Dried blood spot (DBS) technology is an expanding tool for trace element determination; however, quantification by X-ray fluorescence (XRF) faces challenges due to matrix effects, surface irregularities, and blood volume variability. This paper presents a novel methodology for the quantitative determination of lead (Pb) in DBS samples using Secondary Target Energy-Dispersive X-ray Fluorescence Spectroscopy (ST-EDXRF). Potassium (K) was employed as an internal reference to compensate for hematocrit variability. A Mo X-ray tube equipped with interchangeable secondary targets was used: vanadium (V) to excite K and strontium chloride (SrCl2) to optimize excitation of the Pb-Lα line. Normalization strategies were implemented to minimize the effects of sample volume variations and surface roughness: K intensities were normalized to the atmospheric Ar-Kα line, whereas Pb intensities were normalized to the incoherent scattering peak. These normalization procedures significantly improved analytical performance, increasing the coefficients of determination (R2) of the calibration curves to 0.996 for K and 0.999 for Pb. A detection limit of 0.04 ppm (w/w) Pb in blood was achieved with a 15 min acquisition time, comparable to that of state-of-the-art commercial EDXRF systems. As a preliminary evaluation, the methodology was applied to a single DBS prepared from venous blood collected from a healthy volunteer. Pb was not detected, as expected, whereas K was successfully quantified within the physiological range. In addition, the method accurately quantified Pb in an artificially spiked blood sample, demonstrating its capability for quantitative analysis. The developed ST-EDXRF approach effectively compensates for substrate irregularities, sample volume variations, and hematocrit effects, providing a low-cost and sensitive methodology suitable for field monitoring of populations at risk of lead exposure.
In the field of vaccines, adjuvants have been optimized to enhance immune responses while reducing adverse effects. Transferring these advances to antivenom production could improve antibody quality and animal welfare. Here, we evaluated a novel adjuvant platform, CpG-ODN/Coa-ASC16, which combines immunostimulatory CpG-ODN oligodeoxynucleotides with the biodegradable nanostructure Coa-ASC16, co-formulated with a hemorrhagic antigenic model based on Bothrops diporus venom (B.dV). Immunization with B.dV/CpG-ODN/Coa-ASC16 elicited IgG titers equivalent to those induced by Freund’s adjuvant, with significantly higher IgG1 levels. Antibodies generated with both formulations exhibited progressively higher avidity, reflecting effective affinity maturation. Sera from B.dV/CpG-ODN/Coa-ASC16-immunized mice recognized major venom proteins and neutralized key toxic activities—proteolytic, coagulant, and indirect hemolytic—at levels comparable to Freund’s-induced sera. Functionally, sera from B.dV/CpG-ODN/Coa-ASC16-immunized mice conferred protection against a lethal venom challenge, resulting in survival of a fraction of animals and prolonged time to death in non-survivors. Importantly, histopathological analyses revealed minimal tissue alterations in the mice treated with CpG-ODN/Coa-ASC16, in sharp contrast to the severe abscesses and granulomas caused by Freund’s adjuvant. Overall, this study provides new evidence that CpG-ODN/Coa-ASC16 can be effectively combined with hemorrhagic Bothrops venom to generate robust, high-affinity antibodies that efficiently neutralize the major venom toxins while markedly reducing local tissue damage. These findings position CpG-ODN/Coa-ASC16 as a safer and ethical alternative platform for antivenom production.
An ideal vaccine adjuvant should be biocompatible, enable antigen storage and controlled release, and effectively stimulate the immune system. In this study, we evaluated the dual functionality of ascorbyl palmitate (ASC16): as an adjuvant additive in its dispersed state (120 µM), and as a coagel formed at low temperatures and high ASC16 concentrations (Coa-ASC16PEG), for use in the antivenom development. Coa-ASC16PEG was prepared by mixing ASC16 with polyethylene glycol 400 (PEG400) and solubilising the mixture at 64 °C for 2 min; subsequently, the formulation was allowed to cool to 40 °C and whole Crotalus durissus terrificus venom, which naturally contains a complex mixture of proteins, was incorporated for 10 s, including major toxins such as crotoxin, serine proteinases, L-amino acid oxidase, and phosphodiesterases. To produce experimental antivenom, mice were immunized with Coa-ASC16PEG or Freund’s adjuvant, with or without the addition of dispersed ASC16. The physicochemical analyses showed that the Coa-ASC16PEG exhibited a semi-crystalline, viscoelastic structure capable of effectively encapsulating and releasing proteins. The addition of dispersed ASC16 enhanced the immune response induced by both adjuvants (Freund’s and Coa-ASC16PEG), yielding up to a 1.2-fold increase in venom-specific IgG titres and an approximately 2.7-fold increase in IgG antibody avidity. ASC16-based formulations were associated with reduced local adverse reactions compared with Freund’s adjuvant–based formulations. In conclusion, we propose an alternative ASC16-based formulation obtained at low temperatures that preserves key physicochemical properties and shows minimal impact at the inoculation site. Moreover, the incorporation of dispersed ASC16 as an additive enhances the humoral immune response. Taken together, our results obtained in a murine model position ASC16 as a promising adjuvant, demonstrating reduced local reactogenicity compared with Freund’s adjuvant.
In response to high densities of red-winged blackbird (Agelaius phoeniceus) in the northern Great Plains, natural resource managers have proposed population management plans such as reducing breeding adult numbers. However, data on compensatory effects of variation in nesting densities on the reproductive performance of red-winged blackbirds that could counteract effects of breeding population reductions is lacking. Maternally derived hormones in egg yolks could act as a compensatory mechanism by which adult social interactions affect offspring success. We conducted simulated territorial intrusions by placing caged blackbird females near nesting female red-winged blackbirds to quantify effects of increased density and associated social interactions on behavior, yolk hormones, and reproductive output. We measured aggressive behavior, maternally derived testosterone and corticosterone present in the yolks of eggs and other measures of reproductive performance. Aggressive behavior was greatest in females receiving simulated territorial intrusions compared to females not receiving simulated intrusions but otherwise we observed few differences in reproduction among the experimental groups. Maternally derived testosterone and corticosterone concentrations in eggs were not significantly different among experimental groups. However, brown-headed cowbird (Molothrus ater) parasitism was greater in, and survival probability was lower for, nests of females receiving simulated territorial intrusions. We conclude that maternally-derived testosterone and corticosterone in eggs resulting from increased social interactions among nesting females are not likely density dependent regulatory mechanisms for red-winged blackbird populations.
This study quantifies the hydraulic conductivity k of compacted loess–bentonite mixtures (0–10
