Assessment of combined α-GAL enzyme activity and lyso-GL3 for Fabry disease screening in women with chronic kidney disease.

INTRODUCTION: The spectrum of clinical presentation of Fabry disease (FD) in women is broad and challenging. The aim is to evaluate the effectiveness of an alternative screening method for FD in women. METHODS: A collaborative multicenter cross-sectional study to evaluate the sensitivity and specificity of the combination of two tests (α-GAL enzyme activity assay and lyso-GL3 assay) for the diagnosis of FD in women. We included women with chronic kidney disease (CKD) stages 3 to 5, receiving conservative treatment or on dialysis programs, from different nephrology services in Brazil. RESULTS: We evaluated 1874 patients that underwent blood collection for α-GAL and lyso-GL3 assays. Isolated decreased α-GAL enzyme activity was found in 64 patients (3.5%), while isolated increased lyso-GL3 levels were found in 67 patients (3.6%), with one patient presenting alterations in both tests. All cases with low α-GAL enzyme activity and/or increased lyso-GL3 levels underwent genetic analysis for FD variants (132 performed GLA genetic test). Low α-GAL enzyme activity had higher sensitivity and specificity to detect FD compared to the other measures (elevated lyso-GL3 alone or both altered). The negative predictive value (NPV) of α-GAL activity was 99%, and the positive predictive value (PPV) was 9.2%. For lyso-GL3 assay, the specificity was 99.7% and the PPV was 2.9%, therefore considered inferior to α-GAL assay. Both assays altered, had higher PPV (100%) and higher NPV (99.7%) considered the best method. We found 7 cases of GLA gene variants found, resulting in an initial prevalence of 0.37% for FD in this sample female population. CONCLUSION: This study contributes to the diagnostic value of the biomarkers α-GAL and lyso-GL3 in the context of FD in women with CKD. The combination of these biomarkers was an effective approach for the diagnosis of the disease, with high PPV and NPV.

Multinational experience with next-generation sequencing: opportunity to identify transthyretin cardiac amyloidosis and Fabry disease.

Background: Sarcomeric hypertrophic cardiomyopathy (HCM) must be differentiated from phenotypically similar conditions because clinical management and prognosis may greatly differ. Patients with unexplained left ventricular hypertrophy require an early, confirmed genetic diagnosis through diagnostic or predictive genetic testing. We tested the feasibility and practicality of the application of a 17-gene next-generation sequencing (NGS) panel to detect the most common genetic causes of HCM and HCM phenocopies, including treatable phenocopies, and report detection rates. Identification of transthyretin cardiac amyloidosis (ATTR-CA) and Fabry disease (FD) is essential because of the availability of disease-specific therapy. Early initiation of these treatments may lead to better clinical outcomes. Methods: In this international, multicenter, cross-sectional pilot study, peripheral dried blood spot samples from patients of cardiology clinics with an unexplained increased left ventricular wall thickness (LVWT) of ≥13 mm in one or more left ventricular myocardial segments (measured by imaging methods) were analyzed at a central laboratory. NGS included the detection of known splice regions and flanking regions of 17 genes using the Illumina NextSeq 500 and NovaSeq 6000 sequencing systems. Results: Samples for NGS screening were collected between May 2019 and October 2020 at cardiology clinics in Colombia, Brazil, Mexico, Turkey, Israel, and Saudi Arabia. Out of 535 samples, 128 (23.9%) samples tested positive for pathogenic/likely pathogenic genetic variants associated with HCM or HCM phenocopies with double pathogenic/likely pathogenic variants detected in four samples. Among the 132 (24.7%) detected variants, 115 (21.5%) variants were associated with HCM and 17 (3.2%) variants with HCM phenocopies. Variants in MYH7 (n=60, 11.2%) and MYBPC3 (n=41, 7.7%) were the most common HCM variants. The HCM phenocopy variants included variants in the TTR (n=7, 1.3%) and GLA (n=2, 0.4%) genes. The mean (standard deviation) ages of patients with HCM or HCM phenocopy variants, including TTR and GLA variants, were 42.8 (17.9), 54.6 (17.0), and 69.0 (1.4) years, respectively. Conclusions: The overall diagnostic yield of 24.7% indicates that the screening strategy effectively identified the most common forms of HCM and HCM phenocopies among geographically dispersed patients. The results underscore the importance of including ATTR-CA (TTR variants) and FD (GLA variants), which are treatable disorders, in the differential diagnosis of patients with increased LVWT of unknown etiology.

XAANTAL1 Reveals an Additional Level of Flowering Regulation in the Shoot Apical Meristem in Response to Light and Increased Temperature in Arabidopsis.

Light and photoperiod are environmental signals that regulate flowering transition. In plants like Arabidopsis thaliana, this regulation relies on CONSTANS, a transcription factor that is negatively posttranslational regulated by phytochrome B during the morning, while it is stabilized by PHYA and cryptochromes 1/2 at the end of daylight hours. CO induces the expression of FT, whose protein travels from the leaves to the apical meristem, where it binds to FD to regulate some flowering genes. Although PHYB delays flowering, we show that light and PHYB positively regulate XAANTAL1 and other flowering genes in the shoot apices. Also, the genetic data indicate that XAL1 and FD participate in the same signaling pathway in flowering promotion when plants are grown under a long-day photoperiod at 22 °C. By contrast, XAL1 functions independently of FD or PIF4 to induce flowering at higher temperatures (27 °C), even under long days. Furthermore, XAL1 directly binds to FD, SOC1, LFY, and AP1 promoters. Our findings lead us to propose that light and temperature influence the floral network at the meristem level in a partially independent way of the signaling generated from the leaves.

Relevance of Fluorodopa PET Scan in Dopamine Responsive Dystonia and Juvenile Parkinsonism: A Systematic Review.

BACKGROUND: Dopamine Responsive Dystonia (DRD) and Juvenile Parkinsonism (JP) are two diseases commonly presenting with parkinsonian symptoms in young patients. Current clinical guidelines offer a diagnostic approach based on molecular analysis. However, developing countries have limitations in terms of accessibility to these tests. We aimed to assess the utility of imaging equipment, usually more available worldwide, to help diagnose and improve patients' quality of life with these diseases. METHODS: We performed a systematic literature review in English using the preferred reporting items for systematic reviews and meta-analyses (PRISMA) and meta-analysis of observational studies in epidemiology (MOOSE) protocols. We only used human clinical trials about dopamine responsive dystonia and juvenile parkinsonism patients in which a fluorodopa (FD) positron emission tomography (PET) scan was performed to identify its use in these diseases. RESULTS: We included six studies that fulfilled our criteria. We found a clear pattern of decreased uptake in the putamen and caudate nucleus in JP cases. At the same time, the results in DRD were comparable to normal subjects, with only a slightly decreased marker uptake in the previously mentioned regions by the FD PET scan. CONCLUSIONS: We found a distinctive pattern for each of these diseases. Identifying these findings with FD PET scans can shorten the delay in making a definitive diagnosis when genetic testing is unavailable, a common scenario in developing countries.

Chemical typicity of tropical tannat red wines from sub-middle São Francisco Valley, Brazil.

Tannat is a Vitis vinifera cultivar with typically high phenolic compound contents, showing intense coloration, well-bodied, and great aging potential. However, even with this great potential, this variety is still commercially underexplored in the Sub-middle São Francisco Valley (SSFV). This work aimed to characterize the typicity of Tannat red wines from Sub-middle São Francisco Valley (SSFV), Brazil. In addition, the present work represents the first study featuring phenolic compounds quantification and antioxidant activity of Tannat in tropical climate wine-producing regions. Considering the condition of a short-applied maceration time during the winemaking, the tropical Tannat wine showed significant antioxidant activity and high phenolic contents. Trans-caftaric, malvidin-3-O-glucoside, and procyanidin B1 stood out among the phenolic compounds quantified, presenting Tannat with the potential to be an important grape variety to tropical wine-producing regions in Brazil, containing high contents of bioactive compounds. Previously results to compounds (-)-epigallocatechin gallate, procyanidin B2, quercetin-3-ß-D-glucoside, pelargonidin-3-O-glucoside, chlorogenic acid, and piceatannol were not found in Tannat wines. Further studies are necessary to make the Tannat grape's adaptation better in tropical climate conditions, including investigating the phenolic profile and antioxidant activity of Tannat red wines with longer maceration times during the winemaking.

Spectroscopic study of the 4fn-15dtransitions of LaPO4doped with Pr3+ or co-doped with Pr3+ and Gd3+ in the vacuum ultra violet region.

The energy off-dtransitions depends on the crystalline field in which the lanthanide ion is inserted. Depending on the experimental setup, these transitions could occur at high energy, so several studies regarding theoretical data have been conducted. Here, we present the experimental determination of the energy of interconfigurational 4fn â†’ 4fn-15d (f-d)transitions from Pr3+ions to the lanthanum orthophosphate LaPO4matrix; we have also determined the bandgap value for this host. The experiments were carried out at the Synchrotron setup of the Brazilian LNLS laboratory. Specifically, we synthesized LaPO4:Pr3+and LaPO4:Pr3+/Gd3+by the hydrothermal method under different pH conditions or by spray pyrolysis. The particles resulting from hydrothermal synthesis had different morphologies and the influence of pH value was showed: the reaction medium was controlled along the process, which changed the surface potential. On the basis of Raman spectroscopy and x-ray diffraction analyses, we found that the crystalline phase was monoclinic monazite for all the samples. We studied the 4f5dlevel and bandgap transitions at high energy by absorption analysis in the VUV range. The experimental results were 7.5 eV (LaPO4bandgap) and 5 eV (4fn→ 4fn-15dtransition of the Pr3+ion), which were close to the theoretical values reported in the literature for this ion and this matrix.

Ion channels and pain in Fabry disease.

Fabry disease (FD) is a progressive, X-linked inherited disorder of glycosphingolipid metabolism due to deficient or absent lysosomal α-galactosidase A (α-Gal A) activity which results in progressive accumulation of globotriaosylceramide (Gb3) and related metabolites. One prominent feature of Fabry disease is neuropathic pain. Accumulation of Gb3 has been documented in dorsal root ganglia (DRG) as well as other neurons, and has lately been associated with the mechanism of pain though the pathophysiology is still unclear. Small fiber (SF) neuropathy in FD differs from other entities in several aspects related to the perception of pain, alteration of fibers as well as drug therapies used in the practice with patients, with therapies far from satisfying. In order to develop better treatments, more information on the underlying mechanisms of pain is needed. Research in neuropathy has gained momentum from the development of preclinical models where different aspects of pain can be modelled and further analyzed. This review aims at describing the different in vitro and FD animal models that have been used so far, as well as some of the insights gained from their use. We focus especially in recent findings associated with ion channel alterations -that apart from the vascular alterations-, could provide targets for improved therapies in pain.

Depression, sleep disturbances, pain, disability and quality of LIFE in Brazilian Fabry disease patients.

BACKGROUND: Fabry disease (FD) is a lysosomal disease in which mutations affect the GLA gene located on the X chromosome. The defective product, the enzyme alpha-galactosidase A, causes accumulation of substrate and contributes to the disruption of cell function in several organs, with variable severity and consequent damage of tissue or organ function. Patient reported outcomes (PROs) enable patients to provide information regarding the consequences of their disease and its treatment and are often recognized as the most important outcomes for them. OBJECTIVES: To evaluate pain, depression, sleep disturbances, disability and disease impact on quality of life in a cohort of Brazilian FD patients and compare between groups stratified by the Mainz Symptom Severity Index (MSSI) Methods: Thirty-seven genotype confirmed classic FD patients - 16 male and 21 female - (mutations: C142R, A156D, L180F, R227X, W262X, G271A, P293S, Y264SX) were evaluated and answered the following questionnaires: Brief Pain Inventory (BPI), Hamilton Depression Rating Scale (HAM-D), Pittsburgh Sleep Quality Index (PSQI), Health Assessment Questionnaire Disability Index (HAQ-DI), Short-Form Health Survey 36 (SF-36). RESULTS: In FD patients, mean ± SD BPI severity result was 2.78 ± 2.66 for severe; 2.80 ± 2.55 for moderate and 1.55 ± 2.38 for mild severity patients. Mean ± SD BPI interference result was 2.55 ± 2.44 for severe; 2.80 ± 3.18 for moderate and 1.36 ± 2.83 for mild patients. BPI severity and interference values correlated with MSSI scores (r = 0.24; p < .001 / r = 0.25; p < .001). Application of HAM-D indicated depression in 21 patients (56.8%). HAM-D results had positive correlation with MSSI values (r = 0.21; p < .001), with BPI severity (r = 0.54; p < .001) and interference (r = 0.65; p < .001). PSQI depicted sleep disturbances in 22 patients (59.5%). PSQI values correlated with MSSI values (r = 0.25; p < .001), with HAM-D results (r = 0.65; p < .001) and BPI severity (r = 0.47; p < .001) and interference (r = 0.66; p < .001). Mean HAQ-DI result was 0.490 for severe; 0.274 for moderate and 0.157 for mild severity patients. CONCLUSIONS: Depression, sleep disturbances and disability were under-recognized in FD patients. HAQ-DI revealed worse disability according to MSSI severity status. The lowest raw scores from the SF-36 questionnaire were for the domains general health perception and physical role functioning. Standardized assessments should be routine care and started as early as diagnosis of Fabry disease is made.

Vaccination With Recombinant Filamentous fd Phages Against Parasite Infection Requires TLR9 Expression.

Recombinant filamentous fd bacteriophages (rfd) expressing antigenic peptides were shown to induce cell-mediated immune responses in the absence of added adjuvant, being a promising delivery system for vaccination. Here, we tested the capacity of rfd phages to protect against infection with the human protozoan Trypanosoma cruzi, the etiologic agent of Chagas Disease. For this, C57BL/6 (B6) and Tlr9-/- mice were vaccinated with rfd phages expressing the OVA257-264 peptide or the T. cruzi-immunodominant peptides PA8 and TSKB20 and challenged with either the T. cruzi Y-OVA or Y-strain, respectively. We found that vaccination with rfd phages induces anti-PA8 and anti-TSKB20 IgG production, expansion of Ag-specific IFN-γ, TNF-α, and Granzyme B-producing CD8+ T cells, as well as in vivo Ag-specific cytotoxic responses. Moreover, the fd-TSKB20 vaccine was able to protect against mortality induced by a high-dose inoculum of the parasite. Although vaccination with rfd phages successfully reduced both parasitemia and parasite load in the myocardium of WT B6 mice, Tlr9-/- animals were not protected against infection. Thus, our data extend previous studies, demonstrating that rfd phages induce Ag-specific IgG and CD8+ T cell-mediated responses and confer protection against an important human parasite infection, through a TLR9-dependent mechanism.

Morphological, molecular, and toxin analysis of field populations of Alexandrium genus from the Argentine Sea.

In the Argentine Sea, blooms of toxigenic dinoflagellates of the Alexandrium tamarense species complex have led to fish and bird mortalities and human deaths as a consequence of paralytic shellfish poisoning (PSP). Yet little is known about the occurrence of other toxigenic species of the genus Alexandrium, or of their toxin composition beyond coastal waters. The distribution of Alexandrium species and related toxins in the Argentine Sea was determined by sampling surface waters on an oceanographic expedition during austral spring from ~39°S to 48°S. Light microscope and SEM analysis for species identification and enumeration was supplemented by confirmatory PCR analysis from field samples. The most frequent Alexandrium taxon identified by microscopy corresponded to the classical description of A. tamarense. Only weak signals of Group I from the A. tamarense species complex were detected by PCR of bulk field samples, but phylogenetic reconstruction of rDNA sequences from single cells from one station assigned them to ribotype Group I (Alexandrium catenella). PCR probes for Alexandrium minutum and Alexandrium ostenfeldii yielded a positive signal, although A. minutum morphology did not completely match the classical description. Analysis of PSP toxin composition of plankton samples revealed toxin profiles dominated by gonyautoxins (GTX1/4). The main toxic cyclic imine detected was 13-desMe-spirolide C and this supported the association with A. ostenfeldii in the field. This study represents the first integrated molecular, morphological and toxinological analysis of field populations of the genus Alexandrium in the Argentine Sea.

Approaches to segment stent area from Intravascular Optical Coherence Tomography

Abstract Introduction Cardiovascular diseases (CVD) have been the focus of research in recent years due to its high mortality rate. It is estimated that 17.5 million people died of CVD in 2012, from which 7.4 million were due to coronary heart disease (CHD). In order to monitor CHD patients and avoid waste of specialists' time, this study proposes the development of a method that segments the area contained by stent struts from Frequency Domain Intravascular Optical Coherence Tomography (the latest technology to view vessels internally) of coronary arteries. Methods The novelty of this study is to find areas comprised by stent struts using two optimal strategies that are robust even with false positives and false negatives detection of stent struts. The first one uses an ellipse fitting algorithm and the other uses a cylinder fitting algorithm. Results Both strategies obtained similar accuracy results close to 98% of true positives, but the cylinder technique showed a run time of at least 50 times higher than the ellipse technique. Conclusion The methods were executed on 443 images with different characteristics showing robustness and usefulness in the medical area.

Monitoring dihydrodiol polyaromatic hydrocarbon metabolites produced by the freshwater microalgae Selenastrum capricornutum.

We found that microalgae exposed to a mixture of polycyclic aromatic hydrocarbons (PAHs) did not show growth inhibition. Thus, we assumed that they could metabolize these compounds. In this study, the dihydrodiol-type PAH metabolites of benzo(a)pyrene (BaP), benzo(a)anthracene (BaA), benzo(b)fluoranthene (BbF) and benzo(k)fluoranthene (BkF) produced by the freshwater microalgae Selenastrum capricornutum were monitored and quantified using high-performance liquid chromatography with fluorescence detection (HPLC-FD) techniques. Exposure bioassays with S. capricornutum were performed using a 266 ng mL(-1) mixture of PAHs at different exposure times (0.75, 1, 3, 8, 16, 24 and 48 h) under controlled temperature (25 °C); the dihydrodiol metabolites formed in the liquid medium and the biomass were quantified. Metabolite identities were confirmed using HPLC-mass spectrometry; most of the metabolites formed were derived from BaA degradation. At 48 h after exposure 5,6-dBaA and 8,9-dBaA/10,11-dBaA were present in the liquid medium at 20% and 67% of the initial mass of BaA, respectively. Three metabolites of BaP were monitored in the liquid medium and biomass and, at 24 h, 4,5-dBaP accounted for 19%; , 7,8-dBaP, 5%; and 9,10-dBaP, 5% relative to the initial BaP mass. Microalgae exposed to BbF showed the presence of 1,2-dBbF and 9,10-dBbF (at 0.3% and 0.1% of the initial BbF mass, respectively) and those exposed to BkF produced 8,9-dBkF (6.5% of the initial BkF mass) in the liquid medium. Seven unknown compounds were formed after exposure; two compounds were identified as the metabolites of BaA and BaP. The results could facilitate the elucidation of the controversial biodegradation mechanism in microalgae.

Desenvolvimento e validação de método para quantificação simultânea de ofloxacina, norfloxacina e ciprofloxacina em urina humana / Development and validation of an analytical method for the simultaneous quantification of ofloxacin, norfloxacin and ciprofloxacin in human urine

Processos de tratamentos têm sido alvo de estudos com a finalidade de promover a remoção dos fármacos na urina humana de forma a garantir a segurança ambiental da utilização dessa como fertilizante na agricultura. Neste artigo foi desenvolvido e validado um método de CLAE-DF para a determinação simultânea de ofloxacina, norfloxacina e ciprofloxacina na urina humana. O método proposto descreve uma alternativa eficaz para a determinação de resíduos de fluoroquinolonas, eliminando o passo de limpeza. Além disso, o método proposto demonstrou ser seletivo, com boa linearidade (r>0,99), sensibilidade, precisão (80% a 107%) e repetitividade. Este método mostra ser adequado para análises de rotina (de baixo custo, simples, e utilizando pequeno volume de solventes) para abordagens ecológicas.

Treatment processes have been the focus of studies aiming at promoting the removal of drugs in human urine in order to ensure environmental safety using it as a fertilizer in agriculture. So, an HPLC-FD method for the simultaneous determination of ofloxacin, norfloxacin and ciprofloxacin in human urine was developed and validated. The proposed method describes an efficient alternative for the determination of fluoroquinolone residues by eliminating the clean-up step. Moreover, the proposed method proved to be selective, with good linearity (r>0.99), sensitivity, accuracy (80% to 107%) and repeatability. This method shows to be suitable for routine analyses (simple, low price and using a small volume of solvents) for ecological approaches.

Emprego de casca de arroz como adsorvente para execução de técnica de MSPD para determinar aflatoxinas em cebolas / Use of rice husk as an adsorbent for performing MSPS technique for determining the aflatoxins occurrence in onion

Foi estudada a extração simultânea de aflatoxinas (AFLAs) B1, B2, G1 e G2 em cebolas por meio de técnica de dispersão da matriz em fase sólida (MSPD), utilizando-se a casca de arroz como adsorvente. A identificação e quantificação das aflatoxinas foram realizadas empregando-se cromatografia em camada delgada de alta eficiência (CCDAE) e cromatografia líquida de alta eficiência acoplada ao detector de fluorescência (CLAE-FL). O melhor adsorvente foi constituído de mistura de casca de arroz: terra diatomácea (1:1) (p/p), empregado na proporção de 1:1 (p/p) com a massa de amostra e a mistura clorofórmio:metanol (5:25) (v/v) como eluente. A metodologia mostrou limites de detecção que variaram de 0,05 a 1 µg.kg-1, de exatidão entre 78 e 93%, e coeficientes de variação compreendidos entre 11 e 14%. Após a validação, a metodologia foi testada quanto à sua aplicabilidade para determinar a ocorrência das aflatoxinas em amostras de cebola, as quais foram classificadas segundo a norma do MAPA. A contaminação com aflatoxina foi verificada em 43% das amostras analisadas com teor máximo de 90µg.kg-1 de AFLAB2.(AU)

The simultaneous determination of aflatoxins (AFLA) B1, B2, G1 and G2 was investigated in onions by means of matrix solid-phase dispersion (MSPD) extraction methodology, and using rice husk as adsorbent element. The AFLA identification and quantification were carried out by using high efficiency thin layer chromatography (HPTLC) and high efficiency liquid chromatography coupled to a fluorescence detector (HPLC-FD). The best adsorbent was the compound constituted by the rice husk:celite mixture (1:1) in a 1:1 ratio with the mass of the sample, and employing the mixture chloroform:methanol (5:25) as eluent. The methodology showed the limit of detection ranging from 0.05 to 1 µg.kg-1, and of the accuracy from 78 to 93%; and variation coefficients ranged from 11 to 14%. After being validated, the methodology was tested on its applicability in determining the aflatoxins occurrence in onion samples, which were classified according to the standard MAPA. Aflatoxin contamination was found in 43% of analyzed samples with a maximum content of 90 µg.kg-1 of AFLAB2.(AU)

An in vivo system involving co-expression of cyanobacterial flavodoxin and ferredoxin-NADP(+) reductase confers increased tolerance to oxidative stress in plants.

Oxidative stress in plants causes ferredoxin down-regulation and NADP(+) shortage, over-reduction of the photosynthetic electron transport chain, electron leakage to oxygen and generation of reactive oxygen species (ROS). Expression of cyanobacterial flavodoxin in tobacco chloroplasts compensates for ferredoxin decline and restores electron delivery to productive routes, resulting in enhanced stress tolerance. We have designed an in vivo system to optimize flavodoxin reduction and NADP(+) regeneration under stress using a version of cyanobacterial ferredoxin-NADP(+) reductase without the thylakoid-binding domain. Co-expression of the two soluble flavoproteins in the chloroplast stroma resulted in lines displaying maximal tolerance to redox-cycling oxidants, lower damage and decreased ROS accumulation. The results underscore the importance of chloroplast redox homeostasis in plants exposed to adverse conditions, and provide a tool to improve crop tolerance toward environmental hardships.