Applied Biochemistry and Biotechnology最新文献

Non-small cell lung cancer (NSCLC) remains a leading cause of cancer-related deaths worldwide, necessitating the identification of novel therapeutic targets. Mesoderm posterior bHLH transcription factor 1 (MESP1) has been implicated in various developmental processes, but its role in cancer, particularly NSCLC, is poorly understood. Given the emerging evidence linking MESP1 to cellular processes relevant to cancer biology, investigating its regulatory mechanisms in NSCLC could provide critical insights for developing new therapies. This study employed quantitative real-time PCR (qRT-PCR) to assess the mRNA levels of MESP1, ubiquitin specific peptidase 7 (USP7), and clusters of differentiation 163 (CD163). Western blotting was used to analyze the protein expression of MESP1 and USP7. Cellular proliferation was evaluated through colony-forming assays, while apoptosis was quantified using flow cytometry. Mitochondrial membrane potential was measured by JC-1 staining, and reactive oxygen species (ROS) levels were also analyzed via flow cytometry. Additionally, colorimetric assays were utilized to determine malondialdehyde (MDA), total iron, and Fe²⁺ levels. The in vivo effects of MESP1 silencing on NSCLC progression were examined using a xenograft mouse model. GST-pull down assay, Co-immunoprecipitation (Co-IP) assay, and ubiquitination assay were conducted to explore the interaction between USP7 and MESP1. The expression of both MESP1 and USP7 was found to be upregulated in NSCLC tissues and cells when compared with normal lung tissues and normal human bronchial epithelial cells. Knockdown of MESP1 significantly inhibited NSCLC cell proliferation, induced apoptosis and promoted features associated with ferroptosis. Moreover, MESP1 silencing suppressed M2 macrophage polarization and tumor formation. Mechanistically, USP7 was identified to stabilize MESP1 protein expression through its deubiquitinating activity. Overexpression of MESP1 attenuated the inhibitory effects of USP7 silencing on NSCLC cell proliferation and M2 macrophage polarization and also mitigated the promoting effects of USP7 knockdown on apoptosis and the induction of features associated with ferroptosis. USP7 stabilized MESP1 to promote the malignant progression of NSCLC. The findings highlight the potential of targeting the USP7-MESP1 axis as a novel therapeutic strategy for NSCLC.

Keliu Pill (KLW) is a Chinese medicine formula that has been shown to be effective in treating non-small cell lung cancer (NSCLC) patients. However, the potential molecular mechanism remains unclear. To explore the underlying mechanism of Keliu Pill (KLW) in treating NSCLC, the network pharmacology was applied to explore the potential mechanism of KLW in the treatment of lung cancer. With oral bioavailability (OB) ≥ 30% and drug-like index (DL) ≥ 0.18 as the filter criteria, the compounds of 6 traditional Chinese medicines (TCMs) from KLW were retrieved on Traditional Chinese Medicine Systematic Pharmacology Database and Analysis Platform (TCMSP) and supplemented by Traditional Chinese Medicine Comprehensive Database (TCMID). The potential targets corresponding to the active components of the TCMs mentioned above were obtained from TCMSP. Subsequently, the potential underlying action mechanisms of KLW on NSCLC predicted by the network pharmacology analyses were experimentally validated in Lewis lung cancer cell inoculation mice models and co-culture models with Lewis lung cancer cell and tumor-associated macrophages (TAMs). After database search and screening, a total of 20 active ingredients of Astragalus Membranaceus, 21 of Sophare Tonkinensis Radix et Rhizoma, 1 of Mylabris, 3 of Eupolyphaga, 1 of Leech, and 3 of Gecko were obtained. After deleting duplicates, 49 active ingredients were obtained. Thirty important active ingredients in KLW were sorted out by referring to relevant literature. The KEGG pathway enrichment analysis based on the DAVID platform showed that the VEGF-C signaling pathway may be the core signaling pathway associated with KLW in the treatment of NSCLC. The network pharmacological analysis demonstrated that the various components of KLW acted on NSCLC through 29 potential targets such as VEGF-C, VEGF-D, MAPK8, MAPK1, and AKT1. The experiments in vivo indicated KLW could inhibit the transcription and translation of VEGF-C and VEGF-D in tumor tissues. KLW could reduce the proportion of activated TAMs in the tissue. KLW-contained serum may decrease the expression level of VEGF-C and VEGF-D and inhibit the lymphatic lumen formation of human lymphatic endothelial cells. VEGFC and VEGFD were confirmed as the potential KLW-associated targets for NSCLC. KLW may inhibit lymphatic angiogenesis of lung cancer by regulating the function of TAMs.

In recent years, bacterial quorum quenching (QQ) has emerged as an effective strategy for reducing biofouling in membrane bioreactors (MBRs). Understanding microbial community dynamics is crucial for developing effective QQ strategies, as changes in these communities can significantly influence the risk of biofouling in the sludge. This study systematically investigates the formation, mechanisms, and regulatory strategies related to biofouling in MBRs. It offers a comprehensive analysis of quorum sensing (QS) mechanisms within microbial communities and their biofouling tendencies. Moreover, the interactions between quorum sensing, extracellular polymerization, and membrane biofouling are discussed. Additionally, the short-term addition of exogenous QQ was found to temporarily cause a reduction in the sludge's QQ capabilities, thereby increasing its susceptibility to membrane biofouling. The study concludes with future perspectives on managing biofouling in membrane bioreactors and provides recommendations for further research on leveraging QS-MBR systems to mitigate membrane biofouling.

Mosquitoes rely heavily on olfactory cues for locating suitable oviposition sites, with microbial communities in aquatic habitats playing a crucial role in producing volatile organic compounds (VOCs) that influence mosquito behaviour. In this study, we isolated Bacillus subtilis DHB13 from the breeding habitat of Culex quinquefasciatus, a major vector of several human diseases. The partial 16S rRNA gene sequence of the isolate has been submitted to NCBI GenBank with the accession number PV698100. The identity and resistance profile of the strain was confirmed through biochemical and antibiotic susceptibility tests. The bacterial suspension demonstrated a notable oviposition activity index (OAI) of 0.77 ± SE, with moderate variation among treatments (F(3, 8) = 3.631, p = 0.0642). Multiple comparison analysis (Tukey's test) showed that OAI values for DHB13-treated media did not differ significantly from natural habitat water but were significantly higher than the sterile control, indicating a biologically relevant attraction of gravid female mosquitoes. LC-MS analysis of the bacterial culture supernatant revealed the presence of three cresol derivatives: diisopropyl-m-cresol, 3-ethyl-p-cresol, and 6-ethyl-o-cresol. These compounds were evaluated through molecular docking against Cx. quinquefasciatus Odorant Binding Protein 1 (CxOBP1), a protein known to mediate olfactory-driven oviposition behaviour. However, mosquito olfaction involves several OBPs, receptors, and enzymes, so interaction with CxOBP1 represents only part of this complex sensory system. Molecular docking revealed strong binding of CxOBP1 with diisopropyl-m-cresol (-6.7 kcal/mol), 3-ethyl-p-cresol (-6.2 kcal/mol), and 6-ethyl-o-cresol (-5.9 kcal/mol), indicating potential oviposition attractant activity. All three ligands were found to bind within a conserved binding pocket of CxOBP1, behavioural assays confirmed the oviposition-stimulant properties of the bacterial suspension, indicating that the detected compounds mimic natural semio-chemicals such as p-cresol, previously recognized as an oviposition cue. These findings reinforce the role of microbiota in shaping mosquito reproductive behaviour through the production of volatile attractants. Moreover, they highlight the potential of using microbial VOCs as environmentally sustainable tools for mosquito surveillance and vector control. This integrative approach linking microbial ecology, chemical analysis, and mosquito behaviour provides novel insights for the development of attractant-based control strategies.

Lysophosphatidic acid acyltransferase (LPAAT) is a pivotal enzyme in the de novo biosynthesis of phosphatidic acid (PA), playing a central role in glycerophospholipid assembly and triacylglycerol (TAG) accumulation. Myrmecia incisa is a green microalga notable for its high content of arachidonic acid (ArA), yet the molecular mechanism underlying ArA enrichment in TAG remains unclear. In this study, a putative LPAAT gene from M. incisa, designated MiLPAAT, was identified and cloned, followed by systematic structural and functional characterization. Sequence analysis revealed that MiLPAAT contains a conserved PlsC domain and the characteristic H(X)₄D and EGTR motifs. Bioinformatic predictions identified at least one transmembrane domain at the N-terminus, supporting its identity as an integral membrane protein. This was further confirmed by membrane fractionation and Western blot analysis, which demonstrated its association with the membrane fraction. Phylogenetic analysis further demonstrated its close evolutionary relationship to LPAAT homologs in other green algae. Heterologous expression in Escherichia coli, coupled with in vitro enzymatic assays, confirmed that the recombinant MiLPAAT protein possesses LPAAT activity, catalyzing the acylation of LPA with various acyl-CoAs. Among the substrates tested, MiLPAAT exhibited the highest catalytic efficiency toward ArA-CoA (104.8 ± 3.2 nmol/mg/min), followed by oleoyl-CoA (81.5 ± 2.7 nmol/mg/min) and palmitoyl-CoA (68.4 ± 2.1 nmol/mg/min), consistent with the ArA-rich TAG composition observed in M. incisa. Immunogold labeling and immunohistochemical localization experiments revealed that MiLPAAT is predominantly localized at the plasma membrane. Findings of the present study suggest that MiLPAAT plays a critical role in PA biosynthesis and assembly of ArA into TAG in M. incisa, providing a novel target for microalgal lipid metabolic engineering.

Aegiceras corniculatum (AC), a mangrove species widely recognized for its diverse pharmacological properties, has attracted significant attention recently due to its antioxidant, anti-inflammatory, and antimicrobial activities. Despite this, the neuroprotective potential of AC, particularly in the context of neurodegenerative disorders such as Parkinson's disease (PD), remains largely unexplored. Due to the growing interest in plant-derived compounds for the management of PD, investigating the therapeutic relevance of AC could provide new insights into alternative strategies for neuroprotection. The present study aimed to evaluate the neuroprotective ability of the leaf extract of AC against PD. Preliminary phytochemical analyses were conducted to identify the presence of bioactive compounds in the AC extract (ACE). The antioxidant capacity of ACE was evaluated using three standard assays: 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, ferric reducing antioxidant power (FRAP) assay, and hydrogen peroxide (H₂O₂) scavenging assay. We used the MPTP-induced PD model established in an SH-SY5Y human neuroblastoma cell line as well as in the U87-MG glioblastoma cell line. MTT assay was conducted to measure the cell viability, and further, 2',7'-dichlorofluorescin diacetate (DCFDA) assay was used to measure the reactive oxygen species (ROS). Glutathione peroxidase (GPx) enzyme activity was determined by a chromogenic reaction-based assay. Western blot analysis was used to assess the expression levels of SNCA protein. The ACE was found to contain a diverse range of phytochemicals, including polyphenols, flavonoids, and terpenoids. Among the ethanolic, hydroethanolic, and aqueous extracts evaluated, ethanolic ACE (eACE) exhibited the highest antioxidant activity. The SH-SY5Y cell line demonstrated significantly higher neuroprotective potential to eACE treatment compared to the U87-MG cell line. Moreover, eACE markedly attenuated intracellular ROS levels and enhanced glutathione peroxidase (GP_X) enzymatic activity. In line with these effects, eACE also significantly downregulated the expression of the SNCA gene, suggesting its potential modulatory role in oxidative stress-related neurodegeneration. AC exhibited notable neuroprotective effects in an in vitro model of PD. These effects are likely mediated through the attenuation of oxidative stress and neuroinflammation, inhibition of apoptosis, and preservation of cellular energy metabolism. The results suggest that the extract of AC may provide important insights for developing therapies that modify the progression of PD. Elucidating the underlying mechanisms could inform the creation of innovative treatments designed to slow or alter the trajectory of PD. The present study emphasizes the potential benefits of AC with contemporary scientific investigation to address the unmet needs of patients suffering from PD.