Alterations of ATP-sensitive K+ channels in human umbilical arterial smooth muscle during gestational diabetes mellitus.

We investigated the alterations of ATP-sensitive K+ (KATP) channels in human umbilical arterial smooth muscle cells during gestational diabetes mellitus (GDM). The amplitude of the KATP current induced by application of the KATP channel opener pinacidil (10 µM) was reduced in the GDM group than in the control group. Pinacidil-induced vasorelaxation was also predominant in the normal group compared with the GDM group. Reverse transcription polymerase chain reaction and Western blot analysis suggested that the expression of KATP channel subunits such as Kir6.1, Kir6.2, and SUR2B were decreased in the GDM group relative to the normal group. The application of forskolin and adenosine, which activates protein kinase A (PKA) and thereby KATP channels, elicited KATP current in both the normal and GDM groups. However, the current amplitudes were not different between the normal and GDM groups. In addition, the expression levels of PKA subunits were not altered between the two groups. These results suggest that the reduction of KATP current and KATP channel-induced vasorelaxation are due to the decreased expression of KATP channels, not to the impairment of KATP-related signaling pathways.

Enhancement of biomass and lipid productivity by overexpression of a bZIP transcription factor in Nannochloropsis salina.

Microalgae are considered as excellent platforms for biomaterial production that can replace conventional fossil fuel-based fuels and chemicals. Genetic engineering of microalgae is prerequisite to maximize production of materials and to reduce costs for the production. Transcription factors (TFs) are emerging as key regulators of metabolic pathways to enhance production of molecules for biofuels and other materials. TFs with the basic leucine zipper (bZIP) domain have been known as stress regulators and are associated with lipid metabolism in plants. We overexpressed a bZIP TF, NsbZIP1, in Nannochloropsis salina, and found that transformants showed enhanced growth with concomitant increase in lipid contents. The improved phenotypes were also notable under stress conditions including N limitation and high salt. To understand the mechanism underlying improved phenotypes, we analyzed expression patterns of predicted target genes involved in lipid metabolism via quantitative RT-PCR, confirming increases transcript levels. NsbZIP1 appeared to be one of type C bZIPs in plants that has been known to regulate lipid metabolism under stress. Taken together, we demonstrated that NsbZIP1 could improve both growth and lipid production, and TF engineering can serve as an excellent genetic engineering tool for production of biofuels and biomaterials in microalgae.

Blockade of voltage-dependent K+ current in rabbit coronary arterial smooth muscle cells by the tricyclic antidepressant clomipramine.

We investigated the effect of the tricyclic antidepressant clomipramine on voltage-dependent K+ (Kv) channels in native rabbit coronary arterial smooth muscle cells. Our results showed that clomipramine inhibited vascular Kv channels in a concentration-dependent manner, with an IC50 value of 8.61 ± 4.86 µM and a Hill coefficient (n) of 0.58 ± 0.07. The application of 10 µM clomipramine did not affect the activation curves of the Kv channels; however, the inactivation curves of the Kv channels were shifted toward a more negative potential. The clomipramine-induced inhibition of Kv currents was not changed by the application of train pulses (1 or 2 Hz), which demonstrated that clomipramine inhibited Kv current in a state (use)-independent manner. Pretreatment with the Kv1.5 and Kv2.1 inhibitors, DPO-1 and guangxitoxin, respectively, partially reduced the clomipramine-induced inhibition of Kv currents. Therefore, we concluded that clomipramine inhibited vascular Kv channels in a concentration-dependent, but state (use)-independent manner, regardless of its own function.

Inhibitory effect of the tricyclic antidepressant amitriptyline on voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

Amitriptyline, a tricyclic antidepressant (TCA) drug, is widely used in treatment of psychiatric disorders. However, the side effects of amitriptyline on vascular K+ channels remain to be determined. Therefore, we investigated the effect of the tricyclic antidepressant and serotonin reuptake inhibitor amitriptyline on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells, using the whole-cell patch clamp technique. The Kv current amplitudes were inhibited by amitriptyline in a concentration-dependent manner, with an apparent IC50 value of 2.2 ± 0.14 µmol/L and a Hill coefficient of 0.87 ± 0.03. Amitriptyline shifted the activation curve to a more positive potential, but had no significant effect on the inactivation curve, suggesting that amitriptyline altered the voltage sensitivity of Kv channels. Pretreatment with Kv1.5 and Kv1.2 channel inhibitors did not alter the inhibitory effect of amitriptyline on Kv channels. Additionally, application of train pulses (1 and 2 Hz) did not affect amitriptyline-induced inhibition of Kv currents, which suggested that the action of amitriptyline on Kv channels was not use (state)-dependent. From these results, we concluded that amitriptyline inhibited the channels in a concentration-dependent, but state-independent manner.

The selective serotonin reuptake inhibitor dapoxetine inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

We investigated the inhibitory effect of dapoxetine, a selective serotonin reuptake inhibitor (SSRI), on voltage-dependent K+ (Kv) channels using native smooth muscle cells from rabbit coronary arteries. Dapoxetine inhibited Kv channel currents in a concentration-dependent manner, with an IC50 value of 2.68±0.94 µmol/L and a slope value (Hill coefficient) of 0.63±0.11. Application of 10 µmol/L dapoxetine accelerated the rate of inactivation of Kv currents. Although dapoxetine did not modify current activation kinetics, it caused a significant negative shift in the inactivation curves. Application of train step (1 or 2 Hz) progressively increased the inhibitory effect of dapoxetine on Kv channels. In addition, the recovery time constant was extended in its presence, suggesting that the longer recovery time constant from inactivation underlies a use-dependent inhibition of the channel. From these results, we conclude that dapoxetine inhibits Kv channels in a dose-, time-, use-, and state (open)-dependent manner, independent of serotonin reuptake inhibition.

Escitalopram, a selective serotonin reuptake inhibitor, inhibits voltage-dependent K+ channels in coronary arterial smooth muscle cells.

We investigated the inhibitory effect of escitalopram, a selective serotonin reuptake inhibitor (SSRI), on voltage-dependent K+ (Kv) channels in freshly separated from rabbit coronary arterial smooth muscle cells. The application of escitalopram rapidly inhibited vascular Kv channels. Kv currents were progressively inhibited by an increase in the concentrations of escitalopram, suggesting that escitalopram inhibited vascular Kv currents in a concentration-dependent manner. The IC50 value and Hill coefficient for escitalopram-induced inhibition of Kv channels were 9.54±1.33 µM and 0.75±0.10, respectively. Addition of escitalopram did not alter the steady-state activation and inactivation curves, suggesting that the voltage sensors of the channels were not affected. Pretreatment with inhibitors of Kv1.5 and/or Kv2.1 did not affect the inhibitory action of escitalopram on vascular Kv channels. From these results, we concluded that escitalopram decreased the vascular Kv current in a concentration-dependent manner, independent of serotonin reuptake inhibition.

Nortriptyline, a tricyclic antidepressant, inhibits voltage-dependent K+ channels in coronary arterial smooth muscle cells.

We demonstrated the effect of nortriptyline, a tricyclic antidepressant drug and serotonin reuptake inhibitor, on voltage-dependent K+ (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Nortriptyline inhibited Kv currents in a concentration-dependent manner, with an apparent IC50 value of 2.86±0.52 µM and a Hill coefficient of 0.77±0.1. Although application of nortriptyline did not change the activation curve, nortriptyline shifted the inactivation current toward a more negative potential. Application of train pulses (1 or 2 Hz) did not change the nortriptyline-induced Kv channel inhibition, suggesting that the effects of nortiprtyline were not use-dependent. Preincubation with the Kv1.5 and Kv2.1/2.2 inhibitors, DPO-1 and guangxitoxin did not affect nortriptyline inhibition of Kv channels. From these results, we concluded that nortriptyline inhibited Kv channels in a concentration-dependent and state-independent manner independently of serotonin reuptake.

Cisapride, a selective serotonin 5-HT4-receptor agonist, inhibits voltage-dependent K(+) channels in rabbit coronary arterial smooth muscle cells.

We investigated the effect of cisapride, a selective serotonin 5-HT4-receptor agonist, on voltage-dependent K(+) (Kv) channels using freshly isolated smooth muscle cells from the coronary arteries of rabbits. The amplitude of Kv currents was reduced by cisapride in a concentration-dependent manner, with an IC50 value of 6.77 ± 6.01 µM and a Hill coefficient of 0.51 ± 0.18. The application of cisapride shifted the steady-state inactivation curve toward a more negative potential, but had no significant effect on the steady-state activation curve. This suggested that cisapride inhibited the Kv channel in a closed state by changing the voltage sensitivity of Kv channels. The application of another selective serotonin 5-HT4-receptor agonist, prucalopride, did not affect the basal Kv current and did not alter the inhibitory effect of cisapride on Kv channels. From these results, we concluded that cisapride inhibited vascular Kv current in a concentration-dependent manner by shifting the steady-state inactivation curve, independent of its own function as a selective serotonin 5-HT4-receptor agonist.

Y-27632, a Rho-Associated Protein Kinase Inhibitor, Inhibits Voltage-Dependent K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells.

We examined the effects of the Rho-associated protein kinase (ROCK) inhibitor Y-27632 on voltage-dependent K+ (Kv) channels in rabbit coronary arterial smooth muscle cells using the whole-cell patch clamp technique. Y-27632 reduced the amplitude of the Kv current in a concentration-dependent manner, with an IC50 of 0.87 ± 0.06 µmol/l and a Hill coefficient of 1.48 ± 0.06. Y-27632 did not affect the steady-state activation or inactivation curves, suggesting that the drug does not affect the voltage sensitivity of Kv channels. Another ROCK inhibitor, H-1152, did not affect the Kv current and had no significant effect on the Y-27632-induced inhibition of Kv channels, indicating that the inhibitory effect of Y-27632 on the Kv current is independent of ROCK signaling. From these results, we conclude that Y-27632 inhibits the Kv channel current in a dose-dependent and ROCK signaling-independent manner.

The Effects of the Selective Serotonin Reuptake Inhibitor Fluvoxamine on Voltage-Dependent K(+) Channels in Rabbit Coronary Arterial Smooth Muscle Cells.

We demonstrated the inhibitory effect of fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), on voltage-dependent K(+) (Kv) channels in freshly isolated rabbit coronary arterial smooth muscle cells using a whole-cell patch clamp technique. Fluvoxamine reduced the amplitude of Kv currents in a concentration-dependent manner with an IC50 value of 3.71±1.09 µM and a Hill coefficient of 0.62±0.14. Although fluvoxamine did not significantly affect the steady-state activation curve, it shifted the steady-state inactivation curve toward a more negative potential. Pretreatment with another SSRI, paroxetine, did not affect the basal Kv current and did not alter the inhibitory effect of fluvoxamine on Kv channels. We concluded that fluvoxamine inhibits the Kv current in a concentration-dependent manner and in a closed (inactivated) state of the Kv channels independent of serotonin reuptake inhibition.

Increasing lipid production in Chlamydomonas reinhardtii through genetic introduction for the overexpression of glyceraldehyde-3-phosphate dehydrogenase.

Microalgae, valued for their sustainability and CO2 fixation capabilities, are emerging as promising sources of biofuels and high-value compounds. This study aimed to boost lipid production in C. reinhardtii by overexpressing chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a key enzyme in the Calvin cycle and glycolysis, under the control of a nitrogen-inducible NIT1 promoter, to positively impact overall carbon metabolism. The standout transformant, PNG#7, exhibited significantly increased lipid production under nitrogen starvation, with biomass rising by 44% and 76% on days 4 and 16, respectively. Fatty acid methyl ester (FAME) content in PNG#7 surged by 2.4-fold and 2.1-fold, notably surpassing the wild type (WT) in lipid productivity by 3.4 and 3.7 times on days 4 and 16, respectively. Transcriptome analysis revealed a tenfold increase in transgenic GAPDH expression and significant upregulation of genes involved in fatty acid and triacylglycerol synthesis, especially the gene encoding acyl-carrier protein gene (ACP, Cre13. g577100. t1.2). In contrast, genes related to cellulose synthesis were downregulated. Single Nucleotide Polymorphism (SNP)/Indel analysis indicated substantial DNA modifications, which likely contributed to the observed extensive transcriptomic and phenotypic changes. These findings suggest that overexpressing chloroplast GAPDH, coupled with genetic modifications, effectively enhances lipid synthesis in C. reinhardtii. This study not only underscores the potential of chloroplast GAPDH overexpression in microalgal lipid synthesis but also highlights the expansive potential of metabolic engineering in microalgae for biofuel production.

Heterologous synthesis of chlorophyll b in Nannochloropsis salina enhances growth and lipid production by increasing photosynthetic efficiency.

BACKGROUND: Chlorophylls play important roles in photosynthesis, and thus are critical for growth and related metabolic pathways in photosynthetic organisms. They are particularly important in microalgae, emerging as the next generation feedstock for biomass and biofuels. Nannochloropsis are industrial microalgae for these purposes, but are peculiar in that they lack accessory chlorophylls. In addition, the localization of heterologous proteins to the chloroplast of Nannochloropsis has not been fully studied, due to the secondary plastid surrounded by four membranes. This study addressed questions of correct localization and functional benefits of heterologous expression of chlorophyllide a oxygenase from Chlamydomonas (CrCAO) in Nannochloropsis. RESULTS: We cloned CrCAO from Chlamydomonas, which catalyzes oxidation of Chla producing Chlb, and overexpressed it in N. salina to reveal effects of the heterologous Chlb for photosynthesis, growth, and lipid production. For correct localization of CrCAO into the secondary plastid in N. salina, we added the signal-recognition sequence and the transit peptide (cloned from an endogenous chloroplast-localized protein) to the N terminus of CrCAO. We obtained two transformants that expressed CrCAO and produced Chlb. They showed improved growth under medium light (90 µmol/m2/s) conditions, and their photosynthetic efficiency was increased compared to WT. They also showed increased expression of certain photosynthetic proteins, accompanied by an increased maximum electron-transfer rate up to 15.8% and quantum yields up to 17%, likely supporting the faster growth. This improved growth resulted in increased biomass production, and more importantly lipid productivity particularly with medium light. CONCLUSIONS: We demonstrated beneficial effects of heterologous expression of CrCAO in Chlb-less organism N. salina, where the newly produced Chlb enhanced photosynthesis and growth. Accordingly, transformants showed improved production of biomass and lipids, important traits of microalgae from the industrial perspectives. Our transformants are the first Nannochloropsis cells that produced Chlb in the whole evolutionary path. We also succeeded in delivering a heterologous protein into the secondary plastid for the first time in Nannochloropsis. Taken together, our data showed that manipulation of photosynthetic pigments, including Chlb, can be employed in genetic improvements of microalgae for production of biofuels and other biomaterials.

Advanced multigene expression system for Nannochloropsis salina using 2A self-cleaving peptides.

Even though there has been much interest in genetic engineering of microalgae, its progress has been slow due to the difficulty and limitation of available techniques. Currently, genetic modification in most microalgal strains is confined to single gene transformation. Here, a multigene expression system for the oleaginous model strain Nannochloropsis salina was developed with glycine-serine-glycine spacer linked 2A self-cleaving peptides (2A) for the first time. An efficiency test of the four most widely used 2As revealed that two different types of 2As T2A and E2A have the best performance in N. salina with a maximum cleavage rate of nearly 45%. The system was able to express the linked sequence of the selection marker shble and the fluorescence protein sfCherry with intact functions. Because 2A enabled multigene expression in the single cassette form, the use of 2A also reduced the vector size, which along with the stronger promoter resulted in a 9-fold increase in the transformation efficiency. Furthermore, confirmative screening accuracy of more than 90% was observed. Hence, the 2A applied vector system is expected to be beneficial in microalgal research field because it enables multigene expression as well as offering improved transformation and screening efficiency.

The vasorelaxant effect of antidiabetic drug nateglinide via activation of voltage-dependent K+ channels in aortic smooth muscle.

AIMS: We investigated the vasorelaxant effect of nateglinide and its related mechanisms using phenylephrine (Phe)-induced precontracted aortic rings. METHODS: Arterial tone measurement was performed in aortic smooth muscle. RESULTS: The application of nateglinide induced vasorelaxation in a concentration-dependent manner. Pretreatment with the large-conductance Ca2+ -activated K+ (BKCa ) channel inhibitor paxilline, the inwardly rectifying K+ (Kir) channel inhibitor Ba2+ , and ATP-sensitive K+ (KATP ) channel inhibitor glibenclamide did not affect the vasorelaxant effect of nateglinide. However, pretreatment with the voltage-dependent K+ (Kv) channel inhibitor 4-aminopyridine (4-AP) effectively reduced the vasorelaxant effect of nateglinide. Pretreatment with the Ca2+ inhibitor nifedipine and the sarcoplasmic/endoplasmic reticulum Ca2+ -ATPase inhibitor thapsigargin did not change the vasorelaxant effect of nateglinide. Additionally, the vasorelaxant effect of nateglinide was not altered in the presence of an adenylyl cyclase, a protein kinase A, a guanylyl cyclase, or a protein kinase G inhibitor. The vasorelaxant effect of nateglinide was not affected by the elimination of the endothelium. In addition, pretreatment with a nitric oxide synthase inhibitor, L-NAME, and a small-conductance Ca2+ -activated K+ (SKCa ) channel inhibitor, apamin, did not change the vasorelaxant effect of nateglinide. CONCLUSION: Nateglinide induced vasorelaxation via the activation of the Kv channel independent of other K+ channels, Ca2+ channels, intracellular Ca2+ ([Ca2+ ]i ), and the endothelium.

Inhibition of the Voltage-Dependent K+ Current by the Tricyclic Antidepressant Desipramine in Rabbit Coronary Arterial Smooth Muscle Cells.

We describe the effect of a tricyclic antidepressant drug desipramine on voltage-dependent K+ (Kv) currents in freshly isolated rabbit coronary arterial smooth muscle cells using a conventional whole-cell patch clamp technique. Application of desipramine rapidly decreased the Kv current amplitude in a concentration-dependent manner, with an IC50 value of 5.91 ± 0.18 µM and a Hill coefficient of 0.61 ± 0.09. The steady-state inactivation curves of the Kv channels were not affected by desipramine. However, desipramine shifted the steady-state inactivation curves toward a more negative potential. Application of train pulses (1 or 2 Hz) slightly reduced the Kv current amplitude. Such reduction in the Kv current amplitude by train pulses increased in the presence of desipramine. Furthermore, the inactivation recovery time constant was also increased in the presence of desipramine, suggesting that desipramine-induced inhibition of the Kv current was use-dependent. Application of a Kv1.5 inhibitor (DPO-1) and/or a Kv2.1 inhibitor (guangxitoxin) did not change the inhibitory effect of desipramine on Kv currents. Based on these results, we concluded that desipramine directly inhibited the Kv channels in a dose- and state-dependent manner, but the effect was independent of norepinephrine/serotonin reuptake inhibition.

The anti-diabetic drug dapagliflozin induces vasodilation via activation of PKG and Kv channels.

AIM: Considering the clinical efficacy of dapagliflozin in patients with type 2 DM and the pathophysiological relevance of Kv channels for vascular reactivity. We investigate the vasodilatory effect of dapagliflozin and related mechanisms using phenylephrine (Phe)-induced contracted aortic rings. MATERIAL AND METHODS: Arterial tone measurement was performed in aortic smooth muscle. KEY FINDINGS: Application of dapagliflozin induced vasodilation in a concentration-dependent manner. Pre-treatment with the BKCa channel inhibitor paxilline, the KATP channel inhibitor glibenclamide, and the Kir channel inhibitor Ba2+ did not change dapagliflozin-induced vasodilation. However, application of the Kv channels inhibitor 4-AP effectively inhibited dapagliflozin-induced vasodilation. Application of the Ca2+ channel inhibitor nifedipine and the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor thapsigargin did not alter the vasodilatory effect of dapagliflozin. Moreover, the adenylyl cyclase inhibitor SQ 22536 and the protein kinase A (PKA) inhibitor KT 5720 had no effect on dapagliflozin-induced vasodilation. Although guanylyl cyclase inhibitors, NS 2028 and ODQ, did not reduce the vasodilatory effect of dapagliflozin, the protein kinase G (PKG) inhibitor KT 5823 effectively inhibited dapagliflozin-induced vasodilation. The vasodilatory effect of dapagliflozin was not affected by elimination of the endothelium. Furthermore, pretreatment with the nitric oxide synthase inhibitor L-NAME or the small-conductance Ca2+-activated K (SKCa) channel inhibitor apamin did not change the vasodilatory effect of dapagliflozin. SIGNIFICANCE: We concluded that dapagliflozin induced vasodilation via the activation of Kv channels and PKG, and was independent of other K+ channels, Ca2+ channels, intracellular Ca2+, and the endothelium.

Functional expression and pharmaceutical efficacy of cardiac-specific ion channels in human embryonic stem cell-derived cardiomyocytes.

Cardiomyocytes differentiated from human pluripotent stem cells provide promising tools for screening of cardiotoxic drugs. For evaluation of human pluripotent stem cell-derived cardiomyocytes for cardiotoxicity test, in the present study, human embryonic stem cells (hESCs) were differentiated to cardiomyocytes, followed by metabolic selection to enrich the differentiated cardiomyocytes. The highly purified hESC-derived cardiomyocytes (hESC-CMs) expressed several cardiomyocyte-specific markers including cTnT, MLC2a, and α-SA, but not pluripotency markers, such as OCT4 and NANOG. Patch clamp technique and RT-PCR revealed the expression of cardiomyocyte-specific Na+, Ca2+, and K+ channels and cardiac action potential in hESC-CMs. To explore the potential use of hESC-CMs as functional cardiomyocytes for drug discovery and cardiotoxicity screening, we examined the effects of bisindolylmaleimide (BIM) (I), which inhibits native cardiac Ca2+ channels, on the Ca2+ channel activity of hESC-CMs. We observed a similar response for the BIM (I)-induced modulation of Ca2+ channels between hESC-CMs and native cardiomyocytes through L-type Ca2+ channel current. These results suggest that hESC-CMs can be useful for evaluation of pharmaceutical efficacy and safety of novel drug candidate in cardiac research.

Isolation, phenotypic characterization and genome wide analysis of a Chlamydomonas reinhardtii strain naturally modified under laboratory conditions: towards enhanced microalgal biomass and lipid production for biofuels.

BACKGROUND: Microalgal strain development through genetic engineering has received much attention as a way to improve the traits of microalgae suitable for biofuel production. However, there are still some limitations in application of genetically modified organisms. In this regard, there has been recent interest in the isolation and characterization of superior strains naturally modified and/or adapted under a certain condition and on the interpretation of phenotypic changes through the whole genome sequencing. RESULTS: In this study, we isolated and characterized a novel derivative of C. reinhardtii, whose phenotypic traits diverged significantly from its ancestral strain, C. reinhardtii CC-124. This strain, designated as CC-124H, displayed cell population containing increased numbers of larger cells, which resulted in an increased biomass productivity compared to its ancestor CC-124. CC-124H was further compared with the CC-124 wild-type strain which underwent long-term storage under low light condition, designated as CC-124L. In an effort to evaluate the potential of CC-124H for biofuel production, we also found that CC-124H accumulated 116 and 66% greater lipids than that of the CC-124L, after 4 days under nitrogen and sulfur depleted conditions, respectively. Taken together, our results revealed that CC-124H had significantly increased fatty acid methyl ester (FAME) yields that were 2.66 and 1.98 times higher than that of the CC-124L at 4 days after the onset of cultivation under N and S depleted conditions, respectively, and these higher FAME yields were still maintained by day 8. We next analyzed single nucleotide polymorphisms (SNPs) and insertion/deletions (indels) based on the whole genome sequencing. The result revealed that of the 44 CDS region alterations, 34 resulted in non-synonymous substitutions within 33 genes which may mostly be involved in cell cycle, division or proliferation. CONCLUSION: Our phenotypic analysis, which emphasized lipid productivity, clearly revealed that CC-124H had a dramatically enhanced biomass and lipid content compared to the CC-124L. Moreover, SNPs and indels analysis enabled us to identify 34 of non-synonymous substitutions which may result in phenotypic changes of CC-124H. All of these results suggest that the concept of adaptive evolution combined with genome wide analysis can be applied to microalgal strain development for biofuel production.

Current status and perspectives of genome editing technology for microalgae.

Genome editing techniques are critical for manipulating genes not only to investigate their functions in biology but also to improve traits for genetic engineering in biotechnology. Genome editing has been greatly facilitated by engineered nucleases, dubbed molecular scissors, including zinc-finger nuclease (ZFN), TAL effector endonuclease (TALEN) and clustered regularly interspaced palindromic sequences (CRISPR)/Cas9. In particular, CRISPR/Cas9 has revolutionized genome editing fields with its simplicity, efficiency and accuracy compared to previous nucleases. CRISPR/Cas9-induced genome editing is being used in numerous organisms including microalgae. Microalgae have been subjected to extensive genetic and biological engineering due to their great potential as sustainable biofuel and chemical feedstocks. However, progress in microalgal engineering is slow mainly due to a lack of a proper transformation toolbox, and the same problem also applies to genome editing techniques. Given these problems, there are a few reports on successful genome editing in microalgae. It is, thus, time to consider the problems and solutions of genome editing in microalgae as well as further applications of this exciting technology for other scientific and engineering purposes.

The PPARα activator fenofibrate inhibits voltage-dependent K+ channels in rabbit coronary arterial smooth muscle cells.

We examined the effects of the PPARα activator fenofibrate on voltage-dependent K+ (Kv) channels using a patch clamp technique in native rabbit coronary arterial smooth muscle cells. Kv current was inhibited by application of fenofibrate in a concentration-dependent manner, with an apparent IC50 value of 6.39 ± 0.53µM and a slope value (Hill coefficient) of 1.63 ± 0.10. Fenofibrate accelerated the decay rate of Kv channel inactivation. The rate constants of association and dissociation for fenofibrate were 0.81± 0.05µM-1s-1 and 4.70 ± 0.47s-1, respectively. Although fenofibrate did not affect the steady-state activation curves, fenofibrate shifted the inactivation curves toward a more negative potential. Application of train pulses (1 or 2Hz) progressively increased the fenofibrate-induced inhibition of the Kv channel, and the recovery time constant from inactivation was increased in the presence of fenofibrate, which suggested that the inhibitory effect of fenofibrate is use-dependent. Another PPARα activator, bezafibrate and PPARα inhibitor, GW 6471, did not affect the Kv current and also did not change the inhibitory effect of fenofibrate on the Kv current. From these results, we suggest that fenofibrate inhibited Kv current in a state-, time-, and use-dependent manner, completely independent of PPARα activation.