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Malaria is a parasitic disease, caused by protozoa of the genus Plasmodium, and is transmitted to humans through the bites of infected Anopheles mosquitoes. More than 200 million cases of malaria are reported annually and about 400,000 deaths worldwide. Currently, the use of antimalarial drugs has been efficient in most cases, however, resistance to these drugs is increasing, making it necessary and essential to have a new range of possible drugs or medicines to combat this disease. Scorpion venom contains peptides whose functions are now intensively studied. Some of these peptides are known as Scorpine-like, which have anti-bacterial and antiplasmodial properties as they have been described to inhibit the development of parasites responsible for malaria. Scorpine-like peptides are composed of two structural domains: one α-helical N-terminal domain, and a C-terminal domain with the cysteine-stabilized α/β motif, which confers the peptide the function of blocking potassium channels and/or anti-bacteria activity. In this work, two C-terminal domains from Scorpine-like peptides were constructed and expressed in Escherichia coli, and their function was analyzed. We were able to demonstrate that the recombinant C-terminal domains rCterVm and rCterHg showed antiplasmodial activity producing 60% and 90% inhibition, respectively, of Plasmodium berghei development at 1 µM and 0.15 µM concentration, which makes these peptides promising candidates against Malaria.

The use of bacteriocins holds great promise in different areas such as health, food, nutrition, veterinary, nanotechnology, among others. Many research groups worldwide continue to advance the knowledge to unravel a novel range of therapeutic agents and food preservatives. This review addresses the advances of bacteriocins and their producer organisms as biocontrol agents for applications in the medical industry and agriculture. Furthermore, the bacteriocin mechanism of action and structural characteristics will be reviewed. Finally, the potential role of bacteriocins to modulate the signaling in host-associated microbial communities will be discussed.

Growing global viral infections have been a serious public health problem in recent years. This current situation emphasizes the importance of developing more therapeutic antiviral compounds. Hepatitis C virus (HCV) and dengue virus (DENV) belong to the Flaviviridae family and are an increasing global health threat. Our previous study reported that the crude venom of Scorpio maurus palmatus possessed anti-HCV and anti-DENV activities in vitro. We report here the characterization of a natural antiviral peptide (scorpion-like peptide Smp76) that prevents HCV and DENV infection. Smp76 was purified from S. m. palmatus venom and contains 76 amino acids with six residues of cysteine. Smp76 antiviral activity was evaluated using a cell culture technique utilizing Huh7it-1, Vero/SLAM, HCV (JFH1, genotype 2a) and DENV (Trinidad 1751, type 2). A potential antiviral activity of Smp76 was detected in culture cells with an approximate IC50 of 0.01 μg/ml. Moreover, Smp76 prevents HCV infection and suppresses secondary infection, by inactivating extra-cellular infectious particles without affecting viral replication. Interestingly, Smp76 is neither toxic nor hemolytic in vitro at a concentration 1000-fold higher than that required for antiviral activity. Conclusively, this report highlights novel anti-HCV and anti-DENV activities of Smp76, which may lay the foundation for developing a new therapeutic intervention against these flaviviruses.

Inhibitory substances can be obtained and purified by different methodologies. Free cell supernatants obtained by centrifugation and filtration are commonly used for evaluating antimicrobial metabolites produced by bacteria. In this work, a new methodology for obtaining antimicrobial metabolites is proposed, especially implemented for bacteria which are not able to produce these compounds in liquid media; based on the use of nitrocellulose filter membranes and solid culture media.

Inhibitory substances can be obtained and purified by different methodologies. Free cell supernatants obtained by centrifugation and filtration are commonly used for evaluating antimicrobial metabolites produced by bacteria. In this work, a new methodology for obtaining antimicrobial metabolites is proposed, especially implemented for bacteria which are not able to produce these compounds in liquid media; based on the use of nitrocellulose filter membranes and solid culture media.

Bacteria produce antimicrobial compounds to compete for nutrients and space in a particular habitat. Antagonistic interactions can be evaluated by several methodologies including the double-layer agar and simultaneous inhibition assays. Among the well-known inhibitory substances produced by bacteria are the broad-spectrum antibiotics, organic acids, siderophores, antifungal, and bacteriocins. The most studied bacterial genera able to produce these inhibitory substances are Enterococcus, Lactococcus, Streptomyces, Bacillus, Pseudomonas, Klebsiella, Escherichia, and Burkholderia. Some beneficial bacteria can promote plant growth and degrade toxic compounds in the environment representing an attractive solution to diverse issues in agriculture and soil pollution, particularly in fields with damaged soils where pesticides and fertilizers have been indiscriminately used. Beneficial bacteria may increase plant health by inhibiting pathogenic microorganisms; some examples include Gluconacetobacter diazotrophicus, Azospirullum brasilense, Pseudomonas fluorescens, Pseudomonas protegens, and Burkholderia tropica. However, most studies showing the antagonistic potential of these bacteria have been performed in vitro, and just a few of them have been evaluated in association with plants. Several inhibitory substances involved in pathogen antagonism have not been elucidated yet; in fact, we know only 1 % of the bacterial diversity in a natural environment leading us to assume that many other inhibitory substances remain unexplored. In this review, we will describe the characteristics of some antimicrobial compounds produced by beneficial bacteria, the principal methodologies performed to evaluate their production, modes of action, and their importance for biotechnological purposes. RESUMEN Las bacterias producen compuestos antimicrobianos para competir por nutrientes y espacio en un hábitat particular. Las interacciones antagónicas pueden evaluarse mediante varias metodologías, incluido el agar de doble capa y los ensayos de inhibición simultánea. Las sustancias inhibidoras mejor conocidas producidas por bacterias incluyen antibióticos, ácidos orgánicos, sideróforos, antifúngicos y bacteriocinas. Entre los géneros bacterianos más estudiados que producen sustancias inhibidoras se incluyen Enterococcus, Lactococcus, Streptomyces, Bacillus, Pseudomonas, Klebsiella, Escherichia y Burkholderia. Algunas bacterias beneficiosas tienen la capacidad de promover el crecimiento de las plantas y degradar compuestos tóxicos en el ambiente, por lo que podrían incrementar el rendimiento de los cultivos y disminuir problemas de contaminación del suelo, especialmente donde los pesticidas y fertilizantes han sido utilizados indiscriminadamente. Algunas bacterias beneficiosas pueden aumentar la salud de las plantas al inhibir microorganismos patógenos, por ejemplo, Gluconacetobacter diazotrophicus, Azospirullum brasilense, Pseudomonas fluorescens, Pseudomonas protegens y Burkholderia tropica. Sin embargo, la mayoría de los estudios que muestran el potencial antagónico de estas bacterias se han realizado in vitro, y pocos de ellos se han evaluado en asociación con plantas. Varias sustancias inhibitorias implicadas en el antagonismo de los patógenos aún son desconocidas; de hecho, sabemos que solo se ha aislado el 1 % de la diversidad bacteriana en un ambiente natural, lo que sugiere que hay muchas otras sustancias inhibitorias que no han sido exploradas. En esta revisión describimos las características de algunos compuestos antimicrobianos producidos por bacterias beneficiosas, las principales metodologías usadas para evaluar su producción, modos de acción y su importancia para fines biotecnológicos.

Purpose: Pseudomonas putida KT2440 is a desiccation-sensitive bacterium that loses culturability after 15 days of air desiccation. We have previously shown that P. putida KT2440 can develop a viable but nonculturable (VBNC) state after being exposed to desiccation stress and eventually recover when desiccated cells are rehydrated for at least 24 h. Methods: To determine which genes of transport, oxidation-reduction, and transcription processes could be involved in the return of P. putida KT2440 to the culturable state, a transcriptome analysis was carried out comparing the gene expression of non-desiccated samples with samples subjected to desiccation followed by 20 min of rehydration or desiccation followed by 24 h of rehydration. Results: Desiccation stress triggered a VBNC state of P. putida. The major response was detected after 24 h of rehydration with 148 upregulated and 42 downregulated genes. During the VBNC state, P. putida activated transmembrane transport processes like that of siderophores through a TonB-dependent transporter and putative polyhydric alcohol transport systems. Prolonged rehydration with distilled water resuscitated P. putida KT2440 cells activating the catabolism of phenylalanine/tyrosine to provide energy and carbon for ubiquinone biosynthesis while maintaining a reduced protein synthesis. On the other hand, the interruption of the TonB-dependent receptor gene (PP_1446) increased desiccation survival of the mutant strain. Conclusion: The activation of the iron transport system (TonB-dependent siderophore receptor) and alcohol transport can be helping the VBNC state of P. putida. Activation of catabolism of phenylalanine/tyrosine and reduced protein synthesis was needed for resuscitation from the VBNC state., Funding was provided by the intramural program of the Vicerrectoria de Investigación y Estudios de Posgrado, BUAP through grants PV16-ID00450, PV17-ID00337. Lilia Isela Lopez Lara received a scholarship (number 273274) from the National Council of Science and Technology (CONACyT).

Background Arthropod-borne diseases remain a leading cause of human morbidity and mortality and exact an enormous toll on global agriculture. The practice of insecticide-based control is fraught with issues of excessive cost, human and environmental toxicity, unwanted impact on beneficial insects and selection of resistant insects. Efforts to modulate insects to eliminate pathogen transmission have gained some traction and remain future options for disease control. Results Here, we report a paratransgenic strategy that targets transmission of Xylella fastidiosa, a leading bacterial pathogen of agriculture, by the Glassy-Winged Sharpshooter (GWSS), Homalodisca vitripennis. Earlier, we identified Pantoea agglomerans, a bacterial symbiont of the GWSS as the paratransgenic control agent. We genetically engineered P. agglomerans to express two antimicrobial peptides (AMP)-melittin and scorpine-like molecule (SLM). Melittin and SLM were chosen as the effector molecules based on in vitro studies, which showed that both molecules have anti-Xylella activity at concentrations that did not kill P. agglomerans. Using these AMP-expressing strains of P. agglomerans, we demonstrated disruption of pathogen transmission from insects to grape plants below detectable levels. Conclusion This is the first report of halting pathogen transmission from paratransgenically modified insects. It is also the first demonstration of paratransgenic control in an agriculturally important insect vector. Electronic supplementary material The online version of this article (10.1186/s12896-018-0460-z) contains supplementary material, which is available to authorized users.

Background Scorpion venoms contain toxins that modulate ionic channels, among which are the calcins, a small group of short, basic peptides with an Inhibitor Cystine Knot (ICK) motif that target calcium release channels/ryanodine receptors (RyRs) with high affinity and selectivity. Here we describe the heterologous expression of Intrepicalcin, identified by transcriptomic analysis of venomous glands from Vaejovis intrepidus. Methods Recombinant Intrepicalcin was obtained in Escherichia coli BL21-DE3 (periplasm) by fusing the Intrepicalcin gene to sequences coding for signal-peptide, thioredoxin, His-tag and enterokinase cleavage site. Results [3H]Ryanodine binding, used as a functional index of RyR activity, revealed that recombinant Intrepicalcin activates skeletal RyR (RyR1) dose-dependently with Kd = 17.4 ± 4.0 nM. Intrepicalcin significantly augments the bell-shaped [Ca2 +]-[3H]ryanodine binding curve at all [Ca2 +] ranges, as is characteristic of the calcins. In single channel recordings, Intrepicalcin induces the appearance of a subconductance state in RyR1 with a fractional value ∼ 55% of the full conductance state, very close to that of Vejocalcin. Furthermore, Intrepicalcin stimulates Ca2 + release at an initial dose = 45.3 ± 2.5 nM, and depletes ~ 50% of Ca2 + load from skeletal sarcoplasmic reticulum vesicles. Conclusions We conclude that active recombinant Intrepicalcin was successfully obtained without the need of manual oxidation, enabling it to target RyR1s with high affinity. General significance This is the first calcin heterologously expressed in the periplasma of Escherichia coli BL21-DE3, shown to be pharmacologically effective, thus paving the way for the generation of Intrepicalcin variants that are required for structure-function relationship studies of calcins and RyRs.

It is widely known that targeting a variety of antigens to the DEC-205 receptor on dendritic cells (DCs) significantly potentiate immunity. This communication reports the development of a new murine monoclonal antibody (mAb) against the chicken DEC-205, using as immunogen the carbohydrate recognition domain-2 (CRD-2) heterologously expressed. This mAb recognizes a protein band of 250kDa by immunoprecipitation analysis and shows strong cross-reactivity with human and pig DEC-205. Furthermore, the hemagglutinin (HA) of avian influenza H5N2 virus was cloned and expressed using insect cell-baculovirus expression system. We chemically conjugated the anti-chicken DEC-205 antibody with the highly purified HA to direct the antigen to the dendritic cells and evaluate the immune response elicited in vivo by this conjugate. A single dose of chemical conjugate was sufficient to elicit a strong immune response in chickens as early as fourteen days after priming. In addition, the conjugate induced an earlier and higher response compared to unconjugated HA. These results suggest that the strategy described here has potential to be used in the future design and development of successful vaccines against different chicken infectious diseases with direct impact in biotechnology and veterinary fields.

This manuscript describes the design of plasmids containing the genes coding for four main mammalian toxins of scorpions from the genus Centruroides (C.) of Mexico. The genes that code for toxin 2 of C. noxius (Cn2), toxin 2 from C. suffusus (Css2) and toxins 1 and 2 from C. limpidus (Cll1 and Cll2) were included into individual plasmids carrying the genetic construction for expression of fusion proteins containing a leader peptide (pelB) that directs the expressed protein to the bacterial periplasm, a carrier protein (thioredoxin), the cleavage site for enterokinase, the chosen toxin and a poly-histidine tag (6xHis-tag) for purification of the hybrid protein by immobilized metal ion affinity chromatography after expression in Escherichia coli strain BL21 (DE3). The purified hybrid proteins containing the recombinant toxins (abbreviated Thio-EK-Toxin) were used for immunization of three independent groups of ten mice and four rabbits. Challenging the first group of mice, immunized with recombinant Thio-EK-Css2, with three median lethal doses (LD50) of C. suffusus soluble venom resulted in the survival of all the test animals without showing intoxication symptoms. All control mice (none immunized) died. Similar results were obtained with mice previously immunized with Thio-EK-Cn2 and challenged with C. noxius venom. The third group of mice immunized with both Thio-EK-Cll1 and Thio-EK-Cll2 showed an 80% survival ratio when challenged with only one LD50 of C. limpidus venom, all showing symptoms of intoxication. The sera from rabbits immunized with a combination of the four recombinant toxins were collected separately and used to assess their neutralization capacity in vitro (pre-incubating the serum with the respective scorpion venom and injecting the mixture into mice), using six mice for each serum/venom combination tested. The venoms from the six most dangerous scorpion species of Mexico were assayed: C. noxius, C. suffusus, C. limpidus, C. elegans, C. tecomanus and C. sculpturatus. Two hundred and 50 μL of serum from any of the immunized rabbits were enough to neutralize three LD50 of any of the tested venoms, with mice showing no symptoms of intoxication. These results confirm that the recombinant forms of the main toxins from the most dangerous scorpions of Mexico are excellent immunogens for the production of antivenoms to treat scorpion intoxications.

Scorpion venom peptides represent a novel source of antimicrobial peptides (AMPs) with broad-spectrum activity. In this study, we determined the minimum bactericidal concentration (MBC) of three scorpion AMPs, Uy234, Uy17, and Uy192, which are found in the venomous glands of the Urodacus yaschenkoi scorpion, against the clinical isolates of multidrug-resistant (MDR) bacteria. In addition, we tested the activity of a consensus AMP designed in our laboratory based on some previously reported IsCT-type (cytotoxic linear peptide) AMPs with the aim of obtaining higher antimicrobial activity. All peptides tested showed high antimicrobial activity against MDR clinical isolates, with the highest activity against β-hemolytic Streptococcus strains. The hemolytic activity was determined against human red blood cells and was significantly lower than that of previously reported AMPs. The α-helical structure of the four AMPs was confirmed by circular dichroism (CD). These results suggest that the four peptides can be valuable tools for the design and development of AMPs for use in the inhibition of MDR pathogenic bacteria. A clear index of synergism and additivity was found for the combination of QnCs-BUAP + Uy234, which makes these peptides the most promising candidates against pathogenic bacteria.

Plant growth-promoting bacteria (PGPB) have been extensively studied, because of different mechanisms to perform phytostimulation, as well as the abilities to colonize plants. The number of crop types and hectares of agricultural land, where PGPR are applied is low compared with the total crops or farming area. However, the PGPB application in crop fields is increasingly becoming more accepted due to the advantages to crops and environment such as the increment in productivity, the diminution in the use of chemical fertilizers and toxic compounds such as pesticides and herbicides. These traits make beneficial bacteria formulations the ‘right choice’ in healthy agriculture since they are highly compatible with sustainable crop production. The co-inoculation of plants with two or more beneficial bacteria apparently provides greater phytostimulation than mono-inoculation, perhaps because of the synergistic and multifarious effects occurring when two or more microorganisms are co-interacting. There is a consensus that bacterial mixtures provide greater benefits to the plants, the number of formulations containing more than three species of microorganisms in consortium is still limited. Therefore, we believe that more research and investment is needed to design and formulate multi-species inoculants containing compatible bacteria and other beneficial microorganisms in order to be capable of coexisting both in the designed formulation and associated with plants for sustainable benefits.

The bacterial strain, EMM-1, was isolated from the rhizosphere of red maize ("Rojo Criollo") and identified as Pseudomonas protegens EMM-1 based on phylogenetic analysis of 16S rDNA, rpoB, rpoD, and gyrB gene sequences. We uncovered genes involved in the production of antimicrobial compounds like 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin, and lectin-like bacteriocins. These antimicrobial compounds are also produced by other fluorescent pseudomonads alike P. protegens. Double-layer agar assay showed that P. protegens EMM-1 inhibited the growth of several multidrug-resistant (MDR) bacteria, especially clinical isolates of the genera Klebsiella and β-hemolytic Streptococcus. This strain also displayed inhibitory effects against diverse fungi, such as Aspergillus, Botrytis, and Fusarium. Besides, a crude extract of inhibitory substances secreted into agar was obtained after the cold-leaching process, and physicochemical characterization was performed. The partially purified inhibitory substances produced by P. protegens EMM-1 inhibited the growth of Streptococcus sp. and Microbacterium sp., but no inhibitory effect was noted for other bacterial or fungal strains. The molecular weight determined after ultrafiltration was between 3 and 10 kDa. The inhibitory activity was thermally stable up to 60°C (but completely lost at 100°C), and the inhibitory activity remained active in a wide pH range (from 3 to 9). After treatment with a protease from Bacillus licheniformis, the inhibitory activity was decreased by 90%, suggesting the presence of proteic natural compounds. All these findings suggested that P. protegens EMM-1 is a potential source of antimicrobials to be used against pathogens for humans and plants.

Centruroides tecomanus is a medically important scorpion of the state of Colima (Mexico). This communication reports the identification of venom components of this scorpion with biological activity over insects/crickets (Acheta domestica), crustaceans/fresh water shrimps (Cambarellus montezumae), and mammalians/mice (Mus musculus, strain CD1). It also describes the pharmacological effects on cell lines in culture (L5178Y cells, HeLa cells, HuTu cells and Jurkat E6-1 cells), as well as on several types of bacteria (see below). The soluble venom of this scorpion was fractionated by high-performance liquid chromatography (HPLC) and collected separately in twelve independent fractions collected over 60 min run (5 min time apart each other). The HPLC components of fraction VII were lethal to all three species used for assay. The IVth fraction had a toxic effect on freshwater shrimps. In this species, fractions VI, VII and VIII were all lethal. For crickets, fractions V and VI were toxic and fraction VII was lethal. In mouse, the lethal components were found in fraction VII, whereas fraction VIII was toxic, but not lethal, at the doses assayed. The molecular weight of peptides from the various group of fractions were identified by mass spectrometry determination. Components lethal to mice showed molecular weights from 7013 to 7487 Da. Two peptides were obtained in homogeneous form and shown to be lethal to the three species of animal used for assay. The soluble venom tested on L5178Y cell line survival was shown to be cytotoxic, at 10-100 μg/mL concentration, when compared to control murine splenocytes (p = 0.007). The soluble venom applied to Hela, Hutu and Jurkat cell lines did not show cytotoxic effects at these concentrations. On the contrary, it seems to have a proliferative effect. However the HPLC fractions I, III, VI and XII do have a cytotoxic effect on Jurkat E06-1 cells in culture at 200 μg/mL concentration. The antimicrobial activity of the venom fractions on Staphylococcus aureus (gram-positive), Escherichia coli, Pseudomonas aeruginosa y Salmonella spp (gram-negative) was measured, using the liquid inhibition growth system. The four strains of bacteria used were susceptible to fractions III and IV, affecting all four bacterial strains at concentrations below 5 μg/mL.

Bacteria from rhizosphere have the potential to promote the growth of plants, and could be used as inoculants to increase the crop profitability. However, under drought stress conditions, the number of bacteria associated to seeds could decrease below the minimal number of bacteria required to obtain a positive plant response. At the present work, the capability of 28 rhizospheric bacterial strains to tolerate 18 days of air desiccation stress (at 30oC and 50% of relative humidity) was evaluated. Results showed different levels of bacterial tolerance and five categories were proposed based on the bacterial survival ratio to air desiccation (BSRad): highly tolerant bacteria (BSRad˃80), tolerant (60 (40 bacteria were selected to inoculate maize seeds. After inoculation, seeds were subjected to desiccation conditions for 18 days and then sown in sterile vermiculite and watered with MS solution. Only highly tolerant strains were able to survive associated to seeds under desiccation conditions keeping their beneficial traits such as, bacterial adherence, colonization and plant growth promotion. In contrast, the very-low tolerant bacterial strain was not able to colonize the rhizosphere nor promote the growth of the plants. Tolerant bacteria to desiccation have desirable traits in comparison to non-tolerant bacteria, because they could be more stable in powder presentations of inoculants without need of protectants, they could survive with higher numbers in soils with low water availability keeping their ability to promote the plant-growth until the end of adverse conditions; when the seed germination take place., {"references":["Morales-García Y-E, Duque E, Rodríguez-Andrade O, et al. (2010) Bacterias preservadas, una fuente importante de recursos biotecnológicos. Bio Tecnol 14:11-29.","Lin W, Bazylinski DA, Xiao T, et al. 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Appl Soil Ecol 108: 371-380.","Velázquez-Hernández ML, Baizabal-Aguirre VM, Cruz- Vázquez F et al. (2011) Gluconacetobacter diazotrophicus levansucrase is involved in tolerance to NaCl, sucrose and desiccation, and in biofilm formation. Arch Microbiol 193: 137- 149.","Alpert P (2006) Constraints of tolerance : why are desiccationtolerant organisms so small or rare ? J Exp Biol 209: 1575-1584.","Elliott J, Deryng D, Müller C, et al. (2014) Constraints and potentials of future irrigation water availability on agricultural production under climate change. Proc Natl Acad Sci 111: 3239- 3244.","Talbi C, Sánchez C, Hidalgo-Garcia A, et al. (2012) Enhanced expression of Rhizobium etli cbb3 oxidase improves drought tolerance of common bean symbiotic nitrogen fixation. J Exp Bot 63: 5035-5043.","Thakur R, Sharma KC, Gulati A, et al. (2017) Stress-tolerant Viridibacillus arenosi strain IHB B 7171 from Tea rhizosphere as a potential broad-spectrum microbial inoculant. Indian J Microbiol 57: 195-200.","Sandra C-P, Rebeca BR (2015) Polymers selection for a liquid inoculant of Azospirillum brasilense based on the Arrhenius thermodynamic model. African J Biotechnol 33: 2547-2553.","Bashan Y, De-Bashan LE, Prabhu SR (2016) Superior Polymeric Formulations and Emerging Innovative Products of Bacterial Inoculants for Sustainable Agriculture and the Environment BT - Agriculturally Important Microorganisms: Commercialization and Regulatory Requirements in Asia. In: Singh HB, Sarma BK, Keswani C, editors. Singapore: Springer Singapore; 15-46."]}

Plant growth-promoting rhizobacteria (PGPR) increase plant growth and crop productivity. The inoculation of plants with a bacterial mixture (consortium) apparently provides greater benefits to plant growth than inoculation with a single bacterial strain. In the present work, a bacterial consortium was formulated containing four compatible and desiccation-tolerant strains with potential as PGPR. The formulation had one moderately (Pseudomonas putida KT2440) and three highly desiccation-tolerant (Sphingomonas sp. OF178, Azospirillum brasilense Sp7 and Acinetobacter sp. EMM02) strains. The four bacterial strains were able to adhere to seeds and colonize the rhizosphere of plants when applied in both mono-inoculation and multi-inoculation treatments, showing that they can also coexist without antagonistic effects in association with plants. The effects of the bacterial consortium on the growth of blue maize were evaluated. Seeds inoculated with either individual bacterial strains or the bacterial consortium were subjected to two experimental conditions before sowing: normal hydration or desiccation. In general, inoculation with the bacterial consortium increased the shoot and root dry weight, plant height and plant diameter compared to the non-inoculated control or mono-inoculation treatments. The bacterial consortium formulated in this work had greater benefits for blue maize plants even when the inoculated seeds underwent desiccation stress before germination, making this formulation attractive for future field applications.

The αX I-domain of the horse integrin CD11c was successfully expressed in Escherichia coli, purified, biochemically characterized and used as immunogen to generate murine monoclonal antibodies against horse CD11c, which are not yet commercially available. One monoclonal antibody mAb-1C4 against the αX I-domain, is an IgG2a able to interact with the recombinant I-domain, showing an EC50 = 2.4 ng according to ELISA assays. By western blot with horse PBMCs lysates the mAb-1C4 recognized a protein of 150 kDa which corresponds well with the CD11c molecule. Using immunohistochemistry in horse lymph node tissue sections, mAb-1C4 marked cells in situ, some with apparent dendritic morphology. Thus the mAb generated to a recombinant epitope from horse CD11c identified the molecule in intact cells within horse lymphoid tissue. By the labelling intensity, the histological location (paracortical and interfollicular areas) and the apparent morphology of the marked cells, we can say that these are putative horse dendritic cells (DCs). The development of a mAb to horse CD11c provides a new tool to better study the horse DC biology and opens other biotechnological avenues, such as DC targeting-based vaccines.

Here we describe two methods for assessing the ability of one bacteria to inhibit the growth of another through the production of antimicrobial compounds. In the first assay, the double agar layer method, only the secreted substances but not the candidate antagonist bacteria interact with the sensitive microorganisms. In the second assay, the simultaneous inhibition method, both microorganisms are grown together allowing the competitive exclusion.

We describe a method to evaluate bacterial tolerance to air desiccation at 30 °C and 50% relative humidity.

This communication reports the structural and functional characterization of urotoxin, the first K(+) channel toxin isolated from the venom of the Australian scorpion Urodacus yaschenkoi. It is a basic peptide consisting of 37 amino acids with an amidated C-terminal residue. Urotoxin contains eight cysteines forming four disulfide bridges with sequence similarities resembling the α-potassium channel toxin 6 (α-KTx-6) subfamily of peptides; it was assigned the systematic number of α-KTx-6.21. Urotoxin is a potent blocker of human voltage-gated potassium channel (Kv)1.2 channels, with an IC50 of 160 pM, whereas its affinity for other channels tested was in the nanomolar range (hKv1.1, IC50 = 253 nM; hKv1.3, IC50 = 91 nM; and hKCa3.1, IC50 = 70 nM). The toxin had no effect on hKv1.4, hKv1.5, human ether-à-go-go-related gene type 1 (hERG1), or human ether-à-go-go-like (hELK2) channels. Multiple sequence alignments from the venom gland transcriptome showed the existence of four other new peptides similar to urotoxin. Computer modeling of urotoxin's three-dimensional structure suggests the presence of the α/β-scaffold characteristic of other scorpion toxins, although very likely forming an uncommon disulfide pairing pattern. Using molecular dynamics, a model for the binding of this peptide to human Kv1.2 and hKv1.1 channels is presented, along with the binding of an in silico mutant urotoxin (Lys25Ala) to both channels. Urotoxin enriches our knowledge of K(+) channel toxins and, due to its high affinity for hKv1.2 channels, it may be a good candidate for the development of pharmacologic tools to study the physiologic functions of K(+) channels or related channelopathies and for restoring axonal conduction in demyelinated axons.

The number and types of venom components that affect ion-channel function are reviewed. These are the most important venom components responsible for human intoxication, deserving medical attention, often requiring the use of specific anti-venoms. Special emphasis is given to peptides that recognize Na(+)-, K(+)- and Ca(++)-channels of excitable cells. Knowledge generated by direct isolation of peptides from venom and components deduced from cloned genes, whose amino acid sequences are deposited into databanks are nowadays in the order of 1.5 thousands, out of an estimate biodiversity closed to 300,000. Here the diversity of components is briefly reviewed with mention to specific references. Structural characteristic are discussed with examples taken from published work. The principal mechanisms of action of the three different types of peptides are also reviewed. Na(+)-channel specific venom components usually are modifier of the open and closing kinetic mechanisms of the ion-channels, whereas peptides affecting K(+)-channels are normally pore blocking agents. The Ryanodine Ca(++)-channel specific peptides are known for causing sub-conducting stages of the channels conductance and some were shown to be able to internalize penetrating inside the muscle cells.

Due to the medical importance played in Turkey by stings of the scorpion Androctonus crassicauda, its venom has been studied with more attention. In this communication we report a new toxic peptide, named Acra4, because it is the fourth peptide completely characterized from venom of this scorpion. The peptide contains 64 amino acid residues stabilized by four disulfide bridges, with a molecular weight of 6937 Da. Purification of the lethal peptide was performed by three steps of high performance liquid chromatography (HPLC) separations, and the molecular weight was determined by mass spectrometry analysis and the full amino acid sequence was obtained by direct Edman degradation in conjunction with gene cloning. The LD50 of Acra4 was 50.5 ng/20 g mouse body weight (95% confidence intervals from 48.8 to 52.2 ng/20 g mouse body weight). Additionally, from a sample of cDNA of A. crassicauda four genes were cloned displaying sequence similarities to known scorpion toxins, and are reported here as potentially toxic peptides, named Acra5 to Acra8. Electrophysiological studies of Acra4 were performed using Na+-channels expressed in F11 cell culture, by patch-clamp recordings. This is the first time that such peptide from A. crassicauda having a specific Na+-channel α-type effect is reported. Its affinity toward Na+-channels in F11 cell line is in the order of 1 μM concentration.

The Urodacidae scorpions are the most widely distributed of the four families in Australia and represent half of the species in the continent, yet their venoms remain largely unstudied. This communication reports the first results of a proteome analysis of the venom of the scorpion Urodacus yaschenkoi performed by mass fingerprinting, after high performance liquid chromatography (HPLC) separation. A total of 74 fractions were obtained by HPLC separation allowing the identification of approximately 274 different molecular masses with molecular weights varying from 287 to 43,437 Da. The most abundant peptides were those from 1 K Da and 4-5 K Da representing antimicrobial peptides and putative potassium channel toxins, respectively. Three such peptides were chemically synthesized and tested against Gram-positive and Gram-negative bacteria showing minimum inhibitory concentration in the low micromolar range, but with moderate hemolytic activity. It also reports a transcriptome analysis of the venom glands of the same scorpion species, undertaken by constructing a cDNA library and conducting random sequencing screening of the transcripts. From the resultant cDNA library 172 expressed sequence tags (ESTs) were analyzed. These transcripts were further clustered into 120 unique sequences (23 contigs and 97 singlets). The identified putative proteins can be assorted in several groups, such as those implicated in common cellular processes, putative neurotoxins and antimicrobial peptides. The scorpion U. yaschenkoi is not known to be dangerous to humans and its venom contains peptides similar to those of Opisthacanthus cayaporum (antibacterial), Scorpio maurus palmatus (maurocalcin), Opistophthalmus carinatus (opistoporines) and Hadrurus gerstchi (scorpine-like molecules), amongst others.

In this Crystal Ball we describe the negative effects of the scheme of intensive agriculture of the green revolution technology. To recover the contaminated soils derived from intensive farming is necessary introduce new successful technologies to replace the use of chemical fertilizer and toxic pesticides by organic fertilizers and biological control agents. Our principal speculation is that in a short time authors in the field of PGPB and bioremediation will be expanding the knowledge on the development of different formulations containing super-bacteria or a mixture of super-bacteria able to provide beneficial effect for agriculture and bioremediation.

From the cDNA libraries made from the venom glands of two scorpions belonging to the Vaejovidae family, four different putative non disulfide-bridged antimicrobial peptides were identified: VmCT1 and VmCT2 from Vaejovis mexicanus smithi plus VsCT1 and VsCT2 from Vaejovis subcristatus . These short peptides (with only 13 amino acid residues each) share important amino acid sequence similarities among themselves and with other reported antimicrobial peptides, but their biological activities vary dramatically. This communication reports the cloning, chemical synthesis and characterization of these peptides. Two peptides, VmCT1 and VmCT2 showed broad-spectrum antibacterial activity with minimum inhibitory concentrations MICs in the range of 5–25 μM and 10–20 μM respectively, whereas their hemolytic activity at these concentrations was low. Structure–function relationships that might determine the differences in activities are discussed.

The single-chain antibody fragment (scFv) 6009F, obtained by directed evolution, neutralizes the effects of the Cn2 toxin, which is the major toxic component of Centruroides noxius scorpion venom. In this work we compared the neutralization capacity and the thermodynamic stability of scFv 6009F with those of two other derived formats: Fab 6009F and diabody 6009F. Additionally, the affinity constants to Cn2 toxin of the three recombinant antibody fragments were determined by means of BIAcore. We found a correlation between the thermodynamic stability of these antibody fragments with their neutralization capacity. The order of thermodynamic stability determined was Fab ≫ scFv > diabody. The Fab and scFv were capable of neutralizing the toxic effects of Cn2 and whole venom but the diabody was unable to fully neutralize intoxication. In silico analysis of the diabody format indicates that the reduction of stability and neutralization capacity could be explained by a less cooperative interface between the heavy and the light variable domains.

Scorpions belonging to the Buthidae family have traditionally drawn much of the biochemist's attention due to the strong toxicity of their venoms. Scorpions not toxic to mammals, however, also have complex venoms. They have been shown to be an important source of bioactive peptides, some of them identified as potential drug candidates for the treatment of several emerging diseases and conditions. It is therefore important to characterize the large diversity of components found in the non-Buthidae venoms. As a contribution to this goal, this manuscript reports the construction and characterization of cDNA libraries from four scorpion species belonging to the Vaejovis genus of the Vaejovidae family: Vaejovis mexicanus, V. intrepidus, V. subcristatus and V. punctatus. Some sequences coding for channel-acting toxins were found, as expected, but the main transcribed genes in the glands actively producing venom were those coding for non disulfide-bridged peptides. The ESTs coding for putative channel-acting toxins, corresponded to sodium channel β toxins, to members of the potassium channel-acting α or κ families, and to calcium channel-acting toxins of the calcin family. Transcripts for scorpine-like peptides of two different lengths were found, with some of the species coding for the two kinds. One sequence coding for La1-like peptides, of yet unknown function, was found for each species. Finally, the most abundant transcripts corresponded to peptides belonging to the long chain multifunctional NDBP-2 family and to the short antimicrobials of the NDBP-4 family. This apparent venom composition is in correspondence with the data obtained to date for other non-Buthidae species. Our study constitutes the first approach to the characterization of the venom gland transcriptome for scorpion species belonging to the Vaejovidae family.

Previous studies have indicated differences in the specificity-determining residues (SDRs) of antibodies that recognize haptens, peptides, or proteins. Here, we designed a VH repertoire based on the human scaffold 3-23/JH4 and diversification of high and medium-usage SDRs of anti-protein and anti-peptide antibodies. The repertoire was synthesized by overlapping polymerase chain reaction (PCR) and combined with the VL chain of the anti-hen egg-white lysozyme (HEL) antibody D1.3. The resulting chimeric single-chain Fv fragments (scFvs) phage-displayed library was panned in HEL-coated immunotubes. After two rounds of selection under non-stringent conditions, that is, trypsinization after 2 h of incubation at room temperature, 63 of 167 clones analyzed (38%) were found to express scFvs specific to HEL. Twenty clones were characterized by DNA sequencing resulting in 10 unique scFvs. Interestingly, the panel of unique scFvs was highly diverse, with VH sequences differing in 16 of the 17 positions variegated in the repertoire. Thus, diverse chemico-physical and structural solutions were selected from the library, even when the VH repertoire was constrained by the VL chain of D1.3 to yield binders against a definite region of HEL surface. The more often selected scFvs, namely H6-1 and B7-1, which differed in eight SDRs, showed levels of expression in E. coli TG1 strain, 6 and 10 times higher than the parental D1.3 Fv fragment, respectively. Dissociation constants (KDs) measured in the BIAcore were 11 and 6.6 nM for H6-1 and B7-1, respectively. These values compared well to the KD of 4.7 nM measured for D1.3, indicating that the VH repertoire here designed is a valuable source of diverse, well-expressed and high affinity VH domains. Copyright © 2006 John Wiley & Sons, Ltd.

BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.

Australian scorpion venoms have been poorly studied, probably because they do not pose an evident threat to humans. In addition, the continent has other medically important venomous animals capable of causing serious health problems. Urodacus yaschenkoi belongs to the most widely distributed family of Australian scorpions (Urodacidae) and it is found all over the continent, making it a useful model system for studying venom composition and evolution. This communication reports the whole set of mRNA transcripts produced by the venom gland. U. yaschenkoi venom is as complex as its overseas counterparts. These transcripts certainly code for several components similar to known scorpion venom components, such as: alpha-KTxs, beta-KTxs, calcins, protease inhibitors, antimicrobial peptides, sodium-channel toxins, toxin-like peptides, allergens, La1-like, hyaluronidases, ribosomal proteins, proteasome components and proteins related to cellular processes. A comparison with the venom gland transcriptome of Centruroides noxius (Buthidae) showed that these two scorpions have similar components related to biological processes, although important differences occur among the venom toxins. In contrast, a comparison with sequences reported for Urodacus manicatus revealed that these two Urodacidae species possess the same subfamily of scorpion toxins. A comparison with sequences of an U. yaschenkoi cDNA library previously reported by our group showed that both techniques are reliable for the description of the venom components, but the whole transcriptome generated with Next Generation Sequencing platform provides sequences of all transcripts expressed. Several of which were identified in the proteome, but many more transcripts were identified including uncommon transcripts. The information reported here constitutes a reference for non-Buthidae scorpion venoms, providing a comprehensive view of genes that are involved in venom production. Further, this work identifies new putative bioactive compounds that could be used to seed research into new pharmacological compounds and increase our understanding of the function of different ion channels.

Centruroides tecomanus is a Mexican scorpion endemic of the State of Colima, that causes human fatalities. This communication describes a proteome analysis obtained from milked venom and a transcriptome analysis from a cDNA library constructed from two pairs of venom glands of this scorpion. High perfomance liquid chromatography separation of soluble venom produced 80 fractions, from which at least 104 individual components were identified by mass spectrometry analysis, showing to contain molecular masses from 259 to 44,392 Da. Most of these components are within the expected molecular masses for Na(+)- and K(+)-channel specific toxic peptides, supporting the clinical findings of intoxication, when humans are stung by this scorpion. From the cDNA library 162 clones were randomly chosen, from which 130 sequences of good quality were identified and were clustered in 28 contigs containing, each, two or more expressed sequence tags (EST) and 49 singlets with only one EST. Deduced amino acid sequence analysis from 53% of the total ESTs showed that 81% (24 sequences) are similar to known toxic peptides that affect Na(+)-channel activity, and 19% (7 unique sequences) are similar to K(+)-channel especific toxins. Out of the 31 sequences, at least 8 peptides were confirmed by direct Edman degradation, using components isolated directly from the venom. The remaining 19%, 4%, 4%, 15% and 5% of the ESTs correspond respectively to proteins involved in cellular processes, antimicrobial peptides, venom components, proteins without defined function and sequences without similarity in databases. Among the cloned genes are those similar to metalloproteinases.

Proteomic analysis of the scorpion venom Scorpio maurus palmatus was performed using reverse-phase HPLC separation followed by mass spectrometry determination. Sixty five components were identified with molecular masses varying from 413 to 14,009 Da. The high percentage of peptides (41.5%) was from 3 to 5 KDa which may represent linear antimicrobial peptides and KScTxs. Also, 155 expressed sequence tags (ESTs) were analyzed through construction the cDNA library prepared from a pair of venomous gland. About 77% of the ESTs correspond to toxin-like peptides and proteins with definite open reading frames. The cDNA sequencing results also show the presence of sequences whose putative products have sequence similarity with antimicrobial peptides (24%), insecticidal toxins, β-NaScTxs, κ-KScTxs, α-KScTxs, calcines and La1-like peptides. Also, we have obtained 23 atypical types of venom molecules not recorded in other scorpion species. Moreover, 9% of the total ESTs revealed significant similarities with proteins involved in the cellular processes of these scorpion venomous glands. This is the first set of molecular masses and transcripts described from this species, in which various venom molecules have been identified. They belong to either known or unassigned types of scorpion venom peptides and proteins, and provide valuable information for evolutionary analysis and venomics.