Evaluation of the Analytical Performance of the Fully Automated Gene Analyzer µTASWako g1 for the Detection of SARS-CoV-2.

BACKGROUND: The worldwide spread of coronavirus disease 2019 (COVID-19) has led to an urgent need for nucleic acid amplification test (NAAT) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Because NAAT has many manual processes, results may vary depending on the operator. Therefore, it has been required to develop a fully automated testing device and reagent that detects genetic material from SARS-CoV-2. The µTASWako g1 system (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), a genetic analyzer, provides results in 75 minutes by performing a fully automated PCR process. METHODS: We evaluated the analytical and clinical performance of the µTASWako g1 system for the detection of SARS-CoV-2 RNA. RESULTS: The µTASWako g1 system had the limit of detection at 2,000 copies/mL using a known concentration of RNA. In clinical samples, the µTASWako g1 system had a sensitivity of 88.0% and 100% specificity compared to conventional RT-PCR. The µTAS Wako g1 system could detect three variants of concern carrying spike mutations including N501Y, E484K, and L452R. CONCLUSIONS: As the assay on the µTASWako g1 system is highly accurate for the detection of SARS-CoV-2 regardless of the experience of operator, it can be widely applicable in clinical laboratories.

Immortalization of cells derived from domestic dogs through expressing mutant cyclin-dependent kinase 4, cyclin D1, and telomerase reverse transcriptase.

Dog is the first animal that was established as a close partner of human beings. Based on the vast genetic diversity and breeding, dogs exhibit unique genetic evolution and diversity from Chihuahua to St. Bernard. The safety tests of the pharmacological products also included domestic dogs as the test subjects. Although the safety confirmation test of chemicals for human use is important, the welfare of experimental animals requires special consideration. In this study, we cultured domestic dog-derived primary fibroblasts isolated from their muscle tissues. Furthermore, we successfully immortalized them through lentivirus-mediated gene transfer of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomere reverse transcriptase (TERT). We further demonstrated that the established immortalized domestic dog-derived fibroblasts retained the characteristics of the original parental cells. These cells might act a suitable in vivo model system to replace the implication of animals for evaluating the potential toxicity of pharmacological chemicals. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10616-021-00504-0.

Detailed chromosome analysis of wild-type, immortalized fibroblasts with SV40T, E6E7, combinational introduction of cyclin dependent kinase 4, cyclin D1, telomerase reverse transcriptase.

Cell immortalization enables us to expand the cultured cell infinitely. However, the process of immortalization sometimes changes the nature of the original cell. In this study, we established immortalized embryonic fibroblasts with oncogenic SV40T and human papilla virus-derived E6E7, combinational expression of mutant cyclin-dependent kinase 4 (CDK4), cyclin D1, and telomerase reverse transcriptase (TERT) from identical primary wild-type human embryonic fibroblasts (HE16). After the establishment of immortalized cells, we compared the details of chromosome condition with the G-banding and Q-banding methods. There is no example of detailed analysis so far about chromosome abnormalities, such as trisomy, ring chromosome, reciprocal translocation, and dicentric chromosomes. The detailed chromosome analysis revealed that immortalized cells with SV40T and E6E7 showed intensive chromosome abnormalities, such as gain or loss of the chromosomes all through the genome. Furthermore, we detected that the incidence of chromosome abnormities in the immortalized cell with the combinational introduction of R24C mutant of CDK4, cyclin D1, and TERT is almost identical to that of wild-type cell. Furthermore, short tandem repeat analysis demonstrated that the origin of K4DT cell is primary HE16. These results showed that cellular immortalization with CDK4, cyclin D1, and TERT is more advantageous in keeping the chromosome's original condition than oncogenic immortalization methods.

Requirement for C-mannosylation to be secreted and activated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4).

BACKGROUND: C-mannosylation is a unique type of glycosylation. A disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4) is a multidomain extracellular metalloproteinase that contains several potential C-mannosylation sites. Although some ADAMTS family proteins have been reported to be C-mannosylated proteins, whether C-mannosylation affects the activation and protease activity of these proteins is unclear. METHODS: We established wild-type and mutant ADAMTS4-overexpressing HT1080 cell lines. Recombinant ADAMTS4 was purified from the conditioned medium of the wild-type ADAMTS4-overexpressing cells, and the C-mannosylation sites of ADAMTS4 were identified by LC-MS/MS. The processing, secretion, and intracellular localization of ADAMTS4 were examined by immunoblot and immunofluorescence analyses. ADAMTS4 enzymatic activity was evaluated by assessing the cleavage of recombinant aggrecan. RESULTS: We identified that ADAMTS4 is C-mannosylated at Trp404 in the metalloprotease domain and at Trp523, Trp526, and Trp529 in the thrombospondin type 1 repeat (TSR). The replacement of Trp404 with Phe affected ADAMTS4 processing, without affecting secretion and intracellular localization. In contrast, the substitution of Trp523, Trp526, and Trp529 with Phe residues suppressed ADAMTS4 secretion, processing, intracellular trafficking, and enzymatic activity. CONCLUSIONS: Our results demonstrated that the C-mannosylation of ADAMTS4 plays important roles in protein processing, intracellular trafficking, secretion, and enzymatic activity. GENERAL SIGNIFICANCE: Because C-mannosylation appears to regulate many ADAMTS4 functions, C-mannosylation may also affect other members of the ADAMTS superfamily.