RNA sequencing and gene co-expression network of in vitro matured oocytes and blastocysts of buffalo.

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.

EasySSR: a user-friendly web application with full command-line features for large-scale batch microsatellite mining and samples comparison.

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.

Immunoexpression of stem cell markers SOX-2, NANOG AND OCT4 in ameloblastoma.

Background: Ameloblastoma (AME) is characterized by a locally invasive growth pattern. In an attempt to justify the aggressiveness of neoplasms, the investigation of the role of stem cells has gained prominence. The SOX-2, NANOG and OCT4 proteins are important stem cell biomarkers. Methodology: To verify the expression of these proteins in tissue samples of AME, dentigerous cyst (DC) and dental follicle (DF), immunohistochemistry was performed and indirect immunofluorescence were performed on the human AME (AME-hTERT) cell line. Results: Revealed expression of SOX-2, NANOG and OCT4 in the tissue samples and AME-hTERT lineage. Greater immunostaining of the studied proteins was observed in AME compared to DC and DF (p < 0.001). Conclusions: The presence of biomarkers indicates a probable role of stem cells in the genesis and progression of AME.

In silico approach to identify microsatellite candidate biomarkers to differentiate the biovar of Corynebacterium pseudotuberculosis genomes.

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.

Viral Metagenomics Reveals Widely Diverse Viral Community of Freshwater Amazonian Lake.

Despite the importance of understanding the ecology of freshwater viruses, there are not many studies on the subject compared to marine viruses. The microbiological interactions in these environments are still poorly known, especially between bacteriophages and their host bacteria and between cyanophages and cyanobacteria. Lake Bologna, Belém, capital of the Brazilian State of Pará, is a water source that supplies the city and its metropolitan region. However, it remains unexplored regarding the contents of its virome and viral diversity composition. Therefore, this work aims to explore the taxonomic diversity of DNA viruses in this lake, especially bacteriophages and cyanophages, since they can act as transducers of resistance genes and reporters of water quality for human consumption. We used metagenomic sequencing data generated by previous studies. We analyzed it at the taxonomic level using the tools Kraken2, Bracken, and Pavian; later, the data was assembled using Genome Detective, which performs the assembly of viruses. The results observed here suggest the existence of a widely diverse viral community and established microbial phage-regulated dynamics in Lake Bolonha. This work is the first ever to describe the virome of Lake Bolonha using a metagenomic approach based on high-throughput sequencing, as it contributes to the understanding of water-related public health concerns regarding the spreading of antibiotic resistance genes and population control of native bacteria and cyanobacteria.

HIF-1α Is Associated with Resistance to Hypoxia-Induced Apoptosis in Ameloblastoma.

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.

Co-Expression Networks for Causal Gene Identification Based on RNA-Seq Data of Corynebacterium pseudotuberculosis.

Corynebacterium pseudotuberculosis is a Gram-positive bacterium that causes caseous lymphadenitis, a disease that predominantly affects sheep, goat, cattle, buffalo, and horses, but has also been recognized in other animals. This bacterium generates a severe economic impact on countries producing meat. Gene expression studies using RNA-Seq are one of the most commonly used techniques to perform transcriptional experiments. Computational analysis of such data through reverse-engineering algorithms leads to a better understanding of the genome-wide complexity of gene interactomes, enabling the identification of genes having the most significant functions inferred by the activated stress response pathways. In this study, we identified the influential or causal genes from four RNA-Seq datasets from different stress conditions (high iron, low iron, acid, osmosis, and PH) in C. pseudotuberculosis, using a consensus-based network inference algorithm called miRsigand next identified the causal genes in the network using the miRinfluence tool, which is based on the influence diffusion model. We found that over 50% of the genes identified as influential had some essential cellular functions in the genomes. In the strains analyzed, most of the causal genes had crucial roles or participated in processes associated with the response to extracellular stresses, pathogenicity, membrane components, and essential genes. This research brings new insight into the understanding of virulence and infection by C. pseudotuberculosis.

Characterization of pharmacogenetic markers related to Acute Lymphoblastic Leukemia toxicity in Amazonian native Americans population.

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.

Bacterial diversity in deep-sea sediments under influence of asphalt seep at the São Paulo Plateau.

Here we investigated the diversity of bacterial communities from deep-sea surface sediments under influence of asphalt seeps at the Sao Paulo Plateau using next-generation sequencing method. Sampling was performed at North São Paulo Plateau using the human occupied vehicle Shinkai 6500 and her support vessel Yokosuka. The microbial diversity was studied at two surficial sediment layers (0-1 and 1-4 cm) of five samples collected in cores in water depths ranging from 2456 to 2728 m. Bacterial communities were studied through sequencing of 16S rRNA gene on the Ion Torrent platform and clustered in operational taxonomic units. We observed high diversity of bacterial sediment communities as previously described by other studies. When we considered community composition, the most abundant classes were Alphaproteobacteria (27.7%), Acidimicrobiia (20%), Gammaproteobacteria (11.3%) and Deltaproteobacteria (6.6%). Most abundant OTUs at family level were from two uncultured bacteria from Actinomarinales (5.95%) and Kiloniellaceae (3.17%). The unexpected high abundance of Alphaproteobacteria and Acidimicrobiia in our deep-sea microbial communities may be related to the presence of asphalt seep at North São Paulo Plateau, since these bacterial classes contain bacteria that possess the capability of metabolizing hydrocarbon compounds.

Metallothionein Expression and its Influence on the In Vitro Biological Behavior of Mucoepidermoid Carcinoma.

Mucoepidermoid carcinoma (MEC) is the most common tumor in the salivary glands, often presenting with recurrence and metastasis due to its high invasive capacity. Metallothionein (MT), a zinc storage protein that supplies this element for protease activity, is probably related to mucoepidermoid carcinoma behavior. This prompted us to characterize a cell line derived from mucoepidermoid carcinoma and to correlate metallothionein expression with transforming growth factor-α (TGF-α), tumor necrosis factor-α (TNF-α) and matrix metalloproteinases (MMPs). Transcriptomic analysis and cytogenetic assays were performed to detect the expression of genes of interest and cellular chromosomal alterations, respectively. MEC cells with a depleted metallothionein 2A (MT2A) gene were subjected to Western blot to correlate metallothionein expression with growth factors and MMPs. Additionally, cells with depleted MT were subjected to migration and invasion assays. The transcriptomic study revealed reads mapped to cytokeratins 19 and AE1/AE3, α-smooth muscle actin, vimentin, and fibronectin. Cytogenetic evaluation demonstrated structural and numerical alterations, including the translocation t(11;19)(q21;p13), characteristic of MEC. Metallothionein depletion was correlated with the decreased expression of TGF-α and MMP-9, while TNF-α protein levels were augmented. Migration and invasion activity were diminished after metallothionein silencing. Our findings suggest an important role of MT in MEC invasion, through the regulation of proteins involved in this process.

Re-sequencing and optical mapping reveals misassemblies and real inversions on Corynebacterium pseudotuberculosis genomes.

The number of draft genomes deposited in Genbank from the National Center for Biotechnology Information (NCBI) is higher than the complete ones. Draft genomes are assemblies that contain fragments of misassembled regions (gaps). Such draft genomes present a hindrance to the complete understanding of the biology and evolution of the organism since they lack genomic information. To overcome this problem, strategies to improve the assembly process are developed continuously. Also, the greatest challenge to the assembly progress is the presence of repetitive DNA regions. This article highlights the use of optical mapping, to detect and correct assembly errors in Corynebacterium pseudotuberculosis. We also demonstrate that choosing a reference genome should be done with caution to avoid assembly errors and loss of genetic information.

Polymorphisms of ADME-related genes and their implications for drug safety and efficacy in Amazonian Amerindians.

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.

PhageWeb - Web Interface for Rapid Identification and Characterization of Prophages in Bacterial Genomes.

This study developed a computational tool with a graphical interface and a web-service that allows the identification of phage regions through homology search and gene clustering. It uses G+C content variation evaluation and tRNA prediction sites as evidence to reinforce the presence of prophages in indeterminate regions. Also, it performs the functional characterization of the prophages regions through data integration of biological databases. The performance of PhageWeb was compared to other available tools (PHASTER, Prophinder, and PhiSpy) using Sensitivity (Sn) and Positive Predictive Value (PPV) tests. As a reference for the tests, more than 80 manually annotated genomes were used. In the PhageWeb analysis, the Sn index was 86.1% and the PPV was approximately 87%, while the second best tool presented Sn and PPV values of 83.3 and 86.5%, respectively. These numbers allowed us to observe a greater precision in the regions identified by PhageWeb while compared to other prediction tools submitted to the same tests. Additionally, PhageWeb was much faster than the other computational alternatives, decreasing the processing time to approximately one-ninth of the time required by the second best software. PhageWeb is freely available at http://computationalbiology.ufpa.br/phageweb.

A novel cell line derived from pleomorphic adenoma expresses MMP2, MMP9, TIMP1, TIMP2, and shows numeric chromosomal anomalies.

Pleomorphic adenoma is the most common salivary gland neoplasm, and it can be locally invasive, despite its slow growth. This study aimed to establish a novel cell line (AP-1) derived from a human pleomorphic adenoma sample to better understand local invasiveness of this tumor. AP-1 cell line was characterized by cell growth analysis, expression of epithelial and myoepithelial markers by immunofluorescence, electron microscopy, 3D cell culture assays, cytogenetic features and transcriptomic study. Expression of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) was also analyzed by immunofluorescence and zymography. Furthermore, epithelial and myoepithelial markers, MMPs and TIMPs were studied in the tumor that originated the cell line. AP-1 cells showed neoplastic epithelial and myoepithelial markers, such as cytokeratins, vimentin, S100 protein and smooth-muscle actin. These molecules were also found in vivo, in the tumor that originated the cell line. MMPs and TIMPs were observed in vivo and in AP-1 cells. Growth curve showed that AP-1 exhibited a doubling time of 3.342 days. AP-1 cells grown inside Matrigel recapitulated tumor architecture. Different numerical and structural chromosomal anomalies were visualized in cytogenetic analysis. Transcriptomic analysis addressed expression of 7 target genes (VIM, TIMP2, MMP2, MMP9, TIMP1, ACTA2 e PLAG1). Results were compared to transcriptomic profile of non-neoplastic salivary gland cells (HSG). Only MMP9 was not expressed in both libraries, and VIM was expressed solely in AP-1 library. The major difference regarding gene expression level between AP-1 and HSG samples occurred for MMP2. This gene was 184 times more expressed in AP-1 cells. Our findings suggest that AP-1 cell line could be a useful model for further studies on pleomorphic adenoma biology.

Draft Genome Sequence of Corynebacterium ulcerans FRC58, Isolated from the Bronchitic Aspiration of a Patient in France.

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.

Brazilian Microbiome Project: revealing the unexplored microbial diversity--challenges and prospects.

The Brazilian Microbiome Project (BMP) aims to assemble a Brazilian Metagenomic Consortium/Database. At present, many metagenomic projects underway in Brazil are widely known. Our goal in this initiative is to co-ordinate and standardize these together with new projects to come. It is estimated that Brazil hosts approximately 20 % of the entire world's macroorganism biological diversity. It is 1 of the 17 countries that share nearly 70 % of the world's catalogued animal and plant species, and is recognized as one of the most megadiverse countries. At the end of 2012, Brazil has joined GBIF (Global Biodiversity Information Facility), as associated member, to improve the access to the Brazilian biodiversity data in a free and open way. This was an important step toward increasing international collaboration and clearly shows the commitment of the Brazilian government in directing national policies toward sustainable development. Despite its importance, the Brazilian microbial diversity is still considered to be largely unknown, and it is clear that to maintain ecosystem dynamics and to sustainably manage land use, it is crucial to understand the biological and functional diversity of the system. This is the first attempt to collect and collate information about Brazilian microbial genetic and functional diversity in a systematic and holistic manner. The success of the BMP depends on a massive collaborative effort of both the Brazilian and international scientific communities, and therefore, we invite all colleagues to participate in this project.

Whole genome sequencing of environmental Vibrio cholerae O1 from 10 nanograms of DNA using short reads.

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.

An alternative genotyping method using dye-labeled universal primer to reduce unspecific amplifications.

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.