Showing papers by "Fanny Guzmán published in 2018"

Functionalization of stable fluorescent nanodiamonds towards reliable detection of biomarkers for Alzheimer's disease.

Abstract: Stable and non-toxic fluorescent markers are gaining attention in molecular diagnostics as powerful tools for enabling long and reliable biological studies. Such markers should not only have a long half-life under several assay conditions showing no photo bleaching or blinking but also, they must allow for their conjugation or functionalization as a crucial step for numerous applications such as cellular tracking, biomarker detection and drug delivery. We report the functionalization of stable fluorescent markers based on nanodiamonds (NDs) with a bifunctional peptide. This peptide is made of a cell penetrating peptide and a six amino acids long β-sheet breaker peptide that is able to recognize amyloid β (Aβ) aggregates, a biomarker for the Alzheimer disease. Our results indicate that functionalized NDs (fNDs) are not cytotoxic and can be internalized by the cells. The fNDs allow ultrasensitive detection (at picomolar concentrations of NDs) of in vitro amyloid fibrils and amyloid aggregates in AD mice brains. The fluorescence of functionalized NDs is more stable than that of fluorescent markers commonly used to stain Aβ aggregates such as Thioflavin T. These results pave the way for performing ultrasensitive and reliable detection of Aβ aggregates involved in the pathogenesis of the Alzheimer disease.

Massive Gene Expansion and Sequence Diversification Is Associated with Diverse Tissue Distribution, Regulation and Antimicrobial Properties of Anti-Lipopolysaccharide Factors in Shrimp.

Abstract: Anti-lipopolysaccharide factors (ALFs) are antimicrobial peptides with a central β-hairpin structure able to bind to microbial components. Mining sequence databases for ALFs allowed us to show the remarkable diversity of ALF sequences in shrimp. We found at least seven members of the ALF family (Groups A to G), including two novel Groups (F and G), all of which are encoded by different loci with conserved gene organization. Phylogenetic analyses revealed that gene expansion and subsequent diversification of the ALF family occurred in crustaceans before shrimp speciation occurred. The transcriptional profile of ALFs was compared in terms of tissue distribution, response to two pathogens and during shrimp development in Litopenaeus vannamei, the most cultivated species. ALFs were found to be constitutively expressed in hemocytes and to respond differently to tissue damage. While synthetic β-hairpins of Groups E and G displayed both antibacterial and antifungal activities, no activity was recorded for Group F β-hairpins. Altogether, our results showed that ALFs form a family of shrimp AMPs that has been the subject of intense diversification. The different genes differ in terms of tissue expression, regulation and function. These data strongly suggest that multiple selection pressures have led to functional diversification of ALFs in shrimp.

Identification of Peptides in Flowers of Sambucus nigra with Antimicrobial Activity against Aquaculture Pathogens.

Abstract: The elder (Sambucus spp.) tree has a number of uses in traditional medicine. Previous studies have demonstrated the antimicrobial properties of elderberry liquid extract against human pathogenic bacteria and also influenza viruses. These properties have been mainly attributed to phenolic compounds. However, other plant defense molecules, such as antimicrobial peptides (AMPs), may be present. Here, we studied peptide extracts from flowers of Sambucus nigra L. The mass spectrometry analyses determined peptides of 3 to 3.6 kDa, among them, cysteine-rich peptides were identified with antimicrobial activity against various Gram-negative bacteria, including recurrent pathogens of Chilean aquaculture. In addition, membrane blebbing on the bacterial surface after exposure to the cyclotide was visualized by SEM microscopy and SYTOX Green permeabilization assay showed the ability to disrupt the bacterial membrane. We postulate that these peptides exert their action by destroying the bacterial membrane.

Understanding the antimicrobial properties/activity of an 11-residue Lys homopeptide by alanine and proline scan

Abstract: Previous work demonstrated that lysine homopeptides adopt a polyproline II (PPII) structure. Lysine homopeptides with odd number of residues, especially with 11 residues (K11), were capable of inhibiting the growth of a broader spectrum of bacteria than those with an even number. Confocal studies also determined that K11 was able to localize exclusively in the bacterial membrane, leading to cell death. In this work, the mechanism of action of this peptide was further analyzed focused on examining the structural changes in bacterial membrane induced by K11, and in K11 itself when interacting with bacterial membrane lipids. Moreover, alanine and proline scans were performed for K11 to identify relevant positions in structure conformation and antibacterial activity. To do so, circular dichroism spectroscopy (CD) was conducted in saline phosphate buffer (PBS) and in lipidic vesicles, using large unilamellar vesicles (LUV), composed of 2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or bacterial membrane lipid. Antimicrobial activity of K11 and their analogs was evaluated in Gram-positive and Gram-negative bacterial strains. The scanning electron microscopy (SEM) micrographs of Staphylococcus aureus ATCC 25923 exposed to the Lys homopeptide at MIC concentration showed blisters and bubbles formed on the bacterial surface, suggesting that K11 exerts its action by destabilizing the bacterial membrane. CD analysis revealed a remarkably enhanced PPII structure of K11 when replacing some of its central residues by proline in PBS. However, when such peptide analogs were confronted with either DMPG-LUV or membrane lipid extract-LUV, the tendency to form PPII structure was severely weakened. On the contrary, K11 peptide showed a remarkably enhanced PPII structure in the presence of DMPG-LUV. Antibacterial tests revealed that K11 was able to inhibit all tested bacteria with an MIC value of 5 µM, while proline and alanine analogs have a reduced activity on Listeria monocytogenes. Besides, the activity against Vibrio parahaemolyticus was affected in most of the alanine-substituted analogs. However, lysine substitutions by alanine or proline at position 7 did not alter the activity against all tested bacterial strains, suggesting that this position can be screened to find a substitute amino acid yielding a peptide with increased antibacterial activity. These results also indicate that the PPII secondary structure of K11 is stabilized by the interaction of the peptide with negatively charged phospholipids in the bacterial membrane, though not being the sole determinant for its antimicrobial activity.

Detection of peroxiredoxin-like protein in Antarctic sea urchin ( Sterechinus neumayeri ) under heat stress and induced with pathogen-associated molecular pattern from Vibrio anguillarum

Abstract: Antarctic marine organisms have developed in an environment of low temperatures and high levels of stability. Consequently, these species have lost the ability to adapt to sudden changes in temperature. Rising ocean temperatures could make the Antarctic sea urchin Sterechinus neumayeri vulnerable to pathogens, triggering responses that increase oxidative stress. In order to understand how the immune system reacts, we can analyze the expression of anti-oxidant molecules such as the peroxiredoxins (Prxs). Prxs are an anti-oxidant protein family with conserved catalytic redox-active cysteine residues. In S. neumayeri, one full-length cDNA of the gene that encodes Prx (Sn-Prx) was characterized. The Sn-Prx cDNA contains a 786-bp open reading frame, which encodes 262 amino acids, including two conserved cysteine residues that are characteristic of the typical 2-Cys subgroup of the Prx family. An in silico analysis was performed to establish epitope molecules, which were then chemically synthesized and used to obtain antibodies. The antibodies were validated by indirect ELISA against a synthetic peptide and western blot against S. neumayeri proteins. In different tissues, the expression of Sn-Prx protein was increased after Vibrio anguillarum challenge. Heat stress also increased expression of Sn-Prx in coelomocytes after 7 days, but the availability of the protein decreased in digestive gland tissues after 2 and 3 weeks of heat stress. This may indicate the involvement of Prx in antioxidant response in S. neumayeri. The evidence presented in this study proposes anti-Prx antibody as an experimental evaluation tool that can be used to establish a baseline of the ability of S. neumayeri antioxidant response.

Dipeptidyl peptidase 3, a novel protease from Leishmania braziliensis.

Abstract: The increase of leishmaniasis cases worldwide and the emergence of Leishmania strains resistant to current treatments make necessary to find new therapeutic targets. Proteases are appealing drug targets because they play pivotal roles in facilitating parasite survival and promoting pathogenesis. Enzymes belonging to the dipeptidyl peptidase 3 (DPP3) group have been described in different organisms such as mammals, insects and yeast, in which these enzymes have been involved in both protein turnover and protection against oxidative damage. The aim of this work was to characterize the structure and function of the Leishmania braziliensis DPP3 (LbDPP3) protein as the first step to elucidate its suitability as a potential drug target. Sequence alignment showed 43% of identity between LbDPP3 and its human orthologous (hDPP3) enzyme. Although the modeled protein adopted a globally conserved three-dimensional (3D) structure, structural differences were found in the vicinity of the active site and the substrate binding-cleft. In addition, the Leishmania protein was expressed as a soluble recombinant protein and its kinetics parameters were determined using the z-Arginine-Arginine-AMC substrate. The LbDPP3 activity was maximal at pH values between 8.0-8.5. Interestingly, classical enzyme inhibitors such as the tynorphin and its derivative peptide IVYPW were found to actively inhibit the LbDPP3 activity. Moreover, these DPP3 inhibitors showed a detrimental effect upon parasite survival, decreasing the viability of promastigotes by up to 29%. Finally, it was observed that LbDPP3 was equally expressed along the in vitro differentiation from promastigotes to axenic amastigotes. In conclusion, these findings suggest that the L. brazileinsis DPP3 could be a promising drug target.

Synthetic Peptides Derived from Ribosomal Proteins of Leishmania spp. in Mucocutaneous Leishmaniasis: Diagnostic Usefulness.

Abstract: Universidad Nacional de Colombia’s Bogota Research Division (DIB) project 16015, as well as a grant from the Colombian Science, Technology and Innovation Department’s (Colciencias) Young Researchers and Innovators programme "Virginia Gutierrez de Pineda" (2012) and Colciencias Grant (contract number 03092013, code 57635945- project 6).

Peptide Synthesis Using Proteases as Catalyst

Abstract: Proteolytic enzymes (proteases) comprise a group of hydrolases (EC 3.4, NC-IUBMB) which share the common feature of acting on peptide bonds. Proteases are among the best-studied enzymes in terms of structure–function relationship (Krowarsch et al. 2005). Proteases, by catalyzing the cleavage of other proteins and even themselves, have an enormous physiological significance, their coding genes representing as much as 2% of the total human genome (Schilling and Overall 2008).

Enzymatic and chemical synthesis of new anticoagulant peptides.

Abstract: In this study we report the enzymatic synthesis of N-α-[Carbobenzyloxy]-Tyr-Gln-Gln (Z-YQQ), a new anticoagulant tripeptide. It was obtained using phytoproteases from the stems and petioles of Asclepias curassavica L. as catalyst in an aqueous-organic biphasic system formed by 50% (v/v) ethyl acetate and 0.1 M Tris-HCl buffer pH 8. The resulting peptide was compared with the analogous peptide Tyr-Gln-Gln (YQQ) produced by solid-phase chemical synthesis. The in vitro anticoagulant activity of the aforementioned peptides was determined using Wiener Lab Test (Wiener, Argentina). The toxicological activity of the peptides was also determined. The enzymatically synthesized Z-YQQ peptide acted on the extrinsic pathway of the coagulation cascade, delaying the conversion time of prothrombin to thrombin and fibrinogen to fibrin by 136 and 50%, respectively, with respect to the controls. The chemically synthesized YQQ peptide acted specifically on the intrinsic pathway of the coagulation cascade, affecting factors VIII, IX, XI, and XII from such cascade, and increasing the coagulation time by 105% with respect to the control. The results suggest that two new anticoagulant peptides (Z-YQQ and YQQ) can be useful for safe pharmaceutical applications. Nevertheless, some aspects related to peptide production should be optimized. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 2018 © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1093-1101, 2018.

Design of Bactericidal Peptides Against Escherichia coli O157:H7, Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus.

Abstract: Background Antimicrobial peptides are on the first line of defense against pathogenic microorganisms of many living beings. These compounds are considered natural antibiotics that can overcome bacterial resistance to conventional antibiotics. Due to this characteristic, new peptides with improved properties are quite appealing for designing new strategies for fighting pathogenic bacteria. Methods Sixteen designed peptides were synthesized using Fmoc chemistry; five of them are new cationic antimicrobial peptides (CAMPs) designed using a genetic algorithm that optimizes the antibacterial activity based on selected physicochemical descriptors and 11 analog peptides derived from these five peptides were designed and constructed by single amino acid substitutions. These 16 peptides were structurally characterized and their biological activity was determined against Escherichia coli O157:H7 (E. coli O157:H7), and methicillin-resistant strains of Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa (P. aeruginosa) were determined. Results These 16 peptides were folded into an α-helix structure in membrane-mimicking environment. Among these 16 peptides, GIBIM-P5S9K (ATKKCGLFKILKGVGKI) showed the highest antimicrobial activity against E. coli O157:H7 (MIC=10µM), methicillin-resistant Staphylococcus aureus (MRSA) (MIC=25µM) and Pseudomonas aeruginosa (MIC=10 µM). Peptide GIBIM-P5S9K caused permeabilization of the bacterial membrane at 25 µM as determined by the Sytox Green uptake assay and the labelling of these bacteria by using the fluoresceinated peptide. GIBIM-P5S9K seems to be specific for these bacteria because at 50 µM, it provoked lower than 40% of erythrocyte hemolysis. Conclusion New CAMPs have been designed using a genetic algorithm based on selected physicochemical descriptors and single amino acid substitution. These CAMPs interacted quite specifically with the bacterial cell membrane, GIBIM-P5S9K exhibiting high antibacterial activity on Escherichia coli O157:H7, methicillin-resistant strains of Staphylococcus aureus and P. aeruginosa.

Conformationally Restricted Peptides from Rice Proteins Elicit Antibodies That Recognize the Corresponding Native Protein in ELISA Assays

Abstract: The rice hoja blanca virus (RHBV), transmitted by the planthopper insect Tagosodes orizicolus, is a disease that attacks rice and generates significant production losses in Colombia. Fedearroz 2000 and Colombia I commercial rice varieties, which have different resistance levels to the disease, were selected in this study. To identify proteins associated to the insect and virus signaling, a comparative proteomics study was performed. By comparing proteomic profiles, between virus-infected and control group plants in two-dimensional electrophoresis, proteins exhibiting significant changes in abundance were found. In another test, peptide dendrimers containing sequences conformationally restricted to α-helix from four of those rice proteins were synthesized. In the experiment, sera from mice inoculated with peptide dendrimers could recognize the corresponding native protein in ELISA assays. Reported comparative proteomic results provide new insights into the molecular mechanisms of plant response to the RHBV and comprehensive tools for the analysis of new crop varieties. Besides, results from conformational peptide dendrimer approach are promising and show that it is feasible to detect proteins as markers, and may have biological applications by decreasing the susceptibility to proteolytic degradation.