Importance of CD8 Tex cell-associated gene signatures in the prognosis and immunology of osteosarcoma.

As a highly aggressive bone malignancy, osteosarcoma poses a significant therapeutic challenge, especially in the setting of metastasis or recurrence. This study aimed to investigate the potential of CD8-Tex cell-associated genes as prognostic biomarkers to reveal the immunogenomic profile of osteosarcoma and guide therapeutic decisions. mRNA expression data and clinical details of osteosarcoma patients were obtained from the TCGA database (TARGET-OS dataset). The GSE21257 dataset (from the GEO database) was used as an external validation set to provide additional information on osteosarcoma specimens. 84 samples from the TARGET-OS dataset were used as the training set, and 53 samples from the GSE21257 dataset served as the external validation cohort. Univariate Cox regression analysis was utilized to identify CD8 Tex cell genes associated with prognosis. The LASSO algorithm was performed for 1000 iterations to select the best subset to form the CD8 Tex cell gene signature (TRS). Final genes were identified using the multivariate Cox regression model of the LASSO algorithm. Risk scores were calculated to categorize patients into high- and low-risk groups, and clinical differences were explored by Kaplan-Meier survival analysis to assess model performance. Prediction maps were constructed to estimate 1-, 3-, and 5 year survival rates for osteosarcoma patients, including risk scores for CD8 Texcell gene markers and clinicopathologic factors. The ssGSEA algorithm was used to assess the differences in immune function between TRS-defined high- and low-risk groups. TME and immune cell infiltration were further assessed using the ESTIMATE and CIBERSORT algorithms. To explore the relationship between immune checkpoint gene expression levels and the two risk-defined groups. A CD8 Tex cell-associated gene signature was extracted from the TISCH database and prognostic markers including two genes were developed. The high-risk group showed lower survival, and model performance was validated by ROC curves and C-index. Predictive plots were constructed to demonstrate survival estimates, combining CD8 Tex cell gene markers and clinical factors. This study provides valuable insights into the molecular and immune characteristics of osteosarcoma and offers potential avenues for advances in therapeutic approaches.

Novel Sophoridine Derivatives as Potential Larvicidal Agents against Aedes albopictus: Synthesis, Biological Evaluation, Acetylcholinesterase Inhibition, and Morphological Study.

Two series of novel sophoridine derivatives were designed, synthesized, and evaluated for their anti-mosquito activity. SOP-2g, SOP-2q, and SOP-2r exhibited potential larvicidal activity against Aedes albopictus larva with LC50 values of 330.98, 430.53, and 411.09 ppm, respectively. Analysis of structure-activity relationships indicated that the oxime ester group was beneficial for improving the larvicidal biological activity, whereas the long-chain aliphatic group and fused-ring group were introduced. Furthermore, the larvicidal mechanism was also investigated based on the inhibition assay of acetylcholinesterase (AChE) and the morphological observation of dead larva treated with derivatives. Results indicated that the AChE inhibitory activity of the preferred three derivatives were 63.16%, 46.67%, and 35.11%, respectively, at 250 ppm concentration. Additionally, morphological evidence demonstrated that SOP-2q and SOP-2r induced changes in the larva's intestinal cavity, caudal gill, and tail, thereby displaying larvicidal action against Ae. albopictus together with AChE inhibition. Therefore, this study implied that sophoridine and its novel derivatives could be used to control the population of mosquito larva, which may also be effective alkaloids to reduce the mosquito population density.

Synthesis and Biological Evaluation of Novel Fusidic Acid Derivatives as Two-in-One Agent with Potent Antibacterial and Anti-Inflammatory Activity.

Fusidic acid (FA), a narrow-spectrum antibiotics, is highly sensitive to various Gram-positive cocci associated with skin infections. It has outstanding antibacterial effects against certain Gram-positive bacteria whilst no cross-resistance with other antibiotics. Two series of FA derivatives were synthesized and their antibacterial activities were tested. A new aromatic side-chain analog, FA-15 exhibited good antibacterial activity with MIC values in the range of 0.781-1.563 µM against three strains of Staphylococcus spp. Furthermore, through the assessment by the kinetic assay, similar characteristics of bacteriostasis by FA and its aromatic derivatives were observed. In addition, anti-inflammatory activities of FA and its aromatic derivatives were evaluated by using a 12-O-tetradecanoylphorbol-13-acetate (TPA) induced mouse ear edema model. The results also indicated that FA and its aromatic derivatives effectively reduced TPA-induced ear edema in a dose-dependent manner. Following, multiform computerized simulation, including homology modeling, molecular docking, molecular dynamic simulation and QSAR was conducted to clarify the mechanism and regularity of activities. Overall, the present work gave vital clues about structural modifications and has profound significance in deeply scouting for bioactive potentials of FA and its derivatives.

Nonviral mcDNA-mediated bispecific CAR T cells kill tumor cells in an experimental mouse model of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide and the adoptive immunotherapy of which is worth studying. CD133, a kind of cancer stem cell (CSC) antigen, together with glypican-3 (GPC3) has been proved to be highly expressed in HCC cells and both of them are used as targets to generate chimeric antigen receptor (CAR) T cells. But there are limitations like "off-target" toxicity, low transfection efficacy and weak antitumor ability in CAR T cells treatment. METHODS: The peripheral blood was acquired from healthy donors and T cells were separated by density-gradient centrifugation. We used an electroporation system to deliver anti-CD133 and anti-GPC3 single chain Fragment variable (scFv) structures as target genes into the T cells. The cell membrane was opened by the momentary electric current effect, and the target gene was delivered into the cell by non-viral minicircle DNA (mcDNA) vector. The flow cytometry and western blot assays were used to detect whether the two scFv were simultaneously transfected and the transfection efficacy of this bispecific CAR T cell generation method. We respectively detected the in vitro and in vivo tumor-suppression efficacy of CAR T cells through the CCK-8 assays and the HCC xenograft mice models. The CoG133-CAR T cells containing both CD133 and GPC3 antigen recognition sites were the effector cells. CD133-CAR T cells and GPC3-CAR T cells were defined as single-targeted control groups, normal T and mock T cells were defined as blank control groups. RESULTS: The mcDNA vector accommodated two target gene structures successfully transfected to generate bispecific CAR T cells. The detection methods on gene level and protein level confirmed that CoG133-CAR T cells had considerable transfection efficiency and exhibited both antigen-binding capacity of CD133 and GPC3. Compared to single-targeted CAR T cells or control T cells, CoG133-CAR T cells performed enhanced eliminated efficacy against CD133 and GPC3 double-positive HCC cell line in vitro and HCC xenograft mice in vivo. Hematoxylin and eosin (H&E) staining indicated no fatal "off-target" combination existed on CoG133-CAR T cells and major organs. CONCLUSION: Our study suggests that it is with higher efficiency and more safety to prepare bispecific CAR T cells through non-viral mcDNA vectors. CoG133-CAR T cells have enhanced tumor-suppression capacity through dual antigen recognition and internal activation. It provides an innovative strategy for CAR T therapy of HCC, even solid tumors.

Arabidopsis SKU5 Similar 11 and 12 play crucial roles in pollen tube integrity, growth and guidance.

Pollen tube integrity, growth and guidance are crucial factors in plant sexual reproduction. Members of the plant Skewed5 (SKU5) Similar (SKS) family show strong similarity to multicopper oxidases (MCOs), but they lack conserved histidines in MCO active sites. The functions of most SKS family members are unknown. Here, we show that Arabidopsis pollen-expressed SKS11 and SKS12 play important roles in pollen tube integrity, growth and guidance. The sks11sks12 mutant exhibited significantly reduced male fertility. Most of the pollen from sks11sks12 plants burst when germinated, and the pollen tubes grew slowly and exhibited defective growth along the funiculus and micropyle. SKS11-GFP and SKS12-mCherry were detected at the cell wall in pollen tubes. The contents of several cell wall polysaccharides and arabinogalactans were decreased in the pollen tube cell walls of sks11sks12 plants. Staining with a reactive oxygen species (ROS)-sensitive dye and use of the H2 O2 sensor HyPer revealed that the ROS content in the pollen tubes of sks11sks12 plants was remarkably reduced. SKS11444His-Ala , in which the last conserved histidine was mutated, could restore the mutant phenotypes of sks11sks12. Thus, SKS11/12 are required for pollen tube integrity, growth and guidance possibly by regulating the ROS level and cell wall polysaccharide deposition or remodeling in pollen tubes.

Preparation and Corrosion Performance of PPy/Silane Film on AZ31 Magnesium Alloy via One-Step Cyclic Voltammetry.

PPy/silane composite film on a magnesium alloy surface was prepared by one-step cycle voltammetry. The mixed solution of methanol and water was used as the hydrolysis solvent of a γ-(2,3-glycidoxypropyl) trimethoxysilane coupling agent (KH-560). The surface morphology of the PPy/silane film, the electro-polymerization progress of KH-560 and PPy, the influence of the silane coupling agent and the corrosion behavior of the coated AZ31 Mg alloy were all investigated. The results indicated that the PPy/silane film on AZ31 Mg alloy via one-step cyclic voltammetry could provide better corrosion protection for an Mg alloy when the volume fraction of KH-560 in the hydrolysis solution was 15% and the time span of hydrolysis was 24 h with the 5.935 × 10-10 A cm-2 corrosion current density.

Band-pass filter-assisted ratiometric fluorescent nanoprobe composed of N-(2-aminoethyl-1,8-naphthalimide)-functionalized gold nanoclusters for the determination of alkaline phosphatase using digital image analysis.

A smartphone-based dual-wavelength digital imaging platform containing red (539-695 nm) and blue (389-511 nm) band-pass filters was developed for point-of-care (POC) testing of alkaline phosphatase (ALP) activity. The platform was based on dual-emitting fluorescent nanohybrids (AuNC@NAN), the ratiometric probe, which had a fluorescence "on-off-on-off" response. The probe comprised red-emitting gold nanoclusters (AuNCs) acting as the signal report units and blue-emitting N-(2-aminoethyl-1,8-naphthalimide) (NAN) acting as an internal reference. The different responses of the ratiometric probes resulted in a continuous color-multiplexing change from pink-red to dark-purple upon exposure to ALP. The dual-wavelength digital imaging platform was employed to acquire images of AuNC or NAN fluorescence signals without the influence of background light. Unlike the classical one-time digital imaging mode, the accurate red (R) and blue (B) channel values of the generated images can help to directly judge or eliminate the disturbance from unavoidable interfering factors. The R/B values were successfully employed for determining the ALP activity at a range 2.0 to 35.0 mU·mL-1 with the detection limit of 1.04 mU·mL-1. Such sensing imaging platform is also successful in determining ALP activity in human serum with 94.9-105% recoveries and relative standard deviation in the range 4.2-5.6%. A novel dual-wavelength smartphone-based digital imaging platform was proposed for simultaneous readout of the reporting and internal reference signals from dual-emitting ratiometric fluorescence probes, which allowed us to the accurate, reliable, and highly sensitive assay of ALP activity in complex samples.

Potential of natural products as radioprotectors and radiosensitizers: opportunities and challenges.

Natural products can be used as natural radiosensitizers and radioprotectors, showing promising effects in cancer treatments in combination with radiotherapy, while reducing ionizing radiation (IR) damage to normal cells/tissues. The different effects of natural products on irradiated normal and tumor cells/tissues have attracted more and more researchers' interest. Nonetheless, the clinical applications of natural products in radiotherapy are few, which may be related to their low bioavailability in the human body. Here, we displayed the radiation protection and radiation sensitization of major natural products, highlighted the related molecular mechanisms of these bioactive substances combined with radiotherapy to treat cancer, and critically reviewed their deficiency and improved measures. Lastly, several clinical trials were presented to verify the clinical application of natural products as radiosensitizers and radioprotectors. Further clinical evaluation is still needed. This review provides a reference for the utilization of natural products as radiosensitizers and radioprotectors.

Synthesis and biological evaluation of pentacyclic triterpenoid derivatives as potential novel antibacterial agents.

A series of ursolic acid (UA), oleanolic acid (OA) and 18ß-glycyrrhetinic acid (GA) derivatives were synthesized by introducing a range of substituted aromatic side-chains at the C-2 position after the hydroxyl group at C-3 position was oxidized. Their antibacterial activities were evaluated in vitro against a panel of four Staphylococcus spp. The results revealed that the introduction of aromatic side-chains at the C-2 position of GA led to the discovery of potent triterpenoid derivatives for inhibition of both drug sensitive and resistant S. aureus, while the other two series derivatives of UA and OA showed no significant antibacterial activity even at high concentrations. In particular, GA derivative 33 showed good potency against all four Staphylococcus spp. (MIC = 1.25-5 µmol/L) with acceptable pharmacokinetics properties and low cytotoxicity in vitro. Molecular docking was also performed using S. aureus DNA gyrase to rationalize the observed antibacterial activity. This series of GA derivatives has strong potential for the development of a new type of triterpenoid antibacterial agent.

Oleanolic acid indole derivatives as novel α-glucosidase inhibitors: Synthesis, biological evaluation, and mechanistic analysis.

Research efforts have been directed to the development of oleanolic acid (OA) based α-glucosidase inhibitors and various OA derivatives showed improved anti-α-glucosidase activity. However, the inhibitory effects of indole infused OA derivatives on α-glucosidase is unknown. Herein, we synthesized a series of indole-OA (2a-2o) and -OA methyl ester (3a-3 l) derivatives with various electron withdrawing groups inducted to indole benzene ring and evaluated their anti-α-glucosidase activity. Indole OA derivatives (2a-2o) exhibited superior α-glucosidase inhibitory effects as compared to OA methyl ester derivatives (3a-3l) and OA (with IC50 values of 4.02 µM-5.30 µM v.s. over 10 µM and 5.52 µM, respectively). In addition, mechanistic studies using biochemical (kinetic assay), biophysical (circular dichroism), and computational (docking) methods revealed that OA-indole derivatives (2a and 2f) are mixed type of α-glucosidase inhibitors and their inhibitory effects were attributed to their capacity of forming the ligand-enzyme complex with α-glucosidase enzyme. Findings from this study support that OA indole derivatives are promising α-glucosidase inhibitors as a potential management of diabetes mellitus.

EGCG enhances cancer cells sensitivity under 60Coγ radiation based on miR-34a/Sirt1/p53.

Ionizing radiation (IR) resistance and toxicity to normal cells are the main problems in radiotherapy for cancer. In this study, we demonstrated that epigallocatechin gallate (EGCG) could inhibit effectively IR-induced damage to mouse normal hepatic cells AML-12, and improve dramatically the radiosensitivity of mouse hepatoma cells H22 to 60Coγ. In addition, the different effects of EGCG and underlying molecular mechanisms based on microRNA-34a (miR-34a) and apoptosis-related proteins were investigated by cells viability analysis, quantitative realtime PCR (qRT-PCR), Western blot and cells transfection. The results indicated EGCG played the key role of radiosensitization on H22 cells by activating the miR-34a/Sirt1/p53 signaling pathway. Besides, EGCG could down-regulate the expression of anti-apoptotic protein Bcl-2, and up-regulate the expression of pro-apoptotic proteins Bax and Caspase-3 in H22 cells. Interestingly, EGCG showed contrary results on AML-12 cells. Therefore, radiation protection and radiosensitization of EGCG were associated with apoptosis regulated by miR-34a/Sirt1/p53 signaling pathway.

Potential applications of polyphenols on main ncRNAs regulations as novel therapeutic strategy for cancer.

In the recent years, plant polyphenols have gained significant attention in oncotherapy. Accumulating evidence indicates that polyphenols have potential antitumor properties for multiple types of cancer. But their regulatory mechanisms are still elusive. Noncoding RNAs (ncRNAs) were identified involving in regulating tumorigenesis and tumor progression. Recent evidence has suggested that a number of ncRNAs, including main small ncRNAs (microRNA, miRNA) and long ncRNAs (lncRNAs), play crucial roles concerning the anticancer effects of polyphenols. Indeed, targeting the miRNAs or lncRNAs by polyphenols will be a novel and promising strategy in anticancer chemotherapy. Herein, we displayed the effects of plant polyphenols in different cancers, highlighted the double role of main ncRNAs as oncogenes or tumor suppressor genes involved in different cancer developments, and critically reviewed the potential applications of polyphenols on main ncRNAs regulations involved in oncogenic and tumor suppressor ncRNAs, which implied that polyphenols regulating ncRNAs to exert antitumor effects may be a new strategy for tumor treatment.

Short communication: Global transcriptome analysis of Lactococcus lactis ssp. lactis in response to gradient freezing.

Lactic acid bacteria are often preserved as starter cultures by freezing to extend shelf stability as well as maintain cell viability and acidification activity. Previous studies showed that the endocyte extracted from gradient-freezing pretreated cells could act as lyoprotectant in the lyophilization process of Lactococcus lactis ssp. lactis. In this study, the molecular mechanisms of L. lactis in response to gradient freezing exposure are described using high-throughput sequencing. Nineteen of 56 genes were upregulated after gradient freezing, whereas 37 genes were downregulated. Further validation results of quantitative real-time PCR experiments were consistent with the RNA sequencing. Gene Ontology (http://www.geneontology.org/) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG; https://www.genome.jp/kegg/) pathway were used to analyze the differentially expressed genes. Several pathways, such as glutathione metabolism, ATP-binding cassette transport, metabolism of cell wall and cell membrane components, and stress response-related pathways, were affected by gradient freezing. Six genes relevant to freezing stress response were selected for quantitative real-time PCR, including 3 upregulated genes (hisK, eutD, dukA) and 3 downregulated genes (als, yedF, pepN). The Gene Ontology enrichment and KEGG pathway analyses showed these genes may influence stress response-related pathways, improving the survival of the L. lactis under freezing stress. The identification of these genes deepened an understanding about their response under freezing stress, helping us find potential genes or pathways related to gradient freezing for further research on lyoprotectants.

Pollen-Expressed Leucine-Rich Repeat Extensins Are Essential for Pollen Germination and Growth.

During pollen tube growth, the walls of the tube provide the mechanical strength resisting turgor pressure to protect two sperm cells. Cell wall proteins may play an important role in this process. Pollen tube cell wall proteins known as leucine-rich repeat extensins (LRXs) harbor a leucine-rich repeat domain and an extensin domain. In this study, the functions of four pollen-expressed LRXs, LRX8, LRX9, LRX10, and LRX11 (LRX8-11), were characterized in Arabidopsis (Arabidopsis thaliana). LRX8-11 displayed a consistent expression pattern in mature pollen grains and pollen tubes. In a phenotypic analysis of four single mutants, six double mutants, four triple mutants, and a quadruple mutant, the triple and quadruple mutant plants displayed markedly reduced seed set and decreased male transmission efficiency accompanied by compromised pollen germination and pollen tube growth. GFP-fused LRX8, LRX10, and LRX11 were found to be localized to pollen tube cell walls. An immunohistochemical analysis of pollen tube cell wall polysaccharides showed an increase in the amount of rhamnogalacturonan I in the subapical walls of pollen tubes of the lrx9 lrx10 lrx11 and lrx8 lrx9 lrx11 mutants and a decrease in the content of fucosylated xyloglucans in lrx8 lrx9 lrx11 compared with wild-type plants. Moreover, the callose content in the apical walls of pollen tubes increased in the lrx8 lrx9 lrx11 mutant. In conclusion, we propose that LRX8-11 function synergistically to maintain pollen tube cell wall integrity; thus, they play critical roles in pollen germination and pollen tube growth.

Roles of coinhibitory molecules B7-H3 and B7-H4 in esophageal squamous cell carcinoma.

The coinhibitory molecules, B7-H3 and B7-H4, have shown negative regulation in T cell activation and tumor-associated macrophage (TAM) polarization in tumor-specific immunity. Here, we investigated the expression of B7-H3 and B7-H4 in human and murine esophageal squamous cell carcinoma (ESCC) tissues to define their clinical significance and mechanism in a tumor microenvironment. In the present study, B7-H3 and B7-H4 were expressed in 90.6 and 92.7 % samples, respectively. High B7-H3 and B7-H4 expression was associated with advanced TNM stage and lymph node metastasis (p < 0.05, respectively). Patients with both B7-H3 and B7-H4 high-expressed tumors had the poorest prognosis (26.7 months), whereas those with both low-expressed tumors had the best survival (56.7 months). B7-H3 and B7-H4 expression were inclined to be positively related to the infiltration intensity of Treg cells and TAMs (p < 0.05, respectively), and B7-H3 expression is negatively associated with the intensity of CD8(+) T cells (p < 0.05). In 4-nitroquinoline 1-oxide (4-NQO)-induced murine models, high B7-H3 expression could only be detected at carcinoma stage, but abnormal B7-H4 expression appeared a little earlier at dysplasia stage. In vitro studies revealed that knockdown of B7-H3 on tumor cells suppressed ESCC cell migration and invasion, while knockdown of B7-H4 could inhibit ESCC cell growth. Overall, B7-H3 and B7-H4 are involved in ESCC progression and development and their coexpression could be valuable prognostic indicators. Interference of these negative regulatory molecules might be a new strategy for treating ESCC.

B7-H3 is overexpressed in patients suffering osteosarcoma and associated with tumor aggressiveness and metastasis.

B7-H3 is a member of the B7-family of co-stimulatory molecules, which has been shown to be broadly expressed in various tumor tissues, and which plays an important role in adaptive immune responses. The role of B7-H3 in osteosarcoma, however, remains unknown. In this study we used immunohistochemistry to analyze B7-H3 expression in 61 primary osteosarcoma tissues with case-matched adjacent normal tissues, and 37 osteochondroma and 20 bone fibrous dysplasia tissues. B7-H3 expression was expressed in 91.8% (56/61) of the osteosarcoma lesions, and the intensity of B7-H3 expression in osteosarcoma was significantly increased compared with adjacent normal tissues, osteochondroma and bone fibrous dysplasia tissues (p<0.001). Patients with high tumor B7-H3 levels had a significantly shorter survival time and recurrence time than patients with low tumor B7-H3 levels (p<0.001). Moreover, tumor B7-H3 expression inversely correlated with the number of tumor-infiltrating CD8(+) T cells (p<0.05). In vitro, increasing expression of B7-H3 promotes osteosarcoma cell invasion, at least in part by upregulating matrix metalloproteinase-2 (MMP-2). In conclusion, our study provides the first evidence of B7-H3 expression in osteosarcoma cells as a potential mechanism controlling tumor immunity and invasive malignancy, and which is correlated with patients' survival and metastasis.