His-tagged lactate oxidase production for industrial applications using fed-batch fermentation.

L-lactate oxidase (LOX) is a biotechnologically important enzyme used in biosensors and colorimetric kits to detect lactate, a key biomarker in clinical diagnostics, sports medicine and the food industry. In this work, we produced a recombinant His-tagged Aerococcus viridans LOX (rLOX) in Escherichia coli and carried out its functional characterization for industrial applications. Our rLOX was evaluated in a colorimetric kit for human diagnostics and in an amperometric biosensor to measure the lactic acid in food products. The rLOX was fully functional for both applications, with a performance comparable to commercial untagged LOXs. As the industrial use of LOX enzyme requires a large-scale production, we scaled up the rLOX production in a fed-batch bioreactor culture and obtained a yield approximately ten times higher than that of the Erlenmeyer scale. The His-tag allowed an easy and highly efficient purification process, and a high-purity rLOX was recovered after this one-step affinity purification. In this study, we described a simple, rapid and cost-competitive approach for the production of a recombinant His-tagged LOX enzyme suitable for industrial use.

Validation of a continuous flow method for the determination of soluble iron in atmospheric dust and volcanic ash.

Iron is an essential micronutrient for phytoplankton growth and is supplied to the remote areas of the ocean mainly through atmospheric dust/ash. The amount of soluble Fe in dust/ash is a major source of uncertainty in modeling-Fe dissolution and deposition to the surface ocean. Currently in the literature, there exist almost as many different methods to estimate fractional solubility as researchers in the field, making it difficult to compare results between research groups. Also, an important constraint to evaluate Fe solubility in atmospheric dust is the limited mass of sample which is usually only available in micrograms to milligrams amounts. A continuous flow (CF) method that can be run with low mass of sediments (<10mg) was tested against a standard method which require about 1g of sediments (BCR of the European Union). For validation of the CF experiment, we run both methods using South American surface sediment and deposited volcanic ash. Both materials tested are easy eroded by wind and are representative of atmospheric dust/ash exported from this region. The uncertainty of the CF method was obtained from seven replicates of one surface sediment sample, and shows very good reproducibility. The replication was conducted on different days in a span of two years and ranged between 8 and 22% (i.e., the uncertainty for the standard method was 6-19%). Compared to other standardized methods, the CF method allows studies of dissolution kinetic of metals and consumes less reagents and time (<3h). The method validated here is suggested to be used as a standardized method for Fe solubility studies on dust/ash.

Activation of Toll-like receptor 4 on tumor cells in vitro inhibits subsequent tumor growth in vivo.

Although an eruption of information on the role of Toll-like receptor 4 (TLR4), the main receptor for bacterial lipopolysaccharide, in activating macrophages and dendritic cells has emerged, very little is known about the role of TLR4 present on epithelial cells from sterile environments like tumors. The main goal of this work was to investigate the consequences of TLR4 activation present on tumor cells in two different animal models of cancer: the Dunning rat prostate cancer and the B16 murine melanoma models. We show that (a) activating TLR4 signaling in two different tumor cell lines in vitro modifies the tumor outgrowth in vivo; (b) this effect is not due to a direct consequence of TLR4 signaling on the proliferation/apoptosis balance of the tumor cells; (c) the T-cell compartment is somehow involved in the described phenomenon because the inhibitory effect observed is not seen in athymic nude mice; and (d) tumor-infiltrating lymphocytes purified from tumors induced by TLR4-activated cells show strong induction of IFN gamma transcript in detriment of interleukin-10 transcript, suggesting a change in their functionality. We hypothesize that TLR4 signaling in tumor cells in vitro induces the expression of proinflammatory mediators, which could dramatically alter the maturation state of dendritic cells present at the site of inoculation, switching the type of immune response elicited against the tumor. These results open up new avenues for understanding the role of TLR4 in tumor cells and for identifying potential new therapy strategies for cancer.