BMC Biotechnology最新文献

Macrophages serve both as a first line of defense against invading pathogens and mediate tissue homeostasis. These cells are inherently responsive and heterogeneous and lie on a spectrum of activation states book-ended by M1-like (inflammatory) and M2-like (anti-inflammatory) extremes. The study of human macrophages is necessary to unravel the complex signals and environmental cues that these cells integrate to create their varied phenotypes. In vitro protocols to differentiate human monocytes into macrophages use many distinct activation stimuli at variable concentrations and for differing durations of treatment that can impact macrophage fate. These variations can make it challenging to reproduce findings and compare datasets across research environments. Additionally, few protocols to date have performed rigorous characterization with input material from frozen peripheral blood mononuclear cells (PBMCs). This is important since the availability of fresh blood can often be limiting and can lead to a loss of standardized procedures, particularly for cell therapy applications. Here, we have developed a comprehensive protocol to generate human macrophages from monocytes where we rigorously characterize the impact of differentiation conditions and polarization conditions on human macrophages. We compared 4 conditions for M1-like (50 ng/mL LPS, 50 ng/mL IFNγ, 20 ng/mL IFNγ + 10 ng/mL LPS and 20 ng/mL IFNγ + 100 ng/mL LPS) and for M2-like (10 ng/mL IL-4, 20 ng/mL IL-4, 20 ng/mL IL-13 and 20ng/mL IL-4 + 20 ng/mL IL-13). We provide a detailed protocol for their characterization using several 'omics readouts, including their cytokine production and transcriptomes. We also perform depolarization experiments to determine durability of macrophage immunophenotype post-removal of polarizing stimuli for 0 to 72 hours. Finally, we demonstrate that nuclei can be isolated and profiled by snRNA-seq directly from macrophages in culture, alleviating the need to detach these adherent cells for downstream multi-ome analyses. Taken together, we provide a comprehensive, detailed and streamlined procedure for the differentiation and characterization of human macrophages from monocytes isolated from frozen PBMCs. This is important for enabling the study of macrophages in a more systematic way from biobanked material.

The growing demand for medicinal plants in herbal medicine highlights their therapeutic value, yet heavy metal contamination, such as cobalt (Co), poses potential health risks. Cobalt's dual role as an essential micronutrient and a toxic pollutant necessitates a more profound understanding of its impact on medicinal plants like Adhatoda vasica. We investigated how varying Co concentrations affect A. vasica shoot growth, leaf anatomy, antioxidant enzyme activity, and secondary metabolite profiles. Additionally, molecular docking was performed to assess the interaction of the metabolites with the skin cancer-related protein anti-ssDNA antigen-binding fragment (PDB code: 1P7K). Low Co concentrations (50 µM) enhanced shoot dry weight by 41.45%, while higher levels (100-1000 µM) reduced it by up to 66.86%. Cobalt exposure increased hydrogen peroxide (H₂O₂) and lipoxygenase (LOX) activity, indicating reactive oxygen species (ROS)-induced oxidative stress. Higher Co levels increased superoxide dismutase (SOD), catalase (CAT), phenylalanine ammonia-lyase (PAL), and polyphenol oxidase (PPO) but decreased peroxidase (POD) and ascorbate peroxidase (APX) activity. HPLC-UV and GC-MS data showed that Co altered the secondary metabolites of A. vasica, including phenolics, flavonoids, alkaloids, and terpenoids, in both qualitative and quantitative ways. Molecular docking shows that naringin has a higher binding affinity (-9.2 kcal/mol) to PDB: 1P7K than phenolics (-4.8 to -6.4 kcal/mol). Cobalt stress significantly impacts A. vasica, altering its leaf structure, growth, and antioxidant defenses. These effects extend to secondary metabolites in a dose-dependent manner. These findings highlight the plant's potential for Co tolerance and its metabolites' therapeutic promise, particularly naringin, for skin cancer applications.