CD151-enriched migrasomes mediate hepatocellular carcinoma invasion by conditioning cancer cells and promoting angiogenesis.

BACKGROUND: The tetraspanin family plays a pivotal role in the genesis of migrasomes, and Tetraspanin CD151 is also implicated in neovascularization within tumorous contexts. Nevertheless, research pertaining to the involvement of CD151 in hepatocellular carcinoma (HCC) neovascularization and its association with migrasomes remains inadequate. METHODS: To investigate the correlation between CD151 and migrasome marker TSPAN4 in liver cancer, we conducted database analysis using clinical data from HCC patients. Expression levels of CD151 were assessed in HCC tissues and correlated with patient survival outcomes. In vitro experiments were performed using HCC cell lines to evaluate the impact of CD151 expression on migrasome formation and cellular invasiveness. Cell lines with altered CD151 expression levels were utilized to study migrasome generation and in vitro invasion capabilities. Additionally, migrasome function was explored through cellular aggregation assays and phagocytosis studies. Subsequent VEGF level analysis and tissue chip experiments further confirmed the role of CD151 in mediating migrasome involvement in angiogenesis and cellular signal transduction. RESULTS: Our study revealed a significant correlation between CD151 expression and migrasome marker TSPAN4 in liver cancer, based on database analysis of clinical samples. High expression levels of CD151 were closely associated with poor survival outcomes in HCC patients. Experimentally, decreased CD151 expression led to reduced migrasome generation and diminished in vitro invasion capabilities, resulting in attenuated in vivo metastatic potential. Migrasomes were demonstrated to facilitate cellular aggregation and phagocytosis, thereby promoting cellular invasiveness. Furthermore, VEGF-enriched migrasomes were implicated in signaling and angiogenesis, accelerating HCC progression. CONCLUSIONS: In summary, our findings support the notion that elevated CD151 expression promotes migrasome formation, and migrasomes play a pivotal role in the invasiveness and angiogenesis of liver cancer cells, thereby facilitating HCC progression. This finding implies that migrasomes generated by elevated CD151 expression may constitute a promising high-priority target for anti-angiogenic therapy in HCC, offering crucial insights for the in-depth exploration of migrasome function and a renewed comprehension of the mechanism underlying liver cancer metastasis.

A Kazal-type serine protease inhibitor mediates innate immunity in wild silkworm Actias selene Hübner.

The prophenoloxidase (PPO) activation system is an important innate immune defense mechanism in arthropods. Actias selene is a rare and important wild silk insect that can spin high-quality cocoon silk, but, other than its morphology, its molecular mechanism is rarely reported. Here, we report the purification and characterization of a novel KSPI gene from A. selene (AsKSPI, which can negatively regulate PPO activation. Its open reading frame (ORF) was 291 bp, encoding 96 amino acids. Real-time quantitative PCR (RT-qPCR) showed that AsKSPI mRNA was significantly expressed in the fat body. Immunostimulatory tests showed that the mRNA levels of AsKSPI in the fat body were up-regulated following injection of Micrococcus luteus, Escherichia coli, Beauveria bassiana, and nuclear polyhedrosis virus (NPV). Enzyme activity experiments showed that the purified recombinant AsKSPI could inhibit the activation of PPO in hemolymph of A. selene, but did not affect phenoloxidase (PO) activity after PPO had been activated. So, AsKSPI could regulate the innate immunity of A. selene through the PPO cascade. These findings will contribute to the understanding of the immune mechanism of wild silkworm and provide a basis for better protection and utilization of special economic insect resources.

Subtilisin-like serine protease gene TghSS42 from Trichoderma ghanense ACCC 30153 was successfully expressed in Escherichia coli and recombinant protease rTghSS42 exhibited antifungal ability to five phytopathogens.

　The subtilisin-like serine protease gene TghSS42 was cloned from biocontrol agent Trichoderma ghanense ACCC 30153. Its coding region is 1302 bp in length, encoding 433 aa with a predicted protein molecular weight of 42.5 kDa and pI of 5.53. The accession number of cDNA sequence of TghSS42 gene is KJ740359. Furthermore, the transcription of the TghSS42 gene was all up-regulated under nine different treatments by RT-qPCR analysis, and the highest transcription level of TghSS42 approached 177.29-fold at 4 h under induction with 1% (w/v) Alternaria alternata cell walls, indicating that TghSS42 could be induced by the plant or phytopathogen. Furthermore, Escherichia coli recombinant strain BL21-TghSS42 was constructed. The recombinant protease rTghSS42, with an expected molecular weight of approximately 68.5 kDa (containing 26.0 kDa GST tag), has been successfully expressed and purified from BL21-TghSS42. The purified protease rTghSS42 activity reached a peak of 18.7 U/mL at 4 h following 1.0 mM IPTG induction. The optimal enzyme reaction temperature was 40℃ and the optimal pH was 7.0. The recombinant protease rTghSS42 exerted broad-spectrum antifungal ability against Rhizoctonia solani, Fusarium oxysporum, A. alternata, Sclerotinia sclerotiorum and Cytospora chrysosperma. The inhibition rate of mycelial growth varied between 21.2% and 50.0%.

Penehyclidine ameliorates acute lung injury by inhibiting Toll-like receptor 2/4 expression and nuclear factor-κB activation.

The aim of the present study was to investigate the effect of penehyclidine (PHC) on endotoxin-induced acute lung injury (ALI), as well as to examine the mechanism underlying this effect. A total of 60 rats were randomly divided into five groups, including the control (saline), LPS and three LPS + PHC groups. ALI was induced in the rats by injection of 8 mg lipopolysaccharide (LPS)/kg body weight. The rats were then treated with or without PHC at 0.3, 1 or 3 mg/kg body weight 1 min following LPS injection. After 6 h, serum levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6 were determined by ELISA. In addition, the mRNA expression levels of toll-like receptor (TLR)2 and TLR4 were examined by reverse transcription-quantitative polymerase chain reaction in the lung tissue samples, and nuclear factor (NF)-κB p65 protein expression levels were examined by western blot analysis. The results demonstrated that lung injury was ameliorated by treatment with PHC (1 and 3 mg/kg body weight) as compared with treatment with LPS alone. Injection of LPS significantly increased the mRNA expression levels of TLR2 and TLR4, as well as the protein expression levels of NF-κB p65 in the lung tissue samples. Serum levels of TNF-α and IL-6 were also upregulated by LPS injection. Treatment of the rats with PHC following LPS injection suppressed the LPS-induced increase in TLR2/4 mRNA and NF-κB p65 protein expression levels. PHC also inhibited the increase in TNF-α and IL-6 serum levels. In addition, PHC reduced LPS-induced ALI and decreased the serum levels of TNF-α and IL-6, possibly by downregulating TLR2/4 mRNA expression and inhibiting NF-κB activity, and consequently alleviating the inflammatory response.

Use of a Heterologous Bridge Coating Antigen for the Immunoassay of 3-Acetyldeoxynivalenol in Barley.

The study describes procedures that were used to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA) for the quantitation of a trichothecene mycotoxin, 3-acetyldeoxynivalenol (3-AcDON), in barley. Polyclonal antibodies were produced in rabbits immunized with a conjugate of 3-AcDON and human serum albumin linked by an ester linkage (hemisuccinate bridge). High anti-3-AcDON titers were obtained after multiple immunizations. However, only a negligible degree of inhibition was obtained with 3-AcDON in a competitive ELISA when the coating conjugate contained the same ester linkage group (hemiglutarate bridge) as the immunogen. The use of a conjugate containing a heterologous ether linkage (O-methylcarboxyl bridge) compared to that of the immunogen yielded an inhibition curve for 3-AcDON that was highly sensitive (IC50 = 0.21 ng/ml) with essentially no interference from the bridging group. This conjugate was synthesized using iodoacetate and 1,1'-carbonyldiimidazole chemistries. The assay showed low cross-reactivity with other trichothecenes including several analogs of deoxynivalenol (DON) with the exception of acetylated DON. The ELISA developed on the basis of this new conjugate was able to detect low concentrations of 3-AcDON (16 ppb) in spiked barley without any cleanup treatments.