Deciphering the Clinical Significance and Kinase Functions of GSK3α in Colon Cancer by Proteomics and Phosphoproteomics.

GSK3α and GSK3ß are two GSK3 isoforms with 84% overall identity and 98% identity in their catalytic domains. GSK3ß plays important roles in the pathogenesis of cancer, while GSK3α has long been considered a functionally redundant protein of GSK3ß. Few studies have specifically investigated the functions of GSK3α. In this study, unexpectedly, we found that the expression of GSK3α, but not GSK3ß, was significantly correlated with the overall survival of colon cancer patients in 4 independent cohorts. To decipher the roles of GSK3α in colon cancer, we profiled the phosphorylation substrates of GSK3α and uncovered 156 phosphosites from 130 proteins specifically regulated by GSK3α. A number of these GSK3α-mediated phosphosites have never been reported before or have been incorrectly identified as substrates of GSK3ß. Among them, the levels of HSF1S303p, CANXS583p, MCM2S41p, POGZS425p, SRRM2T983p, and PRPF4BS431p were significantly correlated with the overall survival of colon cancer patients. Further pull-down assays identified 23 proteins, such as THRAP3, BCLAF1, and STAU1, showing strong binding affinity to GSK3α. The interaction between THRAP3 and GSK3α was verified by biochemical experiments. Notably, among the 18 phosphosites of THRAP3, phosphorylation at S248, S253, and S682 is specifically mediated by GSK3α. Mutation of S248 to D (S248D), which mimics the effect of phosphorylation, obviously increased cancer cell migration and the binding affinity to proteins related to DNA damage repair. Collectively, this work not only discloses the specific function of GSK3α as a kinase but also suggests GSK3α as a promising therapeutic target for colon cancer.

Dnajb11-Kidney Disease Develops from Reduced Polycystin-1 Dosage but not Unfolded Protein Response in Mice.

SIGNIFICANCE STATEMENT: Heterozygous DNAJB11 mutation carriers manifest with small cystic kidneys and renal failure in adulthood. Recessive cases with prenatal cystic kidney dysplasia were recently described. Our in vitro and mouse model studies investigate the proposed disease mechanism as an overlap of autosomal-dominant polycystic kidney disease and autosomal-dominant tubulointerstitial kidney disease pathogenesis. We find that DNAJB11 loss impairs cleavage and maturation of the autosomal-dominant polycystic kidney disease protein polycystin-1 (PC1) and results in dosage-dependent cyst formation in mice. We find that Dnajb11 loss does not activate the unfolded protein response, drawing a fundamental contrast with the pathogenesis of autosomal-dominant tubulointerstitial kidney disease. We instead propose that fibrosis in DNAJB11 -kidney disease may represent an exaggerated response to polycystin-dependent cysts. BACKGROUND: Patients with heterozygous inactivating mutations in DNAJB11 manifest with cystic but not enlarged kidneys and renal failure in adulthood. Pathogenesis is proposed to resemble an overlap of autosomal-dominant polycystic kidney disease (ADPKD) and autosomal-dominant tubulointerstitial kidney disease (ADTKD), but this phenotype has never been modeled in vivo . DNAJB11 encodes an Hsp40 cochaperone in the endoplasmic reticulum: the site of maturation of the ADPKD polycystin-1 (PC1) protein and of unfolded protein response (UPR) activation in ADTKD. We hypothesized that investigation of DNAJB11 would shed light on mechanisms for both diseases. METHODS: We used germline and conditional alleles to model Dnajb11 -kidney disease in mice. In complementary experiments, we generated two novel Dnajb11-/- cell lines that allow assessment of PC1 C-terminal fragment and its ratio to the immature full-length protein. RESULTS: Dnajb11 loss results in a profound defect in PC1 cleavage but with no effect on other cystoproteins assayed. Dnajb11-/- mice are live-born at below the expected Mendelian ratio and die at a weaning age with cystic kidneys. Conditional loss of Dnajb11 in renal tubular epithelium results in PC1 dosage-dependent kidney cysts, thus defining a shared mechanism with ADPKD. Dnajb11 mouse models show no evidence of UPR activation or cyst-independent fibrosis, which is a fundamental distinction from typical ADTKD pathogenesis. CONCLUSIONS: DNAJB11 -kidney disease is on the spectrum of ADPKD phenotypes with a PC1-dependent pathomechanism. The absence of UPR across multiple models suggests that alternative mechanisms, which may be cyst-dependent, explain the renal failure in the absence of kidney enlargement.

NR3C2 inhibits the proliferation of colorectal cancer via regulating glucose metabolism and phosphorylating AMPK.

We aim to investigate the roles and mechanisms of NR3C2 in colorectal cancer (CRC). The expression of NR3C2 in CRC tumours and paired paracancerous tissues of 71 CRC patients and five CRC cell lines was detected by western blotting, immunohistochemistry and real-time reverse-transcription PCR. Moreover, NR3C2 was overexpressed or knocked down in CRC cells by lentiviral vector transfection. The proliferation of cells was measured by MTT, colony formation assay and flow cytometry. Glucose metabolism was assessed by detecting lactate production, glucose consumption and ATP production. Western blotting and real-time reverse-transcription PCR were used to detect the expression of AMPK, LDHA and HK2. The expression of NR3C2 was significantly decreased in CRC tumours compared to paracancerous tissues, which was correlated with distant metastasis, poor prognosis and advanced stages of CRC patients. Overexpressing NR3C2 suppressed the proliferation and promoted the G2/M cell cycle arrest of CRC cells. Furthermore, NR3C2 inhibited glucose metabolism by decreasing the expression of HK2 and LDHA. The phosphorylation of AMPK was also downregulated in CRC cells overexpressing NR3C2. This study demonstrated that NR3C2 inhibited the proliferation of CRC by inhibiting glucose metabolism and phosphorylation of AMPK which may serve as a therapeutic target for CRC.

Age-Associated Proteomic Signatures and Potential Clinically Actionable Targets of Colorectal Cancer.

The occurrence and prevalence of colorectal cancer (CRC) is closely associated with age. More than 90% of patients with CRC are diagnosed after 50 years of age. However, CRC incidence of young individuals has been increasing since 1990s, whereas the overall CRC frequency is declining. Distinct overall survival rates between young and aged patients with CRC have been established. Tremendous efforts have been made to clarify the underlying mechanisms of age-dependent clinical differences, but it still remains elusive. Here, we performed proteomic profiling of 50 patients with CRC and revealed proteomic signatures of CRC across age groups. Gene set enrichment analysis showed that distinct age-dependent clinical outcomes might mainly attribute to varied MYC targets V1/V2, E2F targets and G2M checkpoint gene sets, which were associated with cancer cell proliferation, cell apoptosis, tumor growth, and tumor metastasis. Multiple linear regression analysis revealed a large number of functional proteins, such as NOP2, CSE1L, NHP2, NOC2L and CDK1, with adjusted expression significantly correlated with age (p < 0.05). Among them, NHP2 is a core component of the telomerase complex associated with age. High NHP2 expression predicted poor overall survival, with a more significant correlation in aged patients with CRC. Knockdown of NHP2 significantly suppressed cancer cell proliferation. In addition, we revealed some age-related potential clinically actionable targets, such as PSEN1, TSPO, and CDK1, which might be more suitable for patients with late-onset CRC. Collectively, this study identifies age-associated proteomic signatures and potential therapeutic targets of CRC and may help make a precise decision on CRC treatment.

Ultrasensitive DNA Methylation Ratio Detection Based on the Target-Induced Nanoparticle-Coupling and Site-Specific Base Oxidation Damage for Colorectal Cancer.

DNA methylation analysis holds great promise in the whole process management of cancer early screening, diagnosis, and prognosis monitoring. Nevertheless, accurate detection of target methylated DNA, especially its methylation ratio in the genome, remains challenging. Herein, we report for the first time an integrated strategy of target-induced nanoparticle-coupling and site-specific base oxidation damage for DNA methylation analysis with the assistance of well-designed nanosensors. The ultrahigh sensitivity for detecting target methylated DNA as low as 32 × 10-17 M and high specificity for distinguishing 0.001% methylation ratio are achieved by this proposed strategy without amplification operations. Notably, the precise quantification of target DNA methylation ratio has been achieved for the first time. Through quantitative detection of target methylated DNA and methylation ratio, this proposed strategy could reliably diagnose and monitor cancer progression and treatment responses for colorectal cancer, which is superior to the clinical Septin 9 kit. It is anticipated that the proposed strategy has attractive application prospects in early diagnosis and monitoring for colorectal cancer and other various diseases.

Proteomic Maps of Human Gastrointestinal Stromal Tumor Subgroups.

Gastrointestinal stromal tumor (GIST) is a common sarcoma of gastrointestinal tract (GIT) with high metastatic and recurrence rates, but the proteomic features are still less understood. Here we performed systematic quantitative proteome profiling of GIST from 13 patients classified into very low/low, intermediate and high risk subgroups. An extended cohort of GIST (n = 131) was used for immunohistochemical validation of proteins of interest. In total, 9177 proteins were quantified, covering 55.9% of the GIT transcriptome from The Human Protein Altas. Out of the 9177 quantified proteins, 4930 proteins were observed in all 13 cases with 517 upregulated and 187 downregulated proteins in tumorous tissues independent of risk stage. Pathway analysis showed that the downregulated proteins were mostly enriched in metabolic pathway, whereas the upregulated proteins mainly belonged to spliceosome pathway. In addition, 131 proteins showed differentially expressed patterns among GIST subgroups with statistical significance. The 13 GIST cases were classified into 3 subgroups perfectly based on the expression of these proteins. The intensive comparison of molecular phenotypes and possible functions of quantified oncoproteins, tumor suppressors, phosphatases and kinases between GIST subgroups was carried out. Immunohistochemical analysis of the phosphatase PTPN1 (n = 117) revealed that the GIST patients with high PTPN1 expression had low chances of developing metastasis. Collectively, this work provides valuable information for understanding the inherent biology and evolution of GIST.

CINP is a novel cofactor of KLF5 required for its role in the promotion of cell proliferation, survival and tumor growth.

Krüppel-like factor 5 (KLF5) both suppresses and promotes tumor growth depending on cellular context. The mechanisms underlying tumor promotion could be targetable for therapy. Although a number of transcriptional targets of KLF5 have been identified and implicated in KLF5-mediated tumor growth, how KLF5 regulates these genes remains to be addressed. Here we performed coimmunoprecipitation (co-IP) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) in the TSU-Pr1 bladder cancer cell line, in which KLF5 is shown to promote tumor growth, to identify KLF5-interacting nuclear proteins that are necessary for KLF5's tumor promoting function. LC-MS/MS revealed 122 potential KLF5 binding proteins in the nuclear proteins precipitated by the KLF5 antibody, and the top nine candidates included AHNAK, TFAM, HSDL2, HNRNPC, CINP, IST1, FBL, PABPC1 and SNRNP40. SRB assays of these nine proteins indicated that silencing CINP had the most potent inhibitory effect on cell growth in KLF5-expressing cells but did not affect parental TSU-Pr1 cells. Further analyses not only confirmed the physical interaction between KLF5 and CINP, also demonstrated that knockdown of CINP attenuated the effects of KLF5 on cell cycle progression, apoptosis and tumorigenesis. Silencing CINP also attenuated the effect of KLF5 on the expression of a number of genes and signaling pathways, including cell cycle regulator Cyclin D1 and apoptosis-related Caspase 7. These results suggest that CINP is a cofactor of KLF5 that is crucial for the promotion of tumor growth, and that the KLF5-CINP interaction could be a novel therapeutic target for inhibiting KLF5-promoted tumor growth.

Effects of miR-200b-3p inhibition on the TRPC6 and BKCa channels of podocytes.

Transient receptor potential canonical 6 (TRPC6) and large-conductance Ca2+-activated K+ channels (BKCa), two of the key ion channels for blood filtration function of podocytes, have been implicated in the pathogenesis of kidney diseases. Moreover, it has been reported that miR-200 b plays an important role in regulating the biological processes of podocytes. In this study, we aimed to examine whether there was a relationship between miR-200 b-3p and the two ion channels. It was suggested that miR-200 b-3p down-regulation inhibited the currents of TRPC6 and BKCa channels. It also showed that miR-200 b-3p inhibition reduced the levels of protein expression and mRNA transcription of TRPC6 and BKCa channels. Moreover, the down-regulation of miR-200 b-3p resulted in the decrease of the intracellular Ca2+ concentration. It was also suggested that the decrease of BKCa currents resulting from miR-200 b-3p inhibition could be regulated by TRPC6 channels. TRPC6 blockage also inhibited BKCa currents and reduced the level of BKCa expression. These results together suggested that miR-200 b-3p inhibition reduced the currents of TRPC6, which led to the decrease of intracellular Ca2+ concentration. The decrease of Ca2+ source required for BKCa activation may result in the inhibition of BKCa currents.

Nano-TiO2 induces autophagy to protect against cell death through antioxidative mechanism in podocytes.

Autophagy is a cellular pathway involved in degradation of damaged organelles and proteins in order to keep cellular homeostasis. It plays vital role in podocytes. Titanium dioxide nanoparticles (nano-TiO2) are known to induce autophagy in cells, but little has been reported about the mechanism of this process in podocytes and the role of autophagy in podocyte death. In the present study, we examined how nano-TiO2 induced authophagy. Besides that, whether autophagy could protect podocytes from the damage induced by nano-TiO2 and its mechanism was also investigated. Western blot assay and acridine orange staining presented that nano-TiO2 significantly enhanced autophagy flux in podocytes. In addition, AMP-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were involved in such process. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that upregulated level of autophagy induced by rapamycin in high concentration nano-TiO2-treated podocytes could significantly reduce the level of oxidative stress and alleviate podocyte death. Downregulating the level of autophagy with 3-methyladenine had the opposite effects. These findings indicate that nano-TiO2 induces autophagy through activating AMPK to inhibit mTOR in podocytes, and such autophagy plays a protecting role against oxidative stress on the cell proliferation. Changing autophagy level may become a new treatment strategy to relieve the damage induced by nano-TiO2 in podocytes.

MiRNA expression profile and miRNA-mRNA integrated analysis (MMIA) during podocyte differentiation.

The podocyte is a prominent cell type, which encases the capillaries of glomerulus. Podocyte-selective deletion of Dicer or Drosha was reported to induce proteinuria and glomerulosclerosis, suggesting the essential role of microRNA (miRNA) in podocytes for renal function. However, no comprehensive miRNA expression or miRNA-mRNA integrated analysis (MMIA) can be found during podocyte differentiation. Herein, miRNA and mRNA microarrays are presented, which were carried out in differentiated and undifferentiated mouse podocyte cell lines (MPC5). A total of 50 abnormal miRNAs (26 down-regulated and 24 up-regulated) were identified in differentiated and undifferentiated podocytes. Using MMIA, 80 of the 743 mRNAs (>twofold change) were predicted for potential crosstalk with 30 miRNAs of the 50 abnormal miRNAs. In addition, the gene ontology of mRNAs and the pathway analysis of miRNAs revealed a new potential-regulated network during podocyte differentiation. The expressions of three remarkably changed miRNAs (miR-34c, miR-200a and miR-467e) and four mRNAs (Runx1t1, Atp2a2, Glrp1, and Mmp15), were randomly chosen for further validation by the quantitative real-time polymerase chain reaction, and their expression trends were consistent with the microarray data. Reference searching was also conducted to confirm our data and to find potential new molecules and miRNA-target pairs involved in the podocyte differentiation. The dual luciferase reporter assay for miR-200a/GLRX and let-7b/ARL4D confirmed the prediction of MMIA. The results of this study provide a detailed integration of mRNA and miRNA during podocyte differentiation. The molecular integration mode will open up new perspectives for a better understanding of the mechanism during podocyte differentiation.

Crosstalk between protective autophagy and NF-κB signal in high glucose-induced podocytes.

Despite a great deal of recent studies focused on the pivotal role of autophagy in maintaining podocyte energy homeostasis, the mechanisms of autophagy in regulating transcriptional factors under high glucose (HG) condition are not fully understood. Here, we evaluated the effect of HG on nuclear factor-kappa B (NF-κB) signaling and autophagic process. The results showed that HG promoted autophagy in podocytes. Bafilomycin A1 (Baf A1) further enhanced this effect, but 3-methyadenine (3-MA) inhibited it. The proautophagic effects of HG manifested in the form of enhanced podocyte expression of light chain 3 (LC3)-II. In these cells, blockade of NF-κB signal by ammonium pyrrolidinethiocarbamate constrained in effectively reducing LC3-II up-regulation and increasing podocyte apoptosis. Furthermore, the autophagy inhibitors, such as Baf A1 and 3-MA, significantly enhanced HG-induced NF-κB activation and increased apoptosis. Thus, we conclude that the accumulation of autophagosomes results from enhancement of the autophagic flux, but not the blockage of autophagosome-lysosome fusion by HG. We also prove that HG-induced apoptosis, autophagy, and NF-κB signal are in a close crosstalk through a yet undetermined mechanism in podocytes.

High glucose inhibits autophagy and promotes the proliferation and metastasis of colorectal cancer through the PI3K/AKT/mTOR pathway.

BACKGROUND: Colorectal cancer (CRC) ranks among the most prevalent malignancies worldwide, characterized by its complex etiology and slow research progress. Diabetes, as an independent risk factor for CRC, has been widely certified. Consequently, this study centers on elucidating the intricacies of CRC cells initiation and progression within a high-glucose environment. METHODS: A battery of assays was employed to assess the proliferation and metastasis of CRC cells cultured under varying glucose concentrations. Optimal glucose levels conducive to cells' proliferation and migration were identified. Western blot analyses were conducted to evaluate alterations in apoptosis, autophagy, and EMT-related proteins in CRC cells under high-glucose conditions. The expression of PI3K/AKT/mTOR pathway-associated proteins was assessed using western blot. The effect of high glucose on xenograft growth was investigated in vivo by MC38 cells, and changes in inflammatory factors (IL-4, IL-13, TNF-α, IL-5, and IL-12) were measured via serum ELISA. RESULTS: Our experiments demonstrated that elevated glucose concentrations promoted both the proliferation and migration of CRC cells; the most favorable glucose dose is 20 mM. Western blot analyses revealed a decrease in apoptotic proteins, such as Bim, Bax, and caspase-3 with increasing glucose levels. Concurrently, the expression of EMT-related proteins, including N-cadherin, vimentin, ZEB1, and MMP9, increased. High-glucose cultured cells exhibited elevated levels of PI3K/AKT/mTOR pathway proteins. In the xenograft model, tumor cells stimulated by high glucose exhibited accelerated growth, larger tumor volumes, and heightened KI67 expression of immunohistochemistry. ELISA experiments revealed higher expression of IL-4 and IL-13 and lower expression of TNF-α and IL-5 in the serum of high-glucose-stimulated mice. CONCLUSION: The most favorable dose and time for tumor cells proliferation and migration is 20 mM, 48 h. High glucose fosters CRC cell proliferation and migration while suppressing autophagy through the activation of the PI3K/AKT/mTOR pathway.

Comprehensive analysis of m6A RNA methylation regulators in esophageal carcinoma.

Background: N6-methyladenosine (m6A) is the most pervasive modification of RNA methylation in eukaryotic cells. m6A modification plays a pivotal role in tumorigenesis and progression in many types of cancers. Until now, the role of m6A modification in esophageal carcinoma (ESCA) has remained obscure. The aim of the study was to construct and validate prognostic signatures based on m6A regulators for ESCA. Methods: Transcriptomic data, somatic mutations and clinical information were obtained from The Cancer Genome Atlas (TCGA). Copy number variations were obtained from the UCSC (University of California, Santa Cruz) Xena database. We curated 21 m6A regulators and performed consensus clustering analysis to quantify the m6A modification pattern. Results: Of the 184 patients, 23 (12.5%) were genetically altered in m6A regulators, with the highest frequency of mutations in ZC3H13 and LRPPRC. We constructed a m6A score system to investigate the prognosis of ESCA. The m6A score was closely related to immune cell infiltration in the tumor immune microenvironment. Patients with a high m6A score had an unfavorable prognosis. The combination of tumor mutation burden and m6A score would improve the prognostic value. Conclusions: Our study established and validated a strong prognostic signature based on m6A regulators. This can be used to accurately predict the prognosis of ESCA.

Efficacy and safety of ketamine as an adjuvant to regional anesthesia: A systematic review and meta-analysis of randomized controlled trials.

STUDY OBJECTIVE: To identify whether adding ketamine to the local anesthetics (LA) in the regional anesthesia could prolong the duration of analgesia. DESIGN: A Systematic review and meta-analysis of randomized controlled trials. SETTING: The major dates were obtained in the operating room and the postoperative recovery ward. PATIENTS: A total of 1011 patients at ASA physical status I and II were included in the analysis. Procedure performed including cesarean section, orthopedic, radical mastectomy, urological or lower abdominal surgery and intracavitary brachytherapy implants insertion. INTERVENTIONS: After an extensive search of the electronic database, patients received regional anesthesia combined or not combined general anesthesia and with or without adding ketamine to LA were included in the analysis. The regional anesthesia includes spinal anesthesia, brachial plexus block, pectoral nerve block, transversus abdominis plane block and femoral and sciatic nerve block. MEASUREMENT: The primary outcome was the duration of analgesia. Secondary outcomes were the duration and onset time of motor and sensory block as well as the ketamine-related adverse effect. Data are expressed in mean differences in continuous data and odds ratios (OR) for dichotomous data with 95% confidence intervals. The risk of bias of the included studies was evaluated using the revised Cochrane risk of bias tool for randomized trials. The quality of evidence for each outcome was rated according to the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) Working Group system. MAIN RESULT: Twenty randomized controlled trials were included in the analysis. When ketamine was used as an adjuvant to LA, the duration of analgesia could be prolonged(172.21 min, 95% CI, 118.20 to 226.22; P<0.00001, I2 = 98%), especially in the peripheral nerve block(366.96 min, 95% CI, 154.19 to 579.74; P = 0.0007, I2 = 98%). Secondary outcomes showed ketamine could prolong the duration of sensory block(29.12 min, 95% CI, 10.22 to 48.01; P = 0.003, I2 = 96%) but no effect on the motor block(6.94 min, 95% CI,-2.65 to 16.53;P = 0.16, I2 = 84%), the onset time of motor and sensory block (motor onset time, -1.17 min, 95% CI, -2.67 to 0.34; P = 0.13, I2 = 100%; sensory onset time, -0.33 min, 95% CI,-0.87 to 0.20; P = 0.23, I2 = 96%) as well as the ketamine-related adverse effect(OR, 1.97, 95% CI,0.93 to 4.17;P = 0.08, I2 = 57%). CONCLUSION: This study indicates that ketamine could be an ideal adjuvant to local anesthetics regardless of the types of anesthesia. Overall, the quality of the evidence is low.

Efficacy of Transcutaneous Electrical Acupoint Stimulation on Chronic Postsurgical Pain After Video-Assisted Thoracoscopic Lobectomy: Study Protocol for a Prospective Randomized Controlled Trial.

INTRODUCTION: Patients undergoing video-assisted thoracoscopic lobectomy (VATL) often experience chronic postsurgical pain (CPSP). Postoperative pain can affect the recovery of postoperative lung function, prolong postoperative recovery time, and increase patient hospitalization expenses. Transcutaneous electrical acupoint stimulation (TEAS) is an alternative therapy based on acupuncture that has shown promise in postoperative recovery and pain management across various medical fields. However, research specifically focused on the improvement of CPSP after VATL is currently lacking. The purpose of this study is to evaluate whether TEAS can effectively reduce the severity and occurrence of chronic postsurgical pain in patients undergoing VATL. By investigating the potential benefits of TEAS in mitigating CPSP after VATL, this study aims to provide valuable clinical evidence to support the integration of TEAS into postoperative care protocols for patients undergoing VATL. METHODS: This study is a prospective, single-center, double-blinded, randomized controlled trial to be conducted at the 920th Hospital of Joint Logistics Support Force. Eighty patients undergoing VATL will be randomly divided into an experimental group (TEAS group) and a control group (sham group). The experimental group will receive TEAS at bilateral PC6, LI4, LR3, LU5, TE5, and LI11. The control group will not receive TEAS at the same acupoints. Both groups will receive TEAS or no TEAS before anesthesia induction and 1-7 days after surgery, with each session lasting 30 min. PLANNED OUTCOMES: The primary outcome will be the incidence of CPSP at 3 months after surgery. Secondary outcomes will include the incidence of CPSP at 6 months after surgery, the numerical rating scale (NRS) scores at 3 and 6 months after surgery, as well as the NRS scores at 24, 48, and 72 h after surgery, remifentanil consumption during general anesthesia, demand for rescue analgesics, number and duration of indwelling chest tubes, incidence of postoperative nausea and vomiting, and changes of norepinephrine (NE), cortisol (Cor), tumor necrosis factor (TNF- α), and interleukin 6 (IL-6) in serum. TRIAL REGISTRATION: ChiCTR2300069458. Registered on March 16, 2023.

The role of glycogen synthase kinase-3ß in glioma cell apoptosis induced by remifentanil.

The aim of malignant glioma treatment is to inhibit tumor cell proliferation and induce tumor cell apoptosis. Remifentanil is a clinical anesthetic drug that can activate the N-methyl-D-aspartate (NMDA) receptor. NMDA receptor signaling activates glycogen synthase kinase-3ß (GSK-3ß). Discovered some 32 years ago, GSK-3ß was only recently considered as a therapeutic target in cancer treatment. The purpose of this study was to assess whether remifentanil can induce the apoptosis of C6 cells through GSK-3ß activation. 3-(4,5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was used to detect cell viability. Hoechst 33342 staining and flow cytometry were used to detect cell apoptosis. The effect of GSK-3ß activation was detected using a GSK-3ß activation assay kit and 4-benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (TDZD-8), a potent and selective small molecule inhibitor of GSK-3ß. The MTT assay indicated that remifentanil induced C6 cell death in a concentration- and time-dependent manner. Hoechst 33342 staining and flow cytometry showed that remifentanil significantly induced C6 cell apoptosis. The measurement of GSK-3ß activation showed that remifentanil increased the cellular level of GSK-3ß. All of these toxic effects can be attenuated by treatment with TDZD-8. These results suggest that remifentanil is able to induce C6 cell apoptosis through GSK-3ß activation, which provides a basis for its potential use in the treatment of malignant gliomas.

Nanosized copper oxide induces apoptosis through oxidative stress in podocytes.

Nanosized copper oxide (nano-CuO) has been widely used in many fields. Recent studies have shown that nano-CuO has toxic effects on various organs, but the effects of nano-CuO on kidney remain unclear. The aim of this study was to assess whether nano-CuO can induce the apoptosis of podocytes. The result of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay indicated that nano-CuO decreased podocyte viability in a concentration-dependent and time-dependent manner. The apoptotic assay by flow cytometry showed that nano-CuO induced podocyte apoptosis significantly. Meanwhile, the result of flow cytometric assay gave a clear indication that nano-CuO increased reactive oxygen species (ROS) level. The measurement of superoxide dismutase (SOD) and malondialdehyde (MDA) showed that nano-CuO decreased SOD and increased MDA levels in podocytes. These results may provide basic information for the safe application of nano-CuO in the future.

TOMM34 serves as a candidate therapeutic target associated with immune cell infiltration in colon cancer.

Background: Colon cancer represents one of the most pervasive digestive malignancies worldwide. Translocase of the outer mitochondrial membrane 34 (TOMM34) is considered an oncogene and is implicated in tumor proliferation. However, the correlation between TOMM34 and immune cell infiltration in colon cancer has not been investigated. Materials and methods: Based on multiple open online databases, we performed integrated bioinformatics analysis of TOMM34 to evaluate the prognostic value of TOMM34 and its correlation with immune cell infiltration. Results: TOMM34 gene and protein expression levels were elevated in tumor tissues compared with normal tissues. Survival analysis revealed that upregulation of TOMM34 was significantly associated with poorer survival time in colon cancer. High TOMM34 expression was dramatically related to low levels of B cells, CD8+ T cells, neutrophils, dendritic cells, PD-1, PD-L1 and CTLA-4. Conclusions: Our results confirmed that high expression of TOMM34 in tumor tissue correlates with immune cell infiltration and worse prognosis in colon cancer patients. TOMM34 may serve as a potential prognostic biomarker for colon cancer diagnosis and prognosis prediction.

Guideline- Versus Non-Guideline-Based Neoadjuvant Management of Clinical T4 Rectal Cancer.

(1) Background: Practice guidelines recommend neoadjuvant treatment for clinical T4 rectal cancer. The primary objective of this retrospective study was to assess whether compliance with guidelines correlates with patient outcomes. Secondarily, we evaluated predictors of adherence to guidelines and mortality. (2) Methods: A total of 397 qualified rectal cancer (RC) patients from 2017 to 2020 at West China Hospital of Sichuan University were included. Patients were divided into two groups depending on adherence to neoadjuvant treatment guidelines. The main endpoints were overall survival (OS) and disease special survival (DSS). We analyzed factors associated with guideline adherence and mortality. (3) Results: Compliance with guidelines was only 39.55%. Patients' neoadjuvant therapy treated not according to the guidelines for clinical T4 RC was not associated with an overall survival (95.7% vs. 88.9%) and disease special survival (96.3% vs. 91.1%) benefit. Patients were more likely to get recommended therapy with positive patient compliance. Staging Ⅲ, medium/high differentiation and objective compliance were associated with increased risk of mortality. (4) Conclusions: Guideline adherence for clinical T4 RC in our system is low. Compliance with the relevant guidelines for neoadjuvant therapy seems not to lead to better overall survival for patients with clinical T4 RC.

HWJMSC-derived extracellular vesicles ameliorate IL-1ß-induced chondrocyte injury through regulation of the BMP2/RUNX2 axis via up-regulation TFRC.

Articular osteochondral injury is a common and frequently occurring disease in orthopedics that is caused by aging, disease, and trauma. The cytokine interleukin-1ß (IL-1ß) is a crucial mediator of the inflammatory response, which exacerbates damage during chronic disease and acute tissue injury. Human Wharton's jelly mesenchymal stem cell (HWJMSC) extracellular vesicles (HWJMSC-EVs) have been shown to promote cartilage regeneration. The study aimed to investigate the influence and mechanisms of HWJMSC-EVs on the viability, apoptosis, and cell cycle of IL-1ß-induced chondrocytes. HWJMSC-EVs were isolated by Ribo™ Exosome Isolation Reagent kit. Nanoparticle tracking analysis was used to determine the size and concentration of HWJMSC-EVs. We characterized HWJMSC-EVs by western blot and transmission electron microscope. The differentiation, viability, and protein level of chondrocytes were measured by Alcian blue staining, Cell Counting Kit-8, and western blot, respectively. Flow cytometer was used to determine apoptosis and cell cycle of chondrocytes. The results showed that HWJMSCs relieved IL-1ß-induced chondrocyte injury by inhibiting apoptosis and elevating viability and cell cycle of chondrocyte, which was reversed with exosome inhibitor (GW4869). HWJMSC-EVs were successfully extracted and proven to be uptake by chondrocytes. HWJMSC-EVs ameliorate IL-1ß-induced chondrocyte injury by inhibiting cell apoptosis and elevating viability and cycle of cell, but these effects were effectively reversed by knockdown of transferrin receptor (TFRC). Notably, using bone morphogenetic protein 2 (BMP2) pathway agonist and inhibitor suggested that HWJMSC-EVs ameliorate IL-1ß-induced chondrocyte injury through activating the BMP2 pathway via up-regulation TFRC. Furthermore, over-expression of runt-related transcription factor 2 (RUNX2) reversed the effects of BMP2 pathway inhibitor promotion of IL-1ß-induced chondrocyte injury. These results suggested that HWJMSC-EVs ameliorate IL-1ß-induced chondrocyte injury by regulating the BMP2/RUNX2 axis via up-regulation TFRC. HWJMSC-EVs may play a new insight for early medical interventions in patients with articular osteochondral injury.