At concentrations of up to 100 µM, asunaprevir, atazanavir, daclatasvir, darunavir, elbasvir, etravirine, grazoprevir, ledipasvir, lopinavir, rilpivirine, ritonavir, saquinavir, and velpatasvir inhibited [3H]-digoxin transport in Caco-2 cells and/or in precision-cut intestinal slices prepared from the human jejunum (hPCIS). However, abacavir, dolutegravir, maraviroc, sofosbuvir, tenofovir disoproxil fumarate, and zidovudine had no inhibitory effect. We thus found that most of the tested antivirals have a high potential to cause drug–drug interactions on intestinal ABCB1. Comparing the Caco-2 and hPCIS experimental models, we conclude that the Caco-2 transport assay is more sensitive, but the results obtained using hPCIS agree better with reported in vivo observations. More inhibitors were identified when using digoxin as the ABCB1 probe substrate than when using rhodamine123. However, both approaches had limitations, indicating that inhibitory potency should be tested with at least these two ABCB1 probes.The inhibition of P-glycoprotein (ABCB1) could lead to increased drug plasma concentrations and hence increase drug toxicity. The evaluation of a drug’s ability to inhibit ABCB1 is complicated by the presence of several transport-competent sites within the ABCB1 binding pocket, making it difficult to select appropriate substrates. Here, we investigate the capacity of antiretrovirals and direct-acting antivirals to inhibit the ABCB1-mediated intestinal efflux of [ Human immunodeficiency virus (HIV) and hepatitis C virus (HCV) infections are major global health problems. Over 100 million people are currently living with HIV or HCV ,3, almosMost antivirals are thought to be substrates and/or inhibitors of P-glycoprotein (ABCB1) ,17,18. AHuman-derived precision-cut intestinal slices (hPCIS) are miniature models of the intestine with a physiological 3D architecture that can be used to study the effects of intestinal metabolism and transporter activity on drug pharmacokinetics ,33. By c3H]-digoxin alone, for which the efflux ratio (rPapp) was 9.53 ± 2.22. Adding the model ABCB1 inhibitor CP100356 monohydrochloride (CP100356) (2 µM) reduced the rPapp of [3H]-digoxin to 1.49 ± 0.11. These values are comparable to those reported previously, confirming the functional expression of ABCB1 in the Caco-2 cells [app of [3H]-digoxin to values in the range of 1.11 to 1.91, making their effects comparable to that of the model inhibitor. Atazanavir (20 µM), darunavir (50 µM), etravirine (20 µM), lopinavir (5 µM), and ritonavir (20 µM) and DAA asunaprevir (20 µM), elbasvir (5 µM), grazoprevir (20 µM), and ledipasvir (50 µM) also significantly inhibited [3H]-digoxin, albeit to a lesser extent, giving rPapp values of 2.75 to 5.88. Etravirine, elbasvir, and ledipasvir exhibited low solubility, so higher concentrations that could potentially inhibit ABCB1 more strongly were not tested. No significant effect on [3H]-digoxin transport was observed for the antiretrovirals abacavir, dolutegravir, maraviroc, tenofovir DF, and zidovudine, or the DAAs sofosbuvir and velpatasvir. Whereas abacavir, maraviroc, and tenofovir DF were tested at the highest chosen concentration of 100 µM, dolutegravir and velpatasvir were only tested at concentrations of 10 and 5 µM, respectively, due to their limited solubility. These results are summarized in We initially performed bidirectional transport experiments using [-2 cells . The ant3H]-digoxin  together with antiretrovirals, DAAs, or CP100356 on the ATP content of hPCIS prepared from intestine samples collected from four donors. There were no statistically significant differences between the ATP contents of the tested samples  were 6.1 and 6.6 pmol × µg−1, respectively. It thus appears that the model inhibitor and antivirals did not affect PCIS viability, even at the highest tested concentrations.Because the validity of accumulation studies in hPCIS strongly depends on hPCIS viability, we investigated the effect of 2.5 h treatments with [ samples . The med3H]-digoxin 12-fold. As shown in 3H]-digoxin increased when hPCIS were treated with atazanavir , darunavir , lopinavir , ritonavir , and saquinavir . The observed increases were comparable to those observed for CP100256 (2 µM). In contrast to the results obtained in Caco-2 cells, atazanavir (20 µM), darunavir (50 µM), etravirine (20 µM), and rilpivirine (20 µM) did not inhibit [3H]-digoxin efflux from hPCIS. Abacavir (100 µM), dolutegravir (10 µM), lopinavir (20 µM), maraviroc (100 µM), saquinavir (5 µM), tenofovir DF (100 µM), and zidovudine (100 µM) also did not induce any detectable inhibition of [3H]-digoxin efflux, in accordance with the results obtained using Caco-2 cells.To investigate the inhibitory effect of antivirals on ABCB1 in the intestine, we used hPCIS prepared from jejunal tissue obtained from five donors. The model ABCB1 inhibitor CP100356 (2 µM) increased the accumulation of [3H]-digoxin uptake by factors of 9.3, 15.2, and 13.2, respectively, whereas sofosbuvir (100 µM) had no effect. However, in contrast to the results obtained in Caco-2 cells, grazoprevir increased [3H]-digoxin uptake only at the highest tested concentration (50 µM); the other compounds showing inhibitory activity in vitro, i.e., elbasvir (5 µM) and ledipasvir (20 and 50 µM), caused no inhibition of [3H]-digoxin efflux from hPCIS. Conversely, velpatasvir (5 µM) caused a significant (9.3-fold) increase in [3H]-digoxin accumulation in hPCIS.In keeping with the observation in Caco-2 cells, the DAAs asunaprevir (at both tested concentrations of 20 and 50 µM) and daclatasvir (at 20 µM) increased the -digoxin was purchased from Moravek Biochemicals . Abacavir, atazanavir, etravirine, lopinavir, maraviroc, rilpivirine, ritonavir, saquinavir, tenofovir DF, and zidovudine were obtained from the NIH AIDS Reagent Program. Asunaprevir, daclatasvir, darunavir, dolutegravir, elbasvir, grazoprevir, ledipasvir, sofosbuvir, and velpatasvir were acquired from MedChemExpress LLC . The model ABCB1 inhibitor CP100356 -digoxin at an activity level of 0.12 µCi/mL, corresponding to a low non-saturating concentration of 6 nM [3H]-digoxin was quantified using a Tri-Carb 2900TR liquid scintillation analyzer . Its concentration in the samples collected during Caco-2 experiments was measured after adding 1 mL of Ultima GoldTM Cocktail. A to B and B to A transport were evaluated in terms of an apparent permeability coefficient (Papp), calculated using the equation in -digoxin  and/or the antivirals. The ATP contents before and after incubation were then compared.To evaluate whether antivirals or -digoxin  and the compound being tested. Both incubation steps were conducted in a humidified atmosphere of 80% O2 and 5% CO2 at 37 °C [3H]-digoxin was stopped after 2 h of incubation by washing the slices twice in the Krebs–Henseleit cold buffer (4 °C). Slices were transferred to 2 mL microvials containing 600 µL of acetonitrile solution  and approximately 300 mg of glass minibeads , and homogenized with a FastPrep24 5G minibead beater . The samples were then centrifuged . Concentrations of [3H]-digoxin in the supernatant samples (300 µL) were assessed after mixing them with 1.5 mL of Ultima Gold™ via scintillation counting . The pellets obtained during centrifugation were dried overnight at 37 °C and then solubilized in 200 µL of 5 M NaOH for 24 h. Milli-Q water was then added to the samples to achieve a NaOH concentration of 1 M. The protein content was measured using a bicinchoninic acid protein kit . The measured [3H]-digoxin concentrations were normalized against the protein content.Ex vivo accumulation assays were performed as previously described . Directlat 37 °C ,34,35. T3H]-digoxin accumulation in hPCIS was evaluated using the non-parametric paired Friedman test followed by Dunn’s test. Values differing from controls at the p < 0.05, p < 0.01, and p < 0.001 levels are indicated with the labels *, **, and ***, respectively.Statistical analysis of bidirectional transport across Caco-2 cells was performed using an ordinary one-way ANOVA with Dunnett’s post hoc multiple comparisons test. The statistical significance of differences in the measured ATP contents of hPCIS after different treatments was assessed using the non-parametric Kruskal–Wallis test followed by Dunn’s test. The effect of antivirals on [We have shown that asunaprevir, atazanavir, daclatasvir, darunavir, grazoprevir, lopinavir, ritonavir, and saquinavir inhibit ABCB1 in both Caco-2 monolayers and hPCIS. Therefore, we conclude that these drugs have a high potential to cause DDIs on intestinal ABCB1. Our hPCIS data also suggest that velpatasvir may inhibit intestinal ABCB1. There is supporting clinical evidence that most of these antivirals increase the AUC of ABCB1 substrates. Our findings suggest that the inhibition of intestinal ABCB1 contributes to this increase in AUC and should, therefore, be taken into account when establishing new antiviral combination regimens or when considering polypharmacy in HIV- and/or HCV-positive patients, especially in cases involving drugs whose absorption is significantly reduced by ABCB1, such as dabigatran etexilate and digoxin ,15. On t


ALFPm3), encoding an antimicrobial peptide from Penaeus monodon with a super activity was expressed in Chlamydomonas reinhardtii, which would develop a microalga strain that can be used for the antimicrobial peptide production. To construct the expression cluster, namely pH2A-Pm3, the codon optimized ALFPm3 gene was fused with the ble reporter by 2A peptide and inserted into pH124 vector. The glass-bead method was performed to transform pH2A-Pm3 into C. reinhardtii CC-849. In addition to 8 μg/mL zeocin resistance selection, the C. reinhardtii transformants were further confirmed by genomic PCR and RT-PCR. Western blot analysis showed that the C. reinhardtii-derived ALFPm3 (cALFPm3) was successfully expressed in C. reinhardtii transformants and accounted for 0.35% of the total soluble protein (TSP). Furthermore, the results of antibacterial assay revealed that the cALFPm3 could significantly inhibit the growth of a variety of bacteria, including both Gram-negative bacteria and Gram-positive bacteria at a concentration of 0.77 μM. Especially, the inhibition could last longer than 24 h, which performed better than ampicillin. Hence, this study successfully developed a transgenic C. reinhardtii strain, which can produce the active ALFPm3 driven from P. monodon, providing a potential strategy to use C. reinhardtii as the cell factory to produce antimicrobial peptides.Antimicrobial peptides are a class of proteins with antibacterial functions. In this study, the anti-lipopolysaccharide factor isoform 3 gene ( Limulus polyphemus [Penaeus monodon [Escherichia coli [Pichia pastoris [E. coli possibly because of its strong antimicrobial activity [ALFPm3 gene could be expressed in P. pastoris and its protein extracts showed a broad-spectrum antimicrobial activity, especially on Gram-negative bacteria [2 releasement, and the challenge of rALFPm3 extraction and purification. Additionally, P. pastoris expression system will cause excessive glycosylation, which would restrain the activity of ALFPm3 [Chlamydomonas reinhardtii can overcome these problems. Currently, C. reinhardtii has established the stable genetic transformation systems in nucleus, chloroplast, and mitochondrion [C. reinhardtii has been sequenced [C. reinhardtii. As a platform for protein production, C. reinhardtii has certain advantages, including rapid growth, capability of protein post-translational processing, and expressing a large amount of foreign protein in chloroplasts. Furthermore, it is capable of photoautotrophy using low-cost medium and it would not release environment- or human-unfriendly metabolites and endotoxins [Nowadays, people are suffering from the multidrug-resistant (MDR) microorganisms caused by the overuse of antibiotics ,2,3. Thelyphemus . ALFs ha monodon ,17,18. A monodon ,20,21,22hia coli ,25,26,27pastoris ,29 due tbacteria . Howeverf ALFPm3 ,32,33,34hondrion ,37,38,39equenced  and manyequenced , antigenequenced , antibodequenced , and vacequenced  have beedotoxins ,46,47.C. reinhardtii. In this study, the nucleotides of ALFPm3 gene were optimized according to the codon bias of C. reinhardtii and fused with the ble gene mediated by FMDV self-cleavage peptide 2A sequence, and then introduced into C. reinhardtii by a glass-bead method. Transformants were screened through zeocin resistance, confirmed by PCR detection, and evaluated by antimicrobial activity analysis. Our results showed that ALFPm3 could be efficiently expressed in transgenic chlamy. It is more important that extracts from transgenic chlamy exhibited a strong antimicrobial activity to bacteria, including some pathogenic bacteria and its performance even better than ampicillin. Our results demonstrated that C. reinhardtii is suitable for AMPs production, showing its potentiality to be used as a replacement of antibiotics on aquaculture.Considering the benefits of using chlamy to produce AMPs, it would be attractive if we can produce ALFPm3 in ALFPm3 gene from P. monodon (Genbank accession: JQ256520) was optimized according to the codon bias of C. reinhardtii. The signal peptide of ALFPm3 composed of 25 amino-acid residues in its N-terminal was removed. After optimization, the GC content of ALFPm3 gene was increased from 55.89% to 68.37% (ALFPm3 gene by Foot and Mouth Disease Virus (FMDV) self-cleavage 2A peptide. FMDV 2A peptide can cleavage between Glycine and Proline to form two independent peptides  extracted from T-2APm3 transformants . The molALFPm3 gene did not affect the normal growth of C. reinhardtii. After 6 months of cultivation, ALFPm3 gene did not lose or silence, indicating that the target gene could be stably expressed in C. reinhardtii  and the Gram-positive bacteria at a higher concentration (between 50 mM and 100 mM) [P. pastoris-derived ALFPm3, the cALFPm3 showed a higher antimicrobial activity and a long-time effect on the tested bacteria within 24 h at a lower concentration of 0.77 μM. More encouragingly, most of the tested bacteria hardly grew when incubated with 0.77 μM cALFPm3 within 24 h, which is different from the bacteria that were recovered after 9 h when treated with ampicillin. Thus, it is possible to reduce the number of antibacterial substances and obtain better antimicrobial effects by using cALFPm3. Moreover, microalgae are easy to cultivate and processing and is recognized as a safe feed additive, showing its great application in the future.It is reported that the ALFPm3 derived from  100 mM) . Here, cC. reinhardtii with high antimicrobial activity. It provides a new way for producing AMPs with high activity and the potential use of AMPs in the aquaculture industry.In this work, an expression cassette of ALFPm3 was designed and expressed in C. reinhardtii cell wall-deficient strain CC-849 was purchased from the Chlamydomonas Resource Center (USA), and was cultivated mixotrophically in Tris-Acetate-Phosphate (TAP) liquid medium or TAP agar medium contained 100 μg/mL ampicillin  at 25 °C under a continuous light intensity of 100 μE·m−2·s−1. Transgenic Chlamy was selected on the TAP agar plate supplemented with 8 μg/mL zeocin  and 100 μg/mL ampicillin. Algal cells were cultivated until the cell density reached 1 × 106 cell/mL. For the heat shock induction, cells were incubated at 40 °C for 20 min. For high light stress, cells were cultivated under the light intensity of 100, 200, 300, and 400 μE·m−2·s−1 for 30 min. After each treatment, cells were harvested by centrifugation at 5000× g for 5 min.C. reinhardtii. The optimized nucleotides were synthesized by AnHui General Biosystem Company  and then inserted into pH124 vector to obtain the plasmid pH2A-Pm3  was optimized according to the codon bias of pH2A-Pm3 . This veC. reinhardtii transformation was processed using glass-bead method according to recommendations [−2·s−1 until the cell density was reached to 1~2 × 106 cell/mL. Cells were harvested by centrifugation at 5000× g for 5 min and resolved with 300 μL TAP liquid medium to keep the final density of 2 × 108 cell/mL. Cell cultures were transferred to a new 1.5 mL microcentrifuge tube containing 300 mg glass beads (0.5 mm in diameter) and 2 μg linearized pH2A-Pm3 plasmid. The glass beads were blown gently and vortexed at the highest speed, 2500 rpm, for 25 s. Afterward, the supernatant was transferred to a new microcentrifuge tube with 10 mL TAP liquid medium, cultivated at 25 °C with a light intensity of 90 μE·m−2·s−1, and shaken continuously at 100 rpm for 22 h. Finally, cells were harvested by centrifugation at 3000× g for 5 min, resolved with 100 μL TAP liquid medium, and spread on the TAP agar medium containing 8 μg/mL zeocin and 100 μg/mL ampicillin. They were cultivated at 25 °C in an incubator under a continuous light intensity of 100 μE·m−2·s−1 until the green colonies were visible. The number of colonies was recorded and counted as the transformation frequency. Transformation frequency = numbers of colonies/total recipient cells.The ndations . Brieflyble gene and ALFPm3 gene [5 cells were harvested and then resuspended in 50 μL EDTA solution , after which they were incubated in boiling water for 8 min, cooled down on ice for 1 min, and pelleted at 14,000× g for 1 min. Finally, one microliter of the extract was used in the 20 μL PCR reaction containing a set of primers . Two microliter cDNA was used in a 20-μL PCR reaction containing a set of primers  on the ALFPm3 production, total protein was extracted and processed on immunoblot analysis. Algal cells were cultivated in TAP liquid medium under constant light intensity of 100 μE·m−2·s−1 until a cell density of 1~2 × 106 cell/mL was reached. Cells were harvested from one milliliter culture and resuspended with 100 μL lysis buffer  . After being shaken at 1200 rpm under room temperature for 10 min, cells were frozen-thawed three times in liquid nitrogen with 3 min between each repetition. The supernatant was collected at 14,000× g for 10 min at 4 °C for the following western blot analysis. Briefly, 20 μg total protein was separated by 4–20% SDS-PAGE  and then transferred to a PVDF membrane  activated by 100% methanol. The membrane was then blocked with blocking buffer ) for 2 h at room temperature. The membrane was incubated with the mouse anti-His tag antibody (1:8000)  in blocking buffer at 4 °C overnight right after washed three times with TBST. Afterward, the membrane was incubated with HRP-conjugated goat anti-mouse IgG antibody   at room temperature for 2 h, and washed with TBST for 3 times. Finally, the membrane was incubated in ECL substrate buffer   at room temperature for 3 min. The chemiluminescence signal was detected by Odyssey·Fc . The α-tublin was used as the internal control. The gray values referring protein content were analyzed by Image J software . For calculating the content of target protein, a protein contained 6 × His tag at the concentration of 3.4 μg/mL was used as the calibrator. The content of the target protein was calculated by comparing its gray value with that of calibrator.To evaluate the performance of transgenic −2·s−1 until the cell density reached 1~2 × 106 cell/mL. Cultures were centrifuged at 5000× g at 4 °C for 5 min, the supernatant was discarded, and pellets were resuspended with 10 mL lysis buffer   and shaken at 1200 rpm under room temperature for 10 min. Then cells were frozen and thawed three times in liquid nitrogen with 3 min between each repetition. The supernatant contained ALFPm3 peptide was obtained by centrifugation at 14,000× g for 10 min at 4 °C, followed by filtration with 0.22 μm filter tip. To investigate the antibacterial activity of peptide extracts, a total of six Gram-negative bacteria  and three Gram-positive bacteria  were tested. Before the assay, the tested bacteria were cultivated in 5 mL LB liquid medium under 37 °C with shaking at 200 rpm overnight. The 500 μL cultures were added into 5 mL fresh LB liquid medium and cultivated at 37 °C with shaking at 200 rpm for 1 h. Then, the bacteria cultures were diluted by 1000 times using fresh LB medium and 150 μL diluted cultures were mixed with 50 μL ALFPm3 peptide in 96-well plate. The final concentration of ALFPm3 peptide was 0.7669 μM. The extracts from wide type C. reinhardtii and 5 mg/mL ampicillin acted as negative and positive control, respectively. Finally, the mixture of bacteria and ALFPm3 was incubated at 37 °C and the OD600 was determined at 0 h, 3 h, 6 h, 9 h, 12 h, and 24 h, respectively. The growth rate (ΔOD600) of the bacteria under different treatments was obtained by comparing the OD600 value with that at 0 h.For the anti-bacterial assay, the ALFPm3 peptide was prepared as follows. The transgenic Chlamydomonas T-2APm3 was cultivated in 800 mL TAP liquid medium under a continuous light intensity of 100 μE·mC. reinhardtii, the transgenic Chlamydomonas T-2APm3 was kept constant for 6 months and the total RNA of T-2APm3 was isolated and followed by RT-PCR with primers ALF3-F and ALF3-R. PCR products were analyzed by 1% agarose gel electrophoresis. The growth curves of non-transgenic C. reinhardtii and transgenic C. reinhardtii were measured to analyze the effect of ALFPm3 on algae growth. They were cultivated in 100 mL TAP liquid medium under continuous light intensity of 100 μE·m−2·s−1 with shaking. Cell numbers were counted every day via a hemocytometer measurement.To observe the expression of ALFPm3 in p < 0.05 was considered as showing significant differences.Each experiment was conducted with three independent replicates. GraphPad Prism 5 was used to perform the statistical analysis and generate figures and tables. Student’s t-test was used to calculate the statistical difference between treatments and


L12– (5-((4-carboxyphenyl)ethynyl)picolinate)and L22– dipicolinate) have been usedin the synthesis of the highly robust and luminescent europium(III)coordination polymers [(CH3)2NH2][Eu(H2O)2(L1)2] (1) and [(CH3)2NH2][Eu(L2)2]·H2O·CH3COOH (2). Both 1 and 2 exhibit high selectivityfor detection of nitroaromatic compounds since they act as quenchersof the Eu3+ emission. Stern–Volmer plots, usingnitrobenzene as a quencher, yielded values of KSV = 150 M–1 and 160 M–1 for 1 and 2, respectively. Luminescencestudies in the presence of different metal ions indicate a high selectivityfor Fe3+ detection, with KSV values of 471 M–1 and 706 M–1 for 1 and 2, respectively. Both 1 and 2 possess extremely robust extended structures,leading to emissive properties that are stable in a wide pH range.Picolinate-basedsegmented dianionic ligands  3+ ions in aqueous medium through luminescence quenching. Furthermore,they present a high stability over a wide pH range that allows theirpractical use in harsh conditions.Luminescent europium(III) coordinationpolymers exhibitinga remarkable robustness have been prepared from ethynyl-bridged picolinateligands. These compounds can selectively detect nitrobenzene and Fe Supramolecular interactionsof luminescent CPs with the analyte may increase or decrease the luminescenceresponse, resulting in a turn-on or turn-off display, respectively.2+, Cd2+, Hg2+, Al3+, and Fe3+ is highlyrecommended, due to their accumulation as pollutants in water. Fe(III)is one of the most common and relevant components in the Earth’scrust and is also present in biological systems, playing an importantrole in metabolic processes. Its presence in an appropriate amountis essential for regular growth; nevertheless, an excess of iron(III)can lead to health problems. Recently, Ln-CPs have been found to bepromising candidates in this regard, their main drawback being theirpoor stability in aqueous medium. The first example of a europium(III)fluorescent sensor for Fe3+ detection was developed byDang et al. in 2012. Nonetheless, the selective quenching mechanismconsisted of cation exchange between the original metal ion and Fe3+ cations rather than in a specific interaction.25 Later, more Eu-CPs have been applied for thesensing of Fe3+ ions, and Table S1 contains some examples with corresponding KSV (Stern–Volmer constant) and limit of detection (LOD)values. The quenching mechanisms are based on cation exchange betweenFe3+ and Eu3+, competitive absorption of excitationenergy, and the interaction between Fe3+ and the organicligand.33Early detection of metal ions such as PbKSV values among the highestknown for CPs.34Table S2 contains a list of selected metallic complexes used fordetection of nitrobenzene (chosen as a representative nitroaromaticcompound), together with the KSV and LODachieved. Zn(II)-CPs are those which present the best parameters (higher KSV and lower LOD) in this context.Nitroaromatic compounds (NACs) represent also serious sourcesofpollution of soils and groundwater, mainly because of their high toxicity.Such materials are also frequently used as explosive materials interrorism. Highly sensitive and efficient materials able to detecttrace amounts of NACs in water are thus in strong demand. It was notuntil 2015 that Ln-CPs were applied as fluorescent sensors for NACsdetection, exhibiting quenching in the presence of nitro explosiveswith estimated 35 One is the combinationof carboxylate-based ligands and high-valent metal ions. Alternatively,soft azolate ligands in conjunction with soft divalent metal ionshave also been used.Mostmetal–organic frameworks are however easily hydrolyzed.In order to construct stable complexes, strong coordination bondsare needed, and several strategies are currently being developed forthis purpose.38 and catecholates.39 In this context, the picolinate (pic) ligandis a promising chelating anionic fragment for the design of robustCPs with high stability. On the other hand, ethynylene bridges havebeen incorporated in the design of some organic linkers because theyprovide rigidity, linear connectivity, and electron delocalization.41 Despite the fact that the picolinate ligand is a universal chelatinganionic entity, only a few segmented polytopic picolinate ligandshave been reported45 and their use in metal–organic frameworks is unexplored.Further, picolinate ligands are known to act as photosensitizers,transferring the absorbed energy in the UV region to lanthanoid cations,resulting in an enhanced lanthanoid luminescence (antenna effect).46 For example, a 104-fold enhancementof Tb(III) luminescence by using dipicolinate ligands as photosensitizershas been reported.47Another approach is focused on chelatinganionic ligands, whichis an almost unexplored alternative to that based on high-valent complexes.There are only a few examples related to tetraanionic bisbidentatedioxidobenzenedicarboxylate ligandsL12–) and5,5′-dipicolinate (L22–). L12– is a heteroditopic ligand containing apicolinate chelating unit and a benzoate anion connected by an ethynylenelinker. L22– is homoditopic, the ethynylene rigid spacer connecting two picolinatechelating units. Comparison is given on the emission properties ofthese Eu3+ compounds and their use in sensing of metalions and nitroaromatic pollutants under extreme pH conditions.We report hereinon the synthesis, structural characterization,and optical properties of highly robust europium(III) coordinationpolymers based on the segmented ligands 1 5-3·6H2O  and 5-((4-carboxyphenyl)ethynyl)picolinic acid  in 2.5mL of DMF/H2O (3:2 ratio) was placed in a glass vial andstirred for 30 min. Next, two drops of nitric acid  wereadded, and the resulting suspension was placed in an oven. The mixturewas heated at 130 °C for 3 days and then cooled to room temperatureat a cooling rate of 0.2 K·min–1. Colorlessspearhead-shaped crystals were obtained, filtered, and air-dried togive compound 1. Yield: 15.1 mg (79%). Elem anal. calcdfor C32H26EuN3O10: C,50.27; H, 3.43; N, 5.50. Found: C, 49.86; H, 3.38; N, 5.36. IR (cm–1): 3097, 3075, 3019, 2963, 2801, 2221, 1631, 1587,1373, 1356, 1244, 782, 724, 693, 662, 627, 560, 400.A solution of Eu(NO3)3·6H2O  and 5,5′-dipicolinicacid  in H2O (0.5 mL) and DMF (4 mL)was stirred in a glass vial for 30 min until a solution was formed.Then, acetic acid (1100 μL) was added, and the mixture was placedin an oven at 130 °C for 3 days. A cooling rate of 0.2 K·min–1 was applied to cool down the sample to room temperature.After the liquid was decanted, colorless aggregated crystals of 2 were obtained. The crystals were washed with water and driedin the air. Yield: 14.3 mg (60%). Elem. anal. calcd for C32H26EuN5O11·2H2O:C, 45.51; H, 3.58; N, 8.29. Found: C, 45.22; H, 3.11; N, 8.29. IR(cm–1): 3383, 3033, 2761, 2461, 1606, 1556, 1361,1244, 1033, 806, 700, 661, 628, 389.A mixture of Eu at 120 K. The data collectionroutines, unit cell refinements, and data processing were carriedout using the CrysAlis software package.49 The structures were solved using SHELXT 2018/2 via the WinGX graphicalinterface50 and refined using SHELXL-2018/3.51 All non-hydrogen atoms were refined anisotropically. H atoms on carbon atoms were included at calculated positionsand refined with a riding model with relative isotropic displacementparameters. Instead, H atoms on solvent molecules and amine H atomson dimethylammonium cations were found in Fourier difference maps,except for H atoms of O1W in compound 2. CCDC 2068840 and CCDC 2068875 contain the supplementary crystallographic datafor 1 and 2, respectively. These data areprovided free of charge by The Cambridge Crystallographic Data Centre.Suitable crystalsof 1 and 2 were collected using CuKα radiation (λ = 1.54056 Å) at room temperatureand in a 2θ range from 2 to 40°. Polycrystalline sampleswere lightly ground in an agate mortar and filled into a 0.5 mm borosilicatecapillary prior to being mounted and aligned on an Empyrean PANalyticalpowder diffractometer. For pH-dependent measurements, the sampleswere soaked in aqueous solutions with pH values ranging from 1 to14 for 2 h. Then, the samples were filtered and air-dried prior totheir analysis. Simulated diffractograms were obtained from single-crystalX-ray data using the CrystalDiffract software.PXRD measurements ofcompounds 1 and 2 and for the ligandsin their acid forms on a JASCO V-670 absorption spectrometer. Also,the absorption spectra of solvent samples and 0.01 M aqueous solutionsof different metal ions were registered at room temperature. Measurementswere performed in the 240–1000 nm range. Photoluminescent dataof both complexes in aqueous suspensions were obtained with a VarianCary Eclipse spectrometer, in quartz cuvettes (1 cm path length).For pH-sensing measurements, the solid materials (1 mg) were vigorouslydispersed in aqueous solutions (1 mL) at different pH values. Emissionspectra were registered after 2 h of sample preparation. The rangeof study was 540–720 nm with excitation wavelengths of 344and 340 nm for compounds 1 and 2, respectively.For titration experiments, 25 mM or 50 mM solution of Fe(NO3)3 in water  or 0.1 M nitrobenzene in EtOH were incrementallyadded to a cuvette containing 1 mg of the sensor in 1 mL of Milli-Qwater, and emission spectra were recorded at each point.UV–visible absorptionspectra were recorded at room temperaturefor complexes 1 and 2. Luminescence decay measurements were measured using anEdinburgh FLS 1000 spectrometer setup and Fluoracle software. Thesamples were excited by a 375 nm laser (CNI MLL-III-375-100 mW) ata frequency of 20 Hz. The luminescence was collected via two monochromatorsat 614 nm with a bandwidth of 1 nm in the multi-channel-scaling mode.Thephotoluminescence characteristics were studied in the solid stateusing a Xe lamp coupled to a monochromator as the excitation sourceand an integrated sphere coupled to a spectrometer  in order to quantitativelydetermine the quantum yield for complexes –1. C, H, and N elemental analyses were performedusing a CE INSTRUMENTS 1110 EA elemental analyzer . Thermogravimetric analyses of salts 1 and 2 were performed on a Mettler-Toledo TGA/SDTA/851eapparatus under N2 atmosphere at a scan rate of 10 K·min–1. Analysis of the proportion of metals in the sampleswas performed on a Philips XL30 ESEM scanning microscope equippedwith an EDAX microprobe .IR transmission measurementsof both complexes were performed directly from the powdered samplesat room temperature in a FT-IR spectrometer  equippedwith an attenuated total reflection (ATR) accessory in the range 400–4000cm1 and 2 were preparedby the reaction of Eu(NO3)3·6H2O with the appropriate picolinic acid under hydrothermal conditions.The addition of an acid modulator (nitric acid or acetic acid) wasnecessary in order to obtain single crystals suitable for structuralcharacterization. Both compounds could also be obtained from the correspondingdiesters in similar conditions.Crystallineeuropium(III)coordination polymers Figure S1). The experimental diffractograms recorded at roomtemperature for compounds 1 and 2 comparewell with the simulated patterns obtained from single-crystal dataat 120 K.The phase purity of the bulkmaterials was confirmed by PXRD measurements . For 1, the TGA curveshows threeseparated steps of weight loss. The first one takes place between416 and 435 K and is ascribed to the release of two coordinated watermolecules . The second and third stepsoccur above 540 K and are attributed to the decomposition of dimethylammoniumcations and L12– ligands. These results are consistent with the formulationdeduced from single-crystal X-ray diffraction measurements that showthe presence of two water molecules per lanthanoid ion. For 2, the TGA plot exhibits also a weight loss in multiple steps.Between RT and 390 K, a first weight loss associated with the releaseof water present in the voids of the structure takes place. A secondstep ascribed to the loss of acetic acid is detected between 440 and580 K. The total weight loss for these first two steps is 9.26% . Then, above 620 K decomposition of dimethylammonium cationsand L22– ligands occurs. These observations are consistent with therelease of two different types of solvent molecules and are in agreementwith the formulation deduced from single-crystal X-ray diffraction.Clearly, 1 shows a higher dehydration temperature, indicatingthat water is more tightly bound to the anionic lattice as comparedto 2. This is expected for the presence of H2O molecules coordinating to the Eu3+ cation in 1.Thermogravimetric analyses (TGA) of 1 2NH2]+). This cation isrequired in order to balance the negative charge of the host frameworkand originates from the hydrolysis of dimethylformamide (DMF) solventmolecules.53Single-crystal X-ray analysisreveals that 1 1 crystal3+ cation sitson a two-fold axis and displaysan eight-coordinated distorted square antiprism geometric configuration.58Continuous shape measure(CShM) calculations using SHAPE 2.1 confirmthat the coordination geometry is a distorted square antiprism rhombus grid layer that presents channels (6.685 × 24.582 Å)along the a axis . These voids are occupied by dimethylammonium cations (one cationper channel) thanks to the establishment of hydrogen bonds with noncoordinatedpicolinate oxygen atoms (N2···O4: 2.7254(10) Å).The ditopic ligand intramolecular H-bondbetween water molecules and benzoate anions coordinating to the sameEu3+ cation, whereas the weaker intermolecular H-bond interaction involving the picolinate moiety O1W···O3bridges complexes of consecutive layers, leading to a 3D doubly H-bondednetwork with an AA′AA′ arrangement  Å,O1W···O2: 2.6092(14)) are established. The O1W···O2interaction is a very strong angement 2a. In adangement 2b.The anion exhibits an almost planar conformation, with a smalldihedral angle between the pyridine and benzene rings (12.2(4)°)that allows coordination to two different metal cations. In addition,the carboxylate group involved in chelation presents a low torsionangle of 6.0(4)°, and the geometry of the triple bond is onlyslightly distorted (−C–C–C– bond anglesof 178.0(4)° and 177.6(4)°, in comparison with the idealvalue of 180°).1 with [(CH3)2NH2][Ln(H2O)2(CPA)2], a related family of lanthanoidcomplexes basedon 5-(4-carboxyphenyl)picolinate dianions (CPA2–).54 Although the crystal structure ofthese compounds was reported in the C2/c space group, this could be converted to I2/a by the choice of a conventional unit cell59 with parameters similar to 1. The lanthanoidcoordination sphere is analogous to that described for 1, and CPA2– ligands adopt the same coordinationmode to form an equivalent 2D rhombus grid layer. Even more, theselayers are assembled into 3D networks via similar H-bonding and π–πstacking interactions. As expected, the size of the channels in 1 along its longest direction is notably higher in comparisonwith this family of compounds (7.278 Å × 19.161 Å forthe Eu complex), due to the presence of the ethynylene spacer. However,when countercations are removed, the “SQUEEZE” optionof PLATON60 indicates an empty volume ofthe anionic framework of 558.8 Å3 per unit cell for[(CH3)2NH2][Eu(H2O)2(CPA)2], corresponding to 20% of the totalcrystal volume. In compound 1, a significantly lowervalue of 442.4 Å3, corresponding to a percent volumeof 14%, is obtained indicating a more compact structure despite thepresence of two additional carbon atoms in the ligand.It is interesting to compare 2 crystallizes in the orthorhombic Pna21 space group . The Eu3+ cation is located in a general positionand exhibits a nine-coordinated tricapped trigonal prismatic (TTP)structure as determined by continuous shape measure (CShM) calculations 1, Eu–O bond distances  Å)are shorter than Eu–N  Å). Thus,the nitrogen atoms N2, N3, and N4 define the capping positions ofthe tricapped trigonal prism, the two triangular faces being definedby O3O6N2 and O1O5O7 atoms, respectively. The angle between the trigonalfaces is 175.50°, close to the parallel arrangement expectedfor a TTP structure.Compound ce group 1, and itL22– ligands (AL22– and BL22–) that adopt differentcoordination modes. Dianion AL22– employs the two picolinatechelating subunits (N3O6 and N4O7) incoordination: one of them (N3O6) acts as a tridentate ligand in a;O mode, connecting two lanthanoid metal ions by anti, anti-carboxylate (O5O6) bridges. This yields zigzagchains running parallel to the c axis, with a relativelyshort intrachain distance between adjacent Eu3+ cationsof 6.6891(7) Å . The otherpicolinate subunit (N4O7) coordinates in a terminal chelating manner,bridging neighboring chains related by a translation along the b axis. Thus, this first independent ligand defines layersthat lie parallel to the yz plane being bound in a terminal chelating manner to two Eu3+ cationsseparated by a long metal–metal distance of 12.8369(6) Å.There are two independentbispicolinate 891(7) Å S4. The oThis results in a 3D anionic lattice showing triangular cavities4b. ChargAL22–, the two pyridine rings are almost perpendicularto each other °),whereas BL22– shows a more planar transoid conformation, with a smaller dihedral angle of 27.2(9)°. Inturn, the distortion of the triple bond is higher for BL22– (−C–C–C– bond angles of 175.5(9)°and 171.5(9)°) than for AL22– (−C–C–C–bond angles of 177.5(9)° and 179.7(9)°). As expected, thepicolinate subunits, involved in chelation, are nearly planar, withthe highest deviation from planarity being observed for the bridgingtridentate picolinate anion (torsion angle between carboxylate andpyridine moieties of 12.3(9)°).The two independent ligands adopt markedly different conformations:in 2L1 and H2L2 andtheir Eu3+ complexes were measured in the solid state atroom temperature . The UV absorptionspectra of both complexes show an intense absorption band at about250–255 nm and a second absorption band at about 331–334nm with the same intensity. These features are almost identical tothose observed for the free ligands and are attributed to π→ π* and n → π* electronictransitions of the pyridine and benzoate rings. The similarity observedbetween these patterns indicates that absorption takes place on theligand rather than directly at the metal center.The electronic absorptionspectra of free ligands H3)2NH2][Eu(H2O)2(L1)2] (1) and [(CH3)2NH2][Eu(L2)2]·H2O·CH3COOH (2) and ligands H2L1 and H2L2 were investigated in water at room temperature   2.615D0 → 7F2 transition,which is responsible for the strong red emission. In fact, the intensityratio of the 5D0 → 7F2 transition and the 5D0 → 7F1 transition gives an indication about the symmetry ofthe first coordination environment of the Eu(III) ions and has a valueof 3.6 for 1 and 3.8 for 2. These values,together with the very low intensity of the 5D0 → 7F0 transitionband, suggest a non-centrosymmetric environment for the metal center.61 The values of this ratio are very similar tothose obtained for previous europium(III) complexes. For instance,[EuL(HL)H2O]·6H2O (being H2L bis(5-(pyridine-2-yl)-1,2,4-triazol-3- yl)methane ligand) presentsa ratio of 3.4.62In both complexes, the emissionspectrum is dominated by the mostintense band at 615 nm ascribed to 1 and 2. Therefore, both ligands have provento be suitable for absorbing light efficiently and transferring thisenergy to the europium cations with high efficiency. In fact, theexcitation spectra of both complexes  monitored within the Eu3+ most intense transition (5D0 → 7F2) are dominated by a broad band from 310 to390 nm and from 220 to 375 nm, respectively, corresponding to intraligandtransitions. There is also a weak signal at 395 nm attributed to atransition between the 7F0 andexcited states of Eu3+. The very low relative intensityof this line compared with that of the ligand-based broad band suggestsa more efficient ligand-sensitization process than direct intra-4f6 excitation.It is worth notingthat there is a negligible contribution below500 nm coming from the ligand in the emission spectrum of complexes 1 and 2 inthe solid state were measured at room temperature by monitoring thetime dependence of the strongest 5D0 → 7F2 emission, were obtained. This correspondsto an average lifetime τav = 1116 μs. Luminescencelifetimes on the micro- to millisecond time scale are typical forf–f transitions that involve long-lived excited states, andthe values obtained for 1 and 2 are withinthis range (Table S5).68 However, 2 shows clearly higher lifetimevalues than 1. This can be explained taking into accountthat 1 presents two H2O molecules in the coordinationsphere of the Eu3+ cation. It is well-known that nonradiativedecay of the excited states of Eu3+ takes place via vibroniccoupling with the vibrational modes of O–H bonds of coordinatedwater molecules, leading to a significant reduction of excited statelifetimes.69 This is why the luminescenceof lanthanoid-based complexes is markedly influenced by the denticityof the ligand, polydentate ligands being useful for increasing thestability of the complexes and allowing the metal center to be protectedfrom solvent molecules.Instead, for compound 1 and 2, respectively.These values are typical for Eu3+ coordination polymers(Table S5). The slightly higher value obtainedfor 2 parallels the lifetime measurements and is dueto the lack of coordinating water molecules. This behavior has beenwell documented for many hydrated europium diketonate complexes.70 Nonetheless, a recent report on [EuKL4(H2O)2]·H2O  presents an ultrahigh quantum yield value of 92%, which is muchhigher than for most lanthanoid complexes.71Photoluminescence quantum yields (PLQY)were measured by placingthe samples into an integrating sphere. Values of 23% and 29% wereobtained for complexes Figure S7). The intensity of the strongest peak that correspondsto the 5D0 → 7F2 transition was taken for comparison.The luminescence signal of 1 /I(H2O) ≈ 1.72). For other solvent molecules, the emissionintensity was slightly reduced. Remarkably, nitrobenzene (PhNO2) could effectively quench the luminescence of 1. For 2, a general enhancement of the luminescence signalwas observed when a solvent different to water was used, except forPhNO2, that exhibited an almost complete luminescence quenching(I(DMSO)/I(H2O) ≈ 3.6) was observed in DMSO suspensions.The luminescencesensing response of both europium derivatives for different solventmolecules was studied at room temperature. The as-synthesized crystals(1 mg) were ground and suspended in different solvents (1 mL), andthe corresponding emission spectra were recorded S7. The iFigure S8), powder X-ray diffraction(PXRD) measurements of both Eu3+ coordination polymerswere carried out after the corresponding solid samples were suspendedin DMSO and PhNO2. A perfect agreement between these diffractogramsand those corresponding to the as-synthesized materials was obtained,indicating that their crystal structures are retained after interactionwith these solvent molecules.In order to investigate theseluminescence enhancement and quenchingeffects , discarding this hypothesis. Thus, a possible explanationfor this fact relies on the exchange of inner-sphere coordinatingwater molecules or hydrogen-bonded water molecules by DMSO on the surface of the particles, which would preserve theircrystal integrity. In this way, nonradiative decay of the excitedstates through vibronic coupling with the vibrational modes of O–Hbonds would be reduced, with the corresponding enhancement in theeuropium emission.An increase of emission intensityafter soaking in DMSO has beenpreviously noted in Eu2 was compared to the emission spectraof 1 and 2 . The lack of spectral overlap between absorption and emission patternsdiscards a resonance energy transfer process between Eu3+ ions and PhNO2. Nevertheless, there is a spectral overlapbetween the absorption spectrum of PhNO2 and the emissionof ligands H2L1 andH2L2, indicating thata Förster resonance energy transfer (FRET) might be at theorigin of the quenching process. However, since PhNO2 canabsorb energy at the excitation wavelength used in these experiments, a competitive absorption mechanismcannot be excluded. Energy competition between Eu complexes and nitroaromaticcompounds has been already noted as a possible mechanism of luminescencequenching.74Regarding nitrobenzene quenching, the disruptionof the crystalstructure can also be excluded since the diffraction patterns remainunaltered after dispersing the solids in this solvent. The absorptionspectrum of PhNO1 and 2 could be used as specific luminescentsensors for nitroaromaticmolecules. Thus, titration experiments with PhNO2 (0.1M in EtOH) were undertaken on aqueous suspensions of 1 and 2. Both complexes exhibited a very similar gradualdecrease of the luminescence intensity as the concentration of PhNO2 was increased  were 1.50 ×102 M–1 and 1.60 × 102 M–1 for 1 and 2, respectively,indicating that both compounds show a moderate nitrobenzene sensingability in comparison to other Eu(III) luminescent sensors (Table S2). Moreover, the limit of PhNO2 detection (LOD) was calculated based on the following equation:s is theslope of theplot of luminescence intensity against PhNO2 concentrationin the linear region  and δis the standard deviation for 10 repeated luminescence measurementsof the blank solution. The results show detection limits of 2.05 ×10–5 M and 3.03 × 10–5 Mfor 1 and 2, respectively. These valuescompare well with those reported for similar Eu(III) complexes (Table S2).The above results suggestthat ncreased 8a,b. Theninsets 8a,b:75,71 and 2 (1 mg) were dispersed in 1 mL aqueoussolutions containing 0.01 M of MCln orM(NO3)n metal salts , and then the luminescence spectra were recorded. Again, in order to analyzethe data, the strongest peak, corresponding to the 5D0 → 7F2 transition, was taken for comparison. For 1,all metal ions exhibited luminescence quenching effects . On theother hand, metals like Li+, Na+, Mg2+, and Ca2+ increased the emission intensity of 2, the remaining metal ions resulting in different degrees of luminescencequenching  ligand, shows a better stabilityin these metal-sensing experiments than 1.Coordination polymers recordedS11. AgairecordedS11. AgaiFigure S12) in order to check the stability of the crystalline frameworksafterinteraction with Fe3+ and Pb2+ cations. PXRDpatterns of 1 and 2 suspended in aqueoussolutions of Fe3+ cations are consistent with the originalframeworks, whereas the pattern of 1 suspended in anaqueous solution of Pb2+ cations is completely different.This indicates that collapse of the original network takes place,possibly resulting from the interaction between Pb2+ cationsand the uncoordinated carboxylate groups in the channel. Energy-dispersiveX-ray (EDX) microanalysis carried out on a scanning electron microscope(SEM) showed that this sample contains both Pb2+ and Eu3+ ions, with a Pb2+/Eu3+ ratio of 1.3.The same analysis for the compounds soaked in aqueous solutions ofFe3+ ions indicates that these cations are not presentin the composition of the soaked materials. Therefore, the collapseof the framework of 1 induces the variation of the luminescenceintensity after immersion in a Pb2+ aqueous solution, butthis can be ruled out for Fe3+ sensing. In order to getmore insight into the iron(III) quenching mechanism, electronic absorptionspectra were recorded for aqueous solutions of the different metalions and compared to the emission spectra of 1 and 2, together with those of corresponding ligands . The lack of overlap between theabsorption spectra of the different metallic ions and the emissionspectra of the complexes discards a resonance energy transfer mechanismfor luminescence quenching. Nonetheless, there is a considerable overlapbetween the absorption spectrum of Fe3+ ions and the emissionspectra of ligands H2L1 and H2L2. Energy transfer from excitedlevels of the ligand to the Fe3+ ions could be a possiblequenching mechanism. On the other hand, the wide absorption band ofFe3+ ions ranging from 430 to 260 nm covers the wavelengthused for excitation of 1 and 2 , while other metal ions such as Pb2+ have no absorption in this range. Therefore, competitive absorptionby Fe3+ ions could also account for the quenching process.PXRD measurements were conducted . A suspension of each complex (1mg in 1 mL H2O) was then titrated with Fe3+ aqueoussolutions of concentrations 25 mM and 50 mM, respectively, for 1 and 2. The luminescence of the Eu3+ polymers was gradually quenched . There is a clear difference between the intensities recorded inthe presence and in the absence of Fe3+ ions, verifyingthat a quenching process is present.Selectivity and sensitivity of both complexes toward Fe species S14. A su3+ being the quencher species. The Stern–Volmerplots present again an upward bending at a high concentration of quencher,characteristic of the existence of static and dynamic quenching mechanisms.76 From the fit of the experimental data at lowFe3+ concentrations  to theStern–Volmer equation .55The quenching efficiency was quantitativelyevaluated using theStern–Volmer s (s is the slope of luminescence intensity vs [Fe3+] plot in the linear region), giving a value of 5.82 ×10–6 M and 3.16 × 10–6 Mfor 1 and 2, respectively . Both values are very similar and within the rangereported for other sensors based on Eu3+ coordination polymers(Table S1) but still far from the 10–7 M value obtained for [(CH3)2NH2][Eu(CPA)2(H2O)2] (CPA2– = 5-(4-carboxyphenyl)picolinate dianion),one of the highest reported to date.54Furthermore, the limit of detection (LOD) was calculated from3δ/1, it was found (max = 396–432 nm), which is attributed to the decomposition ofthe complex and release of the free ligand in its protonated or fullydeprotonated forms.62 The remarkable stabilityof 1 was demonstrated after soaking the coordinationpolymer in an aqueous solution at pH = 12 for 12 h . The powder X-ray diffractogram of the solid obtainedwas identical to that of the pristine material. This is in contrastwith previous reports showing a lack of stability in most cases.71 There are only two previous reports showinga similar robustness in these conditions,78 but these compounds exhibit higher LOD in comparison with 1. Instead, 2 displays the strong emissive propertiesin a shorter pH range (3 < pH < 8)  confirm the collapseof the crystal structure at pH = 1 and pH = 14 for both compounds.The lower stability of 2 outside the 3–8 pH rangewas also confirmed by the presence of minor diffraction peaks in theX-ray diffractogram.Luminescence studies as a function of pH were carried out for bothcomplexes in water suspensions. The emission was registered at differentpH values ranging from 1 to 14. The pH of each aqueous solution wasadjusted by the addition of HCl or NaOH solutions. For as found 11a that for 12 h S17. The 3+ coordinationpolymers 1 and 2 have been synthesized fromethynylene-bridged ditopic picolinate ligands by the hydrothermalmethod. Suspensions of these compounds in water exhibited a strongEu3+ emission under UV light irradiation, characteristicof effective ligand-to-metal energy transfer (antenna effect). Luminescencecould be quenched by nitrobenzene, with limits of detection of 2.05× 10–5 M and 3.03 × 10–5 for 1 and 2, respectively. These coordinationpolymers were also able to act as luminescent sensors for Fe3+ cations, with limits of detection in the micromolar region . Noteworthyis the very high stability of these coordination polymers under extremepH conditions.To summarize, two water-stable luminescent Eu


This hormonotoxin has a significant antiproliferative effect on the PC3 tumor cell line, with an IC50 value close to that of dermaseptin-B2. Its antitumor activity has been confirmed in vivo in a xenograft mouse model with PC3 tumors and appears to be better tolerated than dermaseptin-B2. Biophysical experiments showed that the addition of LHRH to dermaseptin-B2 did not alter its secondary structure or biological activity. The combination of different experimental approaches indicated that this hormonotoxin induces cell death by an apoptotic mechanism instead of necrosis, as observed for dermaseptin-B2. These results could explain the lower toxicity observed for this hormonotoxin compared to dermaseptin-B2 and may represent a promising targeting approach for cancer therapy.Prostate cancer is the most common cancer in men. For patients with advanced or metastatic prostate cancer, available treatments can slow down its progression but cannot cure it. The development of innovative drugs resulting from the exploration of biodiversity could open new therapeutic alternatives. Dermaseptin-B2, a natural multifunctional antimicrobial peptide isolated from Amazonian frog skin, has been reported to possess antitumor activity. To improve its pharmacological properties and to decrease its peripheral toxicity and lethality we developed a hormonotoxin molecule composed of dermaseptin-B2 combined with d-Lys Prostate cancer (PCa) is the second most frequent cancer diagnosis made in men and was the fifth leading cause of death worldwide in 2018   [Circular dichroism (CD) spectroscopy of peptides. The helical structures of HB2 and DRS-B2 were analyzed by CD spectroscopy using a Jobin Yvon CD6 dichrograph linked to a PC microprocessor as described in . Briefly 222 nm) .Fluorescence of tryptophan-containing peptides was performed as previously described by Dos Santos et al. . Emissio2O/D2O (90:10 v/v) in the presence of 85 mM DPC-d38  and using a peptide concentration of 1 mM. The pH was set to 4.1 using microliter amounts of NaOH 0.1 M. DSS was added at a concentration of 0.11 mM for chemical shift calibration. The NMR spectra were acquired at 45 °C on a 500 MHz (11.7 T) Avance III Bruker® spectrometer equipped with a TCI cryoprobe. The following experiments were recorded: 2D 1H-1H TOCSY (66 ms mixing time), 2D 1H-1H NOESY (150 ms mixing time), 2D natural abundance 15N-1H and 2D 13C-1H HSQC experiments, as described in [Nuclear Magnetic Resonance (NMR) Spectroscopy. The NMR sample was prepared in 550 μL of Hribed in .2 in a humidified atmosphere. All culture reagents were purchased from Life Technologies . All experiments were performed as previously described by Dos Santos et al. [Cell culture. Human PCa cell lines, PC3, DU145 and 22Rv1 were purchased from ATCC (American Type Culture Collection) and were cultured in RPMI supplemented with 10% of fetal bovine serum (FBS). The human hyperplasic cell line BPH1 was purchased from the German Collection of Cell Cultures . BPH1 were maintained in RPMI 1640 supplemented by 10% FBS, 20 ng/mL testosterone and 1% insulin-transferrin-selenium. Cell cultures were maintained at 37 °C and 5% CO3 cells/well in 96-multiwell plates in complete medium and incubated for 24 h at 37 °C in a controlled humidified 7% CO2 environment. Cells were then treated with DRS-B2 or H-B2 as indicated, for 48 h. Cell viability was measured using the 3--diphenyltetrazolium bromide (MTT) dye method  according to the manufacturer’s instructions. Each experiment was performed in triplicate with at least three independent experiments, as previously described by Dos santos et al. [50 values were determined by GraphPad Prism 5.0 .Cell viability assays. Cells were seeded at a density of 5 × 10g for 10 min at 4 °C to separate plasma from erythrocytes. The pellet was then rinsed with PBS pH 7.4 and centrifuged at 900× g for 10 min at 4 °C. After counting and making a red cell solution at 4 × 108 erythrocytes/mL (diluted in PBS pH 7.4), 50 µL of a diluted peptide solution was added in a cascade to which 50 µL of the erythrocyte solution was added (made in triplicate). After 1 h of incubation at 37 °C, the tubes were centrifuged at 12,000 rpm for 15 s at 4 °C. The supernatant was then recovered. The hemoglobin present in the erythrocytes was determined in the supernatants via a plate reader at 450 nm. A parallel incubation in the presence of 0.2% (v/v) Triton was carried out to determine the absorbance associated with 100% hemolysis.Hemolysis assessment. The hemolytic activity of H-B2 and DRS-B2 was determined using fresh human erythrocytes from a healthy donor that was prepared as follows: 4 mL of whole blood was collected and centrifuged at 900× 6) cells were injected subcutaneously into the right flank of 4-week-old male NMRI nude mice . When the tumor volume reached approximately 100 mm3, the mice were randomly divided into four groups (n = 6): control (PBS), DRS-B2 (2.5 mg/kg), H-B2 (2.5 mg/kg), H-B2 (5 mg/kg) by intraperitoneal injection twice a week. Tumor size was measured two times per week with a caliper and the tumor volume was calculated with the formula: V = 4/3π × R12 × R2 with radius 1 (R1), and radius 2 (R2). At the end of the experiment, the mice were sacrificed, and their body weight was measured. The tumors were isolated, weighted and then fixed in formalin.Tumor xenograft studies were performed as previously described by Van Zoggel et al. . PC3 .Lactate dehydrogenase (LDH) release assay. The cytoplasm LDH release assay was performed as previously described in . Briefly6 cells per mL. One milliliter of this cell extract was centrifuged, suspended in 200 μL PBS, transferred to a microtiterplate with a round bottom and centrifuged again. The resulting cell pellet was resuspended in 200 μL of Binding Buffer 1x (BD PharmingenTM), containing 5 μL of FITC-Annexin-V (BD PharmingenTM) and incubated for 10 min in the dark at room temperature. The cells were washed with PBS and incubated with 200 μL of Binding Buffer 1x containing PI  (BD Pharmin-genTM)) for 5 min in the dark at room temperature. Flow cytometry analysis was performed with an LSR Fortessa X20 analyzer  and FlowJo V10 software.Apoptosis analysis by flow cytometry. For the apoptosis assay, an FITC-Annexin-V (A-V) and Propidium Iodide (PI) double staining method was used. PC3 cells were grown in a 12-well plate and treated or not with H-B2, DRS-B2 or hydrogen peroxide as described previously . At 24 h®. In addition to the fluorescein labeling, a second labeling with DAPI, intercalating DNA was performed by incubating for 15 min at room temperature with a 1 µg/mL solution. The different labeling steps were performed avoiding any exposure to light.Terminal transferase-mediated dUTP Nick End-Labeling (TUNEL) assay. PC3 cells were cultured in 6-well plates at 150,000 cells per well in complete medium and treated or not with 1 or 2.5 µM of DRS-B2 or H-B2 and with LHRH (5 μM) or Taxotere (10 nM) and Staurosporine (1 μM) as a positive control. After 24 h of treatment, the supernatant was aspirated and the cells trypsinized and plated on a Superfrost PLUS slide and fixed with a 4% formaldehyde solution without methanol. The cells were then permeabilized with a 0.2% Triton X-100 solution in 1x PBS for 5 min at room temperature. Terminal transferase-mediated dUTP nick end-labeling (TUNEL) was performed according to the DeadEnd™ Fluorometric TUNEL System kit from PromegaImaging was performed with an Axiolmager M2 epifluorescence microscope. The images obtained by microscopy were recorded with Zen 2012 software and analyzed with ImageJ© software. Each image underwent a thresholding of fluorescence calibrated on the positive control, and which was preserved for all the other images. The software then analyzed the number of DAPI and fluorescein fluorescence events to calculate the ratio between the total number of DAPI-labeled cell nuclei and the number of fluorescein-labeled nuclei with fragmented DNA. The experiment was performed in triplicate.t-test. The statistical significance of the differences is given as * p < 0.05; ** p < 0.01; *** p < 0.001; ns: not significant.Statistical analysis. The statistical analyses were performed using GraphPad PrismTM version 4.00 software from GraphPad Software Inc. . The results are expressed as the means ± standard deviation (SD) or standard error of the mean (SEM) of at least three determinations for each test from three independent experiments. Statistical analyses were carried out using the unpaired


Kv1.5) and protein levels of Kv1.5 in the plasma membrane. Mechanistically, PKC activation led to monoubiquitination and degradation of the mature Kv1.5 proteins. Overexpression of Vps24, a protein that sorts transmembrane proteins into lysosomes via the multivesicular body (MVB) pathway, accelerated, whereas the lysosome inhibitor bafilomycin A1 completely prevented PKC-mediated Kv1.5 degradation. Kv1.5, but not Kv1.1, Kv1.2, Kv1.3, or Kv1.4, was uniquely sensitive to PMA treatment. Sequence alignments suggested that residues within the N terminus of Kv1.5 are essential for PKC-mediated Kv1.5 reduction. Using N-terminal truncation as well as site-directed mutagenesis, we identified that Thr15 is the target site for PKC that mediates endocytic degradation of Kv1.5 channels. These findings indicate that alteration of protein levels in the plasma membrane represents an important regulatory mechanism of Kv1.5 channel function under PKC activation conditions.The voltage-gated potassium channel Kv1.5 plays important roles in the repolarization of atrial action potentials and regulation of the vascular tone. While the modulation of Kv1.5 function has been well studied, less is known about how the protein levels of Kv1.5 on the cell membrane are regulated. Here, through electrophysiological and biochemical analyses of Kv1.5 channels heterologously expressed in HEK293 cells and neonatal rat ventricular myocytes, as well as native Kv1.5 in human induced pluripotent stem cell (iPSC)-derived atrial cardiomyocytes, we found that activation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate  diminished Kv1.5 current (I KCNA5, is a member of the Shaker superfamily of Kv channels. Kv1.5 channels are expressed in a number of cell types such as atrial myocytes (+ current (IKur), which is important for the repolarization of atrial action potentials  (Voltage-gated potassium (Kv) channels play important roles in excitable cells and their malfunction can lead to various diseases. Kv1.5, encoded by myocytes , smooth myocytes , 4, and tentials . Alteration (AF) , 7, and ion (AF) , 9.Kur in human atrial myocytes via protein kinase C (PKC) activation  as a PKC activator, we investigated the effects of PKC activation on the expression and function of Kv1.5 channel expressed in human embryonic kidney (HEK) cells as well as neonatal rat ventricular myocytes. Our data revealed that PKC activation by PMA (10 nM) induces endocytic degradation of mature Kv1.5 channels through a lysosomal pathway. Using N-terminal truncation as well as site-directed mutagenesis, we identified that the threonine residue at position 15 in the N terminus is the target site for PKC-mediated endocytic degradation of Kv1.5 channels.A, CTL). The 75-kDa band represents the mature fully glycosylated form localized in the plasma membrane, while the 68-kDa band is the immature core-glycosylated form of the Kv1.5 channel residing in the endoplasmic reticulum (ER). It is the plasma membrane localized, mature protein that conducts Kv1.5 current (IKv1.5)  . As showKv1.5 using whole-cell patch clamp method. As shown in Figure 1B, consistent with the decrease in mature channel protein expression, PMA treatment for 3 h also reduced IKv1.5 in a concentration-dependent manner.To determine the effects of PMA treatment on Kv1.5 activity, we recorded IC). The IKv1.5 showed similar changes at various periods following PMA treatment (D). One possible explanation for these results is that while short periods of PMA treatment  activate PKC, long periods  lead to a depletion of certain PKC isoforms such as PKCα as we demonstrated recently  did not cause any further reduction, instead, mature Kv1.5 protein expression recovered partially at 6 h and almost fully at 24 h C. The IKreatment D. One porecently , which aA).To validate that the PMA-induced effects on Kv1.5 are indeed though PKC activation, the PKC inhibitor, bisindolylmaleimide I (BIM-1), was used to prevent PKC activation , 23. WhiFig. 2B, the left lane, in control cells, the majority of the Kv1.5 channels were present in the plasma membrane. In PMA-treated  cells, Kv1.5 channels displayed a diminished membrane expression and punctate intracellular localization . In cells treated with both PMA  and BIM-1 , Kv1.5 channels remained in the plasma membrane .To visualize the fate of Kv1.5 channels in the plasma membrane under PMA treatment, Kv1.5 was labeled with an anti-Kv1.5 primary antibody and Alexa Fluor 488-conjugated (green) secondary antibody. The cell membrane was labeled using Texas Red X-conjugated wheat germ agglutinin (WGA). As illustrated in Figure 2Kv1.5 reduction. The effects of PMA  on IKv1.5 with or without BIM-1 (10 μM) were evaluated. As shown in Figure 2C, similar to the effect on Kv1.5 surface protein expression, BIM-1 (10 μM) abolished the PMA-induced IKv1.5 reduction.BIM-1 also abolished PMA-induced ID). Taken together, these findings indicate that the effects of PMA on Kv1.5 are mediated by PKC activation.In addition to BIM-1, we also examined the effects of other PKC inhibitors Sotrastaurin and Staurosporine on PMA-induced reduction in Kv1.5 mature channel expression. Both Sotrastaurin (200 nM) and Staurosporine (200 nM) abolished the PMA-induced reduction of the mature Kv1.5 channel expression D. Taken A and B). To determine the structural determinants involved in the unique PMA-induced Kv1.5 reduction, we first compared the amino acid residue differences among these Kv channels using COBALT: constraint-based alignment tool (NIH). A single Kv1.5 α-subunit contains six transmembrane domains (S1-S6), a cytoplasmic N-terminal and a C-terminal domain. We found that the greatest amount of differences is located in the N terminus among Kv channels (C), while the rest of the protein sequences among Kv channels are much more conserved. We hypothesized that residues within the N terminus in Kv1.5 channel are essential for PMA-induced reduction in Kv1.5 expression and current.Kv1.5 belongs to Shaker superfamily of potassium channels. To test whether PKC activation also affects other Shaker superfamily members, we used PMA (10 nM) to treat HEK293 cells stably expressing Kv1.1, Kv1.2, Kv1.3, or Kv1.4 for 3 h. Our results showed that both protein expression and currents of these four channels were not affected by PMA treatments , A and Bchannels C, while A–C). HEK293 cell lines stably expressing these respective truncation mutants were also established. We used PMA (10 nM) to treat Kv1.5-ΔN209-HEK cells and Kv1.5-Δ2-19-HEK cells, respectively, for 3 h and then performed western blots and patch clamp recordings. As shown in A–C, while PMA treatment decreased WT Kv1.5 expression and current, it affected neither expression nor function of these two mutant channels.To test this hypothesis, we truncated the N terminus of Kv1.5 up to residue 209 or deleted amino acid residues 2–19 to create Kv1.5-ΔN209 or Kv1.5-Δ2-19 mutants , A–C. HED). When expressed in HEK293 cells, this mutation (Kv1.5-T15A), like the ΔN209 and Δ2-19, abolished the PMA-induced reduction of mature Kv1.5 expression and IKv1.5 (Kv1.5.The fact that Δ2-19 deletion abolished the PMA-induced mature Kv1.5 reduction indicates that residues within the first 19 amino acid residues are involved in this process. PKC is involved in regulating the function of proteins through the phosphorylation of hydroxyl groups of the amino acids serine (S) and threonine (T) . There iFig. 4KCNA5 gene in control and PMA  treated Kv1.5-HEK cells. As illustrated in Figure 5A, PMA did not decrease KCNA5 mRNA level.The homeostasis of Kv1.5 protein at the surface membrane is a balance between synthesis/anterograde trafficking and retrograde degradation. Mature Kv1.5 expression can be decreased through two mechanisms: reduced protein synthesis and/or accelerated Kv1.5 degradation. We examined the mRNA levels of B). As the molecular mass of single Ub is 8.5-kDa, the 83-kDa band is most likely to be the monoubiquitinated membrane-localized 75-kDa Kv1.5. Monoubiquitination is known to trigger internalization and degradation of various channels  also mediates channel protein degradation . To addrFigure 5Monoubiquitinated proteins are sorted into the luminal vesicles of multivesicular bodies (MVBs) by endosomal sorting complexes required for transport (ESCRT) machinery . When MVA, overexpression of hVps24 enhanced PMA-induced reduction in the expression level of the 75-kDa Kv1.5 protein. To further examine the role of Vps24 in PMA-induced Kv1.5 reduction, we used Vps24 siRNA transfection to knock down the endogenous Vps24 protein expression. As shown in Figure 6B, Vps24 siRNA transfection notably reduced the endogenous Vps24 expression level and partially impeded the reduction of 75-kDa Kv1.5 expression induced by 10 nM PMA treatment for 3 h. Thus, Vps24 plays a role in the PMA-induced degradation of Kv1.5 channels.We examined the effects of Vps24 overexpression on Kv1.5 degradation. Twenty-four hours after transfection of Kv1.5-HEK cells with Myc-tagged hVps24, cells were cultured in 10 nM PMA for 1 h, and Kv1.5 expression levels were examined. As shown in Figure 6A). Similar results were obtained in patch clamp recordings; while Baf completely prevented the PMA-induced IKv1.5 reduction, MG132 only partially prevented PMA-induced IKv1.5 reduction .Monoubiquitination is associated with the endocytosis and lysosomal sorting of plasma membrane proteins, while the formation of lysine 48 (K48)-linked polyubiquitin chains is the primary signal targeting proteins to the 26S proteasome for degradation . To testFig. 7Kur in neonatal rat ventricular myocytes is very small , or lysosomal inhibitor Baf (1 μM), prevented PMA-induced IKv1.5 reduction. These results are consistent with those observed in Kv1.5-HEK cells. Thus, PKC activation decreases IKv1.5 in both the heterologous expression system (HEK293 cells) and a more physiological environment (cardiomyocytes).HEK293 cells have a different cellular environment from native cardiomyocytes. To determine whether PKC activation has a similar effect on Kv1.5 channels expressed in cardiomyocytes, we transfected Kv1.5 plasmid into isolated neonatal rat ventricular myocytes. Such transfection was necessary as Kv1.5 channels are primarily expressed in atria but not ventricles, the native Iry small . In addiKv1.5, human iPSC-derived atrial cardiomyocytes were used. Most cells during culture from day 3 to day 6 displayed the K+ current with biophysical and pharmacological characteristics of IKv1.5. The activation–voltage relationships of IKv1.5 revealed a half-maximal activation voltage (V1/2) of -13.2 ± 3.6 mV with a slope factor of 5.0 ± 1.2 (n = 12) in human iPSC-derived atrial cardiomyocytes and a V1/2 of -7.0 ± 2.5 mV with a slope factor of 5.6 ± 0.6 (n = 12) in Kv1.5-HEK cells. As well, the current displayed slow inactivation during 200-ms depolarizing steps between 30 and 70 mV. Furthermore, as shown in Figure 9A, the current was sensitive to the Kv1.5 channel blocker 4-aminopyridine (4-AP); 0.1 mM 4-AP blocked the current by more than a half (A); and the block was reversible (data not shown). We then paired control and PMA-treated  myocytes each time and performed whole-cell voltage clamp recordings. As shown in Figure 9B, treatment with PMA  reduced the IKv1.5 by 67% (38.0 ± 18.8 pA/pF in CTL (n = 15), 12.3 ± 4.0 pA/pF in PMA (n = 13), p < 0.01). There are two possibilities regarding the remaining currents in PMA-treated myocytes: PMA at 10 nM did not completely inhibit IKv1.5; or, a small amount of PMA-insensitive currents other than IKv1.5 may contribute to currents in these cells.To further confirm the effect of PKC activation on native In a half A; and thKv1.5 channels expressed in HEK293 cells as well as in rat cardiomyocytes and native IKv1.5 in human iPSC-derived atrial cardiomyocytes. Further detailed analyses in WT and various mutant Kv1.5 channels expressed in HEK293 cells revealed that PKC activation enhanced cell-surface Kv1.5 internalization and degradation through the ubiquitination/MVB/lysosome pathway, which thereby led to decrease of Kv1.5 current and surface protein expression. Additionally, the intact Threonine residue at position 15 (T15) in the N terminus of Kv1.5 is required for this process.Kv1.5 is important for atrial action potential repolarization, and its dysfunction caused by gene mutations has been linked to atrial fibrillation . In the + currents derived from coexpression of Kvβ1.2 with Kv1.5 were markedly reduced by PMA, and the reduction was associated with a small depolarizing shift in the voltage dependence of channel activation (A) and confocal analysis (B) demonstrated that the PKC-induced decrease in Kv1.5 activity is due to downregulation of the cell surface Kv1.5 channels. These findings are consistent with a recently published report    and KATP channel .B) and the reduction effect of PMA on Kv1.5 was not weakened by UbKO overexpression, which disrupts polyubiquitination of target proteins . These results indicate the monoubiquitination of the mature Kv1.5 channels under PKC activation conditions. Monoubiquitination acts as a sorting signal for internalization and lysosomal degradation of membrane proteins. Once protein cargo is tagged by a Ub molecule, it is recognized and sorted into luminal vesicles of MVBs, which then fuse with lysosome through the ESCRT sorting machinery (A), whereas knockdown of endogenous Vps24 with siRNA transfection markedly impeded the degradation (B). These results suggest that the ESCRT component, Vps24, is involved in transport of the Kv1.5 channels from early endosomes to lysosomes for degradation under PKC activation conditions.The internalized membrane proteins are either recycled back to the plasma membrane or subjected to degradation . There aFig. 5achinery , 46. Vpsachinery A, wherearadation B. These Ultimately, Kv1.5 channels were degraded in the lysosome. This is supported by our data that lysosomal inhibitor (bafilomycin A1) completely prevented PMA-induced reduction in Kv1.5 expression and current . PreventA and B). Sequence alignment results suggested that residues within the N terminus in Kv1.5 channel are essential for PKC activation-induced Kv1.5 reduction (C). We then utilized mutant channel Kv1.5-ΔN209 in which amino acid residues up to 209 in the N terminus has been truncated (B), we further searched the target region for PKC activation. We focused on the proline-rich sequence existing at position 65–82 in Kv1.5 N terminus. Because Src tyrosine kinase targets this proline-rich sequence that is important for Kv1.5 endocytosis . It has been demonstrated that phosphorylation of the norepinephrine transporter (NET) at T258 and S259 is linked to PKC-mediated transporter internalization  TVR (residues 15–17) in the N terminus. Previous studies showed that SAP97 (synapse-associated protein 97) increases Inhibitor , 54. In nhibitor D. It haslization . Furtherlization  and glutlization .Kv1.5 occurred earlier than the reduction of mature protein (C and D). At least two possibilities exist: (1) PMA induces a loss of Kv1.5 function, which triggers protein internalization; (2) PMA induces Kv1.5 protein internalization, which results in a loss of Kv1.5 function, and internalized channels then degrade at rate slower than internalization. Further study will be needed to distinguish these possibilities. As well, T15A mutant displays a higher current density than WT Kv1.5 probably due to the disruption of the basal level of PKC-mediated degradation. However, although it would be expected this mutant to have higher surface expression, our western blots show that the apparent ratio of mature to immature channels is lower than wild-type (WT) channel (Our results showed that acute application of PMA (10–100 nM) to cells during whole-cell clamp recordings for up to 30 min did not cause a reduction in current (data not shown). This indicates that an intact cellular environment is required for PKC-mediated modulation of the channel. We believe that T15 residue serves as a PKC phosphorylation site. It would be interesting to investigate how the phosphorylation of T15 residue by PKC activation causes the subsequent Kv1.5 channel ubiquitination and endocytosis. In this regard, our results showed that PMA-treatment-induced reduction in I protein , C and D channel . The exaPKC activation occurs in various physiological and pathophysiological conditions . For insvia endocytic degradation. This process relies on the presence of T15 residue at the Kv1.5 N terminus. To the best of our knowledge, this is the first study providing detailed insight into the intracellular pathways that regulate Kv1.5 channels via PKC activation, and this finding extends our understanding of ion channel biology and provides a potential mechanism for the development of atrial arrhythmias in multiple cardiac pathologies.In summary, we have demonstrated that PKC activation leads to a reduction in cell surface expression of the voltage-gated potassium channel Kv1.5 WT Kv1.5 cDNA was provided by Dr Michael Tamkun . For generating plasmid Δ2-19 (deletion of 2–19), ΔN209 (truncation of 1–209), and T15A, PCR cloning of the corresponding inserts from WT Kv1.5 template was constructed into pcDNA3 using BamH1 and EcoR1 restriction enzymes. The constructs were verified through sequencing across the inserts (GENEWIZ). The human lysineless mutant Ub  was obtained from Addgene. Human Vps24 cDNA open reading frame (ORF) clone with Myc tag in pCMV6-entry vector  was obtained from OriGene Technologies. FLAG-tagged Kv1.1, Kv1.2, Kv1.3, and Kv1.4 cDNAs were purchased from GenScript.Lipofectamine 2000 was used to transfect WT Kv1.5 and mutant constructs with GFP plasmid into human embryonic kidney (HEK) 293 cells. Cell lines stably expressing Kv1.5-WT (Kv1.5-HEK), Kv1.5-ΔN209 (Kv1.5-ΔN209-HEK), Kv1.5-Δ2-19 (Kv1.5-Δ2-19-HEK), and Kv1.5-T15A (Kv1.5-T15A-HEK) were established using G418 for selection (1 mg/ml) and maintenance (0.4 mg/ml). HEK cells were cultured in Minimum Essential Medium (MEM) supplemented with 10% fetal bovine serum (FBS), 1 mM sodium pyruvate, and 1× nonessential amino acids (Thermo Fisher Scientific). Twenty-four hours after transfection, cells were collected for biochemical and patch clamp experiments. GFP-positive cells were used for whole-cell patch clamp recordings.To activate PKC, PMA (Sigma-Aldrich) at various concentrations in culture media was used in Kv1.5-HEK cells for various periods at 37 °C. In total, 10 μM Bisindolylmaleimide I , 200 nM Sotrastaurin , and 200 nM Staurosporine  were used to inhibit PKC. Cells were incubated with a proteasomal inhibitor MG132  or a lysosomal inhibitor bafilomycin A1  in culture media with PMA to block the proteasome and lysosome.Experiments using rats were approved by the Queen's University Animal Care Committee and conducted in conformity with the Canadian Council on Animal Care. Sprague-Dawley neonatal rats of either sex at 1 day of age were sacrificed by decapitation followed by heart removal. Ventricular myocytes were isolated by enzymatic dissociation as described in detail recently . Myocyteg for 4 min. Next, the cell pellets were lysed using high-frequency sonication in ice-cold lysis buffer containing 1% PMSF and 4% protease inhibitor cocktail. The lysates were centrifuged at 10,000g for 10 min, and supernatants containing proteins were collected. The protein concentrations were determined using a DC Protein Assay kit (Bio-Rad). Appropriate amounts of double-distilled water and loading buffer containing 5% β-mercaptoethanol were added to the protein to make 0.3 μg/μl samples. Protein samples of 15 μg were loaded and separated on SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore). The BLUeye Prestained Protein Ladder was used to identify the mass of proteins. The membranes were blocked to prevent nonspecific protein interactions with 5% nonfat skim milk and 0.1% Tween-20 in Tris-buffered saline (TBST) for 1 h at room temperature. Next, membranes were incubated with appropriate primary antibodies for 1 h at room temperature. Actin was detected as a loading control. The membranes were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies  for 1 h at room temperature. An ECL (enhanced chemiluminescence) detection kit  was used to visualize the protein bands on X-ray films (Fujifilm).Whole-cell lysates from cultured cells were used for western blot analysis . Cells w2, 2 mM CaCl2, 10 mM glucose, and 10 mM HEPES (pH 7.4 with NaOH). The pipette solution consisted of 135 mM KCl, 5 mM MgATP, 5 mM EGTA, and 10 mM HEPES (pH 7.2 with KOH). Patch clamp experiments were performed at room temperature (22 ± 1 °C).Currents were recorded using whole-cell patch clamp method. Cells were settled on the bottom of a 0.5 ml perfusion chamber in the bath solution. Patch glass pipettes were pulled using thin-walled borosilicate glass (World Precision Instruments). The pipettes had inner diameters of 1.5 μm and resistances of 2 MΩ when filled with solution. An Axopatch 200B amplifier and pCLAMP10 (Molecular Devices) were used for data acquisition and analysis. Data were sampled at 20 kHz and filtered at 5 kHz. Series resistance (Rs) was compensated by 80%, and leak subtraction was not used. All currents were elicited from a holding potential of –80 mV by depolarizing steps to voltages between –70 and +70 mV in 10 mV increments for 200 ms. A repolarizing step to −30 mV was applied for 250 ms before returning to the holding potential. To construct the voltage–current (I-V) relationships, the maximal currents during depolarizing steps were plotted against depolarizing voltages in each cell and summarized in each of the groups. The bath solution contained 135 mM NaCl, 5 mM KCl, 1 mM MgClHEK cells expressing WT Kv1.5 were grown on glass coverslips, then were exposed to MEM with or without PMA under various conditions. For membrane staining, the cells were incubated with Texas Red X-conjugated wheat germ agglutinin  for 1 min at room temperature prior to cell fixation. Cells were washed with PBS and fixed using 4% ice-cold paraformaldehyde in PBS for 15 min, permeabilized with 0.1% Triton X-100 for 10 min, and blocked with 5% bovine serum albumin in PBS for 1 h. Kv1.5 was labeled with rabbit anti-Kv1.5 primary antibody  and Alexa Fluor 488-conjugated donkey anti-rabbit secondary antibody . The coverslips were mounted onto glass slides using Prolong Gold Antifade reagent. Images were acquired using a Quorum Wave Effects Spinning Disc Confocal microscope at Queen's University Cancer Research Institute Image Centre.Endogenous Vps24 in Kv1.5-HEK cells was knocked down using the Vps24 siRNA (Sigma). Scrambled siRNA was used as the control. Cells were grown in 60-mm dishes at 60∼70% confluence and 100 pmoL of siRNA was transfected into cells using Lipofectamine 2000 (Invitrogen). Forty-eight hours after transfection, cells were subsequently cultured in MEM with 10 nM PMA for 3 h and were harvested for further experiments.β-actin was used. The primers used for KCNA5 gene including forward sequence (5′ to 3′): GTTCCGCATCTTCAAGCTCTCC, and reverse sequence (5′ to 3′): CGAAGTAGACGGCACTGGAGAA; for β-actin gene including forward sequence (5′ to 3′): TGGCACCCAGCACAATGAA, and reverse sequence (5′ to 3′): CTAAGTCATAGTCCGCCTAGAAGCA. The real-time PCR protocol was: 2 min at 50 °C, then 10 min at 95 °C, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. Each PCR reaction was performed in duplicate. The cycle threshold (Ct) values of β-actin mRNA levels were not changed by PMA treatment. The transcription levels of KCNA5 gene were calculated using the 2−ΔΔCt method . RNA concentration was measured using an ND-1000 Spectrophotometer (NanoDrop Technologies). After treatment with DNase I , the Omniscript Reverse Transcription Kit (Qiagen) was used for reverse transcription of total RNA (1 μg) to cDNA. Quantitative real-time PCR was performed with SensiFAST SYBR Hi-ROX Kit  and a Model 7500 thermal cycler (Applied Biosystems). For the internal control, a housekeeping gene t method  using β-g for 5 min, the supernatant was discarded, and the pellet was resuspended in 1 ml plating medium for cell count to determine density. Cells were seeded at 1000 cells/cm2 in plating medium onto the prepared coverslips in 24-well plates after removing excess fibronectin from the wells. Cells were cultured at 37 °C, 5% CO2. Twenty-four hours after initial plating, the plating medium was replaced with maintenance medium  to remove any dead cells; the final cell density was around 500 cells/cm2. The culture medium was replaced with fresh maintenance medium every 48 h. Electrophysiology recordings were performed from day 3 to day 9 of culture. Most cells from day 3 to day 6 displayed the K+ current with biophysical characteristics of IKv1.5. IKv1.5 recorded from day 7 to day 9 became small and was not used. IKv1.5 in human iPSC-derived atrial cardiomyocytes was further confirmed through its pharmacological properties . 4-AP was applied after control currents were recorded. Cells treated with PMA (10 nM) for 3 h were matched to control cells each time for whole-cell voltage clamp recordings. The currents were recorded with the standard external and pipette solutions used in HEK293 cells described above at room temperature (22 ± 1 °C).Human iPSC-derived atrial cardiomyocytes (ax2518) were obtained from Axol Bioscience (Little Chesterford) and grown on glass 18 mm circular coverslips following the manufacturer's instructions. Briefly, coverslips in 24-well plates were coated using 1× fibronectin coating solution  overnight at 37 °C in a humidified incubator. Cells were quickly thawed in a 37 °C water bath, then transferred to a sterile centrifuge tube, and diluted slowly with 9 ml plating medium . Cells were centrifuged at 200Scrambled siRNA, primary antibodies against Vps24 , N-terminal specific mouse anti-Kv1.5 antibody , rabbit anti-Kv1.5 antibody , goat anti-mouse (sc-2005), and goat anti-rabbit (sc-2004) IgG-HRP secondary antibodies were purchased from Santa Cruz Biotechnology Inc. The rabbit C-terminal-specific anti-Kv1.5 antibody (APC-004) was purchased from Alomone Labs. The horse anti-mouse (7076S) and goat anti-rabbit (7074S) HRP-linked secondary antibodies were purchased from Cell Signaling Technology. MEM, FBS, trypsin, sodium pyruvate, minimal essential amino acids, hVps24 siRNA (ID: SASI_Hs01_00160243), Lipofectamine 2000, Opti-MEM, Hank's Balanced Salt Solution were purchased from Invitrogen. G418, PMSF, protease inhibitor cocktail, β-mercaptoethanol, Triton X-100, BSA, Prolong Gold Antifade mountant, monoclonal mouse anti-actin antibody (A4700), mouse anti-FLAG antibody (F3165), and all chemicals/electrolytes used were obtained from Sigma-Aldrich. The BLUeye Prestained Protein Ladder (GeneDirex) was purchased from FroggaBio. X-ray films were from Fujifilm. Paraformaldehyde was obtained from Alfa Aesar.versus each voltage and shown as mean + SD. For experiments with multiple groups, a one-way analysis of variance (ANOVA) with Tukey's post-hoc test was used. For experiments where multiple groups were being compared with control, a one-way ANOVA with Dunnett's post-hoc test was used. For experiments between two groups, a two-tailed paired or unpaired Student's t-test was used. A p-value ≤ 0.05 was considered statistically significant.All data are expressed as the mean ± the standard deviation (SD). For western blots, box plots superimposed with original data points are shown, in which the line in the box shows the median, the small square shows the mean, and the bars show SD. For electrophysiological data, current densities (pA/pF) are plotted All the data are in the article.The authors declare that they have no conflicts of interest with the contents of this article.