rJararacin, a recombinant disintegrin from Bothrops jararaca venom: Exploring its effects on hemostasis and thrombosis.

Integrins are a family of heterodimeric transmembrane receptors which link the extracellular matrix to the cell cytoskeleton. These receptors play a role in many cellular processes: adhesion, proliferation, migration, apoptosis, and platelet aggregation, thus modulating a wide range of scenarios in health and disease. Therefore, integrins have been the target of new antithrombotic drugs. Disintegrins from snake venoms are recognized by the ability to modulate the activity of integrins, such as integrin αIIbß3, a fundamental platelet glycoprotein, and αvß3 expressed on tumor cells. For this reason, disintegrins are unique and potential tools for examining integrin-matrix interaction and the development of novel antithrombotic agents. The present study aims to obtain the recombinant form of jararacin and evaluate the secondary structure and its effects on hemostasis and thrombosis. rJararacin was expressed in the Pichia pastoris (P. pastoris) expression system and purified the recombinant protein with a yield of 40 mg/L of culture. The molecular mass (7722 Da) and internal sequence were confirmed by mass spectrometry. Structure and folding analysis were obtained by Circular Dichroism and 1H Nuclear Magnetic Resonance spectra. Disintegrin structure reveals properly folded with the presence of ß-sheet structure. rJararacin significantly demonstrated inhibition of the adhesion of B16F10 cells and platelets to the fibronectin matrix under static conditions. rJararacin inhibited platelet aggregation induced by ADP (IC50 95 nM), collagen (IC50 57 nM), and thrombin (IC50 22 nM) in a dose-dependent manner. This disintegrin also inhibited 81% and 94% of the adhesion of platelets to fibrinogen and collagen under continuous flow, respectively. In addition, rjararacin efficaciously prevents platelet aggregation in vitro and ex vivo with rat platelets and thrombus occlusion at an effective dose (5 mg/kg). The data here provides evidence that rjararacin possesses the potential as an αIIbß3 antagonist, capable of preventing arterial thrombosis.

Pharmacological Screening of Venoms from Five Brazilian Micrurus Species on Different Ion Channels.

Coral snake venoms from the Micrurus genus are a natural library of components with multiple targets, yet are poorly explored. In Brazil, 34 Micrurus species are currently described, and just a few have been investigated for their venom activities. Micrurus venoms are composed mainly of phospholipases A2 and three-finger toxins, which are responsible for neuromuscular blockade-the main envenomation outcome in humans. Beyond these two major toxin families, minor components are also important for the global venom activity, including Kunitz-peptides, serine proteases, 5' nucleotidases, among others. In the present study, we used the two-microelectrode voltage clamp technique to explore the crude venom activities of five different Micrurus species from the south and southeast of Brazil: M. altirostris, M. corallinus, M. frontalis, M. carvalhoi and M. decoratus. All five venoms induced full inhibition of the muscle-type α1ß1δε nAChR with different levels of reversibility. We found M. altirostris and M. frontalis venoms acting as partial inhibitors of the neuronal-type α7 nAChR with an interesting subsequent potentiation after one washout. We discovered that M. altirostris and M. corallinus venoms modulate the α1ß2 GABAAR. Interestingly, the screening on KV1.3 showed that all five Micrurus venoms act as inhibitors, being totally reversible after the washout. Since this activity seems to be conserved among different species, we hypothesized that the Micrurus venoms may rely on potassium channel inhibitory activity as an important feature of their envenomation strategy. Finally, tests on NaV1.2 and NaV1.4 showed that these channels do not seem to be targeted by Micrurus venoms. In summary, the venoms tested are multifunctional, each of them acting on at least two different types of targets.

Protective factors in mature human milk: a look into the proteome and peptidome of adolescent mothers' breast milk.

The characterisation of proteome and peptidome of adolescent mothers' breast milk brings important information to both mother's and infant's health; however, it has not been investigated. Bioactive peptides derived from milk proteins have numerous functions. The bioactivity of breast milk peptides includes anti-inflammatory and antimicrobial activities and regulation of gastrointestinal function. We aimed to characterise the proteome and peptidome of mature breast milk of adolescent mothers and investigate whether it is affected by lactational period. We used a combination of electrophoretic and nano-scale LC-quadrupole time-of-flight MS/MS (nLC-Q-TOF-MS/MS) techniques and bioinformatics to explore the proteome of human skimmed milk expressed by lactating adolescents in two groups according to postpartum period (up to 3 and over 5 weeks postpartum). This is the first study that analysed the proteome of adolescent mothers' breast milk produced during two periods of lactation using 1D-electrophoresis combined with nLC-Q-TOF-MS/MS analysis. Our results showed that the protein composition of adolescent milk varies independently of lactation stage and showed high inter-individual variation. A total of 424 proteins were identified in skimmed milk, of which 137 proteins were common to both groups. Most of the peptides found in adolescents' breast milk were not derived from major proteins in milk. Association maps showed several interactions between groups of peptides that pointed to the relevance of breast milk peptides to neonatal defensive system.

The costa of trichomonads: A complex macromolecular cytoskeleton structure made of uncommon proteins.

BACKGROUND INFORMATION: The costa is a prominent striated fibre that is found in protozoa of the Trichomonadidae family that present an undulating membrane. It is composed primarily of proteins that have not yet been explored. In this study, we used cell fractionation to obtain a highly enriched costa fraction whose structure and composition was further analysed by electron microscopy and mass spectrometry. RESULTS: Electron microscopy of negatively stained samples revealed that the costa, which is a periodic structure with alternating electron-dense and electron-lucent bands, displays three distinct regions, named the head, neck and body. Fourier transform analysis showed that the electron-lucent bands present sub-bands with a regular pattern. An analysis of the costa fraction via one- and two-dimensional electrophoresis and liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) allowed the identification of 54 hypothetical proteins. Fourteen of those proteins were considered to be major components of the fraction. CONCLUSIONS: The costa of T. foetus is a complex and organised cytoskeleton structure made of a large number of proteins which is assembled into filamentous structures. Some of these proteins exhibit uncharacterised domains and no function related according to gene ontology, suggesting that the costa structure may be formed by a new class of proteins that differ from those previously described in other organisms. Seven of these proteins contain prefoldin domains displaying coiled-coil regions. This propriety is shared with proteins of the striated fibres of other protozoan as well as in intermediate filaments. SIGNIFICANCE: Our observations suggest the presence of a new class of the cytoskeleton filaments in T. foetus. We believe that our data could auxiliate in determining the specific locations of these proteins in the distinct regions that compose the costa, as well as to define the functional roles of each component. Therefore, our study will help in the better understanding of the organisation and function of this structure in unicellular organisms.

New insights on the Golgi complex of Tritrichomonas foetus.

Tritrichomonas foetus is a protist that causes bovine trichomoniasis and presents a well-developed Golgi. There are very few studies concerning the Golgi in trichomonads. In this work, monoclonal antibodies were raised against Golgi of T. foetus and used as a tool on morphologic and biochemical studies of this organelle. Among the antibodies produced, one was named mAb anti-Golgi 20.3, which recognized specifically the Golgi complex by fluorescence and electron microscopy. By immunoblotting this antibody recognized two proteins with 60 and 66 kDa that were identified as putative beta-tubulin and adenosine triphosphatase, respectively. The mAb 20.3 also recognized the Golgi complex of the Trichomonas vaginalis, a human parasite. In addition, the nucleotide coding sequences of these proteins were identified and included in the T. foetus database, and the 3D structure of the proteins was predicted. In conclusion, this study indicated: (1) adenosine triphosphatase is present in the Golgi, (2) ATPase is conserved between T. foetus and T. vaginalis, (3) there is new information concerning the nucleic acid sequences and protein structures of adenosine triphosphatase and beta-tubulin from T. foetus and (4) the mAb anti-Golgi 20.3 is a good Golgi marker and can be used in future studies.

Anti-hemostatic, antithrombotic, and chemical profiles of a curly-leaf variety of Petroselinum crispum (Apiaceae), a food and medicinal aromatic herb.

Thrombosis is currently among the major causes of morbidity and mortality in the World. New prevention and therapy alternatives have been increasingly sought in medicinal plants. In this context, we have been investigating parsley, Petroselinum crispum (Mill.) Nym, an aromatic herb with two leaf varieties. We report here the in vitro, in vivo, and ex vivo anti-hemostatic and antithrombotic activities of a parsley curly-leaf variety. Aqueous extracts of aerial parts (PCC-AP), stems (PCC-S), and leaves (PCC-L) showed significant in vitro antiplatelet activity. PCC-AP extract exhibited the highest activity (IC50 2.92 mg/mL) when using ADP and collagen as agonists. All extracts also presented in vitro anticoagulant activity (APTT and PT) and anti-thrombogenic activity. PCC-S was the most active, with more significant interference in the factors of the intrinsic coagulation pathway. The oral administration of PCC-AP extract in rats caused a greater inhibitory activity in the deep vein thrombi (50%; 65 mg/kg) than in arterial thrombi formation (50%; 200 mg/kg), without cumulative effect after consecutive five-day administration. PCC-AP extract was safe in the induced bleeding time test. Its anti-aggregating profile was similar in ex vivo and in vitro conditions but was more effective in the extrinsic pathway when compared to in vitro results. Apiin and coumaric acid derivatives are the main compounds in PCC-AP according to the HPLC-DAD-ESI-MS/MS profile. We demonstrated for the first time that extracts from different parts of curly parsley have significant antiplatelet, anticoagulant, and antithrombotic activity without inducing hemorrhage, proving its potential as a source of antithrombotic compounds.

Potential Biotechnological Applications of Venoms from the Viperidae Family in Central America for Thrombosis.

Central America is home to one of the most abundant herpetofauna in the Americas, occupying only 7% of the continent's total area. Vipers and lizards are among the most relevant venomous animals in medical practice due to the consequences of envenomation from the bite of these animals. A great diversity of biomolecules with immense therapeutic and biotechnological value is contained in their venom. This paper describes the prominent leading representatives of the family Viperidae, emphasizing their morphology, distribution, habitat, feeding, and venom composition, as well as the biotechnological application of some isolated components from the venom of the animals from these families, focusing on molecules with potential anti-thrombotic action. We present the leading protein families that interfere with blood clotting, platelet activity, or the endothelium pro-thrombotic profile. In conclusion, Central America is an endemic region of venomous animals that can provide many molecules for biotechnological applications.

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.

Pyruvate decarboxylase activity is regulated by the Ser/Thr protein phosphatase Sit4p in the yeast Saccharomyces cerevisiae.

Deletion of SIT4 phosphatase decreased the pyruvate decarboxylase activity, which is essential for directing the glucose flux to ethanol production. Concomitantly, a reduction in the fermentative capacity was observed. As pyruvate decarboxylase expression was not altered, its post-translational phosphorylation was studied. Immunoblot analyses using anti-phosphoserine antibodies against the affinity-purified Pdc1p showed that Pdc1p is a phosphoenzyme. Dephosphorylation of Pdc1p by alkaline phosphatase inhibited activity by 50%. Moreover, phosphorylation of Pdc1p was dependent on the growth phase, being hyperphosphorylated in the logarithmic phase, which showed to be dependent on the presence of SIT4. A comparison of the kinetic parameters of pyruvate decarboxylase in total protein extracts from WT yeast and the Δsit4 mutant revealed that the apparent K(m) values of the cofactor thiamin pyrophosphate (TPP) were 81 and 205 µM, respectively, with V(max) values of 0.294 and 0.173 µmol mg⁻¹ min⁻¹, respectively. Treatment of the purified enzyme with alkaline phosphatase increased the K(m) for TPP from 20 to 84 µM and for pyruvate from 2.3 to 4.6 mM, while the V(max) changed from 0.806 to 0.673 µmol mg⁻¹ min⁻¹. These results suggest that the Pdc1p phosphorylation dependent on SIT4 occurs at residues that change the apparent affinity for TPP and pyruvate.

Keratinases and sulfide from Bacillus subtilis SLC to recycle feather waste.

The aim of this study is to investigate the culture conditions of chicken feather degradation and keratinolytic enzyme production by the recently isolated Bacillus subtilis SLC and to evaluate the potential of the SLC strain to recycle feather waste discarded by the poultry industry. The SLC strain was isolated from the agroindustrial waste of a poultry farm in Brazil and was confirmed to belong to Bacillus subtilis by rDNA gene analysis. There was high keratinase production when the medium was at pH 8 (280 U ml(-1)). Activity was higher using the inoculum propagated for 72 h on 1% whole feathers supplemented with 0.1% yeast extract. In the enzymatic extract, the keratinases were active in the pH range from 2.0 to 12.0 with a maximum activity at pH 10.0 and temperature 60°C. For gelatinase the best pH was 5.0 and the best temperature was 37°C. All keratinases are serine peptidases. The crude enzymatic extract degraded keratin, gelatin, casein, and hemoglobin. Scanning electron microscopy showed Bacillus cells adhered onto feather surfaces after 98 h of culture and degraded feather filaments were observed. MALDI-TOF mass spectrometric analysis showed multiple peaks from 522 to 892 m/z indicating feather degradation. The presence of sulfide was detected on extracellular medium probably participating in the breakdown of sulfide bridges of the feather keratin. External addition of sulfide increased feather degradation.

15N, 13C, and 1H resonance assignments of Jarastatin: a disintegrin of Bothrops jararaca.

Disintegrins are a group of cysteine-rich proteins found in a wide variety of snake venoms. These proteins selectively bind to integrins, which play a fundamental role in the regulation of many physiological and pathological processes. Here, we report the NMR chemical shift assignments for 1H, 15N, and 13C nuclei in the backbone and side chains of recombinant disintegrin Jarastatin (rJast), which was further validated by secondary structure prediction using the TALOS-N server. Taken together, these data are essential to perform NMR-based experiments, including structure determination, backbone dynamics, mapping ligand sites and enabling a deeper understanding of the effect of hydrophobic surface clusters, which are important elements to stabilize some 3D proteins structure/folding.

The recombinant disintegrin, jarastatin, inhibits platelet adhesion and endothelial cell migration.

Integrins are transmembrane heterodimeric glycoproteins, present in most cell types that act as mechanoreceptors, connecting extracellular matrix proteins to the cytoskeleton of the cell, mediating several physiological and pathological processes. The disintegrins are peptides capable of modulating the activity of integrins, such as αIIbß3, responsible for the platelet aggregation and αvß3, related to angiogenesis. The aim of this study was to produce the recombinant disintegrin jarastatin (rJast), to evaluate its secondary structure and biological activity. rJast was expressed in the yeast Komagataella phaffii (earlier Pichia pastoris) purified using molecular exclusion chromatography and the internal sequence and molecular mass were confirmed by mass spectrometry. The yield was approximately 40 mg/L of culture. rJast inhibited platelet aggregation induced by 2-4 µM ADP, 10 nM thrombin, and 1 µg/mL collagen (IC50 of 244.8 nM, 166.3 nM and 223.5 nM, respectively). It also blocked the adhesion of platelets to collagen under continuous flow in approximately 60% when used 1 µM. We also evaluated the effect of rJast on HMEC-1 cells. rJast significantly inhibited the adhesion of these cells to vitronectin, as well as cell migration (IC50 1.77 µM) without changing the viability. Conclusions: rJast was successfully expressed with activity in human platelets aggregation identical to the native molecule. Also, rJast inhibits adhesion and migration of endothelial cells. Thus, being relevant for the development of anti-thrombotic and anti-angiogenic drugs.

Monoclonal-Based Antivenomics Reveals Conserved Neutralizing Epitopes in Type I PLA2 Molecules from Coral Snakes.

For over a century, polyclonal antibodies have been used to treat snakebite envenoming and are still considered by the WHO as the only scientifically validated treatment for snakebites. Nevertheless, moderate innovations have been introduced to this immunotherapy. New strategies and approaches to understanding how antibodies recognize and neutralize snake toxins represent a challenge for next-generation antivenoms. The neurotoxic activity of Micrurus venom is mainly due to two distinct protein families, three-finger toxins (3FTx) and phospholipases A2 (PLA2). Structural conservation among protein family members may represent an opportunity to generate neutralizing monoclonal antibodies (mAbs) against family-conserved epitopes. In this work, we sought to produce a set of monoclonal antibodies against the most toxic components of M. altirostris venom. To this end, the crude venom was fractionated, and its major toxic proteins were identified and used to generate a panel of five mAbs. The specificity of these mAbs was characterized by ELISA and antivenomics approaches. Two of the generated mAbs recognized PLA2 epitopes. They inhibited PLA2 catalytic activity and showed paraspecific neutralization against the myotoxicity from the lethal effect of Micrurus and Naja venoms' PLA2s. Epitope conservation among venom PLA2 molecules suggests the possibility of generating pan-PLA2 neutralizing antibodies.

Extracellular Vesicles from Bothrops jararaca Venom Are Diverse in Structure and Protein Composition and Interact with Mammalian Cells.

Snake venoms are complex cocktails of non-toxic and toxic molecules that work synergistically for the envenoming outcome. Alongside the immediate consequences, chronic manifestations and long-term sequelae can occur. Recently, extracellular vesicles (EVs) were found in snake venom. EVs mediate cellular communication through long distances, delivering proteins and nucleic acids that modulate the recipient cell's function. However, the biological roles of snake venom EVs, including possible cross-organism communication, are still unknown. This knowledge may expand the understanding of envenoming mechanisms. In the present study, we isolated and characterized the EVs from Bothrops jararaca venom (Bj-EVs), giving insights into their biological roles. Fresh venom was submitted to differential centrifugation, resulting in two EV populations with typical morphology and size range. Several conserved EV markers and a subset of venom related EV markers, represented mainly by processing enzymes, were identified by proteomic analysis. The most abundant protein family observed in Bj-EVs was 5'-nucleotidase, known to be immunosuppressive and a low abundant and ubiquitous toxin in snake venoms. Additionally, we demonstrated that mammalian cells efficiently internalize Bj-EVs. The commercial antibothropic antivenom partially recognizes Bj-EVs and inhibits cellular EV uptake. Based on the proteomic results and the in vitro interaction assays using macrophages and muscle cells, we propose that Bj-EVs may be involved not only in venom production and processing but also in host immune modulation and long-term effects of envenoming.

BmooMPα-I, a Metalloproteinase Isolated from Bothrops moojeni Venom, Reduces Blood Pressure, Reverses Left Ventricular Remodeling and Improves Cardiac Electrical Conduction in Rats with Renovascular Hypertension.

BmooMPα-I has kininogenase activity, cleaving kininogen releasing bradykinin and can hydrolyze angiotensin I at post-proline and aspartic acid positions, generating an inactive peptide. We evaluated the antihypertensive activity of BmooMPα-I in a model of two-kidney, one-clip (2K1C). Wistar rats were divided into groups: Sham, who underwent sham surgery, and 2K1C, who suffered stenosis of the right renal artery. In the second week of hypertension, we started treatment (Vehicle, BmooMPα-I and Losartan) for two weeks. We performed an electrocardiogram and blood and heart collection in the fourth week of hypertension. The 2K1C BmooMPα-I showed a reduction in blood pressure (systolic pressure: 131 ± 2 mmHg; diastolic pressure: 84 ± 2 mmHg versus 174 ± 3 mmHg; 97 ± 4 mmHg, 2K1C Vehicle, p < 0.05), improvement in electrocardiographic parameters (Heart Rate: 297 ± 4 bpm; QRS: 42 ± 0.1 ms; QT: 92 ± 1 ms versus 332 ± 6 bpm; 48 ± 0.2 ms; 122 ± 1 ms, 2K1C Vehicle, p < 0.05), without changing the hematological profile (platelets: 758 ± 67; leukocytes: 3980 ± 326 versus 758 ± 75; 4400 ± 800, 2K1C Vehicle, p > 0.05), with reversal of hypertrophy (left ventricular area: 12.1 ± 0.3; left ventricle wall thickness: 2.5 ± 0.2; septum wall thickness: 2.3 ± 0.06 versus 10.5 ± 0.3; 2.7 ± 0.2; 2.5 ± 0.04, 2K1C Vehicle, p < 0.05) and fibrosis (3.9 ± 0.2 versus 7.4 ± 0.7, 2K1C Vehicle, p < 0.05). We concluded that BmooMPα-I improved blood pressure levels and cardiac remodeling, having a cardioprotective effect.

Widow spiders in the New World: a review on Latrodectus Walckenaer, 1805 (Theridiidae) and latrodectism in the Americas.

Humankind has always been fascinated by venomous animals, as their toxic substances have transformed them into symbols of power and mystery. Over the centuries, researchers have been trying to understand animal venoms, unveiling intricate mixtures of molecules and their biological effects. Among venomous animals, Latrodectus Walckenaer, 1805 (widow spiders) have become feared in many cultures worldwide due to their extremely neurotoxic venom. The Latrodectus genus encompasses 32 species broadly spread around the globe, 14 of which occur in the Americas. Despite the high number of species found in the New World, the knowledge on these spiders is still scarce. This review covers the general knowledge on Latrodectus spp. from the Americas. We address widow spiders' taxonomy; geographical distribution and epidemiology; symptoms and treatments of envenomation (latrodectism); venom collection, experimental studies, proteome and transcriptome; and biotechnological studies on these Latrodectus spp. Moreover, we discuss the main challenges and limitations faced by researchers when trying to comprehend this neglected group of medically important spiders. We expect this review to help overcome the lack of information regarding widow spiders in the New World.

Three-Finger Toxins from Brazilian Coral Snakes: From Molecular Framework to Insights in Biological Function.

Studies on 3FTxs around the world are showing the amazing diversity in these proteins both in structure and function. In Brazil, we have not realized the broad variety of their amino acid sequences and probable diversified structures and targets. In this context, this work aims to conduct an in silico systematic study on available 3FTxs found in Micrurus species from Brazil. We elaborated a specific guideline for this toxin family. First, we grouped them according to their structural homologue predicted by HHPred server and further curated manually. For each group, we selected one sequence and constructed a representative structural model. By looking at conserved features and comparing with the information available in the literature for this toxin family, we managed to point to potential biological functions. In parallel, the phylogenetic relationship was estimated for our database by maximum likelihood analyses and a phylogenetic tree was constructed including the homologous 3FTx previously characterized. Our results highlighted an astonishing diversity inside this family of toxins, showing some groups with expected functional similarities to known 3FTxs, and pointing out others with potential novel roles and perhaps structures. Moreover, this classification guideline may be useful to aid future studies on these abundant toxins.

Oral treatment with a chemically characterized parsley (Petroselinum crispum var. neapolitanum Danert) aqueous extract reduces thrombi formation in rats.

Petroselinum crispum var. neapolitanum Danert (Apiaceae) (PC), popularly known as parsley, is an herb native to the Mediterranean region widely cultivated around the world for culinary and ethnomedicinal purposes. The herb is traditionally used in various parts of the world to treat arterial hypertension, hemorrhoid, nose bleeding, hyperlipidemia, and pain, among other indications. The aim of this study was to evaluate the antithrombotic activity of an aqueous extract PC in rats. Aerial parts of a flat-leaf variety of parsley were extracted by decoction. In vivo thrombosis in rat models as well as ex vivo assays were used in the evaluation of PC antithrombotic effects. Intravenous administration of PC (25 mg/kg.b.w), 5 min before thrombosis induction, reduced the venous thrombus formation by 98.2%, while oral administration (125 mg/kg.b.w) impaired it by 76.2%. In the arterial thrombosis model, the oral administration of PC at 15 or 25 mg/kg.b.w, 60 min before thrombosis induction, increased the carotid artery occlusion time by 150% (37.0 ± 6.44 min) and 240% (more than 60 min), respectively. A HPLC-DAD-MS/MS profile of PC extract used in this study was provided. Apiin showed to be the most abundant phenolic compound in the extract. It also revealed the presence of many coumaric acid derivatives. Our results indicate that PC is a potential candidate for the development of a phytotherapeutic drug in the treatment of thromboembolic diseases and provide a detailed chemical profile useful for controlling PC extract production in view of phytotherapy.

Cellular Growth Arrest and Efflux Pumps Are Associated With Antibiotic Persisters in Streptococcus pyogenes Induced in Biofilm-Like Environments.

Streptococcus pyogenes (group A Streptococcus-GAS) is an important pathogen for humans. GAS has been associated with severe and invasive diseases. Despite the fact that these bacteria remain universally susceptible to penicillin, therapeutic failures have been reported in some GAS infections. Many hypotheses have been proposed to explain these antibiotic-unresponsive infections; however, none of them have fully elucidated this phenomenon. In this study, we show that GAS strains have the ability to form antimicrobial persisters when inoculated on abiotic surfaces to form a film of bacterial agglomerates (biofilm-like environment). Our data suggest that efflux pumps were possibly involved in this phenomenon. In fact, gene expression assays by real-time qRT-PCR showed upregulation of some genes associated with efflux pumps in persisters arising in the presence of penicillin. Phenotypic reversion assay and whole-genome sequencing indicated that this event was due to non-inherited resistance mechanisms. The persister cells showed downregulation of genes associated with protein biosynthesis and cell growth, as demonstrated by gene expression assays. Moreover, the proteomic analysis revealed that susceptible cells express higher levels of ribosome proteins. It is remarkable that previous studies have reported the recovery of S. pyogenes viable cells from tissue biopsies of patients presented with GAS invasive infections and submitted to therapy with antibiotics. The persistence phenomenon described herein brings new insights into the origin of therapeutic failures in S. pyogenes infections. Multifactorial mechanisms involving protein synthesis inhibition, cell growth impairment and efflux pumps seem to play roles in the formation of antimicrobial persisters in S. pyogenes.

Investigation of the ability of the oviposition-stimulant lectin from Moringa oleifera seeds (WSMoL) to bind with membrane proteins present in the legs of Aedes aegypti.

The mosquito Aedes aegypti L. is a vector transmitting diseases such as dengue, chikungunya and Zika virus fever. The water-soluble lectin from Moringa oleifera Lam. seeds (WSMoL) is larvicidal, ovicidal and can stimulate oviposition in A. aegypti. This study aimed to investigate whether WSMoL could bind to membrane proteins from A. aegypti legs. Initially, proteins from the legs were extracted using sodium deoxycholate, digitonin, dodecyl sodium sulfate (SDS) or Triton X-100. The protein concentration was found to be higher in the extract obtained using Triton X-100, which was applied to a WSMoL-Sepharose column. The adsorbed proteins were evaluated using gel filtration chromatography and polyacrylamide gel electrophoresis (PAGE) in presence of SDS. The similarity in the sequences of adsorbed proteins with those available in databases was determined. The proteins adsorbed on the matrix were eluted forming a single peak. Gel filtration chromatography and SDS-PAGE revealed the presence of proteins with molecular masses of approximately 20 kDa and polypeptide bands of 17.0 and 23.7 kDa, respectively. MS/MS analysis indicated similarity between these proteins and ABC carriers, which are expressed in the legs of mosquitos. WSMoL could bind to membrane proteins in the legs of A. aegypti females and induce oviposition through these interactions.