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.     

Photochemistry and Photoinduced Proton-Transfer by Pharaonis Phoborhodopsin

Phoborhodopsin (pR or sensory rhodopsin II, sRII) is a photoreceptor of the negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. The photocycle of ppR is essentially as follows: ppR(498) ppR_K(540) ppR_KL(512) ppR_L(488) ppR_M(390) ppR_O(560) ppR (numbers in parenthesis denote the maximum absorbance). The photocycle is very similar to that of bacteriorhodopsin, but the rate of initial pigment recovery is about two-orders of magnitude slower. By low-temperature spectroscopy, two K-intermediates were found but the L intermediate was not detected. The lack of L indicates extraordinary stability of K at low temperature. ppR_M is photoactive similar to M of bR. The ground state ppR contains only all-trans retinal whereas ppR_M and ppR_O contain 13-cis and all-trans, respectively. ppR has the ability of lightinduced proton transport from the inside to the outside. Proton uptake occurs at the formation of ppR_O and the release at its decay. ppR associates with its transducer and this complex transmits a signal to the cytoplasm. The proton transport ability is lost when the complex forms, but the proton uptake and release still occur, suggesting that the proton movement is non-electrogenic (release and uptake occur from the same side). The stoichiometry of the complex between ppR and the transducer is 1 : 1. ppR or pR has absorption maximum at 500 nm, which is blue-shifted from those of other archaeal rhodopsins. The molecular mechanism of this color regulation is not yet solved.     

Five cases of Diphyllobothrium nihonkaiense infection with discovery of plerocercoids from an infective source, Oncorhynchus masou ishikawae

Five persons from 2 families residing at Miyama Town, Mie Prefecture, Japan, ingested fresh raw fish Oncorhynchus sp. on 9 May 1999 that was caught at Owase district in Mie. They all expelled diphyllobothriid cestodes 11-37 days after ingesting the fish. The parasites were morphologically identical to Diphyllobothrium nihonkaiense Yamane et al., 1986. Five plerocercoids were detected from a portion of the fish. Nucleotide sequence of a region of the cytochrome c oxidase subunit I gene of mitochondrial DNA from an adult worm was identical with that from the plerocercoid. The fish was identified as Oncorhynchus masou ishikawae according to the nucleotide sequence of the nuclear ribosomal second internal transcribed spacer region II gene. This is the first record of D. nihonkaiense plerocercoids from O. m. ishikawae.     

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.