Protein modelling using a chimera reference protein derived from exons

Bovine pancreatic /S-trypsin (PDB ID-code: 1TPO) which is registeredin the Brookhaven Protein Data Bank (PDB) consists of four exons.The results of homology searches for each exon in the PDB showedthat homologous proteins were tonin (PDB ID-code: 1TON), ratmast cell protease (PDB ID-code: 3RP2_A), kaffikrein A (PDBID-code: 2PKA_B) and kallikrein A (2PKA_B) respectively. Thus,for the three-dimensional structure prediction of 1TPO, a chimeraprotein was constructed from the three proteins mentioned aboveand the 3-D structure prediction was performed using this chimerareference protein. The modelled structure of 1TPO was energeticallyoptimized by molecular mechanics and molecular dynamics simulationand was compared with its X-ray crystal structure registeredin the PDB. The root mean square deviations (r.m.s.d.) of mainchain atoms and the neighbouring active site (5 sphere fromHis57, AsplO2 and Serl95) between the modelled structure andthe X-ray structure were 1.66 and 0.94 respectively. Porcinepancreatic elastase (PDB ID-code: 3EST) which is registeredin the PDB was used as the reference protein and the modelledstructure from 3EST was also compared with the X-ray data. Ther.m.s.d. of main chain atoms and that of the active site were2.14 and 1.18 respectively. These results dearly support thepropriety of this method using the chimera reference protein.     

Development of the avian lymphatic system

Recently, highly specific markers of the lymphatic endothelium have been found enabling us to reinvestigate the embryonic origin of the lymphatics. Here we present a review of our studies on the development of the lymphatic system in chick and quail embryos. We show that the lymphatic endothelium is derived from two sources: the embryonic lymph sacs and mesenchymal lymphangioblasts. Proliferation studies reveal a BrdU-labeling index of 11.5% of lymph sac endothelial cells by day 6.25, which drops to 3.5% by day 7. Lymphangioblasts are able to integrate into the lining of lymph sacs. Lymphatic endothelial cells express the vascular endothelial growth factor (VEGF) receptors-2 and -3. Their ligand, VEGF-C, is expressed almost ubiquitously in embryonic and fetal tissues. Elevated expression levels are found in the tunica media of large blood vessels, which usually serve as major routes for growing lymphatics. The homeobox gene, Prox1, is expressed in lymphatic but not in blood vascular endothelial cells throughout all stages examined, namely, in developing lymph sacs of day 6 embryos and in lymphatics at day 16. Experimental studies show the existence of lymphangioblasts in the mesoderm, a considerable time before the development of the lymph sacs. Lymphangioblasts migrate from the somites into the somatopleure and contribute to the lymphatics of the limbs. Our studies indicate that these lymphangioblasts already express Prox1.     

Cellular automata and integrodifferential equation models for cell renewal in mosaic tissues

Mosaic tissues are composed of two or more genetically distinct cell types. They occur naturally, and are also a useful experimental method for exploring tissue growth and maintenance. By marking the different cell types, one can study the patterns formed by proliferation, renewal and migration. Here, we present mathematical modelling suggesting that small changes in the type of interaction that cells have with their local cellular environment can lead to very different outcomes for the composition of mosaics. In cell renewal, proliferation of each cell type may depend linearly or nonlinearly on the local proportion of cells of that type, and these two possibilities produce very different patterns. We study two variations of a cellular automaton model based on simple rules for renewal. We then propose an integrodifferential equation model, and again consider two different forms of cellular interaction. The results of the continuous and cellular automata models are qualitatively the same, and we observe that changes in local environment interaction affect the dynamics for both. Furthermore, we demonstrate that the models reproduce some of the patterns seen in actual mosaic tissues. In particular, our results suggest that the differing patterns seen in organ parenchymas may be driven purely by the process of cell replacement under different interaction scenarios.     

Sustained attention in the mouse: A study of the relationship with the cerebellum.

To explore the role of the cerebellum in sustained attention, the authors tested lurcher, wildtype, and lurcher chimeric mice with zero, normal, and variable numbers of Purkinje cells, respectively, in a previously validated task of sustained attention. Results indicate that lurcher mice had a deficit in performance likely related to their motor disability, whereas lurcher chimeras performed similarly to wildtype controls. Presentation of auditory or visual distracters caused deficits in the performance of all mice that were specific to either signal (auditory) or nonsignal (visual) events. The authors' results do not support a role of the cerebellum in sustained attention, instead indicating that behavioral changes are an indirect result of motor deficits. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

超声速下栅格舵展开过程数值模拟

栅格舵从折叠到展开的过程中气动特性变化剧烈,对展开可靠性和导弹整体气动特性的影响都比较大。针对栅格舵这种复杂的构造形式,生成了带有棱柱层的非结构网格,再结合重叠网格技术对栅格舵导弹超声速绕流流场进行了数值模拟,计算结果与风洞试验结果吻合较好。在此基础上,对超声速下栅格舵动态展开过程的非定常流场进行了数值模拟,分析了栅格舵导弹动态气动特性的变化规律。     

CFD simulation of a floating offshore wind turbine system using a variable-speed generator-torque controller

Prediction and control of rotor rotational velocity is critical for accurate aerodynamic loading and generator power predictions. A variable-speed generator-torque controller is combined with the two-phase CFD solver CFDShip-Iowa V4.5. The developed code is utilized in simulations of the 5 MW floating offshore wind turbine (FOWT) conceptualized by the National Renewable Energy Laboratory (NREL) for the Offshore Code Comparison Collaboration (OC3). Fixed platform simulations are first performed to determine baseline rotor velocity and developed torque. A prescribed platform motion simulation is completed to identify effects of platform motion on rotor torque. The OC3’s load case 5.1, with regular wave and steady wind excitation, is performed and results are compared to NREL’s OC3 results. The developed code is shown to functionally control generator speed and torque but requires controller calibration for maximum power extraction. Generator speed variance is observed to be a function of unsteady stream-wise platform motions. The increased mooring forces of the present model are shown to keep the turbine in a more favorable variable-speed control region. Lower overall platform velocity magnitudes and less rotor torque are predicted corresponding to lower rotor rotational velocities and a reduction in generated power. Potential improvements and modifications to the present method are considered.     

花鲈胚胎干细胞移植及嵌合体的构建

旨在通过花鲈胚胎干细胞(LJES1)移植构建嵌合体,证实LJES1细胞的体内分化能力和发育全能性.采用脂质体介导法将线性化pCMV-EGFP质粒导入到长期培养的LJES1细胞内,细胞绿色荧光蛋白(GFP)的表达率为5%～10%,以此为供体,通过显微注射方法把20～50个LJES1细胞移植到花鲈囊胚中,共移植囊胚478枚,获得了20枚表达GFP的嵌合体胚胎,其中的15枚胚胎发育成鱼苗.荧光显微观察和PCR检测显示了LJES1细胞可在宿主胚胎内片状嵌合和单细胞分散嵌合,嵌合部位可分布于胚体的三个胚层;同时也证明GFP是一种优良的遗传标记.本实验结果为证实LJES1细胞的发育全能性提供了有力证据.     

New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing

Despite recent progress, the application of CRISPR/Cas9 in perennial plants still has many obstacles to overcome. Our previous results with CRISPR/Cas9 in apple and pear indicated the frequent production of phenotypic and genotypic chimeras, after editing of the phytoene desaturase (PDS) gene conferring albino phenotype. Therefore, our first objective was to determine if adding an adventitious regeneration step from leaves of the primary transgenic plants (T0) would allow a reduction in chimerism. Among hundreds of adventitious buds regenerated from a variegated T0 line, 89% were homogeneous albino. Furthermore, the analysis of the target zone sequences of twelve of these regenerated lines (RT0 for “regenerated T0” lines) indicated that 99% of the RT0 alleles were predicted to produce a truncated target protein and that 67% of RT0 plants had less heterogeneous editing profiles than the T0. Base editors are CRISPR/Cas9-derived new genome-editing tools that allow precise nucleotide substitutions without double-stranded breaks. Hence, our second goal was to demonstrate the feasibility of CRISPR/Cas9 base editing in apple and pear using two easily scorable genes: acetolactate synthase—ALS (conferring resistance to chlorsulfuron) and PDS. The two guide RNAs under MdU3 and MdU6 promoters were coupled into a cytidine base editor harboring a cytidine deaminase fused to a nickase Cas9. Using this vector; we induced C-to-T DNA substitutions in the target genes; leading to discrete variation in the amino-acid sequence and generating new alleles. By co-editing ALS and PDS genes; we successfully obtained chlorsulfuron resistant and albino lines in pear. Overall; our work indicates that a regeneration step can efficiently reduce the initial chimerism and could be coupled with the application of base editing to create accurate genome edits in perennial plants.     

Efficient Targeted Delivery of Bifunctional Circular Aptamer-ASO Chimera to Suppress the SARS-CoV-2 Proliferation and Inflammation

Inhibition of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and excessive inflammation is the current task in the prevention and treatment of corona vireus disease 2019 (COVID-19). Here, a dual-function circular aptamer-ASO chimera (circSApt-NASO) is designed to suppress SARS-CoV-2 replication and inflammation. The chemically unmodified circSApt-NASO exhibits high serum stability by artificial cyclization. It is also demonstrated that the SApt binding to spike protein enables the chimera to be efficiently delivered into the host cells expressing ACE2 along with the infection of SARS-CoV-2. Among them, the SApt potently inhibits spike-induced inflammation. The NASO targeting to silence N genes not only display robust anti-N-induced inflammatory activity, but also achieve efficient inhibition of SARS-CoV-2 replication. Overall, benefiting from the high stability of the cyclization, antispike aptamer-dependent, and viral infection-mediate targeted delivery, the circSApt-NASO displays robust potential against authentic SARS-CoV-2 and Omicron, providing a promising specific anti-inflammatory and antiproliferative reagent for therapeutic COVID-19.     

Comparing cytosolic expression to peroxisomal targeting of the chimeric L1/L2 (ChiΔH-L2) gene from human papillomavirus type 16 in the methylotrophic yeasts Pichia pastoris and Hansenula polymorpha

The chimeric ChiΔH‐L2 gene from human papillomavirus type 16, consisting of structural proteins L1 and L2, was successfully expressed in the cytosol of both Pichia pastoris and Hansenula polymorpha during methanol induction. In addition, a novel approach was employed whereby ChiΔH‐L2 was targeted to the peroxisome using peroxisomal targeting sequence 1 (PTS1) to compare ChiΔH‐L2 yields in the peroxisome vs the cytosol. The ChiΔH‐L2 gene was yeast‐optimized and cloned into plasmids aimed at genomic integration. Levels of intracellular ChiΔH‐L2 accumulation in the cytosol were highest in P. pastoris KM71 strain KMChiΔH‐L2 (1.43 mg/l), compared to the maximum production level of 0.72 mg/l obtained with H. polymorpha. ChiΔH‐L2 targeting to the peroxisome was successful; however, it appeared to negatively affect ChiΔH‐L2 production in both P. pastoris and H. polymorpha. Copyright © 2012 John Wiley & Sons, Ltd.