Quantitative Phenotyping-Based In Vivo Chemical Screening in a Zebrafish Model of Leukemia Stem Cell Xenotransplantation

Zebrafish-based chemical screening has recently emerged as a rapid and efficient method to identify important compounds that modulate specific biological processes and to test the therapeutic efficacy in disease models, including cancer. In leukemia, the ablation of leukemia stem cells (LSCs) is necessary to permanently eradicate the leukemia cell population. However, because of the very small number of LSCs in leukemia cell populations, their use in xenotransplantation studies (in vivo) and the difficulties in functionally and pathophysiologically replicating clinical conditions in cell culture experiments (in vitro), the progress of drug discovery for LSC inhibitors has been painfully slow. In this study, we developed a novel phenotype-based in vivo screening method using LSCs xenotransplanted into zebrafish. Aldehyde dehydrogenase-positive (ALDH+) cells were purified from chronic myelogenous leukemia K562 cells tagged with a fluorescent protein (Kusabira-orange) and then implanted in young zebrafish at 48 hours post-fertilization. Twenty-four hours after transplantation, the animals were treated with one of eight different therapeutic agents (imatinib, dasatinib, parthenolide, TDZD-8, arsenic trioxide, niclosamide, salinomycin, and thioridazine). Cancer cell proliferation, and cell migration were determined by high-content imaging. Of the eight compounds that were tested, all except imatinib and dasatinib selectively inhibited ALDH+ cell proliferation in zebrafish. In addition, these anti-LSC agents suppressed tumor cell migration in LSC-xenotransplants. Our approach offers a simple, rapid, and reliable in vivo screening system that facilitates the phenotype-driven discovery of drugs effective in suppressing LSCs.     

Studies on Phyto-peroxidase

Peroxidase c was isolated and purified from Japanese-radish roots by means of a chromatographic technique with carboxymethyl cellulose. Two or more components exhibiting the absorption spectrum of peroxidase c were separated chromatographically, and the most basic component was crystallized from ammonium sulphate solution. The Reinheit Zahl and the purpurogallin number of the crystalline preparation were found to be 3.55 and 1100 respectively. The absorption maxima were found at 420 and 540 mμ, for the oxidized form and at 425 and 560 mμ for the reduced form. The crystalline preparation contained 1.57% protohematin as the prosthetic group, and then the minimum molecular weight of peroxidase c was found to be 41500.     

Changes in Lipoxygenase Components of Rice Seedlings during Germination

Changes in lipoxygenase (LOX) activity were followed duringthe germination of rice seeds. The enzyme activity of 3-day-oldseedlings was 20 times higher than that of ungerminated seeds.Sixty per cent of the increased activity was found in shoots.The increase in LOX activity was mainly due to an increase inlipoxygenase-2 (LOX-2), a minor component in ungerminated seeds;this increase was inhibited by cycloheximide. LOX-2 was isolatedfrom the 3-day-old seedlings and compared for its enzymologicalproperties with rice lipoxygenase-3 (LOX-3), a major componentin ungerminated seeds. Both LOX-2 and LOX-3 were stable at pH5 to 8, but LOX-2 was more heatstable than LOX-3. Apparent K_mvalues of LOX-2 and LOX-3 for linoleic acid were 170 and 59µM, and those for linolenic acid were 5,300 and 88 µM,respectively. Both LOXs were inhibited by some metal ions andantioxidants. (Received February 5, 1986; Accepted May 9, 1986)     

High production of a class III lantipeptide AmfS in Streptomyces griseus

AmfS, a class III lantipeptide serves as a morphogen in Streptomyces griseus. Here, we constructed a high production system of AmfS in S. griseus. We isolated S. griseus Grd1 strain defective in glucose repression of aerial mycelium formation and found it suitable for the overproduction of AmfS. Two expression vectors carrying the strong and constitutive ermE2 promoter were constructed using a multicopy number plasmid, pIJ702. The use of the Grd1 strain combined with the expression vectors enabled high production of AmfS by S. griseus into its culture broth. The expression system was also effective for the generation of abundant AmfS derived from Streptomyces avermitilis. In addition, site-directed mutagenesis revealed the amino acid residues essential for the morphogen activity of AmfS. These results indicate that the constructed system enables efficient production of class III lantipeptides by Streptomyces.     

Smoking enhances the expression of angiotensin-converting enzyme 2 involved in the efficiency of severe acute respiratory syndrome coronavirus 2 infection

Smoking is one of the risk factors most closely related to the severity of coronavirus disease 2019 (COVID-19). However, the relationship between smoking history and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infectivity is unknown. In this study, we evaluated the ACE2 expression level in the lungs of current smokers, ex-smokers, and nonsmokers. The ACE2 expression level of ex-smokers who smoked cigarettes until recently (cessation period shorter than 6 months) was higher than that of nonsmokers and ex-smokers with a long history of nonsmoking (cessation period longer than 6 months). We also showed that the efficiency of SARS-CoV-2 infection was enhanced in a manner dependent on the angiotensin-converting enzyme 2 (ACE2) expression level. Using RNA-seq analysis on the lungs of smokers, we identified that the expression of inflammatory signaling genes was correlated with ACE2 expression. Notably, with increasing duration of smoking cessation among ex-smokers, not only ACE2 expression level but also the expression levels of inflammatory signaling genes decreased. These results indicated that smoking enhances the expression levels of ACE2 and inflammatory signaling genes. Our data suggest that the efficiency of SARS-CoV-2 infection is enhanced by smoking-mediated upregulation of ACE2 expression level.     

ATM regulates Cdt1 stability during the unperturbed S phase to prevent re-replication

Ataxia-telangiectasia mutated (ATM) plays crucial roles in DNA damage responses, especially with regard to DNA double-strand breaks (DSBs). However, it appears that ATM can be activated not only by DSB, but also by some changes in chromatin architecture, suggesting potential ATM function in cell cycle control. Here, we found that ATM is involved in timely degradation of Cdt1, a critical replication licensing factor, during the unperturbed S phase. At least in certain cell types, degradation of p27^Kip1 was also impaired by ATM inhibition. The novel ATM function for Cdt1 regulation was dependent on its kinase activity and NBS1. Indeed, we found that ATM is moderately phosphorylated at Ser1981 during the S phase. ATM silencing induced partial reduction in levels of Skp2, a component of SCF^Skp2 ubiquitin ligase that controls Cdt1 degradation. Furthermore, Skp2 silencing resulted in Cdt1 stabilization like ATM inhibition. In addition, as reported previously, ATM silencing partially prevented Akt phosphorylation at Ser473, indicative of its activation, and Akt inhibition led to modest stabilization of Cdt1. Therefore, the ATM-Akt-SCF^Skp2 pathway may partly contribute to the novel ATM function. Finally, ATM inhibition rendered cells hypersensitive to induction of re-replication, indicating importance for maintenance of genome stability.     

Interleukin-6 as an independent predictor of future cardiovascular events in high-risk Japanese patients: comparison with C-reactive protein

Inflammation is associated with the development of atherosclerotic vascular lesions and some inflammatory parameters are used as cardiovascular (CV) risk markers. The present study was designed to assess the predictive power of interleukin (IL)-6 for future CV events. In 121 Japanese patients with multiple CV risk factors and/or disease, serum concentrations of IL-6 and high sensitive C-reactive protein (hs-CRP) were measured. During follow-up periods (mean, 2.9 years) after the baseline assessment, 50 patients newly experienced CV events such as stroke/transient ischemic attack (n=10), heart failure hospitalization (n=6), acute coronary syndrome (n=7), and revascularization for coronary artery disease (n=15) and peripheral arterial disease (n=12). The serum level of IL-6, but not hs-CRP, was significantly higher in patients who had CV events than in event-free subjects (3.9±2.6 and 3.0±2.2 pg/mL, P=0.04). When the patients were divided into three groups by tertiles of basal levels of IL-6 (<1.85, 1.85-3.77, and ≥3.77 pg/mL), cumulative event-free rates by the Kaplan-Meier method were decreased according to the increase in basal IL-6 levels (65%, 50%, and 19% in the lowest, middle, and highest tertiles of IL-6, respectively; log-rank test, P=0.002). By univariate Cox regression analysis, previous CV disease, creatinine clearance, and serum IL-6 levels were significantly associated with CV events during follow-up. Among these possible predictors, the highest tertile of IL-6 was only an independent determinant for the morbidity in the multivariate analysis (hazard ratio 2.80 vs. lowest tertile, P=0.006). These findings indicate that IL-6 is a powerful independent predictor of future CV events in high-risk Japanese patients, suggesting its predictive value is superior to that of hs-CRP.