Development of Artificial-Neural-Network-Based Permanent Deformation Prediction Model of Unbound Granular Materials Subjected to Moving Wheel Loading.

The majority of existing regression models for unbound granular materials (UGMs) consider only the effects of the number of loading cycles and stress levels on the permanent deformation characteristics and are thus unable to account for the complexity of plastic deformation accumulation behavior influenced by other factors, such as dry density, moisture content and gradation. In this study, research efforts were made to develop artificial-neural-network (ANN)-based prediction models for the permanent deformation of UGMs. A series of laboratory repeated load triaxial tests were conducted on UGM specimens with varying gradations to simulate realistic stress paths exerted by moving wheel loads and study permanent deformation characteristics. On the basis of the laboratory testing database, the ANN prediction models were established. Parametric sensitivity analyses were then performed to evaluate and rank the relative importance of each factor on permanent deformation behavior. The results indicated that the developed ANN prediction model is more accurate and reliable as compared to previously published regression models. The two major factors influencing the magnitude of accumulated plastic deformation of UGMs are the shear stress ratio (SSR) and the number of loading cycles, of which the calculated influence coefficients are 38% and 41%, respectively, while the degree of influence of gradation is twice that of the confining pressure.

Characterizing Particle-Scale Acceleration of Mud-Pumping Ballast Bed of Heavy-Haul Railway Subjected to Maintenance Operations.

Fouling and mud-pumping problems in ballasted track significantly degrade serviceability and jeopardize train operational safety. The phenomenological approaches for post hoc forensic investigation and remedies of mud pumps have relatively been well studied, but there still lacks studies on inherent mechanisms and ex ante approaches for early-age detection of mud pumps. This paper was aimed to exploring the feasibility of using particle acceleration responses to diagnose and identify early-age mud-pumping risks in real-world field applications. The innovative wireless sensors with 3D-printed shells resembling real shape of ballast particles were instrumented in the problematic railway section to monitor ballast particle movement prior to, during, and after maintenance operations, respectively. The real-time particle-scale acceleration data of ballast bed under both degraded and maintenance-restored clean conditions were recorded. The time histories, power spectra, and marginal spectra of 3D acceleration were comparatively analyzed. The results showed the 3D acceleration of ballast particles underneath rail-supporting tie plates displayed relatively clear periodicity of about 0.8 s with adjacent bogies regarded as a loading unit. The tamping operation was effective for compacting ballast bed laterally and improving the lateral interlocking of ballast particles, whereas the stabilizing operation was effective mainly in the lateral direction and for ballast particles underneath the sleepers. The mud pumps caused intensive particle-scale acceleration, and ballast particles underneath the sleepers were affected more severely than those in between adjacent sleepers. The ballast particles directly underneath tie plates exhibit dramatic acceleration variations due to maintenance operations as compared to those in other positions studied; hence, it seems promising to use particle-scale acceleration underneath tie plates as readily-implementable indicators for smart in-service track health monitoring.

Discrete Element Modeling of Instability Mechanisms of Unbound Permeable Aggregate Base Materials in Triaxial Compression.

Unbound permeable aggregate base (UPAB) materials with strong load-transmitting skeleton yet adequate inter-connected pores are desired for use in the sponge-city initiative. However, the micro-scale fabric evolution and instability mechanism of macroscopic strength behavior of such UPAB materials still remain unclear. In this study, virtual monotonic triaxial compression tests were conducted by using the discrete element method (DEM) modeling approach on specimens with different gradations quantified by the parameter of gravel-to-sand ratio (G/S). The realistic aggregate particle shape and inter-particle contact behavior were properly considered in the DEM model. The micromechanical mechanisms of the shearing failure of such UPAB materials and their evolution characteristics with G/S values were disclosed from contact force chains, microstructures, and particle motion. It was found that the proportion of rotating particles in the specimens decreased and the proportion of relative sliding between particles increased as the content of fine particles decreased. The plastic yielding of the specimens originated from the failure of contact force chains and the occurrence of the relative motion between particles, while the final instability was manifested by the large-scale relative motion among particles along the failure plane (i.e., changes in the internal particle topology). By comparing the macroscopic strength, microstructure evolution, and particle motion characteristics of the specimens with different G/S values, it was found that the specimens with G/S value of 1.8 performed the best, and that the G/S value of 1.8 could be regarded as the threshold for separating floating dense and skeletal gap type packing structures. The variation of Euler angles of rotating particles was significantly reduced in the particle size range of 4.75 mm to 9.50 mm, indicating that this size range separates most of the particles from rolling and sliding. Since particle rolling and sliding behavior are directly related to shear strength, this validates the rationality of the parameter G/S for controlling and optimizing gradations from the perspective of particle movement. The findings could provide theoretical basis and technical guidance for the effective design and efficient utilization of UPAB materials.

Myostatin regulates fatty acid desaturation and fat deposition through MEF2C/miR222/SCD5 cascade in pigs.

Myostatin (MSTN), associated with the "double muscling" phenotype, affects muscle growth and fat deposition in animals, whereas how MSTN affects adipogenesis remains to be discovered. Here we show that MSTN can act through the MEF2C/miR222/SCD5 cascade to regulate fatty acid metabolism. We generated MSTN-knockout (KO) cloned Meishan pigs, which exhibits typical double muscling trait. We then sequenced transcriptome of subcutaneous fat tissues of wild-type (WT) and MSTN-KO pigs, and intersected the differentially expressed mRNAs and miRNAs to predict that stearoyl-CoA desaturase 5 (SCD5) is targeted by miR222. Transcription factor binding prediction showed that myogenic transcription factor 2C (MEF2C) potentially binds to the miR222 promoter. We hypothesized that MSTN-KO upregulates MEF2C and consequently increases the miR222 expression, which in turn targets SCD5 to suppress its translation. Biochemical, molecular and cellular experiments verified the existence of the cascade. This novel molecular pathway sheds light on new targets for genetic improvements in pigs.

ФC31 Integrase-Mediated Isolation and Characterization of Novel Safe Harbors for Transgene Expression in the Pig Genome.

Programmable nucleases have allowed the rapid development of gene editing and transgenics, but the technology still suffers from the lack of predefined genetic loci for reliable transgene expression and maintenance. To address this issue, we used ФC31 integrase to navigate the porcine genome and identify the pseudo attP sites suitable as safe harbors for sustained transgene expression. The combined ФC31 integrase mRNA and an enhanced green fluorescence protein (EGFP) reporter donor were microinjected into one-cell zygotes for transgene integration. Among the resulting seven EGFP-positive piglets, two had transgene integrations at pseudo attP sites, located in an intergenic region of chromosome 1 (chr1-attP) and the 6th intron of the TRABD2A gene on chromosome 3 (chr3-attP), respectively. The integration structure was determined by TAIL-PCR and Southern blotting. Primary fibroblast cells were isolated from the two piglets and examined using fluorescence-activated cell sorting (FACS) and enzyme-linked immunosorbent assay (ELISA), which demonstrated that the chr1-attP site was more potent than chr3-attP site in supporting the EGFP expression. Both piglets had green feet under the emission of UV light, and pelleted primary fibroblast cells were green-colored under natural light, corroborating that the two pseudo attP sites are beneficial to transgene expression. The discovery of these two novel safe harbors for robust and durable transgene expression will greatly facilitate the use of transgenic pigs for basic, biomedical and agricultural studies and applications.

Isozygous and selectable marker-free MSTN knockout cloned pigs generated by the combined use of CRISPR/Cas9 and Cre/LoxP.

Predictable, clean genetic modification (GM) in livestock is important for reliable phenotyping and biosafety. Here we reported the generation of isozygous, functional myostatin (MSTN) knockout cloned pigs free of selectable marker gene (SMG) by CRISPR/Cas9 and Cre/LoxP. CRISPR/Cas9-mediated homologous recombination (HR) was exploited to knock out (KO) one allele of MSTN in pig primary cells. Cre recombinase was then used to excise the SMG with an efficiency of 82.7%. The SMG-free non-EGFP cells were isolated by flow cytometery and immediately used as donor nuclei for nuclear transfer. A total of 685 reconstructed embryos were transferred into three surrogates with one delivering two male live piglets. Molecular testing verified the mono-allelic MSTN KO and SMG deletion in these cloned pigs. Western blots showed approximately 50% decrease in MSTN and concurrent increased expression of myogenic genes in muscle. Histological examination revealed the enhanced myofiber quantity but myofiber size remained unaltered. Ultrasonic detection showed the increased longissimus muscle size and decreased backfat thickness. Precision editing of pig MSTN gene has generated isozygous, SMG-free MSTN KO cloned founders, which guaranteed a reliable route for elite livestock production and a strategy to minimize potential biological risks.

Pseudo attP sites in favor of transgene integration and expression in cultured porcine cells identified by Streptomyces phage phiC31 integrase.

Phage PhiC31 integrase integrates attB-containing plasmid into pseudo attP site in eukaryotic genomes in a unidirectional site-specific manner and maintains robust transgene expression. Few studies, however, explore its potential in livestock. This study aims to discover the molecular basis of PhiC31 integrase-mediated site-specific recombination in pig cells. We show that PhiC31 integrase can mediate site-specific transgene integration into the genome of pig kidney PK15 cells. Intramolecular recombination in pig PK15 cell line occurred at maximum frequency of 82% with transiently transfected attB- and attP-containing plasmids. An optimal molar ratio of pCMV-Int to pEGFP-N1-attB at 5:1 was observed for maximum number of cell clones under drug selection. Four candidate pseudo attP sites were identified by TAIL-PCR from those cell clones with single-copy transgene integration. Two of them gave rise to higher integration frequency occurred at 33%. 5' and 3' junction PCR showed that transgene integration mediated by PhiC31 integrase was mono-allelic. Micro- deletion and insertion were observed by sequencing the integration border, indicating that double strand break was induced by the recombination. We then constructed rescue reporter plasmids by ABI-REC cloning of the four pseudo attP sites into pBCPB + plasmid. Transfection of these rescue plasmids and pCMV-Int resulted in expected intramolecular recombination between attB and pseudo attP sites. This proved that the endogenous pseudo attP sites were functional substrates for PhiC31 integrase-mediated site-specific recombination. Two pseudo attP sites maintained robust extracellular and intracellular EGFP expression. Alamar blue assay showed that transgene integration into these specific sites had little effect on cell proliferation. This is the first report to document the potential use of PhiC31 integrase to mediate site-specific recombination in pig cells. Our work established an ideal model to study the position effect of identical transgene located in diverse chromosomal contexts. These findings also form the basis for targeted pig genome engineering and may be used to produce genetically modified pigs for agricultural and biomedical uses.

An asymmetric PCR-based, reliable and rapid single-tube native DNA engineering strategy.

BACKGROUND: Widely used restriction-dependent cloning methods are labour-intensive and time-consuming, while several types of ligase-independent cloning approaches have inherent limitations. A rapid and reliable method of cloning native DNA sequences into desired plasmids are highly desired. RESULTS: This paper introduces ABI-REC, a novel strategy combining asymmetric bridge PCR with intramolecular homologous recombination in bacteria for native DNA cloning. ABI-REC was developed to precisely clone inserts into defined location in a directional manner within recipient plasmids. It featured an asymmetric 3-primer PCR performed in a single tube that could robustly amplify a chimeric insert-plasmid DNA sequence with homologous arms at both ends. Intramolecular homologous recombination occurred to the chimera when it was transformed into E.coli and produced the desired recombinant plasmids with high efficiency and fidelity. It is rapid, and does not involve any operational nucleotides. We proved the reliability of ABI-REC using a double-resistance reporter assay, and investigated the effects of homology and insert length upon its efficiency. We found that 15 bp homology was sufficient to initiate recombination, while 25 bp homology had the highest cloning efficiency. Inserts up to 4 kb in size could be cloned by this method. The utility and advantages of ABI-REC were demonstrated through a series of pig myostatin (MSTN) promoter and terminator reporter plasmids, whose transcriptional activity was assessed in mammalian cells. We finally used ABI-REC to construct a pig MSTN promoter-terminator cassette reporter and showed that it could work coordinately to express EGFP. CONCLUSIONS: ABI-REC has the following advantages: (i) rapid and highly efficient; (ii) native DNA cloning without introduction of extra bases; (iii) restriction-free; (iv) easy positioning of directional and site-specific recombination owing to formulated primer design. ABI-REC is a novel approach to DNA engineering and gene functional analysis.

Construction of Early Human Embryo cDNA Libraries and Screening Objective Genes.

The construction, evaluation, and application of cDNA libraries from 3-, 4-, and 5-week-old human embryos are described. Total RNAs were extracted from whole embryos using a modified single-step method. mRNA purified by two passes through oligo (dT) columns was reverse-transcripted into single-stranded cDNA. Alkaline agarose electrophoresis showed that the double-strand cDNA fragments ranged from 0.4 9.0 kb and most of them were in the range of 1.0 2.0 kb. After separation on SizeSep 400 Spun columns to eliminate excess adaptors and small cDNA fragments(less than 400 bp), the cDNAs were ligated into pSPORT1 plasmid and lambdaZipLox phage. The plasmid libraries have complexities of 2.6x10(5), 1.7x10(5) and 2.1x10(5) clones and the phage cDNA libraries have complexities of 3.4x10(6), 3.7x10(6) and 2.3x10(6) clones, respectively. Three whole length cDNAs encoding human CD59, MCP and DAF were amplified by PCR using 3-week-old phage library as templates, and human tPA gene with whole length cDNA was screened from 4-week-old plasmid library by hybridization. It was shown that these libraries are of high quality and are suitable to screen rarely expressed genes. The libraries are a valuable source for the study of novel gene expression during human development.