Inntags: small self-structured epitopes for innocuous protein tagging.

Protein tagging is widely used in approaches ranging from affinity purification to fluorescence-based detection in live cells. However, an intrinsic limitation of tagging is that the native function of the protein may be compromised or even abolished by the presence of the tag. Here we describe and characterize a set of small, innocuous protein tags (inntags) that we anticipate will find application in a variety of biological techniques.

Antigen-Antibody Affinity for Dry Eye Biomarkers by Label Free Biosensing. Comparison with the ELISA Technique.

The specificity and affinity of antibody-antigen interactions is a fundamental way to achieve reliable biosensing responses. Different proteins involved with dry eye dysfunction: ANXA1, ANXA11, CST4, PRDX5, PLAA and S100A6; were validated as biomarkers. In this work several antibodies were tested for ANXA1, ANXA11 and PRDX5 to select the best candidates for each biomarker. The results were obtained by using Biophotonic Sensing Cells (BICELLs) as an efficient methodology for label-free biosensing and compared with the Enzyme-Linked Immuno Sorbent Assay (ELISA) technique.

Quantifying the potential seepage from porphyry copper tailing impoundments using a multi-isotopic approach.

Porphyry-style copper deposits are characterized by low Cu grades and high tonnages, resulting in large mine tailing volumes disposed in impoundments. Due to the mining tailing sizes, waterproofing techniques cannot be applied along the dam base. Therefore, to minimize seepage towards the aquifers, pumping wells are usually installed as hydraulic barriers. Currently, there is a controversy over whether or not the water extracted from hydraulic barriers should be counted as the use of new water rights. Consequently, a growing interest to develop tools to trace and quantify the tailing impacts in groundwater and to determine the water pumped amount subjected to water rights exist. In the present study, isotope data (δ2H-H2O, δ18O-H2O, δ34S-SO42- and δ18O-SO42-) are proposed as a tool to quantify tailings seepage towards groundwater and to assess hydraulic barriers effectiveness. To illustrate this approach usefulness, the Quillayes porphyry Cu tailing impoundment (Chile) case study is presented. The multi-isotopic approach revealed that tailing waters are highly evaporated showing high SO42- content (~1900 mg L-1) derived from primary sulfate ore dissolution, whereas freshwaters, derived from recharge water, have low SO42- contents (10-400 mg L-1) resulting from the interaction with geogenic sulfides from barren host rock. The δ2H and δ18O values of groundwater samples collected downstream from the impoundment suggest a mixing at different proportions of highly evaporated water from the mine tailing waters and non-evaporated regional fresh groundwater. Cl-/SO42-, δ34S-SO42-/δ18O-SO42-, δ34S-SO42-/ln(SO42-) and δ2H-H2O/δ18O-H2O mixing models allowed to determine that groundwater located closer to the impoundment had a mine tailing water contribution from 45 to 90 %, whereas those located farther away had lower contribution (5-25 %). Results confirmed the stable isotope usefulness to determine the water origin and to calculate the hydraulic barrier efficiencies and the pumped water proportions unrelated to the mining tailing subject to the water rights.

The peroxiredoxin Tpx1 is essential as a H2O2 scavenger during aerobic growth in fission yeast.

Peroxiredoxins are known to interact with hydrogen peroxide (H(2)O(2)) and to participate in oxidant scavenging, redox signal transduction, and heat-shock responses. The two-cysteine peroxiredoxin Tpx1 of Schizosaccharomyces pombe has been characterized as the H(2)O(2) sensor that transduces the redox signal to the transcription factor Pap1. Here, we show that Tpx1 is essential for aerobic, but not anaerobic, growth. We demonstrate that Tpx1 has an exquisite sensitivity for its substrate, which explains its participation in maintaining low steady-state levels of H(2)O(2). We also show in vitro and in vivo that inactivation of Tpx1 by oxidation of its catalytic cysteine to a sulfinic acid is always preceded by a sulfinic acid form in a covalently linked dimer, which may be important for understanding the kinetics of Tpx1 inactivation. Furthermore, we provide evidence that a strain expressing Tpx1.C169S, lacking the resolving cysteine, can sustain aerobic growth, and we show that small reductants can modulate the activity of the mutant protein in vitro, probably by supplying a thiol group to substitute for cysteine 169.

C-terminal truncation of the peroxiredoxin Tpx1 decreases its sensitivity for hydrogen peroxide without compromising its role in signal transduction.

Peroxiredoxins (Prxs) participate in hydrogen peroxide (H2O2) scavenging. Eukaryotic Prxs suffer H2O2-dependent inactivation, due to the oxidation of its catalytic cysteine to sulfinic acid, a modification which can be enzymatically reversed. This substrate-mediated reversible inactivation has been suggested to allow eukaryotic Prxs to act as floodgates, permitting high levels of H2O2 to trigger signal transduction. To test this hypothesis, we used the fission yeast Prx Tpx1, which acts as a H2O2 scavenger during aerobic metabolism and also participates in peroxide-induced signal transduction pathways. High concentrations of peroxide reversibly inactivate Tpx1.Here, we describe the characterization of a Tpx1 derivative, which lacks a carboxy-terminal extension present only in eukaryotic Prxs. This mutant protein is not inactivated by high doses of H2O2. Exclusive expression of this truncated version of Tpx1 is deleterious for aerobic growth, but H2O2-dependent signal transduction is not impaired in this strain. Instead, the ability of Tpx1.DeltaCTD to detect and detoxify peroxides is impaired. Our results indicate that inactivation of Tpx1 by excess peroxides is not required for H2O2 signaling towards the Sty1 pathway, as expected from the floodgate model, and that the carboxy-terminal extension of Tpx1 concomitantly improves H2O2 scavenging and increases susceptibility to inactivation.

Differences in LexA regulon structure among Proteobacteria through in vivo assisted comparative genomics.

The LexA regulon encompasses an ensemble of genes involved in preserving cell viability under massive DNA damage and is present in most bacterial phyla. Up to date, however, the scope of this network had only been assessed in the Gamma Proteobacteria. Here, we report the structure of the LexA regulon in the Alpha Proteobacteria, using a combined approach that makes use of in vitro and in vivo techniques to assist and validate the comparative genomics in silico methodology. This leads to the first experimentally validated description of the LexA regulon in the Alpha Proteobacteria, and comparison of regulon core structures in both classes suggests that a least common multiple set of genes (recA, ssb, uvrA and ruvCAB) might be a defining property of the Proteobacteria LexA network.

Enfermedad de Devic con respuesta favorable a rituximab: reporte de un caso / Devic disease with favorable response to rituximab: report of a case

La enfermedad de Devic, también llamada neuromielitis óptica, es una entidad autoinmune desmielinizante del Sistema Nervioso Central, poco común que compromete los nervios ópticos y la médula espinal, ocasio-nando pérdida de la agudeza visual y afección motora. A continuación, se presenta el caso clínico de una mujer de 25 años, que presenta signos de mielopatía y amaurosis súbitas, cumpliendo criterios para neuromielitis óptica. Se inicia tratamiento con bolos de metilprednisolona sin mejoría, decidiéndose administrar Rituximab con respuesta favorable temprana. El Rituximab pertenece al grupo de anticuerpos monoclonales anti ­ CD20, una opción importante ante la falta de respuesta a la primera línea de tra-tamiento.

Devic's disease, is also called neuromyelitis optica, it is a demyelinating autoimmune entity of the central nervous system, it is uncommon and it compromises the optic nerves and the spinal cord, causing loss of visual acuity and motor impairment. A clinical case of a 25-year-old woman with signs of sudden myelopathy and amaurosis is presented. It is fulfilling crite-ria for neuromyelitis optica. The treatment with methylprednisolone boluses started without any improvement, and Rituximab was applied with an early favorable response. Rituximab belongs to the group of anti-CD20 monoclo-nal antibodies, it was an important option in the absence of response to the first line of treatment.

Oxidative stress in Schizosaccharomyces pombe: different H2O2 levels, different response pathways.

Schizosaccharomyces pombe triggers different signalling pathways depending on the severity of the oxidative stress exerted, the main ones being the Pap1 and the Sty1 pathways. The Pap1 transcription factor is more sensitive to hydrogen peroxide (H(2)O(2)) than the MAP kinase Sty1 pathway, and is designed to induce adaptation, rather than survival, responses. The peroxiredoxin Tpx1 acts as a H(2)O(2) sensor and the upstream activator of the Pap1 pathway. Therefore, sensitivity to H(2)O(2) depends on this thioredoxin peroxidase. In order to achieve maximal activation of the MAP kinase pathway, the concentration of H(2)O(2) needs to be at least fivefold higher than that to fully activate Pap1. Tpx1 is a H(2)O(2) scavenger, thus its peroxidase activity is essential for aerobic growth. As described for other eukaryotic peroxiredoxins, high doses of H(2)O(2) temporarily inactivate Tpx1 and delay Pap1 activation, whereas the Sty1 pathway remains fully functional under these conditions. As part of the Sty1-dependent transcriptional response, the expression of Srx1 is induced and this reductase re-activates the over-oxidised Tpx1. Therefore, the antioxidant pathways of the fission yeast are perfectly designed so that the transcriptional programs triggered by the different signalling pathways never overlap.

Widespread distribution of a lexA-regulated DNA damage-inducible multiple gene cassette in the Proteobacteria phylum.

The SOS response comprises a set of cellular functions aimed at preserving bacterial cell viability in front of DNA injuries. The SOS network, negatively regulated by the LexA protein, is found in many bacterial species that have not suffered major reductions in their gene contents, but presents distinctly divergent LexA-binding sites across the Bacteria domain. In this article, we report the identification and characterization of an imported multiple gene cassette in the Gamma Proteobacterium Pseudomonas putida that encodes a LexA protein, an inhibitor of cell division (SulA), an error-prone polymerase (DinP) and the alpha subunit of DNA polymerase III (DnaE). We also demonstrate that these genes constitute a DNA damage-inducible operon that is regulated by its own encoded LexA protein, and we establish that the latter is a direct derivative of the Gram-positive LexA protein. In addition, in silico analyses reveal that this multiple gene cassette is also present in many Proteobacteria families, and that both its gene content and LexA-binding sequence have evolved over time, ultimately giving rise to the lexA lineage of extant Gamma Proteobacteria.

Intracellular cyclic AMP concentration is decreased in Salmonella typhimurium fur mutants.

It is known that the Fur protein negatively regulates iron-uptake systems in different bacterial species, including Salmonella typhimurium. In this study it has been shown that the intracellular concentration of cyclic AMP (cAMP) is lower in a knockout S. typhimurium fur mutant than in the wild-type strain. According to this, the expression of two cAMP-regulated genes, such as pepE (encoding an alpha-aspartyl dipeptidase) and the Escherichia coli lac operon, is decreased in S. typhimurium fur cells in comparison with wild-type cells. Introduction of an additional mutation in cpdA, encoding a cyclic 3',5'-cAMP phosphodiesterase, recovers wild-type intracellular cAMP concentration in the S. typhimurium fur mutant. Likewise, expression of pepE and the E. coli lac operon was the same in the S. typhimurium fur cpdA double mutant and the wild-type strain. Moreover, these results also demonstrate that the S. typhimurium Fur protein positively regulates the expression of the flhD master operon governing the flagellar regulon. This positive control must be mediated by binding of the S. typhimurium Fur protein to the flhD promoter as indicated by the fact that this promoter tests positive in a Fur titration assay.

Geobacter sulfurreducens has two autoregulated lexA genes whose products do not bind the recA promoter: differing responses of lexA and recA to DNA damage.

The Escherichia coli LexA protein was used as a query sequence in TBLASTN searches to identify the lexA gene of the delta-proteobacterium Geobacter sulfurreducens from its genome sequence. The results of the search indicated that G. sulfurreducens has two independent lexA genes designated lexA1 and lexA2. A copy of a dinB gene homologue, which in E. coli encodes DNA polymerase IV, is present downstream of each lexA gene. Reverse transcription-PCR analyses demonstrated that, in both cases, lexA and dinB constitute a single transcriptional unit. Electrophoretic mobility shift assays with purified LexA1 and LexA2 proteins have shown that both proteins bind the imperfect palindrome GGTTN(2)CN(4)GN(3)ACC found in the promoter region of both lexA1 and lexA2. This sequence is also present upstream of the Geobacter metallireducens lexA gene, indicating that it is the LexA box of this bacterial genus. This palindrome is not found upstream of either the G. sulfurreducens or the G. metallireducens recA genes. Furthermore, DNA damage induces expression of the lexA-dinB transcriptional unit but not that of the recA gene. However, the basal level of recA gene expression is dramatically higher than that of the lexA gene. Likewise, the promoters of the G. sulfurreducens recN, ruvAB, ssb, umuDC, uvrA, and uvrB genes do not contain the LexA box and are not likely to bind to the LexA1 or LexA2 proteins. G. sulfurreducens is the first bacterial species harboring a lexA gene for which a constitutive expression of its recA gene has been described.

Role of the high-affinity zinc uptake znuABC system in Salmonella enterica serovar typhimurium virulence.

The Salmonella enterica serovar Typhimurium znuABC genes encoding a high-affinity zinc uptake system and its regulatory zur gene have been cloned. Salmonella serovar Typhimurium zur and znuC knockout mutants have been constructed by marker exchange. The 50% lethal dose of the znuC mutant increased when either orally or intraperitoneally inoculated in BALB/c mice, while virulence of the zur mutant decreased only when mice were intraperitoneally challenged.

Acaro-test: un método simple para el diagnóstico de sarna en niños / Acaro-test: a simple method for the diagnosis of scabies in children

Se describe una técnica sencilla para el diagnóstico de certeza de sarna humana a Sarcoptes scabiei var. hominis usando portaobjetos con un trozo de scotch-tape, el cual es aplicado sobre la piel en zonas con lesiones sospechosas antes de volverlo a adherir a la lámina de vidrio. Al examen microscópico de estas muestras se observan los ácaros en sus diferentes estados evolutivos. Por su simplicidad este método es especialmente aplicable en niños. Permite un sistema rápido e inocuo para establecer el diagnóstico y controlar la efectividad del tratamiento médico. Mediante su aplicación se ha observado que los ácaros tienen una capacidad de sobrevida de al menos 48 horas a temperatura ambiente, lo cual induciría a pensar en que los mecanismos de transmisión indirecta pueden tener importancia, aparte del contacto directo piel a piel