Search Results (6)

The thymidylate kinase (tmk) gene is indispensable for the proliferation and survival of phytoplasma. To reveal the molecular variation and phylogeny of the tmk genes of Candidatus phytoplasma ziziphi, in this study, the tmk genes of 50 phytoplasma strains infecting different resistant and susceptible jujube cultivars from different regions in China were amplified and analyzed. Two sequence types, tmk-x and tmk-y, were identified using clone-based sequencing. The JWB phytoplasma strains were classified into three types, type-X, type-Y, and type-XY, based on the sequencing chromatograms of the tmk genes. The type-X and type-Y strains contained only tmk-x and tmk-y genes, respectively. The type-XY strain contained both tmk-x and tmk-y genes. The type-X, type-Y, and type-XY strains comprised 42%, 12%, and 46% of all the strains, respectively. The type-X and type-XY strains were identified in both susceptible and resistant jujube cultivars, while type-Y strain was only identified in susceptible cultivars. Phylogenetic analysis indicated that the tmk genes of the phytoplasmas were divided into two categories: phylo-S and phylo-M. The phylo-S tmk gene was single-copied in the genome, with an evolutionary pattern similar to the 16S rRNA gene; the phylo-M tmk gene was multi-copied, related to PMU-mediated within-genome transposition and between-genome transfer. Furthermore, the phylogenetic tree suggested that the tmk genes shuttled between the genomes of the Paulownia witches’ broom phytoplasma and JWB phytoplasma. These findings provide insights into the evolutionary and adaptive mechanisms of phytoplasmas. Full article

RET-kinase-activating gene rearrangements occur in approximately 1–2% of non-small-cell lung carcinomas (NSCLCs). Their reliable detection requires next-generation sequencing (NGS), while conventional methods, such as immunohistochemistry (IHC), fluorescence in situ hybridization (FISH) or variant-specific PCR, have significant limitations. We developed an assay that compares the level of RNA transcripts corresponding to 5′- and 3′-end portions of the RET gene; this test relies on the fact that RET translocations result in the upregulation of the kinase domain of the gene and, therefore, the 5′/3′-end expression imbalance. The present study included 16,106 consecutive NSCLC patients, 14,449 (89.7%) of whom passed cDNA quality control. The 5′/3′-end unbalanced RET expression was observed in 184 (1.3%) tumors, 169 of which had a sufficient amount of material for the identification of translocation variants. Variant-specific PCR revealed RET rearrangements in 155/169 (91.7%) tumors. RNA quality was sufficient for RNA-based NGS in 10 cases, 8 of which carried exceptionally rare or novel (HOOK1::RET and ZC3H7A::RET) RET translocations. We also applied variant-specific PCR for eight common RET rearrangements in 4680 tumors, which emerged negative upon the 5′/3′-end unbalanced expression test; 33 (0.7%) of these NSCLCs showed RET fusion. While the combination of the analysis of 5′/3′-end RET expression imbalance and variant-specific PCR allowed identification of RET translocations in approximately 2% of consecutive NSCLCs, this estimate approached 120/2361 (5.1%) in EGFR/KRAS/ALK/ROS1/BRAF/MET-negative carcinomas. RET-rearranged tumors obtained from females, but not males, had a decreased level of expression of thymidylate synthase (p < 0.00001), which is a known predictive marker of the efficacy of pemetrexed. The results of our study provide a viable alternative for RET testing in facilities that do not have access to NGS due to cost or technical limitations. Full article

Our previous research suggests an important regulatory role of CK2-mediated phosphorylation of enzymes involved in the thymidylate biosynthesis cycle, i.e., thymidylate synthase (TS), dihydrofolate reductase (DHFR), and serine hydroxymethyltransferase (SHMT). The aim of this study was to show whether silencing of the CK2α gene affects TS and DHFR expression in A-549 cells. Additionally, we attempted to identify the endogenous kinases that phosphorylate TS and DHFR in CCRF-CEM and A-549 cells. We used immunodetection, immunofluorescence/confocal analyses, reverse transcription–quantitative polymerase chain reaction (RT-qPCR), in-gel kinase assay, and mass spectrometry analysis. Our results demonstrate that silencing of the CK2α gene in lung adenocarcinoma cells significantly increases both TS and DHFR expression and affects their cellular distribution. Additionally, we show for the first time that both TS and DHFR are very likely phosphorylated by endogenous CK2 in two types of cancer cells, i.e., acute lymphoblastic leukaemia and lung adenocarcinoma. Moreover, our studies indicate that DHFR is phosphorylated intracellularly by CK2 to a greater extent in leukaemia cells than in lung adenocarcinoma cells. Interestingly, in-gel kinase assay results indicate that the CK2α’ isoform was more active than the CK2α subunit. Our results confirm the previous studies concerning the physiological relevance of CK2-mediated phosphorylation of TS and DHFR. Full article

There is a continued need to understand varicella-zoster virus (VZV) pathogenesis and to develop more effective antivirals, as it causes chickenpox and zoster. As a human-restricted alphaherpesvirus, the use of human skin in culture and mice is critical in order to reveal the important VZV genes that are required for pathogenesis but that are not necessarily observed in the cell culture. We previously used VZV-expressing firefly luciferase (fLuc), under the control of the constitutively active SV40 promoter (VZV-BAC-Luc), to measure the VZV spread in the same sample. However, the fLuc expression was independent of viral gene expression and viral DNA replication programs. Here, we developed robust reporter VZV viruses by using bacterial artificial chromosome (BAC) technology, expressing luciferase from VZV-specific promoters. We also identified two spurious mutations in VZV-BAC that were corrected for maximum pathogenesis. VZV with fLuc driven by ORF57 showed superior growth in cells, human skin explants, and skin xenografts in mice. The ORF57-driven luciferase activity had a short half-life in the presence of foscarnet. This background was then used to investigate the roles for ORF36 (thymidine kinase (TK)) and ORF13 (thymidylate synthase (TS)) in skin. The studies reveal that VZV-∆TS had increased sensitivity to brivudine and was highly impaired for skin replication. This is the first report of a phenotype that is associated with the loss of TS. Full article

Interferon-induced protein with tetratricopeptide repeats 2 (IFIT2) is a member of the interferon-stimulated gene family that contains tetratricopeptide repeats (TPRs), which mediate protein–protein interactions in various biological systems. We previously showed the depletion of IFIT2 enhanced cell migration and metastatic activity in oral squamous cell carcinoma (OSCC) cells via the activation of atypical PKC signaling. In this study, we found that IFIT2-knockdown cells displayed higher resistance to 5-fluorouracil (5-FU) than control cells. The comet assay and annexin V analysis showed decreased DNA damage and cell death in IFIT2-knockdown cells compared to control cells treated with 5-FU. Cell cycle progression was also perturbed by 5-FU treatment, with the accumulation of IFIT2-depleted cells in S phase in a time-dependent manner. We further observed the overexpression of thymidylate synthase (TS) and thymidine kinase (TK) in IFIT2-knockdown cells. Inhibition of TS alone or double inhibition of TS and TK1 using the siRNA technique increased susceptibility to 5-FU in IFIT2-knockdown cells. We further identified that suberanilohydroxamic acid (SAHA) treatment decreased the expression of TS in IFIT2-knockdown cells and demonstrated that pretreatment with SAHA sensitized IFIT2-knockdown cells to 5-FU in vitro and in vivo. In conclusion, IFIT2 knockdown enhances TS expression, which mediates 5-FU resistance, and SAHA pretreatment suppresses TS expression and hence sensitizes cells to 5-FU. SAHA will be an effective strategy for the treatment of OSCC patients with 5-FU resistance. Full article

Baculoviruses are capable of infecting a wide diversity of insect pests. In the 1990s, the Dione juno nucleopolyhedrovirus (DijuNPV) was isolated from larvae of the major passionfruit defoliator pest Dione juno juno (Nymphalidae) and described at ultrastructural and pathological levels. In this study, the complete genome sequence of DijuNPV was determined and analyzed. The circular genome presents 122,075 bp with a G + C content of 50.9%. DijuNPV is the first alphabaculovirus completely sequenced that was isolated from a nymphalid host and may represent a divergent species. It appeared closely related to Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) and other Choristoneura-isolated group I alphabaculoviruses. We annotated 153 open reading frames (ORFs), including a set of 38 core genes, 26 ORFs identified as present in lepidopteran baculoviruses, 17 ORFs unique in baculovirus, and several auxiliary genes (e.g., bro, cathepsin, chitinase, iap-1, iap-2, and thymidylate kinase). The thymidylate kinase (tmk) gene was present fused to a dUTPase (dut) gene in other baculovirus genomes. DijuNPV likely lost the dut portion together with the iap-3 homolog. Overall, the genome sequencing of novel alphabaculoviruses enables a wide understanding of baculovirus evolution. Full article