COPMAN: A novel high-throughput and highly sensitive method to detect viral nucleic acids including SARS-CoV-2 RNA in wastewater.

During the coronavirus disease 2019 (COVID-19) pandemic, wastewater-based epidemiology (WBE) attracted attention as an objective and comprehensive indicator of community infection that does not require individual inspection. Although several severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection methods from wastewater have been developed, there are obstacles to their social implementation. In this study, we developed the COPMAN (Coagulation and Proteolysis method using Magnetic beads for detection of Nucleic acids in wastewater), an automatable method that can concentrate and detect multiple types of viruses from a limited volume (∼10 mL) of wastewater. The COPMAN consists of a high basicity polyaluminum chloride (PAC) coagulation process, magnetic bead-based RNA purification, and RT-preamplification, followed by qPCR. A series of enzymes exhibiting a high tolerance to PCR inhibitors derived from wastewater was identified and employed in the molecular detection steps in the COPMAN. We compared the detectability of viral RNA from 10-mL samples of virus-spiked (heat-inactivated SARS-CoV-2 and intact RSV) or unspiked wastewater by the COPMAN and other methods (PEG-qPCR, UF-qPCR, and EPISENS-S). The COPMAN was the most efficient for detecting spiked viruses from wastewater, detecting the highest level of pepper mild mottle virus (PMMoV), a typical intrinsic virus in human stool, from wastewater samples. The COPMAN also successfully detected indigenous SARS-CoV-2 RNA from 12 samples of wastewater at concentrations of 2.2 × 104 to 5.4 × 105 copies/L, during initial stages of an infection wave in the right and the left bank of the Sagami River in Japan (0.65 to 11.45 daily reported cases per 100,000 people). These results indicate that the COPMAN is suitable for detection of multiple pathogens from small volume of wastewater in automated stations.

Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.

Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei.

Highly accurate synthesis of the fully 2'-fluoro-modified oligonucleotide by Therminator DNA polymerases.

Oligonucleotides composed of natural nucleotides are inapplicable for biotechnical and therapeutic use due to its instability under biological conditions. Therminator DNA polymerases, mutant DNA polymerases of thermophilic marine archaea, show that they can efficiently synthesize fully 2'-fluoro-modified (2'F-) oligonucleotides. Furthermore, the sequence analysis reveals that the oligonucleotide sequence is highly accurate, especially the fidelity of a 2'F-oligonucleotide synthesized by Therminator II is more accurate than that of natural RNA synthesized by conventional RNA polymerase. These finding would be helpful for the synthesis of chemically modified oligonucleotides, for the use of biotechnical or medical applications.

Prostanoid DP receptor antagonists suppress symptomatic asthma-like manifestation by distinct actions from a glucocorticoid in rats.

While inhaled glucocorticoids are the best treatment for the majority of chronic asthmatics, there is a small group who do not respond to these drugs or whose disease can only be controlled by high doses of oral glucocorticoids with risks of severe side effects. Therefore, a safe novel anti-asthmatic agent which has a different mechanism from that of glucocorticoids is needed for the management of asthma. We have previously shown that an orally active prostanoid DP receptor antagonist, S-5751, had potent anti-inflammatory effects in guinea pig and sheep asthma models. In this study, using a rat asthma like model, we found that lung neutrophilia and proinflammatory cytokine secretion as well as bronchial hyperresponsiveness and lung eosinophilia were induced by repeated antigen-inhalations after antigen-sensitization. These symptoms are similar to the pathogenesis of symptomatic asthma. Orally-administered prostanoid DP receptor antagonists S-5751 and pinagladin significantly suppressed not only bronchial hyperresponsiveness and lung eosinophilia but also neutrophilia and mucus secretion in the lung, while oral prednisolone inhibited only bronchial hyperresponsiveness and eosinophil infiltration. In addition, prostanoid DP receptor antagonists significantly suppressed interleukin (IL)-1ß, IL-6 and CXCL1 mRNA in contrast to suppression of IL-4 and CCL11 mRNA by prednisolone. The majority of prostanoid DP receptor-expressing cells in both rat and human asthmatic lungs are infiltrative macrophages and/or monocytes. These results suggest that prostanoid DP receptor antagonists utilize different mechanisms from glucocorticoids, and that they would be a novel alternative and/or combination drug for asthma therapy.

Irbesartan enhances GLUT4 translocation and glucose transport in skeletal muscle cells.

Irbesartan, an angiotensin II type 1 receptor blocker has been reported to alleviate metabolic disorder in animal studies and human clinical trials. Although this effect may be related to the ability of irbesartan to serve as a partial agonist for the peroxisome proliferator-activated receptor (PPAR)-γ, the target tissues on which irbesartan acts remain poorly defined. As muscle glucose transport plays a major role in maintaining systemic glucose homeostasis, we investigated the effect of irbesartan on glucose uptake in skeletal muscle cells. In C2C12 myotubes, 24-h treatment with irbesartan significantly promoted both basal and insulin-stimulated glucose transport. In L6-GLUT4myc myoblasts, irbesartan caused a significant increase in glucose transport and GLUT4 translocation to the cell surface in a concentration-dependent manner. Valsartan, another angiotensin II type 1 receptor blocker had no effect on either glucose uptake or GLUT4 translocation, implying that these actions on glucose transport are independent of angiotensin II receptor blockade. Moreover, irbesartan exerted these effects in an additive manner with insulin, but not with acute treatment for 3 h, suggesting that they may require the synthesis of new proteins. Finally, in insulin-resistant Zucker fatty rat, irbesartan (50 mg/kg/day for 3 weeks) significantly ameliorated insulin resistance without increasing weight gain. We conclude that irbesartan has a direct action, which can be additive to insulin, of promoting glucose transport in skeletal muscle. This may be beneficial for ameliorating obesity-related glucose homeostasis derangement.

A single nucleotide polymorphism within the acetyl-coenzyme A carboxylase beta gene is associated with proteinuria in patients with type 2 diabetes.

It has been suggested that genetic susceptibility plays an important role in the pathogenesis of diabetic nephropathy. A large-scale genotyping analysis of gene-based single nucleotide polymorphisms (SNPs) in Japanese patients with type 2 diabetes identified the gene encoding acetyl-coenzyme A carboxylase beta (ACACB) as a candidate for a susceptibility to diabetic nephropathy; the landmark SNP was found in the intron 18 of ACACB (rs2268388: intron 18 +4139 C > T, p = 1.4x10(-6), odds ratio = 1.61, 95% confidence interval [CI]: 1.33-1.96). The association of this SNP with diabetic nephropathy was examined in 9 independent studies (4 from Japan including the original study, one Singaporean, one Korean, and two European) with type 2 diabetes. One case-control study involving European patients with type 1 diabetes was included. The frequency of the T allele for SNP rs2268388 was consistently higher among patients with type 2 diabetes and proteinuria. A meta-analysis revealed that rs2268388 was significantly associated with proteinuria in Japanese patients with type 2 diabetes (p = 5.35 x 10(-8), odds ratio = 1.61, 95% Cl: 1.35-1.91). Rs2268388 was also associated with type 2 diabetes-associated end-stage renal disease (ESRD) in European Americans (p = 6 x 10(-4), odds ratio = 1.61, 95% Cl: 1.22-2.13). Significant association was not detected between this SNP and nephropathy in those with type 1 diabetes. A subsequent in vitro functional analysis revealed that a 29-bp DNA fragment, including rs2268388, had significant enhancer activity in cultured human renal proximal tubular epithelial cells. Fragments corresponding to the disease susceptibility allele (T) had higher enhancer activity than those of the major allele. These results suggest that ACACB is a strong candidate for conferring susceptibility for proteinuria in patients with type 2 diabetes.

The interaction of monocytes with rheumatoid synovial cells is a key step in LIGHT-mediated inflammatory bone destruction.

Formation of osteoclasts and consequent joint destruction are hallmarks of rheumatoid arthritis (RA). Here we show that LIGHT, a member of the tumour necrosis factor (TNF) superfamily, induced the differentiation into tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) of CD14(+) monocytes cocultured with nurse-like cells isolated from RA synovium, but not of freshly isolated CD14(+) monocytes. Receptor activator of nuclear factor-kappaB ligand (RANKL) enhanced this LIGHT-induced generation of TRAP-positive MNCs. The MNCs showed the phenotypical and functional characteristics of osteoclasts; they showed the expression of osteoclast markers such as cathepsin K, actin-ring formation, and the ability to resorb bone. Moreover, the MNCs expressed both matrix metalloproteinase 9 (MMP-9) and MMP-12, but the latter was not expressed in osteoclasts induced from CD14(+) monocytes by RANKL. Immunohistochemical analysis showed that the MMP-12-producing MNCs were present in the erosive areas of joints in RA, but not in the affected joints of osteoarthritic patients. These findings suggested that LIGHT might be involved in the progression of inflammatory bone destruction in RA, and that osteoclast progenitors might become competent for LIGHT-mediated osteoclastogenesis via interactions with synoviocyte-like nurse-like cells.

Transcriptomic analysis of nephrotoxicity induced by cephaloridine, a representative cephalosporin antibiotic.

Cephaloridine (CER) is a classical beta-lactam antibiotic that has long served as a model drug for the study of cephalosporin antibiotic-induced acute tubular necrosis. In the present study, we analyzed gene expression profiles in the kidney of rats given subtoxic and toxic doses of CER to identify gene expression alterations closely associated with CER-induced nephrotoxicity. Male Fischer 344 rats were intravenously injected with CER at three different dose levels (150, 300, and 600 mg/kg) and sacrificed after 24 h. Only the high dose (600 mg/kg) caused mild proximal tubular necrosis and slight renal dysfunction. Microarray analysis identified hundreds of genes differentially expressed in the renal cortex following CER exposure, which could be classified into two main groups that were deregulated in dose-dependent and high dose-specific manners. The genes upregulated dose dependently mainly included those involved in detoxification and antioxidant defense, which was considered to be associated with CER-induced oxidative stress. In contrast, the genes showing high dose-specific (lesion-specific) induction included a number of genes related to cell proliferation, which appeared to reflect a compensatory response to CER injury. Of the genes modulated in both manners, we found many genes reported to be associated with renal toxicity by other nephrotoxicants. We could also predict potential transcription regulators responsible for the observed gene expression alterations, such as Nrf2 and the E2F family. Among the candidate gene biomarkers, kidney injury molecule 1 was markedly upregulated at the mildly toxic dose, suggesting that this gene can be used as an early and sensitive indicator for cephalosporin nephrotoxicity. In conclusion, our transcriptomic data revealed several characteristic expression patterns of genes associated with specific cellular processes, including oxidative stress response and proliferative response, upon exposure to CER, which may enhance our understanding of the molecular mechanisms behind cephalosporin antibiotic-induced nephrotoxicity.

Genetic variations in the gene encoding ELMO1 are associated with susceptibility to diabetic nephropathy.

To search for a gene(s) conferring susceptibility to diabetic nephropathy (DN), we genotyped over 80,000 gene-based single nucleotide polymorphisms (SNPs) in Japanese patients and identified that the engulfment and cell motility 1 gene (ELMO1) was a likely candidate for conferring susceptibility to DN, in view of the significant association of an SNP in this gene with the disease (intron 18+9170, GG vs. GA+AA, chi(2) = 19.9, P = 0.000008; odds ratio 2.67, 95% CI 1.71-4.16). In situ hybridization (ISH) using the kidney of normal and diabetic mice revealed that ELMO1 expression was weakly detectable mainly in tubular and glomerular epithelial cells in normal mouse kidney and was clearly elevated in the kidney of diabetic mice. Subsequent in vitro analysis revealed that ELMO1 expression was elevated in cells cultured under high glucose conditions (25 mmol/l) compared with cells cultured under normal glucose conditions (5.5 mmol/l). Furthermore, we identified that the expression of extracellular matrix protein genes, such as type 1 collagen and fibronectin, were increased in cells that overexpress ELMO1, whereas the expression of matrix metalloproteinases was decreased. These results indicate that ELMO1 is a novel candidate gene that both confers susceptibility to DN and plays an important role in the development and progression of this disease.

Wnt5b partially inhibits canonical Wnt/beta-catenin signaling pathway and promotes adipogenesis in 3T3-L1 preadipocytes.

To elucidate the functional roles of Wnt5b in adipogenesis, we characterized gene expression profiles in Wnt5b overexpressing 3T3-L1 cells using microarray analysis. Of the approximately 20,000 genes screened, we found that 85 genes were up-regulated and 211 genes were down-regulated in 3T3-L1 cells overexpressing Wnt5b. Among the genes regulated by Wnt5b, the expressions of insulin like growth factor-1 (IGF-1), vascular endothelial growth factor-C (VEGF-C), and WNT1 inducible signaling pathway protein 1 (WISP-1), which were known to be up-regulated by Wnt1/beta-catenin signaling, were decreased in the Wnt5b overexpressing cells. This result was subsequently confirmed by real-time quantitative RT-PCR (IGF-1; 0.74+/-0.08 and 0.56+/-0.08, WISP-1; 0.71+/-0.03 and 0.56+/-0.08, and VEGF-C; 0.67+/-0.01 and 0.80+/-0.07, mean+/-SEM, compared with the control at zero and two days after induction of differentiation, respectively). We also found that Wnt5b overexpression in 3T3-L1 preadipocytes was able to partially prevent the inhibitory effect of Wnt3a on adipogenesis. Furthermore, the overexpression of Wnt5b was able to inhibit Wnt3a-induced activation of the canonical Wnt/beta-catenin pathway as evidenced by the reduced translocation of beta-catenin into the nucleus. These findings indicate that Wnt5b may promote adipogenesis in 3T3-L1 cells, at least in part, by antagonizing the canonical Wnt/beta-catenin pathway.