Functional and comparative analysis of THI1 gene in grasses with a focus on sugarcane.

De novo synthesis of thiamine (vitamin B1) in plants depends on the action of thiamine thiazole synthase, which synthesizes the thiazole ring, and is encoded by the THI1 gene. Here, we investigated the evolution and diversity of THI1 in Poaceae, where C4 and C3 photosynthetic plants co-evolved. An ancestral duplication of THI1 is observed in Panicoideae that remains in many modern monocots, including sugarcane. In addition to the two sugarcane copies (ScTHI1-1 and ScTHI1-2), we identified ScTHI1-2 alleles showing differences in their sequence, indicating divergence between ScTHI1-2a and ScTHI1-2b. Such variations are observed only in the Saccharum complex, corroborating the phylogeny. At least five THI1 genomic environments were found in Poaceae, two in sugarcane, M. sinensis, and S. bicolor. The THI1 promoter in Poaceae is highly conserved at 300 bp upstream of the start codon ATG and has cis-regulatory elements that putatively bind to transcription factors associated with development, growth, development and biological rhythms. An experiment set to compare gene expression levels in different tissues across the sugarcane R570 life cycle showed that ScTHI1-1 was expressed mainly in leaves regardless of age. Furthermore, ScTHI1 displayed relatively high expression levels in meristem and culm, which varied with the plant age. Finally, yeast complementation studies with THI4-defective strain demonstrate that only ScTHI1-1 and ScTHI1-2b isoforms can partially restore thiamine auxotrophy, albeit at a low frequency. Taken together, the present work supports the existence of multiple origins of THI1 harboring genomic regions in Poaceae with predicted functional redundancy. In addition, it questions the contribution of the levels of the thiazole ring in C4 photosynthetic plant tissues or potentially the relevance of the THI1 protein activity.

Associated substitution and complementation patterns of processed discretionary foods and drinks on total energy and added sugar intake.

BACKGROUND: Processed discretionary foods and drinks (industrialised sugary drinks, sweet and savoury snacks, and grain-based sweets) are often target of policies aimed at regulating the food environment. We aimed to understand if a lower intake of processed foods or drinks is associated with substitution or complementation patterns and overall intake. METHODS: We analysed a subsample with two 24-h dietary recalls of the Mexican National Health and Nutrition Survey 2012 (358 children, 253 adolescents and 278 adults). We compared within-person, energy and added sugar intakes between days with and without consumption of each food group with fixed-effects regressions. We estimated the relative change (change in intake when not consumed/average intake when consumed × 100). RESULTS: Processed discretionary foods were not fully substituted, as total energy was 200-400 kcal/day lower when these foods were not consumed. The change in total intake was larger than the intake when consumed (i.e., complemented) for industrialised sugary drinks in adolescents (-136%) and adults (-215%), and sweet, savoury snacks for children (-141%). The change was lower (i.e., partially substituted) for grain-based sweets among children (-78%) and adolescents (-73%). For added sugars, most processed discretionary groups were complemented. CONCLUSIONS: Days without intake of processed discretionary foods were associated with lower total energy and lower added sugar intake compared to days when those foods were consumed. This suggests that regulatory policies to reduce the intake of processed foods could have a meaningful impact on improving the overall diet.

Bioinformatics and expression analysis of the Xeroderma Pigmentosum complementation group C (XPC) of Trypanosoma evansi in Trypanosoma cruzi cells / Análises de bioinformática e da expressão do gene Xeroderma Pigmentosum complementation group C (XPC) de Trypanosoma evansi em células de Trypanosoma cruzi

Nucleotide excision repair (NER) acts repairing damages in DNA, such as lesions caused by cisplatin. Xeroderma Pigmentosum complementation group C (XPC) protein is involved in recognition of global genome DNA damages during NER (GG-NER) and it has been studied in different organisms due to its importance in other cellular processes. In this work, we studied NER proteins in Trypanosoma cruzi and Trypanosoma evansi, parasites of humans and animals respectively. We performed three-dimensional models of XPC proteins from T. cruzi and T. evansi and observed few structural differences between these proteins. In our tests, insertion of XPC gene from T. evansi (TevXPC) in T. cruzi resulted in slower cell growth under normal conditions. After cisplatin treatment, T. cruzi overexpressing its own XPC gene (TcXPC) was able to recover cell division rates faster than T. cruzi expressing TevXPC gene. Based on these tests, it is suggested that TevXPC (being an exogenous protein in T. cruzi) interferes negatively in cellular processes where TcXPC (the endogenous protein) is involved. This probably occurred due interaction of TevXPC with some endogenous molecules or proteins from T. cruzi but incapacity of interaction with others. This reinforces the importance of correctly XPC functioning within the cell.(AU)

O reparo por excisão de nucleotídeos (NER) atua reparando danos no DNA, como lesões causadas por cisplatina. A proteína Xeroderma Pigmentosum complementation group C (XPC) está envolvida no reconhecimento de danos pela via de reparação global do genoma pelo NER (GG-NER) e tem sido estudada em diferentes organismos devido à sua importância em outros processos celulares. Neste trabalho, estudamos proteínas do NER em Trypanosoma cruzi e Trypanosoma evansi, parasitos de humanos e animais, respectivamente. Modelos tridimensionais das proteínas XPC de T. cruzi e T. evansi foram feitos e observou-se poucas diferenças estruturais entre estas proteínas. Durante testes, a inserção do gene XPC de T. evansi (TevXPC) em T. cruzi resultou em crescimento celular mais lento em condições normais. Após o tratamento com cisplatina, T. cruzi superexpressando seu próprio gene XPC (TcXPC) foi capaz de recuperar as taxas de divisão celular mais rapidamente do que T. cruzi expressando o gene TevXPC. Com base nesses testes, sugere-se que TevXPC (sendo uma proteína exógena em T. cruzi) interfere negativamente nos processos celulares em que TcXPC (a proteína endógena) está envolvida. Isso provavelmente ocorreu pois TevXPC é capaz de interagir com algumas moléculas ou proteínas endógenas de T. cruzi, mas é incapaz de interagir com outras. Isso reforça a importância do correto funcionamento de XPC dentro da célula.(AU)

Bioinformatics and expression analysis of the Xeroderma Pigmentosum complementation group C (XPC) of Trypanosoma evansi in Trypanosoma cruzi cells / Análises de bioinformática e da expressão do gene Xeroderma Pigmentosum complementation group C (XPC) de Trypanosoma evansi em células de Trypanosoma cruzi

Nucleotide excision repair (NER) acts repairing damages in DNA, such as lesions caused by cisplatin. Xeroderma Pigmentosum complementation group C (XPC) protein is involved in recognition of global genome DNA damages during NER (GG-NER) and it has been studied in different organisms due to its importance in other cellular processes. In this work, we studied NER proteins in Trypanosoma cruzi and Trypanosoma evansi, parasites of humans and animals respectively. We performed three-dimensional models of XPC proteins from T. cruzi and T. evansi and observed few structural differences between these proteins. In our tests, insertion of XPC gene from T. evansi (TevXPC) in T. cruzi resulted in slower cell growth under normal conditions. After cisplatin treatment, T. cruzi overexpressing its own XPC gene (TcXPC) was able to recover cell division rates faster than T. cruzi expressing TevXPC gene. Based on these tests, it is suggested that TevXPC (being an exogenous protein in T. cruzi) interferes negatively in cellular processes where TcXPC (the endogenous protein) is involved. This probably occurred due interaction of TevXPC with some endogenous molecules or proteins from T. cruzi but incapacity of interaction with others. This reinforces the importance of correctly XPC functioning within the cell.

O reparo por excisão de nucleotídeos (NER) atua reparando danos no DNA, como lesões causadas por cisplatina. A proteína Xeroderma Pigmentosum complementation group C (XPC) está envolvida no reconhecimento de danos pela via de reparação global do genoma pelo NER (GG-NER) e tem sido estudada em diferentes organismos devido à sua importância em outros processos celulares. Neste trabalho, estudamos proteínas do NER em Trypanosoma cruzi e Trypanosoma evansi, parasitos de humanos e animais, respectivamente. Modelos tridimensionais das proteínas XPC de T. cruzi e T. evansi foram feitos e observou-se poucas diferenças estruturais entre estas proteínas. Durante testes, a inserção do gene XPC de T. evansi (TevXPC) em T. cruzi resultou em crescimento celular mais lento em condições normais. Após o tratamento com cisplatina, T. cruzi superexpressando seu próprio gene XPC (TcXPC) foi capaz de recuperar as taxas de divisão celular mais rapidamente do que T. cruzi expressando o gene TevXPC. Com base nesses testes, sugere-se que TevXPC (sendo uma proteína exógena em T. cruzi) interfere negativamente nos processos celulares em que TcXPC (a proteína endógena) está envolvida. Isso provavelmente ocorreu pois TevXPC é capaz de interagir com algumas moléculas ou proteínas endógenas de T. cruzi, mas é incapaz de interagir com outras. Isso reforça a importância do correto funcionamento de XPC dentro da célula.

Bioinformatics and expression analysis of the Xeroderma Pigmentosum complementation group C (XPC) of Trypanosoma evansi in Trypanosoma cruzi cells

Abstract Nucleotide excision repair (NER) acts repairing damages in DNA, such as lesions caused by cisplatin. Xeroderma Pigmentosum complementation group C (XPC) protein is involved in recognition of global genome DNA damages during NER (GG-NER) and it has been studied in different organisms due to its importance in other cellular processes. In this work, we studied NER proteins in Trypanosoma cruzi and Trypanosoma evansi, parasites of humans and animals respectively. We performed three-dimensional models of XPC proteins from T. cruzi and T. evansi and observed few structural differences between these proteins. In our tests, insertion of XPC gene from T. evansi (TevXPC) in T. cruzi resulted in slower cell growth under normal conditions. After cisplatin treatment, T. cruzi overexpressing its own XPC gene (TcXPC) was able to recover cell division rates faster than T. cruzi expressing TevXPC gene. Based on these tests, it is suggested that TevXPC (being an exogenous protein in T. cruzi) interferes negatively in cellular processes where TcXPC (the endogenous protein) is involved. This probably occurred due interaction of TevXPC with some endogenous molecules or proteins from T.cruzi but incapacity of interaction with others. This reinforces the importance of correctly XPC functioning within the cell.

Resumo O reparo por excisão de nucleotídeos (NER) atua reparando danos no DNA, como lesões causadas por cisplatina. A proteína Xeroderma Pigmentosum complementation group C (XPC) está envolvida no reconhecimento de danos pela via de reparação global do genoma pelo NER (GG-NER) e tem sido estudada em diferentes organismos devido à sua importância em outros processos celulares. Neste trabalho, estudamos proteínas do NER em Trypanosoma cruzi e Trypanosoma evansi, parasitos de humanos e animais, respectivamente. Modelos tridimensionais das proteínas XPC de T. cruzi e T. evansi foram feitos e observou-se poucas diferenças estruturais entre estas proteínas. Durante testes, a inserção do gene XPC de T. evansi (TevXPC) em T. cruzi resultou em crescimento celular mais lento em condições normais. Após o tratamento com cisplatina, T. cruzi superexpressando seu próprio gene XPC (TcXPC) foi capaz de recuperar as taxas de divisão celular mais rapidamente do que T. cruzi expressando o gene TevXPC. Com base nesses testes, sugere-se que TevXPC (sendo uma proteína exógena em T. cruzi) interfere negativamente nos processos celulares em que TcXPC (a proteína endógena) está envolvida. Isso provavelmente ocorreu pois TevXPC é capaz de interagir com algumas moléculas ou proteínas endógenas de T.cruzi, mas é incapaz de interagir com outras. Isso reforça a importância do correto funcionamento de XPC dentro da célula.

Bioinformatics and expression analysis of the Xeroderma Pigmentosum complementation group C (XPC) of Trypanosoma evansi in Trypanosoma cruzi cells / Análises de bioinformática e da expressão do gene Xeroderma Pigmentosum complementation group C (XPC) de Trypanosoma evansi em células de Trypanosoma cruzi

Abstract Nucleotide excision repair (NER) acts repairing damages in DNA, such as lesions caused by cisplatin. Xeroderma Pigmentosum complementation group C (XPC) protein is involved in recognition of global genome DNA damages during NER (GG-NER) and it has been studied in different organisms due to its importance in other cellular processes. In this work, we studied NER proteins in Trypanosoma cruzi and Trypanosoma evansi, parasites of humans and animals respectively. We performed three-dimensional models of XPC proteins from T. cruzi and T. evansi and observed few structural differences between these proteins. In our tests, insertion of XPC gene from T. evansi (TevXPC) in T. cruzi resulted in slower cell growth under normal conditions. After cisplatin treatment, T. cruzi overexpressing its own XPC gene (TcXPC) was able to recover cell division rates faster than T. cruzi expressing TevXPC gene. Based on these tests, it is suggested that TevXPC (being an exogenous protein in T. cruzi) interferes negatively in cellular processes where TcXPC (the endogenous protein) is involved. This probably occurred due interaction of TevXPC with some endogenous molecules or proteins from T.cruzi but incapacity of interaction with others. This reinforces the importance of correctly XPC functioning within the cell.

Resumo O reparo por excisão de nucleotídeos (NER) atua reparando danos no DNA, como lesões causadas por cisplatina. A proteína Xeroderma Pigmentosum complementation group C (XPC) está envolvida no reconhecimento de danos pela via de reparação global do genoma pelo NER (GG-NER) e tem sido estudada em diferentes organismos devido à sua importância em outros processos celulares. Neste trabalho, estudamos proteínas do NER em Trypanosoma cruzi e Trypanosoma evansi, parasitos de humanos e animais, respectivamente. Modelos tridimensionais das proteínas XPC de T. cruzi e T. evansi foram feitos e observou-se poucas diferenças estruturais entre estas proteínas. Durante testes, a inserção do gene XPC de T. evansi (TevXPC) em T. cruzi resultou em crescimento celular mais lento em condições normais. Após o tratamento com cisplatina, T. cruzi superexpressando seu próprio gene XPC (TcXPC) foi capaz de recuperar as taxas de divisão celular mais rapidamente do que T. cruzi expressando o gene TevXPC. Com base nesses testes, sugere-se que TevXPC (sendo uma proteína exógena em T. cruzi) interfere negativamente nos processos celulares em que TcXPC (a proteína endógena) está envolvida. Isso provavelmente ocorreu pois TevXPC é capaz de interagir com algumas moléculas ou proteínas endógenas de T.cruzi, mas é incapaz de interagir com outras. Isso reforça a importância do correto funcionamento de XPC dentro da célula.

Nucleopolyhedrovirus Coocclusion Technology: A New Concept in the Development of Biological Insecticides.

Nucleopolyhedroviruses (NPV, Baculoviridae) that infect lepidopteran pests have an established record as safe and effective biological insecticides. Here, we describe a new approach for the development of NPV-based insecticides. This technology takes advantage of the unique way in which these viruses are transmitted as collective infectious units, and the genotypic diversity present in natural virus populations. A ten-step procedure is described involving genotypic variant selection, mixing, coinfection and intraspecific coocclusion of variants within viral occlusion bodies. Using two examples, we demonstrate how this approach can be used to produce highly pathogenic virus preparations for pest control. As restricted host range limits the uptake of NPV-based insecticides, this technology has recently been adapted to produce custom-designed interspecific mixtures of viruses that can be applied to control complexes of lepidopteran pests on particular crops, as long as a shared host species is available for virus production. This approach to the development of NPV-based insecticides has the potential to be applied across a broad range of NPV-pest pathosystems.

An interactome analysis reveals that Arabidopsis thaliana GRDP2 interacts with proteins involved in post-transcriptional processes.

The Arabidopsis thaliana glycine-rich domain protein 2 (AtGRDP2) gene encodes a protein of unknown function that is involved in plant growth and salt stress tolerance. The AtGRDP2 protein (787 aa, At4g37900) is constituted by three domains: a DUF1399 located at the N-terminus, a potential RNA Recognition Motif (RRM) in the central region, and a short glycine-rich domain at the C-terminus. Herein, we analyzed the subcellular localization of AtGRDP2 protein as a GFP translational fusion and found it was localized in the cytosol and the nucleus of tobacco leaf cells. Truncated versions of AtGRDP2 showed that the DUF1399 or the RRM domains were sufficient for nuclear localization. In addition, we performed a yeast two-hybrid split-ubiquitin assay (Y2H) to identify potential interactors for AtGRDP2 protein. The Y2H assay identified proteins associated with RNA binding functions such as PABN3 (At5g65260), EF-1α (At1g07920), and CL15 (At3g25920). Heterodimeric associations in planta between AtGRDP2 and its interactors were carried out by Bimolecular Fluorescence Complementation (BiFC) assays. The data revealed heterodimeric interactions between AtGRDP2 and PABN3 in the nucleus and AtGRDP2 with EF-1α in the cytosol, while AtGRDP2-CL15 associations occurred only in the chloroplasts. Finally, functional characterization of the protein-protein interaction regions revealed that both DUF1399 and RRM domains were key for heterodimerization with its interactors. The AtGRDP2 interaction with these proteins in different compartments suggests that this glycine-rich domain protein is involved in post-transcriptional processes.

Natural Defect of a Plant Rhabdovirus Glycoprotein Gene: A Case Study of Virus-Plant Coevolution.

Seven isolates of a putative cytorhabdovirus (family Rhabdoviridae, order Mononegavirales) designated as citrus-associated rhabdovirus (CiaRV) were identified in citrus, passion fruit, and paper bush from the same geographical area in China. CiaRV, bean-associated cytorhabdovirus (Brazil), and papaya virus E (Ecuador) should be taxonomically classified in the species Papaya cytorhabdovirus. Due to natural mutations, the glycoprotein (G) and P4 genes were impaired in citrus-infecting isolates of CiaRV, resulting in an atypical rhabdovirus genome organization of 3' leader-N-P-P3-M-L-5' trailer. The P3 protein of CiaRV shared a common origin with begomoviral movement proteins (family Geminiviridae). Secondary structure analysis and trans-complementation of movement-deficient tomato mosaic virus and potato virus X mutants by CiaRV P3 supported its function in viral cell-to-cell trafficking. The wide geographical dispersal of CiaRV and related viruses suggests an efficient transmission mechanism, as well as an underlying risk to global agriculture. Both the natural phenomenon and experimental analyses demonstrated presence of the "degraded" type of CiaRV in citrus, in parallel to "undegraded" types in other host plant species. This case study shows a plant virus losing the function of an important but nonessential gene, likely due to host shift and adaption, which deepened our understanding of course of natural viral diversification.

Functional characterisation and in silico modelling of MdPSY2 variants and MdPSY5 phytoene synthases from Malus domestica.

Carotenoids are plastid isoprenoid pigments that play critical roles in light harvesting, photoprotection, and phytohormone biosynthesis. They are also vitamin-A precursors and antioxidant molecules important for human nutrition. Apples (e.g. Malus x domestica Borkh), one of the most widely consumed fruits with high nutrient levels, have a very low carotenoid concentration in flesh, compared with other fruits and vegetables. This could be explained by a deficiency in carotenoid synthesis/accumulation and/or accelerated degradation. We analysed the contribution of M. domestica cv. 'Fuji' phytoene synthase (PSY) in the biosynthesis of carotenoids and determined that among four MdPSY genes present in the organism, MdPSY2 and MdPSY5 are highly expressed in leaves and during fruit ripening in line with an increment in carotenoid content in fruits. Furthermore, two representative polymorphic MdPSY2 variants were found, one with a Tyr358Phe substitution (MdPSY2_F) and the other that additionally has a six-amino-acid deletion in the signal peptide (MdPSY2_CG). MdPSY2, MdPSY5, MdPSY2_F and MdPSY2_CG are all localised in plastids. Interestingly, the polymorphic MdPSY2_F and MdPSY2_CG variants show lower enzymatic activity than the wild-type form in a heterologous complementation assay, which could be attributed to the Tyr358Phe substitution close to the active-site pocket, as was suggested by 3-D modelling analysis. The presence of polymorphic MdPSY2 variants with lower enzymatic activity could be partially responsible for the low carotenoid content in Fuji apple fruits.

Solanum lycopersicum (tomato) possesses mitochondrial and plastidial lipoyl synthases capable of increasing lipoylation levels when expressed in bacteria.

Lipoic acid (LA) and its reduced form (dihydrolipoic acid, DHLA) have unique antioxidant properties among such molecules. Moreover, after a process termed lipoylation, LA is an essential prosthetic group covalently-attached to several key multi-subunit enzymatic complexes involved in primary metabolism, including E2 subunits of pyruvate dehydrogenase (PDH). The metabolic pathway of lipoylation has been extensively studied in Escherichia coli and Arabidopsis thaliana in which protein modification occurs via two routes: de novo synthesis and salvage. Common to both pathways, lipoyl synthase (LIP1 in plants, LipA in bacteria, EC 2.8.1.8) inserts sulphur atoms into the molecule in a final, activating step. However, despite the detection of LA and DHLA in other plant species, including tomato (Solanum lycopersicum), no plant LIP1s have been characterised to date from species other than Arabidopsis. In this work, we present the identification and characterisation of two LIPs from tomato, SlLIP1 and SlLIP1p. Consistent with in silico data, both are widely-expressed, particularly in reproductive organs. In line with bioinformatic predictions, we determine that yellow fluorescent protein tagged versions of SlLIP1 and SlLIP1p are mitochondrially- and plastidially-localised, respectively. Both possess the molecular hallmarks and domains of well-characterised bacterial LipAs. When heterologously-expressed in an E. coli lipA mutant, both are capable of complementing specific growth phenotypes and increasing lipoylation levels of E2 subunits of PDH in vivo, demonstrating that they do indeed function as lipoyl synthases.

Radiotherapy modulates expression of EGFR, ERCC1 and p53 in cervical cancer

Cervical cancer is a public health problem and the molecular mechanisms underlying radioresistance are still poorly understood. Here, we evaluated the modulation of key molecules involved in cell proliferation, cell cycle and DNA repair in cervical cancer cell lines (CASKI and C33A) and in malignant tissues biopsied from 10 patients before and after radiotherapy. The expression patterns of epidermal growth factor receptor (EGFR), excision repair cross-complementation group 1 (ERCC1) and p53 were evaluated in cancer cell lines by quantitative PCR and western blotting, and in human malignant tissues by immunohistochemistry. The mutation status of TP53 gene was evaluated by direct sequencing. Among cell lines, absent or weak modulations of EGFR, ERCC1 and p53 were observed after exposure to 1.8 Gy. Conversely, increased expressions of p53 (5/10 patients; P=0.0239), ERCC1 (5/10 patients; P=0.0294) and EGFR (4/10 patients; P=0.1773) were observed in malignant tissues after radiotherapy with the same radiation dose. TP53 mutations were found only in one patient. Here we show that a single dose of radiotherapy induced EGFR, ERCC1 and p53 expression in malignant tissues from cervical cancer patients but not in cancer cell lines, highlighting the gap between in vitro and in vivo experimental models. Studies on larger patient cohorts are needed to allow an interpretation that an upregulation of p53, EGFR and ERCC1 may be part of a radioresistance mechanism.

Azospirillum brasilense: Laboratory Maintenance and Genetic Manipulation.

Bacteria of the genus Azospirillum, including the most comprehensively studied Azospirillum brasilense, are non-pathogenic soil bacteria that promote the growth of diverse plants, making them an attractive model to understand non-symbiotic, beneficial plant-bacteria associations. Research into the physiology and genetics of these organisms spans decades and a range of molecular tools and protocols have been developed for allelic exchange mutagenesis, in trans expression of genes, and fusions to reporter genes. © 2017 by John Wiley & Sons, Inc.

Functional sensorial complementation during host orientation in an Asilidae parasitoid larva.

Changes in environmental conditions influence the performance of organisms in every aspect of their life. Being capable of accurately sensing these changes allow organisms to better adapt. The detection of environmental conditions involves different sensory modalities. There are many studies on the morphology of different sensory structures but not so many studies showing their function. Here we studied the morphology of different sensory structures in the larva of a dipteran parasitoid. We occluded the putative sensory structures coupling the morphology with their function. First, we could develop a non-invasive method in which we occluded the putative sensorial structures annulling their function temporarily. Regarding their functionality, we found that larvae of Mallophora ruficauda require simultaneously of the sensilla found both in the antennae and those of the maxillary palps in order to orient to its host. When either both antennae or both maxillary palps were occluded, no orientation to the host was observed. We also found that these structures are not involved in the acceptance of the host because high and similar proportion of parasitized hosts was found in host acceptance experiments. We propose that other sensilla could be involved in host acceptance and discuss how the different sensilla in the antennae and maxillary palps complement each other to provide larvae with the information for locating its host.

A family 13 thioesterase isolated from an activated sludge metagenome: Insights into aromatic compounds metabolism.

Activated sludge is produced during the treatment of sewage and industrial wastewaters. Its diverse chemical composition allows growth of a large collection of microbial phylotypes with very different physiologic and metabolic profiles. Thus, activated sludge is considered as an excellent environment to discover novel enzymes through functional metagenomics, especially activities related with degradation of environmental pollutants. Metagenomic DNA was isolated and purified from an activated sludge sample. Metagenomic libraries were subsequently constructed in Escherichia coli. Using tributyrin hydrolysis, a screening by functional analysis was conducted and a clone that showed esterase activity was isolated. Blastx analysis of the sequence of the cloned DNA revealed, among others, an ORF that encodes a putative thioesterase with 47-64% identity to GenBank CDS reported genes, similar to those in the hotdog fold thioesterase superfamily. On the basis of its amino acid similarity and its homology-modelled structure we deduced that this gene encodes an enzyme (ThYest_ar) that belongs to family TE13, with a preference for aryl-CoA substrates and a novel catalytic residue constellation. Plasmid retransformation in E. coli confirmed the clone's phenotype, and functional complementation of a paaI E. coli mutant showed preference for phenylacetate over chlorobenzene as a carbon source. This work suggests a role for TE13 family thioesterases in swimming and degradation approaches for phenyl acetic acid. Proteins 2017; 85:1222-1237. © 2017 Wiley Periodicals, Inc.

Stable expression of Mycobacterium bovis antigen 85B in auxotrophic M. bovis bacillus Calmette-Guérin

BACKGROUND Bovine tuberculosis (TB) is a zoonotic disease caused by Mycobacterium bovis, responsible for causing major losses in livestock. A cost effective alternative to control the disease could be herd vaccination. The bacillus Calmette-Guérin (BCG) vaccine has a limited efficacy against bovine TB, but can improved by over-expression of protective antigens. The M. bovis antigen 85B demonstrates ability to induce protective immune response against bovine TB in animal models. However, current systems for the construction of recombinant BCG expressing multiple copies of the gene result in strains of low genetic stability that rapidly lose the plasmid in vivo. Employing antibiotic resistance as selective markers, these systems also compromise vaccine safety. We previously reported the construction of a stable BCG expression system using auxotrophic complementation as a selectable marker. OBJECTIVES The fundamental aim of this study was to construct strains of M. bovis BCG Pasteur and the auxotrophic M. bovis BCG ΔleuD expressing Ag85B and determine their stability in vivo. METHODS Employing the auxotrophic system, we constructed rBCG strains that expressed M. bovis Ag85B and compared their stability with a conventional BCG strain in mice. Stability was measured in terms of bacterial growth on the selective medium and retention of antigen expression. FINDINGS The auxotrophic complementation system was highly stable after 18 weeks, even during in vivo growth, as the selective pressure and expression of antigen were maintained comparing to the conventional vector. MAIN CONCLUSION The Ag85B continuous expression within the host may generate a stronger and long-lasting immune response compared to conventional systems.

Functional characterization of thiolase-encoding genes from Xanthophyllomyces dendrorhous and their effects on carotenoid synthesis.

BACKGROUND: The basidiomycetous yeast Xanthophyllomyces dendrorhous has been described as a potential biofactory for terpenoid-derived compounds due to its ability to synthesize astaxanthin. Functional knowledge of the genes involved in terpenoid synthesis would create opportunities to enhance carotenoid production. A thiolase enzyme catalyzes the first step in terpenoid synthesis. RESULTS: Two potential thiolase-encoding genes were found in the yeast genome; bioinformatically, one was identified as an acetyl-CoA C-acetyltransferase (ERG10), and the other was identified as a 3-ketoacyl Co-A thiolase (POT1). Heterologous complementation assays in Saccharomyces cerevisiae showed that the ERG10 gene from X. dendrorhous could complement the lack of the endogenous ERG10 gene in S. cerevisiae, thereby allowing cellular growth and sterol synthesis. X. dendrorhous heterozygous mutants for each gene were created, and a homozygous POT1 mutant was also obtained. This mutant exhibited changes in pigment composition and higher ERG10 transcript levels than the wild type strain. CONCLUSIONS: The results support the notion that the ERG10 gene in X. dendrorhous is a functional acetyl-CoA C-acetyltransferase essential for the synthesis of mevalonate in yeast. The POT1 gene would encode a functional 3-ketoacyl Co-A thiolase that is non-essential for cell growth, but its mutation indirectly affects pigment production.

Analysis of ERCC1, BRCA1, RRM1 and TUBB3 as predictors of prognosis in patients with non-small cell lung cancer who received cisplatin-based adjuvant chemotherapy: A prospective study.

Although adjuvant platinum-based chemotherapy has been demonstrated to improve survival in patients with completely resected non-small cell lung cancer (NSCLC), individualized approaches to therapy are urgently required to improve the treatment efficacy and reduce unnecessary toxicity. It was hypothesized in the present study that the protein levels of excision repair cross-complementation group 1 (ERCC1), breast cancer 1 (BRCA1), ribonucleotide reductase M1 (RRM1) and class III ß-tubulin (TUBB3) may influence the therapeutic effect of adjuvant cisplatin-based chemotherapy. The expression of ERCC1, BRCA1, RRM1 and TUBB3 in tissues obtained from 84 patients with NSCLC was analyzed in the present non-interventional study by immunohistochemistry prior to adjuvant chemotherapy. All patients received adjuvant cisplatin-based chemotherapy. The primary endpoint in the present study was disease free survival (DFS). Out of the 84 tumors, the expression of ERCC1, BRCA1, RRM1 and TUBB3 was identified in 46 (55%), 11 (13%), 73 (87%) and 76 (90%) tissues, respectively. A beneficial response to adjuvant cisplatin-based chemotherapy in DFS was associated with the absence of the expression of ERCC1 [hazard ratio (HR), 2.166; 95% confidence interval (CI), 1.049-4.474; P=0.037] and BRCA1 (HR, 2.419; 95% CI, 1.127-5.193; P=0.023), but not with the expression status of RRM1 (HR, 0.568; 95% CI, 0.234-1.379; P=0.212) or TUBB3 (HR, 1.874; 95% CI, 0.448-7.842; P=0.39). In addition, patients lacking the expression of ERCC1 and BRCA1 benefited more from adjuvant cisplatin-based chemotherapy compared with patients that expressed either ERCC1 or BRCA1 (HR, 3.102; 95% CI, 1.343-7.163; P=0.008). The expression of ERCC1 and BRCA1 was significantly associated with the DFS time in patients with NSCLC treated with adjuvant cisplatin-based chemotherapy, respectively. The combination of the ERCC1 and BRCA1 expression levels may be a promising prognostic prediction for adjuvant cisplatin-based chemotherapy.

Characterization of maize spermine synthase 1 (ZmSPMS1): Evidence for dimerization and intracellular location.

Polyamines are ubiquitous positively charged metabolites that play an important role in wide fundamental cellular processes; because of their importance, the homeostasis of these amines is tightly regulated. Spermine synthase catalyzes the formation of polyamine spermine, which is necessary for growth and development in higher eukaryotes. Previously, we reported a stress inducible spermine synthase 1 (ZmSPMS1) gene from maize. The ZmSPMS1 enzyme differs from their dicot orthologous by a C-terminal extension, which contains a degradation PEST sequence involved in its turnover. Herein, we demonstrate that ZmSPMS1 protein interacts with itself in split yeast two-hybrid (Y2H) assays. A Bimolecular Fluorescence Complementation (BiFC) assay revealed that ZmSPMS1 homodimer has a cytoplasmic localization. In order to gain a better understanding about ZmSPMS1 interaction, two deletion constructs of ZmSPMS1 protein were obtained. The ΔN-ZmSPMS1 version, where the first 74 N-terminal amino acids were eliminated, showed reduced capability of dimer formation, whereas the ΔC-ZmSPMS1 version, lacking the last 40 C-terminal residues, dramatically abated the ZmSPMS1-ZmSPMS1 protein interaction. Recombinant protein expression in Escherichia coli of ZmSPMS1 derived versions revealed that deletion of its N-terminal domain affected the spermine biosynthesis, whereas C-terminal ZmSPMS1 truncated version fail to generate this polyamine. These data suggest that N- and C-terminal domains of ZmSPMS1 play a role in a functional homodimer.

Quantitation of yeast cell-cell fusion using multicolor flow cytometry.

Mating of haploid Saccharomyces cerevisiae cells of opposite sex provides a powerful model system to study the cell-cell fusion. However, a rapid and standardized method is much needed for quantitative assessment of fusion efficiency. The gold standard method relies on counting mating pairs in fluorescence microscopy images. This current method is limited by expectancy bias and it is time consuming, restricting the number of both cell-cell fusion events and strains that can be analyzed at once. Automatic approaches present a solution to these limitations. Here, we describe a novel flow cytometric approach that is able to quickly both identify mating pairs within a mixture of gametes and quantify cell fusion efficiency. This method is based on staining the cell wall of yeast populations with different Concanavalin A-fluorophore conjugates. The mating subpopulation is identified as the two-colored events set and fused and unfused mating pairs are subsequently discriminated by green fluorescent protein bimolecular complementation. A series of experiments was conducted to validate a simple and reliable protocol. Mating efficiency in each sample was determined by flow cytometry and compared with the one obtained with the current gold standard technique. The results show that mating pair counts using both methods produce indistinguishable outcomes and that the flow cytometry-based method provides quantitative relevant information in a short time, making possible to quickly analyze many different cell populations. In conclusion, our data show multicolor flow cytometry-based fusion quantitation to be a fast, robust, and reliable method to quantify the cell-cell fusion in yeast.