Dimer-assisted mechanism of (un)saturated fatty acid decarboxylation for alkene production.

The enzymatic decarboxylation of fatty acids (FAs) represents an advance toward the development of biological routes to produce drop-in hydrocarbons. The current mechanism for the P450-catalyzed decarboxylation has been largely established from the bacterial cytochrome P450 OleTJE. Herein, we describe OleTPRN, a poly-unsaturated alkene-producing decarboxylase that outrivals the functional properties of the model enzyme and exploits a distinct molecular mechanism for substrate binding and chemoselectivity. In addition to the high conversion rates into alkenes from a broad range of saturated FAs without dependence on high salt concentrations, OleTPRN can also efficiently produce alkenes from unsaturated (oleic and linoleic) acids, the most abundant FAs found in nature. OleTPRN performs carbon-carbon cleavage by a catalytic itinerary that involves hydrogen-atom transfer by the heme-ferryl intermediate Compound I and features a hydrophobic cradle at the distal region of the substrate-binding pocket, not found in OleTJE, which is proposed to play a role in the productive binding of long-chain FAs and favors the rapid release of products from the metabolism of short-chain FAs. Moreover, it is shown that the dimeric configuration of OleTPRN is involved in the stabilization of the A-A' helical motif, a second-coordination sphere of the substrate, which contributes to the proper accommodation of the aliphatic tail in the distal and medial active-site pocket. These findings provide an alternative molecular mechanism for alkene production by P450 peroxygenases, creating new opportunities for biological production of renewable hydrocarbons.

Predictive model for temporomandibular disorder in adolescents: Decision tree.

BACKGROUND: Temporomandibular disorders (TMD) do not only occur in adults but also in adolescents, with negative impacts on their development. AIM: To propose a predictive model for TMD in adolescents using a decision tree (DT) analysis and to identify groups at high and low risk of developing TMD in the city of Recife, PE, Brazil. DESIGN: This cross-sectional study was conducted in Recife on 1342 schoolchildren of both sexes aged 10-17 years. The analyses were performed using Pearson's chi-squared test and Fisher's exact test, as well as the CHAID algorithm for the construction of the DT. The SPSS statistical program was used. RESULTS: The prevalence of TMD was 33.2%. Statistically significant associations were observed between TMD and sex, depression, self-reported orofacial pain, and orofacial pain on clinical examination. The DT consisted of self-reported orofacial pain, orofacial pain on physical examination, and depression, with an overall predictive power of 73.0%. CONCLUSION: The proposed tree has a good predictive capacity and permits to identify groups at high risk of developing TMD among adolescents, such as those with self-reported orofacial pain or orofacial pain on examination associated with depression.

Paradigm shift in xylose isomerase usage: a novel scenario with distinct applications.

Isomerases are enzymes that induce physical changes in a molecule without affecting the original molecular formula. Among this class of enzymes, xylose isomerases (XIs) are the most studied to date, partly due to their extensive application in industrial processes to produce high-fructose corn sirups. In recent years, the need for sustainable initiatives has triggered efforts to improve the biobased economy through the use of renewable raw materials. In this context, D-xylose usage is crucial as it is the second-most abundant sugar in nature. The application of XIs in biotransforming xylose, enabling downstream metabolism in several microorganisms, is a smart strategy for ensuring a low-carbon footprint and producing several value-added biochemicals with broad industrial applications such as in the food, cosmetics, pharmaceutical, and polymer industries. Considering recent advancements that have expanded the range of applications of XIs, this review provides a comprehensive and concise overview of XIs, from their primary sources to the biochemical and structural features that influence their mechanisms of action. This comprehensive review may help address the challenges involved in XI applications in different industries and facilitate the exploitation of xylose bioprocesses.

Electrostatic interaction optimization improves catalytic rates and thermotolerance on xylanases.

Understanding the aspects that contribute to improving proteins' biochemical properties is of high relevance for protein engineering. Properties such as the catalytic rate, thermal stability, and thermal resistance are crucial for applying enzymes in the industry. Different interactions can influence those biochemical properties of an enzyme. Among them, the surface charge-charge interactions have been a target of particular attention. In this study, we employ the Tanford-Kirkwood solvent accessibility model using the Monte Carlo algorithm (TKSA-MC) to predict possible interactions that could improve stability and catalytic rate of a WT xylanase (XynAWT) and its M6 xylanase (XynAM6) mutant. The modeling prediction indicates that mutating from a lysine in position 99 to a glutamic acid (K99E) favors the native state stabilization in both xylanases. Our lab results showed that mutated xylanases had their thermotolerance and catalytic rate increased, which conferred higher processivity of delignified sugarcane bagasse. The TKSA-MC approach employed here is presented as an efficient computational-based design strategy that can be applied to improve the thermal resistance of enzymes with industrial and biotechnological applications.

Inferior pancreaticoduodenal artery aneurysm: endovascular approach.

Aneurysms of the pancreaticoduodenal arteries are a rare condition. In the majority of cases, diagnosis is made in emergency situations due to complications such as rupture, which is associated with high mortality rates (21-26%). Embolization of the aneurysm sac is the treatment of choice, because of its high efficacy and lower mortality. This article presents and discusses a case of inferior pancreaticoduodenal artery aneurysm that was diagnosed during investigation of gastrointestinal symptoms. The treatment provided was microcoil embolization, with complete exclusion of the aneurysm and a good clinical course.

Lactation performance of dairy cows fed rehydrated and ensiled corn grain differing in particle size and proportion in the diet.

Rehydrated and ensiled mature ground corn has high ruminal starch digestibility, but particle size (PS) and dietary starch proportion (ST) can affect starch digestion and lactating cow performance. We evaluated the effect of rehydrated and ensiled corn (REC), PS, and ST on intake, lactation performance, nutrient digestibility, ruminal fermentation profile, and chewing behavior of dairy cows. Kernels from an 84% vitreousness hybrid were finely (FN) or coarsely (CS) ground, yielding geometric mean particle sizes of 1,591 and 2,185 µm, respectively. Ground kernels were rehydrated [60% dry matter (DM)] and ensiled in 200-L buckets for ≥205 d. The grinding rate (t/h) was 3.9 for FN and 11.7 for CS. The PS did not affect DM loss (11.3% of ensiled) or silage pH (3.8). Samples of each bucket (n = 15/PS) before and after silage fermentation were incubated in situ for 0, 3, 6, 18, and 48 h in 4 rumen-cannulated lactating cows. Ensiling increased the effective ruminal in situ DM degradation (63.7 vs. 34.1%), regardless of PS. Sixteen Holstein cows (152 ± 96 d in milk) in 4 × 4 Latin squares (21-d periods) were individually fed a 2 × 2 factorial combination of low (LO) or high (HI) starch diets with FN or CS. Cows were fed the same REC incubated in situ. Varied concentration of starch in the diet (29.2 vs. 23.5% of DM) was achieved by partial replacement of REC (22.0 vs. 14.2% of DM) with citrus pulp (0 vs. 8.2% of DM). Milk, protein, fat, and lactose yields did not differ. Milk fat percentage was reduced and protein percentage was increased by HI. Treatment FN increased feed efficiency (energy-corrected milk/digestible organic matter intake) when fed with HI. Total-tract starch digestibility tended to be reduced by CS (96.4 vs. 97.2% of starch intake). Serum ß-hydroxybutyrate was increased by LO. High-starch diet reduced the molar proportions of acetate and butyrate in ruminal fluid and increased propionate and isoacids. Particle size did not affect ruminal fermentation profile. Coarse grinding reduced plasma d-lactate concentration with HI. Diet HI reduced the proportion of daily intake from 1900 to 0700 h and induced preferential intake of feed particles <8 mm and greater refusal of particles >19 mm in the morning. Fine REC reduced rumination time per day and increased eating time per DM intake. Milk and plasma urea-N did not differ. Ensiling of mature flint corn for >200 d largely eliminated the effect of the PS of REC on the studied outcomes. The proportion of REC in the diet affected ruminal fermentation profile and milk solids concentration, but did not affect short-term performance and digestibility. Coarse grinding of REC may allow increasing the grinding rate and thus save labor and energy during ensiling.

Detoxification Mechanism of 8,8-Dimethyl-3-[(R-phenyl)amino]-1,4,5(8H)-naphthalentrione Derivatives by Botrytis cinerea.

The effect of 8,8-dimethyl-3-[(R-phenyl)amino]-1,4,5(8H)-naphthalentrione derivatives (compounds 1⁻13) on the mycelial growth of Botrytis cinerea was evaluated. The fungitoxic effect depended on the substituent and its position in the aromatic ring. Compounds substituted with halogens in meta and/or para positions (compounds 3, 4, 5 and 7), methyl (compounds 8 and 9), methoxyl (compounds 10 and 11), or ethoxy-carbonyl groups (compound 12) presented higher antifungal activity than compound 1, which had an unsubstituted aromatic ring. In addition, compounds with halogens in the ortho position, such as compounds 2 and 6, and a substitution with an acetyl group in the para position (compound 13) were less active. The role of the ABC efflux pump Bctr B-type as a defense mechanism of B. cinerea against these naphthalentrione derivatives was analyzed. This pump could be involved in the detoxification of compounds 2, 6, and 13. On the contrary, this mechanism would not participate in the detoxification of compounds 1, 7, 9 and 12. Finally, the biotransformation of compound 7 by B. cinerea was studied. A mixture of two biotransformed products was obtained. One of them was compound 7A, which is reduced at C1 and C4, compared to compound 7. The other product of biotransformation, 7B, is oxidized at C7.

An overview of Bacillus proteases: from production to application.

Proteases have a broad range of applications in industrial processes and products and are representative of most worldwide enzyme sales. The genus Bacillus is probably the most important bacterial source of proteases and is capable of producing high yields of neutral and alkaline proteolytic enzymes with remarkable properties, such as high stability towards extreme temperatures, pH, organic solvents, detergents and oxidizing compounds. Therefore, several strategies have been developed for the cost-effective production of Bacillus proteases, including optimization of the fermentation parameters. Moreover, there are many studies on the use of low-cost substrates for submerged and solid state fermentation. Other alternatives include genetic tools such as protein engineering in order to obtain more active and stable proteases and strain engineering to better secrete recombinant proteases from Bacillus through homologous and heterologous protein expression. There has been extensive research on proteases because of the broad number of applications for these enzymes, such as in detergent formulations for the removal of blood stains from fabrics, production of bioactive peptides, food processing, enantioselective reactions, and dehairing of skins. Moreover, many commercial proteases have been characterized and purified from different Bacillus species. Therefore, this review highlights the production, purification, characterization, and application of proteases from a number of Bacillus species.

New Heterofunctional Supports Based on Glutaraldehyde-Activation: A Tool for Enzyme Immobilization at Neutral pH.

Immobilization is an exciting alternative to improve the stability of enzymatic processes. However, part of the applied covalent strategies for immobilization uses specific conditions, generally alkaline pH, where some enzymes are not stable. Here, a new generation of heterofunctional supports with application at neutral pH conditions was proposed. New supports were developed with different bifunctional groups (i.e., hydrophobic or carboxylic/metal) capable of adsorbing biocatalysts at different regions (hydrophobic or histidine richest place), together with a glutaraldehyde group that promotes an irreversible immobilization at neutral conditions. To verify these supports, a multi-protein model system (E. coli extract) and four enzymes (Candidarugosa lipase, metagenomic lipase, ß-galactosidase and ß-glucosidase) were used. The immobilization mechanism was tested and indicated that moderate ionic strength should be applied to avoid possible unspecific adsorption. The use of different supports allowed the immobilization of most of the proteins contained in a crude protein extract. In addition, different supports yielded catalysts of the tested enzymes with different catalytic properties. At neutral pH, the new supports were able to adsorb and covalently immobilize the four enzymes tested with different recovered activity values. Notably, the use of these supports proved to be an efficient alternative tool for enzyme immobilization at neutral pH.

In vitro and in vivo evaluation of the antioxidant and prooxidant activity of phenolic compounds obtained from grape (Vitis vinifera) pomace.

The antioxidant and/or prooxidant ability of extracts obtained from wine waste were analyzed using in vitro and in vivo assays. Cyclic voltammetry was used as the in vitro assay to determine the antioxidant and/or prooxidant properties and, the in vivo effect on mycelial growth of the fungus Botrytis cinerea was evaluated. In addition, the prooxidant activity was evaluated by intracellular oxidation of compound 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) in B. cinerea. The extracts used in this study were obtained from grape pomace of Cabernet Sauvignon, Carménère and Syrah varieties from the Misiones de Rengo Vineyard by simple extraction, using methanol/HCl 1% (v/v), ethanol 70% (v/v), or Soxhlet extraction. According to the results obtained, gallic acid was the most represented phenolic compound independent of grape variety and extraction method. In addition, vanillic acid; protocatechuic acid, syringic acid, quercetin and kaempferol were found in the extracts. From this study it was possible concluded that, depending of the method of extraction of the grape residues and the grape variety (Cabernet Sauvignon, Carménère and Syrah), the extracts showed antioxidant and/or prooxidant activity. However, no correlation can be established between the anodic oxidation potentials of the extracts and their effect on the fungus B. cinerea.

[Anorexia and Weight Loss as First Symptoms of a Solitary Fibrous Tumor of the Pleura]. / Anorexia e Perda Ponderal, Os Primeiros Sintomas de Um Tumor Fibroso Solitário da Pleura.

We report the clinical case of a 66 year-old woman with anorexia and weight loss, in whom a giant mass was found in the left pulmonary field. A computorized tomography guided fine-needle biopsy established the diagnosis of a solitary fibrous tumor of the pleura. Surgical resection of a 2655 grams of tumor mass was performed and the pathological examination confirmed the diagnosis. Nine months after surgery, the patient remained free of symptoms and with no evidence of disease recurrence. Solitary fibrous tumor of the pleura is a rare entity with mesenchymal origin. Although most solitary fibrous tumors of the pleura are benign, they possess a malignant potential and thus should be totally excised. Diagnosis is generally incidental, being the majority of the patients asymptomatic. However, extrinsic compression of the solitary fibrous pleural tumor on lung parenchyma may cause symptoms, from which the commonest are cough, dyspnea, and chest pain. The clinical presentation, suggestive of a consumptive and malignant disease reinforced the heterogeneity of this disease and the singularity of this clinical case, thus justifying its presentation.

Farnesol induces apoptosis-like phenotype in the phytopathogenic fungus Botrytis cinerea.

This study demonstrates that the isoprenoid farnesol produces a toxic effect on the phytopathogenic fungus Botrytis cinerea in solid and liquid media. In solid media farnesol retarded 72 h the beginning of mycelial growth. Also, it was demonstrated that the toxic effect is due to farnesol triggers apoptosis in B. cinerea because ROS accumulation, DNA fragmentation and phosphatidylserine externalization were detected in farnesol-treated mycelium. Therefore, compounds that increase the intracellular farnesol or induce apoptosis could have a potential application as fungicide against B. cinerea.

Learning from Health Professionals: A User-Centred Approach to Design a Wound Monitoring Platform.

Health professionals are able to improve the care quality of chronic wounds by monitoring and reporting the wound status. Resorting to visual representations of wound status enhances comprehension by facilitating knowledge transfer to all stakeholders. However, selecting appropriate healthcare data visualisations is a critical challenge and healthcare platforms must be designed to meet their users' needs and constraints. This article describes the methods used to identify the design requirements and inform the development of a wound monitoring platform through a user-centred approach.

Nurses' Work Methods Assessment Scale: a study of content validation.

OBJECTIVE: To develop and validate the content of the Nurses' Work Methods Assessment Scale. METHODS: Methodological study conducted between January and February 2022, based on the Quality Standards for Nursing Care and Imogene King's framework. A literature review was previously undertaken to design the scale. The content validation was carried out by 23 experts. RESULTS: The initial version consisted of 40 items organized in four work methods identified in the literature. In the first dimension, from ten items, seven were considered, and one was reformulated. Seven of the initial ten items were considered in the second version. The third dimension consisted of seven items. In the fourth dimension, three items were reformulated, and three were excluded, leaving seven items. The final version ended with 28 items, whose Content Validity Index ranged between 0.83 and 1. CONCLUSIONS: The involvement of experts has become pivotal in the development and validation of the items, providing confidence to the continuity of psychometric procedures.

Removal of Methylene Blue from a synthetic effluent by ionic flocculation.

Methylene Blue (MB) is a dye widely used in the industrial sector and in human and veterinary pharmacology. This dye, if improperly disposed of, can cause a significant environmental impact due to its low biodegradability, as it is a stable and complex substance. Additionally, it may affect human health and generate highly toxic byproducts. Hence, the purpose of this work is to assess the removal efficiency of MB from a synthetic effluent using a ionic flocculation process. Such a process consists of the dissolution of a biodegradable anionic surfactant (obtained from soybean oil used for frying food) in the synthetic effluent and the subsequent addition of calcium to the system. The addition of Ca leads to the formation of insoluble surfactant flocs with a high capacity to adsorb organic pollutants. The FTIR testing showed the presence of OH- and C=O groups in the surfactant flocs, which favor the removal of MB by an adsorption process. The maximum adsorption capacity of MB was 101.38 mg g-1. The process is in fact a chemisorption and has an exothermic nature. Desorption studies showed a desorption efficiency of up to 47.81% using an ethanol 1:2 solution. An MB removal efficiency of up to 93.71% was attained in just 0.5 min for an initial MB concentration of 100 mg L-1, showing that ionic flocculation is a very fast and effective process for the treatment of effluents.

Characterization of the Fungitoxic Activity on Botrytis cinerea of N-phenyl-driman-9-carboxamides.

A total of 12 compounds were synthesized from the natural sesquiterpene (-) drimenol (compounds 4 to 15). The synthesized compounds corresponded to N-phenyl-driman-9-carboxamide derivatives, similar to some fungicides that inhibit the electron-transport chain. Their structures were characterized and confirmed by 1H NMR, 13C NMR spectroscopy, and mass spectrometry. Compounds 5 to 15 corresponded to novel compounds. The effect of the compounds on the mycelial growth of Botrytis cinerea was evaluated. Methoxylated and chlorinated compounds in the aromatic ring (compounds 6, 7, 12, and 13) exhibited the highest antifungal activity with IC50 values between 0.20 and 0.26 mM. On the other hand, the effect on conidial germination of B. cinerea of one methoxylated compound (6) and one chlorinated compound (7) was analyzed, and no inhibition was observed. Additionally, compound 7 decreased 36% the rate of oxygen consumption by germinating conidia.

Influence of Gender and Age of Brown Seaweed (Fucus vesiculosus) on Biochemical Activities of Its Aqueous Extracts.

Fucus vesiculosus L. is a common coastal brown seaweed associated with various benefits to human health due to its phenolic content and nutrients and is used as food through different methods of consumption. This study aims to evaluate the influence of the seaweed's gender and growth stage on different types of biological activities as well as its chemical constitution and elements present. Akin to food preparation, aqueous extracts of the seaweed were prepared at 25 °C (salad) and 100 °C (soup). Biological activities were determined by measuring total phenol content (TPC), antioxidant activity and inhibition of acetylcholinesterase (AChE). Liquid Chromatography High Resolution Mass Spectrometry (LC-HRMS/MS) was used for compound identification, and elemental analysis was carried out by using Total Reflection X-ray Fluorescence Spectrometry (TXRF). Older females and males had higher TPC compared to the new ones at 100 °C. Antioxidant activity depended on the extraction temperature but was higher for the youngest male at 100 °C. AChE inhibitory activity was higher for older males at 25 °C, but at 100 °C it was higher for older females. Primary metabolites and various phloroglucinol were the main compounds identified. Additionally, since this seaweed is often harvested in estuarine systems with high anthropogenic impacts, its safety was evaluated through the evaluation of the sample's metal content. The heavy metals detected are within the limits established by various regulating entities, pointing to a safe food source.

Patient and caregiver productivity loss and indirect costs associated with cardiovascular events in Portugal.

OBJECTIVES: To present the Portuguese results of a multi-country cross-sectional survey aiming to estimate productivity loss in the first year after an acute coronary syndrome (ACS) or stroke. METHODS: Patients previously hospitalized for ACS or stroke were enrolled during a routine cardiology/neurology visit 3-12 months after the index event and ≥4 weeks after returning to work. Productivity loss for the patient and the caregiver in the previous four weeks were reported by the patient using the validated iMTA Productivity Cost Questionnaire (iPCQ). Hours lost were converted into eight-hour work days and prorated to one year, combined with initial hospitalization and sick leave, and valued according to Portuguese labor costs. RESULTS: The analysis included 39 employed patients with ACS (mean age 51 years, 80% men, 95% with myocardial infarction, mean left ventricular ejection fraction 55%) and 31 with stroke (mean age 50 years, 80% men, all ischemic, 77% with modified Rankin Scale 0-1); 41% of ACS and 10% of stroke patients had a history of cardiovascular disease. Mean (SD) productivity loss for patients and caregivers was 47 (62) work days for ACS and 76 (101) work days for stroke. ACS patients lost 37 (39) and caregivers lost 10 (42) work days. Stroke patients and caregivers lost 65 (78) and 12 (38) work days, respectively. Total mean indirect cost per case was €5403 (€7095) and €8726 (€11558) for employed patients with ACS and stroke, respectively. CONCLUSIONS: The annual proportions of productive time lost by employed patients due to ACS and stroke in Portugal were 17% and 27%, respectively. Caregivers of these patients lost about 5% of their annual productive time.

A novel fungal metal-dependent α-L-arabinofuranosidase of family 54 glycoside hydrolase shows expanded substrate specificity.

Trichoderma genus fungi present great potential for the production of carbohydrate-active enzymes (CAZYmes), including glycoside hydrolase (GH) family members. From a renewability perspective, CAZYmes can be biotechnologically exploited to convert plant biomass into free sugars for the production of advanced biofuels and other high-value chemicals. GH54 is an attractive enzyme family for biotechnological applications because many GH54 enzymes are bifunctional. Thus, GH54 enzymes are interesting targets in the search for new enzymes for use in industrial processes such as plant biomass conversion. Herein, a novel metal-dependent GH54 arabinofuranosidase (ThABF) from the cellulolytic fungus Trichoderma harzianum was identified and biochemically characterized. Initial in silico searches were performed to identify the GH54 sequence. Next, the gene was cloned and heterologously overexpressed in Escherichia coli. The recombinant protein was purified, and the enzyme's biochemical and biophysical properties were assessed. GH54 members show wide functional diversity and specifically remove plant cell substitutions including arabinose and galactose in the presence of a metallic cofactor. Plant cell wall substitution has a major impact on lignocellulosic substrate conversion into high-value chemicals. These results expand the known functional diversity of the GH54 family, showing the potential of a novel arabinofuranosidase for plant biomass degradation.

Identification of a cold-adapted and metal-stimulated ß-1,4-glucanase with potential use in the extraction of bioactive compounds from plants.

Cold-adapted endo-ß-1,4-glucanases hold great potential for industrial processes requiring high activity at mild temperatures such as in food processing and extraction of bioactive compounds from plants. Here, we identified and explored the specificity, mode of action, kinetic behavior, molecular structure and biotechnological application of a novel endo-ß-1,4-glucanase (XacCel8) from the phytopathogen Xanthomonas citri subsp. citri. This enzyme belongs to an uncharacterized phylogenetic branch of the glycoside hydrolase family 8 (GH8) and specifically cleaves internal ß-1,4-linkages of cellulose and mixed-linkage ß-glucans releasing short cello-oligosaccharides ranging from cellobiose to cellohexaose. XacCel8 acts in near-neutral pHs and in a broad temperature range (10-50 °C), which are distinguishing features from conventional thermophilic ß-1,4-glucanases. Interestingly, XacCel8 was greatly stimulated by cobalt ions, which conferred higher conformational stability and boosted the enzyme turnover number. The potential application of XacCel8 was demonstrated in the caffeine extraction from guarana seeds, which improved the yield by 2.5 g/kg compared to the traditional hydroethanolic method (HEM), indicating to be an effective additive in this industrial process. Therefore, XacCel8 is a metal-stimulated and cold-adapted endo-ß-1,4-glucanase that could be applied in a diverse range of biotechnological processes under mild conditions such as caffeine extraction from guarana seeds.