Characterization of new anti-IL-6 antibodies revealed high potency candidates for intracellular cytokine detection and specific targeting of IL-6 receptor binding sites

Interleukin-6 (IL-6) expression and secretion, induced by inflammatory processes, stimulate the acute phase response cascade. The overexpression of IL-6 contributes to a variety of inflammatory diseases, e.g. rheumatoid arthritis, Castleman’s disease, multiple myeloma, and prostate cancer. Screening for high amounts of IL-6 in the patients’ blood serum can be crucial for an adequate treatment. In this study, five novel murine monoclonal antibodies (mAbs) reactive to human IL-6 were generated. The mAbs were characterized for potential diagnostic purposes and recombinant antibodies were derived thereof. Initial epitope mapping using a combination of blocking experiments and Hyper-IL-6, a fusion protein consisting of IL-6 and the soluble IL-6 receptor revealed distinct but overlapping binding sites. At least one of the mAbs was found to interact with the region of IL-6/ IL-R complex formation. Three mAbs were applied successfully in intracellular staining by flow cytometry, whereas one of the mAbs showed comparable binding as a reference reagent. Furthermore, the mAbs were tested for applications in various immunological assays such as ELISA, Western blot and surface plasmon resonance spectroscopy (SPR), using IL-6 from commercial sources as well as in-house produced protein (IL-6_IME). The limit of detection was determined by sandwich ELISA (0.5 ng/mL, SD ±0.005). Our results also demonstrated that the recombinant IL- 6 produced was functional and correctly folded. These findings support the use of the generated mAb clones as promising candidates for application in various immunological assays for diagnostic and scientific purposes.     

Fungal utilization of a known and safe macroalga for pigment production using solid-state fermentation

Saccharina (Laminaria) japonica, a safe, cheap, and readily available macroalga can be used as a substrate for various microbial fermentations. This work investigated the feasibility of S. japonica as a substrate for production of pigments by the fungus Talaromyces amestolkiae GT11 in solid-state fermentation without additional salt and/or nitrogen sources. Under optimized conditions, the pigment exhibited maximum absorption spectrum at 410 (yellow) and 510 nm (red), and the pigment yield of 1,153.5 (yellow) and 506.2 (red) OD units g^?1 of dry fermented substrate were achieved with a particle size of 1.0 mm and pH 7, although visually the pigment was reddish in color. The optimum incubation period, pH, moisture, inoculum size, and temperature were observed to be at 192 h, pH 7.0, 80 % (w/w) moisture, 1.8?×?10⁶ spores mL^?1 of inoculum g^-1 of dry substrate and 28 °C. Hence, this study indicates the suitability of utilization of S. japonica as a substrate for natural pigment production by T. amestolkiae GT11 which can be used in food, cosmetics and pharmaceutical industries for various applications.     

Reconstruction of methanol and formate metabolic pathway in non-native host for biosynthesis of chemicals and biofuels

One-carbon feedstock such as methanol and formate has attracted much attention as carbon substrate of industrial biotechnology for production of value-added chemicals and biofuels. Productivity improvement of natural one-carbon metabolic pathways in native hosts such as methanotrophs is somewhat difficult due to inefficient genetic tools and low specific growth rate. As an alternative, metabolic engineering can create new and efficient metabolic pathways of one-carbon substrate that can be readily transferred to non-native hosts. In this paper, recent progresses in protein and metabolic engineering for creation of methanol and formate-utilizing synthetic pathways based on RuMP cycle and formolase are reviewed. Perspectives on one-carbon metabolic pathway engineering in non-native host are also discussed.     

Nitric oxide inhibits spleen cell proliferative response after burn injury by inducing cytostasis,apoptosis, and necrosis of activated T lymphocytes: role of the guanylate cyclase

We previously showed that an overproduction of nitric oxide (NO) by macrophages was responsible for the collapse of lymphoproliferative responses after burn injury in rats. First, we demonstrate here that 10 days post-burn, the inhibition of splenocyte response to concanavalin-A results from cytostatic, apoptotic, and necrotic effects of NO on activated T cells. This was evidenced by various criteria at the levels of DNA, mitochondria, and plasma membrane. Inhibition of NO synthase by S-methylisothiourea (10 microM) normalized all the parameters. Second, we show that two soluble guanylate cyclase (sGC) inhibitors, LY83583 and ODQ, restored the proliferative response in a concentration-dependent manner. LY83583 (0.5 microM) rescued T cells from apoptosis. Similar results were obtained with KT5823 (5 microM) a specific inhibitor of protein kinase G (PKG). In contrast, neither LY83583 nor KT5823 inhibited NO-induced necrosis. These results suggest that NO blocked T cells in the G1 phase and induced apoptosis through a sGC-PKG-dependent pathway and necrosis through an independent one.     

Flash Infrared Annealing for Antisolvent‐Free Highly Efficient Perovskite Solar Cells

Organic–inorganic perovskites have demonstrated an impressive potential for the design of the next generation of solar cells. Perovskite solar cells (PSCs) are currently considered for scaling up and commercialization. Many of the lab‐scale preparation methods are however difficult to scale up or are environmentally unfriendly. The highest efficient PSCs are currently prepared using the antisolvent method, which utilizes a significant amount of an organic solvent to induce perovskite crystallization in a thin film. An antisolvent‐free method is developed in this work using flash infrared annealing (FIRA) to prepare methylammonium lead iodide (MAPbI₃) PSCs with a record stabilized power conversion efficiency of 18.3%. With an irradiation time of fewer than 2 s, FIRA enables the coating of glass and plastic substrates with pinhole‐free perovskite films that exhibit micrometer‐size crystalline domains. This work discusses the FIRA‐induced crystallization mechanism and unveils the main parameters controlling the film morphology. The replacement of the antisolvent method and the larger crystalline domains resulting from flash annealing make FIRA a highly promising method for the scale‐up of PSC manufacture.     

Expression of caveolin-1 enhances cholesterol efflux in hepatic cells

HepG2 cells were stably transfected with human caveolin-1 (HepG2/cav cells). Transfection resulted in expression of caveolin-1 mRNA, a high abundance of caveolin-1 protein, and the formation of caveolae on the plasma membrane. Cholesterol efflux from HepG2/cav cells was 280 and 45% higher than that from parent HepG2 cells when human plasma and human apoA-I, respectively, were used as acceptors. The difference in efflux was eliminated by treatment of cells with progesterone. There was no difference in cholesterol efflux to cyclodextrin. Cholesterol efflux from plasma membrane vesicles was similar for the two cell types. Transfection led to a 40% increase in the amount of plasma membrane cholesterol in cholesterol-rich domains (caveolae and/or rafts) and a 67% increase in the rate of cholesterol trafficking from intracellular compartments to these domains. Cholesterol biosynthesis in HepG2/cav cells was increased by 2-fold, and cholesterol esterification was reduced by 50% compared with parent HepG2 cells. The proliferation rate of transfected cells was significantly lower than that of non-transfected cells. Transfection did not affect expression of ABCA1 or the abundance of ABCA1 protein, but decreased secretion of apoA-I. We conclude that overexpression of caveolin-1 in hepatic cells stimulates cholesterol efflux by enhancing transfer of cholesterol to cholesterol-rich domains in the plasma membrane.