Development of a Molecular Aptamer Beacon Applied to Magnetic-Assisted RNA Extraction for Detection of Dengue and Zika Viruses Using Clinical Samples.

Limitations in the detection of cocirculating flaviviruses such as Dengue and Zika lead us to propose the use of aptameric capture of the viral RNA in combination with RT-PCR (APTA-RT-PCR). Aptamers were obtained via SELEX and next-generation sequencing, followed by colorimetric and fluorescent characterizations. An APTA-RT-PCR assay was developed, optimized, and tested against the viral RNAs in 108 serum samples. After selection, sequence APTAZC10 was designed as a bifunctional molecular beacon (APTAZC10-MB), exhibiting affinity for the viral targets. APTA-RT-PCR was able to detect Dengue and Zika RNA in 43% and 8% of samples, respectively. Our results indicate that APTAZC10-MB and APTA-RT-PCR will be useful to improve the detection of Dengue and Zika viruses in a fast molecular assay for the improvement of infectious disease surveillance.

Aptamer-associated colorimetric reverse transcription loop-mediated isothermal amplification assay for detection of dengue virus.

Current diagnostic methods for dengue, such as serological tests, have limitations in terms of cross-reactivity with other viruses. To address this issue, we explored the potential of combining the loop-mediated isothermal amplification (LAMP) technique with the affinity of aptamers to develop point-of-care testing. In this study, we utilized 60 serum samples. An aptamer capable of binding to the dengue virus was employed as a platform for capturing genetic material, and its performance was compared to a commercial kit. Dengue virus was detected through RT-PCR and colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP), allowing visual observation of the results without the need for equipment. In the context of the aptamer LAMP assay, our analysis revealed the detection of the dengue virus in 38 out of 60 samples, with 95% sensitivity and 100% specificity compared to RT-PCR and/or APTA-RT-PCR. Importantly, we observed no cross-reaction when assessing samples positive for the zika virus, underscoring the assay's selectivity. This innovative aptameric capture of the viral RNA in combination with the RT-LAMP (APTA-RT-LAMP) method has the potential to offer valuable molecular insights into neglected infectious diseases in a simpler and faster manner. IMPORTANCE: Dengue is a neglected tropical disease of significant epidemiological importance in tropical and subtropical countries. Current diagnostics for this infection present challenges, such as cross-reactivity in serological tests. Finding ways to enhance the diagnosis of this disease is crucial, given the absence of specific treatments. An accurate, simple, and effective diagnosis contributes to the improved management of infected individuals. In this context, our work combines molecular biology techniques, such as isothermal loop amplification, with aptamers to detect the dengue virus in biological samples. Our method produces colorimetric results based on a color change, with outcomes available in less than 2 hours. Moreover, it requires simpler equipment compared to molecular PCR tests.

PCA3 noncoding RNA is involved in the control of prostate-cancer cell survival and modulates androgen receptor signaling.

BACKGROUND: PCA3 is a non-coding RNA (ncRNA) that is highly expressed in prostate cancer (PCa) cells, but its functional role is unknown. To investigate its putative function in PCa biology, we used gene expression knockdown by small interference RNA, and also analyzed its involvement in androgen receptor (AR) signaling. METHODS: LNCaP and PC3 cells were used as in vitro models for these functional assays, and three different siRNA sequences were specifically designed to target PCA3 exon 4. Transfected cells were analyzed by real-time qRT-PCR and cell growth, viability, and apoptosis assays. Associations between PCA3 and the androgen-receptor (AR) signaling pathway were investigated by treating LNCaP cells with 100 nM dihydrotestosterone (DHT) and with its antagonist (flutamide), and analyzing the expression of some AR-modulated genes (TMPRSS2, NDRG1, GREB1, PSA, AR, FGF8, CdK1, CdK2 and PMEPA1). PCA3 expression levels were investigated in different cell compartments by using differential centrifugation and qRT-PCR. RESULTS: LNCaP siPCA3-transfected cells significantly inhibited cell growth and viability, and increased the proportion of cells in the sub G0/G1 phase of the cell cycle and the percentage of pyknotic nuclei, compared to those transfected with scramble siRNA (siSCr)-transfected cells. DHT-treated LNCaP cells induced a significant upregulation of PCA3 expression, which was reversed by flutamide. In siPCA3/LNCaP-transfected cells, the expression of AR target genes was downregulated compared to siSCr-transfected cells. The siPCA3 transfection also counteracted DHT stimulatory effects on the AR signaling cascade, significantly downregulating expression of the AR target gene. Analysis of PCA3 expression in different cell compartments provided evidence that the main functional roles of PCA3 occur in the nuclei and microsomal cell fractions. CONCLUSIONS: Our findings suggest that the ncRNA PCA3 is involved in the control of PCa cell survival, in part through modulating AR signaling, which may raise new possibilities of using PCA3 knockdown as an additional therapeutic strategy for PCa control.

Gene surgery: Potential applications for human diseases.

Gene therapy became in last decade a new emerging therapeutic era showing promising results against different diseases such as cancer, cardiovascular diseases, diabetes, and neurological disorders. Recently, the genome editing technique for eukaryotic cells called CRISPR-Cas (Clustered Regulatory Interspaced Short Palindromic Repeats) has enriched the field of gene surgery with enhanced applications. In the present review, we summarized the different applications of gene surgery for treating human diseases such as cancer, diabetes, nervous, and cardiovascular diseases, besides the molecular mechanisms involved in these important effects. Several studies support the important therapeutic applications of gene surgery in a large number of health disorders and diseases including ß-thalassemia, cancer, immunodeficiencies, diabetes, and neurological disorders. In diabetes, gene surgery was shown to be effective in type 1 diabetes by triggering different signaling pathways. Furthermore, gene surgery, especially that using CRISPR-Cas possessed important application on diagnosis, screening and treatment of several cancers such as lung, liver, pancreatic and colorectal cancer. Nevertheless, gene surgery still presents some limitations such as the design difficulties and costs regarding ZFNs (Zinc Finger Nucleases) and TALENs (Transcription Activator-Like Effector Nucleases) use, off-target effects, low transfection efficiency, in vivo delivery-safety and ethical issues.

Transforming growth factor-beta 1 gene polymorphisms and expression in the blood of prostate cancer patients.

The transforming growth factor beta 1 (TGF-beta1) is a multifunctional cytokine with several regulatory activities in tumor cells affecting growth, differentiation, and function. Alterations in gene expression, secretion, and regulation of TGF-beta1 may lead to a favorable environment for tumor development by angiogenesis stimulation and immune system suppression. We evaluated the influence of the TGFB1 polymorphisms by ARMS-PCR, Leu10Pro, and Arg25Pro, on prostate cancer (PCa) and benign prostatic hyperplasia (BPH). We assessed TGFB1 polymorphisms and their relation to mRNA levels (semi-quantitative RT-PCR) in blood samples as well as the implications in disease occurrence and progression. Peripheral blood samples from 175 patients were analyzed as to 92 BPH and 83 PCa. Samples obtained from 132 healthy males were used as negative controls. PCa patients with a Gleason score greater than 7 presented a higher frequency of the C allele (Leu10Pro). This allele was associated with a higher risk of developing PCa and BPH compared to the population (2.6 and 3.6 times higher, respectively). Patients with TGFB1 transcript levels equal to or more than 70% higher than control levels presented a 5.34 and 2.14-fold higher risk of having PCa and BPH, respectively, relative to the population. No association was detected between polymorphisms and mRNA levels. The C allele of the Leu10Pro polymorphism may predispose men to a more rapid cancer progression. Additionally, higher mRNA levels in the peripheral blood of PCa patients suggest that tumor cells may be disseminated in the circulation and could be used as a biomarker for extra-capsular invasion.

Association of vitamin D receptor variants with clinical parameters in prostate cancer.

PURPOSE: Prostate Cancer (PCa) is one of the most common cancers in men and its early detection can provide a high chance of cure. The detection of Vitamin D Receptor (VDR) gene polymorphisms may be useful as a molecular indicator of clinical outcome, once VDR is implicated in a wide variety of biological processes including modulation of the immune response and inhibition of cancer cell growth, angiogenesis and metastasis. In this study we explored the Single Nucleotide Polymorphisms (SNPs) FokI, BsmI, ApaI and TaqI, to evaluate the susceptibility locus for PCa and verify its correlation with clinical parameters. METHODS: VDR polymorphisms were detected by PCR followed by Restriction Fragment Length Polymorphism (PCR-RFLP). DNA samples were extracted from peripheral blood of 342 patients: 132 PCa, 41 Benign Prostatic Hyperplasia and 169 young healthy volunteers. RESULTS: Statistical analysis showed a noteworthy correlation among SNPs and clinical pathological features. CC genotype (TaqI) was correlated with the age at diagnosis (>58 years old), and GG (BsmI) was associated to lower Prostate-Specific Antigen (PSA) levels (<10 ng/mL). Moreover, when PCa patients were subgrouped, G allele (BsmI) significantly increased the estimated chance for PSA < 10 ng/mL, and GG/GG genotype (BsmI/ApaI) provided a 9.75 fold increased chance of patients with PCa to present lower PSA levels. CONCLUSIONS: The polymorphisms of VDR gene showed a genotype-phenotype association and presented new correlations with different parameters as age and PSA levels.

Androgen receptor: CAG repeat length and transcriptional levels related to prostate cancer

Objectives: The present work evaluates variations of polymorphic [CAG]n repeats present at exon 1 of the AR gene, as well as relative levels of its transcript, in order to investigate associations of these factors with prostatic tumor genesis in the Brazilian male population. Methods: Genomic DNA was extracted from blood samples from patients with prostate cancer (PCa), benign prostatic hyperplasia (BPH), and from a group of young Brazilian males to determine the number of [CAG]n repeats amplified by PCR. Mutation analysis in this amplified fragment was carried out using the LIS-SSCP technique. Total RNA was extracted from prostatic tissue to evaluate the AR gene transcript levels using semi-quantitative multiplex RT-PCR. Results: CAG length varied from 14 to 30, with an average of 21 repeats for PCa and the male group and 20 for the BPH group. No significant difference was found for [CAG]n polymorphism among the analyzed groups and there was no sporadic change in the amplified portion of the AR gene, nor loss of [CAG]n repeats, demonstrating that these do not contribute to the cancer occurrence. Nevertheless, the positive association between short alleles and TNM pT3 staging may indicate that CAG repeats is associated to PCa progression. The transcriptional levels were significantly increased in PCa than in BPH and were associated with serum PSA levels of 5-10 ng/mL. As diagnostic clinical parameter, the levels of AR gene presented 17-fold higher chance for PCa occurrence, 60% of sensibility and 95% of specificity. Conclusion: The data suggest that the highly miscegenated Brazilian male population presents a high frequency of [CAG]n short repeats, which may be associated with the PCa progression, while AR mRNA levels seems to be a good indicator of the incidence of this pathology, being useful in clinical practice for distinguishing patients with PCa from those with BPH.