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.

Halophytic plants have been recognized as a promising source of lignocellulosic biomass for bioconversion into value added products. In this study, cellulosic content of wild biomass from Typha domingensis was saccharified by cellulase from a thermophilic bacterium, Neobacillus sedimentimangrovi UE25. Indeed, this is the first report describing the use of enzymatic saccharification of T. domingensis biomass. Initially, the N. sedimentimangrovi UE25 cellulase was produced in the medium containing wild biomass from the halophyte plant. The data revealed that 159.84 IU mL^- 1 endoglucanase was obtained when T. domingensis biomass was used as a substrate. The result of saccharification showed that 172 mg g^- 1 reducing sugars were obtained when cellulosic content of T. domingensis biomass was saccharified by cellulase. The effect of temperature, cellulase units and incubation time on saccharification was determined by using a statistical tool, Central Composite design. Under optimized conditions of 14.6 endoglucanase units per g of substrate, temperature 60 °C and reaction time 13.4 h, 610.65 mg g^- 1 reducing sugars were obtained after the saccharification of cellulose from T. domingensis biomass. Moreover, Fourier Transform Infrared spectroscopy and Scanning Electron microscopy affirmed structural changes in the substrate. These findings demonstrate that biomass from halophyte such as T. domingensis can serve as an efficient substrate for thermostable cellulase production and saccharification, contributing to sustainable bioresource utilization in saline environments.

The current study aims to assess the efficiency of different irradiated potassium sources as alternatives to ethrel in enhancing the fruit quality and storability of Ruby grapevines. This investigation was conducted on seven- year- old Ruby Seedless grape cultivars that were sprayed as follows: control (water); ethrel at 150 ppm; potassium citrate at 2000 and 4000 ppm; irradiated potassium citrate at 750 and 1500 ppm; potassium silicate at 2000 and 4000 ppm and irradiated potassium silicate at 750 and 1500 ppm. The data revealed that all the potassium sprays used significantly increased cluster weight, berry weight, and berry firmness compared with those in the control and ethrel groups. Moreover, the total soluble solids, sugars and anthocyanin contents increased with decreasing acidity% compared with those of the control group. Storability data revealed that all potassium sprays decreased weight loss, decay, firmness loss, and shattering during the storage period compared with those of the control and ethrel groups. Additionally, the total soluble solids, sugars and anthocyanin contents increased with decreasing acidity compared with those of the control. In conclusion, potassium sprays, especially irradiated potassium silicate, can be a good alternative to ethrel for improving the physical and chemical properties of Ruby Seedless grapes and increasing their storability by improving firmness and reducing weight loss, decay, and shattering.