Partial in vivo protection against Peruvian spider Loxosceles laeta venom by immunization with a multiepitopic protein (rMEPLox).

Loxoscelism is a serious public health problem in Peru, with approximately 2500 accidents reported per year. To envision alternatives to cope with this health problem, the neutralizing humoral immune response against the lethal effects of Peruvian spider Loxosceles laeta venom was evaluated in a mouse model by immunization with a non-toxic multiepitopic protein (rMEPLox). This immunogen contains epitopes from an astacin-like metalloprotease, a hyaluronidase and a sphingomyelinase-D from Loxosceles intermedia and from SMase-I from L. laeta venoms. In vivo protection assays showed that five out of six mice immunized with rMEPLox (after six injections) resisted to 1.4 LD50 of L. laeta venom, whereas only two animals from a control group survived. The present results indicates that this multiepitopic protein can be a promising candidate for anti-loxoscelic antivenom production and experimental vaccination approaches.

Identification of Hox genes and their expression profiles during embryonic development of the emerging model organism, Macrobrachium olfersii.

Hox genes encode transcription factors that specify the body segment identity during development, including crustaceans, such as amphipods and decapods, that possess a remarkable diversity of segments and specialized appendages. In amphipods, alterations of specialized appendages have been obtained using knockout experiment of Hox genes, which suggests that these genes are involved in the evolution of morphology within crustaceans. However, studies of Hox genes in crustaceans have been limited to a few species. Here, we identified the homeodomain of nine Hox genes: labial (lab), proboscipedia (pb), Deformed (Dfd), Sex combs reduced (Scr), fushi tarazu (ftz), Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abdA), and Abdominal-B (AbdB), and evaluated their expression by RT-qPCR and RT-PCR in the ovary, during embryonic development, and at the first larval stage (Zoea I) of the decapod Macrobrachium olfersii. The transcript levels of lab, Dfd, and ftz decreased and transcripts of pb, Scr, Antp, Ubx, abdA, and AbdB increased during embryonic development. Hox genes were expressed in mature ovaries and Zoea I larval stages, except Scr and ftz, respectively. In addition, isoforms of Dfd, Scr, Ubx, and abdA, which have been scarcely reported in crustaceans, were described. New partial sequences of 87 Hox genes from other crustaceans were identified from the GenBank database. Our results are interesting for future studies to determine the specific function of Hox genes and their isoforms in the freshwater prawn M. olfersii and to contribute to the understanding of the diversity and evolution of body plans and appendages in Crustaceans.

A Novel Diselenide-Probucol-Analogue Protects Against Methylmercury-Induced Toxicity in HT22 Cells by Upregulating Peroxide Detoxification Systems: a Comparison with Diphenyl Diselenide.

Methylmercury (MeHg) is a ubiquitous environmental neurotoxicant whose mechanisms of action involve oxidation of endogenous nucleophilic groups (mainly thiols and selenols), depletion of antioxidant defenses, and disruption of neurotransmitter homeostasis. Diphenyl diselenide-(PhSe)2-a model diaryl diselenide, has been reported to display significant protective effects against MeHg-induced neurotoxicity under both in vitro and in vivo experimental conditions. In this study, we compared the protective effects of (PhSe)2 with those of RC513 (4,4'-diselanediylbis(2,6-di-tert-butylphenol), a novel diselenide-probucol-analog) against MeHg-induced toxicity in the neuronal (hippocampal) cell line HT22. Although both (PhSe)2 and RC513 significantly mitigated MeHg- and tert-butylhydroperoxide (t-BuOOH)-cytotoxicity, the probucol analog exhibited superior protective effects, which were observed earlier and at lower concentrations compared to (PhSe)2. RC513 treatment (at either 0.5 µM or 2 µM) significantly increased glutathione peroxidase (GPx) activity, which has been reported to counteract MeHg-toxicity. (PhSe)2 was also able to increase GPx activity, but only at 2 µM. Although both compounds increased the Gpx1 transcripts at 6 h after treatments, only RC513 was able to increase mRNA levels of Prx2, Prx3, Prx5, and Txn2, which are also involved in peroxide detoxification. RC513 (at 2 µM) significantly increased GPx-1 protein expression in HT22 cells, although (PhSe)2 displayed a minor (nonsignificant) effect in this parameter. In agreement, RC513 induced a faster and superior capability to cope with exogenously-added peroxide (t-BuOOH). In summary, when compared to the prototypical organic diaryl diselenide [(PhSe)2], RC513 displayed superior protective properties against MeHg-toxicity in vitro; this was paralleled by a more pronounced upregulation of defenses related to detoxification of peroxides, which are well-known MeHg-derived intermediate oxidant species.

Identification and evaluation of reference genes for expression studies by RT-qPCR during embryonic development of the emerging model organism, Macrobrachium olfersii.

RT-qPCR is a sensitive and highly efficient technique that is widely used in gene expression analysis and to provide insight into the molecular mechanisms underlying embryonic development. The freshwater prawn, Macrobrachium olfersii is an emerging model organism, but, the stable reference genes of this species need to be identified and validated for RT-qPCR analysis. Thus, the aim of this study was to evaluate the expression stability of six genes (ß-act, GAPDH, EF-1α, RpL8, RpS6, AK) in embryos and in adult tissues (cerebral ganglia, muscle and hepatopancreas) of M. olfersii. The expression stabilities of these genes were evaluated using geNorm, NormFinder, BestKeeper, ΔCt method and integrated tool RefFinder. In the general ranking, RpL8 and RpS6 were the most stable genes in embryos, while RpS6 and RpL8 were the most stable in a combined adult tissue analysis. Analysis of the adult tissues revealed that ß-act and AK were the most stable genes in cerebral ganglia, RpL8 and AK in muscle, and RpS6 and ß-act in hepatopancreas. EF-1α and GAPDH were the least stable genes and as normalizer genes in RT-qPCR affected expression of the Distal-less gene during M. olfersii development. This study provides suitable reference genes for RT-qPCR analysis and allows future studies of the gene expression in M. olfersii for understanding the molecular mechanisms of their development. To our knowledge, this is the first published study that identifies and evaluates reference genes for RT-qPCR analysis in M. olfersii and could be useful as basis for evaluations of reference genes in other prawns.

Transcriptional profiling of immune-related genes in Pacific white shrimp (Litopenaeus vannamei) during ontogenesis.

We have performed here a gene expression analysis to determine the developmental stage at the main genes involved in crustacean immune response begin to be expressed and their changes in mRNA abundance during shrimp development. By using a quantitative PCR-based approach, we have measured the mRNA abundance of 24 immune-related genes from different functional categories in twelve developmental stages ranging from fertilized eggs to larval and postlarval stages and also in juveniles. We showed for the first time that the main genes from the RNAi-based post-transcriptional pathway involved in shrimp antiviral immunity are transcribed in all developmental stages, but exhibit a diverse pattern of gene expression during shrimp ontogenesis. On the other hand, hemocyte-expressed genes mainly involved in antimicrobial defenses appeared to be transcribed in larval stages, indicating that hematopoiesis initiates early in development. Moreover, transcript levels of some genes were early detected in fertilized eggs at 0-4 h post-spawning, suggesting a maternal contribution of immune-related transcripts to shrimp progeny. Altogether, our results provide important clues regarding the ontogenesis of hemocytes as well the establishment of antiviral and antimicrobial defenses in shrimp.

Cloning, expression and bioinformatics analysis of ATP sulfurylase from Acidithiobacillus ferrooxidans ATCC 23270 in Escherichia coli.

Molecular studies of enzymes involved in sulfite oxidation in Acidithiobacillus ferrooxidans have not yet been developed, especially in the ATP sulfurylase (ATPS) of these acidophilus tiobacilli that have importance in biomining. This enzyme synthesizes ATP and sulfate from adenosine phosphosulfate (APS) and pyrophosphate (PPi), final stage of the sulfite oxidation by these organisms in order to obtain energy. The atpS gene (1674 bp) encoding the ATPS from Acidithiobacillus ferrooxidans ATCC 23270 was amplified using PCR, cloned in the pET101-TOPO plasmid, sequenced and expressed in Escherichia coli obtaining a 63.5 kDa ATPS recombinant protein according to SDS-PAGE analysis. The bioinformatics and phylogenetic analyses determined that the ATPS from A. ferrooxidans presents ATP sulfurylase (ATS) and APS kinase (ASK) domains similar to ATPS of Aquifex aeolicus, probably of a more ancestral origin. Enzyme activity towards ATP formation was determined by quantification of ATP formed from E. coli cell extracts, using a bioluminescence assay based on light emission by the luciferase enzyme. Our results demonstrate that the recombinant ATP sulfurylase from A. ferrooxidans presents an enzymatic activity for the formation of ATP and sulfate, and possibly is a bifunctional enzyme due to its high homology to the ASK domain from A. aeolicus and true kinases.

Bartonella bacilliformis, endemic pathogen of the Andean region, is intrinsically resistant to quinolones.

OBJECTIVES: To analyze the sequence of the region involved in the development of quinolone resistance of the gyrA and parC genes in a series of Bartonella bacilliformis isolates recovered prior to the introduction of quinolones, as well as one clinical isolate recovered in the 1970s, establishing the susceptibility levels to nalidixic acid and ciprofloxacin. METHODS: Five B. bacilliformis were studied: four isolated before 1957, prior to the introduction of quinolones in clinical practice. The remaining strain was isolated in 1977. A fragment of the gyrA and parC genes was amplified and sequenced. Susceptibility to nalidixic acid and ciprofloxacin was established by the E-test method. RESULTS: All the strains were resistant to nalidixic acid (minimum inhibitory concentration (MIC) >256 mg/l). Three isolates presented decreased susceptibility to ciprofloxacin and two were highly resistant (MIC >32 mg/l). All the strains presented an Ala at position 91 of GyrA and position 85 of ParC. CONCLUSIONS: B. bacilliformis presents a constitutive resistance to quinolones, which may be related to the presence of Ala at position 91 of GyrA and 85 of ParC. These results advise against the current clinical guidelines recommending the use of ciprofloxacin to treat bartonellosis in some countries of the Andean area.