Ethylenediaminetetraacetic acid-based Mg2+ chelation facilitates the dissolution of Mg2+-BP NP, thus enabling the reversion of the MgBNP to the BNP. This convertible nanoassembly incorporating cell-adhesive Mg2+ moieties directs reversible attachment and detachment of macrophages by BP and EDTA, without physical scraping or trypsin that could damage cells. The swift formation of RGD ligand- and Mg2+-bifunctional RGD-Mg2+-BP NP that yields (RGD-Mg2+-BP)-Au dimer (RGDBNP) further stimulates the adhesion and pro-regenerative M2-type polarization of macrophages, both in vitro and in vivo, including rho-associated protein kinase. This swift and non-toxic dimer formation can include diverse bio-functional moieties to regulate host responses to implants.Macrophages regulate host responses to implants through their dynamic adhesion, release, and activation. Herein, we employ bisphosphonate (BP)-coated gold nanoparticle template (BNP) to direct the swift and convertible formation of Mg Control of macrophage adhesion and phenotype is important to biomaterial applications. Here, the authors report on the use of bisphosphonate coated gold nanoparticles by magnesium coordination for the controlled adhesion and polarisation of macrophages in vitro and in vivo and controlled cell release. When macrophages are activated, they can be polarized into different phenotypes that elicit distinct immune functions, which are simply classified as pro-inflammatory M1-type or pro-regenerative M2-type. Macrophages also display a spectrum of activation states involving central transcriptional regulators that govern macrophage activation, and stimulus-specific regulators2. Macrophages play a key role in regulating foreign body response4. Foreign body response to implants is induced primarily by macrophages and foreign body giant cells that regulate the inflammatory and wound healing responses4. Macrophages that polarize into M2 phenotypes typically undergo fusion to form foreign body giant cells4. Macrophages and foreign body giant cells depending on their phenotypes over the time course secrete various cytokines and growth factors that coordinate foreign body response4. Hence, designing biomaterials with their cell-adhesive surface properties that are diversely convertible, can offer a powerful control over the foreign body responses to the implanted biomaterials by modulating the attachment and activation of macrophages5.Macrophages are crucial immune cells that regulate immune systems, disease progression, and wound healing2+ moieties in biomaterials can effectively stimulate integrin binding and cellular attachment8. The RGD motif can act as an integrin-binding ligand to mediate the attachment of macrophages10. The detachment of macrophages has typically been achieved by physically scraping them from culture substrate surfaces13. However, this method can mechanically damage the macrophages, yielding detached macrophages in low viability13. Trypsin-ethylenediaminetetraacetic acid (EDTA) buffer has been commonly employed to detach cells from various substrate surfaces. However, trypsin is a proteolytic enzyme that cleaves cell surface receptors that carry out various cellular functions including cell attachment and spreading14. Thus, developing new biomaterials that can enable the non-toxic, efficient, and reversible attachment and detachment of macrophages without the use of physical scraping or proteolytic enzymes can offer unique advantages over those conventional methods. In addition, adhesive features in macrophages were reported to modulate their polarization phenotypes18. The spatial patterning of pre-assembled nanomaterials presenting various bio-functional motifs were reported to modulate the attachment or activation of immune cells22. Thus, it is desirable to develop materials that can manipulate the attachment and detachment of macrophages as well as their phenotypic polarization by dynamically presenting bio-functional moieties.Macrophages actively associate with the extracellular matrix that modulates their attachment, detachment, and activation. Developing biomaterials with cell-adhesive functional moieties can enable the manipulation of reversible attachment and detachment of macrophages as well as their functional activation. The inclusion of Mg24. Light was used to mediate the conversion between cell-adhesive and non-adhesive biomaterials via the release of RGD-conjugated DNA25 or photo-induced isomerization26 in vitro or to trigger the attachment of host macrophages in vivo by the photocleavage of RGD-caging molecule10. Enzymes were used as chemical trigger to modulate cell attachment by cleaving bio-functional moieties in vitro27. However, to the best of our knowledge, there have been no prior studies demonstrating the regulation of reversible attachment and detachment of macrophages, and their phenotypic polarization by an extrinsic trigger.Developing biomaterials with bio-functional moieties that can be converted by an extrinsic trigger in vivo can manipulate the function of host macrophages to modulate host responses to the implants. Cell–material interactions have been manipulated by various physical and chemical trigger. For example, a magnetic field was used in our own recent studies to manipulate the tether mobility or continuous motion of RGD-coated superparamagnetic nanoparticles to control cell attachment2+-bisphosphonate (BP) coordination by employing small molecules utilized in the clinics as the trigger, to manipulate the attachment, detachment, and polarization of macrophages  on the BP-AuNP template, resulting in the growth of Mg2+-functional (Mg2+-BP)-Au dimer (MgBNP). Chelation of Mg2+ by EDTA disassembles the Mg2+-BP NP in the dimer, resulting in the reversion of the MgBNP to the BNP. This convertible process is performed with macrophages to enable their reversible attachment and detachment. We employ BP and EDTA, non-toxic small molecules, and thus supply RGD-conjugated BP, BP, and Mg2+ to direct the formation of RGD-Mg2+-BP NP, thus generating RGD- and Mg2+-bifunctional (RGD-Mg2+-BP)-Au dimer (RGDBNP), which promotes the attachment and M2-type polarization of macrophages. Biomaterials that can reversibly convert their cell-adhesive and non-adhesive property can culture macrophages in a reversible and non-toxic way or function as implants that direct the attachment and activation of host macrophages to modulate inflammatory or tissue-healing processes.In this study, we utilize the swift and convertible dimer formation driven by the reversible Mgges Fig. . We conj2+-BP-NP on its surface, driven by the coordination between the Mg2+ ion and BP molecule  and BP (1 mM) for enabling the growth of the Mg2+-BP NP on the BP-AuNP surface via Mg2+-BP coordination swiftly in 10 min, thus generating the (Mg2+-BP)-Au dimer (MgBNP). High-resolution TEM (HRTEM) image revealed a single layer of newly formed Mg2+-BP NP on the BP-AuNP surface in a dimeric MgBNP nanostructure  was characterized to be roughly 79 nanoparticles/μm2  and BP (3 mM) and found that the coverage of the BP-AuNP surface by the Mg2+-BP NPs slightly increased to 84%; however, this nano-formation produced the uncontrollable and heterogeneous size and shape of the Mg2+-BP NPs  and BP (1 mM) in this study because it facilitated the homogenous, and controllable formation of the Mg2+-BP NPs. The nanoscale arrangements of bio-functional moieties, including their nanoparticle density and interparticle spacing30, influence cell attachment. Thus, in our study, we controlled the density of the Mg2+-functional Mg2+-BP NPs formed in dimers to direct efficient manipulation of the attachment and phenotypic polarization of macrophages. The MgBNP dimer was found to be stable under culture conditions for 7 days -Au dimer (MgBNP) obtained by the formation of the Mg2+-BP NPs supported the attachment of macrophages, whereas the BNP to which they reverted after the swift dissolution of the Mg2+-BP NPs, effectively detached by roughly 89% of the attached macrophages, and this attachment and detachment were repeatable in two series . The newly formed Ca2+-BP NPs or Sr2+-BP NPs revealed similar size to that of the Mg2+-BP NPs -Au or (Sr2+-BP)-Au dimers. The extent of macrophage adhesion to the (Ca2+-BP)-Au dimers was comparable with that of the (Mg2+-BP)-Au dimers and it was significantly higher than that to the (Sr2+-BP)-Au dimers  was also prepared and the attachment of macrophages to this substrate was significantly less efficient than that to the substrate with higher MgBNP dimer density (64 dimers/μm2), which was thus employed in our study  and BP (1 mM) swiftly in 10 min  and treated the MgBNP substrate with RGD-BP, Mg2+, and BP swiftly in 10 min to induce the swift formation of RGD- and Mg2+-bifunctional RGD-Mg2+-BP NPs that generates (RGD-Mg2+-BP)-Au dimer (RGDBNP)  or RGD-Mg2+-BP NPs (RGDBNP dimer) after 0 h under culture. After 12 h under culture with basal medium, significantly higher density of attached macrophages (72% more adherent cells) was observed in the RGD- and Mg2+-bifunctional RGDBNP dimer than in the Mg2+-functional MgBNP dimer  and then the RGD-Mg2+-BP NP formation after 12 h or 18 h (MgBNP-RGDBNP 12 h or MgBNP-RGDBNP 18 h dimer), to explore the possibility of time-manipulated attachment of macrophages. Strikingly, the delayed formation of the RGD-Mg2+-BP NPs after 12 or 18 h under cell culture condition (MgBNP-RGDBNP 12 h and MgBNP-RGDBNP 18 h dimers) significantly increased the area and elongation factor of attached macrophages after 24 h, when compared with those of the MgBNP dimer . The detached macrophages obtained via the dissolution of Mg2+-BP NPs re-adhered and maintained their polarization capacity as evidenced by their efficient polarization into M1-type or M2-type under the culture with M1-type- or M2-type-polarizing medium, respectively  by reverse transcription-quantitative polymerase chain reaction (RT-qPCR) after 36 h under culture. The levels of both iNOS expression  and CD80 expression  were significantly downregulated in the RGDBNP and MgBNP-RGDBNP dimers, when compared with their expression in the MgBNP dimer  or M2-type marker (Arg-1 expression). The iNOS expression was significantly suppressed in the RGD- and Mgmer Fig. . The inhmer Fig. . We alsoArg-1 expression  and Ym2 expression  were observed in the RGDBNP and MgBNP-RGDBNP dimers, when compared with those in the MgBNP dimer  is a key mediator that regulates adhesive structures, such as the actin cytoskeletal organization and contractility. ROCK2 mediates the M2-type polarization of macrophages and functions as a molecular switch in macrophages between their M2-type and M1-typemer Fig. . We nextmer Fig. . Interesmer Fig. , withoutmer Fig. . In addimer Fig. . These f4. Hence, it is a vital requirement to modulate the attachment and functional states of macrophages to achieve desirable immune responses to the implants. To this end, we explored whether the convertible dimer nano-formation can convert their cell-adhesive and non-adhesive surface property in vivo to regulate the attachment and activation of host macrophages. We employed the non-functional PEGylated BNP substrate as the implant into a subcutaneous pocket and then administered small molecules used in the clinics, such as BP37 or EDTA38, or ions (Mg2+), for non-toxic and reversible conversion between cell-adhesive and non-adhesive implant surface  and CD80 expression (by 174%) were upregulated in the MgBNP dimer, when compared with their expression in the RGDBNP dimer  and Ym2 expression (by 181%) were upregulated in the RGDBNP, when compared with their expression in the MgBNP dimer  than the BNP Fig. . This inmer Fig. . The levors Fig. . With thmer Fig. . The levMacrophages can fuse to form foreign body giant cells, which regulate foreign body response. To explore the fusion of macrophages after longer implantation period (10 days), we prepared and implanted AuNP-conjugated PEGDA-based hydrogels as the BNP substrate -Au dimer (MgBNP) and Mg2+-deficient AuNP monomer (BNP), spatiotemporally manipulated reversible attachment and detachment of macrophages by utilizing small molecules used in the clinics, including BP and EDTA. We showed the reversible attachment and detachment of macrophages manipulated by the convertible nano-formation and dissolution, and this can be used to reversibly and cytocompatibly culture any cell types without the need for physical scraping or enzymes, such as trypsin, which could damage cells. The formation of RGD- and Mg2+-bifunctional (RGD-Mg2+-BP)-Au dimer (RGDBNP) further enhanced the attachment and M2-type polarization of macrophages, including ROCK signaling, whereas the “MgBNP” dimer-stimulated M1-type polarization of macrophages. This convertible dimer formation enables complex presentation of various bio-functional moieties that could potentially facilitate tissue engineering by regulating host responses to the implants.In summary, we demonstrated the convertible dimer formation presenting integrin-binding cell-adhesive Mg4·3H2O (0.88 mM) in deionized (DI) water (20 mL) with vigorous stirring first. To this solution, 1% (w/v) sodium citrate solution (2.4 mL) was rapidly added, which was further boiled for 20 min and then cooled to 25 °C. The resultant product was diluted with 1% sodium citrate solution to obtain AuNP solution (0.1 nM) before they were grafted to the substrate. The distribution in the diameter of the AuNPs was characterized by dynamic light scattering analysis. AuNPs were conjugated to glass coverslip substrate. The glass substrate was cleaned with a mixture of HCl and methanol (1:1) for 30 min, activated with sulfuric acid for 1 h, and thiolated with mercaptopropylsilatrane (0.5 mM) in methanol for 1 h in the dark. The substrate was treated with AuNP solution (0.1 nM) for 15 min to obtain the AuNP-decorated substrate.AuNPs were synthesized by boiling HAuClThe surface of the AuNP on the substrate was coated with 0.2% (w/v) thiolated BP, including N,N-diisopropylethylamine (12 µL) and Tris(2-carboxyethyl)phosphine hydrochloride (TCEP) (10 mM), at 25 °C for 16 h in the dark, and then PEGylated. Thiolated PB was prepared by reacting 1% (w/v) pamidronate disodium  and 1% (w/v) thioglycolic acid with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in NaOH solution (pH = 8.0) at 25 °C for 16 h.2 (1 mM) in tris-buffered saline  for 10 min to obtain MgBNP dimer. The MgBNP dimer was treated with EDTA (1 mM) in TBS for 10 min to dissolve Mg2+-BP NPs, which then reverts to the BNP substrate. The MgBNP substrate was treated with pamidronate disodium , RGD-BP (0.2 mM), and MgCl2 (1 mM) in TBS for 10 min, to obtain RGDBNP dimer. The RGD-BP was prepared by conjugating thiolated RGD peptide  to acryloyl BP (2 mM) with TCEP (5 mM) at 37 °C for 16 h. The acryloyl BP was prepared by reacting 1% (w/v) pamidronate disodium (BP) and 1% (w/v) N-acryloxysuccinimide in NaOH solution at 25 °C for 16 h.The BNP substrate was treated with pamidronate disodium  and MgCl2+-BP NPs was determined with the Image J software. Energy dispersive spectroscopy analysis was performed to characterize the convertible dimer formation for gold (Au) element in the AuNPs as well as magnesium (Mg) and phosphorous (P) element in the Mg2+-BP NPs. The binding of integrin β1 to the substrate was analyzed by incubating the substrate with integrin β1 (50 µg/mL) at 37 °C for 30 min and staining for integrin β1 .Transmission electron microscope (TEM) imaging  and high-resolution TEM imaging (JEOL JEM-ARM200F) were carried out to characterize the convertible dimer formation using TEM grid (Dune Sciences). The approximate diameter and density of the Mg2 and cultured in basal medium [high-glucose DMEM with 10% (v/v) heat-inactivated fetal bovine serum and penicillin/streptomycin] at 37 °C and 5% CO2. Cells were treated in the identical conditions used for MgBNP formation, reversion to BNP and RGDBNP formation, and then replaced with basal medium. The real-time fluorescent confocal microscopy imaging was performed for the cells stained with calcein AM. The viability of the macrophages was determined for the cells stained with calcein AM and propidium iodide.Macrophages  at passage 5 were plated onto the BNP substrate at a density of 50,000 cells/cmMacrophages were cultured under M1-type-polarizing medium [basal medium containing 10 ng/mL lipopolysaccharide (LPS) and 10 ng/mL recombinant interferon-gamma (IFN-γ)] or M2-type-polarizing medium [basal medium containing 20 ng/mL interleukin-4 (IL-4) and 20 ng/mL interleukin-13 (IL-13)] with or without Y27632 . The MgBNP or RGDBNP dimer was formed at the beginning of culture.To quantify secreted levels of cytokines by macrophages, enzyme-linked immunosorbent assay (ELISA) was performed by using the ELISA plate, HRP-conjugate reagent, and chromogen solution for absorbance reading at 450 nm. Flow cytometry was performed with BD LSR Fortessa for iNOS  and Arg-1  and analyzed with FlowJo software. The fusion of macrophages was evaluated after their culture under a medium containing IL-4 and IL-13, stimulators for macrophage fusion. The macrophages were analyzed by immunofluorescent staining. Cells were treated with 4% (w/v) paraformaldehyde and 0.25% (v/v) Triton-X for 10 min and then blocked with 3% (w/v) bovine serum albumin for 30 min. Cells were incubated in primary antibodies against vinculin , iNOS , Arg-1 , ROCK2 , or NIMP-R14  at 4 °C for 16 h. Cells were treated with secondary antibodies , phalloidin , or DAPI  at 25 °C for 45 min. Cell were imaged using confocal microscope (Nikon) and then analyzed with Image J software. Western blotting was performed with ChemiDoc Touch Imaging System (Bio-Rad) for iNOS , Arg-1 , or GAPDH  and presented with molecular weight markers, which were subjected to densitometric analysis using Image J software. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to analyze expression levels of iNOS, CD80, Arg-1, or Ym2 gene after normalization to GAPDH gene, by using TRIzol, RevertAid First Strand cDNA Synthesis Kit and TaqMan assays.2+ (0.1 µg) were administered to form MgBNP dimer. EDTA (1.5 µg) was then administered to revert to the BNP. BP (1 µg), RGD-BP (0.2 µg), and Mg2+ (0.1 µg) were administered to form RGDBNP dimer. IL-4 (100 ng) and IL-13 (100 ng) were administered as M2 stimulators. The mice were housed in the cages and sacrificed at 24 h post implantation. To examine maturation of host macrophages to fuse and thus form foreign body giant cells, PEGDA-based hydrogels containing thiol group were used for AuNP coupling to yield the BNP substrate, which was imaged by scanning electron microscopy. The BNP substrate was subcutaneously implanted and subjected to form the MgBNP or RGDBNP dimer in situ, which then received M2 stimulators. The substrates were analyzed at 10 days post implantation by paraffin embedding and immunohistochemical staining for F4/80  with methyl green counterstaining (Abcam).Forty, 3-month-old male BALB/c mice were used with five mice per group in compliance with all relevant ethical regulations after the approval of the Institutional Animal Care and Use Committee at the Chinese University of Hong Kong. The mice were anesthetized by the intraperitoneal injection of ketamine (100 mg/kg) and xylazine (10 mg/kg). A 2-cm-long incision was made in the back of each mouse. The BNP silicon substrate was implanted into the subcutaneous pocket, and the skin was closed. BP (1.2 µg) and Mgt-test or ANOVA, and p-values <0.05 were considered statistically significant differences between the compared groups, to which different asterisks were assigned .All the experiments in this study were repeated at least two times independently and analyzed with Graphpad Prism 5.00 software. Statistical analyses were carried out with two-tailed Student’s Further information on experimental design is available in the Supplementary InformationDescription of Additional Supplementary FilesSupplementary Movie 1Reporting SummarySource Data


The obtained results reveal that the type of grafting, grafting density, and structure of the grafted species at the silica surface depend strongly on the length of organosiloxane polymer and on the nature of the “green” additive, DMC or DEC. The spectral changes observed by solid-state NMR spectroscopy suggest that the major products of the reaction of various organosiloxanes and their DMC or DEC mixtures with the surface are D (RR’Si(O0.5)2) and T (RSi(O0.5)3) organosiloxane units. It was found that shorter methylhydro (PMHS) and dimethylsiloxane (PDMS) and their mixtures with DMC or DEC form a denser coverage at the silica surface since SBET diminution is larger and grafting density is higher than the longest epoxymethylsiloxane (CPDMS) used for FSNs modification. Additionally, for FSNs modified with short organosiloxane PMHS/DEC and also medium organosiloxane PDMS/DMC, the dense coverage formation is accompanied by a greater reduction of isolated silanols, as shown by solid-state 29Si NMR spectroscopy, in contrast to reactions with neat organosiloxanes. The surface coverage at FSNs with the longest siloxane (CPDMS) greatly improves with the addition of DMC or DEC. The data on grafting density suggest that molecules in the attached layers of FSNs modified with short PMHS and its mixture of DMC or DEC and medium PDMS and its mixture of DMC form a “vertical” orientation of the grafted methylhydrosiloxane and dimethylsiloxane chains, in contrast to the reaction with PDMS/DEC and epoxide methylsiloxane in the presence of DMC or DEC, which indicates a “horizontal” chain orientation of the grafted methyl and epoxysiloxane molecules. This study highlights the major role of solid-state NMR spectroscopy for comprehensive characterization of solid surfaces.Three poly(organosiloxanes)  of different chain lengths and pendant groups and their mixtures of dimethyl (DMC) or diethyl carbonates (DEC) were applied in the modification of fumed silica nanoparticles (FSNs). The resulting modified silicas were studied in depth using The online version of this article (10.1186/s11671-019-2982-2) contains supplementary material, which is available to authorized users. Indeed, only the use of abovementioned method allows a full characterization of the surface species on silylated silica. Some solid-state NMR studies have been already performed on gel and fumed silicas modified with different alkoxysilanes   were purchased from Sigma Aldrich, USA. Commercial, dimethyl carbonate (DMC), diethyl carbonate (DEC), and fumed silica  were purchased from Aladdin Reagents, China. The purity of the reagents, as reported by the manufacturers, was ≥ 99.0 %. The reagents were used as received.For preparation of the modified silica surfaces, poly(methylhydrosiloxane)  dispersion media . The amount of modifier agent was determined to be 17 wt% of silica weight. The modification process was performed in a glass reactor with a stirrer with a rotational speed of 350–500 rpm. The modifying agent was added by means of aerosol-nozzle spray. The samples were subsequently cooled to room temperature after the synthesis.2O and CO2 during heating of the samples in the oxygen flow at 750 °C.The content of grafted organic groups in the synthesized samples was measured a couple of times by a vario MACRO cube analyzer , and average values for carbon content and relative deviations were calculated Table . The ancThe bonding density of the attached layers was calculated using the formula , 12Mw is the molecular weight of the grafted group, %C is the carbon weight percentage of the modified silica, S(BET) is the surface area of the original silica (m2/g), and nс is the number of carbon atoms in the grafted group in each silicone used for modification. Equation 1)O–] repeat units per 1 nm2 of the surface (ρ) (where R is methyl group (CH3); R1 is hydro (H) or methyl (CH3) or epoxy(cyclohexylethyl) group (CH2CH2C6H9)).where 1H MAS NMR spectra were recorded on a Bruker Avance 400 III HD spectrometer  at resonance frequency of 79.49 MHz. The powder samples were placed in a pencil-type zirconia rotor of 4.0 mm o.d. The spectra were obtained at a spinning speed of 10 kHz, with a recycle delay of 1 s. The adamantane was used as the reference of 1H chemical shift.Solid-state 29Si CP/MAS NMR spectra were recorded on a Bruker Avance 400 III HD spectrometer  at resonance frequency of 79.49 MHz for 29Si using the cross-polarization (CP), magic-angle spinning (MAS), and a high-power 1H decoupling. The powder samples were placed in a pencil-type zirconia rotor of 4.0 mm o.d. The spectra were obtained at a spinning speed of 8 kHz (4 μs 90° pulses), a 8-ms CP pulse, and a recycle delay of 4 s. The Si signal of tetramethylsilane (TMS) at 0 ppm was used as the reference of 29Si chemical shift.Solid-state 13C CP/MAS NMR spectra were recorded on a spectrometer  at a resonance frequency of 100.61 MHz for 13C using the cross-polarization (CP), magic-angle spinning (MAS), and a high-power 1H decoupling. The powder samples were placed in a pencil-type zirconia rotor of 4.0 mm o.d. The spectra were obtained at a spinning speed of 5 kHz (4 μs 90° pulses), a 2-ms CP pulse, and a recycle delay of 4 s. The methylene signal of glycine at 176.03 ppm was used as the reference of 13C chemical shift.Solid-state 1H NMR spectra of each initial organosiloxane  of the silicas, the samples were degassed at 150 °C for 300 min. Low-temperature (77.4 K) nitrogen adsorption–desorption isotherms were recorded using a Micromeritics ASAP 2420 adsorption analyzer . The specific surface area (Table SBET) was calculated according to the standard BET method.To analyze the surface area 2, RSi(O0.5)3, and Si4 units, respectively, where R represents aliphatic and/or aromatic substituents or H [29Si NMR spectrum of unreacted silica ,  of C18H37SiH3, C18H37SiCl3, and C18H37P(O)(OH)2 on metals and metal oxides [18H37SH on gold [2  as is the case for SiO2/PMHS+DEC  changes in the length of initial organosiloxane and its fragments after reactions with alkyl carbonate (as shorter polymer fragments will react intensively with silica surface sites due to the lower of steric hindrance of side polymer chains). It is therefore more likely that neat PHMS and the mixture of PHMS/DMC absorb at the SiO2 surface through the formation of adsorption complexes by the binding of hydrogen in the surface silanol group with siloxane oxygen of organosiloxane, while the chemical reaction between the SiO2 surface and PMHS/DEC could be carried out through the formation of chemical bond by the electrophilic substitution of the proton in the silanol group (see Scheme 3) for SiO2/PHMS+DEC  indicates that some of the OH groups were inaccessible to the modifier reagent. These silanols could be located inside SiO2 nanoparticles. Note that these intra-particle silanols and water molecules can be removed upon heating at 550–700 °C, and only a very small amount of residual silanols remains upon heating even at 1000 °C [2 primary particles with a size of 1–2 nm into a finite globule with a diameter of 10–20 nm. In addition, one cannot rule out the possibility of internal hydroxy group formation in the course of diffusion of the water molecules into the SiO2 globules. On the other hand, unreacted silanols play an important role in the stabilization of alkylsilanes layers at the SiO2 surface as considered by other researchers [2 after the modification, as the maximal length of the Si–OH·····HO–Si sequence of bonds is ≈ 0.6 nm, which is notably higher than the Van de Waals diameter of the alkyl chains (≈ 0.46 nm). In the case of the absence of the uncoupled silanols, the attached monolayers form a hexagonal array  a type of organosiloxane bonding  with SiO a type o 1000 °C . The exi 1000 °C . Accordiearchers , 35–43. earchers , graftedray Fig.  where Si29Si CP/MAS NMR spectra of SiO2 modified with organosiloxane of medium chain length (PDMS) and its mixture with DMC or DEC are shown in Fig. 1 and D2 units in cyclotetrasiloxane and dimethylsiloxane species in linear MD4M, respectively 3SiR functionalities  – 7.4 groups/nm2 and the lowest surface area in comparison with other silicas presented in this work  and SiO2/PDMS (curve a) as compared with SiO2/PDMS+DEC (curve c). All the signals in the 13C CP/MAS NMR spectra  and SiO2/PDMS+DMC in comparison to other samples presented here can be explained also by the presence of additional reactive centers at the SiO2 surface, the attached methoxy groups (–OCH3 or OR), which can be formed by the reaction of DMC or DEC with the SiO2 surface  as well as adsorbed water (δ = 3.5 ppm). The presence of grafted siloxane species is confirmed by the emergence of a chemical shift at 0.0 ppm for all the samples  used for modification at the SiO2 surface, while DMC has minimal impact on the chemical interaction of SiO2 with PMHS.Overall, from the solid-state NMR data obtained, it is evident that the addition of DMC to the modifying mixture has a significant effect on the chemical interaction of organosiloxane of a medium length of polymer chain (PDMS) used for modification at the silica surface, while DEC addition has practically no influence on the chemical interaction of SiO29Si CP/MAS NMR spectrum of SiO2 modified with the longest polymer, poly[dimethylsiloxane-co-ethyl)methylsiloxane (CPDMS)  Fig.  a, the rDEC Fig.  c, d—repane Fig.  and D1 uane Fig. , respecttructure , 47 due tructure , and thiDMC Fig.  c and SiDEC Fig.  d are du1H MAS NMR, the spectra of the silicas which were modified by methyl-epoxy siloxane in the presence of DMC 2) and T (RSi(O0.5)3) organosiloxane units. The appearance of grafted siloxane units at SiO2/PHMS+DEC and SiO2/PDMS+DMC surfaces is accompanied by a significant reduction of Q3 signals, while for neat organosiloxanes and some of their mixture with alkyl carbonate used for SiO2 modification, a reduction of Q3 is hardly observable. The small amounts of residual silanols (hardly accessible for modifier reagents used) and physisorbed water remain in all the samples of modified silicas (note that the crude silica was not preheated at high temperatures).An in-depth solid-state NMR study of FSNs functionalized with organosiloxanes of various lengths of polymer chains and their mixtures of DMC or DEC is presented. For better analysis of the length of polymer chain effects, the organosiloxanes studied here are much longer and with a larger difference in the viscosity as well as pendant groups than the organosiloxanes studied before , 48–54. 2 surface. Diethyl carbonate addition somewhat worsens the chemical reaction between medium organosiloxane (PDMS) and SiO2 surface but greatly facilitates the reaction when organosiloxanes at short (PMHS) and long polymer chain (CPDMS) are applied for FSNs modification. Thus, from the technological point of view, for FSNs modification with short organosiloxanes, it is reasonable to use DEC; at medium organosiloxane, the application of DMC is necessary; and at long organosiloxane, it is beneficial to use both DMC and DEC.Addition of DMC to the modifying mixture facilitates the passage of chemical reaction between medium (PDMS) or long (CPDMS) polymer and the SiOρ = 7.2–7.4 groups/nm2) are applied for FSNs modification. The reaction of FSNs with medium and long polymer and its mixture with DEC (PDMS/DEC or CPDMS/DEC) leads to the formation of the “horizontal” chains at the surface (ρ = 0.1–2.5 groups/nm2). The findings open new ways for the preparation of similar materials of the same quality using different substrates such as various silicas—silica gels, porous silicas, and precipitated silica. The comparison of the influence of substrate nature on poly(organosiloxane)/alkyl carbonate modification is of undoubted interest for future study.The data for CP/MAS NMR, BET, and chemical analysis suggest the “vertical” orientation of grafted organosiloxane chains when short and medium polymer or its mixture with DMC


The individual impact of North Atlantic and Pacific Ocean Western Boundary Currents (OWBCs) on the tropospheric circulation has recently been studied in depth. However, their simultaneous role in shaping the hemisphere-scale wintertime troposphere/stratosphere-coupled circulation and its variability have not been considered. Through semi-idealized Atmospheric General-Circulation-Model experiments, we show that the North Atlantic and Pacific OWBCs jointly maintain and shape the wintertime hemispheric circulation and its leading mode of variability Northern Annular Mode (NAM). The OWBCs energize baroclinic waves that reinforce quasi-annular hemispheric structure in the tropospheric eddy-driven jetstreams and NAM variability. Without the OWBCs, the wintertime NAM variability is much weaker and its impact on the continental and maritime surface climate is largely insignificant. Atmospheric energy redistribution caused by the OWBCs acts to damp the near-surface atmospheric baroclinicity and compensates the associated oceanic meridional energy transport. Furthermore, the OWBCs substantially weaken the wintertime stratospheric polar vortex by enhancing the upward planetary wave propagation, and thereby affecting both stratospheric and tropospheric NAM-annularity. Whereas the overall impact of the extra-tropical OWBCs on the stratosphere results mainly from the Pacific, the impact on the troposphere results from both the Pacific and Atlantic OWBCs. In contrast, the understanding of the impact of Northern Hemisphere (NH) OWBCs on the atmospheric circulation has advanced only recently and has focused mainly on the individual impact of the Gulf Stream and Kuroshio/Oyashio Currents on the tropospheric circulation and on small, local and basin scales9. Compared to the Northern hemisphere, a recent idealized aqua-planet model simulations indicate that Southern Hemisphere mid-latitude SST fronts impact the hemispheric-scale atmospheric circulation and its dominant variability14. For the Northern Hemisphere there is emerging evidence that the Gulf Stream in the Atlantic and Kuroshio/Oyashio Currents in the Pacific locally maintain and anchor storm-tracks, precipitation and basin-scale tropospheric circulation15. However, the combined role of the North Atlantic and Pacific OWBCs and their individual contributions in maintaining the hemispheric-scale circulation and NAM variability in stratospheric/troposphere-coupled system remains unclear.The physical understanding of the extra-tropical atmospheric impact on the Ocean Western Boundary Currents (OWBCs) has greatly advanced since the classic pioneering works17. The NAM and NAO exert a big impact on the surface continental climate19, ocean circulation21, and the marine and terrestrial ecosystems23. It has been established that the NAM and NAO are essentially an internal mode of atmospheric variability governed primarily by interactions between midlatitude westerlies and eddies , as it can be simulated in AGCMs with climatological SSTs prescribed at the lower boundary24. The coupling to the ocean is therefore not considered essential for the NAM (NAO)25. Nonetheless, the coupling to the ocean and hence ocean circulation is very important and largely influences the SST-distributions used in those AGCM-studies. In fact, recent idealized AGCM-studies show that organization of those eddies into storm-tracks is influenced by the SST-fronts along the OWBCs12. This influence is understandable from linear wave-theory27, which suggests that cyclones and anticyclones recurrently develop where the sharp gradient of near-surface air-temperature is maintained28. This temperature gradient is directly linked to the SST fronts through turbulent heat fluxes at the ocean surface29.It has been argued that the dynamical coupling with the ocean is not an essential feature of the intrinsic dynamics of North Atlantic Oscillation (NAO) or NAM14.The aim here is (i) to identify the joint impact of the north Atlantic and Pacific OWBCs on the hemispheric-scale circulation in both troposphere and stratosphere using semi-idealized model experiments and (ii) to clarify the implications for the Northern Hemisphere climate, including energy budget and climate variability. We focus on the features of large-scale circulation that can be largely simulated with stratospheric-resolving IPCC-class models: i.e., the mid-latitude tropospheric eddy-driven jets, stratospheric polar vortex, and associated dynamics and variability. By considering the climatological SST and continents, this work can be seen as an extension of the previous highly idealized aqua-planet studies and the studies with idealized continents30; thus it is difficult to isolate the OWBC-impacts through conventional AGCM-experiments or observational analysis. We therefore utilize several sets of semi-idealized AGCM-experiments similar to12, but under more realistic Northern Hemisphere conditions using the Hamburg stratosphere resolving AGCM MAECHAM531 (Method).Unlike its Southern Hemisphere counterpart, the impact of Northern Hemisphere OWBCs on the atmosphere tends to be masked by the corresponding impacts of land-sea thermal contrasts and large–scale orography32 only in the North Atlantic and Pacific  is conducted using observed climatological SSTs with observed extra-tropical SST-frontsfic Fig. . In ordewe removed the tropical SST-asymmetry, the impact of the sea-ice and the mid-latitude SST-front in the Southern Hemisphere  are thus enhanced along the SST-fronts BCs Fig. , respectBCs Fig. 12, which mimic the Southern Hemisphere situation.The overall tropospheric adjustment acts to offset the OWBC-maintained baroclinicity (due to turbulent heat fluxes), in which the horizontal atmospheric circulation advects warmer air into the cooler areas and vice versa Fig. . The tra36, which is accounted for by the reduced poleward transport of sensible heat by transient eddies  and only in the Pacific . Regarding the contributions from the individual ocean basins, the overall strengthening of the tropospheric eddy-driven polar-front jets results from both Pacific and Atlantic OWBCs Fig. , whereas18 can be reproduced simply by the Northern Hemisphere climatological SST-front prescribed in the presence of continents  and 39 vertical levels going up to 0.01 hPa. The lower-boundary condition of the AGCM was taken from the observed climatology of monthly-mean SSTs from 1950 through 2008 (HadISST)32. Here we investigate the impact of the Northern Hemisphere OWBCs on the atmospheric circulation that can be simulated with model resolutions currently used to study climate . The OWBCs maintain local maxima in the SST-gradients that we call SST-fronts : MAECHAM5nts Fig. . The OWBnts Fig. . The effIn order to isolate the impact of the Northern Hemisphere OWBCs on the large-scale atmospheric circulation, we further performed six sensitivity experiments with realistic climatological SST  and more idealized zonally symmetric (or uniform) SST fronts .The first experiment (BCF-experiment) is conducted using observed climatological SSTs with realistic climatological extra-tropical SST fronts in both, the Atlantic and the Pacific Fig. ; otherwiIn all SST front experiments a meridional linear interpolation in the equatorial flank of the imposed frontal area was performed in order to avoid discontinuity in the SST profile between the front and non-front regions. Similar linear interpolations in the polar region were performed but in the zonal direction in ASF, PSF, ACF and PCF experiments, in order to avoid the inter-basin discontinuity. Because of the similarity of the atmospheric response in the realistic and symmetric SST-front experiments SFig. , only th18. The NAM-index (NAMI), defined as the corresponding principal component time series, was regressed locally onto various meteorological quantities, in order to assess the impact of the OWBCs on NAM-associated climate variability.All AGCM experiments were integrated for at least 50 years after 3-year spin-up, and the response is defined as the difference in the January-February-March mean between a given front-experiment and the NF experiment. A two-tailed t-test was performed to assess statistical significance of the response. In order to understand the impact of the OWBCs on the variability of the coupled stratosphere/troposphere-system, the Northern Annular Mode (NAM) is extracted as the leading Empirical Orthogonal Function (EOF) mode of geopotential height north of 20N28 that is proportional to the meridional air temperature gradient and inversely proportional to the static stability. The baroclinic wave-activity (or storm-track activity) is diagnosed with the 3-dimensional wave-activity flux called E-vector28. The storm-track activity is measured as the variance of high-pass filtered geopotential height fluctuations (in periods of 1–12 day), and the E-vector with and35 can be used to diagnose the feedback forcing by baroclinic waves on the background zonal flow. In the definition, u (v) is the zonal  velocity and θ (Θ(p)) potential temperature  and the bar denotes time averaging. Only high-pass-filtered fluctuations (with periods shorter than ~12 day) are used for evaluating the E-vector. Its convergence (divergence) indicates eddy-induced acceleration (deceleration) of the westerlies, and its equatorward component Baroclinic eddies: The baroclinicity is measured locally as a quantity12 as equatorward gradient of the temperature tendency between 1000 and 850 hPa due to the upward heat flux: p = 150 hpa and Cp is the atmospheric specific heat at constant pressure. In order to understand the atmospheric adjustment due to the OWBCs, the lower-tropospheric (1000-850 hPa) horizontal temperature- (T-) advection T-advection into contributions from the zonal T-advection on the low-level baroclinicity was measured as the 1000-850 hPa mean equatorward gradient of the T-tendency due to the T-advection.In this work, the forcing of the baroclinicity in the planetary boundary layer (PBL) by sensible heat flux (SHF) is computed analogously to36. In order to understand the role of the oceanic fronts in modulating the poleward energy flux of the climate system, we computed the zonally averaged meridional energy flux of the atmosphere (FA) and the ocean (FO) based on the energy balance of the Earth-atmosphere system39. Here we assume a steady state, where the net radiative flux at the top of the atmosphere (Rtop) is balanced by the convergence of the meridional heat transport by both the atmosphere and the ocean 39. The meridional energy flux in the atmosphere is computed directly from the atmospheric variables as the sum of potential, latent, sensible and kinetic energy transport. The equivalent oceanic meridional energy transport  is obtained as residual39. The contribution of the kinetic energy is negligible compared to the other forms of energy and will not be presented. The meridional sensible heat flux is decomposed into the contributions from steady mean meridional circulation, transient and stationary eddies39.Bjerknes compensation and poleward energy flux: The Bjerknes compensation states: if the energy flux at the top of the atmosphere and the oceanic heat storage are quasi-stationary, the total energy transport of the climate system should not vary much. This means that the energy transport of the atmosphere and the ocean should compensate each other40). In the quasi-geostrophic approximation, the vertical component of the EP-flux is proportional to the poleward eddy heat transport and its meridional component is proportional to the equatorward eddy zonal momentum transport. Convergence of the EP-flux in the high-latitude stratosphere means weakening of the westerly polar-night jet during NH winter and warming in the stratospheric polar vortex.Stratospheric dynamics: The stratospheric changes through planetary waves can be diagnosed through the Eliassen-Palm (EP) flux in its primitive form (page 128 Eq. 3.5.3(a) and (b) inKey Role of the Ocean Western Boundary currents in shaping the Northern Hemisphere climate.


The findings of recent research on drinking patterns and problems among African-Americans can be summarized as follows: (1) African-Americans report higher abstention rates than do whites; (2) African-Americans and whites report similar levels of frequent heavy drinking; (3) rates of heavy drinking have not declined at the same rate among African-American men and women as among white men; and (4) variables such as age, social class, church attendance, drinking norms, and avoidance coping may be important in understanding differences in drinking and drinking problem rates among African-Americans and whites. Limitations of the research are described and suggestions are made for possible directions for future research. Alcohol studies on African-Americans make up a small but growing body of research. The limited research that does exist has focused on a number of issues, including patterns and determinants of drinking and drinking problems. This article reviews recent studies examining the drinking patterns and problems of adult African-Americans. Studies of African-Americans have, for the most part, involved comparisons of drinking patterns and drinking problems among African-American and white populations. This review therefore focuses primarily on studies of that type. After discussing drinking patterns and problems among African-Americans, the article describes some of the possible determinants of drinking patterns in that population.The term “drinking patterns” refers to various styles of drinking, such as abstention or heavy drinking. Categories of drinking patterns frequently are based on indexes of quantity and frequency or on assessments of the volume of alcohol consumed by a person in a given period of time. Unless otherwise noted, terms describing drinking patterns are based on the definitions used in the studies being discussed.National surveys examining the prevalence of drinking in African-American populations indicate that African-Americans report lower rates of alcohol use than whites. In 1997, according to the National Household Survey on Drug Abuse, 53 percent of African-Americans and 68 percent of whites reported drinking in the past year . The difHeavy drinking increases the risk of drinking problems; researchers therefore often focus on heavy drinking when comparing African-American and white drinkers. Data from the National Alcohol Survey  show thaPanel studiesTwo of the most widely studied indicators of drinking problems include drinking consequences and alcohol dependence symptoms. Drinking consequences are concrete problems that arise in different areas of a person’s life because of drinking . Alcohol dependence symptoms refer to a set of behaviors and experiences associated with alcoholism or addiction, such as withdrawal and blackouts . Some stLongitudinal surveys have focused on changes in drinking-problem indicators and alcohol-related mortality among African-Americans and whites. Rates of alcohol dependence symptoms and drinking consequences remained stable from 1984 to 1995 among African-American and white men and women  see tab, p. 262.Some of the most promising studies on drinking problems have examined the relationships between race, social class, and drinking problems. Studies in this area have focused on two central hypotheses. The first hypothesis is that adjusting for social class may eliminate racial and ethnic differences in drinking problem rates. To summarize, studies comparing self-reported drinking problems in African-American and white populations have yielded mixed results. Some studies have shown that African-Americans report more drinking problems than do whites, whereas other studies have reported no significant differences between the two groups. Higher social class appears to be a protective factor for African-Americans against the effects of race on drinking problems. Continuing research is needed to understand the effects of social class status on drinking problems among African-Americans.For the most part, research on drinking patterns and drinking problems in African-American populations has not been based on theory. Nevertheless, etiological studies of drinking patterns and drinking problems in African-American populations have focused on a wide range of demographic, biological, environmental, and behavioral variables.Sex, age, income, education, and employment status have been shown to be related to drinking patterns and drinking problems in both African-American and white populations a, 1997. In addition to demographic variables, studies also have examined whether a family history of alcohol problems contributes to drinking problems. Two of the studies reviewed for this article reported—and many researchers agree—that a family history of drinking problems is an important correlate of drinking behavior . People Studies have examined the influence of stress, parental and peer attitudes about drinking, drinking norms, and drinking contexts  on drinking patterns and problems among African-Americans. To see how stress influenced drinking, Studies have consistently found a positive relationship between drinking and drinking problems—that is, as drinking increases, drinking-related problems increase . IncreasResearch regarding drinking patterns and drinking problems in African-Americans can be summarized as follows:African-Americans report higher abstention rates than do whites.African-Americans and whites report similar levels of frequent heavy drinking.Rates of heavy drinking have not declined at the same rate among African-American men and women as among white men.Variables such as age, social class, church attendance, drinking norms, and coping behaviors may be important in understanding differences in drinking and drinking problem rates among African-Americans and whites.Studies examining the determinants of drinking patterns and problems suggest that demographic and psychological variables may be important in understanding the differences in drinking patterns and drinking problems among African-American and white men and women. Despite the wide range of variables that have been studied, however, notable gaps exist in the research. None of the studies reviewed examined the effects of illicit drugs and tobacco use, the concentration of stores selling alcohol in a given area , or exposure to alcohol through the mass media on drinking patterns and drinking problems. The scant research on alcohol outlet density and media exposure is surprising, given the large number of alcohol outlets in many African-American inner cities and efforts by the alcohol industry to target African-Americans . Future studies should focus on those variables. In addition, few studies have included other ethnic populations, such as Asians and American Indians; those studies are needed to investigate the reasons behind the different trends in heavy drinking patterns for different ethnic groups.Continuing research is needed on the drinking patterns and drinking problems of African-Americans to address current gaps in knowledge. To ensure that interventions and policies to reduce drinking problems in African-American populations are guided by sound research, future studies will need to be theory based; use a wide range of study designs and variables; and include comparisons with other ethnic groups, such as Hispanics, Asians, and Native Americans. Panel studies also are needed to better understand changes in drinking patterns of African-Americans over time.


Autophagy is an endogenous protective process; the loss of autophagy could destabilize proteostasis and elevate intracellular oxidative stress, which is critically involved in the development of cardiac hypertrophy and heart failure. Oridonin, a natural tetracycline diterpenoid from the Chinese herb Rabdosia, has autophagy activation properties. In this study, we tested whether oridonin protects against cardiac hypertrophy in mice and cardiomyocytes. We implemented aortic banding to induce a cardiac hypertrophy mouse model, and oridonin was given by gavage for 4 weeks. Neonatal rat cardiomyocytes were stimulated with angiotensin II to simulate neurohumoural stress. Both in vivo and in vitro studies suggested that oridonin treatment mitigated pressure overload-induced cardiac hypertrophy and fibrosis, and also preserved heart function. Mice that received oridonin exhibited increased antioxidase activities and suppressed oxidative injury compared with the aortic banding group. Moreover, oridonin enhanced myocardial autophagy in pressure-overloaded hearts and angiotensin II-stimulated cardiomyocytes. Mechanistically, we discovered that oridonin administration regulated myocardial P21, and cytoplasmic P21 activated autophagy via regulating Akt and AMPK phosphorylation. These findings were further corroborated in a P21 knockout mouse model. Collectively, pressure overload-induced autophagy dysfunction causes intracellular protein accumulation, resulting in ROS injury while aggravating cardiac hypertrophy. Thus, our data show that oridonin promoted P21-related autophagic lysosomal degradation, hence attenuating oxidative injury and cardiac hypertrophy. Heart failure (HF) is the final common pathway of chronic cardiovascular disorders, and few therapies, including neurohormonal antagonists and approaches for symptomatic relief or inotropic action, have provided early interventions or positive long-term outcomes to date2. Cardiac hypertrophy has received considerable attention as a crucial pathophysiologic process of HF4. Haemodynamic overload, such as pressure overload, impels the heart to initiate various adaptive mechanisms, including hypertrophy to cope with wall stress increase, and altered protein synthesis to preserve myocyte survival and pump function. Long-lasting stress stimuli may elicit maladaptive alterations, such as disarranged autophagy, impaired protein homeostasis, oxidative stress injury and myocardial fibrosis6. Pharmacological therapies that modulate hypertrophic signalling may intervene in HF at the preliminary stage to avert hard-to-treat conditions and embody great promise for improving the prognosis of HF7.Cardiovascular diseases remain the leading cause of mortality worldwide9. Previous studies showed that autophagy augmentation alleviates intracellular oxidative injury10. Increasing evidence, using genetic or pharmacological interventions, indicates that maintaining autophagy plays a protective role in the heart during cardiac remodelling, which limits myocardial damage in response to pressure overload13 or ischaemia14. Numerous pharmacological agents that can regulate autophagy have been identified. However, clinical applications of maintaining moderate autophagy are challenging due to the proper extent of autophagy activation and the side effects inherent in each intervention15. In view of this, new drugs that are most appropriate for upholding autophagy have yet to be identified in the treatment of cardiac diseases.As a vital mechanism for eliminating cytosolic misfolding proteins and delivering them to lysosomes for degradation and recycling, myocardial autophagy helps maintain the quality of the intracellular environment and conserves the ATP level in response to energy stress16. Oridonin, a natural tetracycline diterpenoid, is a flavonoid compound extracted from the Chinese herb Rabdosia rubescens, which is known for its well-studied, multitargeting properties for antitumour activities17. Studies have also verified the anti-proliferation effects of oridonin18. Furthermore, oridonin exerts stimulative actions on autophagy20. Given the growth-inhibition and autophagy-promotion effects together, we propose the hypothesis that oridonin may play a role in cardiac hypertrophy.Traditional Chinese medicines have been widely and effectively used in treating cardiovascular diseases, and compounds from various herbs play important roles in taming diverse pathophysiological processes such as peroxidation and metabolic abnormalitiesThe present study investigated the effects of oridonin on pressure overload induced cardiac hypertrophy and explored the underlying mechanisms. Our results confirmed that oridonin mitigated cardiac hypertrophy and preserved heart function. Mechanistically, we further discovered that oridonin-prevented maladaptive hypertrophy was partially dependent on P21 signalling-regulated myocardial autophagy.P < 0.05). Cell size was assessed with α-actinin immunostaining. The results suggested that oridonin treatment patently diminished the increase in cardiomyocyte size in the presence of Ang II after 12 or 24 h of culture, and the inhibitory effects were seen in a dose-dependent manner ranging between 5 and 20 μM . No discernible differences between 12 and 24 h at each dose were observed. In addition, oridonin markedly attenuated the increased transcription levels of atrial natriuretic peptide (ANP), B-type natriuretic peptide (BNP), and β-myosin heavy chain (β-MHC) provoked by Ang II, especially in the oridonin-20 μM group . These data implied that oridonin could inhibit Ang II induced isolated cardiaomyocyte hypertrophy.To examine whether oridonin protects cardiomyocytes against stress-induced hypertrophy, we used primary cultured neonatal rat cardiomyocytes (NRCMs) in a well-controlled experimental setting. Cells were treated with Ang II for 12 or 24 h, and oridonin was used at the indicated concentrations that were previously verified to be safe . Moreover, after AB surgery, left ventricular dilation occurs, oridonin could prevent the LV enlargement, presented by limited increase of LV end-diastolic dimension. The increase of interventricular septal thickness at end-diastole and decrease of ejection fraction and fractional shortening (FS), were also prevented by oridonin . In parallel with this, harm in active relaxation was also prevented in oridonin-treated mice, as these mice displayed an increased rate of pressure development or decay (±dp/dt) compared with the AB group .To demonstrate the beneficial role of oridonin in cardiac hypertrophy, we next sought to discover whether oridonin could attenuate the hypertrophic response induced by pressure overload. Mice were subjected to pressure overload by aortic banding (AB) surgery, and 40 mg/kg of oridonin was given orally once a day Fig. S. After 4P < 0.05). Consistently, 4 weeks after AB surgery, the expression levels of the markers related to cardiac fibrosis, including collagen Iα, collagen IIIα and CTGF, were lower in the hearts of oridonin treated mice compared with vehicle hearts .Cardiomyocyte injury during hypertrophy promotes alterations within the extracellular matrix and induces fibrosis. To further define the effect of oridonin on cardiac fibrosis, picrosirius red (PSR) staining and quantitative analysis were performed. The results indicated that hearts subjected to chronic pressure overload developed significantly increased fibrosis in the interstitial and perivascular spaces compared with the sham operated hearts, and this fibrosis was remarkably limited in oridonin-treated hearts . Protein expression detected by WB showed similar alterations of p67phox and SOD2 as mRNA levels, and restored haem oxygenase 1 by oridonin treatment . IHC staining showed the oridonin could raise the expression of myocardial HO-1 while limiting the level of 4-hydroxynonenal after AB surgery . Dihydroethidium fluorescence showed oridonin could restrict the generation of ROS after AB . Quantitative analysis showed enhanced myocardial SOD and GPx with oridonin treatment post AB, while the MDA level was reduced in oridonin-AB group .Cardiac hypertrophy is always accompanied by excessive oxidative stress injury; hence, we evaluated the anti-oxidant effects of oridonin, the extent of which was detected by the expression of oxidase and lipid peroxidation, and the antioxidase activities. We first assessed the transcription levels of the NADPH oxidase subunits gp91phox, p67phox and SOD2. Compared with the AB group, the cardiac mRNA and serum levels of gp91phox, p67phox and SOD2 were all significantly decreased in the presence of oridonin . A similar conclusion was obtained by detection of LC3II accumulation using immunofluorescence staining both in mouse hearts and cardiomyocytes . Transmission electron microscopy revealed infrequency of autophagosomes and autolysosomes in pressure overloaded hearts, whereas oridonin treatment increased the amount of autophagosomes and autolysosomes .Autophagy plays an essential role in maintaining cellular homeostasis in the heart, both under baseline and stress conditions. Exacerbated cardiac oxidative stress could also be the consequence of impaired autophagy. Autophagic activities after AB surgery were analysed by immunoblotting of a panel of autophagy-related genes, staining of protein-LC3 dots, and observing of autophagosomes and autolysosomes. Autophagy was suppressed in AB-induced hypertrophied hearts compared to sham operated hearts at 1-week post-surgery. In AB hearts treated with oridonin, LC3II expression was significantly increased, with increased ATG5-12, ATG7 and Beclin1 but decreased accumulation of P62  of AKT and mTOR was remarkably increased after 1 week of pressure overload, which was blunted by oridonin treatment. Phosphorylation of AMPK, which is critical to autophagic action, was promoted by oridonin . It has been demonstrated that upregulation of P21 is relevant to autophagy, as well as a target of oridonin22. Therefore, we inspected the protein level and distribution of P21. We found that P21 expression was down regulated by 1 week after AB, but promoted by oridonin, especially the alteration of cytoplasmic P21 expression . Oridonin intensified cytoplasmic P21 expression, as shown by immunofluorescence staining . In accordance with the in vivo manifestation, P21 expression was down regulated in NRCMs with the treatment of Ang II . The P21 immunofluorescence of cells also showed cytoplasmic aggregation with the treatment of oridonin, especially after AngII stimulation .To further study the role of oridonin in upholding autophagy, we primarily examined the classical signal pathways and molecules involved. MTOR complex 1 (mTORC1) and AMP-activated protein kinase (AMPK) are well established as upstream kinases that regulate autophagy during cardiac hypertrophyP < 0.05). An mCherry-GFP-LC3 autophagic flux reporter assay was carried out to identify the specific point within the autophagic cascade inhibited by oridonin and P21. Autophagosome formation and autolysosome acidification, as shown by the yellow dots and red dots, respectively, both decreased after Ang II stimulation, which was reversed by oridonin. However, the comparably increased number of autophagosomes and autolysosomes brought by oridonin treatment were mostly reduced upon silencing of P21. We further used bafilomycin A1 (BafA1), which inhibits autophagosome-lysosome fusion, to block autophagic flux by inhibiting autolysosome turnover. Together with the oridonin promoted P21, BafA1 remarkably raised the number of autophagosomes (yellow) while depressed the formation of autolysosomes (red) . These data suggested that oridonin increased myocyte autophagy by promoting autophagosome formation, and P21 was required in this process.To investigate whether the increase of P21 is a phenomenon accompanied by elevated autophagy, or the critical factor account for autophagy enhancement, we evaluated the specific role of P21 in cardiomyocytes by infecting NRCMs with lentivirus harbouring P21 short hairpin RNA  or recombinant adenoviruses expressing murine P21 (AdP21) Fig. S. MyocyteP < 0.05).We next tested autophagy in cells overexpressing P21 and treated with Ang-II. H9C2 cells were infected with AdP21. LC3II immunofluorescence indicated that autophagy was suppressed in Ang II stimulated cells. In Ang II cells protected with oridonin, LC3II expression was significantly increased, showing that P21 activation is sufficient to increase autophagy in Ang II-treated cells .We further tested whether autophagy inhibition attenuates the beneficial effects of oridonin-P21. The cellular surface areas of cardiomyocytes were measured by immunostaining with α-actinin 24 h after Ang II treatment. Autophagy inhibition by 3-MA eliminated the protective effects of oridonin on myocyte enlargement, even in the presence of additional expression of P21 by AdP21 transfection . Consistently, more ROS production was detected with deficient P21 expression, while restrained autophagy partly removed the protection of oridonin on myocyte oxidative stress . A converse variation tendency was found in SOD levels in cell lysate .Previous studies have suggested that promoting autophagy could alleviate oxidation in the heart−/− mice). P21−/− mice and their wild-type littermates were subjected to AB surgery, and 40 mg/kg of oridonin was given orally once a day for 4 weeks. P21 knockout abolished oridonin-induced cardiac hypertrophy and oxidative injury . The P21 dependent protective properties in the heart were also evidenced by increased heart weight and ventricular wall thickness, as well as decreased fractional shortening . The role of increased autophagy mediated by oridonin-P21 was investigated by autophagy inhibitor in vivo. Intraperitoneal injection of 3-MA resulted in an increased HW/BW and left ventricular wall thichness (IVS + PW), and reduced FS in mice . Transcript levels of hypertrophic markers ANP, BNP and βMHC were also elevated in P21−/− mouse hearts . Additionally, P21 deficiency altered AKT/AMPK phosphorylation and LC3 membrane transfer via oridonin therapy .To further confirm whether oridonin exerted antihypertrophic effects through P21 in vivo, we generated viable global P21 knockout mice  facilitated the formation of LC3-positive autophagosomes and (2) coordinated the core molecular machinery ATG proteins covering the fusion and maturation and degradation of autophagosomes; (3) its protective effects on myocyte hypertrophy were eliminated by autophagy inhibition using 3-MA. These results were consistent with previous studies implying the autophagy-inductive action of oridonin29. Moreover, we found that oridonin blunted the phosphorylation of AKT and mTOR while salvaging the phosphorylation of AMPK. Autophagy is regulated by AKT-mTOR and AMPK-ULK1 signalling, which activates the anabolic and catabolic processes respectively, and interact to control autophagosome formation32. Sustained pressure overload induces long-term activation of Akt, which successively activates mTORC1 to accentuate cardiac contractile defects through a loss of autophagic regulation33. AMPK serves as a sensor and regulator of cellular energy status and can promote autophagy34. Our data showing that oridonin treatment attenuated maladaptive hypertrophy via AMPK were consistent with a previous report that AMPK prevents heart failure through enhancing autophagy35. Thus, oridonin exerts its antihypertrophic effect throughout the classic autophagy pathway. However, contrary to our present finding, AMPK was deactivated following oridonin treatment in cancer cells20. Our data implied that oridonin had diverse regulatory effects on AMPK in different cell types. Nevertheless, the molecular target in cardiomyocytes by which oridonin induces autophagy has not yet been identified.Previous studies implied that the beneficial effects of oridonin might be mediated by autophagy activation, which offered an important source of ATP and could inhibit the generation of reactive oxygen species36. Our study probes into the role of P21 in oridonin-induced autophagy. Here, the conceptually important points that emerge from our study are (1) oridonin intensified cytoplasmic P21 expression; (2) oridonin failed to inhibit myocyte hypertrophy in either P21 silenced NRCMs or P21−/− mice 41. Taking all the findings into consideration, we surmise that oridonin-mediated alleviation of cardiac hypertrophy is a consequence of autophagy activation and oxidation inhibition caused by increased P21 expression.Although our study focused on the autophagy promoting effects of P21 with oridonin treatment, as regards cardiomyocyte hypertrophy, P21 may also have other regulatory properties. Notably, 3-MA blocked the protection of oridonin on myocyte enlargement but allowed its alleviation of peroxidation injury. In line with these observations, the role of P21 as a suppressor of cardiac hypertrophy or oxidative stress was highlighted in several studiesThe strengths of our study include the use of oridonin on cardiac hypertrophy in vivo and in vitro, the evaluation of the protective effects of oridonin on the accompanying pathological manifestations, the determination of a possible mechanism of oridonin action, and the identification of a potentially useful molecule that mediates the pharmacological function. The findings from our study provided evidence for the application of oridonin in the treatment of pressure overload-induced cardiac hypertrophy, which may have pragmatic implications in the future. Finally, although we clearly demonstrated that oridonin-P21 induced autophagy are responsible for the protection of cardiac hypertrophy, the gap of how oridonin modificate P21, and lead to activation of autophagy needs to be further filled.Impaired autophagy and oxidative injury are closely involved in cardiac remodelling. We investigated whether the traditional Chinese medicine oridonin could inhibit these hypertrophic responses.We found that oridonin protected the heart from pressure overload induced hypertrophy by promoting the P21-related autophagy pathway.Our study provided evidence for the application of oridonin in the therapy of cardiac hypertrophy.Oridonin  was purchased from Shanghai Winherb Medical Science Co., Ltd. .42. H9C2 cells were obtained from the Cell Bank of the Chinese Academy of Sciences  and were cultured in DMEM  with 10% FBS. Cells were incubated in a humidified incubator  at 37 °C with 5% CO2. To simulate in vitro myocardial hypertrophy, the cells were stimulated with Angiotensin II , in the presence or absence of different concentrations of oridonin , and incubated for another 12 or 24 h.To examine the in vitro anti-hypertrophic effects of oridonin, neonatal rat cardiac myocytes (NRCMs) were isolated and cultured in Dulbecco’s modified Eagle’s/F-12 , supplemented with 15% foetal bovine serum  as previously describedThe cell surface area of the NRCMs was assessed via immunofluorescence staining. Briefly, the cardiomyocytes were fixed with 3.7% formaldehyde, and permeabilized with 0.1% Triton X-100 in PBS, followed by staining with α-actinin  overnight at 4 °C. The cells were subsequently incubated with Alexa Fluor 488 (green) secondary antibodies (1:200) for 60 min at 37 °C, followed by the visualization of nuclei with 4,6-diamidino-2-phenylindole (DAPI). The surface areas were measured using Image-Pro Plus software, version 6.0. At the magnification of 400 times, 6–10 microscopic vision were randomly selected and 5 cells were counted for each vision (30–50 cells were counted for each group). AOI was used to automatically trace the cell boundary, and the cell area was converted by the tool COUNT/SIZE according to the ruler taken under the same magnification.All of the animal care and experimental procedures were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals, published by the US National Institutes of Health , and were approved by the Animal Care and Use Committee of Renmin Hospital of Wuhan University.CIP1/WAF1  were purchased from The Jackson Laboratory .Male, 8–10 weeks old mice in the C57BL/6 background (body weight ranging from 23.5 to 27.5 g) were purchased from the Institute of Laboratory Animal Science, Chinese Academy of Medical Sciences . Mice with targeted knockout mutation of the gene encoding P2143. The next day, mice were administered oridonin suspension (40 mg/kg) or equal volumes of vehicle by gavage. One, 2 and 4 weeks post AB surgery, the mice were sacrificed by cervical dislocation, and the left ventricular tissue was excised for further detection. The hearts and lungs were weighed to calculate the heart weight/body weight ratio , lung weight/body weight ratio  and HW/tibia length  in each group 4 weeks post AB surgery.To establish a mouse model of cardiac hypertrophy, aortic banding (AB) surgery was performed to create pressure overload-induced hypertrophy. Animals were weighed, randomly divided into groups, and then received the surgical procedure, during which mice were anaesthetized  and AB or sham surgery was performed as previously described44. Mice were anaesthetized by 1.5% isoflurane, while the inhalational flow was adjusted to maintain their heart rate at 450–550 beats/min. Transthoracic ultrasonography was performed with a MyLab 30CV ultrasound system . The left ventricle (LV) was assessed by M-mode scanning in both parasternal long- and short-axis views at the mid-papillary muscle level. LV internal dimensions and chamber wall thickness at diastole and systole were measured respectively. Invasive haemodynamic monitoring was implemented with a microtip catheter transducer , which was inserted into the right carotid artery and proceeded into the LV. Pressure signals and volume signals were continuously recorded with a Millar Pressure-Volume System  coupled with a Powerlab/4SP A/D converter and then stored and displayed. The data were processed using PVAN data analysis software.To inspect the cardiac function, echocardiography and haemodynamic monitoring were analysed 4 weeks after AB surgery, as described previouslyTo assess cardiac hypertrophy and fibrosis, we performed morphological analysis on heart sections. Mouse hearts were excised after euthanasia and immediately placed into 10% potassium chloride solution to be arrested in diastole. Subsequently, these hearts were fixed in 10% formalin for 12–24 h, embedded in paraffin, and then sectioned transversely at 3–5 µm. Afterward, the sections were stained with haematoxylin-eosin (HE) or picrosirius red (PSR) to evaluate myocyte cellular hypertrophy or extracellular collagen deposition, respectively. The cross-sectional areas of myocytes and fibrotic areas were measured using a digital image analysis system  from captured images of the stained sections.Immunofluorescence staining was performed in order to determine the expression as well as the localization of LC3/P21 in the mice hearts. Briefly, for the heart section staining, all sections were autoclaved with citrate solution for antigen retrieval. After blocking with 10% goat serum , the sections were incubated in LC3A/B  or P21 antibody  overnight. Heart sections were subsequently incubated with secondary antibodies for 60 min at 37 °C, followed by visualization of the nuclei with DAPI. For immunohistochemistry, the tissue sections were heated for antigen retrieval, and then incubated with anti-heme oxygenase-1  or 4-hydroxynonenal , followed by incubation with goat anti-rabbit EnVisionTM+/HRP reagent, and finally staining with a DAB detection kit. The images were captured using a fluorescence microscope  and then analysed using Image-Pro Plus software.To examine the mRNA expression of cardiac hypertrophy and fibrosis related markers, total mRNA was extracted from the mouse LV using TRIzol reagent  according to the manufacturer’s instructions and subsequently reverse transcribed into cDNA using oligo (DT) primers and the cDNA Synthesis Kit . The indicated genes were amplified using LightCycler 480 SYBR Green 1 Master Mix , and the expression levels of target genes were normalized to GAPDH gene expression.Western blot analysis was performed to evaluate protein expression. Total proteins from mouse hearts or cultured cardiomyocytes were extracted by use of RIPA lysis buffer supplemented by a protease inhibitor cocktail . Cytoplasmic proteins were extracted using an extraction kit . Protein concentration was evaluated using a BCA protein assay kit . Equal amounts (50 μg) of protein were electrophoresed through 10% SDS-PAGE and transferred to PVDF membranes . Next, the membranes were blocked with 5% skimmed milk at room temperature and then incubated overnight at 4 °C with indicated primary antibodies against different antigens, including P21 , SOD2 , HO-1 , P-AKT , T-AKT , P-mTOR , T-mTOR , P-AMPK , T-AMPK , LC3  and P62 . Protein expression levels were normalized to that of GAPDH . Secondary antibodies were incubated at room temperature for 1 h the next morning. Finally, images were obtained by Odyssey Infrared Imaging System  to quantify protein expression.To knock down P21 expression, NRCMs were infected with lentiviral particles containing the short hairpin RNA targeting P21 . To overexpress P21, an adenovirus vector carrying the P21-coding gene (Ad-P21) were generated by iBioscience Company . NCRMs were infected with the virus at a multiplicity of infection of 100 for 8 h. Lenti-shNC and AdNC were used as non-targeting controls.To assess total superoxidase dismutase (SOD) and glutathione peroxidase (GPx) activity and the content of malondialdehyde (MDA) and dihydroethidium (DHE) in the myocardium, a series of commercial assay kits  was obtained and performed according to the manufacturer’s instructions. Briefly, heart tissues were lysed, centrifuged, and then mixed with the detection working dilution. A microplate reader  was used to record the luminescence, and then SOD, GPx and MDA content was calculated. ROS generation was detected by DHE staining in vivo and DCFH-DA staining in vitro respectively. In brief, NRCMs were incubated with DCFH-DA (5 μM) at 37 °C for 30 min in a dark chamber. Fluorescent images were observed with a fluorescence microscope (Olympus DX51).4. After being dehydrated and embedded, tissues were cut into ultrathin sections (70–100 nm) using an ultramicrotome  and stained with uranyl and lead citrate. Finally, image observation was performed using a transmission electron microscope . Micrographs were captured at different magnifications (2000 and 8000-fold).Heart sections were viewed by transmission electron microscopy to detect autophagosomes and autolysosomes. Freshly isolated hearts were fixed with 2.5% glutaraldehyde in PBS (pH 7.0) at 4 °C for 8 h and then post-fixed for 1 h in 1% OsOTo assess autophagic flux in mice heart by Western blot, bafilomycin A1  was administered to mice 2 h before they were euthanized. To assess autophagic flux in NRCMs, mCherry-GFP-LC3 adenovirus  was administered to infect NRCMs, and 3-methyladenine  was used to interfere autophagy initiation while bafilomycin A1  was added to block autophagosome-lysosome fusion. For red fluorescent protein (Cherry)/green fluorescent protein (GFP) analysis, NRVMs were cultured on coverslips; after treatment with Ang II, 3-MA or BafA1 for 2 h, cells were washed with ice-cold PBS, and fixed with 4% paraformaldehyde, and then scanned with a fluorescence microscope.P > 0.05) according to the one‑sample K‑S test. One-way analysis of variance (ANOVA) was used to evaluate differences between multiple groups, followed by a post hoc Tukey test. Comparisons between two groups were assessed by Student’s t-test. P < 0.05 was defined as statistically significant.All statistical analyses were computed using the SPSS 22.0 software and the results are presented as the means ± SD. All measurement data had normal distributions (Supplementary material


The Chieti Affective Action Videos (CAAV) is a new database designed for the experimental study of emotions in psychology. The main goal of the CAAV is to provide a wide range of standardized stimuli based on two emotional dimensions: valence and arousal. The CAAV is the first database to present emotional stimuli through videos of actions filmed and developed specifically for experimental research. 444 young adults were recruited to evaluate this database, which consisted of a sub-set of 90 actions filmed in four versions, for a total of 360 videos. The four versions differ based on the gender of the main actor  and in the perspective in which each action was shot (first-person or third-person). CAAV validation procedure highlighted a distribution of different stimuli based on valence and arousal indexes. The material provided by CAAV can be used in future experimental studies investigating the role of emotions, perception, attention, and memory in addition to the study of differences between gender and perspective taking. Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.11448795 Studies have shown that the use of emotional movie clips could offer additional advantages8, compared to more simple stimuli. In fact, movie clips present more complex visual stimuli than words and images. Along with being highly capable of involving and capturing the observer’s attention9, at the same time, movie clips increase the level of ecological validity, simulating a real-life setting. Furthermore, meta-analyses of emotion induction have shown that movie clips appear to be among the most effective ways to elicit emotions10. Despite the advantages of ecological validity and emotional activation, it has been shown that variation in the camera angle and light exposure, within the same movie clip, may lead the observer to perceive some of the real-life situations as unusual or unrealistic11. Schaefer et al., also has pointed out that most movie clips activate both, the auditory and the visual systems, and that they depict multiple actions using different emotional levels. The multimodal features of most video clips could result in a mixing of different emotional experiences for the observers, confusing the evaluation of the different actions and the recollection of them. Furthermore, the gender of the main actor and the point of view (POV) shot, are not always strictly controlled. The first-person POV allows the observer to take on the perspective of the actor, while in the third-person POV, the observer assumes an external role, watching the action happening. Based on the above rationale, the CAAV (Chieti Affective Action Video) was created with the aim of solving, or at least smoothing-out, some of the issues that affect currently used databases, most of which rely on already existing video clips. Specifically, the CAAV was developed to control for the following critical aspects: the camera angle, the number of stimuli included in the scene, the number of actions presented, the gender of the actors, and the POV with which the actions are carried out.In psychology, a large range of procedures and experimental materials have been used to elicit emotions in a laboratory setting13. This dimensional approach to study emotions is, itself, characterized by the circumplex model14, which establishes valence and arousal to be the two dimensions considered when evaluating an emotion. The valence dimension indicates whether the observer likes or dislikes an event, an object, or a situation. The arousal dimension refers to the level of psychological activation induced by an emotional stimulus. According to the circumplex model, from the linear combination of different levels of valence and arousal (e.g. high/low), it is possible to organize different discrete emotions. Specifically to the CAAV database, an example of an action with high level of valence would be “Finding cash”, while one with low level of valence would be “Poisoning a person”. Regarding arousal, an example of an action of the CAAV with high level of arousal would be “Being threatened with a knife”, while one with low level of arousal would be “Sharpening a pencil”. Research, based on this theoretical framework, has led to the development of databases, which has categorized different types of emotional stimuli, such as pictures or words, on the basis of these fundamental dimensions of emotions2. Therefore, based on this approach, experimental subjects were asked to rate the videos of the CAAV on both, valence and arousal, dimensions. Regarding the CAAV characteristics, controlling for the gender of the actors allows for this database to be used to investigate the role of gender identification. Researchers suggest that gender plays a crucial role in processes related to self-perception, face-recognition, emotion, and memory19. Furthermore, the perspective dimension (POV) is a critical feature in the present database, since the research participant will either watch the actions being executed by someone else (third-person), or will watch them being performed in first-person as if he/she were performing them. The perspective from which an action is viewed has a fundamental role during the imitation of a behavior, thus influencing the way through which the sensory information is transferred between the person performing an action and the imitator20. Several imitation studies have reported that sensory information available from the first-person POV, as if the imitator were observing the model from his/her own perspective, is greater than that viewed from the third-person perspective, where the model is facing the observer23. The first-person perspective model facilitates more accurate imitative behavior than the third-person perspective model25 and it induces greater activity in the mirror neuron system (MNS), which is implicated in the processing of visuomotor information27. Differently, studies investigating the first and third-person through videogames, have highlighted that the playing view influences the presence of emotional responses28. The first-person playing view seems to generate a greater emotional response compared to the third-person playing view. Furthermore, participants are more immersed in a game when they look at it through the eyes of the character (first-person condition), regardless of their preferred perspective29. Considering all the possible advantages that the use of the first-person POV can have on the observer; our movie clips were filmed using both, the third-person and the first-person point of view. Staging these two different perspectives allows to compare and analyze the different emotional aspects arising from both POVs. Finally, in relation to identification processes and emotional responses, the perspective dimension may further interact with the gender. Therefore, using the CAAV also allows to compare and analyze the mixed emotional aspects arising from the manipulation of these two variables.The most innovative aspect of the CAAV consists in being the first database to present emotional stimuli through videos of actions filmed and developed specifically for experimental research. In relation to the emotional aspect, the development of the CAAV database is founded based on the Dimensional Model of Emotions. This model assumes that it is possible to classify emotions through fundamental dimensions that go beyond the different types of emotional responses444 healthy participants took part in the CAAV validation procedure voluntarily. Specifically, the sample consisted of 217 young males and 227 young females between the ages of 18 and 30 years , most of which were university students at G. d’Annunzio University of Chieti. They received no compensation and signed an informed consent before starting the experiment. IRB approval was obtained by the G. d’Annunzio University ethical committee.To select the actions to be included in the CAAV we took into account different criteria. Particularly, the actions had to be perceived as realistic as possible. Furthermore, the actions had to be reproducible within a laboratory setting, kept as controlled as possible in order to: 1) avoid presenting too many stimuli simultaneously, (2) avoid variations in camera angle and light exposure, and (3) keep the same setting and background across the videos. Finally, the goal was to select different emotional actions to obtain a subsequent representative distribution of CAAV videos along the valence and arousal dimensions continuum. All CAAV videos were filmed in an empty laboratory room which was kept as controlled as possible. Only if required by the movie clip setting creation, objects were incorporated into the laboratory room, for example the addition of a table, a chair, or a knife. The actions presented in the database were carried out by either female or male characters. Two 24-year-old amateur actors  were involved in the recording, always wearing a black shirt and blue jeans. Videos were recorded with a GoPro Sony HDR-as50. Each video lasted exactly 15 seconds. In order to make all videos the same duration, for actions that were carried out in less than 15 seconds, filler scenes were added (up to a maximum of 5 seconds). In these scenes, the actors were waiting to perform the action. These filler scenes were included to obtain the same standard duration for all stimuli, even though different actions for their own nature were performed by the actors in different time intervals. The CAAV’s emotional stimuli are only depicted through visual presentation. All the auditory stimuli have been muted from the movie clips. In addition, the CAAV presents just one main action in each video, avoiding the succession of many actions that could carry a different emotional valence. Furthermore, our videos have been created to represent a broad sample of contents across the entire affective space and to contain simple and easily understandable actions. The same conditions of brightness were ensured across filming sessions. For videos where differences were detected at a later stage, the brightness was modified using “VideoPad Video Editor”. Finally, the camera angle and the distance of the camera from the scene were controlled across all conditions. To get an overview of what has been described, frames extracted from some videos of the CAAV are shown in Fig.  avoid pr2. To further control for fatigue, each participant was given the option to interrupt the task at any time if s/he was feeling tired. None of the subjects asked to interrupt the task. The presentation order of the videos was randomized within each list and each video was rated for arousal or valence after its presentation. Participants were divided into 2 groups  first-person POV with male main actor; (2) first-person POV with female main actor; (3) third-person POV with male main actor and (4) third-person POV with female main actor. The resulting 360 videos were divided into 4 different lists . Each list contained all 90 actions but varied based on the gender of the main actor and perspective (first-person/third-person POV). The administration of the 4 different lists was balanced between experimental subjects. This subdivision was needed to reduce the subjects’ fatigue due to the excessive duration of the task first-pe24, each emotion arises from the linear combination of valence and arousal. The valence dimension indicates whether the observer likes or dislikes an event, an object, or a situation. Valence ranges along a continuum that goes from negative valence to positive valence. Differently, arousal is defined as the physiological activation level. Arousal compares the states of low physiological activation, which can usually occur in conditions of sadness and relaxation, with those of high physiological activation which can usually be observed in conditions of anger and happiness. In order to measure the emotional dimensions of valence and arousal in relation to our action video clips, we used the Self-Assessment Manikin (SAM) which is a widely used tool in this research field30. The SAM is a non-verbal assessment technique which uses figures to measure the valence and arousal associated with a person’s emotional reaction to a variety of stimuli. To study the valence dimension, we used a version of the 9-point SAM scale30. In addition, we considered another version of the 9-point SAM scale to analyze the arousal dimension30. Using these tools, the subject can select any of the 9 points on the continuum, with 1 corresponding to the lowest possible rating on each dimension  and 9 corresponding to the highest possible rating .According to Russell & Barrett31. Specifically, all CAAV videos are reported in the file .zip named “CAAV_database”. Furthermore, eight sample videos of the CAAV are available for download on the Figshare platform as an overview of the CAAV stimuli. The eight sample videos include two different actions, “Losing hair”  and “Blowing candles”, both presented in four different conditions (in relation to gender and POV variables). All 360 videos are in.mpg format with a 1920 × 1080 resolution. Additionally, the results of the CAAV validation are reported in a separate Excel file. This file is called “CAAV_dataset” and contains a dataset with the average scores, for both valence and arousal dimensions, for each video. This table is accompanied by a legend that provides a detailed description of all the variables. The table reports the code assigned to each video. Each video was renamed with a corresponding code within the depository. Furthermore, this table reports a brief description of the action contained in each video, the type of perspective used (first-person POV vs. third-person POV), and the gender of the actor . The average values and the standard deviation for both valence and arousal dimensions are also reported. Furthermore, the mean ratings and standard deviation distinguished by the gender of the experimental subjects  are also reported. Finally, in a second Excel file named “CAAV_rawdata” the raw data for all experimental subjects are reported. The table contains: subject ID, age, gender, list of stimuli administered, and the rating for each of the 360 videos.All data and videos are available on Figshare platformIn the present study, participants could select any of the 9 points on the SAM rating scale by pressing the corresponding numeric key on a laptop keyboard. Experimental sessions were conducted individually in a laboratory room under similar lighting conditions. Each trial began with a preparatory slide (“Please rate the next video”) that was presented for 3 seconds. Then, the video was presented for its entire duration (15 seconds), and immediately after the video terminated, a new slide with rating instructions was shown. For the first group, instructions stated “Please rate the video based on valence” while for the second group, instructions stated “Please rate the video based on arousal”. After the video disappeared from the screen, subjects made their ratings of valence or arousal using SAM. A standard 5 seconds rating period was used. In addition to the 90 CAAV videos, 3 practice videos (with different randomized perspective and different randomized gender of the actor) were showed prior to the experimental block . The purpose of these preliminary videos was to train the subjects for subsequent evaluations, informing them of the type of content they would have been exposed to, as well as to familiarize over the use of the rating scales. A laptop computer using E.Prime 2.0 software was used to control the randomization and timing of the stimuli presentation, as well as of the collection of the rating responses. The entire task lasted around 35 minutes. “CAAV_dataset” shows the mean ratings. The interaction between valence and arousal scores of each video is reported in Fig. 32, a further development could be to involve different age groups in the rating of the CAAV stimuli making the database more appropriate in studies on emotions and memory in aging.The present database has the potential to be applied to several fields in psychology. Specifically, since the CAAV stimuli are indexed on both, valence and arousal dimensions, they can be used in experimental studies to investigate the role of emotions. Moreover, the CAAV stimuli can be suitable in cognitive studies to investigate perception, visual attention, and emotional memory. In social psychology, this database and its stimuli can be potentially useful to study morality, responsibility, and empathy. The CAAV can also be used with clinical populations. In this context, for example, video stimuli could be implemented in both emotional and memory training or to investigate reality monitoring and memory distortion. Also, considering further features of the CAAV, it is possible to use this database to investigate the differences related to gender and to perspective taking. Another possibility would be to manipulate these two variables to study the different levels of self-identification and the consequent emotional response. In relation to the limitations and the possible future developments of the CAAV, video stimuli could also be evaluated for other interesting attributes, such as the frequency and typicality of the action in daily life. Finally, since it has been shown that emotional stimuli are processed differently during the life span