Publications by authors named "Gerardo Corzo"

87 Publications

Identification of a crocodylian β-defensin variant from Alligator mississippiensis with antimicrobial and antibiofilm activity.

Peptides 2021 Apr 15;141:170549. Epub 2021 Apr 15.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A.P. 510-3, Cuernavaca, Mor., 62250, Mexico. Electronic address:

β-defensin host defense peptides are important components of the innate immune system of vertebrates. Although evidence of their broad antimicrobial, antibiofilm and immunomodulatory activities in mammals have been presented, β-defensins from other vertebrate species, like crocodylians, remain largely unexplored. In this study, five new crocodylian β-defensin variants from Alligator mississippiensis and Crocodylus porosus were selected for synthesis and characterization based on their charge and hydrophobicity values. Linear peptides were synthesized, folded, purified and then evaluated for their antimicrobial and antibiofilm activities against the bacterial pathogens, Salmonella enterica serovar Typhimurium, Staphylococcus aureus, Enterobacter cloacae and Acinetobacter baumannii. The Am23SK variant (SCRFSGGYCIWNWERCRSGHFLVALCPFRKRCCK) from A. mississippiensis displayed promising activity against both planktonic cells and bacterial biofilms, outperforming the human β-defensin 3 under the experimental conditions. Moreover, Am23SK exhibited no cytotoxicity towards mammalian cells and exerted immunomodulatory effects in vitro, moderately suppressing the production of proinflammatory mediators from stimulated human bronchial epithelial cells. Overall, our results have expanded the activity landscape of crocodylian and reptilian β-defensin in general.
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http://dx.doi.org/10.1016/j.peptides.2021.170549DOI Listing
April 2021

Structural and functional characterization of NDBP-4 family antimicrobial peptides from the scorpion Mesomexovis variegatus.

Peptides 2021 Apr 20;141:170553. Epub 2021 Apr 20.

Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad, 2001, Colonia Chamilpa, Cuernavaca, Morelos, 62210, Mexico. Electronic address:

Six peptides, belonging to the NDBP-4 family of scorpion antimicrobial peptides were structurally and functionally characterized. The sequence of the mature peptides VpCT1, VpCT2, VpCT3 and VpCT4 was inferred by transcriptomic analysis of the venom gland of the scorpion Mesomexovis variegatus. Analysis of their amino acid sequences revealed patterns that are also present in previously reported peptides that show differences in their hemolytic and antimicrobial activities in vitro. Two other variants, VpCT3W and VpCTConsensus were designed to evaluate the effect of sequence changes of interest on their structure and activity. The synthesized peptides were evaluated by circular dichroism to confirm their α-helical conformation in a folding promoting medium. The peptides were assayed on two Gram-positive and three Gram-negative bacterial strains, and on two yeast strains. They preferentially inhibited the growth of Staphylococcus aureus, were mostly ineffective on Pseudomonas aeruginosa, and moderately inhibited the growth of Candida yeasts. All six peptides exhibited hemolytic activity on human erythrocytes in the range of 4.8-83.7 μM. VpCT3W displayed increased hemolytic and anti-yeast activities, but showed no change in antibacterial activity, relative to its parental peptide, suggesting that Trp6 may potentiate the interaction of VpCT3 with eukaryotic cell membranes. VpCTConsensus showed broader and enhanced antimicrobial activity relative to several of the natural peptides. The results presented here contribute new information on the structure and function of NDBP-4 antimicrobial peptides and provides clues for the design of less hemolytic and more effective antimicrobial peptides.
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http://dx.doi.org/10.1016/j.peptides.2021.170553DOI Listing
April 2021

A Novel Insecticidal Spider Peptide that Affects the Mammalian Voltage-Gated Ion Channel hKv1.5.

Front Pharmacol 2020 13;11:563858. Epub 2021 Jan 13.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, México.

Spider venoms include various peptide toxins that modify the ion currents, mainly of excitable insect cells. Consequently, scientific research on spider venoms has revealed a broad range of peptide toxins with different pharmacological properties, even for mammal species. In this work, thirty animal venoms were screened against hK1.5, a potential target for atrial fibrillation therapy. The whole venom of the spider , which is also insecticidal to house crickets, caused voltage-gated potassium ion channel modulation in hK1.5. Therefore, a peptide from the spider venom, named Osu1, was identified through HPLC reverse-phase fractionation. Osu1 displayed similar biological properties as the whole venom; so, the primary sequence of Osu1 was elucidated by both of N-terminal degradation and endoproteolytic cleavage. Based on its primary structure, a gene that codifies for Osu1 was constructed from protein to DNA by reverse translation. A recombinant Osu1 was expressed using a pQE30 vector inside the SHuffle expression system. recombinant Osu1 had voltage-gated potassium ion channel modulation of human hK1.5, and it was also as insecticidal as the native toxin. Due to its novel primary structure, and hypothesized disulfide pairing motif, Osu1 may represent a new family of spider toxins.
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http://dx.doi.org/10.3389/fphar.2020.563858DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7883638PMC
January 2021

Assessment of neutralization of Micrurus venoms with a blend of anti-Micrurus tener and anti-ScNtx antibodies.

Vaccine 2021 Feb 8;39(6):1000-1006. Epub 2021 Jan 8.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología - UNAM, Av. Universidad 2001, Cuernavaca, Morelos 62210, Mexico. Electronic address:

Background: Micrurus venoms contain two main groups of toxic protein components: short-chain α-neurotoxins (SNtx) and phospholipases type A (PLA). In North America, generally, the Micrurus venoms have low abundance of SNtx compared to that of PLAs; however, both are highly toxic to mammals, and consequently both can play a major role in the envenomation processes. Concerning the commercial horse-derived antivenoms against Micrurus from the North America region, they contain a relatively large amount of antibodies against PLAs, and a low content of antibodies against short chain α-neurotoxins. This is mainly due to the lower relative abundance of SNtxs, and also to its poor immunogenicity due to their size and nature. Hence, Micrurus antivenoms made in North America usually present low neutralizing capacity towards Micrurus venoms whose lethality depend largely on short chain α-neurotoxins, such as South American Micrurus species.

Methods: Horses were hyperimmunized with either the venom of M. tener (PLA-predominant) or a recombinant short-chain consensus α-neurotoxin (ScNtx). Then, the combination of the two monospecific horse antibodies (anti-M. tener and anti-ScNtx) was used to test their efficacy against eleven Micrurus venoms.

Results: The blend of anti-M. tener and anti-ScNtx antibodies had a better capacity to neutralize the lethality of diverse species from North, Central and South American Micrurus venoms. The antibodies combination neutralized both the ScNtx and ten out of eleven Micrurus venom tested, and particularly, it neutralized the venoms of M. distans and M. laticollaris that were neither neutralized by monospecific anti-M. tener nor anti-ScNtx.

Conclusions: These results provide a proof-of-principle for using recombinant immunogens to enrich poor or even non-neutralizing antisera against elapid venoms containing short chain α-neurotoxins to develop antivenoms with higher effectiveness and broader neutralizing capacity.
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http://dx.doi.org/10.1016/j.vaccine.2020.12.052DOI Listing
February 2021

Reptilian β-defensins: Expanding the repertoire of known crocodylian peptides.

Peptides 2021 Feb 10;136:170473. Epub 2020 Dec 10.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, A.P. 510-3, Cuernavaca Mor., 62250, Mexico. Electronic address:

One of the major families of host defense peptides (HDPs) in vertebrates are β-defensins. They constitute important components of innate immunity and have remained an interesting topic of research for more than two decades. While many β-defensin sequences in mammals and birds have been identified and their properties and functions characterized, β-defensin peptides from other groups of vertebrates, particularly reptiles, are still largely unexplored. In this review, we focus on reptilian β-defensins and summarize different aspects of their biology, such as their genomic organization, evolution, structure, and biological activities. Reptilian β-defensin genes exhibit similar genomic organization to birds and their number and gene structure are variable among different species. During the evolution of reptiles, several gene duplication and deletion events have occurred and the functional diversification of β-defensins has been mainly driven by positive selection. These peptides display broad antimicrobial activity in vitro, but a deeper understanding of their mechanisms of action in vivo, including their role as immunomodulators, is still lacking. Reptilian β-defensins constitute unique polypeptide sequences to expand our current understanding of innate immunity in these animals and elucidate core biological functions of this family of HDPs across amniotes.
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http://dx.doi.org/10.1016/j.peptides.2020.170473DOI Listing
February 2021

Antimicrobial Peptide against That Activates Autophagy Is an Effective Treatment for Tuberculosis.

Pharmaceutics 2020 Nov 9;12(11). Epub 2020 Nov 9.

Department of Biochemistry and Structural Biology, Institute of Cellular Physiology, National Autonomous University of Mexico (UNAM), 04510 Mexico City, Mexico.

(MTB) is the principal cause of human tuberculosis (TB), which is a serious health problem worldwide. The development of innovative therapeutic modalities to treat TB is mainly due to the emergence of multi drug resistant (MDR) TB. Autophagy is a cell-host defense process. Previous studies have reported that autophagy-activating agents eliminate intracellular MDR MTB. Thus, combining a direct antibiotic activity against circulating bacteria with autophagy activation to eliminate bacteria residing inside cells could treat MDR TB. We show that the synthetic peptide, IP-1 (KFLNRFWHWLQLKPGQPMY), induced autophagy in HEK293T cells and macrophages at a low dose (10 μM), while increasing the dose (50 μM) induced cell death; IP-1 induced the secretion of TNFα in macrophages and killed Mtb at a dose where macrophages are not killed by IP-1. Moreover, IP-1 showed significant therapeutic activity in a mice model of progressive pulmonary TB. In terms of the mechanism of action, IP-1 sequesters ATP in vitro and inside living cells. Thus, IP-1 is the first antimicrobial peptide that eliminates MDR MTB infection by combining four activities: reducing ATP levels, bactericidal activity, autophagy activation, and TNFα secretion.
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http://dx.doi.org/10.3390/pharmaceutics12111071DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7697726PMC
November 2020

and Antibiotic Capacity of Two Host Defense Peptides.

Antimicrob Agents Chemother 2020 06 23;64(7). Epub 2020 Jun 23.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Mexico City, Mexico

Two nonamidated host defense peptides named Pin2[G] and FA1 were evaluated against three types of pathogenic bacteria: two ( UPD13 and UPD3) isolated from diabetic foot ulcer patients, and another ( serovar Typhimurium [ATCC 14028]) from a commercial collection. experiments showed that the antimicrobial performance of the synthetic peptides Pin2[G] and FA1 was modest, although FA1 was more effective than Pin2[G]. In contrast, Pin2[G] had superior anti-infective activity to FA1 in rabbit wound infections by the diabetic foot ulcer pathogens UPD13 and UPD3. Indeed, Pin2[G] reduced bacterial colony counts of both UPD13 and UPD3 by >100,000-fold after 48 to 72 h on skin wounds of infected rabbits, while in similar infected wounds, FA1 had no major effects at 72 to 96 h of treatment. Ceftriaxone was equally effective versus but less effective versus infections. Additionally, the two peptides were evaluated in mice against intragastrically inoculated serovar Typhimurium (ATCC 14028). Only Pin2[G] at 0.56 mg/kg was effective in reducing systemic (liver) infection by >67-fold, equivalent to the effect of treatment with levofloxacin. Pin2[G] showed superior immunomodulatory activity in increasing chemokine production by a human bronchial cell line and suppressing polyinosinic-polycytidylic acid (poly[I:C])-induced proinflammatory IL-6 production. These data showed that the antimicrobial activity of these peptides was not correlated with their anti-infective activity and suggest that other factors such as immunomodulatory activity were more important.
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http://dx.doi.org/10.1128/AAC.00145-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7317995PMC
June 2020

Venom gland transcriptome from Heloderma horridum horridum by high-throughput sequencing.

Toxicon 2020 Jun 10;180:62-78. Epub 2020 Apr 10.

Facultad de Ciencias Químicas, Universidad de Colima, Colima, Mexico.

Lizards of the Helodermatidae (Anguimorpha) family consist of at least two well recognized species: Heloderma horridum horridum and Heloderma suspectum suspectum. They contain specialized glands in their jaws that produce venomous secretions that causes envenoming symptoms to bitten animals. One way to study proteins from such secretions is by RNA-seq; a powerful molecular tool to characterize the transcriptome of such specialized gland, and its protein secretions. The total RNA from venom gland tissues of H. horridum horridum was extracted and a cDNA library was constructed and sequenced. Overall, 114,172 transcripts were found, and 199 were annotated based on sequence similarities to previously described peptides/proteins. Transcripts coding for putative exendins, defensins, natriuretics and serine protease inhibitors were the most highly expressed. Transcripts that code for several putative serine proteases, phospholipases, metalloproteases, lipases, L-amino oxidase and nucleases were also found. Some of the novel identified transcripts were translationally controlled tumor proteins, venom factors, vespryns, waprins, lectins, cystatins and serine protease inhibitors. All these new protein structures may contribute to a better understanding of the venomous secretions of the Helodermatidae family.
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http://dx.doi.org/10.1016/j.toxicon.2020.04.003DOI Listing
June 2020

Key amino acid residues involved in mammalian and insecticidal activities of Magi4 and Hv1b, cysteine-rich spider peptides from the δ-atracotoxin family.

Amino Acids 2020 Mar 17;52(3):465-475. Epub 2020 Feb 17.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Avenida Universidad, 2001, Apartado Postal 510-3, 62210, Cuernavaca Mor., México.

δ-Atracotoxins, also known as δ-hexatoxins, are spider neurotoxic peptides, lethal to both vertebrates and insects. Their mechanism of action involves the binding to of the S3/S4 loop of the domain IV of the voltage-gated sodium channels (Na). Because of the chemical difficulties of synthesizing folded synthetic δ-atracotoxins correctly, here we explore an expression system that is designed to produce biologically active recombinant δ-atracotoxins, and a number of variants, in order to establish certain amino acids implicated in the pharmacophore of this lethal neurotoxin. In order to elucidate and verify which amino acid residues play a key role that is toxic to vertebrates and insects, amino acid substitutes were produced by aligning the primary structures of several lethal δ-atracotoxins with those of δ-atracotoxins-Hv1b; a member of the δ-atracotoxin family that has low impact on vertebrates and is not toxic to insects. Our findings corroborate that the substitutions of the amino acid residue Y22 from δ-atracotoxin-Mg1a (Magi4) to K22 in δ-atracotoxin-Hv1b reduces its mammalian activity. Moreover, the substitutions of the amino acid residues Y22 and N26 from δ-atracotoxin-Mg1a (Magi4) to K22 and N26 in δ-atracotoxin-Hv1b reduces its insecticidal activity. Also, the basic residues K4 and R5 are important for keeping such insecticidal activity. Structural models suggest that such residues are clustered onto two bioactive surfaces, which share similar areas, previously reported as bioactive surfaces for scorpion α-toxins. Furthermore, these bioactive surfaces were also found to be similar to those found in related spider and anemone toxins, which affect the same Na receptor, indicating that these motifs are important not only for scorpion but may be also for animal toxins that affect the S3/S4 loop of the domain IV of the Na.
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http://dx.doi.org/10.1007/s00726-020-02825-4DOI Listing
March 2020

Immunogenic Properties of Recombinant Enzymes from Towards the Generation of Neutralizing Antibodies against Its Own Venom.

Toxins (Basel) 2019 12 2;11(12). Epub 2019 Dec 2.

Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Av. Universidad 2001, Cuernavaca Mor 62209, México.

Bothropic venoms contain enzymes such as metalloproteases, serine-proteases, and phospholipases, which acting by themselves, or in synergism, are the cause of the envenomation symptoms and death. Here, two mRNA transcripts, one that codes for a metalloprotease and another for a serine-protease, were isolated from a venom gland. The metalloprotease and serine-protease transcripts were cloned on a pCR2.1-TOPO vector and consequently expressed in a recombinant way in E. coli (strains Origami and M15, respectively), using pQE30 vectors. The recombinant proteins were named rBamSP_1 and rBamMP_1, and they were formed by an N-terminal fusion protein of 16 amino acid residues, followed by the sequence of the mature proteins. After bacterial expression, each recombinant enzyme was recovered from inclusion bodies and treated with chaotropic agents. The experimental molecular masses for rBamSP_1 and rBamMP_1 agreed with their expected theoretical ones, and their secondary structure spectra obtained by circular dichroism were comparable to that of similar proteins. Additionally, equivalent mixtures of rBamSP_1, rBamMP_1 together with a previous reported recombinant phospholipase, rBamPLA2_1, were used to immunize rabbits to produce serum antibodies, which in turn recognized serine-proteases, metalloproteases and PLA2s from and other regional viper venoms. Finally, rabbit antibodies neutralized the 3LD50 of venom.
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http://dx.doi.org/10.3390/toxins11120702DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6949999PMC
December 2019

Improving the heterologous expression of human β-defensin 2 (HBD2) using an experimental design.

Protein Expr Purif 2020 03 9;167:105539. Epub 2019 Nov 9.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM. Apartado Postal 510-3, Cuernavaca Morelos, 61500, Mexico.

At present, expressing antimicrobial peptides in bacterial models is considered a routine lab bench work. However, low expression yields of these types of proteins are usually obtained. In this work, the antimicrobial peptide human β-defensin 2 (HBD2) was obtained in low expression yields in Escherichia coli BL21(DE3). To improve the expression yields of HBD2, some variables such as cell density, temperature, and length of induction, as well as the inducer concentration, were investigated using a 2-factorial design of experiments (DoE). This approach allowed us to identify the identification of critical variables (main effects and interactions among factors) affecting bacterial HBD2 expression. After the evaluation of 19 different combination, the best condition to express HBD2 had a pre-induction temperature of 37 °C, a cell density of 1.0 U (600 nm), an induction temperature of 20 °C and a 0.1 mM of gene expression inducer (IPTG) over four hours. Under such conditions, the expression yield of the HBD2 peptide was one order of magnitude higher than the peptide expression performed initially.
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http://dx.doi.org/10.1016/j.pep.2019.105539DOI Listing
March 2020

Biophysical characterization of the insertion of two potent antimicrobial peptides-Pin2 and its variant Pin2[GVG] in biological model membranes.

Biochim Biophys Acta Biomembr 2020 02 2;1862(2):183105. Epub 2019 Nov 2.

Instituto de Ciencias Físicas, Universidad Autónoma Nacional de México (ICF-UNAM), Avenida Universidad 2001, Chamilpa, 62210 Cuernavaca, Morelos, Mexico. Electronic address:

The aim of this study was to investigate the factors that govern the activity and selectivity of two potent antimicrobial peptides (AMPs) using lipid membrane models of bacterial, erythrocyte and fungal cells. These models were used in calcein liposome leakage experiments to explore peptide efficiency. The AMPs (Pin2 and its variant Pin2[GVG]) showed highest affinity towards the bacterial models in the nanomolar range, followed by the erythrocyte and fungal systems. The presence of sterols modulated the variant's selectivity, while the wild type was unaffected. Liposome leakage experiments with Fluorescein Isothiocyanate-dextran (FITC)-dextran conjugates indicated that pore size depended on peptide concentration. Dynamic Light Scattering revealed peptide aggregation in aqueous solution, and that aggregate size was related to activity. The interacting peptides did not alter liposome size, suggesting pore forming activity rather than detergent activity. Atomic Force Microscopy showed differential membrane absorption, being greater in the bacterial model compared to the mammalian model, and pore-like defects were observed. Electrophysiological assays with the Tip-Dip Patch Clamp method provided evidence of changes in the electrical resistance of the membrane. Membrane potential experiments showed that liposomes were also depolarized in the presence of the peptides. Both peptides increased the Laurdan Generalized Polarization of the bacterial model indicating increased viscosity, on the contrary, no effect was observed with the erythrocyte and the fungal models. Peptide membrane insertion and pore formation was corroborated with Langmuir Pressure-Area isotherms and Brewster Angle Microscopy. Finally, molecular dynamics simulations were used to get an insight into the molecular mechanism of action.
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http://dx.doi.org/10.1016/j.bbamem.2019.183105DOI Listing
February 2020

Horse immunization with short-chain consensus α-neurotoxin generates antibodies against broad spectrum of elapid venomous species.

Nat Commun 2019 08 13;10(1):3642. Epub 2019 Aug 13.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México - UNAM, Apartado Postal 510-3, Cuernavaca Morelos, 61500, Mexico.

Antivenoms are fundamental in the therapy for snakebites. In elapid venoms, there are toxins, e.g. short-chain α-neurotoxins, which are quite abundant, highly toxic, and consequently play a major role in envenomation processes. The core problem is that such α-neurotoxins are weakly immunogenic, and many current elapid antivenoms show low reactivity towards them. We have previously developed a recombinant consensus short-chain α-neurotoxin (ScNtx) based on sequences from the most lethal elapid venoms from America, Africa, Asia, and Oceania. Here we report that an antivenom generated by immunizing horses with ScNtx can successfully neutralize the lethality of pure recombinant and native short-chain α-neurotoxins, as well as whole neurotoxic elapid venoms from diverse genera such as Micrurus, Dendroaspis, Naja, Walterinnesia, Ophiophagus and Hydrophis. These results provide a proof-of-principle for using recombinant proteins with rationally designed consensus sequences as universal immunogens for developing next-generation antivenoms with higher effectiveness and broader neutralizing capacity.
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http://dx.doi.org/10.1038/s41467-019-11639-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6692343PMC
August 2019

Folding profiles of antimicrobial scorpion venom-derived peptides on hydrophobic surfaces: a molecular dynamics study.

J Biomol Struct Dyn 2020 Jul 2;38(10):2928-2938. Epub 2019 Aug 2.

Instituto de Ciencias Físicas, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, México.

Most helical antimicrobial peptides (AMPs) are usually unfolded in aqueous solution; however they acquire their secondary structure in the presence of a hydrophobic environment such as lipid membranes. Being the biological membranes the main target of many AMPs it is necessary to understand their way of action. Pandinin 2 (Pin2) is an alpha-helical AMP isolated from the venom of the African scorpion which shows high antimicrobial activity against Gram-positive bacteria and it is less active against Gram-negative bacteria, nevertheless, it has strong hemolytic activity. Its chemically synthesized Pin2GVG analog has low hemolytic activity while keeping its antimicrobial activity. With the aim of exploring the partition and subsequent folding of these peptides, in this work we report the results of extensive molecular dynamics simulations of Pin2 and Pin2GVG peptides in the presence of 2 hydrophobic environments such as dodecyl-phosphocholine (DPC) micelle and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocoline (POPC) membrane. Our results indicate that Pin2 folds in DPC with a 79% of alpha-helical content, which is in agreement with the experimental results, while in POPC it has 62.5% of alpha-helical content. On the other hand, Pin2GVG presents a higher percentage of alpha-helical structure in POPC and a smaller content in DPC when compared with Pin2. These results can help to better choose the starting structures in future molecular dynamics simulations of AMPs, because these peptides can adopt slightly different conformations depending on the hydrophobic environment.Communicated by Ramaswamy H. Sarma.
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http://dx.doi.org/10.1080/07391102.2019.1648319DOI Listing
July 2020

Antimicrobial activity and structure of a consensus human β-defensin and its comparison to a novel putative hBD10.

Proteins 2020 01 30;88(1):175-186. Epub 2019 Jul 30.

Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Mexico.

The spread of multidrug resistant bacteria owing to the intensive use of antibiotics is challenging current antibiotic therapies, and making the discovery and evaluation of new antimicrobial agents a high priority. The evaluation of novel peptide sequences of predicted antimicrobial peptides from different sources is valuable approach to identify alternative antibiotic leads. Two strategies were pursued in this study to evaluate novel antimicrobial peptides from the human β-defensin family (hBD). In the first, a 32-residue peptide was designed based on the alignment of all available hBD primary structures, while in the second a putative 35-residue peptide, hBD10, was mined from the gene DEFB110. Both hBDconsensus and hBD10 were chemically synthesized, folded and purified. They showed antimicrobial activity against Escherichia coli, Staphylococcus aureus, and Mycobacterium tuberculosis, but were not hemolytic on human red blood cells. The NMR-based solution structure of hBDconsensus revealed that it adopts a classical β-defensin fold and disulfide connectivities. Even though the mass spectrum of hBD10 confirmed the formation of three disulfide bonds, it showed limited dispersion in H NMR spectra and structural studies were not pursued. The evaluation of different β-defensin structures may identify new antimicrobial agents effective against multidrug-resistant bacterial strains.
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http://dx.doi.org/10.1002/prot.25785DOI Listing
January 2020

Enhanced Tolerance against a Fungal Pathogen and Insect Resistance in Transgenic Tobacco Plants Overexpressing an Endochitinase Gene from .

Int J Mol Sci 2019 Jul 16;20(14). Epub 2019 Jul 16.

Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Av. Universidad 1001, Col. Chamilpa, Cuernavaca, Morelos 62209, Mexico.

In this study we cloned a chitinase gene (), from isolated from the corpse of a lepidopteran, which is an important sugarcane pest. The chitinase gene amplified from the genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco ( L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus and to the pest was evaluated: both the activity of chitinase as well as the resistance against and was significantly higher in transgenic plants compared to the wild-type.
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http://dx.doi.org/10.3390/ijms20143482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6679225PMC
July 2019

Nonenzymatically oxidized arachidonic acid regulates T-type Ca currents in mouse spermatogenic cells.

FEBS Lett 2019 07 6;593(14):1735-1750. Epub 2019 Jun 6.

Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, México, México.

During spermatogenesis, fatty acids play an important role both as structural components and messengers that trigger male germ cell line differentiation. The spontaneous oxidation of fatty acids causes a decrease in mammalian fertility. Here, we examine the effects of nonenzymatically oxidized arachidonic acid (AA ) on mouse spermatogenic T-type Ca currents (I ) due to their physiological relevance during spermatogenesis. AA is 25-fold more potent than AA at inhibiting I and it left shifts the I-V curve peak and both activation and steady-state inactivation curves. In addition, I deactivation kinetics and their recovery from inactivation are slower in the presence of AA . Therefore, the fraction of inactivated Ca channels is increased. AA -induced I inhibition could contribute to male infertility affecting Ca regulation in spermatogenic cells.
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http://dx.doi.org/10.1002/1873-3468.13448DOI Listing
July 2019

Pore-forming spider venom peptides show cytotoxicity to hyperpolarized cancer cells expressing K+ channels: A lentiviral vector approach.

PLoS One 2019 12;14(4):e0215391. Epub 2019 Apr 12.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Cuernavaca, Morelos, Mexico.

Recent studies demonstrated the upregulation of K+ channels in cancer cells. We have previously found that a pore-forming peptide LaFr26, purified from the venom of the Lachesana sp spider, was selectively incorporated into K+ channel expressing hyperpolarized cells. Therefore, it is expected that this peptide would have selective cytotoxicity to hyperpolarized cancer cells. Here we have tested whether LaFr26 and its related peptide, oxyopinin-2b, are selectively cytotoxic to K+ channel expressing cancer cells. These peptides were cytotoxic to the cells, of which resting membrane potential was hyperpolarized. The vulnerabilities of K+ channel-expressing cell lines correlated with their resting membrane potential. They were cytotoxic to lung cancer cell lines LX22 and BEN, which endogenously expressed K+ current. Contrastingly, these peptides were ineffective to glioblastoma cell lines, U87 and T98G, of which membrane potentials were depolarized. Peptides have a drawback, i.e. poor drug-delivery, that hinders their potential use as medicine. To overcome this drawback, we prepared lentiviral vectors that can express these pore-forming peptides and tested the cytotoxicity to K+ channel expressing cells. The transduction with these lentiviral vectors showed autotoxic activity to the channel expressing cells. Our study provides the basis for a new oncolytic viral therapy.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0215391PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461346PMC
February 2020

Use of irradiated elapid and viperid venoms for antivenom production in small and large animals.

Toxicon 2018 Dec 10;155:32-37. Epub 2018 Oct 10.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM, Avenida Universidad 2001, Cuernavaca, Morelos, 62210, Mexico.

This work evaluated the feasibility of using toxoids obtained by gamma radiation in the production of antivenoms in small and large animals. Mixtures of African snake venoms from viperids or elapids were used. The viperid mixture contained the crude venom of five species of the genera Echis and Bitis, while the elapid mixture contained the crude venom of six species of the genera Naja and Dendroaspis. The viperid mixture had an LD of 1.25 mg/kg in mice, and the elapid mixture had an LD of 0.46 mg/kg. Both viper and elapid aqueous mixtures were subjected to Cobalt-60 gamma irradiation in three physical states: lyophilized, frozen and liquid. Radiation doses ranged from 0.5 to 100 kGy. The LDs of the lyophilized and frozen mixtures of both viperid and elapid mixtures remained unaltered with radiation doses as high as 100 kGy; nevertheless, in the liquid state, doses of 3.5 and 5.5 kGy reduced the venom toxicity of both the viperid and elapid mixtures to 7.25 mg/kg and 1.74 mg/kg; less toxic by factors of 5.8 and 3.8, respectively. Groups of four rabbits and three horses were immunized with either irradiated or non-irradiated mixtures. In vitro and in vivo analysis of the rabbit and horse sera revealed that neutralizing antibodies were produced against both irradiated (toxoids) and native venom mixtures. None of the animals used in this study, either immunized with native venom or toxoids, developed severe local effects due to the application of venoms mixtures. Gamma-irradiated detoxified venoms mixtures, under well-controlled and studied conditions, could be a practical alternative for the production of polyvalent equine serum with high neutralization potency against snake venoms.
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http://dx.doi.org/10.1016/j.toxicon.2018.10.001DOI Listing
December 2018

cDNA cloning, heterologous expression, protein folding and immunogenic properties of a phospholipase A from Bothrops ammodytoides venom.

Protein Expr Purif 2019 02 8;154:33-43. Epub 2018 Sep 8.

Centro de Investigación en Biotecnología, Universidad Autónoma del Estado de Morelos, Av. Universidad 2001, Cuernavaca Mor, 62209, Mexico. Electronic address:

A mRNA transcript that codes for a phospholipase (PLA) was isolated from a single venom gland of the Bothrops ammodytoides viper. The PLA transcript was cloned onto a pCR2.1-TOPO vector and subsequently expressed heterologously in the E. coli strain M15, using the pQE30 vector. The recombinant phospholipase was named rBamPLA2_1, and is composed of an N-terminal fusion protein of 16 residues, along with 122 residues from the mature protein that includes 14 cysteines that form 7 disulfide bonds. Following bacterial expression, rBamPLA2_1 was obtained from inclusion bodies and extracted using a chaotropic agent. rBamPLA2_1 had an experimental molecular mass of 15,692.5 Da that concurred with its theoretical molecular mass. rBamPLA2_1 was refolded in in vitro conditions and after refolding, three main protein fractions with similar molecular masses, were identified. Although, the three fractions were considered to represent different oxidized cystine isoforms, their secondary structures were comparable. All three recombinant isoforms were active on egg-yolk phospholipid and recognized similar cell membrane phospholipids to be native PLAs, isolated from B. ammodytoides venom. A mixture of the three rBamPLA2_1 cystine isoforms was used to immunize a horse in order to produce serum antibodies (anti-rBamPLA2_1), which partially inhibited the indirect hemolytic activity of B. ammodytoides venom. Although, anti-rBamPLA2_1 antibodies were not able to recognize crotoxin, a PLA from the venom of a related but different viper genus, Crotalus durissus terrificus, they recognized PLAs in other venoms from regional species of Bothrops.
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http://dx.doi.org/10.1016/j.pep.2018.09.004DOI Listing
February 2019

Short-chain consensus alpha-neurotoxin: a synthetic 60-mer peptide with generic traits and enhanced immunogenic properties.

Amino Acids 2018 Jul 6;50(7):885-895. Epub 2018 Apr 6.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM, Apartado Postal 510-3, 61500, Cuernavaca, Morelos, Mexico.

The three-fingered toxin family and more precisely short-chain α-neurotoxins (also known as Type I α-neurotoxins) are crucial in defining the elapid envenomation process, but paradoxically, they are barely neutralized by current elapid snake antivenoms. This work has been focused on the primary structural identity among Type I neurotoxins in order to create a consensus short-chain α-neurotoxin with conserved characteristics. A multiple sequence alignment considering the twelve most toxic short-chain α-neurotoxins reported from the venoms of the elapid genera Acanthophis, Oxyuranus, Walterinnesia, Naja, Dendroaspis and Micrurus led us to propose a short-chain consensus α-neurotoxin, here named ScNtx. The synthetic ScNtx gene was de novo constructed and cloned into the expression vector pQE30 containing a 6His-Tag and an FXa proteolytic cleavage region. Escherichia coli Origami cells transfected with the pQE30/ScNtx vector expressed the recombinant consensus neurotoxin in a soluble form with a yield of 1.5 mg/L of culture medium. The 60-amino acid residue ScNtx contains canonical structural motifs similar to α-neurotoxins from African elapids and its LD of 3.8 µg/mice is similar to the most toxic short-chain α-neurotoxins reported from elapid venoms. Furthermore, ScNtx was also able to antagonize muscular, but not neuronal, nicotinic acetylcholine receptors (nAChR). Rabbits immunized with ScNtx were able to immune-recognize short-chain α-neurotoxins within whole elapid venoms. Type I neurotoxins are difficult to isolate and purify from natural sources; therefore, the heterologous expression of molecules such ScNtx, bearing crucial motifs and key amino acids, is a step forward to create common immunogens for developing cost-effective antivenoms with a wider spectrum of efficacy, quality and strong therapeutic value.
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http://dx.doi.org/10.1007/s00726-018-2556-0DOI Listing
July 2018

Giant fish-killing water bug reveals ancient and dynamic venom evolution in Heteroptera.

Cell Mol Life Sci 2018 09 9;75(17):3215-3229. Epub 2018 Feb 9.

Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072, Australia.

True Bugs (Insecta: Heteroptera) produce venom or saliva with diverse bioactivities depending on their feeding strategies. However, little is known about the molecular evolution of the venom toxins underlying these biological activities. We examined venom of the giant fish-killing water bug Lethocerus distinctifemur (Insecta: Belostomatidae) using infrared spectroscopy, transcriptomics, and proteomics. We report 132 venom proteins including putative enzymes, cytolytic toxins, and antimicrobial peptides. Over 73% (96 proteins) showed homology to venom proteins from assassin bugs (Reduviidae), including 21% (28 proteins from seven families) not known from other sources. These data suggest that numerous protein families were recruited into venom and diversified rapidly following the switch from phytophagy to predation by ancestral heteropterans, and then were retained over > 200 my of evolution. In contrast, trophic switches to blood-feeding (e.g. in Triatominae and Cimicidae) or reversions to plant-feeding (e.g., in Pentatomomorpha) were accompanied by rapid changes in the composition of venom/saliva, including the loss of many protein families.
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http://dx.doi.org/10.1007/s00018-018-2768-1DOI Listing
September 2018

Cloning and sequencing of three-finger toxins from the venom glands of four Micrurus species from Mexico and heterologous expression of an alpha-neurotoxin from Micrurus diastema.

Biochimie 2018 Apr 31;147:114-121. Epub 2018 Jan 31.

Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad # 2001, Colonia Chamilpa, CP: 62210 Cuernavaca, Morelos, Mexico. Electronic address:

The three-finger toxins (3FTxs) represent an extremely diverse protein family in elapid venoms, where the short chain α-neurotoxins are the most relevant toxin group from the clinical point of view. Essentially, the 3FTxs variability and the low proportions of α-neurotoxins in the venoms of North American coral snakes make it difficult to obtain effective elapid antivenoms against the envenomation symptoms caused mainly by these α-neurotoxins. In this work, thirty 3FTx transcript sequences were obtained from the venom glands of four coral snake species from Mexico (M. diastema, M. laticollaris, M. browni and M. tener). The transcripts were mined using a forward oligonucleotide based on the highly conserved signal peptide from the 3FTxs, and four of these transcripts, named MlatA1, B.D, B.E and D.H, encoded for short-chain α-neurotoxins. Additionally, one isoform of the D.H α-neurotoxin transcript was identified in the venom of M. diastema. The mature α-neurotoxin coded in the D.H transcript was heterologously expressed, and it was found soluble (4.2 mg/l) in the cytoplasm of a bacterial system. The recombinant D.H (rD.H) had an IC value of 31.5 ± 4.4 nM on nicotinic acetylcholine receptors of the muscular type expressed in rhabdomyosarcoma cells (TE671). The rDH also had an LD of 0.15 mg/kg mice, and it was evaluated as a potential immunogen in New Zealand rabbits. The protective capacity of rabbit sera was tested against two native coral snake α-neurotoxins, and against rD.H. One of the anti-rD.H rabbit sera was able to neutralize the lethality of all three neurotoxins when tested in groups of CD1 mice. This work contributes to the increasing understanding of the high diversity of 3FTxs, and shows that recombinant coral snake α-neurotoxins are promising supplements for hyperimmunization protocols for coral snake antivenom production.
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http://dx.doi.org/10.1016/j.biochi.2018.01.006DOI Listing
April 2018

Successful refolding and NMR structure of rMagi3: A disulfide-rich insecticidal spider toxin.

Protein Sci 2018 03 3;27(3):692-701. Epub 2018 Jan 3.

Departamento de Química de Biomacromoléculas, Instituto de Química, Universidad Nacional Autónoma de México, CU, Ciudad de México, 04510, México.

The need for molecules with high specificity against noxious insects leads the search towards spider venoms that have evolved highly selective toxins for insect preys. In this respect, spiders as a highly diversified group of almost exclusive insect predators appear to possess infinite potential for the discovery of novel insect-selective toxins. In 2003, a group of toxins was isolated from the spider Macrothele gigas and the amino acid sequence was reported. We obtained, by molecular biology techniques in a heterologous system, one of these toxins. Purification process was optimized by chromatographic methods to determine the three-dimensional structure by nuclear magnetic resonance in solution, and, finally, their biological activity was tested. rMagi3 resulted to be a specific insect toxin with no effect on mice.
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http://dx.doi.org/10.1002/pro.3363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5818767PMC
March 2018

Venoms of Centruroides and Tityus species from Panama and their main toxic fractions.

Toxicon 2018 Jan 2;141:79-87. Epub 2017 Dec 2.

Centro de Investigación e Información de Medicamentos y Tóxicos (CIIMET), Facultad de Medicina, Universidad de Panamá, Ciudad de Panamá, Panama. Electronic address:

The scorpionism in Panama is notorious for the confluence and coexistence of buthid scorpions from the genera Centruroides and Tityus. This communication describes an overview of the larger representative toxic venom fractions from eight dangerous buthid scorpion species of Panama: Centruroides (C. granosus, C. bicolor, C. limbatus and C. panamensis) and Tityus (T. (A.) asthenes, T. (A.) festae, T. (T.) cerroazul and T. (A.) pachyurus). Their venoms were separated by HPLC and the corresponding sub-fractions were tested for lethality effects on mice and insects. Many fractions toxic to either mice or insects, or both, were found and have had their molecular masses determined by mass spectrometry analysis. The great majority of the lethal components had a molecular mass close to 7000 Da, assumed to be peptides that recognize Na-channels, responsible for the toxicity symptoms observed in other buthids scorpion venoms. A toxic peptide isolated from the venom of T. pachyurus was sequenced by Edman degradation, allowing the synthesis of nucleotide probe for cloning the correspondent gene. The mature toxin based on the cDNA sequencing has the C-terminal residue amidated, contains 62 amino acid packed by 4 disulfide linkages, with molecular mass of 7099.1 Da. This same toxic peptide seems to be present in scorpions of the species T. pachyurus collected in 5 different regions of Panama, although the overall HPLC profile is quite different. The most diverse neurotoxic venom components from the genus Centruroides were found in the species C. panamensis, whereas T. cerroazul was the one from the genus Tityus. The most common neurotoxins were observed in the venoms of T. festae, T. asthenes and T. pachyurus with closely related molecular masses of 7099.1 and 7332 Da. The information reported here is considered very important for future generation of a neutralizing antivenom against scorpions from Panama. Furthermore, it will contribute to the growing interest in using bioactive toxins from scorpions for drug discovery purposes.
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http://dx.doi.org/10.1016/j.toxicon.2017.11.013DOI Listing
January 2018

Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus.

Protein Expr Purif 2017 Aug 15;136:45-51. Epub 2017 Jun 15.

Unidad de Bioquímica y Biología Molecular de Plantas. Centro de Investigación Científica de Yucatán A.C., Calle 43 No. 130, Col. Chuburná de Hidalgo, Mérida, Yucatán 97205, México. Electronic address:

The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa.
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http://dx.doi.org/10.1016/j.pep.2017.06.007DOI Listing
August 2017

Molecular dynamics simulation of the membrane binding and disruption mechanisms by antimicrobial scorpion venom-derived peptides.

J Biomol Struct Dyn 2018 Jun 22;36(8):2070-2084. Epub 2017 Jun 22.

a Instituto de Ciencias Físicas , Universidad Nacional Autónoma de México , Cuernavaca , Morelos 62210 , México.

Pandinin 2 (Pin2) is an alpha-helical polycationic peptide, identified and characterized from venom of the African scorpion Pandinus imperator with high antimicrobial activity against Gram-positive bacteria and less active against Gram-negative bacteria, however it has demonstrated strong hemolytic activity against sheep red blood cells. In the chemically synthesized Pin2GVG analog, the GVG motif grants it low hemolytic activity while keeping its antimicrobial activity. In this work, we performed 12 μs all-atom molecular dynamics simulation of the antimicrobial peptides (AMPs) Pin2 and Pin2GVG to explore their adsorption mechanism and the role of their constituent amino acid residues when interacting with pure POPC and pure POPG membrane bilayers. Starting from an α-helical conformation, both AMPs are attracted at different rates to the POPC and POPG bilayer surfaces due to the electrostatic interaction between the positively charged amino acid residues and the charged moieties of the membranes. Since POPG is an anionic membrane, the PAMs adhesion is stronger to the POPG membrane than to the POPC membrane and they are stabilized more rapidly. This study reveals that, before the insertion begins, Pin2 and Pin2GVG remained partially folded in the POPC surface during the first 300 and 600 ns, respectively, while they are mostly unfolded in the POPG surface during most of the simulation time. The unfolded structures provide for a large number of intermolecular hydrogen bonds and stronger electrostatic interactions with the POPG surface. The results show that the aromatic residues at the N-terminus of Pin2 initiate the insertion process in both POPC and POPG bilayers. As for Pin2GVG in POPC the C-terminus residues seem to initiate the insertion process while in POPG this process seems to be slowed down due to a strong electrostatic attraction. The membrane conformational effects upon PAMs binding are measured in terms of the area per lipid and the contact surface area. Several replicas of the systems lead to the same observations.
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http://dx.doi.org/10.1080/07391102.2017.1341340DOI Listing
June 2018

Proteomic and transcriptomic analysis of saliva components from the hematophagous reduviid Triatoma pallidipennis.

J Proteomics 2017 06 22;162:30-39. Epub 2017 Apr 22.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM, Apartado Postal 510-3, Cuernavaca, Morelos 61500, Mexico. Electronic address:

Species belonging to the Triatominae subfamily are commonly associated with Chagas disease, as they are potential vectors of the parasite Trypanosoma cruzi. However, their saliva contains a cocktail of diverse anti-hemostatic proteins that prevent blood coagulation, vasodilation and platelet aggregation of blood; components with indisputable therapeutic potential. We performed a transcriptomic and proteomic analyses of salivary glands and protein spots from 2DE gels of milked saliva, respectively, from the Mexican Triatoma pallidipennis. Massive sequencing techniques were used to reveal this protein diversity. A total of 78 out of 233 transcripts were identified as proteins in the saliva, divided among 43 of 55 spots from 2DE gels of saliva, identified by LC-MS/MS analysis. Some of the annotated transcripts putatively code for anti-hemostatic proteins, which share sequence similarities with proteins previously described for South American triatomines. The most abundant as well as diverse transcripts and proteins in the saliva were the anti-hemostatic triabins. For the first time, a transcriptomic analysis uncovered other unrelated but relevant components in triatomines, including antimicrobial and thrombolytic polypeptides. Likewise, unique proteins such as the angiotensin-converting enzyme were identified not just in the salivary gland transcriptome but also at saliva proteome of this North American bloodsucking insect.

Biological Significance: This manuscript is the first report of the correlation between proteome and transcriptome of Triatoma pallidipennis, which shows for the first time the presence of proteins in this insect that have not been characterized in other species of this family. This information contributes to a better understanding of the multiple host defense mechanisms that are being affected at the moment of blood ingestion by the insect. Furthermore, this report gives a repertoire of possible therapeutic proteins.
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http://dx.doi.org/10.1016/j.jprot.2017.04.022DOI Listing
June 2017

Selected scorpion toxin exposures induce cytokine release in human peripheral blood mononuclear cells.

Toxicon 2017 Mar 11;127:56-62. Epub 2017 Jan 11.

Baylor Institute For Immunology Research, 3410 Worth Street, Suite 600, Dallas, TX 75246, USA. Electronic address:

A cytokine screening on human peripheral blood mononuclear cells (PBMCs) stimulated with selected scorpion toxins (ScTx's) was performed in order to evaluate their effect on human immune cells. The ScTx's chosen for this report were three typical buthid scorpion venom peptides, one with lethal effects on mammals Centruroides suffussus suffusus toxin II (CssII), another, with lethal effects on insects and crustaceans Centruroides noxius toxin 5 (Cn5), and one more without lethal effects Tityus discrepans toxin (Discrepin). A Luminex multiplex analysis was performed in order to determine the amounts chemokines and cytokines IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12-p40, IL-13, interferon alpha (IFN-α), interferon gamma (IFN-γ), tumor necrosis factor alpha TNF-α, and interferon-inducible protein-10 (IP-10) secreted from human PBMCs exposed to these toxins. Although, the ScTx Cn5 is not lethal for mammals, it was able to induce the secretion of cytokines IL-1β, IL-6, and TNF-α, IL-10 and IP-10 in comparison to the lethal CssII, which was able to induce only IP-10 secretion. Discrepin also was able to induce only IP-10. Interestingly, only low amounts of interferons α and β were induced in the presence of the ScTx's assayed. In a synergic experiment, the combination of Discrepin and Cn5 displayed considerable reverse effects on induction of IL-1β, IL-6, IL-10 and TNF-α, but they had a slight synergic effect on IP-10 cytokine production in comparison with the single effect obtained with the Cn5 alone. Thus, the results obtained suggest that the profile of secreted cytokines promoted by ScTx Cn5 is highly related with a cytokine storm event, and also it suggests that the mammalian lethal neurotoxins are not solely responsible of the scorpion envenomation symptomatology.
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http://dx.doi.org/10.1016/j.toxicon.2017.01.007DOI Listing
March 2017

Synthetic peptide antigens derived from long-chain alpha-neurotoxins: Immunogenicity effect against elapid venoms.

Peptides 2017 02 21;88:80-86. Epub 2016 Dec 21.

Departamento de Medicina Molecular y Bioprocesos, Instituto de Biotecnología, Universidad Nacional Autónoma de México, UNAM, Apartado Postal 510-3, Cuernavaca Morelos 61500, Mexico. Electronic address:

Three-finger toxins (3FTXs), especially α-neurotoxins, are the most poorly neutralized elapid snake toxins by current antivenoms. In this work, the conserved structural similarity and motif arrangements of long-chain α-neurotoxins led us to design peptides with consensus sequences. Eight long-chain α-neurotoxins (also known as Type II) were used to generate a consensus sequence from which two peptides were chemically synthesized, LCP1 and LCP2. Rabbit sera raised against them were able to generate partially-neutralizing antibodies, which delayed mice mortality in neutralization assays against Naja haje, Dendrospis polylepis and Ophiophagus hannah venoms.
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http://dx.doi.org/10.1016/j.peptides.2016.12.006DOI Listing
February 2017