Individuals with the ABCB1 rs1045642 T/T genotype were associated with a higher valsartan tmax compared to those with T/G and G/G genotypes  and with a tendency toward a higher postural dizziness incidence . A higher hydrochlorothiazide dose/weight (DW)-corrected area under the curve (AUC∞/DW) was observed in SLC22A1 rs34059508 G/A volunteers compared to G/G volunteers , and a tendency toward a higher postural dizziness incidence . Sex impacted valsartan and hydrochlorothiazide pharmacokinetics, showing a lower exposure in women, whereas no significant differences were found for olmesartan pharmacokinetics.Drug combination therapy is the most common pharmacological strategy for hypertension management. No pharmacogenetic biomarkers for guiding hypertension pharmacotherapy are available to date. The study population were 64 volunteers from seven bioequivalence trials investigating formulations with valsartan, olmesartan and/or hydrochlorothiazide. Every volunteer was genotyped for 10 genetic variants in different transporters’ genes. Additionally, valsartan-treated volunteers were genotyped for 29 genetic variants in genes encoding for different metabolizing enzymes. Variability in pharmacokinetic parameters such as maximum concentration (C Valsartan is slightly metabolized to an inactive compound by unknown enzymes and it is excreted mainly unaltered in urine and feces, with an elimination half-life (t1/2) of 6 h [1/2 of approximately 13 h [Angiotensin II receptor blockers (ARBs), such as valsartan and olmesartan, are widely-used antihypertensive drugs. Their mechanism of action is based on the selective antagonism of the angiotensin II receptor type 1, which leads to an increase in renin, angiotensin I and angiotensin II plasmatic levels and a decrease in aldosterone plasmatic levels, causing a decrease in blood pressure (BP) . While vmax is variable (1 to 5 h). It is not metabolized; therefore, it is excreted unaltered in urine, with a t1/2 ranging from 5 to 15 h [Diuretic thiazides, such as hydrochlorothiazide, are also used in high-BP (HBP) treatment. They increase urine production by the inhibition of sodium and chloride reabsorption, causing a BP reduction . Hydroch to 15 h .ABCB1, ABCC2, ABCG2, SLCO1B1 and SLC22A1 transporters on valsartan, olmesartan and hydrochlorothiazide pharmacokinetics, safety and BP variability. Secondarily, the impact of genetic variation in the metabolizing enzymes of the cytochrome P450 family (CYP) CYP2A6, CYP2B6, CYP2D6, CYP2C8, CYP2C9, CYP2C19, CYP3A4, CYP3A5 and UGT1A1 on valsartan pharmacokinetics was analyzed. The present work is part of the La Princesa Multidisciplinary Initiative for the Implementation of Pharmacogenetics (PriME-PGx) [Antihypertensive treatment is commonly administered in a combination therapy in order to reach a higher efficacy with lower adverse drug reactions (ADRs) incidence, since a better BP control is achieved through the sum of the different mechanisms of action . Pharmacp = 0.011, p < 0.001, p < 0.001, p = 0.029, respectively). No differences were found between individuals self-reported as Europeans and Latin-Americans or Africans  or among CTs , 64 (27 women and 37 men) gave written informed consent for the candidate gene study and had previously been genotyped in other research [mong CTs . Since h∞) and Cmax were 593.51 ± 133.37 ng*h/mL and 87.67 ± 24.59 ng/mL; and 1203.68 ± 396.52 ng*h/mL and 166.19 ± 48.31 ng/mL for the 25 mg dose, showing linear pharmacokinetics. No significant differences in tmax and t1/2 were found between doses. Hydrochlorothiazide’s dose-corrected AUC∞ (AUC∞/D) was significantly higher in women compared to men  and t1/2 was significantly lower  = 0.006, multivariate p-value (pmv) = 0.008, β = −1.014, R2 = 0.103), with no differences in dose-corrected Cmax (Cmax/D) (6.97 ± 2.19 versus 6.79 ± 1.80 ng/mL*mg) or in dose/weight-corrected AUC∞ (AUC∞/DW), dose/weight-corrected Cmax (Cmax/DW) and tmax   . No sign= 0.051) .∞ and Cmax were 27,359.24 ± 12,523.54 ng*h/mL and 3452.35 ± 1485.89 ng/mL; and 54,443.93 ± 13,513.52 ng*h/mL and 6623.88 ± 1653.98 ng/mL for the 320 mg strength, showing linear pharmacokinetics. No significant differences in tmax and t1/2 were found between doses. Valsartan AUC∞/DW was significantly lower in women compared to men (puv = 0.023), with no differences in AUC∞/D (166.73 ± 79.11 versus 176.19 ± 53.34 ng*h/mL*mg), in Cmax/D (22.47 ± 9.48 versus 19.72 ± 5.92 ng/mL*mg) or in the remaining pharmacokinetic parameters  and in ABCB1 rs1045642 T/T individuals compared to T/C and to C/C individuals   (max was significantly higher in CYP3A5 intermediate metabolizers (IMs) compared to poor metabolizers (PMs) (puv = 0.014). However, differences disappeared in the multivariate analysis. CYP2D6 PMs showed higher valsartan t1/2 compared to IMs, to normal metabolizers (NMs) and to ultrarrapid metabolizers (UMs)   and Cmax/DW  compared to normal function (NF) volunteers  . ABCB1 r= 0.020) . Moreove= 0.342) . Additiolunteers .∞ and Cmax for olmesartan 40 mg were 5879.71 ± 1813.67 ng*h/mL and 775.16 ± 255.19 ng/mL. AUC∞ and Cmax were significantly higher in women compared to men , with no differences after DW adjustment (max was lower compared to the other CTs (puv = 0.006)  (The mean AUCjustment . In CT 5= 0.006) . No diff= 0.006) . A tende= 0.066) .3h and t6h after drug intake, whereas heart rate (HR) significantly increased at t6h . The SBP reduction at t6h was significantly higher in women compared to men . The HR increase at t6h was significantly higher in those CTs with olmesartan  compared to CTs with valsartan . No further significant differences in SBP, DBP, HR or QTc changes were found according to sex, biogeographic origin, CTs, SLCO1B1 or ABCG2 phenotype or ABCB1, ABCC2 and SLC22A1 genotypes.Systolic and diastolic BP  significantly decreased at t n = 64, . The QT ∞ was observed in individuals experiencing ADRs compared to those volunteers without ADRs , with no differences in olmesartan and hydrochlorothiazide pharmacokinetics.A total of 36 ADRs in 28 healthy volunteers were observed. A 45% higher valsartan AUCp = 0.031). A tendency toward a higher postural dizziness incidence was observed in ABCB1 rs1045642 T/T volunteers  compared to T/C and C/C volunteers  and in SLC22A1 rs34059508 G/A individuals  compared to G/G individuals . No further differences in ADR incidence were found according to sex, biogeographic origin, CTs or the remaining transporter genotypes.The most common ADR was headache , followed by dizziness , postural dizziness and nausea . ADRs occurred in 28 out of the 58 (48.3%) individuals treated with triple pharmacotherapy, whereas none of the six individuals treated with double pharmacotherapy suffered from an ADR . Thus, hydrochlorothiazide may interact with transporters involved in olmesartan pharmacokinetics, accelerating its absorption. However, no previous information of this interaction is available in the literature and other factors might be involved, such as differences in the drug formulation.When olmesartan was co-administered with a 25 mg hydrochlorothiazide dose (CT 5), C3h and t6h . The SBP reduction was higher in women compared to men, as reported in other studies [∞/D values of these two drugs compared to men. Thus, none of these genetic variants seem to be useful predictors of antihypertensive treatment efficacy, whereas olmesartan and hydrochlorothiazide exposure might be a useful predictor of BP reduction. Therefore, therapeutic drug monitoring may be a useful tool for the management of at-risk patients. However, as these results derive from healthy volunteers, further research in patients should be conducted. Regarding safety, the ADRs suffered were consistent with the information of the three drugs’ labels [∞ was related to ADR incidence but not that of olmesartan and hydrochlorothiazide, which suggests that the valsartan tolerability profile may be slightly inferior when pharmacotherapy is initiated. Further studies with HBP patients and multiple dose administration are needed to check whether the associations between olmesartan, hydrochlorothiazide and valsartan exposure, efficacy and ADR incidence might be useful in clinical practice.As expected, antihypertensive treatment resulted in a decrease in SBP and DBP, which might explain some of the ADRs observed  . In fact studies , with no’ labels ,2,3. A H’ labels . The dif’ labels . In contABCB1 and SLC22A1 genetic variation, respectively. ABCB1 codes for the P-glycoprotein (P-gp) transporter, located in enterocytes, renal cells, hepatocytes and blood–brain barrier cells [ABCB1 variants analyzed  slowed valsartan absorption. Our findings hence suggest that valsartan is a P-gp substrate, like other drugs of the ARB family, such as losartan or candesartan [max and the ABCB1 rs1045642 T/T genotype was reinforced by its association with a higher postural dizziness incidence. However, the individual impact of each genetic variant on P-gp expression, structure and/or function is unknown, and its study is hampered by the partial linkage disequilibrium between them and the different locations of the transporter [SLC22A1 codifies for the hepatic organic cationic transporter 1 (OCT1), involved in drug uptake into hepatic cells [SLC22A1 rs34059508 G>A A allele, proposed as a decreased function variant [Valsartan and hydrochlorothiazide pharmacokinetics were altered by er cells . In thisdesartan ,24. In tic cells . Carrier variant , showed  variant . Additio1/2 in PMs compared to UMs, NMs and IMs was found, which suggests that this enzyme might be involved in its metabolism. However, no differences were found in exposure, probably because only 20% of valsartan is metabolized and only two CYP2D6 PMs were included in this research. CYP2D6 seem to participate in the metabolism of other antihypertensive drugs, such as diltiazem or amlodipine, as seen in our previous work derived from the same cohort of healthy volunteers [max between PMs and IMs might be explained by the different proportion of ABCB1 rs1128503 T/T volunteers within each phenotype group . Furthermore, the disappearance of this association in the multivariate analysis and the lack of differences in AUC∞/DW and Cmax/DW suggest that CYP3A5 genetic variation might not have any impact on valsartan pharmacokinetics. Thus, our results suggest that CYP2D6 might be involved in valsartan metabolism and its genetic variation may affect its pharmacokinetics, whereas CYP2C9 and CYP3A5 genetic variation may be less relevant.For CYP2D6, an association toward a higher valsartan tlunteers ,29, othelunteers . In this∞/DW value compared to volunteers with the NF and DF phenotypes, respectively. For the only olmesartan-treated volunteer with a PF phenotype, minor differences were observed: a 1.3- and 1.1-fold higher AUC∞/DW value compared to volunteers with the NF and DF phenotypes. Consistently, valsartan and olmesartan were previously described as substrates of the transporter codified by SLCO1B1, the organic anion transporter polypeptide OATP1B1 [SLCO1B1 is a useful pharmacogenetic biomarker already used in treatment with other drugs, such as statins [Lastly, although the threshold for significance was not reached, the SLCO1B1 phenotype seemed to alter valsartan and olmesartan exposure: the only valsartan-treated volunteer with a PF phenotype showed a 1.9- and 1.5-fold higher valsartan AUC OATP1B1 ,32,33,34 statins .Nevertheless, further research with different drug combinations in less controlled conditions  is needed to determine the usefulness of these associations in clinical practice. If replicated, these biomarkers may be implemented in HBP clinical management and may be used to reach a higher efficacy with lower ADRs incidence, increasing treatment adherence and reducing treatment discontinuation or treatment changes.LIMITATIONSABCB1, ABCC2, ABCG2 and SLC22A1) and functionally . In contrast, the present research strictly controlled confounding factors that affect pharmacokinetics, pharmacodynamics and safety in clinical settings with patients .This research has two main limitations. Firstly, the administration of a single dose and the inclusion of healthy subjects do not allow us to conclude on valsartan, olmesartan and hydrochlorothiazide long-term safety, pharmacodynamics and pharmacokinetics. Secondly, the modest and arbitrary sample size available are limiting, since the candidate-gene study was conducted based on the available pharmacogenetic, pharmacokinetic and safety data from seven bioequivalence trials. The limited sample size reduces the statistical power and the chances to encounter biomarkers of interest with a low prevalence within the sample population, such as CYP2C9 PMs. Due to the absence or reduced olmesartan, hydrochlorothiazide and valsartan metabolism, the main candidate genes available were transporters, which are poorly characterized structurally  for CT 1, Exforge HCT®  for CTs 2, 3 and 4, Sevikar HCT®  for CT 5 and 6 and Olmetec Plus®  and Norvas®  for CT 7, all of them at the same dose as the T formulation. In all of the CTs, a single oral dose of each drug was administered under fasting conditions; they were phase I, open-label, single-center, crossover and randomized CTs, with two sequences and two periods for olmesartan and hydrochlorothiazide, and four sequences and four periods for valsartan (replicated design). In each period, volunteers were admitted at UECHUP at 10 pm (day 0) and remained there for at least 12 h after drug administration at 9 am of Day 1. The wash out time for CT 1, 2, 3 and 4 was 14 days between Period 1 and 2, while it was 7 days between Periods 2, 3 and 4. For CTs 5, 6, and 7, the wash out time was 21 days. All the formulations included a 10 mg amlodipine dose and its pharmacogenetic results are already published [The data used in this research were obtained from seven CTs performed at the Clinical Trials Unit of Hospital Universitario de La Princesa (UECHUP), Madrid (Spain), from 2012 to 2018 . In everDuring hospital admission and at additional visits in each period, at least 17 blood samples were extracted from pre-dose to 48 h after drug administration. The blood samples obtained were used for the plasmatic quantification of drug levels, which was conducted in an external laboratory by high-performance liquid chromatography coupled to tandem mass spectrometry. For the blinded plasmatic concentration determination, a method validated according to the European Medicines Agency (EMA) guidelines was used. The lower limit of quantification was established at 1.01 ng/mL for hydrochlorothiazide, 2.51 ng/mL for olmesartan and 20.02 ng/mL for valsartan.2 range, being pregnant or breastfeeding women, smoking or alcoholism, positive drug screening, blood donation in the previous month, inability to collaborate or follow instructions and participation in other studies with investigational drugs in the previous three months. The CT protocols were revised and approved by the Hospital’s Research Ethics Committee and by the Spanish Drugs Agency (AEMPS). CT and data handling were performed complying with the Good Clinical Practice guidelines [Inclusion criteria comprised: women or men between 18 and 55 years old, free from organic or psychological disorders, with normal medical records, physical examination, vital signs, electrocardiogram (ECG) and laboratory tests. Exclusion criteria comprised: having aBMI outside the 18.5–30.0 kg/midelines , the Decidelines  and the max and tmax were directly retrieved from the plasmatic time–concentration curves. The remaining parameters were estimated with WinNonlin Professional Edition version 2 (CT 1) or version 7 (CT 2-7)  using a non-compartmental approach. The area under the time–concentration curve (AUC) from pre-dose to the last observed concentration time (48 h) (AUCt) was calculated according to the trapezoidal rule. For AUC extrapolation to infinity (AUC∞), AUCt→∞ was calculated as Ct/Ke  and added to AUCt. Finally, t1/2 was calculated as −ln2/Ke. For valsartan and hydrochlorothiazide, the T formulation was demonstrated to be bioequivalent to the R formulation in every CT. Hence, the arithmetic mean of the pharmacokinetic parameters of both formulations was calculated for each volunteer. For olmesartan, not all T formulations were demonstrated to be bioequivalent to the R formulation; therefore, only the pharmacokinetic parameters of the R formulation were used for the analysis.C3h) and 5 to 6 h (t6h) after drug intake and ECGs were scheduled before and 5 to 6 h (t6h) after drug intake. HR and QTc prolongation measurements were used for the safety analysis. Since this candidate gene study is focused on antihypertensive drugs, BP measurements were used for the pharmacodynamics analysis.For safety assessment, adverse event (AE) identification was performed by direct question to the volunteers or by spontaneous notification. The algorithm of Spanish pharmacovigilance system  was used® RSC Automated DNA extractor . Volunteers were genotyped for 39 variants located in 14 pharmacogenes in a QuantStudio 12 K Flex qPCR instrument with an OpenArray thermal block  using the PriME-PGx Very Important Pharmacogene Open Array (VIPOA) custom genotyping array, version VIPOA1, VIPOA2 or VIPOA3  and metabolizing enzymes  were analyzed. For olmesartan and hydrochlorothiazide, only variants in ABCB1, ABCC2, ABGC2, SLC22A1 and SLCO1B1 transporters were included in the analysis, as these drugs are not metabolized. CYP2D6 deletion (*5), duplication and the presence of hybrid structures were analyzed using two TaqMan® copy number variation assays targeting exon 9 (Assay ID: Hs00010001_cn) and intron 2 (Assay ID: Hs04083572_cn) , as previously described [https://www.pharmvar.org (accessed on 1 August 2023)). Genotype information was translated into phenotype according to the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines for CYP2B6 [CYP2C19 [CYP2C9 [CYP2D6 [CYP3A5 [SLCO1B1 [UGT1A1 [https://www.pharmgkb.org/page/pgxGeneRef (accessed on 1 August 2023), and the Dutch Pharmacogenetic Working Group (DPWG) guideline for CYP3A4 [Blood samples collected in EDTA-K2 tubes during the CTs were used for DNA extraction with a MagNA Pure instrument  or a Maxwellr VIPOA3 . For val [CYP2C9 , CYP2D6  [CYP2D6 , CYP3A5  [CYP3A5 , SLCO1B1[SLCO1B1  and UGT1 [UGT1A1 , in accor CYP3A4 . For CYPr CYP3A4 .∞ and Cmax were divided by the dose/weight ratio (DW) to correct dose and weight effect on bioavailability. The Shapiro–Wilk test was applied for checking normality in variable distributions. For those variables not following a normal distribution, a logarithmic transformation was applied and normality was re-analyzed. Firstly, in the univariate analysis, pharmacokinetic parameters were analyzed according to sex, biogeographic origin, CT, genotypes and phenotypes. T-tests or ANOVA tests followed by a Bonferroni post-hoc were performed for the comparison of means for variables following a normal distribution with two or with three or more categories, respectively. For those variables not normally distributed, nonparametric tests were used: a Mann–Whitney test or a Kruskal–Wallis test, respectively. The multivariate analysis was performed by means of linear regression, in which those independent variables significantly related to the dependent variable in the univariate analysis  lower than 0.05) were included. The multivariate p-value (pmv), the non-standardized β coefficient (β) and R2 are shown for significant associations. To analyze the effect of treatment on SBP and DBP, QTc interval and HR, paired sample T-tests were performed. Furthermore, ADR incidence was analyzed according to sex, biogeographic origin, CT and genotype or genotype-informed phenotypes by Chi2 or Fisher exact tests, when appropriate.SPSS software  was used to perform the statistical analysis. AUCABCB1 and SLC22A1 genetic variation affect drug exposure and safety, and CYP2D6 may be involved in valsartan metabolism. However, the use of ABCB1 and SLC22A1 as pharmacogenetic biomarkers in clinical practice requires an improved characterization of both genes and the replication of these associations in independent additional clinical contexts. Furthermore, valsartan and olmesartan might be OATP1B1 substrates and their exposure may be conditioned by the SLCO1B1 genotype. To the best of our knowledge, this is the first work to propose genetic variation in transporters as biomarker for valsartan, olmesartan and hydrochlorothiazide pharmacokinetics. Further research with an increased sample size is needed to confirm the relevance of the observed associations.Valsartan and hydrochlorothiazide are proposed to be P-gp and OCT1 substrates, respectively, as


Pituitary adenoma (PA) bone invasion seriously affects patient prognosis, and its mechanism needs more investigation. Through exploring the interaction between PA tumor cells and osteoclasts, we have revealed an essential theory of the progression of PA bone invasion. Localized inflammatory environment in PAs was identified as a feature of bone invasion. In this study, we identified PKCθ as a key signal for PA bone invasion, and PAs release IL-1β via the PKCθ/NF-κB/IL-1β pathway to induce monocyte–osteoclast differentiation in a paracrine manner. Meanwhile, we also found that celastrol, as a natural product, can obviously reduce the secretion of IL-1β as well as alleviate the progression of bone invasion, which is promising for clinical application.Background: Pituitary adenoma (PA) bone invasion results in adverse outcomes, such as reduced rates of complete surgical resection and biochemical remission as well as increased recurrence rates, though few studies have been conducted. Methods: We collected clinical specimens of PAs for staining and statistical analysis. Evaluation of the ability of PA cells to induce monocyte–osteoclast differentiation by coculturing PA cells with RAW264.7 in vitro. An in vivo model of bone invasion was used to simulate the process of bone erosion and evaluate the effect of different interventions in alleviating bone invasion. Results: We found an overactivation of osteoclasts in bone-invasive PAs and concomitant aggregation of inflammatory factors. Furthermore, activation of PKCθ in PAs was established as a central signaling promoting PA bone invasion through the PKCθ/NF-κB/IL-1β pathway. By inhibiting PKCθ and blocking IL1β, we were able to significantly reverse bone invasion in an in vivo study. Meanwhile, we also found that celastrol, as a natural product, can obviously reduce the secretion of IL-1β as well as alleviate the progression of bone invasion. Conclusions: By activating the PKCθ/NF-κB/IL-1β pathway, pituitary tumors are able to induce monocyte–osteoclast differentiation in a paracrine manner and promote bone invasion, which can be alleviated by celastrol. Pituitary adenomas (PAs) are benign tumors originating from the anterior pituitary gland with a prevalence of 0.037–0.116% based on clinical case studies, accounting for approximately 10% of all intracranial tumors . AlthougThe mechanisms of bone invasion in PAs are not well understood, and studies have suggested that the invasive characteristics of tumor cells and the appropriate bone microenvironment are essential for bone invasion ,9. PreviThe PKC family, as serine-threonine kinases, contains 12 members that regulate processes including cell cycle and differentiation, morphogenesis, and cell survival ,14 and tIn the present study, we aimed to investigate the potential role of PKCθ in bone invasion and its impact on monocyte–osteoclast differentiation. In addition, we also evaluated the treatment efficiency of celastrol on PA bone invasion, which can be translated into clinical therapeutical application. https://www.ncbi.nlm.nih.gov/geo, accessed on 20 January 2023) database. Eligibility criteria included mRNA data from samples of PAs containing both invasive and noninvasive phenotypes. GSE120350, GSE169498, GSE22812, GSE26966, GSE51618, and GSE72490, containing 81 samples of invasive PAs and 55 noninvasive PAs, were included in this study. The datasets were merged and removed batch effects using sva and preprocess Core package before further analysis.Clinical data and RNA sequencing data regarding bone-invasive PA patients were acquired from Zhu et al.  and conthttps://www.gsea-msigdb.org/gsea/msigdb, accessed on 20 January 2023). Adjusted p-value < 0.05 and |normalized enrichment score (NES)| >1 were considered the cutoff values for identifying the enriched pathway. The immune cells infiltration in PAs was analyzed by ssGSEA which is in package GSVA package. We collected cytokines associated with osteoclast differentiation from previous studies [https://www.genecards.org, accessed on 20 January 2023). The differentially expressed genes (DEGs) of PAs were screened using the limma package in R 4.2.1. A volcano plot and heatmap are displayed for the DEGs and genes of the PKC family in the GEO expression cohort. Gene set enrichment analysis (GSEA v4.2.3.) of DEGs in invasive Pas through hallmark gene sets and the hallmark gene sets were collected from the Molecular Signatures Database  based on the bone invasion of the PAs. The criteria for classification were as follows: 1. Preoperative computed tomography (CT) and magnetic resonance imaging (MRI) imaging reports suggesting bone infiltration. 2. Intraoperative recording of pituitary tumor invasion of surrounding bone (mainly sphenoid bone). 3. Postoperative histopathological sections suggesting tumor cell infiltration in bone. In our classification, the first item is used as a reference basis only, with the second item as the main criterion and the data for the third item often lacking. The summary information of the collected patient data is displayed in The animal experiments were conducted in accordance with the experimental protocol approved by the Tongji Hospital Committee for the care of animals (process number: TJH-202206015). Male BALB/c athymic nude mice  used for molding were housed in an animal facility under SPF conditions. At 6–7 weeks of age, the mice were used to develop an animal model of bone invasion by PAs . GH3, MMTo deplete monocytes, beginning at day 2 after injected tumor cells in calvaria, we administered clodronate liposomes  intraperitoneal injection (I.P.) on alternating days with a dosage of 0.05 mg/g bodyweight until euthanasia, while PBS liposomes were utilized as a control. In addition, we adopted different drug administration to alleviate pituitary tumor bone invasion, including sotrastaurin , Pyrrolidinedithiocarbamate ammonium , IL-1β monoclonal antibodies , celastrol , and PBS (as control). All drugs were administered via I.P. injection on alternating days beginning on day 7 days after implantation of the tumor cells. For HE staining, immunohistochemistry (IHC), and immunofluorescence (IF), 5 μm sections were made from clinical specimens and xenograft tumor specimens. For IHC, rehydrated tissue sections were blocked with 5% bovine serum albumin  and then were stained with primary antibody at 4 ℃ for 12 h. The primary antibodies used include IL-1β , IL-6 , IL-8 , TNF-α , and PKCθ . The secondary antibodies used for IHC were biotinylated anti-rabbit IgG . Subsequently, streptavidin–biotin complex  and DAB (DAB substrate: DAB chromagen = 1 mL: 20 µL) were added to visualize staining, and hematoxylin was used to stain the nucleus. For IF, after rehydration and blocking, samples were incubated with primary antibodies, including F4/80 and TRAP , at 4℃ for 12 h. Second antibodies used for IF are Cy3–conjugated Goat Anti-Rabbit IgG and Dylight 488-conjugated Goat Anti-Rabbit IgG , and 4’,6-diamidino-2-phenylindole  was used for nuclear staining. All section staining results were observed using microscopy  and analyzed using ImageJ.Tumor and skull specimens obtained from nude mice, after fixation with paraformaldehyde, were scanned using micro-CT  before decalcification processing. Consequently, 3-dimensional images were reconstructed using NRecon software (version 1.7.3). Each calvaria’s area was outlined for quantification, and the amount of bone lost was represented as the proportion of bone volume to tissue volume (BV/TV).A total of five cell lines were used in this experiment: pituitary tumor cell lines , macrophage cell line (RAW264.7), and 293T, which were obtained from American Type Culture Collection . All five cell lines were cultured using DMEM/High Glucose medium  supplemented with 10% fetal bovine serum  and 1% antibiotics . Drugs treated in this study included PMA (100 nM), Sotrastaurin (5 nM), RANKL (30 ng/mL), PDTC (30 µM), IL-1β mAb (100 pM), and celastrol (5 μM).https://rnaidesigner.thermofisher.com, accessed on 20 January 2023) based on the sequence for mouse PKCθ mRNA (NM_008859) and rat PKCθ mRNA (NM_001276721.1). For the mouse PKCθ target sequence, sh1, GCAAGAATGTAGACCTCATCT; sh2, GCAAGATACTTTCTGGAAATG; sh3, GCAAGATACTTTCTGGAAATG. For the rat PKCθ target sequence, sh1, GCAAGATACTTTCTGGAAATG; sh2, GGACCAATTGAAATCAGTTTC; sh3, GCAACTTCTCCTTCATTAACC. After ligating the shRNA to the plko.1 plasmid, the target plasmid and capsids plasmid were transfected into the 293T cell line using the lipo3000 transfection reagent . The viral was collected from the 293T medium after 72 h and used for the culture of target cells, and polybrene was given. Green fluorescence can be observed in fluorescence microscopy after successful shRNA transfection, and WB and qPCR were performed to verify knockdown efficiency.We used short hairpin RNAs (shRNAs) in PA cell lines  to knock down PKCθ. shRNAs were designed through RnaiDesigner  was added, and the optical density (OD) value at 450 nm was measured after 1 h in the incubator at 37 °C.Cells were seeded in 96-well plates at a density of 5 × 10The cells were seeded in 24-well plates, and when the cell density reached 70–80%, they were fixed with 4% paraformaldehyde. Cells were blocked for 1 h by 5% BSA within 0.2% Triton X-100 and were incubated with antibodies against Ki-67 overnight in 4 °C. After incubation with Cy3–conjugated goat anti-rabbit IgG and DAPI in the dark for 2 h, they were photographed under the microscope. Calculation of the proportion of Ki-67 positive cells using ImageJ used a suitable threshold value.2 incubator and photographed at 12 h, 24 h, 36 h, and 48 h, respectively. The widths of the intercellular gaps were detected using ImageJ, and the wound width at 48 h versus 0 h was used as an indicator of cell migration ability.Cells were seeded in 6-well plates, and when the cell density reached 100%, a straight line was drawn vertically using a 200 μL tip. Cells were washed with PBS, and the DMEM medium containing the drug was replaced and photographed using a microscope as the state of the cells at 0 h. The cells were placed in a CO4 cells/chamber were seeded in the upper Transwell chamber after resuspension using serum-free DMEM medium. DMEM medium containing 10% FBS was added to the lower Transwell chambers. The Transwell chambers were incubated in a CO2 incubator for 24 h, fixed with 4% paraformaldehyde for 30 min, and then stained with crystal violet for 15 min. Cells in the upper chamber were carefully wiped off using a cotton swab, followed by microscopic photography. Cells migrating to the lower chamber were identified using ImageJ, and their count represents the invasion ability of the cells.Matrigel matrix gel  pre-placed at 4 °C was 1:8 diluted by PBS; 100 μL was added to the upper Transwell chamber  and then stood for 3 h at room temperature. 103 cells/well into 96-well plates, pituitary-tumor-conditioned medium or drug treatment was given accompanied by a lower level of RANKL (30 ng/mL). The medium was changed every 2 days, and the end point of induction was reached after 6 days of culture. After fixation with 4% paraformaldehyde for 30 min, a TRAP staining kit  was used to evaluate the effect of osteoclast differentiation. TRAP-positive multinucleated cells were considered to differentiate into osteoclasts and were counted under a microscope.RAW264.7 was used to induce osteoclasts. After implantation of RAW264.7 5 × 10Total RNA was extracted from PBS-washed cells using TRIzol (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA-2Na, 1% Triton X-100, 1% Sodium deoxycholate and 0.1% SDS, Servicebio, Wuhan, China). Subsequently, 0.5 μg of RNA was reverse transcribed into cDNA using the Hifair* III 1st Strand cDNA Synthesis Kit  according to the instructions of the manufacturer. The target nucleic acid fragment in cDNA was amplified and detected on the ABI-Prism 7500 Real-Time PCR System  using Hieff* qPCR SYBR Green Master Mix (Low Rox Plus) . The content of GAPDH was used as an internal reference to correct the target mRNA. The primers used are shown in Cellular protein was extracted using RIPA Lysis Buffer  and protease inhibitor cocktail, and the protein concentration was detected using BCA . Loading buffer  was added before boiling for 10 min for use. For electrophoresis, 10% SDS-PAGE gel was used transferred to a polyvinylidene difluoride (PVDF) membrane, and blocked with 5% skimmed milk powder. Primary antibodies used for incubation were PKCθ , IKKα , p-IKKα/β , IKBα , p-IKBα , P65 , p-P65 , IL-1β , and β-Tubulin . After washing off the primary antibody with PBST, the HRP-conjugated anti-rabbit or mouse antibody  was incubated for 2 h at room temperature. After adding ECL Chemiluminescent kit , the target proteins were visualized using the GeneGnome XRQ system .After washing with PBS, cellular proteins were extracted using IP lysis buffer  and a phosphorylated protease inhibitors cocktail. The protein samples were incubated with PKCθ antibodies bound to Protein Magnetic Beads , then use a magnet to screen out proteins that bind to PKCθ and eluted using SDS-PAGE Loading Buffer. Western blotting was performed on the the IP samples using antibodies against IKKα, p-IKKα/β, IKBα, and p-IKBα, respectively.Collection of pituitary tumor cell supernatant and detection of IL-1β in cell supernatants using mouse or rat IL-1β ELISA Kits  were performed according to the manufacturer’s instructions. The ELISA assay is based on an antibody sandwich method using microtiter plates with coated antibodies on surfaces. We added 100μL of sample to each well and incubated at 37 °C for 90 min. Biotinylated anti-rat IL-1β antibody and horseradish-peroxidase-labeled avidin were added sequentially. We added tetramethylbenzidine (TMB) and measured the OD value with a microplate reader at a wavelength of 450 nm. The IL-1β concentration in the samples was calculated by drawing a standard curve and was normalized to the cell content of each group.t-test was used for comparison between two groups.The GraphPad Prism (version 9.0) and R version (4.2.0) were used for statistical analyses. All data for this study will be presented as mean ± standard deviation. One-way ANOVA was used for multisample comparison, and Dunnett’s To investigate potential regulative signals that affect PA bone invasion, in silico analysis was performed on previously published datasets . By compTo understand what signal modulates PA inflammation phenotype, we performed comprehensive in silico meta-analysis based on 6 independent PA transcriptome datasets with a total of 136 patients A. The noNext, these 136 RNA-seq data were divided into “inflammation high” and “inflammation low” groups according to expression level of inflammation signature, using the median level as a cutoff C,D. We tTo investigate the role of PKCθ in invasive Pas, we first constructed PKCθ knock-down in GH3, ATT20, and TtT/GF cell lines  in combiTo understand the potential regulative mechanism of PKCθ in PA bone invasion, we first divided the integrated RNA-seq dataset . Studies have reported that celastrol, as a multisite inhibitor of osteoclast differentiation, can inhibit the NF-κB/IL-1β pathway as well as the activation of the RANK pathway in monocytes ,33. AddiPA bone invasion results in adverse outcomes such as reduced rates of complete surgical resection and biochemical remission, and increased recurrence rates. Through exploring the interaction between PA tumor cells and osteoclasts, we have shown that the release of inflammatory factors, mainly IL-1β, of PAs drives monocyte–osteoclast differentiation and aggravates the progression of bone invasion. Furthermore, activation of PKCθ in PAs was established as a central signaling promoting PA bone invasion through the PKCθ/NF-κB/IL-1β pathway. We also found that celastrol, as a natural product, can obviously reduce the secretion of IL-1β as well as alleviate the progression of bone invasion, which is promising for clinical application.


Acinetobacter baumannii has successfully spread during the last decades as one of the main critically important pathogens. However, many aspects including plasmids, are still under-investigated. Here, we report the complete sequence of an Acinetobacter baumannii strain, belonging to the ST25IP (Institut Pasteur) sequence type recovered in 2012 in Lebanon, using a combination of Illumina MiSeq and Oxford Nanopore sequencing and a hybrid assembly approach. This strain (Cl107) carries a 198 kb plasmid called pCl107 that encodes the MPFI conjugative transfer system. The plasmid carries the aacA1, aacC2, sul2, strAB, and tetA(B) antibiotic resistance genes. pCl107 region encompassing the sul2, strAB, tetA(B) is closely related to AbGRI1 chromosomal resistance islands, which are widespread in A. baumannii strains belonging to Global Clone 2. The resistance region found in pCl107 is one of the missing links in the evolutionary history of the AbGRI1 islands. pCl107 also contains a BREX Type 1 region and represents one of the two main evolution patterns observed in BREX clusters found in plasmids related to pCl107. pCl107 also harbours a ptx phosphonate metabolism module, which plays an ancestral structure compared to other large plasmids in ST25 strains. While the uric acid metabolic module found in pCl107 is incomplete, we identified possible ancestors from plasmids and chromosomes of Acinetobacter spp. Our analyses indicate a complex evolutionary history of plasmids related to pCl107 with many links to multiple antibiotic resistance and metabolic pathways. Acinetobacter baumannii is an important nosocomial pathogen that continues to attract worldwide attention due to its resistance to many antibiotics and its genomic plasticity, enabling it to acquire genetic material   have been reported as an epidemic, endemic and sporadic in many regions around the world  characterized in two Australian ST25 strains are amongst those that carry a large number of genes involved in various functions  . Hence, we completed and analyzed the genome sequences of one ST25 isolate from Lebanon, strain Cl107. We showed that it contains a large 198 kb plasmid with several important modules, including two antibiotic resistance clusters, a ptx operon (involved in phosphate metabolism), a BREX (phage resistance) system, and a uric acid metabolic operon. These regions were analyzed in detail. We also explored publicly available complete plasmids to study related plasmids.In Lebanon, ST25 was one of the commonly identified sequence types in clinical settings, although at a lesser degree than the dominant sequence types (e.g. ST2 strains) . The tested antibiotics are imipenem (10 µg), ampicillin (25 µg), cefotaxime (30 µg), meropenem (10 µg), ceftazidime (30 µg), ampicillin/sulbactum (10/10 µg), tobramycin (10 µg), spectinomycin (25 µg), gentamicin (10 µg), netilmicin (30 µg), neomycin (30 µg), kanamycin (30 µg), streptomycin (25 µg), amikacin (30 µg), sulphamethoxazole (100 µg), rifampicin (30 µg), trimethoprim (5 µg), ceftriaxone (30 µg), nalidixic acid (30 µg), ciprofloxacin (5 µg), florfenicol (30 µg), chloramphenicol (30 µg), and tetracycline (30 µg). Resistance to colistin was measured using ETEST® (https://www.biomerieux.com.au/product/etestr) according to the manufacturer's instructions and interpreted according to CLSI breakpoints. Resistance profiles were interpreted according to the CLSI guidelines for Acinetobacter spp. and CDS disk diffusion assay (http://cdstest.net) when a CLSI breakpoint for Acinetobacter spp. was not available .Antibiotic susceptibility was tested by the disc diffusion method according to the recommendations of the Clinical and Laboratory Standards Institute (CLSI) (50N length of 15.2 kbp and 25-fold coverage. FastQC (v.0.11.9) (https://bioinformatics.babraham.ac.uk/projects/fastqc/) and Filtlong (v.0.2.0) (https://github.com/rrwick/Filtlong) were used to check the quality of Illumina and MinION reads, respectively. Filtlong was used to filter long reads with poor quality and those with <1 Kbp length. Reads were trimmed using the Porechop (v.0.2.3) programs using default parameters. The high-quality Illumina and MinION reads were assembled de novo using a hybrid assembly approach using the Unicycler program (v0.4.7) as previously described  library preparation kit and sequenced using Illumina MiSeq. MinION (Oxford Nanopore) long-read sequencing was done following library preparation using the Rapid Barcoding Sequencing Kit (SQK-RBK004) and sequenced on an FLO-MIN106D Flow cell according to the manufacturer's instructions . Illumina MiSeq generated 1102451 paired-end short reads with 50-fold coverage, an average length of 250 bp, and MinION produced a total of 52680 long reads with an https://usegalaxy.org.au/) and PATRIC (https://www.patricbrc.org/) platforms respectively followed by manual annotations of resistance genes, insertion sequences, CRISPR locus and surface polysaccharide loci using ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/), ISFinder (https://www-is.biotoul.fr/), BLAST (https://blast.ncbi.nlm.nih.gov/), InterPro (https://www.ebi.ac.uk/interpro/search/sequence/), CRISPRCASFINDER (https://crisprcas.i2bc.paris-saclay.fr/CrisprCasFinder/Index) and Kaptive  were determined from the genome sequence data. The blaAmpC allele was determined using the Acinetobacter blaAmpC database available at https://pubmlst.org/. The class B cytochrome P450 was typed using the Cytochrome P450 database available at https://drnelson.uthsc.edu/bacteria/. Figures were drawn to scale using SnapGene® (v6.0.3) and Illustrator (v26.2.1).Protein coding, rRNA, and tRNA gene sequences were annotated using Prokka Seemann  and RASTbrxA-brxL regions were extracted from genomes that showed homology with that of pCl107. Partial regions with incomplete or frameshifted BREX genes were dismissed from the phylogenetic analysis. In total, 90 entries were retained and aligned using the mafft (v7.457) tool (Katoh and Standley http://tree.bio.ed.ac.uk/software/figtree/).The entire DNA sequences of the Cl107 was found resistant to ampicillin, third generation cephalosporins , gentamicin, amikacin, tobramycin, tetracycline, nalidixic acid, ciprofloxacin, florfenicol, chloramphenicol, trimethoprim, and sulfamethoxazole (Table S2). Cl107 is susceptible to colistin (with a MIC of <0.25 µg/ml) and ampicillin-sulbactam, imipenem, and meropenem.The genome sequence of Cl107 was completed using a combination of Illumina MiSeq and Oxford Nanopore (MinION) data following a hybrid assembly approach. The genome assembly consists of a 4056235 bp chromosome and a 198716 bp plasmid named pCl107.OX (Oxford scheme) and ST25IP (Institut Pasteur scheme). It carries the KL 14 and OCL 6 capsular polysaccharide and lipooligosaccharide outer core, respectively. The genome contains a CRISPR locus belonging to CAS-TypeIFb  and 48 spacers (bases 1017221–1020131 of GenBank acc. no. CP098521). Analysis of the spacers compared to the pool of spacers previously identified and catalogued by Karah et al. in 2015  and blaAmpC (99% DNA identity to blaAmpC23), which are not preceded by an ISAba1 copy. Cl107 was originally reported to carry blaNDM-1; however, this gene appears to have been lost in its complete genome, likely during the storage or transport to Australia, explaining thus its susceptibility to imipenem  and parC topoisomerase IV genes (ParC S84L). These mutations are known to be associated with fluoroquinolones resistance in Gram-negative bacteria, including A. baumannii , a csuA/BABCDE locus (chaperone-usher pili assembly system) regulated by bfmRS, an abaI gene encoding an autoinducer synthase part of the quorum-sensing system, ompA, a pglL, a blp2, a 5709 bp sized gene encoding BapA prefix-like domain-containing protein, as well as bap gene. The bap gene of 9315 bp encodes a BAP protein sharing 100% identity with the COOH extremity of type 3 (1169 aa), which is mainly observed in ST25 and differed from type 1 and 2 occurring in GC 1 and 2, respectively, according to De Gregorio et al. , pCl107 contains several genes associated with the conjugative transfer, including traIJYWH, trbA, and trbN with 100% DNA identity making pCl107 a potentially conjugative plasmid. Sequence analysis showed that pCl107 is related to pA297-3, pD4, pD46-4, and pAB3   isolate from a catheter sample in Bolivia in 2015 , sul2 for sulfamethoxazole resistance, and tetA(B) gene for tetracycline resistance. pCl107 also carries multiple complete insertion sequences, including three copies of each of ISAba1, ISAba34 and IS1008, two copies of each of IS1007 and ISAba12 and a single copy of each of ISAcsp1, ISAba11, ISAha2, IS17, and the common region CR2, along with many truncated insertion sequences, e.g. IS1006, ISAba22 and the common region elements CR1, and CR2.Cl107 also carries a large plasmid (198716 bp) called pCl107 with a GC% content of 39.13% and 210 coding reading frames. pCl107 encodes several important modules, including the HigB/A toxin/anti-toxin system, the ParA/B and ParM/StbA partitioning protein systems, a DNA primase, a DNA helicase, a DNA topoisomerase, the UmuCD DNA damage repair system, and the BREX-1 phage resistance system. Similar to previously characterized plasmids, such as pD4 and pD46-4 , were found in pCl107 within a variant of Tn6172, which was previously described in pA297-3 (GenBank acc. no. KU744946) with an additional CR∆-tetAR(B) segment (compared to Tn6172) . We proposed that the ancestral structure of AbGRI1 island (AbGRI1-0), which is frequently found in the chromosomal comM genes of GC2 isolates, has evolved from a close relative of pAB3 with Tn6022 in place of Tn6021 and in which Tn6174 had already evolved to form Tn6172 . Putting together all this information, the resistance region found in pCl107 serves as an important missing link in the complex evolutionary history of AbGRI1 islands as it provides further support for the theory that this segment has transposed as a single unit into the comM gene of the ancestor of the GC2 clone.Here, analysis of the surrounding regions of the TnstrAB-sul2-tetA(B) region, another resistance cluster region encompassing aacC2 and aacA1 genes was found in pCl107. Indeed, these genes are absent in pD4, pA297-3 and pD46-4  but present in several other A. baumannii plasmids such as pMC1.1, p1AR_0088, pA1429c, pHWBA8_1, and p40288  . BREX system has recently been described as a phage resistance system and added to the previously known mechanisms (CRISPR-Cas and restriction-modification systems) in the arsenal of bacterial defence against phages  that are characteristic of this type , an-ATP binding protein (brxC), an unknown protein (brxB), and NusB-like RNA binding antitermination protein (brxA).The pCl107 has a BREX (BacteRiophage Exclusion) system located at bases 125 913 to 139 090  or unrelated (10), fourteen A. baumannii chromosomes, and several other entries from Acinetobacter spp., listed in Table S3, were found. These BREX modules differ by the presence and absence of additional genes between pglZ and pglX  and 59% between those of pA297-3 and DT0544C chromosome (GenBank acc. no. KU744946 and CP053215 respectively).The phylogenetic analysis of BREX regions revealed the presence of many clusters where BREX loci with similar arrangements and identities generally clustered together Fig. . For insups Fig.  where eaWe found 24 plasmids related to pCl107 that include a complete BREX gene cluster. Two gene clustering patterns were generally identified—one group (15 plasmids including pA297-3) included the DUF262 domain-containing HNH endonuclease family protein-encoding gene, and the second group (with nine plasmids including pCl107) did not have this gene (Table A. baumannii plasmid BREX Region (pAb-C63_1) clusters together with BREX regions from Acinetobacter johnsonii, A. indicus, and A. pittii. This indicates the extent to which horizontal gene transfer is possibly taken between Acinetobacter spp. and even between plasmid and chromosomal genomes to mitigate the phage pressure.Many clusters delimited by the phylogenetic tree are diverse in species and genetic backgrounds Fig. . For insEscherichia coli there are more than 30 genes in a regulon called PHO to sense, acquire and process phosphate  for transporting and processing the 2-aminoethylphosphonate (2-AEPn) which is one of the most widely occurring phosphonates in nature  was also found in pCl107. This ptx module consists of five genes, namely ptxABCDE, that are related to those found in Pseudomonas stutzeri . ptxABC encodes an ABC transporter system where PtxA (IPR012693 family with IPR003439 and IPR003593 domains) is an ATP-binding protein, PtxB (IPR005770 family with PF12974 domain) is a periplasmic-binding protein and PtxC (IPR005769 family with domain IPR000515) is a membrane-spanning protein. ptxD encodes a phosphonate  dehydrogenase (IPR006140 and IPR006139 domains) and ptxE encodes a transcriptional regulator (IPR005119 and IPR000847 domains) belonging to the LysR family. Additionally, the Cl107 PtxB has the same phosphite-specific cap residues as the PtxB family that are not found in other homologs as C-P lyase-linked phosphonate binding PhnD of E. coli and HtxB (part of HtxBCDE hypophosphite transporter), highlighting a potential role for the Cl107 PtxABC as a phosphonate  transporter   plasmids were found to carry either complete or partial segments of the ptx module found in pCl107. Seven were in A. baumannii plasmids related to pCl107  are identical to that in pCl107  and which is also directly incorporated into biological molecules , which belongs to the family 153 of the subfamily A, which is also 71% aa identical to CYP153A1 of Acinetobacter calcoaceticus (https://drnelson.uthsc.edu/bacteria/). In plasmids related to pCl107, all the P450 identified belong to the same family, with the majority 99% identical to the A. calcoaceticus P450 and only two almost identical to pCl107 P450 H-dependent reductase’ , P450 , and an iron-sulphurs protein  is present in the vicinity of the ule Fig. . These eule Fig. . The cla50 Table . Similar50 Table .puuE, uao, hiuH, uacT) encoding uric acid metabolism and transport functions are also carried by pCl107 . The hiuH and uao genes encode an hydroxyisourate (HIU) hydrolase (family: IPR014306) and 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (domain: IPR017595) respectively. The HiuH and Uao proteins are responsible for the second (hydrolysis of HIU to OHCU) and third (the decarboxylation of OHCU to (S)-allantoin) steps of uric acid processing, respectively -allantoin to allantoate. While these three encoded enzymes can potentially take an HIU and transform it to allantoate, the urate oxidase  were found, including four plasmids with the hpxO gene and five plasmids without hpxO  include a related sequence (DNA identity 99%) with a complete set of uric acid module genes except for the tsx, which is replaced by ISAha2  were found in multiple Acinetobacter spp. chromosomes and plasmids (Table S5). Among Acinetobacter spp. plasmids, p1DUT-2 (GenBank acc. no. CP014652) shares a similar module and surroundings to pA297-3 with 99.9% DNA identities  have uric acid modules almost identical to that in pCl107 (99.9% DNA identity) ; Acinetobacter lowfii H7 and Al-065 and Acinetobacter spp. ACNIH1  have DNA identities ranging from 99% to 100%, suggesting a chromosomal origin.Additionally, several partial uric acid modules related to that in s Table S. Among As Table S. Among As Table S. Among As Table S. Among AAcinetobacter. baylyi (GenPept acc. no. CAG70179-CAG70185)  that is homologous (amino acid identities 39%–44%) to PucJK proteins , which are involved in uric acid transport in Bacillus subtilis.Cl107 also carries an additional chromosomal gene cluster encoding uric acid catabolism functions. This cluster is distantly related to that in pCl107 ) distributed in two complex regions and many non-antimicrobial resistance clusters. We showed that the pCl107 region encompassing the sul2, strAB, tetA(B) is closely related to AbGRI1 islands, commonly found in the chromosomal comM gene of GC2 strains, with an addition of the insertion of ISAba11 as the only difference  and CP098522 (pCl107). Illumina short reads and MinION long reads are also deposited under the Sequence Read Archive (SRA) accession numbers SRR20613520 and SRR20613519, respectively.Complete genome sequence of Cl107 was deposited in GenBank under accession numbers xtac027_Supplemental_FilesClick here for additional data file.


CDK5) contributes to cancer progression, making it a promising target for treatment. This study examined the efficacy of selectively inhibiting CDK5 in colorectal carcinoma using TP5, a small peptide that selectively inhibits the aberrant and hyperactive CDK5/p25 complex while preserving physiological CDK5/p35 functions. We analyzed TP5’s impact on CDK5 activity, cell survival, apoptosis, the cell cycle, DNA damage, ATM phosphorylation, and reactive oxygen species (ROS) signaling in mitochondria, in CRC cell lines, both alone and in combination with chemotherapy. We also assessed TP5’s efficacy on a xenograft mouse model with HCT116 cells. Our results showed that TP5 decreased CDK5 activity, impaired cell viability and colony formation, induced apoptosis, increased DNA damage, and led to the G1 phase arrest of cell cycle progression. In combination with irinotecan, TP5 demonstrated a synergy by leading to the accumulation of DNA damage, increasing the γH2A.X foci number, and inhibiting G2/M arrest induced by Sn38 treatment. TP5 alone or in combination with irinotecan increased mitochondrial ROS levels and inhibited tumor growth, prolonging mouse survival in the CRC xenograft animal model. These results suggest that TP5, either alone or in combination with irinotecan, is a promising therapeutic option for colorectal carcinoma.Colorectal carcinoma (CRC) is a prevalent cancer worldwide with a high mortality rate. Evidence suggests that increased expression of Cyclin-dependent kinase 5 ( Colorectal cancer (CRC) is a widespread disease and one of the most common cancers globally, with approximately 1 million new diagnoses each year ,3. TodayCyclin-dependent kinase 5 (CDK5) has been increasingly implicated in cancer progression. It is a unique type of cyclin-dependent kinase, predominantly expressed in the brain . CDK5/p2CDK5 was highly expressed and activated in CRC. In tumor tissues, CDK5 was overexpressed compared to normal tissue  ATP in kinase buffer with a total volume of 50 μL. The reaction mixture was then incubated at 30 °C for one hour. To terminate the reaction, Laemmli sample buffer was added, and the mixture was heated at 90 °C for five minutes. The reaction product was subsequently electrophoresed on a 4–20% SDS-PAGE gel. After electrophoresis, the gel was dried, and the dried gel was exposed overnight for autoradiography to detect the radioactive signals.Immunoprecipitation and CDK5 kinase activity were performed as previously described . BrieflyThe cell lysates were prepared with RIPA buffer, which contained a protease inhibitor cocktail  and a phosphatase inhibitor cocktail . The protein lysate of each group (5–20 μg) was electrophoresed on Invitrogen NuPAGE Bis-Tris protein gels (4–12%) and then transferred to nitrocellulose membranes using the iBlot2 gel transfer device . The membranes were blocked with BSA or non-fat dry milk and then incubated with primary antibodies overnight at 4 °C. Afterward, the membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. The Western blot bands were detected using an enhanced chemiluminescence substrate  and imaged using the KwikQuant Imager .The tumor cells were plated in Falcon Chambered Cell Culture Slides (#354118) and treated as indicated in the main text. After treatments, the cells were washed with PBS and fixed in 2% paraformaldehyde (PFA), followed by 70% ethanol. Subsequently, the cells were blocked using 5% BSA and then incubated with primary antibodies overnight at 4 °C, followed by incubation with fluorescent-conjugated secondary antibodies. Finally, the images were captured using a Zeiss LSM710 confocal microscope .HCT116 and HT29 cells were seeded into 6-well plates. Once cells reached 100% confluence, “wounds” were created by scraping lines with a 200 μL pipette tip, and then cells were washed three times in a serum-free medium. The “wounds” were observed at 4, 8, 24, and 48 h and photographed using an EVOS Cell Imaging System . The distances between the two edges of the scratch (wound width) were measured at three sites for each image using the ImageJ v1.53t. The migratory distances were calculated by subtracting the wound width at each time point from the wound width at the time zero point.One million cells of each group were stained with Annexin V-APC/PI . After indicated treatments, the tumor cells were harvested with trypsin/EDTA solution, washed with PBS and Annexin V binding buffer , and then incubated with Annexin V-APC antibody in 100 µL of Annexin V binding buffer at room temperature for 10 min. Following this, 400 µL of Annexin V binding buffer containing 1 µL of propidium iodide  was added to the cells. After 15-min incubation on ice, the stained cells were analyzed within one hour using flow cytometry (BD LSRFortessa SORP). The flow cytometry data were analyzed with FlowJo version 10.9.0, and the gating strategy for early and late apoptotic cells is presented in For cell cycle analysis, one million cells were washed twice with PBS and fixed in 70% ethanol. Subsequently, the cells were incubated with 500 µL of FxCycle PI/RNase Staining Solution (Thermo Fisher Scientific) at room temperature for 30 min and analyzed by flow cytometry (BD LSRFortessa SORP). The cell cycle phases  were quantified by the “Cell Cycle” analysis tool in the FlowJo software (FlowJo version 10.9.0).2 incubator. All assays were analyzed using BD SORP Analyzer .Cells were treated in indicated groups for 24 h before collection. Resuspended cells were incubated with 5 μM MitoSox (Thermo Scientific) for 30 min at 37 °C in a 5% CO3. Mice were divided into four groups: control , TP5 alone (I.P. 50 mg/kg every two days), Sn38 alone (I.P. 5 mg/kg every two days), and TP5 and Sn38 combination. Tumor volumes were assessed every other day by standard caliper measurement (V = L × W2/2). The survival endpoint was defined as either of the following criteria: (1) tumor volume reached 2000 mm3, (2) tumor diameter reached 20 mm.The animal procedures described in this study were carried out by staff affiliated with NCI-CCR and were approved by the NCI Animal Care and Use Committee (ACUC) in accordance with federal regulatory requirements and standards. The intramural NIH Animal Care and Use (ACU) program, including all its components, has been accredited by AAALAC International. Briefly, 1 million HCT116 cells per mouse were injected subcutaneously into the right flanks of NSG mice. We randomized the tumor-bearing mice into the indicated groups when the mean tumor volumes reached 100 mmp < 0.05; **, p < 0.01; ***, p < 0.001. Graph figures were presented as means with SEM. Statistical analysis was performed using one-way ANOVA and Student’s t-test. Mouse survival was analyzed using the Kaplan–Meier and log-rank tests.Statistical analysis was performed using the GraphPad Prism software (version 6.05) and SPSS v22. Significance was defined as *,


Background:Penthorum chinense Pursh (PCP) is widely utilized in China to treat a variety of liver diseases. It has been shown that flavonoids inhibit inflammation and have the potential to attenuate tissue damage and fibrosis. However, the mechanisms underlying how total flavonoids isolated from PCP (TFPCP) exert their anti-fibrotic effects remain unclear.Methods: The chemical composition of TFPCP was determined using UHPLC–Q-Orbitrap HRMS. Subsequently, rats were randomly assigned to a control group (Control), a carbon tetrachloride (CCl4)-induced hepatic fibrosis model group (Model), a positive control group [0.2 mg/(kg∙day)] of Colchicine), and three TFPCP treatment groups . All substances were administered by gavage and treatments lasted for 9 weeks. Simultaneously, rats were intraperitoneally injected with 10%–20% CCl4 for 9 weeks to induce liver fibrosis. At the end of the experiment, the liver ultrasound, liver histomorphological, biochemical indicators, and inflammatory cytokine levels were tested respectively. The underlying mechanisms were assessed using Western blot, immunohistochemistry, immunofluorescence, RT-qPCR, and metabolomics.Results: Fourteen flavonoids were identified in TFPCP. Compared with control animals, CCl4-treated rats demonstrated obvious liver injury and fibrosis, manifested as increases in gray values, distal diameter of portal vein (DDPV) and a decrease in blood flow velocity (VPV) in the ultrasound analysis; increased biochemical index values ; marked increases in the contents of fibrotic markers  and inflammatory factors ; and significant pathological changes. However, compared with the Model group, the ultrasound parameters were significantly improved and the serum levels of inflammatory cytokines were reduced in the TFPCP group. In contrast, the expression of TGF-β1, TLR4, and MyD88, as well as the p-P65/P65 and p-IκBα/IκBα ratios, were considerably reduced following TFPCP treatment. In addition, we identified 32 metabolites exhibiting differential abundance in the Model group. Interestingly, TFPCP treatment resulted in the restoration of the levels of 20 of these metabolites.Conclusion: Our findings indicated that TFPCP can ameliorate hepatic fibrosis by improving liver function and morphology via the inactivation of the TLR4/MyD88-mediated NF-κB pathway and the regulation of liver metabolism. Hepatic fibrosis, the primary pathological characteristic of prolonged liver disease, results from the continuous repair of liver injury and persistent inflammation. During hepatic fibrogenesis, there is a gradual buildup of fibrillar extracellular matrix (ECM) and the formation of nodules in the liver parenchyma . Without4) is extensively used to generate animal models of liver fibrosis owing to the reproducibility and efficiency of its effects. Moreover, the pathological alterations observed in these models closely resemble those seen in chronic hepatitis and hepatic fibrosis in humans  and chlorine radicals (Cl•)  and interleukin 6 (IL-6), which can activate HSCs in a paracrine manner; activated HSCs are subsequently stimulated by both autocrine and paracrine signals pathways thereby driving the development of fibrosis  are the major sources of ECM in liver fibrosis, and both are essential components in hepatic fibrogenesis and targets of pro-inflammatory mediators . Carbon n humans . First, ls (Cl•) . This lemmation” . Specififibrosis . Meanwhifibrosis . Colchicactivity . Secondlactivity . Both incytokine . These oPenthorum chinense Pursh  is a well-known traditional Chinese medicine (TCM) that has been used for the treatment of liver disease since the Ming era (1400s)  . In mode (1400s) . Flavono (1400s) . Studies (1400s) . Flavonoin vivo  was purchased from Luoen Chemical Reagent Co., Ltd. . Olive oil (J2122345) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. . Colchicine (20200908) was bought from Yunnan Phytopharmaceutical Co., Ltd. . D101 macroporous adsorption resin (2020101901) was purchased from Chengdu Kelong Chemical Reagent Factory . The reagents for measuring aspartate aminotransferase , alanine aminotransferase , alkaline phosphatase , total bilirubin , serum albumin , and total protein  levels were obtained from Roche . Rat procollagen III , collagen type Ⅳ , hyaluronidase , TNF-α (CHHNV14X86), IL-6 (2ULD7M9LKF), IL-1β (MKIMB94QHN), and transforming growth factor-beta 1  ELISA Kits were purchased from Wuhan Elabscience Biotech Co., Ltd. . The anti-laminin antibody  was obtained from Shanghai JONLN Reagent Co., Ltd. . Anti-TLR4 (AC220407057) and anti-P65 (AC220429001) antibodies were obtained from Wuhan Servicebio Technology Co., Ltd. . The anti-myeloid differentiation factor 88 antibody  was obtained from Abcam, Inc. . Anti-p-P65 (19u7497), anti-IκBα (80k0141), and anti-p-IκBα (47y8501) antibodies were purchased from Qingke Biotech Co., Ltd. .CClPenthorum chinense Pursh of the genus Penthorum Gronov. ex L in the family Saxifragaceae. TFPCP were obtained as follows: Briefly, desiccated, above-ground portions of PCP were heated with distilled water under reflux for 2 h. After filtering, the residue was extracted a second time under identical conditions. The extracts were combined and condensed under reduced pressure to a concentration of 4 g/mL, applied to D101 macroporous resin, and eluted with 60% aqueous ethanol (aq. EtOH). The 60% aq. EtOH eluate was collected and concentrated in vacuo to yield a residue, with the final residue yield being the TFPCP. The percentage content of total flavonoids in PCP was determined by UV spectrophotometry at 510 nm, with rutin serving as the reference. The chemical components of TFPCP were identified using ultra-high performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry (UHPLC–Q-Orbitrap HRMS). Separation was performed on an Accucore C18 column . The mobile phase was 0.1% formic acid (A) and 0.1% formic acid in acetonitrile (B). The elution gradient was 0–10 min, 6%–18% B; 10–20 min, 18%–35% B; 20–30 min, 35%–55% B; 30–40 min, 55%–85% B; 40–45 min, 85%–95% B; and 45–50 min, 95%–99% B; the flow rate was 0.3 mL/min.PCP herbal slice was provided by Sichuan Gulin Gansu Pharmaceutical Co., Ltd. and was identified by Assoiate Professor Jihai Gao of Chengdu University of Traditional Chinese Medicine as n = 8/group): A control group (Control); a CCl4-induced hepatic fibrosis model group (Model); a CCl4 + colchicine group  , respectively]. Animals in both the Control and Model groups were administered saline intragastrically (10 mL/kg) for 9 weeks, while those in the Colchicine and CCl4 + TFPCP groups were treated intragastrically with the same volume of saline containing the respective treatments continuously for 9 weeks. Meanwhile, olive oil was administered intraperitoneally to rats in the Control group (3 mL/kg), while rats in the other five groups received an intraperitoneal injection of olive oil containing CCl4 (3 mL/kg) of differing concentrations . Olive oil or CCl4 (to induce hepatic fibrosis) was given twice a week in the first 5 weeks and three times a week in the following 4 weeks. After 9 weeks of treatment, the rats were anesthetized with 40 mg/kg sodium pentobarbital, and serum and liver tissue were collected for further experiments. A schematic diagram of the animal and total experimental design is shown in Forty-eight male Sprague–Dawley rats, weighing 180–220 g, were procured from SPF (Beijing) Biotechnology Co., Ltd. (NO. SCXK 2019-0010) and maintained under controlled conditions  and with free access to food and water. All animal experiments were approved by the Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine (No. 2021-69). Following 3 days of acclimation, the rats were randomly divided into the following six groups ((kg∙day) ; and thrOn the last 3 days, to evaluate the status of the liver parenchyma and the lumen of the hepatic veins, rats were sedated through the inhalation of 4% isoflurane  and subjected to ultrasonography. Liver ultrasound was conducted on a Vevo 3100 Preclinical Imaging System  using a MX-250 probe (14–28 MHz). The gray value, the proximal diameter of portal vein (PDPV), the distal diameter of portal vein (DDPV), and the blood flow velocity (vein peak velocity [VPV]) were determined as an assessment of liver function.After macroscopic examination, liver tissues were fixed in 4% paraformaldehyde, desiccated, embedded in paraffin wax, cut into 4-µm-thick sections, and subjected to hematoxylin and eosin (H&E) and Masson staining according to standard procedures. Images were captured using a digital slide scanner . Three random fields were taken and scoring by a blinded experimenter. The relative collagenous fiber areas were detected by Masson staining and quantified with the Image-Pro Plus 6.0 software. The fibrotic area (%) was calculated according to the following formula: DAB staining area/total image area × 100%.To assess liver function, serum levels of ALT, AST, TBIL, ALB, ALP, and TP were measured using an automatic biochemical analyzer (Roche).1, TNF-α, IL-6, and IL-1β and the fibrotic markers PC III, COL4, LN, and HA were assessed using the respective ELISA kits following the manufacturer’s instructions.At the end of 9 weeks, the serum levels of the inflammatory cytokines TGF-βthe autosampler temperature was 5°C. Sample analysis was performed in both negative and positive ionization modes. For electrospray ionization (ESI), the source temperature was 600°C and the IonSpray Voltage Floating (ISVF) was ±5500 V.Metabolomic analysis was carried out using the Agilent 1290 Infinity LC System  and the AB Triple TOF 6600 system . Separation was performed on an ACQUITY UPLC BEH C18 column  . The mobile phase was 25 mM ammonium acetate and 25 mM ammonium hydroxide [v/v] in water (A) and acetonitrile (B) and the separation program was as follows: 0–1 min, 15:85, v/v; 1–11 min, gradient increase from 15:85 to 35:65, v/v; 11–11.1 min, gradient increase from 35:65 to 60:40, v/v; 11.1–15.1 min, 60:40, v/v; 15.1–15.2 min, gradient decrease from 60:40 to 15:85, v/v; 15.2–20.2 min, 15:85, v/v. The flow rate was 0.4 mL/min and For protein extraction, liver tissue was homogenized in RIPA lysis buffer. Protein concentrations were determined using BCA Protein Assay Kits . Protein samples were mixed with SDS sample buffer and heated to 100°C for 10 min. Subsequently, equal amounts of protein were separated by 8%–10% SDS–polyacrylamide gel electrophoresis, electro-blotted onto PVDF membranes, blocked with bovine serum albumin (BSA) for 2 h, and then incubated first with primary antibodies against TLR4, MyD88, P65, IκBα , p-P65, and p-IκBα (1:800) overnight at 4°C and, after washing, with the respective secondary antibodies . The immunoreactive bands were developed using chemiluminescence and the gray values were evaluated using ImageJ software.Total RNA was extracted from liver tissue using Trizol reagent  according to the manufacturer’s instructions. The extracted RNA was dissolved in RNase-free water and the RNA concentration was measured by spectrophotometry. Total RNA (1 μg) was reverse transcribed into cDNA , following which qPCR was performed using standard TB Green Premix Ex Taq  on a real-time PCR detection system from Rocgene . β-actin served as an internal reference. The sequences of the primers used are shown in 1, TLR4 and MyD88 relative IOD in liver were analyzed using ImageJ software. The relative IOD was calculated according to the following formula: lg .Paraffin-embedded sections were dewaxed, rehydrated, subjected to antigen retrieval, and incubated with primary antibodies targeting TLR4  and MyD88  at 4°C overnight. After washing, the samples were incubated with secondary antibody (1:200) at ambient temperature for 1 h, washed, stained with DAB, stained with hematoxylin, dehydrated through a graded ethanol series, cleared with xylene, and mounted with neutral rubber. Images were captured using a digital slide scanner (NanoZoomer-S60). Three random fields were taken and scoring by a blinded experimenter. The TGF-βSections were deparaffinized, rehydrated, subjected to antigen retrieval, blocked in blocking buffer containing 3% BSA for 30 min, and incubated with primary antibodies against P65 (1:200) and p-P65 (1:500) overnight at 4°C. After washing, the sections were incubated with secondary antibody for 1 h at ambient temperature, counterstained with DAPI. Photographs were blindly taken at three random fields under a fluorescence microscope , and scoring by a blinded experimenter.p-values <0.05 were considered significant. In addition, association and pathway analyses of the differentially abundant metabolites were conducted using online databases such as MetaboAnalyst, HMDB, and KEGG.Principal component analysis (PCA) and orthogonal partial least squares discriminant analysis (OPLS-DA) were used to analyze metabolic alterations. Variable importance in projection (VIP) values > 1 and post hoc test was used for comparisons among multiple groups. A p-value <0.05 was considered significant.The data were analyzed in GraphPad Prism 8.0 and the results are presented as means ± SD. One-way ANOVA with Tukey’s MassBank Europe, MassBank of North America [MoNA], mzCloud Best Match, and mzVault Best Match) as well as to references. Then, a manual search and match was performed to determine which compounds belonged to which structural categories based on the precise MS/MS data. Finally, 14 flavonoids with various intensities were identified in TFPCP  (p < 0.01). After the administration of TFPCP or colchicine, the gray values and DPVD were significantly decreased, while the VPV values were significantly increased; no differences in PPVD values were observed among the groups. These results indicated that both TFPCP and colchicine improved CCl4-induced liver fibrosis to some extent.Ultrasound was employed to examine the therapeutic effects of TFPCP on CCly [VPV]) . Meanwhiy [VPV]) . As depi4 + TFPCP groups increased with time. Meanwhile, compared with the Control group, the biochemical index values . In contrast, the TNF-α, IL-6, and IL-1β level in serum were significantly lower in the TFPCP group than in the Model group. While, in addition to TNF mRNA, the expression of IL6 and IL1b mRNA level was also lower in the TFPCP treatment groups than in the Model group, but the difference was not significant , which then initiates a lipid peroxidation cascade, resulting in the destruction of polyunsaturated fatty acids, particularly those associated with phospholipids , as storage deposits for lipid mediators, function as integral membrane proteins, transporters, receptors and ion channels . Therefofibrosis . In animfibrosis , which cfibrosis . The decfibrosis . Also, afibrosis . Consist4-induced liver fibrosis rat model. Furthermore, the level of Thiamine, and its phosphate derivatives, thiamine monophosphate were decreased in the Model group, while TFPCP treatment increased these two biomarker levels.Thiamine is one of the carbohydrates metabolizing co-enzymes stored mainly in the liver . PublishIn the Pantothenate and CoA biosynthesis pathway, the biomarker of pantothenic acid (vitamin B5), is the synthetic materials for coenzyme A. and pantetheine is the cysteamine amide analog of pantothenic acid which plays a central role in energy metabolism . On the Meanwhile, it is well established that metabolic processes involving galactose, nucleotide sugars, and amino sugars are also linked to chronic inflammation . ResearcIn summary, employing a combination of molecular biology and metabolomic techniques, we elucidated the mechanism underlying the anti-hepatic fibrosis effects of TFPCP. We found that they are associated with the inactivation of the TLR4/MyD88/NF-κB signaling pathway and the regulation of lipid, energy, nucleoside, and amino acid metabolism. These findings suggest that TFPCP has potential as a natural therapy for hepatic fibrosis.


Meanwhile, CoO–Co@C/CF delivers an excellent electrochemical activity for the selective conversion from HMF into 2,5-furandicarboxylic acid (FDCA), achieving a conversion of 100%, FDCA yield of 99.4% and faradaic efficiency of 99.4% at the lower oxidation potential, along with an excellent cycling stability. The integrated CoO–Co@C/CF||CoO–Co@C/CF configuration actualizes the H2O–HMF-coupled electrolysis at a satisfactory cell voltage of 1.448 V at 10 mA·cm−2. This work highlights the feasibility of engineering double active sites for the coupled electrolytic system.The renewable-energy-driven integration of hydrogen production and biomass conversion into value-added products is desirable for the current global energy transition, but still a challenge. Herein, carbon-coated CoO–Co heterojunction arrays were built on copper foam (CoO–Co@C/CF) by the carbothermal reduction to catalyze the hydrogen evolution reaction (HER) coupled with a 5-hydroxymethylfurfural electrooxidation reaction (HMFEOR). The electronic modulation induced by the CoO–Co heterojunction endows CoO–Co@C/CF with a powerful catalytic ability. CoO–Co@C/CF is energetic for HER, yielding an overpotential of 69 mV at 10 mA·cm Fiderably ,11,12. T2 and high value-added chemicals at the same time, thus maximizing economic efficiency [Five-hydroxymethylfurfural (HMF) is a common renewable biomass that is produced by the dehydration of carbohydrates such as glucose, fructose, and cellulose ,16,17,18ficiency ,28,29.d-band electronic structure have attracted wide attention recently [Developing the suitable bifunctional catalysts, which should contain two different active sites to drive HER and HMFEOR at the cathode and anode, is the necessary to realize integrated electrolysis ,30,31,32recently ,34,35. Irecently ,32,36,37recently ,39,40. Trecently ,42. Meanrecently ,44,45.−2 occurring on CoO–Co@C/CF is about 69 mV, accompanying a small Tafel slope of 58 mV·dec−1. Specifically, the electrolyzer assembled with CoO–Co@C/CF for the coupled H2 and FDCA production only requires a voltage of 1.448 V at 10 mA·cm−2, lower than that for water splitting (1.655 V at 10 mA·cm−2). We expect that the bifunctional two-electrode electrolyzer can couple HER with many other organic reactions for establishing a valuable electrochemical reforming system.In view of the above considerations, the CoO–Co heterojunction arrays covered with carbon were designed and directly constructed on copper foam (CoO–Co@C/CF), which efficiently achieves the integration of HER and HMFEOR in an alkaline system. In the anode, CoO–Co@C/CF can catalyze HMFEOR to produce FDCA with a satisfying yield and a Faraday efficiency (FE) (>99%) at a low applied potential of 1.34 V vs. the reversible hydrogen electrode (RHE). In the cathode, the HER overpotential at 10 mA·cm2, Co(OH)F translates into CoO (JCPDS no. 48-1719) without the morphology change  is about 0.89, indicating that the carbon located near to the Co species is graphitized to a certain degree during the calcination in N2. The graphitic carbon layer in CoO–Co@C/CF is not only beneficial to improving the electrical conductivity, but also protects the catalytic sites during reactions. As shown in SEM and TEM images . In the XRD patterns . tellites ,46,47. I−2, lower than that required in OER (1.46 V vs. RHE). Moreover, with the concentrations of HMF increasing, the superiority of CoO–Co@C/CF in catalyzing HMFEOR is more significant , which suggests that CoO–Co@C/CF has more rapid reaction kinetics in HMFEOR (the insert in ct). When the applied potential increases to 1.3 V vs. RHE, an incomplete arc appears in the Nyquist plots, signifying the beginning of the Faradaic reaction. Increasing the applied potential to 1.34–1.52 V vs. RHE, the semi-arc gradually completes and the radius of the arc became smaller and smaller, indicating that the Rct value decreases rapidly at lower potentials, which causes a more intensive HMFEOR reaction. Compared with Co@C/CF and CoO@C/CF, CoO–Co@C/CF can arouse HMFEOR at a lower potential and deliver a high current density of HMFEOR at each applied potential, highlighting the superiority of CoO–Co@C/CF towards HMFEOR in the thermodynamics . nificant . As showdynamics c. The Tadynamics c. Meanwhn HMFEOR .Generally, there are two possible pathways for HMFEOR ,49. In tη) of 69 mV and 207 mV to reach 10 mA·cm−2 and 100 mA·cm−2, which is only higher than that of Pt-C/CF, but much lower than that of Co@C/CF (η10 = 100 mV and η100 = 296 mV) and CoO@C/CF (η10 = 189 mV and η100 = 327 mV)  . Meanwhi 327 mV) . Such ca 327 mV) .−1, which is smaller than that of CoO@C/CF (87 mV·dec−1) and Co@C/CF (92 mV·dec−1), demonstrating that CoO–Co@C/CF has the rapid HER reaction kinetics of CoO–Co@C/CF  is far less than that of Co@C/CF (26.4 Ω) and CoO@C/CF (38.3 Ω), which profits from the high conductivity capability of the heterojunction and graphitic carbon layer that accelerates the fast charge transfer between CoO–Co@C/CF and reactants/intermediates in the HER process 2·6H2O, urea, 2-methylimidazole, and potassium hydroxide (KOH) were obtained from Tianjin Kemiou Chemical Reagent Co. Ltd. Hydrochloric acid (HCl), acetone was obtained from Tianjin Fuyu Chemical Reagent Co. Ltd. The 5-hydroxymethylfurfural (HMF), 5-formyl-2-furancarboxylic acid (FFCA), 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), 2,5-furandicarboxylic acid (FDCA), 2,5-diformylfuran (DFF), ammonium formate, ethanediol, methanol, and ethyl alcohol were all purchased from Aladdin Chemical Co. Ltd. Additionally, several large pieces of copper foam (CF) were bought wholesale from Suzhou Jiashide Foam Metal Co. Ltd. All the deionized water  used in this experiment was obtained from purification through a splendid Millipore system. The well-cut size CF  was sonicated in acetone for 15 min to remove the oil contamination layer on the surface. Subsequently, in order to remove the appeared copper oxide on the surface, the CF was soaked in 1 M HCl ultrasound for 10 min, then rinsed with water after being pre-treated, and finally dried in a vacuum oven.NH3)2·6H2O, 12 mmol urea, and 8 mmol NH4F were dissolved into 40 mL DI water and stirred for 1 h to form a uniform solution. Then, the solution was transferred into a 50 mL Teflon-lined autoclave, and the CF (3.0 × 4.0 cm) submerged in the mixture with vigorous ultrasonic treatment for 15 min. The Teflon-lined autoclave was sealed, and heated in the oven at 120 °C for 8 h. After the autoclave was cooled down to room temperature, the reactor was removed from the oven, washed with water to neutralize and collect the specimen, followed by drying at 60 °C to obtain the rosy Co(OH)F line array on the CF, denoted as Co(OH)F/CF. The prepared Co(OH)F/CF sample was then heated to 400 °C with a ramp rate of 2 °C·min−1, and the temperature kept at 400 °C for 2 h, under a N2 atmosphere. After cooling to room temperature, the gray-black material was washed with DI water to remove impurities and dried to obtain a black CoO line array on the CF, named CoO/CF.Synthesis of CoO–Co@C/CF: CoO–Co@C/CF was prepared through the successive four-step process. Typically, 2 mmol Co sample was immersed in the above uniform solution. The weighing bottle was placed in a water bath and kept at a constant temperature of 30 °C for 12 h. The purple precipitates were taken out of the weighing bottle, and washed with DI water and ethanol for several times, then dried in the air. Finally, the violet ZIF-67 rod array was obtained on the CF (denoted as CoO@ZIF-67/CF). The resulting ZIF-67/CF sample was heated to 400 °C in a N2 atmosphere, and maintained for 30 min to obtain the CoO@C/CF. On the other hand, the prepared ZIF-67/CF was calcined in a hydrogen atmosphere at 400 °C for 150 min instead of a nitrogen atmosphere to obtain another comparison sample denoted Co@C/CF.Synthesis of CoO@C/CF and Co@C/CF: The CoO@C/CF and Co@C/CF were also synthesized as a comparison. On one hand, the ZIF-67/CF precursor was heated to 350 °C in a N2.Using the Brockke D8 advanced diffractometer to carry out the X-ray diffraction (XRD) test and using a VG ESCALABMK II device equipped with Mg-Ka radiation (1253.6 eV) to perform the X-ray photon spectroscopy (XPS) analysis. A Hitachi S-4800 instrument with an accelerating voltage at 15 KV was used for scanning electron microscopy (SEM) test to obtain images of the prepared samples. The transmission electron microscopy (TEM) characterization was obtained from a JEM-2100 electron microscope  with an acceleration of 200 kV. The micro-Raman spectrometer in an instrument model of Jobin Yvon HR 800 (λ = 457.9 nm) was used to perform the Raman measurements. The organics were quantitatively tested by high-performance liquid chromatography , which was equipped with an ultraviolet-visible (UV) detector (Set UV absorption wavelength at 265 nm and a 4.6 mm × 250 mm Shim-pack GWS 5 μm C 18 column). Gas chromatography  was used for the quantitative analysis of the amount of HThe OER, HER, and HMFEOR measurements were performed on an electrochemical equipment  equipped with a three-electrode system, which has an H-shaped divided cell. An anion exchange membrane (AEM) was used to separate the anode and cathode. The as-prepared free-standing CoO–Co@C/CF samples were cut into 1.5 cm × 1.0 cm, as the working electrode. The Ag/AgCl electrode (saturated KCl filled) was used as the reference electrode, and the counter electrode was the Pt mesh. The electrochemical OER- and HER-related tests were conducted in 1.0 M KOH solution (pH = 13.9). In addition, 10 mL 1.0 M KOH (pH = 13.9) with 10 mM HMF was used as the electrolyte of HMFEOR. All reported potentials were adjusted to a reversible hydrogen electrode (RHE) following the equation:−1 in the electrolyte to obtain a steady state of electrocatalyst. The linear sweep voltammetry (LSV) was recorded in the electrolyte at a scan of 5 mV·s−1. The 90% iR compensation was employed in all the electrochemical measurements. The electrochemical impedance spectra (EIS) were tested by the AUTOLAB electrochemical workstation in different electrolytes and different potentials from 1 × 10−2 Hz to 1 × 105 Hz. The Tafel slopes were calculated according to the Tafel equation as follows:η is the potential, b is the Tafel slope, j is the current density, and j0 is the exchange current density.Prior to all electrochemistry measurements, the working electrode was scanned by 20 cyclic voltammograms curves (CVs) at 100 mV·s−1 by CVs in the potential region of 0 ~ −1.0 V (vs. RHE), and tested for 72 h at the potential required 10 mA·cm−2. The HMFEOR was carried out at room temperature, with the stirring at different potentials passing 58 C . The stability testing of HMFEOR was performed at 1.37 V, 8 cycles with the addition of HMF to measure conversion rate, yield, and Faraday efficiency. For the two-electrode electrolysis, CoO–Co@C/CF was employed as the electrocatalyst for both anodes and cathodes.To explore the stability of electrocatalysts, the long-time stability test of HER was carried out at 100 mV·s−1; all of the separations lasted for 6 min. All the quantitative analysis of reactants, intermediates, and products was made by an external standard method. In the HMF oxidation process, the conversion (%) of HMF, product yield (%), and the Faraday efficiency  of the product were calculated according to the following equations:F is the Faraday constant  and n is the number of electrons transferred for the final product FDCA formation.In order to analyze the reactant conversion rate, the product yield, and the corresponding Faraday efficiency, the electrochemical oxidation of HMF on account of the three-electrode and two–electrode systems was performed with a potentiostatic method at different potentials , passing 58 C with stirring. During and after the reaction, 20 μL of the anode compartment solution was removed, mixed with 100 μL 0.2 M HCl solution, diluted with ultrapure water to 800 μL, then analyzed by HPLC (Wufeng LC-100C) with a 4.6 mm × 250 mm Shim-pack GWS 5 μm C 18 column. The ultraviolet-visible detector wavelength was set to 265 nm, while the methanol as a mobile phase A was mixed with mobile phase B, which was a 5 mM ammonium formate aqueous solution in a ratio of 3:7, and the flow rate was 1 mL·min2 production with HMF oxidation. Benefiting from the synergistic effect between the CoO–Co heterojunction and graphitic carbon, CoO–Co@C/CF is energetic for HER and HMFEOR in both thermodynamics and kinetics. The two-electrode system assembled by CoO–Co@C/CF features a small cell voltage of 1.448 V for the HER-HMFEOR-coupled electrolysis. The strategy developed herein afforded a facile way to fabricate double catalytic sites for electrocatalysis.In summary, the carbon-coated CoO–Co heterojunction arrays made up of nanoparticles were successfully built on CFs to couple the H