Convergence insufficiencyCentral mini-plication of medial rectusDiplopiaConsecutive esotropiaSurgical overcorrection / Detection of anti-SARS-CoV-2 antibodies in tears: Ocular surface immunity to COVID-19

Objetivos Evaluar la presencia de anticuerpos IgA e IgG específicos del SARS-CoV-2 en lágrima de sujetos no vacunados y vacunados contra la COVID-19 con antecedentes de infección SARS-CoV-2. Correlacionar los resultados en lágrima con los de saliva y sangre, datos clínicos y regímenes de vacunación. Métodos Estudio transversal que incluyó a sujetos con antecedentes de infección SARS-CoV-2, tanto no vacunados como vacunados contra la COVID-19. Se recogieron 3muestras: lágrima, saliva y sangre. Se analizaron IgA e IgG frente a S-1 SARS-CoV-2 con ELISA semicuantitativo. Resultados Treinta sujetos, con una edad media 36,4±10, varones 13/30 (43,3%) con historia de infección SARS-CoV-2 leve; 13/30 (43,3%) habían recibido un régimen de 2 dosis y 13/30 (43,3%) un régimen de 3 dosis de vacunación anti-COVID-19, 4/30 (13,3%) no estaban vacunados. Todos los sujetos con vacunación completa presentaron IgA detectable en los 3biofluidos. Entre los no vacunados, se detectó IgA en 3/4 sujetos en lágrima y saliva, mientras que no se detectó IgG. No se observaron diferencias entre la pauta de vacunación de 2 y 3 dosis según los títulos IgA-IgG. Conclusiones Anticuerpos IgA e IgG del SARS-CoV-2 están presentes en lágrimas de pacientes con antecedentes de COVID-19 leve, lo que destaca el papel de la superficie ocular como primera línea de defensa frente a la infección. La mayoría de los sujetos no vacunados presentaron IgA a largo plazo en lágrima y saliva. La inmunización híbrida (infección natural más vacunación) parece potenciar las respuestas IgG mucosas y sistémicas. No se observaron diferencias entre la pauta de 2 y 3 dosis (AU)

Purpose To evaluate the presence of SARS-CoV-2 specific IgA and IgG antibodies in tears of unvaccinated and anti-COVID-19 vaccinated subjects with previous history of SARS-CoV-2 infection. To compare results in tears with those in saliva and serum and correlate with clinical data and vaccination regimens. Methods Cross-sectional study including subjects with a previous history of SARS-CoV-2 infection, both unvaccinated and vaccinated against COVID-19. Three samples were collected: tears, saliva and serum. IgA and IgG antibodies against S-1 protein of SARS-CoV-2 were analyzed with a semi-quantitative ELISA. Results Thirty subjects, mean age 36.4±10, males 13/30 (43.3%) with history of mild SARS-CoV-2 infection were included. 13/30 (43.3%) subjects had received a 2-dose regimen and 13/30 (43.3%) a 3-dose regimen of anti-COVID-19 vaccine, 4/30 (13.3%) subjects were unvaccinated. All the participants with full anti-COVID-19 vaccination (2-or 3-doses) presented detectable anti-S1 specific IgA in all 3biofluids, tears, saliva and serum. Among unvaccinated subjects, specific IgA was detected in 3/4 subjects in tears and saliva, whereas IgG was not detected. Considering IgA and IgG antibodies titers, no differences were observed between the 2- and 3-dose vaccination regimen. Conclusions SARS-CoV-2-specific IgA and IgG antibodies were detected in tears after mild COVID-19, highlighting the role of the ocular surface as a first line of defense against infection. Most naturally infected unvaccinated individuals exhibit long-term specific IgA in tears and saliva. Hybrid immunization (natural infection plus vaccination) appears to enhance mucosal and systemic IgG responses. However, no differences were observed between the 2- and 3-dose vaccination schedule (AU)

[Detection of anti-SARS-CoV-2 antibodies in tears: Ocular surface immunity to COVID-19]. / Detección de anticuerpos anti-SARS-CoV-2 en lágrimas: inmunidad de la superficie ocular frente a COVID-19.

Purpose: To evaluate the presence of SARS-CoV-2 specific IgA and IgG antibodies in tears of unvaccinated and anti-COVID-19 vaccinated subjects with previous history of SARS-CoV-2 infection. To compare results in tears with those in saliva and serum and correlate with clinical data and vaccination regimens. Methods: Cross-sectional study including subjects with a previous history of SARS-CoV-2 infection, both unvaccinated and vaccinated against COVID-19. Three samples were collected: tears, saliva and serum. IgA and IgG antibodies against S-1 protein of SARS-CoV-2 were analyzed with a semi-quantitative ELISA. Results: Thirty subjects, mean age 36.4 ± 10, males 13/30 (43.3%) with history of mild SARS-CoV-2 infection were included. 13/30 (43.3%) subjects had received a 2-dose regimen and 13/30 (43.3%) a 3-dose regimen of anti-COVID-19 vaccine, 4/30 (13.3%) subjects were unvaccinated. All the participants with full anti-COVID-19 vaccination (2-or 3-doses) presented detectable anti-S1 specific IgA in all 3 biofluids, tears, saliva and serum. Among unvaccinated subjects, specific IgA was detected in 3/4 subjects in tears and saliva, whereas IgG was not detected. Considering IgA and IgG antibodies titers, no differences were observed between the 2- and 3-dose vaccination regimen. Conclusions: SARS-CoV-2-specific IgA and IgG antibodies were detected in tears after mild COVID-19, highlighting the role of the ocular surface as a first line of defense against infection. Most naturally infected unvaccinated individuals exhibit long-term specific IgA in tears and saliva. Hybrid immunization (natural infection plus vaccination) appears to enhance mucosal and systemic IgG responses. However, no differences were observed between the 2- and 3-dose vaccination schedule.

Detection of anti-SARS-CoV-2 antibodies in tears: Ocular surface immunity to COVID-19.

PURPOSE: To evaluate the presence of SARS-COV-2 specific IgA and IgG antibodies in tears of unvaccinated and anti-COVID-19 vaccinated subjects with previous history of SARS-COV-2 infection. To compare results in tears with those in saliva and serum and correlate with clinical data and vaccination regimens. METHODS: Cross-sectional study including subjects with a previous history of SARS-CoV-2 infection, both unvaccinated and vaccinated against COVID-19. Three samples were collected: tears, saliva and serum. IgA and IgG antibodies against S-1 protein of SARS-CoV-2 were analyzed with a semi-quantitative ELISA. RESULTS: 30 subjects, mean age 36.4 ±â€¯10, males 13/30 (43.3%) with history of mild SARS-CoV-2 infection were included. 13/30 (43.3%) subjects had received a 2-dose regimen and 13/30 (43.3%) a 3-dose regimen of anti-COVID-19 vaccine, 4/30 (13.3%) subjects were unvaccinated. All the participants with full anti-COVID-19 vaccination (2-or 3-doses) presented detectable anti-S1 specific IgA in all three biofluids, tears, saliva and serum. Among unvaccinated subjects, specific IgA was detected in 3/4 subjects in tears and saliva, whereas IgG was not detected. Considering IgA and IgG antibodies titers, no differences were observed between the 2- and 3-dose vaccination regimen. CONCLUSIONS: SARS-CoV-2-specific IgA and IgG antibodies were detected in tears after mild COVID-19, highlighting the role of the ocular surface as a first line of defense against infection. Most naturally infected unvaccinated individuals exhibit long-term specific IgA in tears and saliva. Hybrid immunization (natural infection plus vaccination) appears to enhance mucosal and systemic IgG responses. However, no differences were observed between the 2- and 3-dose vaccination schedule.

Antibodies from Rabbits Immunized with HIV-1 Clade B SOSIP Trimers Can Neutralize Multiple Clade B Viruses by Destabilizing the Envelope Glycoprotein.

The high viral diversity of HIV-1 is a formidable hurdle for the development of an HIV-1 vaccine. Elicitation of broadly neutralizing antibodies (bNAbs) would offer a solution, but so far immunization strategies have failed to efficiently elicit bNAbs. To overcome these obstacles, it is important to understand the immune responses elicited by current HIV-1 envelope glycoprotein (Env) immunogens. To gain more insight, we characterized monoclonal antibodies (MAbs) isolated from rabbits immunized with Env SOSIP trimers based on the clade B isolate AMC008. Four rabbits that were immunized three times with AMC008 trimer developed robust autologous and sporadic low-titer heterologous neutralizing responses. Seventeen AMC008 trimer-reactive MAbs were isolated using antigen-specific single B-cell sorting. Four of these MAbs neutralized the autologous AMC008 virus and several other clade B viruses. When visualized by electron microscopy, the complex of the neutralizing MAbs with the AMC008 trimer showed binding to the gp41 subunit with unusual approach angles, and we observed that their neutralization ability depended on their capacity to induce Env trimer dissociation. Thus, AMC008 SOSIP trimer immunization induced clade B-neutralizing MAbs with unusual approach angles with neutralizing effects that involve trimer destabilization. Optimizing these responses might provide an avenue to the induction of trimer-dissociating bNAbs. IMPORTANCE Roughly 32 million people have died as a consequence of HIV-1 infection since the start of the epidemic, and 1.7 million people still get infected with HIV-1 annually. Therefore, a vaccine to prevent HIV-1 infection is urgently needed. Current HIV-1 immunogens are not able to elicit the broad immune responses needed to provide protection against the large variation of HIV-1 strains circulating globally. A better understanding of the humoral immune responses elicited by immunization with state-of-the-art HIV-1 immunogens should facilitate the design of improved HIV-1 vaccine candidates. We identified antibodies with the ability to neutralize multiple HIV-1 viruses by destabilization of the envelope glycoprotein. Their weak but consistent cross-neutralization ability indicates the potential of this epitope to elicit broad responses. The trimer-destabilizing effect of the neutralizing MAbs, combined with detailed characterization of the neutralization epitope, can be used to shape the next generation of HIV-1 immunogens to elicit improved humoral responses after vaccination.

Typhim vi immunization assists to discriminate primary antibody responses in hematological malignancies.

Assessment of specific antibody (Ab) production to polysaccharide antigens is clinically relevant, identifying patients at risk for infection by encapsulated bacteria and thus enabling a more rigorous selection of patients that can benefit of immunoglobulin replacement therapy. Classically, the gold-standard test is the measurement of antibody production to pure polysaccharide pneumococcal (PPV) immunization. Several factors, including introduction of conjugate vaccination schedule, serotyping analysis, high baseline Ab levels, have hindered the evaluation of polysaccharide antigens. This is even more difficult in secondary immunodeficiencies (SID), where patients can show secondary responses despite lack of primary antibody responses and present with recurrent or severe infections. Assessment of specific Ab production to pure Salmonella typhi Vi polysaccharide (TV) immunization has been proposed as a complementary test to PPV, given its low seroprevalence. To set the optimal cut-off value for PPV and TV response in SID, we tested different biostatistical methodologies, including ROC analysis, Youden index, Union index and Closest-topleft in a cohort of 42 SID patients and 24 healthy controls. The statistically chosen cut-offs value pre-post TV Ab ratio was ≥5, (sensitivity of 90%, specificity of 100%) and a postvaccination TV concentration of 28.5 U/mL (sensitivity of 90%, specificity of 95%), showing relevant clinical correlate.

Evaluation of Polysaccharide Typhim Vi Antibody Response as a predictor of Humoral Immunodeficiency in Haematological Malignancies.

An increasing healthcare challenge in the management of haematological malignancy (HM) is secondary immunodeficiency. From January 2019, the EMA included the evaluation of specific antibody (Ab) responses to better select patients for immunoglobulin replacement therapy (IgRT). We evaluated Ab responses to pneumococcal and Salmonella typhi pure polysaccharide immunization in a cohort of 42 HM patients and 24 healthy-controls. Pre-post specific Ab concentrations were measured by ELISA at 4 weeks. Globally, significantly lower Typhim Vi (TV) seroprevalence (9%) compared to 23-valent pneumococcal polysaccharide vaccine (PPV) (76%) (p <0.001) was observed. TV non responders (88%) were higher than PPV non responders (62%) (p <0.0001) and correlated better to infectious history. By ROC analysis, pre-post 5-fold TV increase was the best cut-off to discriminate HM with recurrent infections and controls (sensitivity 91%, specificity 100%). Despite the small sample cohort, our results suggest that specific anti-S typhi Ab response is a useful complementary assay in the diagnosis and management decision of SID to HM.

CGRP blockade by galcanezumab was not associated with reductions in signs and symptoms of knee osteoarthritis in a randomized clinical trial.

OBJECTIVE: This study tested whether galcanezumab, a humanized monoclonal antibody with efficacy against migraine, was superior to placebo for the treatment of mild or moderate osteoarthritis (OA) knee pain. METHOD: In a multicenter, double-blind, placebo- and celecoxib-controlled trial, patients with moderate to severe OA pain were randomized to placebo; celecoxib 200 mg daily for 16 weeks; or galcanezumab 5, 50, 120, and 300 mg subcutaneously every 4 weeks, twice. The primary outcome was change from baseline at Week 8 in Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC) pain subscore measured by 100 mm visual analog scale (VAS). The trial was considered positive if ≥1 dose of galcanezumab demonstrated ≥95% Bayesian posterior probability of superiority to placebo and ≥50% posterior probability of superiority to placebo by ≥9 mm. A planned interim analysis allowed termination of the study if posterior probability of superiority to placebo by ≥9 mm was ≤5%. Secondary endpoints included WOMAC function subscore and Patient Global Assessment (PGA) of OA. Safety and tolerability were also assessed. RESULTS: The study was terminated after interim analysis suggested inadequate efficacy. Celecoxib significantly reduced WOMAC pain subscore compared with placebo [-12.0 mm; 95% confidence interval (CI) -23 to -2 mm]. None of the galcanezumab arms demonstrated clinically meaningful improvement (range: 1.5 to -5.0 mm) or met the prespecified success criteria. No improvement in any secondary objective was observed. Galcanezumab was well tolerated by OA patients. CONCLUSIONS: This study failed to demonstrate sufficient statistical evidence that galcanezumab was efficacious for treating OA knee pain. STUDY IDENTIFICATION: NCT02192190.

Differential effects of once-weekly glucagon-like peptide-1 receptor agonist dulaglutide and metformin on pancreatic ß-cell and insulin sensitivity during a standardized test meal in patients with type 2 diabetes.

This substudy of the AWARD-3 trial evaluated the effects of the once-weekly glucagon-like peptide-1 receptor agonist, dulaglutide, versus metformin on glucose control, pancreatic function and insulin sensitivity, after standardized test meals in patients with type 2 diabetes. Meals were administered at baseline, 26 and 52 weeks to patients randomized to monotherapy with dulaglutide 1.5 mg/week (n = 133), dulaglutide 0.75 mg/week (n = 136), or metformin ≥1500 mg/day (n = 140). Fasting and postprandial serum glucose, insulin, C-peptide and glucagon levels were measured up to 3 h post-meal. ß-cell function and insulin sensitivity were assessed using empirical variables and mathematical modelling. At 26 weeks, similar decreases in area under the curve for glucose [AUCglucose (0-3 h)] were observed among all groups. ß-cell function [AUCinsulin /AUCglucose (0-3 h)] increased with dulaglutide and was unchanged with metformin (p ≤ 0.005, both doses). Dulaglutide improved insulin secretion rate at 9 mmol/l glucose (p ≤ 0.04, both doses) and ß-cell glucose sensitivity (p = 0.004, dulaglutide 1.5 mg). Insulin sensitivity increased more with metformin versus dulaglutide. In conclusion, dulaglutide improves postprandial glycaemic control after a standardized test meal by enhancing ß-cell function, while metformin exerts a greater effect on insulin sensitivity.

Localización atípica de metástasis de carcinoma de ovario detectada con 18F-FDG PET/TC / Atypical location of metastatic ovarian carcinoma detected by 18F-FDG PET/CT

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Interethnic variation of the MMP-9 microsatellite in Amerindian and Mexican Mestizo populations: considerations for genetic association studies.

We studied the interethnic variation of the MMP-9 microsatellite in the Mestizo and Amerindian populations using blood samples collected from 435 healthy unrelated individuals from the Central Valley of Mexico. DNA samples were genotyped using the -90 (CA)12-27 repeat near the MMP transcriptional start site using capillary electrophoresis. Our data were compared with those from African, Asian, and European populations (N = 729). Both Mestizo and Amerindian populations were in Hardy-Weinberg equilibrium (P ≥ 0.05). However, strong genetic heterogeneity was found within the Mestizo population (94%, P ≤ 0.0001), which exhibited the highest frequency of Amerindian, African, and European alleles. Likewise, Amerindians showed 6.7% variation among populations (P ≤ 0.0001), suggesting a genetic substructure potentially associated with linguistic affiliations. These findings were corroborated with principal component and population differentiation analyses, which showed relative proximity among the Mestizos and their historical parental populations: Asian (FST ≥ 0.05), European (FST ≥ 0.09), and African (FST ≥ 0.02). Nevertheless, important differences were found between Mestizo and Nahuas (P ≤ 0.0001), and between Mestizo and Me'Phaas (P ≤ 0.0001). These findings highlight the importance of determining local-specific patterns to establish the population variability of MMP-9 and other polymorphic markers. Validation of candidate markers is critical to identifying risk factors; however, this depends on knowledge of population genetic variation, which increases the possibility of finding true causative variants. We also show that dissimilar ethnic backgrounds might lead to spurious associations. Our study provides useful considerations for greater accuracy and robustness in future genetic association studies.

Photoinduced electron transfer fluorometric Hg(II) chemosensor based on a BODIPY armed with a tetrapod receptor.

From the great variety of BODIPY based-chemosensors able to determine Hg(2+), only a small portion has been applied to its determination in environmental and/or biological samples. The lack of studies on the analytical performance of the latter sensors makes interesting the development of investigations oriented to their possible analytical applications. The synthesis of a BODIPY derivative armed with a tetrapod receptor is described. The procedure is based on a previous publication, and the modifications performed to improve the synthesis include alternative procedures with different objectives, as the consecution of a multigram synthesis, improving the low yields of some of the previously proposed procedure steps, simplifying the experimental steps, achieving the desired purity requirements for use with analytical purposes, and enriching the characterization of the implied structures. The characteristics of its selectivity towards Hg(2+) have been investigated, and the OFF-ON fluorometric response, based on a photo-electron transfer (PET) mechanism, served as the base for the development of a method able to determine Hg(2+) in environmental waters at ng mL(-1) levels. The intrinsic fluorescence of the BODIPY core is inhibited and the probe exhibits a weak fluorescence (i.e. "OFF" state due to the deactivating PET effect). Upon complexation, Hg(2+) interacts with the lone-pair electrons on the nitrogen atoms of the receptor moiety so that the electronic transfer from the receptor to the photo-excited fluorophore is slowed down or switched off (i.e. "ON" state due to the suppression of the deactivating PET effect by coordination of the analyte to the probe). Regarding the complex photostability in aqueous solution, it is mandatory to conduct the experiments at darkness due to its photodegradation. The stoichiometry studies indicated a 1:2 relationship for the BODIPY-Hg(2+) complex. The high selectivity towards mercuric ions is considerably influenced by pH, being necessary to conduct the experiments in a pH value higher than 6. Calibration samples were prepared by adding appropriate amounts of Hg(2+) between 20.0-120.0 ng mL(-1), at a constant BODIPY concentration of 1 µmol L(-1). After agitating for 5 min at darkness, phosphate buffer (pH=7.50) was added, and it was diluted to the mark with water. Fluorescence measurements were carried out at 18 °C, exciting at 515 nm, and obtaining fluorescence emission at 538 nm. The method has been satisfactory applied to Hg(2+) determination in environmental water samples.

Enhanced MCR-ALS modeling of HPLC with fast scan fluorimetric detection second-order data for quantitation of metabolic disorder marker pteridines in urine.

This work presents the development of a liquid chromatographic method based on modeling entire fast scan fluorimetric detection second-order data with the multivariate curve resolution alternating least squares algorithm, for the simultaneous determination of five marker pteridines in urine samples. The modeling strategy involves the building of a single MCR-ALS model composed of matrices augmented in the spectral mode, i.e. time profiles remain invariant while spectra may change from sample to sample. This approach allowed us to separate and determine the whole analytes at once. The developed approach enabled us to determine five of the most important metabolic disorder marker pteridines: biopterin, neopterin, isoxanthopterin, pterin and xanthopterin, three of them presenting emission spectra with the same emission wavelength maxima. In addition, some of these analytes present overlapped time profiles. As a consequence of using the entire data sets, a considerable reduction of the data processing experimental time can be achieved. Results are compared with a previous strategy in which data were split in five different regions, and information about the figures of merit of the new strategy compared with the previously reported strategy is reported.

Determination of marker pteridines in urine by HPLC with fluorimetric detection and second-order multivariate calibration using MCR-ALS.

A liquid chromatographic method has been developed, in combination with the multivariate curve resolution-alternating least squares algorithm (MCR-ALS), for the simultaneous determination of marker pteridines in urine samples. A central composite design has been applied to optimize the factors influencing the separation (buffer concentration, buffer pH, flow rate, oven temperature, mobile-phase composition). A set of 15 calibration samples were randomly prepared, in a concentration range of 0.5-10.5 ng mL(-1) for neopterin, biopterin, and pterin; 4.0-8.0 ng mL(-1) for xanthopterin; and 0.5-4.5 ng mL(-1) for isoxanthopterin. The validation was carried out with fortified urine samples from healthy adults. The optimized conditions were a mobile-phase composition of 10 mM citric buffer at pH 5.44 and acetonitrile (94.5/5.5, v/v), a flow rate of 1.0 mL min(-1), and an oven temperature of 25 °C. The detection system consisted of a fast-scanning spectrofluorimeter, which allows obtaining of second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. In this work, MCR-ALS was used to cope with coeluting interferences, on account of the second-order advantage inherent to this algorithm which, in addition, is able to handle data sets deviating from trilinearity, like the high-performance liquid chromatography data analyzed in the present report. The developed approach enabled us to determine five pteridines, some of them with overlapped profiles, reducing the experimental time and reagent consumption. Ratio values for pteridines/creatinine in urine, for infected children with different pathologies, are reported in this work.

Analysis of antibiotics in fish samples.

The use of antibiotics in food-producing animals has generated considerable interest because the widespread administration of these drugs may lead to the development of resistant human pathogens. A large increase in the demand for seafood products has occurred in the last century. This has led to a concomitant increase in high-intensity aquaculture methods, characterized by high stock density and volume, and the heavy use of formulated feeds containing antibiotics, among other substances. Therefore, accurate and sensitive determination of antibiotic residues is now a necessity. In order to protect human health, the European Union and other regulatory authorities worldwide have established maximum residue limits (MRL) for antibiotic residues in animal products entering the human food chain. This paper reviews the most recent methods for analysis of antibiotic residues in fish.

Second-order multivariate calibration procedures applied to high-performance liquid chromatography coupled to fast-scanning fluorescence detection for the determination of fluoroquinolones.

Different second-order multivariate calibration algorithms, namely parallel factor analysis (PARAFAC), N-dimensional partial least-squares (N-PLS) and multivariate curve resolution-alternating least-squares (MCR-ALS) have been compared for the analysis of four fluoroquinolones in aqueous solutions, including some human urine samples (additional four fluoroquinolones were simultaneously determined by univariate calibration). Data were measured in a short time with a chromatographic system operating in the isocratic mode. The detection system consisted of a fast-scanning spectrofluorimeter, which allows one to obtain second-order data matrices containing the fluorescence intensity as a function of retention time and emission wavelength. The developed approach enabled us to determine eight analytes, some of them with overlapped profiles, without the necessity of applying an elution gradient, and thus significantly reducing both the experimental time and complexity. The study was employed for the discussion of the scopes of the applied second-order chemometric tools. The quality of the proposed technique coupled to each of the evaluated algorithms was assessed on the basis of the figures of merit for the determination of fluoroquinolones in the analyzed water and urine samples. Univariate calibration of four analytes led to limits of detection in the range 20-40 ng mL(-1) and root mean square errors for the validation samples in the range 30-60 ng mL(-1) (corresponding to relative prediction errors of 3-8%). The ranges for second-order multivariate calibration (using PARAFAC and N-PLS) of the remaining four analytes were: limit of detection, 2-8 ng mL(-1), root mean square errors, 3-50 ng mL(-1) and relative prediction errors, 1-5%.

Second-order data obtained by time-resolved room temperature phosphorescence. A new approach for PARAFAC multicomponent analysis.

A second-order multivariate calibration approach, based on a combination of PARAFAC with time-resolved room temperature phosphorescence (RTP), has been applied to resolve a binary mixture of Phenanthrene and 1,10-Phenanthroline, as model compounds. The RTP signals were obtained in aqueous beta-cyclodextrin solutions, in the presence of several heavy atom containing compounds. No deoxygenation was necessary to obtain the phosphorescence signals, which adds simplicity to the method. The resolution of the model compounds was possible in base to the differences in the delay-time of the RTP signals of the investigated analytes, opening a new approach for second-order data generation and subsequent second order multivariate calibration.

Genetic analysis of the Hispano-Breton heavy horse.

Hispano-Breton (HB) is a horse breed with a recent mixed ancestry. It was developed in the 1930s by crossing local mares with Breton draught horses imported from France. Nowadays it is considered to be in a vulnerable situation due to census decline. To genetically characterize the breed and to set up the basis for a conservation programme, we have employed two types of molecular markers: a 347-bp D-loop mitochondrial DNA (mtDNA) fragment and 13 microsatellite loci. A representative sample of 53 HB individuals was analysed together with a sample of 40 Pura Raza Española horses for comparison. Both types of markers revealed a high level of genetic diversity in the HB breed, emphasizing the importance of its conservation. The construction of a phylogenetic network with mtDNA sequences including various Iberian breeds and European heavy horses provided an overall picture of the ubiquitous appearance of HB matrilines with respect to other breeds and revealed the singularity of certain HB maternal lineages. Despite the high allelic richness found in HB horses, microsatellite analysis evidenced a certain degree of inbreeding as a consequence of the type of management generally used for local breeds.

On line photochemically induced excitation-emission-kinetic four-way data: analytical application for the determination of folic acid and its two main metabolites in serum by U-PLS and N-PLS/residual trilinearization (RTL) calibration.

The determination of folic acid and its two main serum metabolites, 5-methyltetrahydrofolic acid and tetrahydrofolic acid, has been accomplished using four-way data modelled by the third-order multivariate calibration methods unfolded and N-dimensional partial least-squares (U-PLS and N-PLS), in combination with the separate procedure known as residual trilinearization (RTL). The four-way data were acquired by following the photochemical reaction of these compounds by on line irradiation with a UV lamp. The excitation-emission matrices (EEMs) were recorded as a function of the irradiation time, using a fast scanning spectrofluorimeter. The method achieves selectivity from the different rates at which the corresponding photoproducts of the folic acid derivatives are formed and degraded. Several N-dimensional chemometric algorithms were used and the method was applied to the determination of these compounds in serum samples. The best algorithms to perform the multivariate calibration were U-PLS and N-PLS in combination with the separate residual trilinearization procedure, achieving the second-order advantage. The approach allows minimizing or eliminating traditionally time-consuming sample pre-treatments and can facilitate quantifying an analyte in its native environment.

Photoinduced fluorimetric determination of folic acid and 5-methyltetrahydrofolic acid in serum using the kinetic evolution of the emission spectra accomplished with multivariate second-order calibration methods.

Second-order multivariate calibration methods in combination with a continuous flow system, which allows for the continuous on-line irradiation of the analytes, have been employed for the determination of folic acid and its main metabolite 5-methyltetrahydrofolic acid in serum samples. An experimental central composite design, together with response surface methodology, has been used to find the optimum instrumental variables to perform the photochemical reaction. The time evolution of the emission spectra of the generated photoproducts, in the range 330-540 nm, after irradiation at 275 nm for 20 min, provided the three-way data set employed. On the basis of the differences on the kinetic rates of the photoreaction of both analytes, direct determination of the compounds in human plasma has been accomplished. The second-order methods assayed were parallel factor analysis (PARAFAC), self-weighted alternating trilinear decomposition (SWATLD), and unfolded partial least-squares (U-PLS), multidimensional partial least-squares (N-PLS), and bilinear least-squares (BLLS), all three in combination with the residual bilinearization procedure (RBL).