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Lower serum levels of transforming growth factor- 1 and disease activity in Takayasu arteritis Takayasu arteritis (TAK) is a chronic inflammatory arteritis that affects large vessels ([1]). Serum TGF- 1 levels were significantly lower in Takayasu arteritis patients presenting active disease compared with healthy controls, while similar levels were observed between patients presenting active disease and those in remission (p = 0.587). [Extracted from the article]

Background: Metastatic melanoma stands out as the most lethal form of skin cancer because of its high propensity to spread and its remarkable resistance to treatment methods., Methods: In this review article, we address the incidence of melanoma worldwide and its staging phases. We thoroughly investigate the different melanomas and their associated risk factors. In addition, we underscore the principal therapeutic goals and pharmacological methods that are currently used in the treatment of melanoma., Results: The implementation of targeted therapies has contributed to improving the approach to patients. However, because of the emergence of resistance early in treatment, overall survival and progression-free periods continue to be limited., Conclusions: We provide new insights into plant serine protease inhibitor therapeutics, supporting high-throughput drug screening soon, and seeking a complementary approach to explain crucial mechanisms associated with melanoma.

Background: SARS-CoV2 virus, responsible for the COVID-19 pandemic, has four structural proteins and 16 nonstructural proteins. S-protein is one of the structural proteins exposed on the virus surface and is the main target for producing neutralizing antibodies and vaccines. The S-protein forms a trimer that can bind the angiotensin-converting enzyme 2 (ACE2) through its receptor binding domain (RBD) for cell entry., Aims: The goal of this study was to express in HEK293 cells a new RBD recombinant protein in a constitutive and stable manner in order to use it as an alternative immunogen and diagnostic tool for COVID-19., Materials & Methods: The protein was designed to contain an immunoglobulin signal sequence, an explanded C-terminal section of the RBD, a region responsible for the bacteriophage T4 trimerization inducer, and six histidines in the pCDNA-3.1 plasmid. Following transformation, the cells were selected with geneticin-G418 and purified from serum-fre culture supernatants using Ni2+-agarand size exclusion chromatography. The protein was structurally identified by cross-linking and circular dichroism experiments, and utilized to immunize mice in conjuction with AS03 or alum adjuvants. The mice sera were examined for antibody recognition, receptor-binding inhibition, and virus neutralization, while spleens were evaluated for γ-interferon production in the presence of RBD., Results: The protein released in the culture supernatant of cells, and exhibited a molecular mass of 135 kDa with a secondary structure like the monomeric and trimeric RBD. After purification, it formed a multimeric structure comprising trimers and hexamers, which were able to bind the ACE2 receptor. It generated high antibody titers in mice when combined with AS03 adjuvant (up to 1:50,000). The sera were capable of inhibiting binding of biotin-labeled ACE2 to the virus S1 subunit and could neutralize the entry of the Wuhan virus strain into cells at dilutions up to 1:2000. It produced specific IFN-γ producing cells in immunized mouse splenocytes., Discussion: Our data describe a new RBD containing protein, forming trimers and hexamers, which are able to induce a protective humoral and cellular response against SARS-CoV2., Conclusion: These results add a new arsenal to combat COVID-19, as an alternative immunogen or antigen for diagnosis., (© 2024 The Author(s). Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Gold nanoparticles (AuNPs) hold promise in biomedicine, but challenges like aggregation, protein corona formation, and insufficient biocompatibility must be thoroughly addressed before advancing their clinical applications. Designing AuNPs with specific protein corona compositions is challenging, and strategies for corona elimination, such as coating with polyethylene glycol (PEG), have limitations. In this study, we introduce a commercially available zwitterionic derivative of glutathione, glutathione monoethyl ester (GSH zwt ), for the surface coating of colloidal AuNPs. Particles coated with GSH zwt were investigated alongside four other AuNPs coated with various ligands, including citrate ions, tiopronin, glutathione, cysteine, and PEG. We then undertook a head-to-head comparison of these AuNPs to assess their behavior in biological fluid. GSH zwt -coated AuNPs exhibited exceptional resistance to aggregation and protein adsorption. The particles could also be readily functionalized with biotin and interact with streptavidin receptors in human plasma. Additionally, they exhibited significant blood compatibility and noncytotoxicity. In conclusion, GSH zwt provides a practical and easy method for the surface passivation of AuNPs, creating "stealth" particles for potential clinical applications.

The peptide derived from E. contortisiliquum trypsin inhibitor (Pep-3-EcTI), peptide derived from kallikrein inhibitor isolated from B. bauhinioides (Pep-BbKI), and B. rufa peptide modified from B. bauhinioides (Pep-BrTI) peptides exhibit anti-inflammatory and antioxidant activities, suggesting their potential for treating asthma-chronic obstructive pulmonary disease (COPD) overlap (ACO). We compared the effects of these peptides with dexamethasone (DX) treatment in an ACO model. In this study, 11 groups of male BALB/c mice were pre-treated under different conditions, including sensitization with intraperitoneal injection and inhalation of ovalbumin (OVA), intratracheal instillation of porcine pancreatic elastase (ELA), sensitization with intraperitoneal injection, and various combinations of peptide treatments with Pep-3-EcTI, Pep-BbKI, Pep-BrTI, dexamethasone, and non-treated controls (SAL-saline). Respiratory system resistance, airway resistance, lung tissue resistance, exhaled nitric oxide, linear mean intercept, immune cell counts in the bronchoalveolar lavage fluid, cytokine expression, extracellular matrix remodeling, and oxidative stress in the airways and alveolar septa were evaluated on day 28. Results showed increased respiratory parameters, inflammatory markers, and tissue remodeling in the ACO group compared to controls. Treatment with the peptides or DX attenuated or reversed these responses, with the peptides showing effectiveness in controlling hyperresponsiveness, inflammation, remodeling, and oxidative stress markers. These peptides demonstrated an efficacy comparable to that of corticosteroids in the ACO model. However, this study highlights the need for further research to assess their safety, mechanisms of action, and potential translation to clinical studies before considering these peptides for human use., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 João, Silva Barbosa, Sales da Silva, Fukuzaki, de Campos, Camargo, Santos, Moreira Bezerra, de Almeida, Saraiva-Romanholo, Lopes, Bonturi, Righetti, Oliva, Tibério and Leick.)

Background: The large quantities of by-products generated in the coffee industry are a problem. Studies related to the biological potential of organic coffee husks are still limited. The aim of this work was to investigate the occurrence of phenolic compounds in organic coffee husks and to evaluate their potential as a source of bioactive dietary components., Results: To achieve this objective, three extracts were prepared, namely extractable polyphenols (EPs), hydrolyzable non-extractable polyphenols (H-NEPs), and non-extractable polyphenols (NEPs). These extracts were characterized and evaluated for their bioactive properties after simulated gastrointestinal digestion. The results show that the extraction process affected the occurrence of phenols from coffee peels, especially for caffeic acid, gallic acid, and chlorogenic acid. The free and bound polyphenols found in the extracts and digests not only showed antioxidant properties against 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), ferric reducing antioxidant power (FRAP), and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals but were also strongly bioavailable and had good anticoagulant potential., Conclusion: These results highlight the potential health benefits of phytochemicals from coffee husks and open new perspectives for the use of such compounds in dietary supplements. © 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry., (© 2023 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.)

Introduction: Trypsin inhibitors (TIs) have the ability to competitively or non-competitively bind to trypsin and inhibit its action. These inhibitors are commonly found in plants and are used in protease inhibition studies involved in biochemical pathways of pharmacological interest., Objectives: This work aimed to purify a trypsin inhibitor from Bauhinia pulchella seeds ( Bpu TI), describing its kinetic mechanism and anticoagulant effect., Methods: Affinity chromatography, protein assay, and SDS-PAGE were used to purify the inhibitor. Mass spectrometry, inhibition assays, and enzyme kinetics were used to characterize the inhibitor. In vitro assays were performed to verify its ability to prolong blood clotting time., Results: Affinity chromatography on a Trypsin-Sepharose 4B column gave a yield of 43.1. Bpu TI has an apparent molecular mass of 20 kDa with glycosylation (1.15%). Protein identification was determined by MS/MS, and Bpu TI showed similarity to several Kunitz-type trypsin inhibitors. Bpu TI inhibited bovine trypsin as an uncompetitive inhibitor with IC50 (3 x 10 -6 M) and Ki (1.05 x 10 -6 M). Additionally, Bpu TI showed high stability to temperature and pH variations, maintaining its activity up to 100ºC and in extreme pH ranges. However, the inhibitor was susceptible to reducing agents, such as DTT, which completely abolished its activity. Bpu TI showed an anticoagulant effect in vitro at a concentration of 33 μM, prolonging clotting time by 2.6 times., Conclusion: Our results suggest that Bpu TI can be a biological tool to be used in blood clotting studies., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Serine proteases play crucial biological roles and have their activity controlled by inhibitors, such as the EcTI, a serine protease inhibitor purified from Enterolobium contortisiliquum seeds, which has anticancer activity. This study aimed to conjugate EcTI with quantum dots (QDs), fluorophores with outstanding optical properties, and investigate the interaction of QDs-EcTI nanoprobe with cancer cells. The conjugation was evaluated by fluorescence correlation spectroscopy (FCS) and fluorescence microplate assay (FMA). EcTI inhibitory activity after interaction with QDs was also analyzed. From FCS, the conjugate presented a hydrodynamic diameter about 4× greater than bare QDs, suggesting a successful conjugation. This was supported by FMA, which showed a relative fluorescence intensity of ca. 3815% for the nanosystem, concerning bare QDs or EcTI alone. The EcTI inhibitory activity remained intact after its interaction with QDs. From flow cytometry analyses, approximately 62% of MDA-MB-231 and 90% of HeLa cells were labeled with the QD-EcTI conjugate, suggesting that their membranes have different protease levels to which EcTI exhibits an affinity. Concluding, the QD-EcTI represents a valuable nanotool to study the interaction of this inhibitor with cancer cells using fluorescence-based techniques with the potential to unravel the intricate dynamics of interplays between proteases and inhibitors in cancer biology., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The synthesized peptide derived from Enterolobium contortisiliquum (pep3-EcTI) has been associated with potent anti-inflammatory and antioxidant effects, and it may be a potential new treatment for asthma-COPD overlap-ACO). Purpose: To investigate the primary sequence effects of pep3-EcTI in an experimental ACO. BALB/c mice were divided into eight groups: SAL (saline), OVA (ovalbumin), ELA (elastase), ACO (ovalbumin + elastase), ACO-pep3-EcTI (treated with inhibitor), ACO-DX (treated with dexamethasone), ACO-DX-pep3-EcTI (treated with dexamethasone and inhibitor), and SAL-pep3-EcTI (saline group treated with inhibitor). We evaluated the hyperresponsiveness to methacholine, exhaled nitric oxide, bronchoalveolar lavage fluid (BALF), mean linear intercept (Lm), inflammatory markers, tumor necrosis factor (TNF-α), interferon (IFN)), matrix metalloproteinases (MMPs), growth factor (TGF-β), collagen fibers, the oxidative stress marker inducible nitric oxide synthase (iNOS), transcription factors, and the signaling pathway NF-κB in the airways (AW) and alveolar septa (AS). Statistical analysis was conducted using one-way ANOVA and t -tests, significant when p < 0.05. ACO caused alterations in the airways and alveolar septa. Compared with SAL, ACO-pep3-EcTI reversed the changes in the percentage of resistance of the respiratory system (%Rrs), the elastance of the respiratory system (%Ers), tissue resistance (%Gtis), tissue elastance (%Htis), airway resistance (%Raw), Lm, exhaled nitric oxide (ENO), lymphocytes, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, TNF-α, INF-γ, MMP-12, transforming growth factor (TGF)-β, collagen fibers, and iNOS. ACO-DX reversed the changes in %Rrs, %Ers, %Gtis, %Htis, %Raw, total cells, eosinophils, neutrophils, lymphocytes, macrophages, IL-1β, IL-6, IL-10, IL-13, IL-17, TNF-α, INF-γ, MMP-12, TGF-β, collagen fibers, and iNOS. ACO-DX-pep3-EcTI reversed the changes, as was also observed for the pep3-EcTI and the ACO-DX-pep3-EcTI. Significance: The pep3-EcTI was revealed to be a promising strategy for the treatment of ACO, asthma, and COPD.

(1) There are several patients with asthma-COPD overlap (ACO). A peptide derived from the primary sequence of a kallikrein inhibitor isolated from Bauhinia bauhinioides (pep-BbKI) has potent anti-inflammatory and antioxidant effects. Purpose: To investigate the effects of pep-BbKI treatment in an ACO model and compare them with those of corticosteroids. (2) BALB/c mice were divided into groups: SAL (saline), OVA (ovalbumin), ELA (elastase), ACO (ovalbumin + elastase), ACO-pep-BbKI (treated with inhibitor), ACO-DX (dexamethasone treatment), ACO-DX-pep-BbKI (both treatments), and SAL-pep-BbKI (saline group treated with inhibitor). We evaluated: hyperresponsiveness to methacholine, bronchoalveolar lavage fluid (BALF), exhaled nitric oxide (eNO), IL-1β, IL-4, IL-5, IL-6, IL-10, IL-13, IL-17, IFN-γ, TNF-α, MMP-9, MMP-12, TGF-β, collagen fibers, iNOS, eNO, linear mean intercept (Lm), and NF-κB in airways (AW) and alveolar septa (AS). (3) ACO-pep-BbKI reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, neutrophils, IL-5, IL-10, IL-17, IFN-γ, TNF-α, MMP-12 (AW), collagen fibers, iNOS (AW), and eNO ( p > 0.05). ACO-DX reversed ACO alterations and was similar to SAL in all mechanical parameters, Lm, total cells and differentials, IL-1β(AS), IL-5 (AS), IL-6 (AS), IL-10 (AS), IL-13 (AS), IFN-γ, MMP-12 (AS), TGF-β (AS), collagen fibers (AW), iNOS, and eNO ( p > 0.05). SAL was similar to SAL-pep-BbKI for all comparisons ( p > 0.05). (4) Pep-BbKI was similar to dexamethasone in reducing the majority of alterations of this ACO model.

Melanoma is difficult to treat with chemotherapy, prompting the need for new treatments. Protease inhibitors have emerged as promising candidates as tumor cell proteases promote metastasis. Researchers have developed a chimeric form of the Bauhinia bauhinioides kallikrein inhibitor, rBbKIm, which has shown negative effects on prostate tumor cell lines DU145 and PC3. Crataeva tapia bark lectin, CrataBL, targets sulfated oligosaccharides in glycosylated proteins and has also demonstrated deleterious effects on prostate and glioblastoma tumor cells. However, neither rBbKIm nor its derived peptides affected the viability of SK-MEL-28, a melanoma cell line, while CrataBL decreased viability by over 60%. Two peptides, Pep. 26 (Ac-Q-N-S-S-L-K-V-V-P-L-NH2) and Pep. 27 (Ac-L-P-V-V-K-L-S-S-N-Q-NH2), were also tested. Pep. 27 suppressed cell migration and induced apoptosis when combined with vemurafenib, while Pep. 26 inhibited cell migration and reduced nitric oxide and the number of viable cells. Vemurafenib, a chemotherapy drug used to treat melanoma, was found to decrease the release of interleukin 8 and PDGF-AB/BB cytokines and potentiated the effects of proteins and peptides in reducing these cytokines. These findings suggest that protease inhibitors may be effective in blocking melanoma cells and highlight the potential of CrataBL and its derived peptides.

Snake venom serine protease (SVSP) interferes with the regulation and control of important biological reactions in homeostasis and can be classified as an activator of the fibrinolytic system and platelet aggregation. Our group has recently isolated a new serine protease from Crotalus durissus terrificus total venom (Cdtsp-2). This protein exhibits edematogenic capacity and myotoxic activity. A Kunitz-like EcTI inhibitor protein with a molecular mass of 20 kDa was isolated from Enterolobium contortisiliquum and showed high trypsin inhibition. Thus, the objective of this work is to verify the possible inhibition of the pharmacological activities of Cdtsp-2 by the Kutinz-type inhibitor EcTI. To isolate Cdtsp-2 from total C. d. terrificus venom, we used three-step chromatographic HPLC. Using the mice paw edema model, we observed an edematogenic effect, myotoxicity and hepatotoxicity caused by Cdtsp-2. In vitro and in vivo experiments showed that the alterations in hemostasis caused by Cdtsp-2 are crucial for the development of marked hepatotoxicity and that EcTI significantly inhibits the enzymatic and pharmacological activities of Cdtsp-2. Kunitz-like inhibitor may be a viable alternative for the development of ancillary treatments against the biological activities of venoms.

Several plant protein inhibitors with anticoagulant properties have been studied and characterized, including the Delonix regia trypsin inhibitor (DrTI). This protein inhibits serine proteases (trypsin) and enzymes directly involved in coagulation, such as plasma kallikrein, factor XIIa, and factor XIa. In this study, we evaluated the effects of two new synthetic peptides derived from the primary sequence of DrTI in coagulation and thrombosis models to understand the mechanisms involved in the pathophysiology of thrombus formation as well as in the development of new antithrombotic therapies. Both peptides acted on in vitro hemostasis-related parameters, showing promising results, prolonging the Partially Activated Thromboplastin Time (aPTT) and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and arachidonic acid. In murine models, for arterial thrombosis induced by photochemical injury, and platelet-endothelial interactions monitored by intravital microscopy, both peptides at doses of 0.5 mg/kg significantly extended the time of artery occlusion and modified the platelet adhesion and aggregation pattern with no changes in bleeding time, demonstrating the high biotechnological potential of both molecules.

The pathogenesis of Takayasu arteritis (TAK) is poorly understood and no previous studies have analyzed monocytes in TAK. This study evaluated monocyte subsets and monocyte-related chemokines in the peripheral blood of TAK patients and healthy controls (HC). Monocyte subsets were identified as classical (CD14 + CD16 - ), intermediate (CD14 + CD16 dim ), and non-classical (CD14 dim CD16 high ) in the peripheral blood. The chemokines CCL (C-C chemokine ligand)2, CCL3, CCL4, CCL5, CCL7, CXCL (C-X-C motif ligand)10, and CX3CL (C-X3-C motif ligand)1 were measured in the sera. Thirty-two TAK patients and 30 HC were evaluated. Intermediate monocytes were higher in TAK than HC [25.0 cells ×10 6 /L (16.7-52.0) vs. 17.2 cells ×10 6 /L (9.2-25.3); p = 0.014]. Active disease was associated with monocytosis (p = 0.004), increased classical (p = 0.003), and intermediate (p < 0.001) subsets than HC. Prednisone reduced the percentage of non-classical monocytes (p = 0.011). TAK patients had lower CCL3 (p = 0.033) and CCL4 (p = 0.023) levels than HC, whereas CCL22 levels were higher in active TAK compared to the remission state (p = 0.008). Glucocorticoids were associated with lower CXCL10 levels (p = 0.012). In TAK, CCL4 correlated with total (Rho = 0.489; p = 0.005), classical and intermediate monocytes (Rho = 0.448; p = 0.010 and Rho = 0.412; p = 0.019). In conclusion, TAK is associated with altered counts of monocyte subsets in the peripheral blood compared to HC and CCL22 is the chemokine with the strongest association with active disease in TAK., (© 2023. The Author(s).)

Background: (p-BthTX-I) 2  K, a dimeric analog peptide derived from the C-terminal region of phospholipase A2-like bothropstoxin-I (p-BthTX-I), is resistant to plasma proteolysis and inhibits severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strains with weak cytotoxic effects. Complications of SARS-CoV-2 infection include vascular problems and increased risk of thrombosis; therefore, studies to identify new drugs for treating SARS-CoV-2 infections that also inhibit thrombosis and minimize the risk of bleeding are required., Objectives: To determine whether (p-BthTX-I) 2  K affects the hemostatic system., Methods: Platelet aggregation was induced by collagen, arachidonic acid, and adenosine diphosphate (ADP) in the Chronolog Lumi-aggregometer. The coagulation activity was evaluated by determining activated partial thromboplastin clotting time (aPTT) and prothrombin time (PT) with (p-BthTX-I) 2  K (5.0-434.5 µg) or 0.9% NaCl. Arterial thrombosis was induced with a 540 nm laser and 3.5-20 mg kg - 1 Rose Bengal in the carotid artery of male C57BL/6J mice using (p-BthTX-I) 2  K. Bleeding time was determined in mouse tails immersed in saline at 37 °C after (p-BthTX-I) 2  K (4.0 mg/kg and 8.0 mg/kg) or saline administration., Results: (p-BthTX-I) 2  K prolonged the aPTT and PT by blocking kallikrein and FXa-like activities. Moreover, (p-BthTX-I) 2  K inhibited ADP-, collagen-, and arachidonic acid-induced platelet aggregation in a dose-dependent manner. Further, low concentrations of (p-BthTX-I) 2  K extended the time to artery occlusion by the formed thrombus. However, (p-BthTX-I) 2  K did not prolong the bleeding time in the mouse model of arterial thrombosis., Conclusion: These results demonstrate the antithrombotic activity of the peptide (p-BthTX-I) 2  K possibly by kallikrein inhibition, suggesting its strong biotechnological potential., (© 2023. The Author(s).)

Trypanosoma cruzi is the causative protozoan of Chagas' Disease, a neglected tropical disease that affects 6-7 million people worldwide. Interaction of the parasite with the host immune system is a key factor in disease progression and chronic symptoms. Although the human immune system is capable of controlling the disease, the parasite has numerous evasion mechanisms that aim to maintain intracellular persistence and survival. Due to the pronounced genetic variability of T. cruzi , co-infections or mixed infections with more than one parasite strain have been reported in the literature. The intermodulation in such cases is unclear. This study aimed to evaluate the co-infection of T. cruzi strains G and CL compared to their individual infections in human macrophages derived from THP-1 cells activated by classical or alternative pathways. Flow cytometry analysis demonstrated that trypomastigotes were more infective than extracellular amastigotes (EAs) and that strain G could infect more macrophages than strain CL. Classically activated macrophages showed lower number of infected cells and IL-4-stimulated cells displayed increased CL-infected macrophages. However, co-infection was a rare event. CL EAs decreased the production of reactive oxygen species (ROS), whereas G trypomastigotes displayed increased ROS detection in classically activated cells. Co-infection did not affect ROS production. Monoinfection by strain G or CL mainly induced an anti-inflammatory cytokine profile by decreasing inflammatory cytokines (IFN-γ, TNF-α, IL-1β) and/or increasing IL-4, IL-10, and TGF-β. Co-infection led to a predominant inflammatory milieu, with reduced IL-10 and TGF-β, and/or promotion of IFN-γ and IL-1β release. Infection by strain G reduced activation of intracellular signal transducer and activator of transcription (STAT) factors. In EAs, monoinfections impaired STAT-1 activity and promoted phosphorylation of STAT-3, both changes may prolong cell survival. Coinfected macrophages displayed pronounced activation of all STATs examined. These activations likely promoted parasite persistence and survival of infected cells. The collective results demonstrate that although macrophages respond to both strains, T. cruzi can modulate the intracellular environment, inducing different responses depending on the strain, parasite infective form, and co-infection or monoinfection. The modulation influences parasite persistence and survival of infected cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Oliveira, Bonturi, Salu, Oliva, Mortara and Orikaza.)

Ultrasmall gold nanoparticles (usNPs) and nanoclusters are an emerging class of nanomaterials exhibiting distinctive physicochemical properties and in vivo behaviors. Although understanding the interactions of usNPs with blood components is of fundamental importance to advance their clinical translation, currently, little is known about the way that usNPs interact with the hemostatic system. This study describes the effects of a model anionic p -mercaptobenzoic acid-coated usNP on the coagulation cascade, with particular emphasis on the contact pathway. It is found that in a purified system, the anionic usNPs bind to and activate factor XII (FXII). The formed usNP-FXII complexes are short-lived (residence time of ∼10 s) and characterized by an affinity constant of ∼200 nM. In human plasma, the anionic usNPs activate the contact pathway and promote coagulation. The usNPs also exhibit anticoagulant activity in plasma by interfering with the thrombin-mediated cleavage of fibrinogen. Taken together, these findings establish that anionic usNPs can disturb the normal hemostatic balance, which in turn may hinder their clinical translation. Finally, it is shown that usNPs can be designed to be nearly inert in plasma by surface coating with the natural peptide glutathione.

Cell adhesion and migration are crucial for cancer progression and malignancy. Drugs available for the treatment of metastatic melanoma are expensive and unfit for certain patients. Therefore, there is still a need to identify new drugs that block tumor cell development. We investigated the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI), a protease inhibitor, on cell viability, cell migration, invasion, cell adhesion, and cell death (hallmarks of cancer) in vitro using human melanoma cells (SK-MEL-28 and CHL-1). Although EcTI did not affect non-tumor cells, it significantly inhibited the proliferation, migration, invasion, and adhesion of melanoma cells. Investigation of the underlying mechanisms revealed that EcTI triggered apoptosis and nuclear shrinkage, increased PI uptake, activated effector caspases-3/7, and produced reactive oxygen species (ROS). Furthermore, EcTI disrupted the mitochondrial membrane potential, altered calcium homeostasis, and modified proteins associated with survival and apoptosis/autophagy regulation. Acridine orange staining indicated acidic vesicular organelle formation upon EcTI treatment, demonstrating a cell death display. Electronic microscopy corroborated the apoptotic pattern by allowing the visualization of apoptotic bodies, mitochondrial cristae disorganization, and autophagic vesicles. Taken together, these results provide new insights into the anti-cancer properties of the natural EcTI protein, establishing it as a promising new therapeutic drug for use in melanoma treatment.

The action of proteases can be controlled by several mechanisms, including regulation through gene expression; post-translational modifications, such as glycosylation; zymogen activation; targeting specific compartments, such as lysosomes and mitochondria; and blocking proteolysis using endogenous inhibitors. Protease inhibitors are important molecules to be explored for the control of proteolytic processes in organisms because of their ability to act on several proteases. In this context, plants synthesize numerous proteins that contribute to protection against attacks by microorganisms (fungi and bacteria) and/or invertebrates (insects and nematodes) through the inhibition of proteases in these organisms. These proteins are widely distributed in the plant kingdom, and are present in higher concentrations in legume seeds (compared to other organs and other botanical families), motivating studies on their inhibitory effects in various organisms, including humans. In most cases, the biological roles of these proteins have been assigned based mostly on their in vitro action, as is the case with enzyme inhibitors. This review highlights the structural evolution, function, and wide variety of effects of plant Kunitz protease inhibitors, and their potential for pharmaceutical application based on their interactions with different proteases.

Chagas' disease is a parasitosis caused by Trypanosoma cruzi , which affects approximately 8 million people worldwide. The balance between pro- and anti-inflammatory cytokines produced during immunological responses contributes to disease prognosis and progression. Parasite tissue persistence can induce chronic inflammatory stimuli, which can cause long-term tissue injury and fibrosis. Chronic Chagas' patients exhibit increased levels of interleukin (IL)-9, an important cytokine in the regulation of inflammatory and fibrogenic processes. Data on the role of IL-9 in other pathologies are sometimes contradictory, and few studies have explored this cytokine's influence in Chagas' disease pathology. Hence, the aim of this study was to evaluate the role of IL-9 in the progression of T. cruzi infection in vivo and in vitro . In vitro infection demonstrated that IL-9 reduced the number of infected cells and decreased the multiplication of intracellular amastigotes in both C2C12 myoblasts and bone marrow-derived macrophages. In myoblasts, the increased production of nitric oxide (NO) was essential for reduced parasite multiplication, whereas macrophage responses resulted in increased IL-6 and reduced TGF-β levels, indicating that parasite growth restriction mechanisms induced by IL-9 were cell-type specific. Experimental infection of BALB/c mice with T. cruzi trypomastigotes of the Y strain implicated a major role of IL-9 during the chronic phase, as increased Th9 and Tc9 cells were detected among splenocytes; higher levels of IL-9 in these cell populations and increased cardiac IL-9 levels were detected compared to those of uninfected mice. Moreover, rIL9 treatment decreased serum IL-12, IL-6, and IL-10 levels and cardiac TNF-α levels, possibly attempting to control the inflammatory response. IL-9 neutralization increased cardiac fibrosis, synthesis of collagens I and III, and mastocyte recruitment in BALB/c heart tissue during the chronic phase. In conclusion, our data showed that IL-9 reduced the invasion and multiplication of T. cruzi in vitro , in both myoblasts and macrophages, favoring disease control through cell-specific mechanisms. In vivo , IL-9 was elevated during experimental chronic infection in BALB/c mice, and this cytokine played a protective role in the immunopathological response during this phase by controlling cardiac fibrosis and proinflammatory cytokine production., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Silva, Orikaza, Santana, dos Santos, Salu, Oliva, Sinigaglia and Mortara.)

The anti-inflammatory effects of the plant protease inhibitor BbCI (Bauhinia bauhinioides cruzipain inhibitor), which blocks elastase, cathepsin G, and L, and proteinase 3 has been demonstrated. Here, we investigated the recombinant rBbCI-His (6) (containing a histidine tail) in an experimental venous thrombosis model of vena cava (VC) ligature in rats, comparing to heparin. We evaluate the effects of the inhibitors (native or recombinant) or heparin on the activated partial thromboplastin time (aPTT) and prothrombin time (PT) in human and rat plasmas. The rats undergoing treatment received a saline solution or increasing concentrations of rBbCI-His (6) , heparin, or a mixture of both. After 4 h of ligature VC, thrombus, if present was removed and weighed. aPTT, PT, and cytokines were measured in blood collected by cardiac puncture. aPTT, PT, and bleeding time (BT) were also measured at the time of VC (vena cava) ligature. rBbCI-His (6) (0.45 or 1.40 mg/kg) does not alter aPTT, PT or BT. No differences in coagulation parameters were detected in rBbCI-His (6) treated rats at the time of VC ligature or when the thrombus was removed. There was a significant decrease in the weight of thrombus in the animals of the groups treated with the rBbCI-His (6) (1.40 mg/kg), with the rBbCI-His (6) mixture (1.40 mg/kg) + heparin (50 IU/kg) and heparin (100 IU/kg) in relation to control group (saline). The growth-related oncogene/keratinocyte chemoattractant (GRO/KC) serum levels in rats treated with rBbCI-His (6) (1.40 mg/kg) or heparin (200 IU/kg) were reduced. In the experimental model used, rBbCI-His (6) alone had an antithrombotic effect, not altering blood clotting or bleeding time.

Proteases play a pivotal role in many signaling pathways; inhibitors of well-established proteases have shown a substantial therapeutic success. This study aimed to examine the in vivo effects of 3 protease inhibitors isolated from Bauhinia species: i) Bauhinia mollis elastase inhibitor, which blocks human neutrophil elastase (Ki app  2.8 nM) and cathepsin G (Ki app  1.0 nM) activities; ii) Bauhinia mollis trypsin inhibitor, a trypsin inhibitor (Ki app  5.0 nM); and iii) Bauhinia bauhinioides cruzipain inhibitor, which inhibits elastase (Ki app  2.6 nM), cathepsin G (Ki app  160.0 nM), and the cysteine proteases cathepsin L (Ki app  0.2 nM). Bauhinia bauhinioides cruzipain inhibitor, Bauhinia mollis elastase inhibitor, and Bauhinia mollis trypsin inhibitor were isolated using acetone and ammonium sulfate fractionations, DEAE-Sephadex, trypsin-Sepharose, and Resource-Q chromatographies. Mice and rats were treated intraperitoneally with 1 dose of inhibitor; gastric mucosal lesions were induced by cold-restraint stress. Oral pretreatment of mice with Bauhinia mollis elastase inhibitor or Bauhinia mollis trypsin inhibitor (1 - 10 mg/kg) did not show anti-ulcer effect, while Bauhinia bauhinioides cruzipain inhibitor (0.1 - 1.0 mg/kg) produced a similar reduction of the index of mucosal damage at all effective doses (30 to 33% < control). In rats at doses lower than those used in mice, Bauhinia mollis elastase inhibitor and Bauhinia bauhinioides cruzipain inhibitor reduced the index of mucosal damage by 66% and 54% of controls, respectively. The results indicate a protective effect against gastric mucosal lesions associated with elastase inhibition but not inhibition of trypsin activities. Moreover, the lack of Bauhinia mollis elastase inhibitor efficacy observed in mice may possibly be related to the reported structural differences of elastase in mice and rats., Competing Interests: The authors declare that they have no conflict of interest., (Thieme. All rights reserved.)

This study focused on the characterization of a novel cysteine proteinase inhibitor from Enterolobium contortisiliquum seeds targeting the inhibition of the growth of Callosobruchus maculatus larvae, an important cosmopolitan pest of the cowpea Vigna unguiculata during storage. The inhibitor was isolated by ion-exchange besides of size exclusion chromatography. EcCI molecular mass is 19,757 Da, composed of two polypeptide chains. It strongly inhibits papain (Ki app 0.036 nM) and proteinases from the midguts of C. maculatus (80 μg mL -1 , 60% inhibition). The inhibitory activity is reduced by 40% after a heat treatment at 100 °C for 2 h. The protein displayed noxious activity at 0.5% and 1% (w/w) when incorporated in artificial seeds, reducing larval mass in 87% and 92%, respectively. Treatment of C. maculatus larvae with conjugated EcCI-FIT and subsequent biodistribution resulted in high fluorescence intensity in midguts and markedly low intensity in malpighian tubules and fat body. Small amounts of labeled proteins were detected in larvae feces. The detection of high fluorescence in larvae midguts and low fluorescence in their feces indicate the retention of the FITC conjugated EcCI inhibitor in larvae midguts. These results demonstrate the potential of the natural protein from E. contortisiliquum to inhibit the development of C. maculatus ., Competing Interests: The authors declare that they have no competing interests., (© 2020 Published by Elsevier B.V.)

A careful balance between active-site and exosite contributions is critically important for the specificity of many proteases, but this balance is not yet defined for some of the serine proteases that serve as coagulation factors. Basavaraj and Krishnaswamy have closed an important gap in our knowledge of coagulation factor X activation by the intrinsic Xase complex by showing that exosite binding plays a critical role in this process, which they describe as a "dock and lock." This finding not only significantly enhances our understanding of this step in the coagulation cascade and highlights parallels with the prothrombinase complex, but will also provide a novel rationale for inhibitor development in the future., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Oliva et al.)

Heparin was immobilized on magnetic chitosan particles to be used as a tool for human plasma protein identification. Chitosan was magnetized by co-precipitation with Fe 2+ /Fe 3+ (MAG-CH). Heparin was functionalized with carbodiimide and N-hydroxysuccinimide and covalently linked to MAG-CH (MAG-CH-hep). X-ray diffraction confirmed the presence of chitosan and Fe 3 O 4 in MAG-CH. This particle exhibited superparamagnetism and size between 100-300 μm. Human plasma diluted with 10 mM phosphate buffer (pH 5.5) or 50 mM Tris-HCl buffer (pH 8.5) was incubated with MAG-CH-hep, and the proteins fixed were eluted with the same buffers containing increasing concentrations of NaCl. The proteins obtained were investigated by SDS-PAGE, LC/MS, and biological activity tests (PT, aPTT, and enzymatic chromogenic assay). Inhibitors of the serpin family, prothrombin, and human albumin were identified in this study. Therefore, MAG-CH-hep can be used to purify these proteins and presents the following advantages: low-cost synthesis, magnetic separation, ion-exchange purification, and reusability., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Breast cancer is the most common malignant tumor among women worldwide, and triple-negative breast cancer is the most aggressive type of breast cancer, which does not respond to hormonal therapies. The protease inhibitor, EcTI, extracted from seeds of Enterolobium contortisiliquum, acts on the main signaling pathways of the MDA-MB-231 triple-negative breast cancer cells. This inhibitor, when bound to collagen I of the extracellular matrix, triggers a series of pathways capable of decreasing the viability, adhesion, migration, and invasion of these cells. This inhibitor can interfere in the cell cycle process through the main signaling pathways such as the adhesion, Integrin/FAK/SRC, Akt, ERK, and the cell death pathway BAX and BCL-2. It also acts by reducing the main inflammatory cytokines such as TGF-α, IL-6, IL-8, and MCP-1, besides NFκB, a transcription factor, responsible for the aggressive and metastatic characteristics of this type of tumor. Thus, the inhibitor was able to reduce the main processes of carcinogenesis of this type of cancer., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Nanomaterials displaying well-tailored sizes and surface chemistries can provide novel ways with which to modulate the structure and function of enzymes. Recently, we showed that gold nanoparticles (AuNPs) in the ultrasmall size regime could perform as allosteric effectors inducing partial inhibition of thrombin activity. We now find that the nature of the AuNP surface chemistry controls the interactions to the anion-binding exosites 1 and 2 on the surface of thrombin, the allosterically induced changes to the active-site conformation, and, by extension, the enzymatic activity. Ultrasmall AuNPs passivated with p -mercaptobenzoic acid ligands (AuMBA) and a peptide-based (Ac-ECYN) biomimetic coat (AuECYN) were utilized in our investigations. Remarkably, we found that while AuMBA binds to exosites 1 and 2, AuECYN interacts primarily with exosite 2. It was further established that AuMBA behaves as a "mild denaturant" of thrombin leading to catalytic dysfunction over time. Conversely, AuECYN resembles a proper allosteric effector leading to partial and reversible inhibition of the activity. Collectively, our findings reveal how the distinct binding modes of different AuNP types may uniquely influence thrombin structure and catalysis. The present study further contributes to our understanding of how synthetic nanomaterials could be exploited in the allosteric regulation of enzymes.

Peptides from protein hydrolysate of a mixture of chicken combs and wattles (CCWs) were obtained through enzymatic hydrolysis, and their anticoagulant and inhibitory effects on angiotensin I-converting enzyme (ACE) were investigated. The protein hydrolysate exhibited anticoagulant capacity by the intrinsic pathway (activated partial thromboplastin time) and potent ACE-inhibitory activity. The peptides were sequenced by LC-MS to identify those with higher inhibitory potential. From the pool of sequenced peptides, the following three peptides were selected and synthesized based on their low molecular weight and the presence of amino acids with ACE-inhibitory potential at the C-terminus: peptide I (APGLPGPR), peptide II (Piro-GPPGPT), and peptide III (FPGPPGP). Peptide III (FPGPPGP) showed the highest ACE-inhibitory capacity among the peptides selected. In conclusion, a peptide (FPGPPGP) of unknown sequence was identified as having potent ACE-inhibitory capacity. This peptide originated from unconventional hydrolysates from poultry slaughter waste, including combs and wattles.

Formation of new blood vessels from preexisting ones, a process known as angiogenesis, is one of the limiting steps for success in treatment of ischemic disorders. Therefore, efforts to understanding and characterize new agents capable to stimulate neovascularization are a worldwide need. Crataeva tapia bark lectin (CrataBL) has been shown to have chemoattractant properties for endothelial cells through the stimulation of migration and invasiveness of human umbilical vein endothelial cells (HUVEC) because it is a positively charged protein with high affinity to glycosaminoglycan. In addition, CrataBL increased the production of chondroitin and heparan sulfate in endothelial cells. These findings orchestrated specific adhesion on collagen I and phosphorylation of tyrosine kinase receptors, represented by vascular endothelial growth factor receptor-2 (VEGFR-2) and fibroblast growth factor receptor (FGFR), whose downstream pathways trigger the angiogenic cascade increasing cell viability, cytoskeleton rearrangement, cell motility, and tube formation. Moreover, CrataBL inhibited the activity of matrix metalloproteases type 2 (MMP-2), a protein related to tissue remodeling. Likewise, CrataBL improved wound healing and increased the number of follicular structures in lesioned areas produced in the dorsum-cervical region of C57BL/6 mice. These outcomes altogether indicate that CrataBL is a pro-angiogenic and healing agent., (Copyright © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Blood-derived autologous products are frequently used in both human and equine medicine to treat musculoskeletal disorders. These products, especially the platelet-rich plasma (PRP), may contain high concentrations of growth factors (GFs), and thus improve healing in several tissues. Nevertheless, the procedures for preparation of PRP are currently non-standardized. Several protocols, which are based on distinct centrifugation patterns (rotation speed and time), result in PRPs with different characteristics, concerning platelet and GFs concentrations, as well as platelet activation. The aim of the present study was to compare two different protocols for PRP preparation: protocol (A) that is based on a single-centrifugation step; protocol (B), which included two sequential centrifugation steps (double-centrifugation). The results here reported show that the double-centrifugation protocol resulted in higher platelet concentration, while leukocytes were not concentrated by this procedure. Although platelet activation and aggregation were increased in this protocol in comparison to the single-centrifugation one, the TGF-β1 concentration was also higher. Pearson's correlation coefficients gave a significant, positive correlation between the platelet counts and TGF-β1 concentration. In conclusion, although the double-centrifugation protocol caused premature platelet aggregation, it seems to be an effective method for preparation of PRP with high platelet and TGF-β1 concentrations.

Currently available drugs for treatment of glioblastoma, the most aggressive brain tumor, remain inefficient, thus a plethora of natural compounds have already been shown to have antimalignant effects. However, these have not been tested for their impact on tumor cells in their microenvironment-simulated cell models, e.g., mesenchymal stem cells in coculture with glioblastoma cell U87 (GB). Mesenchymal stem cells (MSC) chemotactically infiltrate the glioblastoma microenvironment. Our previous studies have shown that bone-marrow derived MSCs impair U87 growth and invasion via paracrine and cell-cell contact-mediated cross-talk. Here, we report on a plant-derived protein, obtained from Crataeva tapia tree Bark Lectin (CrataBL), having protease inhibitory/lectin activities, and demonstrate its effects on glioblastoma cells U87 alone and their cocultures with MSCs. CrataBL inhibited U87 cell invasion and adhesion. Using a simplified model of the stromal microenvironment, i.e., GB/MSC direct cocultures, we demonstrated that CrataBL, when added in increased concentrations, caused cell cycle arrest and decreased cocultured cells' viability and proliferation, but not invasion. The cocultured cells' phenotypes were affected by CrataBL via a variety of secreted immunomodulatory cytokines, i.e., G-CSF, GM-CSF, IL-6, IL-8, and VEGF. We hypothesize that CrataBL plays a role by boosting the modulatory effects of MSCs on these glioblastoma cell lines and thus the effects of this and other natural lectins and/or inhibitors would certainly be different in the tumor microenvironment compared to tumor cells alone. We have provided clear evidence that it makes much more sense testing these potential therapeutic adjuvants in cocultures, mimicking heterogeneous tumor-stroma interactions with cancer cells in vivo. As such, CrataBL is suggested as a new candidate to approach adjuvant treatment of this deadly tumor.

Coagulation factor XII (FXII) is a key initiator of the contact pathway, which contributes to inflammatory pathways. FXII circulates as a zymogen, which when auto-activated forms factor XIIa (FXIIa). Here, the production of the recombinant FXIIa protease domain (βFXIIa His ) with yields of ∼1-2 mg per litre of insect-cell culture is reported. A second construct utilized an N-terminal maltose-binding protein (MBP) fusion (MBP-βFXIIa His ). Crystal structures were determined of MBP-βFXIIa His in complex with the inhibitor D-Phe-Pro-Arg chloromethyl ketone (PPACK) and of βFXIIa His in isolation. The βFXIIa His structure revealed that the S2 and S1 pockets were occupied by Thr and Arg residues, respectively, from an adjacent molecule in the crystal. The Thr-Arg sequence mimics the P2-P1 FXIIa cleavage-site residues present in the natural substrates prekallikrein and FXII, and Pro-Arg (from PPACK) mimics the factor XI cleavage site. A comparison of the βFXIIa His structure with the available crystal structure of the zymogen-like FXII protease revealed large conformational changes centred around the S1 pocket and an alternate conformation for the 99-loop, Tyr99 and the S2 pocket. Further comparison with activated protease structures of factors IXa and Xa, which also have the Tyr99 residue, reveals that a more open form of the S2 pocket only occurs in the presence of a substrate mimetic. The FXIIa inhibitors EcTI and infestin-4 have Pro-Arg and Phe-Arg P2-P1 sequences, respectively, and the interactions that these inhibitors make with βFXIIa are also described. These structural studies of βFXIIa provide insight into substrate and inhibitor recognition and establish a scaffold for the structure-guided drug design of novel antithrombotic and anti-inflammatory agents.

Introduction: Proteinase inhibitors have been associated with anti-inflammatory and antioxidant activities and may represent a potential therapeutic treatment for asthma., Purpose: The aim of the present study was to evaluate the effects of Enterolobium contortisiliquum trypsin inhibitor (EcTI) on pulmonary mechanical function, eosinophilic recruitment, inflammatory cytokines, remodeling and oxidative stress in an experimental model of chronic allergic pulmonary inflammation., Methods: BALB/c mice were divided into 4 groups: C (saline i.p and inhalations with saline), OVA (ovalbumin i.p and inhalations with ovalbumin); C+EC (saline i.p, inhalations with s aline and treatment with EcTI); OVA+EC (ovalbumin i.p, inhalations with ovalbumin and treatment with EcTI). On day 29, we performed the following tests: resistance (Rrs) and elastance (Ers) of the respiratory system; (b) quantify eosinophils, 8-ISO-PGF2α, collagen and elastic fiber volume fractions; (c) IFN-γ, IL-4, IL-5, IL-13, MMP-9, TIMP-1, TGF-β, iNOS and p65-NFκB-positive cells in the airway and alveolar walls., Results: In OVA+EC group, there was an attenuation of the Rrs and Ers, reduction of eosinophils, IL-4, IL-5, IL-13, IFN-γ, iNOS and p65-NFκB-positive cells compared to OVA group. The 8-ISO-PGF2α, elastic and collagen fibers volume fractions as well as the positive cells for MMP-9, TIMP-1 and TGF-β positive cells were decreased in OVA+EC compared to the OVA group., Conclusion: EcTI attenuates bronchial hyperresponsiveness, inflammation, remodeling and oxidative stress activation in this experimental mouse model of asthma.

Two recombinant protease inhibitors from Bauhinia bauhinioides, rBbKI (kallikrein inhibitor) and rBbCI (cruzipain inhibitor) were evaluated for insecticidal activity against workers and soldiers of Nasutitermes corniger (order: Isoptera; family: Termitidae) through the inhibitors' effect on the insect's gut enzymes. The inhibitor rBbKI was more effective than rBbCI in inhibiting the termite's gut enzymes. The kallikrein inhibitor showed termiticidal activity in workers with an LC 50 of 0.9 mg mL -1 after 4 days. Conversely, rBbKI did not affect the survival of soldiers and rBbCI did not show termiticidal activity against N. corniger. The two inhibitors showed different specificity towards the termite's gut enzymes, representing interesting tools to characterize N. corniger enzymes. The different effects of rBbKI and rBbCI on the termite's enzymes and survival may be linked to slight structural differences between these inhibitors., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

The increase in urbanization and industrialization has contributed to the contamination of different environments by means of xenobiotic compounds, such as heavy metals, causing changes in microbial communities. Among these metals, the Mercury (Hg 2+ ) is one the most prevalent toxic metals for the environment The present study aimed to evaluate the effect of mercury on the formation of biofilm by environmental (collected from urban stream water) and clinical isolates of Klebsiella pneumoniae. In addition, antibiotic resistance, virulence factors, and genetic diversity were investigated. Taxonomic identity of eight isolates (one reference, two clinical, and five environmental isolates) was performed by MALDI-TOF-MS, while the antibiotic susceptibility profile was assessed by the disc diffusion method. The ability to form biofilms was evaluated by culture on Congo red agar and by crystal violet staining. Biofilm structure was analyzed by scanning electron microscopy. The hydrophobicity profile and the presence of the virulence genes cps, fimH, and mrkD was investigated. The presence of merA and its relationship with antimicrobial resistance were also assessed. The identity of all isolates was confirmed by MALDI-TOF-MS, and different profiles of resistance to mercury and antibiotics as well as of biofilm formation were identified for the clinical and environmental isolates. All isolates were hydrophilic and positive for the virulence genes cps, fimH, and mrkD; only the clinical isolate K36-A2 was positive for merA. The diversity of the isolates was confirmed by ERIC-PCR, which revealed high heterogeneity among the isolates. In conclusion, the data demonstrate that the investigated isolates present different responses to exposure to Hg 2+ and correspond to distinct populations of K. pneumoniae disseminated in the investigated environment. The data obtained in this work will aid in understanding the mechanisms of survival of this pathogen under adverse conditions., (Copyright © 2018. Published by Elsevier Inc.)

The immobilization of Enterolobium contortisiliquum protease inhibitor, EcTI-Sepharose, as an affinity chromatography matrix is a powerful biotechnological tool to purify targets from Nasutitermes corniger in the investigation of insecticidal properties of natural compounds., (© 2018 Society of Chemical Industry.)

The purpose of antithrombotic therapy is the prevention of thrombus formation and/or its extension with a minimum risk of bleeding. The inhibition of a variety of proteolytic processes, particularly those of the coagulation cascade, has been reported as a property of plant protease inhibitors. The role of trypsin inhibitors (TIs) from Delonix regia (Dr) and Acacia schweinfurthii (As), members of the Kunitz family of protease inhibitors, was investigated on blood coagulation, platelet aggregation, and thrombus formation. Different from Acacia schweinfurthii trypsin inhibitor (AsTI), Delonix regia trypsin inhibitor (DrTI) is a potent inhibitor of FXIa with a K iapp of 1.3 × 10 -9  M. In vitro, both inhibitors at 100 µg corresponding to the concentrations of 21 μM and 15.4 μM of DrTI and AsTI, respectively, increased approximately 2.0 times the activated partial thromboplastin time (aPTT) in human plasma compared to the control, likely due to the inhibition of human plasma kallikrein (huPK) or activated factor XI (FXIa), in the case of DrTI. Investigating in vivo models of arterial thrombus formation and bleeding time, DrTI and AsTI, 1.3 µM and 0.96 µM, respectively, prolonged approximately 50% the time for total carotid artery occlusion in mice compared to the control. In contrast to heparin, the bleeding time in mice treated with the two inhibitors did not differ from that of the control group. DrTI and AsTI inhibited 49.3% and 63.8%, respectively, ex vivo murine platelet aggregation induced by adenosine diphosphate (ADP), indicating that these protein inhibitors prevent arterial thrombus formation possibly by interfering with the plasma kallikrein (PK) proteolytic action on the intrinsic coagulation pathway and its ability to enhance the platelet aggregation activity on the intravascular compartment leading to the improvement of a thrombus.

The catalytic activity of enzymes can be regulated by interactions with synthetic nanoparticles (NPs) in a number of ways. To date, however, the potential use of NPs as allosteric effectors has not been investigated in detail. Importantly, targeting allosteric (distal) sites on the enzyme surface could afford unique ways to modulate the activity, allowing for either enzyme activation, partial or full inhibition. Using p -mercaptobenzoic acid-coated ultrasmall gold NPs (AuMBA) and human α-thrombin as a model system, here we experimentally tested the hypothesis that enzyme activity could be regulated through ultrasmall NP interactions at allosteric sites. We show that AuMBA interacted selectively and reversibly around two positively charged regions of the thrombin surface (exosites 1 and 2) and away from the active site. NP complexation at the exosites transmitted long-range structural changes over to the active site, altering both substrate binding affinity and catalysis. Significantly, thrombin activity was partially reduced - but not completely inhibited - by interactions with AuMBA. These findings indicate that interactions of proteins with ultrasmall NPs may mimic a typical biomolecular complexation event, and suggest the prospect of using ultrasmall particles as synthetic receptors to allosterically regulate protein function.

Genipa americana L., commonly known as genipap, is a plant with economical and medicinal importance, and a promising source of bioactive compounds. Lectins are carbohydrate-binding proteins with several biotechnological applications. This study reports the isolation and characterization of a G. americana bark lectin (GaBL). A single chromatographic procedure on Sephacryl S-100 resulted in isolation of GaBL, a protein with native molecular weight of over 200 kDa and pI 4.02, whose hemagglutinating activity was inhibited by lactose and fetuin, not affected by ions (Ca 2+ and Mg 2+ ), and stable upon heating (303-393 K) as well as over the pH range 5-10. The highest activity was found at a temperature lower than 333 K and pH 5. The secondary structure was analyzed by circular dichroism and showed a prevalence of beta structures and unordered forms. GaBL was able to partially refold in acidic pH conditions when dissolved in PBS buffer at pH 7.4. In conclusion, GaBL was purified in milligram quantities with high stability against different conditions, and is a new biomaterial with potential biotechnological applications., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background. CrataBL is a protein isolated from Crataeva tapia bark. It has been shown to exhibit several biological properties, including anti-inflammatory, analgesic, antitumor, and insecticidal activities. There are no studies evaluating the role of CrataBL in experimental asthma models. Aim . To evaluate the effects of CrataBL on lung mechanics, inflammation, remodeling, and oxidative stress activation of mice with allergic pulmonary inflammation. Materials and Methods . BALB/c mice (6-7 weeks old, 25-30g) were divided into four groups: nonsensitized and nontreated mice (C group, n=8); ovalbumin- (OVA-) sensitized and nontreated mice (OVA group, n=8); nonsensitized and CrataBL -treated mice (C+CR group, n=8); OVA-sensitized and CrataBL -treated mice (OVA+CR group, n=8). We evaluated hyperresponsiveness to methacholine, bronchoalveolar lavage fluid (BALF), pulmonary inflammation, extracellular matrix remodeling, and oxidative stress markers. Results. CrataBL treatment in OVA-sensitized mice (OVA+CR group) attenuated the following variables compared to OVA-sensitized mice without treatment (OVA group) (all p<0.05): (1) respiratory system resistance (Rrs) and elastance (Ers) after methacholine challenge; (2) total cells, macrophages, polymorphonuclear cells, and lymphocytes in BALF; (3) eosinophils and volume fraction of collagen and elastic fibers in the airway and alveolar wall according to histopathological and morphometry analysis; (4) IL-4-, IL-5-, IL-13-, IL-17-, IFN- γ -, MMP-9-, TIMP-1-, TGF- β -, iNOS-, and NF-kB-positive cells and volume of 8-iso-PGF2 α in airway and alveolar septa according to immunohistochemistry; and (5) IL-4, IL-5, and IFN- γ according to an ELISA. Conclusion. CrataBL contributes to the control of hyperresponsiveness, pulmonary inflammation, extracellular matrix remodeling, and oxidative stress responses in an animal model of chronic allergic pulmonary inflammation.

Glioblastoma is the most aggressive brain tumor with poor overall survival bellow 2 years. The natural compounds with anti-cancer properties, are thus gaining attention for possible adjuvant GBM treatment. In various cancer models Enterolobium contortisiliquum Trypsin Inhibitor (EcTI) proved to have anti-cancer effects. Here, we investigated the EcTI effects on GBM U87 cells and on mesenchymal stem cells (MSC) compared to their direct coculture (MSC/U87). MSC are present in tumor stroma, modulating GBM cells phenotype, and also represent potential drug delivery vehicle due to their tumor tropism. We showed that in p53-wild type U87 cells, metabolic activity was less affected by EcTI as in MSC monocuture, but the metabolic rate of mixed coculture was significantly reduced at lower EcTI concentration. Under coculture condition, EcTI potentiated MSC induced cell cycle arrest, possible due to highly increased p53, p21 and lower D1 expression, but there was no effect on apoptosis. Accordingly, in the coculture EcTI also enhanced Ca 2+ signalling mediated via bradykinin receptor 2, being associated with nitric oxide release that highly impaired proliferation and invasion. The mechanism did not seem to involve changes in cell adhesion but rather it down-regulated the β 1 integrin signaling with associated p-FAK in U87 cells, both supporting inhibition of invasion. Finally, some cytokines were down-regulated, indicating that EcTI inhibition of signalling might be mediated by cytokines. In conclusion, these results indicate that in cocultured MSC/U87 cells EcTI impairs the metabolic activity, proliferation, and reduced invasion, possibly associated with observed cytokines secretion. In this context, we confirmed that the plant derived protein potentiated the anticancer effects, induced by MSC, as represented by GBM U87 cell line., Competing Interests: CONFLICTS OF INTEREST The authors declare that they have no conflicts of interest with the contents of this article.

Trypanosoma cruzi is the etiologic agent of Chagas' disease, which affects 6-7 million people worldwide. Different strains of T. cruzi present specific genotypic and phenotypic characteristics that affect the host-pathogen interactions, and thus, the parasite has been classified into six groups (TcI to TcVI). T. cruzi infection presents two clinical phases, acute and chronic, both with distinct characteristics and important participation by the immune system. However, the specific contributions of parasite and host factors in the disease phases are not yet fully understood. The murine model for Chagas' disease is well-established and reproduces important features of the human infection, providing an experimental basis for the study of host lineages and parasite strains. Thus, we evaluated acute and chronic infection by the G (TcI) and CL (TcVI) strains of T. cruzi , which have distinct tropisms and infectivity, in two inbred mice lineages (C57BL/6 and BALB/c) that display variable degrees of susceptibility to different T. cruzi strains. Analysis of the parasite loads in host tissues by qPCR showed that CL strain established an infection faster than the G strain; at the same time, the response in BALB/c mice, although diverse in terms of cytokine secretion, was initiated earlier than that in C57BL/6 mice. At the parasitemia peak in the acute phase, we observed, either by confocal microscopy or by qPCR, that the infection was disseminated in all groups analyzed, with some differences concerning parasite tropism; at this point, all animals responded to infection by increasing the serum concentrations of cytokines. However, BALB/c mice seemed to better regulate the immune response than C57BL/6 mice. Indeed, in the chronic phase, C57BL/6 mice still presented exacerbated cytokine and chemokine responses. In summary, our results indicate that in these experimental models, the deregulation of immune response that is typical of chronic Chagas' disease may be due to control loss over pro- and anti-inflammatory cytokines early in the acute phase of the disease, depending primarily on the host background rather than the parasite strain.

Palythoa caribaeorum is a very common colonial zoanthid in the coastal reefs of Brazil. It is known for its massive production of mucus, which is traditionally used in folk medicine by fishermen in northeastern Brazil. This study identified biologically active compounds in P. caribaerum mucus. Crude mucus was collected during low tides by the manual scraping of colonies; samples were maintained in an ice bath, homogenized, and centrifuged at 16,000 g for 1 h at 4 °C; the supernatant (mucus) was kept at -80 °C until use. The enzymatic (proteolytic and phospholipase A 2 ), inhibitory (metallo, cysteine and serine proteases), and hemagglutinating (human erythrocyte) activities were determined. The results showed high levels of cysteine and metallo proteases, intermediate levels of phosholipase A 2 , low levels of trypsin, and no elastase and chymotrypsin like activities. The mucus showed potent inhibitory activity on snake venom metalloproteases and cysteine proteinase papain. In addition, it showed agglutinating activity towards O + , B + , and A + erythrocyte types. The hemostatic results showed that the mucus prolongs the aPTT and PT, and strongly inhibited platelet aggregation induced by arachidonic acid, collagen, epinephrine, ADP, and thrombin. The antimicrobial activity was tested on 15 strains of bacteria and fungi through the radial diffusion assay in agar, and no activity was observed. Compounds in P. caribaeorum mucus were analyzed for the first time in this study, and our results show potential pharmacological activities in these compounds, which are relevant for use in physiopathological investigations. However, the demonstration of these activities indicates caution in the use of crude mucus in folk medicine. Furthermore, the present or absent activities identified in this mucus suggest that the studied P. caribaeorum colonies were in thermal stress conditions at the time of sample collection; these conditions may precede the bleaching process in zoanthids. Hence, the use of mucus as an indicator of this process should be evaluated in the future., (Copyright © 2018. Published by Elsevier Ltd.)

Synthetic ultrasmall nanoparticles (NPs) can be designed to interact with biologically active proteins in a controlled manner. However, the rational design of NPs requires a clear understanding of their interactions with proteins and the precise molecular mechanisms that lead to association/dissociation in biological media. Although much effort has been devoted to the study of the kinetics mechanism of protein corona formation on large NPs, the nature of NP-protein interactions in the ultrasmall regime is radically different and poorly understood. Using a combination of experimental and computational approaches, we studied the interactions of a model protein, CrataBL, with ultrasmall gold NPs passivated with p-mercaptobenzoic acid (AuMBA) and glutathione (AuGSH). We have identified this system as an ideal in vitro platform to understand the dependence of binding affinity and kinetics on NP surface chemistry. We found that the structural and chemical complexity of the passivating NP layer leads to quite different association kinetics, from slow and reaction-limited (AuGSH) to fast and diffusion-limited (AuMBA). We also found that the otherwise weak and slow AuGSH-protein interactions measured in buffer solution are enhanced in macromolecular crowded solutions. These findings advance our mechanistic understanding of biomimetic NP-protein interactions in the ultrasmall regime and have implications for the design and use of NPs in the crowded conditions common to all biological media.

Blood coagulation and platelet-dependent primary homeostasis are important defense mechanisms against bleeding and novel inhibitors have been researched to obtain pharmacological and clinical applications. In this work, the PpyLL, a lectin obtained from Phthirusa pyrifolia, was characterized in terms of its molecular structure and biological functions (anticoagulant, antiplatelet agreggation and hemagglutinating activities) in presence or absence of Gamma radiation exposure. Results revealed a lectin with secondary-structure content by approximately 49% of β-sheet, 20% of β-turn and 31% of disordered structure. Irradiation effect demonstrated possible different sites of function by lectin on anticoagulant and hemagglutinating activities, once a decrease about 80% was observed when compared the activities under 0.5kGy of exposition to gamma radiation. An emphatic discussion about the use of gamma radiation as a possible modulator of the lectin activity was made, and once the ionizing radiation affected differently the anticoagulation and hemagglutinating activities, we speculated that the results are determined by selective molecular damages in different binding sites. PpyLL biological activities and gamma radiation modulation could be considered for future researches in biomedical field aiming possible medical applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Studies have suggested that soluble factors in plasma from patients with active (aBD) and inactive (iBD) Behçet's disease (BD) stimulate neutrophil function. Soluble CD40 ligand (sCD40L) is an important mediator of inflammation in BD. Its expression and effect on neutrophil oxidative burst and neutrophil extracellular trap (NET) release have not been characterized. In this study, we sought to investigate the role of plasma and the CD40L pathway on NET release and the oxidative burst profile in patients with aBD and iBD., Methods: Neutrophils and peripheral blood mononuclear cells (PBMCs) were obtained from patients with aBD (n = 30), patients with iBD (n = 31), and healthy control subjects (HCs; n = 30). sCD40L plasma concentration was determined in individual samples. A pool of plasma for each group was created. In some experiments, plasma pools were treated with recombinant CD40 (rhCD40-muIg) for sCD40L blockade. NET release and H 2 O 2 /O 2 - production were determined after stimulation with phorbol 12-myristate 13-acetate, sCD40L, or plasma pool. Flow cytometric analysis was performed to evaluate the expression of (1) CD40, Mac-1, and phosphorylated NF-κB p65 on neutrophils and monocytes and (2) CD40L on activated T cells and platelets. CD40L gene expression in PBMCs was determined by qRT-PCR., Results: sCD40L plasma levels were significantly higher in patients with iBD (median 17,234, range 2346-19,279 pg/ml) and patients with aBD (median 18,289, range 413-19,883 pg/ml) than in HCs (median 47.5, range 33.7-26.7 pg/ml; p < 0.001). NET release was constitutively increased in BD compared with HC. NET release and H 2 O 2 /O 2 - were higher after stimulation with sCD40L or BD plasma and decreased after sCD40L blockade. Mac-1 expression was constitutively increased in neutrophils of patients with aBD (88.7 ± 13.2% of cells) and patients with iBD (89.2 ± 20.1% of cells) compared with HC (27.1 ± 18.8% of cells; p < 0.01). CD40 expression on phagocytes and CD40L expression on platelets were similar in the three groups. PBMCs as well as nonactivated and activated CD4 + T cells from patients with BD showed higher CD40L expression., Conclusions: Plasma from patients with aBD exerts a stimulus on NET release and oxidative burst, probably induced by sCD40L.