Genomic and Metabolomic Analyses of Natural Products in Nodularia spumigena Isolated from a Shrimp Culture Pond

The bloom-forming cyanobacterium Nodularia spumigena CENA596 encodes the biosynthetic gene clusters (BGCs) of the known natural products nodularins, spumigins, anabaenopeptins/namalides, aeruginosins, mycosporin-like amino acids, and scytonemin, along with the terpenoid geosmin. Targeted metabolomics confirmed the production of these metabolic compounds, except for the alkaloid scytonemin. Genome mining of N. spumigena CENA596 and its three closely related Nodularia strains—two planktonic strains from the Baltic Sea and one benthic strain from Japanese marine sediment—revealed that the number of BGCs in planktonic strains was higher than in benthic one. Geosmin—a volatile compound with unpleasant taste and odor—was unique to the Brazilian strain CENA596. Automatic annotation of the genomes using subsystems technology revealed a related number of coding sequences and functional roles. Orthologs from the Nodularia genomes are involved in the primary and secondary metabolisms. Phylogenomic analysis of N. spumigena CENA596 based on 120 conserved protein sequences positioned this strain close to the Baltic Nodularia. Phylogeny of the 16S rRNA genes separated the Brazilian CENA596 strain from those of the Baltic Sea, despite their high sequence identities (99% identity, 100% coverage). The comparative analysis among planktic Nodularia strains showed that their genomes were considerably similar despite their geographically distant origin

Global variations in diabetes mellitus based on fasting glucose and haemogloblin A1c

Fasting plasma glucose (FPG) and haemoglobin A1c (HbA1c) are both used to diagnose diabetes, but may identify different people as having diabetes. We used data from 117 population-based studies and quantified, in different world regions, the prevalence of diagnosed diabetes, and whether those who were previously undiagnosed and detected as having diabetes in survey screening had elevated FPG, HbA1c, or both. We developed prediction equations for estimating the probability that a person without previously diagnosed diabetes, and at a specific level of FPG, had elevated HbA1c, and vice versa. The age-standardised proportion of diabetes that was previously undiagnosed, and detected in survey screening, ranged from 30% in the high-income western region to 66% in south Asia. Among those with screen-detected diabetes with either test, the agestandardised proportion who had elevated levels of both FPG and HbA1c was 29-39% across regions; the remainder had discordant elevation of FPG or HbA1c. In most low- and middle-income regions, isolated elevated HbA1c more common than isolated elevated FPG. In these regions, the use of FPG alone may delay diabetes diagnosis and underestimate diabetes prevalence. Our prediction equations help allocate finite resources for measuring HbA1c to reduce the global gap in diabetes diagnosis and surveillance.peer-reviewe

Worldwide trends in hypertension prevalence and progress in treatment and control from 1990 to 2019: a pooled analysis of 1201 population-representative studies with 104 million participants

Background Hypertension can be detected at the primary health-care level and low-cost treatments can effectively control hypertension. We aimed to measure the prevalence of hypertension and progress in its detection, treatment, and control from 1990 to 2019 for 200 countries and territories. Methods We used data from 1990 to 2019 on people aged 30–79 years from population-representative studies with measurement of blood pressure and data on blood pressure treatment. We defined hypertension as having systolic blood pressure 140 mm Hg or greater, diastolic blood pressure 90 mm Hg or greater, or taking medication for hypertension. We applied a Bayesian hierarchical model to estimate the prevalence of hypertension and the proportion of people with hypertension who had a previous diagnosis (detection), who were taking medication for hypertension (treatment), and whose hypertension was controlled to below 140/90 mm Hg (control). The model allowed for trends over time to be non-linear and to vary by age. Findings The number of people aged 30–79 years with hypertension doubled from 1990 to 2019, from 331 (95% credible interval 306–359) million women and 317 (292–344) million men in 1990 to 626 (584–668) million women and 652 (604–698) million men in 2019, despite stable global age-standardised prevalence. In 2019, age-standardised hypertension prevalence was lowest in Canada and Peru for both men and women; in Taiwan, South Korea, Japan, and some countries in western Europe including Switzerland, Spain, and the UK for women; and in several low-income and middle-income countries such as Eritrea, Bangladesh, Ethiopia, and Solomon Islands for men. Hypertension prevalence surpassed 50% for women in two countries and men in nine countries, in central and eastern Europe, central Asia, Oceania, and Latin America. Globally, 59% (55–62) of women and 49% (46–52) of men with hypertension reported a previous diagnosis of hypertension in 2019, and 47% (43–51) of women and 38% (35–41) of men were treated. Control rates among people with hypertension in 2019 were 23% (20–27) for women and 18% (16–21) for men. In 2019, treatment and control rates were highest in South Korea, Canada, and Iceland (treatment >70%; control >50%), followed by the USA, Costa Rica, Germany, Portugal, and Taiwan. Treatment rates were less than 25% for women and less than 20% for men in Nepal, Indonesia, and some countries in sub-Saharan Africa and Oceania. Control rates were below 10% for women and men in these countries and for men in some countries in north Africa, central and south Asia, and eastern Europe. Treatment and control rates have improved in most countries since 1990, but we found little change in most countries in sub-Saharan Africa and Oceania. Improvements were largest in high-income countries, central Europe, and some upper-middle-income and recently high-income countries including Costa Rica, Taiwan, Kazakhstan, South Africa, Brazil, Chile, Turkey, and Iran. Interpretation Improvements in the detection, treatment, and control of hypertension have varied substantially across countries, with some middle-income countries now outperforming most high-income nations. The dual approach of reducing hypertension prevalence through primary prevention and enhancing its treatment and control is achievable not only in high-income countries but also in low-income and middle-income settings

Liquid-chromatography coupled to mass spectrometry: applications for the study of toxins produced by cyanobacteria

A crescente demanda por água doce de boa qualidade, associada ao aumento na frequência de florações tóxicas de cianobactérias em reservatórios utilizados para consumo humano, levou à publicação da Portaria nº. 518/04 pelo Ministério da Saúde. Entre outros parâmetros de potabilidade, as empresas fornecedoras de água tratada devem realizar o monitoramento de cianotoxinas. Para tanto, métodos analíticos rápidos e precisos para a determinação destes compostos são imprescindíveis. Neste contexto, o presente trabalho teve como objetivo empregar a cromatografia líquida acoplada à espectrometria de massas para o estudo das principais cianotoxinas em território nacional: microcistinas, anatoxina-a(s), cilindrospermopsina e saxitoxinas. Os resultados obtidos estão distribuídos em capítulos específicos dedicados a cada grupo de toxinas. Dessa forma, o primeiro capítulo apresenta um estudo de fragmentação na fase gasosa de ânions de microcistinas em um equipamento do tipo orbitrap. É demonstrado que o modo negativo de ionização por electrospray fornece informações estruturais importantes e complementares ao modo positivo de ionização. Uma abertura seletiva do peptídeo cíclico é proposta e mecanismos discutidos, o que facilita a interpretação de resultados durante a caracterização de variantes desconhecidas. O modelo de fragmentação desenvolvido foi utilizado para identificar a variante [Leu1]MC-LR em um extrato de Microcystis spp. O segundo capítulo descreve metodologias qualitativas de LC/MS para o monitoramento e identificação do organofosforado natural anatoxina-a(s), cuja análise é prejudicada pela ausência de padrões comerciais. A cromatografia de interação hidrofílica foi empregada e mecanismos de fragmentação na fase gasosa propostos, discutindo-se os íons característicos desta estrutura química. Tal modelo permitiu a identificação desta toxina nas cepas de Anabaena oumiana ITEP-25 e ITEP-26 pela primeira vez. O terceiro capítulo disserta sobre os mecanismos de fragmentação na fase gasosa da toxina cilindrospermopsina quando ionizada por electrospray na forma de aduto com metais alcalinos. Diferenças nas vias de fragmentação são demonstradas de acordo com o raio atômico do metal formador do aduto, com implicações práticas na sua determinação cromatográfica. Já o quarto capítulo discute os mecanismos de fragmentação de variantes sulfatadas de saxitoxinas (GTX1e4, GTX2e3, dcGTX2e3, GTX5) após ionização por electrospray no modo positivo e negativo. É demonstrado pela primeira vez que uma conformação estrutural específica do grupamento sulfato explica a intensa eliminação de SO3 observada para as variantes GTX1, GTX2 e dcGTX2 no modo positivo de ionização. Por outro lado, o modo negativo de ionização apresenta vantagens uma vez que a dissociação na fonte é insignificante se comparada à dissociação observada no modo positivo. Como resultado, métodos quantitativos no modo negativo podem apresentar maior sensibilidade, permitindo a detecção destas toxinas em amostras ambientais em quantidades mais baixas. De maneira geral, conclui-se que a cromatografia líquida acoplada à espectrometria de massas é ferramenta poderosa para a análise quali e quantitativa das principais cianotoxinas, podendo ser amplamente empregada para o monitoramento de água para consumo humano.The increasing occurrence of toxic cyanobacterial blooms in reservoirs used to supply drinking water for human consumption has prompted the publication of resolution 518/04 by the Brazilian Ministry of Health. Among other quality requirements, the monitoring of cyanotoxins in treated water is mandatory for companies responsible for potable water distribution. Therefore, precise and rapid analytical methods are essential. In this context, the aim of this work is to employ liquid chromatography coupled to mass spectrometry to study the most important cyanotoxins in our country: microcystins, anatoxin-a(s), cylindrospermopsis and saxitoxins. The obtained results are distributed in four chapters, each one dedicated to a single group of toxins. In this way, chapter one presents the gas-phase fragmentation behavior of deprotonated microcystins in an Orbitrap instrument. It is demonstrated that electrospray negative ionization can provide significant structural information about microcystins. These results are complementary to the positive ionization mode. A selective ring opening process is proposed and possible mechanisms are discussed, which may facilitate data interpretation when unknown variants are considered. The general fragmentation model was further applied to the characterization of [Leu1]MC-LR in a Microcystis spp. cell extract. The second chapter describes qualitative analytical methods for the identification of anatoxin-a(s), a natural organophosphate whose determination is hampered by the lack of commercial standards. Hydrophilic interaction liquid chromatography was employed and fragmentation mechanisms proposed, identifying the characteristic product ions of this toxin. The developed methods were further used to identify anatoxin-a(s) for the first time in Anabaena oumiana strains ITEP-25 and ITEP-26. The third chapter presents data related to the gas-phase fragmentation behavior of cylindrospermospin when this toxin is ionized as metal adducts. Different fragmentation pathways are accessed depending on the atomic radius of the metal cation involved. Practical implications for the chromatographic analysis of this toxin are presented. The last chapter describes the fragmentation behavior of sulphate-containing saxitoxin variants (GTX1&4, GTX2&3, dcGTX2&3, GTX5) after electrospray ionization in both the positive and negative modes. A mechanism for the intense SO3 elimination from [M+H]+ ions from GTX1, GTX2 and dcGTX2 is proposed for the first time and relies on a specific structure conformation. On the other hand, the negative ionization mode shows much less in-source dissociation when compared to the positive mode. As a consequence, methods based on negative ionization might be more sensitive for sulfate-containing variants, allowing the detection of lower amounts of these toxins in environmental samples. At the end, it can be concluded that liquid chromatography is a well-suited and powerful technique for the qualitative and quantitative analysis of cyanotoxins, being an invaluable contribution to water safety evaluation

Liquid-chromatography coupled to mass spectrometry: applications for the study of toxins produced by cyanobacteria

A crescente demanda por água doce de boa qualidade, associada ao aumento na frequência de florações tóxicas de cianobactérias em reservatórios utilizados para consumo humano, levou à publicação da Portaria nº. 518/04 pelo Ministério da Saúde. Entre outros parâmetros de potabilidade, as empresas fornecedoras de água tratada devem realizar o monitoramento de cianotoxinas. Para tanto, métodos analíticos rápidos e precisos para a determinação destes compostos são imprescindíveis. Neste contexto, o presente trabalho teve como objetivo empregar a cromatografia líquida acoplada à espectrometria de massas para o estudo das principais cianotoxinas em território nacional: microcistinas, anatoxina-a(s), cilindrospermopsina e saxitoxinas. Os resultados obtidos estão distribuídos em capítulos específicos dedicados a cada grupo de toxinas. Dessa forma, o primeiro capítulo apresenta um estudo de fragmentação na fase gasosa de ânions de microcistinas em um equipamento do tipo orbitrap. É demonstrado que o modo negativo de ionização por electrospray fornece informações estruturais importantes e complementares ao modo positivo de ionização. Uma abertura seletiva do peptídeo cíclico é proposta e mecanismos discutidos, o que facilita a interpretação de resultados durante a caracterização de variantes desconhecidas. O modelo de fragmentação desenvolvido foi utilizado para identificar a variante [Leu1]MC-LR em um extrato de Microcystis spp. O segundo capítulo descreve metodologias qualitativas de LC/MS para o monitoramento e identificação do organofosforado natural anatoxina-a(s), cuja análise é prejudicada pela ausência de padrões comerciais. A cromatografia de interação hidrofílica foi empregada e mecanismos de fragmentação na fase gasosa propostos, discutindo-se os íons característicos desta estrutura química. Tal modelo permitiu a identificação desta toxina nas cepas de Anabaena oumiana ITEP-25 e ITEP-26 pela primeira vez. O terceiro capítulo disserta sobre os mecanismos de fragmentação na fase gasosa da toxina cilindrospermopsina quando ionizada por electrospray na forma de aduto com metais alcalinos. Diferenças nas vias de fragmentação são demonstradas de acordo com o raio atômico do metal formador do aduto, com implicações práticas na sua determinação cromatográfica. Já o quarto capítulo discute os mecanismos de fragmentação de variantes sulfatadas de saxitoxinas (GTX1e4, GTX2e3, dcGTX2e3, GTX5) após ionização por electrospray no modo positivo e negativo. É demonstrado pela primeira vez que uma conformação estrutural específica do grupamento sulfato explica a intensa eliminação de SO3 observada para as variantes GTX1, GTX2 e dcGTX2 no modo positivo de ionização. Por outro lado, o modo negativo de ionização apresenta vantagens uma vez que a dissociação na fonte é insignificante se comparada à dissociação observada no modo positivo. Como resultado, métodos quantitativos no modo negativo podem apresentar maior sensibilidade, permitindo a detecção destas toxinas em amostras ambientais em quantidades mais baixas. De maneira geral, conclui-se que a cromatografia líquida acoplada à espectrometria de massas é ferramenta poderosa para a análise quali e quantitativa das principais cianotoxinas, podendo ser amplamente empregada para o monitoramento de água para consumo humano.The increasing occurrence of toxic cyanobacterial blooms in reservoirs used to supply drinking water for human consumption has prompted the publication of resolution 518/04 by the Brazilian Ministry of Health. Among other quality requirements, the monitoring of cyanotoxins in treated water is mandatory for companies responsible for potable water distribution. Therefore, precise and rapid analytical methods are essential. In this context, the aim of this work is to employ liquid chromatography coupled to mass spectrometry to study the most important cyanotoxins in our country: microcystins, anatoxin-a(s), cylindrospermopsis and saxitoxins. The obtained results are distributed in four chapters, each one dedicated to a single group of toxins. In this way, chapter one presents the gas-phase fragmentation behavior of deprotonated microcystins in an Orbitrap instrument. It is demonstrated that electrospray negative ionization can provide significant structural information about microcystins. These results are complementary to the positive ionization mode. A selective ring opening process is proposed and possible mechanisms are discussed, which may facilitate data interpretation when unknown variants are considered. The general fragmentation model was further applied to the characterization of [Leu1]MC-LR in a Microcystis spp. cell extract. The second chapter describes qualitative analytical methods for the identification of anatoxin-a(s), a natural organophosphate whose determination is hampered by the lack of commercial standards. Hydrophilic interaction liquid chromatography was employed and fragmentation mechanisms proposed, identifying the characteristic product ions of this toxin. The developed methods were further used to identify anatoxin-a(s) for the first time in Anabaena oumiana strains ITEP-25 and ITEP-26. The third chapter presents data related to the gas-phase fragmentation behavior of cylindrospermospin when this toxin is ionized as metal adducts. Different fragmentation pathways are accessed depending on the atomic radius of the metal cation involved. Practical implications for the chromatographic analysis of this toxin are presented. The last chapter describes the fragmentation behavior of sulphate-containing saxitoxin variants (GTX1&4, GTX2&3, dcGTX2&3, GTX5) after electrospray ionization in both the positive and negative modes. A mechanism for the intense SO3 elimination from [M+H]+ ions from GTX1, GTX2 and dcGTX2 is proposed for the first time and relies on a specific structure conformation. On the other hand, the negative ionization mode shows much less in-source dissociation when compared to the positive mode. As a consequence, methods based on negative ionization might be more sensitive for sulfate-containing variants, allowing the detection of lower amounts of these toxins in environmental samples. At the end, it can be concluded that liquid chromatography is a well-suited and powerful technique for the qualitative and quantitative analysis of cyanotoxins, being an invaluable contribution to water safety evaluation

Alternative Isolation Protocol for Desulfo and Zwitterionic Cylindrospermopsin Alkaloids and Comparison of Their Toxicity in HepG2 Cells

The term cylindrospermopsins (CYNs) refers to a structurally related class of cyanobacterial metabolites comprised of a tricyclic guanidine group and a hydroxymethyluracil moiety. Most reports in environmental aquatic samples refer to cylindrospermopsin (CYN), and reports on other CYN alkaloids are scarce, due, in part, to a lack of versatile isolation protocols. Thus, using commercially available solid phase extraction (SPE) cartridges, we optimized an isolation protocol for the complete recovery of CYN, 7-deoxy-cylindrospermopsin (7D-CYN) and 7-deoxy-desulfo-cylindrospermopsin (7D-desulfo-CYN) from the same aliquot. The isolation protocol was adaptable depending on the nature of the sample (solid biomass, culture broth or environmental water sample) and tolerates up to 4 L of dense culture broth or 400 mg of lyophilized biomass. To quantitate the CYN alkaloids, we validated an LC-DAD-MS2 method, which takes advantage of the UV absorption of the uracil group (λ 262 nm). Using electrospray ionization (ESI) in a positive ion mode, the high-resolution MS1 data confirms the presence of the protonated alkaloids, and the MS2 fragment assignment is reported as complementary proof of the molecular structure of the CYNs. We isolated three CYN alkaloids with different water solubility using the same lyophilized sample, with a purity that ranged from 95% to 99%. The biological activity of the purified CYNs, along with a synthetic degradation product of CYN (desulfo-cylindrospermopsin), was evaluated by assessing necrosis and apoptosis in vitro using flow cytometry. CYN’s lethal potency in HepG2 cells was greater than the other analogs, due to the presence of all four functional groups: guanidine, uracil, C-7 hydroxyl and the sulfate residue

Preferência da broca-das-cucurbitáceas [Diaphania nitidalis Cramer, 1782 (Lepidoptera: Pyralidae)] por cultivares de pepineiro em ambiente protegido Pickleworm [Diaphania nitidalis Cramer, 1782 (Lepidoptera: Pyralidae)]preference for cucumber cultivars in greenhouse

A broca-das-cucurbitáceas (Diaphania nitidalis) é uma praga de grande importância em diversas culturas, principalmente do pepineiro. Esta espécie pode apresentar preferência em relação a determinadas cultivares quanto à sua alimentação e até mesmo oviposição. Nesse sentido, o presente estudo objetivou avaliar a ocorrência desta praga em frutos de seis cultivares de pepineiro, em ambiente protegido. As cultivares Patton, Victoria, Premier, Vlaspik e Napoleon apresentaram, respectivamente, 50%, 43,75%, 37,50%, 34,37% e 25,87% dos frutos brocados. A cultivar Marinda apresentou resultado promissor, com apenas 10,37% dos frutos com presença de larvas de D. nitidalis.<br>The pickleworm (Diaphania nitidalis) is a major pest on different crops, particulary on cucumber. This species has shown a preference for determined cultivars to its feeding and oviposition habits. Thus, the aim of this study was to evaluate the occurence of D. nitidalis larvae in fruits of six cucumber cultivars in greenhouse. The cultivars Patton, Victoria, Premier, Vlaspik and Napoleon showed, respectively, 50%, 43.75%, 37.50%, 34.37% and 25.87% of bored fruits. Promising results were obtained with the cultivar Marinda, which showed only 10.37% of bored fruits

Cylindrospermopsin and Saxitoxin Synthetase Genes in <i>Cylindrospermopsis raciborskii</i> Strains from Brazilian Freshwater

<div><p>The <i>Cylindrospermopsis raciborskii</i> population from Brazilian freshwater is known to produce saxitoxin derivatives (STX), while cylindrospermopsin (CYN), which is commonly detected in isolates from Australia and Asia continents, has thus far not been detected in South American strains. However, during the investigation for the presence of <i>cyrA</i>, <i>cyrB</i>, <i>cyrC</i> and <i>cyrJ</i> CYN synthetase genes in the genomes of four laboratory-cultured <i>C. raciborskii</i> Brazilian strains, the almost complete <i>cyrA</i> gene sequences were obtained for all strains, while <i>cyrB</i> and <i>cyrC</i> gene fragments were observed in two strains. These nucleotide sequences were translated into amino acids, and the predicted protein functions and domains confirmed their identity as CYN synthetase genes. Attempts to PCR amplify <i>cyrJ</i> gene fragments from the four strains were unsuccessful. Phylogenetic analysis grouped the nucleotide sequences together with their homologues found in known CYN synthetase clusters of <i>C. raciborskii</i> strains with high bootstrap support. In addition, fragments of <i>sxtA</i>, <i>sxtB</i> and <i>sxtI</i> genes involved in STX production were also obtained. Extensive LC-MS analyses were unable to detect CYN in the cultured strains, whereas the production of STX and its analogues was confirmed in CENA302, CENA305 and T3. To our knowledge, this is the first study reporting the presence of <i>cyr</i> genes in South American strains of <i>C. raciborskii</i> and the presence of <i>sxt</i> and <i>cyr</i> genes in a single <i>C. raciborskii</i> strain. This discovery suggests a shift in the type of cyanotoxin production over time of South American strains of <i>C. raciborskii</i> and contributes to the reconstruction of the evolutionary history and diversification of cyanobacterial toxins.</p></div

Maximum likelihood phylogenetic tree based on the 16S rRNA gene sequences showing the relationships of the studied cyanobacteria (in bold).

<p>Bootstrap test (1,000 resamplings) was performed and values >50% for ML and NJ analyses are shown over the nodes. Branch lengths are proportional to the number of substitutions per site (see scale bar). Taxon name in red or blue denotes STX or CYN producer strains, respectively.</p

LC-UV-MS analysis of a freeze-dried culture sample of <i>C. raciborskii</i> CYP011K.

<p>(A) UV trace at 262 nm and extracted ion chromatograms for CYN (<i>m/z</i> 418) and 7-<i>deoxy</i>-CYN (<i>m/z</i> 400). Product ion spectra and UV absorption spectra are depicted for CYN (B and C) and 7-<i>deoxy</i>-CYN (D and E).</p