Conjugation of Lysozyme and Epigallocatechin Gallate for Improving Antibacterial and Antioxidant Properties.

One of the main interests in the food industry is the preservation of food from spoilage by microorganisms or lipid oxidation. A novel alternative is the development of additives of natural origin with dual activity. In the present study, a chemically modified lysozyme (Lys) with epigallocatechin gallate (EGCG) was developed to obtain a conjugate (Lys-EGCG) with antibacterial/antioxidant activity to improve its properties and increase its application potential. The modification reaction was carried out using a free radical grafting method for the Lys modification reaction, using ascorbic acid and hydrogen peroxide as radical initiators in an aqueous medium. The synthesis of Lys-EGCG conjugate was confirmed by spectroscopic (FT-IR, 1H-RMN, and XPS) and calorimetry differential scanning (DSC) analyses. The EGCG binding to the Lys biomolecule was quantified by the Folin-Ciocalteu method; the antibacterial activity was evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MCB) against Staphylococcus aureus and Pseudomonas fluorescens; the antioxidant activity was evaluated by ABTS, DPPH, and FRAP. The spectroscopic results showed that the Lys-EGCG conjugate was successfully obtained, and the DSC analysis revealed a 20 °C increase (P < 0.05) in the denaturation temperature of Lys due to EGCG modification. The EGCG concentration in Lys-EGCG was 97.97 ± 4.7 µmol of EGCG/g of sample. The antibacterial and antioxidant activity of the Lys-EGCG conjugate was higher (P < 0.05) than pure EGCG and Lys. The chemical modification of Lys with EGCG allows for the bioconjugate with a dual function (antibacterial/antioxidant), broadening the range of Lys and EGCG applications to different areas such as food, cosmetic, and pharmaceutical industries.

The bifunctional 6-phosphofructokinase-2/fructose-2,6-bisphosphatase from the shrimp Litopenaeus vannamei: Molecular characterization and down-regulation of expression in response to severe hypoxia.

Hypoxia is a frequent stressor in marine environments with multiple adverse effects on marine species. The white shrimp Litopenaeus vannamei withstands hypoxic conditions by activating anaerobic metabolism with tissue-specific changes in glycolytic and gluconeogenic enzymes. In animal cells, glycolytic/gluconeogenic fluxes are highly controlled by the levels of fructose-2,6-bisphosphate (F-2,6-P2), a signal metabolite synthesized and degraded by the bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2). PFK-2/FBPase-2 has been studied in vertebrates and some invertebrates, but as far as we know, there are no reports on PFK-2/FBPase-2 from crustaceans. In the present work, we obtained cDNA nucleotide sequences corresponding to two mRNAs for PFK-2/FBPase-2 and named them PFKFBP1 (1644 bp) and PFKFBP2 (1566 bp), from the white shrimp L. vannamei. The deduced PFKFBP1 and PFKFBP2 are 547 and 521 amino acids long, respectively. Both proteins share 99.23% of identity, and only differ in 26 additional amino acids present in the kinase domain of the PFKFBP1. The kinase and phosphatase domains are highly conserved in sequence and structure between both isoforms and other proteins from diverse taxa. Total expression of PFKFBP1-2 is tissue-specific, more abundant in gills than in hepatopancreas and undetectable in muscle. Moreover, severe hypoxia (1 mg/L of DO) decreased expression of PFKFBP1-2 in gills while anaerobic glycolysis was induced, as indicated by accumulation of cellular lactate. These results suggest that negative regulation of PFKFBP1-2 at expression level is necessary to set up anaerobic glycolysis in the cells during the response to hypoxia.

Effect of the drug cyclophosphamide on the activity of porcine kidney betaine aldehyde dehydrogenase.

The enzyme betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the synthesis of glycine betaine (GB), an osmolyte and osmoprotectant. Also, it participates in several metabolic pathways in humans. All BADHs known have cysteine in the active site involved in the aldehyde binding, whereas the porcine kidney enzyme (pkBADH) also has a neighborhood cysteine, both sensitive to oxidation. The antineoplastic and immuno-suppressant pre-drug cyclophosphamide (CTX), and its bioactivation products, have two highly oxidating chlorine atoms. This work aimed to analyze the effect of CTX in the activity of porcine kidney betaine aldehyde dehydrogenase. PkBADH was incubated with varying CTX concentration (0 to 2.0 mM) at 25 °C and lost 50 % of its activity with 2.0 mM CTX. The presence of the coenzyme NAD+ (0.5 mM) decreased 95% the activity in 2.0 mM CTX. The substrate betaine aldehyde (0.05 and 0.4 mM, and the products NADH (0.1-0.5 mM) and GB (1 and 10 mM) did not have an effect on the enzyme inactivation by CTX. The reducing agents, dithiothreitol and ß-mercaptoethanol, reverted the pkBADH inactivation, but reduced glutathione (GSH) was unable to restore the enzyme activity. Molecular docking showed that CTX could enter at the enzyme active site, where its chlorine atoms may interact with the catalytic and the neighboring cysteines. The results obtained show that CTX inactivates the pkBADH due to oxidation of the catalytic cysteine or because it oxidizes catalytic and neighborhood cysteine, forming a disulfide bridge with a concomitant decrease in the activity of the enzyme.

Prevalence of Food-Hypersensitivity and Food-Dependent Anaphylaxis in Colombian Schoolchildren by Parent-Report.

Background and objectives: The epidemiology of food allergy (FA) and food-dependent anaphylaxis remains unknown in Colombia. Our aim was to estimate by parent-report the prevalence of FA and food-dependent anaphylaxis in a Colombian population of schoolchildren. Materials and methods: A printed questionnaire was sent to parents of schoolchildren aged 5-12 years old from Medellín, Colombia in order to collect FA-related data. Results: Nine hundred and sixty-nine (969) parents returned the questionnaire with valid responses (response rate, 52.5%). The estimated prevalence rates (95% CI) were: adverse food reactions 12.79% (10.76-15.07), "perceived FA, ever" 10.93% (9.08-13.08), "physician-diagnosed FA, ever" 4.33% (3.14-5.81), "immediate-type FA, ever" 6.81% (5.30-8.58), "immediate-type FA, current" 3.30% (2.26-4.63), and food-dependent anaphylaxis 1.85% (1.10-2.92). The most frequently reported food allergens were milk (1.44%), fruits (0.41%), meat (0.41%), and peanut (0.3%). Sixty-one percent of "food-dependent anaphylaxis" cases sought medical attention, but only eleven percent of the cases reported the prescription of an epinephrine autoinjector. Conclusions: FA and food-dependent anaphylaxis are not uncommon among schoolchildren from Colombia. The prescription of epinephrine autoinjectors should be encouraged among health personnel for the optimal management of suspected cases of food-dependent anaphylaxis.

Heterogeneity of active sites in recombinant betaine aldehyde dehydrogenase is modulated by potassium.

Betaine aldehyde dehydrogenase (BADH EC 1.2.1.8) catalyzes the irreversible oxidation of betaine aldehyde to glycine betaine using NAD+ as a coenzyme. Porcine kidney BADH (pkBADH) follows a bi-bi ordered mechanism in which NAD+ binds to the enzyme before the aldehyde. Previous studies showed that NAD+ induces complex and unusual conformational changes on pkBADH and that potassium is required to maintain its quaternary structure. The aim of this work was to analyze the structural changes in pkBADH caused by NAD+ binding and the role played by potassium in those changes. The pkBADH cDNA was cloned and overexpressed in Escherichia coli, and the protein was purified by affinity chromatography using a chitin matrix. The pkBADH/NAD+ interaction was analyzed by circular dichroism (CD) and by isothermal titration calorimetry (ITC) by titrating the enzyme with NAD+ . The cDNA has an open reading frame of 1485 bp and encodes a protein of 494 amino acids, with a predicted molecular mass of 53.9 kDa. CD data showed that the binding of NAD+ to the enzyme caused changes in its secondary structure, whereas the presence of K+ helps maintain its α-helix content. K+ increased the thermal stability of the pkBADH-NAD+ complex by 5.3°C. ITC data showed that NAD+ binding occurs with different association constants for each active site between 37.5 and 8.6 µM. All the results support previous data in which the enzyme incubation with NAD+ provoked changes in reactivity, which is an indication of slow conformational rearrangements of the active site.

Role of potassium levels in pkBADH heterogeneity of NAD+ binding site.

Betaine aldehyde dehydrogenase (BADH) catalyzes the oxidation of betaine aldehyde to glycine betaine using NAD+ as a coenzyme. Studies in porcine kidney BADH (pkBADH) suggested that the enzyme exhibits heterogeneity of active sites and undergoes potassium-induced conformational changes. This study aimed to analyze if potassium concentration plays a role in the heterogeneity of pkBADH active sites through changes in NAD+ affinity constants, in its secondary structure content and stability. The enzyme was titrated with NAD+ 1 mM at fixed-variable KCl concentration, and the interaction measured by Isothermal Titration Calorimetry (ITC) and Circular Dichroism (CD). ITC data showed that K+ increased the first active site affinity in a manner dependent on its concentration; KD values to the first site were 14.4, 13.1, and 10.4 µM, at 25, 50, and 75 mM KCl. ΔG values showed that the coenzyme binding is a spontaneous reaction without changes between active sites or depending on KCl concentration. ΔH and TΔSb values showed that NAD+ binding to the active site is an endothermic process and is carried out at the expense of changes in entropy. α-Helix content increased as KCl increased, enzyme (Tm)app values were 2.6 °C and 3.3 °C higher at 20 mM and 200 mM K+. PkBADH molecular model showed three different interaction K+ sites. Results suggested K+ can interact with pkBADH and cause changes in the secondary structure, it provokes changes in the enzyme affinity by the coenzyme, and in the thermostability.

The nuclear encoded subunits gamma, delta and epsilon from the shrimp mitochondrial F1-ATP synthase, and their transcriptional response during hypoxia.

The mitochondrial FOF1 ATP synthase produces ATP in a reaction coupled to an electrochemical proton gradient generated by the electron transfer chain. The enzyme also hydrolyzes ATP according to the energy requirements of the organism. Shrimp need to overcome low oxygen concentrations in water and other energetic stressors, which in turn lead to mitochondrial responses. The aim of this study was to characterize the full-length cDNA sequences of three subunits that form the central stalk of the F1 catalytic domain of the ATP synthase of the white shrimp Litopenaeus vannamei and their deduced proteins. The effect of hypoxia on shrimp was also evaluated by measuring changes in the mRNA amounts of these subunits. The cDNA sequences of the nucleus-encoded ATPγ, ATPδ and ATPε subunits are 1382, 477 and 277 bp long, respectively. The three deduced amino acid sequences exhibited highly conserved regions when compared to homologous sequences, and specific substitutions found in shrimp subunits are discussed through an homology structural model of F1 ATP-synthase that included the five deduced proteins, which confirm their functional structures and specific characteristics from the cognate complex of ATP synthases. Genes expression was evaluated during hypoxia-reoxygenation, and resulted in a generalized down-regulation of the F1 subunits and no coordinated changes were detected among these five subunits. The reduced mRNA levels suggest a mitochondrial response to an oxidative stress event, similar to that observed at ischemia-reperfusion in mammals. This model analysis and responses to hypoxia-reoxygenation may help to better understand additional mitochondrial adaptive mechanisms.

A novel viral thymidylate kinase with dual kinase activity.

Nucleotide phosphorylation is a key step in DNA replication and viral infections, since suitable levels of nucleotide triphosphates pool are required for this process. Deoxythymidine monophosphate (dTMP) is produced either by de novo or salvage pathways, which is further phosphorylated to deoxythymidine triphosphate (dTTP). Thymidyne monophosphate kinase (TMK) is the enzyme in the junction of both pathways, which phosphorylates dTMP to yield deoxythymidine diphosphate (dTDP) using adenosine triphosphate (ATP) as a phosphate donor. White spot syndrome virus (WSSV) genome contains an open reading frame (ORF454) that encodes a thymidine kinase and TMK domains in a single polypeptide. We overexpressed the TMK ORF454 domain (TMKwssv) and its specific activity was measured with dTMP and dTDP as phosphate acceptors. We found that TMKwssv can phosphorylate dTMP to yield dTDP and also is able to use dTDP as a substrate to produce dTTP. Kinetic parameters K M and k cat were calculated for dTMP (110 µM, 3.6 s(-1)), dTDP (251 µM, 0.9 s(-1)) and ATP (92 µM, 3.2 s(-1)) substrates, and TMKwssv showed a sequential ordered bi-bi reaction mechanism. The binding constants K d for dTMP (1.9 µM) and dTDP (10 µM) to TMKwssv were determined by Isothermal Titration Calorimetry. The affinity of the nucleotidic analog stavudine monophosphate was in the same order of magnitude (K d 3.6 µM) to the canonical substrate dTMP. These results suggest that nucleotide analogues such as stavudine could be a suitable antiviral strategy for the WSSV-associated disease.

Binding of green tea epigallocatechin gallate to the arginine kinase active site from the brown recluse spider (Loxosceles laeta): A potential synergist to chemical pesticides.

Loxosceles spp. spiders can cause serious public health issues. Chemical control is commonly used, leading to health and environmental problems. Identifying molecular targets and using them with natural compounds can help develop safer and eco-friendlier biopesticides. We studied the kinetics and predicted structural characteristics of arginine kinase (EC 2.7.3.3) from Loxosceles laeta (LlAK), a key enzyme in the energy metabolism of these organisms. Additionally, we explored (-)-epigallocatechin gallate (EGCG), a green tea flavonoid, as a potential lead compound for the LlAK active site through fluorescence and in silico analysis, such as molecular docking and molecular dynamics (MD) simulation and MM/PBSA analyses. The results indicate that LlAK is a highly efficient enzyme (K m Arg 0.14 mM, K m ATP 0.98 mM, k cat 93 s-1, k cat/K m Arg 630 s-1 mM-1, k cat/K m ATP 94 s-1 mM-1), which correlates with its structure similarity to others AKs (such as Litopenaeus vannamei, Polybetes pythagoricus, and Rhipicephalus sanguineus) and might be related to its important function in the spider's energetic metabolism. Furthermore, the MD and MM/PBSA analysis suggests that EGCG interacted with LlAK, specifically at ATP/ADP binding site (RMSD <1 nm) and its interaction is energetically favored for its binding stability (-40 to -15 kcal/mol). Moreover, these results are supported by fluorescence quenching analysis (K d 58.3 µM and K a 1.71 × 104 M-1). In this context, LlAK is a promising target for the chemical control of L. laeta, and EGCG could be used in combination with conventional pesticides to manage the population of Loxosceles species in urban areas.

Dipeptidyl Peptidase IV Inhibitory Peptides from Chickpea Proteins (Cicer arietinum L.): Pharmacokinetics, Molecular Interactions, and Multi-Bioactivities.

Chickpea (Cicer arietinum L.) peptides can inhibit dipeptidyl peptidase IV (DPP-IV), an important type 2 diabetes mellitus therapeutic target. The molecular interactions between the inhibitory peptides and the active site of DPP-IV have not been thoroughly examined, nor have their pharmacokinetic properties. Therefore, the predictions of legumin- and provicilin-derived DPP-IV inhibitory peptides, their molecular interactions with the active site of DPP-IV, and their pharmacokinetic properties were carried out. Ninety-two unique DPP-IV inhibitory peptides were identified. Papain and trypsin were the enzymes with the highest AE (0.0927) and lowest BE (6.8625 × 10-7) values, respectively. Peptide binding energy values ranged from -5.2 to -7.9 kcal/mol. HIS-PHE was the most potent DPP-IV inhibitory peptide and interacts with residues of the active sites S1 (TYR662) and S2 (GLU205/ARG125 (hydrogen bonds: <3.0 Å)), S2 (GLU205/GLU206 (electrostatic interactions: <3.0 Å)), and S2' pocket (PHE357 (hydrophobic interaction: 4.36 Å)). Most peptides showed optimal absorption (76.09%), bioavailability (89.13%), and were non-toxic (97.8%) stable for gastrointestinal digestion (73.9%). Some peptides (60.86%) could also inhibit ACE-I. Chickpea is a source of non-toxic and bioavailable DPP-IV-inhibitory peptides with dual bioactivity. Studies addressing the potential of chickpea peptides as therapeutic or adjunct agents for treating type 2 diabetes are warranted.

A Food Matrix Triggers a Similar Allergic Immune Response in BALB/c Mice Sensitized with Native, Denatured, and Digested Ovalbumin.

The search for an animal model to evaluate the allergenic potential of processed food products is still ongoing. Both the sensitization to ovalbumin (OVA) in different structural states and the allergic response triggered after intragastric or food challenges were assessed. BALB/c mice were sensitized intraperitoneally to OVA (50 µg) in different structural states (native OVA, N-OVA; denatured OVA, D-OVA; formaldehyde- and lysine-treated OVA, FK-OVA; denatured OVA-FK, OVA-DFK; peptides from pepsin digestion, Pep-OVA). Anti-OVA-specific IgE responses were evaluated using ELISA. Anaphylactic signs and mMCP-1 serum levels were evaluated after intragastric (2.0 mg/OVA) and food (0.41 mg/OVA) challenges. IgE reactivities to N-OVA and D-OVA were similar among groups (p > 0.05). After the challenges, all OVA-sensitized mice developed mild to severe anaphylactic signs (p < 0.05 vs. control). Mice sensitized to N-OVA and D-OVA had the highest mMCP-1 serum levels after challenges (p < 0.05 vs. control). Allergic responses were similar despite the different OVA doses used for the challenges. The N-OVA-sensitized murine model of egg allergy proposed in the present study holds the potential for evaluating the impact of food matrix composition and processing on the threshold of egg-allergic responses.

Prevalence of Parent-Reported Food Allergy in a Mexican Pre-School Population.

The magnitude and relevance of food allergies in the preschool population remain unknown in most regions of Mexico and Latin America. Thus, our aim was to estimate the parent-reported prevalence of food allergies in a Mexican preschool population and to characterize their clinical diagnosis and presentation. A cross-sectional survey was conducted in Culiacán City. A validated questionnaire was utilized. A total of 810 parents responded to the questionnaire (valid response rate, 40.7%). The estimated prevalence rates (95% CI) were: "physician-diagnosed Food Allergy (FA), ever" 5.30% (3.86-7.08); "immediate-type FA, ever" 2.96% (1.91-4.38); "immediate-type FA, current" 1.60% (0.86-2.73); and food-dependent anaphylaxis 1.11% (0.51-2.01). The main food allergens were milk (0.49%), strawberries (0.37%), egg, and soy (0.25% each). Atopic diseases and a family history of allergies were significantly associated with immediate-type FA. Among "immediate-type FA, current" cases, 76.9% required emergency room visits, but the prescription of epinephrine autoinjectors was reported in one case only. The food reactions occurred at home (92.35%), in a relative's house (38.5), and at restaurants (23%). Immediate-type FA reactions requiring emergency room visits are not uncommon among the studied population. Actions like proper anaphylaxis management and the prevention of cross-contamination of foods should be encouraged.

Shrimp oncoprotein nm23 is a functional nucleoside diphosphate kinase.

Biosynthesis of nucleoside triphosphates is critical for bioenergetics and nucleic acid replication, and this is achieved by nucleoside diphosphate kinase (NDK). As an emerging biological model and the global importance of shrimp culture, we have addressed the study of the Pacific whiteleg shrimp (Litopenaeus vannamei) NDK. We demonstrated its activity and affinity towards deoxynucleoside diphosphates. Also, the quaternary structure obtained by gel filtration chromatography showed that shrimp NDK is a trimer. Affinity was in the micro-molar range for dADP, dGDP, dTDP and except for dCDP, which presented no detectable interaction by isothermal titration calorimetry, as described previously for Plasmodium falciparum NDK. This information is particularly important, as this enzyme could be used to test nucleotide analogs that can block white spot syndrome virus (WSSV) viral replication and to study its bioenergetics role during hypoxia and fasting.

Shrimp invertebrate lysozyme i-lyz: gene structure, molecular model and response of c and i lysozymes to lipopolysaccharide (LPS).

The invertebrate lysozyme (i-lyz or destabilase) is present in shrimp. This protein may have a function as a peptidoglycan-breaking enzyme and as a peptidase. Shrimp is commonly infected with Vibrio sp., a Gram-negative bacteria, and it is known that the c-lyz (similar to chicken lysozyme) is active against these bacteria. To further understand the regulation of lysozymes, we determined the gene sequence and modeled the protein structure of i-lyz. In addition, the expression of i-lyz and c-lyz in response to lipopolysaccharide (LPS) was studied. The shrimp i-lyz gene is interrupted by two introns with canonical splice junctions. The expression of the shrimp i-lyz was transiently down-regulated after LPS injection followed by induction after 6 h in hepatopancreas. In contrast, c-lyz was up-regulated in hepatopancreas 4 h post-injection and slightly down-regulated in gills. The L. vannamei i-lyz does not contain the catalytic residues for muramidase (glycohydrolase) neither isopeptidase activities; however, it is known that the antibacterial activity does not solely rely on the enzymatic activity of the protein. The study of invertebrate lysozyme will increase our understanding of the regulatory process of the defense mechanisms.

Metal Ions and Chemical Modification Reagents Inhibit the Enzymatic Activity of Lecithin-Dependent Hemolysin from Vibrio parahaemolyticus.

Lecithin-dependent thermolabile hemolysin (LDH) is a virulence factor excreted by Vibrio parahaemolyticus, a marine bacterium that causes important losses in shrimp farming. In this study, the function of LDH was investigated through its inhibition by metal ions (Mg2+, Ca2+, Mn2+, Co2+, Ni2+ and Cu2+) and chemical modification reagents: ß-mercaptoethanol (ßME), phenylmethylsulfonyl fluoride (PMSF) and diethyl pyrocarbonate (DEPC). LDH was expressed in the Escherichia coli strain BL-21, purified under denaturing conditions, and the enzymatic activity was evaluated. Cu2+, Ni2+, Co2+ and Ca2+ at 1 mmol/L inhibited the LDH esterase activity by 20−95%, while Mg2+ and Mn2+ slightly increased its activity. Additionally, PMSF and DEPC at 1 mmol/L inhibited the enzymatic activity by 40% and 80%, respectively. Dose-response analysis showed that DEPC was the best-evaluated inhibitor (IC50 = 0.082 mmol/L), followed by Cu2+ > Co2+ > Ni2+ and PMSF (IC50 = 0.146−1.5 mmol/L). Multiple sequence alignment of LDH of V. parahaemolyticus against other Vibrio species showed that LDH has well-conserved GDSL and SGNH motifs, characteristic of the hydrolase/esterase superfamily. Additionally, the homology model showed that the conserved catalytic triad His-Ser-Asp was in the LDH active site. Our results showed that the enzymatic activity of LDH from V. parahaemolyticus was modulated by metal ions and chemical modification, which could be related to the interaction with catalytic amino acid residues such as Ser153 and/or His 393.

Assessment of the Route of Exposure to Ovalbumin and Cow's Milk Proteins on the Induction of IgE Responses in BALB/c Mice.

BALB/c mice can be orally sensitized to food proteins under acid suppressive medication, mimicking human exposure and triggering a human-like allergic immune response. However, the reproducibility of such an oral food allergy model remains questionable. Our aim was to evaluate the IgE responses triggered against ovalbumin (OVA) and cow's milk proteins (CMP) after intragastric (IG), either under gastric-acid suppression or not, or intraperitoneal (IP) sensitization in BALB/c mice. OVA (0.2 mg) and different concentrations of CMP were administered with/without the antacid sucralfate by the IG route. For IP sensitization, OVA or CMP (0.5 mg) were administered. ELISA was used to evaluate IgE responses. The IP sensitization protocols triggered more robust and consistent anti-OVA or anti-CMP IgE responses than the intragastric ones (with/without sucralfate) (p < 0.05). 2.7% (1/36), and 5.5% (3/54) of the mice that underwent the sucralfate-assisted IG protocol triggered IgE responses against OVA or CMP, respectively. All the mice were administered OVA or CMP via IP triggered detectable IgE responses. The IP sensitization model is more reliable than the IG one for evaluating the intrinsic sensitizing and/or allergenic potential of food proteins, even if IG immunizations are carried out under gastric-acid suppression.

Prediction of ACE-I Inhibitory Peptides Derived from Chickpea (Cicer arietinum L.): In Silico Assessments Using Simulated Enzymatic Hydrolysis, Molecular Docking and ADMET Evaluation.

Chickpea (Cicer arietinum L.) peptides have shown in vitro potential to inhibit the angiotensin I-converting enzyme (ACE-I). However, the potential molecular interactions between chickpea peptides (CP) and ACE-I as well as their ADMET (absorption/distribution/metabolism/excretion/toxicity) characteristics remain unknown. Thus, our aim was to study the in silico interactions of CP with ACE-I and the CP ADMET characteristics. Legumin and provicilin sequences were submitted to in silico analysis to search for ACE-I inhibitory peptides. Simulated enzymatic hydrolysis was performed using the BIOPEP-UWM database, and the ACE-I inhibitory peptides generated (EC50 ≤ 200 µM) were selected to perform molecular docking and ADMET analysis. After hydrolysis, 59 out of 381 peptides with ACE-I inhibitory potential were released. Based on A and B parameters, the legumin peptides showed better ACE-I inhibitory potential than the provicilin ones. CP mainly interact with residues from pocket S1 (Ala354/Glu384) and S2 (His353/His513) through hydrogen bonds (distances < 3.0 Å) and hydrophobic interactions (binding energy from −5.7 to −9.2 kcal/mol). Through ADMET analysis, CP showed optimal values for inhibiting ACE-I in vivo. ACE-I inhibitory peptides from legumin and provicilin can bind strongly and tightly to the active site of ACE-I. Further studies to evaluate in vivo the antihypertensive effects of CP are warranted.

Calotropin and corotoxigenin 3-O-glucopyranoside from the desert milkweed Asclepias subulata inhibit the Na+/K+-ATPase activity.

Na+/K+-ATPase is an essential transmembrane enzyme found in all mammalian cells with critical functions for cell ion homeostasis. The inhibition of this enzyme by several cardiotonic steroids (CTS) has been associated with the cytotoxic effect on cancer cell lines of phytochemicals such as ouabain and digitoxin. This study evaluated the inhibitory capacity of cardenolides calotropin and corotoxigenin 3-O-glucopyranoside (C3OG) from Asclepias subulata over the Na+/K+-ATPase activity in vitro and silico. The inhibitory assays showed that calotropin and C3OG decreased the Na+/K+-ATPase activity with IC50 values of 0.27 and 0.87 µM, respectively. Furthermore, the molecules presented an uncompetitive inhibition on Na+/K+-ATPase activity, with Ki values of 0.2 µM to calotropin and 0.5 µM to C3OG. Furthermore, the molecular modeling indicated that calotropin and C3OG might interact with the Thr797 and Gln111 residues, considered essential to the interaction with the Na+/K+-ATPase. Besides, these cardenolides can interact with amino acid residues such as Phe783, Leu125, and Ala323, to establish hydrophobic interactions on the binding site. Considering the results, these provide novel evidence about the mechanism of action of cardenolides from A. subulata, proposing that C3OG is a novel cardenolide that deserves further consideration for in vitro cellular antiproliferative assays and in vivo studies as an anticancer molecule.

Analysis of IgG, IgA and IgM antibodies against SARS-CoV-2 spike protein S1 in convalescent and vaccinated patients with the Pfizer-BioNTech and CanSinoBio vaccines.

The SARS-CoV-2 virus was detected for the first time in December 2019 in Wuhan, China. Currently, this virus has spread around the world, and new variants have emerged. This new pandemic virus provoked the rapid development of diagnostic tools, therapies and vaccines to control this new disease called COVID-19. Antibody detection by ELISA has been broadly used to recognize the number of persons infected with this virus or to evaluate the response of vaccinated individuals. As the pandemic spread, new questions arose, such as the prevalence of antibodies after natural infection and the response induced by the different vaccines. In Mexico, as in other countries, mRNA and viral-vectored vaccines have been widely used among the population. In this work, we developed an indirect ELISA test to evaluate S1 antibodies in convalescent and vaccinated individuals. By using this test, we showed that IgG antibodies against the S1 protein of SARS-CoV-2 were detected up to 42 weeks after the onset of the symptoms, in contrast to IgA and IgM, which decreased 14 weeks after the onset of symptoms. The evaluation of the antibody response in individuals vaccinated with Pfizer-BioNTech and CanSinoBio vaccines showed no differences 2 weeks after vaccination. However, after completing the two doses of Pfizer-BioNTech and the one dose of CanSinoBio, a significantly higher response of IgG antibodies was observed in persons vaccinated with Pfizer-BioNTech than in those vaccinated with CanSinoBio. In conclusion, these results confirm that after natural infection with SARS-CoV-2, it is possible to detect antibodies for up to 10 months. Additionally, our results showed that one dose of the CanSinoBio vaccine induces a lower response of IgG antibodies than that induced by the complete scheme of the Pfizer-BioNTech vaccine.

White spot syndrome virus Orf514 encodes a bona fide DNA polymerase.

White spot syndrome virus (WSSV) is the causative agent of white spot syndrome, one of the most devastating diseases in shrimp aquaculture. The genome of WSSV includes a gene that encodes a putative family B DNA polymerase (ORF514), which is 16% identical in amino acid sequence to the Herpes virus 1 DNA polymerase. The aim of this work was to demonstrate the activity of the WSSV ORF514-encoded protein as a DNA polymerase and hence a putative antiviral target. A 3.5 kbp fragment encoding the conserved polymerase and exonuclease domains of ORF514 was overexpressed in bacteria. The recombinant protein showed polymerase activity but with very low level of processivity. Molecular modeling of the catalytic protein core encoded in ORF514 revealed a canonical polymerase fold. Amino acid sequence alignments of ORF514 indicate the presence of a putative PIP box, suggesting that the encoded putative DNA polymerase may use a host processivity factor for optimal activity. We postulate that WSSV ORF514 encodes a bona fide DNA polymerase that requires accessory proteins for activity and maybe target for drugs or compounds that inhibit viral DNA replication.