229 registros encontrados - unidade Centro de Biologia Molecular e Engenharia Genética
3D cell-SELEX : development of RNA aptamers as molecular probes for PC-3 tumor cell line
ARTIGO - Inglês
Assunto: Neoplasias da próstata
Human prostate cancer (PCa) is a highly heterogeneous and multifactorial disease. Current clinical biomarkers are not sufficiently accurate, thus...
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Human prostate cancer (PCa) is a highly heterogeneous and multifactorial disease. Current clinical biomarkers are not sufficiently accurate, thus being unable to predict the clinical outcome. Therefore, searching for new biomarkers aiming to improve diagnosis, prognosis and therapy is still required. In this study, we performed 3D Cell-SELEX against PC-3 prostate cancer cell line, a novel strategy to select specific nucleic acid ligands against spheroid cells in 3D cell culture. This original system combines Cell-SELEX, a process that exploits the cellular structure to generate specific ligands, and 3D cell culture, an approach that mimics the tissue microenvironment in vitro. In the first round of 3D Cell-SELEX, a negative selection against RWPE-1, non-tumor cell line, was performed to subtract non-tumor specific aptamers. The supernatant was used in eight additional rounds of selection, which were performed against PC-3 cell line. After nine selection cycles, eight PC-3 specific RNA aptamers were selected and sequenced. The aptamers presented sizes between 20 and 50 nucleotides-long, with low free energy (∆G<−13.6), which contributed for their spontaneous folding and high stability. Furthermore, our results showed the aptamer A4 as a specific ligand to prostate tumor cells, with dissociation constant in the nanomolar scale. Therefore, the novel 3D Cell-SELEX procedure improved the selection of PCa cell-surface ligands and the aptamer A4 has shown potential for the identification of prostate tumor cells, suggesting the application of this molecule in further screening assays for PCa
ARTIGO - Inglês
Assunto: Placenta
Sickle cell disease (SCD) is a complex disease that is characterized by the polymerization of deoxyhemoglobin S, altered red blood cell membrane...
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Sickle cell disease (SCD) is a complex disease that is characterized by the polymerization of deoxyhemoglobin S, altered red blood cell membrane biology, endothelial activation, hemolysis, a procoagulant state, acute and chronic inflammation, and vaso-occlusion. Among the physiological changes that occur during pregnancy, oxygen is consumed by fetal growth, and pregnant women with SCD are more frequently exposed to low oxygen levels. This might lead to red blood cells sickling, and, consequently, to vaso-occlusion. The mechanisms by which SCD affects placental physiology are largely unknown, and chronic inflammation might be involved in this process. This study aimed to evaluate the gene expression profile of inflammatory response mediators in the placentas of pregnant women with sickle cell cell anemia (HbSS) and hemoglobinopathy SC (HbSC). Our results show differences in a number of these genes. For the HbSS group, when compared to the control group, the following genes showed differential expression: IL1RAP (2.76-fold), BCL6 (4.49-fold), CXCL10 (-2.12-fold), CXCR1 (-3.66-fold), and C3 (-2.0-fold). On the other hand, the HbSC group presented differential expressions of the following genes, when compared to the control group: IL1RAP (4.33-fold), CXCL1 (3.05-fold), BCL6 (4.13-fold), CXCL10 (-3.32-fold), C3 (-2.0-fold), and TLR3 (2.38-fold). Taken together, these data strongly suggest a differential expression of several inflammatory genes in both SCD (HbSS and HbSC), indicating that the placenta might become an environment with hypoxia, and increased inflammation, which could lead to improper placental development
A case of 46,XY DSD due to a novel mutation in the HSD17B3 gene
ARTIGO - Inglês
Assunto: Transtorno 46,XY do desenvolvimento sexual
Texto completo: https://www.karger.com/Article/Abstract/451040
A de novo mutation in CYP21A2 gene in a case of in vitro fertilization
ARTIGO - Inglês
Assunto: Proteína CYP21A2 humana
Abstract: Congenital adrenal hyperplasia, one of the most frequent autosome recessive disorders, is caused by defects in steroidogenic enzymes...
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Abstract: Congenital adrenal hyperplasia, one of the most frequent autosome recessive disorders, is caused by defects in steroidogenic enzymes involved in the cortisol biosynthesis. Approximately 95% of the cases are caused by abnormal function of the 21-hydroxylase enzyme. This deficiency leads to androgen excess, consequently, to virilization and rapid somatic growth with accelerated skeletal maturation. Mutations in CYP21A2 are responsible for different forms of 21-hydroxylase deficiency. Mild impairment in the enzymatic activity causes the non-classic or late-onset congenital adrenal hyperplasia that is observed with a prevalence of 1 in 1000 subjects in different populations. The present paper describes a de novo mutation that occurred in the paternal meiosis. The child, who was conceived by in vitro fertilization, presented with precocious puberty and diagnosed with non-classical 21-hydroxylase deficiency. DNA sequencing showed the compound heterozygosis for a de novo CYP21A1P/A2 chimeric gene and the p.Val281Leu mutation inherited from her mother, who was heterozygous for the mutation. The chimeric gene showed pseudogene-derived sequence from 5'-end to intron 3 and CYP21A2 sequences from intron 3 to 3'-end of the gene. Sequencing analysis of the father did not show any mutation. The multiplex ligation-dependent probe amplification (MLPA) assay did not indicate loss of DNA discarding gene deletion but confirmed the chimeric gene. In addition, supernumerary copies of CYP21A1P were observed for both parents and for the affect child. Since paternity has been confirmed, those results suggest that a de novo large gene conversion in the paternal meiosis could have occurred by misalignment of alleles bearing different copy numbers of genes in CYP21 locus
ARTIGO - Inglês
Assunto: Urochloa humidicola
Abstract: We present the highest-density genetic map for the hexaploid Urochloa humidicola. SNP markers expose genetic organization, reproduction, and...
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Abstract: We present the highest-density genetic map for the hexaploid Urochloa humidicola. SNP markers expose genetic organization, reproduction, and species origin, aiding polyploid and tropical forage research. Tropical forage grasses are an important food source for animal feeding, with Urochloa humidicola, also known as Koronivia grass, being one of the main pasture grasses for poorly drained soils in the tropics. However, genetic and genomic resources for this species are lacking due to its genomic complexity, including high heterozygosity, evidence of segmental allopolyploidy, and reproduction by apomixis. These complexities hinder the application of marker-assisted selection (MAS) in breeding programs. Here, we developed the highest-density linkage map currently available for the hexaploid tropical forage grass U. humidicola. This map was constructed using a biparental F1 population generated from a cross between the female parent H031 (CIAT 26146), the only known sexual genotype for the species, and the apomictic male parent H016 (BRS cv. Tupi). The linkage analysis included 4873 single nucleotide polymorphism (SNP) markers with allele dosage information. It allowed mapping of the ASGR locus and apospory phenotype to linkage group 3, in a region syntenic with chromosome 3 of Urochloa ruziziensis and chromosome 1 of Setaria italica. We also identified hexaploid haplotypes for all individuals, assessed the meiotic configuration, and estimated the level of preferential pairing in parents during the meiotic process, which revealed the autopolyploid origin of sexual H031 in contrast to apomictic H016, which presented allopolyploid behavior in preferential pairing analysis. These results provide new information regarding the genetic organization, mode of reproduction, and allopolyploid origin of U. humidicola, potential SNPs markers associated with apomixis for MAS and resources for research on polyploids and tropical forage grasses
PRE-PRINT - Inglês
Assunto: Urochloa humidicola
Abstract: Tropical forage grasses are an important food source for animal feeding, with Urochloa humidicola, also known as Koronivia grass, being one...
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Abstract: Tropical forage grasses are an important food source for animal feeding, with Urochloa humidicola, also known as Koronivia grass, being one of the main pasture grasses for poorly drained soils in the tropics. However, genetic and genomic resources for this species are lacking due to its genomic complexity, including high heterozygosity, evidence of segmental allopolyploidy, and reproduction by apomixis. These complexities hinder the application of marker-assisted selection (MAS) in breeding programs. Here, we developed the highest-density linkage map currently available for the hexaploid tropical forage grass U. humidicola. This map was constructed using a biparental F1 population generated from a cross between the female parent H031 (CIAT 26146), the only known sexual genotype for the species, and the apomictic male parent H016 (BRS cv. Tupi). The linkage analysis included 4,873 single nucleotide polymorphism (SNP) markers with allele dosage information. It allowed mapping of the apospory locus and phenotype to linkage group 3, in a region syntenic with chromosome 3 of Urochloa ruziziensis and chromosome 1 of Setaria italica. We also identified hexaploid haplotypes for all individuals, assessed the meiotic configuration, and estimated the level of preferential pairing in parents during the meiotic process, which revealed the autopolyploid origin of sexual H031 in contrast to H016, which presented allopolyploid behavior in preferential pairing analysis. These results provide new information regarding the genetic organization, mode of reproduction, and allopolyploid origin of U. humidicola, potential SNPs markers associated to apomixes for MAS and resources for research on polyploids and tropical forage grasses
A joint learning approach for genomic prediction in polyploid grasses
ARTIGO - Inglês
Assunto: Artigo original
Abstract: Poaceae, among the most abundant plant families, includes many economically important polyploid species, such as forage grasses and...
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Abstract: Poaceae, among the most abundant plant families, includes many economically important polyploid species, such as forage grasses and sugarcane (Saccharum spp.). These species have elevated genomic complexities and limited genetic resources, hindering the application of marker-assisted selection strategies. Currently, the most promising approach for increasing genetic gains in plant breeding is genomic selection. However, due to the polyploidy nature of these polyploid species, more accurate models for incorporating genomic selection into breeding schemes are needed. This study aims to develop a machine learning method by using a joint learning approach to predict complex traits from genotypic data. Biparental populations of sugarcane and two species of forage grasses (Urochloa decumbens, Megathyrsus maximus) were genotyped, and several quantitative traits were measured. High-quality markers were used to predict several traits in different cross-validation scenarios. By combining classification and regression strategies, we developed a predictive system with promising results. Compared with traditional genomic prediction methods, the proposed strategy achieved accuracy improvements exceeding 50%. Our results suggest that the developed methodology could be implemented in breeding programs, helping reduce breeding cycles and increase genetic gains
A mixed model QTL analysis for sugarcane multiple-harvest-location trial data
ARTIGO - Inglês
Assunto: Cana-de-açúcar
Sugarcane-breeding programs take at least 12 years to develop new commercial cultivars. Molecular markers offer a possibility to study the genetic...
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Sugarcane-breeding programs take at least 12 years to develop new commercial cultivars. Molecular markers offer a possibility to study the genetic architecture of quantitative traits in sugarcane, and they may be used in marker-assisted selection to speed up artificial selection. Although the performance of sugarcane progenies in breeding programs are commonly evaluated across a range of locations and harvest years, many of the QTL detection methods ignore two- and three-way interactions between QTL, harvest, and location. In this work, a strategy for QTL detection in multi-harvest-location trial data, based on interval mapping and mixed models, is proposed and applied to map QTL effects on a segregating progeny from a biparental cross of pre-commercial Brazilian cultivars, evaluated at two locations and three consecutive harvest years for cane yield (tonnes per hectare), sugar yield (tonnes per hectare), fiber percent, and sucrose content. In the mixed model, we have included appropriate (co)variance structures for modeling heterogeneity and correlation of genetic effects and non-genetic residual effects. Forty-six QTLs were found: 13 QTLs for cane yield, 14 for sugar yield, 11 for fiber percent, and 8 for sucrose content. In addition, QTL by harvest, QTL by location, and QTL by harvest by location interaction effects were significant for all evaluated traits (30 QTLs showed some interaction, and 16 none). Our results contribute to a better understanding of the genetic architecture of complex traits related to biomass production and sucrose content in sugarcane
ARTIGO - Inglês
Assunto: Fatores de risco
Age-related macular degeneration (AMD) is a progressive, mutifactorial condition that affects the central retina, and represents an important cause of...
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Age-related macular degeneration (AMD) is a progressive, mutifactorial condition that affects the central retina, and represents an important cause of blindness in the elderly population worldwide. Its pathogenesis is complex, with genetics and environmental factors playing critical roles. The genetic component of AMD has been estimated at 45% to 70%, with at least 34 genomic loci implicated in its etiology. Different variants of complement pathway-associated genes have been associated with the disease, demonstrating the importance of immune pathology and inflammation in the mechanism of AMD. The purpose of this study was to evaluate the role of the variants CFB R32Q (rs641153) and CFH (rs1410996) in the risk of development of AMD in a Brazilian population. This was a case-control study, in which 443 unrelated AMD patients and 471 controls were evaluated for the rs641153 and 451 unrelated AMD patients and 435 controls were evaluated for the rs1410996 SNPs through PCR/direct sequencing. AMD patients were diagnosed according to the International Classification System and subdivided in subgroups: dry and wet; early and advanced disease.Genotype distributions were significantly different when cases and controls were compared. When analyzing rs641153, AG/AA genotypes were more frequent in controls than in cases when compared to GG genotype (p= 0.0008), OR: 0.548 (IC95% 0.385-0.780). When comparing early versus advanced forms, GG genotype was more frequent in patients with advanced AMD (p=0.0006), OR: 1.878 (IC95% 1.081-3.262). No association was observed for the comparison of wet versus dry forms. For rs1410996, GG/AG genotypes were more frequent in cases than controls when compared to AA genotype (p=0.0296), OR: 1.462 (IC95% 1.038-2.059). For the comparison of GG versus AA genotype, OR was 2.030 (IC95% 1.375-2.996) (p=0.0002). No association was observed for the comparisons early versus advanced and dry versus wet AMD.These results are in accordance with the literature and support the role of the A variant of the CFB gene in AMD susceptibility, as a protection factor for the disease and the G variant of the CFH gene as a risk factor for the disease. To our knowledge, this is the first report of the association of these variants with AMD in Brazilians, a highly admixed population
Texto completo: https://iovs.arvojournals.org/article.aspx?articleid=2767534
ARTIGO - Inglês
Assunto: Espectrometria de massa
Mutations in the mitochondrial DNA (mtDNA) have been associated with aminoglycoside-induced and nonsyndromic deafness in different populations. In the...
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Mutations in the mitochondrial DNA (mtDNA) have been associated with aminoglycoside-induced and nonsyndromic deafness in different populations. In the present study, we investigated the contribution of mutations in mitochondrial genes to the etiology of hearing loss in a Brazilian sample. Using mass spectrometry genotyping technology, combined with direct sequencing, 50 alterations previously described in 14 mitochondrial genes were screened in 152 patients with sensorineural hearing loss and in104 normal hearing controls. Fifteen known mitochondrial alterations were detected (G709A, A735G, A827G, G988A, A1555G, T4363C, T5628C, T5655C, G5821A, C7462T, G8363A, T10454C, G12236A, T1291C, G15927A). Pathogenic mutations in MT-RNR1 and MT-TK genes were detected in 3 % (5/152) of the patients with hearing loss. This study contributed to show the spectrum of mitochondrial variants in Brazilian patients with hearing loss. Frequency of A1555G was relatively high (2.6 %), indicating that this mutation is an important cause of hearing loss in our population. This work reports for the first time the investigation and the detection of the tRNALys G8363A mutation in Brazilian patients with maternally inherited sensorineural hearing loss.
ARTIGO - Inglês
Assunto: Cana-de-açúcar
Background: Sugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. Modern cultivars have...
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Background: Sugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. Modern cultivars have polyploid, large complex genomes, with highly unequal contributions from ancestral genomes. Long Terminal Repeat retrotransposons (LTR-RTs) are the single largest components of most plant genomes and can substantially impact the genome in many ways. It is therefore crucial to understand their contribution to the genome and transcriptome, however a detailed study of LTR-RTs in sugarcane has not been previously carried out. Sixty complete LTR-RT elements were classified into 35 families within four Copia and three Gypsy lineages. Structurally, within lineages elements were similar, between lineages there were large size differences. FISH analysis resulted in the expected pattern of Gypsy/heterochromatin, Copia/euchromatin, but in two lineages there was localized clustering on some chromosomes. Analysis of related ESTs and RT-PCR showed transcriptional variation between tissues and families. Four distinct patterns were observed in sRNA mapping, the most unusual of which was that of Ale1, with very large numbers of 24nt sRNAs in the coding region. The results presented support the conclusion that distinct small RNA-regulated pathways in sugarcane target the lineages of LTR-RT elements. Individual LTR-RT sugarcane families have distinct structures, and transcriptional and regulatory signatures. Our results indicate that in sugarcane individual LTR-RT families have distinct behaviors and can potentially impact the genome in diverse ways. For instance, these transposable elements may affect nearby genes by generating a diverse set of small RNA's that trigger gene silencing mechanisms. There is also some evidence that ancestral genomes contribute significantly different element numbers from particular LTR-RT lineages to the modern sugarcane cultivar genome
ARTIGO - Inglês
Assunto: Cana-de-açúcar
Whole genome duplication has played an important role in plant evolution and diversification. Sugarcane is an important crop with a complex hybrid...
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Whole genome duplication has played an important role in plant evolution and diversification. Sugarcane is an important crop with a complex hybrid polyploid genome, for which the process of adaptation to polyploidy is still poorly understood. In order to improve our knowledge about sugarcane genome evolution and the homo/homeologous gene expression balance, we sequenced and analyzed 27 BACs (Bacterial Artificial Chromosome) of sugarcane R570 cultivar, containing the putative single-copy genes LFY (seven haplotypes), PHYC (four haplotypes), and TOR (seven haplotypes). Comparative genomic approaches showed that these sugarcane loci presented a high degree of conservation of gene content and collinearity (synteny) with sorghum and rice orthologous regions, but were invaded by transposable elements (TE). All the homo/homeologous haplotypesof LFY, PHYC, and TOR are likely tobe functional, because they are all under purifying selection (dN/dS << 1). However, they were found to participate in a nonequivalently manner to the overall expression of the corresponding gene. SNPs, indels, and amino acid substitutions allowed inferring the S. officinarum or S. spontaneum origin of the TOR haplotypes, which further led to the estimation that these two sugarcane ancestral species diverged between 2.5 and 3.5 Ma. In addition, analysis of shared TE insertions in TOR haplotypes suggested that two autopolyploidization may have occurred in the lineage that gave rise to S. officinarum, after its divergence from S. spontaneum
DOI: https://doi.org/10.1093/gbe/evw293
Texto completo: https://academic.oup.com/gbe/article/9/2/266/2895932
Ancient DNA sequence revealed by error-correcting codes
ARTIGO - Inglês
Assunto: DNA
A previously described DNA sequence generator algorithm (DNA-SGA) using error-correcting codes has been employed as a computational tool to address...
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A previously described DNA sequence generator algorithm (DNA-SGA) using error-correcting codes has been employed as a computational tool to address the evolutionary pathway of the genetic code. The code-generated sequence alignment demonstrated that a residue mutation revealed by the code can be found in the same position in sequences of distantly related taxa. Furthermore, the code-generated sequences do not promote amino acid changes in the deviant genomes through codon reassignment. A Bayesian evolutionary analysis of both code-generated and homologous sequences of the Arabidopsis thaliana malate dehydrogenase gene indicates an approximately 1 MYA divergence time from the MDH code-generated sequence node to its paralogous sequences. The DNA-SGA helps to determine the plesiomorphic state of DNA sequences because a single nucleotide alteration often occurs in distantly related taxa and can be found in the alternative codon patterns of noncanonical genetic codes. As a consequence, the algorithm may reveal an earlier stage of the evolution of the standard code
DOI: https://doi.org/10.1038/srep12051
Texto completo: https://www.nature.com/articles/srep12051
Androgens by immunoassay and mass spectrometry in children with 46,XY disorder of sex development
ARTIGO - Inglês
Assunto: Testículos
Steroid measurement is a challenge in pediatric endocrinology. Currently, liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered...
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Steroid measurement is a challenge in pediatric endocrinology. Currently, liquid chromatography with tandem mass spectrometry (LC-MS/MS) is considered a gold standard for this purpose. The aim of this study was to compare both LC-MS/MS and immunoassay (IA) for androgens before and after human recombinant chorionic gonadotropin (rhCG) stimulus in children with 46,XY disorders of sex development (DSD). Nineteen patients with 46,XY DSD were evaluated; all of them were prepubertal and non-gonadectomized. Testosterone, dihydrotestosterone (DHT), DHEA and androstenedione were measured by IA and LC-MS/MS before and 7 days after rhCG injection. The correlation between IA and LC-MS/MS was analyzed by the intraclass correlation coefficient (ICC) and Spearman’s rank correlation coefficient (SCC). For concordance analysis the Passing and Bablok (PB) regression and the Bland and Altman (BA) method were used.Testosterone showed excellent correlation (ICC = 0.960 and SCC = 0.964); DHT showed insignificant and moderate correlations as indicated by ICC (0.222) and SCC (0.631), respectively; DHEA showed moderate correlation (ICC = 0.585 and SCC = 0.716); and androstenedione had poor and moderate correlations in ICC (0.363) and SCC (0.735), respectively. Using the PB method, all hormones showed a linear correlation, but proportional and systematic concordance errors were detected for androstenedione, systematic errors for testosterone and no errors for DHEA and DHT. By the BA method, there was a trend of IA to overestimate testosterone and androstenedione and underestimate DHEA and DHT when compared to LC-MS/MS.Traditional IA should be replaced by LC-MS/MS for the androgens measurement in prepubertal children whenever is possible
ARTIGO - Inglês
Assunto: Diabetes Mellitus neonatal permanente
Background: Permanent neonatal diabetes mellitus (PNDM) is a rare disorder, characterized by uncontrolled hyperglycemia diagnosed during the first 6...
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Background: Permanent neonatal diabetes mellitus (PNDM) is a rare disorder, characterized by uncontrolled hyperglycemia diagnosed during the first 6 months of life. In general, PNDM has a genetic origin and most frequently it results from heterozygous mutations in KCNJ11, INS and ABCC8 genes. Homozygous or compound heterozygous inactivating mutations in GCK gene as cause of PNDM are rare. In contrast, heterozygosis for GCK inactivating mutations is frequent and results in the maturity-onset diabetes of young (MODY), manifested by a mild fasting hyperglycemia usually detected later in life. Therefore, as an autosomal recessive disorder, GCK-PNDM should be considered in families with history of glucose intolerance or MODY in first relatives, especially when consanguinity is suspected.
Results: Here we describe two patients born from non-consanguineous parents within a family. They presented low birth weight with persistent hyperglycemia during the first month of life. Molecular analyses for KCNJ11, INS, ABCC8 did not show any mutation. GCK gene sequencing, however, revealed that both patients were compound heterozygous for two missense combined in a novel GCK-PNDM genotype. The p.Asn254His and p.Arg447Gly mutations had been inherited from their mothers and fathers, respectively, as their mothers are sisters and their fathers are brothers. Parents had been later diagnosed as having GCK-MODY. Conclusions: Mutations' in silico analysis was carried out to elucidate the role of the amino acid changes on the enzyme structure. Both p.Asn254His and p.Arg447Gly mutations appeared to be quite damaging. This is the first report of GCK-PNDM in a Brazilian family
Results: Here we describe two patients born from non-consanguineous parents within a family. They presented low birth weight with persistent hyperglycemia during the first month of life. Molecular analyses for KCNJ11, INS, ABCC8 did not show any mutation. GCK gene sequencing, however, revealed that both patients were compound heterozygous for two missense combined in a novel GCK-PNDM genotype. The p.Asn254His and p.Arg447Gly mutations had been inherited from their mothers and fathers, respectively, as their mothers are sisters and their fathers are brothers. Parents had been later diagnosed as having GCK-MODY. Conclusions: Mutations' in silico analysis was carried out to elucidate the role of the amino acid changes on the enzyme structure. Both p.Asn254His and p.Arg447Gly mutations appeared to be quite damaging. This is the first report of GCK-PNDM in a Brazilian family
PRE-PRINT - Inglês
Assunto: Antimaláricos
Abstract: The enzyme deoxyhypusine synthase (DHS) catalyzes the first step in the post-translational modification of the eukaryotic translation factor...
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Abstract: The enzyme deoxyhypusine synthase (DHS) catalyzes the first step in the post-translational modification of the eukaryotic translation factor 5A (eIF5A). This is the only protein known to contain the amino acid hypusine, which results from this modification. Both eIF5A and DHS are essential for cell viability in eukaryotes, and inhibiting DHS can be a promising strategy for the development of new therapeutic alternatives. The human and parasitic orthologous proteins are different enough to render selective targeting against infectious diseases; however, no DHS inhibitor selective for the parasite ortholog has previously been reported. Here, we established a yeast surrogate genetics platform to identify inhibitors of DHS from Plasmodium vivax, one of the major causative agents of malaria. We constructed genetically modified Saccharomyces cerevisiae strains expressing DHS genes from Homo sapiens (HsDHS) or P. vivax (PvDHS) in place of the endogenous DHS gene from S. cerevisiae . This new strain background was ~60-fold more sensitive to an inhibitor of human DHS than the one previously used. Initially, a virtual screen using datasets from the ChEMBL-NTD database was performed. Candidate ligands were tested in growth assays using the newly generated yeast strains expressing heterologous DHS genes. Among these, two showed promise by preferentially reducing the growth of the PvDHS-expressing strain. Further, in a robotized assay, we screened 400 compounds from the Pathogen Box library using the same S. cerevisiae strains, and one compound preferentially reduced the growth of the PvDHS-expressing yeast strain. Western blot revealed that these compounds significantly reduced eIF5A hypusination in yeast. Our study demonstrates that this yeast-based platform is suitable for identifying and verifying candidate small molecule DHS inhibitors, selective for the parasite over the human ortholog
An eye on sickle cell retinopathy
ARTIGO - Inglês
Assunto: Retinopatia falciforme
Angiogenesis‐related genes in endothelial progenitor cells may be involved in sickle cell stroke
ARTIGO - Inglês
Assunto: Expressão gênica
The clinical aspects of sickle cell anemia (SCA) are heterogeneous, and different patients may present significantly different clinical...
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The clinical aspects of sickle cell anemia (SCA) are heterogeneous, and different patients may present significantly different clinical evolutions.Almost all organs can be affected, particularly the central nervous system. Transient ischemic events, infarcts, and cerebral hemorrhage can be observed and affect ≈25% of the patients with SCA. Differences in the expression of molecules produced by endothelial cells may be associated with the clinical heterogeneity of patients affected by vascular diseases. In this study, we investigated the differential expression of genes involved in endothelial cell biology in SCA patients with and without stroke. Endothelial progenitor cells from 4 SCA patients with stroke and 6 SCA patients without stroke were evaluated through the polymerase chain reaction array technique. The analysis of gene expression profiling identified 29 differentially expressed genes. Eleven of these genes were upregulated, and most were associated with angiogenesis (55%), inflammatory response (18%), and coagulation (18%) pathways. Downregulated expression was observed in 18 genes, with the majority associated with angiogenesis (28%), apoptosis (28%), and cell adhesion (22%) pathways. Remarkable overexpression of the MMP1 (matrix metalloproteinase 1) gene in the endothelial progenitor cells of all SCA patients with stroke (fold change: 204.64; P+O46=0.0004) was observed. Our results strongly suggest that angiogenesis is an important process in sickle cell stroke, and differences in the gene expression profile of endothelial cell biology, especially MMP1, may be related to stroke in SCA patients
An open source plant kinase chemogenomics set
ARTIGO - Inglês
Assunto: Artigo original
Abstract: One hundred twenty-nine protein kinases, selected to represent the diversity of the rice (Oryza sativa) kinome, were cloned and tested for...
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Abstract: One hundred twenty-nine protein kinases, selected to represent the diversity of the rice (Oryza sativa) kinome, were cloned and tested for expression in Escherichia coli. Forty of these rice kinases were purified and screened using differential scanning fluorimetry (DSF) against 627 diverse kinase inhibitors, with a range of structures and activities targeting diverse human kinases. Thirty-seven active compounds were then tested for their ability to modify primary root development in Arabidopsis. Of these, 14 compounds caused a significant reduction of primary root length compared with control plants. Two of these inhibitory compounds bind to the predicted orthologue of Arabidopsis PSKR1, one of two receptors for PSK, a small sulfated peptide that positively controls root development. The reduced root length phenotype could not be rescued by the exogenous addition of the PSK peptide, suggesting that chemical treatment may inhibit both PSKR1 and its closely related receptor PSKR2. Six of the compounds acting as root growth inhibitors in Arabidopsis conferred the same effect in rice. Compound RAF265 (CHIR-265), previously shown to bind the human kinase BRAF (B-Raf proto-oncogene, serine/threonine kinase), also binds to nine highly conserved rice kinases tested. The binding of human and rice kinases to the same compound suggests that human kinase inhibitor sets will be useful for dissecting the function of plant kinases
DOI: https://doi.org/10.1002/pld3.460
Texto completo: https://onlinelibrary.wiley.com/doi/10.1002/pld3.460
ARTIGO - Inglês
Assunto: Artigo original
Abstract: Here, we report a bioluminescence resonance energy transfer (BRET) assay as a novel way to investigate the binding of unlabeled ligands to...
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Abstract: Here, we report a bioluminescence resonance energy transfer (BRET) assay as a novel way to investigate the binding of unlabeled ligands to the human transient receptor potential mucolipin 1 (hTRPML1), a lysosomal ion channel involved in several genetic diseases and cancer progression. This novel BRET assay can be used to determine equilibrium and kinetic binding parameters of unlabeled compounds to hTRPML1 using intact human-derived cells, thus complementing the information obtained using functional assays based on ion channel activation. We expect this new BRET assay to expedite the identification and optimization of cell-permeable ligands that interact with hTRPML1 within the physiologically relevant environment of lysosomes
A novel CYP21A2 mutation identified a patient with classical 21-hydroxylase deficiency
ARTIGO - Inglês
Assunto: Mutação (Biologia)
Texto completo: https://www.karger.com/Article/Abstract/451040
A novel image-based approach for soybean seed phenotyping using machine learning techniques
ARTIGO - Inglês
Assunto: Artigo original
Abstract: Soybean is important for protein and oil worldwide, requiring investments in research and production technology, mainly breeding programs,...
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Abstract: Soybean is important for protein and oil worldwide, requiring investments in research and production technology, mainly breeding programs, to meet the increasing demand. Seed morphology, including area, perimeter, and circularity, significantly impacts soybean yield. However, the analysis of these factors has been hindered by the lack of efficient computational approaches that offer accuracy and flexibility to user preferences and needs. This study presents a soybean phenotyping system framework that includes: seed segmentation in soybean images, morphological evaluation, and image-based prediction of hundred-seed weight (HSW). We used genotypes from a partial diallel cross design and collected red-green-blue images of seeds from each plot. We developed an in-house image processing pipeline for seed segmentation, which enabled a full morphological evaluation of the seeds. For predicting the HSW, we compared machine learning algorithms using the obtained morphological characteristics as input and features from state-of-the-art convolutional neural network (CNN) architectures. Our image segmentation methodology correctly identified over 98% of the seeds in the images, even when they were in proximity. Our morphological phenotyping approach's adaptability to other plant species was verified, fully demonstrating the pipeline's generalizability. The morphological measurements were effective in predicting the HSW with an accuracy of 0.71 predictive ability and mean squared error (MSE) of 3.15 (mean HSW of 19.74). The same results were observed for CNN features, highlighting the efficiency of the morphological measurements. ResNet-50 had the most effective feature extraction, with a mean accuracy of 0.60 (MSE of 4.15). Our study can aid automated phenotyping tool development in research and industry. We introduce a novel high-throughput soybean seed phenotyping system. We demonstrate the possibility of predicting an important component of production for the soybean crop. We show the efficiency of morphological measures as extractors of image features. Our results indicate that ML techniques can be easily adapted to precision tasks in plant phenology
Texto completo: https://acsess.onlinelibrary.wiley.com/doi/10.1002/csc2.21032
ARTIGO - Inglês
Assunto: Acidithiobacillus ferrooxidans
The total protein content and activity of the enzymes glutathione reductase (GR), superoxide dismutase (SOD) and thioredoxin reductase (TrxR) were...
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The total protein content and activity of the enzymes glutathione reductase (GR), superoxide dismutase (SOD) and thioredoxin reductase (TrxR) were evaluated in Acidithiobacillus ferrooxidans LR cells maintained in contact with the metal sulfide chalcopyrite for 1 and 10 days. A significant decrease in total protein content was observed in cells maintained for 10 days in the presence of chalcopyrite, suggesting proteolytic breakdown clue to exposure to the metal sulfide. Following 10 clays of contact with chalcopyrite, increases in GR, SOD and TrxR activities were detected, suggesting the formation of reactive oxygen species. After ten clays, there was a fivefold increase in GR activity, of which, isoenzyme IV represented approximately 82% of the total. An increase in Fe-SOD activity following ten days exposure to chalcopyrite was also determined, as measured on non-denaturing polyacrylamide gels. Also, after 10 days. an approximately 31-fold increase was observed for TrxR activity. The presence of oxidative stress when A. ferrooxidans is in the presence of chalcopyrite could have a negative impact on bioleaching. (C) 2010 Elsevier Ltd. All rights reserved