Cutaneous glands of the striped toad, Rhinella crucifer (Wied-Neuwied, 1821) (Amphibia: Bufonidae): Histological study and bioactivities of glandular secretions.

This study investigated the morphology of Rhinella crucifer cutaneous glands, as well as the protein/peptide profiles and bioactivities of body gland secretions (BGS) and parotoid macrogland secretions (PS). The parotoid as well as dorsal and ventral skin fragments of male and female individuals were processed for histological analysis. The protein and peptide profiles of male and female gland secretions were evaluated. Male secretions were also assessed for proteolytic, trypsin inhibiting, hemagglutinating, hemolytic, antimicrobial, and anticoagulant activities. The R. crucifer skin structure presented protuberances that are clearly visible and formed by the integument, which has cutaneous glands throughout the body. An average of 438 and 333 glands were identified in males in females, respectively. No significant differences were observed in the distribution of glands across the body as well as for area and perimeter of glands. Differences were observed in protein composition between the PS and BGS from males and females, and secretions from animals collected from undisturbed and anthropogenically disturbed areas. Proteins with similarities to catalase and elongation factor 1-alpha were detected in the PS. Zymography revealed proteolytic activity in both male BGS and PS. Male BGS showed antibacterial activity against Enterococcus faecalis and Escherichia coli and anticoagulant activity, being able to prolong prothrombin time by 6.34-fold and activated partial thromboplastin time by 2.17-fold. Finally, male PS and BGS caused a maximum hemolysis degree of 1.4%. The data showed that the cutaneous secretions of R. crucifer are potentially promising for biotechnological prospecting.

Efficiency of Experimental Formulation Containing Duddingtonia flagrans and Pochonia chlamydosporia against Moniezia expansa Eggs.

This study aimed to evaluate the effectiveness of the experimental formulation containing chlamydospores of Duddingtonia flagrans and Pochonia chlamydosporia fungi, against Moniezia expansa. Two experiments were carried out. The first experiment evaluated the in vitro efficacy using 1 g of the experimental formulation (V1) added to 100 M. expansa eggs and the control (V2) (without the fungal formulation). Intact eggs or eggs with alterations were counted in order to evaluate their effectiveness. The second experiment evaluated the action of the fungal formulation on M. expansa eggs after passing through the gastrointestinal tract of goats. Three groups were identified as B1, B2, and B3, which received 1.0, 1.5 g of experimental fungal formulation, and placebo, respectively. In experiment 1, all the eggs in V1 were subjected to the predatory action of fungi, while in V2, the eggs remained intact. In experiment 2, the reduction of eggs in groups B1 and B2 were 49% and 57% 24 h after ingestion, 60% and 63% 48 h after, and 48% and 58% 72 h after. The predatory capacity against M. expansa eggs shown in the tests demonstrated that experimental fungal formulation has the potential to be used on integrated helminth control programs.

Purification, Characterization, and Assessment of Antimicrobial Activity and Toxicity of Portulaca elatior Leaf Lectin (PeLL).

Lectins are carbohydrate-binding proteins with several bioactivities, including antimicrobial properties. Portulaca elatior is a species found at Brazilian Caatinga and data on the biochemical composition of this plant are scarce. The present work describes the purification of P. elatior leaf lectin (PeLL) as well as the assessment of its antimicrobial activity and toxicity. PeLL, isolated by chromatography on a chitin column, had native liquid charge and subunit composition evaluated by electrophoresis. Hemagglutinating activity (HA) of PeLL was determined in the presence of carbohydrates or divalent cations, as well as after heating and incubation at different pH values. Changes in the lectin conformation were monitored by evaluating intrinsic tryptophan fluorescence and using the extrinsic probe bis-ANS. Antimicrobial activity was evaluated against Pectobacterium strains and Candida species. The minimal inhibitory (MIC), bactericidal (MBC), and fungicidal (MFC) concentrations were determined. Finally, PeLL was evaluated for in vitro hemolytic activity in human erythrocytes and in vivo acute toxicity in mice (5 and 10 mg/kg b.w. per os). PeLL (pI 5.4; 20 kDa) had its HA was inhibited by mannose, galactose, Ca2+, Mg2+, and Mn2+. PeLL HA was resistant to heating at 100 °C, although conformational changes were detected. PeLL was more active in the acidic pH range, in which no conformational changes were observed. The lectin presented MIC and MBC of 0.185 and 0.74 µg/mL for all Pectobacterium strains, respectively; MIC of 1.48 µg/mL for C. albicans, C. tropicalis, and C. krusei; MIC and MFC of 0.74 and 2.96 µg/mL for C. parapsilosis. No hemolytic activity or signs of acute toxicity were observed in the mice. In conclusion, a new, low-toxic, and thermostable lectin was isolated from P. elatior leaves, being the first plant compound to show antibacterial activity against Pectobacterium.

Usage of Moringa oleifera residual seeds promotes efficient flocculation of Tetradesmus dimorphus biomass.

Bio-flocculation is a sustainable low-cost harvesting technique for microalgae biomass production; however, it is generally less efficient than chemical flocculants. This study aims to investigate the efficiency of Moringa oleifera seeds as a bio-flocculant for harvesting Tetradesmus dimorphus biomass. Four extracts from integral and residual (seeds without lipids) biomass of M. oleifera seeds using salt or aqueous solutions were used at four concentrations (100, 200, 300, and 400 ppm). Flocculation efficiency (FE) increased as the pH decreased. The addition of the extracts reduced the pH of the cultures, dispensing pH adjustment after dosing the flocculating agent. Salt extracts exhibited higher flocculation efficiency than aqueous extracts. The highest flocculation efficiency (~ 98%) was obtained using a salt extract of residual biomass of seeds in any concentration varying from 100 to 400 ppm. The predicted values obtained from a data modeling using response surface methodology approached the real values (r 2 = 0.9382), resulting in an adequate optimization of the flocculant concentration of ~ 335 ppm and pH 5.6 for a predicted FE of ~ 106%. The findings of the present study confirmed that the salt extract from residual biomass of M. oleifera seeds is an effective bio-flocculant for T. dimorphus biomass harvesting.

Mesquite pod (Prosopis juliflora) meal on meat quality of pasture-finishing lambs.

This study was carried out to validate the inclusion of up to 750 g/kg of mesquite pod (Prosopis juliflora) meal in the diet and evaluate the effects on carcass characteristics and meat quality for lambs finished in pasture. Forty male, non-castrated, crossbred Santa Inês lambs, with an initial body weight (24.2 ± 3.1 kg), and approximately 120 days old were used. The animals were kept in a total area of 4 ha, divided in four paddocks of 0.62 ha each (10 animals/paddocks), on pastures of Massai (Panicum maximum cv. Massai) with drinkers and feeders during the finishing phase. Dietary treatments based on mesquite pod meal inclusion levels (g/kg of dry matter): CON, without mesquite pod meal; MPM25, 250 g/kg of mesquite pod meal; MPM50, 500 g/kg of mesquite pod meal; and MPM75, 750 g/kg of mesquite pod meal. No treatment effect were detected (P > 0.05) for carcass measures, carcass characteristics, chemical composition of longissimus thoracis muscle, tissue composition, and lipid oxidation. Lamb meat color values, such as lightness (L*) and yellowness (b*), were not affected (P > 0.05) by mesquite pod meal inclusion on the diets, whereas for redness (a*), HUE-angle, and chroma were influenced (P < 0.05). Palmitic acid had a quadratic effect, while oleic acid, eicosatrienoic acid, saturated fatty acids, monounsaturated fatty acids, and PUFA:SFA had a linear course (P < 0.05). In conclusion, the mesquite pod meal can be used as an energy feed source up to 750 g/kg of dry matter in the diet, without changing the carcass characteristics and meat quality of lambs finished in pasture.

Moderate Physical Exercise Activates ATR2 Receptors, Improving Inflammation and Oxidative Stress in the Duodenum of 2K1C Hypertensive Rats.

Background: In addition to the cardiovascular and renal systems, the gastrointestinal tract also contains angiotensin ATR1a, ATR1b, and ATR2. We previously observed that the 2Kidney-1Clip hypertension model elicits physical exercise and gastrointestinal dysmotility, which is prevented by renin-angiotensin system blockers. Here, we investigate the effect of physical exercise on inflammation, stress biomarkers, and angiotensin II receptors in the duodenum of 2K1C rats. Methods: Arterial hypertension was induced by the 2K1C surgical model. The rats were allocated in Sham, 2K1C, or 2K1C+Exercise groups. One week after surgery, they were submitted to a physical exercise protocol (running 5x/week, 60min/day). Next, we assessed their intestinal contractility, cytokine levels (TNF-α, IL-1ß, and IL-6), oxidative stress levels (MPO, GSH, MDA, and SOD), and the gene expression of angiotensin receptors (ATR1A, ATR1B, and ATR2). Results: In comparison with the Sham group, the 2K1C arterial hypertension decreased (p<0.05) the intestinal contractility. In comparison with 2K1C, the 2K1C+Exercise group exhibited lower (p<0.05) MPO activity (22.04±5.90 vs. 78.95±18.09 UMPO/mg tissue) and higher (p<0.05) GSH concentrations in intestinal tissues (67.63±7.85 vs. 31.85±5.90mg NPSH/mg tissue). The 2K1C+Exercise group showed lower (p<0.05) cytokine levels in the intestine than 2K1C rats. In comparison with the Sham group, the 2K1C+Exercise rats showed higher (p<0.05) gene expression of ATR2 in the duodenum. Conclusion: 2K-1C hypertension elicits an oxidative stress and inflammation process in the duodenum. Physical exercise modulates the expression twice as much of ATR2 receptors, suggesting possible anti-inflammatory and antioxidant effects induced by exercise.

Quantum dots conjugated to lectins from Schinus terebinthifolia leaves (SteLL) and Punica granatum sarcotesta (PgTeL) as potential fluorescent nanotools for investigating Cryptococcus neoformans.

This study reports the development of conjugates based on quantum dots (QD)s and lectins from Schinus terebinthifolia leaves (SteLL) and Punica granatum sarcotesta (PgTeL). Cryptococcus neoformans cells were chosen to evaluate the efficiency of the conjugates. Lectins were conjugated to QDs via adsorption, and the optical parameters (emission and absorption) were monitored. Lectin stability in the conjugates towards denaturing agents was investigated via fluorometry. The conjugation was evaluated using fluorescence microplate (FMA) and hemagglutination (HA) assays. The labeling of the C. neoformans cell surface was quantified using flow cytometry and observed via fluorescence microscopy. The QDs-SteLL and QDs-PgTeL conjugates, obtained at pH 7.0 and 8.0, respectively, showed the maintenance of colloidal and optical properties. FMA confirmed the conjugation, and the HA assay indicated that the lectin carbohydrate-binding ability was preserved after conjugation. SteLL and PgTeL showed stability towards high urea concentrations and heating. Conjugates labeled over 90% of C. neoformans cells as observed via flow cytometry and confirmed through fluorescence microscopy. C. neoformans labeling by conjugates was inhibited by glycoproteins, suggesting specific interactions through the lectin carbohydrate-binding site. Thus, an effective protocol for the conjugation of SteLL or PgTeL with QDs was proposed, yielding new nanoprobes useful for glycobiological studies.

Insecticidal activity of a chemotype VI essential oil from Lippia alba leaves collected at Caatinga and the major compound (1,8-cineole) against Nasutitermes corniger and Sitophilus zeamais.

Lippia alba is an aromatic shrub known to produce a diversity of essential oils, which can be classified into chemotypes. This study reports on the insecticidal activity of essential oil from L. alba leaves collected at Caatinga and its major compound against termite Nasutitermes corniger and maize weevil Sitophilus zeamais. The chromatographic analysis revealed the presence of 19 compounds, with 1,8-cineole being the most common (70.01%). When ingested, the oil promoted the mortality of N. corniger (LC50: 18.25 and 8.4 nL/g for workers and soldiers, respectively). The compound 1,8-cineole was also termiticidal for workers (LC50: 13.7 nL/g). The oil inhibited the activity of N. corniger exoglucanase, xylanase, and proteases. Toxicity by ingestion to S. zeamais was detected for the oil (LC50: 0.297 µL/g) but not for 1,8-cineole; however, both the oil and 1,8-cineole showed anti-nutritional effects. Fumigant effects of the oil and 1,8-cineole against S. zeamais (LC50 of 78.0 and 13.64 µL/L in air, respectively) were detected. This is the first record of a chemotype VI oil from L. alba collected at Caatinga and the first report of the insecticidal activity of a chemotype VI oil. Our study demonstrates that essential oil from L. alba and 1,8 cineole have the potential for the development of natural insecticides.

Renal alterations caused by ligature-induced periodontitis persist after ligature removal in rats.

BACKGROUND AND OBJECTIVE: Periodontitis may crosstalk with renal diseases, yet that remains unclear. We investigated whether the renal alterations caused by induced periodontitis are reversible after removal of the ligatures in experimental ligature-induced periodontitis. MATERIAL AND METHODS: Twenty-four female rats were divided into three groups: control (without periodontitis), periodontitis (20 days of ligature-induced periodontitis), and P20-20 (20 days of ligature-induced periodontitis and 20 days after ligature removal). The following periodontal parameters were assessed: gingival bleeding index, probing pocket depth, myeloperoxidase activity, and alveolar bone height. For renal tissues, histopathology, malonaldehyde (MDA) levels, glutathione (GSH) content, and renal weight were evaluated. In the blood, creatinine, uric acid, albumin, total cholesterol, total protein, and glucose levels were assessed. Total protein and creatinine levels in urine were also investigated. RESULTS: Rat renal tissues did not demonstrate reversal of periodontitis-related changes in the P20-20 group in terms of MDA, GSH, and histopathological evaluations when compared to the periodontitis group. Accordingly, only total cholesterol levels were reversible in the P20-20. CONCLUSION: Renal alterations caused by ligature-induced periodontitis persisted even after removal of ligatures in rats.

Effects of a solid formulation containing lectin-rich fraction of Moringa oleifera seeds on egg hatching and development of Aedes aegypti larvae.

The measures currently used to minimize the spread of arboviruses, comprising dengue, Chikungunya, and Zika virus, involve controlling the size of population of the mosquito Aedes aegypti. However, the search for formulations containing new insecticides is gaining pace due to reports of mosquito populations showing resistance to commonly used compounds. In this study, tablets containing a protein fraction of Moringa oleifera seeds enriched in the WSMoL lectin, known to show larvicidal and ovicidal activities against A. aegypti, were developed. The compatibility between the fraction and the excipients used in obtaining the tablets was evaluated by thermogravimetry (TG), differential scanning calorimetry (DSC), and Fourier transform infrared (FTIR) absorption spectroscopy. The larvicidal and ovicidal activities of the resulting tablets [5%, 10%, and 15% (w/w) of the fraction] were evaluated, as well as their effect on mosquito oviposition. Assays were also performed using a placebo tablet. According to the TG, DSC, and FTIR results, the protein composition of the fraction did not change when mixed with the components of the formulation. Tablets containing 10% and 15% WSMoL-rich fraction caused mortality of 42.5% and 95% of the larvae after 48 h, respectively, with larvae incubated with these tablets showing reduced acetylcholinesterase activity. All tablets inhibited egg hatching after 72 h (36-74%), and tablets containing 15% fraction were found to exert a repellent effect on oviposition. Our results show that the formulation developed in this study interfered with the life cycle of A. aegypti, and thus show potential for use in the control of this mosquito.

Premises among SARS-CoV-2, dysbiosis and diarrhea: Walking through the ACE2/mTOR/autophagy route.

Recently, a new coronavirus (SARS-CoV-2) was discovered in China. Due to its high level of contagion, it has already reached most countries, quickly becoming a pandemic. Although the most common symptoms are related to breathing problems, SARS-CoV-2 infections also affect the gastrointestinal tract culminating in inflammation and diarrhea. However, the mechanisms related to these enteric manifestations are still not well understood. Evidence shows that the SARS-CoV-2 binds to the angiotensin-converting enzyme receptor 2 (ACE2) in host cells as a viral invasion mechanism and can infect the lungs and the gut. Other viruses have already been linked to intestinal symptoms through binding to ACE2. In turn, this medical hypothesis article conjectures that the ACE2 downregulation caused by the SARS-CoV-2 internalization could lead to decreased activation of the mechanistic target of mTOR with increased autophagy and lead to intestinal dysbiosis, resulting in diarrhea. Besides that, dysbiosis can directly affect the respiratory system through the lungs. Although there are clues to other viruses that modulate the ACE2/gut/lungs axis, including the participation of autophagy and dysbiosis in the development of gastrointestinal symptoms, there is still no evidence of the ACE2/mTOR/autophagy pathway in SARS-CoV-2 infections. Thus, we propose that the new coronavirus causes a change in the intestinal microbiota, which culminates in a diarrheal process through the ACE2/mTOR/autophagy pathway into enterocytes. Our assumption is supported by premises that unregulated intestinal microbiota increases the susceptibility to other diseases and extra-intestinal manifestations, which can even cause remote damage in lungs. These putative connections lead us to suggest and encourage future studies aiming at assessing the aforementioned hypothesis and regulating dysbiosis caused by SARS-CoV-2 infection, in order to confirm the decrease in lung injuries and the improvement in the prognosis of the disease.

Polymorphisms in GSTE2 is associated with temephos resistance in Aedes aegypti.

The glutathione S-transferases (GSTs) are enzymes involved in several distinct biological processes. In insects, the GSTs, especially delta and epsilon classes, play a key role in the metabolism of xenobiotics used to control insect populations. Here, we investigated its potential role in temephos resistance, examining the GSTE2 gene from susceptible (RecL) and resistant (RecR) strains of the mosquito Aedes aegypti, vector for several pathogenic arboviruses. Total GST enzymatic activity and the GSTE2 gene expression profile were evaluated, with the GSTE2 cDNA and genomic loci sequenced from both strains. Recombinant GSTE2 and mutants were produced in a heterologous expression system and assayed for enzyme kinetic parameters. These proteins also had their 3D structure predicted through molecular modeling. Our results showed that RecR has a profile of total GST enzymatic activity higher than RecL, with the expression of the GSTE2 gene in resistant larvae increasing six folds. Four exclusive RecR mutations were observed (L111S, I150V, E178A and A198E), which were absent in the laboratory susceptible strains. The enzymatic activity of the recombinant GSTE2 showed different kinetic parameters, with the GSTE2 RecR showing an enhanced ability to metabolize its substrate. The I150V mutation was shown to induce significant changes in catalytic parameters and a 3D modeling of GSTE2 mapped two of the RecR changes (L111S and I150V) near the enzyme's catalytic pocket, also implying an impact on its catalytic activity. Our results reinforce a potential role for GSTE2 in the metabolic resistance phenotype while contributing to the understanding of the molecular basis for the resistance mechanism.

Novel Ocellatin Peptides Mitigate LPS-induced ROS Formation and NF-kB Activation in Microglia and Hippocampal Neurons.

Cutaneous secretions of amphibians have bioactive compounds, such as peptides, with potential for biotechnological applications. Therefore, this study aimed to determine the primary structure and investigate peptides obtained from the cutaneous secretions of the amphibian, Leptodactylus vastus, as a source of bioactive molecules. The peptides obtained possessed the amino acid sequences, GVVDILKGAAKDLAGH and GVVDILKGAAKDLAGHLASKV, with monoisotopic masses of [M + H]± = 1563.8 Da and [M + H]± = 2062.4 Da, respectively. The molecules were characterized as peptides of the class of ocellatins and were named as Ocellatin-K1(1-16) and Ocellatin-K1(1-21). Functional analysis revealed that Ocellatin-K1(1-16) and Ocellatin-K1(1-21) showed weak antibacterial activity. However, treatment of mice with these ocellatins reduced the nitrite and malondialdehyde content. Moreover, superoxide dismutase enzymatic activity and glutathione concentration were increased in the hippocampus of mice. In addition, Ocellatin-K1(1-16) and Ocellatin-K1(1-21) were effective in impairing lipopolysaccharide (LPS)-induced reactive oxygen species (ROS) formation and NF-kB activation in living microglia. We incubated hippocampal neurons with microglial conditioned media treated with LPS and LPS in the presence of Ocellatin-K1(1-16) and Ocellatin-K1(1-21) and observed that both peptides reduced the oxidative stress in hippocampal neurons. Furthermore, these ocellatins demonstrated low cytotoxicity towards erythrocytes. These functional properties suggest possible to neuromodulatory therapeutic applications.

Portulaca elatior root contains a trehalose-binding lectin with antibacterial and antifungal activities.

Lectins are carbohydrate-binding proteins broadly distributed in plants and have several biological functions, including antimicrobial action. Portulaca elatior is a Caatinga plant whose chemical composition and biotechnological potential have not been extensively studied. In this work, a lectin was isolated from P. elatior root extract and evaluated for antimicrobial activity. The P. elatior root lectin (PeRoL) showed native molecular mass of 33 kDa, pI 3.8 and is comprised of two subunits of 15 kDa linked by disulfide bonds. No sequence similarities with Viridiplantae proteins were observed. The PeRoL hemagglutinating activity (HA) was not affected by heating and was detected in a pH ranging from 4.0 to 8.0. Trehalose was identified as an endogenous inhibitor of PeRoL present in the roots. Bacteriostatic activity was detected against Enterococcus faecalis, Pseudomonas aeruginosa and Staphylococcus aureus (minimal inhibitory concentration of 8.1, 32.5 and 4.06 µg/mL, respectively). PeRoL induced the death of Candida albicans, Candida parapsilosis, Candida krusei, and Candida tropicalis cells, with a minimal fungicidal concentration of 16 µg/mL. The lectin (100 µg/mL) was not cytotoxic to human peripheral blood mononuclear cells (PBMCs) and did not show hemolytic activity. In conclusion, the roots of P. elatior contain a trehalose-binding, thermostable, and antimicrobial lectin.

PgTeL, the lectin found in Punica granatum juice, is an antifungal agent against Candida albicans and Candida krusei.

The pomegranate (Punica granatum) sarcotesta contains a chitin-binding lectin (PgTeL) with antibacterial activity against human pathogenic species. In this work, the structural stability of PgTeL was evaluated by fluorimetric analysis and the lectin was evaluated for cytotoxicity to human peripheral blood mononuclear cells (PBMCs) and antifungal activity against Candida albicans and Candida krusei. PgTeL folding was impaired when lectin was incubated at pH≥6.0. On the other hand, the lectin did not undergo unfolding even when heated at 100°C. PgTeL (1, 10, and 100µg/mL) was not cytotoxic to PBMCs. Antifungal activity was detected for C. albicans (MIC: 25µg/mL; MFC: 50µg/mL) and C. krusei (MIC and MFC of 12.5µg/mL). Treatment of yeast cells with PgTeL resulted in decrease of intracellular ATP content even at sub-inhibitory concentrations (½MIC and »MIC) and induced lipid peroxidation. In addition, PgTeL damaged the integrity of fungal cell wall of both species, with more pronounced effects in C. krusei. The lectin showed significant antibiofilm activity on C. albicans at sub-inhibitory concentrations (0.195 and 0.39µg/mL). In conclusion, PgTeL is an anti-Candida agent whose action mechanism involves oxidative stress, energetic collapse, damage to the cell wall and rupture of yeast cells.

Purification and characterization of a protease from the visceral mass of Mytella charruana and its evaluation to obtain antimicrobial peptides.

This work describes purification of a protease from the visceral mass of the mussel Mytella charruana as well as evaluation of its ability to hydrolyze milk casein to generate antimicrobial peptides. The enzyme showed pI of 4.1 and a single polypeptide band of 83.1 kDa after SDS-PAGE. Sequence similarities with tropomyosin and myosin from mollusks were detected. The protease showed a trypsin-like activity with optimal temperature of 40 °C and stability in a wide pH range (3.0-9.0). Km was 4.28 ±â€¯0.34 mM of the synthetic substrate N-benzoyl-dl-arginyl-ρ-nitroanilide, whereas Vmax was 0.056 ±â€¯0.001 nmol min-1. The enzyme hydrolyzed casein, and the hydrolysate inhibited the growth of Escherichia coli, Micrococcus luteus, Bacillus subtilis, and Klebsiella pneumoniae at a minimal inhibitory concentration of 5.0 µg mL-1. In conclusion, the visceral mass of M. charruana contains a trypsin-like protease that can generate peptides from casein that have a bacteriostatic effect.

Ecotoxicity of water-soluble lectin from Moringa oleifera seeds to zebrafish (Danio rerio) embryos and larvae.

The evaluation of ecotoxicity of mosquito larvicidal agents (such as the water-soluble lectin from Moringa oleifera seeds, WSMoL) is an essential step to establish the guidelines for their use. In this sense, this work evaluated the toxicity of WSMoL to Danio rerio embryos and larvae. Embryos were exposed to waterborne WSMoL (0.0125-0.2 mg mL-1) for 96 h and lethal and sub-lethal effects were observed every 24 h. In the bioassays with larvae, the individuals were exposed to the WSMoL (0.025-0.2 mg mL-1), mortality was recorded daily, and larval swimming velocities were analyzed after 72 h and 168 h of exposure. Additionally, acetylcholinesterase (AChE) activity of larvae was determined after 168 h of exposure. WSMoL LC50 values to embryos were 0.190, 0.133 and 0.049 mg mL-1 after 48, 72 and 96 h, respectively. No toxic endpoint was observed after exposure for 24 h. In addition, hatching was delayed and larval length at 96 h was reduced compared to the control. WSMoL LC50 to larvae were 0.21 and 0.135 mg mL-1, after 24 h and 96 h, respectively. Larvae exposed to 0.1 and 0.2 mg mL-1 showed a decrease in swimming speed and a significant reduction in AChE activity. In conclusion, WSMoL at waterborne concentrations needed for its use as a larvicide to A. aegypti causes lethal and sublethal effects to zebrafish embryos and larvae. Therefore, its use in waterbodies where there are non-target organisms is not recommended.

The anti-inflammatory and antinociceptive activity of albumins from Crotalaria retusa seeds.

Seeds of Crotalaria retusa L. are used in popular medicine because of their pharmacological properties. The albumin fraction obtained from its seeds contains lectin, a protein known to have analgesic and anti-inflammatory properties. Thus, albumins extracted from C. retusa were investigated for their anti-inflammatory and antinociceptive effects. The intraperitoneal administration of different doses of albumins (5, 10 or 20mg/kg) significantly inhibited the mice paw edema induced by carrageenan (maximum inhibition rate of 80.9% at four hours, 20mg/kg), and this event was followed by diminishing paw myeloperoxidase measurements. Albumins (20mg/kg) also inhibited neutrophil migration into the peritoneal cavity induced by carrageenan. However, no effect was observed in the dextran-induced paw edema and abdominal contortions induced by acetic acid. Moreover, albumins (20mg/kg) significantly reduced the second (inflammatory) phase of the licking time induced by formalin. The detection of heammaglutinating activity against human erythrocytes in albumins evidences the presence of lectin in seeds of C. retusa. Our data showed that seeds of C. retusa had anti-inflammatory and antinociceptive properties and such activities are probably due to the inhibitory effect on neutrophil migration of lectin present in albumins.

Microgramma vacciniifolia (Polypodiaceae) fronds contain a multifunctional lectin with immunomodulatory properties on human cells.

In this study, we report the purification and characterization of a multifunctional lectin (MvFL) from Microgramma vacciniifolia fronds as well as its immunomodulatory properties on human peripheral blood mononuclear cells (PBMCs). MvFL (pI 4.51; 54kDa) is a glycoprotein able to inhibit trypsin activity and that has sequence similarities (32% coverage) with a plant RNA-binding protein. Hemagglutinating activity of MvFL was not altered by heating at 100°C for 30min, but was reduced in alkaline pH (8.0 and 9.0). Fluorimetric analyses showed that this lectin did not undergo marked conformational changes when heated. However, the MvFL conformation changed depending on the pH. MvFL at 6.25-25µg/mL was not cytotoxic to lymphocytes present among PBMCs. The PBMCs incubated for 24h with the lectin (12.5µg/mL) showed increased TNF-α, IFN-γ, IL-6, IL-10, and nitric oxide production. MvFL also stimulated T lymphocytes from PBMCs to differentiate into CD8+ cells. The activation (indicated by CD28 expression) of these cells was also stimulated. In conclusion, MvFL is a heat-stable and multifunctional protein, with both lectin and trypsin inhibitor activities, and capable of inducing predominantly a Th1 response in human PBMCs as well as activation and differentiation of T lymphocytes.

CasuL: A new lectin isolated from Calliandra surinamensis leaf pinnulae with cytotoxicity to cancer cells, antimicrobial activity and antibiofilm effect.

This work describes the isolation of a lectin (CasuL) from the leaf pinnulae of Calliandra surinamensis and the evaluation of its cytotoxic, antimicrobial and antibiofilm properties. Proteins from pinnulae extract were precipitated with ammonium sulphate (60% saturation) and submitted to Sephadex G-75 chromatography, which yielded isolated CasuL (purification factor: 113). Native CasuL is an acidic protein (pI 5.82) with a relative molecular mass of 48kDa. This lectin is also an oligomeric protein composed of three subunits and mass spectrometry revealed similarities with a Sorghum bicolor protein. CasuL did not undergo unfolding when heated but changes in conformation and hemagglutinating activity were detected at basic pH. CasuL did not reduce the viability of human peripheral blood mononuclear cells but was toxic to leukemic K562 cells (IC50 67.04±5.78µg/mL) and breast cancer T47D cells (IC50: 58.75±2.5µg/mL). CasuL (6.25-800µg/mL) only showed bacteriostatic effect but was able to reduce biofilm formation by Staphylococcus saprophyticcus and Staphylococcus aureus (non-resistant and oxacillin-resistant isolates). CasuL showed antifungal activity against Candida krusei causing alterations in cell morphology and damage to cell wall. In conclusion, the pinnulae of C. surinamensis leaves contain a thermo-stable lectin with biotechnological potential as cytotoxic, antibiofilm, and antifungal agent.