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In reproductive technologies, uncovering the molecular aspects of oocyte and embryo competence under different conditions is crucial for refining protocols and enhancing efficiency. RNA-seq generates high-throughput data and provides transcriptomes that can undergo additional computational analyses. This study presented the transcriptomic profiles of in vitro matured oocytes and blastocysts produced in vitro from buffalo crossbred (Bubalus bubalis), coupled with gene co-expression and module preservation analysis. Cumulus Oophorus Complexes, obtained from slaughterhouse-derived ovaries, were subjected to in vitro maturation to yield metaphase II oocytes (616) or followed in vitro fertilization and culture to yield blastocysts for sequencing (526). Oocyte maturation (72%, ±3.34 sd) and embryo development (21.3%, ±4.18 sd) rates were obtained from three in vitro embryo production routines following standard protocols. Sequencing of 410 metaphase II oocytes and 70 hatched blastocysts (grade 1 and 2) identified a total of 13,976 genes, with 62% being ubiquitously expressed (8,649). Among them, the differentially expressed genes (4,153) and the strongly variable genes with the higher expression (fold-change above 11) were highlighted in oocytes (BMP15, UCHL1, WEE1, NLRPs, KPNA7, ZP2, and ZP4) and blastocysts (APOA1, KRT18, ANXA2, S100A14, SLC34A2, PRSS8 and ANXA2) as representative indicators of molecular quality. Additionally, genes exclusively found in oocytes (224) and blastocysts (2,200) with specific biological functions were identified. Gene co-expression network and module preservation analysis revealed strong preservation of functional modules related to exosome components, steroid metabolism, cell proliferation, and morphogenesis. However, cell cycle and amino acid transport modules exhibited weak preservation, which may reflect differences in embryo development kinetics and the activation of cell signaling pathways between buffalo and bovine. This comprehensive transcriptomic profile serves as a valuable resource for assessing the molecular quality of buffalo oocytes and embryos in future in vitro embryo production assays.
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Microsatellites, also known as SSRs or STRs, are polymorphic DNA regions with tandem repetitions of a nucleotide motif of size 1-6 base pairs with a broad range of applications in many fields, such as comparative genomics, molecular biology, and forensics. However, the majority of researchers do not have computational training and struggle while running command-line tools or very limited web tools for their SSR research, spending a considerable amount of time learning how to execute the software and conducting the post-processing data tabulation in other tools or manually-time that could be used directly in data analysis. We present EasySSR, a user-friendly web tool with command-line full functionality, designed for practical use in batch identifying and comparing SSRs in sequences, draft, or complete genomes, not requiring previous bioinformatic skills to run. EasySSR requires only a FASTA and an optional GENBANK file of one or more genomes to identify and compare STRs. The tool can automatically analyze and compare SSRs in whole genomes, convert GenBank to PTT files, identify perfect and imperfect SSRs and coding and non-coding regions, compare their frequencies, abundancy, motifs, flanking sequences, and iterations, producing many outputs ready for download such as PTT files, interactive charts, and Excel tables, giving the user the data ready for further analysis in minutes. EasySSR was implemented as a web application, which can be executed from any browser and is available for free at https://computationalbiology.ufpa.br/easyssr/. Tutorials, usage notes, and download links to the source code can be found at https://github.com/engbiopct/EasySSR.
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Corynebacterium pseudotuberculosis is the causative bacterial agent of the zoonotic disease known as caseous lymphadenitis, and it presents several mechanisms of response to host defenses, including the presence of virulence factors (VFs). The genomes of these bacteria have several polymorphic markers known as microsatellites, or simple sequence repeats (SSRs), that can be used to characterize the genome, to study possible polymorphisms existing among strains, and to verify the effects of such polymorphic markers in coding regions and regions associated with VFs. In this study, several SSRs were identified within coding regions throughout the 54 genomes of this species, revealing possible polymorphisms associated with coding regions that could be used as strain-specific or serotype-specific identifiers of C. pseudotuberculosis. The similarities associated with SSRs amongst the different serum variants of C. pseudotuberculosis, biovars equi and ovis, were also evaluated, and it was possible to identify SSRs located in coding regions responsible for a VF enrolled in pathogenesis known to mediate bacterial adherence (SpaH-type pili virulence factor). Phylogenetic analyses revealed that strains sharing SSR patterns, including the possible polymorphisms identified in the same position of gene-coding regions, were displayed by strains with a common ancestor, corroborating with the Genome Tree Report of the NCBI. Statistical analysis showed that the microsatellite groups belonging to equi and ovis biovars have a significance of 0.006 (p-value) in similarity, thus indicating them as good biomarker candidates for C. pseudotuberculosis.
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BACKGROUND: Ameloblastoma (AMB) is a benign odontogenic tumour, with an aggressive local behaviour and a high rate of recurrence. Previous studies have demonstrated that hypoxia-induced factor alpha 1 (HIF-1α) and activated caspase-3 contribute to tumour invasiveness and cytogenesis in ameloblastoma. Hypoxia increases HIF-1α levels, which triggers a number of signalling pathways. This paper aimed to present data in the study of hypoxia-activated signalling pathways that modulate proapoptotic and antiapoptotic events in AMB. METHODS: Twenty cases of AMB and ten cases of dental follicle (DF) were used to analyse the immunoexpression of HIF-1α, p53, BNIP3, Bcl-2, IAP-2, GLUT1, and Bax. To contribute to the study, an analysis of expression and genetic interaction was performed using the cell line AME-1. RESULTS: AMB and DF expressed the studied proteins. These proteins showed significantly greater immunoexpression in AMB compared with the DF (p < 0.05). HIF-1α showed an important association with GLUT1, and a positive correlation was observed among p53, Bcl-2, and IAP-2. Transcriptomic analysis showed the significant expression of the studied proteins, and the network generated showed a direct association of HIF-1αF with GLUT1 (SLC2A1), TP53, and LDHA. Interestingly, GLUT1 also exhibited direct interaction with TP53 and LDHA. CONCLUSION: In AMB tumorigenesis, hypoxia is possibly related to antiapoptotic events, which suggests an important role for HIF-1α, GLUT1, Bcl-2, IAP-2, and possibly p53.
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Acute Lymphoblastic Leukemia (ALL) is the most common cancer in children. Differences are found among ethnic groups in the results of the treatment of pediatric ALL. In general, children with a high level of native American ancestry tend to respond less positively to ALL treatments, which may be related to specific genomic variants found in native American groups. Despite the evidence, few data are available on the distribution of the pharmacogenomic variants relevant to the treatment of ALL in traditional Amerindian populations, such the those of the Amazon region. Given this, the present study investigated 27 molecular markers related to the treatment of ALL in Amerindians from Brazilian Amazonia and compared the frequencies with those recorded previously on five continents, that are available in the 1,000 Genomes database. The variation in the genotype frequencies among populations was evaluated using Fisher's exact test. The False Discovery Rate method was used to correct the results of the multiple analyses. Significant differences were found in the frequencies of the majority of markers between the Amerindian populations and those of other regions around the world. These findings highlight the unique genetic profile of the indigenous population of Brazilian Amazonia, which may reflect a distinct therapeutic profile for the treatment of ALL in these populations.
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Antineoplásicos/farmacología , Indígenas Sudamericanos/genética , Variantes Farmacogenómicas , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Antineoplásicos/uso terapéutico , Brasil , Niño , Preescolar , Femenino , Marcadores Genéticos , Humanos , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/genéticaRESUMEN
The variation in the allelic frequencies of polymorphic pharmacogenes among different ethnic groups may be responsible for severe adverse reactions to or altered efficacy of a wide variety of drugs. Amazonian Amerindian populations have a unique genetic profile that may have a fundamental on the efficacy and safety of certain drugs. The genetic characteristics of these populations are poorly known, which can negatively impact the systematic application of treatments guided by pharmacogenomic guidelines. We investigated the diversity of 32 polymorphisms in genes responsible for drug Absorption, Distribution, Metabolism and Excretion (ADME) in Amazonian Amerindians, and compared the findings with populations from other continents available in the 1000 Genomes database. We found significantly different (P ≤ 1.56E-03) allelic frequencies and genotype distributions in many study markers in comparison with African, European, American and Asian populations. Based on FST values, the Amerindian population was also the most distinct (mean FST = 0.09917). These data highlight the unique genetic profile of the indigenous population from the Brazilian Amazon region, which is potentially important from a pharmacogenetic viewpoint. Understanding the diversity of ADME- related genetic markers is crucial to the implementation of individualized pharmacogenomic treatment protocols in Amerindian populations, as well as populations with a high degree of admixture with this ethnic group, such as the general Brazilian population.
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Técnicas de Genotipaje/métodos , Indígenas Sudamericanos/genética , Variantes Farmacogenómicas , Brasil/etnología , Frecuencia de los Genes , Genética de Población , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
Corynebacterium ulcerans is a bacterial species with high importance because it causes infections in animals and, rarely, in humans. Its virulence mechanisms remain unclear. The current study describes the draft genome of C. ulcerans FRC58, which was isolated from the bronchitic aspiration of a patient in France.
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Multiple Displacement Amplification (MDA) of DNA using φ29 (phi29) DNA polymerase amplifies DNA several billion-fold, which has proved to be potentially very useful for evaluating genome information in a culture-independent manner. Whole genome sequencing using DNA from a single prokaryotic genome copy amplified by MDA has not yet been achieved due to the formation of chimeras and skewed amplification of genomic regions during the MDA step, which then precludes genome assembly. We have hereby addressed the issue by using 10 ng of genomic Vibrio cholerae DNA extracted within an agarose plug to ensure circularity as a starting point for MDA and then sequencing the amplified yield using the SOLiD platform. We successfully managed to assemble the entire genome of V. cholerae strain LMA3984-4 (environmental O1 strain isolated in urban Amazonia) using a hybrid de novo assembly strategy. Using our method, only 178 out of 16,713 (1%) of contigs were not able to be inserted into either chromosome scaffold, and out of these 178, only 3 appeared to be chimeras. The other contigs seem to be the result of template-independent non-specific amplification during MDA, yielding spurious reads. Extraction of genomic DNA within an agarose plug in order to ensure circularity of the extracted genome might be key to minimizing amplification bias by MDA for WGS.
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ADN Bacteriano/genética , Microbiología Ambiental , Genoma Bacteriano , Técnicas de Amplificación de Ácido Nucleico/métodos , Vibrio cholerae O1/genética , Límite de Detección , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Vibrio cholerae O1/aislamiento & purificaciónRESUMEN
We proposed a modification the procedure of genotyping based in labeled universal primer and tailed primer. In the standard protocol, three primers are used in the same PCR reaction, a forward primer with tail added at the 5' end of the identical sequence to labeled universal primer with dye-fluorescent and a reverse primer. Unfortunately, the choice of a labeled primer characterized by a large number of complementary sequences in target genomes (which is more probable in larger genomes) result in unspecific amplifications (false positive) can cause absence or decrease amplification of the locus of interest and also false interpretation of the analysis. However, identification of possible homologies between the primer chosen for labelling and the genome is rarely possible from the available DNA data bases. In our approach, cycling is interrupted for the addition of the labeled primer only during the final cycles, thus minimizing unspecific amplification and competition between primers, resulting in the more fidelity amplification of the target regions.