Osteogenic transdifferentiation of vascular smooth muscle cells (VSMCs) is a risk factor associated with vascular diseases. Wnt signaling is one of the major mechanisms implicated in the osteogenic conversion of VSMCs. Since Cdon has a negative effect on Wnt signaling in distinct cellular processes, we sought to investigate the role of Cdon in vascular calcification. The expression of Cdon was significantly downregulated in VSMCs of the aortas of patients with atherosclerosis and aortic stenosis. Consistently, calcification models, including vitamin D3 (VD3)-injected mice and VSMCs cultured with calcifying media, exhibited reduced Cdon expression. Cdon ablation mice (cKO) exhibited exacerbated aortic stiffness and calcification in response to VD3 compared to the controls. Cdon depletion induced the osteogenic conversion of VSMCs accompanied by cellular senescence. The Cdon-deficient aortas showed a significant alteration in gene expression related to cell proliferation and differentiation together with Wnt signaling regulators. Consistently, Cdon depletion or overexpression in VSMCs elevated or attenuated Wnt-reporter activities, respectively. The deletion mutant of the second immunoglobulin domain (Ig2) in the Cdon ectodomain failed to suppress Wnt signaling and osteogenic conversion of VSMCs. Furthermore, treatment with purified recombinant proteins of the entire ectodomain or Ig2 domain of Cdon displayed suppressive effects on Wnt signaling and VSMC calcification. Our results demonstrate a protective role of Cdon in VSMC calcification by suppressing Wnt signaling. The Ig2 domain of Cdon has the potential as a therapeutic tool to prevent vascular calcification. Researchers in South Korea have identified a molecular signaling pathway that suppresses calcium build-up  of muscle tissue in blood vessels, opening a new avenue for preventing health problems such as atherosclerosis, chronic kidney disease and diabetes. Young-Eun Leem, Jong-Sun Kang and colleagues at Sungkyunkwan University, Suwon, investigated the role of a glycoprotein (a protein-carbohydrate combination) called Cdon, a ‘cell adhesion molecule’ involved in binding cells to other cells. In studies in mice, they identified a sequence of molecular interactions by which Cdon can suppress the calcification of smooth muscle cells, the most abundant cells in artery walls. This calcification, commonly known as hardening of the arteries, can cause heart disease and strokes, among other medical problems. The Ig2 domain of Cdon identified by the researchers may have therapeutic potential. VSMCs express a variety of contractile proteins, such as alpha-smooth muscle actin (αSMA), smooth muscle myosin heavy chain (sm-MHC), and smooth muscle 22α (SM22α), ensuring contractile function. In response to diverse physiological and pathological stimuli, VSMCs undergo a process called cellular remodeling2. In homeostatic remodeling, VSMCs dedifferentiate to proliferate and redifferentiate into muscle cells to regain their contractile properties. Upon pathological insults, VSMCs undergo a phenotypic switch from the contractile to synthetic phenotype and eventually transdifferentiate into osteochondrogenic cell types characterized by the loss of VSMC marker proteins and gain of osteoblast cell marker proteins (such as Runt-related transcription factor 2 (Runx2) and osteopontin)4. The resulting cells produce mineralizing matrices, and therefore, the osteochondrogenic transdifferentiation of VSMCs is the key event for vascular calcification associated with diverse pathological conditions, such as aging, diabetes, atherosclerosis, and chronic kidney disease7. Among multiple signaling pathways, canonical Wnt signaling is involved in osteogenic transdifferentiation and vascular calcification. In VSMCs, Wnt signaling is activated by a high level of phosphate and induces Runx2 expression, a key transcription factor for osteochondrogenic transdifferentiation9. A recent study demonstrated that the inhibition of Wnt signaling by magnesium reverses the osteogenic conversion of VSMCs10. Thus, understanding the regulatory mechanisms of Wnt signaling in VSMCs is important to develop therapeutic strategies against vascular calcification.The contractile properties of vascular smooth muscle cells (VSMCs) are critical for the maintenance of vascular function13. A recent study proposed Cdon as a therapeutic target to promote endothelial integrity in response to acute inflammation via inhibition of desert hedgehog-mediated signaling14. In addition to Shh signaling activation by Cdon as a coreceptor, Cdon suppresses Wnt signaling by interacting with the LRP6 coreceptor to promote ventral neuronal cell fates in early forebrain development15. A similar suppressive effect of Cdon on Wnt signaling has also been demonstrated in the prevention of cardiac remodeling and fibrosis16. Considering the importance of the fine control of Wnt signaling in the vascular system, the question arises of whether Cdon plays a role in vascular calcification. The initial open data analysis revealed that Cdon transcripts are decreased in calcified aortas compared to normal aortas. Consistently, Cdon expression showed a negative correlation with osteogenic markers . Therefore, we generated a tamoxifen (tmx)-inducible SM22α-CreERT2;Cdon (Cdonf/f) mouse model to ablate Cdon in smooth muscle upon tmx administration. Using these mice, we examined the role of Cdon in vascular calcification induced by vitamin D. Cdon depletion in VSMCs exacerbated VSMC calcification concurrent with enhanced Wnt signaling. Conversely, Cdon overexpression attenuated the osteogenic conversion of VSMCs by suppressing Wnt signaling. The Cdon deletion mutant of the Ig2 domain, which is responsible for Wnt suppressive activity, failed to attenuate the osteogenic conversion of VSMCs. In addition, treatment with purified Ig2-Fc (Ig2 domain fused to Fc domain of human IgG gamma protein) suppressed the calcification of VSMCs. Collectively, these data demonstrate a protective role of Cdon in vascular calcification via suppression of Wnt signaling. Furthermore, the Ig2 domain of Cdon has the potential as a therapeutic agent to intervene in vascular calcification.Cdon (CAM-related/downregulated by oncogenes) is a member of the immunoglobulin/fibronectin type III superfamily of cell adhesion molecules. Cdon plays critical roles in the development of forebrain and skeletal muscle via regulation of sonic hedgehog (Shh), Wnt, and N-cadherin/cell adhesion signalingCdonf/f) were obtained from EUCOMM and maintained as previously described17. For Cdon deletion in vascular muscle cells, Cdonf/f mice were crossed with SM22a-CreERT2 mice, which express tmx-inducible Cre recombinase under the transcriptional control of the SM22a  promoter. SM22α-CreERT2 mice were obtained from Severance Integrative Research Institute for Cerebral & Cardiovascular Diseases . For deletion of one of the exons of the Cdon gene in VSMCs, approximately 8- or 10-week-old mice were injected intraperitoneally with 100 mg/kg tmx  every two days five times. For the model of vascular calcification in mice, 8- or 10-week-old C57BL/6 male mice and vehicle- or tmx-injected mice were administered VD3, which induces vascular calcification by accelerating calcium levels in the blood . VD3 (14.575 mg) in 70 μl of ethanol was mixed with 500 μl of cremophor for 15 min at room temperature, and this solution was then mixed with 6.2 ml of sterilized water, including 250 mg of dextrose for 15 min at room temperature. The mice were injected with a dose of VD3 (150 μl/25 g per day) subcutaneously for 3 consecutive days18.Mice bearing the Cdon-floxed allele  of Sungkyunkwan University School of Medicine (SUSM) and complied with the animal experiments guidelines of the SUSM Ethics Committee (protocol number: SKKUIACUC 2020-04-14-1).19. PWV was obtained from the B-mode and pulse-waved (PW) Doppler mode of the aortic arch view, calculated as PWV = aortic arch distance/transit time (cm/s). The PW Doppler mode sample volume was placed in the ascending aorta, and the time (T1) from the onset of the QRS complex to the onset of the ascending aortic Doppler waveform was measured. On the same image plane, the PW Doppler mode sample volume was placed as distal as possible in the descending aorta, and the time (T2) from the onset of the QRS complex to the onset of the ascending aortic Doppler waveform was measured. The obtained values for T1 and T2 were averaged over 10 cardiac cycles. The aortic arch distance was measured between the 2 sample volume positions along the central axis of the aortic arch on the B-mode image, and the transit time was calculated by T2 − T1 (ms)20.For measurement of cardiac function and pulse wave velocity (PWV), echocardiographic analysis was performed 1 day before sacrifice. Mice were anesthetized with 1–2% (vol/vol) isoflurane, and body temperature was maintained at 36–38 °C with a heating lamp and a heating platform. Heart rates were monitored and generally maintained at 400–500 beats per minute. Echocardiography was carried out using a Vevo LAZR-X machine (the BIORP of Korea Basic Science Institute) with a 40-MHz probe . Analysis of M-mode images derived from the short-axis view of the left ventricle was carried out to measure the ejection fraction and fractional shortening21. For induction of vascular calcification in vitro, the cells incubated in normal media were switched to calcifying medium (CM) that contains excess levels of phosphate and calcium, leading to mineral deposition in cells. CM was generated by adding 100 nM dexamethasone, 1 mM insulin, 50 μg/ml ascorbic acid, 10 mM β-glycerophosphate, and 8 mM CaCl2 to normal media18. The media were replenished every 48 h for up to 3 days or 7 days, and the first day of culture in CM was defined as Day 0. For overexpression studies, pBABE-puro or pBABE-puro-Cdon was transfected into A7r5 cells by using 1 mg/ml polyethyleneimine . For depletion of Cdon in A7r5 cells, control shRNA (shCont) or Cdon shRNA (shCdon) was expressed by using a lentiviral expression system as previously described17. For analysis of the effects of Cdon on Wnt signaling, cells were treated with 20 ng/ml Wnt3a  or 5 μM XAV939 .A7r5  cells were cultured in a normal medium containing DMEM, 10% FBS, and 1% penicillin‒streptomycin as previously described18. For quantification of the calcium deposition in aortas, the images obtained from TissueFAXS PLUS  were analyzed with ImageJ software .Aortas were isolated from PBS-perfused mice, fixed with 4% PFA, embedded in paraffin, and sectioned at a thickness of 5 μm. For the determination of calcium deposition in the aortas, slide sections were stained with Von Kossa stain. Briefly, deparaffinized sections were incubated with 1% silver nitrate solution under ultraviolet light for 2 h, followed by the removal of unreacted silver with 5% sodium thiosulfate for 5 min. Sections were counterstained with nuclear fast red for 5 min before dehydration and mounting16.For the immunohistochemistry of aortic samples, deparaffinized samples were boiled in Tris-EDTA buffer  for antigen retrieval followed by the standard protocol. For analysis of cell death during vascular calcification, a Click-iT TUNEL assay Alexa imaging assay kit  was utilized on cryosections of aortas that were isolated from PBS-perfused mice and sectioned at a thickness of 5 μm. Fluorescence images were analyzed with an LSM-710 confocal microscope  as previously described16. In brief, cultured cells and homogenized aortic tissues were lysed in RIPA buffer , pH 8.0; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; 10 mM Tris-HCl). The primary antibodies used in this study are listed in Supplementary Table Immunoblot analysis was carried out as previously described16. Total RNA from cells or homogenized tissues was extracted with easy-BLUE  reagent following the manufacturer’s instructions. cDNA samples were generated from 0.5 mg of RNA by PrimeScript RT reagent  according to the manuscript’s protocol. The primer sequences used in this study are listed in Supplementary Table http://software.broadinstitute.org/morpheus/). Global gene expression was assessed by the Reactome with GSEA (http://www.gsea-msigdb.org/gsea/msigdb/index.jsp) using the MSigDB database v7.2 .Quantitative reverse transcription polymerase chain reaction (RT-PCR) and RNA sequencing analysis were performed as previously describedhttp://www.ncbi.nlm.nih.gov/geo) database  were computed for the analysis of gene expression and Pearson’s correlations with GraphPad Prism 7. The single-cell transcriptome of human patients (GSE159677) was utilized to extract each cell type in the aortas, including VSMCs, endothelial cells, macrophages, T-lymphocytes, B-lymphocytes, and NK cells, by marker genes. The genes of VSMCs were aligned and projected through uniform manifold approximation and projection (UMAP) to explore the scRNA-seq data. Visualization of gene expression was carried out in GraphPad Prism 7.The aortic transcriptomes of humans from the NCBI Gene Expression Omnibus .A luciferase reporter assay was performed as previously describedg for 15 min at 4 °C. The supernatant was transferred to a new tube, and 10% ammonium hydroxide was added to the supernatant. Absorbance was read in triplicate at 405 nm using a 96-well plate spectrophotometer18.Cells were washed with PBS and fixed with 4% PFA for 15 min at room temperature. The cells were then washed twice with deionized water and covered with 40 mM Alizarin Red S  at approximately pH 4.2 for 2 h at room temperature with gentle shaking. For removal of the unstained dye, the cells were washed three times with deionized water before images were obtained using a Nikon CELIPS TE-2000U . For quantification of calcification, Alizarin Red S was extracted with 10% acetic acid for 30 min at room temperature, scraped into a microcentrifuge tube, vortexed, and incubated at 85 °C for 15 min. After chilling on ice for 5 min, the mixture was centrifuged at 20,000 × 15. Seventy-two hours after transfection, the cells were pelleted by centrifugation at 2000 × g for 10 min. The supernatant was then filtered (0.45 μm) and subjected to Protein A antibody purification22. In brief, the supernatant was incubated with Protein A agarose  for 1 h at room temperature under constant rotation. Bound proteins were eluted with 0.1 M citric acid (pH 3.0) and immediately neutralized with 1 M Tris-HCl (pH 8.0). Eluates were concentrated using Amicon Ultra4 centrifugal filters  and dialyzed against PBS. Protein concentrations were determined using A280 measurements. Protein purity and integrity were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.HEK293T cells were transfected with recombinant proteins of Cdon fused with the Fc region of human IgG gamma listed as follows: Cdon-Fc; the entire ectodomain fused to Fc, Ig2-Fc; Fc fusion protein with Ig2 domaint-test or one-tailed analysis of variance (ANOVA) followed by Tukey’s test . Differences were considered significant at P < 0.05.Values are the means ± SEMs or SDs, as noted. Statistical significance was calculated by paired or unpaired Student’s Cdon expression , and CD68  obtained from calcified atherosclerotic plaques (AC) and proximal adjacent portions (PA) of three patients . Smooth muscle-specific deletion of Cdon (cKO) was induced by intraperitoneal injection of tmx five times at 2-day intervals  was not significantly altered relative to that of the control-infected cells  staining. The Cdon-depleted VSMCs had an approximately 3.5-fold increase in SA-β-gal-positive cells relative to the control cells , Wnt inhibitory factor 1 (Wif1), and Wnt9a, were altered. Consistently, the Cdon-deficient aortas exhibited altered Wnt activities in response to VD3  and a deletion mutant for the second Ig domain (ΔIg2) on CM-mediated osteogenic conversion. In contrast to the suppressive effect of WT on β-catenin levels in response to CM, the ΔIg2 mutant failed to fully repress the induction of β-catenin and Runx2 levels els Fig. . In addiion Fig. . Furtherion Fig. . Consistion Fig. . Similarase Fig. . Consistase Fig. . CollectAxin2, Runx2, and ALPL expression, while control IgG did not show any effect  or Ig2 domain (Ig2-Fc) fused to the Fc domain of human IgG gamma. The expression of recombinant proteins was validated in the supernatants of the cells transfected with Cdon-Fc or Ig2-Fc by immunoblot analysis Fig. . The treect Fig. . Taken tOur data demonstrate the critical role of Cdon in vascular calcification. Transcriptome data from aortas isolated from patients with atherosclerosis and stenosis revealed a significant decrease in Cdon levels in VSMCs. In agreement, Cdon was reduced in the aortas of mouse models of VD3-induced vascular calcification or in CM-treated VSMCs. Furthermore, VSMC-specific depletion of Cdon exacerbated VD3-induced vascular calcification, although the effect of Cdon needs to be confirmed by performing an in vivo study with Cdon transgenic mice. Likewise, Cdon depletion or overexpression in VSMCs increased or attenuated the Wnt signaling pathway, respectively. Therefore, Cdon is an important regulator of vascular remodeling.26. Since Cdon activates Shh signaling as a coreceptor27, we initially predicted that Cdon depletion might reduce the proliferation of VSMCs and vascular remodeling via reduced Shh signaling. Transcriptome analysis revealed alterations in genes related to cell proliferation and differentiation in Cdon-deficient aortas treated with VD3. However, the involvement of Shh signaling in vascular calcification of Cdon-deficient aortas is unclear. Genes related to Shh signaling were not greatly altered in Cdon-deficient aortas in response to VD3. Unlike the reduced expression of Cdon, the expression of other Shh signaling components, Boc, Gas1, and Shh, was also not significantly changed in the transcriptome data from aortas isolated from patients with atherosclerosis and stenosis. Furthermore, Cdon depletion increased cellular senescence and death in the medial region of the aorta in response to VD3-mediated calcification. These data support an independent role of Cdon in Shh signaling in vascular diseases.Shh signaling induces the proliferation of VSMCs, an essential step for intimal hyperplasia and the pathogenesis of vascular diseases29. BMP2, one of the transforming growth factor-β family members, activates Runx2 in diverse cell types, including VSMCs, and plays a crucial role in bone repair31. Aortic transcriptome analysis revealed a negative correlation between Cdon and the BMP signaling pathway. However, there was no significant difference in BMP signaling in Cdon-overexpressing or Cdon-depleted VSMCs by reporter assays (data not shown). Thus, BMP signaling may not be directly involved in Cdon-mediated VSMC regulation.BMP signaling plays a critical role in the osteogenic differentiation of VSMCs15, Cdon deficiency in VSMCs resulted in Wnt signaling dysregulation and enhanced osteogenic conversion. Our previous study identified the second Ig region of the ectodomain of Cdon to be responsible for Wnt signaling suppression by interacting with LRP615. Accordingly, the deletion of Ig2 in Cdon failed to block osteogenic conversion in response to CM or Wnt3a treatment, suggesting a requirement for Wnt inhibition by the Ig2 domain to prevent VSMC conversion. Consistently, the Fc fusion protein of the Ig2 domain exhibited a suppressive effect on Wnt signaling activity and a protective effect on vascular calcification. Thus, Cdon represents a potential therapeutic tool for vascular calcification via suppression of Wnt signaling activity. Since the Wnt signaling pathway is involved in diverse diseases, including cancers and neurodegenerative diseases, the regulatory mechanisms of Wnt signaling have been continuously investigated for the development of therapeutic strategies34. DKK (Dickkopf) proteins, Wnt regulatory molecules, bind to LRP5/6 coreceptors, thereby acting as functional antagonists of Wnt signaling35. Interestingly, inhibition of Wnt signaling by DKK plays a complex role in cardiovascular diseases. DKK1 is elevated in the plasma and lesions of patients with atherosclerosis and type 2 diabetes with cardiovascular diseases37. Excessive DKK1 can promote endothelial cell dysfunction associated with increased inflammation, likely contributing to atherosclerosis38. However, DKK1 can attenuate calcium deposition and the expression of RUNX2 in calcified aortas from chronic kidney disease in a similar manner to Cdon39. The opposing effects of DKK1 may be attributed to its distinct role dependent on the cellular context and Wnt-independent signaling activities. A recent study on Cdon in the endothelium reported its role as a negative regulator of desert hedgehog-driven endothelial integrity in inflammatory conditions14. Therefore, therapeutic strategies using Wnt signaling regulators will require high specificity to avoid undesirable outcomes. In the case of Cdon, its interaction with Hedgehog proteins is mediated by the third fibronectin domain40. Since the Ig2 domain of Cdon has been shown to be sufficient to distinctively target and suppress Wnt signaling activities, the use of the Ig2 domain of Cdon may serve as a specific suppressor of the Wnt signaling pathway while avoiding the intervention of other signaling pathways.In addition to BMP signaling, an inverse correlation between Cdon and Wnt target genes was observed in the aortic transcriptome of patients with atherosclerosis and stenosis. Wnt signaling is another major mechanism implicated in the osteogenic conversion of VSMCs. Consistent with the negative effects of Cdon on Wnt signaling in forebrain development41. Cellular senescence of VSMCs has been associated with the induction of osteogenic markers, including Runx2 and ALPL42. Thus, cellular senescence is considered a major risk factor for vascular calcification. Cdon-deficient VSMCs exhibit increased cellular senescence in response to calcification stress. Similarly, skeletal muscle stem cells deficient for Cdon display cellular senescence, which contributes to impaired regeneration17. The mechanism by which Cdon depletion causes cellular senescence is currently unclear. Decreased Wnt signaling activity has been generally linked with cellular senescence43, but a recent study reported that the canonical Wnt signaling pathway in chondrocytes induces cellular senescence associated with inflammation44. Further study is required to elucidate the exact role of Wnt signaling and Cdon in VSMC senescence. Here, we report the suppressive effects of the Ig2 domain of Cdon on the Wnt signaling pathway by interacting with LRP6, indicating that the Ig2 domain of Cdon could be a good candidate for therapeutic strategies in vascular diseases.Accumulating evidence suggests that cellular senescence is closely linked with vascular pathogenesisSupplementary material


Focal adhesions also function as mechano-signaling platforms by perceiving and integrating diverse physical and (bio)chemical cues of their microenvironment, and by transducing them into intracellular signaling for the control of cell behavior. The fundamental biological mechanism of creating intracellular signaling in response to changes in tensional forces appears to be tightly linked to paxillin recruitment and binding to focal adhesions. Interestingly, the tension-dependent nature of the paxillin binding to adhesions, combined with its scaffolding function, suggests a major role of this protein in integrating multiple signals from the microenvironment, and accordingly activating diverse molecular responses. This minireview offers an overview of the molecular bases of the mechano-sensitivity and mechano-signaling capacity of core focal adhesion proteins, and highlights the role of paxillin as a key component of the mechano-transducing machinery based on the interaction of cells to substrates activating the  FAs alseceptors  cluster eceptors . The tigeceptors .versus more general functions of FAs  are formed at the level of the integrin-ligand interaction  (via F3-R9) keeps cytosolic talin in a globular, autoinhibited conformation  , connect to R13)   . The intormation   ation  to interact with the talin rod, suggesting that competitive interactions among different LD-motif binding proteins, such as FAK, vinculin and talin can take place within FAs  and the guanine nucleotide exchange factor for Rac1 βPix has been implicated in the regulation of FAs and cell migration . GIT1 isThe gathering of structural and positional data led to the proposal of the slanted fence model of FAs, in which connections among neighbouring integrin-talin1-kindlin-paxillin units stabilize the complex  Figure . The pro


The chloroplast is a complex cellular organelle that not only performs photosynthesis but also synthesizes amino acids, lipids, and phytohormones. Nuclear and chloroplast genetic activity are closely coordinated through signaling chains from the nucleus to chloroplast, referred to as anterograde signaling, and from chloroplast to the nucleus, named retrograde signaling. The chloroplast can act as an environmental sensor and communicates with other cell compartments during its biogenesis and in response to stress, notably with the nucleus through retrograde signaling to regulate nuclear gene expression in response to developmental cues and stresses that affect photosynthesis and growth. Although several components involved in the generation and transmission of plastid-derived retrograde signals and in the regulation of the responsive nuclear genes have been identified, the plastid retrograde signaling network is still poorly understood. Here, we review the current knowledge on multiple plastid retrograde signaling pathways, and on potential plastid signaling molecules. We also discuss the retrograde signaling–dependent regulation of nuclear gene expression within the frame of a multilayered network of transcription factors. Photosynthesis, the process through which plants and algae capture light energy and convert it into chemical energy, is one of the most important energy sources to sustain life on earth. In plants and algae, the photosynthetic process occurs in chloroplasts, intracellular organelles with a size of a few microns . Plant vIt is well accepted that chloroplasts originated more than a billion years ago from cyanobacteria which invaded a primitive eukaryotic cell through endosymbiosis . This evThe components of the photosynthetic complexes are encoded by both the chloroplast and nuclear genomes. As a result, nucleus-encoded components must be imported into chloroplasts after translation in the cytosol , and assPlants and algae harbor many double-membrane organelles, called plastids, which are derived from undifferentiated non-photosynthetic organelles, called proplastids. Plastids are classified based on their biological functions, in amyloplasts, involved in starch storage, etioplasts, formed in dark-grown leaves that accumulate chlorophyll precursors; chloroplasts, responsible for photosynthesis and biosynthesis of metabolites; chromoplasts that produce carotenoid pigments; elaioplasts with high amounts of plastoglobuli that are used for lipid storage .The chloroplast development process is extremely complicated, differing not only between monocotyledonous and dicotyledonous species, but also between developmental stages, organs, and plant tissues, with many underlying mechanisms still unknown . OverallWhen the leaf primordia develop from the shoot apical meristem (SAM) in true leaves, chloroplasts develop directly from proplastids . The numb6f complex, ATP synthase and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) have been detected in etioplasts although generally in much lower amounts compared to mature chloroplasts , which is important for reinforcing the hypocotyl cell wall and decreasing the pace of hypocotyl elongation (HOOKLESS1 (HLS1) to regulate seedling apical hook development (PROTOCHLOROPHYLLIDE OXIDOREDUCTASE (PORA and PORB) (PORA/PORB levels during de-etiolation to protect the seedling from photooxidative damage. EIN3 and PIF3 have been shown to bind to the promoters of LIGHT-HARVESTING CHLOROPHYLL A/B-BINDING (LHC) genes, and to repress numerous LHCA and LHCB genes , cop/det/fus mutants have very clear phenotypes. Their seedlings display light-grown phenotypes in darkness, and they accumulate high levels of anthocyanin  . As a reCB genes . OverallCB genes . In Arabhocyanin . In the hocyanin . Other Chocyanin , 2011.When a seedling emerges from the earth, light triggers a variety of physiological responses, including hypocotyl growth inhibition, chlorophyll accumulation, and chloroplast development. Massive transcriptional reprogramming of both the nuclear and plastid genomes, as well as the onset of a complex signaling interconnecting network, accompany these morphological changes in response to light. This process is called photomorphogenesis and it includes numerous and poorly understood light-sensing mechanisms .Arabidopsis, three cryptochromes (CRY1-CRY3), two phototropins (PHOT1 and PHOT2), and three Zeitlupe family (ZTL) proteins detecting UV-A/blue light, and five phytochromes (PHYA-PHYE) absorbing red (R)/far-red (FR) light  in R) light . These lR) light .Light is primarily sensed by phytochromes and cryptochromes during photomorphogenesis. Phytochromes contain the linear tetrapyrrole phytochromobilin as their chromophore. PHYs are found in the cytosol in their inactive Pr form in the dark. They are photoconverted into the active Pfr form when exposed to light. This light-induced conversion is followed by translocation of PHYs from the cytosol to the nucleus, where they are directed to subnuclear sites known as the photobodies , 2014. PNumerous essential light-signaling components, including many transcription factors such as PIFs, EIN3, and COP1, are regulated by PHYs. All PIFs interact physically with the PHYs in their Pfr forms and are subsequently phosphorylated, ubiquitinated, and degraded. PIF phosphorylation has been linked to casein kinase II (CK2), brassinosteroid insensitive 2 (BIN2), photoregulatory protein kinases (PPK1–PPK4), and phytochromes. The full intricacies of light-induced phosphorylation of PIFs, however, have yet to be uncovered . PIFs arArabidopsis begins with the ground state, oxidized FAD in darkness, and ends with the semi-reduced FADH upon perception of blue light  chromophore . A CRY pue light . Althougue light . CRY2 alue light . CRY1 anal level . CRY2 bial level . CRYs maal level .SIGMA FACTOR 5 (SIG5), CHLOROPHYLL A/B-BINDING PROTEIN (CAB), and RIBULOSE BISPHOSPHATE CARBOXYLASE SMALL SUBUNIT (RBCS), is controlled by CRYs in response to blue light , such as GUN5, PORC, LHCA4, LHCB1.1, and LHCB1.3 , and it also plays a role in plastid transcription by activating the expression of PURPLE ACID PHOSPHATASE 8 (PAP8), an essential subunit of the plastid-encoded plastid RNA polymerase , and DGDG are determined by the structure of the polar head and the fatty acid tails. Moreover, MGDG is present in both photosystem I (PSI), photosystem II (PSII), and the cytochrome b6f complex whereas DGDG is only present in PSII and light-harvesting complex II   proteins, which are part of the GARP superfamily of transcription factors, have also been identified as regulators of chloroplast development. GLKs modulate the expression of directly . GLK1 ane above) . In Arabid grana . This phdundancy . Thus, Gdundancy .The chloroplast DNA (plastome) is around ∼120–160 kb in size and packaged into nucleoids. The nucleoids move from the inner envelope to the thylakoids during chloroplast maturation . The plaRPOTm gene, which encodes the mitochondrial RNA polymerase  in mature chloroplasts  with their prokaryotic counterparts reveals that all of them belong to the σ70 family of transcription factors  (Gel filtration and mass spectrometry studies in es (TAC) . Mutantses (TAC) . These 1es (TAC) . PAP1/pTes (TAC) .Proteins of the second group, PAP4/Fe SUPEROXIDE DISMUTASE 3 (FSD3), PAP6/FRUCTOKINASE-LIKE PROTEIN 1 (FLN1), PAP9/FSD2, and PAP10/THIOREDOXINz (TRXz) are responsible for redox-dependent gene regulation and PEP protection against reactive oxygen species (ROS) .The third category includes the remaining PAPs. The function of PAP11/MurE-like is unknown. PAP3 may interact through its carboxyl-terminal region downstream of the S1 domain with several other PAPs including PAP4, PAP7, PAP9, PAP10, and PAP12. PAP3, on the other hand, does not interact directly with any of the PEP core components, such as RpoA and RpoB . PAP5 anChloroplast gene expression is a complex and highly regulated process, and the molecular relationship between photosynthetic electron transport activity and regulation of plastid gene expression (PGE) has been studied for many years . A majorPhANG induction is dependent on a positive signal from healthy developing plastids  to mediate the redox control of PEP activity . It is tA surprising finding was that redox-inactive versions of TRXz and FLN1 are able to entirely restore PEP function in the corresponding loss of function mutants during early chloroplast development . A possiThe hyperphosphorylated nucleotides guanosine pentaphosphate and tetraphosphate (together referred to as ppGpp) have emerged as likely candidates for regulating chloroplast stress signaling. They are synthesized from ATP and GDP/GTP by enzymes of the RelA SpoT Homolog (RSH) family in chloroplasts . In plan2 fixation enzyme, represents a classic example of gene coordination between nucleus and chloroplast  and SSU proteins in the chloroplasts, allowing the L8S8 holoenzyme to be assembled. Multiple steps of post-transcriptional regulation are required for the coordinated synthesis of LSU and SSU synthesis, including mRNA stability, RNA processing, translational initiation, translational elongation, protein stability, and final assembly of the L8S8 holoenzyme  are encoded by the nucleus . Many nuoroplast . Each plroplasts . The Sma nucleus . As a reloenzyme .8S8, a process which is assisted by the chaperones RbcX, Raf1, and Raf2 , but bind to polysomes when these seedlings are exposed to light, allowing both Rubisco proteins to be synthesized coordinately. Moreover, when light-grown plants are shifted to darkness (dark-shift), the synthesis of both Rubisco subunits is promptly arrested. Although Rubisco production is drastically decreased in dark-shifted amaranth seedlings, rbcL and rbcS transcripts remain bound to polysomes indicating that translation elongation is also regulated in a coordinated manner. These results show that light-induced synthesis of both Rubisco subunits is well synchronized between the two cellular compartments, owing to control at the translational level  and the cytosol influence the photosynthetic crosstalk within plant cells (such as transport) . ProteinIn the plastids of higher vascular plants, four distinct plastid retrograde signaling pathways have been traditionally recognized based on the sources of the signals: Accumulation of tetrapyrrole intermediates, Inhibition of PGE, Changes in plastid redox status, and Production of ROS . Severalgun mutants have provided clues for potential plastid signals  and the FLUORESCENT (FLU) protein which inhibits GluTR thereby preventing accumulation of free Pchlide which is phototoxic  by glutamyl-tRNA reductase (GluTR) and glutamate 1-semialdehyde aminotransferase (GSAT) . In the ototoxic . The steal level . The bioal level .PhANGs) (Arabidopsis gun mutants (genomes uncoupled) that partially recovered LHCB1 expression following norflurazon (NF) treatment  . Furtherreatment . When ex2+ or Fe2+ are inserted into Proto IX by Mg-chelatase (MgCh) or ferrochelatase (FC) to produce Mg-protoporphyrin IX (MgProto) and heme, respectively   . Mg-protpression . When thcription . In addicription . Accordiydomonas . Light i-ProtoIX . Also Mgthe dark .ferrochelatase I (FC1) encoding chloroplast heme synthase/plastid ferrochelatase in a gun mutant called gun6-1D. It has the same phenotype as the gun2-gun5 mutants with higher expression of PhANGs than in WT under photooxidative conditions , the prosthetic group of b-type cytochromes and of proteins like catalase and peroxidase. In Arabidopsis and cucumber, there are two isoforms of FC (FC1 and FC2) which have different tissue-specific and developmental expression profiles: FC2 is light-dependent and mostly expressed in photosynthetic tissues, whereas FC1 is stress-responsive and found throughout the plant  (Ferrochelatase inserts Fehe plant . Other hes (PHY) .Recent studies with Chlamydomonas have revealed novel essential functions of bilins in retrograde signaling, notably in the regulation of the expression of genes involved in the detoxification of ROS , 2017. MPhANG expression  upon LIN treatment  in chloroplasts , hydroxyl radicals and singlet oxygen (1O2)  . Dependi1O2 and initiate a programmed cell death (PCD) response and photobleach (flu mutant in Arabidopsis led to the identification of the chloroplast proteins Executer 1 (EX1) and its homolog EX2. In the ex1 suppressor the 1O2-induced programmed cell death response no longer occurs (1O2 which elicits changes in gene expression (flu line containing a reporter driven by the promoter of an 1O2-responsive AAA-ATPase gene for constitutive high-level expression. One of the mutant isolated is affected in the Pleiotropic Response Locus 1 (PRL1) gene. This gene appears to suppress the expression of 1O2-responsive genes and to play an important role in the response of plants to environmental stress. Additional genetic studies also suggest that 1O2-mediated signals are transmitted to the nucleus through a signaling network and that 1O2-mediated retrograde signaling interconnects to other signaling pathways (Conversion of protochlorophyllide to chlorophyllide is a key step in chlorophyll synthesis and requires light in land plants. Protochlorophyllide is highly photodynamic and its accumulation in the dark causes photo-oxidative damage when plants are shifted from the dark to the light. This is prevented by the FLUORESCENT (FLU) protein which inhibits glu-tRNA reductase, an enzyme which catalyzes one of the early steps of chlorophyll synthesis . After atobleach . Howeverr occurs . EX1 andpression . Furtherpathways ,b.1O2-response mutants  are similarly deficient in mechanisms that impact plastid homeostasis, resulting in an increased 1O2 response . RES signaling, another 1O2–specific response, is likely to be involved in Rose Bengal (RB) acclimation in C. reinhardtii. This 1O2 specific response includes the strong induction of GPXH and maybe other genes at low RB concentrations and during the early high light response , an inhibitor of carotenoid synthesis has been used extensively for studying retrograde signaling. Upon treatment of seedlings with NF and exposure to light, they bleach because photooxidation of chlorophyll in the absence of carotenoids leads to the accumulation of tetrapyrrole intermediates that are highly photodynamic. Under these conditions expression of eedlings . When seciferase . Expressproto IX . Mg-protproto IX . Howeverpression . It is tpression , either pression .Arabidopsis gun6 gain-of-function mutant, heme was found to be a retrograde signaling molecule (Another tetrapyrrole intermediate, heme, affects nuclear gene expression and appears to be involved in retrograde control. In the molecule . MEcPP (molecule . Althougmolecule . PAP appmolecule . As a remolecule . It shou2O2 may be the best candidate whereas 1O2 is thought to have a less important role because of its short half-life, high reactivity and limited diffusion (1O2 oxidation product of carotenoids was identified in Arabidopsis as a messenger in the 1O2 signaling pathway and induces gene expression in response to 1O2 (1O2 (Reactive oxygen species molecules have also been proposed to function as retrograde signaling molecules as they have the ability to move across the subcellular compartments, which is one of the main characteristics of the signaling molecules. Hiffusion . β-Cycloe to 1O2 , 6. As ie to 1O2 , to regu1O2 (1O2 .Plastids are essential compartments of eukaryotic cells. They are involved in the synthesis of many metabolites and are the sites of important metabolic and regulatory pathways. Here we have discussed several retrograde-signaling pathways that are highly complex and diverse. They are not operating in every cell type but restricted to specific cell types and developmental stages. As an example, the singlet-oxygen–mediated pathway occurs mostly in differentiated photosynthetically active mesophyll cells. In the last few decades, significant advances have been achieved in our understanding of plastid-to-nucleus signaling. However, in spite of this progress, there are still a lot of question marks concerning the identity of the authentic retrograde signals and how they work. Possibly, signals could only accumulate transiently and locally and be therefore difficult to detect. It is also not clear how perturbations of chloroplast processes elicit retrograde signals and how these signals are transmitted from the plastids to the nucleus and how they change the expression of nuclear genes. For a long time, the redox state of the plastids and PGE were thought to be the main determinants of retrograde signaling. Yet we still do not know how changes in redox state or impairment of PGE are transduced into signals and their identity remains elusive. At this time, it is not clear whether the signals are proteins, proteolytic products, oxidized lipids, metabolites or ionic fluxes.Most of the cellular biosynthetic pathways occur, at least partly, in the plastids, and specific carriers move different metabolites into and out of them. Different retrograde signaling pathways have been proposed based on different approaches and different experimental models. But it is not yet clear how these signaling networks interact with each other and how some key transcriptional factors like PEND, GLK, WHIRLY1, AP2/EREBP operate within these networks. Clearly, these challenging questions will need to be addressed in the coming years.XS designed the review outline and wrote the “Conclusion and future perspectives” section of the manuscript. MJ, ZL, and J-DR wrote the other sections and designed all figures. All authors contributed to the article and approved the submitted version.


Rapid eye movement (REM) sleep behaviour disorder (RBD) is considered the expression of the initial neurodegenerative process underlying synucleinopathies and constitutes the most important marker of their prodromal phase. This article reviews recent research from longitudinal research studies in isolated RBD (iRBD) aiming to describe the most promising progression biomarkers of iRBD and to delineate the current knowledge on the level of prediction of future outcome in iRBD patients at diagnosis.Longitudinal studies revealed the potential value of a variety of biomarkers, including clinical markers of motor, autonomic, cognitive, and olfactory symptoms, neurophysiological markers such as REM sleep without atonia and electroencephalography, genetic and epigenetic markers, cerebrospinal fluid and serum markers, and neuroimaging markers to track the progression and predict phenoconversion. To-date the most promising neuroimaging biomarker in iRBD to aid the prediction of phenoconversion is striatal presynaptic striatal dopaminergic dysfunction.There is a variety of potential biomarkers for monitoring disease progression and predicting iRBD conversion into synucleinopathies. A combined multimodal biomarker model could offer a more sensitive and specific tool. Further longitudinal studies are warranted to iRBD as a high-risk population for early neuroprotective interventions and disease-modifying therapies. Rapid eye movement (REM) sleep behaviour disorder (RBD) is a parasomnia clinically characterized by active dream enactment, including screaming, flinging, or falling off from bed, that may cause injuries to patients and their bed partners . AccordiIsolated RBD (iRBD) is now considered the expression of the initial neurodegenerative process underlying synucleinopathies, including Parkinson’s disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA)  and consiRBD is considered a marker of neurodegeneration with strong predictive value and scarce sensitivity. The likelihood ratio of PSG-proven iRBD for development of a synucleinopathy has been estimated at 130, more than three times higher than that of detecting striatal dopamine loss on molecular imaging . By contIn the future, iRBD could be considered a condition which is useful to be targeted with new disease-modifying therapies, currently in development aiming to interrupt the pathological processes towards the development of synucleinopathies at an earlier stage. The study of iRBD pathology can allow the understanding of biological alterations preceding the clinical manifestation of a synucleinopathy, thus anticipating personalized treatments at a stage where cellular damage could be reversible . To do sThis article reviews the most relevant results from recent longitudinal research studies in iRBD, with the intent of describing the most promising progression biomarkers of iRBD and to delineate the current knowledge on the level of prediction in iRBD patients at diagnosis and their future outcome Table .Table 1LProdromal PD is a clinical entity characterized by the presence of motor and non-motor symptoms, such as alterations in motor dexterity, autonomic dysfunction, mood, olfaction, and cognition, that reflect the progressing neuronal damage in the brain . Many of123I]FP-CIT SPECT, a marker of presynaptic dopamine transporter (DAT) availability FP-CIT SPECT FP-CIT uptake, and a predictor of cognitive decline in patients with low CSF levels of both α-synuclein and low amyloid-β42 FP-CIT SPECT and transcranial sonography have been shown to detect subclinical changes in iRBD patients, similar to pathology seen in early PD [123I]FP-CIT SPECT, combined with hyperechogenicity of the substantia nigra, using transcranial sonography, had a predictive value of 100%, with 55% specificity, after 2.5 years to predict the conversion of iRBD patients to a neurodegenerative synucleinopathy [123I]FP-CIT SPECT scans show progressive loss of striatal DAT in iRBD patients over three years, with iRBD patients who converted to PD showing the greatest level of nigrostriatal dopaminergic dysfunction at baseline [123I]FP-CIT SPECT greater than 25% in the putamen has been shown to discriminate iRBD patients, with DAT deficits, who converted to a synucleinopathy from iRBD patients who did not convert after three-year follow-up [123I], FP-CIT SPECT had 75% sensitivity and 51% specificity to predict iRBD conversion to a synucleinopathy with a likelihood ratio of 1.54 [99mTC]TRODAT-1 SPECT, in iRBD patients over 5 years with greater DAT deficits in those patients at high risk of progressing to a synucleinopathy [[early PD –44. Fourearly PD –45. Iranbaseline . Furtherollow-up . At a fi of 1.54 . Li and inopathy  therefor99mTc]ECD SPECT study illustrated increased perfusion in the hippocampus of RBD patients who developed a synucleinopathy at a 3-year follow-up compared to those who did not progress [18F]FDG PET scans to measure progressive changes in PD-related brain pattern expression measures as a prodromal PD biomarker to predict phenoconversion in iRBD patients metaiodobenzylguanidine (MIBG) scintigraphy has also been investigated in iRBD patients. While cross-sectional studies have revealed abnormalities [123I]MIBG might not be a sensitive biomarkers for the progression and phenoconversion in RBD patients.Cardiac [malities , 101, a malities . These fStructural and functional MRI techniques have demonstrated changes in deep grey matter, cortical grey matter, microstructural white matter and disrupted functional connectivity networks in patients with RBD which can be associated with clinical symptoms . IsolateDiffusion-weighted MRI has been employed to define longitudinal brain connectome progression scores, using interpretable machine learning algorithm, to evaluate the progression patterns in iRBD patients as a prodromal phase of PD •. The loAs a high-risk population for conversion to synucleinopathies, iRBD offers a valuable therapeutic window for application of early neuroprotective interventions and disease-modifying therapies. Recent longitudinal studies have highlighted a variety of potential biomarkers, including clinical, neurophysiological, genetic, CSF, serum, and neuroimaging, for monitoring disease progression and predicting iRBD conversion into synucleinopathies Fig. . However


Alternatively spliced domain of TnC, fibronectin type III-like repeat D (FnD) strongly affected physiological properties and morphology of both astrocytes and microglia in this injury model. The rate of microglial proliferation in the injury region decreased significantly with the addition of FnD. Additionally, density of microglia also decreased, in part due to reduced proliferation, and possibly due to reduced migration and increased contact inhibition between enlarged FnD-treated cells. Overall morphology of FnD-treated microglia resembled the activated pro-inflammatory cells, and elevated expression of iNOS was in accordance with this phenotype. The effect of FnD on astrocytes was different, as it did not affect their proliferation, but stimulated migration of reactivated astrocytes into the scratched area 48 h after the lesion. Elevated expression and secretion of TNF-α and IL-1β upon FnD treatment indicated the onset of inflammation. Furthermore, on Western blots we observed increased intensity of precursor bands of β1 integrin and appearance of monomeric bands of P2Y12R after FnD treatment which substantiates and clarifies its role in cellular shape and motility changes. Our results show versatile functions of TnC and in particular FnD after injury, mostly contributing to ongoing inflammation in the injury region. Based on our findings, FnD might be instrumental in limiting immune cell infiltration, and promoting astrocyte migration within the injury region, thus influencing spaciotemporal organization of the wound and surrounding area.Understanding processes that occur after injuries to the central nervous system is essential in order to gain insight into how the restoration of function can be improved. Extracellular glycoprotein tenascin-C (TnC) has numerous functions in wound healing process depending on the expression time, location, isoform and binding partners which makes it interesting to study in this context. We used an  Complete recovery from mechanical injuries of the central nervous system (CNS) is challenging goal in regenerative medicine research. Due to the death of neurons within the injury region and small capacity for regeneration of the damaged ones, the injuries always result in a partial or total loss of various sensorimotor functions . CNS injOne of the damage-associated molecules is the extracellular glycoprotein tenascin-C (TnC). This protein is present in the extracellular matrix of vertebrates during embryonic development . In adulTnC interacts with over 25 molecules that are classified into cell receptors, extracellular matrix components, pathogenic components and soluble factors and thus modulates the processes of cell migration, adhesion, proliferation, survival, neurite outgrowth, extracellular matrix reorganization, synthesis of proteases and cytokines . Some ofin vitro injury models lack some of the components present in vivo  mice and constitutively tenascin-C deficient (TnC−/−) mice were housed in the Animal Facility of the Faculty of Biology, University of Belgrade under standard conditions: 21 ± 1°C, 50% humidity, 12:12 h light/dark cycle, water and food ad libitum. TnC−/− mice were derived from the original colony  in total. Three cell cultures per genotype were prepared for each experiment. In these mixed cell cultures of astrocytes and microglia, on average, microglia accounted for 20% of total cells in wild-type (TnC+/+) cultures, and 16% in TnC−/− cultures.Primary astrocyte and microglia mixed cell cultures were prepared for each genotype from cortices of 0–3 days old mice of both sexes. Cortices were isolated in ice-cold phosphate buffered saline (PBS), and tissue was enzymatically dissociated with 1 mg/ml of freshy dissolved Trypsin in PBS for 10 min at 37°C. To inactivate trypsin, four times greater amount of growth medium  was added to the mixture, and cell suspension was centrifuged for 8 min at 500 × g. Cells were then resuspended in supplemented DMEM, passed successively 3–5 times through 21G (ø 0.8 mm) and 23G (ø 0.6 mm) needles and centrifuged again for 8 min at 500 × g. Resuspended cells were again passed through 23G needle and seeded as per experimental requirements: in 6-well plate for Western blot and rtPCR, and on PLO coated glass coverslips  for immunofluorescence labelling. Cultures were maintained in a humidified atmosphere of 5% CO, Scratch wound assay (SW) was performed in all experimental conditions (except in no SW) using sterile 200 μl pipette tip. This was followed by immediate or delayed addition of alternatively spliced domain FnD of protein TnC (10 μg/ml in growth medium). Recombinantly expressed fibronectin type III-like repeat D was generated as described by Dörries et al. ; 6 h SW and 30 h SW (RT PCR).• Control groups in which only SW was performed—24 h SW + FnD and 48 h SW + FnD ; 6 h SW + FnD and 30 h SW + FnD (RT PCR).• Treated groups with immediate addition of FnD upon SW—48 h SW: 24 h + FnD ; 30 h SW: 24 h + FnD (RT PCR).• Treated groups with delayed addition of FnD upon SW—24 h no SW and 48 h no SW ; 6 h no SW and 30 h no SW (RT PCR).• Additional groups without SW: Experimental conditions were as follows:For immunocytochemistry experiments cells were fixed 24 or 48 h after the SW and treatment in 4% formaldehyde solution (PFA), 20 min at RT. If the antibody for the target protein was against extracellular protein domain, the cultures were firstly blocked (5% BSA in PBS) for 1 h, at RT, then incubated with selected antibody of interest, followed by appropriate secondary fluorophore-tagged antibody. List of all used primary and secondary antibodies and their concentrations are given in 2). The same settings were applied for all images.Images were captured using confocal laser-scanning microscope , with Ar Multi-line , HeNe (543 and 643 nm). Objective Plan-Neofluar ×40/1.3 Oil  was used to capture high resolution images  in pre-defined positions.Cell density was calculated per cell type (microglia or astrocyte) as the number of cells  divided by gap or border area. Portion of proliferating cells was calculated per cell type as the number of proliferating nuclei (Ki67 positive) of given cell type divided by the total number of that cell type (OX42 or GFAP positive) in the gap or border area. Gap areas in SW model in cell cultures were easily distinguished from the rest of the confluent parts of the coverslip. At 40x magnification the whole or majority of the gap fitted in the frame, especially at 24 and even more at 48 h, as gaps gradually closed. Gap areas were imaged and processed separately from the border images. Gap area was regarded as the space between the leading edges of astrocytes. Border area was regarded as region of cells directly encompassing the gap area. Twelve frames of gap area as well as twelve frames of border area were imaged for every coverslip  in every genotype . Images were captured using confocal laser-scanning microscope as described in Image Acquisition section.For determining morphology of microglial cells in the gap area, micrographs of cells labelled with OX-42/CD11b, GFAP and Hoechst were used. Four randomly chosen cells per culture  were processed from gap area per treatment group , per genotype .1. Cell area–area of selection (µm2. Cell perimeter—the length of the selection outline (µm).2).3. Convex hull area (CHA)—area of the smallest convex polygon shape encompassing the whole selection (µm4. Convex hull perimeter (CHP)- the length of the outline of the convex hull (µm).5. Roughness—ratio of cell perimeter to convex hull perimeter.6. Fractal dimension (FD)– measure of cell complexity calculated using box counting method.7. Lacunarity—characterizes the distribution of gaps in the selection, calculated using box counting method.8. Aspect ratio (AR)—ratio of major axis/minor axis of selection.2. The circularity value of a circle is 1.9. Cell circularity—(4π × cell area)/(cell perimeter)10. Solidity, also called density—area of the cell divided by its convex hull area.2), or the inverse of the aspect ratio.11. Roundness—4 × area/ in SPSS was ran just to estimate the appropriate number of clusters for data sets including multiple parameters. Data sets were first standardized using Z-scores to obtain values on similar scale and Ward`s method has been used as clustering method. The number of stable clusters was used as an input for K-means cluster analysis that determined the statistical significance of parameter for clustering, assigned the cluster membership and provided distances from cluster centre for each input.Regarding the microglia and astrocyte proliferation and density, images were sorted into three clusters based on values for all four parameters per image. For microglial morphology cells were sorted into two clusters based on all eleven parameters for each cell. Cluster members with the smallest distance from the cluster centre i.e. the most representative images were shown.g and 4°C. Supernatants were collected and the protein concentration was determined using the BCA protein assay kit, according to the manufacturer’s instruction .The cultures were scraped in 150 μl of RIPA buffer, centrifuged for 10 min at 10,000 × The samples (10 μg of proteins) were mixed with the 6× Laemmli sample buffer  containing 5% of β-mercaptoethanol. Denatured proteins were resolved on 10% SDS-PAGE gels and electro-transferred to a PVDF support membrane . Membranes were blocked with 5% BSA in Tris buffer saline/Tween 20 (TBST) for 1 h, at RT. Membranes were then incubated with primary antibody overnight at +4°C, and appropriate secondary HRP-conjugated antibodies for 2 h, at RT . Each me2-fold change of mRNA expression. To determine specificity of the PCR reaction product we performed melting curve analysis. All reactions were performed in duplicates.TRIzol reagent  was used to collect the sample lysates. Further, the total RNA was extracted using phenol/chloroform procedure with ethanol precipitation. RNA concentrations were determined by measuring the absorbance at 260 nm and the purity was estimated from 260/280 nm and 260/230 nm ratios. Total RNA yield range was 0.6–1.0 ug/ml for all three cultures. Volume equivalent to 1 µg of RNA was used for reverse transcription to generate cDNA . The real-time PCR reaction mixture contained 2 μl cDNA , 5 μl QTM SYBR Green PCR Master Mix , 0.5 μl primers  overnight, at RT. The wells were then washed (0.05% Tween in PBS), and incubated with 300 μl of blocking solution (5% BSA in PBS) for 1 h, at RT. After the removal of blocking solutions, 100 μl of each standard dilution point of the recombinant IL-1β/TNF-α  were added alongside 100 μl of samples, and incubated for 2 h, at RT. Since samples were mixed cell culture media, the standard points were diluted in DMEM. After the incubation the wells were washed, and 100 μL of the detection biotinized antibody against IL-1β/TNF-α  was incubated for 2 h, at RT, and washed again. For detection of biotinized antibody 100 μl of avidin-HRP  was used in each well, for 20 min at RT, followed with thorough washing step. As the substrate 100 μl of 3,3′,5,5′-tetramethylbenzidine  was used for 20 min, at RT. The reaction was stopped with 100 μl of 2N Ht-test . Statistically significant interactions, simple main effects, and pairwise comparisons are given in Results section text and in respective tables as p-values, Bonferroni-adjusted within each simple main effect. Data from each experiment is graphically presented as a box and whisker plot, where pairwise comparisons are denoted with letters in such a way that sharing the same letter implies no significant difference between those groups or genotypes within the same group.SPSS 20 software package  was used for performing two-way ANOVA and Independent-samples +/+ than TnC−/− cultures  to understand the dynamics of subsequent events in mixed cultures and decided upon focusing on two time points (24 and 48 h). In the first 24 h only a very small number of cells could be observed in the gap area, and after 48 h the cell density in the gap was so high that individual cells could not be easily distinguished. Microglial cells were the first to enter the gap area, and astrocytes were initially confounded to the border region in control groups, 24 h SW and 48 h SW. Up to 48h, the density of both cell types increased in the gap area, however, significantly more microglial cells were located in the gap area . These rIn order to examine the effects of FnD treatment on microglia and astrocytes in mixed culture upon SW we decided to assess localization, densities and proliferation rates of the two cell types. Coverslips with mixed cell cultures were scratched in cross-like manner, and at 24 and 48 h, cells were fixed and stained with OX42 and GFAP to distinguish microglia and astrocytes respectively. Hoechst was used as nuclear marker and Ki67 as marker of proliferating cells. Region between the two leading edges of astrocytes was considered as gap area. Density and proliferation were analysed for microglia and astrocytes in both gap and border areas. Changes observed in the border area are given as −/− compared to TnC+/+ cultures in both control groups 24 h SW and 48 h SW (p < 0.001), and remained higher even with the addition of FnD within the first 24 h upon SW (p = 0.037). In TnC−/− the addition of FnD lowered density in the first 24 h (p < 0.001) compared to its 24 h SW control. The density of microglia was higher in 48 h SW TnC−/− group compared to 24 h SW (p = 0.001), and FnD treated groups 48 h SW + FnD, and 48 h SW: 24 h + FnD . In group 48 h SW + FnD density was higher than in 24 h SW + FnD (p < 0.001), whereas the delayed addition of FnD (48 h SW: 24 h + FnD), contained densities equal to both other FnD groups. In TnC+/+ density of microglia was the same within 24 and 48 h groups and all of 48 h groups had higher density than at 24 h (p < 0.001). Both the effects of treatment and genotype were significant (p < 0.001), as well as their interaction (p = 0.002). Changes in the border zone were less pronounced than in the gap area. In TnC−/− 24 h SW + FnD lowered density compared to other groups, except for 24 h delayed FnD as in gap area, and in TnC+/+ there were no changes in microglial density , except for 48 h SW: 24 h + FnD. In TnC−/−, 24 h SW + FnD had lower portion of proliferating microglia than 24 h SW control group (p = 0.017). 48 h SW group had statistically higher portion of proliferating microglia compared to 24 h SW and all 48 h FnD treated groups (p < 0.001). Delayed addition of FnD led to even more decreased proliferation rates than in 48 h SW + FnD (p = 0.001) but equal to 24 h SW + FnD. In TnC+/+ the addition of FnD also strongly diminished the portion of proliferating microglia compared to 24 h SW group (p < 0.001) and again 48 h SW group had statistically higher portion of proliferating microglia compared to 24 h SW (p = 0.021) and all 48 h FnD treated groups (p < 0.001). Both the effects of treatment and genotype were statistically significant (p < 0.001). Regarding the microglial proliferation in border region, in TnC−/− only the addition of delayed FnD lowered the proliferation at 48h, and in TnC+/+ no impact was observed (Percentage of proliferating microglia  was highobserved .−/− cultures within the first 24 h addition of FnD did not affect astrocyte density. 48 h SW group had higher densities than 24 h SW (p = 0.024), and even higher values were reached in 48 h SW + FnD and 48 h SW: 24 h + FnD . In TnC+/+ similarly, control and FnD-treated group had equal densities. Astrocyte density was higher, as expected, in 48 h SW group than in 24 h SW (p < 0.001). It was elevated even more in 48 h SW + FnD compared to 24 h SW + FnD, and 48 h SW , but it was similar to 48 h SW: 24 h + FnD. Additionally, FnD had stronger impact on density increase in TnC−/− compared to TnC+/+ cultures both in 24 h SW + FnD (p = 0.007) and 48 h SW: 24 h + FnD group (p = 0.032). Both the effects of treatment and genotype were statistically significant  as well as their interaction (p = 0.037) regarding the density of astrocytes in the gap area. In border area no changes in astrocyte density were observed, regardless of time or treatment . In TnC+/+ cultures, portion of proliferating astrocytes was equal within 24 and 48 h groups. 48 h SW had higher proliferation values than 24 h SW (p = 0.017), and 48 h SW + FnD than 24 h SW + FnD (p = 0.019). Only the effect of treatment was significant (p < 0.001). When it comes to the border region, FnD lowers proliferation rates in both genotypes at 48 h, except for 48 h SW: 24 h + FnD in TnC−/− . At 48 h in control groups cell areas were similar to those in 24 h SW group in both genotypes. In TnC−/− genotype both 48 h SW + FnD and 48 h SW: 24 h + FnD induced bigger cell areas than 48 h SW control group , and all FnD-treated groups were equal to each other. In TnC+/+ 48 h SW + FnD treatment induced bigger cell areas compared to 48 h SW and the other two FnD-treated groups 24 h SW + FnD and 48 h SW: 24 h + FnD . 48 h SW: 24 h + FnD had also bigger cell areas than 48 h SW (p = 0.003), and on average the same as 24 h SW + FnD. On overall only treatment was statistically significant (p < 0.001).Microglial cell areas  were on −/− cultures the addition of FnD did not significantly alter cell perimeter within the first 24h, but increased it compared to 48 h SW control in both 48 h SW + FnD and 48 h SW: 24 h + FnD groups . In all FnD-treated groups in TnC−/− genotype, cell perimeters were the same on average. As was the case with cell area, FnD promoted larger cell perimeters in TnC+/+ at 24 h compared to the 24 h SW (p = 0.023), and at 48 h both 48 h SW + FnD and 48 h SW: 24 h + FnD had bigger perimeters than 48 h SW (p < 0.001). Here also there were no differences amongst FnD-treated groups.Microglial perimeters  were sim+/+ cultures (p = 0.008). FnD had no additional impact in TnC−/− cultures. On the other hand, in TnC+/+ 24 h SW + FnD had higher roughness values than 24 h SW (p = 0.001). 24 h SW and 48 h SW did not differ, but 48 h SW + FnD and 48 h SW: 24 h + FnD had higher roughness values than 48 h SW .Lack of TnC stimulated higher roughness values of microglial cells  at 24 h α and IL-1β were examined at the level of mRNA expression and concentrations of these cytokines released into the medium . In both genotypes the greatest increase was reached in group 30 h SW: 24 h + FnD compared to 6 h SW + FnD and 30 h SW + FnD (p ≤ 0.001). 6 h SW + FnD had also significantly higher increase than 30 h SW + FnD in TnC+/+ and TnC−/− . On overall, both genotype and treatment were statistically significant  regarding the levels of Tnfα mRNA.In order to further investigate the reactivation profile of glial cells upon SW and FnD treatment, Tnf-e medium . In the Il1b were also higher only initially in TnC+/+ groups 6 h no SW and 6 h SW than in TnC−/− . Statistical analysis of Il1b mRNA levels showed that both genotype and treatment were significant .mRNA levels of p < 0.001,p ≤ 0.001) and were equal mutually.Concentrations of cytokines released in medium were assessed by ELISA. Regarding the concentration of Tnf-α, no differences were observed between the genotypes, and only treatment was statistically significant (+/+ between 48 h FnD-treated groups (48 h SW + FnD and 48 h SW: 24 h + FnD) and 48 h SW group since concentrations decreased in untreated and increased in treated groups . The same tendency was seen in TnC−/− cultures, however not statistically significant. On overall, only treatment was significant (p < 0.001).Within the first 24 h in all of the groups in both genotypes, similar concentrations of IL-1β were released into the medium . At 48 hiNOS mRNA expression levels . There were no differences among genotypes. Only treatment was statistically significant (p < 0.001). In group 48 h SW: 24 h + FnD in both genotypes, strong iNOS immunoreactivity was observed on some microglial cells mainly in the gap area labelling cell soma and processes as shown in Regarding the n levels , all no 1 mRNA log2 fold change . This elevation was also significantly higher than in the same group in TnC−/− genotype (p = 0.019). In TnC−/−, in 30 h SW + FnD group Arg1mRNA levels were upregulated also significantly compared to 6 h SW + FnD, 30h SW, 30 h SW: 24 h + FnD . Both genotype and treatment were significant .Relative Argd change  was on a+/+. In TnC+/+, 24 h SW + FnD had higher ratio than 24 h SW (p = 0.002). Both 48 h present D and delayed FnD increased ratio compared to 48 h SW . 48 h SW + FnD had also higher precursor values than 24 h SW + FnD (p < 0.001). In TnC−/− 24 h SW + FnD had higher ratio than 24 h SW (p = 0.005), and both 48 h present D and delayed FnD compared to 48 h SW . In 48 h SW + FnD, higher values were observed in TnC+/+ than in TnC−/− (p < 0.001). Both the genotype and treatment were statistically significant as well as their interaction . TnC, and in particular FnD did not induce any changes regarding β3 integrin in respect to the bands position or intensity (data not shown).β1 Integrin expression was analyzed using Western blot. Both the totally glycosylated mature and partially glycosylated precursor bands were observed at 130 and 115kDa, respectively . The opt+/+ cultures. Our results suggest that upon FnD treatment de novo monomeric P2Y12R bands appear whereas rearrangements in band representation occur in the range of dimer forms. We examined also expression of P2Y13R, but all bands appeared uniform regardless of presence of TnC or FnD (data not shown).The expression of protein P2Y12R was assessed with Western blotting technique. In all groups treated with FnD regardless of time point, the presence of bands at approximately 35 kDa was observed that corresponds to monomer of P2Y12R . AdditioOur results indicate that FnD influences the involvement of microglia and astrocytes in wound healing. FnD treatment decreased density and proliferation of microglia and stimulated migration of astrocytes into the injury. Microglial cells exhibited activated morphology and upregulated levels of proinflammatory markers. Production of proinflammatory cytokines increased in FnD-treated cultures and shifts were observed in the expression levels of isoforms of receptors involved in cell morphology and migration.+/+ cultures up to 24 h after SW. As upregulation of TnC expression is expected after injury, we considered the delayed addition of FnD to be more physiologically relevant approximation of the in vivo situation. This postponed addition induced potentiation of the observed effects in the absence of TnC, while it had no significant impact on wild-type cultures.Expression and secretion of TnC by cultured astrocytes was examined in several studies. Nishio et al., showed biphasic secretion of TnC into the medium in induced astrogliosis model in long-term primary cultures with the first higher peak at 4 h and the second lower at 48 h , and in Cell migration and proliferation are principal mechanisms through which tissue is repaired and repopulated upon injury . TnC hasin vitro even in the presence of mitogenic stimulators such as growth factors and tumor promoters, by inducing early inhibition of the intracellular alkalinization that occurs upon mitogenic stimulation  is one of hallmarks of pro-inflammatory microglial phenotype whereas arginase 1 (Arg1) is present more in pro-resolving phenotype . ActivatiNOS . Our exp−/− cultures, especially in the first 24 h indicating that the absence of tenascin C alone promotes enlarged cells with more processes. The usual role of microglia is the surveillance of the brain parenchyma through continuous processes movement (+/+ cultures. Thus, weather FnD leads to de novo synthesis of P2Y12R, or it has a role in its de-oligomerization resulting in the appearance of new monomeric band, it is clear that FnD affects the expression of P2Y12R isoforms. It has been shown that P2Y12R is essential in microglial sensing of ATP and ADP derived from neuronal activity (We have further demonstrated the development of inflammatory phenotype of microglia in the gap area through morphological changes that occur upon FnD treatment. The addition of FnD promoted formation of swollen-like thick processes and greater cell coverage by thin filopodia that increased significantly cell areas and perimeters and affected cell roughness index. These thin processes were shown to be actin-dependent filopodia responsible for faster cue sensing in the discrete regions . It shoumovement . In respmovement . P2Y12R movement . Nasu-Tamovement . P2Y12 rmovement . Functiomovement . In our activity . Microglactivity . Cserep activity . Taken tin vitro seemed to be regulated by hydrogen peroxide produced by NADPH oxidase upon stimulation with IL-1β and TNF-α, since it was completely abolished with the addition of NADPH oxidase inhibitors while this did not affect astrocyte proliferation (2+ signalling dynamics (FnD treatment exhibited spatiotemporally distinct effects on microglia and astrocytes in the lesion in our current study. In injury microglial cells enter the wound area prior to astrocytes and this was observed in our control groups. The absence of TnC even further potentiated this effect as both density and proliferation of microglia were elevated, whereas the addition of FnD strongly diminished both the density and proliferation of microglia while promoting their activated phenotype. On the other hand, FnD induced migration of the astrocytes into the gap region, but did not affect their proliferation rates. Mander et al. observed that proliferation of rat microglia feration . Additioferation . Even thdynamics . Astrocydynamics .in vivo FnD produced by proteolytic cleavage by MMPs would be confined to specific regions mainly in the injury border zone where astrocytes are located. It is therefore plausible that in the border zone FnD inhibits excessive proliferation of microglia and promotes motile phenotype of astrocytes to functionally separate the injury region from the surrounding tissue. These findings could be even more complex if examined in multicellular models involving other cell types. Our results are offering a new view of the importance of TnC`s multiple roles and, further experiments involving 3D and organotypic culture models as well as in vivo experiments are needed to position this effect precisely in the sequence of events after injury.Finally, the time and place of TnC secretion are vital to its roles. It was shown in epithelial wound that fibrin-fibronectin provisional matrix is first deposited upon injury where fibroblasts are anchored via formation of actin stress fibers and as injury is being repaired, the matrix is being contracted. TnC is however transiently upregulated in boundary regions where it inhibits formation of actin stress fibres and focal adhesion through inhibition of FAK and RhoA activity, thus promoting motile phenotype and preventing premature matrix contraction . In our


No difference in parietal alpha-1 and alpha-2 power was found between groups  = 0.297, p = 0.591, η2p = 0.013; alpha-2: F = 0.118, p = 0.734, η2p = 0.005). No limb differences were presented for any frequency band in selected cortical areas  = 0.149, p = 0.703, η2p = 0.007; alpha-2: F = 0.166, p = 0.688, η2p = 0.007; theta: F = 2.256, p = 0.147, η2p = 0.093). Conclusions: Theta power at the frontal cortex was higher in soccer athletes with CAI during drop-jump landing. Differences in cortical activation provided evidence for an altered neural mechanism of postural control among soccer athletes with CAI.Background: Chronic ankle instability (CAI) is a common peripheral joint injury and there is still no consensus on the mechanisms. It is necessary to investigate electrocortical parameters to provide clinical insight into the functional alterations of brain activity after an ankle sprain, which would greatly affect the implementation of rehabilitation plans. The purpose of this study was to assess cortical activation characteristics during drop-jump landing among soccer athletes with CAI. Methods: A total of 24 participants performed the drop-jump landing task on a force platform while wearing a 64-channel EEG system. The differences of power spectral density (PSD) in theta and alpha  bands were analyzed between two groups (CAI vs. CON) and between two limbs . Results: CAI participants demonstrated significantly higher theta power at the frontal electrode than that in healthy control individuals (F Ankle sprain, the most common lower extremity injury, accounting for 9.4% to 18% of all sports injuries ,3, oftenDespite the theories regarding the development of CAI evolving over decades, there is still no consensus on the mechanisms of CAI. Most traditional models believe that CAI is a musculoskeletal disorder with adaptations typically occurring in the periphery rather than in the brain . HoweverResearch at the reflexive and cortical levels of CNS adaptations in individuals with ligament injury has grown rapidly ,30,31. HThis report is a part of larger explorative investigation of cortical activity among soccer athletes with CAI. In order to examine characteristics in central activation patterns in the CAI population, the current study carried out a groups (CAI vs. CON) × limbs  design. All tests were performed on both injured  and healthy  side and comparisons were made between groups and limbs. Sample size was calculated using G*Power software (v.3.1) with 80% power, 5% type I error, and 55% effect size, and a minimum of 10 participants in each group were recommended .p = 0.000)  . InclusiA single investigator (Y.Z.) performed all the screening procedures for enrolling participants. All participants had normal vision at the time of the experiment and were instructed to refrain from consuming caffeine within 12 h before the EEG test started. Before experiment, the subjects were informed in detail about the procedure and provided written informed consent. This study was approved by the Sports Science Experiment Ethics Committee of Beijing Sport University (2021195H) and registered in Chinese Clinical Trial Registry (ChiCTR2200055738).The test was conducted at the Sport Rehabilitation of Beijing Sport University. Given that this study was part of a larger investigation, the task was evaluated while the athletes were on the force platform . EEG data were collected using 64 Ag/AgCl scalp electrodes  placed in accordance with the international 10/20 system. Cortical activity was recorded continuously via 16 EEG channels , and each electrode impedance was kept lower than 10 KΩ ,38 throuCortical activity was measured while subjects performed the forward drop-jump landing task on the force platform . To reduEEG data were preprocessed using MNE-Python, an open-source software package. Continuous EEG data were band-pass filtered between 3 and 30 Hz (4th order Butterworth FIR) with additional notch filters to reduce line noise (50 Hz). Independent components analysis (ICA) was used to eliminate artifacts and epochs with an absolute difference larger than 150 μV were also excluded as artifacts. The topographic map of ICA components is shown in 2p) were reported to show the effect sizes. Effect sizes of <0.01 were interpreted as small, 0.01 to 0.06 as medium, and ≥0.14 as large effect [p < 0.05.The data were analyzed using SPSS software version 24.0 . Shapiro–Wilk (S-W) test and Levene test were used to determine whether variables fitted normal distribution and variance homogeneity, respectively. The EEG outcome measures were transformed to normal distribution by using natural logarithmic based transformation, if needed. Continuous variables are reported as mean ± standard deviation. Demographics data between two groups were compared using independent-samples t test. To examine cortical activity a mixed-design ANOVA with 2 groups (CAI vs. CON) as between-subject factor, 2 limbs  as within-subject factor was conducted. Partial eta squares  = 7.726, p = 0.011, η2p = 0.260)  = 0.297, p = 0.591, η2p = 0.013; alpha-2: F = 0.118, p = 0.734, η2p = 0.005)  = 0.149, p = 0.703, η2p = 0.007; alpha-2: F = 0.166, p = 0.688, η2p = 0.007; theta: F = 2.256, p = 0.147, η2p = 0.093)  theta power in the CAI group vs. the healthy control group (F= 0.260) , indicat= 0.005) . In addi= 0.093) .To investigate the activation differences on the cerebral cortex in people with chronic ankle instability, we measured and compared the EEG power at different frequencies during injury-related drop-jump landing movement in soccer athletes with and without CAI. Partially consistent with our research hypothesis, the theta band power was increased in soccer athletes with CAI during drop-jump landing, but there were no differences in alpha-1 and alpha-2 activity between groups or between limbs. The main findings in our study were that theta band power in the frontal area (Fz) showed significant difference between groups (CAI vs. CON), but we did not find any limb  differences in the CAI group during drop-jump landing. Theta rhythm is a 4–8 Hz oscillation in the local field potential and has been linked to the encoding of new information into episodic memory and spatial navigation, which was first characterized in rodents in the 1930s . Since tWe did not find any group (CAI vs. CON) or limb  differences in parietal (Pz) alpha  power during drop-jump landing among soccer athletes. Alpha is one of the fundamental functional operators of the brain for signal processing and communication in sensory or cognitive processes in the brain . In addiThere could be several reasons why our results did not match the expectations. At first, there may be a ceiling effect in our study subjects. The ability to focus on sensory information related to performance is one of the key elements for elite athletes . In addiIt is also worth noting that we did not find any differences in alpha  and theta powers between the involved and uninvolved side in CAI patients, which means that we could observe EEG changes during the drop-jump landing task in CAI patients even on their uninvolved side. The result is in line with earlier findings reported by other authors. In an investigation to evaluate corticomotor excitability in individuals with CAI, Pietrosimone  and McLeThere are also some limitations to our study. Firstly, the retrospective study design was unable to determine if the changes in power spectrum occur after unilateral CAI or if the altered cortical activation is a predisposing factor that may lead to CAI. That is, we cannot establish a causal relationship between CAI and cortical changes. Therefore, prospective investigations are needed to verify our speculation. Secondly, although our study investigated the cortical alterations in more sport-relevant and injury-relevant patterns compared with studies assessing in sitting or lying position, we still chose the anticipated task mainly requiring feedback control. Considering athletes are required to quickly adapt to changing environments and cannot just rely on anticipated movements, future studies should include unanticipated jump-landing tasks in order to elucidate its value in predicting injury and monitoring recovery. Thirdly, due to the COVID-19 prevention and control requirements of Beijing Sport University and considering that this experiment is a preliminary study, we failed to recruit enough healthy control subjects, which may have masked cortical differences that could be identified with larger sample sizes. Therefore, we suggest that at least 16 participants be included in each group in future study . Finally, only three trials per participant were required for analysis, and it was difficult to obtain stable EEG signals. Thus, researchers should reconsider the number of tests based on different research objectives. Theta power at the frontal cortex was higher in soccer athletes with CAI than in healthy controls during drop-jump landing. However, no differences existed in parietal alpha-1 and alpha-2 power between groups. We did not find differences in alpha and theta power between the injured and healthy side in soccer athletes with CAI.


In particular, Ni2+‐guided anisotropic growth is observed to generate the nested skeleton structure in PtCu NSCs. Ni2+, though absent from the nanoalloys, not only endows faster Cu reduction kinetics but also acts as a structure‐directing agent. Moreover, it is shown that acetic acid treatment of PtCu NSCs/C exposes Pt‐rich surface with a fine‐tuned Pt d‐band center energy and the reduced Cu leaching, resulting in strikingly high activity and stability. Acid‐treated PtCu NSCs/C shows a remarkable ORR mass activity of 5.13 A mgPt–1, about 26 times higher than commercial Pt/C catalyst. This catalyst also exhibits excellent stability with a lower activity decay of 11.5% and the negligible variation in structure after 10 000 cycles.Structure‐controlled Pt‐based nanocrystals have the great potential to provide a flexible strategy for improving the catalysis of the oxygen reduction reaction (ORR). Here, a new synthetic approach is developed to tune the 3D structure of Pt‐based alloys, and switch a synthetic reaction which produces solid PtCu octahedral stars (OSs) to produce PtCu nested skeleton cubes (NSCs) by simple addition of Ni(acac) 2. An enhanced oxygen reduction reaction (ORR) activity is obtained on PtCu NSC catalysts, which is higher than that of the solid PtCu OS catalysts.An effective strategy is proposed to tune the 3D structure of Pt‐based alloys, and switches a synthetic reaction which produces solid PtCu octahedral stars (OSs) to produce PtCu nested skeleton cubes (NSCs) by simple addition of Ni(acac) The structure and composition of such Pt‐alloys play a very important role in determining their catalytic properties and there has been considerable progress in the tuneability of 3D structure and composition of Pt‐alloys, including the production of solid, hollow, and skeleton structures. Skeleton structures have attracted particular interest for their highly open 3D structures. The accessibility of 3D alloy surfaces in skeleton structures to reactant O2 molecules has been documented to increase the specific activity for ORR. In addition, these nanoscale skeletons are made up of interconnected ultrafine 1D nanostructures, which greatly increases the electrochemical active surface area (ECSA) of the catalyst. Studies on alloy catalysts have also shown that ORR‐favorable activity and stability could be improved by a Pt‐rich surface. For example, in the work on bunched PtNi alloy nanocages by Baoyu Xia et al. the stable catalyst modified to give a Pt‐rich surface showed an ORR mass activity of up to 17 times higher as compared to Pt/C. All of these effects have been explored to create substantial improvement in the mass activity of Pt in ORR. However, the nanoframe field has mostly been focused on the synthesis and formation mechanism of single‐layered Pt‐alloy skeletons. In this work, we show a new method to tune the 3D structure of Pt‐based alloys in synthesis and generate PtCu nested skeleton cubes (NSCs) by simple addition of Ni(acac)2. An enhanced ORR activity was obtained on PtCu NSC catalysts, which was far better than that of the solid PtCu octahedral star‐shaped catalysts generated by the synthesis without Ni(acac)2 and commercially available Pt/C catalyst. Moreover, surface treatment of PtCu NSCs/C catalyst by acetic acid exposed a Pt‐rich surface, leading to a 25.7‐fold enhancement in mass activity compared to Pt/C catalyst. In addition, this catalyst presented a dramatic increase in stability in ORR even after 10 000 reaction cycles compared to commercially available Pt/C and other PtCu alloy catalysts.The high cost and limited availability of commercial Pt catalyst for the cathodic oxygen reduction reaction (ORR) is a bottleneck for the production and use of various types of fuel cells.1 The 22.12, Pt(acac)2, and cetyltrimethyl ammonium bromide (CTAB) molecules at 175 ℃ for 8 h with and without the presence of Ni(acac)2. When the reaction was performed without Ni(acac)2, transmission electron microscope (TEM) imaging showed nanostructures resembling six‐pointed stars of roughly uniform size  solution involving Cu(acac)e Figure 1a. In thr Figure . Upon clFigure 50Cu50 alloys (PDF#48‐1549). The composition of the nanocrystal was probed using energy dispersive X‐ray spectroscopy (EDS) analysis: the overall atomic ratio of Cu/Pt in PtCu A‐OSs/C is measured to be 48.7/51.3 , the nanocrystals were treated with acetic acid to remove surface species including organics and oxides. High resolution TEM (HRTEM) image of a single acid‐treated OS shows a solid star‐shaped structure similar to the untreated nanocrystal Figure 2a. Local3 Figure –j show t3 Figure . The mea3 Figure  and the 3 Figure  of the s . This suggests that although both OSs and NSCs resemble fcc PtCu alloys, the {111} and {100} facets are present in both the spikes and the ridges of the PtCu NSCs in contrast to the {220} facets seen in the PtCu OSs.Nested skeleton cube nanocrystals were also analyzed in the same way: HRTEM image of an acid‐treated skeleton structure reveals a smaller skeleton cube nested entirely within a larger skeleton cube Figure 3a. The e although the metallic form is most common, confirming their presence as an PtCu alloy. On the other hand, Ni can be seen from the Ni2p XPS spectra to be present in Ni2+ form  compared to Cu2+/Cu (0.34 V) led to a faster reduction rate for Pt, forming Pt54.3Cu45.7 alloys which are enriched for Pt. Such thermodynamic arguments have been proposed for the formation of other stable alloy phases, with H. Peng et al. showing that about 7 nm PtCu nanocrystals have a stable composition of 58% Pt and 42% Cu. Initially, energy minimization caused these nanocrystals to form as Wulff polyhedron structures  than that of Cu2+/Cu (0.34 V), kinetic selectivity led to reduction of Cu2+ dominating in the initial nucleation step. Therefore, nanocrystals formed were Cu‐rich Wulff polyhedrons  have been shown to guide the formation of Pt‐rich nanorods, in our case, which can direct the deposition the resulting Pt atoms on the edges of cubes  to form Pt‐rich rods  in 0.1 m HClO4 solution. We also evaluated the ECSAs of these catalysts using CO‐stripping experiments  is much higher than PtCu NSCs/C (52.2 m2 gPt–1). It is also higher than PtCu A‐OSs/C (47.6 m2 gPt–1), which in turn has a higher ECSACO than PtCu OSs/C (35.3 m2 gPt–1). Similarly, we also measured the ECSAHupd  compared to the Pt/C catalyst , 25.7 times higher than Pt/C (0.2 A mgPt–1). PtCu A‐NSCs/C also has 1.3 times greater mass activity than nonacid‐treated PtCu NSCs/C. On the other hand, both acid‐treated and non‐acid‐treated OSs have a substantially lower than PtCu A‐NSCs/C by 5.0 (1.02 A mgPt–1) and 8.7 (0.59 A mgPt–1) times, respectively. Importantly, PtCu NSC catalysts show much higher specific activity than solid PtCu OS catalysts and Pt/C , about 27 times higher than Pt/C (0.27 mA cm–2). Furthermore, we compared the ORR activity of the nested skeleton structures we prepare and the single‐layered Pt‐alloyed skeletons reported recently. Despite their superficially similar hollow structures, our PtCu skeleton cubes with the nested structure show higher mass activity and specific activity  has an intermediate potential between PtCu NSCs/C (0.809 V) and Pt/C (0.876 V), confirming PtCu A‐NSCs has an intermediate Pt–CO binding energy between PtCu NSCs/C and Pt/C 2, solid PtCu OSs were obtained but when Ni(acac)2 was added the product switched to PtCu NSCs. Our studies on growth mechanism suggested that Ni2+ acted as a catalyst, accelerating the reduction of the Cu precursor, and acted as a structure‐directing agent as well, boosting the anisotropic growth of PtCu skeleton cube structures by acting in concert with the thermodynamic effects of surface energy minimization. This anisotropic growth mechanism allowed the formation of nested layers of PtCu skeletons, a novel structure in PtCu alloy nanocrystals. This novel nested structure greatly enhanced ORR activity, far higher than that of solid PtCu OS catalysts and commercially available Pt/C. We revealed that enhanced ORR activity varied depending on the 3D structure of alloy catalysts. The acetic acid treatment could expose more active sites, and thus optimizing the binding strength of the oxygenated species. Moreover, we proposed that the surface protrusions and vertex spikes formed in our nanocrystals can counteract the structural collapse and the agglomeration of catalysts under ORR stability tests and be observed to maintain their 3D structures postexperiment. Finally, our observations suggest that even higher‐order multilayered nesting structures are possible with further optimization of synthesis protocols and this work may offer a starting point for the rational design and synthesis of multilayered alloy skeletons for other electrocatalytic applications.We have developed a one‐pot wet chemical method which can be switched between the production of PtCu nanocrystals of two drastically different 3D structures (solid OSs and NSCs) by the simple addition of Ni(acac)42, Pt 48 wt%), copper acetylacetonate (Cu(acac)2, 97%), nickel acetylacetonate (Ni(acac)2, 95%), cetyltrimethyl ammonium bromide , and Nafion (5 wt%) were purchased from Alfa Aesar. Oleylamine  was purchased from Aladdin. Hexane, ethanol, and Perchloric acid  were purchased from Sinopharm Chemical Reagent Co. Ltd. China. These materials purchased were not purified in this experiments. Ultrapure water (18.2 MΩ cm–1) was used in this experiments.Platinum acetylacetonate (Pt(acac)2, 34 mg Cu(acac)2, 8–14 mg Ni(acac)2, and 100 mg CTAB were added to 7 mL OAm solution in a two‐neck flask and heated to 175 ℃ for 8 h under magnetic stirring in an oil bath after ultrasonic treatment. After the end of the reaction, the product was allowed to cool to room temperature and a mixed solution of hexane and ethanol was used to wash the product obtained. This process was repeated five times with the product being separated by centrifugation. Finally, the black sample was re‐dispersed in hexane.17 mg Pt(acac)2 to OAm solution.The same method was used to prepare solid PtCu OSs, except without the introduction of Ni(acac)PtCu OSs/NSCs were first supported on carbon black (PtCu OSs/C and PtCu NSCs/C), followed by dispersion in an acetic acid solution. This mix solution was heated to 70 ℃ for 10 h under magnetic stirring to obtain final catalysts (PtCu A‐OSs/C and PtCu A‐NSCs/C).α source was used to characterize the crystal pattern of samples. The elemental XPS spectrum of samples was collected on a Thermo Scientific K‐Alpha instrument equipped with an Al Kα source. The ICP‐OES (Agilent 5110) instrument was used to quantify elemental composition in samples.A FEI Tecnai F20 instrument and a Titan Themis G3 ETEM instrument equipped with a spherical‐aberration corrector were used to collect the transmission electron microscope (TEM) images of samples loaded on Mo grids. The spherical‐aberration corrected scanning transmission electron microscope (STEM) images and the energy dispersive X‐ray spectroscopy (EDS) mappings/line‐scans of samples were obtained on a JEM ARM 200F instrument at 200 kV. The XRD instrument with a Cu‐K–2 for PtCu alloy nanoskeleton catalysts, 6.8 µg cm–2 for solid PtCu alloy catalysts, and 10.2 µg cm–2 for commercial Pt/C catalyst). Saturated Ag/AgCl and Pt wire electrodes were acted as the reference and counter electrodes, respectively. Cyclic voltammetry (CV) curves were measured in N2‐purged 0.1 m HClO4 solution at a scan rate of 100 mV s–1. The polarization curves were measured in O2‐purged 0.1 m HClO4 solution at a scan rate of 10 mV s–1 at 1600 rmp. The kinetic current was calculated at 0.9 V versus RHE (reversible hydrogen electrode) according to K–L equation. The ORR stability tests were conducted in O2‐purged 0.1 m HClO4 solution at a scan rate of 100 mV s–1. The CO‐stripping CV curves were measured in N2‐purged 0.1 m HClO4 solution at a scan rate of 50 mV s–1 after CO molecules adsorbed on catalysts. All electrochemical tests were carried out at room temperature.All catalysts were first loaded on glassy carbon dish electrodes with a diameter of 5 mm (Pt loading content: 2.6 µg cmequation.40 TheThe authors declare no conflict of interest.Supporting InformationClick here for additional data file.