Ischemic cerebrovascular accident (iCVA) is a public health issue, whose subjacent events involve the development of nitroxidative distress. Identifying biomarkers that assist in the diagnosis of this disease has clinically relevant implications. The aim of this study was to develop an analytic tool for measuring nitroxidative distress biomarkers, intended for application in clinical practice to enhance patient healthcare. Three enzyme linked immunosorbent assays (ELISA) were developed, with different detection objectives. One of them, in a sandwich format, quantifies the amount of fibrinogen in human plasma, an important glycoprotein involved in the blood coagulation process, contributing to thrombus formation and thereby participating in the mechanism of ischemic stroke. Another ELISA, also in a sandwich format, detects the presence of nitrotyrosine residues in fibrinogen from human plasma, a nitroxidative posttranslational modification resulting from the attack of peroxynitrite by-products on tyrosine residues present in proteins. The third one, in inhibition format, determines human plasma nitrotyrosine total content and was used to analyze human plasma samples from control and iCVA patients. Those two groups of plasma samples were analyzed using inhibition ELISA, revealing statistically significant differences in their nitrotyrosine content and molar ratios of nitrotyrosine to fibrinogen, which were higher in the iCVA group. This study provides evidence that nitroxidative distress occurs in ischemic stroke, as indicated by the detection of the biomarker nitrotyrosine. This finding supports other studies that also identified nitrotyrosine in ischemic stroke, through several different methods. This specific ELISA method is applicable for the rapid analysis of clinical samples, making it a potential clinical tool for assessing iCVA patients.

Purpose The current diagnostic methods for leptospirosis diagnosis are technically complex and expensive, with limited applicability to specialized laboratories. Furthermore, they lack diagnostic accuracy in the acute stage of the disease, which coincides with a period when antibiotics are highly effective. New simple and accurate tests are mandatory to decentralize and improve diagnosis. Here, we introduced a new lateral flow immunoassay (Lepto-LF) for human leptospirosis. Methods We conducted a double-blinded assay using 104 serum samples from patients with confirmed or discarded diagnosis for leptospirosis. The diagnostic performance of Lepto-LF was estimated across different ranges of days from onset of symptoms (dpo), considering the diagnostic algorithm as reference standard. Additionally, it was compared with the screening methods enzyme-linked immunosorbent assay (IgM-ELISA) and the slide agglutination test using temperature-resistant antigen (SATR). Results Lepto-LF exhibited perfect diagnostic performance with a Youdens index J = 1 from 6 dpo in the acute phase. IgM-ELISA gave slightly lower accuracy with J = 0.91 and 95.5% of both sensitivity and specificity; while SATR showed a markedly inferior yield (J = 0.41, sensitivity = 95.5%, specificity = 45.5%). The performances remained consistent in the convalescence phase of the disease (>10 dpo). Conclusion Lepto-LF was found to be a reliable test for simple, rapid and early diagnosis of leptospirosis, resulting a promising tool for decentralizing leptospirosis diagnosis and enabling timely treatment of patients. In addition, Lepto-LF may be employed as confirmatory test, especially in remote areas and vulnerable contexts where the standard MAT is not available.

In the agricultural field, chitosan (CS) is of great interest due to their broad antimicrobial activity and their ability to induce resistance mechanisms in plants against phytopathogens. Likewise, the potential of silver nanoparticles in preventing microbial infections has been demonstrated, including their broad antimicrobial activity against phytopathogens. In this study, the synthesis and characterization of chitosan–silver nanohybrids were performed. Firstly, mycosynthesis of biogenic silver nanoparticles (PchNPs) was carried out. Subsequently, the nanohybrid (CS-PchNHs) synthesis involved the addition of PchNPs, with continuous stirring, into a CS solution. For the first time, stable CS-Pch NHs were produced using biogenic silver nanoparticles from the fungus Phanerochaete chrysosporium , characterized by UV–visible spectroscopy (UV–Vis), Dynamic Light Scattering, (DLS), ζ-Potential, and X- ray diffraction (XRD). Additionally, CS-PchNHs exhibited high antifungal activity at low concentrations against four phytopathogens, suggesting that these nanosystems may be used as an alternative to the current hazardous antifungal agents in the integrated disease management. Graphical abstract

The research, development, and application of nanoparticles composed of two different metals continue to be an area of interest in nanotechnology and materials science due to their versatility and great potential to positively impact a variety of applications, from catalysis to nanomedicine and the electronics. Silver (Ag)-coated iron (Fe) nanoparticles as functional antimicrobial agents have become an important research topic. However, there are synthesis methods and important properties and characteristics that still need to be investigated. In this study, a simple synthesis route for Fe2O3–Ag nanoparticles is presented using the microemulsion reaction (MR) technique, since these systems can be applied as antimicrobial agents in biological processes and subsequently recovered through the use of a magnet. The results obtained demonstrate the potential application of the MR technique in the design and synthesis of nanoparticles composed of different metals.

Adjuvants are essential components of subunit, recombinant, nonreplicating and killed vaccines, as they are substances that boost, shape, and/or enhance the immune response triggered by vaccination. Saponins obtained from the Chilean Q. saponaria tree are used as vaccine adjuvants in commercial vaccines, although they are scarce and difficult to obtain. In addition, tree felling is needed during its extraction, which has ecological impact. Q. brasiliensis leaf-extracted saponins arise as a more sustainable alternative, although its use is still limited to preclinical studies. Despite the remarkable immunostimulating properties of saponins, they are toxic to mammalian cells, due to their intrinsic characteristics. For these reasons they are mostly used in veterinary vaccines, although recently the Q. saponaria purified saponin QS-21 has been included in adjuvant systems for human vaccines, such as Mosquirix and Shingrix (GSK). In order to abrogate the toxicity of the saponins fractions, they can be formulated as immunostimulating complexes (ISCOMs). ISCOM-matrices are cage-like nanoparticles of approximately 40 nm, formulated combining saponins and lipids, without antigen, and are great adjuvants able to promote Th1-biased immune responses in a safe manner. Herein we describe how to formulate ISCOM-matrices nanoparticles using Q. brasiliensis purified saponin fractions (IMXQB) by the dialysis method. In addition, we indicate how to verify the appropriate size and homogeneity of the formulated nanoparticles.

Quillaja saponins have an intrinsic capacity to interact with membrane lipids that self-assembles in nanoparticles (immunostimulating complexes or ISCOM-matrices) with outstanding immunoadjuvant activity and low toxicity profile. However, the expensive and laborious purification processes applied to purify Quillaja saponins used to assemble ISCOM-matrices show an important drawback in the large-scale use of this vaccine adjuvant. Thus, in this study, we describe a protocol to appropriately formulate ISCOM-matrices using the raw aqueous extract (AE) of Quillaja lancifolia leaves. In the presence of lipids, AE was able to self-assemble in nanostructures that resembles immunostimulating complexes (ISCOM). These negatively charged nanoparticles of approximately 40 nm were characterized by transmission electron microscopy and dynamic light scattering. In addition, well-known saponins with remarkable immunoadjuvant activity, as QS-21, were detected into nanoparticles. Thus, the easier, robust, cheaper, and environmentally friendly method developed here may be an alternative to the classical methods for ISCOM-matrices production that use high-purified saponins.

Aptamers are oligonucleotides that have the characteristic of recognizing a target with high affinity and specificity. Based on our previous studies, the aptamer probe Sgc8-c-Alexa647 is a promising tool for molecular imaging of PTK7, which is an interesting biomarker in cancer. In order to improve the delivery of this probe as well as create a novel drug delivery nanosystem targeted to the PTK7 receptor, we evaluate the co-association between the probe and preformed nanostructures. In this work, preformed pegylated liposomes (PPL) and linear and branched pristine polymeric micelles (PMs), based on PEO–PPO–PEO triblock copolymers were used: poloxamer F127® and poloxamines T1307® and T908®. For it, Sgc8-c-Alexa647 and its co-association with the different nanostructures was exhaustively analyzed. DLS analysis showed nanometric sizes, and TEM and AFM showed notable differences between free- and co-associated probe. Likewise, all nanosystems were evaluated on A20 lymphoma cell line overexpressing PTK7, and the confocal microscopy images showed distinctness in cellular uptake. Finally, the biodistribution in BALB/c mice bearing lymphoma-tumor and pharmacokinetic study revealed an encouraging profile for T908-probe. All data obtained from this work suggested that PMs and, more specifically T908 ones, are good candidates to improve the pharmacokinetics and the tumor uptake of aptamer-based probes.

The production of Clostridium sordelli veterinary vaccine is prepared by culturing the microorganism in large industrial bioreactors and purifying the toxin from culture supernatants. Currently, the harvesting time is established as function of bacterial growth, but not always this moment is associated to the maximum concentration of toxin present in the supernatant. Thus, it is easy to find different total amounts of toxin produced batch to batch. Besides, at present the toxin concentration is measured by in vivo LD50 methods, and results can be obtained only after 72h, when the bioreactor was already stopped.Moreover, the method is labour intensive and requires the use of a significant number of experimental animals We describe the development of a latex agglutination reagent for semiquantification of Clostridium sordelli lethal toxin (TcsL) and its use in industrial bioreactors. The reagent was developed and characterized in our laboratory achieving a considerable low detection limit (8ng of toxin per ml of culture supernatant) and then it was validated in actual industrial conditions. The use of such a rapid (i.e, in minutes) and easy to use reagent will allowto followthe culture in real time and thus standardizing the optimal end-point for harvesting in terms of toxin quantity.As an immediate consequence, the efficiency of TcsL industrial production may be optimized.

Micro/nanoencapsulated salts, spices, and seasonings are considered as important agents to develop stable tastes and flavors in food products. The bioactive compounds and flavor ingredients of different herbs and spices are sheltered by loading into encapsulated systems. The main privileges of encapsulated spices are maintaining the quality and quantity of functional compartments, reducing germ count without chemical strategies or irradiation and alleviating enzymatic reactions. Encapsulated salts are also an interesting category applied in a wide range of food products for both dietary reasons and industrial functional applications, for example, meat products, bakery goods, and seasonings to decrease the negative effects on meat-protein interactions, keep down freeze/thaw temperatures, expand the efficiency of microorganisms (yeasts), and postpone staling. This chapter aimed to investigate the mode of action and importance of encapsulated ingredients in salts, spices, and seasoning formulations.

Phagocytosis is a fundamental process for removal of pathogens and for clearance of apoptotic cells. The objective of this work was the preparation of fluorescent microspheres by a simple method and the evaluation of its applicability in phagocytosis assays by using different human derived cells, differentiated THP-1 cell line and blood monocytes, with flow cytometry measurements for functionality assays. Our results show that microparticles are efficiently internalised in a non-opsonised form and in dose-dependent manner by both cellular types. Concerning mechanism we determined that tTG-β3 integrin signaling could be involved in the uptake of these particles.

Nanoencapsulation may improve antimicrobial activity of nisin when applied on food systems such as lean beef. Nisin-loaded nanoparticles were prepared by alginate ionic gelation and further complexation with chitosan. A 3-factor, 3-level Box-Behnken experimental design and response surface methodology were used to optimize the formulation. Nanoparticles had an encapsulation efficiency of 36.1 +/- 0.6%, a z-average of 66.4 +/- 8.9 nm and a zeta potential of 31.7 +/- 2.6 mV. Transmission electron microscopy and atomic force microscopy observations confirmed particle size found by dynamic light scattering. Also, Raman spectroscopy analyses and zeta potential measurements showed ionic interactions between nisin and alginate. In vitro assessment of inhibition of Listeria monocytogenes growth at 4 degrees C indicated sustained antimicrobial activity of nisin-loaded nanoparticles for 21 days. Furthermore, encapsulated nisin was able to inhibit L. monocytogenes growth in vacuum-sealed, refrigerated beef samples for 10 days when applied at 400 IU/g and for 24 days when applied at 800 IU/g, while free nisin controlled microbial growth for only 4 and 17 days respectively. In sum, we describe the application of experimental design tools to produce nisin-loaded nanoparticles that may be used in the food industry to control microbial growth and hence extend the shelf-life of lean beef.

Vitamin A and its esters are commonly found in topical applications because of their advantageous properties, however, have the drawback that are highly sensitive to ultraviolet radiation. The aim of this work was to develop and characterize a novel formulation of solid lipid nanoparticles suitable for topical applications in order to protect vitamin A from degradation. Vitamin A-loaded nanoparticles were successfully prepared by hot homogenization employing Gelucire 44/14 and cetyl alcohol as carrier materials, showing an entrapment efficiency of more than 90%. Particle size, measured by dynamic light scattering, was ca. 40 nm, while transmission electron microscopy images showed that dried nanoparticles were spherical with an average size of about 30-50 nm. Small angle X-ray scattering was used to study their aspect ratio and their physicochemical properties were evaluated by differential scanning calorimetry, infrared spectroscopy and X-ray powder diffraction, additionally, stability of vitamin A was studied by UV-Vis spectroscopy.

This study presents fast and reliable immunoassays to provide appropriate toxin concentration data in 5 min to monitor industrial culture processes. A latex agglutination reagent for semi-quantification of Clostridium novyi B α-toxin was developed, characterized and validated in actual industrial conditions. This reagent represents a useful real-time assay, generating data during toxin preparation process and thus increasing efficiency of α-toxin industrial production from C. novyi B cultures.

El objetivo de este estudio fue desarrollar, caracterizar y comparar nano y microemulsiones estables incorporando quercetina como compuesto bioactivo. Se estudiaron emulsiones aceite:agua con beta-lactoglobulina, utilizando dos procesos de elaboración: homogeneizador de alta velocidad (Ultra-turrax) y homogeneizador de alta presión (HPH, Emulsiflex-C5, 500-1200bar). Se evaluó la variación en la concentración de emulsionante, fracción volumétrica de aceite e incorporación de hidrocoloides. La formación y la estabilidad de las emulsiones se analizaron por su perfil de tamaño de gota (Malvern-Zetasizer Nano ZS) y constantes cinéticas de desestabilización obtenidas a partir del estudio de la retro dispersión de la luz en el tiempo (Turbiscan-Classic). Se determinó que utilizando el Ultra-turrax se logra formar nanoemulsiones con mayor estabilidad a una concentración de 2.2 % (m/v) de proteína y 0.2 % (m/v) de goma guar, mientras que utilizando HPH con una concentración de 1.5 % (m/v) de beta-lactoglobulina y sin necesidad de hidrocoloides se logra una emulsión de adecuado tamaño de gota (d<500nm) y mayor estabilidad (α<0.05). Las nanoemulsiones elaboradas con HPH presentaron mayor porcentaje de encapsulación de quercetina.

Introduction: group B streptococcal infection (GBS) may seriously affect mother and fetuses during pregnancy, and the newborn after delivery. Today, diagnosis of colonization during pregnancy is done by means of microbiological methods of vaginal and rectal exudates.Objective:to develop fast and low cost methods to detect the GBS specific group antigen in vaginal-rectal exudates.Method: we used two EGB strains, one of the (IH23) autochthonous and the reference strain O90R, that only expresses the group specific polysaccharide. We prepared a polyclonal antiserum for each one of them which was used to conduct an immunochromatographic test and a latex agglutination test. We used bacterial culture, EGB purified polysaccharides and vagina,-rectal samples as control.Results: detection limits obtained for the immunochromatographic test were 210 µg/ml and 50 µg/ml for purified polysaccharides and cell wall, respectively, there being no EGB antigens detected in the clinical samples analyzed. Latex detection limit was 65 µg/ml compared to purified polysaccharides of IH23 culture supernatant and 6,5 x 107 UFC/ml of IH23. Sensitivity and specificity for latex was 30% and 90% respectively.Conclusions: the methods used failed to reach the detection limit required for its application in our clinical samples. This agrees with what is described in bibliography about quick tests based on antigen-antibody reactions and indicated the need to add previous extraction and concentration steps or to improve the quality of the immunologic reagents used.

Monodisperse core–shell stable latexes with reactive methylchloride surface functionalities were prepared at two different reaction temperatures. The reaction temperature played an important role in the amount of reactive functional groups. The covalent coupling had an efficiency of more than 50%. Antibodies covalently bound to functionalized polystyrene beads were used to detect corresponding antigens by nephelometry.