No abstract

SARS-CoV-2 is very contagious and has rapidly spread globally. Due to various symptomatic and asymptomatic cases and the possibility of asymptomatic transmission, there is a pressing need for a fast and sensitive detection protocol to diagnose asymptomatic people. Various SARS-CoV-2 diagnostic kits are already available from many companies and national health agencies. However, publicly available information on these diagnostic kits is lacking. In response to the growing need and the lack of information, we developed and made available a low-cost, easy-access, real-time PCRbased protocol for the early detection of the virus in a previous study. During the development of the detection protocol, we found that unoptimized primer sets could inadvertently show false-positive results, raising the possibility that commercially available diagnostic kits might also contain primer sets that produce false-positive results. Here, we provide three-step guidelines for the design and optimization of specific primer sets. The three steps include (1) the selection of primer sets for target genes (RdRP, N, E, and S) in the genome of interest (SARS-CoV-2), (2) the in silico validation of primer and amplicon sequences, and (3) the optimization of PCR conditions (i.e., primer concentrations and annealing temperatures) for specific hybridization between the primers and target genes, and the elimination of spurious primer dimers. Furthermore, we have expanded the previously developed real-time PCR-based protocol to more conventional PCR-based protocols and applied a multiplex PCR-based protocol that allows the simultaneous testing of primer sets for RdRP, N, E, and S all in one reaction. Our newly optimized protocol should be helpful for the large-scale, high-fidelity screening of asymptomatic people, even without any high-specification equipment, for the further prevention of transmission, and to achieve early intervention and treatment for the rapidly propagating virus.

No abstract

In the brain, a reduction in extracellular osmolality causes water-influx and swelling, which subsequently triggers Cl − - and osmolytes-efflux via volume-regulated anion channel (VRAC). Although LRRC8 family has been recently proposed as the pore-forming VRAC which is activated by low cytoplasmic ionic strength but not by swelling, the molecular identity of the pore-forming swelling-dependent VRAC (VRAC swell ) remains unclear. Here we identify and characterize Tweety-homologs (TTYH1, TTYH2, TTYH3) as the major VRAC swell in astrocytes. Gene-silencing of all Ttyh1/2/3 eliminated hypo-osmotic-solution-induced Cl − conductance (I Cl,swell ) in cultured and hippocampal astrocytes. When heterologously expressed in HEK293T or CHO-K1 cells, each TTYH isoform showed a significant I Cl,swell with similar aquaporin-4 dependency, pharmacological properties and glutamate permeability as I Cl,swell observed in native astrocytes. Mutagenesis-based structure-activity analysis revealed that positively charged arginine residue at 165 in TTYH1 and 164 in TTYH2 is critical for the formation of the channel-pore. Our results demonstrate that TTYH family confers the bona fide VRAC swell in the brain.

An ongoing pandemic of coronavirus disease 2019 (COVID-19) is now the greatest threat to global public health. Herbal medicines and their derived natural products have drawn much attention in the treatment of COVID-19, but the detailed mechanisms by which natural products inhibit SARS-CoV-2 have not been elucidated. Here, we show that platycodin D (PD), a triterpenoid saponin abundant in Platycodon grandiflorum (PG), a dietary and medicinal herb commonly used in East Asia, effectively blocks the two main SARS-CoV-2 infection routes via lysosome- and transmembrane protease serine 2 (TMPRSS2)-driven entry. Mechanistically, PD prevents host entry of SARS-CoV-2 by redistributing membrane cholesterol to prevent membrane fusion, which can be reinstated by treatment with a PD-encapsulating agent. Furthermore, the inhibitory effects of PD are recapitulated by the pharmacological inhibition or gene silencing of NPC1, which is mutated in patients with Niemann–Pick type C (NPC) displaying disrupted membrane cholesterol distribution. Finally, readily available local foods or herbal medicines containing PG root show similar inhibitory effects against SARS-CoV-2 infection. Our study proposes that PD is a potent natural product for preventing or treating COVID-19 and that briefly disrupting the distribution of membrane cholesterol is a potential novel therapeutic strategy for SARS-CoV-2 infection.

2) In Quantitative rtPCR section (Page 110, in material and methods) we would like to correct the following sentence from; "In brief, each reaction buffer consisted of a total volume of 20 μl containing 8 μl of 100 μM forward and reverse primers (4 μl for each primer), 2 μl of cDNA, and 10 μl power SYBR Green PCR Master Mix. " to; "In brief, each reaction buffer consisted of a total volume of 20 μl containing 2 μl of 10 μM forward and reverse primers (1 μl for each primer), 2 μl of cDNA, and 10 μl power SYBR Green PCR Master Mix. "

Astrocytes are non-excitable cells in the brain and their activity largely depends on the intracellular calcium (Ca2+) level. Therefore, maintaining the intracellular Ca2+ homeostasis is critical for proper functioning of astrocytes. One of the key regulatory mechanisms of Ca2+ homeostasis in astrocytes is the store-operated Ca2+ entry (SOCE). This process is mediated by a combination of the Ca2+-store-depletion-sensor, Stim, and the store-operated Ca2+-channels, Orai and TrpC families. Despite the existence of all those families in astrocytes, previous studies have provided conflicting results on the molecular identification of astrocytic SOCE. Here, using the shRNA-based gene-silencing approach and Ca2+-imaging from cultured mouse astrocytes, we report that Stim1 in combination with Orai1 and Orai3 contribute to the major portion of astrocytic SOCE. Gene-silencing of Stim1 showed a 79.2% reduction of SOCE, indicating that Stim1 is the major Ca2+-store-depletion-sensor. Further gene-silencing showed that Orai1, Orai2, Orai3, and TrpC1 contribute to SOCE by 35.7%, 20.3%, 26.8% and 12.2%, respectively. Simultaneous gene-silencing of all three Orai subtypes exhibited a 67.6% reduction of SOCE. Based on the detailed population analysis, we predict that Orai1 and Orai3 are expressed in astrocytes with a large SOCE, whereas TrpC1 is exclusively expressed in astrocytes with a small SOCE. This analytical approach allows us to identify the store operated channel (SOC) subtype in each cell by the degree of SOCE. Our results propose that Stim1 in combination with Orai1 and Orai3 are the major molecular components of astrocytic SOCE under various physiological and pathological conditions.

Recently, μ-opioid receptor (MOR), one of the well-known Gi-protein coupled receptors (Gi-GPCR), was reported to be highly expressed in the hippocampal astrocytes. However, the role of astrocytic MOR has not been investigated. Here we report that activation of astrocytic MOR by [D-Ala2,N-MePhe4,Gly-ol]-enkephalin (DAMGO), a selective MOR agonist, causes a fast glutamate release using sniffer patch technique. We also found that the DAMGO-induced glutamate release was not observed in the astrocytes from MOR-deficient mice and MOR-short hairpin RNA (shRNA)-expressed astrocytes. In addition, the glutamate release was significantly reduced by gene silencing of the TREK-1-containing two-pore potassium (K2P) channel, which mediates passive conductance in astrocytes. Our findings were consistent with the previous study demonstrating that activation of Gi-GPCR such as cannabinoid receptor CB1 and adenosine receptor A1 causes a glutamate release through TREK-1-containing K2P channel from hippocampal astrocytes. We also demonstrated that MOR and TREK-1 are significantly co-localized in the hippocampal astrocytes. Furthermore, we found that both MOR and TREK-1-containing K2P channels are localized in the same subcellular compartments, soma and processes, of astrocytes. Our study raises a novel possibility that astrocytic MOR may participate in several physiological and pathological actions of opioids, including analgesia and addiction, through astrocytically released glutamate and its signaling pathway.