Determining factors of functioning in hemodialysis patients using the international classification of functioning, disability and health.

BACKGROUND: Hemodialysis (HD) treatment affects functioning, physical activity level, clinical biomarkers, and body composition. However, the association between these variables with functioning, considering International Classification of Functioning, Disability and Health (ICF) domains remains unclear. Thus, the aim of this study was to investigate the possible association between physical activity, biomarkers, and body composition with functioning in HD patients in reference to the ICF. METHODS: Eighty HD patients performed different tests grouped according to ICF domain: Body structure and function - handgrip strength (HS), 5-repetition sit-to-stand test, and 60-s sit-to-stand test (5-STS, 60-STS, respectively); Activity - short physical performance battery (SPPB); and Participation - participation scale questionnaire. Physical activity [Human Activity Profile questionnaire (HAP)], body composition (Dual-energy X-ray absorptiometry), Parathormone (PTH), and alkaline phosphatase were analyzed as possible variables associated with ICF domains. Data analyses were performed using simple and multiple regression models adjusted for age, duration of HD, and diuresis volume. RESULTS: In the body structure and function domain, appendicular lean mass, PTH level, and age were associated with HS (R2 = 0.558); HAP and PTH were associated with 5-STS (R2 = 0.263); and HAP, PTH, duration of HD, and age were associated with 60-STS (R2 = 0.337). In the activity domain, HAP, PTH, alkaline phosphatase, duration of HD, age, and body fat were associated with SPPB (R2 = 0.689). Finally, only HAP was associated with the participation scale (R2 = 0.067). CONCLUSION: Physical activity and PTH levels are determinant protagonists of functioning in all ICF domains in hemodialysis patients.

Risk factors associated with mammary tumors in female dogs / Fatores de risco para tumores mamários em cadelas de Uberlândia e Patos de Minas, Minas Gerais

Mammary tumors in female dogs are the most frequent and corresponds to half of the canine tumors. The objectives of this study were to determine the risk factors associated with the occurrence of mammary tumors in female dogs and to evaluate the macroscopic characteristics of these neoformations, using 386 dogs from the "Outubro Rosa Pets" events done within the cities of Uberlândia and Patos de Minas, Minas Gerais State, Brazil, in 2015 (n=194), 2016 (n=105) and 2017 (n=87). For the determination of risk factors, the binary logistic regression test (P<0.05) was performed. The occurrence of mammary tumors was 23.6% (91/386). The significant risk factors identified were increased age (P<0.001), overweight (P=0.048) and non-castration (P<0.001) with a chance of, respectively, 1.6, 2.3 and 9.3 times for the development of mammary tumors. In dogs with mammary tumors (n=91), 153 lesions were present, of which 39 female dogs had two or more lesions (42.8%). Most of the lesions were at the caudal abdominal (M4) and inguinal (M5) mammary glands (60.13%, 92/153). Relative to the size of the lesions, it was observed that in 78% of the female dogs the lesions were determined asT1 (<3cm), 16.5% were T2 (3-5cm) and 5.5% T3 (>5cm). At least 15.4% (14/91) of the dogs had one of the regional lymph nodes increased. In conclusion, the occurrence of mammary tumors in the evaluated population was 23.6% and that age, overweight and non-realization of ovariohysterectomy are risk factors associated with the development of mammary tumors.(AU)

Em cadelas os tumores mamários são os mais frequentes e correspondem a aproximadamente metade dos tumores em cães. Este estudo teve os objetivos de determinar os fatores de risco envolvidos na ocorrência de tumores mamários em cadelas e avaliar as características macroscópicas destas neoformações, utilizando 386 cadelas do evento "Outubro Rosa Pets" nos municípios de Uberlândia e Patos de Minas, Minas Gerais, Brasil, em 2015 (n=194), 2016 (n=105) e 2017 (n=87). Para a determinação dos fatores de risco utilizou-se o teste de Regressão logística binária (P<0,05). A ocorrência de tumores mamários foi de 23,6% (91/386). Os fatores de risco significativos identificados foram aumento da idade (P<0,001), sobrepeso (P=0,048) e não-castração (P<0,001) com a chance de, respectivamente, 1,6, 2,3 e 9,3 vezes de desenvolvimento de tumores mamários. Nas cadelas com tumores mamários (n=91), constatou-se a presença de 153 lesões, sendo que 39 cadelas apresentaram duas ou mais lesões (42,8%). A maioria das lesões localizaram-se nas mamas abdominais caudais (M4) e inguinais (M5) (60,13%; 92/153). Em relação ao tamanho das lesões, observou-se que 78% das cadelas eram T1 (<3cm), 16,5% T2 (3-5cm) e 5,5% T3 (>5cm). Pelo menos 15,4% (14/91) das cadelas apresentaram um dos linfonodos regionais aumentados. Conclui-se que a ocorrência dos tumores mamários na população avaliada foi de 23,6% e que a idade, sobrepeso e não ovariohisterectomia são fatores de risco para o desenvolvimento de tumores mamários.(AU)

New genes of Xanthomonas citri subsp. citri involved in pathogenesis and adaptation revealed by a transposon-based mutant library.

BACKGROUND: Citrus canker is a disease caused by the phytopathogens Xanthomonas citri subsp. citri, Xanthomonas fuscans subsp. aurantifolli and Xanthomonas alfalfae subsp. citrumelonis. The first of the three species, which causes citrus bacterial canker type A, is the most widely spread and severe, attacking all citrus species. In Brazil, this species is the most important, being found in practically all areas where citrus canker has been detected. Like most phytobacterioses, there is no efficient way to control citrus canker. Considering the importance of the disease worldwide, investigation is needed to accurately detect which genes are related to the pathogen-host adaptation process and which are associated with pathogenesis. RESULTS: Through transposon insertion mutagenesis, 10,000 mutants of Xanthomonas citri subsp. citri strain 306 (Xcc) were obtained, and 3,300 were inoculated in Rangpur lime (Citrus limonia) leaves. Their ability to cause citrus canker was analyzed every 3 days until 21 days after inoculation; a set of 44 mutants showed altered virulence, with 8 presenting a complete loss of causing citrus canker symptoms. Sequencing of the insertion site in all 44 mutants revealed that 35 different ORFs were hit, since some ORFs were hit in more than one mutant, with mutants for the same ORF presenting the same phenotype. An analysis of these ORFs showed that some encoded genes were previously known as related to pathogenicity in phytobacteria and, more interestingly, revealed new genes never implicated with Xanthomonas pathogenicity before, including hypothetical ORFs. Among the 8 mutants with no canker symptoms are the hrpB4 and hrpX genes, two genes that belong to type III secretion system (TTSS), two hypothetical ORFS and, surprisingly, the htrA gene, a gene reported as involved with the virulence process in animal-pathogenic bacteria but not described as involved in phytobacteria virulence. Nucleic acid hybridization using labeled cDNA probes showed that some of the mutated genes are differentially expressed when the bacterium is grown in citrus leaves. Finally, comparative genomic analysis revealed that 5 mutated ORFs are in new putative pathogenicity islands. CONCLUSION: The identification of these new genes related with Xcc infection and virulence is a great step towards the understanding of plant-pathogen interactions and could allow the development of strategies to control citrus canker.

Bacterial phytopathogens and genome science.

There are now fourteen completed genomes of bacterial phytopathogens, all of which have been generated in the past six years. These genomes come from a phylogenetically diverse set of organisms, and range in size from 870 kb to more than 6Mb. The publication of these annotated genomes has significantly helped our understanding of bacterial plant disease. These genomes have also provided important information about bacterial evolution. Examples of recently completed genomes include: Pseudomonas syringae pv tomato, which is notable for its large repertoire of effector proteins; Leifsonia xyli subsp. xyli, the first Gram-positive bacterial genome to be sequenced; and Phytoplasma asteris, the small genome that lacks important functions previously thought to be essential in a bacterium.

Comparative analyses of Xanthomonas and Xylella complete genomes.

Computational analyses of four bacterial genomes of the Xanthomonadaceae family reveal new unique genes that may be involved in adaptation, pathogenicity, and host specificity. The Xanthomonas genus presents 3636 unique genes distributed in 1470 families, while Xylella genus presents 1026 unique genes distributed in 375 families. Among Xanthomonas-specific genes, we highlight a large number of cell wall degrading enzymes, proteases, and iron receptors, a set of energy metabolism genes, second copy of the type II secretion system, type III secretion system, flagella and chemotactic machinery, and the xanthomonadin synthesis gene cluster. Important genes unique to the Xylella genus are an additional copy of a type IV pili gene cluster and the complete machinery of colicin V synthesis and secretion. Intersections of gene sets from both genera reveal a cluster of genes homologous to Salmonella's SPI-7 island in Xanthomonas axonopodis pv citri and Xylella fastidiosa 9a5c, which might be involved in host specificity. Each genome also presents important unique genes, such as an HMS cluster, the kdgT gene, and O-antigen in Xanthomonas axonopodis pv citri; a number of avrBS genes and a distinct O-antigen in Xanthomonas campestris pv campestris, a type I restriction-modification system and a nickase gene in Xylella fastidiosa 9a5c, and a type II restriction-modification system and two genes related to peptidoglycan biosynthesis in Xylella fastidiosa temecula 1. All these differences imply a considerable number of gene gains and losses during the divergence of the four lineages, and are associated with structural genome modifications that may have a direct relation with the mode of transmission, adaptation to specific environments and pathogenicity of each organism.

Whole-genome expression profiling of Xylella fastidiosa in response to growth on glucose.

Xylella fastidiosa is the etiologic agent of diseases in a wide range of economically important crops including citrus variegated chlorosis, a major threat to the Brazilian citrus industry. The genomes of several strains of this phytopathogen have been completely sequenced enabling large-scale functional studies. In this work we used whole-genome DNA microarrays to investigate the transcription profile of X. fastidiosa grown in defined media with different glucose concentrations. Our analysis revealed that while transcripts related to fastidian gum production were unaffected, colicin-V-like and fimbria precursors were induced in high glucose medium. Based on these results, we suggest a model for colicin-defense mechanism in X. fastidiosa.

Identification of new protein-protein interactions involving the products of the chromosome- and plasmid-encoded type IV secretion loci of the phytopathogen Xanthomonas axonopodis pv. citri.

The recently sequenced genome of the bacterial plant pathogen Xanthomonas axonopodis pv. citri contains two virB gene clusters, one on the chromosome and one on a 64-kb plasmid, each of which codes for a previously uncharacterized type IV secretion system (T4SS). Here we used a yeast two-hybrid assay to identify protein-protein interactions in these two systems. Our results revealed interactions between known T4SS components as well as previously uncharacterized interactions involving hypothetical proteins coded by open reading frames in the two X. axonopodis pv. citri virB loci. Our results indicate that both loci may code for previously unidentified VirB7 proteins, which we show interact with either VirB6 or VirB9 or with a hypothetical protein coded by the same locus. Furthermore, a set of previously uncharacterized Xanthomonas proteins have been found to interact with VirD4, whose gene is adjacent to the chromosomal virB locus. The gene for one member of this family is found within the chromosomal virB locus. All these uncharacterized proteins possess a conserved 120-amino-acid domain in their C termini and may represent a family of cofactors or substrates of the Xanthomonas T4SS.

Identification and genomic characterization of a new virus (Tymoviridae family) associated with citrus sudden death disease.

Citrus sudden death (CSD) is a new disease that has killed approximately 1 million orange trees in Brazil. Here we report the identification of a new virus associated with the disease. RNAs isolated from CSD-affected and nonaffected trees were used to construct cDNA libraries. A set of viral sequences present exclusively in libraries of CSD-affected trees was used to obtain the complete genome sequence of the new virus. Phylogenetic analysis revealed that this virus is a new member of the genus Marafivirus. Antibodies raised against the putative viral coat proteins allowed detection of viral antigens of expected sizes in affected plants. Electron microscopy of purified virus confirmed the presence of typical isometric Marafivirus particles. The screening of 773 affected and nonaffected citrus trees for the presence of the virus showed a 99.7% correlation between disease symptoms and the presence of the virus. We also detected the virus in aphids feeding on affected trees. These results suggest that this virus is likely to be the causative agent of CSD. The virus was named Citrus sudden death-associated virus.

Mapping transposon insertion sites by touchdown PCR and hybrid degenerate primers.

A novel mapping method based on touchdown PCR was developed for identifying a transposon insertion site in genomic DNA using a hybrid consensus-degenerate primer in combination with a specific primer that anneals to the transposon. The method was tested using Xanthomonas citri transposon mutants. PCR products contained adjacent DNA regions that belonged to both X. citri genomic DNA and the transposon. Products were directly sequenced from PCRs using only the specific primer. Different PCR conditions were tested, and the optimized reaction parameters that increased product yields and specificity are described. Best results were obtained with the HIB17 hybrid primer, which is a 25-mer oligonucleotide having degenerate bases at 6 different positions within the last 12 bases at the 3' end. An X. citri mutants library was produced by random transposition using the EZ::TN transposon, and we identified the insertion sites within the genome of 90 mutants. Insertions were found within both the chromosomal and the plasmid DNA in these X. citri mutants. Restriction mapping and Southern blot analysis confirmed the insertion sites for eight randomly chosen mutants. This method is a very useful tool for large-scale characterization of mutants in functional genomics studies.

In silico identification of potential chaperone genes that belong to type III and type IV secretion systems in Xanthomonas axonopodis pv citri

The secretion of bacterial virulence factors and flagellar components requires the assistance of specific type III and flagellar chaperones. Standard computational annotation of the genome of Xanthomonas axonopodis pv citri, a plant pathogen that causes citrus canker, initially did not identify any genes belonging to these chaperone categories since the primary sequence homology between them was very low. However, in a search for hypothetical proteins with characteristics similar to these chaperones, we have now identified 30 chromosomal and 10 plasmidial potential genes encoding chaperones belonging to types III/IV, and flagellar secretion systems in this organism. The significance of these findings is discussed.

New protein-protein interactions identified for the regulatory and structural components and substrates of the type III Secretion system of the phytopathogen Xanthomonas axonopodis Pathovar citri.

We have initiated a project to identify protein-protein interactions involved in the pathogenicity of the bacterial plant pathogen Xanthomonas axonopodis pv. citri. Using a yeast two-hybrid system based on Gal4 DNA-binding and activation domains, we have focused on identifying interactions involving subunits, regulators, and substrates of the type III secretion system coded by the hrp (for hypersensitive response and pathogenicity), hrc (for hrp conserved), and hpa (for hrp associated) genes. We have identified several previously uncharacterized interactions involving (i) HrpG, a two-component system response regulator responsible for the expression of X. axonopodis pv. citri hrp operons, and XAC0095, a previously uncharacterized protein encountered only in Xanthomonas spp.; (ii) HpaA, a protein secreted by the type III secretion system, HpaB, and the C-terminal domain of HrcV; (iii) HrpB1, HrpD6, and HrpW; and (iv) HrpB2 and HrcU. Homotropic interactions were also identified for the ATPase HrcN. These newly identified protein-protein interactions increase our understanding of the functional integration of phytopathogen-specific type III secretion system components and suggest new hypotheses regarding the molecular mechanisms underlying Xanthomonas pathogenicity.

The genome sequence of the gram-positive sugarcane pathogen Leifsonia xyli subsp. xyli.

The genome sequence of Leifsonia xyli subsp. xyli, which causes ratoon stunting disease and affects sugarcane worldwide, was determined. The single circular chromosome of Leifsonia xyli subsp. xyli CTCB07 was 2.6 Mb in length with a GC content of 68% and 2,044 predicted open reading frames. The analysis also revealed 307 predicted pseudogenes, which is more than any bacterial plant pathogen sequenced to date. Many of these pseudogenes, if functional, would likely be involved in the degradation of plant heteropolysaccharides, uptake of free sugars, and synthesis of amino acids. Although L. xyli subsp. xyli has only been identified colonizing the xylem vessels of sugarcane, the numbers of predicted regulatory genes and sugar transporters are similar to those in free-living organisms. Some of the predicted pathogenicity genes appear to have been acquired by lateral transfer and include genes for cellulase, pectinase, wilt-inducing protein, lysozyme, and desaturase. The presence of the latter may contribute to stunting, since it is likely involved in the synthesis of abscisic acid, a hormone that arrests growth. Our findings are consistent with the nutritionally fastidious behavior exhibited by L. xyli subsp. xyli and suggest an ongoing adaptation to the restricted ecological niche it inhabits.

Comparative genomics analyses of citrus-associated bacteria.

Xylella fastidiosa 9a5c (XF-9a5c) and Xanthomonas axonopodis pv. citri (XAC) are bacteria that infect citrus plants. Sequencing of the genomes of these strains is complete and comparative analyses are now under way with the genomes of other bacteria of the same genera. In this review, we present an overview of this comparative genomic work. We also present a detailed genomic comparison between XF-9a5a and XAC. Based on this analysis, genes and operons were identified that might be relevant for adaptation to citrus. XAC has two copies of a type II secretion system, a large number of cell wall-degrading enzymes and sugar transporters, a complete energy metabolism, a whole set of avirulence genes associated with a type III secretion system, and a complete flagellar and chemotatic system. By contrast, XF-9a5c possesses more genes involved with type IV pili biosynthesis than does XAC, contains genes encoding for production of colicins, and has 4 copies of Type I restriction/modification system while XAC has only one.