Targeting the PI3K/AKT/mTOR pathway offer a promising therapeutic strategy for cholangiocarcinoma patients with high doublecortin-like kinase 1 expression.

BACKGROUND: Cholangiocarcinoma (CCA), characterized by high heterogeneity and extreme malignancy, has a poor prognosis. Doublecortin-like kinase 1 (DCLK1) promotes a variety of malignant cancers in their progression. Targeting DCLK1 or its associated regulatory pathways can prevent the generation and deterioration of several malignancies. However, the role of DCLK1 in CCA progression and its molecular mechanisms remain unknown. Therefore, we aimed to investigate whether and how DCLK1 contributes to CCA progression. METHODS: The expression of DCLK1 in CCA patients was detected using Immunohistochemistry (IHC). We established DCLK1 knockout and DCLK1 overexpression cell lines for Colony Formation Assay and Transwell experiments to explore the tumor-promoting role of DCLK1. RT-PCR, Western blot and multiple fluorescent staining were used to assess the association between DCLK1 and epithelial-mesenchymal transition (EMT) markers. RNA sequencing and bioinformatics analysis were performed to identify the underlying mechanisms by which DCLK1 regulates CCA progression and the EMT program. RESULTS: DCLK1 was overexpressed in CCA tissues and was associated with poor prognosis. DCLK1 overexpression facilitated CCA cell invasion, migration, and proliferation, whereas DCLK1 knockdown reversed the malignant tendencies of CCA cells, which had been confirmed both in vivo and in vitro. Furthermore, we demonstrated that DCLK1 was substantially linked to the advancement of the EMT program, which included the overexpression of mesenchymal markers and the downregulation of epithelial markers. For the underlying mechanism, we proposed that the PI3K/AKT/mTOR pathway is the key process for the role of DCLK1 in tumor progression and the occurrence of the EMT program. When administered with LY294002, an inhibitor of the PI3K/AKT/mTOR pathway, the tumor's ability to proliferate, migrate, and invade was greatly suppressed, and the EMT process was generally reversed. CONCLUSIONS: DCLK1 facilitates the malignant biological behavior of CCA cells through the PI3K/AKT/mTOR pathway. In individuals with cholangiocarcinoma who express DCLK1 at high levels, inhibitors of the PI3K/AKT/mTOR signaling pathway may be an effective therapeutic approach.

GGT5: a potential immunotherapy response inhibitor in gastric cancer by modulating GSH metabolism and sustaining memory CD8+ T cell infiltration.

PURPOSE: The variable responses to immunotherapy observed in gastric cancer (GC) patients can be attributed to the intricate nature of the tumor microenvironment. Glutathione (GSH) metabolism significantly influences the initiation and progression of gastric cancer. Consequently, targeting GSH metabolism holds promise for improving the effectiveness of Immune checkpoints inhibitors (ICIs). METHODS: We investigated 16 genes related to GSH metabolism, sourced from the MSigDB database, using pan-cancer datasets from TCGA. The most representative prognosis-related gene was identified for further analysis. ScRNA-sequencing analysis was used to explore the tumor heterogeneity of GC, and the results were confirmed by  Multiplex immunohistochemistry (mIHC). RESULTS: Through DEGs, LASSO, univariate and multivariate Cox regression analyses, and survival analysis, we identified GGT5 as the hub gene in GSH metabolism with the potential to promote GC. Combining CIBERSORT, ssGSEA, and scRNA analysis, we constructed the immune architecture of GC. The subpopulations of T cells were isolated, revealing a strong association between GGT5 and memory CD8+ T cells. Furthermore, specimens from 10 GC patients receiving immunotherapy were collected. mIHC was used to assess the expression levels of GGT5 and memory CD8+ T cell markers. Our results established a positive correlation between GGT5 expression, the enrichment of memory CD8+ T cells, and a suboptimal response to immunotherapy. CONCLUSIONS: Our study identifies GGT5, a hub gene in GSH metabolism, as a potential therapeutic target for inhibiting the response to immunotherapy in GC patients. These findings offer new insights into strategies for optimizing immunotherapy of GC.

Association of preoperative elevated lipoprotein (a) with poor survival in patients with biliary tract cancers.

BACKGROUND: Biliary tract cancers have garnered significant attention due to their highly malignant nature. The relationship between abnormal lipid metabolism and tumor occurrence and development is a research hotspot. However, its correlation with biliary tract cancers is unclear. METHODS: We enrolled 78 patients with biliary tract cancers and obtained data on clinical characteristics, pathological findings, and preoperative blood lipid indices, including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and lipoprotein (a) [Lp(a)]. Receiver operating characteristic (ROC) curves were used to determine the optimal predictive cutoff values of lipid indicators among the participants. Independent risk factors were determined using Cox regression, and survival was predicted using the Kaplan-Meier method. Statistical analyses were performed using SPSS software. RESULTS: Univariate Cox regression analysis revealed that the body mass index (BMI), tumor location, surgical margin, N stage, and abnormally increased LDL-C, TG, and Lp(a) levels were significantly associated with poor prognosis of biliary tract cancers (p < 0.05). Multifactor Cox regression demonstrated that only N stage (HR = 3.393, p < 0.001) and abnormally increased Lp(a) levels (HR = 2.814, p = 0.004) were significantly associated with shorter survival. N stage and Lp(a) were identified as independent prognostic risk factors for patients with biliary tract cancers. CONCLUSION: This study presents Lp(a) as a novel biochemical marker that can guide clinical treatment strategies for patients with biliary tract cancers. More effective treatment options and intensive postoperative testing should be considered to prolong the survival of these patients with preoperative abnormal lipid metabolism.

Identification of genetic modifiers enhancing B7-H3-targeting CAR T cell therapy against glioblastoma through large-scale CRISPRi screening.

BACKGROUND: Glioblastoma multiforme (GBM) is a highly aggressive brain tumor with a poor prognosis. Current treatment options are limited and often ineffective. CAR T cell therapy has shown success in treating hematologic malignancies, and there is growing interest in its potential application in solid tumors, including GBM. However, current CAR T therapy lacks clinical efficacy against GBM due to tumor-related resistance mechanisms and CAR T cell deficiencies. Therefore, there is a need to improve CAR T cell therapy efficacy in GBM. METHODS: We conducted large-scale CRISPR interference (CRISPRi) screens in GBM cell line U87 MG cells co-cultured with B7-H3 targeting CAR T cells to identify genetic modifiers that can enhance CAR T cell-mediated tumor killing. Flow cytometry-based tumor killing assay and CAR T cell activation assay were performed to validate screening hits. Bioinformatic analyses on bulk and single-cell RNA sequencing data and the TCGA database were employed to elucidate the mechanism underlying enhanced CAR T efficacy upon knocking down the selected screening hits in U87 MG cells. RESULTS: We established B7-H3 as a targetable antigen for CAR T therapy in GBM. Through large-scale CRISPRi screening, we discovered genetic modifiers in GBM cells, including ARPC4, PI4KA, ATP6V1A, UBA1, and NDUFV1, that regulated the efficacy of CAR T cell-mediated tumor killing. Furthermore, we discovered that TNFSF15 was upregulated in both ARPC4 and NDUFV1 knockdown GBM cells and revealed an immunostimulatory role of TNFSF15 in modulating tumor-CAR T interaction to enhance CAR T cell efficacy. CONCLUSIONS: Our study highlights the power of CRISPR-based genetic screening in investigating tumor-CAR T interaction and identifies potential druggable targets in tumor cells that confer resistance to CAR T cell killing. Furthermore, we devised targeted strategies that synergize with CAR T therapy against GBM. These findings shed light on the development of novel combinatorial strategies for effective immunotherapy of GBM and other solid tumors.

Effect comparisons of different conditioners and microbial agents on the degradation of estrogens during dairy manure composting.

To investigate the degradation efficiency of conditioners and commercial microbial agents on estrogens (E1, 17α-E2, 17ß-E2, E3, EE2, and DES) in the composting process of dairy manure, seven different treatments (RHB-BF, OSP-BF, SD-BF, MR-BF, MR-FS, MR-EM, and MR-CK) under forced ventilation conditions were composted and monitored regularly for 30 days. The results indicated that the removal rates of estrogens in seven treatments ranged from 95.35% to 99.63%, meanwhile the degradation effect of the composting process on 17ß-Estradiol equivalent (EEQ) was evaluated, and the removal rate of ΣEEQ ranged from 96.42% to 99.72%. With the combined addition of rice husk biochar (RHB) or oyster shell powder (OSP) and bio-bacterial fertilizer starter cultures (BF), namely RHB-BF and OSP-BF obviously promoted the rapid degradation of estrogens. 17ß-E2 was completely degraded on the fifth day of composting in OSP-BF. Microbial agents have some promotional effect and enhances the microbial degradation of synthetic estrogen (EE2, DES). According to the results of RDA, pH and EC were the main environmental factors affecting on the composition and succession of estrogen-related degrading bacteria in composting system. As predominant estrogens-degrading genera, Acinetobacter, Bacillus, and Pseudomonas effected obviously on the change of estrogens contents. The research results provide a practical reference for effective composting of dairy manure to enhancing estrogens removal and decreasing ecological risk.

Preventive Treatment with PD-1 Antibody Increases Tissue-resident Memory T Cells Infiltration and Delays Esophageal Carcinogenesis.

Numerous studies and clinical trials have shown that immune checkpoint inhibitors can effectively prevent tumor growth and metastasis in esophageal squamous cell carcinoma (ESCC) patients. In this study, we aimed to evaluate the anti-tumor effects of PD-1 antibody preventive treatment in patients with early stages ESCC as well as patients with high-grade intraepithelial neoplasia (HGIN). We first established an ESCC model using C57BL/6J mice treated with the chemical carcinogen 4- NQO and observed esophageal lesions at different time points. Second, we compared the antitumor efficacy of PD-1 antibody treatment in mice at the ESCC stage and PD-1 antibody preventive treatment in mice at the HGIN stage. The results showed that PD-1 antibody preventive treatment effectively impeded the progression of 4NQO-induced esophageal tumorigenesis. IHC analysis was performed to observe the infiltration of immune cells into the tumor microenvironment. It has been shown that active tissue-resident memory T cells can be induced and resided into the tumor microenvironment for a long period after treatment with PD-1 antibody. Reexposure to the oncogenic environment colonized by CD8+TRM cells can still exert antitumor effects. These results provide new strategies for the treatment of patients with early stage ESCC and HGIN. PREVENTION RELEVANCE: Immune checkpoint inhibitors have shown promising results in multiple tumor species. However, there is currently no clinical application to evaluate their therapeutic value in cancer preventive treatment. Prophylactic use of immune checkpoint inhibitors in the early stages of ESCC may provide long-term benefits to patients.

[Progress in preparation and application of sodium alginate microspheres].

Sodium alginate (SA) is a kind of natural polymer material extracted from kelp, which has excellent biocompatibility, non-toxicity, biodegradability and abundant storage capacity. The formation condition of sodium alginate gel is mild, effectively avoiding the inactivation of active substances. After a variety of preparation methods, sodium alginate microspheres are widely used in the fields of biomaterials and tissue engineering. This paper reviewed the common methods of preparing alginate microspheres, including extrusion, emulsification, electrostatic spraying, spray drying and coaxial airflow, and discussed their applications in biomedical fields such as bone repair, hemostasis and drug delivery.

Construction of a novel immune response prediction signature to predict the efficacy of immune checkpoint inhibitors in clear cell renal cell carcinoma patients.

Background: Immune checkpoint inhibitor (ICI) treatment has enhanced survival outcomes in clear cell renal cell carcinoma (ccRCC) patients. Nevertheless, the effectiveness of immunotherapy in ccRCC patients is restricted and we intended to develop and characterize an immune response prediction signature (IRPS) to forecast the efficacy of immunotherapy. Methods: RNA-seq expression profile and clinicopathologic characteristics of 539 kidney cancer and 72 patients with normal specimens, were downloaded from the Cancer Genome Atlas (TCGA) database, while the Gene Expression Omnibus (GEO) database was used as the validation set, which included 24 ccRCC samples. Utilization of the TCGA data and immune genes databases (ImmPort and the InnateDB), we explored through Weighted Gene Co-expression Network Analysis (WGCNA), along with Least Absolute Shrinkage and Selection Operator method (LASSO), and constructed an IRPS for kidney cancer patients. GSEA and CIBERSORT were performed to declare the molecular and immunologic mechanism underlying the predictive value of IRPS. The Human Protein Atlas (HPA) was deployed to verify the protein expressions of IRPS genes. Tumor immune dysfunction and exclusion (TIDE) score and immunophenoscore (IPS) were computed to determine the risk of immune escape and value the discrimination of IRPS. A ccRCC cohort with anti-PD-1 therapy was obtained as an external validation data set to verify the predictive value of IRPS. Results: We constructed a 10 gene signature related to the prognosis and immune response of ccRCC patients. Considering the IRPS risk score, patients were split into high and low risk groups. Patients with high risk in the TCGA cohort tended towards advanced tumor stage and grade with poor prognosis (p < 0.001), which was validated in GEO database (p = 0.004). High-risk group tumors were related with lower PD-L1 expression, higher TMB, higher MSIsensor score, lower IPS, higher TIDE score, and enriched Treg cells, which might be the potential mechanism of immune dysfunction and exclusion. Patients in the IRPS low risk group had better PFS (HR:0.73; 95% CI: 0.54-1.0; P = 0.047). Conclusion: A novel biomarker of IRPS was constructed to predict the benefit of immunotherapy, which might lead to more individualized prognoses and tailored therapy for kidney cancer patients.

Injectable antibacterial Ag-HA/ GelMA hydrogel for bone tissue engineering.

Background: Fracture or bone defect caused by accidental trauma or disease is a growing medical problem that threats to human health.Currently, most orthopedic implant materials must be removed via follow-up surgery, which requires a lengthy recovery period and may result in bacterial infection. Building bone tissue engineering scaffolds with hydrogel as a an efficient therapeutic strategy has outstanding bionic efficiency.By combining some bionic inorganic particles and hydrogels to imitate the organic-inorganic characteristics of natural bone extracellular matrix, developing injectable multifunctional hydrogels with bone tissue repair effects and also displaying excellent antibacterial activity possesses attractive advantages in the field of minimally invasive therapy in clinical. Methods: In the present work, a multifunctional injectable hydrogel formed by photocrosslinking was developed by introducing hydroxyapatite (HA) microspheres to Gelatin Methacryloyl (GelMA) hydrogel. Results: The composite hydrogels exhibited good adhesion and bending resistance properties due to the existence of HA. In addition, when the concentration of GelMA is 10% and the concentration of HA microspheres is 3%, HA/GelMA hydrogel system displayed increased microstructure stability, lower swelling rate, increased viscosity, and improved mechanical properties. Furthermore, the Ag-HA/GelMA demonstrated good antibacterial activity against Staphylococcus aureus and Escherichia coli, which could signifificantly lower the risk of bacterial infection following implantation. According to cell experiment, the Ag-HA/GelMA hydrogel is capable of cytocompatibility and has low toxicity to MC3T3 cell. Conclusion: Therefore, the new photothermal injectable antibacterial hydrogel materials proposed in this study will provide a promising clinical bone repair strategy and is expected to as a minimally invasive treatment biomaterial in bone repair fields.

The role of individual exopolysaccharides in antibiotic tolerance of Pseudomonas aeruginosa aggregates.

The bacterium Pseudomonas aeruginosa is involved in chronic infections of cystic fibrosis lungs and chronic wounds. In these infections the bacteria are present as aggregates suspended in host secretions. During the course of the infections there is a selection for mutants that overproduce exopolysaccharides, suggesting that the exopolysaccharides play a role in the persistence and antibiotic tolerance of the aggregated bacteria. Here, we investigated the role of individual P. aeruginosa exopolysaccharides in aggregate-associated antibiotic tolerance. We employed an aggregate-based antibiotic tolerance assay on a set of P. aeruginosa strains that were genetically engineered to over-produce a single, none, or all of the three exopolysaccharides Pel, Psl, and alginate. The antibiotic tolerance assays were conducted with the clinically relevant antibiotics tobramycin, ciprofloxacin and meropenem. Our study suggests that alginate plays a role in the tolerance of P. aeruginosa aggregates toward tobramycin and meropenem, but not ciprofloxacin. However, contrary to previous studies we did not observe a role for Psl or Pel in the tolerance of P. aeruginosa aggregates toward tobramycin, ciprofloxacin, and meropenem.

Multifunctional Fe3O4-PEI@HA nanoparticles in the ferroptosis treatment of hepatocellular carcinoma through modulating reactive oxygen species.

Ferroptosis is a novel form of regulated cell death induced by iron-dependent lipid peroxidation imbalance. It has emerged as a promising antitumor therapeutic strategy in recent years. In this work, we successfully synthesized a complex magnetic nanocube Fe3O4 modified with PEI and HA by the thermal decomposition method. While loading a ferroptosis inducer RSL3 inhibited cancer cells through the ferroptosis signal transduction pathway. The drug delivery system could actively target tumor cells through an external magnetic field and HA-CD44 binding. Zeta potential analysis showed that Fe3O4-PEI@HA-RSL3 nanoparticles were more stable and uniformly dispersed in tumor acidic environment. Moreover, cellular experiments demonstrated that Fe3O4-PEI@HA-RSL3 nanoparticles could significantly inhibit the proliferation of hepatoma cells without a cytotoxic effect on normal hepatic cells. In addition, Fe3O4-PEI@HA-RSL3 played a vital role in ferroptosis by accelerating ROS production. The expression of ferroptosis-related genes Lactoferrin, FACL 4, GPX 4 and Ferritin was significantly suppressed with increasing treatment of Fe3O4-PEI@HA-RSL3 nanocubes. Therefore, this ferroptosis nanomaterial has great potential in Hepatocellular carcinoma (HCC) therapy.

Abundant tannic acid modified gelatin/sodium alginate biocomposite hydrogels with high toughness, antifreezing, antioxidant and antibacterial properties.

The acidity of high tannic acid (TA) content solution can destroy the structure of protein, such as gelatin (G). This causes a big challenge to introduce abundant TA into the G-based hydrogels. Here, the G-based hydrogel system with abundant TA as hydrogen bonds provider was constructed by a "protective film" strategy. The protective film around the composite hydrogel was first formed by the chelation of sodium alginate (SA) and Ca2+. Subsequently, abundant TA and Ca2+ were successively introduced into the hydrogel system by immersing method. This strategy effectively protected the structure of the designed hydrogel. After treatment with 0.3 w/v TA and 0.06 w/v Ca2+ solutions, the tensile modulus, elongation at break and toughness of G/SA hydrogel increased about 4-, 2-, and 6-fold, respectively. Besides, G/SA-TA/Ca2+ hydrogels exhibited good water retention, anti-freezing, antioxidant, antibacterial properties and low hemolysis ratio. Cell experiments showed that G/SA-TA/Ca2+ hydrogels possessed good biocompatibility and could promote cell migration. Therefore, G/SA-TA/Ca2+ hydrogels are expected to be used in the field of biomedical engineering. The strategy proposed in this work also provides a new idea for improving the properties of other protein-based hydrogels.

Hyaluronic acid mediated Fe3O4 nanocubes reversing the EMT through targeted cancer stem cell.

Hepatocellular carcinoma (HCC) is one of the deadliest tumors in the world with a high rate of recurrence and metastasis. Therefore, the most pressing issue today is the development of new drugs, diagnostic and therapeutic approaches for effective cancer treatment. Cancer stem cells (CSCs) play a pivotal role in tumor recurrence, tumor resistance, and tumor metastasis, which provides a new perspective on the development of liver cancer. In the study, a high-temperature thermal breakdown approach was used to create composite magnetic nanocubes modified by polyethyleneimine (PEI) and hyaluronic acid (HA). The Fe3O4 nanocubes can recognize HCC stem cells via receptor-ligand binding of HA and CD44 (HA receptor). While loading a small molecule LDN193189 inhibited the expression of stemness-related genes OCT4 and Nanog. More crucially, the Fe3O4 nanocubes significantly suppressed HCC cell proliferation and migration by regulating the expression of epithelial-mesenchymal transition (EMT) process markers E-cadherin, Vimentin, and N-cadherin. Dual targeting using magnetic and receptor-mediated targeting improved the uptake of the drug delivery system. Our findings imply that the medication delivery method might be a potential therapeutic strategy for HCC.

CAR-Macrophages and CAR-T Cells Synergistically Kill Tumor Cells In Vitro.

Chimeric antigen receptor (CAR)-expressing macrophages (CAR-M) have a great potential to improve cancer therapy, as shown from several recent preclinical studies. However, unlike CAR-T cell therapy, which has been widely studied, the efficacy and limitations of CAR-M cells remain to be established. To address this issue, in the present study, we compared three intracellular signaling domains (derived from common γ subunit of Fc receptors (FcRγ), multiple EGF-like-domains protein 10 (Megf10), and the CD19 cytoplasmic domain that recruits the p85 subunit of phosphoinositide-3 kinase (PI3K), respectively) for their ability to promote primary CAR-M functions, and investigated the potential synergistic effect between CAR-M and CAR-T cells in their ability to kill tumor cells. We found that CAR-MFcRγ exerted more potent phagocytic and tumor-killing capacity than CAR-MMegf10 and CAR-MPI3K. CAR-M and CAR-T demonstrated synergistic cytotoxicity against tumor cells in vitro. Mechanistically, the inflammatory factors secreted by CAR-T increased the expression of costimulatory ligands (CD86 and CD80) on CAR-M and augmented the cytotoxicity of CAR-M by inducing macrophage M1 polarization. The upregulated costimulatory ligands may promote the fitness and activation of CAR-T cells in turn, achieving significantly enhanced cytotoxicity. Taken together, our study demonstrated for the first time that CAR-M could synergize with CAR-T cells to kill tumor cells, which provides proof-of-concept for a novel combinational immunotherapy.

Anterior Quadratus Lumborum Block at the Lateral Supra-Arcuate Ligament versus Transmuscular Quadratus Lumborum Block for Analgesia after Elective Cesarean Section: A Randomized Controlled Trial.

Several studies have shown the effectiveness of trans-muscular quadratus lumborum block (TQLB) in analgesia after cesarean delivery. However, the influence of anterior QLB at the lateral supra-arcuate ligament (QLB-LSAL) in this surgery is unclear. This study aimed to compare the analgesic efficacy of bilateral TQLBs with bilateral QLBs-LSAL following cesarean delivery. Ninety-four parturients scheduled for cesarean delivery under spinal anesthesia were enrolled and randomly allocated to undergo either bilateral TQLBs or bilateral QLBs-LSAL with 0.375% of ropivacaine (20 mL each side) following cesarean delivery. Intravenous sufentanil was administered for patient-controlled analgesia (PCA). The primary outcome was postoperative sufentanil consumption during the initial 24 h post-surgery. Secondary endpoints included pain scores, time to the first PCA request, postoperative rescue analgesia, satisfaction scores, and nausea/vomiting events. Sufentanil consumption was significantly reduced in the QLB-LSAL group in the first 24 h compared with the TQLB group after surgery (29.4 ± 5.7 µg vs. 39.4 ± 9.6 µg, p < 0.001). In comparison with TQLB, the time to the first PCA request in the QLB-LSAL group was significantly longer (10.9 ± 4.1 h vs. 6.7 ± 1.8 h, p < 0.001). No differences were observed between two groups regarding pain scores, rescue analgesia after surgery, satisfaction scores, or nausea/vomiting incidence. The significant reduction in opioid consumption in the first 24 h and prolongation in time to first opioid demand in parturients receiving QLB-LSAL compared with TQLB suggest that the QLB-LSAL is a superior choice for multimodal analgesia after cesarean delivery.

Transcription of the Alginate Operon in Pseudomonas aeruginosa Is Regulated by c-di-GMP.

Overproduction of the exopolysaccharide alginate contributes to the pathogenicity and antibiotic tolerance of Pseudomonas aeruginosa in chronic infections. The second messenger, c-di-GMP, is a positive regulator of the production of various biofilm matrix components and is known to regulate alginate synthesis at the posttranslational level in P. aeruginosa. We provide evidence that c-di-GMP also regulates transcription of the alginate operon in P. aeruginosa. Previous work has shown that transcription of the alginate operon is regulated by nine different proteins, AmrZ, AlgP, IHFα, IHFß, CysB, Vfr, AlgR, AlgB, and AlgQ, and we investigated if some of these proteins function as a c-di-GMP effector. We found that deletion of algP, algQ, IHFα, and IHFß had only a marginal effect on the transcription of the alginate operon. Deletion of vfr and cysB led to decreased transcription of the alginate operon, and the dependence of the c-di-GMP level was less pronounced, indicating that Vfr and CysB could be partially required for c-di-GMP-mediated regulation of alginate operon transcription. Our experiments indicated that the AmrZ, AlgR, and AlgB proteins are absolutely required for transcription of the alginate operon. However, differential radial capillary action of ligand assay (DRaCALA) and site-directed mutagenesis indicated that c-di-GMP does not bind to any of the AmrZ, AlgR, and AlgB proteins. IMPORTANCE The proliferation of alginate-overproducing P. aeruginosa variants in the lungs of cystic fibrosis patients often leads to chronic infection. The alginate functions as a biofilm matrix that protects the bacteria against host immune defenses and antibiotic treatment. Knowledge about the regulation of alginate synthesis is important in order to identify drug targets for the development of medicine against chronic P. aeruginosa infections. We provide evidence that c-di-GMP positively regulates transcription of the alginate operon in P. aeruginosa. Moreover, we revisited the role of the known alginate regulators, AmrZ, AlgP, IHFα, IHFß, CysB, Vfr, AlgR, AlgB, and AlgQ, and found that their effect on transcription of the alginate operon is highly varied. Deletion of algP, algQ, IHFα, or IHFß only had a marginal effect on transcription of the alginate operon, whereas deletion of vfr or cysB led to decreased transcription and deletion of amrZ, algR, or algB abrogated transcription.

S1PR2/RhoA/ROCK1 pathway promotes inflammatory bowel disease by inducing intestinal vascular endothelial barrier damage and M1 macrophage polarization.

Vascular and immune dysfunctions are thought to be related to the pathogenesis of inflammatory bowel disease (IBD), but behind this, the exact mechanism of mucosal vascular endothelial barrier dysfunction and macrophage phenotypic transition is not fully understood. Here, we explored the mechanistic role of sphingosine 1-phosphate receptor 2 (S1PR2) and its downstream G protein RhoA/Rho kinase 1 (ROCK1) signaling pathway in the intestinal endothelial barrier damage and M1 macrophage polarization in IBD. We found that the expression of S1PR2 in intestinal mucosal vascular endothelial cells and macrophages of IBD patients and DSS-induced colitis mice as well as vascular endothelial cells and macrophages treated with LPS in vitro was significantly increased. Knocking down or pharmacologically inhibiting S1PR2 significantly downregulated the expression of RhoA and ROCK1 in vascular endothelial cells and macrophages. Furthermore, inhibition of S1PR2 and ROCK1 reversed the impaired vascular barrier function and M1 macrophage polarization in vivo and in vitro, while reducing ER stress in vascular endothelial cells and glycolysis in macrophages. In addition, inhibition of ER stress or glycolysis reversed LPS-induced impairment of vascular endothelial cell barrier function and M1 macrophage polarization. Collectively, our results indicate that the S1PR2/RhoA/ROCK1 signaling pathway may participate in the pathogenesis of IBD by regulating vascular endothelial barrier function and M1 macrophage polarization.

Establishment of a Competing Risk Nomogram in Patients with Pulmonary Sarcomatoid Carcinoma.

Background and aim: Pulmonary sarcomatoid carcinoma (PSC) is a rare subtype of nonsmall cell lung cancer with a poor prognosis. This study aimed to analyze the clinicopathological characteristics and survival outcomes among patients with PSC, lung squamous cell cancer (SCC), and lung adenocarcinoma (LAC), and to construct a competing risk nomogram for patients with PSC. Method: Data of 3 groups of patients diagnosed with PSC, SCC, or LAC from the surveillance, epidemiology, and end results (SEER) database between 1988 and 2015 were retrospectively reviewed. A 1:1 propensity score matching (PSM) analysis was used to balance the baseline data of patients. Independent risk factors associated with survival outcomes were screened by the least absolute shrinkage and selection operator and further determined by univariate and multivariate Cox proportional risk regression analyses. The overall survival (OS) of patients was evaluated by Kaplan-Meier analysis and compared with a log-rank test. The cumulative incidence function was used to estimate the 5-year probabilities of the cancer-specific mortality of PSC. A nomogram was constructed to illustrate the competing risk model to predict the 3- and 5-year OS, and corresponding concordance indexes (C-indexes) and calibration curves were used to assess and validate the competing risk nomogram. Results: A total of 2285 patients with PSC were included in this study. Compared with SCC and LAC patients, the Kaplan-Meier analysis showed that patients with PSC had a worse prognosis, with a median survival of 5 months (95% confidence interval [CI]: 5-6 months) and a 5-year OS rate of 15.3% (95% CI: 13.9%-16.9%). Similar outcomes were demonstrated after 1:1 PSM. Moreover, the competing risk model showed that age, T stage, M stage, tumor size, lymph node ratio (LNR), surgery, and chemotherapy were associated with PSC-specific mortality. The 5-year C-index of the nomogram was 0.718. Calibration curves illustrated that the nomogram was well-validated and had great accuracy. Conclusions: Patients with PSC had a worse survival outcome compared with SCC or LAC patients. Age, T stage, M stage, tumor size, LNR, surgery, and chemotherapy were associated with PSC-specific mortality. The competing risk nomogram displayed excellent discrimination in predicting PSC-specific mortality.

Human Cancer Cell Membrane-Cloaked Fe3O4 Nanocubes for Homologous Targeting Improvement.

Surface modification of nanoparticles with cellular protein components is a new biomimetic modification strategy, which utilizes the inherent affinity between homologous cells to introduce the same surface molecules into nanoparticles to improve the targeting performance. In this study, oleic acid (OA)-coated Fe3O4 nanocubes were prepared by a high-temperature thermal decomposition method and modified by 3, 4-dihydroxyphenylpropionic acid (DHCA); then, HeLa cell membranes were introduced onto the surface of the nanocubes through mixed coextrusion to try to endow them with the targeting function of natural cells. The results show that the prepared Fe3O4 nanocubes have high monodispersity, excellent water solubility, and biocompatibility. Moreover, the Fe3O4 nanocubes encapsulated by cellular protein show an obvious core-shell structure and the specific targeting property to HeLa cells is improved significantly, which is expected to be used in clinical targeted diagnosis and treatment of cancer.