From haystack to high precision: advanced sequencing methods to unraveling circulating tumor DNA mutations.

Identifying mutations in cancer-associated genes to guide patient treatments is essential for precision medicine. Circulating tumor DNA (ctDNA) offers valuable insights for early cancer detection, treatment assessment, and surveillance. However, a key issue in ctDNA analysis from the bloodstream is the choice of a technique with adequate sensitivity to identify low frequent molecular changes. Next-generation sequencing (NGS) technology, evolving from parallel to long-read capabilities, enhances ctDNA mutation analysis. In the present review, we describe different NGS approaches for identifying ctDNA mutation, discussing challenges to standardized methodologies, cost, specificity, clinical context, and bioinformatics expertise for optimal NGS application.

Exploring the interplay between miRNAs, apoptosis and viral load, in Dengue virus infection.

Dengue virus (DENV) is a major global health concern, causing millions of infections annually. Understanding the cellular response to DENV infection is crucial for developing effective therapies. This study provides an in-depth analysis of the cellular response to Dengue virus (DENV) infection, with a specific focus on the interplay between microRNAs (miRNAs), apoptosis, and viral load across different DENV serotypes. Utilizing a variety of cell lines infected with four DENV serotypes, the research methodically quantifies viral load, and the expression levels of miRNA-15, miRNA-16, and BCL2 protein, alongside measuring apoptosis markers. Methodologically, the study employs quantitative PCR for viral load and miRNA expression analysis, and Western blot for apoptosis and BCL2 detection, with a statistical framework that includes ANOVA and correlation analysis to discern significant differences and relationships. The findings reveal that despite similar viral loads across DENV serotypes, DENV-2 exhibits a marginally higher load. A notable upregulation of miRNA-15 and miRNA-16 correlates positively with increased viral load, suggesting their potential role in modulating viral replication. Concurrently, a marked activation of caspases 3 and 7, along with changes in BCL2 protein levels, underscores the role of apoptosis in the cellular response to DENV infection. Conclusively, the study enhances the understanding of miRNA involvement in DENV pathogenesis, highlighting miRNA-15 and miRNA-16 as potential regulatory agents in viral replication and apoptosis. These findings pave the way for further exploration into miRNA-based therapeutic strategies against DENV infection.

Analysis of Epidemiological Factors and SNP rs3804100 of TLR2 for COVID-19 in a Cohort of Professionals Who Worked in the First Pandemic Wave in Belém-PA, Brazil.

COVID-19 is an infectious disease caused by coronavirus 2 of the severe acute syndrome (SARS-CoV-2). Single nucleotide polymorphisms (SNPs) in genes, such as TLR2, responsible for an effective human immune response, can change the course of infection. The objective of this article was to verify associations between epidemiological factors and TLR2 SNP rs3804100 (Thymine [T] > Cytosine [C]) in professionals from Health Institutions (HI) who worked during the first pandemic wave and COVID-19. A case-control study was conducted with Belém-PA HI workers (Northern Brazil), divided into symptomatology groups (Asymptomatic-AS; n = 91; and Symptomatic-SI; n = 123); and severity groups classified by Chest Computerized Tomography data (symptomatic with pulmonary involvement-SCP; n = 35; symptomatic without pulmonary involvement-SSP; n = 8). Genotyping was performed by Sanger sequencing, and Statistical Analysis was conducted through the SPSS program. Bioinformatics servers predicted the biological functions of the TLR2 SNP. There were associations between the presence of comorbidities and poor prognosis of COVID-19 (especially between symptomatology and severity of COVID-19 and overweight and obesity) and between the sickness in family members and kinship (related to blood relatives). The homozygous recessive (C/C) genotype was not found, and the frequency of the mutant allele (C) was less than 10% in the cohort. No significant associations were found for this SNP in this cohort. The presence of SNP was indicated to be benign and causes a decrease in the stability of the TLR2 protein. These data can help the scientific community and medicine find new forms of COVID-19 containment.

Anticancer Potential of Flavonoids: An Overview with an Emphasis on Tangeretin.

Natural compounds with pharmacological activity, flavonoids have been the subject of an exponential increase in studies in the field of scientific research focused on therapeutic purposes due to their bioactive properties, such as antioxidant, anti-inflammatory, anti-aging, antibacterial, antiviral, neuroprotective, radioprotective, and antitumor activities. The biological potential of flavonoids, added to their bioavailability, cost-effectiveness, and minimal side effects, direct them as promising cytotoxic anticancer compounds in the optimization of therapies and the search for new drugs in the treatment of cancer, since some extensively antineoplastic therapeutic approaches have become less effective due to tumor resistance to drugs commonly used in chemotherapy. In this review, we emphasize the antitumor properties of tangeretin, a flavonoid found in citrus fruits that has shown activity against some hallmarks of cancer in several types of cancerous cell lines, such as antiproliferative, apoptotic, anti-inflammatory, anti-metastatic, anti-angiogenic, antioxidant, regulatory expression of tumor-suppressor genes, and epigenetic modulation.

Experimental Dengue Virus Type 4 Infection Increases the Expression of MicroRNAs-15/16, Triggering a Caspase-Induced Apoptosis Pathway.

The World Health Organization has estimated the annual occurrence of approximately 392 million dengue virus (DENV) infections in more than 100 countries where the virus is endemic, which represents a serious threat to humanity. DENV is a serologic group with four distinct serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) belonging to the genus Flavivirus, in the family Flaviviridae. Dengue is the most widespread mosquito-borne disease in the world. The ~10.7 kb DENV genome encodes three structural proteins (capsid (C), pre-membrane (prM), and envelope (E)) and seven non-structural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The NS1 protein is a membrane-associated dimer and a secreted, lipid-associated hexamer. Dimeric NS1 is found on membranes both in cellular compartments and cell surfaces. Secreted NS1 (sNS1) is often present in patient serum at very high levels, which correlates with severe dengue symptoms. This study was conducted to discover how the NS1 protein, microRNAs-15/16 (miRNAs-15/16), and apoptosis are related during DENV-4 infection in human liver cell lines. Huh 7.5 and HepG2 cells were infected with DENV-4, and miRNAs-15/16, viral load, NS1 protein, and caspases-3/7 were quantified after different durations of infection. This study demonstrated that miRNAs-15/16 were overexpressed during the infection of HepG2 and Huh 7.5 cells with DENV-4 and had a relationship with NS1 protein expression, viral load, and the activity of caspases-3/7, thus making these miRNAs potential injury markers during DENV infection in human hepatocytes.

Replication of Rocio virus in primary cultures of mouse neural cells.

OBJECTIVE: This study verified the replication efficiency of the Rocio virus in a primary culture of mouse neural cells. METHODS: Mixed primary cultures (neurons/glia) obtained from the brains of newborn isogenic BALB/c mice were inoculated with Rocio virus on the 7 th day of culture, and the development of cytopathogenic effects was monitored. The infection was confirmed via immunocytochemistry (anti-ROCV), while viral replication was quantified in infected primary cultures. The titration method used depended on the infection period. RESULTS: Rocio virus efficiently infected primary cultured neural cells, with the highest viral titer causing cytopathic changes was observed at 2 days post infection. The virus-infected primary culture survived for up to 7 days post infection, and viral load quantitation showed viral replication kinetics compatible with the cell death kinetics of cultures. CONCLUSION: The findings of this study suggest that mouse neural cell primary cultures support Rocio virus replication and could be used as an alternative system for studying Flavivirus infection in the central nervous system.

Analysis of associations between the TLR3 SNPs rs3775291 and rs3775290 and COVID-19 in a cohort of professionals of Belém-PA, Brazil.

The objective of this article was to verify associations between the SNPs rs3775291 (Cytosine [C]>Thymine [T]) and rs3775290 (C>T) of TLR3 in professionals from Health Institutions (HI) who worked during the first pandemic wave and COVID-19. A case-control study was carried out with workers from HI in Belém-PA, Brazil, divided into symptomatology groups (Asymptomatic-AS, n=91; and Symptomatic-SI, n=121), and severity groups, classified by Chest CT scan (symptomatic with lung involvement - SCP, n=34; symptomatic without lung involvement - SSP, n=8). Genotyping was performed by Sanger sequencing and statistical analysis was performed using the SPSS program. In the analysis of SNP rs3775291, the homozygous recessive genotype (T/T) was not found and the frequency of the mutant allele (T) was less than 2% in the cohort. For the rs3775290 SNP, the frequency of the mutant allele (T) was greater than 42% in the cohort. No significant associations were found for these SNPs in this cohort (N= 212 individuals). The scientific community and physicians can use these facts to find new methods of managing COVID-19.

Replication of Rocio virus in primary cultures of mouse neural cells

ABSTRACT Objective This study verified the replication efficiency of the Rocio virus in a primary culture of mouse neural cells. Methods Mixed primary cultures (neurons/glia) obtained from the brains of newborn isogenic BALB/c mice were inoculated with Rocio virus on the 7 th day of culture, and the development of cytopathogenic effects was monitored. The infection was confirmed via immunocytochemistry (anti-ROCV), while viral replication was quantified in infected primary cultures. The titration method used depended on the infection period. Results Rocio virus efficiently infected primary cultured neural cells, with the highest viral titer causing cytopathic changes was observed at 2 days post infection. The virus-infected primary culture survived for up to 7 days post infection, and viral load quantitation showed viral replication kinetics compatible with the cell death kinetics of cultures. Conclusion The findings of this study suggest that mouse neural cell primary cultures support Rocio virus replication and could be used as an alternative system for studying Flavivirus infection in the central nervous system.

Anticipating the Next Chess Move: Blocking SARS-CoV-2 Replication and Simultaneously Disarming Viral Escape Mechanisms.

The COVID-19 pandemic initiated a race to determine the best measures to control the disease and to save as many people as possible. Efforts to implement social distancing, the use of masks, and massive vaccination programs turned out to be essential in reducing the devastating effects of the pandemic. Nevertheless, the high mutation rates of SARS-CoV-2 challenge the vaccination strategy and maintain the threat of new outbreaks due to the risk of infection surges and even lethal variations able to resist the effects of vaccines and upset the balance. Most of the new therapies tested against SARS-CoV-2 came from already available formulations developed to treat other diseases, so they were not specifically developed for SARS-CoV-2. In parallel, the knowledge produced regarding the molecular mechanisms involved in this disease was vast due to massive efforts worldwide. Taking advantage of such a vast molecular understanding of virus genomes and disease mechanisms, a targeted molecular therapy based on siRNA specifically developed to reach exclusive SARS-CoV-2 genomic sequences was tested in a non-transformed human cell model. Since coronavirus can escape from siRNA by producing siRNA inhibitors, a complex strategy to simultaneously strike both the viral infectious mechanism and the capability of evading siRNA therapy was developed. The combined administration of the chosen produced siRNA proved to be highly effective in successfully reducing viral load and keeping virus replication under control, even after many days of treatment, unlike the combinations of siRNAs lacking this anti-anti-siRNA capability. Additionally, the developed therapy did not harm the normal cells, which was demonstrated because, instead of testing the siRNA in nonhuman cells or in transformed human cells, a non-transformed human thyroid cell was specifically chosen for the experiment. The proposed siRNA combination could reduce the viral load and allow the cellular recovery, presenting a potential innovation for consideration as an additional strategy to counter or cope COVID-19.

Comparative Genomic Analyses of New and Old World Viscerotropic Leishmanine Parasites: Further Insights into the Origins of Visceral Leishmaniasis Agents.

Visceral leishmaniasis (VL), also known as kala-azar, is an anthropozoonotic disease affecting human populations on five continents. Aetiologic agents belong to the Leishmania (L.) donovani complex. Until the 1990s, three leishmanine parasites comprised this complex: L. (L.) donovani Laveran & Mesnil 1903, L. (L.) infantum Nicolle 1908, and L. (L.) chagasi Lainson & Shaw 1987 (=L. chagasi Cunha & Chagas 1937). The VL causal agent in the New World (NW) was previously identified as L. (L.) chagasi. After the development of molecular characterization, however, comparisons between L. (L.) chagasi and L. (L.) infantum showed high similarity, and L. (L.) chagasi was then regarded as synonymous with L. (L.) infantum. It was, therefore, suggested that L. (L.) chagasi was not native to the NW but had been introduced from the Old World by Iberian colonizers. However, in light of ecological evidence from the NW parasite's enzootic cycle involving a wild phlebotomine vector (Lutzomyia longipalpis) and a wild mammal reservoir (the fox, Cerdocyon thous), we have recently analyzed by molecular clock comparisons of the DNA polymerase alpha subunit gene the whole-genome sequence of L. (L.) infantum chagasi of the most prevalent clinical form, atypical dermal leishmaniasis (ADL), from Honduras (Central America) with that of the same parasite from Brazil (South America), as well as those of L. (L.) donovani (India) and L. (L.) infantum (Europe), which revealed that the Honduran parasite is older ancestry (382,800 ya) than the parasite from Brazil (143,300 ya), L. (L.) donovani (33,776 ya), or L. (L.) infantum (13,000 ya). In the present work, we have now amplified the genomic comparisons among these leishmanine parasites, exploring mainly the variations in the genome for each chromosome, and the number of genomic SNPs for each chromosome. Although the results of this new analysis have confirmed a high genomic similarity (~99%) among these parasites [except L. (L.) donovani], the Honduran parasite revealed a single structural variation on chromosome 17, and the highest frequency of genomic SNPs (more than twice the number seen in the Brazilian one), which together to its extraordinary ancestry (382,800 ya) represent strong evidence that L. (L.) chagasi/L. (L.) infantum chagasi is, in fact, native to the NW, and therefore with valid taxonomic status. Furthermore, the Honduran parasite, the most ancestral viscerotropic leishmanine parasite, showed genomic and clinical taxonomic characteristics compatible with a new Leishmania species causing ADL in Central America.