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.

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.

Precursor-directed Biosynthesis in Tabernaemontana catharinensis as a New Avenue for Alzheimer's Disease-modifying Agents.

Plants produce a high diversity of metabolites that can act as regulators of cholinergic dysfunction. Among plants, the potential of species of the genus Tabernaemontana to treat neurological disorders has been linked to iboga-type alkaloids that are biosynthesized by those species. In this context, precursor-directed biosynthesis approaches were carried out using T. catharinensis plantlets to achieve new-to-nature molecules as promising agents against Alzheimer's disease. Aerial parts of T. catharinensis, cultured in vitro, produced 7 unnatural alkaloids (5-fluoro-ibogamine, 5-fluoro-voachalotine, 5-fluoro-12-methoxy-Nb-methyl-voachalotine, 5-fluoro-isovoacangine, 5-fluoro-catharanthine, 5-fluoro-19-(S)-hydroxy-ibogamine, and 5-fluoro-coronaridine), while root extracts showed the presence of the same unnatural iboga-type alkaloids and 2 additional ones: 5-fluoro-voafinine and 5-fluoro-affinisine. Moreover, molecular docking approaches were carried out to evaluate the potential inhibition activity of T. catharinensis' natural and unnatural alkaloids against AChE and BChE enzymes. Fluorinated iboga alkaloids (5-fluoro-catharanthine, 5-fluoro-voachalotine, 5-fluoro-affinisine, 5-fluoro-isovoacangine, 5-fluoro-corinaridine) were more active than natural ones and controls against AchE, while 5-fluoro-19-(S)-hydroxy-ibogamine, 5-fluoro-catharanthine, 5-fluoro-isovoacangine, and 5-fluoro-corinaridine showed better activity than natural ones and controls against BChE. Our findings showed that precursor-directed biosynthesis strategies generated "new-to-nature" alkaloids that are promising Alzheimer's disease drug candidates. Furthermore, the isotopic experiments also allowed us to elucidate the initial steps of the biosynthetic pathway for iboga-type alkaloids, which are derived from the MEP and shikimate pathways.

Advances in the Biosynthesis of Pyranocoumarins: Isolation and 13C-Incorporation Analysis by High-Performance Liquid Chromatography-Ultraviolet-Solid-Phase Extraction-Nuclear Magnetic Resonance Data.

Citrus sinensis and Citrus limonia were obtained by germination from seeds, and isotopic-labeling experiments using d-[1-13C]glucose were performed with the seedlings. After 60 days, the seedlings were analyzed by high-performance liquid chromatography-ultraviolet-solid-phase extraction-nuclear magnetic resonance, data and the 13C enrichment patterns of xanthyletin and seselin indicated that the pyran ring was formed by the methylerythritol phosphate pathway and that the coumarin moiety was derived from the shikimate pathway in both compounds. This information regarding the biosynthetic pathway can be used to increase resistance against phytopathogens, because xanthyletin and seselin are reported to have antimicrobial activity on the growth of Xylella fastidiosa, which causes citrus variegated chlorosis in orange.

Antioxidant Activity of an Aqueous Leaf Extract from Uncaria tomentosa and Its Major Alkaloids Mitraphylline and Isomitraphylline in Caenorhabditis elegans.

Uncaria tomentosa (Rubiaceae) has a recognized therapeutic potential against various diseases associated with oxidative stress. The aim of this research was to evaluate the antioxidant potential of an aqueous leaf extract (ALE) from U. tomentosa, and its major alkaloids mitraphylline and isomitraphylline. The antioxidant activity of ALE was investigated in vitro using standard assays (DPPH, ABTS and FRAP), while the in vivo activity and mode of action were studied using Caenorhabditis elegans as a model organism. The purified alkaloids did not exhibit antioxidant effects in vivo. ALE reduced the accumulation of reactive oxygen species (ROS) in wild-type worms, and was able to rescue the worms from a lethal dose of the pro-oxidant juglone. The ALE treatment led to a decreased expression of the oxidative stress response related genes sod-3, gst-4, and hsp-16.2. The treatment of mutant worms lacking the DAF-16 transcription factor with ALE resulted in a significant reduction of ROS levels. Contrarily, the extract had a pro-oxidant effect in the worms lacking the SKN-1 transcription factor. Our results suggest that the antioxidant activity of ALE in C. elegans is independent of its alkaloid content, and that SKN-1 is required for ALE-mediated stress resistance.

Identity and role of the non-conserved acid/base catalytic residue in the GH29 fucosidase from the spider Nephilingis cruentata.

α-l-Fucosidases are widely occurring enzymes that remove fucose residues from N- and O-fucosylated glycoproteins. Comparison of amino acid sequences of fucosidases reveals that although the nucleophile is conserved among all α-l-fucosidases, the position of the acid/base residue is quite variable. Although several site-directed mutation studies have previously been performed on bacterial fucosidases, the only eukaryotic fucosidase so studied was the human fucosidase. Recent alignments indicate that human and Arthropoda α-l-fucosidases share at least 50% identity and the acid/base residue seems to be conserved among them suggesting a common acid/base residue in Metazoa. Here we describe the cloning and expression in Pichia pastoris of a very active α-l-fucosidase from the spider Nephilingis cruentata (NcFuc) with a Km value for pNPFuc of 0.4 mM. NcFuc hydrolyzed fucoidan, 2´fucosyllactose and also lacto-N-difucohexaose II. Mutants modified at the conserved residues D214N, E209A, E59A were expressed and characterized. The 500-fold lower kcat of D214N than the wild type was consistent with a role in catalysis, as was the 8000-fold lower kcat value of E59A. This was supported by the 57-fold increase in the kcat of E59A upon addition of azide. A complex pH/rate profile was seen for the wild-type and mutant forms of NcFuc, similar to those measured previously for the Sulfolobus fucosidase. The non-conservative catalytic structure and distinct active site organization reinforce the necessity of structural studies of new fucosidases.

First characterization of fucosidases in spiders.

l-fucose is a constituent of glycoconjugates in different organisms. Fucosidases catalyze the removal of fucose residues, and have been correlated to different physiological and pathological processes, such as fertilization, cancer, fucosidosis, and digestion in molluscs and ticks. An α-l-fucosidase sequence was identified from the transcriptome and proteome from the midgut diverticula of the synanthropic spider Nephilingis cruentata. In this article, we describe the isolation of this α-l-fucosidase and the characterization of its activity using substrates and inhibitors demonstrating different specificities among fucosidases. The enzyme had a Km of 32 and 400 µM for 4-methylumbelliferyl α-l-fucopyranoside and 4-nitrophenyl α-l-fucopyranoside, respectively; and was unable to hydrolyze fucoidan. Nephilingis cruentata α-l-fucosidase was inhibited competitively by fucose and fuconojyrimycin. The fucosidase had two distinct pH optima even in the isolated form, due to oligomerization dependent on pH, as previously described to other fucosidases. Alignment and molecular homology modeling of the protein sequence with other fucosidases indicated that the active sites and catalytic residues were different, including residues involved in acid/base catalysis. Phylogenetic analysis showed, for the first time, gene-duplication events for fucosidases in Arachnida species. All these data reveal that studies on fucosidases in organisms distinct from bacteria, fungi, and humans are important.

High throughput techniques to reveal the molecular physiology and evolution of digestion in spiders.

BACKGROUND: Spiders are known for their predatory efficiency and for their high capacity of digesting relatively large prey. They do this by combining both extracorporeal and intracellular digestion. Whereas many high throughput ("-omics") techniques focus on biomolecules in spider venom, so far this approach has not yet been applied to investigate the protein composition of spider midgut diverticula (MD) and digestive fluid (DF). RESULTS: We here report on our investigations of both MD and DF of the spider Nephilingis (Nephilengys) cruentata through the use of next generation sequencing and shotgun proteomics. This shows that the DF is composed of a variety of hydrolases including peptidases, carbohydrases, lipases and nuclease, as well as of toxins and regulatory proteins. We detect 25 astacins in the DF. Phylogenetic analysis of the corresponding transcript(s) in Arachnida suggests that astacins have acquired an unprecedented role for extracorporeal digestion in Araneae, with different orthologs used by each family. The results of a comparative study of spiders in distinct physiological conditions allow us to propose some digestion mechanisms in this interesting animal taxon. CONCLUSION: All the high throughput data allowed the demonstration that DF is a secretion originating from the MD. We identified enzymes involved in the extracellular and intracellular phases of digestion. Besides that, data analyses show a large gene duplication event in Araneae digestive process evolution, mainly of astacin genes. We were also able to identify proteins expressed and translated in the digestive system, which until now had been exclusively associated to venom glands.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Application of a simple unstructured kinetic and cost of goods models to support T-cell therapy manufacture.

Manufacturing of cell therapy products requires sufficient understanding of the cell culture variables and associated mechanisms for adequate control and risk analysis. The aim of this study was to apply an unstructured ordinary differential equation-based model for prediction of T-cell bioprocess outcomes as a function of process input parameters. A series of models were developed to represent the growth of T-cells as a function of time, culture volumes, cell densities, and glucose concentration using data from the Ambr®15 stirred bioreactor system. The models were sufficiently representative of the process to predict the glucose and volume provision required to maintain cell growth rate and quantitatively defined the relationship between glucose concentration, cell growth rate, and glucose utilization rate. The models demonstrated that although glucose is a limiting factor in batch supplied medium, a delivery rate of glucose at significantly less than the maximal specific consumption rate (0.05 mg 1 × 106  cell h-1 ) will adequately sustain cell growth due to a lower glucose Monod constant determining glucose consumption rate relative to the glucose Monod constant determining cell growth rate. The resultant volume and exchange requirements were used as inputs to an operational BioSolve cost model to suggest a cost-effective T-cell manufacturing process with minimum cost of goods per million cells produced and optimal volumetric productivity in a manufacturing settings. These findings highlight the potential of a simple unstructured model of T-cell growth in a stirred tank system to provide a framework for control and optimization of bioprocesses for manufacture.

Structural and functional characterization of the glutathione peroxidase-like thioredoxin peroxidase from the fungus Trichoderma reesei.

Glutathione peroxidases (GPx) are a family of enzymes with the ability to reduce organic and inorganic hydroperoxides to the corresponding alcohols using glutathione or thioredoxin as an electron donor. Here, we report the functional and structural characterization of a GPx identified in Trichoderma reesei (TrGPx). TrGPx was recombinantly expressed in a bacterial host and purified using affinity. Using a thioredoxin coupled assay, TrGPx exhibited activity of 28 U and 12.5 U in the presence of the substrates H2O2 and t-BOOH, respectively, and no activity was observed when glutathione was used. These results indicated that TrGPx is a thioredoxin peroxidase and hydrolyses H2O2 better than t-BOOH. TrGPx kinetic parameters using a pyrogallol assay resulted at Kmapp = 11.7 mM, Vmaxapp = 10.9 IU/µg TrGPx, kcat = 19 s-1 and a catalytic efficiency of 1.6 mM-1 s-1 to H2O2 as substrate. Besides that, TrGPx demonstrated an optimum pH ranging from 9.0-12.0 and a half-life of 36 min at 80 °C. TrGPx 3D-structure was obtained in a reduced state and non-catalytic conformation. The overall fold is similar to the other phospholipid-hydroperoxide glutathione peroxidases. These data contribute to understand the antioxidant mechanism in fungi and provide information for using antioxidant enzymes in biotechnological applications.