Use of Calotropis procera cysteine peptidases (CpCPs) immobilized on glyoxyl-agarose for cheesemaking.

Calotropis procera cysteine peptidases (CpCPs) have presented several potential biotechnological applications. Here, these enzymes were immobilized on glyoxyl-agarose (glyoxyl-CpCPs) with yields of 90-95 % and the recovered activities ranged from 10 % to 15 %, according to enzyme loadings (5, 10, 20, 40, and 50 mgBSAeq/g). Spectrophotometric assays and SDS-PAGE showed that the casein hydrolysis by glyoxyl-CpCPs was similar to soluble CpCPs. In addition, glyoxyl-CpCPs exhibited similar ratio of milk-clotting activity to proteolytic activity in comparison with soluble CpCPs and chymosin. Even after being stored for six months at 8 °C, the residual proteolytic activity of glyoxyl-CpCPs remained close to 100 %. Atomic force microscopy and dynamic light scattering techniques showed that the process of casein micelle aggregation after treatment with glyoxyl-CpCPs was very similar to its soluble form and chymosin. Glyoxyl-CpCPs performed well after five reaction cycles, producing cheeses with yield, moisture, protein, and fat similar to those produced with chymosin.

Luffa operculata seed proteins: Identification by LC-ESI-MS/MS and biotechnological potential against Candida albicans and C. krusei.

L: operculata is a plant commonly found in the North and Northeast of Brazil. Although the regional population knows its medicinal potential, there are few scientific studies about its antimicrobial potential. Thus, this study aimed to characterize the proteins from L. operculata seeds extracted using different solutions and evaluate their antimicrobial potentials. The protein extracts obtained with NaCl and sodium acetate buffer presented the best inhibitory activities against Candida albicans and C. krusei. The study of the mechanism of action revealed proteins from L. operculata seeds induced pore formation on the membrane and ROS overaccumulation. Scanning Electron Microscopy images also showed severe morphological changes in Candida albicans and C. krusei. Proteins from L.operculata seeds did not show antibacterial activity. The enzymatic assays revealed the presence of proteolytic enzymes, serine and cysteine protease inhibitors, and chitinases in both protein extracts. Proteomic analysis by LC-ESI-MS/MS identified 57 proteins related to many biological processes, such as defense to (a)biotic stress, energetic metabolism, protein folding, and nucleotide metabolism. In conclusion, the L. operculata seed proteins have biotechnological potential against the human pathogenic yeasts Candida albicans and C. krusei.

A compilation of experimental data on the mechanical properties and microstructural features of Ti-alloys.

The present work depicts a compilation of mechanical properties of 282 distinct multicomponent Ti-based alloys and their respective microstructural features. The dataset includes the chemical composition (in at.%), phase constituents, Young modulus, hardness, yield strength, ultimate strength, and elongation. Each entry is associated with a high-quality experimental work containing a complete description of the processing route and testing setup. Furthermore, we incorporated flags to the dataset indicating (a) the use of high-resolution techniques for microstructural analysis and (b) the observation of non-linear elastic responses during mechanical testing. Oxygen content and average grain size are presented whenever available. The selected features can help material scientists to adjust the data to their needs concerning materials selection and discovery. Most alloys in the dataset were produced via an ingot metallurgy route, followed by solubilization and water quench (≈58%), which is considered a standard condition for ß-Ti alloys. The database is hosted and maintained up to date in an open platform. For completeness, a few graphical representations of the dataset are included.

Latex peptidases produce peptides capable of delaying fungal growth in bread.

Antimicrobial peptides (AMPs) have been reported to be promising alternatives to chemical preservatives. Thus, this study aimed to characterise AMPs generated from the hydrolysis of wheat gluten proteins using latex peptidases of Calotropis procera, Cryptostegia grandiflora, and Carica papaya. The three hydrolysates (obtained after 16 h at 37 °C, using a 1: 25 enzyme:  substrate ratio) inhibited the growth of Aspergillus niger, A. chevalieri, Trichoderma reesei, Pythium oligandrum, Penicillium sp., and Lasiodiplodia sp. by 60-90%, and delayed fungal growth on bread by 3 days when used at 0.3 g/kg. Moreover, the specific volume and expansion factor of bread were not affected by the hydrolysates. Of 28 peptides identified, four were synthesised and exhibited activity against Penicillium sp. Fluorescence and scanning electron microscopy suggested that the peptides damaged the fungal plasma membrane. Bioinformatics analysis showed that no peptide was toxic and that the antigenic ones had cleavage sites for trypsin or pepsin.

Serine carboxypeptidases from the carnivorous plant Nepenthes mirabilis: Partial characterization and heterologous expression.

This study aimed to partially characterize the three main serine carboxypeptidases (SCP3, SCP20, and SCP47) from Nepenthes mirabilis. Furthermore, one peptidase (SCP3) was chosen for further heterologous expression in Escherichia coli Shuffle®T7. SCP3 also was characterized in terms of its allergenic potential using bioinformatics tools. SCP3, SCP20, and SCP47 showed very similar 3D structures and mechanistic features to other plant serine peptidases belonging to clan SC and family S10. Although SCP3 was obtained in its soluble form, using 1% ethanol during induction with 0.5 mM IPTG at 16 °C for 18 h, it did not show proteolytic activity by zymography or in vitro analysis. SCP3 presented a few allergenic peptides and several cleavage sites for digestive enzymes. This work describes additional features of these enzymes, opening new perspectives for further studies for characterization and analysis of heterologous expression, as well as their potential biotechnological applications.

Characterization of Three Osmotin-Like Proteins from Plumeria rubra and Prospection for Adiponectin Peptidomimetics.

BACKGROUND: Osmotin-Like Proteins (OLPs) have been purified and characterized from different plant tissues, including latex fluids. Besides its defensive role, tobacco osmotin seems to induce adiponectin-like physiological effects, acting as an agonist. However, molecular information about this agonistic effect on adiponectin receptors has been poorly exploited and other osmotins have not been investigated yet. OBJECTIVE AND METHODS: The present study involved the characterization of three OLPs from Plumeria rubra latex and molecular docking studies to evaluate the interaction between them and adiponectin receptors (AdipoR1 and AdipoR2). RESULTS: P. rubra Osmotin-Like Proteins (PrOLPs) exhibited molecular masses from 21 to 25 kDa and isoelectric points ranging from 4.4 to 7.7. The proteins have 16 cysteine residues, which are involved in eight disulfide bonds, conserved in the same positions as other plant OLPs. The threedimensional (3D) models exhibited the three typical domains of OLPs, and molecular docking analysis showed that two PrOLP peptides interacted with two adiponectin receptors similarly to tobacco osmotin peptide. CONCLUSION: As observed for tobacco osmotin, the latex osmotins of P. rubra exhibited compatible interactions with adiponectin receptors. Therefore, these plant defense proteins (without known counterparts in humans) are potential tools to study modulation of glucose metabolism in type II diabetes, where adiponectin plays a pivotal role in homeostasis.

Biotechnological potential of a cysteine protease (CpCP3) from Calotropis procera latex for cheesemaking.

This article reports the characterization and evaluation of the biotechnological potential of a cysteine protease purified from Calotropis procera (CpCP3). This enzyme was highly stable to different metal ions and was able to hydrolyze κ-casein similarly to bovine chymosin. Atomic force microscopy showed that the process of casein micelle aggregation induced by CpCP3 was similar to that caused by chymosin. The cheeses made using CpCP3 showed higher moisture content than those made with chymosin, but protein, fat, and ash were similar. The sensory analysis showed that cheeses made with CpCP3 had high acceptance index (>80%). In silico analysis predicted the presence of only two short allergenic peptides on the surface of CpCP3, which was highly susceptible to digestive enzymes and did not alter zebrafish embryos' morphology and development. Moreover, recombinant CpCP3 was expressed in Escherichia coli. All results support the biotechnological potential of CpCP3 as an alternative enzyme to chymosin.

Acute effects of ayahuasca in a juvenile non-human primate model of depression

Objective: The incidence rate of major depression in adolescents reaches approximately 14%. This disorder is usually recurrent, without remission of symptoms even after pharmacological treatment, and persists throughout adult life. Since the effects of antidepressants take approximately 2 weeks to begin, new pharmacological therapies are under continuous exploration. Recent evidence suggests that psychedelics could produce rapid antidepressant effects. In this study, we evaluated the potential antidepressant effects of ayahuasca in a juvenile non-human primate model of depression. Methods: While living with their families, juvenile marmosets (8 males; 7 females) were observed on alternate days for four weeks during a baseline phase. This was followed by 8 weeks of an induced depressive state protocol, the social isolated context (IC), in which the animals were monitored in the first and last weeks. Subsequently, five males and four females were randomly selected for treatment, first with a single administration of saline vehicle (1.67 mL/300 g of body weight, via gavage), followed by a single dose of ayahuasca (1.67 mL/300 g of body weight, via gavage). Both phases lasted 1 week and the animals were monitored daily. A third week of sampling was called the tardive-pharmacological effects phase. In all phases the marmosets were assessed for behavior, fecal cortisol levels, and body weight. Results: After IC, the animals presented typical hypocortisolemia, but cortisol recovered to baseline levels 24 h after an acute dose of ayahuasca; this recovery was not observed in vehicle-treated animals. Additionally, in males, ayahuasca, but not the vehicle, reduced scratching, a stereotypic behavior, and increased feeding. Ayahuasca treatment also improved body weight to baseline levels in both sexes. The ayahuasca-induced behavioral response had long-term effects (14 days). Thus, in this translational juvenile animal model of depression, ayahuasca presented beneficial effects. Conclusions: These results can contribute to the validation of ayahuasca as an antidepressant drug and encourage new studies on psychedelic drugs as a tool for treating mood disorders, including for adolescents with early-onset depression.

Allergenicity reduction of cow's milk proteins using latex peptidases.

The present study evaluated four laticifer fluids as a novel source of peptidases capable of hydrolyzing proteins in cow's milk. The latex peptidases from Calotropis procera (CpLP), Cryptostegia grandiflora (CgLP), and Carica papaya (CapLP) were able to perform total hydrolysis of caseins after 30 min at pH 6.5, as confirmed by a significant reduction in the residual antigenicity. Casein hydrolysis by Plumeria rubra latex peptidases (PrLP) was negligible. Moreover, whey proteins were more resistant to proteolysis by latex peptidases; however, heat pretreatment of the whey proteins enhanced the degree of hydrolysis and reduced the residual antigenicity of the hydrolysates. The in vivo assays show that the cow's milk proteins hydrolysed by CgLP and CapLP exhibited no immune reactions in mice allergic to cow's milk, similar to a commercial partially hydrolysed formula. Thus, these peptidases are promising enzymes for the development of novel hypoallergenic formulas for children with a milk allergy.

Close Relationships between NMR J-Coupling Alternation (JCA) and Molecular Properties of Carbon Chains.

We propose a J-coupling alternation (JCA) value that is demonstrated to be a suitable parameter to evaluate the nuclear magnetic resonance (NMR) indirect spin-spin coupling constants (SSCCs) as a function of molecular properties of chains by increasing their length. As an application, we report a theoretical study of the SSCCs for the interactions between neighbor nuclei in increasingly patterned carbon chains within density functional theory. First, we examine the J-coupling constants between 1H and 13C nuclei ( n JHC) considering the separation distance, as well as between two adjacent 13C nuclei (1 JCC) considering their relative positions in polyynes and cumulenes. Further, we define and determine JCA in terms of the differences of 1 JCC, which is investigated as a function of several molecular properties, e.g., cohesive energy, characteristic vibrational frequency, average polarizability, and energy gap of the systems. We also determine JCA for other types of carbon chains, such as diphenyl-capped polyynes, polyacetylene and polythiophene. The behavior of JCA as a function of the energy gap may be related to highly π-conjugated low-band-gap carbon chains. Overall, JCA correlates very well with the electronic properties of these chains, especially with their energy gap, exhibiting positive values for pristine polyyne and polythiophene and negative values for pristine cumulene and plyacetylene. These findings indicate an alternative way to establish an appropriate SSCC descriptor that characterizes the electronic nature of the system, such as the proposed JCA value averaging the whole system, instead of using only the individual J-coupling values to give insights into the properties of large and extended systems.

Investigation of a rapid infrared heating assisted mineralization of soybean matrices for trace element analysis.

A fast sample preparation procedure based on use of infrared (IR) assisted heating for mineralization of soybean derived samples has been developed for their subsequent multielement analysis by inductively coupled plasma optical emission spectrometry (ICP-OES) and flame atomic absorption spectrometry (FAAS). A cold finger was examined for refluxing of acid vapors to determine its impact on efficiency and economy of digestion. The optimized procedure, based on 1 g subsamples, 8 mL of HNO3 (65% w/w) and exposure of the mixture to a 500 W IR source for 5 min without refluxing, permitted accurate determination of all analytes in NIST SRM 1568b (rice flour). Detection limits using ICP-OES were (µg/kg) 97, 1.0, 39, 185, 0.47 and 1200 for Ca, Cu, Fe, K, Mn and P, respectively, and 18 for Zn by FAAS. The IR-assisted digestion approach provided a low cost, easy to use system having great potential for implementation in routine analysis of trace elements in soybean and similar matrices.

Rapid antidepressant effects of the psychedelic ayahuasca in treatment-resistant depression: a randomized placebo-controlled trial.

BACKGROUND: Recent open-label trials show that psychedelics, such as ayahuasca, hold promise as fast-onset antidepressants in treatment-resistant depression. METHODS: To test the antidepressant effects of ayahuasca, we conducted a parallel-arm, double-blind randomized placebo-controlled trial in 29 patients with treatment-resistant depression. Patients received a single dose of either ayahuasca or placebo. We assessed changes in depression severity with the Montgomery-Åsberg Depression Rating Scale (MADRS) and the Hamilton Depression Rating scale at baseline, and at 1 (D1), 2 (D2), and 7 (D7) days after dosing. RESULTS: We observed significant antidepressant effects of ayahuasca when compared with placebo at all-time points. MADRS scores were significantly lower in the ayahuasca group compared with placebo at D1 and D2 (p = 0.04), and at D7 (p < 0.0001). Between-group effect sizes increased from D1 to D7 (D1: Cohen's d = 0.84; D2: Cohen's d = 0.84; D7: Cohen's d = 1.49). Response rates were high for both groups at D1 and D2, and significantly higher in the ayahuasca group at D7 (64% v. 27%; p = 0.04). Remission rate showed a trend toward significance at D7 (36% v. 7%, p = 0.054). CONCLUSIONS: To our knowledge, this is the first controlled trial to test a psychedelic substance in treatment-resistant depression. Overall, this study brings new evidence supporting the safety and therapeutic value of ayahuasca, dosed within an appropriate setting, to help treat depression. This study is registered at http://clinicaltrials.gov (NCT02914769).

Acute effects of ayahuasca in a juvenile non-human primate model of depression.

OBJECTIVE: The incidence rate of major depression in adolescents reaches approximately 14%. This disorder is usually recurrent, without remission of symptoms even after pharmacological treatment, and persists throughout adult life. Since the effects of antidepressants take approximately 2 weeks to begin, new pharmacological therapies are under continuous exploration. Recent evidence suggests that psychedelics could produce rapid antidepressant effects. In this study, we evaluated the potential antidepressant effects of ayahuasca in a juvenile non-human primate model of depression. METHODS: While living with their families, juvenile marmosets (8 males; 7 females) were observed on alternate days for four weeks during a baseline phase. This was followed by 8 weeks of an induced depressive state protocol, the social isolated context (IC), in which the animals were monitored in the first and last weeks. Subsequently, five males and four females were randomly selected for treatment, first with a single administration of saline vehicle (1.67 mL/300 g of body weight, via gavage), followed by a single dose of ayahuasca (1.67 mL/300 g of body weight, via gavage). Both phases lasted 1 week and the animals were monitored daily. A third week of sampling was called the tardive-pharmacological effects phase. In all phases the marmosets were assessed for behavior, fecal cortisol levels, and body weight. RESULTS: After IC, the animals presented typical hypocortisolemia, but cortisol recovered to baseline levels 24 h after an acute dose of ayahuasca; this recovery was not observed in vehicle-treated animals. Additionally, in males, ayahuasca, but not the vehicle, reduced scratching, a stereotypic behavior, and increased feeding. Ayahuasca treatment also improved body weight to baseline levels in both sexes. The ayahuasca-induced behavioral response had long-term effects (14 days). Thus, in this translational juvenile animal model of depression, ayahuasca presented beneficial effects. CONCLUSIONS: These results can contribute to the validation of ayahuasca as an antidepressant drug and encourage new studies on psychedelic drugs as a tool for treating mood disorders, including for adolescents with early-onset depression.

Peptide from thaumatin plant protein exhibits selective anticandidal activity by inducing apoptosis via membrane receptor.

Osmotin- and thaumatin-like proteins (OLPs and TLPs) have been associated with plant defense responses to different biotic stresses. In the present work, several in silico sequences from OLPs and TLPs were investigated by means of bioinformatics tools aiming to prospect for antimicrobial peptides. The peptide sequences chosen were further synthesized and characterized, and their activities and action mechanisms were assayed against some phytopathogenic fungi, bacteria and yeasts of clinical importance. From this survey approach, four peptide sequences (GDCKATSC, CPRALKVPGGCN, IVGQCPAKLKA, and CAADIVGQCPAKLK) were selected considering some chemical parameters commonly attributed to antimicrobial peptides. Antimicrobial assays showed that these peptides were unable to inhibit mycelial growth of phytopathogenic fungi and they did not affect bacterial cell growth. Nevertheless, significant inhibitory activity was found for CPRALKVPGGCN and CAADIVGQCPAKLK against Candida albicans and Saccharomyces cerevisiae. Fluorescence and scanning electron microscopy assays suggested that CAADIVGQCPAKLK did not damage the overall cell structure, or its activity was negligible on yeast membrane and cell wall integrity. However, it induced the production of reactive oxygen species (ROS) and apoptosis. Molecular docking analysis showed that CAADIVGQCPAKLK had strong affinity to interact with specific plasma membrane receptors of C. albicans and S. cerevisiae, which have been described as promoting the induction of apoptosis. The results indicate that CAADIVGQCPAKLK can be a valuable target for the development of a desired antimicrobial agent against the pathogen C. albicans.

Cortisol Modulation by Ayahuasca in Patients With Treatment Resistant Depression and Healthy Controls.

Major depression is a highly prevalent mood disorder, affecting about 350 million people, and around 30% of the patients are resistant to currently available antidepressant medications. Recent evidence from a randomized controlled trial (RCT) supports the rapid antidepressant effects of the psychedelic ayahuasca in treatment-resistant depression. The aim of this study was to explore the effect of ayahuasca on plasma cortisol and awakening salivary cortisol response, in the same group of treatment-resistant patients (MD) and in healthy volunteers (C). Subjects received a single dose of ayahuasca or placebo (dosing session), and both plasma and awakening salivary cortisol response were measured at baseline (before dosing session) and 48 h after the dosing session. Baseline assessment (D0) showed blunted awakening salivary cortisol response and hypocortisolemia in patients, with respect to healthy controls. Salivary cortisol was also measured during dosing session, and we observed higher increases for both C and MD that ingested ayahuasca than placebo. After 48 h from the dosing session with ayahuasca, patients' awakening salivary cortisol response is similar to the ones detected in controls. No significant changes in plasma cortisol levels were observed 48 h after the sessions. Therefore, these findings point to new evidence on the modulation of salivary cortisol levels as a result of an ayahuasca session, both in healthy and depressive volunteers. Considering that cortisol acts in regulation of distinct physiological pathways, emotional and cognitive processes, it is assumed to be critically involved to the etiology of depression and its regulation seems to be important for the treatment and remission of major depression, ayahuasca use as antidepressant should be further investigated. Moreover, this study highlights the importance of psychedelics in the treatment of human mental disorders.

Infrastructure for Integration of Legacy Electrical Equipment into a Smart-Grid Using Wireless Sensor Networks.

At present, the standardisation of electrical equipment communications is on the rise. In particular, manufacturers are releasing equipment for the smart grid endowed with communication protocols such as DNP3, IEC 61850, and MODBUS. However, there are legacy equipment operating in the electricity distribution network that cannot communicate using any of these protocols. Thus, we propose an infrastructure to allow the integration of legacy electrical equipment to smart grids by using wireless sensor networks (WSNs). In this infrastructure, each legacy electrical device is connected to a sensor node, and the sink node runs a middleware that enables the integration of this device into a smart grid based on suitable communication protocols. This middleware performs tasks such as the translation of messages between the power substation control centre (PSCC) and electrical equipment in the smart grid. Moreover, the infrastructure satisfies certain requirements for communication between the electrical equipment and the PSCC, such as enhanced security, short response time, and automatic configuration. The paper’s contributions include a solution that enables electrical companies to integrate their legacy equipment into smart-grid networks relying on any of the above mentioned communication protocols. This integration will reduce the costs related to the modernisation of power substations.

Gut peptidases from a specialist herbivore of latex plants are capable of milk protein hydrolysis: Inputs for hypoallergenic milk formulas.

Transitory allergies to cow milk proteins in infants or adults have become a public health problem. Although extensively or partially hydrolyzed cow milk protein formulas are available, these products are costly. Therefore, studies into innovative enzymes to digest cow milk proteins are needed. Danaus plexippus gut peptidases were purified and examined with regard to cow milk protein hydrolysis. The peptidases hydrolyzed caseins and whey proteins. However, after heat treatment, there was a significant improvement in ß-lactoglobulin hydrolysis. The hydrolyzed cow milk proteins were not recognized by anti-casein antibodies and only reacted slightly with antibodies against whey proteins. This performance was better than that of partially hydrolyzed formulas and similar to that of an extensively hydrolyzed formula. These results suggest that D. plexippus gut peptidases are suitable and innovative enzymes to produce hypoallergenic cow milk protein formulas.

A new approach to mineralization of flaxseed (Linum usitatissimum L.) for trace element analysis by flame atomic absorption spectrometry.

A new approach to the analysis of Cu, Fe, Mn and Zn in flaxseed was developed based on infrared-assisted acid digestion. Quantitation by flame atomic absorption spectrometry yields results in agreement with those arising from aggressive total decomposition using conventional microwave-assisted (MW) digestions. A full factorial design in two levels was applied to evaluate the impact of significant variables for all elements to determine optimal experimental conditions. A desirability function revealed these to be: 2.0g sample mass, 8mL of HNO3 and 8min of heating time in the IR system. Precision better than 10% (RSD) was obtained, superior to that of a combined IR-MW approach. Sample preparation based on IR-assisted digestion provides a rapid and inexpensive alternative to other conventional techniques for the analysis of complex samples and is able to accommodate relatively large masses of sample, alleviating potential homogeneity issues as well as enhancing detection power.