Spatial Distribution and Biochemical Characterization of Serine Peptidase Inhibitors in the Venom of the Brazilian Sea Anemone Anthopleura cascaia Using Mass Spectrometry Imaging.

Sea anemones are known to produce a diverse array of toxins with different cysteine-rich peptide scaffolds in their venoms. The serine peptidase inhibitors, specifically Kunitz inhibitors, are an important toxin family that is believed to function as defensive peptides, as well as prevent proteolysis of other secreted anemone toxins. In this study, we isolated three serine peptidase inhibitors named Anthopleura cascaia peptide inhibitors I, II, and III (ACPI-I, ACPI-II, and ACPI-III) from the venom of the endemic Brazilian sea anemone A. cascaia. The venom was fractionated using RP-HPLC, and the inhibitory activity of these fractions against trypsin was determined and found to range from 59% to 93%. The spatial distribution of the anemone peptides throughout A. cascaia was observed using mass spectrometry imaging. The inhibitory peptides were found to be present in the tentacles, pedal disc, and mesenterial filaments. We suggest that the three inhibitors observed during this study belong to the venom Kunitz toxin family on the basis of their similarity to PI-actitoxin-aeq3a-like and the identification of amino acid residues that correspond to a serine peptidase binding site. Our findings expand our understanding of the diversity of toxins present in sea anemone venom and shed light on their potential role in protecting other venom components from proteolysis.

Spiders' digestive system as a source of trypsin inhibitors: functional activity of a member of atracotoxin structural family.

Spiders are important predators of insects and their venoms play an essential role in prey capture. Spider venoms have several potential applications as pharmaceutical compounds and insecticides. However, transcriptomic and proteomic analyses of the digestive system (DS) of spiders show that DS is also a rich source of new peptidase inhibitor molecules. Biochemical, transcriptomic and proteomic data of crude DS extracts show the presence of molecules with peptidase inhibitor potential in the spider Nephilingis cruentata. Therefore, the aims of this work were to isolate and characterize molecules with trypsin inhibitory activity. The DS of fasting adult females was homogenized under acidic conditions and subjected to heat treatment. After that, samples were submitted to ion exchange batch and high-performance reverse-phase chromatography. The fractions with trypsin inhibitory activity were confirmed by mass spectrometry, identifying six molecules with inhibitory potential. The inhibitor NcTI (Nephilingis cruentata trypsin inhibitor) was kinetically characterized, showing a KD value of 30.25 nM ± 8.13. Analysis of the tertiary structure by molecular modeling using Alpha-Fold2 indicates that the inhibitor NcTI structurally belongs to the MIT1-like atracotoxin family. This is the first time that a serine peptidase inhibitory function is attributed to this structural family and the inhibitor reactive site residue is identified. Sequence analysis indicates that these molecules may be present in the DS of other spiders and could be associated to the inactivation of prey trypsin (serine peptidase) ingested by the spiders.

Digestive enzymes and sphingomyelinase D in spiders without venom (Uloboridae).

Spiders have distinct predatory behaviours selected along Araneae's evolutionary history but are mainly based on the use of venom for prey paralysis. Uloboridae spiders have lost their venom glands secondarily during evolution. Because of this, they immobilise their prey by extensively wrapping, and digestion starts with the addition of digestive fluid. During the extra-oral digestion, the digestive fluid liquefies both the prey and the AcSp2 spidroins from the web fibres. Despite the efficiency of this process, the cocktail of enzymes involved in digestion in Uloboridae spiders remains unknown. In this study, the protein content in the midgut of Uloborus sp. was evaluated through enzymatic, proteomic, and phylogenetic analysis. Hydrolases such as peptidases (endo and exopeptidases: cysteine, serine, and metallopeptidases), carbohydrases (alpha-amylase, chitinase, and alpha-mannosidase), and lipases were biochemically assayed, and 50 proteins (annotated as enzymes, structural proteins, and toxins) were identified, evidencing the identity between the digestive enzymes present in venomous and non-venomous spiders. Even enzymes thought to be unique to venom, including enzymes such as sphingomyelinase D, were found in the digestive system of non-venomous spiders, suggesting a common origin between digestive enzymes and enzymes present in venoms. This is the first characterization of the molecules involved in the digestive process and the midgut protein content of a non-venomous spider.

Spatial distribution and biochemical characterization of serine peptidase inhibitors in the venom of the brazilian sea anemone anthopleura cascaia using mass spectrometry imaging

Sea anemones are known to produce a diverse array of toxins with different cysteine-rich peptide scaffolds in their venoms. The serine peptidase inhibitors, specifically Kunitz inhibitors, are an important toxin family that is believed to function as defensive peptides, as well as prevent proteolysis of other secreted anemone toxins. In this study, we isolated three serine peptidase inhibitors named Anthopleura cascaia peptide inhibitors I, II, and III (ACPI-I, ACPI-II, and ACPI-III) from the venom of the endemic Brazilian sea anemone A. cascaia. The venom was fractionated using RP-HPLC, and the inhibitory activity of these fractions against trypsin was determined and found to range from 59% to 93%. The spatial distribution of the anemone peptides throughout A. cascaia was observed using mass spectrometry imaging. The inhibitory peptides were found to be present in the tentacles, pedal disc, and mesenterial filaments. We suggest that the three inhibitors observed during this study belong to the venom Kunitz toxin family on the basis of their similarity to PI-actitoxin-aeq3a-like and the identification of amino acid residues that correspond to a serine peptidase binding site. Our findings expand our understanding of the diversity of toxins present in sea anemone venom and shed light on their potential role in protecting other venom components from proteolysis.

Spiders digestive system as a source of trypsin inhibitors: functional activity of a member of atracotoxin structural family

Spiders are important predators of insects and their venoms play an essential role in prey capture. Spider venoms have several potential applications as pharmaceutical compounds and insecticides. However, transcriptomic and proteomic analyses of the digestive system (DS) of spiders show that DS is also a rich source of new peptidase inhibitor molecules. Biochemical, transcriptomic and proteomic data of crude DS extracts show the presence of molecules with peptidase inhibitor potential in the spider Nephilingis cruentata. Therefore, the aims of this work were to isolate and characterize molecules with trypsin inhibitory activity. The DS of fasting adult females was homogenized under acidic conditions and subjected to heat treatment. After that, samples were submitted to ion exchange batch and high-performance reverse-phase chromatography. The fractions with trypsin inhibitory activity were confirmed by mass spectrometry, identifying six molecules with inhibitory potential. The inhibitor NcTI (Nephilingis cruentata trypsin inhibitor) was kinetically characterized, showing a KD value of 30.25 nM ± 8.13. Analysis of the tertiary structure by molecular modeling using Alpha-Fold2 indicates that the inhibitor NcTI structurally belongs to the MIT1-like atracotoxin family. This is the first time that a serine peptidase inhibitory function is attributed to this structural family and the inhibitor reactive site residue is identified. Sequence analysis indicates that these molecules may be present in the DS of other spiders and could be associated to the inactivation of prey trypsin (serine peptidase) ingested by the spiders.

Digestive enzymes and sphingomyelinase D in spiders without venom (Uloboridae)

Spiders have distinct predatory behaviours selected along Araneae’s evolutionary history but are mainly based on the use of venom for prey paralysis. Uloboridae spiders have lost their venom glands secondarily during evolution. Because of this, they immobilise their prey by extensively wrapping, and digestion starts with the addition of digestive fluid. During the extra-oral digestion, the digestive fluid liquefies both the prey and the AcSp2 spidroins from the web fibres. Despite the efficiency of this process, the cocktail of enzymes involved in digestion in Uloboridae spiders remains unknown. In this study, the protein content in the midgut of Uloborus sp. was evaluated through enzymatic, proteomic, and phylogenetic analysis. Hydrolases such as peptidases (endo and exopeptidases: cysteine, serine, and metallopeptidases), carbohydrases (alpha-amylase, chitinase, and alpha-mannosidase), and lipases were biochemically assayed, and 50 proteins (annotated as enzymes, structural proteins, and toxins) were identified, evidencing the identity between the digestive enzymes present in venomous and non-venomous spiders. Even enzymes thought to be unique to venom, including enzymes such as sphingomyelinase D, were found in the digestive system of non-venomous spiders, suggesting a common origin between digestive enzymes and enzymes present in venoms. This is the first characterization of the molecules involved in the digestive process and the midgut protein content of a non-venomous spider.

Preditores de impactos negativos da pandemia de covid-19 em universitários brasileiros / Preditors of negative impacts of the covid-19 pandemic on brazilian university undergraduates / Predictores de impactos negativos de la pandemia de covid- 19 en estudiantes universitarios brasileños

Impactos negativos da pandemia de COVID-19 têm sido observados na população em geral e em estudantes. Este estudo transversal objetivou identificar variáveis preditoras de 3 ou mais impactos negativos da pandemia de COVID-19 em graduandos de universidade pública brasileira. A amostra foi composta de 1.579 graduandos que responderam questionário on-line de setembro a outubro de 2020, sendo realizada análise logística univariada e multivariada. 97,2% dos graduandos indicaram aspectos afetados negativamente pela pandemia de COVID-19, e os mais citados foram estudo (80,1%), saúde mental (65,8%) e convívio social (64,9%). 72,9% indicaram impacto em 3 ou mais aspectos da vida, sendo preditores: sexo feminino; idade 18 a 24 anos; consumo de bebida alcoólica; doença crônica; acompanhamento psicológico antes pandemia; teve COVID-19; fez isolamento social; está em isolamento social; cessou/reduziu renda familiar; cessou/reduziu atividade física; cessou/reduziu lazer. Os dados indicaram que a evitação do consumo de álcool e a prática regular de atividade física e lazer podem ser fatores protetivos para os impactos negativos da pandemia de COVID-19, e sugerem reflexões sobre ações de apoio aos graduandos para minorar os impactos pandêmicos.

Negative impacts of the COVID-19 pandemic have been observed in the general population and in students. The present cross-sectional study aimed to identify predictor variables of 3 or more negative impacts of the COVID-19 pandemic on Brazilian public university undergraduates. The sample consisted of 1.579 undergraduates who answered an online questionnaire from September to October 2020, applying univariate and multivariate logistic analysis. 97.2% of undergraduates indicated aspects negatively affected by the COVID-19 pandemic, and the most cited were studies (80.1%), mental health (65.8%) and social interaction (64.9%). 72.9% indicated impact on 3 or more aspects of life, being predictors: female sex; age 18 to 24 years; consumption of alcoholic beverages; chronic disease; had psychological counseling before the pandemic; had COVID-19; did social isolation; is in social isolation; ceased/reduced family income; ceased/reduced physical activity; ceased/reduced leisure. The data indicated that avoidance of alcohol consumption and regular practice of physical activity and leisure may be protective factors for negative impacts of the COVID-19 pandemic and suggest reflections on actions to support undergraduates to mitigate pandemic impacts.

Se han observado impactos negativos de la pandemia de COVID-19 en la población general y en estudiantes. El presente estudio transversal tuvo como objetivo identificar variables predictoras de 3 o más impactos negativos de la pandemia de COVID-19 en estudiantes universitarios públicos brasileños. La muestra consistió en 1.579 estudiantes universitarios que respondieron a un cuestionario en línea de septiembre a octubre de 2020, aplicando análisis logístico univariado y multivariado. El 97,2% de los estudiantes universitarios indicaron aspectos afectados negativamente por la pandemia COVID-19, y los más citados fueron los estudios (80,1%), la salud mental (65,8%) y la interacción social (64,9%). 72,9% indicaron impacto en 3 o más aspectos de la vida, siendo predictores: sexo femenino; edad de 18 a 24 años; consumo de bebidas alcohólicas; enfermedad crónica; tuvo orientación psicológica antes de la pandemia; tuvo COVID-19; hizo aislamiento social; está en aislamiento social; cesó/redujo la renta familiar; cesó/redujo la actividad física; cesó/redujo el ocio. Los datos indicaron que evitar el consumo de alcohol y la práctica regular de actividad física y ocio pueden ser factores de protección para los impactos negativos de la pandemia de COVID-19 y sugieren reflexiones sobre acciones de apoyo a los estudiantes universitarios para mitigar los impactos de la pandemia.

Temporal and spatial analysis of municipal dengue cases in Paraná and social and environmental indicators, 2012 to 2021: ecological study.

OBJECTIVE: Analyze the temporal trend and spatial distribution of the incidence rate of dengue cases in Paraná and its regions between 2012 to 2021 and investigate associated sociodemographic and environmental variables. METHODS: Ecological study with temporal and spatial analysis of the dengue incidence rate reported in the Disease and Notification Information System (SINAN) in the period 2012 to 2021 and investigation of sociodemographic and environmental variables. To identify differences between municipal incidence rates the Mann-Whitney and Kruskal-Wallis test followed by Dunn's test for multiple comparisons were used. Prais-Winsten regression was used for temporal trend analysis and for spatial analysis the univariate and bivariate Local Moran analysis were applied. RESULTS: 548,683 cases of dengue were confirmed in the period, the highest state incidence rate was observed in 2020, with 15 health regions presenting more than 500 cases/100,000 inhabitants. Higher incidences were observed among women, age group of 20-59 years and white color/race. Despite annual variations, a stationary trend was observed for incidence rates according to sex, age group, color/race and macro-region. More than half of the municipalities in Paraná formed spatial clusters (Moran's I=0.679), where 73 (18.3%) municipalities with high incidence rate formed clusters. High-High clusters of dengue incidence rate with urbanization and High-Low clusters of incidence rate with vegetation cover were observed. CONCLUSION: Sociodemographic and environmental determinants were related to the high incidence rates of dengue and heterogeneous spatial distribution in the state of Paraná, indicating the need to strengthen health surveillance actions.

Clinical Manifestations and Complications of Children With COVID-19 Compared to Other Respiratory Viral Infections: A Cohort Inpatient Study From Rio de Janeiro, Brazil.

Introduction: The coronavirus disease-2019 (COVID-19) clinical manifestations in children and adolescents are diverse, despite the respiratory condition being the main presentation. Factors such as comorbidities and other respiratory infections may play a role in the initial presentation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. This study aims to describe the epidemiological aspects, clinical, and laboratory manifestations of pediatric patients admitted to a tertiary pediatric hospital in Rio de Janeiro, diagnosed with COVID-19, and compare these with other viral conditions during the first year of the SARS-CoV-2 pandemic. Methods: All patients under 18 years of age that were admitted with upper airway infection were enrolled and followed up for 30 days. The main dependent variable was the laboratorial diagnosis of SARS-CoV-2, and independent variables were studied through logistic regression. Results: A total of 533 patients were recruited, and 105 had confirmed SARS-CoV-2 infection. Detection of other viruses occurred in 34% of 264 tested participants. Six patients died (two in SARS-CoV-2 infected group). The variables independently associated with COVID-19 were older age (OR = 1.1, 95% CI = 1.0-1.1), lower leukocytes count at entry (OR = 0.9, 95% CI = 0.8-0.9), and contact with suspected case (OR = 1.6, 95% CI = 1.0-2.6). Patients with COVID-19 presented higher odds to be admitted in an intensive care unit (OR = 1.99, 95% CI = 1.08-3.66). Conclusions: Even during the SARS-CoV-2 pandemic, several other respiratory viruses were present in admitted pediatric patients. Variables associated with COVID-19 infection were older age, lower leukocytes count at entry, and a domiciliary suspect contact. Although patients with COVID-19 were more frequently admitted to ICU, we did not observe higher mortality in this group.

Biosynthetic studies through feeding experiments in Eclipta prostrata (L.) L. hairy roots.

Eclipta prostrata (L.) L. is a medicinal plant of the Asteraceae family, and several extracts and isolated compounds of E. prostrata (L.) L. showed a wide range of biological activities such as antimicrobial, anticancer, hepatoprotective, neuroprotective, hair growth promoting activities, and more recently against covid-19. Eclipta prostrata (L.) L. hairy roots produce wedelolactone (WL), demethylwedelolactone (DWL) and 3,5-di-O-caffeoylquinic acid (3,5-diCQA), and there is no data in literature regarding biosynthetic pathways are involved. To verify the biosynthetic route, feeding experiments were carried out using sodium [2-13C]acetate, [3-13C]dl-phenylalanine, and 13C-labeled compounds (WL, DWL and 3,5-diCQA) were detected by ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry (HPLC-QTOF-MS). Analysis showed that the metabolic pathways operative of coumestans (WL and DWL) are derived from acetate and shikimate pathways, while that the phenylpropanoid (3,5-diCQA) biosynthesis is exclusively from shikimate pathway. Supplementary Information: The online version contains supplementary material available at 10.1007/s11240-022-02342-0.

Directed Biosynthesis of New to Nature Alkaloids in a Heterologous Nicotiana benthamiana Expression Host.

Plants produce a wide variety of pharmacologically active molecules classified as natural products. Derivatization of these natural products can modulate or improve the bioactivity of the parent compound. Unfortunately, chemical derivatization of natural products is often difficult or impractical. Here we use the newly discovered biosynthetic genes for two monoterpene indole alkaloids, alstonine and stemmadenine acetate, to generate analogs of these compounds. We reconstitute these biosynthetic genes in the heterologous host Nicotiana benthamiana along with an unnatural starting substrate to produce the corresponding new-to-nature alkaloid product.

Structural Characterization of L-Galactose Dehydrogenase: An Essential Enzyme for Vitamin C Biosynthesis.

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learned concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase [Spinacia oleracea GDH (SoGDH) from spinach], from the D-mannose/L-galactose (Smirnoff-Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homolog from camu camu, a super-accumulator of vitamin C found in the Peruvian Amazon. Both enzymes are monomers in solution and have a pH optimum of 7, and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (ß/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favors the oxidation reaction that ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes toward a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

Bioinformatic analyses to uncover genes involved in trehalose metabolism in the polyploid sugarcane.

Trehalose-6-phosphate (T6P) is an intermediate of trehalose biosynthesis that plays an essential role in plant metabolism and development. Here, we comprehensively analyzed sequences from enzymes of trehalose metabolism in sugarcane, one of the main crops used for bioenergy production. We identified protein domains, phylogeny, and in silico expression levels for all classes of enzymes. However, post-translational modifications and residues involved in catalysis and substrate binding were analyzed only in trehalose-6-phosphate synthase (TPS) sequences. We retrieved 71 putative full-length TPS, 93 trehalose-6-phosphate phosphatase (TPP), and 3 trehalase (TRE) of sugarcane, showing all their conserved domains, respectively. Putative TPS (Classes I and II) and TPP sugarcane sequences were categorized into well-known groups reported in the literature. We measured the expression levels of the sequences from one sugarcane leaf transcriptomic dataset. Furthermore, TPS Class I has specific N-glycosylation sites inserted in conserved motifs and carries catalytic and binding residues in its TPS domain. Some of these residues are mutated in TPS Class II members, which implies loss of enzyme activity. Our approach retrieved many homo(eo)logous sequences for genes involved in trehalose metabolism, paving the way to discover the role of T6P signaling in sugarcane.

Jasmonates promote enhanced production of bioactive caffeoylquinic acid derivative in Eclipta prostrata (L.) L. hairy roots.

Eclipta prostrata (L.) L. is widely used in traditional medicine for treatment of hepatitis, poisoning from snake bites and viral infections. Pharmacological studies confirmed its antioxidant, anti-inflammatory and anticancer activities. The efficacy of E. prostrata (L.) L. extracts has been correlated to phenylpropanoids such as flavonoids, coumestans and caffeoylquinic acid derivatives. In this work, the production of wedelolactone, demethylwedelolactone and 3,5-di-O-caffeoylquinic acid (3,5-diCQA) in hairy root cultures of E. prostrata (L.) L. C19 clone was increased after addition of eliciting agents jasmonic acid (JA) or methyl jasmonate (MeJA) at multiple concentrations. Cultures elicited with 100 µM of JA saw a 5.2 fold increase in wedelolactone (from 0.72 to 3.72 mg/g d.w.), a 1.6 fold increase in demethylwedelolactone (from 5.54 to 9.04 mg/g d.w.) and a 2.47 fold increase in 3,5-diCQA (from 18.08 to 44.71 mg/g d.w.). Obtained data validate the potential of E. prostrata (L.) L. hairy root cultures as a production system of wedelolactone, demethylwedelolactone and especially 3,5-diCQA, which has recently been reported to possess activity against coronavirus disease (Covid-19) by in silico computational studies. Supplementary Information: The online version contains supplementary material available at 10.1007/s11240-021-02201-4.

Genetic diversity among genotypes of Uncaria guianensis (Aubl.) J.F. Gmel. maintained in an in vitro germplasm bank.

Phytotherapeutic preparations from Uncaria guianensis (Aubl.) J.F. Gmel. (Rubiaceae) are marketed worldwide and are mainly used for their anti-inflammatory activity. The species has not yet been domesticated and is threatened by deforestation and overexploitation. It is, therefore, important to preserve and manage this genetic resource in germplasm banks, so that the extractive provision of plant material can be replaced by cultivated production. The aim of this study was to evaluate the genetic diversity among 20 genotypes maintained under in vitro conditions using 9 primers start codon targeted (SCoT) polymorphism, and to determine the concentrations of the pentacyclic oxindole alkaloids (POAs); mitraphylline and isomitraphylline in methanolic extracts by high-performance liquid chromatography (HPLC). Plantlets were cultivated on woody plant medium supplemented with 20 g.L-1 sucrose and 4.4 µM benzylaminopurine and incubated under a 16 h photoperiod for 45 days. SCoT analysis separated the genotypes into four divergent clusters and confirmed significant genetic diversity with up to 70% dissimilarity. Moreover, HPLC revealed considerable chemical variability and allowed the separation of the tested genotypes into high, medium and low producers of mitraphylline/isomitraphylline. Genotypes with the highest concentrations of POAs originated from the state of Acre and Amapá, while those with the lowest levels were from the state of Pará. The results demonstrate that the genetic diversity within the in vitro germplasm bank is sufficient to support breeding studies, selection of elite genotypes and the large-scale multiplication of plants that could serve as feedstock for the industrial-scale production of phytomedicines. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03016-y.

Structural characterization of L-Galactose Dehydrogenase: an essential enzyme for vitamin C biosynthesis

In plants, it is well-known that ascorbic acid (vitamin C) can be synthesized via multiple metabolic pathways but there is still much to be learnt concerning their integration and control mechanisms. Furthermore, the structural biology of the component enzymes has been poorly exploited. Here we describe the first crystal structure for an L-galactose dehydrogenase (SoGDH from spinach), from the D-mannose/L-galactose (Smirnoff Wheeler) pathway which converts L-galactose into L-galactono-1,4-lactone. The kinetic parameters for the enzyme are similar to those from its homologue from camu-camu, a super-accumulator of vitamin C found in the Peruvian amazon. Both enzymes are monomers in solution, have a pH optimum of 7 and their activity is largely unaffected by high concentrations of ascorbic acid, suggesting the absence of a feedback mechanism acting via GDH. Previous reports may have been influenced by changes of the pH of the reaction medium as a function of ascorbic acid concentration. The structure of SoGDH is dominated by a (β/α)8 barrel closely related to aldehyde-keto reductases (AKRs). The structure bound to NAD+ shows that the lack of Arg279 justifies its preference for NAD+ over NADP+, as employed by many AKRs. This favours the oxidation reaction which ultimately leads to ascorbic acid accumulation. When compared with other AKRs, residue substitutions at the C-terminal end of the barrel (Tyr185, Tyr61, Ser59 and Asp128) can be identified to be likely determinants of substrate specificity. The present work contributes towards a more comprehensive understanding of structure-function relationships in the enzymes involved in vitamin C synthesis.

Bioinformatic analyses to uncover genes involved in trehalose metabolism in the polyploid sugarcane

Trehalose-6-phosphate (T6P) is an intermediate of trehalose biosynthesis that plays an essential role in plant metabolism and development. Here, we comprehensively analyzed sequences from enzymes of trehalose metabolism in sugarcane, one of the main crops used for bioenergy production. We identified protein domains, phylogeny, and in silico expression levels for all classes of enzymes. However, post-translational modifications and residues involved in catalysis and substrate binding were analyzed only in trehalose-6-phosphate synthase (TPS) sequences. We retrieved 71 putative full-length TPS, 93 trehalose-6-phosphate phosphatase (TPP), and 3 trehalase (TRE) of sugarcane, showing all their conserved domains, respectively. Putative TPS (Classes I and II) and TPP sugarcane sequences were categorized into well-known groups reported in the literature. We measured the expression levels of the sequences from one sugarcane leaf transcriptomic dataset. Furthermore, TPS Class I has specific N-glycosylation sites inserted in conserved motifs and carries catalytic and binding residues in its TPS domain. Some of these residues are mutated in TPS Class II members, which implies loss of enzyme activity. Our approach retrieved many homo(eo)logous sequences for genes involved in trehalose metabolism, paving the way to discover the role of T6P signaling in sugarcane.

Temporal and spatial analysis of municipal dengue cases in Paraná and social and environmental indicators, 2012 to 2021: ecological study / Análise temporal e espacial dos casos municipais de dengue no Paraná e indicadores sociais e ambientais, 2012 a 2021: estudo ecológico

ABSTRACT Objective: Analyze the temporal trend and spatial distribution of the incidence rate of dengue cases in Paraná and its regions between 2012 to 2021 and investigate associated sociodemographic and environmental variables. Methods: Ecological study with temporal and spatial analysis of the dengue incidence rate reported in the Disease and Notification Information System (SINAN) in the period 2012 to 2021 and investigation of sociodemographic and environmental variables. To identify differences between municipal incidence rates the Mann-Whitney and Kruskal-Wallis test followed by Dunn's test for multiple comparisons were used. Prais-Winsten regression was used for temporal trend analysis and for spatial analysis the univariate and bivariate Local Moran analysis were applied. Results: 548,683 cases of dengue were confirmed in the period, the highest state incidence rate was observed in 2020, with 15 health regions presenting more than 500 cases/100,000 inhabitants. Higher incidences were observed among women, age group of 20-59 years and white color/race. Despite annual variations, a stationary trend was observed for incidence rates according to sex, age group, color/race and macro-region. More than half of the municipalities in Paraná formed spatial clusters (Moran's I=0.679), where 73 (18.3%) municipalities with high incidence rate formed clusters. High-High clusters of dengue incidence rate with urbanization and High-Low clusters of incidence rate with vegetation cover were observed. Conclusion: Sociodemographic and environmental determinants were related to the high incidence rates of dengue and heterogeneous spatial distribution in the state of Paraná, indicating the need to strengthen health surveillance actions.

RESUMO Objetivo: Analisar a tendência temporal e a distribuição espacial da taxa de incidência de casos de dengue no Paraná e suas regiões entre 2012 e 2021 e investigar variáveis sociodemográficas e ambientais associadas. Métodos: Estudo ecológico com análises temporais e espaciais da taxa de incidência da dengue registrada no Sistema de Informação de Agravos de Notificação, de 2012 e 2021, e investigação de variáveis sociodemográficas e ambientais. Para analisar as taxas de incidência municipais foram utilizados os testes de Mann-Whitney e Kruskal-Wallis, seguidos do teste de Dunn para múltiplas comparações. Utilizou-se para análise da tendência temporal a regressão de Prais-Winsten, e para a análise espacial, o índice de Moran Local univariado e bivariado. Resultados: Foram confirmados 548.683 casos de dengue no período, apresentando a maior taxa de incidência estadual em 2020, com 15 regionais de saúde registrando mais de 500 casos/100 mil habitantes. Maiores incidências ficaram entre mulheres, faixa etária de 20-59 anos e cor/raça branca. Apesar de variações anuais, observou-se tendência estacionária para incidência segundo sexo, faixa etária, cor e macrorregião. Mais da metade dos municípios paranaenses formou aglomerados espaciais (Moran's 1=0,679) — 73 (18,3%) municípios com alta taxa de incidência formaram agrupamentos. Foram identificados agrupamentos da taxa de incidência da dengue com o grau de urbanização (alto-alto) e com o percentual de cobertura vegetal natural (alto-baixa). Conclusão: Determinantes sociodemográficos e ambientais relacionaram-se com as altas taxas de incidência da dengue e com a distribuição espacial heterogênea no estado do Paraná, indicando a necessidade do fortalecimento das ações de vigilância em saúde.

Depression, anxiety and stress symptoms in Brazilian university students during the COVID-19 pandemic: Predictors and association with life satisfaction, psychological well-being and coping strategies.

BACKGROUND: The COVID-19 pandemic raises concerns about the mental health of the world population. Protection measures to prevention the disease impacted education and undergraduate students were exposed to additional stressors. OBJECTIVES: Analyze depression, anxiety and stress symptoms in undergraduates, their respective predictors and the association with satisfaction with life, psychological well-being and coping strategies. METHODS: An online cross-sectional study was conducted from September 14 to October 19, 2020, involving undergraduate students enrolled in 33 courses from 5 public university campuses in the state of Parana, Brazil, using: questionnaire with sociodemographic, academic, health and pandemic effects variables; Depression, Anxiety and Stress Scale-21 (DASS-21); Satisfaction with Life Scale (SWLS); Psychological Well-Being (PWB); BriefCOPE. The convenience sample was composed of 1,224 participants, with 18 years old or older, that completed all research instruments. Spearman correlation and logistic analysis (univariate and multivariate) were applied to the collected data. RESULTS: Most of the undergraduates presented symptoms of depression (60.5%), anxiety (52.5%) and stress (57.5%). Depression, anxiety and stress presented significant correlations in common: negative with satisfaction with life, all dimensions of psychological well-being, and 3 adaptive copings (active coping, planning, positive reframing); positive with 5 maladaptive copings (behavioral disengagement, denial, self-blame, self-distraction, substance use). In addition, there were 7 common predictors for symptoms of depression, anxiety and stress: female; age 18-24 years old; having a chronic disease; lower scores in 2 dimensions of psychological well-being (positive relations with others, self-acceptance); higher scores in 2 maladaptive copings (self-blame, substance use). CONCLUSIONS: The data indicate a high prevalence of symptoms of depression, anxiety and stress, and suggest that higher scores of satisfaction with life, psychological well-being dimensions and adaptive copings may present protective effects in undergraduates during a pandemic crisis.