Relapsing-remitting multiple sclerosis (RRMS) is a demyelinating disease in which pathogenesis T cells have a major role. Despite the unknown etiology, several risk factors have been described, including a strong association with human leukocyte antigen (HLA) genes. Recent findings showed that HLA class I-G (HLA-G) may be tolerogenic in MS, but further insights are required. To deepen the HLA-G role in MS inflammation, we measured soluble HLA-G (sHLA-G) and cytokines serum level in 27 patients with RRMS at baseline and after 12 and 24 months of natalizumab (NTZ) treatment. Patients were divided into high (sHLA-G>20 ng/ml), medium (sHLA-G between 10 and 20 ng/ml), and low (sHLA-G <10 ng/ml) producers. Results showed a heterogeneous distribution of genotypes among producers, with no significant differences between groups. A significant decrease of sHLA-G was found after 24 months of NTZ in low producers carrying the +3142 C/G genotype. Finally, 83.3% of high and 100% of medium producers were MRI-activity free after 24 months of treatment, compared to 63.5% of low producers. Of note, we did not find any correlation of sHLA-G with peripheral cell counts or cytokines level. These findings suggest that serum sHLA-G level may partly depend on genotype rather than peripheral inflammation, and that may have impacted on MRI activity of patients over treatment. An interplay of immune mediators contributes to MS pathogenesis, with a crucial role of T lymphocytes . About 2Sardinia .Recently, the non-classical HLA histocompatibility antigen G (HLA-G) has been linked with MS susceptibility, particularly with the RRMS form, in a study performed within the Italian population , 8. HLA-in vivo: it was demonstrated that the genotypes 14 bp insertion/insertion (ins/ins) and +3142 G/G determine a low production of HLA-G compared to 14 bp insertion/deletion (ins/del) or to the genotypes deletion/deletion (del/del) and +3142 C/G or C/C (The human leukocyte antigens G (HLA-G) gene is located on chromosome 6 in the major histocompatibility complex (MHC) locus . The expG or C/C , 25–27. G or C/C . FurtherG or C/C , 29. On G or C/C . These fDespite these interesting findings, our knowledge about the role of different HLA-G genotypes in MS pathogenesis and in MS treatment outcomes is still very limited. With the aim to shed light on this topic, we investigated sHLA-G and its genotypes in the serum of patients with RRMS before and after 12 and 24 months of treatment with natalizumab (NTZ), a monoclonal antibody that blocks the alpha4-integrin, or very late antigen-4 (VLA-4), expressed on the surface of T lymphocytes, preventing their migration into the CNS. NTZ is an effective second-line immunomodulant treatment for RRMS, usually applied when first-line treatments fail. NTZ is known to impact immune cell populations, especially leading to a reversible increase of peripheral cell counts due to its mechanism of action , 33. WitClinicalTrials.gov Identifier: NCT01058005). Patient characteristics are reported in A total of 27 patients were enrolled at the Department of Neurology II at Careggi University Hospital, Florence, Italy. Patients were diagnosed with RRMS according to McDonald criteria  and sharThe study was performed according to the Declaration of Helsinki. Written informed consent was signed by all patients involved in the study, and approved by the Local Ethical Committee (#CEAVC12745).During NTZ, patients with RRMS underwent clinical follow-up every 6 months and at least yearly brain MRI, according to clinical practice. All patients with RRMS were evaluated for the presence of anti-JCV and anti-NTZ antibodies in the serum. Clinical MRI and laboratory data were retrospectively collected; occurrence of relapses and MRI activity (new T2 lesions and/or Gd+ lesions) were recorded. Disability was assessed by EDSS score. Additional clinical evaluations were performed upon patients' request in the event of new neurological symptoms or any other neurological issues.For each patient, the whole peripheral blood (PB) was collected in heparin-containing tubes. Peripheral blood mononuclear cells (PBMCs) were collected by density gradient centrifugation by Pancoll  at 1,500 rpm, RT, for 30 min within 2 h from PB collection. To collect serum, 10 ml of blood were drawn in serum-separated tubes (SSTs) according to the same time schedule as performed for PB. Serum was then separated from blood by centrifugation at 3,000 rpm, RT, for 10 min, then aliquoted and stored in −80°C freezer until used.Immunophenotype of patients with RRMS during NTZ was analyzed as routine from fresh whole blood upon erythrocyte lysis  by labeling cells with the following panel of fluorescent monoclonal antibodies: anti-CD3 FITC (clone: clone SK7), anti-CD16 PE clone B73.1 and anti-CD56 PE (clone NCAM16.2), anti-CD45 PerCP-Cy5.5 (clone 2D1), anti-CD4 PE-Cy7 (clone: SK3), anti-CD19 APC (clone: SJ25C1), and anti-CD8 APC-Cy7 (clone: SK1). Data acquisition was performed using a 3-laser 8-color flow cytometer ; data were analyzed using FACSDiva software version 8.0.1. (BD Biosciences).The concentration of sHLA-G in serum samples was measured in triplicate by ELISA as previously described , 35, 36 Genomic DNA (gDNA) was isolated from PB by the Nucleon Bacc 3 kit  according to the manufacturer's protocol. The HLA-G polymorphism 14 bp ins/del was genotyped by PCR as previously described . BrieflySerum from each patient was analyzed by Bioplex device (Biorad) using Milliplex assay (Merck Millipore) following the manufacturer's protocol for the determination of the following 17 cytokines and chemokines: IL1α, IL1β, IL2, IL4, IL6, IL8, IL10, IL12p40, IL12p70, IL17, IL23, IFNγ, TNFα, GM-CSF, CXCL10, CXCL13, and MMP9. Cytokines and chemokines concentrations were reported in pg/ml. The sensitivity limit was 1.0 pg/ml.post-hoc Tukey's test was used to compare patient subgroups  for peripheral absolute cell counts, serum cytokines level, HLA-G genotype distribution, and sHLA-G level over 24 months of NTZ treatment. Non-parametric Kaplan–Meier estimator or chi-square test were used to compare patient subgroups for MRI disease activity by evaluating the cumulative proportion of survival percentage. Pearson correlation coefficient (r) was calculated to evaluate the correlation between variables. Statistical significance was considered when p-value < 0.05.Statistical analyses were performed by IBM SPSS statistic 20 (IBM Corp) and by GraphPad Prism v.6 . One-way ANOVA followed by p-value = 0.04) and T24  compared to T0; in T lymphocytes at T24 compared to T0 , T6 , and T12 ; and in NK cells at T6 , T12 and T24  compared to T0. The increase of total leukocytes and of CD4 and CD8 absolute counts over time was not statistically significant. CD4/CD8 ratio was not altered. On serum of patients with RRMS, we determined the level of 17 cytokines and chemokines at T0, T12, and T24 of NTZ, to evaluate fluctuations in the peripheral inflammatory profile of patients over treatment. In our analysis, IL1α, IL1β, IL2, IL17, IFNγ, IL10, and GM-CSF were below the detection limit (1.0 pg/ml) at all time points, therefore excluded from the analysis. CXCL10, TNFα, and IL2 showed significant variation over time: specifically, we found a significant decrease of CXCL10 at T24 compared to T0  and T12 ; a significant increase of TNFα at T24 compared to T0  and T12  and a significantly higher level of IL2 at T24 compared to T0 and T12   and at 6, 12, and 24 months of NTZ treatment  and included the evaluation of the percentage and absolute cell count of total T, B, natural killer (NK), lymphokine-activated killer (LAK) cells, and CD4/CD8 ratio. Flow cytometry gating strategy and analysis of cell subsets are reported in  = 0.01) . To sum We evaluated the levels of sHLA-G in serum samples of 27 patients with RRMS at baseline (T0) and at T12 and T24 of treatment with NTZ. Based on sHLA-G concentration (ng/ml), we considered patients as low producers with a serum sHLA-G level below 10 ng/ml at T0 and as high producers with a serum sHLA-G level up to 20 ng/ml at T0. With an sHLA-G level between 10 and 20 ng/ml, patients were classified as medium producers. Therefore, the 27 patients with RRMS were subdivided into 6 high sHLA-G producers, 7 medium producers, and 14 low producers.p-value < 0.0001) higher in high producers compared to low and medium producers at baseline (T0)  increased in high producers compared to low producers. No significant differences were detected between groups at T24, and across timepoints within each group .We found that the serum sHLA-G level is significantly (ine (T0) . At T12,We next genotyped patients with RRMS and divided them according to the results. The group including the 14 bp HLA-G genotype was subdivided based on the polymorphism into ins/ins (I/I), ins/del (I/D), and del/del (D/D) subgroups. The group including the +3142 genotype was subdivided based on the SNP C>G in C/C, C/G, and G/G subgroups.N = 6) are equally divided between patients carrying the 14 bp I/D and D/D polymorphism. Medium producers (N = 7) are mainly represented by the 57.14% of patients carrying the 14 bp I/D genotype, followed by the 28.57% with the D/D genotype and the remaining 14.29% with the I/I one. On the other hand, the 50% of low producers (N = 14) were characterized by the 14 bp I/I genotype, whereas the 21.43% and the 28.57% with the 14 bp I/D and D/D genotypes, respectively  decrease of sHLA-G at T24 compared to T0 in patients carrying the +3142 C/G genotype and a significant (p = 0.038) decrease at T12 of +3142 G/G patients compared to C/G at T0  (Magnetic resonance imaging (MRI) activity-free survival was 83.3% in high sHLA-G producers, 63.5% in low producers, and 100% in medium producers; such difference was not statistically significant (= 0.211) .In this work, we evaluated the sHLA-G production and genotype in patients with RRMS during NTZ treatment. We measured serum sHLA-G level and correlated this feature with treatment outcome in terms of disease relapses and MRI activity. Our data suggest that low sHLA-G producers are more at risk of showing disease activity during treatment compared to high and medium producers.Soluble forms of HLA-G (sHLA-G) molecule is known for contributing to maintaining the immune tolerance in both health and disease  and has The characterization of patients by sHLA-G genotype revealed that I/D and D/D genotypes are mostly represented in high and medium producers, while I/I genotype is not present among high producers , in accoWhen investigating sHLA-G concentration across patients based on their genotypes, we did not observe any significant variation among high and medium producers , whereasThe analysis of serum cytokines and chemokines in the RRMS cohort showed a significant decrease in CXCL10 at T24 and a significant increase in TNFα and IL2 at T24 . The incWe then evaluated the association between serum sHLA-G production and patients' outcomes to NTZ treatment in terms of relapse rate and MRI disease activity over 24 months . ResultsTo sum up, we found that sHLA-G significantly decreases over NTZ treatment in low sHLA-G producers carrying the +3142 C/G genotype  and thatThe raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The study was performed in accordance with the Declaration of Helsinki, reviewed and approved by the Local Ethics Committee of the Tuscany region, Centre Area (#CEAVC12745). The patients/participants provided their written informed consent to participate in this study.RA experiments , manuscript writing, and data analysis. RR, DB, and VG experiments (patients HLA-G typization and sHLA-G quantification) and data analysis. AM manuscript writing and clinical data analysis. EB and AA PBMCs and serum collection, cytokines assay, and data analysis. AC data analysis. BP flow cytometry analysis of patients' immunophenotype. AR clinical data collection. LM critical discussion on clinical data. EF MRI data collection. CB study conceptualization and manuscript writing revision. All authors contributed to the article and approved the submitted version.This work was supported by FISM 2019 grant code 2019/R-Single/004.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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Here we show that i.v. injected anti-HIV duoCAR T cells, generated using a clinical-grade anti-HIV duoCAR lentiviral vector, localized to the site of active HIV infection in the spleen of humanized mice and eliminated HIV-infected PBMCs. CyTOF analysis of preinfusion duoCAR T cells revealed an early memory phenotype composed predominantly of CCR7+ stem cell–like/central memory T cells (TSCM/TCM) with expression of some effector-like molecules. In addition, we show that anti-HIV duoCAR T cells effectively sense and kill HIV-infected CD4+ T cells and monocytes/macrophages. Furthermore, we demonstrate efficient genetic modification of T cells from PWH on suppressive ART into anti-HIV duoCAR T cells that subsequently kill autologous PBMCs superinfected with HIV. These studies support the safety and efficacy of anti-HIV duoCAR T cell therapy in our presently open phase I/IIa clinical trial (NCT04648046).HIV-specific chimeric antigen receptor–T cell (CAR T cell) therapies are candidates to functionally cure HIV infection in people with HIV (PWH) by eliminating reactivated HIV-infected cells derived from latently infected cells within the HIV reservoir. Paramount to translating such therapeutic candidates successfully into the clinic will require anti-HIV CAR T cells to localize to lymphoid tissues in the body and eliminate reactivated HIV-infected cells such as CD4  Chimeric antigen receptor–T cell (CAR T cell) therapy has revolutionized the immunotherapy landscape following the development and clinical successes of 4 FDA-approved CAR T cell therapies to treat refractory B cell malignancies –4. RecenClinical trials of first-generation HIV–specific CAR T cells utilizing the CD4 receptor extracellular region to target the HIV envelope (Env) glycoprotein demonstrated that, while they displayed long-term persistence and safety in PWH, they had limited efficacy in controlling HIV –23. This+ T cells and persists, in part, due to clonal expansion of cells harboring intact provirus and continued infection by low levels of virus, despite effective ART , termed duoCAR  encoding 2 distinct anti-HIV CAR molecules  to that of a WPRE– duoCAR LV that used a virus-derived and modified MND  promoter in lieu of the MSCV promoter  and CD8+ T cells to quantify duoCAR expression on CD4+ and CD8+ T cells, respectively. Overall, LV titers (https://doi.org/10.1172/jci.insight.161698DS1). The FDA-recommended threshold of < 5 vector copy number (VCN)/transduced cell was achieved using the MND-ΔW duoCAR LV and high CAR transduction efficiency in MND-ΔW duoCAR T cells  from the original murine stem cell virus–regulated (MSCV-regulated) D13 LV (MSCV+W) used to generate D13 duoCAR T cells because it contained an intact Protein X open reading frame of unknown function . We comp MND-ΔW; . The MND MND-ΔW;  and driv MND-ΔW; , 35. We V titers  and tran T cells .Renilla luciferase reporter and the HIV-1 Env glycoprotein from either Clade A (isolate 396-F1_F6_20), B (isolate CH077), or C (isolate Du151.2) HIV-1 strains , a surrogate for HIV-infected cells, to determine their ability to persist and durably eliminate Env+GFP+ target cells. The anti-HIV duoCAR T cells displayed a significant and durable ability to eliminate the Env+GFP+ target cells  after repeated challenge during the 17-day coculture period  mouse model of intrasplenic HIV-1 infection (hu-spl-PBMC-NSG) by intrasplenically coinjecting CAR T cells with HIV-1 spinoculated PBMCs (1 × 107), followed by quantitation of HIV infection within the PBMCs of the spleen 7 or 30 days later  were i.v. injected into the tail veins of mice following intrasplenic injection of syngeneic HIV-LucR spinoculated PBMCs (1 × 107 cells)  T cells (< 0.05). The spleens from 5 of 6 untreated mice (HIV+ PBMC cohort) and 4 of 6 UTD T cell–treated mice displayed high levels of cell-associated total HIV-1 DNA  (Anti-HIV duoCAR T cells (2 × 107 cells) . Preinfu T cells . Mice we T cells < 0.05. ctively) . Four ofctively) . We postAll of the anti-HIV duoCAR T cell–treated mice survived the duration of the study (17-18 days) and did not display significant body weight changes or atypical behavior. The engraftment and persistence of circulating MND-ΔW duoCAR T cells in mouse blood and organs after i.v. injection was assessed at the study end point (D17 and D18) using a highly sensitive qPCR assay capable of detecting 1 copy/μL/reaction. MND-ΔW duoCAR T cells engrafted and persisted in the peripheral blood of all the HIV-infected mice evaluated 17–18 days after infection ; this alSCM or TCM phenotype are highly desirable, given their propensity for memory formation, self-renewal, persistence, lower potential to induce cytokine release syndrome (CRS), and improved therapeutic efficacy (10 TU/mL) selected for clinical trials (MND-ΔW) generated under GMP conditions. + T cells , CD4+ and CD8+ T cell subsets , and duoCAR+ CD8+ T cells . The frequency of CD4+ T cells was ~3-fold higher than CD8+ T cells  in the final products, indicating no impact of HIV infection on CD4+ T cells despite using an ARV-free manufacturing process. The product passed all release criteria, and we generated 600–800 million CAR+ T cells with a total cell expansion capacity of > 12-fold . VCN was below the FDA-recommended 5 copies per transduced cell for both donor products . We note that it is not unusual that the lower CAR transduction rate on the T cell’s surface (50% as measured by the less sensitive flow cytometry method) may be the result of transduction of multiple copies of vector DNA per transduced cell (as measured by the more sensitive qPCR method) and/or due to transcriptional or posttranscriptional silencing of a subset of integrated vector transgenes. Using VCN reference standards developed by our group  donor apheresis material using an equivalent manufacturing process and GMP-grade critical reagents  used to manufacture duoCAR T cells on the CliniMACS Prodigy. Among the 5 donors tested , we confirmed that anti-HIV duoCAR T cells were predominately enriched for CCR7+ TSCM and TCM cells  subset of duoCAR-T+ cells  using a metal-conjugated antibody directed against the m36.4-CAR, which reliably detected duoCAR-T+ cells  at simil+ cells . Anti-HICM cells , which istemness , and lowstemness . The difssed PWH . Of noteet cells . Cytolytet cells , with a T+ cells . These r+ T cells, HIV-infected monocytes/macrophages represent a component of the HIV reservoir in ART-suppressed PWH that persist due to their resistance to killing by cytotoxic lymphocytes (CTLs)  (1 × 106 IU/mL). After 3 days of culture to establish infection, the HIV-infected PBMCs, CD4+ T cells, or monocytes (1 × 105 cells/well) were either left untreated or treated with added autologous UTD T cells or anti-HIV duoCAR effector T cells (effector/target [E:T] ratio of 1:1). Three days later, HIV infection was quantified by LucR activity in the cell lysates. As shown in + T cells by 92.6% . This result demonstrates the sensitive recognition and robust killing of HIV-infected monocytes, a component of the HIV reservoir, by anti-HIV duoCAR T cells.In addition to HIV-infected memory CD4s (CTLs)  and are s (CTLs) . Therefoby 92.6% , as comp+ T cells — particularly their cytotoxic capacity (+ T cells), viability (>90% viable T cells), and transduction efficiencies of > 50% for LV-modified CD4+ T cells  and > 30% for LV-modified CD8+ T cells  with less than 5 vector copies per cell.Despite effective control of HIV replication in ART-treated PWH, the chronic activation and inflammation induced by HIV infection compromises the function of CD8capacity . Consequcapacity , anti-HI+ T cell–depleted PBMCs from these donors were superinfected with a HIV-LucR IMC expressing the BaL Env and cocultured with autologous MND-ΔW duoCAR T cells either immediately following the initiation of HIV-LucR infection  than the MSCV promoter to control CAR transgene expression. We demonstrated that the MND-ΔW duoCAR T cells potently eliminated HIV-infected CD4P< 0.05). This finding is consistent with our previous work, which showed durable and potent in vivo suppression of HIV-1 infection and prevention of CD4+ T cell depletion for 30 days in humanized mice treated with anti-HIV duoCAR T cell therapy  even when viremia is controlled by ART  and suppress productive HIV infection in vivo without signs of premature CAR T cell exhaustion or lack of persistence. Our previous studies have established the resistance profile of anti-HIV duoCAR T cells to HIV infection and the lack of off-target effects on human cells that do not express the HIV-1 Env glycoprotein  is designed to investigate anti-HIV duoCAR T cell therapy as a potential HIV remission strategy, using ART interruption to enable reactivation of latent reservoir cells and increased HIV-1 Env expression on their surface to render them susceptible to CAR-mediated elimination. CAR T cells may be limited in their ability to eliminate latent infected cells in the absence of reactivation.+ T cell depletion, and has the ability to rapidly assess anti-HIV CAR T cell efficacy, but it does so without the possible confounding effects of a reconstituted endogenous T cell immune response  except for Guide for the Care and Use of Laboratory Animals .All the mouse studies were approved by the IACUC and the IRB at Albert Einstein College of Medicine in compliance with the human and animal experimentation guidelines of the U.S. Department of Health and Human Services and in adherence to the KAG, AR, and HS contributed equally to this work. KAG, AR, RLR, JWB, HG, and BD conceptualized and planned the study. KAG, AR, RLR, and HG wrote the original draft of the manuscript. KAG designed, constructed, and characterized the new anti-HIV duoCAR vectors. YW characterized the anti-HIV duoCAR vectors in HIV seronegative donors under the supervision of KAG. AR, HS, DL, VC, JW, and AB conducted the in vitro and in vivo HIV challenge/CAR T cell killing studies. CO and JCK provided the replication competent HIV-LucR IMC. SGD supervised recruitment of PWH in the study. RLR led CyTOF experiments performed by LZ and YYW. MDL and JSR provided oversight for anti-HIV duoCAR T cell manufacturing runs performed by SKB. DSD developed the anti-HIV binders (mD1.22 and m36.4) used to generate anti-HIV duoCAR T cells. KAG, HG, YX, SGD, DSD, CO, JCK, JWB, RO, and BD critically reviewed and edited the manuscript.


In mitosis, accurate chromosome segregation depends on kinetochores that connect centromeric chromatin to spindle microtubules. The centromeres of budding yeast, which are relatively simple, are connected to individual microtubules via a kinetochore constitutive centromere associated network (CCAN). However, the complex centromeres of human chromosomes comprise millions of DNA base pairs and attach to multiple microtubules. Here, by use of cryo-electron microscopy and functional analyses, we reveal the molecular basis of how human CCAN interacts with duplex DNA and facilitates accurate chromosome segregation. The overall structure relates to the cooperative interactions and interdependency of the constituent sub-complexes of the CCAN. The duplex DNA is topologically entrapped by human CCAN. Further, CENP-N does not bind to the RG-loop of CENP-A but to DNA in the CCAN complex. The DNA binding activity is essential for CENP-LN localization to centromere and chromosome segregation during mitosis. Thus, these analyses provide new insights into mechanisms of action underlying kinetochore assembly and function in mitosis. In eukaryotic cells, kinetochores, which are sophisticated molecular machines for cell division fate decisions, constitute a dynamic link between kinetochore microtubule attachment and spindle checkpoints, a signaling cascade that prevents chromosome segregation before completion of bi-orientation during cell division. In fact, early electron microscopic (EM) analyses have indicated that the morphology of the outer kinetochore structure changes dynamically during mitotic progression4, suggesting that the kinetochore is dynamically assembled and functionally matured in mitosis. However, the molecular details underlying kinetochore assembly and its functional coupling to chromosome movements remain less characterized.Correct chromosome segregation relies on the centromere, a specialized chromatin domain present throughout the cell cycle that acts as a platform on which transient assembly of the kinetochore occurs during mitosisNuc) is surrounded by a group of 16 proteins, the constitutive centromere-associated network (CCAN) and organized in various stable subcomplexes7. CENP-C and another CCAN subunit, CENP-T, provide a platform for assembly of the outer layer of kinetochores8. The outer kinetochore comprises three subcomplexes, KNL1, MIS12, and NDC80, which are collectively referred to as the KMN network9. In addition to its regulatory functions, the KMN network, through its NDC80 complex, provides a site for accurate segregation of chromosomes to the daughter cells11. Unlike yeast, in which the molecular architecture of the kinetochore has been extensively defined14, the molecular architecture of metazoan CCANs and how multiple CCAN modules constitute a functional human kinetochore are less clear as the regional kinetochores of metazoans are generated from multiple copies of the CCAN and KMN to project large disk-like structures15. In particular, how CCAN components are organized and integrated with centromeric nucleosome and the KMN network for spindle microtubule interactions in mitosis remain unclear16.The CENP-A nucleosome  analysis of a human CCAN complex reconstituted in vitro with purified components. Interestingly, CENP-LN in CCAN complex forms a channel-like structure that binds to centromeric linker DNA. Our structure and biochemical analyses revealed a surprising link between CENP-LN binding to DNA in the context of CCAN and their functional relevance during mitotic chromosome segregation in mitosis.Since metazoans have evolved elaborate regional centromere and spindle checkpoint machinery to ensure faithful chromosome segregation in mitosisNuc wrapped with 147 bp of DNA using the Widom 601 sequence , assessed its impact on nucleosome integrity. As shown in Supplementary Fig. 4E; K270E and K296E of CENP-N2E) followed by SEC. As shown in Supplementary Fig. 4EN2E mutant did not interfere with assembly of CCAN judged by its elution profile which is similar to that of CCAN complex with wild-type CENP-LN. We further assessed CCAN binding to DNA using electrophoretic mobility shift assay (EMSA) and confirmed that the CENP-L4EN2E mutant was insufficient for mobilizing free DNA compared to wild-type CCAN  to assess the binding efficiency of CENP-LN to DNA. As shown in Fig. ΔCT complex with the RG-loop mutant CENP-AR80A-G81A nucleosome was indistinguishable from that for the wild-type CENP-A nucleosome, indicating that CENP-N binds poorly to the RG-loop of the CENP-A nucleosome in the CCAN context. However, the elution profile of DNA binding-deficient CCANΔCT  was distinctly different from that of wild-type CCANΔCT  and CENP-N2E (K270E/K296E) are apparently located to interphase centromere  as previously reported28. Consistent with previous observation, localization of CENP-NE3K/E7K and CENP-NE3A/E7A mutants to the centromere of interphase cells was significantly reduced  in complex with CENP-ANuc revealed that CENP-N recognize CENP-ANuc through the interaction with RG-loop of CENP-A29. Our structural and biochemical analyses show that the RG-loop of CENP-A is required for CCAN loading but not sufficient for CCAN localization to centromere and function in mitosis. Thus, the information from these structures suggests that the interaction of CENP-LN alone with CENP-ANuc is different from that of CENP-LN in CCAN complex.Our study of the human CCAN–DNA interaction defines the molecular contacts between the fundamental module of the kinetochore and DNA. In both CCAN and CCAN–DNA complex structures, CCAN adopts a ‘b’ shaped structure which is different from the structure of yeast CCAN32. Furthermore, CENP-LN adopts a more compact conformation of the human CCAN complex compared to their yeast counterparts. In addition, DNA was bound in the central pore of the closed structure of the human CCAN complex. Although human CENP-TWSX is located adjacent to CENP-OPQUR and CENP-HIKM, it is possible for human CENP-TWSX to form a nucleosome-like structure even in the intact CCAN complex similar to that of chicken CENP-TWSX alone25.Our cryo-EM analyses of the CCAN–DNA complex has revealed structure-functional relationship between the human and yeast CCAN complexes  was cloned into pFastBac1 with a glutathione-S-transferase (GST)-tag in its N-terminus to generate the CENP-LNC180–545 complex by co-infection with CENP-LN P3 virus in Sf9 insect cells. The plasmid constructed for CENP-H and -K was similar to that for CENP-LN. For expression of the CENP-HIKM complex, CENP-HK P3 and CENP-HIKM P3 virus were co-infected into Hi5 insect cells as previously reported15. To generate the CENP-OPQUR subcomplex, CENP-O, CENP-P, and CENP-Q were cloned into Multi-Bac vector pACEBac1 with a 6× His-tag fused to the C-terminus of CENP-Q, and CENP-U and CENP-R were cloned into the pIDC plasmid, then the two plasmids were fused by the Cre-LoxP reaction. For expression of the CENP-TWSX complex, the genes encoding full-length human CENP-T and CENP-W were first cloned into Multi-Bac vector1 pACEBac1 (Invitrogen) with a 6× His-tag fused to the N-terminus of CENP-T, and then CENP-S and CENP-X were cloned into Multi-Bac vector1 pIDC with a 6× His-tag fused to the N-terminus of CENP-S. Subsequently, the two plasmids were fused by the Cre-LoxP reaction. The baculoviruses for expression of CCAN subcomplexes were prepared for expression using the Bac-to-Bac® Baculovirus Expression System. We used the baculoviral stock to infect Sf9 insect cells for expression of the CENP-LN, CENP-OPQUR, and CENP-TWSX subcomplexes at 27 °C for 48~72 h. To obtain more of the CENP-HIKM subcomplex, we co-infected the P3 baculoviruses of CENP-HIKM and CENP-HK with a 4:1 volume ratio into Hi5 insect cells. All plasmids were sequenced for verification.Full-length CENP-N, with a 6× His-tag fused to its C-terminus, and CENP-L were cloned into a Multi-Bac vector pACEBac1 (Invitrogen) or a pIDC vector, respectively. Then, two plasmids were fused by a Cre-LoxP reaction; expression of the CENP-LN complex was assessed using a MultiBacEcoRI and BamHI and cloned into pLVX-EGFP-C1 vector, respectively. CENP-A was cloned by PCR and inserted into p3×FLAG-Myc-CMV-24 expression vector (Sigma). To generate siRNA-resistant CENP-L constructs, two synonymous mutations were generated (R306R/V307V). To generate sgRNA-resistant CENP-N plasmid, the corresponding sgRNA PAM sequence in cDNA was eliminated through a synonymous mutation . All the mutations were generated by Mut Express II Fast Mutagenesis Kit (Vazyme) according to the manufacturer’s instructions. And all plasmids with the desired insertions or mutation were sequenced at General Biosystems.To construct pLVX-EGFP-CENP-N or CENP-L plasmid, the CENP-N/L cDNA was amplified by PCR and digested by 180–545-LN and CENP-LN complexes were purified by size-exclusion chromatography (SEC) using a Superdex 200 (10/300) increase  column with SEC buffer -phosphine (TCEP)). CENP-OPQUR and CENP-TWSX complexes were purified with a Mono S 5/50 GL column  with IEX buffer 1 ; and 50 mM HEPES-Na, pH 7.5; 1 M NaCl; 1 mM DTT), respectively, and further purified by a Superdex 200 (10/300) increase  column with SEC buffer. In contrast, purification of the CENP-HIKM complex was accomplished with a Mono Q 5/50 GL column  and IEX buffer 2  and further purification by SEC using the same column and SEC buffer with other subcomplexes. Peak fractions of all protein samples purified by SEC were collected and stored in liquid nitrogen for complex assembly.Sf9 and Hi5 cells were used for expression , after which the cells were harvested by centrifugation and suspended in lysis buffer. Cells were lysed with high-pressure in lysis buffer ) supplemented with 2 μM pepstatin A, 10 μM leupeptin, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride. All protein samples were purified by nickel-nitriloacetic acid  affinity chromatography. Then CENP-C42. The genes encoding full-length human H2A-H2B and CENP-A-H4 were cloned into a pET28K and a pETDuet-1 vector, respectively. The two plasmids were co-transformed into E. coli Rosetta2 (DE3) cells, which were plated on agar containing 50 μg/mL kanamycin and 100 μg/mL ampicillin and grown overnight at 37 °C. Picked monoclones were inoculated into a 5-mL starter culture of Luria-Bertani (LB) media and grown for 3 h at 37 °C. The starter culture was transferred into 50 mL LB medium and incubated at 37 °C for another 3 h. The second culture (15 mL) was transferred into 1 L of LB medium and incubated at 37 °C to an OD600 of ~0.5. The culture was induced with 0.4 mM isopropyl β-D-1-thiogalactopyranoside and incubated at 37 °C for 12–16 h. Cells were harvested and lysed using high pressure in lysis buffer A . The histone octamer was first purified by Ni-NTA (QIAGEN) affinity chromatography, and the (His)8-tag was removed by TEV protease overnight at 4 °C. It was further purified using a HiLoad Superdex200 (16/60)  column with SEC buffer , and the peaks corresponding to histone octamers were assessed by SDS-PAGE. Purified histone octamers were wrapped with a 147-bp ‘Widom 601’ DNA fragment to reconstitute the CENP-A nucleosome by dialysis and used for further studies43. The sequence of the 147-bp ‘Widom 601’ DNA fragment is shown below:Cloning, expression, and purification of CENP-A nucleosomes were as described previouslyATCGAGAATCCCGGTGCCGAGGCCGCTCAATTGGTCGTAGACAGCTCTAGCACCGCTTAAACGCACGTACGCGCTGTCCCCCGCGTTTTAACCGCCAAGGGGATTACTCCCTAGTCTCCAGGCACGTGTCACATATATACATCCGAT.167) was the same as mentioned above. The sequence of the 167-bp DNA fragment is listed below:In some instances, 167-bp DNA was used for in vitro reconstitution experiments. The reconstitution of CENP-A nucleosome with a 167-bp DNA  for 1 h at 4 °C. The assembled complex was then purified using a Superose 6 (10/300)  column equilibrated with SEC buffer . The purified CCAN-CENP-ANuc complex was cross-linked with 5 mM bissulfosuccinimidyl suberate  for 1 h on ice, quenched with 50 mM Tris, and further subjected to SEC with a Superose 6 (10/300)  column for cryo-EM analysis. A CENP-LN subcomplex was used for assembly of a CCAN (LN)-CENP-ANuc complex in the same way as the CCAN-CENP-ANuc complex. The CCAN complex was reconstituted by mixing purified CENP-LNC, CENP-HIKM, CENP-OPQUR, and CENP-TWSX; the sample preparation was the same as for the CCAN-CENP-ANuc complex.The CENP-LNCNuc complex was applied to a glow-discharged holey carbon grid  in an aliquot of 3 μL of fresh sample at a concentration of 1 mg/mL (measured by Nanodrop at 260 nm). The grids were blotted for 5 s; maintained for 40 sec under 100% humidity at 4 °C; and plunged into liquid ethane using an FEI Vitrobot (FEI Company).The CCAN-CENP-ASerialEM with the under-focus range of 1.7~2.7 μm and at magnification of SA22, 500× in super-resolution mode, corresponding to a pixel size of 0.61 Å on the sample level. The dose rate was set to be ~10 electrons per physical pixel per second and the total exposure time for each movie was 7.68 s, fractioned to 32 frames, resulting in a total dose of 50 electrons/Å2.The Titan Krios G3i cryo electron microscopes operating at 300 kV equipped with a Gatan K2 Summit camera, were used for data collection. The cryo-EM data were collected by 44. Defocus values were estimated by CTFFIND445. After manually checking, good micrographs were selected for automatically particle picking with references generated from published 3D volume of yeast CCAN complex using RELION346. Picked particles were extracted and applied to non-supervised 2D classifications. Selected particles were then imported into cryoSPARC (v.3.2.0) for subsequent image processing47. After ab initio 3D classification into 3 classes, 39,863 particles were selected for non-uniform refinement, which gave an EM map at 4.5 Å. Particles were subjected to 2D classification and good classes were selected as template for automatic particle picking. 2,519,574 particles were auto-picked and subjected to several rounds of 2D classifications to exclude junk particles and contaminations. In total, 1,370,060 particles were selected for 3D reconstruction. Ab initio reconstruction was accomplished with five designated classes. The best class displayed clear features of the complex, and the selected particles were submitted to non-uniform refinement. Finally, a map with an overall resolution of 3.3 Å was achieved. Further ab initio reconstructions into two classes resulted in two maps with various DNA occupancies. The one without DNA density in the binding pocket was refined to 3.5 Å, and the one with DNA was refined to 3.7 Å. All reported resolutions were calculated on the basis of a 0.143 Fourier shell correlation cut-off following gold-standard refinement48. Local resolution variations were estimated using ResMap49.The image processing workflow is illustrated in Supplementary Fig. 51. We combined de novo modeling, homology modeling, and rigid-body fitting of subunits with known structures to generate an atomic model. An initial crude model was auto-built combining the results of software Buccaneer in CCPEM program suit52 with that of phenix.trace_and_build command in the Phenix package54 against the cryo-EM map. Bundles of helices were built automatically. The atomic model of the C terminus of CENP-N, full-length CENP-L, CENP-H (residues 39–241), CENP-I (residues 62–654), and CENP-K (residues 20–268) was built de novo combining the information of these auto-built helices, and secondary-structure prediction information from PSIPred analysis55. Sequence identification and assignments of these subunits were guided mainly by aromatic or bulky residues such as His, Tyr, Phe, and Trp. The crystal structure of the N-terminal domain of human CENP-N 26 and the human CENP-M 23 were directly docked into the cryo-EM map by rigid-body fitting using UCSF Chimera. Then they were refitted and refined according to the good density in Coot. The structure of CENP-O (residues 112–300), which was generated from the full-length CENP-O structure predicted by I-TASSER20, was docked into the cryo-EM map by rigid-body fitting using UCSF Chimera. Then they were refitted and refined according to the good density in Coot. By contrast, CENP-P (residues 76–288) performed the preceding operations as CENP-O. The crystal structure of the chicken CENP-TWSX hetero-tetramer (PDB: 3VH5)25, corresponding to residues CENP-T (458–556), CENP-W (14–88), CENP-S (10–106), and CENP-X (8–81), were overall fitted into the cryo-EM density map, then refitted, built, manually adjusted and mutant to human species sequence according to the density in Coot. For CENP-Q and CENP-U, it was also de novo built while assigned as alanine due to the lack of adequate information of side chain to identify the correct sequence. Maps without post-processing were used to build the unassigned chain. The DNA duplex of the CCAN–DNA complex was created by fitting a 20-bp long DNA from the unwrapped nucleosomal DNA in the S. cerevisiae CCAN-CENP-ANuc complex (PDB: 6QLD)14 into the cryo-EM density map at 3.7 Å resolution.For model building of a CCAN complex without DNA, the Cryo-EM density maps were visualized in Chimera and Coot56. The statistics of the map reconstruction and model refinement are summarized in Supplementary Table 50 and Pymol .The structure model of the overall-CCAN complex and CCAN–DNA complex are refined against the 3.3 Å and 3.7 Å overall map in real space with PHENIX and validated by MolProbity2EN4E, CENP-HIKM and CENP-OPQUR complex were first mixed with a molar ratio of 1:1:1 to form the CCANΔCT or CCAN6E-ΔCT complex. The CCANΔCT was then mixed with either CENP-A167 or CENP-A167-(R80A-G81A) nucleosome with a molar ratio of 2:1 and the CCAN6E-ΔCT was mixed with CENP-A167 nucleosome with the same molar ratio for one hour at 4 °C. Different assembled complexes were then performed using a Superose 6 Increase 5/150 GL  column equilibrated with SEC buffer . Elution of proteins was monitored at 280 nm. Fractions (100 μL/tube) were collected and analyzed by 15% SDS-PAGE gel, then stained with Coomassie Brilliant Blue.CENP-LN or CENP-LΔCT and CCAN6E-ΔCT complexes were respectively incubated with 147 bp DNA in SEC buffer  for 10 min on ice. After adding 6% sucrose in the mixtures, samples were analyzed by 1% agarose (w/v) at 120 V for 25 min in 0.5× TAE buffer. The gels were stained with GelRed and visualized using Tanon-1600 imaging system (Tanon Science & Technology) at UV mode.The purified CCAN31. Thymidine-synchronized cells were released for 7 h before being placed onto temperature-controlled chamber for real-time imaging as previously reported58.HeLa and HEK293T cells were maintained in DMEM (Gibco) with 10% fetal bovine serum  and 100 units/mL penicillin (Gibco) plus 100 μg/mL streptomycin (Gibco). The inducible CRISPR/Cas9-mediated CENP-N knockout HeLa cells from Dr. Iain Cheeseman’s laboratory (MIT) were maintained in DMEM (Gibco) with 10% FBS  and 100 units/mL penicillin (Gibco) plus 100 μg/mL streptomycin (Gibco) supplemented with 50 μg/mL G418 (Sigma) and 2.5 μg/mL puromycin (Sigma). Doxycycline  was used to induce CENP-N knockoutTo generate lentivirus expressing GFP-CENP-N/L, pLVX-EGFP-CENP-N/L (WT and different mutants) was cotransfected into HEK293T cells together with pMD2.G and psPAX2 plasmids. Forty-eight hours after transfection, the supernatant was collected and used to infect HeLa cells. HeLa cells stably expressing GFP-CENP-N/L were selected and maintained in DMEM containing puromycin (2.5 μg/mL).2-independent medium (Gibco).For the purpose of CENP-N WT and 2E mutant rescue experiment, the inducible CRISPR/Cas9-mediated CENP-N knockout HeLa cells were infected with lentivirus expressing sgRNA-resistant GFP-CENP-N-WT/2E and the GFP-CENP-N-WT/2E expressing stable cell lines were isolated by single-cell sorting (BD LSRFortessa). Three days after doxycycline treatment, cells were transfected with mCherry-H2B expressing plasmid. Twenty-four hours later, cells were treated with MG132 for 2 h, then cells were fixed and stained to examine the chromosomal alignment. In case of live cell imaging analysis, cells were cultured in MetTek glassed bottom culture dishes. Before imaging, medium was changed to CO59. To analyze the phenotype of CENP-L WT/4E, GFP-CENP-L WT/4E stable cells were transfected twice with siRNA against CENP-L for 60 h. For live cell imaging, 36 h after siRNA transfection, second siRNA treatment was co-transfected with mCherry-H2B plasmid.Transfection of plasmids and siRNA into cells was performed with Lipofectamine RNAiMAX (Invitrogen) according to the user’s manual. The siRNA against CENP-L (AAGAUUAGUUCGUGUUUCA) was obtained from GenePharma and was previously confirmedThymidine , Nocodazole  and MG132  were from Sigma. Puromycin was from Thermo (A1113802).Anti-α-tubulin  and ACA  were used for immunofluorescence. Anti-CENP-N antibody were kindly gifted by Dr. Iain Cheeseman. Antibodies used for Western blots were anti-α-tubulin , anti-β-Actin , anti-FLAG-tag , anti-GFP . The appropriate secondary antibodies were purchased from Jackson ImmunoResearch Laboratories and used as instructed by the vendor’s instruction.For FLAG-tagged protein immunoprecipitation, transfected cells were collected and lysed in lysis buffer  supplemented with protease inhibitor cocktail (Sigma). Cell lysates were clarified by centrifugation and incubated with FLAG-M2 resin (Sigma) at 4 °C with gentle rotation. After washing with lysis buffer three times, the FLAG beads were boiled and assessed by western blotting as previously reported.2, 4% formaldehyde) at room temperature and were processed for indirect immunofluorescence microscopy. Samples were examined on a DeltaVision microscope (Applied Precision) with a 60× objective lens, NA = 1.42, with optical sections acquired 0.25 μm apart in the z axis. Deconvoluted images from each focal plane were projected into a single picture using Softworx (Applied Precision). Images were taken at identical exposure times within each experiment and were acquired as 16-bit gray-scale images. After deconvolution, the images were exported as 24-bit RGB images and processed in Adobe Photoshop. Images shown in the same panel have been identically scaled. Kinetochore intensities were measured in ImageJ (rsb.info.nih.gov/ij/) on nondeconvoluted images. The levels of kinetochore-associated proteins were quantified as described previously60. In brief, the average pixel intensities from at least 100 kinetochore pairs from five cells were measured, and background pixel intensities were subtracted. The pixel intensities at each kinetochore pair then were normalized against ACA pixel values to account for any variations in staining or image acquisition. Unless otherwise specified, the values for treated cells then were plotted as a percentage of the values obtained from cells of the control groups.HeLa cells grown on coverslips were fixed and permeabilized simultaneously with PTEMF buffer (50 mM Pipes (pH 6.8), 0.2% Triton X-100, 10 mM EGTA, 1 mM MgCl2-independent medium (Gibco) supplemented with 10% FBS and 2 mM glutamine. During imaging, the dishes were placed in a sealed chamber at 37 °C. Images of living cells were taken with DeltaVision microscopy system. For presentation of details of the real-time imaging, projection images were constructed from a 0.5-µm/section for 3 sections within a cell.For time-lapse imaging, HeLa cells were cultured in glass-bottom culture dishes (MatTek) and maintained in COAll experiments were performed and repeated independently with similar results for three times. Statistical analyses were performed with GraphPad Prism 8. All statistics were described in the figure legends. No statistical method was used to predetermine sample size. Images were mounted in figures using Photoshop (Adobe).Supplementary Figure S1-8 and Table S1


Induction chemotherapy (IC) can alleviate locoregionally advanced nasopharyngeal carcinoma (LA-NPC), but effectiveness differs between patients, toxicity is problematic, and effective blood-based IC efficacy predictors are lacking. Here, we aimed to identify biomarkers for early identification of IC beneficiaries.Sixty-four pairs of matched plasma samples collected before and after IC from LA-NPC patients including 34 responders and 30 non-responders, as well as 50 plasma samples of healthy individuals, were tested using data-independent acquisition mass spectrometry. The proteins associated with clinical traits or IC benefits were investigated by weighted gene co-expression network analysis (WGCNA) and soft cluster analysis. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes functional annotations were performed to determine the potential function of the identified proteins. The area under the receiver operating characteristic curve (AUC) was used to evaluate the performance of candidate biomarkers in predicting IC beneficiaries.via immunity, complement, coagulation, glycosaminoglycan and serine. Four proteins differentially expressed in all group comparisons, paraoxonase/arylesterase 1 (PON1), insulin-like growth factor-binding protein 3 (IGFBP-3), rheumatoid factor D5 light chain (v-kappa-3) and RNA helicase (DDX55), were associated with clinical traits or IC benefits. A four-protein model accurately identified potential IC beneficiaries (AUC=0.95) while diagnosing LA-NPC (AUC=0.92), and the prediction performance was verified using the models to confirm the effective IC (AUC=0.97) and evaluate IC outcome (AUC=0.94).Compared with healthy individuals, 1027 differentially expressed proteins (DEPs) were found in the plasma of LA-NPC patients. Based on feedback from IC outcomes, 463 DEPs were identified in the pre-IC plasma between responders and non-responders. A total of 1212 DEPs represented the proteomic changes before and after IC in responders, while 276 DEPs were identified in post-IC plasma between responders and non-responders. WGCNA identified nine protein co-expression modules correlated with clinical traits. Soft cluster analysis identified four IC benefits-related protein clusters. Functional enrichment analysis showed that these proteins may play a role in IC The plasma protein profiles among IC responders and non-responders were different. PON1, IGFBP3, v-kappa-3 and DDX55 could serve as potential biomarkers for early identification of IC beneficiaries for individualised treatment of LA-NPC. Nasopharyngeal carcinoma (NPC) is an aggressive type of head and neck malignant disease with a relatively high rate of morbidity and metastasis . In 2020In recent years, data from several large-scale clinical trials demonstrated that induction chemotherapy (IC) prior to radiotherapy of LA-NPC leads to improved survival outcomes –9. When Proteins control all physiological processes including immunity, material exchange, metabolism and signal pathway regulation . During In the present study, we investigated the plasma protein profiles of LA-NPC patients with different IC efficacy using data-independent acquisition (DIA) mass spectrometry, and conducted in-depth screening for differentially expressed proteins (DEPs), as well as the correlative analysis between pre-treatment plasma proteins, clinical traits and IC benefits. Then, we explored whether pre-treatment plasma proteins have the potential as biomarkers for early identification of IC beneficiaries.We recruited 50 healthy controls (HC group) and 64 patients with non-metastatic stage III−IVa LA-NPC patients (LA-NPC group) at Yuebei People’s Hospital, Shantou University Medical College , between May 2020 and January 2021. The detailed descriptions of participants are shown in 2 docetaxel and 80 mg/m2 cisplatin (DP regimen) were given once every 3 weeks for two cycles. Prior to radiotherapy, we evaluated the tumour response to IC using RECIST (version 1.1)  . Based oion 1.1) , we defiPlasma sampling was performed following the Human Proteome Organization (HUPO) recommendations . A totalBriefly, high abundance and low abundance proteins were separated from the plasma pools using a Multiple Affinity Removal LC Column-Human 14 , and their components were desalted and concentrated using a 5 kDa cut-off ultrafiltration device (Sartorius Corp). Samples were mixed with SDT buffer comprising 4% sodium dodecyl sulphate (SDS), 100 mM dithiothreitol (DTT) and 150 mM Tris-HCl pH 8.0, boiled for 15 min, centrifuged at 14,000 g for 20 min, and the supernatant was quantified using a BCA Protein Assay Kit (Bio-Rad Corp). Samples were stored at -80°C for subsequent experiments.4HCO3 buffer twice. The obtained protein suspensions were digested with 4 μg trypsin (Promega Corp) in 40 μL 25 mM NH4HCO3 buffer overnight at 37°C, and the resulting peptides were collected as a filtrate. The peptide content of each sample was estimated by UV light spectral density at 280 nm after desalting on an Empore SPE C18 cartridge , concentrated by vacuum centrifugation, and reconstituted in 40 µL of 0.1% (v/v) formic acid.For digestion, 200 μg of proteins was repeatedly ultrafiltered with UA buffer  according to the filter-aided sample preparation (FASP) procedure to remove detergent and low-molecular-weight components. Next, 100 μL of 100 mM iodoacetamide (IAA) in UA buffer was added to block reduced cysteine residues and samples were incubated for 30 min in darkness. Filters were washed with 100 μL UA buffer three times then with 100 μL 25 mM NHDigested pool peptides were subsequently fractionated using a High pH Reversed-phase Peptide Fractionation Kit (Thermo Scientific Corp) into 10 fractions. Each fraction was concentrated by vacuum centrifugation and reconstituted in 15 µL of 0.1% (v/v) formic acid. Collected peptides were desalted and reconstituted successively with a C18 cartridge and 40 µL of 0.1% (v/v) formic acid. Finally, iRT-Kits (Biognosys Corp) were added to correct the relative retention time differences between runs at a volume ratio of 1:3 for iRT standard peptides versus sample peptides.Peptide fractions for DDA library generation were analysed using a Q Exactive HF X mass spectrometer (Thermo Scientific Corp) coupled to an Easy nLC 1200 chromatography system (Thermo Scientific Corp). Peptide samples (1.5 μg) were first loaded onto an EASY-Spray C18 Trap column , then separated on an EASY-Spray C18 LC Analytical Column  over a 120 min gradient from buffer B (0.1% formic acid and 84% acetonitrile). The column flow rate was maintained at 250 nL/min. MS detection was performed in positive ion mode, full scans were performed between 300 and 1800 m/z, the resolution for the MS1 scan was 60,000 at 200 m/z, the automatic gain control (AGC) target for the MS scan was set to 3e6, and the maximum injection time (IT) was 25 ms. The dynamic exclusion was set to 30.0s. Each full mass spectrometry (MS)-selected ion monitoring (SIM) scan followed 20 ddMS2 scans. The MS2 scan was performed at 15,000 resolution, the AGC target was 5e4, the maximum IT was 25 ms, and the collision energy was 30 eV.Liquid chromatography-tandem mass spectrometry (LC-MS/MS) running in DIA mode  was used for analysis of sample peptides. Each DIA cycle was comprised of one full MS-SIM scan, and 30 DIA scans covered a mass range of 350−1,800 m/z. For full scans, the resolution was 120,000 at 200 m/z, the AGC target was 3e6, and the maximum IT was 50 ms. DIA scans were performed at 15,000 resolution, and the AGC target and maximum IT were set to 3e6 and auto, respectively. The collision energy was 30 eV and the runtime was 120 min, with a linear gradient of buffer B (84% acetonitrile and 0.1% formic acid) at a flow rate of 250 nL/min. MS performance monitoring was conducted by injection of QC samples  in DIA mode at the beginning of the MS study, and after every six injections throughout the experiment.http://www.uniprot.org. The iRT peptide sequence was added to the database (>Biognosys|iRTKit|Sequence_fusionLGGNEQVTRYILAGVENSKGTFIIDPGGVIRGTFIIDPAAVIRGAGSSEPVTGLDAKTPVISGGPYEYRVEATFGVDESNAKTPVITGAPYEYRDGLDAASYYAPVRADVTPADFSEWSKLFLQFGAQGSPFLK). Regarding parameters, trypsin was used as the digestion enzyme, and max missed cleavages was set to 2. Carbamidomethyl (C) was specified as a fixed modification, and oxidation (M) and acetyl (protein N-term) were specified as dynamic modifications. All reported data were based on 99% confidence for protein identification as determined by false discovery rate [FDR = N(decoy)*2/(N(decoy) + N(target))] ≤1%. Finally, Spectronaut Pulsar X TM_12.0.20491.4 (Biognosys Corp) loaded with the original raw files and DDA searching results was used to construct the spectral library.To process and analyse the raw DDA data, Spectronaut 14.4.200727.47784 (Biognosys Corp) was used for FASTA sequence database searching, and the database was downloaded from the web at http://mfuzz.sysbiolab.eu/) based on the open-source statistical language R, was used for soft clustering analysis to observe changes in the expression of plasma proteins in the EFF cohort before and after IC. For DEPs and proteins related to clinical traits or IC benefits, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional annotation were performed to explore the enriched functions and pathways using the clusterProfiler R package  software packages and SPSS Statistics version 22.0 (IBM Corp.). To mine the proteomic differences between IC responders and non-responders, proteins with either increased or decreased relative abundance in the inter-group comparisons were defined as DEPs according to fold change ≥1.2 or ≤0.833 and p-value <0.05. A weighted protein co-expression network was constructed to identify hub modules related to clinical traits among the identified proteins using the Weighted Gene Co-Expression Network Analysis (WGCNA) package for R software. The fuzzy C-means algorithm, a software package termed Mfuzz . It is worth noting that continuous and dynamic expression detection is one of the necessary conditions to determine whether biomarkers have the potential to predict efficacy, we used post-IC samples to validate the efficacy predictive biomarkers identified in pre-IC samples . The advAlthough this study revealed differences in circulating protein profiles among LA-NPC patients with different IC outcomes, the identified potential biomarkers need to be further verified by more independent clinical cohorts due to the limitations of single-center nature. Furthermore, in addition to methods such as enzyme-linked immunosorbent assays, considering that IC usually involves two or more drugs which are administered to the patients at the same time, functional study of the potential biomarkers also needs to explore a reliable method to establish multidrug resistant cell line models.In summary, the present study comprehensively investigated the plasma protein profiles of LA-NPC patients with different IC outcomes and demonstrated the potential correlation between pre-treatment plasma proteins and the short-term efficacy of IC. The identified four proteins, PON1, IGFBP3, v-kappa-3 and DDX55, can achieve the early identification of IC beneficiaries while diagnosing LA-NPC, and they could confirm whether the identified potential beneficiaries have been effectively treated after IC, and distinguish responders and non-responders. These findings may help to reveal new targets of IC and serve the individualised treatment of LA-NPC. Whether the identified potential biomarkers are specific to the DP regimen remains to be clarified.The original contributions presented in the study are included in the article/The studies involving human participants were reviewed and approved by The Institutional Review Board of Yuebei People’s Hospital, Shantou University Medical College (KY-2020-020). The patients/participants provided their written informed consent to participate in this study.Conceptualization, J-CL, S-QZ and S-MP. Methodology, S-QZ. Software, JL. Formal analysis, all authors. Investigation, all authors. Writing—original draft preparation, S-QZ. Writing—review and editing, J-CL. Supervision, H-BC. Project administration, J-CL. All authors contributed to the article and approved the submitted version.This study was supported by grants from the Shaoguan Science and Technology Plan Projects in 2020, China (Grant no. 200812094530421).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.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


This study aimed to analyze the efficacy of the simultaneous rectification of adjacent asymptomatic lumbar disc herniation (asLDH) of L5-S1 isthmic spondylolisthesis (IS).One hundred and forty-eight patients with L5-S1 IS, and simultaneous L4-5 asLDH, were recruited between January 2012 and December 2017, for this study. Group A: seventy-two patients received PLIF at L5-S1. Group B: seventy-six patients received PLIF at L4-S1. The radiographic outcomes were assessed via the lumbar lordosis (LL), segmental lordosis (SL), sacral slope (SS), pelvic incidence (PI), pelvic tilt (PT), PI-LL and slip degree (SD). The functional outcomes were evaluated via the visual analog scale (VAS), Oswestry disability index (ODI), and reoperation rate. The potential risk hazards for reoperation were identified using both uni- and multivariate logistic regression analyses.P < 0.05). Relative to Group A, Group B exhibited markedly better LL, SL, PT, PI-LL,VAS and ODI scores at the final follow-up (P < 0.05). Group B also achieved better SD values post surgery than Group A (P < 0.05). The reoperation rate was remarkably elevated in Group A, compared to Group B (P < 0.05). The multivariate logistic regression analysis showed the L4-5 asLDH grade was a stand-alone risk hazard for reoperation, whereas, pre-SL and pre-LL offered protection against reoperation (P < 0.05).The postoperative LL, SL, PT, SS, SD, VAS, and ODI exhibited vast improvements (L4-S1 PLIF is recommended to correct asLDH in L5-S1 IS patients, with high-grade disc herniation and abnormal sagittal alignment. Isthmic spondylolisthesis (IS) is a common disorder involving spinal surgery, and it is characterized by the partial or total spondylolisthesis of the upper and lower vertebrae caused by isthmus disconnection. The L5-S1 level is commonly affected by IS, and accounts for about 71–95% of all patients with IS . The mosTo treat L5-S1 IS, PLIF is generally performed at the L5-S1 level. However, several studies demonstrated that the sagittal spine balance recovers poorly following single-level PLIF surgery . The lumHence, the purpose of this study was to assess whether asLDH of L5-S1 IS should be simultaneously rectified, along with PLIF surgery, and the primary endpoints were spino-pelvic sagittal balance and functional outcomes.The inclusion criteria were as follows: (1) X-ray and CT imaging-based diagnosis of L5-S1 IS , and confirmed indication for surgery . (2) MRI-confirmed L4-5 disc herniation , but without the symptoms of nerve compression and physical examination related to the L4-5 herniated disc. (3) The asLDH severity evaluated as ≥ Grade 1, based on a report by Pfirrmann et al. . (4) PLIThe exclusion criteria were as follows: (1) Patients with other lumber disc herniation, except for L4-S1 disc herniation. (2) Patients with a history of spinal surgery or fractures. (3) Patients with intervertebral space infections, spinal tumor or tuberculosis, and congenital spinal deformities.n = 72) received PLIF at L5-S1 level, and Group B (n = 76) received PLIF at the L4-S1 level. The medical ethics committee approved the informed consent forms signed by all participants and their families. We analyzed the postsurgical sagittal balance and functional outcomes between Groups A and B. To examine the hazard risk for reoperation, Group A was further separated into two subpopulations, based on reoperation after the final follow-up. Twenty-five patients were grouped into Group A1, which represented patients who underwent reoperation after the last follow-up, and forty-seven patients comprised Group A2, which represented no reoperation after the final follow-up. Univariate analysis assessed significant differences between the two subpopulations. Subsequently, meaningful indicators were entered into binary multivariate analysis to identify stand-alone hazard risk(s) for reoperation.In line with the aforementioned inclusion and exclusion criteria, one hundred and forty-eight patients with L5-S1 IS and L4-5 asLDH were recruited between January 2012 and December 2017 for our retrospective analysis. The patients were separated into Groups A and B. Group A  was employed to determine the patient’s perception of lower back pain prior to surgery, as well as 1 month, 1 year, and at the final follow-up after surgery  .MoreoverAll patients underwent anteroposterior and lateral radiograph imaging prior to surgery, as well as 1 month, 1 year, and at the final follow-up after surgery. The radiological recordings were conducted by three experienced spinal surgeons, and assessment was carried out via the blinding method. Per patient, the three independent radiological recordings showed a difference of less than 5%, thus, suggesting accurate, stable, and reliable measurements. The mean of each radiographic parameter was used during analysis. The radiographic variables were measured as follows: Segmental lordosis (SL), the angle between the lower endplates of the upper vertebrae and the lower endplates of the responsible vertebrae. Lumbar lordosis (LL), the angle between the upper endplate of the L1 vertebra and the sacral plate. The sacral slope (SS), the angle between the sacral plate and the horizontal line. The pelvic incidence (PI), the angle between the line perpendicular to the midpoint of the sacral plate and the line connecting the femoral head midpoint to the sacral plate midpoint. The pelvic tilt (PT), the angle made by a vertical line of the sacral plate midpoint and the femoral head axis. Slip degree (SD), determined by the Meyerding grade Fig. .Fig. 3Pl2 and Fisher exact tests. Univariate analysis compared between the two groups. Significant indicators from the univariate analysis were entered into multivariate analysis to identify stand-alone reoperation risk factors. P < 0.05 was set as the significance threshold.All data analyses employed the SPSS 26.0 software, and data are presented as the mean ± standard deviation. Inter-group variable comparisons were carried out via the paired sample and independent-simple t tests. Categorical data were assessed via the χP > 0.05).Table Table P < 0.05). Relative to Group A, Group B exhibited markedly better LL, SL PI-LL and PT outcomes at the final follow-up (P < 0.05). Likewise, the postoperative SD was better in Group B versus A (P < 0.05). No discernible differences were observed in other parameters between the two groups.Table P < 0.05). Moreover, the final follow-up VAS and ODI showed considerably elevated scores in Group A versus B (P < 0.05). In fact, twenty-five Group A patients experienced such severe L4-5 LDH-related pain symptoms at the final follow-up that they were indicated for reoperation. Hence, the Group A reoperation rate was 34.72%. Interestingly, no patients from Group B underwent reoperation. Therefore, the Group A reoperation rate was markedly elevated, compared to Group B (P < 0.05).Table P < 0.05, 95%CI = 0.025–0.557), pre-SL , pre-LL , pre-ODI .Table P < 0.05, 95%CI = 0.022–0.708) was a strong stand-alone indicator of reoperation, whereas pre-SL  and pre-LL  were protective factors for reoperation.Table Isthmic spondylolisthesis, brought on by a pars interarticularis fracture, involves the forward movement of one vertebral body relative to adjacent vertebral bodies. Initial treatment includes oral anti-inflammatory drugs and physical therapy. If radiculopathy is predominant, transforaminal epidural injection of corticosteroids may provide temporary relief . It has It is generally accepted that the sagittal positioning of the spine is critical for lumbar degeneration to ensue . In thisThe essential pelvic parameters are PI, PT, and SS. SS is described as the angle between the horizontal and parallel sacral plates S1, and it is roughly 41° ± 8°. PT represents pelvic rotation, which reduces with anteversion and enhances with retroversion , and it In terms of the functional outcomes, we observed marked decreases in postoperative ODI and VAS scores in Groups A and B. No discernible differences were observed in the VAS and ODI scores at 1 month and 1 year post operation. However, these scores were considerably elevated at the final follow-up in Group A versus B. As a result, Group A had substantially elevated reoperation rate than Group B. This was primarily because the L4-5 asLDH was not surgically intervened during the initial operation, and the postoperative back pain and lower limb numbness, caused by L4-5 LDH compression of nerve roots, negatively impacted patient quality of life, and eventually required a repeat surgery. Kepler et al. speculated that a reduced postoperative SL indicates more obvious pain in the lower back and legs, as well as a higher VAS score . In our Our research encountered certain limitations. First, our patient population was relatively small (148 patients). A larger patient population is needed to obtain more meaningful statistical data. Second, there is a necessity to perform future prospective randomized controlled trials to validate our results. Finally, certain pre- and postoperative lumbar radiographs did not include the bilateral femoral head. Hence, we could only estimate the central position of the femoral head by observing the acetabular shape, which can result in measurement errors in pelvic parameters.In case of L4-5 asLDH of L5-S1 IS patients, L4-S1 PLIF can achieve better sagittal balance and functional results post surgery. In contrast, L5-S1 PLIF has a higher postoperative reoperation rate. The results of our multivariate analysis revealed that, in asLDH of IS L5-S1 patients, with high-grade disc herniation and abnormal sagittal alignment, L4-S1 PLIF is more suitable during primary surgery.


The β subunits are not essential to the channel activity but confer different properties through binding the T1 domain or the C-terminal of α subunits. Here, we studied the physiological function of a novel gene, KIbB1, encoding a voltage-gated K+ channel β subunit in sweetpotato. The transcriptional level of this gene was significantly higher in the low-K+-tolerant line than that in the low-K+-sensitive line under K+ deficiency conditions. In Arabidopsis, KIbB1 positively regulated low-K+ tolerance through regulating K+ uptake and translocation. Under high-salinity stress, the growth conditions of transgenic lines were obviously better than wild typr (WT). Enzymatic and non-enzymatic reactive oxygen species (ROS) scavenging were activated in transgenic plants. Accordingly, the malondialdehyde (MDA) content and the accumulation of ROS such as H2O2 and O2− were lower in transgenic lines under salt stress. It was also found that the overexpression of KIbB1 enhanced K+ uptake, but the translocation from root to shoot was not affected under salt stress. This demonstrates that KIbB1 acted as a positive regulator in high-salinity stress resistance through regulating Na+ and K+ uptake to maintain K+/Na+ homeostasis. These results collectively suggest that the mechanisms of KIbB1 in regulating K+ were somewhat different between low-K+ and high-salinity conditions.Voltage-gated K Due to tuations . Potassiy weight . K+ playc stress . The deffunction . Meanwhimbalance ,12. Thert stress ,13. Sincn plants . In plansporters ,16.+ channels consist of two main categories, voltage-gated K+ channels and voltage-independent K+ channels [+ channels have different mechanisms for mediating K+ flux [+ channels are important transmembrane regulators selectively catalyzing the transport of K+ across the membrane [+ channel gene was cloned from Drosophila [+ channels were identified in the genome of Arabidopsis thaliana [+ (Kout) channels, including SKOR and GORK; inward-rectifying K+ (Kin) channels, including KAT1, KAT2, AKT1, SPIK and AKT5; weak-rectifying K+ (Kweak) channels including AKT2; and silent-rectifying K+ (Ksilent) channels including KC1 [silent channels only modulate channel properties and do not form functional channels [in channels are divided into two subclades: the Kin-a clade (KAT1-like) and Kin-b clade (AKT1-like) [in channels have been widely analyzed in many kinds of plant species. In Arabidopsis, the expression level of AtAKT1 was induced by K+ deficiency, and the AtAKT1 mutation significantly reduced K+ acquisition [+ uptake into roots. In rice, a mutation of OsAKT1 resulted in sensitivity to low-K+ stress, and OsAKT1 was also related to salt stress tolerance [HvAKT1 might lead to K+/Na+ homeostasis under high-salinity stress [GmAKT1 enhanced K+ uptake and K+/Na+ balance to regulate tolerance to low-K+ and salt stress in soybean [Puccinelli tenuiflora, could increase K+ content to regulate salt stress tolerance in transgenic Arabidopsis [+ channels contained four α subunits forming an ion-conducting pore [in channels, was found in different species [KputB1 from P. tenuiflora and KOB1 from rice might regulate K+ homeostasis in yeast and Arabidopsis [+ channel β subunits in abiotic stress tolerance was still limited.Kchannels ,18. Thes K+ flux . Voltagemembrane . The firosophila . Since tthaliana ,23. Accoding KC1 . The Ksichannels . The KinT1-like) ,27. The uisition ,29. Thesolerance . In barly stress . The ove soybean . PutAKT1bidopsis . Previouing pore . Subsequ species ,36,37,38 species ,40,41. AIpomoea batatas (L.) Lam) plays an important role in food and bio-energy security [+ were needed for yield and quality during the swelling stage of the tuberous root in this crop [+ play a vital role in sweetpotato. In a previous study, a total of 11 members of the voltage-gated K+ channel family were identified in sweetpotato, among which IbAKT1 (α subunit of Kin channels) was demonstrated to regulate the tolerance of K+ deficiency [+ channel β subunits, which also play important roles in K+ transportation in plants, are scarce in sweetpotato.Sweetpotato (security . High amhis crop . Therefoficiency . Neverth+ channel β subunit was identified from the low-K+-tolerant sweetpotato variety Shangshu19. The expression level of this gene was significantly higher in Shangshu19 (low-K+ tolerant) than that in Yuzi7 (low-K+ sensitive) under low-K+ conditions. The overexpression of KIbB1 enhanced tolerance to K+ deficiency and high-salinity stresses. This study provides preliminary evidence for the function of KIbB1 in the tolerance of high salinity and low K+. Meanwhile, this information will play an important role in the further analysis of the mechanisms of voltage-gated K+ channel β subunits and the functional differences between voltage-gated K+ channel α subunits and β subunits in sweetpotato and other plants.In this study, a novel gene encoding a voltage-gated KKIbB1 were analyzed in the low-K+-tolerant sweetpotato variety Shangshu19 and the low-K+-sensitive sweetpotato variety Yuzi7 under both low-K+ and normal conditions. The low-K+-tolerant sweetpotato variety Shangshu19 was employed for isolating the coding sequence (CDS) of KIbB1. Arabidopsis (Columbia-0) was used to generate KIbB1-overexpression lines. The growth conditions of sweetpotato and Arabidopsis were both set according to Zhu et al. [The expression patterns of u et al. .KIbB1 was amplified with the specific primer KIbB1-F/R (https://web.expasy.org/protparam/ accessed on 25 September 2021) was used to predict the molecular weight, isoelectric point and polarity of KIbB1. Protein sequence alignment of KIbB1 and voltage-gated K+ channel β subunits from other plant species was conducted with Jalview software . The transmembrane domain of KIbB1 was predicted with the online analysis tool TMHM (http://www.cbs.dtu.dk/services/TMHMM-2.0/ accessed on 25 September 2021).Total RNA was extracted from Shangshu19 using the TsingZol Total RNA Extraction Reagent Kit . Primescript™ RT Reagent Kit with gDNA Eraser  was employed to synthesize the first-strand cDNA according to the manufacturer’s protocols. The coding sequence (CDS) of IbB1-F/R . The webKIbB1 expression levels were analyzed in leaf, stem, fibrous root and tuberous root of 90-day-old field-grown Shangshu19. For induction analysis under low-K+ stress, the expression profiles of KIbB1 were detected in roots and shoots of 4-week-old Shangshu19 and Yuzi7 plantlets grown on Murashige and Skoog (MS) medium. These two sweetpotato varieties were treated with Hoagland solution containing 0 or 20 mM K+ for 0, 6, 12, 24 and 48 h. The gene expression analysis was conducted using quantitative real-time polymerase chain reaction (qRT-PCR) with TB Green Premix Ex TaqTM II Kit  and QuantStudio 3  as described by Zhu et al. [IbActin  and KIbB1-q-F/R are listed in u et al. . The priKIbB1 was amplified with the specific primer KIbB-OE-F/R  with restriction enzyme cutting sites of B-OE-F/R . After dh et al. . The posKIbB1 transgenic lines were selected with hygromycin to be T3 homozygous for further analysis. After surface sterilization, seeds from wild-type (WT) and transgenic lines were germinated on 1/2 MS medium. For low-K+ treatment, seedlings with 1 cm root length were treated with low K+ (50 μM) or normal K+ (10 mM) conditions upside down for 10 days. The new downward-curving roots of the tested plantlets were measured [The measured .v/v). WT and transgenic plants were irrigated with H2O or 300 mM NaCl solution once every 3 days for 2 weeks [For high-salinity treatment, WT and transgenic lines were transferred to 1/2 MS medium with 0 or 125 mM NaCl after germination on 1/2 MS medium. After two weeks of treatment, the fresh weight and root length of the tested lines were measured. After being grown on 1/2 MS medium for 10 days, seedlings were transferred to potting soil mixture  was employed to detect the maximal photochemical efficiency of photosystem II (PSII) in the dark (Fv/Fm) and the photochemical efficiency of PSII in the light (Fv’/Fm’).Arabidopsis were performed as described above. The expression profiles of stress-responsive genes in the whole plantlets of transgenic and WT lines under normal and high-salinity conditions were analyzed with qRT-PCR assay according to the method of Zhu et al. [Arabidopsis actin gene, and the specific primers are listed in RNA isolation and first-strand cDNA synthesis of transgenic and WT u et al. . The exp2O2 and O2− under different conditions with 3,3′-diaminobenzidine (DAB) and nitro-blue tetrazolium chloride (NBT) staining, respectively [The leaves of transgenic and WT plants were employed to analyze the accumulation of Hectively ,48. Supe+ and Na+ concentration measurement, samples were collected and washed with double-distilled water. Samples were dried at 106 °C for 24 h and were dissolved in HNO3 and HClO4 for 12 h, subsequently. After dissolving on a temperature-controlled heating plate, K+ and Na+ content was measured using an ICO-OES  according to the manufacturer’s instructions. For shoot/root K+ ratio measurement, the shoots and roots of the tested plants were harvested separately.For Kt-test. The graphs were created with GraphPad Prism 8.0 software. * and ** indicate a significant difference at p < 0.05 and p < 0.01, respectively.Each treatment contained three independent biological replicates. All data are presented as the means ± SE. SPSS 17.0 software was employed to perform statistical analysis with a Student’s KIbB1 was isolated from the sweetpotato variety Shangshu19, which was a low-K+-tolerant variety (unpublished data). The ORF of this gene was 990 bp, encoding 329 amino acids, with a molecular weight of 36.677 kDa and an isoelectric point (pI) of 8.18. Multiple alignments of KIbB1 and its homologs from A. thaliana, Oryza sativa, Puccinellia tenuiflora, Egeria densa, Indosa sinica, Hodeum vulgare and Triticum dicoccoides indicated that voltage-gated K+ channel β subunits were highly conserved among different plant species . As a unit of voltage-gated K+ channel, the expression of this gene in response to low-K+ stress was also analyzed. Under normal conditions, the expression level of KIbB1 increased with treatment time over 48 h and showed no significant difference between Shangshu19 (low-K+ tolerant) and Yuzi7 (low-K+ sensitive)  a,b. Undend Yuzi7 a,b. The nditions a,b. Howenditions a,b. ThesKIbB1 overexpression Arabidopsis lines were obtained for further analysis (KIbB1 transgenic seedlings were cultured upside-down on 1/2 MS medium with different concentrations of K+ (50 μM and 10 mM) for 10 days after germination. Under normal conditions, the new roots of WT and KIbB1 overexpression lines curved downward (due to geotropism) and showed no obvious morphological difference  channels and NSCC (non-selective cation) channels mediating NaCl-induced K+ efflux in leaf mesophyll could be increased in the presence of ROS. For a voltage-gated K+ channel β subunit, Ardie et al. [KputB1 increased K+ content under high-salinity stress. In the present study, the overexpression of KIbB1 improved morphology conditions and photosynthetic characteristics, including Fv/Fm and Fv’/Fm’, under salt stress (KIbB1 positively regulated salt stress tolerance in transgenic Arabidopsis. The expression levels of genes related to ROS scavenging were much higher than those in WT after salt stress treatment (2O2 and O2− was lower in transgenic leaves than in WT (KIbB1 activated ROS scavenging under salt stress. Further ion content measurement showed that the variation in K+ and Na+ concentration and K+/Na+ ratio under high-salinity stress was similar to that under low-K+ conditions; however, the shoot/root ratio of K+ showed no significant difference among the tested lines (KIbB1 may be involved in the salt stress response by enhancing K+ uptake to mediate Na+/K+ homeostasis. This study provides gene resources for improving high-salinity and low-K+ tolerance by genetic engineering in sweetpotato. There are two directions for future research: the different molecular mechanisms of KIbB1 under high-salinity or low-K+ conditions; and the functional differences between voltage-gated K+ channel α subunits and β subunits in K+ transportation.High salinity is a major abiotic stress, and the adverse effects on crop production have been demonstrated in numerous studies ,59,60. Ha+ ratio . Meanwhia+ ratio . Plants a+ ratio . Severala+ ratio . K+ channditions . GmHKT1; soybean . Wu et a soybean  demonstre et al.  found tht stress . This sureatment . The conreatment a–c. Accoan in WT e,f. It hed lines a–d. Thes+ channel β subunit from sweetpotato, named KIbB1. The expression of this gene was significantly higher in the low-K+-tolerant line than that in the low-K+-sensitive line under K+-starvation stress. The overexpression of KIbB1 enhanced resistance to K+ deficiency through decreasing Na+ absorption and improving K+ uptake and translocation in Arabidopsis. Meanwhile, KIbB1 also positively regulated high-salinity tolerance. ROS scavenging was activated to decrease the salt-induced ROS accumulation. The measurement of ion concentration showed that KIbB1 regulated the acquisition of Na+ and K+ but not the translocation. These results indicate that KIbB1 enhanced low-K+ and high-salinity tolerance through maintaining the K+/Na+ balance in plants, although the mechanisms are a little different.In conclusion, we isolated a novel gene encoding a voltage-gated K