Perivascular stromal cells (PSCs) identified within several different organs share characteristics of bone marrow‐derived MSCs (BM‐MSCs). These PSCs may also possess tissue‐specific properties and play a role in local tissue homeostasis. We hypothesized that human kidney‐derived PSCs (hkPSCs) would elicit improved kidney repair in comparison with BM‐MSCs. Here we introduce a novel, clinical‐grade isolation method of hkPSCs from cadaveric kidneys by enriching for the perivascular marker, NG2. hkPSCs show strong transcriptional similarities to BM‐MSCs but also show organotypic expression signatures, including the HoxD10 and HoxD11 nephrogenic transcription factors. Comparable to BM‐MSCs, hkPSCs showed immunosuppressive potential and, when cocultured with endothelial cells, vascular plexus formation was supported, which was specifically in the hkPSCs accompanied by an increased NG2 expression. hkPSCs did not undergo myofibroblast transformation after exposure to transforming growth factor‐β, further corroborating their potential regulatory role in tissue homeostasis. This was further supported by the observation that hkPSCs induced accelerated repair in a tubular epithelial wound scratch assay, which was mediated through hepatocyte growth factor release. In vivo, in a neonatal kidney injection model, hkPSCs reintegrated and survived in the interstitial compartment, whereas BM‐MSCs did not show this potential. Moreover, hkPSCs gave protection against the development of acute kidney injury in vivo in a model of rhabdomyolysis‐mediated nephrotoxicity. Overall, this suggests a superior therapeutic potential for the use of hkPSCs and their secretome in the treatment of kidney diseases. S The present study provides a novel clinical‐grade isolation method of human kidney‐derived perivascular stromal cells (hkPSCs). The hypothesis was that, due to tissue‐specific imprinting, hkPSCs are more potent in renal repair compared to human bone marrow‐derived mesenchymal stromal cells. The present study shows that hkPSCs indeed show distinct functional properties with respect to epithelial wound healing, interstitial integration, and amelioration of kidney injury. Therefore, these results suggest significant potential for hkPSCs as therapeutic candidates for the treatment of kidney diseases.Mesenchymal stromal cells (MSCs) are immune modulatory and antifibrotic cells originally isolated from the bone marrow (BM) and are characterized by their spindle‐shaped morphology and ability to adhere to plastic. BM‐MSCs are able to differentiate into fat, bone, and cartilage and express the stromal markers CD73, CD90, and CD105 while being negative for CD34 and CD45 Previously, it has been shown that perivascular stromal cells positive for NG2, CD146, and PDGF‐R‐β with characteristics similar to BM‐MSCs exist within many different solid organs, including skeletal muscle, pancreas, adipose tissue, and placenta +Lin−CD45− cells showed a unique phenotype and immunomodulatory potential +, and although these kCFU‐F have a comparable stromal marker expression and trilineage differentiation potential in comparison with BM‐MSCs, there is a distinct gene and protein expression profile MSC‐like cells could also be isolated from murine kidneys on the basis of Sca‐1 expression. These renal Sca1We hypothesized that perivascular stromal cells can also be isolated from the human kidney (hkPSCs) in a clinical‐grade manner and that these cells have tissue‐specific functions. Extensive characterization of hkPSCs, both in vitro and in vivo, show a capacity for these cells to stabilize endothelial cells, reduce renal injury, and support tubular repair, the latter to a greater extent than BM‐MSCs, suggesting indeed organotypic properties.Cells were isolated from human transplant‐grade kidneys discarded for surgical reasons by using clinical‐grade protocols, enzymes, and products. Research consent was given for all kidneys, and the study was approved by the local medical ethical committee and the ethical advisory board of the consortium.http://www.bridgetolife.com) containing heparin  directly after surgery and stored on ice. Within 30 hours, kidneys were flushed again with UW, and the perirenal fat and kidney capsule were removed. The renal artery was cannulated, and the kidney was perfused via a pump‐driven  recirculation system at 37° with Dulbecco's modified Eagle medium (DMEM)‐F12  at 300 ml per minute. Afterward the kidney was perfused with collagenase  and DNAse  at 37°C with a flow of 300 ml per minute. After approximately 30 minutes, the tissue was digested, and after gentle massage the resulting cell suspension was collected and washed in DMEM‐F12 containing 10% fetal calf serum. Cells were then either directly put into culture or frozen in liquid nitrogen. The complete standard operation procedure (SOP) for the isolation procedure can be found in the supplemental online data.Kidneys were flushed with University of Wisconsin (UW) cold storage solution . The NG2‐positive fraction was cultured in αMEM containing 5% platelet lysate. The cultures were maintained at 37°C and 5% carbon dioxide. Half the medium was refreshed twice a week. When the cells reached confluence, the cells were collected using trypsin (Lonza) and replated at 4 × 103 cells per cm2. Experiments were performed with fluorescence‐activated cell sorting (FACS)‐confirmed homogeneous NG2‐positive hkPSCs between passages 4 and 8, cultured in αMEM 5% platelet lysate unless stated differently.Kidney cell suspensions were cultured in alphaMEM  containing 5% platelet lysates, glutamine (Lonza), and penicillin/streptomyzine (Lonza), and cells were cultured in tissue culture flasks until confluence was reached. At passage 1, cells were trypsinized, and NG2 cell enrichment was performed using MACS, according to manufacturer's protocol , supplemented with penicillin/streptomycin (Lonza) and 5% platelet lysate. The cultures were maintained at 37°C, 5% carbon dioxide. Half the medium was refreshed twice a week. When the MSC colonies or cultures reached confluence, the cells were collected using trypsin (Lonza) and replated at 4 × 103 cells per cm2. Experiments were performed between passages 4 and 8.Ethical committee approval and written consent from the donors was obtained for the aspiration of human bone marrow. Heparinized bone marrow was aspirated under local or general anesthesia. The mononucleated cell fraction was isolated by Ficoll density gradient separation and plated in tissue culture flasks at a density of 160 × 10http://www2.leicabiosystems.com/). For immunophenotyping, the cells were stained for NG2, PDGF‐R‐β, CD146, CD73, CD90, CD105, CD31, CD34, CD45, CD56, human leukocyte antigen (HLA) class I (ABC), and HLA class II (DR). All specific fluorochrome‐labeled antibodies and isotype controls were purchased from BD Bioscience , except for CD105 .The expanded cell populations were characterized by morphology (spindle‐shaped cells), which was imaged with an inverted bright‐field microscope  and the RNeasy kit , according to the manufacturer's protocol. The quality and quantity of RNA were assessed with a Nanodrop spectrophotometer (Thermo Fisher Scientific Life Sciences) and a Bioanalyzer . Gene expression profiling was performed by Aros Applied Biotechnology . cDNA and cRNA synthesis, labeling, and subsequent hybridization on the human HT12 V4 gene expression beadchips  were performed according to the manufacturer's protocols. The beadchips, targeting more than 47,000 gene transcripts, were scanned with the iSCAN system (Illumina Inc.), and fluorescence intensities were uploaded into GenomeStudio Software (Illumina Inc.). Genes with a detection p value of >.05 for all samples were excluded. Average signals of >200 in either the BM‐MSCs or hKPSCs were considered above background levels. Subsequent data were quantile normalized, and the Pearson's correlation coefficient was calculated (r2). Differential expression was analyzed with the gene expression module of Genome Studio (Illumina). For the table of the top differential expressed genes, we sorted genes on the highest difference score. Differential scores were calculated from the differential p value in Illumina software with the following formula: DiffScore = 10 × sgn (μcond − μref) × log 10 p. False discovery rates were calculated according to Benjamini and Hochberg for a total number of 8,462 transcripts http://www.bio‐rad.com), as per the manufacturer's instructions. The real‐time PCR primers of HoxD10 are AGACAGTTGGACAGATCCGAA(fw) and CGAAATGAGTTTGTTGCGCTTAT (rv) and of HoxD11 are TCGACCAGTTCTACGAGGCA (fw) and AAAAACTCGCGTTCCAGTTCG (rev). The amplification reaction volume was 12.5 µl in total, consisting of 6.25 µl iQ SYBR Green PCR master mix, 0.5 µl primers, 2.5 µl cDNA, and 3.25 µl water. Messenger RNA (mRNA) level was normalized to the housekeeping gene glyceraldehyde‐3‐phosphate dehydrogenase.RNA was isolated from biological triplicates using Trizol reagent . After 3 weeks of culture, in the adipogenic differentiation assay, lipid droplets were stained using Oil Red O, and in the osteogenic differentiation assay, calcium depositions were stained with Alizarin Red. For chondrogenic differentiation, cell pellets were formalin fixed  and embedded in paraffin. Subsequently, 5‐µm sections were deparaffinized, rehydrated, and stained with 1% toluidine blue for 20 minutes. All differentiation assays were analyzed with an inverted bright‐field microscope .hkPSCs and BM‐MSCs of 3 different donors were plated in flat‐bottom 96‐well plates, and after 5 days of culture, supernatants were harvested, and cytokine expression profiles were determined in the supernatant with the Bio‐Plex Human Cytokine 17‐Plex Panel, following the manufacturer's instructions (Bio‐Rad). Cytokines in culture medium were also measured as a negative control.https://www.sigmaaldrich.com) and allowed to attach overnight in DMEM‐F12 with 10% normal human serum (NHS). Culture in 10% NHS was chosen because platelet lysates are able to suppress PBMC proliferation on their own (data not shown). PBMCs were stimulated with anti‐CD3/anti‐CD28 Dynabeads (Thermo Fisher) and were seeded in triplicate at a concentration of 1 × 105 cells per well. Stromal cells were added to the PBMC proliferation assay in a ratio of 1:4 and 1:8. 3H‐thymidine (0.5 mCi) was added after 5 days and 3H‐thymidine incorporation was determined after 16 hours as a measure of proliferation.Peripheral blood mononuclear cells (PBMCs) were isolated from buffy coats of healthy blood donors by density gradient centrifugation using Ficoll‐isopaque and frozen in liquid nitrogen until use. Cultured hkPSCs and BM‐MSCs (passages 6–8) of 3 different donors were plated in flat‐bottom 96‐well plates  after informed consent from the parents.www.paa.com), penicillin/streptomycin (PAA Cell Culture Company), 1,000 IU of heparin (Leo Pharma), and 25 mg bovine pituitary extract (BPE) (Thermo Fisher). HUVECs were used at passages 2–3 Human umbilical vein endothelial cells (HUVECs) were isolated according to Jaffe et al. Stromal cells and HUVECS were cocultured in a 96‐well plate  for 1 week in a 4:1 ratio, as has been described previously https://www.peprotech.com). Cells were subsequently trypsinized, permeabilized with 0.1% saponine, and labeled with α‐smooth muscle actin . α‐SMA expression was analyzed with flow cytometry, and mean fluorescent intensities were calculated .hkPSCs and BM‐MSCs were seeded in a density of 200,000 cells in a 6‐well plate and stimulated for 48 hours with 10 ng/ml transforming growth factor (TGF)‐β1  and cultured for 48 hours. HK2 cells were seeded in proximal tubular epithelial cells medium, consisting of a 1:1 ratio of DMEM and Ham's F‐12 (Lonza), supplemented with insulin (5 µg/ml), transferrin (5 µg/ml), selenium (5 ng/ml), hydrocortisone (36 ng/ml), triiodothyrinine (40 pg/ml), and epidermal growth factor (10 ng/ml) (Sigma‐Aldrich) in a density of 500,000 cells per well in a 6‐well cell culture plate (Costar) and cultured until confluent. A scratch wound was created in the monolayer of HK2 cells using a 200‐µl pipette tip. After the scratch, cells were washed with phosphate‐buffered solution (PBS) and provided either with fresh medium (αMEM 5% PL) or with complete conditioned medium from either hkPSCs or BM‐MSCs. Scratches were imaged at 4, 7, 14, and 28 hours at the same position in duplicates with an inverted bright‐field microscope . The scratch area was measured at each time point using ImageJ software, and the percentage wound closure was calculated.https://www.rndsystems.com). The hepatocyte growth factor (HGF)‐receptor was blocked with an HGF receptor/c‐MET antibody (R&D Systems) in a concentration of 1 µg/ml 1 hour before the wound scratch assay and after adding the hkPSC supernatant. In the control conditions an isotype antibody  was added.Growth factors in the conditioned medium of kPSCs were measured using a custom‐made growth‐factor panel, following the manufacturer's instructions  in a ratio of 1:50 for identification of injection sites in the neonatal kidney. Using an Eppendorf microinjector, we injected cells into the kidney through the muscle layer in a volume of 300 nl, corresponding with 3,000–5,000 cells. Kidneys were harvested at 4 days postinjection. In total, seven mice were analyzed: three mice with hkPSCs from three different donors; three mice with BM‐MSCs from two different donors, and one sham‐operated mouse. In all mice, injection into the kidney was confirmed by fluorescence of the coinjected microspheres.Animal experiments were approved by the University of Queensland animal ethics committee and adhered to the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. Neonates of outbred CD1 mice were used for neonatal injection. hkPSCs and BM‐MSCs of 3 and 2 different donors, respectively, were injected into the neonatal kidneys at postnatal day 1 by using a microinjection pipet in a protocol adapted from the protocol previously described http://www.envigo.com) by intramuscular injection on day 0 with hypertonic glycerol  into the inferior hind limbs, as has been described previously n = 6 for blood urea nitrogen [BUN] measurement, n = 4 for confocal microscopy) were anesthetized with Avertin  and subjected to dorsal incision on the left side to exteriorize the left kidney. A 1‐mm incision was made in the capsule of the kidney, and 750,000 cells were injected into 25‐µl of sterile PBS with a Hamilton syringe equipped with a 27‐G blunt‐ended needle. After cell infusion, the kidney capsule was cauterized with an electric scalpel, and the dorsal incision was sutured. The mouse was rehydrated with subcutaneous injection of 500 μl saline solution and maintained in a warm environment for 2 hours postsurgery. Control mice were injected with saline solution . For intravenous retro‐orbital injection, 4 hours and 24 hours following kidney injury, mice  were anesthetized with isoflurane  and injected retro‐orbitally through the venous plexus with 750,000 cells in 150 µl of sterile PBS each time using a 27‐G needle. Control mice were injected with saline solution .Animal experiments were performed in accordance with institutional, regional, and state guidelines and in adherence to the Italian National Institutes of Health Guide for the Care and Use of Laboratory Animals. Rhabdomyolysis‐induced acute kidney injury was studied in 6‐week‐old male C57Bl/6 mice . Four animals per group were sacrificed at day 6, and kidney, lungs, and liver were harvested for confocal microscopy.Blood samples were obtained from the submandibular venous sinus at days 0, 4, 6, and 14, and BUN levels were measured by Reflotron System . Samples were frozen in liquid nitrogen and stored at −80°C. Ten‐micrometer‐thick sections were cut and postfixed with 4% PFA for 10 minutes at room temperature. Stainings were performed using the manufacturer's protocol . Samples were stained with antibodies against human mitochondria, nuclei, and collagen IV  and analyzed using a TCS SP8 laser confocal microscope (Leica Biosystems). In the rhabdomyolysis‐induced acute kidney injury model, confocal microscopy was performed on 10‐ μm sections of renal frozen tissues using a TCS SP5‐II laser confocal microscope (Leica Biosystems). Staining for fluorescein isothiocyanate (FITC)‐labeled Dolichos biflorus agglutinin and FITC‐labeled Lotus tetragonolobus agglutinin (Vector Laboratories) was performed following manufacturer's instructions. To‐pro‐3 (Thermo Fisher) was used for counterstaining nuclei.In the neonatal injection model, kidney samples were fixed in 4% PFA, followed by 30% sucrose overnight and embedded in TissueTek OCT compound . For statistical analysis of the microarray data, p values were corrected for multiple testing according to Benjamini and Hochberg Differences between two groups were analyzed using an unpaired two‐sample Student In order to evaluate whether hkPSCs are a potential new cell source for use in cell therapy to treat kidney disease in a clinical setting, we chose to develop a clinical‐grade acceptable SOP with the use of clinical‐grade materials and enzymes. This protocol is developed on the basis of the pancreatic islet isolation protocol currently in use for clinical application in our center NG2‐positive hkPSCs showed a bright‐field morphology similar to that of BM‐MSCs  and, as p value. The top 50 up‐ and downregulated genes can be found in supplemental online Table 1. Interestingly, homeobox factor HoxD11 is in the top five most upregulated genes in hkPSCs. Homeobox transcription factors, which are important in anatomical patterning during development, showed hierarchical clustering when comparing BM‐MSCs with hkPSCs , suggesting high similarity between the two cell types . Howeverh hkPSCs . Homeoboh hkPSCs . These rOne important and extensively studied feature of MSCs is their anti‐inflammatory and immunomodulatory potential. MSCs are able to regulate proliferation and cytotoxicity of T cells, macrophages, and B cells, which are also major players in kidney disease and transplantation, as has been reviewed elsewhere To evaluate whether cultured hkPSCs still have pericytic properties, we cocultured cells with human umbilical vein endothelial cells (HUVEC). HUVECs were not able to form endothelial sprouts in monoculture. However, when cocultured with either BM‐MSCs or hkPSCs, vascular plexus formation occurred . There wBecause stromal cells in general have the capacity to become myofibroblasts after stimulation with TGF‐β, thereby contributing to fibrosis, we evaluated whether hkPSCs have this capacity. Interestingly, stimulation of hkPSCs with TGF‐β did not increase α‐SMA expression, suggesting that hkPSCs did not become myofibroblasts , 4I.In order to evaluate the effect of hkPSCs on renal tubular epithelial repair, we made a scratch in a monolayer of human kidney proximal tubular epithelial cells (HK2). Under control culture conditions, at least 28 hours was necessary for 80% wound closure. Interestingly, when conditioned media from hkPSCs were added, significant closure was already observed after 4 hours, with 80% of the wound closed after 7 hours (mean of duplicate experiments from three different donors). Importantly, 14 hours was required to reach an 80% wound closure in parallel experiments with the conditioned media from BM‐MSCs , 5B. ThiTo determine the role of hkPSCs in kidney development, we injected hkPSCs into neonatal mice at postnatal day 1 using microinjection , 6B. IntTo access the effect of hkPSCs in kidney injury, we injected the cells in a glycerol‐induced rhabdomyolysis acute kidney injury model. We evaluated the effect of hkPSCs via two routes of administration: renal subcapsular and intravenous. Blood ureum nitrogen (BUN) measurements show that at the peak of acute kidney injury (AKI), renal function was significantly preserved in the hkPSC‐treated group, independent of route of delivery , 6G. AftIt has previously been reported that perivascular stromal cells from several different human organs share features with MSCs Here we sought to isolate human kidney‐derived perivascular stromal cells and show that these cells have distinct properties in comparison with human BM‐MSCs. In order to evaluate the functionality of these cells, we studied their interaction with several different renal cell types, including endothelial cells, proximal tubular cells, and (infiltrating) immune cells. There were similarities between human BM‐MSCs and hkPSCs; however, hkPSCs showed a distinct mRNA expression profile, did not differentiate into adipocytes, and displayed a more potent kidney epithelial wound‐healing capacity. Furthermore, hkPSCs were able to integrate into the interstitium of the developing kidney, whereas BM‐MSCs did not. Finally, hkPSCs could preserve kidney function in a glycerol‐induced rhabdomyolysis model of acute kidney injury.Because there is a lack of specific markers of MSCs, the International Society for Cellular Therapy has proposed criteria to define MSCs. These include plastic adherence; the marker expression of CD73, CD90, and CD105 while being negative for CD14, CD34, and CD45; and the capacity to differentiate into bone, cartilage, and fat in vitro MSCs are important for tissue homeostasis, most likely via the secretion of several soluble factors and microvesicles containing, among others, mRNAs and miRNAs Although this study is not the first to describe an MSC‐like population in the human kidney + renal cortical stroma, and Hox11 genes are expressed in the metanephric mesenchyme. Loss of either Hox10 or Hox11 gene function results in the loss of ureteric bud induction, reduced branching, and decreased nephrogenesis, phenotypes only described for Hox10 and 11 mutants and not for other Hox mutants The differences between human kPSCs and BM‐MSCs mentioned above may reflect differences in imprinting by the tissue of origin. This was previously also observed for fibroblasts isolated from different organs Interestingly, hkPSCs were able to integrate back into the renal interstitium and reduce the severity of kidney injury in vivo in a rhabdomyolysis model. This was independent of the route of delivery because both subcapsular and intravenously injected hkPSCs were able to preserve renal function. However, with the subcapsular injections, integration of the hkPSCs into the interstitium appeared more pronounced, as did the renoprotective effect.12 hkPSCs after passage 6. Hence, one donor kidney could yield a sufficient number of hkPSCs for the treatment of several different patients as a cell therapy. This makes human kPSCs an interesting new cell source to develop for regenerative medicine for kidney diseases.Whether human kPSCs are more suitable for cell therapy for kidney diseases than BM‐MSCs, which are currently studied in clinical trials, still remains to be further elucidated. Their capacity to reintegrate into the renal stroma, to improve tubular epithelial wound repair, and to preserve function in an AKI model would suggest that hkPSCs may have an organotypic role in maintaining and restoring renal interstitial homeostasis without showing the risk of increasing fibrosis. To be able to compare these cell types for future cell therapy purposes, we chose to isolate the hkPSCs with a SOP in a clinical‐grade manner, with clinical‐grade enzymes, materials, and methods that would allow direct translation into a clinical product. Using this protocol, one can obtain large quantities of clinical‐grade isolated hkPSCs. The yield from one human kidney would be an average of 2.7 × 10These data show that human kPSCs show distinct functional properties in comparison with BM‐MSCs with respect to growth factor excretion, interstitial integration, and amelioration of kidney injury, suggesting significant potential for hkPSCs and their secretome as therapeutic candidates for the treatment of kidney disease.D.G.L.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing, final approval of manuscript; M.E.J.R., P.R., C.v.K., and M.H.L.: conception and design, data analysis and interpretation, manuscript writing; J.L., A.J.P., and M.A.E.: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; E.L.: collection and/or assembly of data, data analysis and interpretation; H.C.d.B.: collection and/or assembly of data, data analysis and interpretation, manuscript writing; W.E.F.: conception and design, manuscript writing; T.J.R.: conception and design, data analysis and interpretation, manuscript writing, financial support.M.H.L. is a patent holder and has received honoraria as an invited speaker for the Amgen Symposium, Melbourne, Australia, 2016. D.G.L., M.A.E., and T.J.R. are on a patent application regarding this subject. The other authors indicated no potential conflicts of interest.Supporting InformationClick here for additional data file.


This superior capacitance is attributed to the ideal surface morphology of the solvent, which allows for easy diffusion of electrolyte ions into the inner region of the electrode. High electrical conduction enables a high rate capability. These results suggest that CoS nanoparticles are highly promising for energy storage applications as well as photocatalysis, electrocatalysis, water splitting and solar cells, among others. These results show that CoS is a promising positive electrode material for practical supercapacitors.In this study, cobalt sulfide (CoS) electrodes are synthesized using various solvents such as water, ethanol and a combination of the two via a facile chemical bath deposition method on Ni foam. The crystalline nature, chemical states and surface morphology of the prepared CoS nanoparticles are characterized using X-ray diffraction, X-ray photoelectron spectroscopy, scanning electron microscopy and transition electron microscopy. The electrochemical properties of CoS electrodes are also evaluated using cyclic voltammetry, galvanostatic charge–discharge and electrochemical impedance spectroscopy. When used as an electrode for a supercapacitor, CoS prepared with ethanol as a solvent exhibits a capacitance of 41.36 F g One oBased on the energy storage mechanism, supercapacitors are divided into three categories: electrical double-layer capacitors (EDLCs), pseudocapacitors and hybrid capacitors . In EDLCThe electrode materials used in pseudocapacitors rely on the presence of a redox-based fast faradaic reaction at the surface of the electrode and near the intercalation of electrolytes, which exhibit relatively large capacitance values from stored energy. They also have higher energy than EDLCs . While a2, NiO, V2O5, Co3O4 and RuO2, as well as hydroxide materials such as Co(OH)2 and Ni(OH)2, exhibit high capacitance values [Therefore, various chemically synthesized pseudocapacitive materials have been evaluated for their potential applicability as supercapacitors. In particular, metal oxides, metal hydroxides and metal sulfides have been extensively investigated owing to their relatively fast redox kinetics and high capacitance –13. The e values .2 has been extensively researched owing to its potential to function as a supercapacitive electrode with metallic conductivity and reversible faradaic reactions; however, RuO2 has presented some difficulties such as high costs, commercial restrictions and the toxicity of ruthenium [The large energy storage capacity of RuOuthenium ,16. Moreuthenium . Hence, uthenium .Recently, transition metal sulfides have attracted attention for their potential as pseudocapacitive materials for supercapacitors . Previou2, Co2S3, Co9S8 and Co4S3 [Among these, cobalt sulfide (CoS) has been considered as one of the most promising and versatile pseudocapacitive electrode materials for supercapacitors, solar cells, catalysts, lithium ion batteries and magnetic materials. CoS has multiple advantages such as low cost, electroactivity, high theoretical specific capacitance and long-term stability. CoS can occur in various phases, including CoS, CoSnd Co4S3 . The sur−1 at 1.5 A g−1 with ethanol as a solvent was higher than that with water and water/ethanol-based solvents . These interesting results highlights the potential of CoS supercapacitor as a high performance energy storage system for practical applications.In this study, a low cost and facile strategy was developed for the design and fabrication of CoS nanostructure on nickel foam using a chemical bath deposition (CBD) method for supercapacitor application. Different chemical methods such as one-step ion exchange, hydrothermal, reflux and CBD have been endorsed in the past for the preparation of CoS. In this study, we report an inexpensive and facile CBD method that we used to obtain different nanoparticles with which to form CoS thin films deposited on a Ni foam substrate. Nanostructured cobalt sulfide was synthesized using cobalt nitrate hexahydrate as the cobalt source and thiourea as the sulfur source. The resulting CoS was deposited using various solvents, and its performance was tested in an aqueous solution of 1 M KOH. Our results show that CoS exhibited good electrochemical properties for supercapacitor applications. Its specific capacitance of 41.36 F g2.2.1.3)2·6H2O), thiourea (CH4N2S), potassium hydroxide (KOH), absolute ethanol (C2H6O) and nickel foam (Ni foam) were purchased from Sigma-Aldrich and used as received.All chemicals, such as cobalt nitrate hexahydrate 2·6H2O and 1 M CH4N2S as cobalt and sulfide sources, respectively, in 50 ml of water, ethanol and water/ethanol. The resulting solutions were stirred vigorously for 15 min to obtain homogeneous solutions. Next, the prepared Ni foams were immersed vertically into the solutions and placed in a hot air oven at 90°C for 4 h. After air-cooling to room temperature, the CoS-coated Ni foams were washed several times with water and ethanol to remove unwanted particles. The foams were then dried with a hair dryer and placed in an oven at 60°C for 2 h. CoS was prepared with three different solvents: water, ethanol and water/ethanol.CoS solutions were prepared using 1 M Co with a hemispherical energy analyser, using monochromatic AlKα radiation. The morphology of the samples was studied using a scanning electron microscope  operated at 10 KV, at the Busan Korea Basic Science Institute (KBSI). The elemental composition of the thin film was characterized using energy-dispersive X-ray spectroscopy (EDX), operated at 10 KV.Films consisting of CoS crystals on the prepared electrodes were characterized via X-ray diffraction (XRD) on a D8 ADVANCE, using a CuKα radiation source operated at 40 kV and 30 mA with 2−1, while the GCD tests were performed within a potential range of −0.2 to 0.5 V, at a current density of 1.5 to 9 A g−1. The specific capacitance, energy and power density were calculated from the GCD curves, according to equations (2.1)–(2.3), where Cs is the specific capacitance (F g−1); Δt is the discharge time difference in seconds; m and ΔV are the mass of the electrode (mg) and potential difference during the discharge process (V), respectively; E is the energy density (W h kg−1); P is the power density (W kg−1).i is the discharge current, t is the discharge time, V is the potential window and m is the mass of the active material.The particle size of CoS was examined using transmission electron microscopy  at the Busan KBSI. The supercapacitive properties of the CoS thin films were studied at room temperature using cyclic voltammetry (CV) and galvanostatic charge–discharge (GCD) on a CHI 600 electrochemical work station. The CV was conducted in a potential window from −0.4 to +0.6 V using scan rates ranging from 10 to 100 mV s3.3.1.a–c. Figure 1a clearly shows that there are no prominent or well-defined peaks in the XRD pattern for the CoS. This clearly indicates that the prepared sample is amorphous or that it shows poor crystallinity. This result is consistent with previous reports [To identify the crystal structure, we performed XRD and XPS analysis, as shown in figure 1 reports .Figure b,c. We observed different elements such as Co2p3 (781.49), S2p (162.03), C1s (284.6), N1s (399.36), Ni2p3 (856.1), O1s (531.34), P2p (132.52) in the surveyed spectrum of the CoS samples prepared using ethanol as a solvent. Thus, the cooperative affiliation between oxygen, carbon and air within the samples caused a Ni peak, and may be responsible for surface absorption. The XPS spectrum of CoS prepared using ethanol showed two major peaks, corresponding to Co2p3/2 and Co2p1/2, at binding energies of 782.64 and 798.49 eV, respectively [The exterior and synthetic states of the Co and S molecules within the prepared CoS were evaluated using XPS, the results of which are shown in figure 1ectively . The bina1,a2)), unfledged nanoflakes formed because of a fast increasing rate and high nucleation. When the material had a large pore size and less adhesion to its substrate, it was difficult for ions to diffuse and electrons to be transferred within the electrode. However, this sample also exhibited less adhesion and a large pore size.The surface morphology, size, and surface structure of CoS were analysed via SEM . The corb1,b2) show the SEM images of CoS prepared using ethanol as the solvent. This sample exhibited greater adhesion to its substrate, and had more, but smaller, pores. Moreover, CoS tapioca sago-like nanoparticles prepared using ethanol were distributed uniformly in comparison to CoS prepared using water and water/ethanol. The uniformly coated nanoparticles on the Ni foam showed many electron pathways owing to their relative proximity. c1,c2) show the SEM images of CoS prepared using water/ethanol. The CoS nanoparticles were dispersed unevenly, with voids between the particles on the surface of the Ni foam. This resulted in poor electrochemical reactivity between the KOH electrolyte and CoS electrode.b1,b2) that individual nanoparticles form large agglomerations. Moreover, nanoparticles of different sizes have a uniform shape, which can help electrolyte ions penetrate into the inner surface of the CoS electrodes. Previous studies suggested that thin electrode films often outperform other materials because the electrolyte ions and electrons can be more easily transferred than in thicker films. As seen in c1,c2), irregular CoS nanoparticles are formed, which results in a low surface area. From figures TEM images  were use3.2.a displays the CV curves of the CoS electrode in 1 M KOH electrolyte. A potential window of 0.4–0.6 V and a scan rate of 30 mV s−1 were used. The oxidation and reduction values for water were 37.80 and −36.85 mA, respectively. Ethanol had values of 45.85 and −41.47 mA, while water/ethanol had values of 18.80 and −17.94 mA, respectively. The CoS electrode prepared using ethanol as a solvent showed the highest oxidation and reduction values among all the electrodes.The electrochemical characteristics of CoS electrodes prepared using different solvents were investigated using CV measurements in a three-electrode system. Figure 5Moreover, the surface area of the CV curves for different solvents can be correlated with the SEM and TEM results, as shown in figures b–d) shows CV measurements with different scan rates, ranging from 10 to 100 mV s−1, for three different CoS electrodes in the same electrolyte. A similar behaviour, for example, two pairs of redox peaks, was observed for all conditions, mainly resulting from pseudocapacitance rather than EDLC. On increasing the scan rate from 10 to 100 mV s−1, the anodic and cathodic peaks shifted to a more positive and negative potential, respectively. This may be because of the electrolyte ions being able to access only the outer surface of the electrodes [ectrodes ,30. At a−1 and potential range between 0.5 and −0.2 V. These measurements were conducted using 1 M KOH electrolyte (a). CoS electrodes prepared with different solvents had a specific capacitance of 31.66 F g−1 (water), 41.36 F g−1 (ethanol) and 21.7 F g−1 (water/ethanol) at a current density of 1.5 A g−1. This excellent capacitive performance was attributed to their tapioca sago-like structure, which allowed the KOH electrolyte to easily diffuse into the inner region of the CoS electrode. This facilitated fast electron transport with a high rate capability, which was consistent with the SEM images.The GCD curves for the CoS electrodes are shown in ctrolyte a. CoS el−1, as shown in b–d). The pseudocapacitance decreased gradually in response to an increase in the current density, owing to insufficient faradaic redox reactions [The three electrodes were further tested at various current densities ranging from 1.5 to 9 A geactions –34. ThisUltimately, CoS electrodes prepared with ethanol exhibited higher specific capacitance, electrical conductivity, energy density and greater surface area when compared with electrodes prepared using water and water/ethanol. Continued progress toward optimizing the concentrations of materials, as well as their deposition time, temperature, fabrication techniques and surface morphology, will enable greater specific capacitance for supercapacitors.−1 ions into the inner region of the electrode and improved faradaic reactions with electrolytes on the electrode surface in comparison to other samples. These results indicate that the CoS electrode can be a good candidate for use in supercapacitors.A CoS tapioca sago-like nanostructured film was prepared with various solvents and successfully deposited on a Ni foam substrate using as a CBD method. CoS prepared with ethanol as the solvent exhibited multiple advantages over other samples such as a higher utilization rate of electrodes in KOH solution, easy penetration of OHThe EIS measurements were further carried out to investigate the electrochemical behaviour of various solvents of the CoS electrode. −1 for 2000 cycles. For the ethanol solvent of the CoS electrode the real capacitance is 31.2 F g−1 in the first cycle, and it gradually decreases to 25.1 F g−1 after 2000 cycles, showing 19.2% loss after 2000 cycles, indicating the good cycle property as a good electrode material for electrochemical supercapacitor. These positive results are mainly due to with the electrolyte gradually penetrating into the electrode, more and more electrode materials become activated and contribute to the increase of specific capacitance.The long-term cycling stability of an electrode is also most important for practical supercapacitor applications. 4.−1 at 1.5 A g−1, as well as excellent conductivity and rate capability in comparison to electrodes prepared with water and water/ethanol . This outstanding performance can be attributed to its unique tapioca sago nanostructure constructed by interlaced nanoparticles. Our future work will focus on improving the cycling stability of the CoS electrode. The overall improved electrochemical performance of the CoS electrode prepared using this simple CBD method makes it a promising material for potential applications in high performance supercapacitors because of its low cost and simple preparation and environmental friendliness.The optimized CoS electrode prepared using ethanol yielded interconnected nanoparticles on a Ni foam substrate. This suggests that the electrode could function well in a supercapacitor owing to its good specific capacitance of 41.36 F g


Father absence has negative consequences for children's behaviour. Yet research has not examined how father absence and child behaviour may influence each other. This study models the cross‐lagged relationship between father absence (non‐residence) and child problem behaviour in the early years.UK's Millennium Cohort Study, at children's ages 3, 5 and 7 years (Sweeps 2–4). The sample was 15 293 families in which both biological parents were co‐resident at Sweep 1, when the child was aged 9 months. Child problem behaviour was assessed using the clinical cut‐offs of the Strengths and Difficulties Questionnaire (SDQ). We also investigated gender differences in the association between father absence and problem behaviour.We used data from the SDQ subscales. Using these subscales, we found few child behaviour effects, mostly during the preschool years: children's severe externalizing and social  problems were associated with a greater probability of the father being absent in the next sweep. All cross‐lagged relationships were similar for boys and girls.Father absence at age 3 predicted a higher probability of the child scoring above cut‐off for total difficulties at age 5, as did father absence at age 5 for total difficulties at age 7. There were no significant effects for total difficulties on father absence. Similar father absence effects were found for individual UK families, and to affect boys and girls similarly. There were some child  behaviour effects on father absence, particularly in the early years.Father absence seems to be mainly the cause rather than the outcome of child problem behaviour in young  There is much research on the relationship between father absence because of divorce or parental separation and child problem behaviour, including externalizing and internalizing problems , we examined the cross‐lagged relations between father absence and problem behaviour at ages 3, 5 and 7 years, controlling for pre‐age 3 measures (at the beginning of our study period) of resources , inter‐parental relationship quality (i.e. conflict) and parental mental health, as well as background characteristics . Low levels of family resources, including income and education, are related to parental separation/divorce and lone parenthood (Kiernan & Mensah http://www.cls.ioe.ac.uk/mcs), a longitudinal survey of children born in the UK during 2000–2002. The MCS was designed to over‐represent areas with high proportions of ethnic minorities in England, areas of high child poverty, and the three smaller UK countries. Ethical approval was gained from National Health Service Multi‐Centre Ethics Committees, and parents gave informed consent before interviews. Sweeps 1–4 took place when children were around 9 months, and 3, 5, and 7 years old. The complete MCS sample consists of 19 244 families. Our analytic sample (n = 15 293) comprised MCS singletons and first‐born twins/triplets who lived in families where both biological parents were co‐resident at Sweep 1. The 692 families who entered MCS at Sweep 2 . The biological father was ‘absent’ if he was not co‐resident with the child's mother. Child problem behaviour was measured with the Strengths and Difficulties Questionnaire : total difficulties (13); conduct problems (2); hyperactivity (5); emotional symptoms (3), and peer problems (2), and coded all problem behaviour variables as at or below/above cut‐off. In our analytic sample, 19, 10 and 12% of children scored above cut‐off for total difficulties at ages 3, 5 and 7, respectively. For the individual problem domains, the percentages of children scoring above cut‐off at ages 3, 5 and 7, respectively, were: 49, 20, and 19% (conduct problems); 22, 16, and 18% (hyperactivity); 8, 10, and 13% ; and 24, 15, and 16% (peer problems). Cronbach's alpha across sweeps ranged, respectively, 0.77–0.82 for total difficulties, 0.54–0.66 for conduct problems, 0.71–0.79 for hyperactivity, 0.52–0.64 for emotional symptoms, and 0.47–0.57 for peer problems.The control variables  were also included as control variables. Of the 15 293 families, 71% were married.The To examine whether father absence predicts child problem behaviour, and vice versa, at ages 3, 5 and 7 years, we estimated cross‐lagged path models with dichotomous manifest variables in Mplus 7.0 (Muthén & Muthén n = 15 293) – differed from families with absent fathers at Sweep 1 (n = 3951) on some of our control variables showing some sample selection bias (results available upon request). These differences are largely reflected in Tables Families with co‐resident parents at Sweep 1 – those in our analytic sample n = 15 93 – diffAs expected, our control variables were generally related to both child problem behaviour and father absence at age 3 Table . The relAround 20% of the analytic sample missed data on father absence and child problem behaviour. Specifically, missingness at Sweeps 2, 3 and 4 was, respectively, 18, 19 and 26% for father absence, and 24, 22 and 28% for child problem behaviour.Figure We then examined the four domain subscales of the SDQ in separate models. These cross‐lagged models' unstandardized regression coefficients and SEs are shown in Table There were similarities and differences by domain in the effects of the age 9 months predictors on the age 3 outcomes. For all domains, child's sibship size and parents' higher education, higher relationship quality and married marital status were positively, and family poverty was negatively, related to the likelihood of the father being present at age 3. Compared with White, Indian and Pakistani/Bangladeshi children were more likely, and Mixed and Black children were less likely to co‐reside with their fathers at age 3. For conduct and peer problems, paternal psychological distress was associated with father absence at age 3, as well. Family poverty, maternal psychological distress and lower inter‐parental relationship quality as reported by the mother were significantly related to the child scoring above cut‐off for all difficulties. There were some distinct (by domain) patterns with ethnicity. Compared with White, Pakistani/Bangladeshi children were more likely and Black children were less likely to score above cut‐off for conduct problems. For hyperactivity, Pakistani/Bangladeshi (compared with White) children were more likely to score above cut‐off. Finally, compared with White, Indian and Pakistani/Bangladeshi children were more like to score above cut‐off for emotional symptoms, and Indian, Pakistani/Bangladeshi and mixed children were more likely to score above cut‐off for peer problems.There were some other differences across domains, as well. Paternal psychological distress was related to child conduct problems, but no other difficulty. Mother's and father's higher education was related to a lower probability of the child having borderline/abnormal levels of hyperactivity, emotional symptoms and peer problems. Furthermore, mother's (but not father's) education was related to child's conduct problems. Having married parents was associated with a lower probability of having severe conduct and emotional problems. Larger sibship size was related to lower hyperactivity, emotional symptoms and peer problems. Lastly, girls were less likely to have borderline/abnormal levels of hyperactivity, peer problems and conduct problems, but not emotional symptoms.P < 0.001; girls: B = 0.04, SE = 0.04, P = 0.33, chi‐square difference = 4.93, P = 0.03).Our multiple‐group analysis revealed no gender differences in the cross‐lagged effects between father absence and child problem behaviour. The only significant gender difference was in the cross‐sectional relationship between father presence and conduct problems at age 3. This relationship was stronger for boys  of family structure following the father's departure. As others have shown  and child problem behaviour in early‐to‐middle childhood.Father absence seems to be mainly the cause rather than the outcome of child problem behaviour, and to affect boys and girls similarly.There were some child  behaviour effects on father absence, particularly in the early years.Father absence appears to carry risks for problem behaviour in early and middle childhood.Despite these limitations, our study has important strengths. Even after taking into account reverse causality and selection into family structure, our study documented longitudinal negative effects of father non‐residence on child well‐being in this large sample of young UK families with initially co‐resident biological parents. Future research should investigate the mechanisms behind these effects.This research was supported by a UK Economic and Social Research Council grant (ES/J001414/1) to EF, and by a personal overseas research grant to MKN from the Research Council of Norway (202438).


Spheniscus demersus in response to 8 years of alternating time–area closures around two pairs of colonies. Our results demonstrate that fishing closures improved chick survival and condition, after controlling for changing prey availability. However, this effect was inconsistent across sites and years, highlighting the difficultly of assessing management interventions in marine ecosystems. Nevertheless, modelled increases in population growth rates exceeded 1% at one colony; i.e. the threshold considered biologically meaningful by fisheries management in South Africa. Fishing closures evidently can improve the population trend of a forage-fish-dependent predator—we therefore recommend they continue in South Africa and support their application elsewhere. However, detecting demographic gains for mobile marine predators from small no-take zones requires experimental time frames and scales that will often exceed those desired by decision makers.Global forage-fish landings are increasing, with potentially grave consequences for marine ecosystems. Predators of forage fish may be influenced by this harvest, but the nature of these effects is contentious. Experimental fishery manipulations offer the best solution to quantify population-level impacts, but are rare. We used Bayesian inference to examine changes in chick survival, body condition and population growth rate of endangered African penguins  About oredators –10. Howeredators , and infredators ,11,12.There is a pressing need to determine definitively whether competition with forage fisheries contributes to the ongoing declines of threatened marine predators and—if so—whether or not marine protected areas (MPAs), or no-take zones (time–area closures), offer a useful mitigation option –15. ManaSardinops sagax and anchovy Engraulis encrasicolus purse-seine fisheries and rapidly declining populations of endemic seabirds [Spheniscus demersus breeding colonies in 2008 [In Southern Africa, there is potential for competition between the sardine seabirds . This le in 2008   by more than 1% were evident. This is the threshold considered indicative of demographic impact in a South African management context [Here, we use data from two pairs of proximate island colonies spanning 8 years (2008–2015) before and after, with and without purse-seine fishing closures in place We used data from two sets of paired islands: Robben Island  and Dassen Island  in South Africa's Western Cape province and St Croix Island (33°47′ S 25°46′ E) and Bird Island  in the Eastern Cape province . Between(b)We measured chick condition at all four islands between 2008 and 2015. Nests were selected at random and chicks were measured for head length  using Vernier calipers, and mass (±10 g) using electronic or spring balances. Measurements were made approximately 5–10 days apart from January to December on Dassen Island (which has an extended breeding season), and between March and November at the other sites. We estimated body condition using a species-specific index based on a cohort of chicks with head lengths greater than 75 mm that survived to fledging ; smallerData on chick survival were collected at the Western Cape islands from 2008 to 2015. Marked nests were checked at target intervals of 4–7 days at Robben Island throughout the main breeding season (March to October), and 5 days at Dassen Island throughout the whole year. We recorded the presence and number of chicks at each visit, calculated the number of days exposed to potential mortality (nestling days) and recorded whether mortality occurred (=1) or not (=0) ,23. Wher(c)We used hydro-acoustic survey estimates of sardine and anchovy biomass in South Africa from 2008 to 2015 ,29 as a (d)l), in month k of year j at island i; α is the intercept; jb denotes the year random effect and jb); the β's are the coefficients to be estimated for the fixed effects; jx is a binary covariate for the island closure effect ; iz is a binary covariate denoting to which island a chick belongs ; jS and jA are sardine and anchovy (respectively) biomass estimates associated to year j (see above for details) and σ) for the random effects and residual error were estimated from the data.For chick condition, we implemented a linear-mixed model structure, with random intercepts for the month in which each chick was measured, nested within the monitoring year. Because access to prey resources differs , we modeFor chick survival, we estimated failure rates (deaths/unit time of exposure or hazard functions) for ‘Open’ or ‘Closed’ years using an exponential error distribution and used an exponential distribution to transform these failure rates into chick survival estimates ,23. We ul), in nest k, in year j, at island i; −7 is precision) and All models were implemented using Markov chain Monte Carlo (MCMC) estimation in JAGS (v. 4.1.0) via the ‘jagsUI’ library (v. 1.3.7) for program R v. 3.2.1. The uninformative priors were fied as  . For chified as  ,23 (but (e)l = 9436 observed chick measurements. Next, we simulated a sample size (number of chicks measured) for each future year and island (syn) using a random draw from a uniform distribution bounded by the observed sample sizes at each island ; Robben Island: U). For each year, and each island, we randomly drew (with replacement) syn chick condition values from the posterior distributions in a stratified manner according to whether that island was scheduled to be ‘Open’ or ‘Closed’ that year. Each sample was therefore a random draw from a posterior distribution (corresponding to each observation l) with a mean equal to the original observation and a variance specified by the data. Each simulated estimate was then assigned to the calendar month of the corresponding observation [β terms (see equation (2.1)) with those from the original model fit. Finally, to confirm that any changes were not an artefact of the sample used in each case, we repeated the resampling process to generate 1000 new datasets for each of 4, 7 and 10 additional years. We then compared the parameter estimates from the respective JAGS model to the mean effect size and 95% quantiles from fitting 1000 frequentist models (using ‘nlme’ v. 3.1-122) to each new dataset .To examine how the uncertainty associated with closure effects might respond to additional years of experimental closures, we simulated an extended time series of chick condition data for the Western Cape (the largest dataset). Data were imputed based on a future sequence of 3 years ‘Closed’, followed by 3 years ‘Open’ at each island . To prodervation . New dat(f)ϕa = 0.743) was deterministic to allow for clear comparisons between different scenarios for juvenile (ϕj) and chick survival (ϕc). We modelled ϕj and ϕc as stochastic using observed means and standard deviations (s.d.) . The baseline run was parameterized to represent ‘Open’ to fishing at both islands; ϕc was set at the mean (±s.d.) value estimated for all ‘Open’ years at both islands, and ϕj = 0.194 (±0.117) based on published estimates [We used a Bayesian projection model with a demographic structure and parameter values based on previous African penguin models ,34 . For Robben Island, we also assessed the impact of the simulated 10 additional experiment years of chick condition data on the uncertainty associated with Δλ. For chick survival (ϕc), we used priors with an island-specific mean and s.d. estimated for all closed years (from equation (2.2)). In the absence of species-specific data to link improvements in chick body condition directly to juvenile (ϕj) or chick survival (ϕc), we used observed relationships between mass at fledging and first-year survival in macaroni penguins Eudyptes chrysolophus [ϕj with changes in body condition. These modified models were run as the baseline model, but we used the individual Robben Island (1216 breeding pairs) and Dassen Island (2140 breeding pairs) populations in 2015 as starting populations to model the size of the breeding populations in 2025 and 2035 (±95% CI) to compare with the baseline model. See electronic supplementary material for model details.To assess the effect of fishery closure, we modified the priors assigned to solophus , and betsolophus  (electro3.(a)(i)Chick condition: Based on the observed data and the full model , mean condition at Dassen Island was 0.284 (95% CI: 0.242–0.325) during ‘Open’ years and 0.257 (0.212–0.302) during ‘Closed’ years at mean fish biomass, or 9% lower without fishing  during ‘Open’ years to 0.383 (0.336–0.430) during ‘Closed’ years  based onIn all cases, the simulated datasets produced appropriate means and distributions relative to the observed data . The simplified model estimated that fisheries closures improved chick survival at Dassen Island by 11.2%, from 0.738 (0.708–0.773) during ‘Open’ years to 0.816 (0.787–0.843) during ‘Closed’ years (at mean prey biomass). Fishery closures also increased chick survival at Robben Island by 10.8% from 0.733 (0.704–0.762) in ‘Open’ years to 0.812 (0.784–0.838) in ‘Closed’ years (d’ years . There wd’ years  and the (ii)Chick condition: Mean condition at St Croix Island was 0.361 (0.298–0.4260) in ‘Open’ years and 0.277 (0.210–0.345) in ‘Closed’ years. At Bird Island, mean chick condition was 0.224 (0.164–0.283) and 0.308 (0.234–0.383) in ‘Open’ and ‘Closed’ years, respectively. Fishery closures appeared to generate weak, opposing effects at these islands; i.e. condition increased  at Bird Island and decreased  at St Croix Island. However, the 95% CI for both sets of scenarios overlapped, suggesting that overall, the closure had no impact in this case (his case  and ther(b)λ) at Dassen and Robben Island, estimated with open fisheries, demographic stochasticity and parameter uncertainty on ϕc and ϕj, was λ = 0.805 (95% CI: 0.754–0.864). This was comparable to the equivalent deterministic Leslie matrix estimate, λ = 0.809 and the projections reproduced the observed population trajectory . When ϕc and ϕj were increased by the observed effect sizes of fishing closure, λ improved at both Dassen Island  and Robben Island ; in the latter case Δλ > 1% compared to the baseline model  at Robben Island and the chick survival effect at Dassen Island were modelled, and approximately 20% higher by 2035 . However, the uncertainty around this demographic effect was high and decreased little with 10 additional years of simulated sampling . For chick body condition at Robben Island, less than 1% of the posterior distribution was negative and this small uncertainty disappeared with an additional 3 years of simulated data . At the other islands, the effects were essentially indistinguishable from zero, or became so with 10 years of simulated data at Dassen Island. The mean effect sizes were also relatively robust to the addition of simulated data , so infesampling . MoreoveOvercoming uncertainty in the potential demographic impact of fisheries closures is likely to remain difficult, even in systems where seabird–fisheries interactions are relatively well understood . It is oFinally, our results highlight the need to carefully consider the value of small-scale protected areas for long-lived, motile marine species where benefits to adult survival may be subtle ,34. In o


Art v 1, Amb a 4, and Par h 1 are allergenic defensin‐polyproline–linked proteins present in mugwort, ragweed, and feverfew pollen, respectively. We aimed to investigate the physicochemical and immunological features underlying the different allergenic capacities of those allergens.E. coli and physicochemically characterized in detail regarding identity, secondary structure, and aggregation status. Allergenic activity was assessed by mediator releases assay, serum IgE reactivity, and IgE inhibition ELISA using sera of patients from Austria, Canada, and Korea. Endolysosomal protein degradation and T‐cell cross‐reactivity were studied in vitro.Recombinant defensin‐polyproline–linked proteins were expressed in 25‐36 was observed.Despite variations in the proline‐rich region, similar secondary structure elements were observed in the defensin‐like domains. Seventy‐four percent and 52% of the Austrian and Canadian patients reacted to all three allergens, while Korean patients were almost exclusively sensitized to Art v 1. This was reflected by IgE inhibition assays demonstrating high cross‐reactivity for Austrian, medium for Canadian, and low for Korean sera. In a subgroup of patients, IgE reactivity toward structurally altered Amb a 4 and Par h 1 was not changed suggesting involvement of linear epitopes. Immunologically relevant endolysosomal stability of the defensin‐like domain was limited to Art v 1 and no T‐cell cross‐reactivity with Art v 1Despite structural similarity, different IgE‐binding profiles and proteolytic processing impacted the allergenic capacity of defensin‐polyproline–linked molecules. Based on the fact that Amb a 4 demonstrated distinct IgE‐binding epitopes, we suggest inclusion in molecule‐based allergy diagnosis. In northern America, ragweed represents the most prevalent Asteraceae pollen source, while in Korea exposure to mugwort and ragweed is observed.Allergy to weed pollen has been extensively documented worldwide, being the third most important cause of pollen allergies, after grasses and trees. Relevant allergenic weeds of the Asteraceae family are mugwort Extract‐based allergy diagnosis of weed pollen is frequently challenging due to (i) polysensitization of patients, (ii) overlapping flowering periods, and (iii) similar allergen profiles.Art v 1 is a major allergen with a sensitization rate of 95% among mugwort pollen‐allergic patients.In the present study, we investigated structural and immunological features of allergenic defensin‐polyproline–linked molecules from mugwort, ragweed, and feverfew pollen. Therefore, recombinant allergens were produced to evaluate intrinsic physicochemical characteristics. IgE sensitization profiles and cross‐reactivity pattern were investigated in patients from three geographically distinct regions. Endolysosomal degradation assays were performed to monitor proteolytic susceptibility, and T‐cell assays were conducted to analyze cross‐reactivity at the T‐cell level.2Detailed description of all experimental procedures is provided in the Data 2.1Weed pollen‐allergic patients from Austria (n = 36), Canada (n = 38), and Korea (n = 24) were selected on the basis of typical case history, that is, rhinitis and/or conjunctivitis during late summer, and allergen‐specific IgE to mugwort and/or ragweed pollen determined by ImmunoCAP or skin prick test Table . Experim2.2E. coli Rosetta‐gamiB (DE3) pLysS as described.In silico analysis of the defensin‐like domain was performed with Clustal Omega, SWISS‐MODEL, and Chimera 1.8. Constructs corresponding to the mature sequences of Art v 1.0101, Amb a 4.0101, and Par h 1.0101 were cloned into the pHis Parallel 2 expression vector.2.3Purified allergens were analyzed by reducing SDS‐PAGE and Coomassie staining. Protein concentrations were determined by amino acid analysis, and identity was verified by intact mass analyses using a Q‐Exactive mass spectrometer. The aggregation behavior of the proteins was analyzed by dynamic light scattering (DLS) and high‐performance liquid chromatography (HPLC). Secondary structure elements were determined by Fourier transform infrared (FTIR) spectroscopy using a Tensor 27 spectrophotometer. Circular dichroism spectra of native and heat‐treated proteins were recorded with a JASCO J‐815 spectropolarimeter.2.4IgE reactivity of weed pollen extracts and purified allergens was evaluated by ELISA using sera from weed pollen‐allergic patients. The biological activity of the allergens was verified in a mediator release assay.2.5The proteolytic stability was studied with the endolysosomal degradation assays.2.625‐36‐specific TCR tg Jurkat T‐cells25‐36‐specific T‐cell lines and clones established from mugwort‐allergic donors were tested for proliferation in response to the allergens or Art v 1‐peptide by 3H‐thymidine uptake.Monocyte‐derived dendritic cells were differentiated as described2.7An unsupervised cluster analysis was performed with ClustVis using IgE reactivity and cross‐inhibition data.33.1E. coli. They shared the prototypical features of defensin‐polyproline–linked proteins with an N‐terminal defensin‐like domain and eight conserved cysteine residues.To investigate the physicochemical and structural features of Art v 1, Amb a 4, and Par h 1, the allergens were expressed as nontagged proteins in h ~2 nm as assessed by DLS, and in HPLC, they eluted as single peaks with comparable retention times and molecular weight ; however, its implication in allergy has not been established.Next, the sensitization profile to weed pollen was investigated in patients from Austria, Canada, and Korea. Patients from Austria were sensitized to ragweed and mugwort due to the concomitant growth of both weeds in this regionUsing recombinant allergens, a dominant IgE reactivity to Art v 1 (92%) among Korean patients was noted. Art v 1 sensitization prevalence in Canada and Austria was only 55% and 53%, respectively, suggesting that those patients were also reactive to Amb a 1.To study reactivity pattern in detail, we investigated whether reduction and alkylation lead to loss of IgE reactivity. In line with previous studies, the appropriate folding of the Art v 1 defensin‐like domain was crucial for IgE reactivity.Several studies showed that protein immunogenicity is linked to endolysosomal processing during antigen presentation.25‐36, while T‐cell cross‐reactivity between other, minor epitopes and HLA class II restrictions other than those by HLA‐DR1 was not investigated.As differences in the primary structure might provide distinct cleavage sites for different proteases, we additionally investigated the proteolytic stability to cathepsin S, a major component of the endolysosomal protease cocktail.In summary, we show that regardless of the structural similarity shared by the allergenic defensin‐polyproline–linked proteins, they differ in their immunological properties. The higher sensitization rate of Art v 1 is consistent with the increased stability to proteolytic degradation, which ensures a steady supply of T‐cell epitopes. A group of patients reacted exclusively to Amb a 4/Par h 1 epitopes which are not dependent on the cysteine‐stabilized fold of the defensin‐like domain. We therefore suggest including Amb a 4 in molecule‐based diagnosis of weed pollen allergies, thus allowing further refinement for therapeutics in allergen immunotherapy.Dr. Pablos Ocampo reports grants from Austrian Research Fund, during the conduct of the study; Dr. Eichhorn reports grants from Austrian Science Fund, during the conduct of the study; Dr. Machado reports grants from Austrian Science Fund, during the conduct of the study; Dr. Neunkirchner reports grants from Austrian Science Fund, during the conduct of the study; Dr. Jahn‐Schmid reports grants from Austrian Science Fund, grants from Austrian National Bank, during the conduct of the study; Dr. Wildner reports grants from Christian Doppler Research Association, during the conduct of the study; Dr. Pickl reports grants from Austrian Science Foundation (FWF), during the conduct of the study; and he holds stocks of Biomay AG, Vienna, Austria and received honoraria from Novartis for expert advise and seminars/talks; Dr. Arora reports grants from Department of Biotechnology, Government of India., during the conduct of the study; Prof. Vieths reports personal fees from Ärzteverband Deutscher Allergologen, personal fees from Swiss Society for Allergy and Immunology, personal fees from Schattauer Allergologie Handbuch, personal fees from Elsevier Nahrungsmittelallergien und Intoleranzen, personal fees from Karger Food Allergy: Molecular Basis and Clinical Practice, non‐financial support from German Research Foundation, non‐financial support from European Directorate for the Quality of Medicines and Health Care, non‐financial support from European Academy of Allergy and Clinical Immunology, non‐financial support from German Chemical Society (GDCh), non‐financial support from AKM Allergiekongress, non‐financial support from International Union of Immunological Societies, personal fees from University Hospital Gießen / Marburg, personal fees from University of Bonn, personal fees from Pharmacon, personal fees from Medical University of Vienna, Austria, personal fees from Gesellschaft für pädiatrische Allergologie und Umweltmedizin, non‐financial support from World Allergy Organization, non‐financial support from Technical University of Munich, non‐financial support from Austrian Society for Dermatology and Venerology, outside the submitted work; Dr. Gadermaier reports grants from Austrian Science Fund, during the conduct of the study; personal fees from Thermo Fisher Scientific, outside the submitted work. The other authors declare that they have no relevant conflict of interests.GG, FF, and SV were responsible for study concept and design. IP, SE, PB, NA, BJS, and SW performed experiments. IP, GG, FF, and YM analyzed and interpreted the data. WTS, CE, JWP, and WP provided reagents and materials. IP and GG drafted the manuscript. All authors provided critical revision of the manuscript for important intellectual content and approved the final version for publication. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Hevea brasiliensis (the Para rubber tree). Here, we characterize the entire H. brasiliensis CDPK and CDPK‐related kinase (CRK) gene families comprising 30 CDPK genes (HbCPK1 to 30) and nine CRK genes (HbCRK1 to 9). Structure and phylogeny analyses of these CDPK and CRK genes demonstrate evolutionary conservation in these gene families across H. brasiliensis and other plant species. The expression of HbCPK and HbCRK genes was investigated via Solexa sequencing in a range of experimental conditions . The results suggest that HbCPK and HbCRK genes are important components in growth, development, and stress responses of H. brasiliensis. Parallel studies on the CDPK and CRK gene families were also extended to five other plant species . The CDPK and CRK genes from different plant species that exhibit similar expression patterns tend to cluster together, suggesting a coevolution of gene structure and expression behavior in higher plants. The results serve as a foundation to further functional studies of these gene families in H. brasiliensis as well as in the whole plant kingdom.Calcium‐dependent protein kinases (CDPKs or CPKs) play important roles in various physiological processes of plants, including growth and development, stress responses and hormone signaling. Although the CDPK gene family has been characterized in several model plants, little is known about this gene family in  CDPK/CPKcalcium‐dependent protein kinaseCRKCDPK‐related kinaseQPCRreal‐time quantitative reverse transcription polymerase chain reactionSRASequence Read Archive2+), functioning as a second messenger of plant cells, plays an essential role in various signaling transduction pathways 2+ concentration are sensed by several Ca2+ sensors or Ca2+‐binding proteins. To date, three major classes of Ca2+‐binding proteins, including calcium‐dependent protein kinases (CDPKs or CPKs), calmodulins (CaMs), and CaM‐like proteins (CMLs) and calcineurin B‐like proteins (CBLs), have been characterized in higher plants 2+ binding Plants have evolved a series of survival mechanisms to adapt to diverse environmental challenges, including drought, salinity, wounding, and low temperatures Arabidopsis thaliana, CPK10 participates in abscisic acid (ABA) and Ca2+‐mediated stomatal regulation in response to drought stress AtCPK4 and AtCPK11, acting as positive regulators in ABA signaling pathways, are involved in seed germination, seedling growth, stomatal movement, and salt stress tolerance CDPK gene, AtCPK12, has been characterized as a negative ABA‐signaling pathway regulator AtCPK6 demonstrates crucial roles in responses to drought and salt stresses Oryza sativa, transgenic plants overexpressing OsCDPK7/OsCPK13 show enhanced resistance to cold, drought, and salt stress OsCDPK13/OsCPK7 and OsCPK21 are involved in responses to cold and salt stress, respectively Nicotiana tabacum, two CDPK genes, NtCDPK2 and NtCDPK3, play an essential role in the defense response, and NtCDPK2 functions together with the stress‐induced MAPKs (mitogen‐activated protein kinases) to control response specificity to abiotic and biotic stresses CDPK genes have been characterized in O. sativa, Zea mays, Populus trichocarpa, and Brassica napus, and they point to important roles in the regulation of abiotic stresses, hormones, and the intrinsic developmental program in plant growth and development Accumulating evidence indicates that CDPKs participate in plant responses to a variety of abiotic and biotic stresses, as well as in plant development cis‐1,4‐polyisoprene, NR) is an important industrial raw material, and the sole commercial source of NR is Hevea brasiliensis (the Para rubber tree), a perennial tropical tree species CDPK gene (HbCDPK1) was shown to be induced by ethephon in H. brasiliensisCDPK genes in stress responses and hormone signaling in other plants CDPK gene family in the rubber tree in relation to the regulation of latex production.Natural rubber  genes, the expression patterns of which were analyzed in different plant tissues in response to various treatments, and at several phases of tissue development. In addition, the gene structure and phylogeny of these genes were also compared, and all the results obtained will help further understanding of the roles of CDPK and CRK genes in the regulation of latex production and regeneration. Data on CDPK and CRK genes and their expression in six plant species would also contribute to the understanding of the structure of these gene families and the functions of their members in the plant kingdom.In this study, we performed a genome‐wide analysis of the CDPK and CRK gene family members in six plant species  from their published genome sequences CDPK and CRK sequences of three model plants of A. thaliana, O. sativa, and P. trichocarpa as queries CDPK genes and 45 CRK genes in the six plant species, including 39 H. brasiliensis CDPK and CRK genes , 39 P. trichocarpa genes , and 34 O. sativa genes . The gene numbers of CDPK and CRK families identified here for the three model plants agree well with those previously reported We identified all 2+‐binding EF‐hands. While CRKs share degenerative Ca2+‐binding EF‐hands compared to CDPKs. The lengths of the CDPK‐ and CRK‐coding regions (CDS) were similar among the six plant species examined, ranging from 1305 to 1893 bp in H. brasiliensis, 1452 to 1938 bp in A. thaliana, 1359 to 1896 bp in O. sativa, 1431 to 1839 bp in P. trichocarpa, 1290 to 1932 bp in M. esculenta, and 1401 to 1923 bp in R. communis  with 11, 8, 9, 2, and 9 isoforms, respectively. Similarly, the CDPK and CRK family genes in the five other species are also clustered into different groups : 10, 13, 8, 3, and 8 isoforms, respectively, in A. thaliana; 11, 8, 8, 2, and 5 in O. sativa; 11, 8, 9, 2, and 9 in P. trichocarpa; 8, 5, 7, 2, and 9 in M. esculenta; 5, 5, 5, 1, and 5 in R. communis  and one CRK gene pair (HbCRK3/6) were identified. In A. thaliana, there were nine CDPK gene pairs , three CRK gene pairs , and one paralogous gene cluster in subgroup CPK IIe . In M. esculenta, nine CDPK  and four CRK  gene pairs were identified. However, in this species the two genes in each pair do not cluster together as closely as in the other plant species examined in this study  database were used for the expression analysis of CDPK and CRK genes in five species, i.e. M. esculenta, R. communis A. thaliana, P. trichocarpa, and O. sativa. In the case of H. brasiliensis, two sets of Solexa sequencing data were used, one from the NCBI SRA database (http://www.ncbi.nlm.nih.gov/nuccore/448814761) and the other from our own data HbCPK genes  and five HbCRK genes  were barely detectable in almost all the tissues and all the treatments examined. Such genes comprise a large portion (~ 1/3) of the total HbCPK and HbCRK gene family. This character seems to be shared by the CDPK and CRK gene families in other plant species. For example, similar expression patterns were observed for eight of 31 CDPK and CRK genes in M. esculenta . As shown in Fig. HbCPK and HbCRK genes were obviously down‐regulated during leaf development, and their expression was mainly restricted to the first three stages . This result is in agreement with the assay of CDPK activity in maize, in which expanding tissues such as rapidly growing tips, leaves, and coleoptiles have particularly high levels of CDPK activity, while mature leaves have much less activity CDPK genes in early developmental processes such as embryogenesis, seed development, and germination has been reported in sandalwood R. communis CDPK gene in subgroup CPK IIc, RcCPK12, was mainly expressed in the early stages of embryo development and the male developing flowers, suggesting a similar function. It is interesting that the other members of this subgroup were either expressed at a low level or they showed no expression.Increasing evidence indicates that H. brasiliensis to stimulate rubber production, but the yield‐stimulating mechanisms are still poorly understood HbCPK and HbCRK genes in latex under ethephon treatment. As shown in Fig. CPK genes, HbCPK9 and 15, were obviously up‐regulated by ethephon treatment, implicating them in the ethylene‐simulated latex production. Further studies on these genes would help to understand the underlying molecular mechanism. Although the expression of one HbCPK gene, HbCDPK1 (HbCPK30 in this study) has been reported to be markedly induced by ethephon P. trichocarpa, most of the CDPK and CRK genes were associated with ABA activity, but several genes were also affected by MeJa (methyl jasmonate), which apparently down‐regulated the expression of PtCDPK1, 4, and 11 , or ABA plus GA. In a previous study, AtCPK4 and AtCPK11 have been demonstrated to be involved in ABA signaling‐mediated regulation of seed germination, seedling growth, stomatal movement, and salt stress tolerance CDPK and CRK gene families are implicated in the regulation of plant growth and development through participation in the signaling pathways of various plant hormones, especially ABA.Ethephon, an ethylene generator, is widely used in et al. 2+ influx during cold treatment, and the change in Ca2+ influx was controlled by COLD1, a regulator of G‐protein signaling. In this study, the expression levels of HbCPK and HbCRK genes (Ca2+ sensors) were examined under cold stress. Many HbCPK and HbCRK genes are regulated by cold stress, and most of them showed depressed expression after low‐temperature treatment , the expression of many CDPK and CRK genes was also regulated by low temperatures, but different expression patterns were displayed by respective genes . To verify the expression patterns of the RNA‐seq analyses, quantitative reverse transcriptase PCR (qPCR) expressional analyses were performed on HbCPK genes, which are among the isoforms somewhat regulated by ethephon treatment and leaf development, or tissue‐specific as revealed by RNA‐seq data , both genes were expressed either at a low level or not at all in all the tissues and treatments examined. Second, in the gene pair of HbCPK5/12 and HbCPK18/19, both genes exhibited a similar pattern of universal expression across all tissues. Third, for the gene pair of HbCRK3/6, the two genes displayed dissimilar patterns of expression, with HbCRK3 showing universal expression but HbCRK6 being expressed at very low levels in all the tissues or treatments. Fourth, for the gene pair of HbCPK24/28, HbCPK28 was barely expressed in all the tissues or treatments, but the expression of HbCPK24 was only restricted in leaf when exposed to the low‐temperature treatment. We also investigated the expression patterns of the genes pairs or clusters that are closely located on chromosomes of A. thaliana, O. sativa, and P. trichocarpa  were expressed at low levels or not at all in all the samples examined. Interestingly, the CDPK and CRK genes from different plant species that exhibited low or no expressions in nearly all the samples examined tended to cluster in the same subgroups, for example, CPK Ib and c, CPK IIa and c, CPK IIIa and CRK a  showed the classification of these genes into five major groups. Members within each group might have recent common evolutionary origins, as they share common protein motifs and exon–intron structures. Solexa sequencing analyses revealed that most H. brasiliensis CDPK and CRK genes exhibit different patterns of expression under a number of experiments, suggesting their distinct roles in developmental and stress responses. Relevant studies of gene evolution and expression have been extended to the five other plant species. The results presented here provide a foundation for further functional investigation of the CDPK and CRK gene families in H. brasiliensis as well as the whole plant kingdom.In this study, we pioneered a genome‐wide analysis of two protein kinase subfamilies (CDPK and CRK) in A. thaliana, P. trichocarpa, and O. sativa CDPKs and their closely related CRK genes were downloaded from the A. thaliana Information Resource (http://www.Arabidopsis.org/), the rice genome annotation database (http://rice.plantbiology.msu.edu/) and GenBank (http://www.ncbi.nlm.nih.gov/genbank), respectively. The genome and protein sequences of A. thaliana, O. sativa, P. trichocarpa, M. esculenta, and R. communis were downloaded from Phytozome v10 (http://www.phytozome.net/). The H. brasiliensis genome and transcriptome data were obtained from GenBank (http://www.ncbi.nlm.nih.gov/nuccore/448814761) and our own data CDPK and CRK sequences form A. thaliana, P. trichocarpa, and O. sativa as queries to search against the deduced proteome of each species for the candidate CDPKs and CRKs from H. brasiliensis, A. thaliana, O. sativa, P. trichocarpa, M. esculenta, and R. communis. All putative candidates were manually verified with the InterProScan server (http://www.ebi.ac.uk/Tools/pfa/iprscan/) to confirm the presence of protein kinase and EF‐hand domains.Sequences of clustal x  program. The phylogenetic tree was constructed with mega6.0 (http://www.megasoftware.net/) by employing the Neighbor‐Joining (NJ) method with a bootstrap test for 1000 replicates. Exon–intron structures of the six species of CDPK and CRK genes were analyzed by comparing the cDNA and their genomic DNA sequences through the web server gsds 2.0 (http://gsds.cbi.pku.edu.cn/).Multiple alignments of the amino acid sequences of CDPKs and CRKs from six species were performed using the H. brasiliensis , R. communis , A. thaliana , O. sativa , and P. trichocarpa  (Table S2). For H. brasiliensis samples, additional datasets were used for expression analysis as described previously For Solexa sequencing‐based expression analyses, SRA data were downloaded from the NCBI database. The sequences included those for HbCPK and HbCRK genes were 5′‐AGATAGAGAGGGTCAACTCTG GAC‐3′ (F) and 5′‐GCTTGTCTTTGTTTTTTGTATGTG‐3′ (R) (HbCPK5), 5′‐TAGTTTCAGCATTG GATTTAGAGA‐3′ (F) and 5′‐AGTTATAAAGGTCTGCCTGCTTCT‐3′ (R) (HbCPK6), 5′‐TTCTTCC CTTTTGTTTTTTCTCTC‐3′ (F) and 5′‐CTTCTTCTTGCGTCTGTGATTTGT‐3′ (R) (HbCPK8), 5′‐C AAGTTCCCAACATTTAACCTTCT‐3′ (F) and 5′‐GAAGAGTCAGCGGCGTTAGC‐3′ (R) (HbCP K9), 5′‐AGAGAAAGATTCAATAGTCTGAGCT‐3′ (F) and 5′‐ACTTGTGATGTATTTACAACCACA TA‐3′ (R) (HbCPK10), 5′‐GGGTATCATCTGAATCTGCTTCTG‐3′ (F) and 5′‐GCAGTATCATTGTA GAGCCGTGG‐3′ (R) (HbCPK11), 5′‐CCATTAACACAAGCAGGTCAAGTC‐3′ (F) and 5′‐CGGTA AGGAAGAACCCA TTTG‐3′ (R) (HbCPK12), 5′‐GAT GGGTCATTAGAACTGAACAGT‐3′ (F) and 5′‐AACCAGGGCTCACATCCTATTTA‐3′ (R) (HbCPK13), 5′‐CTGCAACTCACAGATCATTCCCT C‐3′ (F) and 5′‐AAAGTTTTGCTCACGGCCTTC‐3′ (R) (HbCPK15), 5′‐ACCAACCACTGCTACCA ACG‐3′ (F) and 5′‐TACTTCCAAGACTGAGCAAAAGAT‐3′ (R) (HbCPK16), 5′‐CATTCAACGAT GAGGAAGACGAG‐3′ (F) and 5′‐TTTTTAAAGTGCAAATTTCGTCCC‐3′ (R) (HbCPK19), 5′‐GG AGACAAGTGAGAGAGTTACAGAA‐3′ (F) and 5′‐CATCCTATGGGGAATCCTACC‐3′ (R) (HbCP K21), 5′‐ATTCTTTCAGGTAAGCATGTATGC‐3′ (F) and 5′‐AGAAATTAAAAAGGGTTGTAAT CC‐3′ (R) (HbCPK27), 5′‐GGCACACTGATGGAAAGTTGAGT‐3′ (F) and 5′‐TTTTGGAGCTTCTG CATTAGTTTA‐3′ (R) (HbCRK2), and 5′‐CTGTGAAGAGTGCTGCAAGTTAAA‐3′ (F) and 5′‐GG TAAGAACAAAGAGGAAAAACAT‐3′ (R) (HbCRK3). The H. brasiliensis UBC2b and UBC4 gene were used as internal controls as described previously et al. To verify the data obtained by Solexa sequencing, quantitative RT‐PCR (qPCR) was performed as describe previously CRT conceived and designed the experiments; XHX, MY, JLS, and JYQ performed the experiments; XHX, MY, and YJF analyzed the data; XHX, MY, SNH, and CRT wrote the paper. All authors read and approved the final manuscript.Table S1. Characteristics of CDPK and CRK genes in six plant species .Click here for additional data file.Table S2. Basic information for the Solexa sequencing data of Hevea brasiliensis and five other plant species.Click here for additional data file.


In this study, we developed a modified computational model of human ventricular myocyte to investigate the combined role of CaMKII and β-adrenergic signaling pathway on the occurrence of EADs. Our simulation results showed that (1) CaMKII overexpression facilitates EADs through the prolongation of late sodium current's (INaL) deactivation progress; (2) the combined effect of CaMKII overexpression and activation of β-adrenergic signaling pathway further increases the risk of EADs, where EADs could occur at shorter cycle length (2000 ms versus 4000 ms) and lower rapid delayed rectifier K+ current (IKr) blockage (77% versus 85%). In summary, this study computationally demonstrated the combined role of CaMKII and β-adrenergic signaling pathway on the occurrence of EADs, which could be useful for searching for therapy strategies to treat EADs related arrhythmogenesis.Early afterdepolarization (EAD) plays an important role in arrhythmogenesis. Many experimental studies have reported that Ca The broken balance of Ca2+ cycling may contribute to the occurrence of EADs [β-adrenergic on the occurrence of EADs in canine ventricular myocyte and concluded that cellular Ca2+ overload and spontaneous SR Ca2+ release played important roles in EADs [β-adrenergic agonists activate protein kinase A (PKA), which phosphorylates ICaL, RyR, PLB, SERCA, and IKs, resulting in delayed afterdepolarization (DADs) [β-adrenergic agonists in relation to EADs. Recently, different computational models have been used to investigate these complex interactions. Xie et al. developed a biophysically detailed rabbit model and found that the faster time course of ICaL versus IKs increased ISO-induced transient EADs [β-adrenergic-induced EADs and arrhythmia. However, other targets including INaL have not been investigated thoroughly in their model. It is noted that, although the computational studies of EAD mechanisms have been widely taken, the majority of these published modelling studies have been developed based on nonhuman myocyte. Furthermore, to the best of our knowledge, there is no modelling study considering the combined effect of CaMKII overexpression and β-adrenergic agonists on EADs.It has been known that an (PLB) . ICaL e of EADs –16. Vold in EADs . On the n (DADs) . These nent EADs  and emphβ-adrenergic signaling networks into a modified ORd's dynamic model [β-adrenergic signaling pathway on the occurrence of EADs would be investigated.This study aimed to develop a modified computational model of human ventricular myocyte that integrates CaMKII and ic model , with whSince there is a lack of experimental measurements of human ventricle CaMKII pathway, O'Hara et al. used the Hund-Decker-Rudy's dog model to describe CaMKII kinetics , 22. Our0) was set to 0.05 at the control state. CaMK0 of 0.12 was used to simulate CaMKII overexpression according to the study from Kohlhaas et al. [The fraction of active CaMKII binding sites at equilibrium state  has been previously described in the computational models [INaK for the normal human ventricle [ICaL, PLB, and IKs) is described as follows:The detailed description of aucerman . PKA hasibitor-I , and IK-I [IKs . ICaL, l models , 26, itsentricle . The phoICaL phosphorylation. In (vshift” was increased from 3.94 (no ISO application) to 10.0 (for saturated ISO application), which means that the steady state activation curve of ICaL was moved left by 6.06 mV. The permeability of ion Ca2+ was increased by 10% with saturated ISO application. Equation  was extended by 20% with saturated ISO application Equation  shows thtion. In , the valIKs by PKA, which was used to alter maximum conductance of IKs (GKs) in  in . Meanwhivail” in  and 10)IKs by Pactive that is affected by CaMK0 as shown in . The fraction of SERCAs phosphorylated by CaMKII in , respectively, while β-adrenergic signaling was maintained inactive. Next, CaMKII overexpression was applied with ISO administration. 1 μM ISO was applied in this study to simulate its effect on myocyte action potential and ion currents.In order to determine the potential targets in CaMKII-induced EADs, CaMKII was solely overexpressed by assigning the CaMKIKr blockage of about 85% [The cycle length of 2000 ms was used in this study since it was closer to normal human beat rhythm than the length of 4000 ms used in the experiments from Guo et al. . In Guo bout 85% , which w0 value to INaL of 0.12 and the normal CaMK0 value of 0.05 to other targets, the alternated EADs occur from our simulation with the cycle length (CL) of 2000 ms and IKr blockage of 85%.As shown in NaL amplitudes alternated with overexpressed CaMK0. In beats with EADs, INaL amplitude was about 24% smaller (0.19 μA/μF versus 0.25 μA/μF) than these with normal CaMK0 value to INaL and in beats without EADs, INaL amplitude was also reduced by 32% (0.17 μA/μF versus 0.25 μA/μF). In INaL deactivated, which was about 168% of normal beats (1297 ms versus 773 ms) and about 170% of the control situation in INaL reduces its amplitude and prolongs INaL deactivation process. Furthermore, the results also indicate that the delayed deactivation of INaL, rather than its amplitude variation, contributes to the formation of EADs.As shown in 0 to ICaL of 0.12 and the normal CaMK0 value of 0.05 to other targets, no EADs occur at CL of 2000 ms and IKr blockage of 85%, suggesting that the probability of EADs might have little relation with amplitude of ICaL. With the enhanced CaMK0 value to ICaL, the amplitudes of ICaL only increased by 4.2% (1.74 μA/μF versus 1.67 μA/μF).Similarly, as shown in IKr was blocked by 85%. As shown in 0 = 0.12) to different targets, ICaK increased only by 6.5% (0.66 μA/μF versus 0.62 μA/μF), ICaNa by 8.1% (0.40 μA/μF versus 0.37 μA/μF), and Ito by 3.2% (0.98 μA/μF versus 0.95 μA/μF). No EADs occurred under all these conditions.As above, CL was set to 2000 ms and IKr blockage level are shown in 0 = 0.05). In IKr blocked by 85%, stable EADs occurred with the application of ISO, indicating that β-adrenergic agonist facilitates EADs. The IKr blockage level decreased gradually from 85%, and, when it was decreased to 77%, as shown in The action potentials with different INaL and 1 μM ISO, when IKr was blocked by 85%, EADs were induced. The EADs were still observed when IKr was blocked by 77%, suggesting that INaL phosphorylation by CaMKII and ISO application together increased the probability of EADs. These results are listed from row 4 to row 5 in As shown in ICaL and 1 μM ISO, when IKr was blocked by 85%, EADs were induced, but when IKr blockage was reduced to 77%, EADs disappeared. These results are listed from row 6 to row 7 in As shown in This study developed a modified computational model of human ventricular myocardium cell based on the ORd human model with the integration of regulation mechanism by CaMKII and PKA , with whIKr blocked by 85% [IKr blocked by 85%, alternated EADs occurred with the sole effect of CaMKII overexpression to INaL, suggesting that ventricular myocardial cell with CaMKII overexpression is more susceptible to EADs in normal HR range (CL = 2000 ms). Additionally, previous work has shown that ICaL plays an important role in the occurrence of EADs [ICaL amplitude, but this effect alone did not induce EADs, and CaMKII did not alter the overlap region of ICaL steady state activation and reactivation curves. Zaza et al. reported that [+-Ca2+ exchange current (INaCa) may play certain roles in triggering EADs if amplitude is augmented and falls into the overlap region. Our simulation results suggest that CaMKII enhances these currents but does not induce EADs via this effect individually. Therefore, when CaMKII is overexpressed, the susceptibility to EADs is mainly originated from INaL variation.EADs often occur during bradycardia under the condition of reduced repolarization reserve. O'Hara's group successfully elicited EADs with cycle length reduced to 4000 ms and d by 85% . In our  of EADs . Our simted that , when reβ-adrenergic mediated activation of adenylyl cyclase, which activates PKA, a well-described mediator with targets that promote myocardial performance. In the case where PKA took effect independently, EADs occurred with shorter cycle length (2000 ms versus 4000 ms), indicating that the precondition of EADs is relaxed. Our results were different from that from Xie et al.'s study, where the APD shortening after ISO application was observed without EADs in steady state [ICaL recovery and the prevented spontaneous SR Ca2+ release, limiting the INaCa in forward mode. Therefore, when the cells step into steady state with small inward INaCa, the shortening of APD could be reasonable. In our work, SR Ca2+ release was enhanced by ICaL amplification. With the integration with IKr blockage, INaCa was more likely to work as inward current, contributing to the prolongation of APD and occurrence of EADs. Additionally, the shortening of APD was also obtained without IKr blockage when β-adrenergic pathway was activated. Simulation results showed that when cycle length was chosen at 500 ms, 1000 ms, 1500 ms, 2000 ms, and 4000 ms, the corresponding APD90 shortening was 31.6 ms (233.0 versus 201.4 ms), 30.5 ms (268.6 versus 238.1 ms), 22.3 ms (282.6 versus 260.3 ms), 17.3 ms (289.1 versus 271.8 ms), and 18.6 ms (305.6 versus 287.0 ms). These results were consistent with the experiment measurements from Volders et al. [INaL alone with ISO application, EADs occur at CL = 2000 ms and IKr blockage by 77%, suggesting that the combination of PKA and CaMKII overexpression on INaL relaxes the precondition further (IKr blockage 77% versus 85%) and increases the probability of EADs. With the CaMKII overexpressed on ICaL alone, PKA induced EADs occurrence with 85%  IKr blockage, not 77%  IKr blockage, suggesting that, with the ISO application, INaL phosphorylation by CaMKII has more effect on EADs than ICaL. The steady state of CaMKactive was simulated with and without ISO application when CL = 2000 ms, and their corresponding maximum values were 0.0469 and 0.0421, respectively. This suggests that CaMKII activation increased with the application of ISO.Our study also demonstrated the behavior of ventricular myocardial cell with the integration of CaMKII overexpression and ISO application. With the ISO application, cyclic AMP (cAMP) is formed through dy state . Xie et s et al. . In the β-adrenergic receptor signaling and CaMKII in formation of EADs; some potential limitations need to be addressed. Firstly, the role of CaMKII on RyR function has not been considered. There is growing evidence about its role in modulating RyR function. Increasing Ca2+ leak via RyR would limit SR Ca2+ load and disturb Ca2+ cycling. This should be incorporated into an improved model in a future study. Secondly, CaMKII overexpression may downregulate inward rectifier potassium current (IK1) and increase baseline IK1 amplitude [IK1 in our model. Thirdly, our model only describes the short-term effect of CaMKII on different current targets. It has been suggested that short-term and chronic effects of CaMKII are different [Ito inactivation and accelerate recovery, but chronic overexpression may downregulate Ito,fast and upregulate Ito,slow [INaK with dynamic calcium change during the beat cycle, although it has been published by Gao et al. [NaK increased  or decreased in pump current  depending on the intracellular Ca2+ concentrations. In our model the resting calcium concentration was 0.12 μM and the peak calcium concentration was 1.1 μM. Similar to the simulation work of Heijman et al. [INaK in triggering EADs (results not shown here) has been observed. However, when the continuous experimental data from human ventricular cells is available, the regulation of ISO on INaK could be incorporated into the model to reconfirm this nonsignificant effect. Fifthly, it has been reported that local regulation of cAMP and substrate phosphorylation play important roles in β-adrenergic receptor signaling [α, β, γ, δ) with different distributions, kinetics, and roles in physiological and pathological adjustments. Until now, these differences have not been fully understood. Developing a model with detailed CaMKII isoforms information could be useful when experiment data are available.Our simulation results have shown that our proposed model is useful for exploring the interaction of mplitude . We did ifferent –33. Shorto,slow . Therefoo et al.  that ISOn et al. , by simpignaling , 36–38, In conclusion, our simulation results computationally demonstrated a better understanding of the combinational effect of CaMKII and ISO stimulus on the occurrence of EADs in human ventricular myocyte, which may provide useful tool to research therapeutic methods for the treatment of arrhythmia.