Introgression of the Self-Pruning Gene into Dwarf Tomatoes to Obtain Salad-Type Determinate Growth Lines.

The use of dwarf plants in tomato breeding has provided several advantages. However, there are no identified dwarf plants (dd) containing the self-pruning habit (spsp). The aim of this work was to obtain future generations, characterize the germplasm, and select potential dwarf plants with a determinate growth habit to obtain Salad-type lines. The work was started by carrying out hybridization, followed by the first, second, and third backcrosses. Once F2BC3 seeds became available, the introgression of the self-pruning gene (spsp) into dwarf plants (dd) began. Three strains of normal architecture and a determinate growth habit were hybridized with two strains of dwarf size and an indeterminate growth habit, thus yielding four hybrids. Additionally, donor genotype UFU MC TOM1, the commercial cultivar Santa Clara, and the wild accession Solanum pennellii were used in the experiment. Agronomic traits, fruit quality, metabolomics, and acylsugars content were evaluated, and dwarf plants with a determinate growth habit were selected. Hybrid 3 exhibited the highest yields. Visual differences between determinate and indeterminate dwarf plant seedlings were observed. It is suggested to carry out five self-pollinations of the best dwarf plant determined and subsequent hybridization with homozygous lines of normal plant architecture and determinate growth habit to obtain hybrids.

Coping with Gender Dysphoria in a Rural Environment during Adolescence.

Gender dysphoria is socially more visible and discussed today, but still underdiagnosed. It refers to distress and/or impaired function caused by inconsistency between the sex assigned at birth and gender identification. Clinical manifestations are variable. Lack of training and investment in gender issues make the diagnosis and management in primary care complex, particularly in conservative and isolated communities, with poor access to information and specialized health services. We describe the diagnosis of gender dysphoria and use of a patient centered multidisciplinary and family approach in a 12-year-old rural born adolescent, assigned female at birth. Our aim is to raise awareness of early symptoms and signs of gender dysphoria and problems faced by transgender people and their families during childhood, leading to gender dysphoria, and we hope our successful approach might improve healthcare provision for these patients, particularly in rural areas.

Stabilization of Enzymes by Using Thermophiles.

Manufactured steroid compounds have many applications in the pharmaceutical industry. Due to the chemical complexity and chirality of steroids, there is an increasing demand for enzyme-based bioconversion processes to replace those based on chemical synthesis. In this context, thermostability of the involved enzymes is a highly desirable property as both the increased half-life of the enzyme and the enhanced solubility of substrates and products will improve the yield of the reactions. Metagenomic libraries from thermal environments are potential sources of thermostable enzymes of prokaryotic origin, but the number of expected hits could be quite low for enzymes handling substrates such as steroids, rarely found in prokaryotes. An alternative to metagenome screening is the selection of thermostable variants of well-known steroid-processing enzymes. Here we review and detail a protocol for such selection, where error-prone PCR (epPCR) is used to introduce random mutations into a gene to create a variants library for further selection of thermostable variants in the thermophile Thermus thermophilus. The method involves the use of folding interference vectors where the proper folding of the enzyme of interest at high temperature is linked to the folding of a reporter encoding a selectable property such as thermostable resistance to kanamycin, leading to a life-or-death selection of variants of reinforced folding independently of the activity of the enzyme.

Adequação da assistência pré-natal entre puérperas em uma maternidade do nordeste brasileiro / Adequacy of prenatal care among puerperas in a maternity in the northeast of Brazil / Adecuación de la asistencia prenatal entre los puertos en una maternidad del Brasil noreste

Avaliar a adequação da assistência pré-natal entre puérperas em uma maternidade de referência do nordeste brasileiro. MÉTODO: estudo transversal e descritivo de caráter quantitativo realizado com 205 puérperas. Os dados foram coletados por meio de um formulário estruturado com informações extraídas no cartão pré-natal e recomendações do Ministério da Saúde. O estudo foi submetido e aprovado pelo Comitê de Ética e Pesquisa da Universidade Federal do Piauí sob o CAAE: 81905417.3.0000.5214. RESULTADOS: 69,8% das entrevistadas fizeram sete ou mais consultas pré-natais. A maioria das puérperas apresentou captação precoce com menos de 12 semanas. Verificou-se prevalência minimamente adequada com 5 ou mais registros dos procedimentos clínico-obstétricos, dentre eles a altura uterina obteve a menor taxa de adequação (61,5%). CONCLUSÃO: A assistência pré-natal foi considerada adequada quanto aos parâmetros analisados, porém observou-se a necessidade de melhoria da qualidade nas consultas.

To assess the adequacy of prenatal care among women who have recently given birth in a reference maternity hospital in northeastern Brazil. METHOD: transverse and descriptive study of quantitative character carried out with 205 postpartum women. The data was collected using a structured form with information extracted on the prenatal card and recommendations from the Ministry of Health. The study was submitted and approved by the Ethics and Research Committee of the Federal University of Piauí under the CAAE: 81905417.3.0000.5214. RESULTS: 69.8% of respondents had seven or more prenatal consultations. Most of the postpartum women had early uptake at less than 12 weeks. Minimally adequate prevalence was found with 5 or more clinical-obstetric procedures records, among them uterine height obtained the lowest rate of adequacy (61.5%). CONCLUSION: Prenatal care was considered adequate for the parameters analyzed, but the need for quality improvement was observed in the consultations.

Evaluar la adecuación de la asistencia prenatal entre el parto precoz en una sala de maternidad de referencia en el noreste de Brasil. MÉTODO: Estudio cuantitativo transversal y descriptivo realizado en el año 2005. Los datos se recopilaron mediante un formulario estructurado con información extraída de la tarjeta prenatal y recomendaciones del Ministerio de Salud. El estudio fue presentado y aprobado por el Comité de Ética e Investigación de la Universidad Federal de Piauí bajo el CAAE: 81905417.3.000.5214. RESULTADOS: el 69,8% de los entrevistados celebraron siete o más consultas prenatales. La mayoría de las madres tuvieron un inicio temprano de la lactancia en menos de 12 semanas. Se observó una prevalencia mínima adecuada con 5 o más registros de procedimientos clínico-obstétricos, entre los cuales la talla uterina obtuvo la menor tasa de adecuación (61,5%). CONCLUSIÓN: La asistencia prenatal se consideró adecuada para los parámetros analizados, pero se observó la necesidad de mejorar la calidad de las consultas.

Reaction of biofortified lettuce genotypes to different strains of Xanthomonas campestris pv. vitians

Lettuce bacterial leaf spot caused by Xanthomonas campestris pv. vitians is an aggressive disease that is difficult to control. So far there are no reports of the reaction of biofortified lettuce genotypes to different isolates of the bacteria. Thus, the objective was to evaluate the aggressiveness of X. campestris pv. vitians, as well as the reaction of biofortified lettuce genotypes to bacterial spot. Two experiments were performed in two distinct seasons (winter and summer), in greenhouse at the Vegetable Experimental Station of the Federal University of Uberlândia (UFU). The experimental design in both experiments was a randomized block design, in a factor scheme of 5 × 4 (five genotypes and four strains), with four repetitions. Were evaluated the severity and the area under the disease progress curve. In general, the biofortified lettuce 'Uberlândia 10000' was more resistant to most bacterial strains in the summer cultivation, and in the winter period UFU 'Crespa 206'. The commercial cultivar Robusta was the most susceptible to the strains during both seasons. The UFU E125 strain was the most aggressive for most genotypes in both seasons.

FGF23-klotho axis as predictive factors of fractures in type 2 diabetics with early chronic kidney disease.

BACKGROUND: The aim of our study was to evaluate the relevance of FGF23-klotho axis in the predisposition for bone fractures in type 2 diabetic patients with early chronic kidney disease. METHODS: In a prospective study we included 126 type 2 diabetic patients with CKD stages 2-3 (from 2010 to 2017). We used descriptive statistics, ANOVA and chi-square test. Our population was divided into two groups according to the occurrence of a bone fracture event or not, and the groups were compared considering several biological and laboratorial parameters. We employed a multiple regression model to identify risk factors for bone fracture events and hazard ratios (HR) were calculated using a backward stepwise likelihood ratio (LR) Cox regression. RESULTS: Patients with a fracture event displayed higher levels of FGF-23, Phosphorus, PTH, TNF-α, OxLDL, HOMA-IR, calcium × phosphorus product and ACR and lower levels of Osteocalcin, α-Klotho, 25(OH)D3 and eGFR compared with patients without a fracture event (p < 0.001). The number of patients with a fracture event was higher than expected within inclining CKD stages (χ2, p = 0.06). The occurrence of fracture and the levels of TNF- α, klotho, 25(OH)D3 and OxLDL were found to predict patient entry into RRT (p < 0.05). Age, osteocalcin, α-Klotho and FGF-23 independently influenced the occurrence of bone fracture (p < 0.05). CONCLUSIONS: α-Klotho and FGF-23 levels may have a good clinical use as biomarkers to predict the occurrence of fracture events.

Characterization of a promiscuous cadmium and arsenic resistance mechanism in Thermus thermophilus HB27 and potential application of a novel bioreporter system.

BACKGROUND: The characterization of the molecular determinants of metal resistance has potential biotechnological application in biosensing and bioremediation. In this context, the bacterium Thermus thermophilus HB27 is a metal tolerant thermophile containing a set of genes involved in arsenic resistance which, differently from other microbes, are not organized into a single operon. They encode the proteins: arsenate reductase, TtArsC, arsenic efflux membrane transporter, TtArsX, and transcriptional repressor, TtSmtB. RESULTS: In this work we show that the arsenic efflux protein TtArsX and the arsenic responsive transcriptional repressor TtSmtB are required to provide resistance to cadmium. We analyzed the sensitivity to Cd(II) of mutants lacking TtArsX, finding that they are more sensitive to this metal than the wild type strain. In addition, using promoter probe reporter plasmids, we show that the transcription of TtarsX is also stimulated by the presence of Cd(II) in a TtSmtB-dependent way. Actually, a regulatory circuit composed of TtSmtB and a reporter gene expressed from the TtarsX promoter responds to variation in Cd(II), As(III) and As(V) concentrations. CONCLUSIONS: Our results demonstrate that the system composed by TtSmtB and TtArsX is responsible for both the arsenic and cadmium resistance in T. thermophilus. The data also support the use of T. thermophilus as a suitable chassis for the design and development of As-Cd biosensors.

A multitarget approach to drug discovery inhibiting Mycobacterium tuberculosis PyrG and PanK.

Mycobacterium tuberculosis, the etiological agent of the infectious disease tuberculosis, kills approximately 1.5 million people annually, while the spread of multidrug-resistant strains is of great global concern. Thus, continuous efforts to identify new antitubercular drugs as well as novel targets are crucial. Recently, two prodrugs activated by the monooxygenase EthA, 7947882 and 7904688, which target the CTP synthetase PyrG, were identified and characterized. In this work, microbiological, biochemical, and in silico methodologies were used to demonstrate that both prodrugs possess a second target, the pantothenate kinase PanK. This enzyme is involved in coenzyme A biosynthesis, an essential pathway for M. tuberculosis growth. Moreover, compound 11426026, the active metabolite of 7947882, was demonstrated to directly inhibit PanK, as well. In an independent screen of a compound library against PyrG, two additional inhibitors were also found to be active against PanK. In conclusion, these direct PyrG and PanK inhibitors can be considered as leads for multitarget antitubercular drugs and these two enzymes could be employed as a "double-tool" in order to find additional hit compounds.

A Brief Guide to the High-Throughput Expression of Directed Evolution Libraries.

The process of protein production optimization requires time and labor, constituting one of the main bottlenecks for the downstream utilization of the proteins. However, once through this bottleneck, the protein production process can be easily standardized and multiplexed to find the fittest variants in large libraries created by random mutagenesis. In this chapter, we present an overview of the most important choices to achieve homogeneous and functional expression of directed evolution libraries in microplate format: (1) choice of induction system and host strain, (2) choice of media and growth conditions, and (3) modifications to the genetic sequence.

An ArsR/SmtB family member regulates arsenic resistance genes unusually arranged in Thermus thermophilus HB27.

Arsenic resistance is commonly clustered in ars operons in bacteria; main ars operon components encode an arsenate reductase, a membrane extrusion protein, and an As-sensitive transcription factor. In the As-resistant thermophile Thermus thermophilus HB27, genes encoding homologues of these proteins are interspersed in the chromosome. In this article, we show that two adjacent genes, TtsmtB, encoding an ArsR/SmtB transcriptional repressor and, TTC0354, encoding a Zn2+ /Cd2+ -dependent membrane ATPase are involved in As resistance; differently from characterized ars operons, the two genes are transcribed from dedicated promoters upstream of their respective genes, whose expression is differentially regulated at transcriptional level. Mutants defective in TtsmtB or TTC0354 are more sensitive to As than the wild type, proving their role in arsenic resistance. Recombinant dimeric TtSmtB binds in vitro to both promoters, but its binding capability decreases upon interaction with arsenate and, less efficiently, with arsenite. In vivo and in vitro experiments also demonstrate that the arsenate reductase (TtArsC) is subjected to regulation by TtSmtB. We propose a model for the regulation of As resistance in T. thermophilus in which TtSmtB is the arsenate sensor responsible for the induction of TtArsC which generates arsenite exported by TTC0354 efflux protein to detoxify cells.

Stabilization of Enzymes by Using Thermophiles.

Manufactured steroid compounds have many applications in the pharmaceutical industry. Due to the chemical complexity and chirality of steroids, there is an increasing demand for enzyme-based bioconversion processes to replace those based on chemical synthesis. In this context, thermostability of the involved enzymes is a highly desirable property as both the increased half-life of the enzyme and the enhanced solubility of substrates and products will improve the yield of the reactions. Metagenomic libraries from thermal environments are potential sources of thermostable enzymes of prokaryotic origin, but the number of expected hits could be quite low for enzymes handling substrates such as steroids, rarely found in prokaryotes. An alternative to metagenome screening is the selection of thermostable variants of well-known steroid-processing enzymes. Here we review and detail a protocol for such selection, where error-prone PCR (epPCR) is used to introduce random mutations into a gene to create a variants library for further selection of thermostable variants in the thermophile Thermus thermophilus. The method involves the use of folding interference vectors where the proper folding of the enzyme of interest at high temperature is linked to the folding of a reporter encoding a selectable property such as thermostable resistance to kanamycin, leading to a life-or-death selection of variants of reinforced folding independently of the activity of the enzyme.

Naa50/San-dependent N-terminal acetylation of Scc1 is potentially important for sister chromatid cohesion.

The gene separation anxiety (san) encodes Naa50/San, a N-terminal acetyltransferase required for chromosome segregation during mitosis. Although highly conserved among higher eukaryotes, the mitotic function of this enzyme is still poorly understood. Naa50/San was originally proposed to be required for centromeric sister chromatid cohesion in Drosophila and human cells, yet, more recently, it was also suggested to be a negative regulator of microtubule polymerization through internal acetylation of beta Tubulin. We used genetic and biochemical approaches to clarify the function of Naa50/San during development. Our work suggests that Naa50/San is required during tissue proliferation for the correct interaction between the cohesin subunits Scc1 and Smc3. Our results also suggest a working model where Naa50/San N-terminally acetylates the nascent Scc1 polypeptide, and that this co-translational modification is subsequently required for the establishment and/or maintenance of sister chromatid cohesion.

Thiophenecarboxamide Derivatives Activated by EthA Kill Mycobacterium tuberculosis by Inhibiting the CTP Synthetase PyrG.

To combat the emergence of drug-resistant strains of Mycobacterium tuberculosis, new antitubercular agents and novel drug targets are needed. Phenotypic screening of a library of 594 hit compounds uncovered two leads that were active against M. tuberculosis in its replicating, non-replicating, and intracellular states: compounds 7947882 (5-methyl-N-(4-nitrophenyl)thiophene-2-carboxamide) and 7904688 (3-phenyl-N-[(4-piperidin-1-ylphenyl)carbamothioyl]propanamide). Mutants resistant to both compounds harbored mutations in ethA (rv3854c), the gene encoding the monooxygenase EthA, and/or in pyrG (rv1699) coding for the CTP synthetase, PyrG. Biochemical investigations demonstrated that EthA is responsible for the activation of the compounds, and by mass spectrometry we identified the active metabolite of 7947882, which directly inhibits PyrG activity. Metabolomic studies revealed that pharmacological inhibition of PyrG strongly perturbs DNA and RNA biosynthesis, and other metabolic processes requiring nucleotides. Finally, the crystal structure of PyrG was solved, paving the way for rational drug design with this newly validated drug target.

2-Carboxyquinoxalines kill mycobacterium tuberculosis through noncovalent inhibition of DprE1.

Phenotypic screening of a quinoxaline library against replicating Mycobacterium tuberculosis led to the identification of lead compound Ty38c (3-((4-methoxybenzyl)amino)-6-(trifluoromethyl)quinoxaline-2-carboxylic acid). With an MIC99 and MBC of 3.1 µM, Ty38c is bactericidal and active against intracellular bacteria. To investigate its mechanism of action, we isolated mutants resistant to Ty38c and sequenced their genomes. Mutations were found in rv3405c, coding for the transcriptional repressor of the divergently expressed rv3406 gene. Biochemical studies clearly showed that Rv3406 decarboxylates Ty38c into its inactive keto metabolite. The actual target was then identified by isolating Ty38c-resistant mutants of an M. tuberculosis strain lacking rv3406. Here, mutations were found in dprE1, encoding the decaprenylphosphoryl-d-ribose oxidase DprE1, essential for biogenesis of the mycobacterial cell wall. Genetics, biochemical validation, and X-ray crystallography revealed Ty38c to be a noncovalent, noncompetitive DprE1 inhibitor. Structure-activity relationship studies generated a family of DprE1 inhibitors with a range of IC50's and bactericidal activity. Co-crystal structures of DprE1 in complex with eight different quinoxaline analogs provided a high-resolution interaction map of the active site of this extremely vulnerable target in M. tuberculosis.

Qualidade de assistência em um centro de atenção secundária em saúde / Quality assessment in outpatient center of secondary attention

JUSTIFICATIVA E OBJETIVOS: Este trabalho avaliou a política de humanização ao se considerar a opinião dos usuários, em um ambulatório universitário de especialidades médicas,como indicador de aspectos da assistência integral e integrada à saúde. MÉTODOS: Os membros da Liga Acadêmica de Humanidades Médicas (LAHM-Unifenas BH) realizaram entrevistas de 218 usuários, aos quais foi aplicado um questionário padronizado. RESULTADOS E CONCLUSÕES: Conclui que um bom exemplo para se conduzir a cultura humanizada de atendimento, em conjunto com a formação acadêmica, poderia ser referência de atitude humanística e responsabilidade social em ser seguida por outro serviço universitário de saúde pública...

BACKGROUND AND OBJECTIVES: This study evaluated the policy of humanization by considering the views of users in out patient university medical specialties, as an indicator of health care aspects. METHODS: The members of the League Academic Medical Humanities (LAHM-Unifenas BH) conducted interviews with 218 users, who were administered a standardized questionnaire. RESULTS AND CONCLUSIONS: It concludes that a good example to lead the culture humanized service, together with academic training, reference could be humanistic attitude and social responsibility to be followed byother university public health service...

Rv2466c mediates the activation of TP053 to kill replicating and non-replicating Mycobacterium tuberculosis.

The emergence of multidrug- and extensively drug-resistant strains of Mycobacterium tuberculosis highlights the need to discover new antitubercular agents. Here we describe the synthesis and characterization of a new series of thienopyrimidine (TP) compounds that kill both replicating and non-replicating M. tuberculosis. The strategy to determine the mechanism of action of these TP derivatives was to generate resistant mutants to the most effective compound TP053 and to isolate the genetic mutation responsible for this phenotype. The only non-synonymous mutation found was a g83c transition in the Rv2466c gene, resulting in the replacement of tryptophan 28 by a serine. The Rv2466c overexpression increased the sensitivity of M. tuberculosis wild-type and resistant mutant strains to TP053, indicating that TP053 is a prodrug activated by Rv2466c. Biochemical studies performed with purified Rv2466c demonstrated that only the reduced form of Rv2466c can activate TP053. The 1.7 Å resolution crystal structure of the reduced form of Rv2466c, a protein whose expression is transcriptionally regulated during the oxidative stress response, revealed a unique homodimer in which a ß-strand is swapped between the thioredoxin domains of each subunit. A pronounced groove harboring the unusual active-site motif CPWC might account for the uncommon reactivity profile of the protein. The mutation of Trp28Ser clearly predicts structural defects in the thioredoxin fold, including the destabilization of the dimerization core and the CPWC motif, likely impairing the activity of Rv2466c against TP053. Altogether our experimental data provide insights into the molecular mechanism underlying the anti-mycobacterial activity of TP-based compounds, paving the way for future drug development programmes.

Design, chemical synthesis of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives and biological activity against enoyl-ACP reductase (InhA) and Mycobacterium tuberculosis.

We report here the discovery, synthesis and screening results of a series of 3-(9H-fluoren-9-yl)pyrrolidine-2,5-dione derivatives as a novel class of potent inhibitors of Mycobacterium tuberculosis H37Rv strain as well as the enoyl acyl carrier protein reductase (ENR) InhA. Among them, several compounds displayed good activities against InhA which is one of the key enzymes involved in the type II fatty acid biosynthesis pathway of the mycobacteria cell wall. Furthermore, some exhibited promising activities against M. tuberculosis and multi-drug resistant M. tuberculosis strains.

DprE1, a new taxonomic marker in mycobacteria.

Among the species of the Mycobacterium genus, more than 50 have been recognized as human pathogens. In spite of the different diseases caused by mycobacteria, the interspecies genetic similarity ranges from 94% to 100%, and for some species, this value is higher than in other bacteria. Consequently, it is important to understand the relationships existing among mycobacterial species. In this context, the possibility to use Mycobacterium tuberculosis dprE1 gene as new phylogenetic/taxonomic marker has been explored. The dprE1 gene codes for the target of benzothiazinones, belonging to a very promising class of antitubercular drugs. Mutations in cysteine 387 of DprE1 are responsible for benzothiazinone resistance. The DprE1 tree, obtained with 73 amino acid sequences of mycobacterial species, revealed that concerning the benzothiazinone sensitivity/resistance, it is possible to discriminate two clusters. To validate it, a concatamer obtained from the amino acid sequences of nine mycobacterial housekeeping genes was performed. The concatamer revealed that there is no separation between the benzothiazinone-susceptible and benzothiazinone-resistant species; consequently, this parameter is not linked to the phylogeny. DprE1 tree might represent a good taxonomic marker for the assignment of a mycobacterial isolate to a species. Moreover, the concatamer represents a good reference phylogeny for the Mycobacterium genus.

Synthesis and evaluation of α-ketotriazoles and α,ß-diketotriazoles as inhibitors of Mycobacterium tuberculosis.

Two series of α-ketotriazole and α,ß-diketotriazole derivatives were synthesized and evaluated for antitubercular and cytotoxic activities. Among them, two α,ß-diketotriazole compounds, 6b and 9b, exhibited good activities (minimum inhibitory concentration = 7.6 µM and 6.9 µM, respectively) on Mycobacterium tuberculosis and multi-drug resistant M. tuberculosis strains and presented no cytotoxicity (IC50 > 50 µM) on colorectal cancer HCT116 and normal fibroblast GM637H cell lines. These two compounds represent promising leads for further optimization.

Lack of relevance of HIF-1α polymorphisms in breast cancer in a Portuguese population.

We aimed to study the prevalence of Hypoxia inducible factor-1α (HIF-1α) P582S and A588T polymorphisms in a Portuguese breast cancer population and its effect on the transcriptional activity of HIF-1α in MCF7 breast adenocarcinoma cells. We performed a polymerase chain Reaction--restriction fragment length polymorphism (PCR-RFLP)-based genotyping of a Portuguese breast cancer population for two HIF-1α polymorphisms. Furthermore, by site-directed mutagenesis, we generated a variant form of HIF-1α and compared its transcriptional activity with the wild-type HIF-1α in MCF7 cells. There were no significant differences in genotypic frequencies for P582S and A588T between breast cancer patients and controls, nor between the transcriptional activity of the 582S mutant and the wild-type HIF-1α. In conclusion, there is no association between HIF-1α polymorphisms and incidence of breast cancer in the Portuguese examined population. Furthermore, the P582S mutation does not affect transcriptional activity of HIF-1α in breast cancer cells, contrary to previous findings in other cell lines, suggesting that the impact of this mutation is cell-type specific.