3.704, p<0.0.0208 with Student’s t-test).To examine the effect of different stimulation schemes including stimulation frequency and pulse duration on the dopamine release and influx of Ca−9 C) and the 30 Hz/30 ms pulse (0.314±0.034×10−9 C) of blue light illumination evoked higher background dopamine currents compared to the 10 Hz/10 ms pulse (0.007±0.011×10−9 C). −9 C) was far lower than those of the 30 Hz/30 ms stimulation (1.215±0.135×10−9 C) and CI (1.006±0.128×10−9 C) with artifacts removed.The dopamine release of controlled EGFP- and ChR2-EGFP-expressing cells under various stimulation schemes was also observed. The photoelectrical artifacts of detected currents of EGFP-expressing PC12 cells under various stimulation schemes of blue light illumination were examined. The results are shown in 4 PC12 cells in 100 µL of medium under long period (5 hours) of high K+ stimulation + buffer (60 mM) for PC12 cell membrane depolarization. Our approach overcomes the disadvantages of a long diffusion time and the continuous replacement of K+ buffer, which is infeasible for studies in vivo. The present study validated that the evoked dopamine signals in ChR2-EGFP-expressing cells are significantly higher than those of EGFP-expressing cells . ChR2-mediated Ca2+ influx can active depolarized dopaminergic PC12 cells. An immediate dopamine release signal of around ∼30 pA (artifact-removed) can be recorded within 100 ms. On the other hand, the evoked dopamine signals can reach to 155 pA after 10 seconds of optogenetic stimulation. The signal stops rising immediately and slowly returns to the basal line in around 3 minutes upon dopamine reuptake and diffusion after turn off blue light.It is important to develop a delicate tool for effective control of dynamic process of dopamine, release or reuptake, from cells and real-time monitoring of extracellular dopamine levels for neuroscience investigation or clinical application. The developed microelectrodes at a diameter of 0.1 µm was enhanced with Au-NP sensing surface area for high sensitivity of detection limit of 7 nM for monitoring dopamine release from cells. Previous studies recorded the elevation of environmental dopamine levels at 10 nM/min/5×10+ stimulation passive depolarized cells only elevated 1.8-fold of dopamine level in 20 minutes from 5×105 cells/well of PC12 cells Moreover, the dopamine signal was elevated about 3.5-fold higher than those of cells without optogenetic stimulation, whereas the conventional high Ketal. measured Ca2+ dynamics in PC12 cells upon optogenetic stimulation by fluorescent indicators 2+ influx and the relationship to dopamine release. Single-cell Ca2+ measurement of fura-2/AM was used to characterize the optogenetic stimulation effect on the Ca2+ cascade in PC12 cells. In our findings, blue light did not affect the intracellular Ca2+ level in PC12 cells without ChR2 expression. In contrast, the Ca2+ level was significantly elevated by activation of ChR2 upon blue light illumination. The 340/380 ratio was elevated about 1.6-fold of control upon 10 seconds of optogenetic stimulation. However, the elevation immediately stopped and slowly returned to the basal level after the blue light was turned off. These results show the specific effect and temporal precision of optogenetic stimulation. Moreover, our findings indicate that PC12 cells with ChR2-EGFP expression had a higher basal intracellular Ca2+ level than that of EGFP-expressing cells, which might be caused by the self-leakage of ChR2 itself and/or activation of ChR2 by the ultraviolet light (340 nm and 380 nm) used for Ca2+ recording. Ca2+-free buffer and the L-type VGCC antagonist, Nifedipine, were used to explore the effect of optogenetic stimulation on extracellular Ca2+ influx. Nifedipine is a kind of L-type VGCC inhibitor, which can be a blocker in Ca2+-dependent dopamine release. Abu-Raya etal. showed that Nifedipine can block about 25% of intracellular Ca2+ elevation upon high K+ stimulation 2+ elevation induced by optogenetic stimulation. Additionally, the intracellular Ca2+ level barely changed when cells were immersed in Ca2+-free buffer. These results indicate that extracellular Ca2+ influx is the major source for cytosolic Ca2+ elevation during optogenetic stimulation. Then, the L-type VGCC plays an essential role in optogenetic stimulation-evoked Ca2+ influx. This phenomenon can be explained as ChR2-mediated extracellular Ca2+ influx slightly depolarized PC12 cells, which turns to activate L-type VGCC then following large Ca2+ influx. Compared to those using high K+ buffer, the optogenetic approach is more controllable and can be easily manipulated using Ca2+-free buffer or L-type VGCC blocker.Previous studies have demonstrated that optogenetic stimulation can induce cell membrane depolarization of PC12 cells. The relation between blue light intensity and evoked potential has been examined 2+-free buffer and Nifedipine on the modulation of dopamine release were examined after the cascade of Ca2+ influx was characterized. Our results in 2+-free PBS) and 81% (Nifedipine in 1.8 mM Ca2+ PBS) inhibition of dopamine signals were found in comparison to that of the controlled group (1.8 mM Ca2+-containing PBS). The high correlation between Ca2+ influx and dopamine release indicates that L-type VGCC might play a major role in dopamine release from PC12 during optogenetic stimulation. As shown in 2+ elevation by blue light is in a frequency- and time-dependent manner. The effects of illumination parameters were similar in Ca2+ influx and dopamine release experiments. However, a higher power-level of blue light caused stronger artifacts in the dopamine signal as The effects of Ca2+ imaging (340 nm and 380 nm) and for activation of ChR2 (325–550 nm). It may cause a large portion of ChR2 has already activated by 340 nm and 380 nm before a start of 473 nm pulses. Also, 380 nm is more efficient in activating ChR2 than 340 nm, causing a biased readout on the 340/380 ratio. In order to obtain continuous Ca2+ measurement during 473 nm illumination for ChR2 excitation, the potential solutions to overcome this problem by using red Ca2+ indicator with ChR2, green Ca2+ indicator with red-shifted ChR2, or using the bioluminescent Ca2+ indicator Nano-lantern 2+ indicators are available with the apply on ChR2 stimulation, including synthetic dyes  and protein-based red Ca2+ sensors  The problem and limitation on this study is the overlap between excitation wavelengths for Ca2+ influx and dopamine release under optogenetic stimulation. Furthermore, varied stimulation schemes to modulate ChR2 activation of PC12 cells have been examined. The 30 Hz with 30 ms pulse duration evoked more dopamine release than CI stimulation. The cationic channel ChR2 with sequenced activation and inactivation cycles based on its channel nature. So, the CI stimulation didn’t evoke more dopamine release compared to the 30 Hz with 30 ms pulse duration might due to it unreached the maximal activation of ChR2. The traditional stimulation approaches for dopamine release always limited the therapeutic potential of their studies by used of pharmaceutical or electrical stimulations. Based on our result, novel optogenetic stimulation approach would open powerful modulation scheme for further clinical application and investigation of dopamine regulation.Our results demonstrated that optogenetic stimulation could evoke and precisely control dopamine signals from PC12 cells. The extracellular dopamine signals can be evoked immediately within 100 ms when the ChR2-expressing cells were illuminated by blue light and slowly returned to the baseline after the stimulation was turned off. Optogenetic stimulation could be a powerful tool for activation of PC12 cells and controlling of dopamine release at high temporal resolution, easy manipulation and less side effects. In addition, the L-type VGCC on membrane might play a key role in the evoked Ca