Generation of the Y-chromosome linked red fluorescent protein transgenic mouse model and sexing at the preimplantation stage

In mammals, sexual fate is determined by the chromosomes of the male and female gametes during fertilization. Males (XY) or females (XX) are produced when a sperm containing a Y or X-chromosome respectively fertilizes an X-chromosome-containing unfertilized egg. However, sexing of preimplantation stage embryos cannot be conducted visually. To address this, transgenic male mouse models with the ubiquitously expressed green fluorescent protein (GFP) transgene on X- (X-GFP) or Y-chromosomes (Y-GFP) have been established. However, when crossed with wild-type females, sexing of the preimplantation stage embryos by observing the GFP signal is problematic in some cases due to X-inactivation, loss of Y-chromosome (LOY), or loss of transgene fluorescence. In this study, a mouse model with the ubiquitously expressed red fluorescent protein (RFP) transgene on the Y-chromosome was generated since RFP is easily distinguishable from GFP signals. Unfortunately, the ubiquitously expressed tdTomato RFP transgene on the Y-chromosome (Y-RFP) mouse showed the lethal phenotype after birth. No lethal phenotypes were observed when the mitochondrial locating signal N-terminal of tdTomato (mtRFP) was included in the transgene construct. Almost half of the collected fertilized eggs from Y-mtRFP male mice crossed with wild-type females had an RFP signal at the preimplantation stage (E1.5). Therefore, XY eggs were recognized as RFP-positive embryos at the preimplantation stage. Furthermore, 100% sexing was observed at the preimplantation stage using the X-linked GFP/Y-linked RFP male mouse. The established Y-mtRFP mouse models may be used to study sex chromosome related research.     

IQCELL: A platform for predicting the effect of gene perturbations on developmental trajectories using single-cell RNA-seq data

The increasing availability of single-cell RNA-sequencing (scRNA-seq) data from various developmental systems provides the opportunity to infer gene regulatory networks (GRNs) directly from data. Herein we describe IQCELL, a platform to infer, simulate, and study executable logical GRNs directly from scRNA-seq data. Such executable GRNs allow simulation of fundamental hypotheses governing developmental programs and help accelerate the design of strategies to control stem cell fate. We first describe the architecture of IQCELL. Next, we apply IQCELL to scRNA-seq datasets from early mouse T-cell and red blood cell development, and show that the platform can infer overall over 74% of causal gene interactions previously reported from decades of research. We will also show that dynamic simulations of the generated GRN qualitatively recapitulate the effects of known gene perturbations. Finally, we implement an IQCELL gene selection pipeline that allows us to identify candidate genes, without prior knowledge. We demonstrate that GRN simulations based on the inferred set yield results similar to the original curated lists. In summary, the IQCELL platform offers a versatile tool to infer, simulate, and study executable GRNs in dynamic biological systems.     

Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10(-7) M markedly increased mRNA expression for bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3-E1), while suppressing gene expression for collagenase-1, and collagenase-3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase-digestive proteins, and noncollagenous proteins was increased in the cells treated with 10(-7) M simvastatin, or 10(-8) M cerivastatin. In the culture of MC3T3-E1 cells, statins stimulated mineralization; pretreating MC3T3-E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin-induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.     

Complementation of SOD-deficient Escherichia coli by manganese porphyrin mimics of superoxide dismutase activity

Cationic Mn(III) porphyrins substituted on the methine bridge carbons (meso positions) with N-alkylpyridinium or N,N'-diethylimidazolium groups have been prepared and characterized, both chemically and as SOD mimics. The ortho tetrakis N-methylpyridinium compound was substantially more active than the corresponding para isomer. This ortho compound also exhibited a more positive redox potential and greater ability to facilitate the aerobic growth of a SOD-deficient Escherichia coli. Analogs with longer alkyl side chains and with methoxyethyl side chains, as well as with N,N'-diethylimidazolium and N,N'-dimethoxyethylimidazolium groups on the meso positions, have been prepared in anticipation of greater penetration of the cells due to greater lipophilicity. We now report that the more lipophilic compounds were effective at complementing the SOD-deficient E. coli at lower concentrations than were needed with the less lipophilic compounds. The greater efficacy of the more lipophilic compounds was achieved at the cost of greater toxicity that became apparent when these compounds were applied at higher concentrations.     

Protective effects of quercetin and its metabolites on H2O2-induced chromosomal damage to WIL2-NS cells

We investigated chromosomal damage caused by a typical flavonoid, quercetin, and its two conjugates, quercetin-3-O-sulfate and isorhamnetin, and their protective effects against chromosomal damage induced by H2O2. The chromosomal damage was detected by the cytokinesis-block micronucleus (CBMN) assay using a lymphoblastoid cell line, WIL2-NS. We found that quercetin itself induced chromosomal damage at 10 microM, but quercetin-3-O-sulfate and isorhamnetin did not induce damage up to 30 microM. In the medium used for the CBMN assay, quercetin (at 100 microM) generated a high concentration of H2O2, but the two conjugates did not at the same concentration. On the other hand, pretreatment with quercetin (at 1 microM), quercetin-3-O-sulfate (at 10 microM), and isorhamnetin (at 5 microM) prevented H2O2-induced chromosomal damage to WIL2-NS cells. These findings suggest that the induction and prevention of H2O2-induced chromosomal damage are different between quercetin and its metabolites.