Removal of enrofloxacin using Eichhornia crassipes in microcosm wetlands.

The global consumption of antibiotics leads to their possible occurrence in the environment. In this context, nature-based solutions (NBS) can be used to sustainably manage and restore natural and modified ecosystems. In this work, we studied the efficiency of the NBS free-water surface wetlands (FWSWs) using Eichhornia crassipes in microcosm for enrofloxacin removal. We also explored the behavior of enrofloxacin in the system, its accumulation and distribution in plant tissues, the detoxification mechanisms, and the possible effects on plant growth. Enrofloxacin was initially taken up by E. crassipes (first 100 h). Notably, it accumulated in the sediment at the end of the experimental time. Removal rates above 94% were obtained in systems with sediment and sediment + E. crassipes. In addition, enrofloxacin was found in leaves, petioles, and roots (8.8-23.6 µg, 11-78.3 µg, and 10.2-70.7 µg, respectively). Furthermore, enrofloxacin, the main degradation product (ciprofloxacin), and other degradation products were quantified in the tissues and chlorosis was observed on days 5 and 9. Finally, the degradation products of enrofloxacin were analyzed, and four possible metabolic pathways of enrofloxacin in E. crassipes were described.

Toxicity Evaluation of Anti-cholinesterasic Amphibian Extracts by MTT and an Optimized Artemia salina Test.

The amphibian skin is an important source of bioactive compounds. Recently, our workgroup reported the bioactivity of new extracts from the Hylidae, Microhylidae and Leptodactylidae families against several pathways involved in Alzheimer's disease. However, since cytotoxicity can be a limiting factor for their applicability, we evaluated the toxicity of nine amphibian skin extracts with reported anticholinesterase activity, using the traditional MTT assay and an optimized Artemia salina test. The proposed improvement, guided by experimental design, aims to reduce the amount of biological sample needed. Overall, we proved that the active extracts were non-toxic at effective concentration against cholinesterases (AChE/BChE), positioning the amphibian skin as a promising and preliminary safe source of bioactive compounds in the anti-Alzheimer's treatment. Interestingly, we demonstrated that both toxicity assays can discriminate between toxic and non-toxic samples. We propose the A. salina bioassay as a reliable and cost-effective alternative for early toxicity screening.

Acetylcholine hydrolytic activity of fibrillated ß-amyloid (1-40) peptide.

Alzheimer's disease is characterized by the presence of senile plaques composed of ß-amyloid peptide (Aß) aggregates with toxic effects that are still not fully understood. Recently, it was discovered that Aß(1-42) fibrils possess catalytic activity on acetylcholine hydrolysis. Catalytic amyloids are an emerging and exciting field of research. In this study, we examined the catalytic activity of the fibrils formed by Aß(1-40), the most abundant Aß variant, on acetylcholine hydrolysis. Our findings reveal that Aß(1-40) fibrils exhibit moderate enzymatic activity, indicating that natural peptide aggregates could serve as biocatalysts and provide new insights into the potential role of Aß in neurological disorders.

D- and N-Methyl Amino Acids for Modulating the Therapeutic Properties of Antimicrobial Peptides and Lipopeptides.

Here we designed and synthesized analogs of two antimicrobial peptides, namely C10:0-A2, a lipopeptide, and TA4, a cationic α-helical amphipathic peptide, and used non-proteinogenic amino acids to improve their therapeutic properties. The physicochemical properties of these analogs were analyzed, including their retention time, hydrophobicity, and critical micelle concentration, as well as their antimicrobial activity against gram-positive and gram-negative bacteria and yeast. Our results showed that substitution with D- and N-methyl amino acids could be a useful strategy to modulate the therapeutic properties of antimicrobial peptides and lipopeptides, including enhancing stability against enzymatic degradation. The study provides insights into the design and optimization of antimicrobial peptides to achieve improved stability and therapeutic efficacy. TA4(dK), C10:0-A2(6-NMeLys), and C10:0-A2(9-NMeLys) were identified as the most promising molecules for further studies.

Inhibition of Human Cholinesterases and in†vitro ß-Amyloid Aggregation by Rationally Designed Peptides.

The multifactorial nature of Alzheimer's disease (AD) is now widely recognized, which has increased the interest in compounds that can address more than one AD-associated targets. Herein, we report the inhibitory activity on the human cholinesterases (acetylcholinesterase, hAChE and butyrylcholinesterase, hBChE) and on the AChE-induced ß-amyloid peptide (Aß) aggregation by a series of peptide derivatives designed by mutating aliphatic residues for aromatic ones. We identified peptide W3 (LGWVSKGKLL-NH2 ) as an interesting scaffold for the development of new anti-AD multitarget-directed drugs. It showed the lowest IC50 value against hAChE reported for a peptide (0.99±0.02 µM) and inhibited 94.2 %±1.2 of AChE-induced Aß aggregation at 10 µM. Furthermore, it inhibited hBChE (IC50 , 15.44±0.91 µM), showed no in vivo toxicity in brine shrimp and had shown moderated radical scavenging and Fe2+ chelating capabilities in previous studies. The results are in line with multiple reports showing the utility of the indole moiety for the development of cholinesterase inhibitors.

Development of low-cost cage-like particles to formulate veterinary vaccines.

Low-cost adjuvants are urgently needed for the development of veterinary vaccines able to trigger strong immune responses. In this work, we describe a method to obtain a low-cost cage-like particles (ISCOMATRIX-like) adjuvant useful to formulate veterinary vaccines candidates. The main components to form the particles are lipids and saponins, which were obtained from egg yolk by ethanolic extraction and by dialyzing a non-refined saponins extract, respectively. Lipids were fully characterized by thin layer chromatography (TLC) and gas-chromatography (GC) and enzymatic methods, and saponins were characterized by TLC, HPLC and MALDI-TOF. Cage-like particles were prepared with these components or with commercial inputs. Both particles and the traditional Alum used in veterinary vaccines were compared by immunizing mice with Ovalbumin (OVA) formulated with these adjuvants and assessing IgG1, IgG2a anti OVA antibodies and specific Delayed-type Hypersensitivity (DTH). In the yolk extract, a mixture of phospholipids, cholesterol and minor components of the extract (e.g. lyso-phospholipids) with suitable proportions to generate cage-like particles was obtained. Also, semi-purified saponins with similar features to those of the QuilA® were obtained. Cage-like particles prepared with these components have 40-50 nm and triggers similar levels of Anti-OVA IgG1 and DTH than with commercial inputs but higher specific-IgG2a. Both adjuvants largely increased the levels of IgG1, IgG2a and DTH in relation to the formulation with Alum. The methods described to extract lipids from egg yolk and saponins from non-refined extract allowed us to obtain an inexpensive and highly effective adjuvant.

Rational design and synthesis of modified natural peptides from Boana pulchella (anura) as acetylcholinesterase inhibitors and antioxidants.

The use of acetylcholinesterase (AChE) inhibitors, antioxidants or multitarget compounds are among the main strategies against Alzheimer's disease (AD). Between AChE inhibitors, those targeting the peripheral anionic site (PAS) are of special interest. Here, we describe the rational design and synthesis of peptide analogs of a natural PAS-targeting sequence that we recently discovered, aiming at increasing its activity against AChE. We also tested their radical scavenging and metal chelating properties. Our design strategy was based on the position-specific, computer-aided insertion of aromatic residues. The analog named as W3 showed a 30-fold higher inhibitory activity than the original sequence and an improved antioxidant activity. W3 is the most potent modified natural peptide against Electrophorus electricus AChE ever reported with an IC50 of 10.42 µM (± 1.02). In addition, it showed a radical scavenging activity of 47.00% ± 3.11 at 50 µM and 93.47% ± 1.53 at 400 µM. Since peptides are receiving increasing interest as drugs, we propose the W3 analog as an attractive sequence for the development of new peptide-based multitarget drugs for AD. Besides, this work sheds light on the importance of the aromatic residues in the modulation of AChE activity and their effect on the radical scavenging activity of a peptide.

Natural multi-target modulators of pathological pathways in Alzheimer's disease isolated from the Rhinella arenarum skin.

The skin of anuran amphibians is a rich source of compounds with great medicinal potential. Alzheimer's disease (AD) is a complex disease associated with numerous pathological pathways, making their simultaneous modulation necessary. Nowadays the development of anti-AD drugs is focused on a Multi-Target Directed Ligands strategy. Herein we report the bioactivity of the skin extracts of the toad Rhinella arenarum obtained by an invasive and non-invasive methods, against five AD pathological targets (AChE, BChE, MAO-B, antioxidant and chelating activities). The extract derived from the non-invasive technique showed the highest biological activity, being capable of acting on all or almost all the pathological targets of AD, while also avoiding harm to the animal.

Functional Autoreactive Anti-ß2 Adrenergic Antibodies May Contribute to Insulin Resistance Profile in Patients with Chronic Chagas Disease.

Potential activation of ß2 adrenergic receptors (ß2AR) by specific autoreactive antibodies (Abs) that arise during the host reaction to Trypanosoma cruzi, could contribute to the elevated prevalence of metabolic disturbances described in patients with chronic Chagas disease (CCD). This study aimed to determine the prevalence of anti-ß2AR Abs in patients with CCD, as well as the correlation of these Abs with the presence of glucose and lipid metabolism disturbances, in order to explore their association with an insulin resistance profile. Additionally, we tested the functional effects of anti-ß2AR Abs employing an in vitro bioassay with neuroendocrine cells expressing ß2AR. A clinical and metabolic evaluation including an OGTT was performed in 80 CCD patients and 40 controls. Anti-ß2AR Abs were measured by an in-house-developed ELISA, and the ß2 adrenergic activity of affinity-purified IgG fractions from patient' sera were assayed in CRE-Luc and POMCLuc transfected AtT-20 cells. A higher proportion of dysglycemia (72.5% vs. 37.5%; p = 0.001) was observed in the CCD group, accompanied by increased HOMA2-IR (p = 0.019), especially in subjects with Abs (+). Anti-ß2AR Abs reactivity (7.01 (2.39-20.5); p = 0.0004) and age >50 years (3.83 (1.30-11.25); p = 0.014) resulted as relevant for IR prediction (AUC: 0.786). Concordantly, Abs (+) CCD patients showed elevated metabolic risk scores and an increased prevalence of atherogenic dyslipidemia (p = 0.040), as compared to Abs (-) patients and controls. On functional bioassays, Abs exerted specific and dose-dependent ß2-agonist effects. Our findings suggest that anti-ß2AR Abs may induce the activation of ß2AR in other tissues besides the heart; furthermore, we show that in patients with CCD these Abs are associated with an insulin resistance profile and atherogenic dyslipidemia, providing biological plausibility to the hypothesis that adrenergic activation by anti-ß2AR Abs could contribute to the pathogenesis of metabolic disturbances described in CCD patients, increasing their cardiovascular risk.

Amphibian skin extracts as source of bioactive multi-target agents against different pathways of Alzheimer's disease.

Natural products represent a rich source of bioactive compounds that have been historically used to obtain substances with great medicinal potential. The skin of anuran amphibians is a rich source of compounds with a wide range of biological activity. Alzheimer's disease (AD) is a complex disease associated with all kind of different pathways, making their simultaneous modulation necessary. Nowadays anti-AD treatments are focused on enzymatic inhibitors. Here in we report the activity of skin extracts from nine species from Argentina, belonging to three anuran families, as inhibitors of AChE, BChE and MAO-B enzymes. The extracts also showed antioxidant activities, acting as multi-target on four important pathways of AD.

Cytotoxic and antiproliferative activities of amphibian (anuran) skin extracts on human acute monocytic leukemia cells.

The use of preparations derived from frog skins for curative purposes antedates research history and is perpetuated in current medicine. The skins of anuran's (frogs and toads) are a rich source of compounds with a great importance in the search of antibiotics, analgesics, immunomodulators, enzymatic inhibitors and antitumoral agents applying to human health. Nowadays, cancer is the second most common cause of mortality with more than 8.2 million of deaths worldwide per year. Acute monocytic leukemia is the subtype M5 of acute myeloid leukemia (AML) a cancer type with reduced survival rates in patients. The monocyte to macrophage differentiation plays an essential role increasing the expansion of AML cell lines. Herein we studied the cytotoxic and antiproliferative activities of eleven amphibian species of three families belonging to Argentinean zones, against THP-1 monocytes and THP-1 macrophages acute monocytic leukemia cell lines. The evaluated species showed pronounced deleterious effects on acute monocytic leukemia THP-1 cell lines, reducing cell proliferation and inducing apoptosis, autophagy and in some cases cell aggregation. Being this work of great importance for the study of new natural anti-cancer compounds.

Acetylcholinesterase inhibitory activity of a naturally occurring peptide isolated from Boana pulchella (Anura: Hylidae) and its analogs.

Alzheimer's disease (AD), the most common form of dementia, is a growing problem worldwide, with 10 million incident cases registered every year. The complex etiology of AD has not been clarified yet and represents an active research topic. In this work, we studied the inhibitory properties of Hp-1935, a natural peptide extracted from the skin secretions of an Argentinian frog (Boana pulchella). It was initially isolated as an antimicrobial peptide by our group, but we later discovered its anti-AChE action. Since not many peptides with this activity have been reported, we focused on defining the basis of its inhibitory mechanism against acetylcholinesterase (AChE) and on finding the primary portion for the inhibitory activity in its sequence, through the combination of an experimental strategy of design and synthesis with molecular dynamics simulations. We also tested its cytotoxicity. We found that Hp-1935 is an interesting sequence for the development of new AChE inhibitors. This peptide is a peripheral anionic site inhibitor with an inhibitory activity that collocates it between the most potent natural amino acids peptides against AChE reported. We also demonstrate that its inhibitory activity is concentrated on the central part of the sequence.

Trichogin GA IV Alignment and Oligomerization in Phospholipid Bilayers.

Trichogin GA IV is a short peptaibol with antimicrobial activity. This uncharged, but amphipathic, sequence is aligned at the membrane interface and undergoes a transition to an aggregated state that inserts more deeply into the membrane, an assembly that predominates at a peptide-to-lipid ratio (P/L) of 1:20. In this work, the natural trichogin sequence was prepared and reconstituted into oriented lipid bilayers. The 15 N NMR chemical shift is indicative of a well-defined alignment of the peptide parallel to the membrane surface at P/Ls of 1:120 and 1:20. When the P/L is increased to 1:8, an additional peptide topology is observed that is indicative of a heterogeneous orientation, with helix alignments ranging from around the magic angle to perfectly in-plane. The topological preference of the trichogin helix for an orientation parallel to the membrane surface was confirmed by attenuated total reflection FTIR spectroscopy. Furthermore, 19 F CODEX experiments were performed on a trichogin sequence with 19 F-Phe at position 10. The CODEX decay is in agreement with a tetrameric complex, in which the 19 F sites are about 9-9.5 Šapart. Thus, a model emerges in which the monomeric peptide aligns along the membrane surface. When the peptide concentration increases, first dimeric and then tetrameric assemblies form, made up from helices oriented predominantly parallel to the membrane surface. The formation of these aggregates correlates with the release of vesicle contents including relatively large molecules.

Rational Design of Antiangiogenic Helical Oligopeptides Targeting the Vascular Endothelial Growth Factor Receptors.

Tumor angiogenesis, essential for cancer development, is regulated mainly by vascular endothelial growth factors (VEGFs) and their receptors (VEGFRs), which are overexpressed in cancer cells. Therefore, the VEGF/VEGFR interaction represents a promising pharmaceutical target to fight cancer progression. The VEGF surface interacting with VEGFRs comprises a short α-helix. In this work, helical oligopeptides mimicking the VEGF-C helix were rationally designed based on structural analyses and computational studies. The helical conformation was stabilized by optimizing intramolecular interactions and by introducing helix-inducing Cα,α-disubstituted amino acids. The conformational features of the synthetic peptides were characterized by circular dichroism and nuclear magnetic resonance, and their receptor binding properties and antiangiogenic activity were determined. The best hits exhibited antiangiogenic activity in vitro at nanomolar concentrations and were resistant to proteolytic degradation.

Natural Multi-Target Inhibitors of Cholinesterases and Monoamine Oxidase Enzymes with Antioxidant Potential from Skin Extracts of Hypsiboas cordobae and Pseudis minuta (Anura: Hylidae).

Alzheimer's disease (AD) is the most common cause of dementia, characterized by loss of selective neuronal and normal brain functions. Every year, ten million new cases are diagnosed worldwide. AD is a complex disease associated with all kind of different pathways, making their simultaneous modulation necessary. Nowadays anti-AD treatments are focused on enzymatic inhibitors. The study of the amphibians' skin had acquired great importance in the fields of biology and human health and represents an attractive and novel source for natural compounds with high potential in the development of new drugs. The present work exhibits the power of amphibian skins as a source of bioactive compounds. Herein we report the activity of extracts of two species from Hylidae family (H. cordobae and P. minuta) as reversible inhibitors of acetylcholinesterase and butyrylcholinesterase enzymes. Furthermore, the extracts inhibit MAO-B enzyme and showed antioxidant activities, acting on four important pathways of AD.

Leptodactylus latrans Amphibian Skin Secretions as a Novel Source for the Isolation of Antibacterial Peptides.

Amphibians´ skin produces a diverse array of antimicrobial peptides that play a crucial role as the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, current knowledge about the presence of peptides with antimicrobial properties is limited to a only few species. Here we used LC-MS-MS to identify antimicrobial peptides with masses ranging from 1000 to 4000 Da from samples of skin secretions of Leptodactylus latrans (Anura: Leptodactylidae). Three novel amino acid sequences were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Ll-1577, P2-Ll-1298, and P3-Ll-2085, inhibited the growth of two ATCC strains, namely Escherichia coli and Staphylococcus aureus. P3-Ll-2085 was the most active peptide. In the presence of trifluoroethanol (TFE) and anionic liposomes, it adopted an amphipathic α-helical structure. P2-Ll-1298 showed slightly lower activity than P3-Ll-2085. Comparison of the MIC values of these two peptides revealed that the addition of seven amino acid residues (GLLDFLK) on the N-terminal of P2-Ll-1298 significantly improved activity against both strains. P1-Ll-1577, which remarkably is an anionic peptide, showed interesting antimicrobial activity against E. coli and S. aureus strain, showing marked membrane selectivity and non-hemolysis. Due to this, P1-L1-1577 emerges as a potential candidate for the development of new antibacterial drugs.

Molecular design and synthesis of novel peptides from amphibians skin acting as inhibitors of cholinesterase enzymes.

Cholinesterases are a family of enzymes that catalyze the hydrolysis of neurotransmitter acetylcholine. There are two types of cholinesterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), which differ in their distribution in the body. Currently, cholinesterase inhibitors (ChEI) represent the treatment of choice for Alzheimer's disease (AD). In this paper, we report the synthesis and inhibitory effect on both enzymes of four new peptides structurally related to P1-Hp-1971 (amphibian skin peptide found in our previous work. Sequence: TKPTLLGLPLGAGPAAGPGKR-NH2 ). The bioassay data and cytotoxicity test show that some of the compounds possess a significant AChE and BChE inhibition and no toxic effect. The present work demonstrates that diminution of the size of the original peptide could potentially result in new compounds with significant cholinesterase inhibition activity, although it appears that there is an optimal size for the sequence. We also conducted an exhaustive molecular modeling study to better understand the mechanism of action of these compounds by combining docking techniques with molecular dynamics simulations on BChE. This is the first report about amphibian peptides and the second one of natural peptides with ChE inhibitory activity. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

Insights into peptide-membrane interactions of newly synthesized, nitroxide-containing analogs of the peptaibiotic trichogin GA IV using EPR.

Trichogin GA IV is a short-length (10-amino acid long), mostly hydrophobic, peptaibiotic with an N-terminal fatty acyl chain and a C-terminal 1,2-amino alcohol. A cardinal role of the terminal moieties in the cytotoxic activity of trichogin has been recently found. Previously, peptide orientation and dynamics of trichogin analogs in the membrane were studied using methyl ester derivatives. Therefore, in the present work we synthesized several trichogin analogs with naturally occurring terminal groups to verify whether these moieties have any effect on peptide-membrane interaction. These trichogin analogs, both neutral and carrying a positively charged Lys residue, bear the nitroxide-containing α-amino acid TOAC to study them using EPR spectroscopy. Vesicles were used to investigate orientation and penetration depth of the peptide at room temperature. Bicelles were employed to evaluate the order, dynamics, and orientation of the peptide at a near physiological temperature. In addition, the position of the N-terminal 1-octanoyl chain in the membrane was studied by labeling it with a nitroxide. The secondary structure of the peptides in vesicles was studied by CD spectroscopy showing that they adopt a mostly α-helical structure. In vesicles, the analogs insert below the lipid headgroups with the helix axis oriented parallel to the membrane surface at a peptide-to-lipid (P:L) ratio of 1:100. The presence of the single, positively charged Lys residue does not alter the orientation adopted by the peptides. In bicelles at P:L ratios 1:100 and 1:60, the peptide adopts a transmembrane orientation characterized by a very low orientational order, whereas at a 1:15 P:L ratio it severely disrupts the membrane. Our data shows that overall orientation and insertion in model membranes of the native trichogin GA IV are strictly comparable to those of its methyl ester analogs previously examined.

Combined analysis of cross-reacting antibodies anti-ß1AR and anti-B13 in advanced stages of Chagas heart disease.

OBJECTIVE: Autoantibodies cross-reacting with the ß1 adrenergic receptor (anti-ß1AR and anti-p2ß) and cardiac myosin antigens (anti-B13) have been related to the pathogenesis of chronic Chagas heart disease (CCHD). Studies exploring their levels in different stages are scarce. We aimed to evaluate the relationship of these autoantibodies with the clinical profile of chronic patients, especially regarding their classificatory accuracy in severe presentation with heart failure. METHODS AND RESULTS: We conducted a cross-sectional study of 155 T. cruzi-seropositive patients and 26 age- and gender-matched healthy controls. They were categorised in three stages of CCHD. Serum antibodies were measured by specific immunoassays. Symptomatic individuals showed increased levels of anti-ß1AR and anti-B13, while anti-p2ß antibodies were similar between groups. A composite logistic regression model including anti-B13, anti-ß1AR antibody levels and age was able to predict systolic heart failure yielding an area under the curve of 83% (sensitivity of 67% and specificity of 89%). CONCLUSIONS: In our study, anti-ß1AR and anti-B13 antibodies were higher in individuals with chronic Chagas heart disease stage III, mainly in those with dilated cardiomyopathy associated with systolic heart failure. Logistic regression analysis showed that both antibodies were good predictors of severe CCHD. As well as being involved in disease progression, anti-ß1AR and anti-B13 antibodies may be used as a serum marker of poor prognosis in terms of heart compromise.

Antimicrobial peptides from skin secretions of Hypsiboas pulchellus (Anura: Hylidae).

The skin of many amphibians produces a large repertoire of antimicrobial peptides that are crucial in the first line of defense against microbial invasion. Despite the immense richness of wild amphibians in Argentina, knowledge about peptides with antimicrobial properties is limited to a few species. Here we used LC-MS-MS to analyze samples of Hypsiboas pulchellus skin with the aim to identify antimicrobial peptides in the mass range of 1000 to 2000 Da. Twenty-three novel sequences were identified by MS, three of which were selected for chemical synthesis and further studies. The three synthetic peptides, named P1-Hp-1971, P2-Hp-1935, and P3-Hp-1891, inhibited the growth of two ATCC strains: Escherichia coli (MIC: 16, 33, and 17 µM, respectively) and Staphylococcus aureus (MIC: 8, 66, and 17 µM, respectively). P1-Hp-1971 and P3-Hp-1891 were the most active peptides. P1-Hp-1971, which showed the highest therapeutic indices (40 for E. coli and 80 for S. aureus), is a proline-glycine-rich peptide with a highly unordered structure, while P3-Hp-1891 adopts an amphipathic α-helical structure in the presence of 2,2,2-trifluoroethanol and anionic liposomes. This is the first peptidomic study of Hypsiboas pulchellus skin secretions to allow the identification of antimicrobial peptides.