Binding of green tea epigallocatechin gallate to the arginine kinase active site from the brown recluse spider (Loxosceles laeta): A potential synergist to chemical pesticides.

Loxosceles spp. spiders can cause serious public health issues. Chemical control is commonly used, leading to health and environmental problems. Identifying molecular targets and using them with natural compounds can help develop safer and eco-friendlier biopesticides. We studied the kinetics and predicted structural characteristics of arginine kinase (EC 2.7.3.3) from Loxosceles laeta (LlAK), a key enzyme in the energy metabolism of these organisms. Additionally, we explored (-)-epigallocatechin gallate (EGCG), a green tea flavonoid, as a potential lead compound for the LlAK active site through fluorescence and in silico analysis, such as molecular docking and molecular dynamics (MD) simulation and MM/PBSA analyses. The results indicate that LlAK is a highly efficient enzyme (K m Arg 0.14 mM, K m ATP 0.98 mM, k cat 93 s-1, k cat/K m Arg 630 s-1 mM-1, k cat/K m ATP 94 s-1 mM-1), which correlates with its structure similarity to others AKs (such as Litopenaeus vannamei, Polybetes pythagoricus, and Rhipicephalus sanguineus) and might be related to its important function in the spider's energetic metabolism. Furthermore, the MD and MM/PBSA analysis suggests that EGCG interacted with LlAK, specifically at ATP/ADP binding site (RMSD <1 nm) and its interaction is energetically favored for its binding stability (-40 to -15 kcal/mol). Moreover, these results are supported by fluorescence quenching analysis (K d 58.3 µM and K a 1.71 × 104 M-1). In this context, LlAK is a promising target for the chemical control of L. laeta, and EGCG could be used in combination with conventional pesticides to manage the population of Loxosceles species in urban areas.

Endocarditis due to Candida albicans in an immunocompromised patient: A case report.

BACKGROUND: Fungal endocarditis is a low-frequency disease with a challenging diagnosis, as it can be mistaken with bacterial endocarditis. Fungal endocarditis causes higher mortality rates in immunocompromised patients. In the clinical practice, the endocarditis caused by fungi represents up to 10% of all infectious endocarditis cases and has a mortality rate of nearly 50%. CASE REPORT: Here we present the case of a 53-year-old woman under corticosteroid therapy with a history of rheumatic heart disease, aortic valve replacement, and rheumatoid arthritis, who presented with fungal endocarditis caused by Candida albicans. Even though the patient received 3 years of antifungal prophylaxis with fluconazole, had valve replacement surgery, and received intensive care, the patient finally worsened and died. CONCLUSIONS: Comorbidities and corticosteroid therapy predisposed the patient to acquire fungal endocarditis. This case highlights the importance of implementing procedures for the isolation and identification of fungi, and for carrying out antifungal-susceptibility testing, as well as establishing surveillance programs to identify infection-causing species and drug resistance patterns in hospitals. Moreover, designing and upgrading the algorithm for infectious endocarditis is the key to future improvements in diagnosis.

A Novel Glutathione S-Transferase Gtt2 Class (VpGSTT2) Is Found in the Genome of the AHPND/EMS Vibrio parahaemolyticus Shrimp Pathogen.

Glutathione S-transferases are a family of detoxifying enzymes that catalyze the conjugation of reduced glutathione (GSH) with different xenobiotic compounds using either Ser, Tyr, or Cys as a primary catalytic residue. We identified a novel GST in the genome of the shrimp pathogen V. parahaemolyticus FIM- S1708+, a bacterial strain associated with Acute Hepatopancreatic Necrosis Disease (AHPND)/Early Mortality Syndrome (EMS) in cultured shrimp. This new GST class was named Gtt2. It has an atypical catalytic mechanism in which a water molecule instead of Ser, Tyr, or Cys activates the sulfhydryl group of GSH. The biochemical properties of Gtt2 from Vibrio parahaemolyticus (VpGSTT2) were characterized using kinetic and crystallographic methods. Recombinant VpGSTT2 was enzymatically active using GSH and CDNB as substrates, with a specific activity of 5.7 units/mg. Low affinity for substrates was demonstrated using both Michaelis-Menten kinetics and isothermal titration calorimetry. The crystal structure showed a canonical two-domain structure comprising a glutathione binding G-domain and a hydrophobic ligand H domain. A water molecule was hydrogen-bonded to residues Thr9 and Ser 11, as reported for the yeast Gtt2, suggesting a primary role in the reaction. Molecular docking showed that GSH could bind at the G-site in the vicinity of Ser11. G-site mutationsT9A and S11A were analyzed. S11A retained 30% activity, while T9A/S11A showed no detectable activity. VpGSTT2 was the first bacterial Gtt2 characterized, in which residues Ser11 and Thr9 coordinated a water molecule as part of a catalytic mechanism that was characteristic of yeast GTT2. The GTT2 family has been shown to provide protection against metal toxicity; in some cases, excess heavy metals appear in shrimp ponds presenting AHPND/EMS. Further studies may address whether GTT2 in V. parahaemolyticus pathogenic strains may provide a competitive advantage as a novel detoxification mechanism.

The clinical laboratory evolution in coccidioidomycosis detection: Future perspectives.

Coccidioidomycosis is a systemic disease caused by the fungi Coccidioides immitis and C. posadasii. It is a prevalent disease in arid regions with high temperatures and low precipitations in America. Coccidioidomycosis is a highly endemic disease of US-Mexico border states but commonly underdiagnosed. The diagnosis of coccidiomycosis is not easy due to the lack of specific symptoms; it is usually an integral approach, including clinical laboratory tests as an essential part of the diagnosis. Nevertheless, despite various laboratory tests available, affordability can be a limitation, mainly in developing countries. This review's objectives are 1) to learn the different laboratory approaches that arose and their application for clinical diagnosis; 2) to discuss their advantages and weaknesses, and finally, 3) propose what is on the horizon for future advances in clinical laboratory diagnosis of coccidioidomycosis. It has been a long way in laboratory tests evolution to detect coccidioidomycosis from tissue microscopy to Real-Time PCR. However, there is a delay in technology adoption for Coccidioides spp. detection in the clinical laboratory. The molecular Point of Care Testing (POCT) technology has reached us in our trench while research in PCR variants stills on-going. None of the currently existing scientific literature in coccidioidomycosis research has mentioned it. However, this trend in infectious and non-infectious disease diagnosis will continue in that way in order to offer better options for an easy and fast diagnosis. Undoubtedly, the implementation of molecular POCT for Coccidioides spp. would save resources in health care attention and improve access to diagnostic tools.

Ferulated Pectins and Ferulated Arabinoxylans Mixed Gel for Saccharomyces boulardii Entrapment in Electrosprayed Microbeads.

Ferulated polysaccharides such as pectin and arabinoxylan form covalent gels which are attractive for drug delivery or cell immobilization. Saccharomyces boulardii is a probiotic yeast known for providing humans with health benefits; however, its application is limited by viability loss under environmental stress. In this study, ferulated pectin from sugar beet solid waste (SBWP) and ferulated arabinoxylan from maize bioethanol waste (AX) were used to form a covalent mixed gel, which was in turn used to entrap S. boulardii (2.08 × 108 cells/mL) in microbeads using electrospray. SBWP presented a low degree of esterification (30%), which allowed gelation through Ca2+, making it possible to reduce microbead aggregation and coalescence by curing the particles in a 2% CaCl2 cross-linking solution. SBWP/AX and SBWP/AX+ S. boulardii microbeads presented a diameter of 214 and 344 µm, respectively, and a covalent cross-linking content (dimers di-FA and trimer tri-FA of ferulic acid) of 1.15 mg/g polysaccharide. The 8-5', 8-O-4'and 5-5'di-FA isomers proportions were 79%, 18%, and 3%, respectively. Confocal laser scanning microscopy images of propidium iodide-stained yeasts confirmed cell viability before and after microbeads preparation by electrospray. SBWP/AX capability to entrap S. boulardii would represent an alternative for probiotic immobilization in tailored biomaterials and an opportunity for sustainable waste upcycling to value-added products.

Molecular characterization and expression analysis of the chicken-type and goose-type lysozymes from totoaba (Totoaba macdonaldi).

Lysozymes play a key role in innate immune response to bacterial pathogens, catalyzing the hydrolysis of the peptidoglycan layer of bacterial cell walls. In this study, the genes encoding the c-type (TmLyzc) and g-type (TmLyzg) lysozymes from Totoaba macdonaldi were cloned and characterized. The cDNA sequences of TmLyzg and TmLyzc were 582 and 432 bp, encoding polypeptides of 193 and 143 amino acids, respectively. Amino acid sequences of these lysozymes shared high identity (60-90%) with their counterparts of other teleosts and showed conserved functional-structural signatures of the lysozyme superfamily. Phylogenetic analysis indicated a close relationship with their vertebrate homologues but distinct evolutionary paths for each lysozyme. Expression analysis by qRT-PCR revealed that TmLyzc was expressed in stomach and pyloric caeca, while TmLyzg was highly expressed in stomach and heart. These results suggest that both lysozymes play important roles in defense of totoaba against bacterial infections or as digestive enzyme.

Characterization of the trypsin-III from Monterey sardine (Sardinops caeruleus): Insights on the cold-adaptation from the A236N mutant.

Trypsins (E.C. 3.4.21.4) are digestive enzymes that catalyze the hydrolysis of peptide bonds containing arginine and lysine residues. Some trypsins from fish species are active at temperatures just above freezing, and for that are called cold-adapted enzymes, having many biotechnological applications. In this work, we characterized a recombinant trypsin-III from Monterey sardine (Sardinops caeruleus) and studied the role of a single residue on its cold-adapted features. The A236N mutant from sardine trypsin-III showed higher activation energy for the enzyme-catalyzed reaction, it was more active at higher temperatures, and exhibited a higher thermal stability than the wild-type enzyme, suggesting a key role of this residue. The thermodynamic activation parameters revealed an increase in the activation enthalpy for the A236N mutant, suggesting the existence of more intramolecular contacts during the activation step. Molecular models for both enzymes suggest that a hydrogen-bond involving N236 may contact the C-terminal α-helix to the vicinity of the active site, thus affecting the biochemical and thermodynamic properties of the enzyme.

Immune response of human cultured cells towards macrocyclic Fe2PO and Fe2PC bioactive cyclophane complexes.

Synthetic molecules that mimic the function of natural enzymes or molecules have untapped potential for use in the next generation of drugs. Cyclic compounds that contain aromatic rings are macrocyclic cyclophanes, and when they coordinate iron ions are of particular interest due to their antioxidant and biomimetic properties. However, little is known about the molecular responses at the cellular level. This study aims to evaluate the changes in immune gene expression in human cells exposed to the cyclophanes Fe2PO and Fe2PC. Confluent human embryonic kidney cells were exposed to either the cyclophane Fe2PO or Fe2PC before extraction of RNA. The expression of a panel of innate and adaptive immune genes was analyzed by quantitative real-time PCR. Evidence was found for an inflammatory response elicited by the cyclophane exposures. After 8 h of exposure, the cells increased the relative expression of inflammatory mediators such as interleukin 1; IRAK, which transduces signals between interleukin 1 receptors and the NFκB pathway; and the LPS pattern recognition receptor CD14. After 24 h of exposure, regulatory genes begin to counter the inflammation, as some genes involved in oxidative stress, apoptosis and non-inflammatory immune responses come into play. Both Fe2PO and Fe2PC induced similar immunogenetic changes in transcription profiles, but equal molar doses of Fe2PC resulted in more robust responses. These data suggest that further work in whole animal models may provide more insights into the extent of systemic physiological changes induced by these cyclophanes.

TBRG-1 a Ras-like protein in Trichoderma virens involved in conidiation, development, secondary metabolism, mycoparasitism, and biocontrol unveils a new family of Ras-GTPases.

Ras-GTPases are nucleotide hydrolases involved in key cellular processes. In fungi, Ras-GTPases regulate conidiation, development, virulence, and interactions with other fungi or plants. Trichoderma spp. are filamentous saprophytic fungi, widely distributed along all latitudes, characterized by their rapid growth and metabolic diversity. Many species of this genus interact with other fungi, animals or plants. Furthermore, these fungi are used as biocontrol agents due to their ability to antagonize phytopathogenic fungi and oomycetes, through competence, antibiosis, and parasitism. However, the genetic and molecular regulation of these processes is scarcely described in these fungi. In this work, we investigated the role of the gene tbrg-1 product (GenBank accession number XP_013956100; JGI ID: Tv_70852) of T. virens during its interaction with other fungi and plants. Sequence analyses predicted that TBRG-1 bears the characteristic domains of Ras-GTPases; however, its size (1011 aa) is 3- to 4-times bigger compared with classical GTPases. Interestingly, phylogenetic analyses grouped the TBRG-1 protein with hypothetical proteins of similar sizes, sharing conserved regions; whereas other known Ras-GTPases were perfectly grouped with their respective families. These facts led us to classify TBRG-1 into a new family of Ras-GTPases, the Big Ras-GTPases (BRG). Therefore, the gene was named tbrg-1 (TrichodermaBigRas-GTPase-1). Quantification of conidia and scanning electron microscopy showed that the mutants-lacking tbrg-1 produced less conidia, as well as a delayed conidiophore development compared to the wild-type (wt). Moreover, a deregulation of conidiation-related genes (con-10, con-13, and stuA) was observed in tbrg-1-lacking strains, which indicates that TBRG-1 is necessary for proper conidiophore and conidia development. Furthermore, the lack of tbrg-1 affected positively the antagonistic capability of T. virens against the phytopathogens Rhizoctonia solani, Sclerotium rolfsii, and Fusarium oxysporum, which was consistent with the expression patterns of mycoparasitism-related genes, sp1 and cht1, that code for a protease and for a chitinase, respectively. Furthermore, the antibiosis effect of mycelium-free culture filtrates of Δtbrg-1 against R. solani was considerably enhanced. The expression of secondary metabolism-related genes, particularly gliP, showed an upregulation in Δtbrg-1, which paralleled an increase in gliotoxin production as compared to the wt. These results indicate that TBRG-1 plays a negative role in secondary metabolism and antagonism. Unexpectedly, the biocontrol activity of Δtbrg-1 was ineffective to protect the tomato seeds and seedlings against R. solani. On the contrary, Δtbrg-1 behaved like a plant pathogen, indicating that TBRG-1 is probably implicated in the recognition process for establishing a beneficial relationship with plants.

Genome-Wide Identification of Mango (Mangifera indica L.) Polygalacturonases: Expression Analysis of Family Members and Total Enzyme Activity During Fruit Ripening.

Mango (Mangifera indica L.) is an important commercial fruit that shows a noticeable loss of firmness during ripening. Polygalacturonase (PG, E.C. 3.2.1.15) is a crucial enzyme for cell wall loosening during fruit ripening since it solubilizes pectin and its activity correlates with fruit softening. Mango PGs were mapped to a genome draft using seventeen PGs found in mango transcriptomes and 48 bonafide PGs were identified. The phylogenetic analysis suggests that they are related to Citrus sinensis, which may indicate a recent evolutive divergence and related functions with orthologs in the tree. Gene expression analysis for nine PGs showed differential expression for them during post-harvest fruit ripening, MiPG21-1, MiPG14, MiPG69-1, MiPG17, MiPG49, MiPG23-3, MiPG22-7, and MiPG16 were highly up-regulated. PG enzymatic activity also increased during maturation and these results correlate with the loss of firmness observed in mango during post-harvest ripening, between the ethylene production burst and the climacteric peak. The analysis of PGs promoter regions identified regulatory sequences associated to ripening such as MADS-box, ethylene regulation like ethylene insensitive 3 (EIN3) factors, APETALA2-like and ethylene response element factors. During mango fruit ripening the action of at least these nine PGs contribute to softening, and their expression is regulated at the transcriptional level. The prediction of the tridimensional structure of some PGs showed a conserved parallel beta-helical fold related to polysaccharide hydrolysis and a modular architecture, where exons correspond to structural elements. Further biotechnological approaches could target specific softening-related PGs to extend mango post-harvest shelf life.

Comparison of Single and Combined Use of Catechin, Protocatechuic, and Vanillic Acids as Antioxidant and Antibacterial Agents against Uropathogenic Escherichia Coli at Planktonic and Biofilm Levels.

The objective of this study was to evaluate the effect of combining catechin, protocatechuic, and vanillic acids against planktonic growing, adhesion, and biofilm eradication of uropathogenic Escherichia coli (UPEC), as well as antioxidant agents. The minimum inhibitory concentrations (MIC) of protocatechuic, vanillic acids and catechin against the growth of planktonic bacteria were 12.98, 11.80, and 13.78 mM, respectively. Mixing 1.62 mM protocatechuic acid + 0.74 mM vanillic acid + 0.05 mM catechin resulted in a synergistic effect acting as an MIC. Similarly, the minimum concentrations of phenolic compounds to prevent UPEC adhesion and biofilm formation (MBIC) were 11.03 and 7.13 mM of protocatechuic and vanillic acids, respectively, whereas no MBIC of catechin was found. However, combinations of 1.62 mM protocatechuic acid + 0.74 mM vanillic acid + 0.05 mM catechin showed a synergistic effect acting as MBIC. On the other hand, the minimum concentrations to eradicate biofilms (MBEC) were 25.95 and 23.78 mM, respectively. The combination of 3.20 mM protocatechuic acid, 2.97 mM vanillic acid, and 1.72 mM catechin eradicated pre-formed biofilms. The antioxidant capacity of the combination of phenolics was higher than the expected theoretical values, indicating synergism by the DPPH•, ABTS, and FRAP assays. Effective concentrations of catechin, protocatechuic, and vanillic acids were reduced from 8 to 1378 times when combined. In contrast, the antibiotic nitrofurantoin was not effective in eradicating biofilms from silicone surfaces. In conclusion, the mixture of phenolic compounds was more effective in preventing cell adhesion and eradicating pre-formed biofilms of uropathogenic E. coli than single compounds and nitrofurantoin, and showed antioxidant synergy.

Insights into ligand binding to a glutathione S-transferase from mango: Structure, thermodynamics and kinetics.

We studied a mango glutathione S-transferase (GST) (Mangifera indica) bound to glutathione (GSH) and S-hexyl glutathione (GSX). This GST Tau class (MiGSTU) had a molecular mass of 25.5 kDa. MiGSTU Michaelis-Menten kinetic constants were determined for their substrates obtaining a Km, Vmax and kcat for CDNB of 0.792 mM, 80.58 mM min-1 and 68.49 s-1 respectively and 0.693 mM, 105.32 mM min-1 and 89.57 s-1, for reduced GSH respectively. MiGSTU had a micromolar affinity towards GSH (5.2 µM) or GSX (7.8 µM). The crystal structure of the MiGSTU in apo or bound to GSH or GSX generated a model that explains the thermodynamic signatures of binding and showed the importance of enthalpic-entropic compensation in ligand binding to Tau-class GST enzymes.

Mango (Mangifera indica L.) cv. Kent fruit mesocarp de novo transcriptome assembly identifies gene families important for ripening.

Fruit ripening is a physiological and biochemical process genetically programmed to regulate fruit quality parameters like firmness, flavor, odor and color, as well as production of ethylene in climacteric fruit. In this study, a transcriptomic analysis of mango (Mangifera indica L.) mesocarp cv. "Kent" was done to identify key genes associated with fruit ripening. Using the Illumina sequencing platform, 67,682,269 clean reads were obtained and a transcriptome of 4.8 Gb. A total of 33,142 coding sequences were predicted and after functional annotation, 25,154 protein sequences were assigned with a product according to Swiss-Prot database and 32,560 according to non-redundant database. Differential expression analysis identified 2,306 genes with significant differences in expression between mature-green and ripe mango [1,178 up-regulated and 1,128 down-regulated (FDR ≤ 0.05)]. The expression of 10 genes evaluated by both qRT-PCR and RNA-seq data was highly correlated (R = 0.97), validating the differential expression data from RNA-seq alone. Gene Ontology enrichment analysis, showed significantly represented terms associated to fruit ripening like "cell wall," "carbohydrate catabolic process" and "starch and sucrose metabolic process" among others. Mango genes were assigned to 327 metabolic pathways according to Kyoto Encyclopedia of Genes and Genomes database, among them those involved in fruit ripening such as plant hormone signal transduction, starch and sucrose metabolism, galactose metabolism, terpenoid backbone, and carotenoid biosynthesis. This study provides a mango transcriptome that will be very helpful to identify genes for expression studies in early and late flowering mangos during fruit ripening.

The Epl1 and Sm1 proteins from Trichoderma atroviride and Trichoderma virens differentially modulate systemic disease resistance against different life style pathogens in Solanum lycopersicum.

Fungi belonging to the genus Trichoderma, commonly found in soil or colonizing plant roots, exert beneficial effects on plants, including the promotion of growth and the induction of resistance to disease. T. virens and T. atroviride secrete the proteins Sm1 and Epl1, respectively, which elicit local and systemic disease resistance in plants. In this work, we show that these fungi promote growth in tomato (Solanum lycopersicum) plants. T. virens was more effective than T. atroviride in promoting biomass gain, and both fungi were capable of inducing systemic protection in tomato against Alternaria solani, Botrytis cinerea, and Pseudomonas syringae pv. tomato (Pst DC3000). Deletion (KO) of epl1 in T. atroviride resulted in diminished systemic protection against A. solani and B. cinerea, whereas the T. virens sm1 KO strain was less effective in protecting tomato against Pst DC3000 and B. cinerea. Importantly, overexpression (OE) of epl1 and sm1 led to an increase in disease resistance against all tested pathogens. Although the Trichoderma WT strains induced both systemic acquired resistance (SAR)- and induced systemic resistance (ISR)-related genes in tomato, inoculation of plants with OE and KO strains revealed that Epl1 and Sm1 play a minor role in the induction of these genes. However, we found that Epl1 and Sm1 induce the expression of a peroxidase and an α-dioxygenase encoding genes, respectively, which could be important for tomato protection by Trichoderma spp. Altogether, these observations indicate that colonization by beneficial and/or infection by pathogenic microorganisms dictates many of the outcomes in plants, which are more complex than previously thought.

Molecular modeling and expression analysis of a MADS-box cDNA from mango (Mangifera indica L.).

MADS-box genes are a large family of transcription factors initially discovered for their role during development of flowers and fruits. The MADS-box transcription factors from animals have been studied by X-ray protein crystallography but those from plants remain to be studied. In this work, a MADS-box cDNA from mango encoding a protein of 254 residues was obtained and compared. Based on phylogenetic analysis, it is proposed that the MADS-box transcription factor expressed in mango fruit (MiMADS1) belongs to the SEP clade of MADS-box proteins. MiMADS1 mRNA steady-state levels did not changed during mango fruit development and were up-regulated, when mango fruits reached physiological maturity as assessed by qRT-PCR. Thus, MiMADS1 could have a role during development and ripening of this fruit. The theoretical structural model of MiMADS1 showed the DNA-binding domain folding bound to a double-stranded DNA. Therefore, MiMADS1 is an interesting model for understanding DNA-binding for transcriptional regulation.

Pectin-derived oligosaccharides increase color and anthocyanin content in Flame Seedless grapes.

BACKGROUND: Grapes grown in warm weather do not develop a desirable red color and require the use of products to enhance berry color. Pectin-derived oligosaccharides (PDOs) have been shown to have a role in various responses including plant defense, growth and development. In this work a mixture of PDOs with 3-20 degrees of polymerization was applied to Vitis vinifera cv. Flame Seedless grapes under field conditions and compared to the effects of ethephon (an ethylene-releasing compound). The effect of treatments on grape color, anthocyanin content and phenylalanine ammonia lyase (PAL) mRNA levels was evaluated. RESULTS: PDOs treatment increased berry color measured by the Color Index of Red Grapes (CIRG) and anthocyanin content, compared to ethephon and untreated berries (control); 1.5, 1 and 0.5 mg mL⁻¹ PDOs increased berry color by 30%, 27% and 26%, respectively, when compared to control berries. Levels of PAL mRNA accumulating in berries treated with PDOs were elevated within the first 24 h of treatment. CONCLUSIONS: PDOs enhanced the color and anthocyanin content of Flame Seedless grape berries possibly due by the induction of PAL mRNA expression. The results demonstrated that PDOs can be used to improve fruit quality aspects such as berry skin color.

Molecular modeling and expression of the Litopenaeus vannamei proliferating cell nuclear antigen (PCNA) after white spot syndrome virus shrimp infection.

Proliferating cell nuclear antigen (PCNA) is the eukaryotic sliding clamp that tethers DNA polymerase to DNA during replication. The full-length cDNA of the Pacific white shrimp Litopenaeus vannamei PCNA (LvPCNA) was cloned and encoded a protein of 260 amino acids that is highly similar to other Crustacean PCNAs. The theoretical shrimp PCNA structure has all the domains that are necessary for its interaction with template DNA and DNA polymerase. RT-PCR analysis showed that LvPCNA is expressed mainly in muscle and hemocytes and much less in hepatopancreas and gills. LvPCNA mRNA levels are not statistically different in muscle from healthy and challenged shrimp with the white spot syndrome virus (WSSV). In contrast, the mRNA levels of the viral DNA polymerase show a biphasic pattern with expression at 6 h post-infection and later at 24 and 48 h. These results suggest that in shrimp muscle LvPCNA levels are steadily kept to allow viral replication and that WSSV DNA polymerase (WSSV-DNApol) is more responsive towards later stages of infection. More knowledge of the DNA replication machinery would result in a better understanding of the mechanism and components of viral replication, since the WSSV genome does not have all the components required for assembly of a fully functional replisome.

Regulación del inicio de la esporulación e histidina cinasas: un análisis comparativo entre bacillus subtilis y el grupo bacillus cereus / Sporulation start and hystidine kinase regulation: a comparative analysis of bacillus subtilis the group of bacillus cereus / Regulamentação do início da esporulação e histidina-cinase: uma análise comparativa entre bacillus subtilis e o grupo bacillus cereus

La esporulación, que es una respuesta de quorum sensing, es un proceso de diferenciación celular mediado por moléculas de señalización, señales fisiológicas y ambientales. Se sabe que Bacillus subtilis detecta las señales metabólicas y ambientales y éstas son integradas a un sistema de transferencia secuencial de fosfatos. Las señales son detectadas por histidina cinasas que se autofosforilan y fosforilan, a su vez, a proteínas que actúan como reguladores de respuesta y activan la expresión de genes específicos de esporulación. Dada la importancia de B. cereus desde el punto de vista epidemiológico, el potencial para bioterrorismo de B. anthracis y la importancia en biotecnología agrícola de B. thuringiensis, la investigación sobre los mecanismos moleculares de señalización y la regulación del inicio de la esporulación en estas bacterias del grupo B. cereus reviste especial interés. En esta revisión se discute la literatura sobre este tema, haciendo hincapié en las histidina cinasas y en el análisis comparativo de los genomas de B. subtilis y del grupo de B. cereus, en cuanto a las secuencias de posibles histidina cinasas y reguladores de respuesta. Cabe destacar que en los genomas del grupo B. cereus hay mayor número de histidina cinasas (10 a 14) y de reguladores de respuesta (7 a 11) putativos que en B. subtilis (6 histidina cinasas y 6 reguladores de respuesta), lo cual sugiere una mayor capacidad para responder a estímulos ambientales y metabólicos en estas bacterias.

Sporulation is a quorum sensing response and a cellular differentiation process regulated by signalling molecules and physiological and environmental signals. The regulation of sporulation initiation has been extensively studied in Bacillus subtilis and occurs through phosphorelay. B. subtilis detects metabolic and environmental signals through histidine kinases that are autophosphorylated and then transfer the phosphate group to response regulators, activating the expression of sporulation genes. However, there are other important sporulated bacilli like those from the B. cereus group. B. cereus toxins are related to food-borne intoxication, B. anthracis may be used as biological weapon in bioterrorism, and B. thuringiensis is an excellent biological control agent. Therefore, it is critical to understand the signalling processes that control sporulation initiation and the toxin synthesis. This review summarizes known literature about regulation of initiation of sporulation in the B. cereus group focusing in the role of histidine kinases and the putative open reading frames of these sensors in B. subtilis and B. thuringiensis. The genomes of the B. cereus group have 10 to 14 putative histidine kinases and 7 to 11 response regulators, compared to 6 histidine kinases and 6 response regulators in B. subtilis, implying that this last bacteria should have a lower capacity to respond to environmental and metabolic signals.

A esporulação, que é uma resposta de quorum sensing, é um processo de diferenciação celular mediado por moléculas de sinalização, sinais fisiológicas e ambientais. Sabe-se que Bacillus subtilis detecta os sinais metabólicos e ambientais e estes são integrados a um sistema de transferência sequencial de fosfatos. Os sinais são detectados por histidina cinase que, por sua vez, se autofosforilam e fosforilam, em proteínas que atuam como reguladores de resposta e que ativam a expresão de genes específicos de esporulação. Devido à importância de B. cereus do ponto de vista epidemiológico, o potencial para bioterrorismo de B. anthracis e a importância em biotecnologia agrícola de B. thuringiensis, a investigação sobre os mecanismos moleculares de sinalização e a regulamentação do início da esporulação em estas bactérias do grupo B. cereus revestem especial interesse. Nesta revisão se discute a literatura sobre este tema, colocando especial atenção nas histidina cinases, e na análise comparativa dos genomas de B. subtilis e do grupo de B. cereus, em relação às sequências de posíveis histidina cinases e reguladores de resposta. Cabe destacar que nos genomas do grupo B. cereus há maior número de histidina cinases (10 a 14) e de reguladores de resposta (7 a 11) putativos que en B. subtilis (6 histidina cinases e 6 reguladores de resposta), o que sugere uma maior capacidade para responder a estímulos ambientais e metabólicos nestas bactérias.

Recombinant expression of marine shrimp lysozyme in Escherichia coli

Shrimp Lysozyme (Lyz) is a key component of the antibacterial response as part of the innate defense in Crustacea; however, it has not been possible to purify this protein because of the very low amount present in the shrimp blood cells (hemocytes). In an effort to produce enough protein to study its function and biochemical properties we have overexpressed Lysozyme from marine shrimp (Penaeus vannamei) in E. coli. A bacterial protein expression system based on the T7 polymerase promoter was used. Although Lyz was produced as insoluble protein in inclusion bodies, its refolding led to an active protein with a yield of ~10 percent. Details of the protein recombinant expression techniques applied to this shrimp protein are presented.