In an alternative process, azure A or toluidine blue O were grafted to poly(acrylic acid), which was subsequently grafted to nanofiber-based and microfiber-based fabrics. The fabrics grafted with photosensitizers induced antifungal effects on all seven types of fungi in the order of rose bengal > phloxine B > toluidine blue O > azure A, which follows the quantum yield production of singlet oxygen for these photoactive dyes. Their inhibition rates for inactivating fungal spores decreased in the order of P. cinnamomi, T. viride, A. niger, A. fumigatus, C. globosum, P. funiculosum, and M. grisea, which is associated with lipid composition in membrane and the morphology of fungal spores. The antifungal activity was also correlated with the surface area of fabric types which grafted the photosensitizer covalently on the surface as determined by the bound color strength.Indoor fungi growth is an increasing home health problem as our homes are more tightly sealed. One thing that limits durability of the antifungal agents is the scarcity of reactive sites on many surfaces to attach these agents. In order to increase graft yield of photosensitizers to the fabrics, poly(acrylic acid- Candida albicans for up to 28 days [C. albicans. Furthermore, various PS have been shown to be effective against fungi and yeast such as Aspergillus fumigatus and Kluyveromyces fragilis, Kluyveromyces marxianus and C. albicans [Antimicrobial fabrics based on natural or synthetic polymers treated with silver ions and copper nanoparticle have been used often ,2. Recen 28 days . However 28 days . Hence,  28 days , since t 28 days . Further 28 days . Azure Aalbicans . Hong analbicans . However0 which means that there are no unpaired electron spins [1 or S2, with higher energy [1 or T2, via intersystem crossing, which involves a change in the spin of an electron [Photodynamic therapy (PDT) is a treatment with PS, which can be activated by exposure to visible light in a specific wavelength range. PS has a stable electronic configuration, which is in a singlet state in their lowest or ground energy level, Son spins . Upon abr energy . The exielectron . The tri1O2) is the first excited electronic energy state of molecular oxygen species. It is one of the most active intermediates involved in chemical and biochemical reactions. Hence, 1O2 can react with many kinds of biological molecules, such as DNA, proteins and lipids, resulting in chemical reactions due to the production of this reactive oxygen species [Singlet oxygen  are plant pathogens inducing, respectively, rice blast resulting in serious crop loss globally [Eucalyptus [y humans ,17. Magnglobally  and rootcalyptus .In this study, photosensitizers rose bengal (RB), phloxine B (PB), azure A (AA), and toluidine blue O (TBO), were either copolymerized with acrylic acid or grafted to poly(acrylic acid), and then grafted to fabrics consisting of nanofiber-based fabrics or microfiber-based fabrics. The antifungal effects of the fabrics were then evaluated against seven types of fungi and analyzed antifungal effect.MV ~450,000), 4-vinyl-benzyl chloride, 4-styrene sulfonic acid, acrylic acid (AC), nylon 6,6 pellets, formic acid (reagent grade > 95%), phloxine B (PB), azure A (AA), toluidine blue O (TBO), potato dextrose agar (PDA), potato dextrose broth, phosphate buffer saline (pH 7.0), Tween® 20, RPMI 1640 medium, sterile water, 3-(N-morpholino)-propanesulfonic acid (MOPS), and 4--4-methylmorpholinium chloride (DMTMM) were purchased from Sigma Aldrich Chemical Co. . RB and meltspun nylon 6.6, Cerex Spectramax® were donated by LaamScience , while 0.5 McFarland standard for turbidity measurements was purchased from Fisher Scientific Co. . Aspergillus niger (ATCC 6275), Trichiderma viride (ATCC 28020), Penicillium funiculosum (ATCC 10509), Aspergillus fumigatus (ATCC 13073) and Chaetomium globosum (ATCC 6205) were donated from the United States Department of Agriculture, Agricultural Research Service, Peoria, IL, USA. Magnaporthe grisea and Phytophthora cinnamomi (fungus-like organism) were donated from the North Carolina State University, Plant Pathogens Department, Raleigh, NC, USA.Poly(acrylic acid) , was filtered and washed. Next the VBXD was dissolved in water and acetone at the volume ratio of 1:1 and polymerized with 4-styrene sulfonic acid (SAA) and acrylic acid (AC) at 1:40:140 molar ratio of VBXD: SSA:AC to increase water solubility to the polymers and to confer functional groups with which to graft to nylon [co-styrene sulfonic acid-co-vinyl benzyl rose bengal or phloxine B) were obtained. A condensing agent, 4--4-methylmorpholinium chloride (DMTMM), was used to promote chemical reactions between –COOH groups of PAA of polymerized dye solution to NH2 end-groups of nylon 6,6 fabrics to produce amide linkages as shown in The xanthene dyes (RB or PB) were incorporated into poly(acrylic acid) (PAA) via polymerization. Briefly, 3 g of RB or 2.5 g of PB was stirred in a 70-mL solution of 50:50 to nylon ,21. Aftew/v PAA solution in distilled water was prepared at room temperature; 100 µmol/L of AA or TBO was added to the PAA solution. After stirring for 1 h, 0.3 g DMTMM was added to the mixture [The thiazine dyes were also grafted to PAA as follows. A 5%  mixture . After sIn order to graft the photosensitizer containing polymers to the fabric, each polymerized solution was prepared at 100 µmol/L concentration in a flat-bottomed dish, which had been covered with aluminum foil to exclude light. Electrospun nanofiber-based fabrics and microfiber-based fabrics were immersed into the solution bath. The condensing agent, 0.3 g DMTMM, was dissolved in the solution bath. After 12 h, the fabric was removed from the solution and placed into an oven  for 1 min at the temperature of 170 °C. A. fumigatus, A. niger, T. viride, and P. funiculosum, C. globosum, P. cinnamomi and M. grisea were cultured at 35 °C on potato dextrose agar (PDA) plates for seven days. Broth medium, RPMI-1640 which contains 0.2% glucose and 0.165 mol/L MOPS (3-N-morpholino propanesulfonic acid) at pH 7.0, was used as medium for five fungi, while the potato dextrose broth (PDB) was used as medium for P. cinnamomi and M. grisea. Next, 10 mL broth medium with 0.01% wetting agent Tween® 20 was added to the cultures and then scraped to separate spores. The scraped solution in the culture was filtered through Millipore® polytetrafluoroethylene film to remove hyphae. 6 CFU/mL was transferred to a PDA plate and then a 5 × 5 cm piece of the fabrics prepared above were placed onto the inoculated PDA plate. The plates were placed into a tray and the tray was placed under the lamp. A water-filled pyrex glass dish was placed between the tray and the lamp to absorb infrared light from a photoflood lamp  to avoid heating the tray. The lamp was placed 35 cm above the glass dish and the inoculated PDA plates were placed below the glass dish. The light intensity was measured using a digital illuminance meter  at 15,500 Lux. The plates were kept under this exposure for 5 h at 24 °C and then incubated for seven days in the dark at 35 °C. The inhibition zone test of fabrics grafted with photosensitizers RB, PB, TBO and AA was performed as follows. A 100 µL inoculum of each strain prepared above at 2 × 106 CFU/mL using 0.5 McFarland standard as a reference and a hemocytometer . Then 100 µL inoculum of fungal spores was deposited into each well containing the free photosensitizer solutions prepared above. The 96-well plates were then illuminated for 3 h at 24 °C. Finally, a 96-well plate was incubated at 35 °C for all fungi for 48 h. All tests were conducted three times and then MIC was selected as the highest concentration.In order to test the effectiveness of the free photosensitizer dyes, minimum inhibitory concentration (MIC) was conducted according to the CLSI M38-A standard ; 100 μL 5 CFU/mL. The prepared inoculum (50 mL) was added into each 125 mL Erlenmeyer flask and then treated or untreated circular fabric samples with 4.8 cm in diameter were immersed in the flask, which was then covered with a cap with a hole in it. The flasks were placed under the light as above for 3 h at 24 °C and the flask was shaken at 200 strokes/minute throughout the test. Then a100 µL aliquot of inoculum from each flask was transferred to individual wells of a 96-well tray every 30 min; 96-well trays were incubated in the dark at 35 °C for 48 h. The optical density (OD) was measured at 405 nm using Biotek® Synergy HT multi-mode reader. The ODs of the treated fabric with inoculum (B) was subtracted from the ODs of the untreated fabric (A) with inoculum [A is the OD at 405 nm of the solution in the well containing the inoculum exposed to the untreated fabric sample and B is the OD of the solution in the well corresponding to each treated fabric after the specified contact time between the treated and illuminated fabric sample. A quantitative antifungal assay was also conducted following a modified ASTM E2149-01 method . The inoThe specific surface areas of untreated nanofiber-based fabric and microfiber-based fabrics were obtained from the Brunauer-Emmett-Teller (BET) analysis using liquid nitrogen as the adsorbate and a Gemini VII 2390p physiosorption analyzer from Micrometrics coupled with SmartPrep 065 degassing unit . The chopped fabric sample (5 × 5 cm) consisting of nanofibers (0.109 g) or microfibers (0.249 g) was used for BET analysis. Field emission scanning electron microscopy (JEOL 6400)  was used to examine the morphology of spores from the seven pathogens and to examine the nylon fibers. The amounts of RB, PB, AA and TBO grafted to the nanofiber-based and microfiber-based fabrics were obtained from reflectance measurements over the spectral range 360–700 nm using a Datacolor SF600X  spectrometer. The fabrics were folded to ensure optical opacity, which required at least 16 layers thick for microfiber-based fabrics and at least five layers for nanofiber-based fabrics. Each measurement was repeated two times in four different spots of the samples. Antifungal agents must be on the surface of fibers in order to be able to inhibit the microbes. However, typical treatments with antifungal agents often wash off easily. Thus, it is advantageous to permanently attach the antifungal agents. The amino groups of nylon 6,6 can react with carboxylic acid groups from the polymerized, PAA-based photosensitizer dyes. This enabled the permanent grafting of the photosensitizer dyes that had been either copolymerized with AC-(xanthene dyes) or that had been grafted onto PAA-(thiazine dyes), thus producing amide linkages.2/g for the nanofiber-based fabric, while the microfiber-based fabric had a specific surface area of 1.41 m2/g. Thus, the BET analysis gives a specific surface area ratio of 20:1 for the nanofiber-based fabrics to the microfiber-based fabrics. K/S value as shown in Equation (2) [K and S are absorbance and scattering coefficients (cm−1), and ∞R is the reflectance for infinite thickness. If the fabric is sufficiently thick so that it is opaque, λR = ∞R, where the reflectance is measured at the wavelength of maximum absorbance [The amount of photosensitizer dye on the fabric surface should be proportional to the color strength of the fabric. The fabric color strength was calculated using reflectance percentage and tion (2) .(2)KS= using Equation (3) [The tion (3) .(3)RelaK/S value and the color strengths for each of the fabrics used in this study. The results in A. niger and A. fumigatus, P. funiculosum, and C. globosum. For T. viride, RB displayed the MIC of 31.2 µmol/L and 15.6 µmol/L for P. cinnamomi. For PB, P. funiculosum, and C. globosum showed the largest resistance with MIC at 125 µmol/L, while A. niger and A. fumigatus had MIC at 62.5 µmol/L. TBO and AA both had much higher MICs than RB or PB. TBO has an MIC of 62.5 µmol/L on A. niger, A. fumigatus, and T. viride. For AA, below 125 µmol/L visible growth and turbidity was observed for A. niger and A. fumigatus, P. funiculosum and C. globosum, but not T. viride, giving MIC of 62.5 µmol/L. RB, PB, TBO. AA showed the lowest MIC on T. viride while exhibiting the largest MIC on P. funiculosum or C. globosum.Before testing the antifungal activities of fabrics with the photosensitizer dyes attached, the antifungal effectiveness of the free dyes was tested using the MIC test. The results shown in P. cinnamomi, the MIC was very low, with an MIC of 15.6 (RB) and 31.25 (PB) µmol/L. However, M. grisea was quite resistant to inhibition with an MIC of 125 µmol/L for RB while PB inhibited M. grisea at 250 µmol/L. AA and TBO inhibited fungal growth of M. grisea above 250 µmol/L and P. cinnamomi at 62.5 µmol/L. M. grisea showed the largest resistance to photosensitizers showing the highest MIC at 250 µmol/L.For To evaluate the antifungal effectiveness of the surface-bond photosensitizers, two types of antifungal assays were conducted against seven fungi types. The first method examined visible growth of hyphae and germination on the fabric to identify the zone of inhibition. This test is a qualitative test that relies on the ability of the antifungal agent to diffuse away from the treated fabric in combination with the antifungal activity of the agent. Large inhibition zones indicate that the agent is quite mobile while small inhibition zones indicate either little mobility or a lack of effectiveness.a) P. cinnamomi; (b) T. viride; and (c) M. grisea on nanofiber-based and microfiber-based fabrics. In general, the fabrics exhibited less growth of P. cinnamomi and T. viride on the surface, compared to the growth of M. grisea. The nanofiber-based fabrics grafted with RB and PB showed no growth of P. cinnamomi or T. viride on the fabric, but nanofiber-based fabrics grafted with AA showed P. cinnamomi growth around the edge of the fabric and substantial growth of T. viride on the fabric. The microfiber-based fabric with RB and PB showed little P. cinnamomi and T. viride colony growth on the fabric while the control was completely covered by growth and germination. Most microfiber-based fabrics grafted with AA and TBO exhibited no inhibition of fungal growth (not shown). On the other hand, M. grisea exhibited substantial growth on all fabrics except for the RB-grafted nanofiber-based fabric. T. viride, while the OD reduction of C. globosum and P. funiculosum was much less than for the others. The plant pathogens, M. grisea, displayed the highest resistance to all types of photosensitizers, while P. cinnamomi showed the least resistance to fabrics grafted with each of the dyes. In order to compare inhibition quantitatively among various variables, the four types of photosensitizers and all seven types of pathogens, we analyzed the inhibition rates and fitted them using Equation (4) developed by Kim and Michielsen [ODt is the optical density of the fungal suspensions at 405 nm. These fungal suspensions were obtained by growing the fungi that survived after t min exposure to the treated fabrics under illumination, as described in the experimental section; ks is the inhibition rate of the growth, D0 is the initial dye concentration, t is the exposure time, and kD is the reduction rate of concentration of dye by photobleaching.The ability of the dyes RB, PB, TBO, and AA immobilized on the fabrics was investigated as functions of the photo-irradiation time, up to a maximum of 180 min. All the fabrics grafted with RB, PB, AA, and TBO reduced growth of all seven types of pathogens as illustrated by the reduction of the ODs. The largest inhibition among the human pathogens tested in this study was on chielsen .lnODt t-test and ANOVA.Equation (4) was used to compare the inhibition rate of each fabric grafted with RB, PB, TBO and AA against seven pathogens and the photobleaching rate of the grafted dyes. The significance of the data was evaluated using the student P. cinnamomi and T. viride with the inhibition rates of 3.7 × 10−2 and 3.5 × 10−2 L/µmol·min, respectively. M. grisea had the lowest inhibition rate of 1.4 × 10−2 L/µmol·min for the RB-treated fabric. The next highest inhibition rates were seen with PB, showing 2.5 × 10−2 and 2.4 × 10−2 L/µmol·min against P. cinnamomi and T. viride. In general, the inhibition rates for the treated fabrics decreased in the order RB > PB > TBO > AA. Likewise, the inhibition rates for the fungi decreased in the order P. cinnamomi > T. viride > A. niger > A. fumigatus > C. globosum > P. funiculosum > M. grisea. The results also show that the inhibition rate for the nanofiber-based fabrics was always higher than for the microfiber-based fabrics, as shown in The nanofiber-based fabric grafted with RB exhibited the largest OD reduction on A. niger and A. fumigatus are spherical and present individually or as strings, and the diameters were 2.4 µm (σ = 0.34) and 2.7 (σ = 0.58), respectively. The average diameters of T. viride and P. cinnamomi spores are 1.8 µm (σ = 0.28) and 2.5 (σ = 0.43). Conidia of P. funiculosum are 3.2 µm (σ = 0.52) with globule shapes. C. globosum and M. grisea spores have larger diameters of 3.7 µm (σ = 0.46) and 3.8 (σ = 0.27), as shown in M. grisea, which has a larger size than T. viride. Prokaryotic bacteria appear to be inhibited in a single-hit process by PDT. This occurs at much lower concentration of photosensitizers than for fungi because of their lack of a nuclear membrane [C. albicans is more difficult to inhibit by PDT, requiring a higher concentration at around 2–5 mg/mL of TBO and stronger light doses because eukaryotic fungus is more complex and a larger organism, requiring multiple hits to induce cell death [M. grisea requires higher dye concentration to be inhibited because bigger spore size requires a multi-hit process by singlet oxygen. The photosensitizer cannot easily reach inside spores to inhibit it. Using SEM images, the sizes of fungal spores were measured from 20 to 50 randomly selected spores. The shape of conidia of membrane . It has ll death . The larP. cinnamomi seems to be an outlier. It shows more antifungal activity as compared to its size. This is because the initial attack of singlet oxygen occurs mainly on lipids, especially at the carbon–carbon double bonds in unsaturated fatty acids in biological molecules to form hydroperoxides [1O2 is very electrophilic, which is capable of directly oxidizing electron-rich double bonds in cells, as shown in P. cinnamomi has been reported to include the high amount of arachidonic acid (AR) and eicosapentaenoic acid (EPA)  were polymerized and AA or TBO was polymerized with poly(acrylic acid). All four types of copolymers were successfully grafted to microsize- or nanosize-based fabrics. The inhibition zone test showed that the nanofibers grafted with RB and PB displayed no growth on the fabrics, but micro fabrics grafted with AA and TBO had little effectiveness against the seven types of fungi used in this study. The quantitative method was also used to compare inhibition rate for the different photosensitizers against the different fungi.In order to increase graft yield of photosensitizers on the surface of fabrics, poly(acrylic acid-ks) on P. cinnamomi, up to 3.7 × 10−2 and 3.5 × 10−2 L/µmol·min, while on M. grisea exhibited the lowest inhibition rate of 1.4 × 10−2 L/µmol·min. This activity was related to fungal morphologies and lipid composition of the cell membranes. P. cinnamomi had a high amount of arachidonic acid and eicosapentaenoic acid, which were good targets for photooxidation by singlet oxygen, while filamentous ascomycota fungi had ergosterol instead. M. grisea had the largest spore size with diameters of 3.9 µm and exhibited the highest resistance to singlet oxygen due to inhibition by a multi-hit process with singlet oxygen. Finally, it was concluded that the results showed the increasing inhibition percentage in the order of AA < TBO < PB < RB. The nano fabrics had higher color strength of photosensitizers and were more effective on fungi because of a larger surface area at 29 m2/g, while micro fabrics had 1.4 m2/g. The inhibition rates for RB, PB, TBO and AA for all fabrics had efficiency in the decreasing order of P. cinnamomi, T. viride, A. niger, A. fumigatus, C. globosum, P. funiculosum, and M. grisea, which was associated to lipid composition in membrane and morphology of fungal spores. In the results, nano fabrics grafted with RB resulted in up to a 92% reduction in growth with a high inhibition rate. Nano fabric grafted with RB had the largest inhibition rate (


The majority of foods that are consumed in our developed society have been processed. Processing promotes a non-enzymatic reaction between proteins and sugars, the Maillard reaction (MR). Maillard reaction products (MRPs) contribute to the taste, smell and color of many food products, and thus influence consumers’ choices. However, in recent years, MRPs have been linked to the increasing prevalence of diet- and inflammation-related non-communicable diseases including food allergy. Although during the last years a better understanding of immunogenicity of MRPs has been achieved, still only little is known about the structural/chemical characteristics predisposing MRPs to interact with antigen presenting cells (APCs). This report provides a comprehensive review of recent studies on the influence of the Maillard reaction on the immunogenicity and allergenicity of food proteins. Most of the food consumed nowadays by developed societies is processed. The diversity of processed food products increased exponentially during the last century, along with the need for more safe, convenient and varied food products. Methods used for food processing can be categorized into two processing types: conventional thermal methods, including pasteurization, sterilization, drying and roasting ,5 and noNative proteins are folded into specific and compact 3D structures. This is determined by primary structure (sequence of amino acids), secondary structure (formation of α-helixes and β-sheets) and tertiary structure. The formation of α-helixes and β-sheets is driven by interactions between polypeptide chains linked together by hydrophobic and hydrophilic interactions, electrostatic interactions and disulfide bonds . All theOne of the best-known interactions between proteins and sugars occurring during heat processing of food is the Maillard reaction (MR), also known as glycation. During the MR, sugars are linked to proteins by a covalent bond between free amino groups of amino acids (mostly lysine and arginine) and the carbonyl groups of a reducing sugar  ,16,17. HMoreover, the type and amounts of MRPs formed are under the control of factors such as the structural diversity of (poly)saccharides and proteins, reaction temperature and time, ratio of amino group and reducing sugar, pH and water activity ,20. MRPsIn order to elicit an allergic immune response, food proteins (or peptides thereof) must survive digestion and remain in the gastrointestinal tract for a period of time that is sufficient to induce sensitization. From that perspective, the susceptibility of food proteins to enzymatic hydrolysis seems to be an important factor determining their allergenicity ,27,28. EHeating itself causes unfolding of protein and exposure of linear epitopes and may result in enhanced susceptibility to enzymatic proteolysis as described for β-lactoglobulin heated at the temperature of 90 °C . HoweverThe number of studies involving in vivo experiments investigating an effect of MR on digestibility of food proteins is limited to date. A recently published study performed on humans compared the effects of diets with different MRP contents on dietary protein utilization in adolescent males. The study revealed that a diet high in the MRPs limits the digestibility of proteins since 47% higher fecal nitrogen excretion, 12% lower apparent nitrogen absorption and 6% lower nitrogen digestibility was observed in the group consuming diet high in MRPs . HellwigThe question if the glycated protein/peptide can have immunological effect in vivo depends on whether these molecules are available in the gastrointestinal mucosa to be absorbed into the circulation and subsequently to get in contact with the immune system. Dietary MPRs were shown to appear in the circulation and/or urine of human subjects after consumption of MRPs rich diet ,50,51,52Cross-linking of proteins seems to reduce an epithelial uptake of proteins although promote an uptake through Peyer’s patches as was shown for crossed-linked β-lactoglobulin and α-lactalbumin ,55. In aEven though the role of AGEs in chronic inflammatory diseases is more well-known and studied ,60,61, eAllergy is commonly divided in two phases. An initial phase of allergic sensitization represents principally a particular immune reaction leading to the formation of allergen-specific IgE antibodies. IgE antibodies can crosslink adjacent cell-bound IgE molecules on basophils and mast cells upon repeated exposure to allergens, leading to degranulation and the release of mediators, including histamine, prostaglandins, leukotriens and tromboxanes. These mediators cause the typical symptoms of a type 1 (within 20 min) IgE-mediated allergy, including rhinitis, atopic dermatitis, allergic asthma, and occasionally even anaphylaxis .Immunogenicity of proteins is thus dependent on their ability to eventually induce adaptive T-and B-lymphocyte responses during the allergic sensitization phase see . This isTo induce an allergic immune response the MR-modified protein need to be recognized and taken up by antigen presenting cells (APCs) and subsequently presented to T-cells . RecentlRAGE is the most studied receptor that can recognize and bind dietary AGEs ,86,87,88The recent study of Liu and colleagues revealed that whey proteins glycated by dry heating (at 130 °C) interacts with sRAGE. The strength of binding was positively correlated with the time of heating and the formation of agglomerates and was more prominent in the samples with lower water activity . These fInteraction of AGEs with receptors present on APCs may result in internalization and therefore presentation of antigen to T-cells. Ilchman and colleagues demonstrated that AGE-modified OVA was taken up much more efficiently by bone marrow-derived murine myeloid dendritic cells (mDCs) than native OVA, and enhanced activation of OVA-specific CD4+ T cells . ScavengHeilmann and colleagues aimed to identify glycation structures enhancing T-cell immunogenicity of a food allergen by modification of OVA with different AGEs such as CML, CEL and pyrraline. To assess the T-cell immunogenicity of glycated OVAs, murine OVA-specific CD4+ T-cells were co-cultured with bone marrow-derived DCs in the presence of differently processed OVA samples. Pyrraline modified OVA enhanced CD4+ T-cell immunogenicity, as evidenced by increased IL-2 production, higher production of IFN-γ and IL-17A when compared with native OVA. Moreover, pyrraline-OVA and AGE-OVA were efficiently taken up by BMDCs via SR-A. In addition to antigen uptake, cell maturation is required for DCs to gain their full T-cell stimulatory capacity. However, no differences in expression of co-stimulatory molecules CD40, CD80, CD86, and MHC class II on the cell surface of DC were seen suggesting that pyrraline modification does not induce BMDC maturation. This is in line with observations of Moghaddam and colleagues , who sugThese results raise the question which structural changes triggered by MRPs can explain the immunogenicity of AGEs and capabilities to initiate the polarization of allogenic naive CD4+ T-cells into Th2 cells. Maillard reaction caused agglomeration of proteins was excluded as a major contributor to increased immunogenicity of proteins in the studies of Moghaddam and colleagues  and HeilThe research outcomes discussed above provide strong evidence that some dietary AGEs can bind to receptors on antigen presenting cells and thus modify T-cell immunogenicity  of naïve B cells come in contact with specific dietary antigens, and are activated by ligation of the surface molecule CD40 to CD40L on activated Th2 cells that produce IL-4 and IL-13 , the B cells are induced to switch to produce IgE. Naïve B cells further differentiate and proliferate into activated plasma cells synthesizing and secreting antigen-specific IgE see . LymphocMR induced during food processing may also modulate binding potential of specific IgE to food allergens. This can be induced by: (a) disruption of the conformational and linear epitopes accompanied with the changes of the tertiary and secondary structure that impair the IgE binding potential of the protein ,106,107;The MR was shown to either reduce or enhance IgG and/or IgE binding capacities of some food allergens ,112,113.The study of Vissers and colleagues showed reduced allergenicity of MR-modified peanut allergen Ara h 1 in IgE binding test while enhanced β-hexosaminidase release from basophils upon incubation with the same MR-modified allergen . The autBox 1Allergens are generally proteins that, when exposing genetically susceptible individuals frequently and in low doses (<1 µg) on a mucosal surface are able to induce a Th2 response associated with the production of IL-4, IL-5, and IL-13 leading to the synthesis of allergen-specific IgE antibodies. At present, no identified antibody characteristics and no identified structural features of IgE binding epitopes seem to be associated with the phenotype of the food allergic disease. Potential allergens must necessarily contain B-cell epitopes to which IgE can bind, and T-cell epitopes capable of inducing a Th2 type response. Peptide sequences of T-cell epitopes (10–15 amino acid residues long) show no general homology across allergen families and thus it has been proven impossible to identify a consensus sequence for an allergenic epitope .IgE binding allergen epitopes generally comprise conformational and more hydrophobic patches present on the allergen. A significant proportion of the IgE is directed against the glycosylated B-cell epitopes . This miAn epitope is identified by its ability to bind antibodies and is suggested to consist of a recognizable sequence of 6–15 amino acids (covering a 11–13 nm distance) contributing to the binding between epitope and antibody molecule . Two of Box 24 times less than that of IgG. IgE molecules have a 12% carbohydrate content with oligosaccharides asparagine-linked at six places all in the first three of the four constant region domains, they have a limited segmental flexibility . Therefoa ≈ 1010 M−1). This exceptionally high affinity is mainly a reflection of the very slow dissociation rate with a half-life of about 20 h for circulating IgE. The residence time on mast cells in tissues is further extended to more than 14 days by restricted diffusion and rebinding to cell receptors [These IgE antibodies have the capacity to bind to IgE Fc epsilon receptors with high affinity . Principally, this elicitation phase requires a multivalent interaction between (mostly linear) allergen multi-epitopes and multiple Fab fragments on IgE antibodies. This allergen capacity can be analyzed ex vivo with blood-derived basophils or in vitro by using e.g., the rat basophilic leukemia (RBL)-HE3 cell line and is generally called allergenicity of the allergen. To analyze the basophil degranulation capacity sensitive assays are required as these cells comprise only 0–2% of leukocytes equaling 1–8 × 10cells/mL . This dicells/mL . Upon IgIn conclusion, the MR can alter immunoreactivity towards food proteins, and MR-modified proteins may enhance the immune response by selective interaction with APCs carrying receptors for AGE. In addition, it has become clear that the presentation of MR-modified allergens to T-cells may skew the subsequent T-cell differentiation into Th2 cells producing IL-4, IL-5 and IL-13, which are responsible for the initiation of IgE antibody production. Some studies also observed enhanced mediator release from basophils incubated with MR-modified proteins that may be resulting from: (a) the immunogenic potential of MRPs enhancing the sensitization; and/or (b) agglomeration leading to more efficient cross-linking and therefore mediator release. These findings reveal a need for better understanding of the influence of MR on both the sensitization phase as well as the development of the symptoms of the allergy. Understanding the mechanisms involved in immunoreactivity of AGEs would help to improve the diagnostics of food allergy as well as develop optimized conditions for food processing to control the rate of MR.


PGI1), enolase (ENO2), triosephosphate isomerase (TPI1), or folate biosynthesis enzyme (FOL3) significantly reduced H3T11 phosphorylation. Further study demonstrated that glycolysis regulates H3T11 phosphorylation by fueling the substrate, phosphoenonylpyruvate and the coactivator, FBP to Pyk1. Thus, our results provide a comprehensive view of how glycolysis modulates H3T11 phosphorylation.Cancer cells prefer aerobic glycolysis, but little is known about the underlying mechanism. Recent studies showed that the rate-limiting glycolytic enzymes, pyruvate kinase M2 (PKM2) directly phosphorylates H3 at threonine 11 (H3T11) to regulate gene expression and cell proliferation, revealing its non-metabolic functions in connecting glycolysis and histone modifications. We have reported that the yeast homolog of PKM2, Pyk1 phosphorylates H3T11 to regulate gene expression and oxidative stress resistance. But how glycolysis regulates H3T11 phosphorylation remains unclear. Here, using a series of glycolytic enzyme mutants and commercial available metabolites, we investigated the role of glycolytic enzymes and metabolites on H3T11 phosphorylation. Mutation of glycolytic genes including phosphoglucose isomerase ( Glycolysis is the fundamental metabolism highly conserved in most organisms, which comprises a series of enzymatic steps that sequentially convert glucose to pyruvate. In the presence of oxygen, most pyruvate undergoes oxidative phosphorylation to generate ATP in mitochondria; while in the absence of oxygen, pyruvate is converted to lactate with few ATP produced ,2. HowevCCDN1(encoding cyclin D1) and MYC, leading to their activation, tumor cell proliferation, cell-cycle progression, and brain tumorigenesis [serine responsive SAM-containing metabolic enzyme complex) with other metabolic enzymes, including Sam1, Sam2, Ser33, Shm2 and Acs2 [Emerging evidence showed that most glycolytic enzymes are deregulated in cancer cells and plays important roles in tumorigenesis ,5. All eigenesis . Previouigenesis . Similarigenesis . Specifiand Acs2 . Furtherand Acs2 .+/NADH, which in turn regulate the activity of histone acetyltransferases and histone deacetylases as well as the chromatin structure [Cellular metabolism regulates histone modifications and many metabolites serve as essential cofactors for chromatin-modifying enzymes to control the transcription or translation processes ,10,11. Ftructure –14. We htructure ; howeverAll yeast strains used in this study are described in Histones were extracted from yeast cells as described previously ,15. BrieProtein samples were separated by 15% SDS-PAGE and transferred to Immobilon-P PVDF membrane (Merck Millipore). The blots were probed with antibodies against H3  and H3pT11  followed by incubation with horseradish peroxidase-labeled IgG secondary antibodies . The specific proteins were visualized by using the ECL Chemiluminescence Detection Kit (Amersham Biosciences). Western blots were quantified with ImageJ software.600 of 0.6–0.8 and treated with doxycycline for 0.5 hour. Total RNA was isolated and Real-Time RT-PCR was performed with SYBR green and gene specific primers as described previously [ACT1 transcript. Primers used were listed in 10 ml cultures were grown in YPD to an ODeviously . All tra600 of ~1.0 and treated with 50 μg/μl doxycycline for 3 hours. Cells were harvested and lysed with glass beads. The intracellular FBP concentrations were determined using FBP analysis kit according to the protocol recommended by the manufacturer .50 ml cells were grown in YPD to an ODcdc19-1) when grown at 37°C to demonstrated that Pyk1 phosphorylates H3T11 in vivo. Here, we employed the strains that display different PYK1 expression and activity by expressing PYK1 under the control of either a strong (TEF1) or a weak (CYC1) constitutive promoter [TEF1pr-PYK1, CYC1pr-PYK1) in rich media and examined the global levels of H3T11 phosphorylation by western blot analysis. As shown in CYC1pr-PYK1 strain  was significantly reduced in H3T11A mutant  strain defective in Pyk1  with different carbon sources (glucose and potassium acetate) and then examined their effects on global H3T11 phosphorylation. In contrast to glucose as the sole carbon source, the global levels of H3pT11 were comparable between SESAME mutants and its parental wild type strain when potassium acetate was used as the carbon source  is an important cofactor for the pyruvate kinase activity of both PKM2 and Pyk1 and depletion of glucose immediately reduces the intracellular level of FBP –20. As a for FBP . We emplycycline . We treapromoter . We alsoreatment , P<0.05.et cells , indicatTo directly investigate whether FBP stimulates the protein kinase activity of Pyk1, we treated cells with different amounts of FBP and examined the global levels of H3T11 phosphorylation. FBP significantly stimulated Pyk1-catalyzed H3T11 phosphorylation . Thus, lENO1, ENO2, which encode enolase converting 2-phosphoglycerate to PEP. The expression of ENO2 was down-regulated in TetO7-ENO2 mutant upon doxycycline treatment  . Tpi1 capromoter . The levet cells , suggestsistance , we thus1 mutant , suggestPyruvate kinase catalyzes the last step of glycolysis, which is also the rate-limiting and irreversible step. We next investigated whether pyruvate as the product of pyruvate kinase , can feepdc1∆ mutant (pdc5∆ mutant (data not shown). Together, these data indicate that pyruvate decarboxylase and pyruvate do not regulate the activity of SESAME to phosphorylate H3T11.We also examined the impact of in vivo accumulation of pyruvate by deleting genes encoding pyruvate decarboxylase, which encodes pyruvate decarboxylase that converts pyruvate to acetaldehyde. H3T11 phosphorylation was not significantly affected in either ∆ mutant  or in pdMET6 and MET13 specifically reduced both H3K4me3 and H3pT11 [FOL3 specifically reduced global H3K4me3 [7-FOL3 mutant and treated it with doxycycline to down-regulate FOL3 transcription from the Tet promoter MET6 and  H3K4me3 . Since Hpromoter . The lev3 strain , indicatGlycolysis is required for global histone acetylation and mono-ubiquitination of H2B at K123 ,24. Our PYK1 expression and confers cells the resistance to oxidative stress [PYK1 expression, which in turn stimulates flux towards pentose phosphate pathway. As a consequence, redox metabolism is enhanced to prevent the accumulation of reactive oxygen species (ROS)  . NeverthGlucose metabolism is required for SESAME-mediated H3T11 phosphorylation. Depletion of glucose or inhibition of glycolysis by 2-Deoxy-D-glucose significantly reduced H3T11 phosphorylation . When ceENO2 reduced the global levels of H3T11 phosphorylation , only ENO2 is required for H3T11 phosphorylation. The role of Eno2 in regulating H3T11 phosphorylation is probably related to their abundance with 20-fold higher Eno2 than Eno1 when glucose is used as the carbon source [Km) of Pyk1 to PEP is very low, approximately 0.3 mM [One contribution of glycolysis to H3T11 phosphorylation is providing the substrate, PEP for Pyk1. Down-regulating the expression of rylation . Althougn source . HoweverAnother contribution of glycolysis to H3T11 phosphorylation is supplying the cofactor FBP for Pyk1. It is well-known that FBP stimulates Pyk1 to convert PEP to pyruvate . FBP hasde novo synthesis of serine. On one hand, serine derived from glycolysis contributes to H3T11 phosphorylation by acting as a coactivator for Pyk1; on the other hand, serine can be fueled to SAM synthesis and facilitate H3K4me3, which then enhanced the ability of Pyk1 to phosphorylate H3T11 via a cross-talk between Set1 and SESAME [Together, we identified three major contribution of glucose metabolism to H3T11 phosphorylation: 1. Glycolysis provides the substrate PEP; 2. Glycolysis supplies cofactor FBP; 3. Glycolysis promotes the d SESAME . Given td SESAME ,27, undeS1 FigPYK1 was expressed at a lower level in TEFpr-PYK1 than CYC1pr-PYK1. Actin was used as an internal control. Data represent the mean ± SE (n = 3). *, P<0.05. (B) The expression of PGI1 was significantly down-regulated in TetO7-PGI1 mutant by doxycycline. (C) The expression of ENO2 was significantly down-regulated in TetO7-PGI1 mutant by doxycycline. (D) ENO1 was deleted in eno1∆ mutant. The genome of WT and eno1∆ mutant were extracted. The deletion of ENO1 was confirmed by PCR using ENO1 specific primers. ACT1 primers were used as an internal control. (E) The expression of PGI1 was significantly down-regulated in TetO7-PGI1 mutant by doxycycline. (F) The expression of PGI1 was significantly down-regulated in TetO7-PGI1 mutant by doxycycline. All experiments except (A) (EPS)Click here for additional data file.S2 FigActin was used as an internal control. Data represent the mean ± SE (n = 3). *, P<0.05.(EPS)Click here for additional data file.S3 FigPGI1 was significantly down-regulated in TetO7-PGI1 mutant by doxycycline treatment. (B) Down-regulated PGI1 leads to reduced intracellular FBP concentrations. Data represent the mean ± SE (n = 3). *, P<0.05; ***, P<0.001.(A) The expression of (EPS)Click here for additional data file.S4 FigPYK1 expression. Reduced PYK1 confers cells resistance to oxidative stress by stimulating pentose phosphate pathway, which increased antioxidative metabolism and prevents ROS accumulation. (B) Model explains oxidative stress resistance in CYC1pr-PYK1 mutant. In CYC1pr-PYK1 mutant, the protein level of Pyk1 was low, which increased flux towards pentose phosphate pathway to gain resistance to oxidative stress. (C) Model explains oxidative stress resistance in H3T11A mutant. In H3T11A mutant, PYK1 expression was up-regulated, which attenuates flux towards pentose phosphate pathway and reduces resistance to oxidative stress.(A) Around 1.9% Pyk1 is localized in nucleus and this nucleus Pyk1 catalyzed H3T11 phosphorylation [(EPS)Click here for additional data file.S1 Table(DOC)Click here for additional data file.


Structure-based virtual screening is an in-silico method to screen a target receptor against a virtual molecular library. Applying docking-based screening to large molecular libraries can be computationally expensive, however it constitutes a trivially parallelizable task. Most of the available parallel implementations are based on message passing interface, relying on low failure rate hardware and fast network connection. Google’s MapReduce revolutionized large-scale analysis, enabling the processing of massive datasets on commodity hardware and cloud resources, providing transparent scalability and fault tolerance at the software level. Open source implementations of MapReduce include Apache Hadoop and the more recent Apache Spark.We developed a method to run existing docking-based screening software on distributed cloud resources, utilizing the MapReduce approach. We benchmarked our method, which is implemented in Apache Spark, docking a publicly available target receptor against https://github.com/mcapuccini/spark-vs).Our method enables parallel Structure-based virtual screening on public cloud resources or commodity computer clusters. The degree of scalability that we achieve allows for trying out our method on relatively small libraries first and then to scale to larger libraries. Our implementation is named Spark-VS and it is freely available as open source from GitHub ( Identification of new drug leads is a key process in drug development. In the pharmaceutical industry, a widely established approach for this is High-Throughput Screening (HTS), where large molecular libraries are screened against a bioassay in fully automated environments . HoweverIn recent decades, methods in high-throughput structural biology allowed the production of massive virtual molecular libraries. ZINC  represenGoogle pioneered Big Data analytics on inexpensive hardware with its MapReduce (MR) programming model (and implementation) . In MR aGoogle’s MR has some limitations. MR is based on an acyclic data flow model, which penalizes many popular applications where the same dataset needs to be accessed in multiple iterations  . In factResilient Distributed Datasets (RDDs) represent the core component of Spark . An RDD The applicability of MR-oriented frameworks to virtual screening has already been investigated. For instance, Ahmed et al.  implemenIn this paper we introduce a method for large-scale SBVS on public cloud resources. This represents an advancement over the earlier work by Ellingson and Baudry, as they acknowledged that further refinement in AutoDockCloud was needed in order to run their method on public cloud providers . FurtherWe developed a method for parallel SBVS following the MR approach, which enables the screening of large molecular libraries on public cloud resources or on commodity hardware clusters. The method is implemented in Apache Spark and it is distributed as an open source library, named Spark-VS, along with some example ready-to-run SBVS pipelines.Using Spark-VS, the user can define custom SBVS pipelines with a high level API . Despitchunk size parameter (default: 30) that controls the number of molecules to be loaded into a single RDD record.When loading a text file to an RDD, Spark normally splits it line-by-line. Nevertheless, most molecular 3D representations consist of multiple lines. The Structure Data File (SDF) is a handy, multiline format that can be used to store 3D molecular structures along with some metadata  . Spark-VaddFile primitive) and produces a chunk of poses (with the relative score) in the standard output. Using the standard input and the standard output in this way is very convenient. In fact, Spark-VS uses the map RDD primitive to pipe each RDD record to a docking executable instance via standard input and takes the results back via standard output. This contrasts to the implementations by Zhao et al. [OEDocking TK  was usedo et al.  and by Eo et al.  where thsaveAsTextFile primitive to handle persistence on a distributed storage. However, the built-in sortBy primitive is not suitable for SBVS. The Spark primitive assumes small RDD records so shuffling the relatively large SDF representations through the network would lead to a large overhead. Fortunately, we figured out a simple workaround to avoid this. Instead of performing distributed sorting, Spark-VS collects ID/Score tuples for each pose and efficiently sorts them serially. Then, using the RDD filter primitive, Spark-VS retrieves the top scoring molecules by ID.RDDs offer some convenient primitives to do the post processing part of SBVS. For instance, Spark-VS uses the With the aim of testing our parallel implementation, we ran OEDocking TK serially over 1000 molecules that were randomly drawn from the benchmark dataset. We observed that the output for the parallel implementation did not differ from the one that we got in the serial execution. We have provided this validation procedure along with Spark-VS and it can be easily reproduced by running in local mode.We deployed a standalone Spark cluster, along with HDFS, on the CityCloud  public cWe benchmarked our method and the Spark-VS implementation, screening HIV-1 protease receptor against the whole SureChEMBL library  download of data . NeverthFinally, it is worth mentioning that the target receptor was converted in the OEDocking TK format starting from an HIV-1 protease receptor representation available in the literature .N processing elements to process N work units , and then scale to larger libraries. Furthermore, being relieved form the up-front investments, hardware configuration and maintenance costs is certainly an advantage too.The method, along with its source code and unit tests, is free to use and publicly available on GitHub . Even th


Development of MMP-resistant analogues of kisspeptins may provide better therapeutic outputs. In the present study, two kisspeptin phosphinic peptides were designed and synthesized, and their ability to induce phosphorylation of ERK1/2 through kisspeptin receptor and their inhibition on MMP-2 and MMP-9 whose activity correlates with cancer metastasis were assessed. The results showed that one analogue, phosphinic kisspeptin R isomer (PKPR), exhibited kisspeptin receptor-agonistic activity and also inhibitory activity on MMP-2, indicating that PKPR may serve as a lead for the further development of kisspeptin analogues for therapeutic purpose.Kisspeptins acting on their cognate G protein-coupled receptor, kisspeptin receptor, play important roles in the suppression of cancer cell metastasis and regulation of the reproductive system, and therefore are important for therapeutic intervention. All native functional human kisspeptins  share the 10 amino acids of kisspeptin-10 at their C-terminus (45–54). However, they are inactivated rapidly by matrix metalloproteinases (MMPs) through the cleavage of the peptide bond between glycine The full enzyme activity (control) was measured without addition of any inhibitor or tested peptides. The reaction velocity (V) was determined by calculating the slope of the fitted line to the linear portion of the data collected. The slope of blank (without addition of MMP enzyme) was subtracted from all samples and the remaining activity of the enzyme was calculated using following formula.The means ± SD are shown for values obtained for the numbers of independent experiments indicated in the figure legends. GraphPad Prism software 6.0  was used to analyse the data for statistical significance. The statistical significance was calculated by one-way or two-way ANOVA.51 and Leu52 with the phosphinate (-P(O)(OH)CH2) bond which is resistant to hydrolysis of MMPs (50 side-chain of KP-10. The synthesis is described in section 2.2. The KP phosphinic peptides have S isomer (PKPS) and R isomer (PKPR) which are diastereoisomers  in the phosphorylation of ERK1/2 (P < 0.01)  , indicatP < 0.001) phosphorylation of ERK1/2 (P < 0.05) in the phosphorylation of ERK1/2, similar to the result obtained above.To examine whether the KP phosphinic peptides exhibit antagonistic activity on the kisspeptin receptor, their ability to inhibit KP-10-induced phosphorylation of ERK1/2 was tested. MCF-7 cells transiently expressing kisspeptin receptor were used as model cells. The cells were first treated with 10 μM of PKPS or PKPR respectively for 30 minutes. After that, KP-10  was added for a further 10-minute stimulation which itself elicited a significant  inhibition (P < 0.005) on Sulfo-Cy5-KP-18-induced internalisation of the kisspeptin receptor (OH)CH2]~ group at the cleavage site of KPs leads to inhibition of the peptides to zinc metalloproteases, their ability to inhibit the activity of MMP-2 and MMP-9 was further evaluated. A significant inhibition of PKPR at 10 μM on MMP-2 enzyme activity was observed  while PK51 and Leu52 with a phosphinic acid moiety. PKPR may serve as a lead for further development of a metabolically stable peptide analogue for hormone replacement therapies and/or of a potent anti-metastasis agent targeting both kisspeptin receptor and MMPs.In conclusion, our studies propose a new option to develop novel KP phosphinic analogues, such as PKPR, which activate kisspeptin receptor and inhibit the activity of MMPs, by replacing the peptide bond between GlyS1 FigTime-course of the phosphorylation of ERK1/2 induced by KP-10 in HEK293 (A) and MCF-7 (B) cells. Cells transiently expressing kisspeptin receptor were starved for 20 hours. The cells were then treated with vehicle (0.02% (v/v) propylene glycol) or 100 nM KP-10 for different times as indicated. Western blot analyses were carried out using monoclonal anti-phospho-ERK1/2 and anti-β-actin antibodies. Representative western blot and densitometric analysis are shown. The data represent the mean fold-change over basal (vehicle treated control) ± SD of three independent experiments. **, P < 0.01; ****, P < 0.001, compared with the basal.(TIF)Click here for additional data file.S2 FigHEK293 cells expressing kisspeptin receptor were stimulated by PKPS (A) or PKPR (B) and the phosphorylation of ERK1/2 was measured by western blotting.(TIF)Click here for additional data file.S3 FigEffect of PKPS and PKPR on KP-10-induced phosphorylation of ERK1/2.(TIF)Click here for additional data file.S4 FigPhosphorylation of ERK1/2 stimulated by KP-10 in HEK293 (A) and MCF-7 (B) cells.(TIF)Click here for additional data file.


Elevated CaMKK2 activity is strongly associated with prostate and hepatic cancers, whereas reduced CaMKK2 activity has been linked to schizophrenia and bipolar disease in humans. Here we report the functional effects of nine rare-variant point mutations that were detected in large-scale human genetic studies and cancer tissues, all of which occur close to two regulatory phosphorylation sites and the catalytic site on human CaMKK2. Four mutations  cause a marked decrease in Ca2+-independent autonomous activity, however S137L and P138S mutants displayed increased autonomous and Ca2+-CaM stimulated activities. Furthermore, the G87R mutant is defective in Thr85-autophosphorylation dependent autonomous activity, whereas the A329T mutation rendered CaMKK2 virtually insensitive to Ca2+-CaM stimulation. The G87R and R139W mutants behave as dominant-negative inhibitors of CaMKK2 signaling in cells as they block phosphorylation of the downstream substrate AMP-activated protein kinase (AMPK) in response to ionomycin. Our study provides insight into functionally disruptive, rare-variant mutations in human CaMKK2, which have the potential to influence risk and burden of disease associated with aberrant CaMKK2 activity in human populations carrying these variants.The Ca CaMKK2 expression is up-regulated in hepato-cellular carcinoma (HCC) and negatively correlates with HCC patient survivalession rs63843, as2+-CaM and multisite phosphorylation.Nine rare-variant point mutations that occur in close proximity to regulatory phosphorylation sites and the catalytic site on human CaMKK2 have been detected in large-scale genetic studies1416171819212+-independent autonomous activity and Ca2+-CaM stimulated activity of WT, G87R and R91W mutant CaMKK2. Autonomous activity was first demonstrated in CaMKII, and is defined as the kinase activity that is measured when the Ca2+ stimulus (but not CaM) is removed with the chelator EGTA272+ in the CaM preparation  that occur adjacent to the Thr85 autophosphorylation site  have bee15231925paration . Ca2+-Capression . We obsepression . This ispression . Similarpression .2+-CaM and MgATP  at a fixed concentration (50 μM) of Ca2+. Neither mutation had a significant effect on the concentration of CaM required for half-maximal stimulation, indicating that CaM sensitivity was unaltered (Although the G87R and R91W mutations are distantly located from the CaM-binding site (residues 475–500) in the primary sequence of CaMKK2 , intra-mnaltered ; Table 1naltered ; Table 12+-CaM2+-CaM stimulated activity in the presence of either 10 μM or 50 μM Ca2+. Consistent with loss of phosphorylation of Ser129/Ser133/Ser137, both the S137L and P138S mutants exhibited a 2.3- and 4.5-fold increase in autonomous activity relative to WT CaMKK2, respectively  that were detected in two human genetic variation studies15ectively . MoreoveT CaMKK2 , indicat2+-CaM, as shown by the lower concentration of CaM required for half-maximal stimulation compared with WT CaMKK2  that occur close to the ATP-binding site of the catalytic domain of human CaMKK2 , have be1518sitivity ; Table 1t manner .The small-molecule CaMKK2 inhibitor STO-609 has been shown to regress tumor burden in mouse models of prostate and hepatic cancers672+-CaM stimulated activities among the mutants tested, we examined whether they can behave as dominant-negative inhibitors of CaMKK2 signaling in cells. For these experiments, we over-expressed WT CaMKK2, G87R and R139W mutants in HeLa cells to test whether they interfere with downstream phosphorylation of AMPK on Thr172 in response to ionomycin (a Ca2+-ionophore). The advantage of using AMPK phosphorylation as a read out of CaMKK2 signaling is that CaMKK2 is the sole upstream kinase for AMPK Thr172 in HeLa cellsSince the G87R and R139W mutations gave rise to the largest decrease in autonomous and Ca2+-CaM and sensitivity to the small-molecule inhibitor STO-609. Rare genetic variants are thought to contribute to complex disease risk, since large genome-wide studies of common variants have explained only a small proportion of the known heritability of most complex disordersIn this study, we report the effects of nine rare-variant point mutations on the regulation of human CaMKK2 by multi-site phosphorylation, CaThe G87R mutation significantly reduced CaMKK2 autonomous activity, and rendered the enzyme incapable of further increasing autonomous activity via Thr85 autophosphorylation. This is similar to the effect of the T85S mutation in CaMKK2 that is linked to bipolar and anxiety disorder in humans1133112+-CaM stimulated activity of WT CaMKK2 measured at 10 μM Ca2+, indicating that the P138S mutation confers gain-of-function and will likely be in an activated state under resting conditions in the cell. Elevated CaMKK2 activity has been shown to play an adverse role in HCC as well as prostate cancer. CaMKK2 is generally increased in tumors from HCC patients compared with adjacent normal tissue, and these elevated levels negatively correlate with HCC patient survival36Analysis of the S137L and P138S mutants revealed they lacked phosphorylation on Ser129, but unlike the R139W and R142W mutants, had significantly increased autonomous activity compared with WT CaMKK2. Notably, the autonomous activity of the P138S mutant is comparable in magnitude to the CaThe dichotomy between decreased CaMKK2 activity and schizophrenia, and increased CaMKK2 activity and prostate cancer, is borne out by an inverse co-morbidity between the two diseases402+-CaM regulation. The E268K mutant had significantly lower autonomous and Ca2+-CaM stimulated activity, similar to the effect of the G87R and R139W mutants, and could be considered a risk marker for disorders associated with decreased CaMKK2 activity. Consistent with previous studies2+-CaM stimulation despite being able to bind CaM. Ala329 is located immediately N-terminal to the DFG motif at the start of the activation loop, but is likely to be distally located from the CaM binding site based on the crystal structures of other CaMK family members 43442+-CaM is thought to stimulate CaMKK2 activity by sequestering the autoinhibitory sequence from the active siteOf the three mutations that occur in the catalytic domain, the E268K and A329T mutants had the greatest effect on CaMKK2 activity and CaIn summary, we propose that several of the rare-variant CaMKK2 mutations  have a functional basis for being risk markers for susceptibility to diseases associated with aberrant CaMKK2 activity. As these mutations are rare with a global allele frequency of <1%, this may indicate that there is evolutionary pressure on the coding region of CaMKK2, particularly around these important regulatory sites, to prevent accumulation of deleterious variations within the human population.Plus SV Miniprep DNA Purification Kits (Promega) and quantified by absorbance at 260 nm.A plasmid construct for full length C-terminal Flag-tagged human CaMKK2 isoform-1 was cloned into pcDNA3(−) using XhoI/HindIII restriction sites for expression in COS7 cells. Point mutations were generated using Quikchange site-directed mutagenesis (Stratagene), and all plasmid constructs were verified by sequencing the entire open reading frame. DNA for transfection was prepared using Wizard 2. Cells were transfected at 60% confluency with 2 μg of pcDNA3 containing C-terminal Flag-tagged human or various point mutants using FuGene HD (Roche). CaMKK2 expressed under standard culturing conditions exists primarily as a triply phosphorylated species, which we showed previously is due to constitutive phosphorylation of Ser129/Ser133/Ser1372+-CaM dependent, and can readily undergo Thr85 autophosphorylation in the presence of Ca2+-CaM and MgATP. Transfected cells were harvested after 48 hr by washing with ice-cold phosphate-buffered saline (PBS) followed by rapid lysis in situ using 1 ml of lysis buffer  containing Complete protease inhibitor cocktail (Roche). Cellular debris was removed by centrifugation and total protein was determined using the Bradford protein assay (Pierce). Recombinant CaMKK2 was purified from 2 μg of cell lysate using 10 μl of anti-Flag M2 agarose (50% v/v) (Sigma) pre-equilibrated in lysis buffer, followed by successive washes in lysis buffer containing 1 M NaCl, and finally into 50 mM HEPES [pH 7.4]. The immobilized CaMKK2 was then sedimented by centrifugation and used for kinase assays, or the CaMKK2 was eluted from the beads by incubating overnight at 4 °C with two bead volumes of Flag peptide (1 mg/ml), and used for the CaM overlay and MS/MS phosphopeptide analysis.COS7 cells were grown in DMEM (Sigma) media with 10% fetal calf serum at 37 °C with 5% CO2, 1 μM CaM (Sigma), 40 μM EGTA, 200 μM [γ-32P]-ATP (Perkin Elmer) and 5 mM MgCl2. All the kinase reactions were buffered with 40 μM EGTA, which we determined by titration to chelate trace amounts of Ca2+ present in the CaM preparations. This was essential to measure the stimulation of CaMKK2 activity at the 10 μM Ca2+ concentration2, 50 μM CaCl2 and 1 μM CaM in the presence and absence of 200 μM ATP (Sigma). Reactions were incubated at 30 °C for various times, after which the beads were washed successively in lysis buffer containing 1 M NaCl, and finally re-suspended in 50 mM HEPES [pH 7.4] to achieve a 50% slurry. A 10 μl aliquot (50% slurry) was removed and kinase activity measured in assay buffer containing 200 μM CaMKKtide, 50 μM CaCl2, 1 μM CaM, 200 μM [γ-32P]-ATP and 5 mM MgCl2, in the presence or absence of 1 mM EGTA using the CaMKKtide peptide substrate assay described above. Based on CaMKK2 having a specific activity of 1 μmol.min−1.mg−1 the concentration of CaMKK2 in the pull down assays is approximately 0.5 nM.CaMKK2 activity was measured using a synthetic peptide substrate (CaMKKtide) as previously describedCell lysates or immuno-precipitated CaMKK2 were denatured in SDS sample buffer, separated by SDS-PAGE and transferred to Immobilon PVDF membranes (Millipore). Membranes were blocked for 1 hr in PBS/1% Tween-20 (PBS-T) supplemented with 2% non-fat milk. Primary antibodies were diluted in PBS-T containing 1% non-fat milk at the following dilution: rabbit or mouse anti-Flag , rabbit anti-pThr85 (300 ng/ml)2. The membranes were briefly washed in PBS-T containing 10 mM CaCl2, and then incubated with IR680 dye labeled streptavidin (Li-Cor) for 1 hr. After successive washing with PBS-T containing 10 mM CaCl2, the membranes were then scanned with an Odyssey Infrared Imager. The membranes were then incubated in PBS-T containing 15 mM EGTA overnight at 4 °C in order to determine if the biotinylated CaM bound in a Ca2+ dependent manner.1 μg of purified WT and A329T mutant CaMKK2 were spotted onto nitrocellulose membrane, which was then blocked for 1 hr in PBS/1% Tween-20 (PBS-T) supplemented with 2% non-fat milk. The membrane was incubated overnight at 4 °C with biotinylated CaM  diluted in PBS-T containing 1% non-fat milk and 10 mM CaCl2, 50 μM CaCl2 and 1 μM CaM in the presence and absence of 200 μM ATP for 40 min, then digested with trypsin and analyzed by reversed-phase HPLC-ESI-MS/MS using an UltiMate 3000 Nano LC HPLC system (Dionex) directly connected to a Triple-TOF 5600 mass spectrometer (AB SCIEX) in direct injection mode. Peptide mixtures were resolved on an analytical nanocapillary HPLC column (100 μm i.d. × 15 cm) packed with C18 Acclaim PepMap100  using a 1–75% elution gradient of 98% acetonitrile/2% of 0.1% formic acid (v/v) in water at a flow rate of 250 nl/min. Mass spectrometric data were analyzed using the database search engine ProteinPilot and the Paragon algorithm (AB Sciex).In order to analyse Thr85 autophosphorylation in the G87R mutant, 1 μg of WT CaMKK2 or G87R mutant was incubated with 5 mM MgClResults are expressed as the mean ± standard error of mean (SEM). Statistical analysis, where indicated, was performed using two-way analysis of variance (ANOVA), with the alpha level set at 0.05 for each test. P-values of less than 0.05 were considered statistically significant.How to cite this article: O’Brien, M. T. et al. Impact of Genetic Variation on Human CaMKK2 Regulation by Ca2+-Calmodulin and Multisite Phosphorylation. Sci. Rep.7, 43264; doi: 10.1038/srep43264 (2017).Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


TQ-6 (0.3 µM) exhibited extremely strong inhibitory activity against platelet aggregation, Src, and Syk phosphorylation stimulated by agonists in human platelets. In collagen-activated platelets, TQ-6 also inhibited ATP-release, [Ca+2]i, P-selectin expression, FITC-PAC-1 binding, and hydroxyl radical formation, as well as the phosphorylation of phospholipase Cγ2, protein kinase C, mitogen-activated protein kinases, and Akt. Neither FITC-JAQ1 nor FITC-triflavin binding or integrin β3 phosphorylation stimulated by immobilized fibrinogen were diminished by TQ-6. Furthermore, TQ-6 had no effects in cyclic nucleotide formation. Moreover, TQ-6 substantially prolonged the closure time in whole blood, increased the occlusion time of thrombotic platelet plug formation and bleeding time in mice. In conclusion, TQ-6 has a novel role in inhibiting platelet activation through the inhibition of the agonist receptors-mediated inside-out signaling such as Src-Syk-PLCγ2 cascade and subsequent suppression of granule secretion, leading to disturb integrin αIIbβ3-mediated outside-in signaling, and ultimately inhibiting platelet aggregation. Therefore, TQ-6 has potential to develop as a therapeutic agent for preventing or treating thromboembolic disorders.Arterial thrombosis plays a key role in cardiovascular diseases. Hence, developing more effective antithrombotic agents is necessary. We designed a ruthenium (II)-derived complex, [Ru(η The involvement of platelets in hemostasis is associated with their adherence and aggregation ability; storage granule content release; adsorption, deposition, and transportation of biologically active substances; and endothelial supporting functions2. Under high shear conditions in arterial blood vessels, the vascular endothelium is disrupted, and platelets then adhere to the damaged intima and undergo activation. During platelet activation, the release of several mediators  occurs, coupled with intracellular calcium mobilization, thus drawing additional platelets toward the injured endothelium and consequently causing the initial platelet monolayer to thicken. Finally, fibrinogen binds to its specific platelet receptor glycoprotein (GP) IIb/IIIa complex , thus completing the final common pathway for platelet aggregation. The complex is formed through calcium-dependent association of GP IIb and GP IIIa, a required step in normal platelet aggregation and endothelial adherence.Platelets are anucleate blood cells that originate from megakaryocytes and play a central role in hemostatic processes. Platelets have an established role in the pathogenesis of atherosclerosis-related diseases including coronary artery diseases and stroke. Under normal conditions, platelets circulate in an inactive form, without significant interactions with the vessel walls. Rupture of an atherosclerotic plaque promotes the activation of platelets and initiates the coagulation cascade2, a crucial secondary mediator for further platelet activation. The cyclooxygenase-1 mediated conversion of arachidonic acid (AA) to TxA2 can be irreversibly inhibited by aspirin, a standard drug has been used for preventing cardiovascular diseases (CVDs)3. Several studies have been focused on searching drugs that target the TxA2 pathway in platelets, including various TxA2 receptor antagonists 4, TxA2 synthase inhibitors 5, and compounds act on these two functions 6. Moreover, although aspirin, clopidogrel, and tirofiban are considered as well-established antiplatelet agents on treating thromboembolic diseases, they have still more substantial restrictions when using in clinical settings8. Hence, it is important for developing more effective and safe antithrombotic agents to meet the clinical requirements.Activated platelets are produced TxA11. In addition, antiangiogenic therapy is considered a promising strategy for treating cancers. Lai et al.12 reported that ruthenium complexes have potent antiangiogenic effects through the activation of distinct antiangiogenic signaling pathways. On account of the preceding observations, to discover new biologically active ruthenium derivatives, we developed a new ruthenium compound, TQ-6  values of TQ-6 for platelet aggregation induced by collagen, U46619, and thrombin were approximately 0.3, 5, and 60 μM, respectively  under equivalent conditions Fig. , preliminel Fig. ,Figure 2IIbβ3 is the final step of the common pathway for agonist-induced platelet aggregation. Therefore, we further evaluated whether TQ-6 influences platelet integrin αIIbβ3 activation, leading to the inhibition of platelet aggregation. As shown in the Fig. IIbβ3 stimulated by collagen (1 μg/ml). The relative fluorescence intensity of binding FITC-PAC-1 in activated platelets was significantly higher than that of the resting platelets  and binding numbers16. As shown in Fig. n = 4), and it significantly decreased in the presence of 5 mM EDTA   Fig. . Taken t17. We further evaluated whether TQ-6 inhibits platelet activation by specifically binding to GP VI on the platelet membrane. As shown in Fig. GP VI is the major collagen receptor that mediates platelet adhesion and aggregation1 - and 10 μM NTG-treated platelets, respectively  and 1 μM PGE1 Fig. . Neither1 Fig. . As show18. The Src family kinases are activated through phosphorylation of tyrosine based activation and subsequently leading to binding and activation of the tyrosine kinase Syk. TQ-6 manifestly reduced both SFK  and Syk phosphorylation, respectively, in washed platelets stimulated by collagen, thrombin, and U46619  and diacylglycerol (DAG). IP3 triggers intracellular Ca2+ mobilization and DAG activates protein kinase C (PKC) activation, inducing a protein with an apparent molecular weight of approximately 47 kDa, which is predominantly phosphorylated  and causes the ATP-release reaction19. As presented in the Fig. 2+ mobilization. We further investigated the influence of TQ-6 on the phosphorylation of the PLCγ2-PKC signaling cascade. At 0.3 and 0.5 µM, TQ-6 evidently reduced both PLCγ2 phosphorylation and PKC activation (pleckstrin phosphorylation), respectively, in collagen-activated platelets  play a central role in mediating the rapid response of platelet activation. Ligand-mediated of diverse receptors triggers an increase in SFK activity and downstream tyrosine phosphorylation of enzymes, adaptors, and cytoskeletal proteins that collectively propagate the signal and coordinate platelet activation619 Fig. . Additioets Fig. . In addiets Fig. , signify20. Notably, 0.5 μM TQ-6 significantly inhibited the phosphorylation of ERK2, JNK1, and p38 MAPK , c-Jun N-terminal kinases 1 and 2 (JNK1/2), and p38 MAPK, control major cellular responses in eukaryotic organisms and contribute to cell proliferation, migration, differentiation, and apoptosis. ERK2, JNK1, and p38 MAPK have been identified in plateletsion Fig. , indicat·) formation was observed in collagen-activated platelets, but not in resting platelets  and long lived g = 2.005 radical detectable by the spin trapped DMPO  was 93.2 ± 5.5 s (n = 6)   Fig. . Treatme01) Fig. . After i01) Fig.  a,b. On 01) Fig.  c,d. The01) Fig.  b.In the tail transection model of mice, after 30 min of administering 150 mg/kg aspirin intraperitoneally, the bleeding time considerably increased from 229.2 ± 20.5 s (PBS-treated control group) to 438.1 ± 22.6 s. The bleeding time of the 0.4 mg/kg TQ-6-treated mice was slightly longer than that of the solvent control (0.5% DMSO)-treated mice (218.7 ± 22.5 s vs. 291.2 ± 17.8 s) Fig. . In this22. Notably, our results show, for the first time, that in addition to its antitumor activity, TQ-6, a novel synthetic ruthenium (II)-derived compound, exhibits highly potent antiplatelet activity  ex vivo and effectively inhibits arterial thrombogenesis (0.4 mg/kg) in vivo. Platelets are activated by various physiological stimuli . These agonists are considered to exert their effects by interacting with specific receptors on platelet membranes. According to the IC50 values, TQ-6 had the stronger inhibitory activity against collagen-stimulated platelet aggregation than did U46619; it also presented relatively weak activity against thrombin stimulation, indicating that TQ-6 inhibits platelet aggregation through a considerable SFK-Syk-PLCγ2/PKC-dependent mechanism. Aspirin has been clinically used for treating and preventing CVDs. Our evaluation demonstrated that under identical conditions, TQ-6 was more potent than aspirin, indicating its potential clinical applications.Ruthenium complexes are an attractive alternative for platinum because they have several favorable properties suitable for rational anticancer drug design and biological applications. Thus, ruthenium metal complexes are considered one of the most promising anticancer agents+2, and P-selectin). Collagen, present in the vascular subendothelium and vessel wall, acts as a platelet adhesion substrate as well as an endogenous platelet activator. Among the platelet receptors interacting directly with collagen, integrin α2β1 (GP Ia/IIa) and GP VI might contribute to the overall processes of platelet adhesion and activation23. Signals  are activated in platelets expressing integrin α2β1 after cell adhesion to collagen24. GP VI ligands, including collagen and convulxin, induce receptor clustering, which facilitates the phosphorylation of the tandem tyrosine found in the immunoreceptor tyrosine-based activation motifs of the noncovalently associated Fcγ-chain receptor adaptor by SFKs 25. TQ-6 inhibited platelet aggregation at different degrees, depending on the agonists , thus indicating that TQ-6 was potentially not effective on specific/individual receptors of these agonists . Thus, TQ-6 probably acts through a common signal cascade against stimulated platelets. A major component of the platelet aggregation response is the binding of fibrinogen to integrin αIIbβ3 on activated platelets. Triflavin acts as a specific antagonist for integrin αIIbβ3, which interferes with the fibrinogen–integrin αIIbβ3 interaction15. In this study, TQ-6 significantly affected the binding efficiency of FITC-PAC-1 to activated integrin αIIbβ3 without influencing upon the FITC-triflavin binding. Besides, integrin β3 phosphorylation stimulated by immobilized fibrinogen was not alleviated by TQ-6, which infers that the antiplatelet activity of TQ-6 is not directly interference the binding of fibrinogen toward to integrin αIIbβ3 and its mediated outside-in signaling. Furthermore, TQ-6 inhibited ATP release and [Ca+2]i mobilization from intracellular granules, corroborating the concept that changes in ATP and cytosolic Ca+2 play a major role in platelet activation.Platelets adhere to subendothelial matrix proteins , thus changing their shape and causing the release of granular contents  activation that are either associated with or in close proximity to their cytoplasmic tails, to transmit signals. Downstream effectors of SFK include adaptors, enzymes , and cytoskeletal proteins that collectively coordinate cytoskeletal remodeling, degranulation, and integrin activation, which is referred to as inside-out signaling29. Therefore, selective inhibitors of Akt isoforms or of proteins contributing to its activation, such as individual PI3K isoforms, may be attractive targets for antithrombotic therapy21. In addition, MAPKs are activated by specific MAPK kinases (MEKs); specifically, MEK1/2 activates ERK1/2, MEK3/6 activates p38 MAPK, and MEK4/7 activates JNK1/230. ERK2 activation was reported to be involved in platelet aggregation induced by low-dose collagen, requiring prior ATP release, which triggers P2 X1-mediated Ca2+ influx and activates ERK2, thereby increasing myosin light-chain kinase phosphorylation31. Cytosolic phospholipase A2 (cPLA2) is a substrate of p38 MAPK activity induced by various agonists such as von Willebrand factor (VWF), thrombin, and collagen31. Thus, p38 MAPK is essential for the stimulation of cPLA2 and release of AA, leading to TxA2 production in platelets32. JNK1 is the most recently identified MAPK in platelets, and its activation or role is therefore poorly defined. JNK1 is activated by several agonists such as thrombin, VWF, collagen, and ADP31, and JNK1 activation was reported to trigger integrin αIIbβ3 activation31. In the current study, ERK2, JNK1, and p38 MAPK, appear to play a crucial role in TQ-6-mediated inhibition of platelet activation.Akt is a downstream effector of phosphoinositide 3-kinase (PI3K); Akt-knockout mice have been reported to exhibit defects in agonist-induced platelet activation, suggesting that Akt regulates platelet activation, with potential consequences for thrombosis·) might affect cells they come in contact with, such as endothelial cells; this can amplify platelet reactivity during thrombus formation. Free radical species act as secondary signals that increase [Ca2+]i levels during the initial phase of platelet activation, and PKC is involved in receptor-mediated free radical production in platelets33. In addition, hydrogen peroxide produced by platelets is converted into OH· because platelet aggregation can be inhibited by OH· scavengers33. Our ESR spectrometry results provide direct evidence that TQ-6 diminished OH· formed through collagen stimulation in platelets. Thus, the in vivo TQ-6-mediated inhibition of thrombogenesis may involve free radical scavenging. After vascular endothelial cell injury, exposure to subendothelial collagen is the major trigger initiating platelet adhesion and aggregation at the site of injury, followed by arterial thrombus formation. The PFA-100 system records the time required for platelet aggregation to occlude an aperture in a collagen-coated membrane. Platelet adhesion to collagen depends on flow conditions; in this study, platelets prolonged adhesion to collagen under flow conditions. In a thrombosis study, the mesenteric venules were continuously irradiated by fluorescein sodium throughout the experimental period, leading to strong damage to the endothelial cells34. TQ-6 significantly prolonged both closure times and occlusion times; these effects may be mediated, at least partly, by the inhibition of platelet activation. In addition, the tail transection model of mice was used to examine the effect of TQ-6 on bleeding time in vivo. Although aspirin is the most effective antiplatelet drug prescribed for preventing or treating cardiovascular and cerebrovascular diseases, it causes an unwanted prolongation of bleeding time. In the tail transection model, the slight prolongation of the bleeding time may be caused by the antiplatelet activity of TQ-6, at least partly. Furthermore, these results may serve basically as a general example of how TQ-6 increases bleeding time compares to prominent bleeding phenotypes.Reactive oxygen species derived from platelet activation , leading to disturb integrin αIIbβ3-mediated outside-in signaling , and ultimately inhibiting platelet aggregation. However, we could not rule out the involvement of other mechanisms, which are yet to be identified, in TQ-6-mediated inhibition of platelet activation. Thus, our findings suggest a novel ruthenium derivative, TQ-6, as a potential therapeutic agent for preventing or treating thromboembolic disorders.In conclusion, the findings of this study reveal that TQ-6 has a novel role in inhibiting platelet activation through the inhibition of the agonist receptors-mediated inside-out signaling such as SFK-Syk-PLCγ2-PKC cascade and subsequent suppression of Akt and MAPKs activation. These alterations reduce granule secretion , aspirin, 5,5-dimethyl-1-pyrroline N-oxide (DMPO), SQ22536, 1H-oxadiazoloquinoxalin-1-one (ODQ), bovine serum albumin (BSA), fibrinogen, and thrombin were purchased from Sigma . Fura-2AM, an intracellular calcium indicator, was purchased from Molecular Probes . Convulxin, anti-phospho-p38 mitogen-activated protein kinase (MAPK) Ser182, anti-integrin β3 monoclonal antibodies (mAbs), and anti-phospho-integrin β3 (Tyr759) polyclonal antibody (pAb) were purchased from Santa Cruz Biotechnology . Anti-p38 MAPK, anti-phospho-JNK (Thr183/Tyr185), anti-Syk (D3Z1E) XP®, anti-phospho-Src family (Tyr416), and anti-p44/42 ERK (1/2) mAbs, as well as anti-PLCγ2, anti-phospho (Tyr759) PLCγ2, anti-phospho-(Ser) PKC substrate , anti-JNK, anti-phospho-Syk (Tyr525/526), anti-Src family, and anti-phospho-p44/p42 ERK (Thr202/Tyr204) pAbs, were purchased from Cell Signaling . Anti-phospho-Akt (Ser473) and anti-Akt mAbs were purchased from Biovision . An anti-pleckstrin (p47) pAb was purchased from GeneTex . A Hybond-P PVDF membrane, an enhanced chemiluminescence Western blotting detection reagent, horseradish peroxidase (HRP)-conjugated donkey anti-rabbit immunoglobulin G (IgG), and sheep anti-mouse IgG were purchased from Amersham . Fluorescein isothiocyanate (FITC)-labelled anti-GP VI (JAQ1) mAb was obtained from Emfret Analytics . FITC anti-human CD42P (P-selectin) and FITC anti-human CD41/CD61 (PAC-1) mAbs were obtained from BioLegend . Cyclic AMP and cyclic GMP enzyme immunoassay (EIA) kits were obtained from Cayman . The Dade Behring PFA-100 collagen/epinephrine (CEPI) test cartridge was obtained from Siemens Healthcare .Collagen, luciferin-luciferase, U46619, phorbol 12,13-dibutyrate (PDBu), heparin, prostaglandin E2SO4, and concentrated through rotary evaporation. The resulting crude product was subjected to silica gel column chromatography and eluted with DCM/MeOH (99:1) to afford the pure title compound L: pale yellow solid ; mp 218 °C–220 °C; 1H NMR  δ 10.97 , 8.50–8.48 , 8.26–8.24 , 8.05–8.02 , 7.83–7.79 , 7.71–7.66 , 7.53–7.49 , 7.43–7.41 , 7.25–7.21 ; ESI-MS m/z 244 (M+).2-Methyl-quinoline  was added to a Schlenk flask containing 2-nitro-phenylamine , elemental sulfur , and iron powder . After the flask was flushed with argon, the contents were stirred at 160 °C under an atmosphere of argon for 24 h. The reaction mixture was then allowed to cool down, excess iron powder was removed by filtration through a celite pad, and the solvent was evaporated under reduced pressure. Next, the residue was dissolved in dichloromethane, washed with water, dried over anhydrous Na2]2  and L  were suspended in methanol (20 ml) and stirred at room temperature for 2 h. NH4BF4  in MeOH (10 ml) was added to the initially yellow solution, thus changing its color to orange. After 24 h, the solution was evaporated, and the resulting solid was filtered out. The residue was washed with diethyl ether (40 ml) and dried under vacuum. The desired products were recrystallized from DCM:hexane solvent mixture to give orange microcrystals: 1H NMR  δ 15.46 , 8.98–8.96 , 8.83–8.80 , 8.66–8.64 , 8.29–8.27 , 8.19–8.13 , 7.97–7.94 , 7.90–7.87 , 6.33–6.32 , 6.31–6.29 , 6.11–6.09 , 2.28 , 2.21–2.15 , 0.73 ; ESI-MS: m/z = 516 [M-BF4]+ (Cl)4]+ Fig. . The fre34. Human blood samples were obtained from adult volunteers who refrained from the use of drugs or other substances that could interfere with the experiment for at least 14 days before collection; the collected blood samples were mixed with an acid-citrate-dextrose solution. After centrifugation, the platelet-rich plasma was supplemented with 0.5 μM PGE1 and 6.4 IU/ml heparin. Tyrode’s solution containing 3.5 mg/mL BSA was used to prepare the final suspension of washed human platelets. The final Ca2+ concentration in Tyrode’s solution was 1 mM. The platelet aggregation test was performed using a lumiaggregometer  as described previously34. Various concentrations of TQ-6 or a solvent control (0.5% DMSO) were preincubated with platelet suspensions (3.6 × 108 cells/ml) for 3 min before the addition of various concentrations of agonists . The extent of platelet aggregation was calculated as a percentage of the control (absence of TQ-6) of light-transmission units, after the reaction proceeded for 6 min. For ATP release assay tests, 20 μl of luciferin-luciferase was added 1 min before the addition of the agonist; the amount of ATP released was compared with that released by the control.This study was approved by the Institutional Review Board of Taipei Medical University (TMU-JIRB-N201612050) and conformed to the directives of the Declaration of Helsinki. All human volunteers involved in this study provided informed consent. Human platelet suspensions were prepared as described previously2+]i was determined using Fura-2AM as described previously34. Briefly, citrated whole blood was centrifuged at 120 × g for 10 min; the supernatant was collected and incubated with 5 μM Fura-2AM for 1 h. Human platelets were prepared as described in the previous section. The Fura-2AM-loaded platelets were washed and preincubated with TQ-6 in the presence of 1 mM CaCl2 and then stimulated with collagen. The Fura-2 fluorescence was measured using a spectrofluorometer  at excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm.The amount of intracellular calcium concentration 35.Electron spin resonance (ESR) spectrometry was performed on a Bruker EMX ESR spectrometer  as described previously36. Cartridges containing CEPI-coated membranes were preincubated with various concentrations of TQ-6 or the solvent control (0.5% DMSO) for 2 min. Whole blood aliquots (0.8 ml/cartridge) were applied to the cartridges before the contents were exposed to high-shear-flow conditions (5000–6000/s). Closure time (CT) was defined as the time required for a platelet plug to occlude the aperture in the collagen membrane36.A Dade Behring PFA-100 system  was used to analyze platelet function37. Venules (30–40 µm) were irradiated at wavelengths of <520 nm to produce a microthrombus. Two doses of TQ-6 (0.2 and 0.4 mg/kg) were administered 1 min following sodium fluorescein (15 µg/kg) administration, and the time required for the thrombus to occlude the microvessel (occlusion time) was recorded. In this experiment, the method applied on the thrombogenic animal model conformed to the Guide for the Care and Use of Laboratory Animals  and was approved by Affidavit of Approval of Animal Use Protocol-Taipei Medical University (LAC-2016–0395).Male ICR mice (6 weeks) were anesthetized using a mixture containing 75% air and 3% isoflurane maintained in 25% oxygen; their external jugular veins were cannulated with a PE-10 tube for administering the dye and drugs intravenouslyThe bleeding time was measured through transection of the tail of the mice. In brief, after 30 min of administering either 0.4 mg/kg TQ-6 or 150 mg/kg aspirin intraperitoneally, we sharply cut the tail of the mice at 3 mm from the tip. The tails were immediately placed into a tube filled with saline at 37 °C for measuring bleeding time, which was recorded until the bleeding completely stopped.n). Values of n refer to the number of experiments, and each experiment was conducted using different blood donors. The unpaired Student’s t test was used to determine significant differences in the occlusion times and bleeding time of mice. The differences between the groups in other experiments were assessed using ANOVA. When the ANOVA indicated significant differences among the group means, the groups were compared using the Student–Newman–Keuls method. A p value of <0.05 indicated statistical significance. Statistical analyses were performed using SAS .The experimental results are expressed as means ± standard errors of the means and are accompanied by the number of observations (


The nuclear import receptor karyopherin β1 (KPNB1) is involved in the nuclear import of most proteins and in the regulation of multiple mitotic events. Upregulation of KPNB1 has been observed in cancers including glioblastoma. Depletion of KPNB1 induces mitotic arrest and apoptosis in cancer cells, but the underlying mechanism is not clearly elucidated. Here, we found that downregulation and functional inhibition of KPNB1 in glioblastoma cells induced growth arrest and apoptosis without apparent mitotic arrest. KPNB1 inhibition upregulated Puma and Noxa and freed Mcl-1-sequestered Bax and Bak, leading to mitochondrial outer membrane permeabilization (MOMP) and apoptosis. Moreover, combination of Bcl-xL inhibitors and KPNB1 inhibition enhanced apoptosis in glioblastoma cells. KPNB1 inhibition promoted cytosolic retention of its cargo and impaired cellular proteostasis, resulting in elevated polyubiquitination, formation of aggresome-like-induced structure (ALIS), and unfolded protein response (UPR). Ubiquitination elevation and UPR activation in KPNB1-deficient cells were reversed by KPNB1 overexpression or inhibitors of protein synthesis but aggravated by inhibitors of autophagy-lysosome or proteasome, indicating that rebalance of cytosolic/nuclear protein distribution and alleviation of protein overload favor proteostasis and cell survival. Chronic activation of eIF2α/ATF4 cascade of UPR was responsible for the upregulation of Puma and Noxa, apoptosis and ABT-263 sensitivity. Taken together, our findings demonstrate that KPNB1 is required for proteostasis maintenance and its inhibition induces apoptosis in glioblastoma cells through UPR-mediated deregulation of Bcl-2 family members. Karyopherin β1 (KPNB1), also known as importin β, is a nuclear transport receptor belonging to the karyopherin family that is involved in transporting proteins through the nuclear pore . GeneralKPNB1 concentration correlates with its nuclear import efficiency and speed . Many KPGlioblastoma multiforme (GBM) is the most common malignant brain tumor in adults and remains incurable using current therapies, which urgently needs deeper understanding of its molecular pathology to develop novel therapeutic strategies. In this study, we show that KPNB1 is required for glioblastoma survival. KPNB1 deficiency disturbed proteostasis, caused UPR-mediated deregulation of Bcl-2 family proteins, and ultimately induced apoptosis, which can be potentiated by Bcl-xL inhibitors, lysosome inhibitors or proteasome inhibitors. These data can have translational implication in glioblastoma treatment.http://www.betastasis.com/glioma/rembrandt/) quinoxalin-2-amine) (ZINC identification no. 20547783) .shRNAs targeting human KPNB1, Bax, Bak, Bcl-xL, Mcl-1, ATF4, CHOP, and ATF3, and a scrambled (control) shRNA were cloned into the lentiviral vector pLKD-CMV-GFP-U6-shRNA. Human 3Flag-tagged KPNB1 gene, untagged phospho-defective Bcl-xL (S62A) mutant and non-degradable phospho-defective Mcl-1 (T92A) mutant were generated by PCR amplification. KPNB1 and Bcl-xL (S62A) were cloned into the lentiviral vector pLOV-CMV-eGFP and Mcl-1 (T92A) was cloned into pLOV-EF1a-eGFP. Lentiviruses encoding various shRNA plasmids and expression plasmids were generated as previous described . The seqAfter collection, cells were lysed in RIPA buffer supplemented with PMSF, phosphatase inhibitor, and protease inhibitor cocktail. Western blots were carried out in compliance with the standard protocol.MTT assays and colony formation assays were performed as previously described . Three iCell cycle and mitochondrial membrane potential analysis were carried out using LSR-II. Cell cycle distribution was determined as previously described . MitochoCells were washed by phosphate-buffered saline (PBS) and lysed on ice using cell lysis buffer for Western and IP (to detect interacting proteins) or RIPA (to detect p65 ubiquitination). Lysates were adjusted to equal protein concentrations and incubated with indicated antibodies at 4 °C overnight, followed by incubation with protein A or protein G agarose at 4 °C for 2 h. Precipitates were then washed four times with cell lysis buffer and analyzed by standard Western blots.Cells plated onto glass coverslips were fixed with 4% paraformaldehyde. Cells were incubated with blocking buffer  for 1 h and stained with antibodies and Hoechst 33342 overnight. Fluorescent signals were observed with a Nikon FN1 confocal microscope at ×60 magnification.t-test was used to determine significant differences between the mean values of groups, with statistical significance defined as P < 0.05. The variance is similar between groups that are being statistically compared. Sample size in the cell cycle analysis is calculated by Sample Size Calculator .GraphPad Prism 6.01 software  was used in the study. The two-tailed unpaired supplementary informationsupplementary Figure 1-4supplementary Figure 5Full blots for western blot