Agrobacterium infiltration-mediated transient expression assay indicated that TbCSV V2 can suppress local and systemic RNA silencing and the deletion analyses demonstrated that the amino acid region 1–92 of V2, including the five predicted α-helices, are required for local RNA silencing suppression. Site-directed substitutions showed that the conserved basic and ring-structured amino acids in TbCSV V2 are critical for its suppressor activity. Potato virus X-mediated heteroexpression of TbCSV V2 in Nicotiana benthamiana induced hypersensitive response-like (HR-like) cell death and systemic necrosis in a manner independent of V2′s suppressor activity. Furthermore, TbCSV infectious clone mutant with untranslated V2 protein (TbCSV∆V2) could not induce visual symptoms, and coinfection with betasatellite (TbCSB) could obviously elevate the viral accumulation and symptom development. Interestingly, symptom recovery occurred at 15 days postinoculation (dpi) and onward in TbCSV∆V2/TbCSB-inoculated plants. The presented work contributes to understanding the RNA silencing suppression activity of TbCSV V2 and extends our knowledge of the multifunctional role of begomovirus-encoded V2 proteins during viral infections.V2 proteins encoded by some whitefly-transmitted geminiviruses were reported to be functionally important proteins. However, the functions of the V2 protein of tobacco curly shoot virus (TbCSV), a monopartite begomovirus that causes leaf curl disease on tomato and tobacco in China, remains to be characterized. In our report, an  RNA silencing is a highly conserved mechanism that plays important roles in endogenous gene regulation and primary antiviral response in eukaryotes ,2. To coAn increasing number of reports have revealed that special sequence motifs or amino acids of some VSRs play crucial roles in active RNA silencing suppression. The suppressors p1 of sweet potato mild mottle virus (SPMMV) and p0 of potato leafroll virus (PLRV) contain GW/WG motifs that were reported to be crucial for AGO1 targeting and ultimately RNA silencing suppression ,17. P21 Begomovirus is the largest genus, with more than 400 members [Plant-infecting geminiviruses with circular single-stranded DNA (ssDNA) genome are important causal agents of various viral diseases worldwide, which lead to extensive agriculture production losses, especially in tropical and subtropical regions . Geminiv members . Partial members ,23. Six  members ,25. Incr members ,28,29,30 members ,32,33. To our knowledge, independent studies have identified that V2, C1, C2, C4/AC4 and βC1 encoded by different geminiviruses can suppress PTGS and/or TGS ,38,39,40N. benthamiana, and this function is independent of the VSR activity of V2. The TbCSV mutant with loss-of-function of V2 (TbCSV∆V2) failed to induce any visible symptom throughout the infection stage, and the accumulation of mutant virus was decreased significantly. Coinfection of TbCSV∆V2 with betasatellite (TbCSB) could obviously elevate the viral accumulation and induce symptom at the early infection stage, whereas the symptom recovery on the systemically infected leaves occurred over time.In this study, we showed that TbCSV V2 can suppress positive-sense GFP RNA-induced local and systemic RNA silencing, and the conserved amino acid region 1–92, which contains five predicted α-helices, is required for VSR activity of V2. Site-directed mutagenesis showed that basic and ring-structured amino acids located in the conserved region (aa 1–92) of V2 are crucial for RNA silencing suppression. Notably, R17 and R43, which may be involved in RNA binding and V2′s self-interaction, respectively, are crucial for suppressor activity of V2. We also found that PVX-mediated expression of TbCSV V2 can induce HR and systemic necrosis in Agrobacterium co-infiltration assay with pGD-GFP, as described previously, to test its suppression activity [Nicotiana benthamiana leaf patches agroinfiltrated with pGD-V2/pGD-GFP showed obvious GFP fluorescence at 3 days post-agroinfiltration (dpa), with less intensity of the fluorescence than that emanated from the leaf patches co-infiltrated with p19 encoded by tomato bushy stunt virus (TBSV) and GFP (pGD-p19/pGD-GFP), the positive control  showed that a “Genimi_V2”  domain and a “WCCH” (aa 79–102) motif are contained in V2 (115 amino acids). Secondary structure analysis (http://bioinf.cs.ucl.ac.uk/psipred/) predicted that V2 possesses six α-helices  (N. benthamiana leaf patches coinfiltrated with V2 (1–92), V2 (1–100) or V2 (1–110) and pGD-GFP showed GFP fluorescence, suggesting the retainment of RNA silencing suppression activity  . Based oactivity A. Howeveactivity A. In wesactivity B. Furtheactivity . InteresOur present work suggested that the α-helices and conserved residues should contribute to the VSR activity of V2 . InteresRing-structured amino acids were reported to affect the VSR activity of melon aphid-borne yellows virus (MABYV) p0 and GLRDV-2 p24 proteins with unknown precise mechanism ,54. We gNorthern blot analysis was performed to determine the GFP mRNA accumulations in leaf patches agroinfiltrated with V2 mutants . Our results showed that obviously decreased GFP mRNA levels were detected in leaf patches co-infiltrated with GFP and R17A, R43A, R91A, F9A, Y24A or pGD empty vector. In contrast, for the K95A mutant, which retains the RNA silencing suppression activity, the GFP mRNA level was comparable with that in the wt V2-infiltrated plant C. Seven V2 mutants were subjected to Western blot analyses for assessing the protein accumulations. First, wt V2 and V2 mutants were tagged with a 3xFlag epitope at their N-termini, and the effects of the tag on RNA silencing suppression activity of V2 were investigated. Agroinfiltration assay indicated that 3xFlag-V2 can still suppress positive-sense GFP RNA triggered gene silencing, although with decreased activity . SubsequSimilarly, we created mutations targeting basic or ring-structured amino acids by substituting with isomorphic amino acids. Three basic amino acids  and three amino acids with a ring-structured side chain  located in conserved motifs were selected for further analyses. We substituted the R17, R43 and R91 with lysine (K), another positively charged residue, respectively. Similarly, we generated substitutions targeting F9, Y24 and Y61 with tyrosine (Y), phenylalanine (F) and F, respectively. Agroinfiltration assays showed that leaves co-infiltrated with GFP and R17K displayed weaker GFP fluorescence than that of leaves co-infiltrated with pGD-GFP/pGD-V2. However, fluorescence in leaves co-infiltrated with R43K, R91K, F9Y, Y24F or Y61F and GFP was not easily detectable A. WesterN-V2 and YFPC-V2) or V2 mutants (YFPC-V2(R17A), YFPC-V2(R43A), YFPC-V2(R91A), YFPC-V2(F9A), YFPC-V2(Y24A), YFPC-V2(Y61A) and YFPC-V2(K105A)) for bimolecular fluorescence complementation (BiFC) analyses. GFP fluorescence could be observed in YFPN-V2/YFPC-V2 co-expressed N. benthamiana cells, suggesting the self-interaction of TbCSV V2 in vivo , YFPN-V2/YFPC-V2 (R91A), YFPN-V2/YFPC-V2 (F9A), YFPN-V2/YFPC-V2(Y24A), YFPN-V2/YFPC-V2(Y61A) and YFPN-V2/YFPC-V2(K105A) could be observed, just as that shown in YFPN-V2/YFPC-V2 co-expressed cells. However, co-infiltration with YFPN-V2/YFPC-V2 (R43A) led to an obviously weaker YFP signal, suggesting that the arginine (R) at the position 43 is critical for the self-interaction of V2 in plants  (data not shown). At 9 dpi, the cell death phenotype could be obviously observed on the whole infiltrated leaves, and subsequently, the cell death extended to upper systemically infected leaves until systemic necrosis occurred - and PVX-V2(K95A)-infected plants, whereas only mild local necrosis spots emerged on PVX-V2(K105A)-inoculated plants. Furthermore, necrosis on some of the petioles and leaves developed for PVX-V2-, PVX-V2(F9A)- and PVX-V2(K95A)-infected plants at 13 dpi. In contrast, there were still only denser local necrosis spots and chlorosis on the upper leaves of PVX-V2(K105A)-infected plants  or p19 encoded by tomato bushy stunt virus (TBSV), can induce hypersensitive response-like necrosis of a leaves . As a VSd plants . These rd plants . N. benthamiana plants, and this function of V2 is independent of its suppressor activity as well as the increased accumulation of PVX.Taken together, PVX-mediated heterogenous expression of TbCSV V2 can aggravate the symptoms, ultimately leading to systemic necrosis of infected ∆V2) was constructed by changing the start codon of ATG to ATC. Subsequently, TbCSV, TbCSV∆V2, TbCSV associated with DNAβ satellite (TbCSV/TbCSB) and TbCSV∆V2/TbCSB were inoculated into wild-type N. benthamiana plants using the method previously reported, and inoculation with buffer (mock) served as a control [∆V2 failed to induce visible symptoms for all the inoculated plants until 20 dpi, as that shown on mock-inoculated control plants  and several others [N. benthamiana [N. benthamiana  in l others ,46,50,51thamiana . In thisthamiana . Howeverthamiana . Thus, t∆V2) alone could not induce any symptom on N. benthamiana, whereas coinfection with its satellite TbCSB could significantly increase the virus accumulation and induce typical downward curling symptom on upper new leaves at an early stage. However, the symptom was gradually recovered over time, the leaf curling symptom disappeared and only mild leaf abnormality could be observed at 20 dpi  [∆L2-infected Arabidopsis, and DNA methylation-mediated TGS plays an important role in the recovery phenotype [In our report, infection with V2-mutated TbCSV (TbCSVt 20 dpi . In contms (SAM) ,70. Durims (SAM) ,71. VSRsms (SAM) ,72,73,74ms (SAM) . Furtherhenotype ,77,78. Thenotype ,32. HoweN. benthamiana plants were grown in a growth room at 24 °C under a 16 h/8 h (light/dark) photoperiod cycle.Wild-type and GFP-transgenic16c line To identify the RNA silencing suppression activity of TbCSV V2 by agroinfiltration assay, the coding sequences of V2 were amplified from TbCSV-Y35 (GenBank No. AJ420318) isolate infectious clone and inserted into the BglII-HindIII site of binary expression vector pGD to produce pGD-V2 recombinant construct. The truncated derivatives of V2, including V2 (1–90) (contains amino acids 1–90), V2 (1–92), V2 (1–100), V2 (1–110), V2 (6–115) and V2 (16–115), were amplified using pGD-V2 as template and inserted into the BglII-HindIII site of the pGD vector, respectively. To produce V2 substitution mutants, the V2 coding sequence was inserted into pMD19-Tsimple vector and used as a template to amplify the mutated V2 sequence by reverse PCR as described previously [To test the pathogenicity of TbCSV V2 or V2 mutants, the coding sequences were amplified from pGD-V2, pGD-V2-F9A, pGD-V2-K95A and pGD-V2-K105A, respectively, using a primer pair (PVX-V2-F/PVX-V2-R) and subcloned into the ClaI-SalI site of pGR106 . To examine the self-interaction of V2, the coding sequences were amplified from pGD-V2 using primer pairs YFPN-V2-F/YFPN-V2-R and YFPC-V2-F/YFPC-V2-R, and subcloned into N-terminus of YFP and C-terminus of YFP respectively. To examine the self-interaction of V2 mutants, the coding sequences of V2 mutants were amplified from pGD-V2-R17A, pGD-V2-R43A, pGD-V2-R91A, pGD-V2-F9A, pGD-V2-Y24A, pGD-V2-Y61A and pGD-V2-K105A using primer pair YFPC-V2-F/YFPC-V2-R and cloned into C-terminus of YFP, respectively. To obtain TbCSV V2 mutant infections clone in which potential start codons of V2 were substituted, a construct including tandem 1 and 0.9 copy of TbCSV-Y35 clone was used as the template for PCR. First, a fragment with mutations of the second ATG codon of the first 1 copy and the first ATG codon of the second 0.9 copy was amplified using ΔV2-F1/ΔV2-R1 primer pair. The product was extended using ΔV2-F2/ΔV2-R1 and ΔV2-F3/ΔV2-R1 primer pairs in turn, to produce a fragment designated as P1 with an extension at 5′ terminus overlapping with pBinPLUS vector. Primer pair ΔV2-F1/ΔV2-R2 was used to amplify a fragment with mutation to another potential ATG codon of the second 0.9 copy. Subsequently, the products were extended by PCR, using ΔV2-F2/ΔV2-R2 and ΔV2-F3/ΔV2-R2 primer pairs in turn to produce P2 fragment overlapping with P1 at 5′ terminus and with pBinPLUS vector at 3′ terminus. Eventually, P1 and P2 were inserted into pBinPLUS vector that digested with BamHI-HindIII enzymes, by the homologues recombination method, to produce the TbCSVΔV2 construct for further use.Agrobacterium tumefaciens GV3101, and subsequent infiltration and fluorescence detecting were performed as described previously [For local and systemic RNA silencing suppression activity identification, each plasmid that needed to be tested was transformed into eviously ,79.N. benthamiana leaves and Western blot assay were performed as described previously [N. benthamiana leaf patches using 2xSDS sample buffer and boiled at 100 °C for denaturation. The protein samples were separated by 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and an anti-GFP polyclonal antibody  was used to probe the blots, and then incubating with a horseradish peroxidase (HRP)-conjugated goat antirabbit IgG secondary antibody . Finally, GFP signal was detected with the Super ECL plus Western blotting kit according to the manufacturer’s instructions .The protein extraction from eviously . BrieflyN. benthamiana plants using TRIzol reagent  as instructed by the manufacturer. For accumulation levels analyses of GFP mRNA, probes were prepared using a PCR DIG probe synthesis kit  using specific primers (http://www.gelifesciences.com.cn/CNLS/indexAction.action). The hybridization was performed using a DIG-High Prime DNA labeling and detection starter kit II (Roche) according to the manufacturer’s instructions. GFP mRNA signal was detected using the ChemiDoc™ Touch Imaging System .Total RNA was extracted from the agroinfiltrated leaf patches of  primers . Total RAgrobacterium tumefaciens strain GV3101, respectively. The cultured Agrobacterium cells were resuspended in MMA buffer  to an OD600 of 0.5, and YFP split combinations were prepared for coinfiltration. At 3 dpa, the fluorescence in agroinfiltrated leaves were detected under a confocal laser scanning microscope  at a wavelength of 488 nm. Experiments were repeated three times.Plasmids YFPN-V2, YFPC-V2, YFPC, YFPC-V2(R17A), YFPC-V2(R43A), YFPC-V2(R91A), YFPC-V2(F9A), YFPC-V2(Y24A), YFPC-V2(Y61A) and YFPC-V2(K105A) were transformed into N. benthamiana 25S ribosomal RNA gene (Accession no. KP824745) was examined with primer pair qNbrRNA-F/qNbrRNA-R and used as the internal control , using primers specific to TbCSV V1 gene (qTbCSV-F/qTbCSV-R). An optimal standard curve for TbCSV with a coefficient of regression The constructs and primers used in this study are listed in To better understand the function of TbCSV V2 during viral infection, in the present study, the inoculation assay using TbCSV infectious clone mutant with untranslated V2 showed that V2 protein is critical for viral infection and continuous symptom development. Agroinfiltration assays indicated that V2 can suppress local and systemic gene silencing. Deletion and site-directed substitutions analyses demonstrated that the α-helix regions and the conserved basic or ring-structured amino acids are critical for RNA silencing suppression activity of V2. Further analyses suggested that RNA binding and self-interaction of V2 may contribute to its suppressor activity. As that shown for some other homologues, TbCSV V2 can also exacerbate the symptoms induced by PVX infection, and we first confirmed that this function of V2 is independent of its suppressor activity.


To investigate refractive stability and characterize corneal incision repair up to 3 months after implantation of a new hydrophobic acrylic intraocular lens (IOL) with hydroxyethylmethacrylate using a new automated IOL delivery system.® CNA0T0 IOL using the AutonoMe® automated delivery system in the Department of Ophthalmology, Keio University School of Medicine. The clinical data were collected from 46 eyes of 46 patients preoperatively and 1 day, 1 week, and 1 and 3 months postoperatively. Endothelial-side incision gaping, posterior incision retraction, and Descemet’s membrane detachment were recorded as present or absent using anterior-segment optical coherence tomography postoperatively.This prospective case series included 50 eyes of 50 patients undergoing phacoemulsification and implantation of the ClareonThe uncorrected distance and corrected distance visual acuities improved and stabilized 1 week postoperatively. The anterior chamber depth was stable from 1 week postoperatively. The subjective refraction was stable from 1 day postoperatively. Descemet’s membrane detachments and endothelial-side wound gaping were seen in 19 (41.3%) eyes and 34 (73.9%) eyes 1 day postoperatively and decreased gradually. Posterior incision retraction was seen in eight eyes (17.4%) on day 1 and increased to 19 eyes (41.3%) 3 months postoperatively.The Clareon IOL had excellent refractive stability from day 1 postoperatively. The AutonoMe automated delivery system enables safe IOL implantation through a 2.4-mm corneal incision, although the wound required longer than 1 month to heal postoperatively. As a result, the water content of the Clareon IOL is higher than that of the AcrySof IOL (1.5% versus 0.4% at 35°C), although it is still classified as a hydrophobic IOL.® automated delivery system [Several experimental and clinical studies have reported that the new lens had the lowest levels of surface and bulk inhomogeneities and optical characteristics including surface light scattering, glistenings, and chromatic aberration compared to the commercially available IOLs, those resulted in good clinical outcomes –10. Sevey system , 11; howThe Keio University School of Medicine Ethics Committee approved the study protocol . Each patient provided written informed consent before entry into the study, which adhered to the tenets of the Declaration of Helsinki.This prospective single-site observational study included 50 eyes of 50 patients who underwent cataract surgery with Clareon IOL implantation using the AutonoMe automated delivery system  from FebOne experienced surgeon (K.N.) performed all surgeries using a standard technique of sutureless phacoemulsification through a 2.4-mm corneal incision using the Centurion Vision System (Alcon). An anterior capsulorhexis about 5.0 mm in diameter was created and the Clareon CNA0T0 IOL was implanted into the capsular bag using the AutonoMe automated delivery system. The cumulative dissipated energy was recorded at the end of the surgery.The foldable, single-piece monofocal Clareon CNA0T0 IOL was implanted in all cases. This IOL is made of a hydrophobic copolymer with a hydrophilic copolymer, 2-hydroxyethylmethacrylate, with water content of 1.5% at 35°C, a refractive index of 1.55, and a glass transition temperature of 9.1°C.The uncorrected distance visual acuity (UDVA), corrected distance visual acuity (CDVA), subjective refraction , and subjective astigmatism were evaluated preoperatively, and at 1 day, 1 week, and 1 and 3 months postoperatively. The corneal endothelial cell density was evaluated using specular microscopy  preoperatively and 1 month postoperatively.AS-OCT imaging  was performed using the cornea and angle analysis mode. The anterior chamber depth (ACD) was defined as the distance from the corneal endothelial surface to the crystalline lens or the IOL parallel to the fixation axis. The AS-OCT images of the main incision were acquired on 1 day, 1 week, and 1 and 3 months postoperatively. The time-wise changes in the corneal incision site were evaluated using AS-OCT, according to a previous report . The gapThe presence of recession of the edge of the posterior wound surface was considered as posterior incision retraction.The values are expressed as the means ± standard deviations unless otherwise specified. The data obtained in this study were analyzed using the SPSS 24 statistical software package . The study included four time-factor levels  at which the UDVA, CDVA, and ACD were measured. The Kolmogorov–Smirnov test was used to verify the distribution for normality. The Wilcoxon signed-rank test with Bonferroni correction was used to analyze the UDVA and CDVA. The paired t-test with Bonferroni correction was used to analyze the ACD. McNemar test with Bonferroni correction was used to analyze endothelial-side wound gaping, Descemet’s membrane detachment, and posterior incision retraction. The level of significance was set at P < 0.05.2) were 2,767 ± 224 and 2,601 ± 346, respectively.Fifty patients (50 eyes) were included in this study. Phacoemulsification with IOL implantation was performed successfully in all patients without any intraoperative complications. Four eyes were excluded from the analysis; three patients were lost to follow-up, and one patient developed postoperative lens-induced uveitis due to the nuclear remnant. Ultimately, data from 46 eyes of 46 patients were evaluated. The patient demographic and preoperative data are shown in The postoperative UCVAs in 19 eyes with a postoperative target refraction of emmetropia at 1 day, 1 week, and 1 and 3 months postoperatively were 0.11 ± 0.18, 0.03 ± 0.12, 0.01 ± 0.13, and -0.02 ± 0.11, respectively. The postoperative CDVA at 1 day, 1 week, and 1 and 3 months postoperatively were -0.04 ± 0.07, -0.07 ± 0.07, -0.09 ± 0.07, and -0.09 ± 0.05, respectively, in all eyes. The UDVA and CDVA improved 1 day postoperatively, increased at 1 week postoperatively, and stabilized thereafter . The sphThe changes in the ACD are shown in This study showed that the Clareon IOL provided good postoperative visual and refractive outcomes, both of which were very stable from day 1 postoperatively. The AutonoMe automated delivery system enabled safe IOL implantation through a 2.4-mm temporal clear corneal incision, although the wound required more than 1 month to heal.The stability of the implanted IOL affects the postoperative refraction, optical performance, and development of posterior capsular opacification , 14. TheIn an experimental study, the Clareon CNA0T0 had the lowest levels of axial displacement and corresponding simulated dioptric power shift over all tested compression diameters compared with other one-piece acrylic IOLs, i.e., the MX60 , ZCB00 , and XY1  IOLs . The curRegarding the postoperative wound repair, Li et al. reported a detailed comparison of the wound repair and remodeling between different incision sizes (2.2 mm versus 2.85 mm) after cataract surgery using AS-OCT . AccordiThe cartridge used for the Clareon IOL is compatible with a 2.40-mm clear corneal incision, because the recommended incision size is 2.2 mm and over according to the manufacturer. Several factors affect stretching of the incision, such as the design of the cartridge tip and insertion methods . The cirNanavaty and Kubrak-Kisza reported that the maximal post-IOL implantation percentages of stretching of the external and internal wounds using the Ultrasert delivery system were over 15% and 11% for a 2.2-mm limbal incision in porcine eyes . They meImportantly, the current results showed that there were no significant differences in the UCVA and CDVA after 1 week postoperatively, which indicated that the damage was not relevant to the clinical outcome. However, the damage should be reduced in the future with improvements in the devices and techniques to increase safety and earlier recovery.The endothelial-side wound gaping is thought to reflect wound distortion and incision trauma . Li et. Based on previous reports, the posterior incisional retraction increased over time independent of incision size, presence of a Descemet’s membrane detachment, and wound gaping. This retraction may be associated with incisional remodeling  that conThis study had some limitations, in that it was a single-center, prospective, observational study with a small number of cases. However, these limitations also had some merits because of the consistency of the data and differences that can be ascribed only to differences in the patients. The data obtained from this study provided new information about the refractive stability of the Clareon IOL in the early postoperative stage and safety after cataract surgery using the new AutonoMe automated delivery system.S1 Data(XLSX)Click here for additional data file.


Since the discovery that the pro-inflammatory cytokine tumor necrosis factor-α (TNF-α) mediates some forms of HSP, it has been possible to probe some of the in vivo contribution of TNF-mediated HSP. Work from our lab and others has found roles for TNF-HSP in a variety of functions, including the developmental plasticity of sensory systems, models of drug addiction, and the response to psychiatric drugs.Since it was first described almost 30 years ago, homeostatic synaptic plasticity (HSP) has been hypothesized to play a key role in maintaining neuronal circuit function in both developing and adult animals. While well characterized  The maintenance of neural circuit function is a dynamic balance of several different types of synaptic plasticity. Synaptic strength can be modified by two broad types of plasticity mechanisms: Hebbian and non-Hebbian. Long term potentiation (LTP) and long term depression (LTD) are examples of Hebbian plasticity, where the strength of a given synapse is adjusted in response to synchronous activity . The HSP at the Drosophila NMJ appears mechanistically distinct from HSP in the mammalian CNS, and therefore will not be covered here  serves to keep neuronal activity levels in a range optimal for neurotransmission. It was first described as a response to prolonged perturbations in overall activity levels: when firing rates decrease, it serves to augment excitatory synaptic strength to normalize activity , and when firing rates increase, the opposite occurs . This phenomenon has been described in a variety of systems, including the mammalian central nervous system and the For the mammalian system, since being first described in the late nineties  was considered a site that was kept separate from the peripheral immune system, with immune signaling molecules excluded from the CNS by the blood-brain barrier  with activity blockade or other manipulations  frequency decreases with TNF treatment of hippocampal cultures , and their abundance at the synapse largely determines the neuronal response to a given stimulus. They are therefore a frequent point of regulation for the expression of synaptic plasticity  and large-scale trafficking of AMPARs (doubling) to the surface of pyramidal neurons, as determined by immunocytochemistry  are the main mediators of fast inhibitory transmission in the brain  of the striatum . In expeThe exogenous application of TNF has clear effects on synapses, so it is, critical to consider the biological conditions that lead to TNF release in the CNS. Examining the role of TNF in various forms of synaptic plasticity, therefore, gives context to the effects on neurotransmitter receptor trafficking observed by TNF administration.TNF is critical to the process of scaling up excitatory synaptic strength in response to prolonged activity blockade  in neurons, but typically only in pathological conditions. Which cells secrete the low level of TNF regulating HSP is currently unclear. For many years, glia were assumed to merely provide physical and trophic support for neuronal function. The finding that glial TNF is required for HSP lends weight to the more recent observation that glia are capable of being active players at the synapse, shaping properties of neurotransmission through the secretion of modulatory factors.While dissociated culture is a valuable tool for the dissection of neural function, it is important to verify the biological relevance of the information gleaned from them. As such, performing experiments in more intact systems is necessary to examine whether findings in dissociated culture hold true outside of that system. In that vein, HSP has been studied in entorhino-hippocampal slice cultures.The entorhinal cortex is the major input and output structure for the hippocampus and is often thought of as a gateway between the hippocampus and cortex. Projections from the entorhinal cortex to the dentate gyrus termed the perforant path, have been studied extensively in terms of both structure and plasticity for many years  in adult mice  as a result of dysregulation of the striatal circuit responsible for movement. These symptoms are accompanied by both increased TNF levels as well as increased AMPA binding, raising the possibility of HSP-type mechanisms contributing to this pathology  of the striatum. More specifically, repeated administration of cocaine to rodents results in an initial decrease in AMPA/NMDA ratio in the NAc 24 h after the last injection, followed by a gradual increase in AMPA/NMDA ratio during a period of abstinence after that (Kourrich et al., A behavioral readout of responses to cocaine administration is the extent of sensitization to cocaine exposure. When given repeatedly, cocaine causes an increasingly large locomotor response, termed behavioral sensitization, and its expression depends on AMPAR content in the NAc Kalivas, . In a stTNF appears to play a role in the response to other drugs of abuse in addition to cocaine. In morphine withdrawal models, TNF plays a role in synaptic adaptations after cessation of drug administration (Valentinova et al., in vitro and in vivo. Disrupting TNF signaling can, therefore, be a route to investigating the role of HSP in vivo. However, TNF-mediated changes can also be driving changes in circuit function, as seen during morphine withdrawal. TNF-mediated HSP could also become dysregulated under pathological conditions, leading to TNF driving maladaptive changes in circuit function. Whether TNF is acting in an adaptive or maladaptive manner must be assessed for individual circuits in response to particular situations.TNF is well known to have pleiotropic effects, allowing it to coordinate many functions under different circumstances and conditions. Within the immune system, various cell types will respond in distinct ways to coordinate the inflammatory response. We suggest that TNF may play a similar pleiotropic role in regulating neuronal circuit function. It is clear that the effects of TNF on neurotransmission are neuronal subtype-specific, and that it can lead to several different outcomes at the level of synapses. However, the common thread is that these changes still appear to normalize circuit output in response to perturbing stimuli, which is consistent with TNF being a mediator of HSP. Thus, TNF-induced trafficking of neurotransmitter receptors in the CNS may be a general mechanism by which circuit homeostasis and function are maintained both RH and DS wrote the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Trypanosoma cruzi, Leishmania spp., and Trypanosoma brucei. In this context, it is important to consider the evasion mechanisms of these protozoa in the host and the antigens involved in the mechanisms of the parasite–host interaction. Thus, the immunostimulatory properties of VLPs can be part of an important strategy for the development and evaluation of new vaccines. This work aims to highlight the potential of VLPs as vaccine adjuvants for the development of immunity in complex diseases, specifically in the context of tropical diseases caused by trypanosomatids.Research on vaccines against trypanosomatids, a family of protozoa that cause neglected tropical diseases, such as Chagas disease, leishmaniasis, and sleeping sickness, is a current need. Today, according to modern vaccinology, virus-like particle (VLP) technology is involved in many vaccines, including those undergoing studies related to COVID-19. The potential use of VLPs as vaccine adjuvants opens an opportunity for the use of protozoan antigens for the development of vaccines against diseases caused by  Trypanosoma cruzi, Trypanosoma brucei, and Leishmania spp. are flagellate parasites that belong to the Trypanosomatidae family and have a complex life cycle between invertebrate and mammalian hosts, where they cause Chagas disease, African trypanosomiasis, and leishmaniasis, respectively  has become an interesting tool for the development of vaccines in the context of infections caused by trypanosomatids, since VLPs have the immunoadjuvant ability to manipulate immune response polarization .VLPs used as vaccine adjuvants provide an alternative to current chemical adjuvants, along with an ability to carry foreign epitopes; they constitute platforms for the development of new prototypes and vaccine protocols. VLPs’ immunostimulatory properties are similar to natural infections of the viruses from which they are derived, and the preservation of the native conformation is, in addition to the primary sequence, a major factor responsible for this property ,7. ThereThe structural conformation of VLPs mimics the morphology and structures of viral particles without the presence of the virus genome, and therefore, they are not infectious particles. Thus, the main advantages of using VLPs as vaccine prototypes are safety, since they are not infectious viral particles, and the ability to induce a robust immune response that is highly similar to that elicited by a natural viral infection. These two characteristics allow VLPs to be used in the development of vaccines .Additionally, there are several ways to produce recombinant VLPs, mainly using gene expression systems in bacteria, yeasts, insect cells, and mammalian cells, among others . The str® [®, both against hepatitis B [® against human papillomavirus (HPV) [Regarding VLP-based vaccines, about a dozen are currently approved and available in Europe and the USA. These include Recombivax HB®  and Engeatitis B , and Garus (HPV) . Cervarius (HPV) ,15. Reguus (HPV) . During the past few decades, studies on different VLP systems used against important human diseases—such as Ebola , ChikungToxoplasma gondii, the etiologic agent of toxoplasmosis [Plasmodium, the presence of lasting immunity is observed when different vaccine prototypes are produced using antigens associated with VLPs [In the context of the use of VLPs for the development of vaccines against protozoa, it was observed that the immunostimulatory property of VLPs with targeted epitopes is able to induce the production of protective antibodies and a high survival rate of animals infected with lasmosis ,26. In tith VLPs ,28,29.The main challenge in the development of trypanosomatid vaccines is the adequate identification of important antigenic candidates for the generation and maintenance of protective immunity. A second challenge is related to the type and nature of the immune response induced during the immunization process. However, several studies have been performed in order to identify potential antigenic candidates for these protozoa, as well as mechanisms of activation of immunity for the development of vaccines (reviewed in Reference ). TherefCurrently, several tools are being used in the development of vaccines in the context of trypanosomatids, such as immunoinformatic, genomic, and proteomic techniques that mainly contribute to the identification of new antigenic targets and epitopes of these parasites ,32, as wAdditionally, for the development of vaccines against these trypanosomatids of medical importance, VLPs are presented as alternatives to induce immunity in a polarized way, since the mechanisms of resistance and susceptibilities of these infections are related to the immune response polarization that occurs during a natural infection. Thus, due to their constitution, they may have the ability to express specific components in amounts comparable to high quantities of these parasites and, furthermore, can present other immunogenic components, thus allowing a stronger general immune response of the organism .+ T helper cells) is important for controlling infections caused by trypanosomatids, mainly due to the production of interferon-γ (IFN-γ) and tumor necrosis factor α (TNF-α). These cytokines induce the activation of macrophages and, consequently, an increase in the production of reactive oxygen species, which control the parasitic load during infection. Trypanosomatid parasites have the ability to reduce Th1-type immune response polarization by enhancing the production of IL-10 and IL-17 cytokines due to the release of numerous virulence factors [The immune response polarization of the Th1 type  used in experiments are necessary to understand, for example, whether the formulation of specific antigens with VLP technology helps or not in the survival of those infected. After pre-clinical trials proceed as expected, clinical trials have a significant step in producing results that confirm a specific, lasting, and harmless response in humans.According to the illustration, there are some essential steps in the checklist:Due to the time spent in pre-clinical and clinical trials in the context of safety for humans, the ideal model at the present moment would be based on known and/or safe marketed VLPs that could be directed against these three diseases in a formulation system. Based on the already-known mechanisms of VLP technology and the deep understanding of the immune response to these parasites, it is still a utopian model of how this strategy can act in practice. + T-helper lymphocytes, which will activate B cells and, in turn, stimulate the production of specific IgG antibodies.Here, we propose VLPs that carry antigens of a single individual parasite, thereby becoming chimeric VLPs. According to some studies, intramuscular application has the highest efficiency , as it p+ T cells, a cytotoxic induction will be observed. Another expected response would be after the recognition of these antigens by CD4+ T cells: there will be an intensification in the production of cytokines, such as interferon-γ and TNF-α, which will induce a more aggressive response by macrophages with a high production of ROS and activate CD8+ T cells [When processed by APCs and presented by MHC class I to CD8 T cells .As presented above, VLPs have contributed to the development of new vaccines to control numerous pathogens of medical importance. Therefore, we believe that this technology is promising as a vaccine adjuvant for the manipulation of the immune response during the induction of immunity, mainly in the context of complex diseases, such as infections caused by trypanosomatids. Finally, VLP-based vaccines, when properly designed and formulated, represent a promising platform for chemical or genetic conjugation of parasitic antigens and the development of multivalent vaccines against protozoa.


Spermidine has been known to inhibit the production of pro-inflammatory cytokines. However, there are no reports about anti-inflammatory effects of spermidine on osteoarthritis (OA). Herein, we examined whether OA progression could be delayed by intraperitoneal injection (i.p.) of spermidine in the anterior cruciate ligament transection (ACLT) and TNF-α induced arthritis (TIA) mouse models. During the process, human FLS cells (H-FLS) were used to investigate the potential ubiquitination mechanism of spermidine-mediated RIP1 in TNF-α-induced NF-κB/p65 signaling. We found that spermidine attenuated synovitis, cartilage degeneration and osteophyte formation, resulting in substantially lower OARSI scores and TNF-α scores in spermidine-treated ACLT and TIA mice. In terms of the mechanism, 9 μM spermidine did not affect the viability, proliferation, cell cycle and apoptosis of H-FLS, and exerted inhibitory effects by activating CYLD-mediated RIP1 deubiquitination on TNF-α-induced NF-κB/p65 signaling in H-FLS. From these data, we can conclude that spermidine attenuates OA progression by the inhibition of TNF-α-induced NF-κB pathway via the deubiquitination of RIP1 in FLS. Therefore, intake of spermidine could be a potential therapy for preventing OA. OA is a disease of complex pathological mechanism that could be caused by various factors, including age, obesity, joint trauma, and instability2. However, due to the inherent complexity of biological factors that consists of joint loading, aging, obesity with metabolic syndrome and inflammation3, current treatments which cannot prevent the progression of OA, leading to sustained structural damage of the knee, are deemed ineffective.Osteoarthritis (OA) is one of the most common form of arthritis and is the leading cause of joint pain and disability4. Inflamed joints bring about increasing pro-inflammatory cytokines such as TNF-α5 produced by fibroblast-like synoviocytes (FLS). Previous studies have shown that TNF-α is highly expressed in synovial tissue of osteoarthritic joints8. In the pro-inflammatory cascade, TNF-α would ultimately activate the most important NF-κB/p65 transcriptional pathways, which in turn secretes IL-6 and IL-8 leading to the aggravation of OA10. However, until now, the greatest drawback for anti-TNF-α agents is its potent systemic toxicity, which poses some potential safety issues, including infection and autoimmune diseases11. In light of this, nutraceuticals and naturally occurring compounds represents another safe opportunity for anti-TNF-α treatment of OA.During the onset of OA, the most common symptom is synovial inflammation of the diseased joint12 and further increase the bioavailability of NO through the reduction of oxidative stress in cardiomyocytes13. In another study, intraperitoneal injection (i.p.) of spermidine analog, spermine, reduces TNF-α in the plasma of septic mice via the inhibition of high mobility group protein-1 (HMGB1)-induced release of inflammatory cytokines in macrophages14. However, there are no reports on whether spermidine could inhibit TNF-α-induced inflammation in OA. Herein, the focus of our research is to explore the role of spermidine in TNF-α-induced inflammation in OA, determine safe and effective drug concentrations, and explain its specific location and molecular targets, which could provide a safe and reliable alternative treatment for OA.The natural polyamine spermidine was reported to have cardioprotective effects, dietary spermidine can reduce TNF-α in plasmaTNF-α , Collagen 10 , MMP3 , MMP13 , iNOS , UBA1  Adamts4  and Adamts5  were purchased from Abcam , Adalimumab  and Glucosamine  were purchased from Selleckchem , UBE2N , phospho-RIP1 , NF-κB phospho-p65 , NF-κB p65 , RIP1 , Anti-rabbit IgG (H + L) Alexa Fluor 555  and Anti-mouse IgG (H + L) Alexa Fluor 488  were purchased from Cell Signaling Technology , Vimentin , HA-Tag , IκB  and phospho-IκB  were purchased from Santa Cruz Biotechnology , Spermidine  and Aggrecan  were purchased from Sigma , CLYD  and TRIM21  were purchased from PeproTech .n = 150): (1) ACLT operation group (n = 100), where mice underwent anterior cruciate ligament transection (ACLT) surgery to destabilize the joint and induce post-traumatic OA (PTOA), (2) sham operation group (n = 50), where a sham operation was performed with a similar incision at the right joint capsule. On the next day, ACLT and sham groups were further administered with: (i) phosphate-buffered saline (PBS), (ii) 0.3 mM spermidine, (iii) 3 mM spermidine, (iv) 6 mM spermidine, and (v) 100 mg/kg DMSO, i.p. injection per day. Mice were euthanized for articular cartilage, synovial tissue (infrapatellar area), and osteophyte analysis after 4 weeks or 8 weeks of treatment.Male C57BL/6J mice aged 12 weeks were purchased from the Experimental Animal Centre of SiBeiFu  and housed in a pathogen-free animal facility at the Sun Yat-Sen University. Then the mice were randomly divided into two main groups (n = 15): (i) control group, (ii) DMSO treatment group, or (iii) spermidine treatment group. For each treatment, the mice were further divided into three subgroups: (i) 4 weeks treatment (n = 5), (ii) 8 weeks treatment (n = 5), and (iii) 16 weeks treatment (n = 5). After respective treatments, the mice were euthanized and their knees were harvested for TNF-α immunohistochemistry analysis.The C57BL/6J mice were treated with TNF-α (10 μg/μl/week) by intra-articular injection to stimulate TNF-α-induced arthritis (TIA). The specific operation was as follows: TNF-α was injected into both knee cavities. Then the mice were randomly divided into three groups  and were performed in accordance with the Committee’s guidelines.After the knee specimens were isolated, the surrounding soft tissues were removed and fixed with 4% PFA for 48 h. Micro-CT scan was performed for the knees at 100 kV, 98 μA, 12 μm resolution on a Viva CT40 Micro CT Scanner . The entire joint was chosen as the region of interest (ROI) for three-dimensional (3D) reconstruction, osteophyte score and the volume of ROI were analyzed.15.The knee joints were subjected to formalin fixation and was then decalcified with 0.5 M EDTA at pH 8.0 for 4 weeks, and was subsequently dehydrated and embedded in paraffin. The tissue sections were cut at 4–6 μm thickness in a sagittal orientation. H&E and Safranin O-Fast Green staining was performed, and the OARSI OA cartilage histopathology assessment system was chosen to assess the cartilage degeneration severity as described2O2 to quench endogenous peroxidase activity. Primary antibodies were applied overnight at 4 °C or incubated for 2 h at 37 °C. After being washed three times with PBS and incubated with goat-anti-rabbit HRP-conjugated secondary antibody for 1 h at 37 °C, an immunohistochemical staining signal was developed with 3, 3′-diamino-benzidine . The numbers of positive cells and relative intensity were counted using Image J software .For immunostaining, the sections were deparaffinized, briefly washed with 0.1 M PBS (pH 7.4), and incubated for 10 min in 3% HSynovial tissues were obtained from sham and PTOA mice and incubated overnight in PBS with 1 mg/mL Collagenase I  at 37 °C for 5–6 h to isolate normal synoviocytes and OA–FLS , respectively.16. This study was approved by the Medical Ethics Committee of Sun Yat-sen Memorial Hospital (SYSEC-KY-KS-158).Human FLS (H-FLS) were taken from patients with no osteoarthritic characteristics who underwent arthroscopic meniscus repair surgery. H-FLS were used between passages 4 and 9, as previously describedMouse OA–chondrocytes were obtained from cartilage, briefly, cartilage tissue was obtained from the knees of PTOA mice. Dissected cartilage pieces were incubated overnight in PBS with 1 mg/mL Collagenase D  at 37 °C for 5–6 h. The solution was then centrifuged at 1000 rpm for 5 min to discard the supernatant, the remaining precipitate that contains primary OA–chondrocytes was mixed with 5 mL medium and was then inoculated in a culture flask.17.Since the procedure of obtaining normal cartilage from patients were not approved by the Medical Ethics Committee of Sun Yat-sen Memorial Hospital, we opted to use a chondrogenic cell line (ATDC5) purchased from Riken BioResource Center . The cells were induced into normal chondrocytes by the addition of insulin, transferrin, and selenous acid  according to previous studiesCells were collected by scraping with 1 mL of PBS and centrifuged for 10 min at 3000 rpm to collect cell pellets, and total RNA was isolated from cell pellets using TRIzol reagent . cDNA was transcribed from RNA samples by using reverse transcription reagents  and quantitative real-time polymerase chain reaction (qRT-PCR) assays were carried out to quantify the levels of mRNA expression of these genes. GAPDH was used as the internal loading control using qRT-PCR Mix  and the Light Cycler (Roche Switzerland). All primer sequences used in this manuscript are listed in Supplementary Table The TNF-α, IL-6, IL-8, and IL-4 level of inflammatory cytokines in serum and culture medium were measured using ELISA kits  according to the manufacturer’s instructions.18 was used as the positive control drug for TNF-α pathways. Glucosamine showed no anti-TNF-α activity in OA, we used glucosamine as a negative control.In order to identify the effective concentration of spermidine on TNF-α in OA–FLS, OA–FLS cells were treated with 3, 6, 9, 12, and 15 μM of spermidine, or 7 nM adalimumab or 5 nM glucosamine for 24 h. In our experiments, adalimumab50) is the most widely used and informative measure of a drug’s efficacy, where less than 5% cell activity inhibition rate is the safe range of the drug19. According to the protocol of the drug IC50 experiment, the viability of cells were measured by the cell counting kit-8 , then, IC1, IC3, and IC5 were obtained.Half-maximal inhibitory concentration  assay. EdU staining was conducted using a Cell-Light EdU DNA Cell Proliferation Kit  according to the manufacturer’s protocol and images were captured using an Olympus laser scanning microscope system .5 cells per well were seeded into 6-well culture plates and incubated with either spermidine, adalimumab, or glucosamine for 24 h. Cell-cycle and Apoptosis experiment was conducted according to the manufacturer’s protocol . Cell-cycle and apoptosis were quantified using the FACS Caliber flow cytometer .Briefly, 1 × 10H-FLS cells were treated with TNF-α (10 ng/mL) in the presence or absence of spermidine (9 μM). After 1 h, cells were immersed in 4% paraformaldehyde (PFA) for 15 min. The cells were treated with 0.1% Triton-100 for permeabilization and blocked in 1% bovine serum albumin in PBS for 30 min, followed by incubation with primary antibodies overnight at 4 °C. The cells were then followed by incubation with appropriate secondary immunofluorescent antibodies for 60 min. After washing three times with PBS, cells were added with Anti-fade Fluorescence Mounting Medium with DAPI , and were imaged by confocal microscopy .The shRNA-CYLD and GFP that were packaged as lentivirus were purchased from Gene Copoeia . H-FLS was divided into two groups (i) shRNA-CYLD group and (ii) shRNA-Scrambled Control (shRNA-SC) group. Transfection efficiency of >80% was considered successful and the cells were then used for subsequent experiments.Plasmids were constructed for RIP1 ubiquitination overexpression. Briefly, the coding regions of the genes were amplified from the Homo lung cDNA library via the PCR technique. Then the resulting fragments were cut with restriction enzymes, and inserted into pcDNA3.1-Myc and pcDNA3.1-HA-Ubiquitin (Invitrogen) to form pcDNA3.1-RIP1(WT)-myc and pcDNA3.1(+)-HA-Ub (RIP1 plasmid and HA-Ub plasmid) for overexpressing RIP1(WT) gene and ubiquitination, respectively. All constructs were sequenced for confirmation.To determine the role of spermidine effects on RIP1 ubiquitination, the RIP1 plasmid and HA-Ub plasmid were co-transfected overexpression of RIP1 (WT) and ubiquitinated plasmid (2 μg/μL) for 48 h. The transfection efficiency was determined by using Western blot analysis.To determine the role of spermidine effects on RIP1 deubiquitination by the activation of CLYD, H-FLS cells were divided into two groups: (i) shRNA-CYLD group and (ii) shRNA-SC group. Both groups were then co-transfected with RIP1 (WT) and ubiquitinated plasmid. After transfection of shRNA-CYLD lentivirus and RIP1 (WT) and HA-Ub plasmid, the transfected H-FLS cells were stimulated with 10 ng/mL of TNF-α for 2 min in the presence or absence of 9 μM spermidine for 1 h, then, cells were resuspended in 1 mL of cold PBS, and were subsequently directly lysed by RIPA lysis buffer . The lysate was immediately incubated with anti-RIP1 and A/G Plus Agarose beads. Ubiquitinated RIP1 proteins were detected by immunoblotting with an HA antibody and were subsequently probed with anti-RIP1 as a loading control.In the experiment of inflammation signaling pathway of H-FLS, H-FLS cells were treated with TNF-α with or without spermidine for 24 h, cells lysates were then separated by SDS-PAGE and transferred to PVDF membranes. Membranes were blotted with primary antibodies recognizing Adamts4, iNOS, MMP3, MMP13, NF-κB p65, NF-κB phospho-p65, IκBα, phospho-IκBα, RIP, phospho-RIP1, CYLD, Iκκ-β, and GAPDH, respectively. All blots were probed with horseradish peroxidase (HRP)-conjugated secondary antibodies , and immunoreactive proteins were revealed using the enhanced chemiluminescence  to detect the targeted proteins.The Western Blot membrane was scanned  and calculated the numerical intensity by Image J 4.5 analysis system . Intensity of each protein was normalized with GAPDH and was represented as a ratio to the control.t test. Differences among three groups were analyzed by one-way analysis of variance (ANOVA) and Dunnett’s multiple comparison tests. The level of significance was set at *p < 0.05, **p < 0.01, ***p < 0.001. All statistical analyses were performed with GraphPad Prism software version 7.0 .Data are presented as mean ± standard deviation (mean ± S.D). Differences between two groups were statistically analyzed by unpaired, two-tailed Student’s p < 0.01) and 8 weeks (p < 0.01), OARSI scores were not significantly different between 3 mM spermidine- and 6 mM spermidine-treated ACLT mice    Fig. . Besides01) Fig. .Fig. 1Spp < 0.001)  Fig. . These f1 = 3 μM, IC3 = 6 μM, and IC5 = 9 μM drug concentration of spermidine (IC50 = 102.557 μM)  Fig.  at diffeμM) Fig.  and WestμM) Fig. . These r21. Consistently, the levels of serum TNF-α, IL-6, and IL-8 were also significantly reduced in spermidine-treated ACLT mice compared to DMSO-treated ACLT mice at 8 weeks    Fig. . We disc01) Fig. . These fTo elucidate the effect of spermidine on synovial inflammation-mediated cartilage degeneration, ADTC5 cells were induced by selenous acid and then co-cultured with the supernatant of DMSO-treated primary OA–FLS  or spermidine-treated primary OA–FLS (CM + spermidine) for 24 h. Western blot and qRT-PCR analysis showed the expression of Aggrecan, Collagen10, Adamts4, Adamts5, MMP3, and MMP13 were significantly reduced in the spermidine-treated primary OA–FLS compared to DMSO-treated primary OA–FLS Fig. . Taken t23, the IC50 of Mouse-OA–FLS and H-FLS were determined by CCK-8 assay  drug concentration of spermidine inhibited the production of TNF-α. ELISA analysis showed that spermidine treatment significantly reduced the production of TNF-α, where 9 μM of spermidine had the same anti-TNF-α effect as adalimumab (7 nM) in mouse-OA–FLS (p = 0.998) and TNF-α stimulated H-FLS (p = 0.999)  Fig. . In addi99) Fig. . Here, w99) Fig. . The TNF99) Fig. . In addi99) Fig. . These fThe expression of NF-κB/p65 and IκBα were significantly increased, while the phosphorylation of NF-κB/p65 (NF-κB/p-p65) and IκBα (p-IκBα) were significantly reduced in TNF-α and spermidine-treated H-FLS than that of TNF-α-treated H-FLS alone, which indicated that spermidine treatment inhibited TNF-α-induced NF-κB/p65 activation Fig. . ImmunofIn order to determine which enzyme was activated by spermidine treatment to suppress the RIP1 ubiquitination in H-FLS, (E1)-UBA1, (E2)-UBE2N, (E3)-TRIM21, and CYLD enzyme were detected by Western blot after H-FLS were treated by TNF-α for 2 min and spermidine treatment for 1 h. The result showed that spermidine treatment significantly increased the expression of CYLD enzyme compared to the absence of treatment or TNF-α treatment Fig. . This rep < 0.0001), whereas RIP1 mRNA levels were unaffected (p > 0.05)  Fig. . We founIn this study, we found that i.p. administration of spermidine could effectively reduce the expression of TNF-α in FLS and serum in OA mice model and inhibit the deubiquitination of RIP1 in TNF-α-mediated NF-κB/p65 activation, by activating CYLD in FLS. Taken together, all these resulted in significant inhibition of synovial inflammation, while cartilage degeneration and osteophyte formation were significantly ameliorated (Fig. 24. However, spermidine analog putrescine was highly expressed in RA–FLS compared with OA–FLS25, and polyamine metabolism were positively contributes to RA-FLS intrinsic activation26. These results indicated that the polyamine metabolism process is the target of RA inhibition27, and spermidine supplementation may not work for RA. So far, it has been reported that oral intake of spermidine could reduce the expression of TNF-α in plasma28, which indicated a potential association between spermidine and anti-TNF-α inflammation. The theory of TNF-α-induced RIP1 ubiquitination signal of NF-κB/p65 pathway leading to inflammatory response in OA–FLS has been widely confirmed23. RIP1 ubiquitination displayed important recruitment and activation of the IKK complex that improved NF-κB activation10. Loss of RIP1 ubiquitination allows RIP1 to bind caspase-8, and thereby activates apoptosis9. Based on the above results, we speculate that the ubiquitination of RIP1 may determine whether its downstream product could activate NF-κB or apoptosis signaling. Interestingly, spermidine could prevent or reduce the apoptotic response triggered by TNF-α stimuli in non-tumoral cells31. Therefore, we estimate that RIP1 ubiquitination independent of apoptosis may be an important target for spermidine against TNF-α-mediated inflammation, and our results also confirmed the role of spermidine in RIP1 deubiquitination in TNF-α-driven NF-κB activation (Fig. 32. However, spermidine is also a double-edged sword, which could also activate apoptosis33. In our results, increased apoptosis of H-FLS cells was found when spermidine concentration was greater than 12 μM (Fig. As contrasted with RA, there are clear drug guidelines for anti-rheumatic drugs for anti-RA treatmention Fig. . This se36. CYLD is a NEMO interacting protein that removes the polyubiquitin chain linked to the K63 position of RIP1, thereby inhibiting IKK and NF-κB activity38. However, previous studies have not applied a safe stimulant to activate the endogenous CYLD deubiquitination system that effects the target protein of RIP1, which is not suitable for clinical application. Importantly, in this study, CYLD could be enhanced by spermidine and promoted the deubiquitination of RIP1 in TNF-α-induced NF-κB activation (Fig. 40. It seems that the ubiquitination of RIP1 protein by CYLD still via apoptosis signaling. We did not detect apoptosis after overexpression of the CYLD gene in H-FLS, because the non-apoptotic dosage of spermidine in cytotoxicity experiments have been determined. In summary, spermidine deubiquitinates RIP1 in TNF-α-induced NF-κB activation by upregulating CYLD, thereby reducing synovial inflammation and cartilage degeneration (Fig. Accumulating evidence highlights the vital role of deubiquitinating enzymes in the pathogenesis and progression of arthritision Fig. . NotablyIn summary, our study provides compelling evidence supporting the role of ubiquitination-regulated inflammation of spermidine in the treatment of OA. The therapeutic effect of spermidine on OA is dependent on FLS rather than direct regulation of chondrocytes. This work suggests that spermidine could be a promising drug candidate for OA intervention.Supplementary Table 1Figure S1Figure S2Figure S3Figure S4


The main aim of this study was the lexical analysis of the literature on this topic considering the most cited words and the relationship between the words and the papers. Furthermore, a geographical analysis was performed to evaluate the worldwide prevalence. (2) Methods: We performed a literature search on PubMed, and we calculated the occurrence of the words indicating nerves and the body parts. Furthermore, we calculated the number of papers for each country, considering the affiliation of the first author. Finally, to describe the relationships between the words and the papers, we used the 30 most cited words, and we built a matrix describing in which papers a word was cited. This matrix was used to create a network based on the graph theory using Gephi 0.9.2 software. (3) Results: The most cited nerves were median and ulnar ones, and the most cited body parts were hand, wrist and elbow. The United States of America was the most productive country, with 80 papers. The graph of the network showed the importance of ultrasound as support for therapy. (4) Conclusions: The study represents a lexical analysis of the literature and shows information about subjects, authors and relationships of the papers. This may be helpful for better understanding and evaluation of the situation of the current literature.  The application of ultrasound (US) in medicine is well known and relatively old. The first research papers about this technique for diagnostic purposes were published in the 1960s . At presUS provides a tool to image and see the organs and can clearly measure and locate the nerve damage . This isBesides diagnosis, US shows effectiveness for the whole management of neuropathy, particularly important for more fragile patients frequently presenting multimorbidity and polipharmacology ,22,23,24The literature on US application in peripheral neuropathies has increased during recent decades, with robust evidence. A large number of scientific papers support clinical activity, providing evidence-based guidelines for diagnosis and treatment. Despite the growth in published studies, how US is used in clinical and research practice remains poorly understood . FurtherThe aim of our study was the evaluation of the present literature on nerve US through the following points of view: (1) the most studied nerves and body parts; (2) the geographical origin of the authors; (3) lexical analysis based on the most used words in the abstracts. With the application of the listed approaches, we aimed to analyze the literature in order to provide information useful for the comprehension of the literature evidence. In particular, with the lexical analysis based on this novel approach, we intended to show which words are mainly used in scientific literature on nerve US and how the same words are connected with the papers.The literature search was performed on PubMed using the following Medical Subject Headings (MeSH) terms: “peripheral neuropathies”, “ultrasound” and “diagnosis”, associated with the Boolean operator “AND”. The following filters were used: article type , language (English) and publication date (the last 10 years). The complete results, including the abstracts, were exported as a text file using the PubMed function. This file was imported into the freeware software TXM 0.8.0 , able to count the frequency of a word in texts (in our case the titles and the abstracts of the papers) . In partThe total number of papers on PubMed, found with the described search, was 282 (on 1 November 2020). The absolute number of publications, visible in the graph visualizing the results per year, was growing with a peak in 2018. The analysis of the words revealed the clear dominance of the median nerve (52.7% of the cases), followed by the ulnar nerve (21.7%) and the plexus (10.2%). The other nerves were present with very low percentages (<5.0%) a. Among Considering the geographical origin of the literature production, the United States of America (USA) was the most productive country (28.4% of the papers), followed by Italy and Turkey . However, with this search, every continent showed its own contribution to the literature production, albeit with dissimilar impact. Emblematically, the Arabic Republic of Egypt was the unique African State, while Europe showed not only a large number of countries but also a large number of papers .Considering the LENGTH approach, the graph of the network showed some peculiarities. In particular, the whole network was separable into two main parts: on the left side, the topics about the diagnosis were visible , while on the right the topics about the treatment could be seen   38]. In. In38]. Our study shows that the current scientific research on nerve US is largely focused on upper limb nerves and is mainly produced by a few countries, although from every continent and independent of the country population. Furthermore, the lexical network, presented with the LENGTH approach, shows the most frequent words in the papers (mainly belonging to diagnostic and therapeutic semantic fields).The information obtained by the described approaches illustrates the literature analysis from novel points of view. They reveal some features of the literature useful for speculation. Furthermore, they can disclose some messages conveyed by the words used in the published studies. Based on our results, we can highlight how the most assessed peripheral nerve structures by US are median and ulnar nerves. This consideration is supported by the evaluation of the most treated single body parts: hand, wrist and elbow. This is expectable, especially because of the relative ease in accessing these nerves and the common diseases involving these structures ,41,42,43Finally, the geographic analysis of the literature shows the very widespread interest in the matter all over the world. There are different levels of literature production independent of the population. The high number of papers (80) published by the USA confirms the high level of production from this country. However, the USA generally represents a very prolific country in literature production, and further analysis should be conducted to better understand the real meaning of this point. Further speculation is possible from the other data. Notably, in our study, the two other productive countries are Italy and Turkey. Indeed, this demonstrates a growing interest in nerve US for these two countries in recent years. Interestingly, two other very populated countries  do not present a comparable level of literature production, and this may be related to their relatively recent interest in the topic analyzed in this study.The study obviously shows some limitations. First, the search was performed on PubMed using specific filters . This probably caused a simplification of the data collection with a possible lack of some literature studies. However, we chose these filters in order to analyze the recent studies with the highest evidence values. Another limitation is linked to the method itself, which was developed to assess the words in the abstracts and not in the whole paper. This can sound like an important restriction for the analysis. However, the abstracts can be used everywhere without the necessity to download the whole papers, and this allows a general lexical evaluation of the considered studies. Finally, the choice of the words is related to researchers’ experience and should be properly adapted (in number and type) for each lexical analysis.In conclusion, the presented approaches shed light on some aspects of the literature. The study is based on a lexical analysis of the literature and may be useful for the comprehension of the lexicon used for the definition of specific topics. The data support reflections about subjects, authors and relationships of the papers. With our analysis, we found that evidence of US application for the diagnosis and treatment of peripheral nerve diseases is large. However, there is a certain unbalance considering the studied body segments; upper limbs, median nerve and ulnar nerve are the most studied, while the nerves in the lower limbs are relatively rare in the literature. Additionally, the geographical assessment reveals a large diffusion of the literature on nerve US all over the world but with important inhomogeneity among the countries, independent of the population. Finally, the LENGTH approach displays the relationships between the words and the papers, showing the importance of US for diagnosis in clinical practice and for the guiding of treatment, especially in carpal tunnel syndrome. With the proposed deep analysis, we can see the potential and limitations of the current literature in a new way. We hope this approach can support the definitions of new research studies for better use in clinical practice.


Introduction. Parkinson's disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease (AD). With the ageing of population, the frequency of PD is expected to increase dramatically in the coming decades. L-DOPA  is the most effective drug in the symptomatic treatment of PD. Nonmotor symptoms in PD include sleep problems, depression, and dementia, which are not adequately controlled with dopaminergic therapy. Here, we report the efficacy of oral administration of scallop-derived ether phospholipids to some nonmotor symptoms of PD.  Ten (10) patients received oral administration of 1 mg/day of purified ether phospholipids derived from scallop for 24 weeks. Clinical symptoms and blood tests were checked at 0, 4, 12, 24, and 28 weeks. The blood levels of plasmalogens in patients with PD were compared with those of 39 age-matched normal controls.  Initial levels of plasma ethanolamine ether phospholipids in PD and ethanolamine plasmalogen of erythrocyte from PD were lower than those of age-matched normal controls. Oral administration of 1 mg/day of the purified ether phospholipids increased plasma ether phospholipids in PD and increased the relative composition of ether phospholipids of erythrocyte membrane in PD. The levels of ether phospholipids in peripheral blood reached to almost normal levels after 24 weeks. Furthermore, some clinical symptoms of PD improved concomitantly.  1 mg/day of oral administration of purified ether phospholipids derived from scallop can increase ether phospholipids in peripheral blood and concomitantly improve some clinical symptoms of PD. PD afia nigra . With thia nigra –3. The hia nigra –3. Thoseia nigra –3. Curreia nigra . Nonmotoia nigra , 5–9, whEther phospholipids represent a specific subclass of glycerophospholipids characterized by the presence of an ether bond at the sn-1 position of glycerol backbone –13. TherWe previously reported efficacy of oral administration of purified ether phospholipids derived from scallop to cognitive functions in patients with mild Alzheimer's disease (AD) and mild cognitive impairment (MCI) by a multicenter, randomized, double-blind, placebo-controlled trial , 16. In Previously, we observed that many patients with PD showed decreased levels of ether phospholipids in peripheral blood as compared to those of age-matched normal controls. Therefore, we carried out a trial, in which the purified ether phospholipids derived from scallop were orally given to 10 patients with PD for 24 weeks. The oral administration of the ether phospholipids increased ether phospholipids in peripheral blood and showed a concomitant improvement in some clinical symptoms of PD.One soft capsule contained 0.28 mg of ethanolamine ether phospholipids (ePE), 0.22 mg of choline ether phospholipid (ePC), 0.08 mg of cholesterol, and 0.07 mg of ceramide aminoethylphosphonate (CAEP). Lipid composition of the capsule and fatty acid composition of each ether phospholipid are listed in Tables Fourteen (14) patients with PD were recruited, of which 7 patients had a history of deep brain stimulation (DBS) therapy. Diagnosis of PD was done by clinical symptoms. Some baseline characteristics of the patients are shown in The study was approved by the Institutional Review Boards of Fukuoka University School of Medicine and the Review Board of BOOCS Clinic . The study was implemented in compliance with the Declaration of Helsinki.Venous blood of the subjects who had fasted overnight was drawn into a tube containing heparin. The blood was cooled in an ice bath and kept in the refrigerator for 48 h at maximum. After centrifugation at 1000 ×g for 5 min at 4°C, plasma was kept at −30°C until measurement. The packed erythrocytes were washed three times with physiological saline solution, and residual plasma and buffy coats were removed at each washing. The washed erythrocytes were lysed in 40 vol. of hypotonic buffer  and were centrifuged at 25000 ×g for 20 min at 4°C. This procedure was repeated four times for removing hemoglobin, and the erythrocyte membranes (white in color) were obtained . The mem1 (PLA1) hydrolyzes ester (acyl) bond at the sn-1 position of glycerophospholipids, but PLA1 does not act on ether bond at the sn-1 position. Diacyl phospholipids completely disappeared after treatment of plasma with PLA1, but ether phospholipids remain intact [Measurement of ether phospholipids and sphingomyelin (SM) in plasma was done by our method which were described recently . Brieflyn intact . After tn intact  and weren intact . The PLAn intact .Lipids from erythrocyte membranes were extracted with chloroform/methanol  method , and totPDQ-39 is a 39-item self-report questionnaire which assesses PD-specific health-related quality of life , 23. We t-test for between-group differences and paired t-test for the within-group differences. Statistical significance was declared when two-sided p < 0.05.Data were analyzed by using unpaired Our previous study confirmed by acid hydrolysis of lipids after PLA1 treatment that chromatographic peaks of ether phospholipids in human plasma contained some alkyl acyl phospholipids in addition to alkenyl acyl phospholipids  ; therefoAt the beginning of oral administration of plasmalogens, mean level of plasma ePE in PD was significantly low as compared to those of normal elderly controls. However, the level of plasma ePE increased rapidly after the ingestion of the capsules , and it As ether phospholipid, only ethanolamine ether phospholipid was detected in human erythrocyte membrane . Acid hyInitially, the relative composition of plsPE in erythrocyte membrane from PD was significantly low as compared to that of normal elderly . RelativThe oral administration of the ether phospholipids improved some clinical symptoms of PD concomitantly with increase of ether phospholipids in peripheral blood . The impThe present study showed increase of ether phospholipids in peripheral blood (plasma and erythrocyte membrane) after ingestion of purified ether phospholipids derived from scallop. The ether phospholipid in the erythrocyte membrane is confirmed to be ethanolamine plasmalogen . Plasmalα-synuclein dimerization in erythrocyte in PD has been indicated [There are indications that high oxidative stress may present in peripheral blood of PD . The incndicated . The decThe present trial showed that oral administration of purified ether phospholipids derived from scallop increased plasma and erythrocyte ether phospholipids in PD. Furthermore, some clinical symptoms of PD were improved concomitantly. Beneficial effects of the same ether phospholipids ingestion were also observed in patients with AD . The oraPhysiological mechanisms of the efficacy of such a small amount (1 mg/day) of ether phospholipid to AD and PD are not clear. One hypothesis is that newly administered plasmalogens and/or ether phospholipids may work through some receptors on cell membrane like hormones. Lipid rafts, cholesterol, and SM-rich microdomains on the cell membrane are considered to be associated with cell signaling . There aOn the other hand, there have been many reports that docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) relate closely to brain functions , 35. ScaThe capsule used in the present study contains alkyl acyl choline phospholipid (ePC) and CAEP in addition to ethanolamine plasmalogen (ePE) . It is pEffectiveness of plasmalogens to symptoms of PD was suggested by animal models of PD. It has been reported that plasmalogen precursor analog treatment reduced levodopa-induced dyskinesia in parkinsonian monkeys , and theIn summary, ethanolamine ether phospholipids (ePE) in plasma from PD and relative composition of ethanolamine plasmalogen (plsPE) of erythrocyte membrane in PD were significantly low as compared to those of age-matched normal controls. Oral administration of purified ether phospholipids derived from scallop for 24 weeks increased plasma ePE and erythrocyte plsPE to almost the normal levels and concomitantly improved some clinical symptoms of patients with PD. The results indicate the efficacy of oral administration of purified ether phospholipids derived from scallop to some nonmotor symptoms of PD.


Tumor necrosis factor-α (TNF) is a pro-inflammatory cytokine upregulated in many inflammatory diseases, and a potent inducer of endothelial cell-derived microparticle (EMP) formation. In this study, we identified the protein kinase PAK4 as a key regulator of the TNF-induced EMP release from human umbilical vein endothelial cells (HUVECs). TNF induces dose- and time-dependent EMP release and downregulation of PAK4 and upstream cdc42 in HUVECs. PAK4 suppression or inhibition of its kinase activity increases TNF-induced EMP release and apoptosis in HUVECs, while PAK4 overexpression reduces EMP release and apoptosis in TNF-stimulated cells. Collectively, these data indicate that PAK4 suppresses TNF-induced EMP generation occurring during apoptosis, and suggest that modulation of PAK4 activity may represent a novel approach to suppress the TNF-induced EMP levels in pro-inflammatory disorders and other pathological conditions. Microparticles (MP) are small pro-inflammatory vesicles released by various cells. MP can be generated by nearly every cell type during activation, injury or apoptosis , 2. TheyIn response to apoptotic and other stimuli, vascular endothelial cells can release endothelial microparticles (EMP) . Althougin vivo and in vitro EMP formation.Tumor necrosis factor-α (TNF) is a pro-inflammatory cytokine that has important roles in regulating cell proliferation and apoptosis, inflammation responses, and immune functions , 12. InjIn murine models of lung injury, increased levels of circulating EMP are accompanied by an inhibition of the cytoskeleton regulating protein PAK4 , 15, andIn this study, we analyzed the relationship between PAK4 and EMP formation in human umbilical vein endothelial cells (HUVECs). Our results indicate that PAK4 suppresses TNF-induced EMP generation that occurs during apoptosis in HUVECs.in vitro and in vivo. In vitro, the ability of TNF to induce EMP was analyzed in human umbilical vein endothelial cells (HUVECs) by flow cytometry using annexin V-FITC, which binds to phosphatidylserine exposed on EMP. As shown in First, we analyzed the ability of TNF to induce the EMP release Next, we investigated whether TNF induces EMP release also in vivo. Male C57BL/6 mice (10 weeks old) were randomly assigned to 5 groups (n=10) and treated 24 h with PBS or increasing doses of TNF . As shown in EMP shed by endothelial cells are associated with cytoskeleton disorders . Thus, PTo investigate how TNF down-regulates the PAK4 levels, HUVECs were treated with TNF alone, or with TNF + soluble TNFR1, the soluble part of TNF receptor 1 that interacts with TNF and inhibits its binding to TNFR1 on the cell membrane. After 6 h stimulation with TNF, the PAK4 expression decreased, both at protein and mRNA levels . HoweverTo investigate the role of PAK4 in TNF-induced EMP generation, we first inhibited PAK4 function in TNF-stimulated (100 ng/ml) HUVECs by PAK4 inhibitor PF-3758309 or PAK4 siRNAs. PF-3758309 is an ATP-competitive, small-molecule pyrrolopyrazole inhibitor, blocking the PAK4 kinase activity and downstream pathways . HUVECs Without TNF stimulation, PF-3758309 or PAK4 siRNAs treatment did not show any significant effect on the basal EMP release, compared to siRNA control  belongs to the serine/threonine kinase family, and serves as a target for the Rho GTPase cdc42 . PAK4 haAttenuated NF-κB survival signals may lead to a decrease in PAK4 expression. Our results indicate that NF-kB signaling might be involved in the TNF-induced downregulation of PAK4 in HUVECs. This is supported by previous studies that have suggested that PAK4 may act as a switch between NF-κB survival signaling and caspase-8-mediated apoptosis induced by TNF in hepatocarcinoma cells . DecreasIn conclusion, our data provide evidence that TNF induces a dose and time dependent release of EMP in HUVECs, by cdc42/PAK4 dependent pathway. Using gene expression and gene silencing, we identified PAK4 as a critical regulator of EMP generation by TNF. TNF downregulates the cdc42/PAK4 levels. Our data indicate that PAK4 suppresses the TNF-induced EMP generation and apoptosis in HUVECs through the TNFR1 signaling pathway. Since the increased EMP levels have been reported in multiple pro-inflammatory and pathologic conditions including sepsis , cardiovMale C57BL/6 mice  were kept under a conventional 12 hour light-dark cycle in a temperature-controlled room with free access to water and food. Mice were randomly assigned to 5 groups (10 mice per group) and treated with PBS or different doses of TNF  for 24 hours. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the Shanghai Tongji University and performed in accordance with the National Institutes of Health Guidelines for the use of experimental animals.The primary human umbilical vein endothelial cells (HUVECs) were purchased from ATCC (PCS-100-010) and were grown in vascular cell basal media supplemented with endothelial cell growth kit components.Primary antibody against PAK4 was purchased from Proteintech. IκBα antibody was from Cell Signalling. Antibodies against tubulin, cdc42, β-actin, and IκBα, phalloidin-FITC, and horseradish peroxidase (HRP)-conjugated secondary antibody were purchased from Sigma. Alexa 488 and 568-conjugated secondary antibodies were from Molecular Probes. Caspase-8 and active caspase-8 antibodies were from Cell Signalling. Purified TNF protein and sTNFR1 (soluble part of TNFR1) were purchased from Immunotools ; thrombin was from Sigma. The PAK4 inhibitor PF-3758309 (Pfizer) was used at 10 μM.5 cells in 12-well plates) were collected by centrifugation (5000g for 5 minutes). EMP was measured in supernatants by flow cytometry . 10 μl of centrifuged media or sera were labelled by annexin V-FITC and 1 μm latex beads  were used as gating parameters. Particles less than or equal to 1 μm were defined as EMPs. Number of EMP was calculated as described5.Untreated or TNF treated HUVECs (7x10PAK4 short interfering (si) RNA duplexes  or a nonsilencing siRNAs as negative controls (Life Technologies). At 48 hours after siRNA transfection, cells were harvested to determine the level of PAK4 proteins by immunoblotting, or were stimulated with 100 ng/mL TNF.For transfection, HUVEC cells were seeded at 85%~90% confluency and allowed to adhere overnight. Small interfering RNA (siRNA), GFP-PAK4, or GFP-PAK4KM plasmid were transfected using Lipofectamine 2000 (Invitrogen) as described by the manufacturers. For studies of RNA interference, transfections were performed with Human PAK4 DNA fragment was amplified from cDNA bank by PCR and cloned into pGEMTeasy (Promega) vector. GFP-PAK4 was cloned into the EcoRI/SalI site of EGFP-C2 vector. The GFP fusion protein was generated to carry GFP at its N-terminus. GFP-PAK4KM was cloned into the EcoRI/SalI site of EGFP-C2 vector. This PAK4 kinase mutant contains a lysine to methionine mutation at amino acid 350. The mutation was generated using site-directed mutagenesis kit (Stratagene).HUVECs were cultured to subconfluence on 8 chamber glass slides . After treatment, they were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) and permeabilized with 0.1% Triton X-100. Actin cytoskeleton was stained with Phalloidin-FITC. Cells were incubated with 1:400 diluted polyclonal anti-PAK4 antibody, followed by a sequential incubation with a 1:1000 diluted Alexa 488 or 568-conjugated secondary antibody. Nuclei were counter stained with DAPI (Sigma-Aldrich). The cells were imaged using a ZEISS 780 inverted confocal laser scanning microscope  and analyzed by using Zen software.Cells were washed with cold PBS and lysed with 1 ml RIPA buffer  containing protease inhibitor cocktail (Roche) on ice. Homogenized samples were microcentrifuged at full speed for 15 min at 4°C. The supernatants were subjected to SDS-PAGE. Separated proteins were transferred onto nitrocellulose membrane and probed with primary antibodies and HRP-conjugated secondary antibodies.-ΔΔCt method with GAPDH as the internal reference. Samples were analysed in triplicate. The primer sequences used were the following. PAK4-F: ATGTGGTGGAGATGTACAACA, PAK4-R: GTTCATCCTGGTGTGGGTGAC, GAPDH-F: GGAGCGAGATCCCTCCAAAAT, GAPDH-R: GGCTGTTGTCATACTTCTCATGGTotal RNA was extracted from cultured HUVECs using Trizol solution (Invitrogen) and reverse transcribed into cDNA with a Reverse Transcriptase kit (Invitrogen) according to manufacturer’s instructions. The expression of PAK4 were detected using a TagMan mRNA assay kit (Biosystems). The relative expression was determined by the 2Cells were fixed with the Cytofix/Cytoperm fixation and permeabilization solution kit (BD Pharmingen), stained with FITC-conjugated anti-caspase-3 antibody (BD Pharmingen), and analyzed using CyAn flow cytometer . Data were evaluated using Flowjo .Results are expressed as mean ± SD. The statistical significance was assessed using analysis of variance (ANOVA), followed by Student’s t-test. A P value of <0.05 was considered significant.


ACM1 transgenic plants displayed albino cotyledons but normal true leaves, while knock-out of the ACM1 gene in seedlings was lethal. Fluorescent protein analysis showed that ACM1 was specifically localized within chloroplasts. PEP-dependent plastid transcript levels and splicing efficiency of several group II introns were seriously affected in cotyledons in the RNAi line. Furthermore, denaturing gel electrophoresis and Western blot experiments showed that the accumulation of chloroplast ribosomes was probably damaged. Collectively, our results indicate ACM1 is indispensable in early chloroplast development in Arabidopsis cotyledons.Chloroplasts cannot develop normally without the coordinated action of various proteins and signaling connections between the nucleus and the chloroplast genome. Many questions regarding these processes remain unanswered. Here, we report a novel P-type pentatricopeptide repeat (PPR) factor, named Albino Cotyledon Mutant1 (ACM1), which is encoded by a nuclear gene and involved in chloroplast development. Knock-down of  Chloroplasts are well known as the most important organelle in higher plants. Beyond photosynthesis, chloroplasts are also the main sites of the biosynthesis of fatty acids, hormones, amino acids, and metabolites, and the assimilation of nitrate ,2. Becausoc1 and soc2 . AC. (2008) .2, 5 mM DTT, 0.5 M sucrose, 50 mM Tris MES, 10 mM EDTA, and protease inhibitor cocktail, pH 8.0). Total protein samples were separated by 10% SDS-PAGE and then transferred onto PVDF membranes. The antibodies used in this study were according to Xiao et al. (2012) [Total protein was separated from 7-day-old cotyledons and 14-day-old true leaves of WT and RNAi plants with NB1 buffer (1 mM MgCl. (2012) , and the. (2012) .The signp-value of 0.05 was considered significant .Data were analyzed using Graphpad Prism  and Microsoft Excel 2016. Values are expressed as mean ± SD. Two-tailed unpaired Student’s t-tests were used to evaluate differences between WT and mutant or RNAi line. A Arabidopsis. ACM1 has 26 PPR motifs and belongs to the P subfamily, which is localized within the chloroplast. Down-regulation of ACM1 led to the abnormal chloroplast structure and reduction of chlorophyll. Compared with WT, the transcription levels of PEP-dependent genes were decreased in cotyledons of the ACM1-RNAi line, and splicing of several group II introns was disturbed. Furthermore, the accumulation of chloroplast ribosomes was probably defective in the ACM1-RNAi cotyledons.Functional chloroplast development, together with gene expression and regulation, are fundamental cellular activities that require coordination between the nucleus and the chloroplast. In this study, we identified a novel factor that affected the chloroplast development in cotyledons of