Novel Additive Manufactured Multielectrode Electrochemical Cell with Honeycomb Inspired Design for the Detection of Methyl Parathion in Honey Samples.

The development and increase in the number of crops recently have led to the requirement for greater efficiency in world food production and greater consumption of pesticides. In this context, the widespread use of pesticides has affected the decrease in the population of pollinating insects and has caused food contamination. Therefore, simple, low-cost, and quick analytical methods can be interesting alternatives for checking the quality of foods such as honey. In this work, we propose a new additively manufactured (3D-printed) device inspired by a honeycomb cell, with 6 working electrodes for the direct electrochemical analysis of methyl parathion by reduction process monitoring in food and environmental samples. Under optimized parameters, the proposed sensor presented a linear range between 0.85 and 19.6 µmol L-1, with a limit of detection of 0.20 µmol L-1. The sensors were successfully applied in honey and tap water samples by using the standard addition method. The proposed honeycomb cell made of polylactic acid and commercial conductive filament is easy to construct, and there is no need for chemical treatments to be used. These devices based on 6 working electrodes array are versatile platforms for rapid, highly repeatable analysis in food and environment, capable of performing detection in low concentrations.

Low-cost, facile droplet modification of screen-printed arrays for internally validated electrochemical detection of serum procalcitonin.

This manuscript presents the design and facile production of screen-printed arrays (SPAs) for the internally validated determination of raised levels of serum procalcitonin (PCT). The screen-printing methodology produced SPAs with six individual working electrodes that exhibit an inter-array reproducibility of 3.64% and 5.51% for the electrochemically active surface area and heterogenous electrochemical rate constant respectively. The SPAs were modified with antibodies specific for the detection of PCT through a facile methodology, where each stage simply uses droplets incubated on the surface, allowing for their mass-production. This platform was used for the detection of PCT, achieving a linear dynamic range between 1 and 10 ng mL-1 with a sensor sensitivity of 1.35 × 10-10 NIC%/ng mL-1. The SPA produced an intra- and inter-day %RSD of 4.00 and 5.05%, with a material cost of £1.14. Internally validated human serum results (3 sample measurements, 3 control) for raised levels of PCT (>2 ng mL-1) were obtained, with no interference effects seen from CRP and IL-6. This SPA platform has the potential to offer clinicians vital information to rapidly begin treatment for "query sepsis" patients while awaiting results from more lengthy remote laboratory testing methods. Analytical ranges tested make this an ideal approach for rapid testing in specific patient populations (such as neonates or critically ill patients) in which PCT ranges are inherently wider. Due to the facile modification methods, we predict this could be used for various analytes on a single array, or the array increased further to maintain the internal validation of the system.

Highly efficient production of rabies virus glycoprotein G ectodomain in Sf9 insect cells.

In the present study, we developed a complete process to produce in insect cells a high amount of the ectodomain of rabies virus glycoprotein G (GE) as suitable antigen for detecting anti-rabies antibodies. Using the baculovirus expression vector system in Sf9 insect cells combined with a novel chimeric promoter (polh-pSeL), the expression level reached a yield of 4.1 ± 0.3 mg/L culture, which was significantly higher than that achieved with the standard polh promoter alone. The protein was recovered from the cell lysates and easily purified in only one step by metal ion affinity chromatography, with a yield of 95% and a purity of 87%. Finally, GE was successfully used in an assay to detect specific antibodies in serum samples derived from rabies-vaccinated animals. The efficient strategy developed in this work is an interesting method to produce high amounts of this glycoprotein.

Paenibacillus xylanivorans sp. nov., a xylan-degrading bacterium isolated from decaying forest soil.

A xylanolytic bacterial strain, named A59T, was isolated from a forest soil consortium in southern Argentina. Strain A59T is a Gram-stain-positive, facultative anaerobic, endospore-forming and rod-shaped bacterium. Its optimal growth conditions are 30 °C (range, 28-37 °C), pH 7 (range, pH 5-10) and it tolerates up to 7 % of NaCl (range, 2-7 %). Chemotaxonomic analysis revealed that strain A59Tpossesses meso-diaminopimelic acid in the cell wall. It contains menaquinone MK-7 as the predominant isoprenoid quinone and the major fatty acid is anteiso-C15 : 0 (35.1 %), with a moderate amount of C16 : 0 (6.9 %). According to 16S RNA gene sequence analysis, the isolate is phylogenetically placed in the same cluster as Paenibacillus taichungensis BCRC 17757T (99.7 % nucleotide sequence identity) and Paenibacillus pabuli NBRC 13638T (99.1 %) and is closely related to Paenibacillus tundrae A10bT (98.8 %). However, phylogenetic studies based on the housekeeping gyrB gene placed A59T in a separate branch from all other related type strains. Furthermore, the results of whole genome average nucleotide identity analysis (gANI) with related type strains was lower than 91.10 % and the digital DNA-DNA hybridization value was lower than 44.30 %, which are below the threshold values for separating two species. The DNA G+C content was estimated as 46.09 mol%, based on genome sequencing. On the basis of these results, A59T represents a new species of the genus Paenibacillus, and we propose the name Paenibacillusxylanivorans sp. nov. The type strain is A59T (=DSM 107920T=NCIMB 15123T).

Detection of Sarcocystis aucheniae in blood of llama using a duplex semi-nested PCR assay and its association with cyst infestation.

The protozoon Sarcocystis aucheniae is the causative agent of South American camelid (SAC) sarcocystosis. Infections are characterized by the presence of cysts in muscles which are in size and appearance similar to rice grains. As consumption of insufficiently cooked infected meat produces gastroenteritis, cyst-containing SAC meat is confiscated by sanitary authorities or depreciated with serious economic consequences for SAC breeders. In this work, a duplex semi-nested PCR was designed to simultaneously detect parasite and llama DNA in host blood samples. Species-specific regions of S. aucheniae 18S rRNA gene and Lama glama 16S mitochondrial gene were amplified, yielding bands of 583 and 257 bp, respectively, and separated by gel electrophoresis. The method proved to be highly sensitive, with a detection limit lower than one parasite per milliliter blood, and the inclusion of primers to detect llama-specific DNA resulted useful as a methodological control. Blood samples collected from llamas of Argentina and Bolivia (n = 225) were analyzed using this method, and 18.7 % resulted positive for S. aucheniae. No correlation was found between PCR results and llama age, sex or the finding of macroscopic cysts in meat after slaughter. Lack of molecular detection in the blood of some llamas harboring macrocysts suggests that parasite circulation in the bloodstream after encystment is under the detection threshold of the test or even absent, while PCR positive results in cyst-infested animals suggests that prior exposure to the parasite does not impede subsequent infections. The described method can be useful to detect active foci of infection, to assess the effectiveness of parasiticide treatments, and for the surveillance and tracing of definitive hosts.

Identification of Lama glama as Reservoirs for Acinetobacter lwoffii.

South American Camelids have an increasing relevance in local economies, worldwide. These animals are bred for their meat, fur and as companion and therapy animals. Thus, their sanitary status should be well-established. According to the OIE (World Organization for Animal Health), respiratory infections mainly produced by Pasteurella spp. have been reported for camelids. It has been stated that this microorganism causes a mild disease, although many authors report it is an important cause of mortality among alpacas. Nevertheless, the incidence of infection by Pasteurella spp. in camelids still needs to be investigated. The aim of the present study was to analyze the occurrence of nasopharyngeal colonization of Lama glama by respiratory bacteria, and to assess the usefulness of serological tests for clinical diagnosis. The colonization was studied by culture techniques carried out with material taken by nasopharyngeal swabs. Bacterial isolates were first phenotypically characterized and then identified by MALDI/TOF-MS. The presence of specific serum antibodies was studied by ELISA and Western blot. In the present work Pasteurella spp. was not found. Nevertheless, we report for the first time, the colonization of L. glama by bacteria of the Acinetobacter lwoffii, at a reliable level in 19.4% of the animals. Acinetobacter species are found in different environmental sources, as well as vegetables, animals, and humans, and their role in infections has recently gained relevance. The results presented herein contribute to a better understanding of the respiratory microbiota in camelids, and increase the knowledge about environmental distribution of Acinetobacter non-baumanii species. Given that these respiratory bacteria might be the cause of infection among cattle, and even humans, this report highlights the need for further research.

Production of a monoclonal antibody against serum immunoglobulin M of South American camelids and assessment of its suitability in two immunoassays.

A monoclonal antibody (mAb) was produced against immunoglobulin M (IgM) of South American camelids. A single radial immunodiffusion (SRID) assay and a competitive enzyme-linked immunosorbent assay (ELISA) were developed to measure IgM in serum samples. Isotype and specificity of the mAb were assessed. The performance of the SRID assay was preliminarily evaluated in terms of working range, plate stability over a 4-week period, and initial intra- and interassay variation. The concentration of IgM was determined in 55 samples by SRID assay and ELISA, and results were not significantly different by t-test (0.64 ± 0.19 mg/ml for the SRID assay, and 0.58 ± 0.24 mg/ml for ELISA; P = 0.1489). The mAb was shown to be stable over the 4-week evaluation period, and the SRID assay was reproducible when tested in triplicate for intra-assay variability and in quadruplicate for interassay variability, with a percentage coefficient of variation of less than or equal to 5%. Also, the SRID assay proved to be sensitive enough to measure IgM levels in undiluted serum samples, and had a good correlation with ELISA. The current study is intended to submit a preliminary report of a mAb against IgM of South American camelids, and suggest the future potential of the mAb developed for diagnostic application, including use in the SRID assay.

Antibody labeling with Remazol Brilliant Violet 5R, a vinylsulphonic reactive dye.

Colloidal gold is the first choice for labeling antibodies to be used in Point Of Care Testing. However, there are some recent reports on a family of textile dyes-named "reactive dyes"-being suitable for protein labeling. In the present article, protein labeling conditions were optimized for Remazol Brilliant Violet 5R, and the sensitivity of the labeled antibodies was assessed and compared with that of colloidal-gold labeled antibodies. Also, the accelerated stability was explored. Optimal conditions were pH 10.95, dye:Ab molar ratio of 264 and an incubation time of 132 min. Labeled antibodies were stable, and could be successfully used in a slot blot assay, detecting as low as 400 ng/mL. Therefore, the present work demonstrates that vinylsulphonic reactive dyes can be successfully used to label antibodies, and are excellent candidates for the construction of a new generation of Point of Care Testing kits.

Lipoteichoic acid from Lactobacillus rhamnosus GG as an oral photoprotective agent against UV-induced carcinogenesis.

Probiotics are live micro-organisms that when administered in adequate amounts confer a health benefit on the host. Cell surface molecules of these micro-organisms are being studied in relation to their ability to interact with the host. The cell wall of lactobacilli possesses lipoteichoic acids (LTA) which are molecules with immunomodulatory properties. UV radiation (UVR) has been proposed as the main cause of skin cancer because of its mutagenic and immunosuppressive effects. Photoprotection with some nutrition interventions including probiotics has recently been shown. The aim of the present study was to investigate whether the oral administration of purified LTA from Lactobacillus rhamnosus GG can modulate the immune-suppressive effect of UVR and skin tumour development in female Crl:SKH-1-hrBR mice. For this purpose, two irradiation models were studied: (1) a chronic irradiation scheme consisting of daily irradiations during twenty consecutive days and (2) a long-term irradiation schedule, irradiating the animals three times per week, during 34 weeks for tumour development. The results showed that T-cells in the inguinal lymph node of LTA-treated mice produced higher levels of (1) interferon-γ and (2) a number of total, helper and cytotoxic T-cells compared with non-treated mice. Moreover, a significant delay in tumour appearance was found in LTA-treated mice. An increased IgA⁺ cell number was found in the small intestine together with a higher number of activated dendritic cells in the mesenteric lymph nodes. The latter results might be indicative of a direct effect of LTA in the gut, affecting the cutaneous immune system and restoring homeostasis through the gut-skin axis.

Skin exposure to chronic but not acute UV radiation affects peripheral T-cell function.

Ultraviolet (UV) radiation (UVR) produces deleterious effects that may finally lead to carcinogenesis. These adverse effects include tissue inflammation, free radical formation with consequent oxidation of proteins and lipids, DNA damage, and immune function suppression. The aim of this study was to evaluate the effects of UVR at the local and systemic levels following acute (4 consecutive days with 0.5 minimal erythema dose [MED]) or chronic (20 consecutive days with 0.25 MED) exposure. Locally, histological alterations and epidermal T-cell populations were studied. Systemically, inguinal lymph-node and spleen T cells were analyzed with respect to proliferative response and cytokine production against a nonspecific mitogen. Lymph-node T-cell populations were also characterized. Our results indicated that while both acute and chronic UVR produced epidermal hyperplasia and a decrease in epidermal T-cell density, acute UVR increased T-cell proliferative response, while chronic UVR produced the opposite effect, shifting the cytokine production toward a Th2/Treg profile. Therefore, even though acute irradiation produced a direct effect on skin, it did not correlate with a marked modification of overall T-cell response, which is in contrast to marked effects in chronically irradiated animals. These findings may contribute to understanding the clinical relevance of occupational UVR exposure, typically related to outdoor activities, which is associated with nonmelanoma skin carcinogenesis.

Anti-ribonucleoproteins autoantibodies in patients with systemic autoimmune diseases. Relation with cutaneous photosensitivity.

A common feature between patients with a certain group of systemic autoimmune pathologies (SAPs) with rheumatic component, such as lupus erythematosus (LE) in all its forms, is the presence of cutaneous photosensitivity (CP) as well as the existence of autoantibodies (Aabs). These Aabs have also high incidence in other SAPs that do not present CP, like primary Sjögren's syndrome and rheumatoid arthritis. Cutaneous photosensitivity is a condition that consists of an exacerbated skin reaction to solar radiations; its incidence can reach 90% in systemic LE. The mechanisms involved in the development of CP have been extensively studied focusing on different approaches; however, the exact mechanism has not been fully elucidated yet. There are many theories that relate specifically the presence of circulating anti-Ro/SS-A Aabs with the CP phenomenon, though there are several studies which are in disagreement. In this study, we evaluated the Aabs profile (anti-Ro/SS-A 52 kDa, anti-Ro/SS-A 60 kDa, anti-La/SS-B, anti-Sm and ANAs) as well as their titer or reactivity, in a local cohort of 169 patients with SAPs. We related those Aabs profiles and titers with the presence or absence of CP, and we found that there was no significant association between the presence of anti-Ro/SS-A Aabs and the occurrence of CP. On the other hand, a statistically significant positive association was found between CP and high reactivity anti-Sm Aabs, though this fact could be biased by the incidence of both events in SLE patients. To sum up, in the particular population studied, there is no direct relationship between anti-Ro/SS-A Aabs and CP, which is in agreement with some authors and in disagreement with many others, contributing to the endless discussion of this issue.

Alterations in skin immune response throughout chronic UVB irradiation-skin cancer development and prevention by naproxen.

Skin exposure to UVB radiation has been reported to produce both a significant inflammatory response and marked immunosuppression. This work was aimed to evaluate whether the response of murine skin to an acute UVB dose was modified by pre-exposure to chronic UVB irradiation and by topical treatment with naproxen, a nonsteroidal anti-inflammatory drug. Moreover, the effect of naproxen on the incidence of UV-induced skin tumors was studied. Prostaglandin E(2) (PGE(2)) and tumor necrosis factor alpha (TNF-alpha) levels were increased 96 h post-UVB in acutely irradiated animals and both mediators were modified by topical naproxen application-PGE(2) was decreased while TNF-alpha was increased. Such inflammatory response was suppressed when mice were chronically irradiated. Naproxen application on chronically irradiated mice reduced the incidence of tumor lesions. Taken together, our data suggest that chronic UVB irradiation generates an immunosuppressive state that prevents skin cells from responding normally to an acute irradiation challenge, thus impairing the protective effect of TNF-alpha against skin tumor development. Furthermore, reduction in the incidence of tumor lesions by naproxen may be due to its ability to increase TNF-alpha levels as well as to decrease PGE(2).

Development of ELISAs for the measurement of IgM and IgG subclasses in sera from llamas (Lama glama) and assessment of the humoral immune response against different antigens.

Members of the Camelidae family possess a functional class of antibodies devoid of light chains (known as heavy chain antibodies, HCAbs). Three IgG isotypes have been identified (IgG(1), IgG(2) and IgG(3)); IgG(2) and IgG(3) are HCAbs whereas the IgG(1) has the conventional structure. Different subtypes of IgG(1) (IgG(1a) and IgG(1b)) and IgG(2) (IgG(2a), IgG(2b) and IgG(2c)) have been classified according to variations in the amino acids sequence of the hinge region. The single variable domain of HCAbs has been referred as VHH. Until now, the relative amount of each subclass has been inferred, but the lack of highly specific antibodies against HCAbs has been a limitation for their quantification. In a previous work, we produced specific polyclonal antibodies against IgG(2a), IgG(2b), IgG(2c) and IgG(3) by immunizing rabbits with synthetic and recombinant peptides corresponding to their hinge region. In this work we produced specific antisera against llama IgM and IgG(1). The anti-IgG(1) serum was obtained by immunizing rabbits with a recombinant fusion protein formed by GST fused to the CH(1) domain of the IgG(1). The anti-IgM serum was obtained by immunizing rabbits with IgM heavy chain. All these antisera were useful for the development of ELISAs for the measurement of IgM, total IgG and IgG subclasses. Sera from llamas (n=20) analyzed by ELISA gave the following values of immunoglobulins: IgG(1)=6.168+/-1.628 mg/ml; IgG(2)=0.684+/-0.310 mg/ml; IgG(3)=1.232+/-0.410 mg/ml; total IgG=8.933+/-1.815 mg/ml and IgM=1.027+/-0.308 mg/ml. These results indicate that HCAbs represent almost 25% of total IgG and the IgG(3) subtype is the predominant HCAb. We also analyzed the primary humoral immune response after immunization llamas with different antigens (BSA, BSA-DNP and dextran). Although it has been described that a few VHH clones are very efficient in the interaction with haptens, in this case the response against DNP was characterized by a delayed appearance of HCAbs in comparison with that of IgG(1). No anti-dextran response was observed in any of the isotypes analyzed.

Time-course evaluation and treatment of skin inflammatory immune response after ultraviolet B irradiation.

Skin exposure to high doses of ultraviolet B (UVB) radiation generates a severe inflammatory skin response. In the present study we aim to investigate, using in vitro and in vivo models, the time-course of the inflammatory skin immune response after an acute exposure to UVB irradiation, as well as its modulation by a topical non-steroidal anti-inflammatory drug (NSAID) treatment, naproxen. PGE2 production and TNF-alpha levels increase in a post-irradiation time-dependent manner both in vivo and in vitro. This production pattern is also reflected in the iNOS expression levels in vivo and in the IL-6 levels in vitro. Changes observed in these mediators are correlated with histological alterations and dermal infiltration after the acute UVB irradiation. Naproxen treatment notably reduces PGE2 production and iNOS expression, reflecting the COX-NOS crosstalk already reported, although it causes an important increment in TNF-alpha synthesis in the epidermis of irradiated mice. Taken together, our data indicates that the epidermis is severely damaged by UVB radiation but then it is able to fully recover, and that the immune response is modulated by the NSAID treatment, since it is able to reduce the levels of some mediators as well as it can increase others.

Skin damage and mitochondrial dysfunction after acute ultraviolet B irradiation: relationship with nitric oxide production.

BACKGROUND: Ultraviolet (UV) radiation is the main environmental carcinogen. It is able to induce injury in the keratinocytes, which triggers mechanisms in order to protect the skin against molecular alterations that may lead to the development of skin cancer. UVB is capable of producing genotoxic damage, directly or indirectly through reactive oxygen species, inducing DNA alterations and mutations. UVB radiation has also been associated with the generation of nitric oxide (NO), which is able to induce many physiological and physiopathological processes. The aim of the current study was to investigate the effect of UVB irradiation in hairless mice skin. METHODS: We evaluated the effect of an acute dose (200 mJ/cm(2)) of UVB irradiation correlating with histological alterations, nitric oxide synthase expression and activity, mitochondrial respiratory function, superoxide anion production and lipid peroxidation, 0, 6, 17 and 24 h post-irradiation treatment. RESULTS: Morphological analysis showed disruption of the epidermal stratum corneum and basale after UVB irradiation. The results indicated that skin UVB irradiation was associated with an increased cytosolic inducible nitric oxide synthase (iNOS) expression, inversely related to lipid peroxidation processes. An increase in mitochondrial superoxide anion (O(2) (*-)) and NO production 17 h post-irradiation was correlated with a mitochondrial dysfunction, all of them integrating the skin response to acute UVB irradiation. CONCLUSIONS: UVB irradiation of the skin produces morphological alterations as a consequence of the induction of molecular mechanisms associated with mitochondrial respiratory dysfunction and O(2) (*-) production, probably mediated by the increased mitochondrial NO production. On the other hand lipid peroxidation decrease inversely correlates with cytosolic iNOS expression, suggesting a protective role for the inflammatory response.

[Isotype switch in the response against T independent antigens by co-inoculation with T dependent antigens]. / Cambio del isotipo en la respuesta a antígenos T independientes por co-inoculación con antígenos T dependientes.

A classical paradigm in immunology establishes that for the isotype switch to take place in antibodies, it is a sine qua non condition that the antigen is presented by an antigen presenting cell to a helper T cell. In the present study an animal model of the immune response against two typical antigens was designed in BALB/c mice. Dextran was chosen as a T independent antigen (TIAg), and bovine seroalbumin (BSA) as a T dependant antigen (TDAg), and the response was studied, analyzing the isotypes of the specific antibodies produced. The results show that the response against dextran, in the presence of BSA, takes place with isotype switch, essentially from IgM to IgG1. These experiments suggest that BSA generates a switch inductor biochemical environment in its own processing pathway as well as in the dextran's. These results indicate that the exclusive association of TDAgs with isotype switch responses is inaccurate. Considering the proposed model, it seems unlikely the finding of a spontaneous in vivo case in which TIAgs enter the organism isolated; instead, it is much more probable that they would enter together with TDAgs, and in consequence the isotype switch would take place.

Cambio del isotipo en la respuesta a antígenos T independientes por co-inoculación con antígenos T dependientes. / [Isotype switch in the response against T independent antigens by co-inoculation with T dependent antigens]

A classical paradigm in immunology establishes that for the isotype switch to take place in antibodies, it is a sine qua non condition that the antigen is presented by an antigen presenting cell to a helper T cell. In the present study an animal model of the immune response against two typical antigens was designed in BALB/c mice. Dextran was chosen as a T independent antigen (TIAg), and bovine seroalbumin (BSA) as a T dependant antigen (TDAg), and the response was studied, analyzing the isotypes of the specific antibodies produced. The results show that the response against dextran, in the presence of BSA, takes place with isotype switch, essentially from IgM to IgG1. These experiments suggest that BSA generates a switch inductor biochemical environment in its own processing pathway as well as in the dextrans. These results indicate that the exclusive association of TDAgs with isotype switch responses is inaccurate. Considering the proposed model, it seems unlikely the finding of a spontaneous in vivo case in which TIAgs enter the organism isolated; instead, it is much more probable that they would enter together with TDAgs, and in consequence the isotype switch would take place.