Preparation and Evaluation of Paclitaxel-Loaded PEGylated Niosomes Composed of Sorbitan Esters.

Niosomes are non-ionic surfactant (NIS)-based bilayer vesicles and, like liposomes, have great potential as drug-delivery systems. Our previous study revealed that polyethylene glycol (PEG) niosomes using different sorbitan ester (Span) surfactants (sorbitan monoester, Span 20, 40, 60, 80; sorbitan triester, Span 65) distributed within tumors similarly to PEG liposomes. The aim of this study was to encapsulate efficiently an anti-cancer drug, paclitaxel (PTX) into Span PEG niosomes, and evaluate PTX release profiles and anti-tumor efficacy of PTX-loaded Span PEG niosomes. Niosome sizes ranged between 100-150 nm, and the PTX encapsulation efficiency was more than 50%. All niosomes examined, in the presence of serum, yielded sustained PTX-release profiles. PTX release at 24 and 48 h from Span 80 PEG niosomes was significantly the highest among the other Span PEG niosomes examined. In C26 tumor-bearing mice, PTX-loaded Span 40 PEG niosomes (the lowest PTX release in vitro) suppressed tumor growth while PTX-loaded Span 80 PEG niosomes (the highest PTX release in vitro) did not. Thus, we succeeded in the control of PTX release from Span PEG niosomes by modifying the component of niosomes, and it influenced the effects of drugs loaded into niosomes. This demonstrates that the excellent NIS physicochemical properties of Spans make them an ideal candidate for anti-cancer drug-carrier niosomes.

2-thiouridine is a broad-spectrum antiviral nucleoside analogue against positive-strand RNA viruses.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections are causing significant morbidity and mortality worldwide. Furthermore, over 1 million cases of newly emerging or re-emerging viral infections, specifically dengue virus (DENV), are known to occur annually. Because no virus-specific and fully effective treatments against these or many other viruses have been approved, there is an urgent need for novel, effective therapeutic agents. Here, we identified 2-thiouridine (s2U) as a broad-spectrum antiviral ribonucleoside analogue that exhibited antiviral activity against several positive-sense single-stranded RNA (ssRNA+) viruses, such as DENV, SARS-CoV-2, and its variants of concern, including the currently circulating Omicron subvariants. s2U inhibits RNA synthesis catalyzed by viral RNA-dependent RNA polymerase, thereby reducing viral RNA replication, which improved the survival rate of mice infected with DENV2 or SARS-CoV-2 in our animal models. Our findings demonstrate that s2U is a potential broad-spectrum antiviral agent not only against DENV and SARS-CoV-2 but other ssRNA+ viruses.

[Analysis of Ciguatoxins in the Spotted Knifejaw, Oplegnathus punctatus from the Waters of Japan].

Ciguatera fish poisoning (CFP) is recognized as the most frequent seafood poisoning due to the consumption of fish containing the principal toxins, ciguatoxins (CTXs). In Japan, CFP events have been reported annually from Okinawa and Amami Islands, locating subtropical regions. In addition, there have been reported several outbreaks due to consumption of the fish caught from the Pacific coast of the Mainland and they were often caused by the matured spotted knifejaw, Oplegnathus punctatus. As part of our research on CFP in Japan, we investigated CTXs analysis by LC-MS/MS on 176 individuals of O. punctatus (weight: 100-6,350 g, standard length: 13-60 cm) from the coast of the Mainland (Honshu, Shikoku, and Kyushu), Amami, Okinawa, and Ogasawara (Bonin) Islands. CTXs were detected from only two specimens collected from Okinawa. Total CTXs levels of the two specimens were at 0.014 and 0.040 µg/kg, respectively, exceeding FDA guidance level at 0.01 µg CTX1B equivalent/kg. However, they might be little risk of CFP because consuming over 1.5 kg of flesh is needed to develop intoxication. The toxins consisted of CTX1B analogs including CTX1B, 52-epi-54-deoxyCTX1B, CTX4A, and CTX4B, and no CTX3C analogs, supporting the finding that ciguatoxic fishes in Okinawan Waters containing only CTX1B analogs.

Clinical feasibility of remote intermittently scanned continuous glucose monitoring in coronavirus disease 2019 patients with and without diabetes during dexamethasone therapy.

The clinical utility of intermittently scanned continuous glucose monitoring (isCGM) in patients with coronavirus disease 2019 (COVID-19) is unclear. Hence, we investigated the accuracy of isCGM in COVID-19 patients during dexamethasone therapy. We evaluated the accuracy of the FreeStyle Libre via smartphone isCGM device compared to point-of-care (POC) fingerstick glucose level monitoring in 16 patients with COVID-19 (10 with and 6 without diabetes, 13 men; HbA1c 6.9 ± 1.0%). Overall, isCGM correlated well with POC measurements (46.2% and 53.8% within areas A and B of the Parkes error grid, respectively). The overall mean absolute relative difference (MARD) for isCGM compared to POC measurements was 19.4%. The MARDs were 19.8% and 19.7% for POC blood glucose measurements ranging from 70 to 180 mg/dL and >180 mg/dL, respectively. When divided according to the presence and absence of diabetes, both groups of paired glucose measurements showed a good correlation (56.3% and 43.7%, and 27.1% and 72.9% within the A and B areas in patients with and without diabetes, respectively), but the MARD was not significant but higher in patients without diabetes (16.5% and 24.2% in patients with and without diabetes). In conclusion, although isCGM may not be as accurate as traditional blood glucose monitoring, it has good reliability in COVID-19 patients with and without diabetes during dexamethasone therapy.

Sensor histidine kinases mediate ABA and osmostress signaling in the moss Physcomitrium patens.

To survive fluctuating water availability on land, terrestrial plants must be able to sense water stresses, such as drought and flooding. The plant hormone abscisic acid (ABA) and plant-specific SNF1-related protein kinase 2 (SnRK2) play key roles in plant osmostress responses. We recently reported that, in the moss Physcomitrium patens, ABA and osmostress-dependent SnRK2 activation requires phosphorylation by an upstream RAF-like kinase (ARK). This RAF/SnRK2 module is an evolutionarily conserved mechanism of osmostress signaling in land plants. Surprisingly, ARK is also an ortholog of Arabidopsis CONSTITUTIVE RESPONSE 1 (CTR1), which negatively regulates the ethylene-mediated submergence response of P. patens, indicating a nexus for cross-talk between the two signaling pathways that regulate responses to water availability. However, the mechanism through which the ARK/SnRK2 module is activated in response to water stress remains to be elucidated. Here, we show that a group of ethylene-receptor-related sensor histidine kinases (ETR-HKs) is essential for ABA and osmostress responses in P. patens. The intracellular kinase domain of an ETR-HK from P. patens physically interacts with ARK at the endoplasmic reticulum in planta. Moreover, HK disruptants lack ABA-dependent autophosphorylation of the critical serine residue in the activation loop of ARK, leading to loss of SnRK2 activation in response to ABA and osmostress. Collectively with the notion that ETR-HKs participate in submergence responses, our present data suggest that the HK/ARK module functions as an integration unit for environmental water availability to elicit optimized water stress responses in the moss P. patens.

Hypervirulent clade 2, ribotype 019/sequence type 67 Clostridioides difficile strain from Japan.

BACKGROUND: Clostridioides difficile ribotype (RT) 019/sequence type (ST) 67 strains belong to a hypervirulent lineage closely related to RT027/ST1; however, limited data are available for hypervirulent clade 2 lineages in Japan. Herein, we report the draft genome of a C. difficile strain B18-123 belonging to clade 2, RT019/ST67 for the first time in Japan. RESULTS: The pathogenicity locus carried by B18-123 (19.6 kb) showed higher homology (97.29% nucleotide identity) with strain R20291 (RT027/ST1) than the reference strain 630 (RT012/ST54), and B18-123 harbored 8-nucleotide substitutions in tcdC. However, it did not contain an 18-base pair (bp) deletion or a single-bp deletion at position 117 in tcdC, which was identified in the previous strain R20291. A cytotoxicity assay revealed similar cytotoxicity levels between strains B18-123 and ATCC BAA-1870 (RT027/ST1). The B18-123 strain was found to be susceptible to metronidazole and vancomycin. CONCLUSION: Our findings contribute to the further understanding of the characteristics of hypervirulent clade 2 including RT019/ST67 lineages.

Metabolic effects of Tofogliflozin are efficiently enhanced with appropriate dietary carbohydrate ratio and are distinct from carbohydrate restriction.

Sodium-glucose cotransporter 2 inhibitors (SGLT2i) exert their antidiabetic effects by promoting urinary glucose excretion. Nutrition therapy is obviously important, but little is known about the interactions between SGLT2i agents and carbohydrate restriction. Therefore, we studied these interactions using an obese diabetic animal model. KK-Ay mice were pair-fed normal chow [NC; carbohydrate: fat: protein = 65:15:20], low carbohydrate [LC; 43:42:15] or severely carbohydrate restricted diets [SR; 12:45:43] for 12 weeks. Tofogliflozin (Tofo) was administered as the SGLT2i in the NC and LC diet groups. Blood glucose levels were significantly increased in the SR group. Tofo reduced blood glucose levels significantly in the NC group during the experiment and in the LC group at 2-6 weeks. Plasma triglycerides were markedly elevated in the SR group without Tofo, but decreased in response to Tofo administration. Hepatic triglyceride contents were not changed by the LC or the SR diet alone. However, Tofo ameliorated hepatosteatosis in NC-fed animals. Consistent with the downregulation of stearoyl-CoA desaturase 1, the ratio of plasma monounsaturated to saturated fatty acids was significantly reduced in the LC with Tofo and in the SR alone groups, but was not altered in the NC with Tofo group. In summary, metabolism of glucose and lipids was improved by Tofo but not by the SR diet. Furthermore, Tofo improved these parameters more effectively in the NC than in the LC diet group. These data suggest that the effects of SGLT2i are distinct from those of carbohydrate restriction and that a nonrestricted dietary carbohydrate composition is essential for SGLT2i treatment to be effective.

Mechanism of lipid-lowering action of the dipeptidyl peptidase-4 inhibitor, anagliptin, in low-density lipoprotein receptor-deficient mice.

AIMS/INTRODUCTION: Dipeptidyl peptidase-4 inhibitors are used for treatment of patients with type 2 diabetes. In addition to glycemic control, these agents showed beneficial effects on lipid metabolism in clinical trials. However, the mechanism underlying the lipid-lowering effect of dipeptidyl peptidase-4 inhibitors remains unclear. Here, we investigated the lipid-lowering efficacy of anagliptin in a hyperlipidemic animal model, and examined the mechanism of action. MATERIALS AND METHODS: Male low-density lipoprotein receptor-deficient mice were administered 0.3% anagliptin in their diet. Plasma lipid levels were assayed and lipoprotein profile was analyzed using high-performance liquid chromatography. Hepatic gene expression was examined by deoxyribonucleic acid microarray and quantitative polymerase chain reaction analyses. Sterol regulatory element-binding protein transactivation assay was carried out in vitro. RESULTS: Anagliptin treatment significantly decreased the plasma total cholesterol (14% reduction, P < 0.01) and triglyceride levels (27% reduction, P < 0.01). Both low-density lipoprotein cholesterol and very low-density lipoprotein cholesterol were also decreased significantly by anagliptin treatment. Sterol regulatory element-binding protein-2 messenger ribonucleic acid expression level was significantly decreased at night in anagliptin-treated mice (15% reduction, P < 0.05). Anagliptin significantly suppressed sterol regulatory element-binding protein activity in HepG2 cells (21% decrease, P < 0.001). CONCLUSIONS: The results presented here showed that the dipeptidyl peptidase-4 inhibitor, anagliptin, exhibited a lipid-lowering effect in a hyperlipidemic animal model, and suggested that the downregulation of hepatic lipid synthesis was involved in the effect. Anagliptin might have beneficial effects on lipid metabolism in addition to a glucose-lowering effect.

Latent tri-lineage potential of adult hippocampal neural stem cells revealed by Nf1 inactivation.

Endogenous neural stem cells (NSCs) in the adult hippocampus are considered to be bi-potent, as they only produce neurons and astrocytes in vivo. In mouse, we found that inactivation of neurofibromin 1 (Nf1), a gene mutated in neurofibromatosis type 1, unlocked a latent oligodendrocyte lineage potential to produce all three lineages from NSCs in vivo. Our results suggest an avenue for promoting stem cell plasticity by targeting barriers of latent lineage potential.

Tangential migration of neuronal precursors of glutamatergic neurons in the adult mammalian brain.

In a classic model of mammalian brain formation, precursors of principal glutamatergic neurons migrate radially along radial glia fibers whereas GABAergic interneuron precursors migrate tangentially. These migration modes have significant implications for brain function. Here we used clonal lineage tracing of active radial glia-like neural stem cells in the adult mouse dentate gyrus and made the surprising discovery that proliferating neuronal precursors of glutamatergic granule neurons exhibit significant tangential migration along blood vessels, followed by limited radial migration. Genetic birthdating and morphological and molecular analyses pinpointed the neuroblast stage as the main developmental window when tangential migration occurs. We also developed a partial "whole-mount" dentate gyrus preparation and observed a dense plexus of capillaries, with which only neuroblasts, among the entire population of progenitors, are directly associated. Together, these results provide insight into neuronal migration in the adult mammalian nervous system.

Circulating level of microRNA-126 may be a potential biomarker for recovery from smoking-related vascular damage in middle-aged habitual smokers.

BACKGROUND: Cigarette smoking promotes vascular endothelial damage and accelerates progression of atherosclerosis. The purpose of this study was to examine whether the circulating level of vascular endothelium-enriched microRNA-126 (miR-126), which is highlighted as a regulator of gene expression, would serve as a novel biomarker for recovery from smoking-related vascular damage. METHODS: Middle-aged male smokers (n = 30) were enrolled and instructed to stop smoking. Their clinical profiles and laboratory findings including expression of miR-126 were investigated before and after 8 weeks of smoking cessation. Serum levels of cotinine, metabolites of nicotine, were measured to confirm smoking cessation. Endothelial function for peripheral small vessels was assessed and expressed as reactive hyperemia peripheral arterial tonometry (RH-PAT) index. The expression of miR-126 in plasma was analyzed by quantitative real-time PCR. RESULTS: At baseline, serum cotinine levels were inversely correlated with RH-PAT index (r = - 0.48, P < 0.01) and positively correlated with levels of metabolic parameters such as non-HDL cholesterol (r = 0.53, P < 0.01) and HOMA-IR (r = 0.52, P < 0.01). The RH-PAT index was not significantly changed after 8 weeks in all subjects, because only 13 subjects could attain smoking cessation. However, changes in the RH-PAT index showed a significant correlation with those in systolic blood pressure (r = - 0.54, P < 0.01). In smokers who completely attained smoking cessation (n = 13), RH-PAT index and plasma levels of miR-126 were significantly increased (P < 0.05, respectively). CONCLUSIONS: Endothelial damage was improved and plasma levels of circulating miR-126 were increased after 8 weeks of smoking cessation. These findings suggested a potential use of miR-126 as a biomarker for recovery from smoking-induced vascular damage.

TNF-α production in NKT cell hybridoma is regulated by sphingosine-1-phosphate: implications for inflammation in atherosclerosis.

OBJECTIVES: Natural killer T (NKT) cells are unique T lymphocytes that recognize glycolipid antigen and produce various cytokines. NKT cells accelerate atherosclerosis in mice. Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid and regulates T-lymphocyte trafficking. We aimed to determine the effects of S1P on the production of proinflammatory cytokine, tumor necrosis factor (TNF)-α, in NKT cell hybridomas and mouse NKT cells. MATERIALS AND METHODS: NKT cell hybridomas and sorted mouse NKT cells were stimulated with S1P and α-galactosylceramide (α-GalCer), the major ligand to produce cytokines in NKT cells. TNF-α mRNA expression and protein production were determined by real-time PCR and ELISA, respectively. Cell migration was assayed using chemotaxicell. Plasma S1P was measured using HPLC. RESULTS: Hybridomas expressed S1P receptors, S1P1, S1P2, and S1P4. S1P and α-GalCer increased TNF-α mRNA expression and protein production. S1P enhanced TNF-α induction by α-GalCer. S1P receptor antagonists decreased the TNF-α mRNA expression induced by S1P. FTY720, an immunosuppressive S1P receptor modulator, also decreased the TNF-α mRNA expression. The migration of NKT cell hybridomas was increased by S1P. FTY720 reduced the migration induced by S1P. S1P also increased the TNF-α mRNA expression in mouse NKT cells. Plasma TNF-α levels in patients with high plasma S1P (≥500 nmol/l) were higher than those in patients with low S1P (<500 nmol/l). CONCLUSION: S1P binds to S1P receptors in NKT cells and enhances TNF-α production. TNF-α overproduction may induce atherogenic inflammatory responses. S1P may serve as a novel therapeutic target for amelioration of vascular inflammatory diseases.

Increased plasma sphingosine-1-phosphate in obese individuals and its capacity to increase the expression of plasminogen activator inhibitor-1 in adipocytes.

OBJECTIVES: Concentrations of plasminogen activator inhibitor-1 (PAI-1) are increased in obese individuals. One source of PAI-1 is adipocytes. Hypoxia develops within adipose tissue as it expands, presumably contributing to increased levels of sphingosine-1-phosphate (S1P). S1P is a breakdown product of sphingosine, ubiquitous in cell membranes. We have shown previously that S1P increases the expression of PAI-1 in human liver-derived cell line. In the present study, we aimed to determine whether hypoxia induces S1P in adipocytes, thereby potentially contributing to an increase in PAI-1 and hence constraints on fibrinolysis associated with obesity. MATERIALS AND METHODS: Mouse 3T3-L1 adipocytes were exposed to CoCl2 to simulate hypoxia. Assays were performed for PAI-1 mRNA (quantitative PCR) and S1P (high-performance liquid chromatography). RESULTS: The physiologic concentration of S1P increased PAI-1 mRNA expression. The S1P2 receptor antagonist attenuated the increase in PAI-1. Adipocytes expressed sphingosine kinase 1/2 (SPHK1/2) and S1P lyase, key enzymes involved in S1P production and degradation. Hypoxia increased SPHK activity and decreased S1P lyase mRNA. Hypoxia reduced cytosolic sphingosine and increased S1P release into conditioned medium. Inhibitors of ABCA1 and ABCC1 reduced the release of S1P into conditioned media. In obese patients with uncomplicated dyslipidemia and hypertension, plasma S1P was increased compared with that in nonobese and lean individuals. CONCLUSION: Hypoxia in adipose tissue of obesity can promote elaboration of S1P that binds to S1P2 receptors in an autocrine or a paracrine manner. S1P potentially contributes toward increased expression of PAI-1 and consequent constraints on fibrinolysis. S1P production and extracellular transport provide an attractive target for therapy to attenuate impaired fibrinolysis associated with obesity.

Circulating microRNA-126 in patients with coronary artery disease: correlation with LDL cholesterol.

BACKGROUND: Coronary artery disease (CAD) is a major problem worldwide. Atherosclerosis and thrombosis underlying CAD involve multiple cell types. New and useful diagnostic markers are required. MicroRNAs (miRNAs) are a class of noncoding RNAs that posttranscriptionally regulate the gene expressions involved in various cellular processes. Endothelial dysfunction is implicated in early processes of athero-thrombosis. Thus, it was hypothesized that the level of vascular endothelium-enriched miRNAs would be altered in plasma samples of CAD patients. METHODS: Vascular endothelium-enriched miRNA (miR-126) level was analyzed in plasma from 31 patients with CAD and 36 patients without CAD (qRT-PCR analysis). RESULTS: MiR-126 was not significantly down-regulated or up-regulated in CAD patients. Interestingly, the level of miR-126 was significantly decreased in patients with CAD and high low-density lipoprotein (LDL) cholesterol level. In contrast, the level of miR-126 was significantly increased when LDL cholesterol was high in patients who had risk factors for CAD but did not have angiographically significant CAD. CONCLUSION: MiR-126 was not significantly down-regulated or up-regulated in CAD patients and was not suitable for discriminating CAD patients from patients without CAD. The oppositely-directed relationship between miR-126 and LDL cholesterol in patients with or without CAD may have significant implications for identifying a potential role of miR-126 in cholesterol metabolism.

Analytical evaluation of plasma serotonin and sphingosine 1-phosphate and their clinical assessment in early atherosclerosis.

OBJECTIVES: Serotonin stored in platelets is released into plasma on aggregation and activation in atherosclerotic diseases. Sphingosine 1-phosphate (S1P) in plasma is mainly derived from red blood cells and is responsible for the production of nitric oxide in endothelial cells and protects vasculature. The purpose of this study was to investigate the plasma levels of serotonin, S1P, and their clinical relationships with vascular endothelial function in patients with early atherosclerosis. METHODS: Blood was withdrawn from patients with low-to-moderate risks of atherosclerotic diseases (n=49, 39 ± 7 years). Platelet-poor plasma was immediately centrifuged. Serotonin levels in plasma were measured with high-performance liquid chromatography. S1P levels in plasma were measured by high-performance liquid chromatography after fluorescent derivatization with o-phthaldialdehyde. Endothelial function was assessed by endothelium-dependent flow-mediated dilation (FMD) and endothelium-independent dilation was measured by glycerol trinitrate-induced dilation using an ultrasound system. RESULTS: Plasma serotonin was inversely correlated with the FMD value (r=-0.287, P<0.05). Fourteen patients with dyslipidemia, who had not shown improvements after lifestyle modifications, were subsequently treated with rosuvastatin (2.5 mg/day). After 4 weeks of treatment, rosuvastatin improved lipid profiles. Rosuvastatin increased FMD, whereas glycerol trinitrate-induced dilation was unchanged. Notably, percentage decrease in plasma serotonin was inversely correlated with percentage increase in plasma S1P (r=-0.557, P<0.05). CONCLUSION: Plasma serotonin was inversely correlated with FMD and a decrease in plasma serotonin was inversely correlated with an increase in plasma S1P after statin treatment. The results suggested that plasma levels of serotonin and S1P may be useful for the assessment of endothelial function of patients with low-to-moderate risks of atherosclerotic diseases.