Moreover, in the FcεRI cascade, KIOM-MA128 not only inhibited activation of LYN, FYN and SYK, known as the rate-limiting proteins of the FcεRI cascade, but also suppressed the phosphorylation of ERK, p38 and JNK, which is related to cytokine expression. Finally, 50 to 100 mg/kg KIOM-MA128 significantly attenuated the Ag/IgE-induced PCA reaction in mice. These findings provide novel information and improve our understanding of the anti-allergic effects of KIOM-MA128 on allergic diseases.KIOM-MA128, a novel herbal medicine, has been reported to exert some beneficial effects on various biological events, such as atopic dermatitis, inflammation and cancer. The aim of this study is to investigate how KIOM-MA128 regulates the allergic response. We measured the activity of β-hexosaminidase and the levels of allergic mediators in the conditioned media of antigen/IgE (Ag/IgE)-activated RBL-2H3 mast cells. We examined the levels of proteins associated with both the FcεRI and arachidonate cascades. Finally, we established the passive cutaneous anaphylaxis (PCA) model in mice to confirm the anti-allergic effects of KIOM-MA128 in vivo. KIOM-MA128 dose-dependently inhibited degranulation and the production of the allergic mediators described above, with no significant cytotoxicity. In the arachidonate cascade, KIOM-MA128 significantly reduced both cytosolic phospholipase A Cross-linking FcεRI and the Ag/IgE complex activates LYN, which then activates SYK. These processes cause the activation of a signaling cascade, including LAT, phospholipase C (PLC) γ, and MAP kinases (MAPKs). Additionally, activation of mast cells by the Ag/IgE complex is associated with the secretion of granules containing various allergic mediators, such as β-hexosaminidase, histamines, eicosanoids, serotonin, and pro-inflammatory cytokines and chemokines. In particular, tumor necrosis factor-α, a pro-inflammatory cytokine, induces inflammation during degranulation and recruits various immune cells, causing severe inflammation [Mast cells play a critical role in the allergic response, including early- and late-phase reactions . A high-ammation ,4.Glycyrrhizae radix, Polygoni cuspidati Rhizoma, Sophorae radix, Cnidii rhizoma and Arctii fructus have been used in traditional oriental medicine in several Asian countries, including China and Korea. We formulated a new herbal medicine formula called KIOM-MA using these herbs. Our previous studies showed that KIOM-MA possessed anti-inflammatory properties in RAW 264.7 macrophages [rophages  as well rophages  and antirophages  than KIO2 (PGD2), were analyzed using enzyme immunoassay (EIA) and enzyme-linked immunosorbent assay (ELISA) kits to evaluate the anti-allergic effects of KIOM-MA128. The FcεRI signaling pathway was investigated by immunoblot analysis to confirm the anti-allergic mechanisms of KIOM-MA128. Finally, we performed Ag/IgE-mediated passive cutaneous anaphylaxis (PCA) reaction in mice to demonstrate the anti-allergic action of KIOM-MA128 in in vivo system. Here, we report that KIOM-MA128 suppresses Ag/IgE-induced allergic responses in RBL-2H3 cells. Furthermore, our findings support the clue to understanding the anti-allergic action of KIOM-MA128 in allergic diseases.In the present study, we hypothesized that KIOM-MA128 might prevent allergic reactions in Ag/IgE-activated mast cells. We investigated the degranulation of Ag/IgE-stimulated RBL-2H3 cells by measuring β-hexosaminidase activity to examine the anti-allergic effects of KIOM-MA128. The levels of inflammatory mediators, such as tumor necrosis factor-α (TNF-α), histamine, interleukin-4 (IL-4), IL-6 and prostaglandin DWe measured the effects of KIOM-MA128 on the viability of RBL-2H3 cells. RBL-2H3 cell viability was measured following treatment with various concentrations of KIOM-MA128 (250–2000 µg/mL) for 24 h. The findings indicated that KIOM-MA128 did produce cytotoxicity in RBL-2H3 cells. The results indicated that KIOM-MA128 has not significant cytotoxicity .We measured both β-hexosaminidase activity and the histamine concentrations in media from IgE-sensitized mast cells that were stimulated with antigen (0.1 µg/mL DNP-HSA) and various concentrations of KIOM-MA128 to investigate the modulatory effects of KIOM-MA128 on Ag/IgE-mediated degranulation and histamine release in RBL-2H3 cells. KIOM-MA128 significantly inhibited degranulation in Ag/IgE-induced RBL-2H3 cells in a dose-dependent manner A. MoreovWe measured the levels of cytokines such as TNF-α, IL-6 and IL-4 in IgE-sensitized RBL-2H3 cells using ELISA to determine the effects of KIOM-MA128 on Ag/IgE-induced pro-inflammatory cytokine production in RBL-2H3 cells. Activated mast cells secreted various cytokines, which play critical roles in allergic responses, such as the chemoattractant response that leads to the recruitment of other immune cells and the regulation of severe allergic reaction ,10. When2, p-cPLA2 and COX-2 levels in Ag/IgE-activated RBL-2H3 cells using immunoblot analysis and ELISA kits to determine the effects of KIOM-MA128 on the levels of pro-inflammatory mediators in Ag/IgE-induced RBL-2H3 cells. cPLA2 is a rate-limiting enzyme in eicosanoid synthesis, and COX-2 converts arachidonic acid to prostaglandin. Therefore, cPLA2 and COX-2 are very important enzymes in the allergic response [2, COX-2 and PGD2 levels in dose-dependent manner . We analyzed the expression of FcεRI cascade-related proteins to investigate the effects of KIOM-MA128 on the IgE-mediated FcεRI cascade. KIOM-MA128 reduced the levels of p-SYK and p-LYN, whereas it increased the phosphorylation levels of FYN (Tyr531), which is an inactive form [We used the passive cutaneous anaphylaxis (PCA) animal model to confirm that KIOM-MA128 exhibited anti-allergic effects in vivo. The ears of ICR mice were locally injected with IgE and then administered KIOM-MA128 for 1 h. A mixture of the antigens and Evans blue was then injected into the tail vein. At 50 mg/kg and 100 mg/kg, KIOM-MA128 significantly inhibited the mast cell-mediated PCA reaction in mice, and it seemed like that the anti-allergic actions were better than dexamethasone .Previously, we reported that KIOM-MA possessed beneficial effects against allergic diseases, such as inflammation , atopic 2, in Ag/IgE-activated mast cells, and Ag/IgE-induced PCA reaction in mice. Moreover, KIOM-MA128 also regulates the activation of the FcεRI and arachidonate cascades in these cells. Therefore, the inhibitory effects of KIOM-MA128 may be closely associated with its ability to reduce the activation of Ag/IgE-stimulated mast cells. In support of this hypothesis, some components of KIOM-MA and KIOM-MA128, such as Polygonum cuspidatum [Sophora flavescents [, and Arctium lappa fruit [In this study, we show that KIOM-MA128 exerts anti-allergic actions in both Ag/IgE-activated RBL-2H3 cells and Ag/IgE-mediated PCA reaction in mice. These anti-allergic actions of KIOM-MA128 result from its ability to suppress both the degranulation process and the production of allergic mediators, such as histamine, IL-4, IL-6, TNF-α, and PGDspidatum , Sophoravescents , and Arcpa fruit , are knopa fruit ,19. All 2+ levels, activation of MAPKs, which is associated with cytokine expression [2 levels were increased in the conditioned media of the cells. In contrast, KIOM-MA128 decreased these responses as well as the activation of LYN, FYN and SYK. Consequently, the phosphorylation of MAPKs including ERK, p38 and JNK, PLCγ1 and PKCδ is also reduced by KIOM-MA128. Therefore, an important role of KIOM-MA128 may be to regulate the activation of LYN, FYN or/and SYK to exert its anti-allergic properties.One possible mechanism for the anti-allergic actions of KIOM-MA128 in Ag/IgE-activated mast cells may be the direct suppression of the activation of the FcεRI cascade, because mast cells express FcεRI receptors on the extracellular face of the plasma membrane . Actuallpression , and thapression . As a repression ,26. In s2 is able to induce bronchoconstriction, increase in capillary permeability, mucous production, and vasodilation in asthma [4 as a strong spasmogenic and chemotactic biochemical can enhance the permeability of small vessels including capillaries, and play both promotion and maintenance of allergic inflammation by activating cysteinyl-LT receptors in allergic diseases such as asthma and allergic rhinitis [2 and LTC4 can sustain, and induce severe inflammation in allergic diseases. Therefore, the regulation of eicosanoid production is another important role of KIOM-MA128 in Ag/IgE-activated mast cells. In fact, in our data, KIOM-MA128 not only reduced the production of PGD2 but also decreased cPLA2 phosphorylation, which is a rate-limiting enzyme in the arachidonate cascade [2 and COX-2 may be targets of KIOM-MA128 and may mediate its anti-allergic properties.Another possible mechanism may be associated with the inhibition of the arachidonate cascade in Ag/IgE-activated mast cells. Ag/IgE-activated mast cells can produce and liberate inflammatory prostaglandins and leukotrienes . In partn asthma ,29. Besirhinitis ,29. Over cascade , and COX cascade . All the2, phospho-ERK, phospho-JNK, phospho-LYN, phospho-p38, phospho-PKCδ, phospho-PLCγ1, phospho-SYK and COX-2 were purchased from Cell Signaling Technology, Inc. . A specific antibody against phospho-FYN was obtained from Biorbyt Ltd. . A specific antibody against α-tubulin was purchased from Santa Cruz Biotechnology, Inc. . An enzyme immunoassay (EIA) kit for PGD2 was obtained from Cayman Chemical Co. . Enzyme-linked immunosorbent assay (ELISA) kits for TNF-α, IL-4, and IL-6 were purchased from e-Bioscience, Inc. . 4-Nitrophenyl-N-acetyl-β-d-glucosaminide (p-NAG), dinitrophenyl-human serum albumin (DNP-HSA) and DNP-immunoglobulin E (DNP-IgE) were obtained from Sigma-Aldrich Co. . All other chemicals were of analytical grade.MEM-α medium, 1 × DPBS, penicillin, streptomycin and fetal bovine serum (FBS) were purchased from GE Healthcare Life Sciences . The EZ-Cytox cell viability assay kit was obtained from Daeillab Service Co. . Specific antibodies against phospho-cPLAGlycyrrhizae radix, Polygoni cuspidati Rhizoma, Sophorae radix, Cnidii rhizoma, Arctii fructus, etc. [Lactobacillus rhamosus  provided by the Korea Food Research Institute, the pH of which was adjusted to 7.0 with 1 N NaOH and then autoclaved for 5 min, and then incubated for 48 h at 37 °C. KIOM-MA128 was filtered through a nylon net filter , and then deposited overnight. The supernatant was lyophilized, and then the dried pellet  was stored at −20°C until use. KIOM-MA128 were dissolved in a 10% DMSO solution or deionized water for in vitro or in vivo studies, respectively.KIOM-MA and KIOM-MA128 were prepared according to previously described methods ; the herus, etc. ,14, was ICR mice, known as Swiss CD-1 mice  . The absorbance at 620 nm was measured using a SpectraMax i3 microplate reader .The Ag/IgE-mediated PCA reaction was evaluated using a previous method . ICR micv/v) FBS, 100 U/mL penicillin and 100 µg/mL streptomycin at 37 °C in a humidified atmosphere of 5% CO2. All the experiments contained a vehicle control group that was treated with 0.1% DMSO.RBL-2H3 cells, a mast cell line originating from rat basophilic leukemia , were cu4 cells/well) in MEM-α medium with 10% FBS and incubated overnight at 37 °C. These cells were washed with 1× DPBS and then incubated with 1 μg/mL DNP-IgE for 24 h. The IgE-sensitized cells were preincubated with KIOM-MA128 (0–2000 μg/mL) in MEM-α medium containing 1% FBS for 1 h, and then simultaneously treated with 0.1 μg/mL DNP-HSA and 10 μL of the EZ-Cytox reagent and incubated for an additional 4 h. Cell viability was determined by measuring the absorbance at 450 nm using a microplate reader.Cell viability was determined by measuring the mitochondria-dependent reduction of WST-1 to the water-soluble tetrazolium salt . Brieflyβ-Hexosaminidase activity assay was evaluated using a previously reported method . Superna2 or TNF-α in the conditioned media. All conditioned media were centrifuged  for 10 min, and the samples were stored at −80 °C until use. The histamine, IL-4, IL-6, PGD2 and TNF-α levels were detected using ELISA kits according to the manufacturer’s instructions.The IgE-sensitized cells were preincubated with KIOM-MA128 in MEM-α medium with 1% FBS for 1 h, spiked with DNP-HSA, and then incubated for an additional 4 h to determine the amounts of histamine, IL-4, IL-6, PGDThe immunoblot analysis was evaluated according to a previously described method . The memAll the experimental results are listed as the mean ± SD or SEM. One-way analysis of variance (ANOVA) was used for multiple comparisons . The Dunnett test was applied when there was a significant variation between the treated groups.The present study shows that KIOM-MA128 exerts its anti-allergic properties by inhibiting both the FcεRI and arachidonate cascades in Ag/IgE-activated mast cells in vitro and in vivo. These findings provide the clue regarding the anti-allergic actions of KIOM-MA128 in asthma and atopic dermatitis. The anti-allergic mechanisms in Ag/IgE-activated mast cells may include several targets, such as LYN, FYN, SYK, MAPKs, cPLA2 and COX-2. Moreover, the anti-allergic effects of KIOM-MA128 may result from the combination of the herbal components, including the anti-allergic actions produced by the fermentation process. Therefore, further study is necessary to reveal the major component of KIOM-MA128 that is responsible for its anti-allergic actions.


The results of 15 years of surveying (1980/81–1994/95) on ambient levels of these pollutants at two sampling sites are given in this work. Site 1 is located in the city, opposite the old petroleum refinery facilities, while Site 2 is located in the settlement 25 km from the city, opposite the eastern industrial zone. Annual means of NO2 varied between 34 and 60 μg/m3 at Site 1 and between 14 and 26 μg/m3 at Site 2, but do not follow the 40% reduction in industrial emissions of this pollutant, probably due to the dominant impact of other minor sources, like traffic. Yearly averages of NH3 were in the range of 13 to 26 μg/m3 at Site 1 and 7 to 16 μg/m3 at Site 2, and are practically constant during the period studied.The determination of ambient levels of nitrogen dioxide (NO


Angelica species were screened by angiotensin-converting enzyme (ACE) assay in vitro. Among the crude extracts, the methanol extract of Angelica decursiva whole plants exhibited potent inhibitory effects against ACE. In addition, the ACE inhibitory activity of coumarins 1–5, 8–18 was evaluated, along with two phenolic acids  obtained from A. decursiva. Among profound coumarins, 11–18 were determined to manifest marked inhibitory activity against ACE with IC50 values of 4.68–20.04 µM. Compounds 12, 13, and 15 displayed competitive inhibition against ACE. Molecular docking studies confirmed that coumarins inhibited ACE via many hydrogen bond and hydrophobic interactions with catalytic residues and zinc ion of C- and N-domain ACE that blocked the catalytic activity of ACE. The results derived from these computational and in vitro experiments give additional scientific support to the anecdotal use of A. decursiva in traditional medicine to treat cardiovascular diseases such as hypertension.The bioactivity of ten traditional Korean  Hypertension is one of the most common diseases worldwide, with many associated risk factors such as stroke, heart disease, chronic renal failure, and aneurysm ,2. GlobaAngelica decursiva Fr. et Sav (Umbelliferae) is a perennial herb that grows throughout Japan, China, and Korea. It is widely employed in traditional Korean medicine to cure diseases as an antitussive, analgesic, antipyretic, and cough remedy -amino}ethyl)phosphinyl]-2-[(3-phenylisoxazol-5-yl)methyl]-1-oxo-propyl}amino)-3-(4-hydroxy-phenyl) propanoic acid] (FII)  (FII) . To provs of ACE . As showractions a. Similadetected c. For co and 15. b. The ox12 and 13 interacted with hydrogen atoms of His331 and His491 residues included in the S1′ pocket of N-ACE. These hydrogen-bonding interactions and hydrophobic interactions between 3′-substituents of inhibitors (12 and 13) and His and Phe residues of N-ACE make a favorable orientation to interact with zinc ion via van der Waals interaction. However, 15 interacted with His331 via carbon–hydrogen interaction, which is a relatively weak force compared to conventional hydrogen bond interaction (such as H‒O and H–N); thus 15, could not get close enough to interact with zinc ions  exhibited inhibitory effects against ACE. Recently, it was reported that another Angelica species, A. gigas, and its coumarin constituents showed potential antihypertensive activity through ACE inhibition -Phe-Gly-Gly), and captopril were purchased from Sigma Chemical Company . All chemicals and solvents used for column chromatography were of reagent grade and purchased from commercial sources, unless otherwise stated.ACE , FAPGG  was registered and deposited in the Department of Food Science and Nutrition, Pukyong National University, Busan, Korea  for future reference.MeOH extracts of whole plants of 1), nodakenetin (2), isorutarine (3), umbelliferone (4), umbelliferone 6-carboxylic acid (5), para-hydroxy benzoic acid (6), vanillic acid (7), 2′-isopropyl psoralene (8), 3′(R)-O-acetyl-4′(S)-O-tigloylkhellactone (9), cis-3′-acetyl-4′-angeloylkhellactone (10), decursinol (11), 4′-hydroxy Pd-C-III (12), Pd-C-I (13), decursidin (14), (+)-trans-decursidinol (15), Pd-C-II (16), Pd-C-III (17), and 4′-methoxy Pd-C-I (18) [The following coumarins were previously isolated and identified in our laboratory: Nodakenin  ,21,22,53The ACE inhibitory activity of 18 compounds was conducted according to Hyun et al. [12 ; 13 ; 15 . Inhibition constants (iK) were determined by interpretation of the secondary plots from Lineweaver–Burk plot.To determine the inhibition mechanisms, ACE inhibition was evaluated by monitoring the effects of different concentrations of substrate (0.1–1 mM). The reaction mixture consisted of different concentrations of FAPGG (0.1–0.5 mM), as the substrate, and ACE in 50 mM Tris–HCl buffer. Several concentrations of each sample were added to the reaction mixture. The tested concentrations of coumarins are as follows: The X-ray crystallographic structures of the C-domain and N-domain human angiotensin I-converting enzyme complex were obtained from the RCSB Protein Data Bank  . The prot-test . All results are presented as mean ± SEM.Statistical significance was analyzed by one-way ANOVA and Student’s A. decursiva and screened their ACE inhibitory activity using an in vitro ACE assay. Among these natural coumarins, 11–18 showed remarkable IC50 values of 4.68–20.04 µM. Therefore, screening and developing new ACE inhibitors from A. decursiva could be beneficial in the treatment of cardiovascular diseases such as hypertension. In addition, the present data indicate that linear pyranocoumarins inhibit ACE activity in vitro, and that activity against ACE and mode of action depend on the class and structure of the coumarins. These structure–function relationships could be useful for designing new ACE inhibitors based on coumarins.In this study, we isolated 16 coumarins and two phenolic compounds from


Background and objectives: Pollens of weeds are relevant elicitors of type I allergies. While many Artemisia species occur worldwide, allergy research so far has only focused on Artemisia vulgaris. We aimed to characterize other prevalent Artemisia species regarding their allergen profiles. Materials and Methods: Aqueous extracts of pollen from seven Artemisia species were characterized by gel electrophoresis and ELISA using sera from mugwort pollen-allergic patients (n = 11). The cDNA sequences of defensin–proline-linked proteins (DPLPs) were obtained, and purified proteins were tested in a competition ELISA, in rat basophil mediator release assays, and for activation of Jurkat T cells transduced with an Art v 1-specific TCR. IgE cross-reactivity to other allergens was evaluated using ImmunoCAP and ISAC. Results: The protein patterns of Artemisia spp. pollen extracts were similar in gel electrophoresis, with a major band at 24 kDa corresponding to DPLPs, like the previously identified Art v 1. Natural Art v 1 potently inhibited IgE binding to immobilized pollen extracts. Six novel Art v 1 homologs with high sequence identity and equivalent IgE reactivity were identified and termed Art ab 1, Art an 1, Art c 1, Art f 1, Art l 1, and Art t 1. All proteins triggered mediator release and cross-reacted at the T cell level. The Artemisia extracts contained additional IgE cross-reactive molecules from the nonspecific lipid transfer protein, pectate lyase, profilin, and polcalcin family. Conclusions: Our findings demonstrate that DPLPs in various Artemisia species have high allergenic potential. Therefore, related Artemisia species need to be considered to be allergen elicitors, especially due to the consideration of potential geographic expansion due to climatic changes.  Pollen allergies are one of the most common causes of type I hypersensitivity reactions, affecting up to 30% of the population in industrialized countries. Besides grasses and trees, weeds are considered relevant elicitors of allergic reactions . Weeds aArtemisia vulgaris) belongs to the Asteraceae plant family, which is comprised of about 350 Artemisia species. It was originally native to Russia, but is now found across Europe, North America, and parts of Asia. Pollen of Artemisia was found to be the predominant species for Asteraceae pollen counts in a study involving 13 pollen monitoring stations across Europe. In Europe, flowering typically starts mid-July and lasts until the end of September (www.pollenwarndienst.at), with a mean duration ranging from 40 to 115 days in Lithuania and Great Britain, respectively [Artemisia has been drastically increasing, among other factors due to the commercial cultivation of Artemisia annua as a source of artemisinin, a compound identified to possess antimalarial properties and recommended by the World Health Organization as first-line treatment for uncomplicated malaria [The herb mugwort  and tree (2.2%) pollen is comparably low in these geographic regions [Artemisia vulgaris was 58.3% among Chinese patients with confirmed intradermal reactivity to pollen allergens [patients . In part regions ,14. In allergens . In Korellergens . 2–4 repeats. Seven isoallergens of Art v 1 differing in 1–6 residues all located in the C-terminal proline-rich region have been identified [Escherichia coli revealed no differences in isoallergens in terms of IgE binding or mediator release potency. Structural integrity and IgE binding capacity were, however, dependent on the cysteine-stabilized fold of the defensin-like domain [The major mugwort pollen allergen Art v 1 belongs to the gamma-thionine family and is a defensin-like protein linked to a polyproline-rich region (defensin–polyproline-linked protein (DPLP)) ,18,19. Ientified . Recombie domain ,21. Epite domain . 25–36, and demonstrates an HLA-DRB1*01 restriction [The natural allergen presents two types of plant-specific O-glycosylations, a large hydroxyproline-linked arabinogalactan and single (but adjacent) β-arabinofuranoses linked to hydroxyprolines, comprising about 30%–40% of molecular mass . While striction ,25,26. Btriction . Recentltriction ,29. Ambrosia artemisiifolia (Amb a 4) and Parthenium hysterophorus (Par h 1) [Allergenic defensin-like proteins linked to a polyproline-rich region have so far been exclusively found in the pollen of the Asteraceae family. Apart from Art v 1, homologs have been identified in Par h 1) ,31. ThosPar h 1) . AlthougPar h 1) . FurtherPar h 1) ,35. Artemisia spp. They belong to the protein families of pectate lyases [Artemisia annua [Artemisia allergens described to date have originated from A. vulgaris, while other Artemisia species have been less investigated.Apart from Art v 1, a number of other allergens have been described in e lyases ,37, nonse lyases ,38,39, pe lyases ,41, and e lyases  and PR-1ia annua ,43. MostArtemisia species. Therefore, pollens from seven relevant Artemisia species were investigated, and DPLPs were identified as major IgE cross-reactive allergens. Purified DPLPs presented highly similar protein sequences, IgE binding, mediator release, and T cell reactivity in comparison to Art v 1. As a result, six novel allergens were officially acknowledged by the WHO/IUIS Allergen Nomenclature Sub-Committee. The presence of IgE cross-reactive allergens from the pectate lyase, nsLTP, profilin, and polcalcin protein family was demonstrated in the investigated pollen sources. The aim of this study was to evaluate the allergological relevance of allergenic molecules in Eleven mugwort pollen-allergic patients from Vienna (Austria) with previously determined IgE reactivity to Art v 1 were selected on the basis of typical case histories (recurrent rhinitis/conjunctivitis during late summer), positive skin prick test (≥3 mm) to mugwort pollen extract and allergen-specific IgE (ImmunoCAP ≥ 3)  . For sinA. absinthium, A. annua, A. ludoviciana, and A. vulgaris were purchased from Allergon AB ; A. californica, A. frigida, and A. tridentata were obtained from Greer Laboratories   . Pollen 3. Bound antibodies were detected by rabbit antimouse IgG and IgM alkaline phosphatase conjugates . Detection was performed using nitroblue tetrazolium chloride in combination with 5-bromo-5-chloro-3-indolyl phosphate as a substrate.Protein extracts and purified molecules were separated by reducing gel electrophoresis and visualized using GelCode Blue Safe Protein Stain . To analyze interactions with Art v 1-specific antibodies, purified natural proteins were transferred to a nitrocellulose membrane . Briefly, membranes were incubated with a monoclonal anti-Art v 1 antibody produced by genetic immunization  at a dilFor purification of natural Art v 1 and homologous molecules, total protein extracts were adjusted to 50 mM triethylamine acetate (pH 5.0) and subjected to ion exchange chromatography using the Äkta prime system  with a 5-mL QFF anion exchange precolumn and a 5-mL CMFF cation exchange column . Fractions containing the target proteins were obtained by gradient elution to 50 mM ammonium bicarbonate (pH 7.9). Proteins were stored at −20 °C.Mass spectrometry-based analyses were performed with a Micromass QTof Global Ultima instrument , as previously described . Intact Artemisia spp. pollen was prepared using TRIzol LS Reagent  according to the manufacturer’s protocol. Briefly, pollen grains were ground with glass sand under liquid nitrogen. Pollen RNA was extracted by the addition of TRIzol and chloroform and precipitated from the aqueous phase. Full-length DNA sequences of Art v 1 homologs were obtained by reverse transcription from cDNA using a SuperScriptTM One-Step RT-PCR with a Platinum Taq kit  with specific 5′-GGAATTCCATATGGCTGGTTCAAAGTTGTGTGAAAAG-3′ forward and 5′-GAGAGAATTC(T)21-3′ reverse primer. In addition, cDNA and protein sequences were aligned and analyzed using the online tool ClustalW2 . Total RNA from Maxisorp plates  were coated with 200 ng of pollen extracts or purified molecules in PBS overnight at 4 °C. Unspecific binding was blocked with TBS (pH 7.4), 0.05% (v/v) Tween, and 1% (w/v) BSA and incubated with patients’ sera (1:4) overnight at 4 °C. Bound IgE was detected with alkaline phosphatase-conjugated monoclonal antihuman IgE antibodies . In addition, 4-nitrophenylphosphate  was used as a substrate, and optical density was measured at 405/492 nm. Values were considered positive when exceeding 3 times the standard deviation of buffer control vials. For inhibition experiments, patients’ sera were pre-incubated overnight at 4 °C with increasing concentrations (0.005–5 µg/mL) of purified natural Art v 1.Artemisia spp. was evaluated using a rat basophil degranulation leukemia (RBL) assay, as described in Reference [n = 4). Cells were stimulated with increasing concentrations of the purified proteins (0.001–100 µg/mL), and the release of β-hexosaminidase was measured by the enzymatic cleavage of the fluorogenic substrate 4-methylumbelliferyl N-acetyl-β-D-glucosaminide . Total release (100%) was obtained when cells were treated with 10% of Triton X-100, and mediator release of DPLPs is given as a percentage.Mediator release of purified DPLPs from eference . RBL-2H36) were generated by transient cotransfection with HLA-DRA*01:01, HLA-DRB1*01:01, CD80::GPI, CD54::GPI, and cathepsin S  and then pre-incubated at 5 × 104/well in 96-well flat-bottom culture plates with indicated concentrations of purified DPLP proteins (range: 6–0.01 × 10−6 M), Art v 125–34 peptides (range: 96–0.01 × 10−6 M), or medium alone in a total volume of 100 µl for two hours. Art v 1 TCR transgenic (tg) Jurkat T cells expressing an IL-2 enhancer/promoter-driving luciferase were cultured as described . Antigen5) were added to the artificial APCs and cocultured for 6 h. PMA (10−7 M) plus PHA (5 µg/mL) served as a positive control, and medium alone was a negative control. Cells were lysed, and luciferase activity was determined  on an Infinite M200 luminometer .Subsequently, TCRtg Jurkat cells (1 × 10n = 5) with specific IgE to Art v 1 and other relevant sensitizations were pooled. Fifteen microliters of sera were pre-incubated with an equal volume of Artemisia spp. pollen extract (0.5 mg/mL) and incubated overnight. IgE binding to Art v 1, nsLTPs , pectate lyases , profilins  and polcalcins (Bet v 4 and Phl p 7) on an ISAC 103 microarray  were evaluated, comparing samples incubated with pollen extracts and buffer as a control. Results are reported as percentage of IgE inhibition. Nonspecific IgE inhibition was evaluated by results obtained from other nonrelevant allergens.Single-point highest inhibition achievable assays (SPHIAs) were performed, as previously reported . BrieflyArtemisia spp. extracts was performed using a one-way ANOVA and Tukey’s multiple comparisons test. An analysis of purified DPLPs was conducted using the Friedman test and Dunn’s multiple comparison test: p-values < 0.05 were considered statistically significant.ELISA analysis of Artemisia species differently spread throughout the world was selected for this study . Thus, a forward primer corresponding to the first eight amino acids of the mature protein was used to obtain the cDNA sequence. As allergenicity of the molecules was subsequently demonstrated, they were officially acknowledged by the WHO/IUIS allergen nomenclature subcommittee with the following allergen names (GenBank entries in brackets): Art ab 1 (KC700032.1), Art an 1 (KC700033.1), Art c 1 (KC700034.1), Art f 1 (KC700035.1), Art l 1 (KC700036.1), and Art t 1 (KC700037.1). Obtained clones showed highly similar DNA sequences, with identities ranging from 95.4% to 100% . Those sequences translated into amino acid sequences of at least 95.4% identity and seven residues that were subject to variations in comparison to Art v 1.0101. Identical protein sequences were obtained for the DPLPs from h domain . InteresArtemisia species was evaluated in an ELISA using the sera of 11 mugwort pollen-allergic patients sensitized to Art v 1  were used [25–34 peptide, dose-related responses were observed ). IgE reactivity to Art v 1 was entirely inhibited by all Artemisia spp. extracts. In addition, very high IgE cross-inhibition to Art v 3, the nsLTP of A. vulgaris, was observed, and to a lesser extent to representatives from hazelnut (Cor a 8) and peach (Pru p 3). IgE cross-inhibition to the pectate lyase Amb a 1 was high in A. annua and A. vulgaris, but only moderate regarding residual pollen extracts. Negligible cross-reactivity with pectate lyses from Japanese cedar (Cry j 1) and cypress (Cup a 1) was noticed. Regarding the panallergens profilin and polcalcin, diverse patterns were observed, ranging from high IgE cross-reactivity to moderate/low cross-reactivity, especially when A. californica extracts were used as an inhibitor. To evaluate the relevance of allergens belonging to families other than the DPLP family, ISAC inhibition studies were performed . TherefoArtemisia species has focused on A. vulgaris, which grows in temperate climate zones and is known to cause allergic reactions in late summer and autumn. In this study, we extended our allergological research to additional species, i.e., A. absinthium , A. frigida , A. annua (cultivated for artemisinin production), A. californica, and A. tridentata (specific occurrence in some parts of the United States). For comparison and due to the fact that our patient cohort was primarily sensitized to A. vulgaris, its pollen was included as a reference. Those representatives are, e.g., termed mugwort, wormwood, and sagebrush, and several are used as herbal medicines and for treatments for malaria [Artemisia are present in many regions of the world, and due to climatic changes, expansion to geographic regions beyond the current habitats is expected [Pollens from weeds represent important contributors to allergic reactions. The Asteraceae plant family comprises relevant allergenic plants such as mugwort, ragweed, feverfew, and sunflower. So far, molecular allergology in the various  malaria ,10. Arteexpected ,3. Artemisia pollen presented similar migration patterns in gel electrophoresis, with a dominant band migrating around 24 kDa (corresponding to DPLPs). Previously, seven Art v 1 isoallergens were identified, which contribute to different proline and thus varying O-glycosylation patterns [A. absinthium and A. frigida presented analogous DPLP patterns, while others localized in one band. Representatives of this allergen family seem to generally constitute highly diverse glycosylation patterns [We found that aqueous protein extracts of patterns ,23. Conspatterns . A. absipatterns , and furA. californica and A. ludoviciana, which corresponded to a less prominent appearance of the DPLPs in gel electrophoresis. In addition, there was a lack of protein bands in the lower molecular weight region where panallergens are typically detectable. Results from the SPHIA experiments showed a very low IgE inhibition of A. californica toward profilin, indicating a substantially lower amount of this allergen in this extract. Even though extraction protocols were consistent for all pollens and equivalent protein amounts were loaded on the gel, there was a certain degree of variation between the extracts. This is explainable by the different environmental conditions those plants were exposed to. Fluctuation levels of allergens can be explained by the fact that many of them are pathogenesis-related proteins that directly respond to external (stress) factors. This also highlights (again) why the standardization of allergen extracts is a challenging task. To verify the presence of a DPLP, we performed IgE inhibition ELISAs using purified natural Art v 1. Indeed, we observed very high IgE inhibitions up to 90.5%. Interestingly, inhibition against A. vulgaris did not achieve the highest levels, which was explained by further IgE reactivity to additional mugwort pollen allergens, which were not inhibited by Art v 1. The results, however, clearly indicate that Art v 1-homologous molecules represented major allergens in all of the investigated pollen sources.Using sera from mugwort pollen-allergic patients, similar IgE reactivity was observed with slightly lower reaction toward Artemisia species, which were detected by a monoclonal anti-Art v 1 antibody [Artemisia representatives [To this end, we chromatographically purified DPLPs from seven antibody . As thisantibody . As inveantibody . All prontatives ,31. UsinWe further assessed allergen binding and mediator release capacities using the sera of Art v 1-sensitized mugwort pollen-allergic patients. Generally, highly similar IgE binding capacities were observed, and all allergens were able to trigger a significant β-hexosaminidase release, corroborating functional activity at individual and patient-specific release levels. While in the ELISAs reduced IgE binding toward Art t 1 was noted, this interestingly did not translate to lower mediator release capacity in the four tested patients’ sera. The presence of tryptophan or tyrosine at residue 13 did not lead to any significant differences in the IgE binding or mediator release patterns. In fact, the residues shown to be directly involved in antibody binding were S3, K4, S16, R40, E41, E45, S46, K55, and A63, and all of those were conserved in all newly identified sequences. However, glycan moieties might play a role in the Art v 1 fold, as the transitional region in the polyproline-rich region can interact with the defensin-like domain . This mi25–34 [To assess T cell cross-reactivity, we used Jurkat T cells with a receptor specific for the single, immunodominant T cell epitope of Art v 1, located in the sequence Art v 125–34 ,27. Iden25–34 .Artemisia species. For this, we used a serum pool of mugwort pollen-allergic patients with broad allergen recognition and evaluated IgE cross-reactivity using all seven extracts [Artemisia pectate lyases [A. californica could be explained by a low quantity of panallergens, as was observed in the gel electrophoresis (around 14 kDa). In summary, we confirmed the presence of IgE cross-reactive molecules from the DPLP, nsLTP, pectate lyase, profilin, and polcalcin family. The results also demonstrated heterogeneity in terms of protein and allergen amounts. We thus propose the use of purified allergen molecules for diagnosis, as quality and quantity can be standardized [Based on the results obtained with the DPLPs, we evaluated the presence of other IgE reactive molecules in the selected extracts ,46. The extracts . Diversee lyases ,47. It se lyases . In conte lyases . High Igdardized .Artemisia species. Thus, exposure and clinical relevance go beyond the primarily investigated species A. vulgaris. Because of climatic changes and further expansions of weed habitats, an increase in sensitization and symptoms due to Artemisia spp. pollen may be encountered. This study demonstrated the high allergological and immunological similarity of DPLPs in pollen from seven


The promigration and proproliferation effects of MAP4 phosphorylation depended on microtubule rearrangement and could be abolished by MAP4 dephosphorylation. We also identified p38/MAPK as an upstream regulator of MAP4 phosphorylation in keratinocytes. Our findings provide new insights into the molecular mechanisms underlying wound-associated keratinocyte migration and proliferation and identify potential targets for the remediation of defective wound healing.Both cell migration and proliferation are indispensable parts of reepithelialization during skin wound healing, which is a complex process for which the underlying molecular mechanisms are largely unknown. Here, we identify a novel role for microtubule-associated protein 4 (MAP4), a cytosolic microtubule-binding protein that regulates microtubule dynamics through phosphorylation modification, as a critical regulator of epidermal wound repair. We showed that MAP4 phosphorylation was induced in skin wounds. In an aberrant phosphorylated MAP4 mouse model, hyperphosphorylation of MAP4 (S737 and S760) accelerated keratinocyte migration and proliferation and skin wound healing. Data from both primary cultured keratinocytes and HaCaT cells Wound healing is a complex and precisely regulated process that mainly involves the following three phases: inflammation, reepithelialization and tissue remodeling. Keratinocyte proliferation and migration are crucial steps in the reepithelialization process Microtubules (MTs), critical components of the cytoskeleton, play an important role in regulating various processes, including cell migration and proliferation Previously, we reported that p38/MAPK activation could lead to MAP4 phosphorylation with MT disassembly in cardiomyocytes during hypoxia In this study, we aimed to investigate the role of MAP4 phosphorylation in wound repair. We found that MAP4 phosphorylation accelerated wound healing by promoting epidermal cell migration and proliferation. We also showed that the induction of MAP4 phosphorylation in response to wound stimuli was dependent on p38/MAPK, a kinase that had previously been shown to be required for efficient wound healing. Mechanistically, MAP4 phosphorylation promoted cell migration and proliferation by modulating MT dynamics in keratinocytes. Our combined data delineate a critical role for MAP4 and MT dynamics in promoting wound healing.All animal experiments were carried out according to the guidelines of the Care and Use of Laboratory Animals published by the National Institutes of Health  and authorized by the Animal Experiment Ethics Committee of the Third Military Medical University in Chongqing, China.The KI mice were constructed and identified by the Shanghai Biomodel Organism Science & Technology Development Co., Ltd. Cas9 mRNA, guide RNAs and the donor vector were microinjected into fertilized eggs (C57BL/6J) before the eggs were implanted into surrogate C57BL/6J females as described in our previous study For wounding experiments, 8- to 12-week-old male/female KI or littermate control mice were anesthetized with intraperitoneal administration of sodium pentobarbital, and full-thickness wounds were made on the mid-dorsal skin with 5-mm disposable biopsy punches. Digital photographs were taken of the injury site, which was circumscribed and identified by standard-sized dot that was placed beside the wound. Wound size was defined as the ratio of the wound area to the dot measurement. For histological examination, skin was harvested and fixed in 4% paraformaldehyde at the indicated time points: frozen sections were used to detect the phosphorylation levels of MAP4; paraffin sections were stained with hematoxylin and eosin (HE). Rabbit anti-p-MAP4 primary antibody was used for immunofluorescence staining of p-MAP4 in wound specimens.2, and 95% humidity.Immortalized human keratinocyte HaCaT cells were obtained from the Cell Bank of the Chinese Academy of Sciences in Beijing, China and cultured in RPMI 1640 medium  supplemented with 10% fetal bovine serum , 100 U/ml penicillin, and 100 μg/ml streptomycin . Primary mouse keratinocytes (MKs) were cultured as previously reported 2, 5% CO2 and 93% N2 were created by a constant flow of nitrogen using a Forma Series II Water Jacket CO2 incubator , which maintained the desired temperature (37 °C) and O2 level. A p38 inhibitor  was added to the cultures and incubated at 37 °C for 30 min prior to hypoxic treatment.Hypoxic conditions of 2% O2 overnight, explants were submerged in the prepared medium. To inhibit cell proliferation, we subjected skin explants to mitomycin C  48 h after being plated on 24-well plates. Then, cellular outgrowth from each explant was monitored with the Zeiss imaging system , and the proximal distance extending between the explant biopsy and the distal edge of the cellular migration was measured using NIH ImageJ software (http://rsb.info.nih.gov/ij/).Circular skin biopsies were obtained from the dorsal skin of neonatal mice of the same litter using 4-mm biopsy punches and were plated on 24-well plates with 200 μl of medium containing the following additives  The recombinant adenovirus that expressed the constitutively activated mitogen-activated protein kinase 6 (MKK6(Glu)), which selectively and constitutively activates p38/MAPK signaling, was produced by Shanghai GeneChem, Co. Ltd. . The CMV-null adenovirus was used as the negative control.TM system  according to the manufacturer's instructions as reported previously The site-directed mutagenesis of MAP4(Ala) recombinant adenovirus was prepared using the AdMax2, 20% glycerol and 2 mM ethylenediaminetetraacetic acid (EDTA) was used to wash the cells cultured in 6-well plates. Cells were incubated with MTSB containing 0.1% Triton X-100, protease inhibitor cocktail, phenylmethylsulfonyl fluoride, and phosphatase inhibitor cocktail (Sigma-Aldrich). Then, Western blotting was performed to analyze the polymerized and free tubulin fractions.The free and polymerized tubulin fractions were isolated as previously described Whole keratinocyte extracts and mouse skin specimens were prepared in RIPA lysis buffer  for Western blotting and centrifuged at 14,000 rpm for 15 min at 4 °C. The supernatants were then obtained and protein concentrations were detected using the Bradford Protein Quantification Kit . The protein extracts were loaded and separated by SDS-PAGE and then transferred to PVDF membranes (Millipore). The membranes were incubated overnight at 4 °C with specific primary antibodies. Then, membranes were incubated with secondary antibodies and visualized using the ChemiDoc XRS System (Bio-Rad Laboratories). The primary antibodies used for immunoblotting were as follows: p-p38 , p38 , MAP4 , p-MAP4(S768) , p-MAP4(S696) , p-MAP4(S787) , p-MAP4(S737) , β-Actin , PCNA , and Ki67 . The rabbit polyclonal antibodies against p-MAP4(S696), p-MAP4(S787) and p-MAP4(S737) were developed in house as described and validated in our previous report Cells cultured on glass coverslips were fixed in 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100 in phosphate buffer saline (PBS) for 25 min, and then blocked with 10% goat serum for 1 h. To obtain the MT structure, rabbit anti-a-tubulin primary antibodies  were diluted with PBS, and the coverslips were incubated with antibodies at 4 °C overnight. The coverslips were washed in PBS and then incubated with goat anti-rabbit secondary antibody conjugated to cyanine 3  for 1 h at 37 °C. The cells were imaged using confocal microscopy .TM EdU imaging detection kit according to the manufacturer's instructions . EdU is a thymidine analog that can be incorporated to label cells undergoing DNA replication. EdU-positive cells are defined as proliferating cells 5-Ethynyl-2'-deoxyuridine (EdU) is a thymidine analog that can be incorporated to label cells undergoing DNA replication 3/wells of 96-well plates and preincubated for 24 h in a humidified incubator with 5% CO2 at 37 °C before the CCK-8 solution (10 μl) was added to each well of the plate. Then, the cells were incubated for 1 h, and the absorbance was measured at 450 nm with a microplate reader .Cell Counting Kit-8  was used as previously described http://rsb.info.nih.gov/ij/).Monolayers of cells plated in 12-well plates were wounded by a 10-μl plastic pipette tip after being incubated at 37 °C for 2 h with mitomycin C  to inhibit cell proliferation and then rinsed with medium to remove any cell debris 4/cm2 in RPMI 1640 medium. Then, time-lapse imaging was performed with a Zeiss imaging system , which was equipped with a CO2- and temperature-controlled chamber. The images were acquired every 3 min for 3 h. Later, cell trajectories were obtained by tracing the position of the cell nucleus at frame intervals of 6 min using NIH ImageJ software, and the velocity (μm/min) of each cell was defined as the total length (μm) of the trajectory divided by time (min), which reflected cell motility.Cells were seeded into 24-well plates at a density of 0.5 × 10P < 0.05 was considered to be significant.All data are expressed as the means ± SEM. Comparisons between two groups were performed using a two-tailed unpaired t-test. Statistical significance among three or more groups was performed by a one-way analysis of variance (ANOVA). Considering the potential role of MAP4 phosphorylation in the reepithelialization process, we first examined the level of MAP4 phosphorylation at the wound margin following full-thickness skin wounding (5 mm in diameter) in 8- to 12-week-old mice. MAP4 phosphorylation was markedly upregulated at the wound margin compared with the normal epidermis and then fell, with the maximal expression level measured at day 5   induced elevated phosphorylation at both residues and p-MAP4 in a time-dependent manner, with the MAP4 levels unchanged.Previously, we and others determined that hypoxia promoted keratinocyte migration in wound repair 2, 24 h) in both the WT and KI group was dramatically restored by MAP4(Ala) transfectants compared with cells transfected with CMV-null, as assessed by the wound healing assay or single-cell motility assay ). HA-tagged MAP4(Ala) and CMV-null were overexpressed at comparable levels in epidermal keratinocytes as verified by Western blot analysis  resulted in significant MT depolymerization represented by a thin and disrupted residual network in keratinocytes. MAP4(Ala) transfection alleviated the hypoxia-induced MT depolymerization in both the WT and KI. Similarly, Western blots showed that MAP4(Ala) transfection reversed the reduced levels of polymerized tubulin and increased the levels of free tubulin in hypoxic cells from the WT and KI groups  overexpression on the MT arrangement in keratinocytes was first observed using confocal microscopy  in epidermal keratinocytes and found that p38/MAPK was significantly activated from 6 h to 24 h after hypoxic treatment (2% O2)  Fig. A.2). Western blots demonstrated that the MAP4 phosphorylation at both S737 and S760 in the MKK6(Glu) group increased significantly, and SB203580 (5 μM) decreased the MAP4 phosphorylation induced by hypoxic treatment , the persistently activated mutant of MKK6, was used to activate p38/MAPK Fig. B, and a Furthermore, to clarify the role of MAP4 phosphorylation in p38/MAPK-induced keratinocyte proliferation and migration, keratinocytes were transiently transfected with MAP4(Ala), MKK6(Glu) or both. MKK6(Glu)-induced p38/MAPK activation was not influenced by MAP4(Ala) cotransfection Fig. J, which 2) using Western blotting. As shown in Fig. 2) induced a marked phosphorylation of both MAP4 and p38/MAPK in a consistent, time-dependent manner, with MAP4 and p38/MAPK levels unchanged. These data suggested a potential role for p38/MAPK signaling in regulating MAP4 phosphorylation in HaCaT cells under hypoxic conditions.Considering the widely known differences in physiology and pathology between mouse and human keratinocytes, we set out to confirm the above hypothesis with immortalized human HaCaT cells. To this end, we first observed MAP4 phosphorylation and p38/MAPK activation in HaCaT cells after hypoxic treatment  was overexpressed to activate p38/MAPK, and SB203580 (5 μM) was used to inhibit p38/MAPK activation in HaCaT cells  or CMV-null was overexpressed at comparable levels in HaCaT cells and analyzed by Western blotting Fig. I. Then, Moreover, we investigated the role of p38/MAPK-induced MAP4 phosphorylation in MT rearrangement in HaCaT cells. The Western blots showed that MKK6(Glu) induced MT disassembly, while the SB203580 protected against MT depolymerization under hypoxia (supplementary Fig. To enhance the wound healing process, it is essential to understand the molecular mechanisms underlying the steps responsible for wound repair. MAP4 was primarily identified in mouse neuroblastoma cells in 1984 The limited oxygen supply and excessive oxygen consumption after acute injury leads to a hypoxic microenvironment in the wounded tissue MTs are dynamic polymers of tubulin and are involved in multiple functions, including vesicle and organelle transport, spindle formation and cell migration. The balance between depolymerization and polymerization in MTs has been attributed to the actions of cellular factors that destabilize MTs, such as Op18, and the opposing action of factors that stabilize MTs, such as MAPs Another important question addresses which signaling pathway is responsible for MAP4 phosphorylation in epidermal keratinocytes under hypoxia. Accordingly, we found that hypoxia activates the p38/MAPK pathway, which is positively related to MAP4 phosphorylation in hypoxic HaCaT cells and primary mouse keratinocytes. In addition, growing evidence indicates that p38/MAPK is an important pathway involved in diverse biological processes under hypoxia Together, these data suggest a model that defines a molecular genetic axis in wound healing in which p38/MAPK is required for the induction of MAP4 phosphorylation, which leads to MT disassembly to promote wound healing through a cell migration and proliferation mechanism. Our results show a novel role for MAP4 phosphorylation in skin wound healing and provide a potential target for the development of new treatments for defective wound repair.Supplementary figures and tables.Click here for additional data file.


The non-secretory platelet proteome was more severely affected during inhibition than during activation. Strikingly, while VASP reached a 1.3-fold increase in phosphorylation levels in inhibited platelets, other protein kinase A targets showed several-fold stronger inhibition-induced phosphorylation levels, including LIM and SH3 domain protein 1 (6.7-fold), Src kinase-associated phosphoprotein 2 (4.6-fold), and Ras-related protein Rap1b (4.1-fold). Moreover, phosphorylation of integrin-linked protein kinase (ILK) and pleckstrin (PLEK) species was associated with P-selectin surface expression. The discrimination power between activation and inhibition was more pronounced for dephosphorylated ILK (3.79 Cohen’s d effect size) and phosphorylated PLEK (3.77) species than for P-selectin (2.35). These data reveal new insights into the quantitative changes of the platelet reactivity proteome and suggest powerful alternatives to characterise their activation and inactivation potential.There is a need to assess platelet activation in patients with thrombotic disorders. P-selectin and activated integrin αIIbβ3 are usually quantified by flow cytometry to measure platelet activation. Monitoring changes in vasodilator-stimulated phosphoprotein (VASP) phosphorylation is an established method to determine the platelet-reactivity status. To study disruptions of platelet reactivity more comprehensively, we compared the human non-secretory platelet proteome after  In the circulation platelets are preserved in a sensitive balance between upholding adequate blood-flow and promoting blood clotting in the case of bleeding. Upon vessel injury, platelets instantly become activated, leading to granule release, aggregation, and clot formation. Although much is known about factors resulting in platelet activation, the molecular characteristics maintaining platelets in their quiescent state are less extensively characterized. Since dysregulation of platelet reactivity represents such a serious health hazard, it is necessary to explore biomarkers reflecting platelet activation status for their monitoring in health and disease.Platelets represent a decisive factor in (cardio)vascular diseases, the leading cause of death in the industrialised world. Indeed, inappropriate platelet reactivity and subsequent thrombus formation contribute to as many as 610,000 deaths per year in the USAin-vivo triggers for activation include binding of sub-endothelial collagen as well as other platelet agonists, like adenosine-diphosphate (ADP) and thrombin, to their cognate receptors3. At balanced physiological conditions, endothelial cells produce prostacyclin (PGI2) and nitric oxide, thereby keeping platelets silenced. Both mediators induce the synthesis of cyclic nucleotides , which activate protein kinases A and G  in platelets. These serine/threonine-kinases phosphorylate particular proteins to counteract the activation of platelets4. Platelet activation and thrombus formation can, therefore, be regarded as the predominance of either pro-thrombotic factors or a reduction of endogenous inhibitors5. An impairment6 of platelet-inhibition mechanisms, or resistance7 to them, may result in hyper-reactive platelets and spontaneous generation of platelet-dependent thrombosis9. Accordingly, typical platelet activation markers like P-selectin (CD62P) and activated integrin αIIbβ3 (CD41/CD61) are significantly changed under these conditions and are thus used to assess the platelet activation status12. However, whether increased platelet activation results from an endogenous activation or from an insufficient inhibition remains largely unknown.Major in-vitro platelet proteomic studies have shed light on the complexity of signal propagation induced upon activation with thrombin receptor activating peptide-6 (TRAP-6)13, ADP14, collagen-related peptide15 or via the C-type lectin-like receptor 216. Although an insufficient inhibition of platelets is also a significant feature in platelet-related thrombotic disorders, the first proteomic studies addressing this issue have been published just recently18. There, the dynamics of the phospho-proteome was analysed after short-term inhibition via selective stimulation of the sGC/cGMPIß (cyclic guanosine monophosphate/protein kinase GIß) pathway18, cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) pathway17 or with simultaneous activation by ADP19. Nevertheless, a direct comparison of the platelet proteome response upon both activation and inhibition might uncover differential pathophysiological signs for hemostatic disorders. Protein profiles that reflect the activation status may enable us to target platelets pharmaceutically according to the underlying perturbations and thus to identify new therapeutic options.To investigate how the platelet proteome is affected by activation and inhibition in a comprehensive and unbiased manner, proteomics may be applied. Several 20. Especially, the generation of protein species may be of great interest in the proteome of anucleate platelets during activation and inhibition in the circulation.Therefore, the aim of the present platelet proteome study was to reveal specific features of activated, as well as inhibited platelets, compared to their untreated controls within one biochemical method using fluorescence two-dimensional differential gel electrophoresis (2D-DIGE). This proteomics technique enables reliable quantitative results on differential protein expressions by displaying thousands of proteins, their isoforms and post-translational modifications at the same time. Previously, the terminology “protein species” has emerged to describe proteins which arise from the same amino acid sequence but have been post-translationally modified. The related term “proteoform” defines distinct protein forms originating from a single gene24. For platelet activation TRAP-6 (15 µM), was chosen to provoke a strong PAR1-mediated activation response while ADP (5 µM) induced a moderate one. For inactivation we used the unstable PGI2 (0.4 µM) and the more stable CTAD formulation . Both inhibitory treatments especially increase cAMP to prevent platelet activation25.Importantly, the concentrations of platelet activators and inhibitors needed to address these two main regulatory conditions were selected from typical protocols of functional platelet studiesin-vitro treated gel-filtered platelet suspension were extracted by TCA-precipitation26 to “freeze” them in their particular activation status. It was thanks to this method that we quantified the non-secretory platelet proteome changes since the granule release was not separated by this protein extraction protocol. Using 2D-DIGE, most of the affected proteoforms were identified to be specific to the either activated or inhibited platelet status. By quantifying their abundances, some of these proteins were found to be more powerful than established biomarkers in defining platelet reactivity.Proteomic snapshots were taken after 15 minutes of activation/inhibition treatments to reveal long-lasting changes in platelet protein profiles, which may be useful for translation of these biomarkers into diagnostic applications. Notably, instead of the usual centrifugation step, proteins of in-vitro activated and inhibited platelets, CD62P, a surface marker specific for platelet activation, was quantified by flow cytometry. A strong platelet activation (>90% CD62P-positive platelets) was induced by TRAP-6 (15 µM) and a moderate response was provoked by ADP , which is comparable to published data of platelet activation studies28. Accordingly, we found a 7.36-fold and 2.53-fold increase of CD62P-positive platelets, as compared to untreated controls, for TRAP-6 (p = 0.009) and ADP (p = 0.031), respectively  treatment. Inhibited gel-filtered platelets showed a reduced CD62P positivity with 0.67-fold after CTAD- (p = 0.005) and of 0.83-fold after PGI2 treatment (p = 0.016) compared to untreated gel-filtered platelets.To assure that our platelet proteome analysis was assessed from well-defined y TRAP-6 5 µM and tot = CVbio + CVtech)29. In the present study, we determined a median CVtot of 12.3% in the untreated control platelet proteome  from all included volunteers, plus a single CVtot of 5.7% for 14-3-3γ, a low biological variation protein, confirming a solid-technical quality when compared to our previous platelet proteomic studies30.We also monitored the quality of our 2D-DIGE proteome analysis regarding the biological and technical coefficient of variation   Fig. . In contts) Fig. . In summts) Fig. . Hence, To uncover the plethora of proteome changes seen in inhibited platelets compared to activated ones, the differentially regulated protein spots were identified by mass spectrometry. These results showed that several identified proteins appeared in more than one spot, giving a total of 31 distinct proteins Tables . These p2 and CTAD-treated platelets were treated with λ-phosphatase (λ-PPase)31. Phosphorylated proteins lose their phospho-groups after λ-PPase treatment (treated + PPase/treated < 0.8), thereby causing a pI-shift towards an alkaline pH accompanied by an increase in the protein abundance of the corresponding “native” protein spots (treated + PPase/treated > 1.2). Using this approach, we were able to identify phosphorylation of protein species due to pI-dependent abundance changes  was applied to identify the most probable underlying biological processes. Knowledge-based Ingenuity Pathway Analysis alongside literature research revealed that one key node of this network was PKA upon inactivation. Since almost all proteins with the strongest changes in abundance  belonged to the cAMP/PKA-pathway  indicating a sequence and different combinations of multi-site phosphorylation events. A hypothesis for VASP multisite phosphorylation chronology is delineated in Supplementary Fig. Protein spot identities and phosphorylation profiles of VASP and LASP1 were further characterised using pan and phosphosite-specific antibodies by 2D Western blots. The pan VASP antibody detected spots at five pI positions in the untreated control Fig.  and at sets Fig. . Thus, a2 treatment (pI 6.15 and 6.34)  induced PKA phosphorylation were verified by the phosphosite-specific antibody pLASP-Ser146  Fig. . These t146 Fig. . No signing Fig. . Phosphoing Fig. .). Thus, potential PKC-mediated phosphorylation of these spots could also be identified by λ-PPase treatment  pathway . CTAD treatment, on the other hand, did not significantly change the abundance of phosphorylated VASP protein species  , SKAP2 , RAP1B , and ZYX  (Table 146 (pI 6.34), RAP1B (pI 5.64) and SKAP2 (pI 4.46), these changes were still present additional 25 minutes after PGI2 addition to platelet rich plasma and gel-filtration. . Likewise, these quantitative differential phosphorylation profiles were also apparent at the expense of the corresponding unphosphorylated proteoforms. For example, the FC of unphosphorylated VASP spot at pI 8.76 was 0.60 after PGI2 treatment while LASP1 (pI 6.60) exhibited an FC of 0.32  Table  and flow2) Table , which i32 Table . Thus, wFinally, we were also interested in identifying platelet protein markers for activation and comparing them to the established activation marker CD62P.After ADP or TRAP-6-mediated platelet activation, a considerably lower number of seven significantly changed protein spots were detected in the non-secretory proteome compared to platelet inhibition Fig. . Howeverin-vitro activation and inhibition, we attempted to uncover discriminative signs for these modulations in platelet activation state. Here, we expose for the first time that platelet inhibition was associated with more pronounced alterations in the non-secretory platelet proteome as compared to activation. Moreover, we showed that these changes were mainly caused by phosphorylation, suggesting a predominant role of this post-translational modification in maintaining platelets in their resting state. Novel diagnostic options were revealed, since PGI2 or CTAD inhibited platelets showed stronger phosphorylation changes in PKA targets than the benchmark platelet reactivity marker pVASP. Additionally, phosphorylated species of PLEK and ILK were associated with CD62P surface expression and showed an even larger effect size than this established activation marker.Increased platelet reactivity may result in thrombosis and other vascular diseases. By investigating the platelet proteome after 17, are not captured based on our study design. On a physiological basis, the disparity between inhibition and activation induced changes in the proteome could be due to platelets being more responsive to inhibitory stimuli. Potent platelet inhibition is constantly needed in vascular system due to the exposure to shear stress, acting as a strong platelet activator33. Shear stress is permanently present under physiological conditions and is much more pronounced in pathophysiological conditions, e.g. carotid or coronary vessel stenosis35. Accordingly, the present findings demonstrate that the addition of PGI2 or CTAD, to obtain resting platelets, strongly influences the non-secretory platelet proteome. Thus, after inhibition, 15 out of 26 proteins were altered in their phosphorylation levels. Knowledge-based pathway analysis demonstrated that the unbiased 2D-DIGE proteomics technology specifically accentuated the cAMP-induced PKA-signaling pathway in PGI2 or CTAD-treated platelets. Finally, we characterised multiple proteoforms at seven pI positions of the well-known PKA phosphorylation target VASP – a negative regulator of platelet activation36 and a clinically established platelet reactivity marker38. Remarkably, CTAD-treated platelets did not show a significant phosphorylation in any 2D-DIGE detected VASP species. Intriguingly, other spots showed substantially stronger abundance changes in their phosphorylation levels than phosphorylated VASP species: LASP1 6.7-fold, RAP1B 4.1-fold, SKAP2 4.6-fold, ZYX 1.7-fold, and RGS18 1.5-fold compared to VASP with 1.3-fold. Previously, a 6.7-fold stronger phosphorylation of RAP1B compared to VASP was reported in platelets treated with cAMP-elevating reagents39, which supports the reliability of our data. This latter study showed that VASP phosphorylation was reversible with maximum phosphorylation after 10 minutes, dropping to baseline after 60 minutes. Conversely, RAP1B phosphorylation was irreversible with constant levels over 2 hours39. Beyond RAP1B, the stability of PGI2- and CTAD-induced changes in LASP1 and SKAP2 phosphorylation was confirmed for at least 40 minutes in our study  in comparison to phosphorylated pVASP-Ser239. Phosphorylated peptides from RAP1B were not detected and RGS18 phosphorylation decreased. However, these discrepancies regarding the phosphorylation degree of these PKA targets may be related to their earlier measuring points  in addition to the completely different methodology employed. Compared to the number of 26 inhibition-affected platelet proteins detected in our study, a considerably higher number with 300 altered phosphorylated proteins from iloprost-inactivated platelets was described in the study by Beck et al.17. This higher count of identified potential PKA targets was achieved since the used TiO2 affinity-based method enriches phospho-peptides and enables the detection of phosphorylated low-abundant proteins17. With this enrichment step, however, a direct quantitative assessment of phosphorylation events compared to the global platelet proteome is lost17. Our 2D-DIGE snapshot, on the other hand, displayed the relationship of protein phosphorylation to the overall proteome and, thereby also highlights the specificity of platelet inhibition pathways.The phosphorylated protein species of LASP1, RAP1B and RGS1846, our experimental design could only measure non-secretory proteome changes but cannot dissect alterations based on granula and microvesicle release. However, our statistical stringent platelet proteome analysis by Benjamini-Hochberg correction showed in total only seven protein spots changed after platelet activation. Without correction for multiple comparisons (FDR), we would have detected 32 additional proteins upon ADP- and TRAP-6 activation. Nevertheless, these changes in spot counts are quite comparable to the explorative 2D-DIGE based proteomics study of gel-filtrated platelets from Schulz et al. with 13 differentially regulated protein spots after activating the glycoprotein VI receptor47. In this study, the degree of platelet stimulation was also quantified by CD62P upregulation47 and is comparable to our in-vitro platelet activation CD62P profiles. Evidence for the specific stimulation of platelet activation pathways in our study is the phosphorylation of well-known PKC substrates such as PLEK and MYL9, proteins that were also detected in most of the published 2D gel-based proteomics studies of in-vitro activated platelets48. Recently, Beck et al.19 characterized 302 phosphorylated proteins following ADP-treatment with kinetics between 10- and 60 seconds. Despite the high sensitivity of TiO2 enrichment, some important PKC substrates in platelet activation, such as MYL9, have not been detected19. These differences in results indicate that each technical proteomic approach has distinct strengths and limitations. For the present proteomics study by 2D-DIGE analysis the following limitations need to be considered: under-representation of low abundant proteins, very hydrophobic proteins and proteins with a high molecular weight. However, 2D-DIGE analysis also provides several advantages like highly reliable qualitative and quantitative accurate profiling of differentially abundant proteins by the inclusion of an internal standard which enables a low technical variation with a CV of 7%29. Finally, only protein-based “top-down” proteomics as 2D-DIGE can provide reliable quantitative results from different proteoforms49. For LC-MS/MS mass spectrometry-based proteome analysis tryptic digestion of the protein samples is necessary but causes a loss of information for the correct assignment of peptides and post-translational modifications to the appropriate proteoform49.Despite a prominent CD62P surface upregulation, the actual 2D-DIGE based analysis revealed unexpected weaker changes in the non-secretory proteome of activated platelets compared to inhibited platelets. In contrast to previous studies investigating the activated platelet proteome by two-dimensional gel electrophoresisi, the central enzyme in the inhibitory cAMP/PKA pathway50. PKC-dependent phosphorylation of PLEK is a sensitive marker for platelet activation51. Although ILK is described as a substrate for PKC and PI3K52 to induce platelet adhesion53, we found an explicit dephosphorylation pattern for ILK (pI 7.10) in activated platelets that has not been reported before. Despite a potent effect size of 2.35 (Cohen’s d) for the established platelet activation marker CD62P, the quantitative discrimination power between activation and inhibition was even more pronounced for phosphorylated PLEK  and ILK  species. As platelet CD62P expression may decrease in severe clinical states of platelet activation54, potentially due to receptor- and microvesicle-shedding and/or platelet adhesion to endothelial cells and leukocytes, phosphorylated ILK and PLEK species may represent further more robust diagnostic targets for platelet activation.In the present study, PLEK (pI 6.60) and ILK (pI 7.10) species were the only two proteins that were altered by both platelet activation and inhibition. Apart from that, a pronounced overlap in the affected proteome was not found. This seems plausible since ADP and thrombin inhibit adenylyl cyclase activity via GThese stronger phosphorylation changes of the non-secretory proteome from inhibited compared to activated platelets may provide fundamental new insights into quantitative functional regulations of platelets. From there, new and more robust biomarkers for platelet inhibition and activation as well as new therapeutic options could follow, which may enable to treat abnormal platelet function more targeted in accordance with underlying pathophysiological mechanisms.51. Blood tubes were centrifuged at 120 xg for 20 minutes to obtain platelet-rich plasma (PRP). A graphical experimental design for in-vitro treatments is displayed in the Supplementary Fig. 2  before gel-filtration, while PRP from CTAD blood tubes was left untreated. A PGI2 concentration of 0.1–0.5 μM is normally used to generate washed platelets24. To gently purify platelets from plasma proteins by size-exclusion chromatography25, 1 ml PRP was applied onto a 12 cm sepharose-2B packed column pre-equilibrated and eluted in PBS buffer. Gel-filtered platelets were caught at the end of the column and precipitated by mixing 3:1 (v/v) with 6.1 N TCA solution (containing 80 mM DTT). Thus, the inhibition of platelets by PGI2 was stopped after altogether 15 minutes and by CTAD after 40 minutes. Alternatively, PGI2 was added not to the PRP, but to the gel-filtered platelet suspension, which induced comparable changes in the proteome as seen in PRP + PGI2 , who had not taken any antiplatelet medication for at least 14 days prior to blood sampling. Individuals had a 1:1 gender ratio and were all normal weight (BMI 23.6) non-smokers. The study was approved by the Ethics Committee of the Medical University of Vienna (1076/2016). Each participant was advised of the purpose of the study and written informed consent was obtained prior in accordance with the principles of the Declaration of Helsinki. Blood was drawn from the antecubital vein (21 G needle) without stasis into sodium-citrate tubes (0.129 mM citrate) as well as CTAD tubes , a mixture of cAMP-elevating agents. These commercially available CTAD blood tubes are used to prevent platelet activation during blood sampling and subsequent processing steps29. Briefly, the TCA-precipitated and acetone-washed platelet proteins were re-solubilised in urea-sample buffer  and fluorescence-labeled prior to 2D-separation. For the normalisation process, an internal standard (IS) of platelet proteins from all treatment conditions was prepared. This IS, labelled in Cy2 and added to every 2D-gel, enables a highly accurate qualitative and quantitative comparison of all samples and treatments53. The 2D-DIGE analysis was performed in the pH-ranges 4–7 and 6–9, respectively. For the acidic side, 36 μg protein  were passively rehydrated on 24 cm pH 4–7 IPG-Dry-Strips in a solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 70 mM DTT and 0.5% ampholyte pH 4–7. For the alkaline side, 24 cm pH 6–9 IPG-Dry-Strips  were soaked in a rehydration solution containing 7 M urea, 2 M thiourea, 4% CHAPS, 150 mM DTT and 2% ampholytes pH 6–9 prior to isoelectric focusing. Afterward, 3 × 12 μg of labeled protein samples were applied by “cup-loading” via the acidic side and the isoelectric focusing was carried out until 30 kVh was reached. The following SDS-PAGE was performed with an 11.5% acrylamide gel at 35 V for 1 hour, 50 V for 1.5 hours and 110 V for 16.5 hours at 10 °C in an Ettan DALTsix electrophoresis chamber . The gels were scanned afterward with a resolution of 100 μm using a Typhoon 9410 imager . The experimental study design of the 2D-DIGE analysis is delineated in Table Resolubilisation of precipitated proteins, determination of protein concentration, preparation of the internal standard and labelling with fluorescent cyanine dyes  were performed as described previously8. Spots differentially regulated on the basis of 2D-DIGE analysis were excised with a scalpel, destained, reduced, alkylated and tryptically digested. Peptides were applied onto a Dionex Ultimate 3000 RSLC nano-HPLC system (Thermo Scientific) equipped with a pre-concentration and desalting cartridge where 0.1% TFA was used as transport liquid. Peptides were separated on a Acclaim PepMap RSLC column  through elution with a 59 minute linear gradient from 4 to 45% of solvent B using flow rate of 500 nL/minute ) and directly subjected to the mass spectrometer. In-line bottom-up proteomics was performed on a QqTOF mass spectrometer oTOF compact from Bruker Daltonics  equipped with nano-flow CaptiveSpray ionization device and using the oTOF control software v3.4 (build16). High mass accuracy was achieved by using an internal calibrant -phosphazine (Agilent Technologies)) during the runs. The cycle time for MS1 and MS/MS fragmentation was 3 seconds, scan range for precursor recording was set to 50−2,200 m/z, spectra rate was set to 2 Hz, dry gas was set to 3 L/minute and the dry temperature was set to 150 °C. The peptide assignment to proteins was conducted with the Proteinscape software v.3.1.5 474  which used the Mascot algorithm version 2.5  and peak lists generated with the Compass Data Analysis software v4.2 . Parameters for the protein search were: enzyme specificity: trypsin; species: human; database: UniProtKB/Swiss-Prot; peptide tolerance ± 10 ppm; MS/MS tolerance ±0.05 Da; number of considered 13 C atoms 1; charge states 1 + –3+; up to two missed cleavage; fixed modifications carbamidomethylation (C), variable modifications: deamidation , oxidation (M), phosphorylation , acetylation . The search algorithm probability score was set at p < 0.05.For MS-based identifications, 250 µg unlabeled proteins were separated on a preparative two-dimensional gel. Proteins were visualised by MS-compatible silver staining30. Briefly, precipitated gel-filtered platelets aliquots were resolubilised in lysis buffer , followed by a sonication step , overnight incubation , and determination of the protein concentration using a Coomassie Plus assay kit . Subsequently, two aliquots à 200 µg of each sample condition (PGI2/CTAD) were mixed with 5 µl of 10% SDS. Thereafter, samples were filled up to 500 µl with a reaction mix containing 5 mM DTT, 2 mM MnCl2, and 1 × λ-PPase buffer. One PGI2 and CTAD aliquot were incubated overnight with 100 units of λ-PPase . Subsequently, all samples (±λ PPase) were precipitated with TCA/DTT.To determine phosphorylated protein species, we employed λ-PPase treatment as described previouslyin-vitro platelet inhibition and activation, 2D-DIGE image analysis, flow cytometry analysis, VASP phosphorylation assay, biological pathway analysis and Western blot analysis is provided in the Supplemental Material and Methods.Detailed description of blood sampling, platelet isolation, 2, CTAD, ADP, TRAP-6) and the untreated control group. Protein spots were considered relevant if 1) spots were matched in more than 90% of all 2D images (n = 24) and 2) if FC were >20% as detected between untreated control and at least one of the intervention groups PGI2, CTAD, ADP, TRAP-6. After this filtering, 495 of 5,906 protein spots in the pH range 4–9 were considered relevant. Log-transformed data of 495 spots were evaluated by a repeated ANOVA conducted as a linear mixed model with “group”  as repeated factor. The multiplicity was controlled by the Benjamini-Hochberg procedure (FDR < 0.05). For a direct comparison of different parameters the effect size was calculated by Cohen’s D = (mean1-mean2)/standard deviationpooled. SPSS Statistics 20.0  was used for the statistical analysis. Unless stated otherwise, all fold changes represent means.Fold changes (FC) of standardised protein values (treatment/IS) were calculated between intervention groups (PGISupplementary Informations


Cannulated screws with an anterior wire are currently used for managing transverse patellar fracture. However, the addition of anterior wiring with various types of screws via open surgery to increase the mechanical stability is yet to be determined. Hence, this study aimed to compare the mechanical behaviors of a fractured patella fixed with various screws types and at various screw locations with and without the anterior wire. The present study hypothesized that using the anterior wire reduces the fracture gap formation.A finite element (FE) model containing a fractured patella fixed with various types of cannulated screws and anterior wiring was created in this study. Three types of screws, namely partial thread, full thread, and headless compression screws, and two screw depths, namely 5 and 10 mm away from the anterior surface of the patella, were included. The effect of the anterior wire was clarified by comparing the results of surgical fixation with and without the wire. Two magnitudes and two loading directions were used to simulate and examine the mechanical responses of the fractured patella with various fixation conditions during knee flexion/extension.Compared with partial thread and headless compression screws, the full thread screw increased the stability of the fractured patella by reducing fragment displacement, fracture gap formation, and contact pressure while increasing the contact area at the fracture site. Under 400-N in the direction 45°, the full thread screw with 5-mm placement reduced the gap formation by 86.7% (from 2.71 to 0.36 mm) and 55.6% (from 0. 81 to 0. 36 mm) compared with the partial thread screw with 10-mm placement, respectively without and with the anterior wire.The anterior wire along with the full thread screw is preferentially recommended for maintaining the surgical fixation of the fractured patella. Without the use of anterior wiring, the full thread screw with 5-mm placement may be considered as a less invasive alternative; however, simple screw fixation at a deeper placement (10 mm) is least recommended for the fixation of transverse patellar fracture. Patellar fracture accounts for 1% of all skeleton fractures , with anDue to the inherent design and enhanced strength, a screw with threading works better than the smooth Kirschner wires as a supportive device while preventing fragment separation , 15. RecOn the other hand, to create an effective tension band, the anterior wire in a figure of eight is often applied via an open approach which may increase the surgical trauma and compromise the following biological healing. Although fixation using anterior wire increases the stability of the fractured patella more substantially than that without anterior wire, a less invasive fixation alternative using screws without the anterior wire was thus proposed based on the supporting strength from screws. Without the anterior tension wiring, these minimally invasive surgeries (MIS) were expected to reduce early postoperative pain, results in higher mobility angles of the injured knee, and decreases the incidence of complications . HoweverIn addition to the mechanical stability, the contact status including the contact area and pressure at the fracture site is a concern critical for surgeons to choose the suitable fixation strategy. However, it is difficult to determine this issue under current laboratory setting due to the difficult embedding minute sensors without disruption the bone and wires. To solve the mentioned problem, the finite element (FE) analysis, a numerical method that does not require sensors, provides a practical method for calculating the contact pressure and area as well as internal stress –22. By uAn FE model comprising a fractured patella fixed with various types of cannulated screws with and without the anterior wire was developed based on our previous model . FurtherFirst, an intact patellar model was created on the basis of computed tomography images obtained from the Visible Human Project of the National Institutes of Health . These images were taken at 1-mm intervals. The bony contours in each image were retrieved by thresholding gray values by using Avizo . A 3D bony model was rendered using the retrieved bone contours. Subsequently, the model was imported into the CAD software SolidWorks 2014  for fracture creation and screw and wire implantation. In addition, based on the image, a 1-mm sheet was sectioned from the tail edge of the patella to serve as cartilage. A virtual transverse fracture without any gap was created at the midline of the patella (AO/OTA 34-C1 classification). Subsequently, three types of cannulated screws, namely partial thread, full thread, and headless compression screws, were created based on commercial screws  and were then inserted from the apex to the base of the fractured patella through “Boolean Operation” in SolidWorks 2014. Two screws were parallelly placed at the middle third of the patella and at 5 or 10 mm away from the anterior surface of the patella  to simulate patella loading during knee flexion/extension. High-order quadratic tetrahedral elements were used to mesh all parts, including the bones, screws, and wire. The mesh density of the screws and wire was locally refined by reducing the length of the element edge by using the command “sizing” in Workbench. The lengths of the global element edge and the refined element edge (the wire and screw) were set to 0.5 and 0.25 mm, respectively. The contact behaviors between the bone and screw, screw and wire, wire and bone, and bone and bone (fracture site) were all set to frictional surface to surface contact (contact 174 and target 170 in ANSYS Workbench), and the frictional coefficients of metal-to-metal, metal-to-bone, and bone-to-bone were set to 0.2, 0.3, and 0.45, respectively . To simuMaterial properties of the bone were defined from literature. The elastic moduli of cortical bone, trabecular bone, and cartilage were set to 1000, 207, and 50 MPa, respectively. Their Poisson ratios were set to 0.3, 0.3, and 0.45, respectively , 26. TheA tension force was applied to the apex of the patella to simulate the worst case condition of the patella during knee flexion/extension. Loading with two magnitudes was applied because the fractured patella fixed without the anterior wire would not be able to take the same load as the fractured patella fixed with the anterior wire. Therefore, 800-N loading force was applied to the fractured patella fixed with the anterior wire, whereas half loading (400 N) was applied to the fractured patella fixed without the wire –29. The To validate the present patellar FE model, the results of the fractured patella fixed with a partial thread cannulated screw without the anterior wire were compared with the results of Dargel’s experimental test . In addiIn a previous mechanical test of a fractured patella fixed with a metallic implant, the relationship between gap formation and the applied load was analyzed using an optical tracker , howeverAmong the three screw types investigated in this study, the full thread screws helped to achieve the smallest gap formation and fragment displacement, particular in the setting of a deep screw placement (10-mm) and the absence of the anterior band wiring Fig. . Additio2 in the full thread screw and partial thread screw, respectively. The full thread screw yield lower contact pressure at the fracture site than the partial thread screw, particular with 10-mm screw placement and without the anterior wire  for the mechanical stability and contact area and pressure when the transverse fractured patella was fixed using screws and a wire. Moreover, the anterior wire is critical to maintain the mechanical stability of the fractured patella when screws were placed far away from the anterior surface of the patella; particularly the partial thread screw was chosen. However, the contribution of the anterior wire to stability became less prominent with the decreasing of the screw proximity. These results provide a biomechanical basis for clinicians to make appropriate decision to treat the transverse patellar fractures with MIS or open techniques based on the devices available.The fractured patella fixed with the screw and wire showed different deformation modes under different loading directions. When the load was applied parallel and close to the screws (5-mm proximity), the whole construction sustained an axial traction force and the fragments separated from each other. In this situation, the full thread screw helps to prevent the fragment from sliding against the screw shaft and led to smaller displacement than the partial thread screw. When the screw was placed far away (10-mm proximity) from the loading site, the loading force generated a torque to the screw, and the fragment demonstrated rotation. When the loading force was applied 45° to the long axis of the patella, both a torque and an axial traction force developed from the loading on each screw location and screw type.Although more and different screw types were considered in this study, screw proximity affects the stability of the fixation of the fractured patella, including gap formation and fragment displacement. This finding is consistent with the result of our previous study, though only partial thread screw was used in that work: the screw located near the anterior surface of the patella provided higher stability than that located near the posterior surface (the articular surface) ; the lowThe anterior wire is important for maintaining the reduction of the fractured patella and contact at the fracture site to prevent separation when the screws are placed at 10 mm and sustain a torque (under a loading force in a direction 45° to the long axis of the patella). While the stabilizing effect of the anterior wire on the fractured patella decreased with the screw proximity to the anterior surface of patella. Inserting the anterior wire at the edge of the patella and on the tension side can efficiently reduce the migration of the fractured patellar fragment when it rotates. In addition, such wire insertion increases the moment of inertia of the fractured patella fixed with screws and wire because of its distant placement from the rotation center; subsequently, stability increased. Thus, once surgeons choose the use of deeper screws (10-mm placement) to fix the transverse patellar fracture without anterior wiring, which means the screws are located closer to the rotation center without any resisting device against the bending moment on the patella. Hence, fragment displacement as well as the gap formation becomes obvious, and the contact area decreases under this fixation. When the screw is with 5-mm placement, it shows higher resistance ability to the bending load than the screw with 10-mm placement because of the higher moment of inertia.In clinical practice, partial thread screws are frequently used to fix the transverse patellar fractures; and the bone fragments slide along the middle shaft of the screw when a force is applied to the patella, resulting in the formation of gap. By contrast, with the use of full thread cannulated screw, the pass of shaft with full threads through the fracture line may reduce the sliding of the bone fragments along the screw; hence, gap formation and fragment displacement obviously decrease. The effect of full threading of screw on the mechanical stability becomes obvious in the setting of deep screw placement and without the anterior wire. Because of no wire sharing the loads, the implanted screws sustain all the loading forces on the fractured patella. Similar to the anterior banding wire, the effect of the screw threading on the stability decreases with the screw proximity to the anterior surface of patella.The present study has some limitations. First, only the patella bone was modeled, but its articulation with the distal femur condyles was not. Thus, the sliding of the patella on the distal femur was not simulated. Second, in this study, just one fracture type was considered in the present simulation. The results with different fractures types may change. Third, the elastic modulus of bone was simplified as linear elastic, isotropic, and homogeneous, and the morphology of the trabecular bone was not considered. Fourth, both the headless compression and full/partial thread screw was set as a uniform thread shape at the screw shaft; however, this simplification helps to excluding the disturbance from thread shape.Recently, MIS techniques had been suggested with fewer healing problems than conventional open ones in the literature , 31 focuTo surgically treat a transverse patellar fracture, all three factors, the screw type, proximity to the anterior surface and the use of anterior wiring, contribute the mechanical stability of internal fixation. Based on our results, the use of anterior wire along with the full thread screw is preferentially recommended for maintaining the reduction of the transverse patellar fracture. To reduce the surgical trauma during the open anterior wiring, the use of full thread screws at a 5-mm placement is suggested as a MIS fixation alternative based on the similar biomechanical behaviors under loads. At all events, the deep placement of screws without the anterior banding wire is least suggested for the surgical fixation in transverse patellar fractures.