A collaborative study to establish the national standard for SARS-CoV-2 RNA nucleic acid amplification techniques (NAAT) in Taiwan.

The conventional PCR remains a valuable method to detect the newly emergent coronavirus rapidly and accurately. Our investigation aimed to establish the standard materials of SARS-CoV-2 for NAAT detection. We provided formalin-inactivated SARS-CoV-2 and confirmed RNA copy numbers. In addition, the virus genome was confirmed with whole-genome sequencing and identified as Wuhan/WI04/2019. Seven laboratories were invited for this collaborative study, according to the reporting data, we determined the SARS-CoV-2 with the unit of 6.35 Log10 copies/mL as the national standard. The availability of the national standard (NS) of SARS-CoV-2 will facilitate the standardization and harmonization of SARS-CoV-2 NAAT assays.

Effects of lipopolysaccharide on the expression of plasma membrane monoamine transporter (PMAT) at the blood-brain barrier and its implications to the transport of neurotoxins.

Plasma membrane monoamine transporter (PMAT) is a polyspecific organic cation transporter that is highly expressed in the central nervous system. This study aimed to investigate the effect of lipopolysaccharide on PMAT expression at the blood-brain barrier and the interaction between PMAT and neurotoxins. As a result, PMAT mRNA was identified in brain microvessels (BMVs), brain microvascular endothelial cells (BMECs), astrocytes, and pericytes isolated from C57BL/6 mice and/or Wistar rats using RT-qPCR. The immunofluorescence staining confirmed the expression of PMAT protein in BMVs and striatum of C57BL/6 mice. Western blotting demonstrated its localization at the luminal and abluminal sides of BMECs. In C57BL/6 mice, PMAT protein was significantly increased in BMVs 24 h after an intraperitoneal injection of 3 mg/kg lipopolysaccharide. Lipopolysaccharide treatment also significantly increased PMAT expression in cerebral cortex and the striatum in a time-dependent manner, as well as the brain-to-plasma ratio of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-benzyl-TIQ). In isolated cells, lipopolysaccharide treatment significantly increased PMAT mRNA in brain astrocytes and the BMECs co-cultured with astrocytes. In addition to 1-methyl-4-phenylpyridinium, the kinetic study indicated that both 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 1-benzyl-TIQ are substrates of human PMAT. These findings suggest that inflammation can change PMAT expression at the blood-brain barrier, which may affect PMAT-mediated transport of neurotoxins. We demonstrated the expression of plasma membrane monoamine transporter (PMAT; mRNA or protein) at several subunits of the blood-brain barrier. Lipopolysaccharide treatment can significantly increase the expression of PMAT in vivo (in brain microvessels, cerebral cortex, and the striatum of C57BL/6 mice) and in vitro (in brain astrocytes and brain microvascular endothelial cells co-cultured with astrocytes). Lipopolysaccharide treatment also increased the brain-to-plasma ratio of 1-benzyl-1,2,3,4-tetrahydroisoquinoline (1-benzyl-TIQ) in mice, where 1-benzyl-TIQ competitively inhibited 1-methyl-4-phenylpyridinium (MPP(+)) uptake in MDCK-human PMAT (hPMAT) cells and its uptake in MDCK-hPMAT is concentration dependent.

Decreased expression of organic cation transporters, Oct1 and Oct2, in brain microvessels and its implication to MPTP-induced dopaminergic toxicity in aged mice.

This study was to investigate the influence of age on the expression of organic cation transporters (OCTs) that belong to the SLC22 family in brain microvessels (BMVs) and its implications for 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic toxicity in mice. Here, we showed that Oct1 and Oct2, but not Oct3, mRNAs were detected and enriched (compared with cerebral cortex) in BMVs of C57BL/6 (B6) mice using reverse transcription-quantitative PCR (RT-qPCR), and immunofluorescence analysis further revealed that Oct1 and Oct2 proteins were colocalized with endothelial markers. Both the mRNA and protein levels of Oct1 and Oct2 were reduced in aged mice. After an intraperitoneal administration of MPTP, brain extracellular levels of MPTP and 1-methyl-4-phenyl-pyridinium (MPP(+)) were much lower in aged mice and in Oct1/2(-/-) mice compared with younger mice and wild-type control mice, respectively. Knockout of Oct1/Oct2 protected Oct1/2(-/-) mice from MPTP-induced neurotoxicity, whereas the loss of tyrosine hydroxylase (TH)-positive neurons was slightly greater in aged than in younger mice. However, intrastriatal infusion of low-dose MPTP caused more severe dopaminergic toxicity in the substantia nigra of both aged mice and Oct1/2(-/-) mice. These findings show that age-dependent downregulation or knockout of Oct1/Oct2 in BMVs may reduce the transport of MPTP, which, in part, affects its dopaminergic toxicity.