Atrial fibrillation (AF) is the most prevalent cardiac arrhythmia. Diabetes mellitus (DM) is one of the risk factors for the development of stroke and thromboembolism in patients with AF. Early identification may reduce the incidence of complications and mortality in AF patients.AF patients with DM have different pattern of small extracellular vesicle (sEV) levels and sEV‐derived microRNA (miRNA) expression compared with those without DM.We compared sEV levels and sEV‐miRNA expression in plasma from AF patients with and without DM using nanoparticle tracking analysis and droplet digital polymerase chain reaction, respectively.p = .004) and a significant decrease in sEV‐miR‐126 level (p = .004) in AF patients with DM. Multivariate logistic regression analysis revealed a positive association between total sEV levels and AF with DM (p = .019), and a negative association between sEV‐miR‐126 level and AF with DM (p = .031). The combination of clinical data, total sEVs, and sEV‐miR‐126 level had an area under the curve of 0.968 (p < .0001) for discriminating AF with DM, which was shown to be significantly better than clinical data analysis alone (p = .0368).We observed a significant increase in total sEV levels  and sEV‐microRNA (miRNA) expression in atrial fibrillation (AF) coexisting with diabetes mullites (DM). We analyzed plasma from age‐ and gender‐matched samples of 2 cohorts (AF with DM and AF without DM). Enriched sEVs were isolated from plasma using PEG precipitation and purified by size exclusion chromatography. sEV levels were measured by nano tracking analysis (NTA) and expression of sEV‐miRNAs was determined by droplet digital PCR. The results showed that increased levels of total sEV and a decreased sEV‐miR‐126 level were associated with the AF coexisting with DM, suggesting that these may play a potential role in the pathophysiology and complications of AF with DM, especially endothelial dysfunction. AF results from abnormal electrical conduction and remodeling of atrial tissues, which influences irregular and fast atrial contractions. The trends in AF prevalence and incidence are rising globally, and they are expected to continue increasing in the future.MicroRNA (miRNAs) are single‐stranded noncoding RNAs that are approximately 22 nucleotides in length that play a crucial role in post‐transcriptional gene regulation by inhibiting mRNA translation or by interfering with mRNA stability. miRNAs control several biological processes, including cell proliferation, cell apoptosis, and cell differentiation. Many miRNAs have been implicated in the pathogenesis of diabetes and its related cardiovascular complications. miR‐320 was reported to be an antiangiogenic and proapoptotic miRNA in the cardiovascular system of diabetes patients.EVs are nanoparticle lipid bilayers that are released from most cells into the extracellular space. EVs act as vehicles that transport bioactive molecules, such as lipids, proteins, metabolites, and nucleic acids, in cell‐to‐cell communication.In this study, we set forth to investigate the difference in sEV levels and sEV‐derived miRNA expression between AF patients with and without DM. We hypothesized that our findings would reveal the basic knowledge to apply sEV and sEV‐miRNAs to be potential biomarkers for AF with DM.22.1Nonvalvular AF patients were prospectively recruited from the Division of Cardiology of the Department of Medicine and from the Department of Preventive and Social Medicine, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand during the November 2019 to February 2022 study period. The protocol for this study was approved by the Siriraj Institutional Review Board (SIRB) (COA no. Si 489/2019). Eighty‐five AF patients were enrolled and then nine AF patients were excluded following the exclusion criteria. The exclusion criteria were refusal to provide written informed consent to participate, history of myeloproliferative disorders, thrombocytopenia, ischemic stroke within the previous 3 months, malignancy, rheumatic mitral valve disease, acute infectious or inflammatory disease, and pregnancy. The sample size was calculated based on information from the previous study using nQuery software.2.2g) for 15 minutes to isolate platelet‐poor plasma (PPP) followed by storage at −80°C until analysis. sEV isolation was modified from a previously described method.g for 2 minutes at 4°C and the large EVs were then removed via centrifugation at 17 000g for 45 minutes at 4°C. The large EV‐free plasma was diluted with an equal volume of phosphate‐buffered saline (PBS) and the mixture was filtered through 0.22 μm syringe filters (Corning Life Sciences). The diluted plasma was mixed with 20% weight/volume (w/v) polyethylene glycol 6000 (PEG6000) (Sigma‐Aldrich Corporation) in diluent buffer  and rotated end‐over‐end at 4°C for 24 h. The samples were then centrifuged at 4000g for 30 minutes and the pellets were resuspended in 500 µL PBS. sEV‐enriched samples were purified using size exclusion chromatography (SEC) columns  following the manufacturer's instructions. sEV‐enriched samples were applied on top of qEV columns and 0.5 mL fractions were collected. EV‐rich fractions (7−12) were pooled and concentrated using an Amicon Ultra‐4 10 kDa centrifugal filter device (Merck Millipore). sEVs were characterized using Nanoparticle tracking analysis (NTA), Transmission electron microscopy (TEM), and Western blot (WB) analysis .EDTA‐anticoagulated peripheral blood was centrifuged at 1500 times gravity . sEV‐RNA was extracted using TRIzol LS® Reagent (Life Technologies) following the manufacturer's protocol. sEV‐RNA concentration was measured using a NanoDrop™ 8000 Spectrophotometer (Thermo Fisher Scientific). Five microliter of total RNA was reverse‐transcribed using a miRCURY LNA™ Universal RT miRNAs PCR, according to the manufacturer's instructions. Detailed information about the primers and thermal cycling conditions used for the evaluated miRNAs is presented in Supporting Information: Table 2.5t‐test, and the results are presented as the mean plus/minus standard deviation (SD). Non‐normally distributed continuous data were compared using Mann−Whitney U test. Categorical variables were compared using χ2 test. Univariate logistic regression analysis was performed to evaluate for significant association between clinical parameters, total sEV levels, sEV smaller than 100 nm and sEV‐miR‐126 levels and AF with DM. Multivariate logistic regression analysis was then performed to identify independent predictors of AF with DM. Total sEV, sEV smaller than 100 nm, and sEV‐miR‐126 were then evaluated for association with AF with DM by adjusting for possible confounding variables. The independent factors identified from multivariate analysis were then used to generate four additional prediction models  using logistic regression analysis. Models 2−5 included model 1 and one or more of the identified independent variables. The data from each of the 5 models specific to each model's ability to predict AF with DM was then used to generate receiver operating characteristic (ROC) curves. A significant difference was defined as a p‐value less than .05.The data were analyzed using PASW Statistics version 18  and MedCalc® Statistical Software version 20 . Normally distributed continuous variables were analyzed using unpaired 33.1n = 28) and the AF without DM group (n = 28) are shown in Table p = .009). AF patients with DM had a significantly higher mean fasting blood glucose than AF patients without DM . HDL‐C and calculated LDL‐C were both significantly lower in AF patients without DM than in patents with DM . All other variables were nonsignificantly different between groups.The baseline demographic and clinical characteristics compared between the AF with DM group    Figure . sEVs we) Figure . There w) Figure . Investi) Figure .3.3p = .004). The expression levels of the other miRNAs were not significantly different between groups, as shown in Figure To study the difference in the expression of sEV‐miRNAs between AF patients with and without DM, we measured the expression of these miRNAs using ddPCR, and the results showed the levels of miR‐126‐3p expression to be significantly decreased in AF patients with DM compared to AF patients without DM , total sEV levels , and sEV smaller than 100 nm . AF with DM was shown to be significantly negatively associated with sEV‐miR‐126‐3p levels , HDL‐C levels , and calculated LDL‐C levels , as shown in the forest plot in Figure p < .0001 for model 1; AUC: 0.945 (95% CI: 0.850−0.988), p < .0001 for model 2; AUC: 0.931 (95% CI: 0.830−0.982), p < .0001 for model 3; AUC: 0.926 (95%CI: 0.824‐0.979), p < .0001 for model 4; and AUC: 0.968 (95% CI: 0.883−0.997), p < .0001 for model 5. Model 5 or the combination of model 1 with sEV and sEV‐miR‐126 had the highest AUC, and showed a significant increase in AUC compared to model 1 alone (p = .038). The sensitivity and specificity were 92.86% and 71.43% for model 1; 78.57% and 100% for model 2; 75.0% and 100% for model 3; 96.43% and 75.0% for model 4; and, 89.29% and 92.86% for model 5.Univariate logistic regression analysis was employed to evaluate for associations between possible risk factors and AF with DM. That analysis revealed significant positive associations between AF with DM as the outcome and fasting blood sugar levels  and its corresponding ligand (CD40L), which influence EC activation and monocyte recruitment leading to the acceleration of atherosclerosis development, have been observed in diabetic patients.In addition to DM, the pathophysiology of AF contributes to sEV release and sEVs can promote AF development. Prolonged rapid atrial pacing in canine model increased the release of plasma sEVs and induced atrial fibrosis.The role of EVs in thrombogenicity has been described in AF and is associated with a risk of thromboembolic complications, including ischemic stroke and systemic thromboembolism. EV subtypes  were found to be significantly higher in patients with AF along with an increased risk of stroke as compared to matched controls.miR‐126, also called angiomiR‐126, is highly expressed in ECs and platelets, and acts as an important regulator for the maintenance of endothelial homeostasis and vascular integrity. Circulating miR‐126 acts as a potential biomarker for vascular damage and cardiac injury in cardiovascular diseases, including myocardial infarction (AMI),Endothelial dysfunction is an important factor in the development of vascular complications in patients with DM. The mechanisms underlying this phenomenon involve hyperglycemia, insulin resistance, and oxidative stress.There were no differences in miR‐320a‐3p, miR‐146a‐5p, or miR‐30c‐5p levels in sEVs between AF patients with DM and without DM, which may be due to the effect of drug treatment. Over half (53%) of AF patients with DM in our cohort were treated with anti‐diabetes drugs, which were shown to influence the generation, release, and composition of EVs in type 2 DM patients. Furthermore, anti‐diabetes drugs were found to restore the levels of many miRNAs in EVs close to those observed in healthy controls.4.1This study has some mentionable limitations. First, although our sample size per group satisfied the minimum required by our sample size calculation, further evaluation of our findings needs to be conducted in a much larger and more racially diverse patient population to verify that total sEV level and sEV‐miR‐126 levels accurate predict AF with DM. Second, we only investigated miRNAs that were reported to have known functions related to the pathophysiology of DM and cardiovascular diseases. An investigation to identify new miRNAs should be conducted using small RNA sequencing. Third, all 56 of the AF patients enrolled in this study were Asian (Thai), which could limit the generalizable of our findings to AF patients of other races. Fourth, the role(s) of miR‐126‐3p in the development and complications of AF and DM need to be explored in future studies. Last, the prospective cohort study (such as the incidence of AF in patients with DM or vice versa) needed to be investigated in the future to understand the pattern of sEV levels and sEV‐miRNA expression in parallel with the development of coexisting DM with AF.5The results of this study strongly suggest that the levels of total sEV and sEV‐miR‐126 associated with AF and coexisting DM. We observed a significant increase in total sEV levels and a significant decrease in sEV‐miR‐126 levels in AF patients with DM. This alteration may be the one of mediators that accelerates the development of pathophysiology and complications in AF and coexisting with DM, especially endothelial dysfunction.Conceptualization: Panjaree Siwaponanan and Rungroj Krittayaphong. Methodology: Panjaree Siwaponanan and Nusara Chomanee. Formal analysis: Panjaree Siwaponanan and Suthipol Udompunturak. Investigation: Panjaree Siwaponanan and Nusara Chomanee. Validation: Panjaree Siwaponanan Resources, Panjaree Siwaponanan, Pontawee Kaewkumdee, Kamol Udol and Rungroj Krittayaphong. Data Curation: Panjaree Siwaponanan and Pontawee Kaewkumdee. Writing—Original Draft: Panjaree Siwaponanan; Writing—review & editing: Panjaree Siwaponanan, Kovit Pattanapanyasat and Rungroj Krittayaphong. Visualization: Panjaree Siwaponanan Supervision, Rungroj Krittayaphong. Project administration: Panjaree Siwaponanan; Funding acquisition: Panjaree Siwaponanan.The authors declare no conflict of interest.Supplementary Table 1. miRCURY LNA™ PCR primers and the thermal cycling conditions used for ddPCR.Click here for additional data file.


Reporting the clinicopathological information of thyroid cancer (TC) patients from a central medical center of east China, and constructing the nomogram predicting lymph node metastasis (LNM).We collected the patients who underwent thyroid cancer surgery in our institute from July 1, 2019 to July 31, 2021, a total of 253 subjects were enrolled. We used HiPure FFPE DNA Kit to extract DNA and RNApure FFPE Kit to extract RNA from the paraffin sections of tumor tissue, the extracted DNA samples and RNA samples were used for NGS sequencing. The clinical and pathological information of TCGA‐THCA cohort was obtained as the validation cohort. Multivariate logistic regression analysis was performed to identify the independent prognostic factor, and the nomogram was subsequently constructed by “rms” R package.p = 0.0031) and contained advanced TI‐RADS levels , as well as more RET genetic alteration . We chose age, tumor diameter, RET fusion, and gender to construct the LNM predicting nomogram. Calibration plot, DCA curve, and the clinical impact plot verified the preferable prognostic value of the nomogram, with an AUC value of 0.724 (0.656–0.792). We successfully validated the prognostic value of the nomogram in TCGA‐THCA cohort. RET fusion might impact the process of protein digestion and absorption, cytokine‐cytokine receptor interaction, ECM‐receptor interaction, focal adhesion.Secondary cases contained more mutation of BRAF (90.48% vs. 62.07%) and TERT (33.0% vs. 3.0%), as compared with primary cases. Primary patients with positive lymph node were younger (40.9 ± 10.8 vs. 45.3 ± 11.8, We provide a novel nomogram to predict the LNM for TC patients, including the features of patient's age, gender, tumor diameter, and RET alteration. Further studies from multiple medical centers are essential to validate the nomogram. All the patients were diagnosed with unilateral or bilateral thyroid nodules of category 3 or higher by ultrasound, CT or MRI. The samples were further double checked with TC by intraoperative frozen section examination and postoperative pathological examination. We recorded the clinicopathological parameters of age, gender, tumor diameter, ultrasound TI‐RADS stage, with or without ultrasound abnormal node, with or without ultrasound abnormal calcification, tumor pathological type, tumor TNM stage, and with or without lymph node metastasis. The TNM staging was based on the staging definition criteria in the eighth edition of the American Joint Committee on Cancer (AJCC).2.2All the selected patients should meet the following criteria: (1) did not receive radionuclide iodine‐131 therapy or radiofrequency ablation before surgery; (2) between 18 and 75 years old; (3) without chronic diseases of hypertension, heart disease, diabetes, etc.; (4) cooperate with the follow‐up. Exclusion criteria: (1) without certain pathological diagnosis of TC; (2) with severe dysfunction of liver, kidney, heart, and lung; (3) not agree to receive NGS testing; (4) others who are considered inappropriate by the researchers. This study was approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University, and written informed consent was obtained from the participants and the data involved were anonymous without any identifiable private information.2.3We used HiPure FFPE DNA Kit to extract DNA and RNApure FFPE Kit to extract RNA from the paraffin sections of tumor tissue, the extracted DNA samples and RNA samples were used for library construction, and then the Qsep100 automatic nucleic acid and protein analysis system was used to assess the quality of the constructed library. For NGS sequencing, the steps are as follows: (1) thawing the sequencing reagent; (2) cleaning the instrument before sequencing; (3) cleaning the Flowcell; (4) establishing a Sample Sheet; (5) dilution and mixing of the library; (6) adding the library; (7) run sequencing; (8) clean the instrument after sequencing. SNV and Indel mutations were filtered with the follow criteria: (1) mutation frequency > 0.01; (2) population frequency (refer to the East Asian population database of 1000G and ExAC) less than 0.01; (3) retain the sites of exons or alternatively spliced regions, and retain the sites of the target UTR region (such as the TERT promoter region); (4) remove the sites of synonymous mutation; (5) remove background mutations and low‐frequency mutations in repetitive regions.2.4https://xenabrowser.net/datapages/?cohort=GDC%20TCGA%20Thyroid%20Cancer%20(THCA)&removeHub=https%3A%2F%2Fxena.treehouse.gi.ucsc.edu%3A443). The heatmap for the clinical information and waterfall plot for the distribution of gene mutation was completed with “ComplexHeatmap” R package.The clinical and pathological information of TCGA‐THCA cohort was obtained from UCSC Xena, as well as the gene expression profile and gene mutation matrix . A two‐tailed p value <0.05 was recognized statistically significant.Student's 33.1p = 5.048e‐06), and only one patient contained three types of mutant gene, BRAF, TERT, and PIK3CA, belonged to secondary group . The basic clinical pathological information of all the patients listed in Table p Figure . In addip Figure .3.2p = 0.0031, Figure p = 1.878e‐03, Figure p = 9.197e‐05, Figure p = 2.566e‐03, Figure As we talked in the introduction part, lymph node metastasis is tightly with the prognosis of TC, therefore it is essential to identify the novel factors to help the identification of lymph node metastasis in clinical work. We analyzed the diverse distribution of clinical and pathological features among patients with and without lymph node metastasis from AHMU‐TC cohort Table . The dis3.3p = 0.002), tumor diameter (p = 0.034), and RET fusion (p = 0.032) still as the prognostic factor to the presence of positive lymph node, while patient gender also showed a marginal prognostic value (p = 0.08). Therefore, we enrolled age, gender, tumor diameter, and RET alteration to construct the lymph node metastasis predict nomogram  and 19–45 years  had a higher risk of lateral LNM than patients aged >60 years.Drawing on the data from the 232 patients, we observed that age, gender, and tumor diameter exhibited predictive value for LNM, with early age, male gender, and a diameter greater than 1 cm appearing to be risk factors. Evidence from other studies supports our findings. A study from central China discovered that patients aged ≤18 years ; investigation ; visualization ; writing – original draft . Rui Wang: Data curation ; formal analysis . Wanxue Zhang: Resources ; software ; visualization . Jing Zhang: Investigation ; validation ; visualization . Qiang Fang: Resources ; supervision ; validation ; writing – original draft . zheng fang: Investigation ; methodology . Bin Liu: Investigation ; supervision ; writing – review and editing . Yongxiang Li: Conceptualization ; data curation ; writing – review and editing .The research contents and research programs were reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Anhui Medical University (Quick‐PJ‐2022‐13‐44).Figure S1.Click here for additional data file.


Multiple myeloma (MM) is the second common hematologic malignancy manifesting as a clonal proliferation of plasma cells in the bone marrow. In recent years, high expression and activity of pim kinase have been found to be associated with both the progression and prognosis of a significant proportion of malignant diseases. Therefore, pim kinase has become a potential therapeutic target in the treatment of MM and some pim kinase inhibitors have demonstrated good efficacy in clinical trials. Based on nearly the entire literature searched from PubMed in the field of pim kinase in MM, the paper concluded how pim kinase got involved in the proliferation of myeloma cells, the progression of bone disease infiltration, and even in the regulation of the immune microenvironment. Next as a very promising drug, the effectiveness of pim kinase inhibitors as single agents or in combination with other drugs in the treatment of MM was also summarized. Our analysis will guide the clinical use of pim kinase inhibitors for managing tumor load and bone disease in MM patients. In multiple myeloma, pim kinase inhibitors inhibit tumor growth and reduce bone destruction while modulating the immune microenvironment. Such a promising drug requires more clinical trials and basic research to provide a more comprehensive treatment option for myeloma. Tumor load, bone disease, and immunity are the three fundamental issues that have drawn clinical attention in MM. The family of pim kinases comprises three isozymes: pim‐1, pim‐2, and pim‐3, all of which are reported to be overexpressed in several hematologic malignancies.2First identified in 1986, pim‐1 is a putative oncogene that is activated in murine leukemia virus‐induced T‐cell lymphomas as a result of provirus insertion.3+ myeloma cells. pim‐1 levels were also higher than pim‐3 levels.Pim‐1 is overexpressed in hematologic diseases and solid tumors, pim‐2 in myeloma, lymphoma, and leukemia, and pim‐3 in substantial organ tumors.3.1The cell cycle is the process by which cells reproduce.Pim‐1 phosphorylates p21Cip1/Waf1 at Thr‐145, exerting an inhibitory effect on the G1/S phase3.2Pim, acting as an oncogene, causes differences in a variety of tumors.Pim‐1 can increase the stability and transcriptional activity of c‐Myc via its phosphorylation at serine 62 (S62).Based on the current data, pim‐2 was expressed at a higher level in myeloma cells than pim‐1 and pim‐3, suggesting a more important role for pim‐2 in MM biology.Pim‐3 has been noted in adenocarcinomas4MM is highly likely to cause bone diseases.4.1RANKL is capable of inducing pim‐1 expression and calcium oscillations, the latter of which is required for NFATc1 induction and osteoclastogenesis.2+]i oscillations.2+ ([Ca2+]i) oscillations.2+]i oscillations of RANKL and upregulates NFATc1.Osteoclasts (OCs) upregulated Pim‐2 expression in MM cells mainly through the IL‐6/STAT3 and NF‐κB pathways, respectively. Pim‐2 short interfering RNA reduced the viability of MM cells when co‐cultured with BMSCs or OCs.Calcium matrix‐coated slides were used to observe resorptive pits after pim447 treatment. A dose‐dependent reduction was detected. Pim447 reduced not only NFATc1, but also the level of a protease (cathepsin K) responsible for the degradation of the organic bone matrix. Other molecules involved in matrix resorption, such as MMP9 and ATP6V1A, were also substantially reduced following therapy, suggesting a role for pan‐pim kinase inhibitors in preventing bone resorption.4.2Pim‐2 kinase is also known as a common downstream molecule for the inhibition of osteoblastogenesis in myeloma5Transgenic mouse models and overexpression studies in cell lines have confirmed that pim kinase is involved in the transduction of many cellular signaling pathways, thereby promoting tumorigenesis, especially in blood tissues. This supports the hypothesis that pim kinase inhibitors are small molecular inhibitors for MM patient therapy.5.15.1.1Pim447 (LGH447) is an oral, potent pim kinase inhibitor that has entered clinical trials according to a study first performed in MM patients NCT01456689) , 77 Caucasian patients were included. The overall response rate (ORR) was 8.9%, with disease control and clinical benefit rates of 72.2% and 25.3%, respectively. A dose intake of 200 mg resulted in one patient (1.3%) achieving the desired PR and 10.9 months as median progression‐free survival.+ cells <10%; however, a 70% reduction in DNA synthesis was observed at 3 μM in U266 cells. Autophagy was induced in all types of cells .SGI‐1776 and AZD1208 are potent ATP‐competitive inhibitors and are in phase 1 clinical trials for the treatment of refractory prostate cancer, RR NHL patients,INCB053914, another selective ATP‐competitive inhibitor of pim kinase, inhibited pro‐apoptotic protein BAD phosphorylation in KMS‐12‐BM cells (IC50 = 134 nM), which is similar to mice bearing MM tumors (IC50 = 145 nM). Growth suppression of 43%, 71%, 77%, and 88% of KMS‐12‐BM tumor was observed with doses of 25, 50, 75, and 100 mg/kg BID, respectively.LGB321 is a small‐molecule selective ATP‐competitive pim kinase inhibitor. MM cells were extensively sensitive to LGB321, of which 14 MM cell lines had a GI50 lower than 1 mmol/L compared to that of NHL, CML, ALL, and AML cell lines. Tumor stasis got the maximal in vivo effect in the KMS‐111 luciferase xenograft model (100 mg/kg QD).SMI‐16a and SMI‐4a are compounds from the thiazolidine‐2,4‐dione‐family, that can reduce the pim‐2 protein activity without affecting its mRNA. Acidic (pH = 6.8) enhanced anti‐MM actions and decreased pim‐2 protein levels in MM cells, but AZD1208 and pim447 did not have similar results. SMI‐16a also restores Dox's anti‐MM effects in acidic conditions.5.1.2JP11646, a non‐ATP competitive inhibitor with selectivity for pim‐2, shows great anti‐proliferative effects, especially in MF‐signature cell lines (MM1.S) with a GI50 of 5 nM. It inhibits MM proliferation and has viability 4–760 times greater than other ATP‐competitive pim inhibitors. JP11646 progressively decreased pBAD (S112) and p4EBP1 (S65) with time in MM1.S and U266 cells and dose dependently minimized phosphorylation of the anti‐apoptotic factor MCL1 at Ser159 or Thr163 to 10% in MM1.S and 30% in U266 cells, because of its pim‐2 inhibitory effect.5.1.3A class of quinoxaline‐pyrrolodihydropiperidinones, pim‐1, and pim‐2 kinase inhibitors, exemplified by 9C, exhibited double‐digit nanomolar activity and potently reduced the viability of KMS‐12‐BM cells (IC50 = 151 nM). In the KMS‐12‐BM tumor mouse model, orally administered 9C at doses of 10–100 mg/kg resulted in a remarkable decline in tumors. Furthermore, the dose of 100 mg/kg completely silted tumor growth.A potent and selective series of quinazolinone–pyrrolopyrrolone compounds have been discovered. Compound 1 is efficient at the molecular (IC50 = 0.3 nM for pim‐1 and pim‐2) and cellular level (IC50 = 23 nM in KMS‐12 BM), and is also an efficient therapeutic in the myeloma model of mice from KMS‐12 BM with 78% TGI at 50 mg/kg QD.Compound 22 m, a sub‐nanomolar inhibitor of the pim‐1 and pim‐2 isoforms , effectively inhibited the phosphorylation of BAD in KMS‐12‐BM, a cell line that expresses high levels of all pim isoforms, to regulate myeloma cell proliferation and apoptosis.5.2Recently, several new drugs for MMs, such as immunomodulatory drugs (IMiDs), proteasome inhibitors (PIs), monoclonal antibodies, as well as several other small molecule inhibitors, have emerged.5.2.1The IMiDs currently used in clinical practice of MM management are lenalidomide, pomalidomide, and thalidomide. which are also among the mainstays in the management of MM.Pim‐2 can be degraded by proteasomes rather than ubiquitination.SMI‐16a could still downregulate pim‐2 kinase in KMS11 and RPMI8226 cells. Bortezomib, an NF‐κB pathway inhibitor, when combined with SMI‐16a,LY294002 is not only an inhibitor of the PI3K/Akt pathway,Aberrant JAK2 signaling and myeloproliferative neoplasms are closely related; however, the data suggest that tumor cells are insensitive to anti‐JAK2 treatment regimens; therefore, effective treatments are needed.In an in vitro experiment, a highly synergistic effect (CI = 0.096) was observed between pim447 (200 nM) and dexamethasone (10 nM) in MM1S. Similar results were observed in RPMI‐8226 cell lines.5.2.2Pim‐Pd refers to a combination of the pim kinase inhibitor IMiD and PI. Paíno combined pim447 with lenalidomide + dexamethasone and pomalidomide + dexamethasone in RPMI‐8226 cell lines. The results showed that the combination of the three drugs led to the death of approximately all myeloma cells at low doses .6The survival of tumor cells is closely related to the immune microenvironment of tumor tissues.6.1+ T cell memory requires both high levels of Eomesa expression and a continuum of intracellular NF‐κB signaling. pim‐1 kinase supports memory fitness in CD8+ T cells by supporting the expression of Eomes, and thus the memory of CD8+ T cells.+ cells from human cord blood differentiated toward Th1 cells, but not in cells toward Th2 cells.β‐selectin is one of the earliest management points in the process by which immature naive thymocytes differentiate, mature, and expand the required c‐Myb protein downstream of pim‐1.6.2Pim‐1 deficiency causes a marked reduction in IgM (−) B‐cell precursors.6.3IL‐12 and IFN‐α (two Th1‐specific cytokines) temporarily upregulate the mRNA levels of pim‐1 and pim‐2 in NK cells from the human peripheral blood.Immunomodulation is an important issue in MM research.7Pim kinase plays a vital role in cellular pathways, including JAK/STAT, c‐Myc, and BAD/Bcl‐2, and acts as a promoter of cell proliferation by phosphorylating several molecules including mTORC1, 4EBP1, and BAD. All these factors have been proven to be important in tumor cell growth and development. Recently, pim kinases have also been shown to regulate the cell cycle, for example, by activating CDC25A/CDC25C and changing cyclin‐dependent kinases such as CDK2, CDK4, and CDK6. Studies on pim kinase in MM bone disease have shown that pim kinase interacts with OCs and OBs via the NF‐κB and RANKL pathways, increasing osteoclastic activity and suppressing osteoblastic differentiation, which causes devastating bone destruction and rapid loss of bone. Pim kinase also regulates the differentiation and proliferation of lymphatic B cells and T cells and NK cells via activation of the NF‐κB, MYC, and mTOR pathways. All these effects of pim kinase indicate pim kinase as a comprehensive therapy target in MM.With more pim kinase inhibitors being discovered, the therapeutic options for patients with MM are increasing. All current myeloma drugs have certain side effects, in the case of pim, this may manifest as an increase in pim kinase levels. Based on the available experimental findings, we can say that this side effect can be largely reduced or even eliminated if pim kinase inhibitors are used in combination.8The pim kinase family was only discovered and defined in the 1990s, but its role in tumors, particularly hematologic tumors, had not received attention until 2010. Nevertheless, the role of pim kinases cannot be underestimated, as evidenced by the fact that a number of pim kinase inhibitors have entered phase III clinical trials in a short time. However, this emerging field still has many unknowns to explore.Current studies have failed to delve into and elucidate the role of pim kinase in the clonal proliferation of tumor cells, for example, there are two opposing views on the regulation of the cell cycle in myeloma cells. Tumor cells generally obtain energy rapidly through anaerobic glycolysis, as do myeloma cells. If pim kinase is able to regulate myeloma cell metabolism then it has the opportunity to influence all cellular activities and even the production of monoclonal immunoglobulins. On the other hand, the involvement of regulatory factors in the transcriptional and translational processes of pim kinases in myeloma cells is an area for future research. The above is focused on the tumor itself, but as the second most common hematologic tumor, myeloma lesion is in the patient's bone marrow, so myeloma inherently has an immune microenvironment containing a large number of naïve immune cells. It has been shown that myeloma cells can secrete cytokines to alter the bone marrow microenvironment to suit themselves. Whether pim kinase, which has been shown to regulate the proliferation and differentiation of immune cells, can regulate the immune microenvironment of myeloma remains to be investigated.Although single‐agent clinical trials such as pim447 have been beneficial for patients with MM, more clinical data are urgently needed. Bone disease is an important complication of myeloma, and osteolytic bone destruction is also involved in determining disease staging. We know that pim kinase is able to modulate osteogenesis and osteolysis, However, the current clinical trials were conducted in patients with relapsed/refractory MM and did not provide a good view of the effect of inhibitors on bone disease progression. Conducting trials in patients with newly diagnosed MM while tracking patients' blood calcium and bone destruction will help determine the efficacy of the drug. Combining pim kinase inhibitors with existing myeloma therapeutic agents to assess treatment efficacy and the occurrence of side effects will provide a reliable basis for the integrated management of myeloma patients in the future.Zhaoyun Liu: Methodology . Yunhe Zhang: Conceptualization . Yixuan Guo: Investigation . Hao Wang: Investigation . Rong Fu: Project administration .None.Figure S1. Supporting informationClick here for additional data file.


Ovarian metastasis from gastric cancer (GC) is characterized by aggressive biological behavior and poor outcome. Currently, there is no standard treatment mode for such patients. Thus, we evaluated the efficacy of conversion therapy in patients with synchronous ovarian metastasis from GC in this study.About 219 GC patients with ovarian metastasis in 2011–2020 were enrolled. Two groups were established based on the different treatment: the conversion therapy group  and the non‐conversion therapy group (NCS group). Propensity score matching (PSM) was used to analyze the efficacy of different treatment modes on the prognosis of these patients.p < 0.001). Twenty‐six patients (56.52%) in the CS group achieved R0 resection, and they had a better prognosis (p = 0.003). Compared with patients who underwent simultaneous gastrectomy and ovarian metastasectomy (CSb group), those who underwent ovarian metastasectomy before systemic chemotherapy (CSa group) had a higher R0 resection rate (p = 0.016) and longer survival time (p = 0.002). A total of 38 patients (41.30%) across both groups received hyperthermic intraperitoneal chemotherapy (HIPEC), and these patients had a better survival (p = 0.043).Ninety‐two patients were included according to PSM results, with 46 patients each in CS and NCS groups. The median overall survival (OS) in the CS group was notably better than that in the NCS group (The conversion therapy is safe and effective for patients with synchronous ovarian metastasis from GC and can improve their prognosis. However, our results need to be confirmed by more randomized controlled clinical studies. After a comprehensive treatment like chemotherapy, the primary lesions and metastases of GC can be well controlled, and this may present an opportunity for the use of radical surgery, which would prolong the survival of these patients.In this study, propensity score matching (PSM) was used to balance the baseline characters and analyzed the efficacy of different treatment modalities and their influence on the prognosis of patients in pursuit of identifying a scientific and effective treatment model for patients with synchronous ovarian metastasis from GC.22.1The clinicopathologic data were retrospectively reviewed in 219 GC patients with ovarian metastasis who underwent gastrectomy and metastasectomy or systemic chemotherapy as the initial treatment in Zhejiang Cancer Hospital  from January 2011 to December 2020. The study design is presented in Figure Patients were enrolled according to the following criteria: (1) histologically confirmed primary gastric/esophagogastric junction adenocarcinoma; (2) age 18–75 years; (3) synchronous ovarian metastasis from GC (ovarian metastases were diagnosed simultaneously with GC or confirmed within 6 months after GC diagnosis) without peritoneal metastasis or with limited peritoneal metastasis  with extensive peritoneal dissemination or other distant metastasis; (2) metachronous ovarian metastasis from GC (ovarian metastases were diagnosed more than 6 months after GC diagnosis or surgery); (3) combined with other malignancies; and (4) lack of complete clinicopathologic data.According to the above criteria, 101 cases were excluded, and 118 cases were finally included in the analysis  and non‐conversion therapy group (NCS group). Before the initial treatment, imaging examination was performed in all patients to assess the severity of disease and resectability. Surgical resection was performed according to consensual opinion after comprehensive assessment of patient by a multidisciplinary team.The NCS group received palliative chemotherapy with or without exploratory surgery . The CS group received chemotherapy plus surgery (gastrectomy and metastasectomy). In addition, we divided the CS group into CSa group (ovarian metastases were resected before systemic chemotherapy) and CSb group (ovarian metastases were resected after systemic chemotherapy).2.2.12; day 1) plus S‐1 dose was calculated on the basis of body surface area  for a 21‐day cycle.PS regimen: intravenous paclitaxel  with 3 L of 0.9% saline was heated to 43.0°C ± 0.3°C and infused into the peritoneal cavity via an automatic HIPEC device  for a circulation about 60 min.Some patients received hyperthermic intraperitoneal chemotherapy (HIPEC) after the initial exploratory surgery: paclitaxel . Overall survival (OS) was defined as the time of pathological diagnosis of GC and the cutoff date was June 30, 2022.2.4PSM was calculated based on a logistic regression model to balance the baseline characters between the groups. According to the analysis results and prognostic factors reported already,2.5p < 0.05. Student's t‐test was used for continuous variables and chi‐squared test was used to assess discrete variables. The Kaplan–Meier method and log‐rank test were used for survival analysis. The Cox regression model was used to estimated hazard ratio (HR) and 95% confidence interval (95% CI) in univariable and multivariable analysis.All data were analyzed by SPSS 26.0 (IBM Corporation) and statistical significance was defined as a 33.1p = 0.889).Ninety‐two patients were enrolled according to the PSM result, including 46 patients in the NCS group while 46 patients in the CS group . The median age at participation was 44.9 years ; the primary tumor was poorly differentiated adenocarcinoma in 87 cases (94.57%) and well‐ or moderately differentiated adenocarcinoma in five cases (5.43%). Primary tumor invasion was T1–T2 in 11 cases (11.96%) and T3–T4 in 81 cases (88.04%). Ovarian metastases were bilateral in 69 cases (75.00%) and unilateral in 23 cases (25%). The maximum diameter of Krukenberg tumors was <5 cm in 20 cases (21.74%) and ≥5 cm in 72 cases (78.26%). Peritoneal metastasis was noted in 61 cases (66.30%) and was absent in 31 cases (33.70%). Baseline characteristics in two groups were presented in Table 3.2Patients in the NCS group received an average of 3.9 cycles of chemotherapy , with 16 patients undergoing exploratory surgery and 14 patients (30.43%) receiving an average of 1.7 cycles of HIPEC .χ2 = 6.669, p = 0.016) and preoperative N0/1  were associated with better R0 resection rate. Additionally, 24 patients had tumor degeneration ≤TRG 2 . The clinicopathologic factors and treatment outcomes of the CS group are presented in Table Patients in the CS group received an average of 2.9 cycles of chemotherapy  before gastrectomy and 3.8 cycles  after the operation. Twenty‐four patients (52.17%) received an average of 2.0 cycles of HIPEC . Among the enrolled patients, 26 patients underwent total gastrectomy while 20 patients underwent distal gastrectomy. Five patients (10.9%) received combined visceral resections, which included pancreatectomies in three cases, splenectomy in one case, and colectomy in one case. Notably, 26 patients (56.52%) achieved R0 resection, and our analysis showed that ovarian metastasectomy before systemic chemotherapy (3.3The median follow‐up duration was 17.6 (range 3–72) months. The median overall survival (mOS) of the 92 patients was 14.0 (95% CI 11.5–16.5) months, and the 1‐year survival rates was 57.0% while the 2‐year survival rates was 24.3%.p < 0.001; Figure p = 0.002; Figure p = 0.003; Figure The mOS in the CS group was 19.0 95%CI 15.4–22.5) months, which was notably longer than that of the NCS group  received HIPEC treatment. The HIPEC group had a notable survival improvement . Among these, leucopenia/neutropenia (11.96%) and thrombocytopenia (2.17%) were the most frequent hematological toxic effects, while elevated serum AST levels (8.70%) and nausea (6.52%) were the most common non‐hematological toxic effects.In this study, 26 out of 92 patients (28.26%) developed Grade 3–4 adverse events  in the CS group developed postoperative complications (Clavien‐Dindo grade II–III), which included four cases of pneumonia, three cases of intestinal obstruction, and one case of anastomotic stricture. All complications were successfully alleviated by conservative treatment.3.5p = 0.002) and peritoneal metastasis  were identified as independent predictors of OS , R0 resection  and peritoneal carcinomatosis  were identified as independent predictors of OS in patients underwent conversion therapy . Additionally, a total of 26 patients (56.52%) in the CS group achieved R0 resection, and the prognosis of them was significantly improved compared with those without R0 resection . Our findings show that the prognosis of these patients can be significantly improved by the conversion therapy model of chemotherapy combined with gastrectomy and ovarian metastasectomy, particularly in R0 resection cases.In this study, 92 patients with synchronous ovarian metastasis from GC were matched using a 1:1 PSM analysis. The mOS in the CS group shows a significant difference with which in the NCS group . These patients are often present with ascites or peritoneal metastasis, which are associated with a large tumor burden and poor chemosensitivity.χ2 = 5.839, p = 0.028). Thus, for GC patients with synchronous ovarian metastases, particularly cases with large metastases and massive ascites, the conversion therapy mode of ovarian metastasectomy‐chemotherapy‐gastric tumor resection may be a better choice; however, the results need to be confirmed by further clinical studies.Moreover, we compared the differences between conversion therapy modalities. Patients who underwent ovarian metastasectomy before systemic chemotherapy (CSa group) had a better R0 resection rate and longer OS than those who underwent simultaneous resection of primary GC and ovarian metastases after chemotherapy , and further analysis presented that ovarian metastasectomy before systemic chemotherapy can achieve a better prognosis in such patients . Moreover, previous studies have shown that HIPEC can exert a potent antitumor effect by increasing contact between tumor lesions and chemotherapeutic agents while enhancing cytotoxicity through its thermal effect.p = 0.043), and further analysis revealed that the difference in prognosis was more significant in patients with peritoneal metastasis . Therefore, HIPEC can prolong the survival time of GC patients with synchronous ovarian metastasis, especially for patients with peritoneal metastases. However, more large sample researches are still required to further verify these results.Analysis of prognostic factors shows that peritoneal carcinomatosis is one of the independent predictors of OS. Ovarian metastasis from GC is often present with varying degrees of peritoneal metastasis, which often leads to ascites and ileus, and is associated with a poor prognosis.Our study also had some limitations. Above all, it was a single‐center retrospective study; PSM can reduce the selection bias, but it cannot be completely eliminated. Differences in some factors between the two groups, such as peritoneal metastasis, preoperative serum levels of CA199 and CA125 may affect the final results. Second, the overall sample size was small, and the conclusions need to be verified by more prospective clinical researches. However, as far as we know, this is the first PSM study on conversion therapy for GC patients with synchronous ovarian metastasis, which has important implications for future exploration of effective treatment models for such patients.In conclusion, the conversion therapy mode of chemotherapy combined with surgical resection for patients with synchronous ovarian metastasis from GC is safe and effective, and can improve the prognosis of these patients. Moreover, resection of ovarian metastases before systemic chemotherapy and combined treatment with HIPEC could improve the therapeutic effect in such patients. In the future, more randomized controlled researches are needed to provide a basis for optimizing the treatment mode for GC patients with synchronous ovarian metastasis.Jingquan Fang: Conceptualization ; data curation (lead); formal analysis (lead); investigation ; methodology ; project administration (lead); resources ; software (lead); supervision ; writing – original draft (lead); writing – review and editing (lead). Xingmao Huang: Data curation ; software ; writing – review and editing . Xiangliu Chen: Data curation (supporting); software (supporting). Qi Xu: Data curation (supporting). Tengjiao Chai: Data curation (supporting). Ling Huang: Data curation (supporting). Han Chen: Data curation (supporting); software (supporting). Hang Chen: Data curation (supporting); software (supporting). Zeyao Ye: Data curation (supporting). Yian Du: Data curation (supporting). Pengfei Yu: Conceptualization (lead); data curation ; formal analysis ; funding acquisition (lead); investigation ; methodology (lead); project administration ; resources (lead); supervision (lead); writing – review and editing .The study was approved by the Institutional Ethics Committee of Zhejiang Cancer Hospital (no. IRB‐2022‐279) and informed consent has been waived by the Ethic Committee.


Rhizobiaceae, known as agrobacteria, are responsible for crown and cane gall diseases of various crops worldwide. Tumorigenic agrobacteria are commonly found in the genera Agrobacterium, Allorhizobium, and Rhizobium. In this study, we analyzed a distinct “tumorigenes” clade of the genus Rhizobium, which includes the tumorigenic species Rhizobium tumorigenes, as well as strains causing crown gall disease on rhododendron. Here, high‐quality, closed genomes of representatives of the “tumorigenes” clade were generated, followed by comparative genomic and phylogenomic analyses. Additionally, the phenotypic characteristics of representatives of the “tumorigenes” clade were analyzed. Our results showed that the tumorigenic strains isolated from rhododendron represent a novel species of the genus Rhizobium for which the name Rhizobium rhododendri sp. nov. is proposed. This species also includes additional strains originating from blueberry and Himalayan blackberry in the United States, whose genome sequences were retrieved from GenBank. Both R. tumorigenes and R. rhododendri contain multipartite genomes, including a chromosome, putative chromids, and megaplasmids. Synteny and phylogenetic analyses indicated that a large putative chromid of R. rhododendri resulted from the cointegration of an ancestral megaplasmid and two putative chromids, following its divergence from R. tumorigenes. Moreover, gene clusters specific for both species of the “tumorigenes” clade were identified, and their biological functions and roles in the ecological diversification of R. rhododendri and R. tumorigenes were predicted and discussed.Tumorigenic members of the family  Rhizobium for which the name Rhizobium rhododendri sp. nov. is proposed. Both species of the “tumorigenes” clade (R. rhododendri and R. tumorigenes), contain multipartite genomes. Synteny and phylogenetic analyses indicated that a large putative chromid of R. rhododendri resulted from the cointegration of an ancestral megaplasmid and two putative chromids, following its divergence from R. tumorigenes.This study revealed additional genomic and taxonomic diversity of tumorigenic agrobacteria. Tumorigenic strains isolated from rhododendron represent a novel species of the genus  Accordingly, Rhizobiaceae members exhibit remarkably diverse lifestyles, ranging from plant symbionts (rhizobia) and pathogens (agrobacteria) to opportunistic human pathogens, to free‐living species in soils, sediments, and water  into plant host cells Gelvin, . ConsequRhizobiaceae. To date, strains carrying Ti plasmids and able to cause crown gall and hairy root diseases (agrobacteria) have been primarily identified within the genera Agrobacterium, Allorhizobium, and Rhizobium. Additionally, a Neorhizobium strain carrying a Ti plasmid and able to cause tumors on multiple host plants was identified recently  was the only tumorigenic Rhizobium species. However, another member of this genus, Rhizobium tumorigenes, was recently isolated from cane gall tumors on thornless blackberries   used in this study was isolated in 2017 from crown gall tumors on rhododendron originating from a nursery in Lower Saxony, Germany  and 932 ( = DSM 104878 = CFBP 8566) reported in our previous study  at 28°C for 48 h. Cultures were stored in a −80°C freezer in nutrient broth with 20% glycerol for long‐term preservation.2.2T and 1078T was assessed on different agar media: yeast mannitol agar (YMA)   following the instructions provided by the manufacturer.The growth of bacterial strains rho‐6.2For the FAME analysis, strains were cultured on R2A medium at 25°C for 3 days. The cellular fatty acids were analyzed using the Microbial Identification System , according to instructions provided by the manufacturer Sasser, . Combine2.3Genomic DNA was extracted from bacterial strains using a Qiagen Genomic DNA Buffer Set  and Qiagen genomic tip 100/G gravity‐flow, anion exchange columns (Cat. No. 10243). The purity and approximate concentration of DNA were determined by spectrophotometry using the NanoDrop instrument. Genomic DNA integrity was assessed by agarose gel electrophoresis.2.4Rhizobium strains rho‐6.2T, 1078T, and 932 was analyzed by the modified method of Eckhardt  and samples were incubated for 15 min on ice. An agarose gel was prepared during the previous incubation steps by loading 25 μL of 10% sodium dodecyl sulfate  into empty wells, followed by gently flooding the gel with 1× TBE buffer and running electrophoresis at 4 V cm−1 for 15 min from positive to negative polarity (opposite direction to standard DNA gel electrophoresis). Next, 10 µL lysing solution in TE buffer containing 0.4 mg mL–1 RNase A, 1 mg mL–1 bromophenol blue, and 1.5 mg mL–1 lysozyme (freshly prepared aqueous solution) was added to each cell sample after incubation on ice. A 30 µL aliquot of the mixture was loaded immediately into wells in the gel. Electrophoresis was run first at 1.5 V cm–1 for 1 h and then at 4 V cm–1 for 20 h (standard DNA gel electrophoresis from negative to positive polarity). The gel was stained in ethidium bromide solution (1 μg/mL) and the plasmids were visualized under UV light. As markers, “Agrobacterium fabrum” C58T, Allorhizobium ampelinum S4T, and R. rhizogenes K84 carrying replicons of known size were used.The plasmid content of Eckhardt . This me2.5T and 932 was performed using an Illumina NextSeq 500 platform in PE75 mode. For strain rho‐6.2T, paired‐end 151 bp reads previously generated on an Illumina NextSeq 500 platform  with modifications according to Baym et al. . Genome 2.6SMRTbell template libraries were prepared according to the instructions from Pacific Biosciences, following the Procedure & Checklist—Greater Than 10 kb Template Preparation document. Briefly, for the preparation of 15 kb libraries, 8 µg genomic DNA was sheared using g‐tubes from Covaris according to the manufacturer's instructions. DNA was end‐repaired and ligated overnight to hairpin adapters applying components from the DNA/Polymerase Binding Kit P6 from Pacific Biosciences. Reactions were carried out according to the instructions of the manufacturer. BluePippin Size‐Selection to greater than 4 kb was performed according to the manufacturer's instructions (Sage Science). Conditions for annealing of the sequencing primers and binding of polymerase to purified SMRTbell template were assessed with the Calculator in RS Remote (Pacific Biosciences). Single‐molecule real‐time (SMRT) sequencing was carried out on the PacBio RSII (Pacific Biosciences) taking one 240‐min movie on one SMRT cell per sample using the P6 Chemistry.2.7dnaA (chromosomes) or repA (chromids and megaplasmids) as the first gene.SMRT Cell data were assembled using the “RS_HGAP_Assembly.3” protocol included in SMRT Portal version 2.3.0 using default parameters. Assembled replicons were circularized and adjusted to T, and 2 × 75 bp for strains 932 and 1078T) onto the PacBio assemblies using BWA 0.6.2    Seemann,  were use2.8Bacterial replicons were classified using an approach similar to that described previously  with the orthologous replicons of related strains (query sequences). The BRIG analysis was done by using the BLASTn option.Synteny between genomes of the “tumorigenes” clade was explored using circos. First, blast bidirectional best hits (Blast‐BBHs) were identified using BLASTn version 2.10.1+  , and edited using Inkscape version 1.2.1 (https://inkscape.org/).To assess the evolutionary relationships among the extrachromosomal replicons of the “tumorigenes” clade, phylogenetic analysis based on the RepA and RepB protein sequences was conducted. Protein sequence alignments for each set of orthologs were generated using MAFFT version 7 . In particular, finished genomes of Rhizobium strain rho‐6.2T and R. tumorigenes strains 1078T and 932 obtained in this study were used. We also included the previously‐reported draft genome sequences of two additional Rhizobium strains associated with rhododendron crown gall, rho‐1.1 and rho‐13.1  searches against the nucleotide collection (nr/nt) and whole‐genome shotgun contigs (wgs) databases using 16 S rRNA and recA housekeeping gene sequences as a query, with default parameters (last accessed on November 2022).The data set comprised 119 genomes, including 116 Core‐genome‐ and pan‐genome‐based phylogenies were inferred using GET_HOMOLOGUES Version 11042019 . For the delineation of species, we computed overall genome relatedness indices (OGRIs), in particular, average nucleotide identity (ANI)   were considered  using the aai_wf command with default parameters. For the calculation of cpAAI, the cpAAI_Rhizobiaceae pipeline (https://github.com/flass/cpAAI_Rhizobiaceae) was employed to generate a concatenated protein alignment of a reference set of 170 marker proteins from 97 reference strains, using the prealigned reference protein files (option –A) as described in Kuzmanović, Fagorzi, et al. (https://github.com/flass/cpAAI_Rhizobiaceae) that relied on the “dist.aa” function from the “ape” package   and proteome (cpAAI and wpAAI) relatedness values were generated and plotted onto the reference core‐proteome phylogenetic tree by the “phylo.heatmap” function in the R package phytools Revell, .2.11R. tumorigenes strains (1078T and 932) and 12 strains comprising the new species R. rhododendri . The analysis was performed using the GET_HOMOLOGUES software and its auxiliary scripts as described before  in the other species, we performed BLASTp  were generated using a combination of long‐ (PacBio) and short‐read (Illumina) sequencing technologies (see Table A2 for summary statistics of the generated sequencing data: https://doi.org/10.6084/m9.figshare.22193809). Genome assembly and polishing resulted in gapless, circular replicons for all sequenced strains, with high average sequencing depths   was confirmed by a modified Eckhardt agarose gel electrophoresis technique, although some of the replicons of similar size could not be clearly differentiated  and differed by 10 SNPs from those of strain rho‐6.2T.For all three strains, four rRNA operons  were identified on the largest replicon. Unlike strains rho‐6.23.2Whole‐genome sequencing revealed that all strains in the “tumorigenes” clade contain multipartite genomes Figure . The larFigure 1Rhizobium rhododendri rho‐6.2T (a), and strains of Rhizobium tumorigenes 1078T (b) and 932 (c). Each replicon is presented by a circular plot containing five rings. Genetic coordinates of the reference sequences are shown within the thin inner ring. The next two rings portray GC content (black ring) and GC skew (purple/green). The next ring shows core (red) and species‐specific (blue) genes. Core genes (364) were identified from a data set of 119 strains using GET_HOMOLOGUES software. The accessory genes (R. rhododendri vs. R. tumorigenes) were identified with the same software. The outermost ring highlights prophage regions identified with PHASTER (intact prophages are shown in green and incomplete in orange) and insertion sequence (IS) elements identified using ISEscan (shown in gray). As in some cases IS elements were identified within the prophage regions, borders of the latter regions are highlighted with the corresponding color. The Figure was generated using BRIG software and edited with Inkscape (see M&M for details).Circular maps of the complete genomes of strain vir operons virD , virBGCE , and virA , as well as T‐DNA  that comprises genes coding for an unknown opine. The pTi1078 plasmid also carried multiple copies of the vir genes , and two T‐DNAs . The first T‐DNA includes genes encoding synthesis of the opines agrocinopine and nopaline, and the second contains genes associated with the production of an unknown opine. The pTi932 plasmid was closely related to pTi1078, and their vir and T‐DNA regions were highly similar or identical.In accordance with our previous work demonstrating the pathogenicity of the “tumorigenes” clade  and three putative chromids (302–433 kb) (Table https://doi.org/10.6084/m9.figshare.22193809), whose gene contents were highly conserved between strains 1078T and 932  was found on putative chromid 2 of strain 932 (orthologous to putative chromid 3 of strain 1078T). Likewise, a 25‐gene cluster of putative chromid 3 of strain 1078 was found on putative chromid 1 of strain 932  and two putative chromids (orthologous to putative chromids 1 and 3 of 1078T)  contains three repABC cassettes, which are orthologous to the repABC cassettes of pRt1078 and putative chromids 1 and 3 of strain 1078T  Figure . ConsistT displayed high conservation with putative chromid 2 of strain 1078T  and they shared less than 92% identity with all other RepA proteins from the family Rhizobiaceae. A comparison of the R. tumorigenes chromids with the most closely related replicons from other Rhizobiaceae species using d‐Geneies , coupling protein (traG), and T4SS proteins (trb). Likewise, the large cointegrant of rho‐6.2T carries genes for conjugation. Interestingly, however, these genes were divergent from those carried on pRt932 and pRt1078. For instance, the VirB4 protein sequences of pRt1078 and the cointegrant of rho‐6.2 shared only 40.8% identity.We could not identify genes associated with mobilization or conjugation on the chromids of strains 10783.3Onobrychidicola muellerharveyae TH2T, and we, therefore, excluded this marker from the analysis.The core genome of the 119 strains included in the analysis was identified using GET_HOMOLOGUES and comprised 364 homologous gene clusters. Phylogeny was inferred from 253 DNA and 191 protein markers that were selected using the GET_PHYLOMARKERS software. Moreover, we also inferred phylogeny from the protein alignment outputted by the cpAAI pipeline, which represented the concatenated sequence of a reference set of 169 protein markers. Although the original data set included 170 protein markers, one marker gene was missing in Rhizobiaceae genera and major Rhizobiaceae clades (data not shown). The only difference was the position of the genus Xaviernesmea, which was an outgroup of the clade containing the genera Ensifer, Pararhizobium, and Sinorhizobium in the DNA‐based phylogenetic tree, while it was grouped with Pararhizobium spp. in the protein‐based phylogenetic trees. Additionally, the phylogenetic position of several taxa within some subclades differed slightly between trees. Regardless, the phylogenetic positions of the taxa that are the subject of this work were identical across trees, and we, therefore, show only the core‐proteome phylogenetic tree based on 191 protein markers  and strains isolated from rhododendron in Germany  clustered within two sister subclades in the clade we previously defined as “tumorigenes” , while the second clade consisted of eight Rhizobium strains isolated from Himalayan blackberry in Oregon   . These results suggest that the two “tumorigenes” subclades represent distinct species, and we propose the name R. rhododendri (see the protologue below) for the sub‐clade containing the strains originating from rhododendron, blueberry, and Himalayan blackberry. During writing this article, genomes of eight additional strains  became available in GenBank (BioProject Accession No. PRJNA762915) that also belong to the species R. rhododendri, based on ANIb comparisons (>99% ANI with rho‐6.2T). These strains were isolated from cane galls of blueberry in Oregon (USA) in 2019. However, as these genomes were unpublished, they were not included in further analyses.For species delineation, we relied on ANI and dDDH computations. The threshold for species delineation was set at ~95%–96% for ANI .Phenotypic characteristics of strains https://doi.org/10.6084/m9.figshare.22193809. Similar to the other phenotypic characteristics that were measured, strains R. rhododendri rho‐6.2T and R. tumorigenes 1078T exhibited highly similar FAME profiles. The only notable difference was in C18:1 w7c 11‐methyl, which was ~2.5‐fold more abundant in rho‐6.2T than in 1078T. Overall, the major fatty acids (>5%) identified in each of these strains are C18:1ω7c (~50%), C19:0 cyclo ω7c (~18‐22%), and C16:0 (~5‐7%).The results of the fatty acid analysis are summarized in Table A6 at Rhizobium species within the clade “tropici‐rhizogenes” that is associated with crown gall disease. This potential new species included strains 17‐2069‐2b and 17‐2069‐2c isolated from blackberry, which were reported to carry Ti plasmids . Primarily, we relied on cpAAI calculated on the marker proteins selected in our former work  and “tropici‐rhizogenes” (86.81%–87.51%) clades, although this taxon was phylogenetically more closely related to the former clade. The wpAAI‐based approach suggested the same unclear delineation of R. tubonense  were highly congruent, with a few differences observed within the genera Hoeflea, Martelella, and Pararhizobium  were identified. These genes were present in all 12 strains of R. rhododendri and absent in both R. tumorigenes strains. More than half (138) of these genes were located on putative chromid 1. Of the remaining genes, 108 were located on the chromosome, 27 on putative chromid 2, and 1 on pTi6.2 . Among those that were functionally annotated, based on COG categories, the most represented functional categories were K (Transcription), G (Carbohydrate metabolism and transport), and M , comprising 32, 31, and 15 genes, respectively (Table A8a: https://doi.org/10.6084/m9.figshare.22193809). Among the Rr‐specific genes or gene clusters with predicted biological functions was the gene cluster Rr62_02696‐Rr62_02698, predicted to be involved in the production of cellulose (Table A8a: https://doi.org/10.6084/m9.figshare.22193809); however, homologous, but divergent gene clusters with the same predicted function were also present in both R. tumorigenes strains (At1078_ 03513‐At1078_ 03517 in strain 1078T), and in another copy in strain rho‐6.2T (Rr62_03520‐Rr62_03524). All putative gene clusters putatively associated with cellulose synthesis were located on chromosomes. Furthermore, the Rr‐specific gene clusters Rr62_04000‐Rr62_04012 and Rr62_05395‐Rr62_05405 are annotated as being involved in the processing of various simple sugars (d‐psicose/d‐tagatose/l‐ribulose) and sugar alcohols , respectively (Table A8a: https://doi.org/10.6084/m9.figshare.22193809).Based on the pan‐genome analysis, 272 genes specific to 2 Figure . Most of3.6.2R. tumorigenes (Rt‐specific), meaning they are present in both R. tumorigenes strains and absent from all R. rhododendri strains. In strain 1078T, one of these 322 genes was present in three copies, while there were two copies of five other genes. In strain 932, nine genes were present in two copies. Of the 326 genes specific for R. tumorigenes strain 1078T, including genes present in multiple copies, 159 were located on pRt1078, 58 on the chromosome, 57 on putative chromid 1, 31 on pTi1078, 3 on putative chromid 2, and 21 on putative chromid 3 . As for the Rr‐specific genes, the function of the majority of the Rt‐specific genes could not be precisely determined (Table A8b: https://doi.org/10.6084/m9.figshare.22193809). Based on COG classification, annotated Rt‐specific genes of strain 1078T were primarily annotated as belonging to the functional categories P , L , K , and E  (Table A8b: https://doi.org/10.6084/m9.figshare.22193809).Exploration of the pan‐genome of the “tumorigenes” clade resulted in 322 genes that are specific to 3 Figure . Of the 3 Figure . DiffereR. tumorigenes strains carried a gene cluster  associated with the type VI secretion system (T6SS). This gene cluster was encoded on putative chromid 1 of strains 1078T and 932 (Table A8b: https://doi.org/10.6084/m9.figshare.22193809). Although they were not identified as species‐specific by GET_HOMOLOGUES, R. tumorigenes strains carried multiple copies of vgrG and single copies of paar genes, which are also associated with T6SS machinery (data not shown). R. tumorigenes strains also carried a gene cluster (At1078_04243‐At1078_04247) annotated as being involved in the synthesis of pseudaminic acid.Most interestingly, T, or on putative chromid 2 of strain 932, a putative gene encoding polygalacturonase (glycoside hydrolase family 28) was identified (Table A8b: https://doi.org/10.6084/m9.figshare.22193809). The polygalacturonase protein sequence of R. tumorigenes 1078T shared only 20.7% amino acid identity (69% of query coverage) with the polygalacturonase protein of A. ampelinum S4T (Avi_1489), which was previously described  . The species R. rhododendri comprised tumorigenic strains isolated from aerial tumors on rhododendron , we could not identify additional strains belonging to the “tumorigenes” clade. Nevertheless, we assume that members of this clade are distributed more widely and their genetic diversity remains to be explored.In this study, we conducted polyphasic characterization of “tumorigenes” clade representatives and described a novel species R. rhizogenes and the “tumorigenes” clade, agrobacteria also occur within other Rhizobium clades. In particular, the “tropici‐rhizogenes” clade comprises at least two additional species that include tumorigenic strains. The first one corresponds to Rhizobium sp. AB2/73 which was isolated from Lippia canescens in the USA .Apart from 4.2Rhizobiaceae consist of a multipartite architecture , we classified this replicon as a putative chromid as parts of the cointegrant are derived from chromid‐like replicons. Although not feasible to test experimentally, it would be interesting to observe whether the megaplasmid portion of the cointegrant evolves chromid‐like properties over time.4.3R. rhododendri and R. tumorigenes by examining the pan‐genome of the “tumorigenes” clade. Our objective was to identify potentially adaptive features among species‐specific genes, to gain a better understanding of the ecological differentiation of these species. We recognize, however, that the availability of genomes for only two R. tumorigenes strains is a limitation of this analysis, and that the number of species‐specific genes will likely decrease as more genomes become available. Nevertheless, based on the available genomes, the majority of species‐specific genes are encoded on putative chromids and megaplasmids, which is in line with previous studies analyzing the A. tumefaciens species complex  associated with nodulation efficiency in some hosts  or nitrogen fixation , suggesting that they do not have N2‐fixing symbiotic capacities.Species 4.4Rhizobium superclade should be split into at least three genera. Besides the clade “leguminosarum‐etli” (Rhizobium sensu stricto), which includes the type species of the genus Rhizobium (R. leguminosarum), clades “tropici‐rhizogenes,” and “tumorigenes” represent candidates for new Rhizobiaceae genera. In our opinion, such a division of Rhizobium species would require additional genomic or phenotypic evidence, thus revealing factors relevant to the biological and ecological diversification of these clades. However, this taxonomic revision was not an objective of this work, and we followed the taxonomic scheme preserving the current structure of the genus Rhizobium.Based on genus demarcation thresholds defined in our previous study  in both cases  contain multipartite genomes, including a chromosome, putative chromids, and megaplasmids. Interestingly, these two species showed distinct genome architecture. Our investigation indicated that the large putative chromid of R. rhododendri is a cointegrant of an R. tumorigenes‐like ancestral megaplasmid and two putative chromids. Moreover, evidence of interreplicon DNA exchange between putative chromids of one R. tumorigenes lineage was detected. Furthermore, we examined the pan‐genome of members of the “tumorigenes” clade and identified genes specific to each of the species R. rhododendri and R. tumorigenes. For some of the genes and gene clusters, it was possible to determine the putative function and possible role in the ecological adaptation of the studied bacterial species. The predicted functions are found to be primarily associated with plant‐bacterial interactions, bacterial competitiveness in plant tissue or rhizosphere, and uptake of specific nutrient sources.This study revealed additional genomic and taxonomic diversity of tumorigenic agrobacteria. OGRIs and phylogenomic analyses clearly showed that tumorigenic strains isolated from rhododendron represent a novel species of the genus 6R. rhododendri . Bacterial cells are Gram‐negative, motile, and nonspore‐forming. They are aerobic, and oxidase and catalase positive. Bacteria grow well on YMA, TY, PDA‐CaCO3, and R2A media, whereas weak growth was observed on King's medium B. Colonies on YMA medium had a diameter of 1‐2 mm after 72 h of growth at 28°C. They were white to cream‐colored, circular, convex, and glistening. Growth was observed at a temperature range between 5°C and 30°C. Nitrate reduction, indole production, and glucose fermentation are negative. Arginine dihydrolase and gelatin hydrolysis tests are negative. Esculin hydrolysis and b‐galactosidase tests are positive. d‐glucose, d‐mannose, and d‐mannitol are assimilated. A weak assimilation was observed for l‐arabinose and d‐maltose. Potassium gluconate, caprate, adipate, malate, trisodium citrate, and phenylacetate are not assimilated. Strain forms clear zones on PDA‐CaCO3, but does not produce 3‐ketolactose from lactose. The major fatty acids (>5%) are C18:1ω7c (~50%), C19:0 cyclo ω7c (~19%), C16:0 (~7%), and C18:1ω7c 11‐methyl (~6%). R. rhododendri strains rho‐6.2T, rho‐1.1, and rho‐13.1 caused tumors on inoculated rhododendron, sunflower, and tomato plants, and were proven to carry a Ti plasmid  is 5.96 Mb. The genome is composed of a circular chromosome (3.71 Mb) and 3 extrachromosomal replicons that are 1.53 Mb, 382 kb, and 337 kb in size. The GC content of the total genomic DNA is 59.98%. R. rhododendri can be distinguished from other Rhizobium spp. based on OGRIs  calculations.The genome size of the type strain (rho‐6.2T ( = DSM 110655T = CFBP 9067T) was isolated from an aerial tumor on Rhododendron sp. in Germany in 2017. The DDBJ/ENA/GenBank accession numbers for the genome sequence are CP117267 to CP117270.The type strain, rho‐6.2Nemanja Kuzmanović: Conceptualization (lead); data curation (lead); formal analysis (lead); funding acquisition (lead); investigation (lead); visualization ; writing—original draft (lead); writing—review and editing . George C. diCenzo: Conceptualization ; data curation ; formal analysis ; visualization ; writing—original draft ; writing—review and editing . Boyke Bunk: Data curation (supporting); formal analysis (supporting); writing—review and editing . Cathrin Spröer: Investigation (supporting). Anja Frühling: Investigation (supporting). Meina Neumann‐Schaal: Investigation ; writing—review and editing . Jörg Overmann: Resources (supporting); writing—review and editing . Kornelia Smalla: Conceptualization (supporting); funding acquisition (supporting); resources ; supervision (supporting); writing—review and editing .None declared.None required.


Spinal cord injury (SCI) causes blood‐spinal cord barrier (BSCB) disruption, leading to secondary damage, such as hemorrhagic infiltration, inflammatory response, and neuronal cell death. It is of great significance to rebuild the BSCB at the early stage of SCI to alleviate the secondary injury for better prognosis. Yet, current research involved in the reconstruction of BSCB is insufficient. Accordingly, we provide a thermosensitive hydrogel‐based G protein‐coupled receptor 124 (GPR124) delivery strategy for rebuilding BSCB. Herein, we firstly found that the expression of GPR124 decreased post‐SCI and demonstrated that treatment with recombinant GPR124 could partially alleviate the disruption of BSCB post‐SCI by restoring tight junctions (TJs) and promoting migration and tube formation of endothelial cells. Interestingly, GPR124 could also boost the energy metabolism of endothelial cells. However, the absence of physicochemical stability restricted the wide usage of GPR124. Hence, we fabricated a thermosensitive heparin‐poloxamer (HP) hydrogel that demonstrated sustained GPR124 production and maintained the bioactivity of GPR124 (HP@124) for rebuilding the BSCB and eventually enhancing functional motor recovery post‐SCI. HP@124 hydrogel can encapsulate GPR124 at the lesion site by injection, providing prolonged release, preserving wounded tissues, and filling injured tissue cavities. Consequently, it induces synergistically efficient integrated regulation by blocking BSCB rupture, decreasing fibrotic scar formation, minimizing inflammatory response, boosting remyelination, and regenerating axons. Mechanistically, giving GPR124 activates energy metabolism via elevating the expression of phosphoenolpyruvate carboxykinase 2 (PCK2), and eventually restores the poor state of endothelial cells. This research demonstrated that early intervention by combining GPR124 with bioactive multifunctional hydrogel may have tremendous promise for restoring locomotor recovery in patients with central nervous system disorders, in addition to a translational approach for the medical therapy of SCI. In the primary phase, structural disturbance, especially blood‐spinal cord barrier (BSCB) disrupted caused by the trauma, which is one of the most crucial triggers of secondary phase.BSCB comprises nonfenestrated endothelial cells (ECs), pericytes, astrocytic end‐feet processes, and the basement membrane, which restricts paracellular and transcellular trafficking in the central nervous system. The BSCB function is similar to the blood–brain barrier in protecting the brain from toxins and infections and stimulating neuronal function. Yet, BSCB has unique structural and functional features such as glycogen deposits, different permeability, and protein expressions, which give it a unique vulnerability to pathological insults.G protein‐coupled receptor 124 (GPR124) is an orphan G‐protein coupled receptor (GPCR),Considering that GPR124 could be combined with heparin,In recent years, biomaterials‐based drug/cell delivery systems for restoring SCI have aroused wild interest, which is due to the unique space structures, easily modified characteristics and multifunctional properties of biomaterials, such as nanoparticles, liposomes, micelles as well as hydrogels.Poloxamer, as one of the classic hydrogels, has the mainly characteristics of hydrogels, and also owns unique merits: First, it could covalently bind to heparin through several chemical modification steps to form a hydrogel with three‐dimensional networks. Second, it is certified by the Food and Drug Administration (FDA), which may speed clinical translation. Next, the excellent effect on temperature sensitivity according to the concentration makes it more suitable for filling injured tissue cavities in situ, resulting in the protection of lesion site. Last, but not least, it has the ability of sustained release and injectability. Accordingly, we enabled poloxamer (P) as the host of our biomaterial. Heparin is used as a bridge between GPR124 and poloxamer. Taken together, an injectable and thermosensitive heparin‐poloxamer (HP) hydrogel was prepared here to load GPR124 (HP@124), which protects GPR124 from degradation and delivery in a sustained manner.Herein, we demonstrated the role of GPR124 on BSCB reconstruction post‐SCI, together with the synergistic effect of HP hydrogels, taking it further in vivo and in vitro. Moreover, we also found that adding GPR124 could attenuate the tight junctions (TJs) loss of endothelial cells, together with boosting migration and tube formation by restoring energy metabolism. Meanwhile, various comprehensive evaluations on function, morphology, and histology were carried out to assess the long‐term effect of the HP@124 hydrogel. In general, a thermosensitive hydrogel‐based GPR124 delivery strategy for early intervention of SCI was designed with notable physicochemical effects, which inhibits the disruption of BSCB and promotes motor function post‐SCI.22.13 mg/mL) was obtained by dissolving the lyophilized powder in fresh saline (4°C), and then GPR124 solution  was added under the aseptic conditions. The final concentration of GPR124 and HP was 50 μg/mL and 160 mg/mL respectively.First, we used 1‐ethyl‐3‐(3‐dimethylaminopropyl) carbodiimide (EDC)  and N‐hydroxylsuccinimide (NHS)  approach to synthesize HP. In brief, a mono amine‐terminated P (MATP) was prepared through the reaction between Poloxamer 407  and 4‐nitrophenyl chloroformate  under the condition of diamino ethylene . Next, the reaction of heparin  and MATP in 2‐(N‐morpholine) sulphonic acid  was performed by the coupling of EDC and NHS at room temperature for 24 h. After dialysis and lyophilization, the HP powder was obtained. Next, the HP hydrogel solution (1.6 × 102.2The HP hydrogel's structure was characterized by Fourier transform infrared spectroscopy (FTIR) . And the thermo‐sensitivity was tested by observing the hydrogel state from 4°C to 37°C and back to 4°C. In addition, dynamic rheology was carried out on a rheometer  by recording the storage and loss moduli, supported by a parallel plate (25 mm). The frequency was set to 10 rad/s; fixed strain was set to 1%.2.3The P hydrogel and HP@124 hydrogel were prepared by adding with 1 μg GPR124, respectively. Then, these two hydrogels were added into tubes, respectively, followed with equal volume of normal saline and incubated at 37°C. The supernatant was accumulated and replaced by the equivalent volume of normal saline at specific time points. The total concentrations of released GPR124 were measured using a micro bicinchoninic acid (BCA) protein assay kit according to the manufacturer's instruction .2.4−1) before the operation. A T9 laminectomy was implemented subsequently to fully expose the spinal cord, followed with a moderate crushing injury of spinal cord performed by a vascular clip for 1.5 min .6 unit dose of penicillin solution was injected intraperitoneally in each animal within 1‐week post‐surgery. Bladder emptying was also undertaken twice a day.One hundred and eight mature female SD rats (for facilitating urination management and eliminating gender differences in SCI repair) were prepared under regulated environmental conditions for the subsequent in vivo evaluations, which were purchased from the SLAC Laboratory Animal Company . The weight of rats was controlled from 220 to 240 g. All the animal procedures were approved by the Institutional Animal Care and Use Committee of Zhejiang University. Rats were anesthetized by intraperitoneal injection of 1% (w/v) pentobarbital sodium , 1 cm segment of the harvested spinal cord including lesion site was anatomized, followed by fixation with 4% paraformaldehyde (PFA) overnight. Paraffin embedment was performed subsequently. For the western blot (WB) experiment, 0.5 cm segment of the spinal cord including lesion epicenter was dissected and preserved at −80°C after harvested.2.6The BSCB permeability was assessed by Evans Blue (EB) staining assay according to previous reports.2.7Paraffin sections were arranged as described above. Hematoxylin and eosin (H&E) staining  of sections was performed for the evaluation of histopathology. Immunofluorescence test was implemented using primary antibodies targeting the corresponding proteins at 4°C overnight. After adequately washed by phosphate‐buffered saline with 0.05% Tween‐20 (PBST), the sections were incubated with secondary antibodies for 1 h at 37°C. Next, phosphate‐buffered saline (PBS)  was used to rinse the sections for three times, followed with 4′, 6‐diamidino‐2‐phenylindole  for 6 min. At last, the sections were sealed with a coverslip after washed with PBS. All primary and secondary antibodies used here are listed in Table 2.8Basso–Beattie–Bresnahan (BBB) locomotion scale was carried out to assess the recovery of motor function, of which the points range from 0 to 21 depending on the extent and coordination of hindlimb joints' movements.2.9Human cerebral microvascular endothelial cell line (HCMEC/D3) and human umbilical vein endothelial cell (HUVEC)  were cultured in high glucose Dulbecco modified Eagle medium (DMEM) medium  with 10% fetal bovine serum . For further detection of the effect of GPR124 and HP@124 hydrogel, we divided cells into four groups: sham group (DMEM), tert‐butyl hydroperoxide (TBHP) group ),TBHP + GPR124 group (add with 100 μM TBHP and 50 μg/mL GPR124), TBHP + HP@124 group .2.9.15 cells/mL after pretreatment, and then seeded into the superior chamber with high glucose DMEM medium only, whereas 600 μL high‐glucose DMEM medium containing 10% fetal bovine serum was added into the inferior chamber. The cells on the inferior surface were fixed by 4% formaldehyde, followed with staining of 0.1% crystal violet .Vertical migration test of HCMEC/D3s was performed by using transwell chamber with 8 μm filter inserts .2.9.2Pretreated HCMEC/D3s were seeded into a six‐well plate until grown into a confluence ratio of over 95%. Afterward a straight shape was scratched by a 200 μL tip. After observation and record, cells were rinsed with PBS and replaced with equal volume of the medium.2.9.3Pretreated HUVECs were seeded in a μ‐Slide Angiogenesis  pre‐coated with 10 μL Matrigel  per well and incubated at 37 °C. After 12 h, the vascular network was recorded by brightfield microscope for following quantitative analysis.2.9.4−1) in the superior chamber, meanwhile, 500 μL high glucose DMEM medium was filled in the inferior chamber. At last, collected the medium in the inferior chamber after 2 h and evaluated by fluorescence.The permeability experiment of HCMEC/D3s was performed using a transwell chamber with 0.4 μm filter inserts . Here, fluorescein isothiocyanate (FITC)‐dextran  was employed to examine the penetration of monolayer barrier of HCMEC/D3s. Pretreated cells were filled in the superior chamber to reach the confluence ratio of 100%, subsequently, adding with 100 μL FITC‐dextran solution  for 3 days. Next, lysed by Trizol , total RNA extraction was collected for the following RNA sequencing analysis . In order to elucidate the role of genes modulated by the gel, gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis were carried out.2.9.6d‐glucose (2‐DG) . Next, cells were incubated with normal medium for 1 h before the mitochondrial stress evaluation by detecting the oxygen consumption rate (OCR) of cells. And detection was carried out per 5 min before and after sequentially adding with oligomycin, carbonyl cyanide 4‐(trifluoromethoxy)phenylhydrazone (FCCP) and rotenone/antimycin A . Wave software and Graphpad were employed to process and analyze the data obtained.Pretreated HCMEC/D3s were seeded in Seahorse XF96 plates  with the amount of 20,000 cells per well. Cells were incubated with nonglucose medium for 1 h before undergoing the glycolysis stress evaluation  by detecting the extracellular acidification rate (ECAR) of cells. Detection was carried out per 5 min before and after sequentially adding with glucose, oligomycin and 2‐dexoy‐2.10g for 15 min at 4°C. The supernatant was needed as subsequent experimental protein samples, of which concentration was examined by BCA protein assay kit . After separated by 10% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), polyvinylidene difluoride membranes  were used to receive the electro‐transferred protein samples. Subsequently, the membranes were blocked by 10% nonfat milk dissolved in PBST for 1 h. Then, the membranes were incubated with the corresponding primary antibodies at 4°C overnight. After washing three times by tris buffered saline with 0.05% tween‐20 (TBST), the samples were incubated with horseradish peroxidase‐conjugated specific antibodies for 60 min at room temperature. Immunoreactive bands were visualized by Chemi DocXRS + Imaging System (Bio‐Rad), and ImageJ software was applied to calculate gray levels. All experiments were performed in triplicate. All primary and secondary antibodies used here are listed in Table Protein extracts were obtained by cells and spinal cord tissues mentioned before. After lysis by radioimmunoprecipitation assay (RIPA) lysis buffer  that contains 1% phenylmethanesulfonyl fluoride (PMSF) , the solutions were centrifuged at 12,0002.11−1) and transcardially perfused with normal saline, followed by 4% PFA and 2% glutaraldehyde in 0.1 M PBS. The samples containing the lesion site of the spinal cord were harvested and post‐fixed with 2.5% glutaraldehyde in 0.1 M PBS before fixation with 1% osmium tetroxide. After rinsing with sterilized double distilled water, the samples were dehydrated with a graded series of ethanol and then embedded in pure epoxy. Ultrathin sections (70–90 nm) were obtained with an ultramicrotome  and doubly stained with lead citrate and uranyl acetate before observation under a transmission electron microscopy (TEM) .The animals were anesthetized by intraperitoneal injection of 1% sodium pentobarbital  test with post hoc Tukey's testing. The significance threshold was set at a *p value < 0.05 and **p < 0.01. Values with different superscripts are significantly different (p < 0.05).All the data are shown as the mean ± SD. All data was subject to outlier (ROUT method) and normality (Shapiro–Wilk) testing. Parametric, normal datasets were analyzed using the Student's 33.1Differential expression profiles of GPCR in different types of endothelial cell (EC) were obtained from the research carried out by Kaur et al. and their uploaded database in the National Center for Biotechnology Information Gene Expression Omnibus database (GSE97955).To indicate the potential role of GPR124 during SCI, we detected the alterations of GPR124 and TJ proteins in the spinal cord post‐SCI by western blot (WB) Figure . We foun3.2First, our experiments revealed that the protein of GPR124 degraded within 2 h in PBS Figure . Interes−1, a new absorption peak belonging to the carbonyl vibration of HP complex could be identified as one of the characteristics of HP. Meanwhile, compared with P, hydroxyl groups of conjugated heparin could also be observed at 3300–3600 cm−1. According to the spectral results, we successfully synthesized HP hydrogels by EDC/NHS method. Besides, the gelation temperature could be modulated by changing the concentration of HP  was employed to detect the chemical conformation of HP. As illustrated in FTIR spectra Figure , at abouP Figure . In viewP Figure . Both deP Figure . An initTogether, we demonstrated that the HP@124 hydrogel is thermosensitive and suitable for the spinal cord. Besides, containing heparin could immobilize and preserve GPR124 from degradation, which enables the release of GPR124 sustainably.3.3To further investigate the potential role of HP@124 hydrogel on rebuilding BSCB, we use HUVECs, and HCMEC/D3s, induced by TBHP to simulate the microenvironment post‐SCI. Wound healing and migration experiments indicated that HP@124 hydrogel notably elevated the horizontal and vertical migration of HCMEC/D3 after injury Figure . In addiRestoring the energy of cells post‐SCI could promote the prognosis of SCI.To obtain insight into the mechanism by which HP@124 acts on HCMEC/D3s, we performed mRNA sequencing analysis of HCMEC/D3s cultivated on different substrates for 3 days Figure . InteresFurthermore, the expression of key genes, especially several respiratory enzymes associated with glucose metabolism, was evaluated to verify the mechanism underlying the ameliorated energy metabolism. Compared with the TBHP group, GPR124 and HP@124 group exhibited an obvious effect on glucose metabolism‐associated genes. Among all tested genes, the expression of phosphoenolpyruvate carboxykinase 2 (PCK2) elevates the most, which is consistent with the sequencing result Figure . PCK2, a3.4To evaluate whether the favorable effects of HP@124 were involved in the BSCB conservation in vivo, we detected the degrees of extravasation post‐SCI. EB staining was decreased in the free GPR124 group and HP@124 group contrasted to the HP group and SCI group at 72 h post‐injury, implying maintenance of BSCB integrity Figure . BesidesMoreover, in vitro BSCB model composed of a monolayer of HCMEC/D3s showed similar results. The addition of GPR124 significantly attenuated the TBHP‐induced endothelial hyperpermeability to dextran Figure . Taken tThe TJ disruption between BSCB may explain the increased permeability of the BSCB. We investigated whether HP@124 hydrogel diminishes the rised permeability by preventing the loss of TJ proteins post‐SCI. Immunofluorescence showed that the fluorescence intensity of TJs, including Claudin 5, delta 1 Catenin (P120), and ZO‐1, was greatly elevated in the HP@124 group, as compared to GPR124, HP, and SCI groups Figure . In addi3.5Although we find that GPR124 could restore the function of BSCB post‐SCI in the acute phase, considerable research regarded it as preventing the disruption of BSCB failed to promote motor recovery in the chronic phase post‐SCI.H&E staining revealed the histological structure of spinal cord on 28 days post‐SCI Figure . CompareTogether, we indicate that HP@124 hydrogel could recover the function of locomotion in the chronic phase of SCI.3.6Next, we evaluate the effect on various aspects on day 28 post‐SCI treating with HP@124 hydrogel. Restoration of nerve conduction, especially axonal regeneration, is one of the potential approaches for the regeneration of SCI. The effect of axonal rehabilitation by HP@124 hydrogel was examined by performing immunofluorescence. First, Immunofluorescence was employed to calculate the distance between the closest neurons (GAP43‐positive cells) and the epicenter using Growth associated protein‐43 (GAP43) Figure . CompareScars proliferate rapidly after the acute phase of SCI, especially fibrous scar, which impedes the regeneration of axons.In conclusion, our results indicated that HP@124 hydrogel might stimulate axon growth and inhibit the establishment of fibrous scars.3.7The relationship between remyelination and inflammation has been widely reported.4The concept of neurovascular units has attached sufficient attention of researchers in recent years, which consists of BSCB and related neurons.In our study, we demonstrated the effect of GPR124 on BSCB reconstruction post‐SCI. GPR124, known as TEM5, was first discovered in the vasculature of human colorectal cancer,The medicinal usage of free proteins to remedy SCI is restricted owing to their short half‐life, fast degradation, and adverse outcomes at high concentrations.In recent years, HP hydrogel has been synthesized and used in different fields, such as SCI regeneration,At a molecular level, oxidative stress and free radicals play an important part in BSCB disruption.As an orphan receptor of G protein, currently, there are no ligands or agonists that fully adapt to GPR124. However, in the studies of GPR124 related to the function of vascular regeneration and adhesion, its mechanism may not lie on the function of G protein receptor.Seahorse test showed that the elevated metabolic level with the treatment of HP@124 rescues the poor state of ECs, and may promote migration and restore TJs. However, further research is required to verify the association between energy metabolism and BSCB restoration. We demonstrated that PCK2 might be the switch of this restoration of energy metabolism, which needs more testing to confirm. Whether the elevated energy metabolism will influence other cells, such as neuronal cells and astrocytes, remains unknown. Together, these findings are of great significance in helping explain the independent mechanism of GPR124.Spinal cord crushing injury model of rats was established here to verify the effect of HP@124 hydrogel on the reconstruction of BSCB in the early stage post‐SCI in vivo. The volume of the injected contents is referenced to the previous research.BSCB breakdown post‐SCI occurs within 5 min,In the acute phase of SCI, simple HP hydrogel had limited effects on the rebuild of BSCB, while the addition of GPR124 and the synergistic effect between GPR124 and HP hydrogel enable this delivery system to provide more BSCB protection, including structural and functional repair, which is detected by EB staining and the protein level of TJs, respectively.Treatment strategies for the long‐term recovery of SCI include controlling inflammation,Balancing the relationship between inflammation and remyelination is important for the regeneration of SCI. None or immoderate inflammatory response will restrict remyelination.Furthermore, the inhibitory microenvironment for axonal regrowth in the chronic phase of SCI is mainly resulted by fibrotic scar.Although there are still differences between rodents and humans in spinal cord size, anatomical structure, function, and inflammatory response,5In summary, we verified the decrease of GPR124 post‐SCI and the important role of recombinant GPR124 protein in restoring BSCB in vivo and in vitro. Meanwhile, a thermosensitive hydrogel has been prepared to conserve GPR124 bioactivity and restraint its release. The HP@124 hydrogel notably ameliorated the SCI regeneration when administered via in situ injections in vivo. HP@124 hydrogel exerted neuroprotective effects and created promising conditions for functional rehabilitation due to its BSCB hyper‐permeability attenuation, TJ disruption suppression, fibrotic scars restraintion, inflammatory response attenuation, remyelination and axonal reconstruction by protecting BSCB from disruption, restoring TJ proteins and energy metabolism. Combined with the moderate regeneration effects post‐SCI treated by free GPR124 alone, HP@124 hydrogel could facilitate and prolong GPR124 delivery to the lesion site.Together, the marked effect of GPR124 in BSCB restoration at the early stage of SCI and the neuro‐preservation benefit of this delivery strategy in easy fabrication, fast gelation, good biocompatibility, and biodegradability give rise to a clinically feasible therapeutic manner for patients with SCI.Jiawei Shu: Conceptualization (lead); data curation (lead); formal analysis (lead); investigation (lead); methodology (lead); project administration (lead); writing – original draft (lead). Chenggui Wang: Conceptualization ; data curation ; formal analysis ; investigation ; methodology ; writing – original draft . Yiqing Tao: Conceptualization ; formal analysis ; funding acquisition ; investigation ; methodology ; writing – original draft . Shaoke Wang: Formal analysis ; methodology ; software . Feng Cheng: Software ; supervision ; writing – review and editing . Yuang Zhang: Formal analysis ; writing – review and editing . Kesi Shi: Conceptualization ; supervision . Kaishun Xia: Conceptualization ; supervision . Ronghao Wang: Conceptualization ; supervision . Jingkai Wang: Conceptualization ; supervision . Chao Yu: Supervision . Jiangjie Chen: Methodology . Xianpeng Huang: Methodology . Haibin Xu: Methodology . Xiaopeng Zhou: Methodology . Haobo Wu: Writing – review and editing . Chengzhen Liang: Writing – review and editing . Qixin Chen: Conceptualization ; funding acquisition . Shigui Yan: Funding acquisition ; supervision . Fangcai Li: Funding acquisition (lead); supervision (lead).All authors declare that they have no conflict of interest.Data S1: Supporting Information.Click here for additional data file.