Wide reference databases for typing Trypanosoma cruzi based on amplicon sequencing of the minicircle hypervariable region.

BACKGROUND: Trypanosoma cruzi, the etiological agent of Chagas Disease, exhibits remarkable genetic diversity and is classified into different Discrete Typing Units (DTUs). Strain typing techniques are crucial for studying T. cruzi, because their DTUs have significant biological differences from one another. However, there is currently no methodological strategy for the direct typing of biological materials that has sufficient sensitivity, specificity, and reproducibility. The high diversity and copy number of the minicircle hypervariable regions (mHVRs) makes it a viable target for typing. METHODOLOGY/PRINCIPAL FINDINGS: Approximately 24 million reads obtained by amplicon sequencing of the mHVR were analyzed for 62 strains belonging to the six main T. cruzi DTUs. To build reference databases of mHVR diversity for each DTU and to evaluate this target as a typing tool. Strains of the same DTU shared more mHVR clusters than strains of different DTUs, and clustered together. Different identity thresholds were used to build the reference sets of the mHVR sequences (85% and 95%, respectively). The 95% set had a higher specificity and was more suited for detecting co-infections, whereas the 85% set was excellent for identifying the primary DTU of a sample. The workflow's capacity for typing samples obtained from cultures, a set of whole-genome data, under various simulated PCR settings, in the presence of co-infecting lineages and for blood samples was also assessed. CONCLUSIONS/SIGNIFICANCE: We present reference databases of mHVR sequences and an optimized typing workflow for T. cruzi including a simple online tool for deep amplicon sequencing analysis (https://ntomasini.github.io/cruzityping/). The results show that the workflow displays an equivalent resolution to that of the other typing methods. Owing to its specificity, sensitivity, relatively low cost, and simplicity, the proposed workflow could be an alternative for screening different types of samples.

Hydrophobicity-Driven Increases in Editing in Mitochondrial mRNAs during the Evolution of Kinetoplastids.

Kinetoplastids are a diverse group of flagellates which exhibit editing by insertion/deletion of Us in the mitochondrial mRNAs. Some mRNAs require editing to build most of their coding sequences, a process known as pan-editing. Evidence suggests that pan-editing is an ancestral feature in kinetoplastids. Here, we investigate how the transition from nonedited to pan-edited states occurred. The mitochondrial mRNAs and protein sequences from nine kinetoplastids and related groups (diplonemids, euglenids, and jakobids) were analyzed. RNA editing increased protein hydrophobicity to extreme values by introducing Us in the second codon position, despite the absence of editing preferences related to codon position. In addition, hydrophobicity was maintained by purifying selection in species that lost editing by retroposition of the fully edited mRNA. Only a few hydrophobic to hydrophilic amino acid changes were inferred for such species. In the protein secondary structure, these changes occurred spatially close to other hydrophilic residues. The analysis of coevolving sites showed that multiple changes are required together for hydrophobicity to be lost, which suggest the proteins are locked into extended hydrophobicity. Finally, an analysis of the NAD7 protein-protein interactions showed they can also influence hydrophobicity increase in the protein and where editing can occur in the mRNA. In conclusion, our results suggest that protein hydrophobicity has influenced editing site selection and how editing expanded in mRNAs. In effect, the hydrophobicity increase was entrenched by a neutral ratchet moved by a mutational pressure to introduce Us, thus helping to explain both RNA editing increase and, possibly, persistence.

BIRC5/Survivin Expression as a Non-Invasive Biomarker of Endometriosis.

The etiology of endometriosis is highly complex, and although it is a benign disease, it has several biological behaviors similar to malignant lesions, including cell invasion, neo-angiogenesis, and decreased apoptosis. Survivin is a protein encoded by the BIRC5 gene that plays a role in cell division by inhibiting apoptosis and regulating the process of mitosis in embryonic and cancer cells. Therefore, we aimed to evaluate the expression of BIRC5 in samples of peripheral blood of women with and without endometriosis. This study comprised of 40 women with endometriosis and 10 healthy women as controls. Peripheral blood samples were collected in the three phases of the menstrual cycle (follicular, ovulatory, and luteal). The expression of the BIRC5 gene was evaluated by RT-qPCR using the TaqMan methodology. The BIRC5 expression was significantly higher in all phases of the menstrual cycle in women with endometriosis, regardless of the disease stage. The accuracy of BIRC5 expression in the peripheral blood for the diagnosis endometriosis presented AUC of 0.887 (p < 0.001), with 97.2% of sensitivity and specificity of 65.5% considering the overall endometriosis group. Regarding the minimal/mild endometriosis group, the AUC presented a value of 0.925 (p < 0.001), with 100% of sensitivity and 79.3% of specificity, whereas in the moderate/severe endometriosis group the AUC was 0.868 (p < 0.001), with a sensitivity of 95.8% and specificity of 65.5%. These findings suggest that the expression of BIRC5 may be a potential noninvasive biomarker for the diagnosis of endometriosis.

Evaluation of the frequency of G-765C polymorphism in the promoter region of the COX-2 gene and its correlation with the expression of this gene in the endometrium of women with endometriosis.

OBJECTIVE: To evaluate the frequency of polymorphism G-765C (rs20417) of the COX-2 gene and the expression of this gene in the endometrium of women with endometriosis. STUDY DESIGN: This is a case-control study of 365 women with endometriosis (251 infertile and 114 fertile) submitted to laparoscopy/laparotomy with histological confirmation of endometriosis. The control group was composed of 522 fertile women without endometriosis. Of these, 37 patients from the endometriosis group and 47 from the control group were submitted to biopsy of the endometrium for analysis of the expression of the COX-2 gene. The genotypes were determined using analysis by High-Resolution Melt. Gene expression was measured by qRT-PCR with TaqMan methodology using the GAPDH gene as normalizer of the reactions. RESULTS: The distribution of the genotypes and alleles in the group of fertile women with moderate/severe endometriosis showed a statistically significant difference, demonstrating association of the ancestral allele, -765G, with increased risk of endometriosis (p = 0.028; OR 0.53; CI 0.32-0.90). The mean expression of the COX-2 gene (mRNA PTGS2) in the group of women with endometriosis was statistically higher compared to the control group (3.85 versus 2.84, p = 0.028). CONCLUSION: The present study identified that in Brazilian women the presence of the ancestral allele, -765G, of the COX-2 gene is associated with an increased risk for development of moderate/severe endometriosis associated with fertility, and that the eutopic endometrium of women with endometriosis showed increased expression of COX-2 when compared to the control group.