Evaluation and normalization of a set of reliable reference genes for quantitative sgk-1 gene expression analysis in Caenorhabditis elegans-focused cancer research.

Multiple signaling pathways have been discovered to play a role in aging and longevity, including the insulin/IGF-1 signaling system, AMPK pathway, TOR signaling, JNK pathway, and germline signaling. Mammalian serum and glucocorticoid-inducible kinase 1 (sgk-1), which has been associated with various disorders including hypertension, obesity, and tumor growth, limits survival in C. elegans by reducing DAF-16/FoxO activity while suppressing FoxO3 activity in human cell culture. C. elegans provides significant protection for a number of genes associated with human cancer. The best known of these are the lin-35/pRb (mammalian ortholog pRb) and CEP-1 (mammalian ortholog p53) genes. Therefore, in this study, we aimed to investigate the expression analyzes of sgk-1, which is overexpressed in many types of mammalian cancer, in mutant lin-35 and to demonstrate the validation of reference genes in wild-type N2 and mutant lin-35 for C. elegans-focused cancer research. To develop functional genomic studies in C. elegans, we evaluated the expression stability of five candidate reference genes (act-1, ama-1, cdc-42, pmp-3, iscu-1) by quantitative real-time PCR using five algorithms (geNorm, NormFinder, Delta Ct method, BestKeeper, RefFinder) in N2 and lin-35 worms. According to our findings, act-1 and cdc-42 were effective in accurately normalizing the levels of gene expression in N2 and lin-35. act-1 and cdc-42 also displayed the most consistent expression patterns, therefore they were utilized to standardize expression level of sgk-1. Furthermore, our results clearly showed that sgk-1 was upregulated in lin-35 worms compared to N2 worms. Our results highlight the importance of definitive validation using mostly expressed reference genes.

Systems-level temporal immune-metabolic profile in Crimean-Congo hemorrhagic fever virus infection.

Crimean-Congo hemorrhagic fever (CCHF) caused by CCHF virus (CCHFV) is one of the epidemic-prone diseases prioritized by the World Health Organisation as public health emergency with an urgent need for accelerated research. The trajectory of host response against CCHFV is multifarious and remains unknown. Here, we reported the temporal spectrum of pathogenesis following the CCHFV infection using genome-wide blood transcriptomics analysis followed by advanced systems biology analysis, temporal immune-pathogenic alterations, and context-specific progressive and postinfection genome-scale metabolic models (GSMM) on samples collected during the acute (T0), early convalescent (T1), and convalescent-phase (T2). The interplay between the retinoic acid-inducible gene-I-like/nucleotide-binding oligomerization domain-like receptor and tumor necrosis factor signaling governed the trajectory of antiviral immune responses. The rearrangement of intracellular metabolic fluxes toward the amino acid metabolism and metabolic shift toward oxidative phosphorylation and fatty acid oxidation during acute CCHFV infection determine the pathogenicity. The upregulation of the tricarboxylic acid cycle during CCHFV infection, compared to the noninfected healthy control and between the severity groups, indicated an increased energy demand and cellular stress. The upregulation of glycolysis and pyruvate metabolism potentiated energy generation through alternative pathways associated with the severity of the infection. The downregulation of metabolic processes at the convalescent phase identified by blood cell transcriptomics and single-cell type proteomics of five immune cells (CD4+ and CD8+ T cells, CD14+ monocytes, B cells, and NK cells) potentially leads to metabolic rewiring through the recovery due to hyperactivity during the acute phase leading to post-viral fatigue syndrome.

Investigating the effect of ribavirin treatment on genetic mutations in Crimean-Congo haemorrhagic fever virus (CCHFV) through next-generation sequencing.

Crimean-Congo haemorrhagic fever (CCHF) is the most widespread tick-borne viral haemorrhagic fever affecting humans, and yet a licensed drug against the virus (CCHFV) is still not available. While several studies have suggested the efficacy of ribavirin against CCHFV, current literature remains inconclusive. In this study, we have utilised next-generation sequencing to investigate the mutagenic effect of ribavirin on the CCHFV genome during clinical disease. Samples collected from CCHF patients receiving ribavirin treatment or supportive care only at Sivas Cumhuriyet University Hospital, Turkey, were analysed. By comparing the frequency of mutations in each group, we found little evidence of an overall mutagenic effect. This suggests that ribavirin, administered at the acute stages of CCHFV infection (at the World Health Organization-recommended dose) is unable to induce lethal mutagenesis that would cause an extinction event in the CCHFV population and reduce viremia.

The investigation of host genetic variants of toll-like receptor 7 and 8 in COVID-19.

Toll-like receptors (TLRs) recognize infectious agents and play an important role in the innate immune system. Studies have suggested that TLR single nucleotide polymorphisms (SNPs) are associated with poor antiviral responses against SARS-CoV-2. Therefore, we aimed to investigate the relationship of TLR7 and TLR8 (SNPs) with COVID-19 disease prognosis. A total of 120 COVID-19 patients, 40 outpatients, 40 clinical ward patients and 40 intensive care unit (ICU) patients were included in the study. TLR7 (rs179009), TLR8-129 C/G (rs3764879) and TLR8 Met1Val (rs3764880) SNPs were genotyped using the PCR-RFLP method. In female patients, individuals carrying AG genotype and G allele for TLR8 Met1Val SNP were found at a higher frequency in patients hospitalized in the ICU than in patients followed in the clinical ward (p < 0.05). In terms of the other two SNPs, no significant difference was found between the groups in females. Furthermore, in male patients, A allele of TLR7 rs179009 SNP was at a higher frequency in patients who have at least one comorbidity than in patients who have no comorbidity (p < 0.05). Our results suggest that TLR8 Met1Val SNP is important in the COVID-19 disease severity in females. Furthermore, TLR7 rs179009 SNP is important in male patients in the presence of comorbid diseases.