Taken together, our findings reveal, for the first time, that CK2 is essential for promoting mitochondrial fusion in a Wnt/β-catenin-dependent manner and suggest that pharmacologically targeting CK2 is a promising therapeutic strategy for ischemic stroke.Mitochondrial fusion/fission dynamics plays a fundamental role in neuroprotection; however, there is still a severe lack of therapeutic targets for this biological process. Here, we found that the naturally derived small molecule echinacoside (ECH) significantly promotes mitochondrial fusion progression. ECH selectively binds to the previously uncharacterized casein kinase 2 (CK2) α′ subunit (CK2α′) as a direct cellular target, and genetic knockdown of CK2α′ abolishes ECH-mediated mitochondrial fusion. Mechanistically, ECH allosterically regulates CK2α′ conformation to recruit basic transcription factor 3 (BTF3) to form a binary protein complex. Then, the CK2α′/BTF3 complex facilitates β-catenin nuclear translocation to activate TCF/LEF transcription factors and stimulate transcription of the mitochondrial fusion gene Mfn2. Strikingly, in a mouse middle cerebral artery occlusion (MCAO) model, ECH administration was found to significantly improve cerebral injuries and behavioral deficits by enhancing Mfn2 expression in wild-type but not CK2α′ Given the essential biological roles of CK2 in signaling pathways related to neurological function, whether CK2 is a potential target for mitochondrial fusion/fission dynamics control in neuronal cells deserves considerable attention.Casein kinase 2 (CK2), a highly evolutionarily conserved serine/threonine kinase, is a stable tetrameric complex consisting of two catalytic (α and α′) subunits and two regulatory (β) subunits.Mfn2, resulting in remarkable neuroprotection against cerebral ischemia.Notably, in this study, we found that the natural small molecule echinacoside (ECH) noticeably promotes the mitochondrial fusion process by directly targeting the CK2 α′ subunit (CK2α′). Mechanistic studies showed that ECH selectively binds to CK2α′ to recruit basic transcription factor 3 (BTF3) as a substrate to form a binary protein complex. Then, the CK2/BTF3 complex promotes Wnt/β-catenin signal activation and subsequent transcription of the mitochondrial fusion-related gene In summary, for the first time, we have built a conceptual framework to explain the fundamental role of CK2 in promoting the mitochondrial fusion process in a Wnt/β-catenin-dependent manner and have shed light on CK2 as a previously undiscovered therapeutic target for ischemic stroke.We screened a natural product library containing 1150 small molecules for their activity against oxygen-glucose deprivation/reperfusion (OGD/R) insult and ultimately found that ECH exerted a significant neuroprotective effect on PC12 cells by increasing cell viability and inhibiting lactate dehydrogenase (LDH) release Fig. . Cell orMfn2 gene encodes a mitochondrial membrane protein that plays a central role in regulating mitochondrial fusion.25 As shown in Fig. Mfn2 gene transcription, increasing its mRNA levels  to capture binding proteins of ECH and then performed silver staining and liquid chromatography (LC)–tandem mass spectrometry (MS/MS) analysis. As shown in Fig. KD) of 1.06 μM , indicating that ECH strongly interacts with CK2α′  technology. As shown in Supplementary Fig. KD values of CK2α (45.80 μM) and CK2β (48.60 μM) with ECH were more than 20 times larger than that of CK2α′ with ECH  assay. As shown in Supplementary Fig. ECH Fig. . Collect27 As shown in Fig. KD values of ECH to CK2α′ (R173A) was 413 nM. However, no binding of ECH to CK2α′ (K171A)   Fig. , indicatFurthermore, to explore whether ECH influences CK2 function, we performed tryptophan fluorescence scanning of CK2α′. As shown in Fig. +/+ cell viability under OGD/R insult, while viability was significantly suppressed in CK2α′−/− cells  Fig. . We captent Fig. . Mito-trent Fig. , indicatent Fig. . Notablyent Fig. , suggest30 thus, we further explored whether CK2α′ selectively interacts with either BTF3 isoform. His-tagged CK2α′ effectively captured both BTF3α and BTF3β; however, ECH promoted only the interaction of BTF3α with CK2α′. This observation was confirmed by pulldown analysis using BTF3α or BTF3β gene-overexpressing cells, indicating that CK2α′–BTF3α binding specifically responded to ECH treatment  sequencing (ChIP-seq) analysis. As shown in Supplementary Fig. oci Fig. , indicatTCF1, TCF3, TCF4, LEF1, HLF, and TFAP2A, which were all highly associated with Wnt/β-catenin signaling  assay. We found that ECH inhibited BTF3α binding to TCF1/7 and LEF1 under OGD/R conditions Fig. , suggest+/− heterozygous C57/BL6 mice to test the neuroprotective effect of ECH. The results showed that MCAO wild-type (CK2α′+/+) mice showed obvious neurological deficits, such as hemianesthesia and hemiparesis, that were effectively relieved by ECH administration. However, we observed that ECH-mediated neurological recovery was significantly attenuated in CK2α′+/− mice . We observed that Mfn2 and COXIV expression was strongly induced by ECH treatment; however, this effect was disrupted in CK2α′ice Fig. , indicatβ-catenin acts as an intracellular signal transducer in transcriptional regulation and chromatin interactions. In addition, β‐catenin in the nucleus is mediated via T‐cell factor (TCF)/lymphoid enhancer-binding factor (LEF) transcription factors, which in association with β-catenin trigger Wnt-mediated transcription. Thus, TCF1/7 were determined by IHC staining as the major Wnt signaling players. Results showed that TCF1/7 significantly translocated into nucleus upon ECH treatment, suggesting that ECH activated Wnt signaling pathway in vivo Fig. . Moreove36 Therefore, it is important to develop feasible strategies to regulate this process. Although accumulating evidence has revealed several proteins critical for the control of mitochondrial dynamics, such as Mfn1/2, Fis1, Mff, and Drp1,36 novel druggable targets that modulate this crucial biological process still urgently need to be identified. Our study reveals, for the first time, that CK2 is a fundamental molecular switch that promotes mitochondrial fusion process to provide neuroprotection against ischemia. Therefore, CK2 is a previously unrevealed therapeutic target for ischemic stroke that acts by controlling mitochondrial homeostasis.Mitochondrial fusion/fission dynamics widely influences neuronal functions, including neuronal survival and plasticity.Notably, we observed that CK2α′ subunit plays a crucial role in mitochondrial fusion by inducing BTF3α cytoplasmic translocation to form a CK2α′/BTF3α complex. We found this complex further promotes mitochondrial fusion by inducing Mfn2 transcription and expression through Wnt/β-catenin-dependent mechanism. Therefore, our findings provide a conceptual framework for pharmacologically targeting CK2 to facilitate mitochondrial fusion via a Wnt/β-catenin-related mechanism. Additionally, to the best of our knowledge, this is the first report of a covalent small-molecule regulator that targets CK2 to exert a neuroprotective effect. Our study thus reveals crucial information supporting future structure-based drug discovery for cerebral ischemia protection.37 In this study, we identified an unexpected pharmacological binding domain containing K171 covalent site, which is far from the previously identified catalytic pocket. The three-dimensional structure of ECH-bound CK2α′ subunit showed a distinctive ligand-binding pocket containing R65, I95, E115, Q169, and G92 residues; this pocket provides a molecular basis for hydrogen bond formation between ECH and CK2α′. Moreover, such hydrogen bond formation may constitute a key precondition for further covalent binding of ECH to K171. Thus, the ligand-binding pocket may involve diverse intermolecular forces and guide novel CK2 small-molecule regulator discovery. In addition, ECH did not inhibit CK2 catalytic activity at classic phosphorylation motif but rather regulated a previously undiscovered phosphorylation site on BTF3. We propose that ECH may exert this effect on CK2α′ via a dynamic allosteric mechanism that enables selectivity for different target motifs. Further studies will be required to understand the precise mechanism by which ECH regulates CK2 with substrate specificity.The currently available small-molecule regulators targeting CK2 family mainly bind to CK2 catalytic pocket.40 In the presence of ligands, Wnt signal activation induces the accumulation of β-catenin through a destructive complex of casein kinase 1 (CK1), APC, Axin, and GSK3β, further stimulating β-catenin nuclear translocation and Wnt target gene transcription.43 CK2 has been previously reported to phosphorylate β-catenin at Thr393,44 which increases β-catenin expression via ubiquitin–proteasome system and activates Wnt signaling pathway.45 Intriguingly, our current study reveals a previously unknown mechanism of CK2-dependent Wnt/β-catenin signal activation, which depends on transcription factor BTF3 as a scaffold protein. For the first time, we report that CK2α′/BTF3α complex promotes β-catenin nuclear translocation and subsequent transcriptional activation of mitochondrial fusion gene Mfn2. We speculate that ECH may enable a CK2α′ conformational change to promote BTF3α-binding domain exposure and recruit BTF3α in the cytoplasm. In addition, we hypothesized that the co-localization may be attributed to ECH-dependent conformational regulation of CK2α′, which further induces BTF3 recruitment to cytoplasm. BTF3 translocation allows easier access of Wnt-related transcription factors to their target loci, such as TCF/LEF1. Thus, CK2α′ may serve as a scaffold or “sponge” for inactivating BTF3 transcriptional functions.Wnt/β-catenin signaling is highly involved in cell survival.46 However, we discovered that BTF3 works as an adaptor by interacting with CK2α′ to form a protein–protein binary complex in the cytoplasm. Thus, our observation indicates that BTF3 plays diverse biological roles and expands our understanding of general transcription factor functions. Importantly, in canonical Wnt/β-catenin pathway, CK1 is involved in regulating β-catenin activation;48 however, the biological function of CK2 in Wnt/β-catenin signaling is largely unexplored. Our findings indicate that CK2 also contributes to β-catenin activation, revealing a previously undiscovered mechanism of Wnt/β-catenin signaling regulation. Moreover, CK2 has been found to be overexpressed in several human tumors such as malignant peripheral nerve sheath tumors, acute myeloid leukemia, and human prostate cancer.49 In particular, recent data has shown that CK2 and β-catenin can be co-precipitated with Wnt signaling intermediate Dvl, suggesting a crucial role of CK2 in Wnt-driven cancer progression.50 Thus, CK2 seems to be a promising therapeutic target in cancers. However, the potential function of CK2-mediated Wnt/β-catenin signal in neuroprotection during cerebral ischemia still remains unclear. In our research, ECH has been discovered to activate Wnt signaling through CK2/BTF3α to exert neuroprotection, which indicates a previously unknown function of CK2/Wnt signal axis in treatment of cerebral ischemia.Interestingly, BTF3 has long been considered to be a general transcription factor for gene transcriptional regulation.Collectively, our findings provide, for the first time, a proof of concept that CK2 is a promising druggable target that promotes mitochondrial fusion via a Wnt/β-catenin-dependent mechanism. In addition, our study demonstrates the feasibility of using the small molecule ECH as a novel molecular template to target CK2 for future clinical therapies for ischemic stroke.35H46O20) was purchased from Shengshi Technology . MTT and DAPI were purchased from Sigma . l-glutathione (reduced) was obtained from Solarbio Science & Technology . Protein A/G-agarose was obtained from Biogot Technology . Lipofectamine RNAiMAX and Lipofectamine 2000 were obtained from Thermo . Pronase was obtained from Roche Diagnostics GmbH . Human recombinant CK2α′ protein was purchased from Sino Biological . An LDH Cytotoxicity Detection Kit, a JC-1 assay kit, Lyso-tracker, Golgi-tracker, and Mito-tracker were purchased from Beyotime Biotechnology . ER-tracker and Super ECL Detection Reagents were purchased from Yeasen . TWS119 was obtained from Selleck Chemicals . IWP-2 was obtained from Bide Medical Technology . Primary antibodies and secondary antibodies were obtained from Cell Signaling Technology  and Abcam . A SuperLight Dual Luciferase Reporter Gene Assay Kit was obtained from Lablead . A StarPure Endo-free Plasmid Maxiprep Spin Kit was purchased from Genstar Biotechnology . SiRNA was obtained from GenePharma . Recombinant plasmids were constructed by Generay Biotech .ECH  and streptomycin (100 μg/ml).Rat pheochromocytoma (PC12) and human embryonic kidney 293T (HEK293T) cells were obtained from the Peking Union Medical College Cell Bank . The cells were cultured in a cell incubator at 37 °C under 5% COCells were cultured with DMEM for 12 h to induce adherence, and then the DMEM was replaced with EBSS. The cells were placed in a sealed incubator  with a Bio-Bag  for 4 h. Then, the EBSS was replaced with DMEM containing different concentrations of ECH , and the cells were cultured under normal culture conditions for 24 h.To test cell viability, cells were first inoculated into 96-well plates. After treatment, the culture medium was removed, and MTT working solution (0.5 mg/ml) was added to the 96-well plates (100 μl/well). Formazan formed after 4 h at 37 °C. Then, the formazan was solubilized in DMSO, and the optical density (OD) was detected at 570 nm by a microplate reader .LDH analysis was performed in accordance with the instructions of an LDH kit (Beyotime Biotechnology). After treatment, the medium from each well was collected and centrifuged at 13,000 rpm for 5 min. Then, 60 μl of LDH detection solution was mixed with 120 μl of supernatant from each well, and the mixture was incubated at 37 °C in the dark for 30 min. The OD was measured at 490 nm by a microplate reader (Tecan).2 incubator for 30 min. The JC-1-treated cells were washed with PBS three times for 2 min each time, and images were obtained with a fluorescence microscope  at 514 nm (excitation) and 590 nm (emission).After treatment, cells were treated with JC-1 working solution (10 μg/ml) and incubated at 37 °C in a 5% CO2 incubator for 40–60 min. Images were obtained by fluorescence microscopy  at 490 nm (excitation) and 516 nm (emission).Cells were cultured in confocal dishes for 24 h. After treatment, Mito-tracker solution (200 nM) was added to the cells, and the cells were incubated at 37 °C in a 5% COCells were collected and placed in 2% glutaraldehyde at 4 °C for 8 h. The fixing solution was removed, 1% osmic acid was added, and the cells were incubated at 4 °C for 2 h. The cells were then dehydrated with gradient acetone solutions, soaked, embedded, polymerized with Epon 812 epoxy resin, and sliced with an ultrathin slicer (0.5 μm) . Then, 0.25% lead citrate and 5% uranium dioxide-acetate were added, and the cells were incubated at 25 °C for 2 min. After PBS washes were performed, TEM  was used for observation. Images were randomly obtained for each group from more than five fields.Cells were collected at 3000 rpm for 3 min after experimental treatment. RIPA lysis buffer with protease inhibitors was used to lyse the cells on ice for 30 min. The cell supernatant was collected for 8–15% SDS–PAGE separation, and the proteins were electrotransferred onto PVDF membranes. Then, the PVDF membranes were incubated with primary antibodies overnight at 4 °C and with HRP-conjugated secondary antibodies for 1 h at room temperature. The membranes were developed with Super ECL Detection Reagent and scanned with a Tanon 5200 Imaging Analysis System .30 Cell supernatant was mixed with vehicle or ECH beads and then incubated at 4 °C overnight. The targeted proteins were captured by the ECH beads. Subsequently, the proteins were denatured by SDS, separated by 10% SDS–PAGE and visualized by silver staining. The targeted proteins were detected by LC–MS/MS analysis using a Nano-HPLC-tandem LTQ-Orbitrap Velos Pro mass spectrometer .31The cellular targets of ECH were determined in a pulldown experiment according to previously published methods, with some modifications.The biomolecular interaction between ECH and CK2α′ was analyzed by SPR using a Biacore T200 system  at 25 °C. First, a CM5 sensor chip was activated by using N-hydroxysulfosuccinimide (sulfo-NHS)/1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). After that, the activated CM5 chip was coupled with recombinant human CK2α′ protein. During coupling, acetic acid  was used to dilute the CK2α′ protein, and blocking was then performed with ethanolamine. ECH was dissolved in PBS+Tween 20 (PBST); the final concentration of ECH was 50 μM at the time of the SPR test. The results were analyzed with Biacore evaluation software (T200 Version 1.0).V = 280 µl) was prefilled with 30 μM CK2α′ protein, and ECH (0.5 mM) was preinjected into the instrument. During titration, an initial injection of ECH (0.4 µl) was followed by 29 successive injections of 1.2 µl with a 150 s interval.The affinities and thermodynamics of CK2α′ protein binding with ECH were determined using a MicroCal PEAQ-ITC . In ITC analysis, 30 μM CK2α′ protein was prepared in PBS (pH 7.4). ECH was dissolved in the same buffer as the CK2α′ protein . The sample well  was added to HEK293T cells in Opti-MEM culture medium. After 6 h, the Opti-MEM was replaced with DMEM. Forty-eight hours later, the plasmid was expressed successfully and could be used for further experiments. The cells were divided into control and ECH groups. The cells in the ECH group were treated with ECH (10 μM) for 6 h. The proteins in the supernatant were crosslinked with 1% formaldehyde for 10 min in PBS, and the reaction was terminated with 125 mM glycine for 5 min. DNA was extracted by using a Pierce Magnetic ChIP Kit. DNA quality was evaluated with an Agilent Bioanalyzer 2100 system . Subsequently, the DNA fragments were sequenced on an Illumina HiSeq platform.Human CK2α′, BTF3α, and BTF3β cDNA sequences were cloned into a pCMV-HA vector containing an HA tag sequence at the N-terminal region. Moreover, human CK2α′, CK2α, and CK2β were cloned into a pGEX-4T-1 vector containing a GST tag sequence at the N-terminal region. All sequences were obtained from NCBI.−/− mice. Lung tissues were isolated, transferred to cell culture dishes and cut to a size of 1 mm3. The medium was high-glucose DMEM with 10% FBS, penicillin (100 U/ml), and streptomycin (100 μg/ml). After the primary LFs were allowed to attach for 72 h, the lung tissues were removed. Cell passage was performed every 3 days. LFs were cultured in a cell incubator at 37 °C and 5% CO2 for further experiments.Primary LFs were isolated from CK2α′2 incubator. Forty-eight hours later, the siRNA was expressed successfully and could be used for further experiments. The siRNAs used are listed in Supplementary Table A mixture of Lipofectamine RNAimax and siRNA  was added to HEK293T cells in Opti-MEM. The medium was changed to DMEM after 4–6 h of culture, and the cells were incubated at 37 °C in a 5% CO2 incubator for 48 h prior to further experiments.The constructed plasmids  were premixed with Lipofectamine 2000 in Opti-MEM (Thermo) and then applied to the cells. The medium was changed to DMEM after 4–6 h. The cells were incubated at 37 °C in a 5% COCells were transiently transfected with the indicated plasmids for 48 h. After experimental treatment, the cells were fixed in 4% paraformaldehyde for 30 min and then permeabilized with 0.5% Triton X-100 (in PBS) for 30 min at room temperature. After washing the permeabilized cells three times with PBS, 5% BSA (in PBST) was added to the cells for 30 min. After that, the cells were incubated with primary antibodies overnight at 4 °C. Subsequently, the cells were incubated with a rabbit Dylight-594-conjugated or mouse Alexa Fluor 488-conjugated secondary antibody for 1 h at room temperature. Finally, the cells were incubated with DAPI (20 μg/ml). Images were obtained  using fluorescence microscopy (Zeiss).Homo sapiens TCF/LEF1 response element with Lipofectamine 2000 for 48 h. An OGD/R model was established and treated with ECH for 24 h. After that, the cells were lysed, and a dual luciferase reporter gene assay  was used to detect TCF/LEF1 reporter gene luciferase activity with a fluorescence spectrophotometer .HEK293T cells were transiently transfected with a mammalian luciferase reporter vector of the +/− mice were generated by disrupting CK2α′ gene expression via homologous recombination in a C57BL/6 background. CK2α′+/− mice were generated using the CRISPR/Cas9 system . The genotyping primers for CK2α′+/− were as follows: forward, 5-AGATGGCCACAATGATTCGC-3′; and reverse, 5′-TCTCACCATCCTCCCAACTC-3′. During the experiment, all mice were kept under a 12 h/12 h light/dark cycle. All mice had access to water and food ad libitum throughout the study period. The procedures for daily animal care and experiments were approved by the Institutional Animal Care and Use Committee of Peking University (License no. LA2017011).CK2α′An MCAO model was established according to the Longa line embolism method with a minor modification. Briefly, the external carotid artery (ECA), common carotid artery (CCA), internal carotid artery (ICA), and pterygopalatine artery (PPA) were separated, and a filament was threaded into the ICA until the blood supply of the middle cerebral artery was blocked. Then, 1.5 h after occlusion, the filament was removed to induce reperfusion. The body temperature was kept at ~37 °C. The sham group was treated similarly to the MCAO group except that no filament was inserted.The Zea-Longa scoring system  was used to estimate the degree of cerebral injury caused by ischemic insult. The neurological score was evaluated after 9 days of ECH (50 mg/kg) administration. The neurological scoring system was as follows: 0, exhibits no obvious neurological deficits; 1, unable to extend the right foreleg normally; 2, walks in circles; 3, exhibits walking imbalance; 4, exhibits numbness and cannot walk.Water was filled with high-purity nitrogen until the dissolved oxygen in the water was below 0.3 mg/L. Anoxic environment was simulated and 5 dpf juvenile fish was added. The juvenile fish sank to the bottom and stood still for 3 min as anoxic end point. Fish were taken out and randomly divided into four groups with 10 fishes in each group. Eight of them were randomly selected from each group, and moved into 48-well plates. The swimming track and data were obtained by zebra fish behavior detection system .P < 0.05 was considered to indicate statistical significance.The results are shown as the means ± SDs. GraphPad Prism 5.0 software was used to carry out one-way analysis of variance (ANOVA) with the least significant difference post hoc test to analyze the data. Supporting information


Given that all in-situ analytical techniques have a non-zero beam size, all measured profiles, resulting from diffusion or otherwise, will be artefactually elongated to some degree. Profiles where the total length over which the concentration changes approaches the resolution of the analytical technique likely suffer from serious convolution; the measured profiles may be considerably elongated relative to the true profile. Resolving this effect is non-trivial, except for some specific combinations of profile type and beam geometry. In this study, a versatile method for numerically deconvoluting diffusion profiles acquired using techniques with Gaussian, Lorentzian, (pseudo-)Voigt, circular/elliptical or square/rectangular interaction volumes, is presented. A MATLAB code, including a user-friendly interface (PACE-the Program for Assessing Convolution Effects in diffusion studies), is also provided, and applied to several experimental and natural profiles interpreted as resulting from diffusion, showing various degrees of convolution. Diffusion modelling is now commonplace in the earth sciences, given its ability to determine timescales of geological processes regardless of their absolute age –5. CurreThe error function, which is fundamental to several analytical solutions of Fick’s second law, has a geometry that corresponds to the cumulative distribution function of the normal distribution. This means that analysing a simple step function, using an analytical method where the interaction volume can be described as Gaussian, will yield an error function. Likewise, analysing an error function-shaped profile with any Gaussian beam  will lead to some smoothing-out/lengthening of the error function, whilst maintaining its form. The effect of beam convolution, in the simple case a Gaussian beam, has been derived both by Ganguly et al. and Arnould and Hild , 12 as:g shapes .a priori, or can be estimated and 3) where an assumption can be made regarding the true nature, i.e. the geometry, of the profile. A second program- PACE-IC  is also provided, this is based on PACE, but allows the user to input initial and boundary conditions. In addition, a third program—PACE-GD (Get Dimensions) is provided for estimating beam sizes from profiles made across known step functions, with any of the interaction types listed above.Whilst this convolution effect has been recognised for several decades, it is still often omitted from diffusion studies. Therefore, to enable the fast, reproducible assessment of convolution effects, PACE (the Program for Assessing Convolution Effects in diffusion studies) is presented . PACE isThe software does not assume knowledge of programming or diffusion past simple recognition of profile types. The use of PACE is demonstrated on several diffusion profiles with different beam geometries, showing both cases where the convolution effects are negligible, and where they may become important.The methodology used in PACE only employs analytical solutions to the diffusion equation—it does not incorporate any numerical diffusion modelling. Therefore, PACE can only be used in cases where the boundary conditions are fixed, and the initial conditions are describable using step functions. Numerical modelling is incorporated into PACE-IC (described below). This precludes the use of PACE in certain situations including 1) changing temperature, leading to partition/distribution coefficients changing over time and 2) loss of the original core composition following a prolonged period of diffusion.2s-1).For simulating sigmoidal shaped profiles that include flat plateaus on the left and right, 1 and C2 represent boundary and core concentrations, and X is the length of the system, i.e. the position of the right-hand boundary, where the left-hand boundary is at x = 0 by default.For U and upside-down-U shaped profiles, 10Dt, along with the respective concentrations. From this, D or t can be readily extracted when the other is known, or, because Dt = ∫D(t)dt ) and Lorentzian (Γ[L]) lines:Likewise, where the beam is Gaussian, Lorentzian or Voigt, the size can be estimated or directly measured. For a Gaussian beam, a measured profile over a step function is fitted to an equation with the form:Γ)[L](9). FWHM (Γ85 and Fo90 (i.e. Mg/(Mg+Fe) = 0.85 and 0.90, respectively) was simulated using CASINO v2 at a series of accelerating voltages, then the resulting profiles fitted using PACE-GD, both assuming a Gaussian beam and assuming a pseudo-Voigt shape. According to the simulation, the assumption of a Gaussian interaction is valid up to ~15 kV, after which the interaction develops wider tails and becomes more Lorentzian in shape, reflected in the relative FWHMs of the Lorentzian and Gaussian components of the pseudo-Voigt interaction volumes . For ex volumes . The advAside from convolution effects associated with interaction volumes, there are also effects associated with secondary fluorescence in EPMA. This is associated with measuring low concentrations of elements in phases adjacent to a phase with a much higher concentration of the same element , 26, sucThese tools have applicability both where experimental diffusion profiles are measured  and for natural diffusion profiles . Some example applications are presented below.Ti diffusion in quartz has had wide ranging applications, including determining timescales of 1) magma chamber processes , 28–30; 10Dt (m2) = -13.41(±0.01) (deconvoluted), with the original fit giving log10D*t (m2) = -13.31(±0.01). As t = 89.5 h, log10t (s) = 5.51 so log10D (m2s-1) = -18.92. Interestingly, this diffusion coefficient is lower than extrapolations of both previous experimental determinations of Ti diffusion in quartz [n quartz , 36. ThiVolcanic quartz crystals often preserve zoning in Ti content, interpreted to be formed by changes in titania activity or temperature, or be the consequence of disequilibrium during growth –41. In a10D*t (m2) = -10.86  = -17.65 ± 0.03. Following deconvolution (10D (m2s-1) = -17.76 ± 0.04, resolvably different from the convoluted data. The advantage with this method is that, because the length scale of a diffusion profile is broadly proportional to the square root of time, if such short profiles can be deconvoluted, then this opens up the possibility for running shorter experiments and/or experiments at relatively low temperatures.Slit-shaped beams are now routinely used in laser ablation inductively-coupled plasma mass spectrometry (LA-ICP-MS) to measure concentration profiles in minerals and glasses –47. In tvolution , log10D An accurate determination of the diffusivity of Pb in zircon is essential for determining the closure temperature associated with U-Pb dating. Cherniak and Watson  presente10Dt units in this case, far smaller than the uncertainty from curve fitting. That this effect is so small, even when using a 25 kV beam, is due to a combination of the high stopping power of zircon and the measurements having been done on a non-oblique section.Deconvoluting the measured profile  shows thdiffusion profile versus no measurable diffusion profile distinction to be made. Such tools will become more important as diffusion modelling is used to determine increasingly short timescales.The PACE software packages allow rapid, simple determinations of analytical convolution artefacts associated with measuring diffusion profiles, either experimental or natural. Whilst the majority of experimental and natural profiles are comfortably much longer than the beam size used to measure them, profiles approaching analytical spatial resolution are becoming more common in the literature. PACE simplifies the extraction of quantitative data from such profiles, and also enables a rapid S1 File(ZIP)Click here for additional data file.S2 File(ZIP)Click here for additional data file.S3 File(PDF)Click here for additional data file.S4 File(ZIP)Click here for additional data file.


The activation of hypoxia-inducible factor (HIF) is an important event for mediating the adaptive response to myocardial ischemia/reperfusion (MI/R) injury. The ubiquitin-conjugating enzyme E2S (Ube2s) catalyzes ubiquitin conjugation to target proteins. Here, we report the positive regulation of HIF-1α signaling by Ube2s via stabilizing β-catenin, by which Ube2s acts to protect against MI/R injury. We show that Ube2s expression is upregulated in the hearts of mice subjected to MI/R injury. Functionally, Ube2s depletion exacerbates and its overexpression ameliorates MI/R injury. In addition, Ube2s augments the activation of HIF-1α and reduces myocardial apoptosis. Moreover, Ube2s induces the accumulation of β-Catenin through increasing its stabilization. Importantly, β-Catenin knockdown abrogates Ube2s-augmented HIF-1α activation, and meanwhile, diminishes the protective effect of Ube2s on MI/R injury, thus establishing a causal link between Ube2s-stabilized β-catenin and HIF-1α-mediated myocardial protection. Altogether, this study identifies the Ube2s/β-catenin/HIF-1α axis as a novel protective regulator involved in MI/R injury, and also implies that it might represent a potential therapeutic target for ameliorating MI/R injury. Myocardial infarction (MI) is a common cause of death and disability across the world, for which the most effective therapeutic intervention is timely myocardial reperfusion . HoweverOn the other hand, accumulating evidence has suggested that myocardial cell survival during MI/R injury could be improved by modulating the expression of genes participating in promoting glycolysis, reducing ROS production and proapoptotic protein expression and limiting mitochondrial metabolism . The hypThe ubiquitin-conjugating enzyme E2S (Ube2s) is a K11 linkage-specific E2, which catalyzes the elongation of K11-linked polyubiquitin chain on substrates to promote proteasome-mediated degradation . HoweverAlthough the well-established role of Ube2s is cell cycle regulation , 17, 18,The upregulation of Ube2s expression after MI/R injury implies that it may play a functional role in this pathological condition. To test this possibility, we performed Ube2s expression knockdown in the mouse heart using the technique of siRNA transfection in vivo . WesternThe effects of Ube2s expression knockdown shown above conversely imply that Ube2s per se may play a protective role against MI/R injury. To verify this, we overexpressed Ube2s via lentivirus infection in the heart after MI/R injury . The oveAmong the protective mechanisms against MI/R injury, the adaptive response in the heart initiated by the activation of transcriptional complex hypoxia-inducible factor 1α (HIF-1α) has emerged as a key cardioprotective factor for mediating the survival of cardiomyocytes , 11, 23.We asked whether HIF-1α activation mediates Ube2s function in MI/R injury. As shown, HIF-1α knockdown via siRNA transfection significantly reversed the Ube2s-induced reduction of both Bax/Bcl-2 –4B and cUbe2s overexpression was found to increase HIF-1α stability , 27. HowOf note, the stabilization of β-Catenin by Ube2s seems irrelevant to GSK-3β, an important upstream regulator , since wLastly, we asked whether Ube2s-stabilized β-Catenin contributes to augmented HIF-1α activation and protective role of Ube2s against MI/R injury. For this purpose, we depleted β-Catenin in the heart using siRNA transfection. Western blotting analysis showed that Ube2s overexpression in the heart consistently resulted in enhanced expression of HIF-1α and β-Catenin after MI/R injury compared with vector control –6B. HoweIn addition to the upstream activated Wnt signaling that leads to the accumulation and nuclear translocation of β-Catenin, whereby it further activates the transcription complex T-cell factor/lymphoid enhancer factor (TCF/LEF) , severalGiven the above clues, our study was designed to investigate whether Ube2s, a K11 linkage-specific Ub-conjugating enzyme previously shown to regulate β-Catenin stabilization in embryonic stem (ES) cells , has a fUbe2s expression is induced following MI/R injury at both transcript level and protein level, suggesting that the condition of MI/R injury may activate Ube2s transcription. Of note, the upregulated expression of Ube2s peaks at 24 h and then declines thereafter following MI/R injury, implying that there may exist a feed-back mechanism that controls Ube2s induction under this condition. Further studies are needed to discover how Ube2s expression is regulated at an extended period of time following MI/R injury. On the other hand, we speculate that the induced expression of Ube2s may be derived from the stimulation of hypoxia during the procedure of ischemia/reperfusion. This is very possible, because Ube2s gene promoter includes hypoxia-response elements, which have been demonstrated to be induced by hypoxia through HIF1α . If thisIt’s well-established that the stabilization of β-Catenin by Ube2s is through modification of K11-linked polyubiquitination . ConsistAll animal experiments were performed according to the protocols approved by The Second Affiliated Hospital of Air Force Medical University. Eight- to 10-week-old wild-type C57BL/6J male mice were purchased from the Jackson Laboratory and maintained in pathogen-free facilities and utilized for developing the MI/R model as reported in previous studies , 35. EigFor extracting the total protein, the whole mouse heart tissues were minced and then lysed completely using the RIPA Lysis and Extraction Buffer  supplemented with the protease inhibitor cocktail . The supernatants of whole lysates were collected, and the protein concentration was quantified via the BCA Protein Assay Kit . Protein samples were denatured for 5 min in SDS loading buffer at 100 °C. The Western blotting was conducted as described previously . BrieflyThe total mRNA in the whole mouse heart tissues were isolated using the TRIzol reagent , and the synthesis of cDNA was performed using the First Strand cDNA Synthesis Kit  according to the manufacturer's instructions. The quantitative reverse transcription PCR (qRT-PCR) was conducted with cDNA template, QuantiTect SYBR Green PCR Kit  and Real-Time PCR Detection System . β-Actin was used as a reference control. The sequence of primers is accessible upon request.The knockdown of Ube2s and β-Catenin in mouse heart was performed using siRNA transfection in vivo. The specific siRNA targeting mouse Ube2s (siUbe2s) or β-Catenin (siβ-Catenin) (Invitrogen) or non-specific scrambled siRNA (siCtrl) (Invitrogen) were mixed with the in vivo-jetPEI reagent (Polyplus Transfection) and then delivered through intra-myocardial injections using the 32.5-gauge needle 48 h prior to MI/R surgery as performed previously . The seqFollowing 24 h of reperfusion, 1% Evans blue (Sigma-Aldrich) was injected through the jugular vein into the aorta. The heart was then removed, washed with PBS and horizontally sectioned into 5-6 slices, which were stained for 15 min with 1 ml 1% 2, 3, 5-triphenyl tetrazolium chloride (TTC) (Sigma-Aldrich) at 37 °C. The slices were photographed with a photomicroscope. The size of left ventricular (LV), area at risk (AAR), and infarct area (IA) was quantified using ImageJ software.The myocyte injury was evaluated by the release level of serum creatine phosphokinase (CPK) as described before . The bloThe apoptosis was measured by TUNEL positive cells stained using the In Situ Cell Death Detection Kit  following the manufacturer’s instructions. In brief, the hearts of sham or MI/R mice were perfused with ice-cold PBS and fixed with 4% paraformaldehyde (PFA). The hearts were embedded in paraffin and cut into 5 μm thick sections. Each section was stained with TUNEL reaction reagents in the dark and finally counterstained with DAPI. The images were captured under the A1 laser scanning confocal microscopy (Nikon). TUNEL positive cells were counted at 5 random fields of 3 different sections. The results are expressed as the percentage of TUNEL positive cells among total cells in the field, which were stained blue with DAPI staining.The apoptosis was reflected by the caspase-3 activity measured using the Ac-DEVD-AFC Caspase-3 Fluorogenic Substrate  according to the manufacturer’s instructions. Briefly, heart tissues from sham or MI/R mice were lysed on ice. After centrifugation, the supernatants were collected and the protein concentration was quantified using BCA method. A total of 50 μg proteins were pipetted and mixed with assay buffer supplemented with 10 mM dithiothreitol (DTT). The fluorescence emission of the AFC (400 nm) was measured via the Spectra Max-Plus Microplate Spectrophotometer (Molecular Devices). The caspase-3 activity is expressed as nmol AFC/h/mg protein.2. For Ube2s overexpression, H9c2 cells were transfected with plasmids using the Lipofectamine 2000 (Invitrogen) based on the manufacturer's protocol. After 2 days, cycloheximide (CHX) chasing assay was then performed with H9c2 cells treated with or without 50 μM CHX for increasing time periods.The cardiomyocyte cell line H9c2 was purchased from the American Type Culture Collection . H9c2 cells were cultured in complete Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) and 50 U/mL penicillin/streptomycin (Invitrogen) at 37 °C in a humidified atmosphere with 5% COt-test. The data among more than two groups were compared by one-way analysis of variance (ANOVA). The values with P < 0.05 were defined as statistically significant.Data are expressed as the mean ± SD. The data between two groups were analyzed by unpaired Student’s Supplementary Figures


Abelmoschus esculentus (L.) Moench, a delicious vegetable known as okra, has been used worldwide as a functional food and folk medicine for many years [6 to 5.94 × 106 Da [ny years . It is nny years ,3. Previny years , antioxiny years , anti-hyny years . Generalny years . Okra po× 106 Da ,8. The rGenerally, extraction methods have noticeable influences on the physicochemical characteristics and bioactivities of OPPs . UltrasoTherefore, in order to well understand the detail chemical structures and chain conformation of OPPs and improve the applications of OPPs in the functional food and pharmaceutical industries, the purification, structural characteristics, chain conformation, and bioactivities of OPPs extracted by UAE were investigated.The extraction yield of OPPs extracted by UAE was 13.99% ± 0.52%, which was comparable to the extraction yields of OPPs extracted by microwave-assisted extraction (MAE), hot water extraction (HWE), and pressurized water extraction (PWE) previously reported . The extMoreover, the chemical compositions of the purified OPP (OPP-D) were determined. The content of total polysaccharides in OPP-D was determined to be 92.85% ± 2.27%, and the content of proteins was 0.33% ± 0.07%, which suggested that polysaccharides were the main biological components in OPP-D. Moreover, the content of uronic acids in OPP-D was determined as 33.35% ± 2.38%. In general, such high content of uronic acids in natural polysaccharides might lead to strong antioxidant activities, digestive enzyme inhibitions, and binding properties ,16. HoweMonosaccharides are the natural basic units that determine the unique structures and properties of polysaccharides . The mon−1 corresponds to the hydroxyl group stretching vibration [−1 is attributed to the stretching vibration of C-H asymmetric [−1 and 1628.48 cm−1 are owing to esterified groups of the C=O and COO- stretching vibration, which indicates the presence of uronic acids [−1 and 1147.97 cm−1 are owing to the C-H or O-H and C-O-C bending vibration, which indicates the existence of -OCH3 [In order to determine the structural features of OPP-D, the FT–IR spectra were measured. As shown in ibration . The absymmetric . Then, tic acids . Resultsof -OCH3 , while tof -OCH3 ,21. Furtof -OCH3 .1H and 13C analyses, were shown in 1H signal at 4.80 ppm belongs to D2O. In combination with the constituent monosaccharides of OPP-D, the abroad peaks at 1.26 and 1.35 ppm are attributed to the H-6 of 1,2-α-l-Rha and 1,2,4-α-l-Rha, respectively [l-Rha [β-d-Gal and 1,4-β-d-Gal [l-GalA [NMR spectra were recorded in order to reveal the precise structural information of OPP-D. 1D NMR spectra, including ectively . The sigy [l-Rha . The sigy [l-Rha . The exi-β-d-Gal . The abr [l-GalA .13C NMR spectra -α-l-Rhap-, the radius of gyration (gR), and chain conformation of OPP-D were investigated. 5 (±0.40%) Da, and the gR of OPP-D was measured at 27.0% ± 3.9% nm. The molecular weight of OPP-D was significantly lower than those of polysaccharides extracted by HWE, PWE, and MAE [Usually, the bioactivities of polysaccharides are connected with their molecular weight and chain conformation . Thus, t and MAE , which m and MAE ,17. In agR) is a constant which can characterize the molecular size of the polymer, and gR can be calculated by the equation gR = kwMv [gR) to log (wM), and v = 0.33 means spherical molecules, ν = 0.5–0.6 means a random string, while ν = 1 means a rigid rod [The radius of gyration , and  KT/6πηr . As show = Rg/Rh . For a s = Rg/Rh .−1, while nearly Newtonian flow behavior at 50–100 s−1. For such shear-thinning behavior may mainly be associated with the untangling of the molecular chains in solution [It is well believed that the apparent viscosity of polysaccharide is closely related to molecular characteristics . The appsolution ,29. Moresolution . All the50 values of ABTS and NO radical scavenging activities of OPP-D were evaluated as 2.47 mg/mL and 0.98 mg/mL, respectively. Additionally, compared to the positive control (vitamin C), OPP-D demonstrated good ABTS and NO radical scavenging activities. Moreover, the antioxidant activities of OPP-D were similar to OPPs extracted by MAE, but relatively higher than those of polysaccharides extracted by HWE and PWE from our previous study [wM, uronic acids, and constituent monosaccharides [Previously reported studies have already reported that polysaccharides from okra possess remarkable antioxidant activities ,11. Therus study . The antcharides ,32. Thus50 values of in vitro α-amylase and α-glucosidase inhibitions of OPP-D were determined as 223.77 μg/mL and 215.70 μg/mL, respectively. Compared with acarbose (IC50 = 2020.41 μg/mL), the in vitro α-glucosidase inhibition of OPP-D was significantly stronger. Furthermore, the in vitro digestive enzyme inhibitions of OPP-D were slightly lower than OPPs extracted by HWE, PWE, and MAE [Annona squamosa [wM, high DE, and high content of uronic acids [The major strategies for counteracting metabolic changes associated with hyperglycemia and type 2 diabetes are the inhibitions of α-amylase and α-glucosidase , and our and MAE ,11. The squamosa  and bittsquamosa , which mic acids ,20. Moreic acids . Resultsβ-glucans from Qingke [wM, and molecular weight distributions [Excessive absorption of bile acids, cholesterol, and fat may lead to obesity problems, which are related to diabetes cancer and cardiovascular disease , and ourm Qingke , and higm Qingke . The in ibutions ,20. Moreibutions . ResultsLactobacilli strains, including L. acidophilus CICC 6089, L. rhamnosus CICC 6133, and L. rhamnosus CICC 6151 were investigated to determine whether OPP-D was a potential substrate to be metabolized by colon microbiota. As shown in Lactobacilli strains treated with OPP-D and the total SCFAs were significantly increased, respectively. These meant that OPP-D was not toxic to the assayed probiotics, whereas it was a good substrate for facilitating probiotic growth, and OPP-D could be utilized by probiotic bacteria to sustain survival and metabolic activities. Polysaccharides comprised of galactose may have typically displayed greater prebiotic activity [600 were observed which ranged from 0.28 to 0.35, from 0.33 to 0.40, and from 0.32 to 0.40, respectively. Furthermore, similar results could be found in the production of SCFAs, and the total SCFAs ranged from 16.17 to 31.32 mM, from 10.28 to 20.81 mM, and from 54.38 to 90.98 mM, respectively. Results showed that all tested Lactobacilli strains exhibited a significantly dose-dependent manner, where a higher content of polysaccharides could have a better proliferative effect [wM, better water solubility, and lower viscosity may exert better prebiotic effect [Previous studies have reported that polysaccharides exert their health benefits called prebiotic activities in the human intestine, regulating the production of short-chain fatty acids (SCFAs) . Short-cactivity ,39, and activity ,40. Addie effect . Moreovec effect . All theβ-d-glucopyranoside (pNPG) were all purchased from Sigma-Aldrich . A free cholesterol assay kit and DEAE Cellulose-52 were purchased from Solarbio . Lactobacillus acidophilus CICC 6089, Lactobacillus rhamnosus CICC 6133, and Lactobacillus rhamnosus CICC 6151 were purchased from China Center of Industrial Culture Collection. All other chemicals and reagents used were of analytical grade.Okra fruits were harvested at a commercial orchard in Chengdu, Sichuan, China. The samples were washed, hot-air-dried (75 °C and 12 h), smashed, screened, and stored at −20 °C. Acarbose, α-amylase (1000 U/mg), α-glucosidase (10 U/mg), arabinose (Ara), galactose , galacturonic acid , glucose (Glc), glucuronic acid (GlcA), mannose (Man), rhamnose (Rha), xylose (Xyl), 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulphonic acid) (ABTS), 1-phenyl-3-methyl-5-pyrazolone (PMP), and 4-nitrophenyl v/v) ethanol overnight at 4 °C. Subsequently, the precipitations were redissolved, dialyzed for 3 days , freeze-dried, and stored at −20 °C.Extraction and preparation of OPPs were performed by the formerly reported method with minor modifications ,19. In bOPPs were purified by using a DEAE Cellulose-52 column (5 × 50 cm). Briefly, OPPs were dissolved in 40.0 mL of deionized water at the concentration of 25.0 mg/mL and filtered through a 0.45 µm syringe filter . The solution was then loaded onto a DEAE Cellulose-52 column and successively eluted by the deionized water and the 0.1 mol/L of NaCl solution at a flow rate of 1.0 mL/min. Each tube (5 mL/tube) was checked at 490 nm by the phenol-sulfuric acid method. Finally, one purified polysaccharide fraction named OPP-D was obtained (about 0.62 g), and the purity of OPP-D was checked by high-performance size-exclusion chromatography.m-hydroxydiphenyl assay, respectively [The contents of total polysaccharides, total proteins, and total uronic acids of OPP-D were evaluated by the phenol-sulfuric acid assay, Bradford’s method, and the ectively .Constituent monosaccharides of OPP-D were investigated by HPLC analysis  based on the previous method . Ara  spectroscopy analysis of OPP-D was also investigated by the previous method . The Nic methods ,19.2O overnight for NMR analysis. 1D NMR spectra, including 1H and 13C analyses, were recorded on a Bruker Ascend 600 MHz spectrometer  with a z-gradient probe with proton and carbon frequencies of 600.13 and 150.90 MHz, respectively.The OPP-D sample (20 mg) was dissolved in 0.5 mL of DwM), polydispersity (wM/nM), and radius of gyration (gR) of OPP-D were determined by high-performance size-exclusion chromatography coupled with multi-angle laser light scattering and refractive index detector  on the basis of the previous method [The purity, absolute molecular weight (s method .hR) of OPP-D was measured by using a dynamic light scattering  according to a previous study with some modifications [The hydrodynamic radius (ications . The samw/v) of OPP-D were selected and dissolved in distilled water, respectively. The apparent viscosity was determined by a Discovery Hybrid Rheometer-1  equipped with a 40 mm diameter parallel steel plate with a 1.0 mm gap. Flow curves of OPP-D were determined at 25 °C with the shear rate range from 0.01 to 100 s−1.The apparent viscosity of OPP-D was measured by the previous method . In brie50 values (mg/mL) of OPP-D could be determined.Both ABTS and nitric oxide (NO) radical scavenging activities of OPP-D were evaluated by the formerly reported methods . In brie50 values (mg/mL) of OPP-D could be calculated.The inhibitory activities of OPP-D against α-amylase and α-glucosidase were evaluated by the previously reported methods . The acaThe in vitro binding properties of OPP-D, including fat, cholesterol, and bile acid-binding capacities, were evaluated by the previously reported methods ,11. In bL. acidophilus CICC 6089, L. rhamnosus CICC 6133, and L. rhamnosus CICC 6151, were selected to determine whether OPP-D was the potential substrate for promotion of the growth of Lactobacillus strains. Inulin and the basal MRS were used as the positive and blank controls, respectively. The samples were prepared as concentrations of 1.0, 2.0, and 3.0% (w/v), filter-sterilized, and then added into the MRS broth. Each Lactobacillus strain was transferred into MRS broth medium at a concentration of 1 × 107 CFU/mL. Afterwards, L. acidophilus CICC 6089 and L. rhamnosus CICC 6151 were incubated at 37 °C for 24 h, and L. rhamnosus CICC 6133 was incubated at 30 °C for 24 h, respectively. Finally, the optical density values of samples were measured at 600 nm.The in vitro prebiotic activity of OPP-D was evaluated by a previous study with minor modifications . Carbohyg for 10 min. Then, 0.4 mL of supernatants were mixed with 0.4 mL of internal standard (0.05 M of 2-ethylbutyric acid), and filter through a 0.22 μm membrane filter. The mixed solution was analyzed by Agilent 7890 series GC system  with an HP-INNOWAX column .SCFAs were determined by gas chromatography according to a previous study with minor modifications . In briep < 0.05 as significant to Duncan’s multiple range test.All experiments were conducted in triplicate, and data were expressed in means ± standard deviations. Statistical significances were carried out by one-way analysis of variance (ANOVA), taking a level of wM, DE, content of uronic acids, and highly branched globular conformation. Results suggested that OPP-D had great potential applications to be further explored in the food and medical industries. Based on the favorable effects of OPP-D on prebiotic activities, further studies about the effects of OPP-D on intestinal microorganisms through the in vitro fermentation model and in vivo mice model could be considered.In this study, the chemical structure, chain conformation, and biological activities of a purified polysaccharide (OPP-D) were investigated. Results showed that OPP-D was a purified pectic-polysaccharide mainly composed of Rha, GalA, and Gal, and the proposed structure of OPP-D was Rha and GalA units as the backbone with galactan side chains. The chain conformation of OPP-D in aqueous solution was determined as an air-core sphere with branching chains for the first time. In addition, OPP-D exhibited remarkable in vitro antioxidant activities, anti-diabetic activity, and binding capacities, as well as prebiotic effect, which might be associated with its


In vitro cytotoxicity study showed dose-dependent activity of MNS-APTES-NTD for human lung cancer cells L-132. About 75% reduction in cellular viability for a 100 μg/mL concentration of nanoformulation was observed. The nanoformulation designed using MNS and monolayer coverage of APTES has potential in cancer therapy as well as in other nanobiological applications.The use of an appropriate delivery system capable of protecting, translocating, and selectively releasing therapeutic moieties to desired sites can promote the efficacy of an active compound. In this work, we have developed a nanoformulation which preserves its magnetization to load a model anticancerous drug and to explore the controlled release of the drug in a cancerous environment. For the preparation of the nanoformulation, self-assembled magnetic nanospheres (MNS) made of superparamagnetic iron oxide nanoparticles were grafted with a monolayer of (3-aminopropyl)triethoxysilane (APTES). A direct functionalization strategy was used to avoid the loss of the MNS magnetization. The successful preparation of the nanoformulation was validated by structural, microstructural, and magnetic investigations. X-ray photoelectron spectroscopy (XPS) and Fourier transform infrared spectroscopy (FTIR) were used to establish the presence of APTES on the MNS surface. The amine content quantified by a ninhydrin assay revealed the monolayer coverage of APTES over MNS. The monolayer coverage of APTES reduced only negligibly the saturation magnetization from 77 emu/g (for MNS) to 74 emu/g (for MNS-APTES). Detailed investigations of the thermoremanent magnetization were carried out to assess the superparamagnetism in the MNS. To make the nanoformulation pH-responsive, the anticancerous drug Nintedanib (NTD) was conjugated with MNS-APTES through the acid liable imine bond. At pH 5.5, which mimics a cancerous environment, a controlled release of 85% in 48 h was observed. On the other hand, prolonged release of NTD was found at physiological conditions ( Even though chemotherapy is the most common treatment in practice, the imprecision in chemotherapeutic drug delivery at the tumour site results in adverse side effects due to the internalization of drugs by healthy cells along with tumour cells6. Researchers around the globe have addressed this issue and have offered different strategies to accomplish site-specific drug release9. A targeted drug delivery (TDD) system is currently considered to be the best alternative for systemic therapy. TDD can significantly improve the potency of the therapeutic drug11. Recently numerous drug carriers have emerged with their potential applicability as nanovectors in cancer treatment14. These drug carriers are comprised of nanoscale and supramolecular drug delivery systems which includes purely organic free radicals, nano-antioxidants, hydrophilic-hydrophobic polymeric nanoparticles (NPs), micelles, liposomes and dendrimers18. Besides some inorganic multifunctional nanoformulations such as magnetic-layered double hydroxide, multicore-shell nanostructure, or disk-shaped magnetic NPs (MNPs), magnetic nano-cubes with a magneto-mechanical approach have also been explored20. Among these carriers, different magnetic nanostructures receive growing attention as a promising theranostic tool, as their unique superparamagnetic nature allows the translocation of anticancer drugs at a chosen site through an applied magnetic field while colloidal stability of carriers in the body fluid is maintained22. However, to use these magnetic nanostructures in a TDD system, they must hold several properties such as biocompatibility, high surface area, high colloidal stability, superparamagnetism, and high saturation magnetization (Ms)23. In this work, we used self-assembled magnetic nanospheres (MNS) made of iron oxide NPs as drug carriers, which exhibit superparamagnetic nature with a decent magnetic response compared to other magnetic nano-formulations and to other magnetic nanostructures24.The contemporary treatments for cancer are usually based on invasive surgery, chemotherapy, radiotherapy, immunotherapy, or a combination of these methods25, fatty acids25, polyacrylic acid26, polyvinylpyrrolidone27, polyethylene glycol28, polyvinyl alcohol29 were used for the surface modification. However, most of them are high molecular weight biomolecules (polymers), and all these multilayered coatings cause a decrease in magnetization Ms after the functionalization process30. Landarani-Isfahani et al.31 reported a decreasing magnetization of MNPs with an increase in the thickness of the polymer coating. Reduced magnetization deteriorates the MNP functionality as a drug carrier due to the lower response to an external magnetic force. On the other hand, MNPs with higher magnetization can aid effective delivery. Huang et al.32 demonstrated the delivery of theranostic agents across the blood–brain barrier in rats using MNPs under an external magnetic field. They observed a significant accumulation of MNPs in the cortex near the magnet and higher magnetization of functionalized MNPs could facilitate the accumulation of MNPs at a target site.For TDD applications, the surface modification of MNPs is essential to load the drug on the MNP surface, restrict the interaction of MNPs with the host cells, and diminish the interparticle interaction. Several surface modification strategies have been employed for the functionalization of MNP surfaces. Different organic and inorganic coatings such as chitosan36. 3-aminopropyl triethoxysilane (APTES) is an amino-silane linker frequently used in surface modification of MNPs40. Several studies reported the use of nonmagnetic SiO2 coating over the MNP surface for APTES surface modification, which in turn reduces the magnetization42. In this regard, it is necessary to adapt the optimal functionalization strategy that does not affect the MNP magnetic properties. In this work, we demonstrate the direct functionalization (without SiO2 shell) and monolayer coverage of APTES as an effective approach to retain the magnetic properties of MNS. The formation of a monolayer of amino silane eases the linking of a specific drug through the tale side amino groups (–NH2). These amino groups form the imine bond (C=N) between the carbonyl group of drug and amine group of MNS-APTES and assist the drug conjugation process.Silane derivatives-based functionalization strategies possess several advantages over other linkers. They improve the structural stability of MNPs by hindering oxidative damage through inert silane linkage coating, besides modify hydrophilicity or hydrophobicity for drug conjugation with functional groups like amine, sulfonyl, and carboxylic moieties44. Inhibition of the signalling pathway at the kinase domain may result in the induction of endothelial cell apoptosis, reduction in tumor vasculature, tumor cell migration, and proliferation. NTD, as an anticancer agent in the treatment of non-small-cell lung carcinoma (NSCLC), was proved in clinical studies45. Although Nintedanib was developed and already launched as Ofev in an oral capsule formulation for the treatment of idiopathic pulmonary fibrosis (IPF), metastatic NSCLC, ovarian, prostate and colorectal cancer, the drug is also under the process of clinical trial studies in various other cancer types46. Since the drug has been minimally explored for the TDD application, the present work provides insights into TDD systems containing the poorly water-soluble drug NTD. We present the nanoformulation for TDD application prepared by the surface modification of MNS through monolayer coverage of APTES and subsequent loading of the anticancer drug NTD (see Scheme Nintedanib (NTD) is a receptor tyrosine kinase (RTK) inhibitor with antiangiogenic and antineoplastic potential which can target three angiogenesis related transmembrane receptors, namely the vascular endothelial growth factor receptor (VEGFR), the fibroblast growth factor receptor (FGFR), and the platelet-derived growth factor receptor (PDGFR) tyrosine kinases3⋅6H2O), ethylene glycol (CH2(OH)⋅CH2OH), and anhydrous sodium acetate (CH3COONa) were procured from S D Fine-Chem India. APTES was purchased from Sigma-Aldrich. Spectroscopic grade organic solvents such as ethanol, dimethyl sulfoxide (DMSO), toluene, and methanol were purchased from Thomas Baker  Pvt. Ltd. Thermo Fisher Scientific India Pvt. Ltd. Cipla Ltd., Mumbai, kindly gifted the Nintedanib. Dulbecco’s modified eagle’s medium (DMEM), L-glutamine, antibiotics (streptomycin-penicillin solution), fetal calf serum (FCS), trypan blue, trypsin–EDTA, phosphate-buffered saline solution (PBS), and 3--2,5-diphenyltetrazoliumbromide (MTT) were purchased from HiMedia Laboratories. Throughout the experiments, ultra-high purity Milli Q water was used.All the chemical precursors used for the synthesis of MNS were of analytical grade without any further purifications. Ferric chloride hexahydrate  was added after the complete dissolution of the iron precursor to get a yellow coloured precipitated solution, which was stirred at 50 °C for 30 min. Afterwards, the solution was transferred into a Teflon liner, fitted in a stainless-steel mould, and baked for 6 h at 200 °C in a furnace. The prepared MNS were removed from the Teflon liner and thoroughly rinsed with methanol and water in the presence of a permanent magnet and dried in a vacuum desiccator. For the functionalization of MNS by APTES, the MNS (1.0 gm) was dispersed in 100 mL methanol/toluene mixture 1:1 (V/V) by sonicating for 30 min. The resultant solution was heated at 95 °C until half of the solution was evaporated. The volume of the reaction mixture was adjusted to 100 mL by adding methanol. This procedure was repeated three times so that the solution becomes anhydrous. Then, 2% (V/V) APTES was added to the solution and kept in the shaking incubator for 24 h at 70 °C49. A washing step similar to that of MNS was applied to rinse the MNS-APTES.The MNS was synthesized by the solvothermal methodDifferent concentrations of leucine (20 − 100 µg/mL) in 50% ethanol (1 mL) were pipetted into a series of eppendorf tubes containing 1 mL ninhydrin solution in 2% ethanol. The above mixture was sonicated and then heated in a water bath at 100 °C for 5 min and the colour change (formation of Ruhemann’s purple) was measured at 570 nm. A suspension of MNS in 50% ethanol  was prepared and used for the ninhydrin assay, as described above. The concentration of the amino groups (from APTES) on the MNS was estimated using the standard calibration curve for leucine. The assay was performed in three replicate experiments.The NTD loading was performed by stirring 10 mg of MNS-APTES in 10 ml ethanol with different concentrations of NTD. Details regarding the optimization of drug loading are discussed in the supplementary information. In vitro drug release study of optimum NTD-conjugated MNS was carried out at 37 °C in a 75 mL dissolution medium (pH 5.5 and pH 7.4 phosphate buffers (PBS)) in a shaking incubator for 42 h.50 and briefly discussed in the supplementary information.Early passage human lung cancer cell line L-132 was procured from National Centre for Cell Sciences . The cytotoxicity assays were performed as per our previous reportα radiation (λ = 1.54184 Å) in the 2θ range from 10° to 80° with a step size of 0.02°. XRD patterns were analyzed by the Rietveld refinement method using the FullProf.2 k program. During refinement, the shape of the peaks was assumed to be a pseudo-Voigt function with asymmetry. The backgrounds of the patterns are fitted to a fourth-degree polynomial function. The size and shape of MNS were evaluated by a JEOL field emission scanning electron microscope (FE-SEM) JSM-7600F. A CM 20 FEG transmission electron microscope (TEM) from Philips was employed to record micrographs and energy-dispersive X-ray spectroscopy (EDX) patterns. Fourier transform infrared (FTIR) spectra were recorded at room temperature in the range of 400–4000 cm−1 using a Shimadzu FTIR spectrophotometer. The magnetic properties of the MNS and MNS-APTES were investigated using superconducting quantum interference device-vibrating sample magnetometry (SQUID-VSM) from Quantum Design. The magnetic hysteresis loop, zero-field cooled (ZFC), and field cooled (FC) measurements were performed over a temperature range of 2–400 K and a magnetic field up to 60 kOe. The experimental procedure for thermoremanent magnetization (TRM) measurements consisted of cooling the sample down to 2 K in the presence of a 40 kOe magnetic field, followed by magnetization measurements at increasing temperatures. For each data point at a given temperature, the sample was magnetized in the magnetic field of 40 kOe for 5 min. Afterward, the field was switched off, and the sample was allowed to relax for 1 min, followed by a magnetization measurement. Thus, the recorded magnetization is the measure of remanent magnetization of all the MNS in the blocked state at a given temperature. The X-ray photoelectron spectroscopy (XPS) analysis was performed with an ESCALAB 250Xi photoelectron spectrometer from Thermo Scientific in an ultra-high vacuum (UHV) using a monochromatic Al-Kα (1486.6 eV) X-ray source and a beam diameter of 300 µm. The hemispherical electron deflection analyzer operated in the constant analyzer energy (CAE) mode at pass energy of 200 eV for survey spectra and 20 eV for high-resolution spectra. The binding energies of all spectra were referenced to the binding energy of C1s (284.4 eV).X-ray diffraction (XRD) measurements were performed on a PANalytical Empyrean diffractometer using Cu-K3O4) phase. The characteristic diffraction peaks for the lattice planes (220), (311), (400), (422), (511), and (440) of a cubic structure with space group Fd-3m were observed (JCPDS card No. 01-088-0866). The detailed parameters extracted after the refinement of diffraction spectra are tabulated in Table 3 are comparable with the reference value for stoichiometric magnetite. The average crystallite size determined by the Debye–Scherrer formula for the most intense peak (311) was about 23 nm. Representative FE-SEM and TEM micrographs displaying the spherical shape of MNS are presented in Fig. 51. Decuzzi et al.52 studied NPs in the range of 50 nm–10 μm and observed that particles having a size less than 500 nm support the Brownian motion and can have colloidal stability. Depending on application perspectives, different sizes and shapes of NPs are recommended. The particles having spherical morphology and critical size less than 200 nm can be used for TDD applications56.The structural and morphological investigations of MNS were performed using XRD, FE-SEM, and HR-TEM. The XRD pattern with Rietveld refinement of an MNS is shown in Fig.  (220), 31, (400),−1 assigned to Fe–O stretching vibrations from iron oxide can be observed for MNS, MNS-APTES, and MNS-APTES-NTD57. Additionally, the spectrum in Fig. −1 overlaying with Fe–O vibrations from iron oxide50. The peak corresponding to Si–O–Si at 1015 cm−1, and the bending and stretching vibrational modes of NH at 1637 cm−1 and 3400 cm−1, respectively, confirm the coverage of silane with a free amine group of APTES on the MNS surface59. A band observed at 2920 cm−1 is due to C-H stretching vibrations of the anchored propyl group61. The TEM–EDX spectra , C = O stretching (from ester), and C = O stretching (from amide) in the NTD are also observed in Fig. 64. This appearance of characteristic NTD bands in MNS-APTES-NTD nanoformulation indicates the successful conjugation of drugs.The FTIR spectra of MNS, MNS-APTES, NTD conjugated MNS, and NTD are shown in Fig. 2 groups estimated were found to be around 41.5 ± 3 µmol/g of MNS. The calculation of fractional monolayer coverage was performed as reported by Sun et al.65, assuming four aminopropylsilanes/nm2 for monolayer coverage of APTES and using the surface area (6.44 m2/g) calculated from the mean MNS diameter (180 nm). The details regarding the calculation of surface area (m2/g) of MNS is provided in SI.The quantification of the amine groups existing on the MNS was evaluated by ninhydrin colourimetric assay. The nucleophilic displacement reaction among the primary amines and ninhydrin occurs, which includes the displacement of a hydroxyl group from ninhydrin by amine. It produces a colour complex known as Ruhemann's purple. The free − NH66. Besides, the monolayer coverage can overcome the problems associated with polymerization of the initial silane, possible formation of heterogeneous multilayers on the surface in the presence of excess silane and the lack of stability of the aminated surfaces67.These results reveal monolayer coverage of APTES over MNS. It was reported that primary amine-modified monolayers on the NPs surface significantly improved the cellular delivery68. The high resolution XPS spectrum of Fe2p 70. The peaks at 533.4 eV and 531.7 eV can be assigned to oxygen associated with SiO271 and surface hydroxyl or carboxyl groups (OH/CO) on MNS-APTES72. The presence of the SiO2 peak in the Si2p spectrum at 102.6 eV and Si–C at 100.2 eV  of MNS and MNS-APTES are about 77 emu/g and 74 emu/g. The observed MS values are smaller than the corresponding bulk value (92 emu/g), which is attributed to surface effects in MNS75. A relatively small or negligible reduction in the saturation magnetization MS value of MNS was observed after APTES functionalization. The nominal reduction in MS after functionalization is occurred due to the monolayer coverage of APTES on the MNS surface. The temperature-dependent magnetic behaviour was investigated by ZFC–FC measurements , as shown in Fig. 78. The derivative curve can be divided into three regions depending on the observed change in slopes, namely region-1 (T < 10 K), region-2 (10 K < T < 50 K), and region-3 (T > 50 K). Region-1 for the range T < 10 K corresponds to extremely fine MNPs with blocking temperatures below 10 K still being in the paramagnetic state. Region-2, between 10 and 50 K, represents particles with a moderate size, which constitute the majority of the sample. The third region T > 50 K is for the MNPs in the samples with blocking temperature considerably above room temperature.To get the information on the magnetic phases present in the samples by thermoremanent magnetization, several hysteresis loops were recorded at different temperatures. The normalized thermoremanent magnetization (TRM) values measured for MNS are shown in Fig. S) and coercive field (HC) response of MNS and MNS-APTES. MNS has a higher saturation magnetization compared to MNS-APTES, which scales linearly with increasing temperature. The scaling of MS with temperature can be explained using Bloch’s law79, which is related to low energy collective spin wave excitations (magnons).Figure S(T) and MS0 are the saturation magnetization at any given temperature (T) and absolute zero temperature (0 K), respectively. “β” is the Bloch constant, which depends on the Curie temperature (β ∝ 1/TC). The values of MS0 of 90.2 emu/g and 89.7 emu/g were obtained for MNS and MNS-APTES, respectively.Here, MC) is observed to decrease monotonously with the square root of temperature and reaches zero at blocking temperature. The temperature dependence of HC can be expressed with Kneller’s law81,In general, MNS exhibits hysteretic behaviour for the temperature below the blocking temperature and non-hysteretic behaviour above the blocking temperature. The coercive field  indicates superparamagnetic response.Here, TTo achieve optimum drug loading on MNS, the ratio of MNS:NTD was varied. The drug loading efficiency and loading capacity for different MNS:NTD ratios are summarized in Table The drug release from the MNS-APTES-NTD nanoformulation at 37 °C at pH 7.4 and 5.5 is shown in Fig. 2, − COOH, − SO3H, − PO3H2, − B(OH)2) at support may get converted into charged moieties depending on the different pH conditions82. The linkages like imine bond are mostly stable under physiological conditions (pH ≈ 7.4), whereas they get hydrolyzed in acidic surroundings. The observed different pH response is ascribed to the protonation state of terminal primary amino groups under different pH conditions83.The functional polymers which exhibit ionizable groups (i.e., − NHThe MTT assay was performed to examine the in vitro cytotoxicity of MNS-APTES, MNS-APTES-NTD, and free NTD. Figure The docking studies were performed among FGFR-4 and NTD; they reveals that amino acid residue Met524 involves in the binding of NTD with the protein. Further detailed surface representation of the docked complex and discussion is given in Table 84. Thermoremanent magnetization studies reveal the superparamagnetism and qualitative distribution of magnetic properties in the MNS. Functionalization of APTES and loading of the anticancerous drug NTD is confirmed by FTIR. A drug loading capacity of 23.6% is obtained. Monolayer coverage of aminosilane is established by quantifying the amine groups with the ninhydrin assay. NTD is conjugated with MNS-APTES through the acid liable imine bond. At physiological conditions, the MNS-APTES-NTD nanoformulation maintains high stability and inhibits drug release. In contrast, the cleavage of imine bonds in an acidic environment would lead to the drug release on demand. The MNS-APTES-NTD nanoformulation exhibits dose-dependent cytotoxicity for the L-132 cell line. Further magnetic studies of the MNS with a different size distribution of the constituent particles are underway to investigate the interparticle interaction and to explore them for various applications. The higher magnetization of functionalized MNS owing to monolayer coverage can be useful in many nanobiotechnology applications such as magnetoresistive biosensors, nanobiocatalysis, magnetic cell separation, etc.This work demonstrates the pH-responsive, controlled release of poorly water-soluble model drug NTD from the nanoformulation prepared by monolayer coverage of APTES over MNS. The use of a direct functionalization strategy and monolayer coverage of APTES prevents the loss in the magnetization of MNS reported for several functionalization approachesSupplementary Information


The goal of this study is the preparation of safer coated microneedles so that tips remaining after the initial use are less likely to be reinserted on a second use. Twelve groups of uncoated microneedles (u-MNs) were prepared from the combination of three different aspect ratios (height to base width) and four kinds of polymer (polyethylene (PE), polypropylene (PP), nylon and polylactic acid (PLA)). After coating the u-MNs with polyvinyl alcohol formulation to make coated MNs (c-MNs), the force displacement of the u-MNs and the c-MNs was measured. The aspect ratio was reduced from 2.2, 2.5 and 3.0 with u-MNs to 1.3, 1.4 and 1.6 with c-MNs, respectively, after the coating formulation was applied to the MNs. All PLA MNs had a puncture performance of more than 95%. However, the puncture performance of u-MNs made of PE and of PP with a 3.0 aspect ratio was only 8% and 53%, respectively, whereas the rates of c-MNs made of PE and of PP were 82% and 95%, respectively. In animal experiments with PP MNs with a 3.0 aspect ratio, the 59% rate of puncture performance with u-MNs increased to above 96% with c-MNs and fell to 13% for r-MNs. Safe c-MNs can overcome the disadvantages of standard c-MNs by reducing the probable contamination of remaining tips after use. Safe c-MNs have advantages over standard c-MNs in terms of humidity resistance, reasonable cost, sterilization process and short processing time through the separate process of u-MN preparation and simple dip-coating. Microneedles (MNs) deliver active pharmaceutical ingredients (API) through the stratum corneum, the outermost layer of skin, regardless of molecular weight and API polarity ,2,3,4. TThis study aims to develop safer c-MNs. Safe c-MNs, like standard c-MNs, will penetrate the skin only when the formulation is coated on the tips of u-MNs, and they will also feature the advantage of dissolving MNs, which cannot insert into skin after dissolution of tips. Skin puncture performance is controlled by the physical parameters of u-MNs, such as the mechanical properties of the polymer used for u-MNs and the aspect ratio of the tips ,29,30. WThe puncture performance of MNs is controlled by the following four variables: (a) kind of polymer used—which determines the mechanical strength of the u-MNs; (b) aspect ratio of u-MNs tips; (c) addition of a coating layer; and d) dissolution of the coating layer of c-MNs. Puncture performance is measured according to these variables. In this study, in vivo skin puncture performance was conducted to compare with in vitro skin puncture performance.Polylactic acid (PLA) was purchased from Green Chemical . Polyethylene (PE) and polypropylene (PP) were purchased from Goodfellow . nylon and polyvinyl alcohol (PVA) were obtained from Sigma-Aldrich .Master structures of microneedles were fabricated by micro-milling of aluminum alloy with milling system . Polydimethylsiloxane  was poured into the microneedle master structures. The PDMS solution was cured for 3 h in an oven of 70 °C . The inner cavity of a polydimethylsiloxane (PDMS) mold was investigated using VHX-6000 digital microscope . Twelve kinds of u-MN arrays were manufactured using a micromolding process, with four groups of u-MNs made from four different polymers: polyethylene (PE), polypropylene (PP), nylon (N) or polylactic acid (PLA). Each group of u-MNs included three subgroups of different aspect ratios of height to width (HM/WM) (from the position 500 μm apart from tip base to tip apex): 2.2 (HM/WM: 300/135), 2.5 (300/118), and 3.0 (300/100) see . Each MNTwelve kinds of c-MN arrays were manufactured using a dip-coating process. The coating solution had 20% (w/w) of polyvinyl alcohol (PVA) in distilled water. The coating solution was loaded into the coating well to a depth of 600 μm. The ozone-treated uncoated microneedles were dipped into the reservoir at a rate of 20 mm/s in the coating reservoir. Coated samples were dried at room temperature. Samples were coated with platinum and then the geometries of the samples were observed using scanning electron microscopy (SEM). Thirty-six MNs  are shown in In order to measure the mechanical strength of PE, PP, nylon and PLA used in this study, specimens of each polymer were prepared in accordance with ASTM D638 Type 1 (for tensile strength) and ASTM D790 . The fabricated specimens were measured for flexural strength, tensile strength and tensile modulus using the universal testing machine .2 for 30 min after pressing them with the fingers for 10 s, and then trypan blue solution was applied on the treated area in the same way to check the puncture performance. After insertion and removal of the c-MNs, r-MNs were re-administered into the porcine skin. The dyeing process and the calculation process were repeated for the reinserted r-MNs. The means and standard deviations were calculated for three samples in each group.Evaluation of in vitro puncture performance was carried out for each group of MNs described in The force displacement of the MNs was observed using a uniaxial force analyzer . Force–displacement curves were obtained by applying force to four MNs on an array for each sample until the force of 2 N was reached.2 for 30 min. Trypan blue solution was applied on the treated skin for 10 min to stain the holes generated by successful puncture. Puncture performance was carried out in the same manner for the r-H-PP MNs. Images of stained holes in the skin were taken using a camera  and puncture performance was calculated for samples.Evaluation of in vivo puncture performance with u-H-PP MNs, c-H-PP MNs and r-H-PP MNs was conducted in the same way as in vitro puncture performance, as explained in The u-MNs were prepared by molding polymer; four kinds of polymer can be replicated from a mold after melting. As a medical device of u-MNs, polymer MNs can be mass produced, easily sterilized and made ready-to-use ,32. ThusOne of the critical concerns regarding polymer MNs is their mechanical stability. The mechanical failure of the MNs was determined by the aspect ratio of height to base width, Young’s modulus and the tensile strength of the polymer ,28,33. TWhen coating a structure having a 2.5 aspect ratio, the coating thickness is about 35 μm, regardless of the kind of polymer used, because of the surface treatment with ozone plasma.Twelve kinds of u-MNs with three kinds of aspect ratio were prepared from four kinds of polymer . All MNs have the same height (800 μm). The u-MNs had different widths  at 300 μm from the top and different aspect ratios , as shown in illustrations 1–3 of The maximum depth of the coating solution could be 600 μm, but the actual coating depth did not exceed 500 μm, due to the surface property of the polymer and the high viscosity of the polyvinyl alcohol solution. In the case of u-M-MNs and u-H-MNs, which was step pyramid, it was difficult for the coating solution to pass the step due to the surface tension generated at the edge of the step. Therefore, for all u-MNs, the solution was coated up to a distance of 500 μm from the tip end of the microneedles. Inkjet printing technology was utilized to coat metal microneedles with the drug formulation, and the formulation was distributed across the surface of fully coated needles . HoweverThe information on the geometries of the samples is summarized in Tensile modulus and flexural strength of PE, PP, nylon, and PLA polymers used as MN material in this study were measured. As shown in The mechanical properties of each group of MNs were evaluated by force–displacement pattern analysis. The slope of the force displacement increased significantly after coating in all kinds of MNs, as shown in As shown in 2 for 30 min and then removed. A range of force from 1 kg/cm2 to 4 kg/cm2 was used to conduct the clinical study of MNs; this range can be generated by thumb pressure [2 on the skin close to bone to induce deformation of tips in order to prevent re-administration. In this study, MNs were pressed onto the full thickness of porcine skin placed on a hard surface with a force in the range of 3–4 kg/cm2. In a clinical study, MNs were pressed against the wrist, whose skin was close to bone [The c-MNs were applied to the porcine skin in vitro at a pressure of 3–4 kg/cmpressure ,39. In apressure ,41. Whenpressure ,39. The  to bone . If MNs For each group of MNs, the relationship between the initial force–displacement slope and puncture performance was shown according to the polymer type in 2 of force. When the MNs were removed after 30 min of administration and then re-administered, the skin puncture performance dropped to 13%, thereby minimizing the chance of contamination. The 13% rate of r-H-PP MNs was lower than that of u-H-PP MNs because deformation occurred at the tips of the MNs during insertion. Therefore, in general, when a hard tissue such as a wrist bone was encountered after MNs were pushed into wrist skin , the MN tips were deformed and thus the puncture performance was drastically reduced.Based on the in vitro experiments, skin puncture was performed on mice in vivo to confirm whether the results obtained from the in vitro study were applicable to the in vivo experiment. Among the various polymers, PP was selected because puncture performance was greatly affected by the aspect ratio change occurring before and after coating and after dissolution of the coating formulation. The MNs used in the in vivo experiments were u-H-PP, c-H-PP and r-H-PP. As shown in The aim of this study was to develop safer coated MNs that reduce the occurrence of punctures in the skin after the MNs are used. Before a solid formulation was coated to u-MNs, physical parameters were found that were not sufficient for the MNs to penetrate the skin. However, adding the coating formulation increased the mechanical strength of the MNs, thereby improving the puncture performance and achieving the desired drug delivery. Then, the dissolution of the coating formulation in the skin after insertion lessened the mechanical stability of the MNs and the rate of skin puncture was lowered as a result.The newly introduced safe c-MNs have the advantages of both c-MNs and dissolving MNs. Safe c-MNs are not affected by moisture, and they can deliver a variety of API regardless of environmental condition. Safe c-MNs leave no biohazardous residue like dissolving microneedles, and the low-temperature process can be used to load thermally sensitive API on the c-MNs. Safe c-MNs are a microneedle system that can be safely and effectively used for the delivery of various vaccines and drugs.


Background: Determining the incidence, progression, and patterns of multimorbidity are important for the prevention, management, and treatment of concurrence of multiple conditions. This study aimed to analyze major multimorbidity patterns and the association of the onset of a primary condition or combinations of a primary and a secondary condition with the progression to subsequent conditions.Methods: We included 53,867 participants aged 45–64 years from the 45 and Up Study who were free of 10 predefined chronic conditions at baseline (2006–2009). The incidence of multimorbidity  was identified using the claims database until December 31, 2016. The primary, secondary, tertiary, and quaternary condition for each participant was defined according to its temporal order of onset.Results: During a mean 9-years follow-up, the cumulative incidence of primary, secondary, tertiary, and quaternary conditions was 49.6, 23.7, 9.0, and 2.9%, respectively. The time to develop a subsequent condition decreased with the accumulation of conditions (P < 0.0001). Two concurrent cardiometabolic disorders  and CMDs clustered with musculoskeletal disorders (15.2%), mental disorders (13.5%), asthma (12.0%), or cancer (8.7%) were the five most common multimorbidity patterns. CMDs tended to occur prior to mental or musculoskeletal disorders but after the onset of cancers or asthma. Compared with all participants who developed cancer as a primary condition, individuals who experienced mental disorders/neurodegenerative disorders and a comorbidity as cardiovascular disease, hypertension, dyslipidemia, diabetes, asthma, or osteoarthritis were 3.36–10.87 times more likely to develop cancer as a tertiary condition. Individuals with neurodegenerative disorders and a comorbidity as hypertension, dyslipidemia, osteoarthritis, or asthma were 5.14–14.15 times more likely to develop mental disorders as a tertiary condition.Conclusions: A high incidence of multimorbidity in middle-aged adults was observed and CMDs were most commonly seen in multimorbidity patterns. There may be accelerated aging after a primary condition occurs. Our findings also reveal a potential preventative window to obviate the development of secondary or tertiary conditions. In Australia, 91% of the total mortality was attributed to chronic conditions in 2016 . PhysiolAn increasing number of studies have investigated the prevalence and patterns of multimorbidity –14, howeWe examined the incidence of multimorbidity with multiple chronic conditions  and the time to develop a subsequent condition. We also identified multimorbidity patterns by computing all combinations and permutations of multiple conditions and investigated the associations between the onsets of multiple chronic conditions.Additional File 1:The Sax Institute's 45 and Up Study is a prospective study of 266,896 participants aged 45 years and over in New South Wales (NSW), Australia between 2006 and 2009 . ParticiThe 45 and Up study has ethical approval from the UNSW Human Research Ethics Committee. Approval to use data from the 45 and Up Study for the current study was received from the Royal Victorian Eye and Ear Hospital Human Research Ethics Committee. Participants provided consent to follow-up and link their data to routine health datasets.Additional File 1:The main outcomes of interest were the incidence of the 10 chronic conditions, which were identified by the date of record in PBS or MBS. Several definitions of multimorbidity from a combination of multiple conditions  out of the 10 chronic conditions were tested. We regarded the chronic conditions as primary, secondary, tertiary, and quaternary according to its temporal onset order. We further categorized the 10 conditions into six groups including CMDs , cancer, asthma, mental disorders, neurodegenerative disorders, and musculoskeletal disorders (hip replacement and osteoarthritis) for the multimorbidity pattern analysis. PBS and MBS codes for each chronic condition were listed in http://www.saxinstitute.org.au/our-work/45-up-study/questionnaires/. Body mass index (BMI) was calculated based on self-reported height and weight. We created an additional “missing” category for each covariate for those with missing values.Information including age, gender, ethnicity, income, education, health insurance, lifestyle, diets, and family history of conditions was assessed using an administrative questionnaire, which is available at Descriptive data were summarized as frequency and percentage according to age and gender. Cox regression models were used to compare the incidence of multimorbidity between genders and between age groups after adjustment for the country of birth, income, education, BMI and health insurance, and mutual adjustment for gender and age.We used the Wilcoxon rank-sum test to examine whether the median time to develop a subsequent condition differed between genders or age groups. The trend of the time to develop a subsequent condition for the accumulation of chronic conditions was examined using the Mann-Kendall test.We analyzed all combinations and permutations of 2–4 conditions to identify the major multimorbidity patterns. Chi-square test was used to examine the difference in the contribution of each condition between primary and secondary onsets while the Cochran-Armitage test was used to assess the trend in the contribution of each condition with the accumulation of conditions.Additional File 1:The associations of the onset of a primary condition with progression to a secondary condition and a combination of a primary and a secondary condition with progression to a tertiary condition were assessed using Poisson regression models with a robust variance for relative risks (RRs)  calculation. The reference was the incidence of the corresponding primary condition within 5 years in all participants who were free of any of 10 chronic conditions at baseline. For example, to examine whether hypertension as the primary condition would increase the risk of cancer as the secondary condition, the comparison was conducted between the incidence of cancer as the second condition in participants with hypertension as the primary condition, and cancer as the primary condition in the total population  and all P < 0.001, As shown in P < 0.001] than women after adjustment for confounders. The difference in the incidence of multimorbidity between genders increased with the accumulation of conditions.Men (49.7%) and women (49.5%) had a similar incidence of a primary condition. Men had a higher incidence of secondary [multivariable-adjusted HR (95%): 1.07 (1.03–1.11)], tertiary [1.20 (1.14–1.27)] and quaternary condition , secondary [1.75 (1.66–1.85)], tertiary [2.02 (1.86–2.19)], and quaternary conditions [2.18 (1.89–2.53)] than those aged 45–54 years . The median (interquartile range) time to develop a new condition as the primary, secondary, tertiary and quaternary condition was 4.7 (2.3–6.7), 1.4 (0.3–3.3), 1.0 (0.3–2.4), and 0.8 (0.2–1.9) years, respectively, in men and 4.6 (2.1–6.6), 1.7 (0.6–3.5), 1.1 (0.4–2.3), and 0.9 (0.2–1.9) years, respectively, in women. Younger individuals had 1.0 years longer to develop a primary condition (P < 0.001) but a similar time to develop a secondary, tertiary, and quaternary condition compared with their older counterparts , CMDs-musculoskeletal disorders (17.5%), CMDs-asthma (10.3%), CMDs-mental disorders (10.0%), and CMDs-cancer (8.5%) were the five leading multimorbidity patterns in men. The five leading multimorbidity patterns in women were the same whereas a lower proportion (24.3%) of two CMDs and a higher proportion of CMDs-mental disorders (16.5%) were observed in women .Additional File 1:As shown in Additional File 1:Additional File 1:Additional File 1:Five most frequent patterns contributed to 60.4% of 112 combinations of three conditions ], 6.58 (5.16–8.40), 7.23 (5.75–9.10), 3.37 (1.84–6.19), 8.50 (6.39–11.31), 5.31 (1.66–16.96), and 7.70 (5.95–9.96) times more likely to progress to cancer as a tertiary condition, respectively, compared with the total population who developed cancer as a primary condition. While individuals with neurodegenerative disorders and comorbidity as CVD, hypertension, dyslipidemia, asthma, osteoarthritis or mental disorders were 10.87 (1.79–66.21), 3.97 (1.65–9.56), 3.88 (1.84–8.20), 9.47 (3.89–23.09), 6.15 (1.95–19.40) and 4.31 (2.03–9.16) times more likely to progress to cancer as a tertiary condition, respectively, compared with the total population who developed cancer as a primary condition.Individuals with two conditions as cancer-asthma, cancer-osteoarthritis, cancer-mental disorders, and asthma-hip replacement were 2.41–3.98 times to progress to CVD as a tertiary condition, compared with the total population who developed CVD as a primary condition.Individuals with neurodegenerative disorders and comorbidity as hypertension [multivariable-adjusted RR (95%): 5.14 (2.07–12.79)], dyslipidemia [6.11 (2.88–12.99)], asthma [14.15 (5.76–34.77)], or osteoarthritis [10.56 (3.33–33.48)] had a higher risk of progression to mental disorders as a tertiary condition, compared with total participants who developed mental disorders as a primary condition .The prevalence of one, two, three, and four conditions was 65.4, 18.3, 9.7, and 4.4%, respectively. The leading multimorbidity patterns defined by the combination of two conditions were two-CMDs (28.1%), CMDs-mental disorders (18.2%), CMDs-asthma (10.0%), CMDs-cancer (8.8%), and asthma-mental disorders (8.2%). Three-CMDs (20.3%) were the most frequent pattern defined by the combination of three prevalent conditions and two CMDs plus one other chronic condition contributed to 33.5% of all combinations.Additional File 1:These patterns were consistent with those defined by the incidence of conditions in our longitudinal analysis  of total death in Australia in 2016 . The genIn conclusion, we found a high incidence of multimorbidity and CMDs were most often clustered with other conditions in middle-aged men and women, highlighting the importance of early intervention. There may be accelerated aging after a primary condition occurs. Our findings also reveal a potential preventative window to obviate the development of secondary or tertiary conditions. These findings may help target priority interventions for the prevention and management of multimorbidity at an early stage and guide the screening of subsequent conditions in primary care.https://www.saxinstitute.org.au/our-work/45-up-study.The datasets presented in this article are not readily available because the data that support the findings of this study are available from The Sax Institute but restrictions apply to the availability of these data, which were used under license for the current study, and so are not publicly available. Data are however available from the authors upon reasonable request and with permission of The Sax Institute. Requests to access the datasets should be directed to The studies involving human participants were reviewed and approved by The 45 and Up study has ethical approval from the UNSW Human Research Ethics Committee. Approval to use data from the 45 and Up Study for the current study was received from the Royal Victorian Eye and Ear Hospital Human Research Ethics Committee. Participants provided consent to follow-up and link their data to routine health datasets. The patients/participants provided their written informed consent to participate in this study.XS, LZ, and MH conceived and designed the research. XS and LZ conducted data analysis and interpretation. XS wrote the initial draft of the manuscript. XS, LZ, CS, WP, EH, and MH revised the manuscript. All authors read and approved the final manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.