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Evaluation of slope stability under rainfall is an important topic of Geotechnical Engineering. In order to study the influence of anisotropy ratio (kr = kx/ky) and anisotropy direction (&alpha, ) on the seepage and stability of a slope, the SEEP/W and SLOPE/W modules in Geo-studio were utilized to carry out the numerical analysis of a homogeneous slope in Luogang District, Guangzhou City, China, which is based on the theory of unsaturated seepage and stability. Two kinds of soils (clay and sand) were included. Results show that: For sandy soil slope, the increase of kr promotes the rainfall infiltration, and the decrease of &alpha, prevents the rainfall infiltration. The maximum water content of the surface (MWCS) reaches maximum with the increase of kr and &alpha, The rising height of groundwater (RHG) is &minus, 3&ndash, 4 m and the safety factor (SF) is 1.3&ndash, 1.7. For clayey soil slope, variations of kr and &alpha, have little impact on the seepage characteristics and slope stability. The MWCS remains almost the same. The rainfall infiltration depth (RID) is 0.5&ndash, 1 m and the SF is about 1.7. Therefore, for sandy soil slope, it is not only necessary to consider the influence of kr, but also the influence of &alpha, For clayey soil slope, it can be treated as isotropic material to simplify calculation.

With the rapid growth of car ownership, traffic congestion has become one of the most serious social problems. For us, accurate real-time travel time predictions are especially important for easing traffic congestion, enabling traffic control and management, and traffic guidance. In this paper, we propose a method to predict urban road travel time by combining XGBoost and LightGBM machine learning models. In order to obtain a relatively complete data set, we mine the GPS data of Beijing and combine them with the weather feature to consider the obtained 14 features as candidate features. By processing and analyzing the data set, we discussed in detail the correlation between each feature and the travel time and the importance of each feature in the model prediction results. Finally, the 10 important features screened by the LightGBM and XGBoost models were used as key features. We use the full feature set and the key feature set as input to the model to explore the effect of different feature combinations on the prediction accuracy of the model and then compare the prediction results of the proposed fusion model with a single model. The results show that the proposed fusion model has great advantages to urban travel time prediction.

To study the effect law of cold molten salt temperature on both the thermal energy storage and mechanical performances of a thermocline molten salt tank, a numerical model of the tank which includes a multi-layer wall is established. The tank wall consists of three layers, which are firebrick, steel, and ceramic, respectively. Fluent code is employed to carry out the simulations of five different complete operation cycles of the thermocline tank. An integrated thermal and mechanical performance analysis of the effect of cold molten salt temperature for the thermocline tank is carried out. The analysis results reveal that for all the simulated cases, the thermocline is steady, and both the charging and discharging processes of the tank are stable. The thermocline thickness of the charging process can be smaller than that of the discharging one. The axial temperature of steel wall does not change synchronously with the central axial temperature of molten salt during both charging and discharging processes. As the cold molten salt temperature decreases, the thermocline thickness as well as the maximum mechanical stress of the steel wall increases. Hence, it can be drawn that increasing the cold molten salt temperature properly will be of benefit to both the energy storage performance and stability of the thermocline tank structure.

In the age of industry 4.0 and smart manufacturing, a large volume of sensor data is produced from cyber-physical systems (CPS) and prediction of remaining useful life (RUL) of a machine or system becomes crucial for prognostics and health management (PHM). Several linear regression and deep learning models have been studied to extract features from segmented time windows and learn the degradation patterns. However, distributions of features are varying between source learning domain and target test domains, due to different working conditions and environments. Thus, the generalization of traditional methods will be influenced, which leads to performance degradation. This paper develops a domain adaptive CNN-LSTM (DACL) model to predict the RUL of a system based on the multi-dimensional sensor data. The DACL model combines the CNN and LSTM with domain adaptive transfer mechanism and takes the operating conditions into consideration. The features extracted by CNN of both source and target data are transformed to a higher dimensional space by reproducing kernel Hilbert space (RKHS) and the loss function is compensated by using maximum mean discrepancy (MMD) to reduce the distributions discrepancy. The model is evaluated on C-MAPSS dataset and demonstrate its performance improvement by comparing with previous methods.

In order to study the thallus changes on microscopic morphology and mechanical properties ofCandida albicansantagonized byStreptococcus sanguinisbacteriocin, the adhesion ability and Young’s modulus of thalli and hypha ofCandida albicanswere measured by the relative measurement method using atomic force microscope’s (AFM) tapping model. The results showed that the average adhesion ability and Young’s modulus of thalli were7.35±0.77 nN and7.33±1.29 Mpa, respectively; the average adhesion ability and Young’s modulus of hypha were9.82±0.39 nN and4.04±0.76 Mpa, respectively. After being antagonized byStreptococcus sanguinisbacteriocin, the adhesion ability was decreased along with the increasing of deformation in reaction region and Young’s modulus followed the same changes. It could be concluded that the adhesion ability of hypha was greater than thalli, Young’s modulus of hypha was less than thalli, and adhesion ability and Young’s modulus ofCandida albicanswere decreased significantly after being antagonized byStreptococcus sanguinisbacteriocin.

Differentiation of effector and memory CD8+ T cells is accompanied by extensive changes in the transcriptome and histone modifications at gene promoters; however, the enhancer repertoire and associated gene regulatory networks are poorly defined. Using histone mark chromatin immunoprecipitation coupled with deep sequencing, we mapped the enhancer and super-enhancer landscapes in antigen-specific naive, differentiated effector, and central memory CD8+ T cells during LCMV infection. Epigenomics-based annotation revealed a highly dynamic repertoire of enhancers, which were inherited, de novo activated, decommissioned and re-activated during CD8+ T cell responses. We employed a computational algorithm to pair enhancers with target gene promoters. On average, each enhancer targeted three promoters and each promoter was regulated by two enhancers. By identifying enriched transcription factor motifs in enhancers, we defined transcriptional regulatory circuitry at each CD8+ T cell response stage. These multi-dimensional datasets provide a blueprint for delineating molecular mechanisms underlying functional differentiation of CD8+ T cells.

Nano-antibodies possess great potential in many applications. However, they are naturally derived from heavy chain-only antibodies (HcAbs), which lack light chains and the CH1 domain, and are only found in camelids and sharks. In this study, we investigated whether the precise genetic removal of the CH1 exon of the γ1 gene enabled the production of a functional heavy chain-only IgG1 in mice. IgG1 heavy chain dimers lacking associated light chains were detected in the sera of the genetically modified mice. However, the genetic modification led to decreased expression of IgG1 but increased expression of other IgG subclasses. The genetically modified mice showed a weaker immune response to specific antigens compared with wild type mice. Using a phage-display approach, antigen-specific, single domain VH antibodies could be screened from the mice but exhibited much weaker antigen binding affinity than the conventional monoclonal antibodies. Although the strategy was only partially successful, this study confirms the feasibility of producing desirable nano-bodies with appropriate genetic modifications in mice.

Spaceflight leads to health risks including bone demineralization, skeletal muscle atrophy, cardiovascular dysfunction, and disorders of almost all physiologic systems. However, the impacts of microgravity on blood lineage cells and hematopoietic stem cells (HSCs) in vivo are largely unknown. In this study, we analyzed peripheral blood samples from 6 astronauts who had participated in spaceflight missions and found significant changes in several cell populations at different time points. These dynamic alterations of lineage cells and the role of HSCs were further studied in a mouse model, using hindlimb unloading (HU) to simulate microgravity. Large reductions in the frequency of NK cells, B cells, and erythrocyte precursors in the bone marrow of the HU mice were observed, together with an increased frequency of T cells, neutrophils, and HSCs. T cell levels recovered faster than those of B cells and erythrocyte precursors, whereas the recovery rates of NK cells and granulocytes were slow. In addition, competitive reconstitution experiments demonstrated the impaired function of HSCs, although these changes were reversible. Deep sequencing showed changes in the expression of regulatory molecules important for the differentiation of HSCs. This study provides the first determination of altered HSC function under simulated microgravity in vivo. The impairment of HSC function and differentiation provides an explanation for the immune disorders that occur under simulated microgravity. Thus, our findings demonstrated that spaceflight and simulated microgravity disrupt the homeostasis of immune system and cause dynamic alterations on both HSCs and lineage cells.-Cao, D., Song, J., Ling, S., Niu, S., Lu, L., Cui, Z., Li, Y., Hao, S., Zhong, G., Qi, Z., Sun, W., Yuan, X., Li, H., Zhao, D., Jin, X., Liu, C., Wu, X., Kan, G., Cao, H., Kang, Y., Yu, S., Li, Y. Hematopoietic stem cells and lineage cells undergo dynamic alterations under microgravity and recovery conditions.

The numerical model of a molten-salt-based thermocline tank with multi-layer wall structure is established. A comprehensive parametric study on integrated thermal and mechanical performances of the tank is conducted by using the CFD approach. The effects of eight factors are investigated. For all factors, complete operation cycle simulations of the tank as well as mechanical calculations are launched. The effect laws are revealed and briefly summarized. The results indicate that the inlet molten salt velocity, cold molten salt temperature, porosity of porous bed, diameter, thermal conductivity and specific heat of solid filler particle all have relatively obvious impacts on the discharging performance of the tank. The thermocline thickness during the discharging process can be decreased by increasing the inlet molten salt velocity, cold molten salt temperature or specific heat of filler particle, or by reducing the porosity of porous bed, diameter or thermal conductivity of solid filler particle. The maximum mechanical stress of the steel wall can be decreased by increasing the inlet molten salt velocity, cold molten salt temperature or firebrick thickness, or by decreasing the steel wall thickness, porosity of porous bed, diameter, thermal conductivity or specific heat of solid filler particle.

Predictive maintenance plays an important role in modern Cyber-Physical Systems (CPSs) and data-driven methods have been a worthwhile direction for Prognostics Health Management (PHM). However, two main challenges have significant influences on the traditional fault diagnostic models: one is that extracting hand-crafted features from multi-dimensional sensors with internal dependencies depends too much on expertise knowledge; the other is that imbalance pervasively exists among faulty and normal samples. As deep learning models have proved to be good methods for automatic feature extraction, the objective of this paper is to study an optimized deep learning model for imbalanced fault diagnosis for CPSs. Thus, this paper proposes a weighted Long Recurrent Convolutional LSTM model with sampling policy (wLRCL-D) to deal with these challenges. The model consists of 2-layer CNNs, 2-layer inner LSTMs and 2-Layer outer LSTMs, with under-sampling policy and weighted cost-sensitive loss function. Experiments are conducted on PHM 2015 challenge datasets, and the results show that wLRCL-D outperforms other baseline methods.

The transcription factors TCF-1 and LEF-1 are essential for early T cell development, but their roles beyond the CD4(+)CD8(+) double-positive (DP) stage are unknown. By specific ablation of these factors in DP thymocytes, we demonstrated that deficiency in TCF-1 and LEF-1 diminished the output of CD4(+) T cells and redirected CD4(+) T cells to a CD8(+) T cell fate. The role of TCF-1 and LEF-1 in the CD4-versus-CD8 lineage 'choice' was mediated in part by direct positive regulation of the transcription factor Th-POK. Furthermore, loss of TCF-1 and LEF-1 unexpectedly caused derepression of CD4 expression in T cells committed to the CD8(+) lineage without affecting the expression of Runx transcription factors. Instead, TCF-1 physically interacted with Runx3 to cooperatively silence Cd4. Thus, TCF-1 and LEF-1 adopted distinct genetic 'wiring' to promote the CD4(+) T cell fate and establish CD8(+) T cell identity.

Tracheobronchial submucosal glands (SMGs) are derived from one or more multipotent glandular stem cells that coalesce to form a placode in surface airway epithelium (SAE). Wnt/β-catenin-dependent induction of lymphoid enhancer factor ( Lef-1) gene expression during placode formation is an early event required for SMG morphogenesis. We discovered that Sox2 expression is repressed as Lef-1 is induced within airway SMG placodes. Deletion of Lef-1 did not activate Sox2 expression in SMG placodes, demonstrating that Lef-1 activation does not directly inhibit Sox2 expression. Repression of Sox2 protein in SMG placodes occurred posttranscriptionally, since the activity of its endogenous promoter remained unchanged in SMG placodes. Thus we hypothesized that Sox2 transcriptionally represses Lef-1 expression in the SAE and that suppression of Sox2 in SMG placodes activates Wnt/β-catenin-dependent induction of Lef-1 during SMG morphogenesis. Consistent with this hypothesis, transcriptional reporter assays, ChIP analyses, and DNA-protein binding studies revealed a functional Sox2 DNA binding site in the Lef-1 promoter that is required for suppressing β-catenin-dependent transcription. In polarized primary airway epithelium, Wnt induction enhanced Lef-1 expression while also inhibiting Sox2 expression. Conditional deletion of Sox2 also enhanced Lef-1 expression in polarized primary airway epithelium, but this induction was significantly augmented by Wnt stimulation. Our findings provide the first evidence that Sox2 acts as a repressor to directly modulate Wnt-responsive transcription of the Lef-1 gene promoter. These studies support a model whereby Wnt signals and Sox2 dynamically regulate the expression of Lef-1 in airway epithelia and potentially also during SMG development.

In order to study the thallus changes on microscopic morphology and mechanical properties of Candida albicans antagonized by Streptococcus sanguinis bacteriocin, the adhesion ability and Young's modulus of thalli and hypha of Candida albicans were measured by the relative measurement method using atomic force microscope's (AFM) tapping model. The results showed that the average adhesion ability and Young's modulus of thalli were 7.35 ± 0.77 nN and 7.33 ± 1.29 Mpa, respectively; the average adhesion ability and Young's modulus of hypha were 9.82 ± 0.39 nN and 4.04 ± 0.76 Mpa, respectively. After being antagonized by Streptococcus sanguinis bacteriocin, the adhesion ability was decreased along with the increasing of deformation in reaction region and Young's modulus followed the same changes. It could be concluded that the adhesion ability of hypha was greater than thalli, Young's modulus of hypha was less than thalli, and adhesion ability and Young's modulus of Candida albicans were decreased significantly after being antagonized by Streptococcus sanguinis bacteriocin.

Differentiation of effector and memory CD8+ T cells is accompanied by extensive changes in the transcriptome and histone modifications at gene promoters; however, the enhancer repertoire and associated gene regulatory networks are poorly defined. Using histone mark chromatin immunoprecipitation coupled with deep sequencing, we mapped the enhancer and super-enhancer landscapes in antigen-specific naïve, differentiated effector, and central memory CD8+ T cells during LCMV infection. Epigenomics-based annotation revealed a highly dynamic repertoire of enhancers, which were inherited, de novo activated, decommissioned and re-activated during CD8+ T cell responses. We employed a computational algorithm to pair enhancers with target gene promoters. On average, each enhancer targeted three promoters and each promoter was regulated by two enhancers. By identifying enriched transcription factor motifs in enhancers, we defined transcriptional regulatory circuitries at each CD8+ T-cell response stage. These multi-dimensional datasets provide a blueprint for delineating molecular mechanisms underlying functional differentiation of CD8+ T cells.

Hematopoietic and leukemic stem cells (HSCs and LSCs) have self-renewal ability to maintain normal hematopoiesis and leukemia propagation, respectively. Tcf1 and Lef1 transcription factors are expressed in HSCs, and targeting both factors modestly expanded the size of the HSC pool due to diminished HSC quiescence. Functional defects of Tcf1/Lef1-deficient HSCs in multi-lineage blood reconstitution was only evident under competitive conditions or when subjected to repeated regenerative stress. These are mechanistically due to direct positive regulation of Egr and Tcf3 by Tcf1 and Lef1, and significantly, forced expression of Egr1 in Tcf1/Lef1-deficient HSCs restored HSC quiescence. In a preclinical CML model, loss of Tcf1/Lef1 did not show strong impact on leukemia initiation and progression. However, when transplanted into secondary recipients, Tcf1/Lef1-deficient LSCs failed to propagate CML. By induced deletion of Tcf1 and Lef1 in pre-established CML, we further demonstrated an intrinsic requirement for these factors in LSC self-renewal. When combined with imatinib therapy, genetic targeting of Tcf1 and Lef1 potently diminished LSCs and conferred better protection to the CML recipients. LSCs are therefore more sensitive to loss of Tcf1 and Lef1 than HSCs in their self-renewal capacity. The differential requirements in HSCs and LSCs thus identify Tcf1 and Lef1 transcription factors as novel therapeutic targets in treating hematological malignancies, and inhibition of Tcf1/Lef1-regulated transcriptional programs may thus provide a therapeutic window to eliminate LSCs with minimal side effect on normal HSC functions.

Summary The TCF-1 and LEF-1 transcription factors are known to play critical roles in normal thymocyte development. Unexpectedly, we found that TCF-1-deficient ( Tcf7 −/− ) mice developed aggressive T cell malignancy, resembling human T cell acute lymphoblastic leukemia (T-ALL). LEF-1 was aberrantly upregulated in premalignant Tcf7 −/− early thymocytes and lymphoma cells. We further demonstrated that TCF-1 directly repressed LEF-1 expression in early thymocytes and that conditional inactivation of Lef1 greatly delayed or prevented T cell malignancy in Tcf7 −/− mice. In human T-ALLs, an early thymic progenitor (ETP) subtype was associated with diminished TCF7 expression, and two of the ETP-ALL cases harbored TCF7 gene deletions. We also showed that TCF-1 and LEF-1 were dispensable for T cell lineage commitment but instead were required for early thymocytes to mature beyond the CD4 − CD8 − stage. TCF-1 thus has dual roles, i.e., acting cooperatively with LEF-1 to promote thymocyte maturation while restraining LEF-1 expression to prevent malignant transformation of developing thymocytes.

In order to investigate the potential involvement of PTPN22 R620W in the pathogenesis of myasthenia gravis (MG), we performed a case-control study including 409 Swedish MG patients and 1557 normal controls. The W620 variant was significantly overrepresented in patients (odds ratio, 1.52; 95% confidence interval, 1.21-1.90; p=0.00027). Incubation of patient (n=100) derived PBMC cells with the autoantigen, the acetylcholine receptor, resulted in a significantly higher number of cells producing anti-AChR antibodies and IL-2 in W620 carriers, suggesting that PTPN22 W620 may be a loss-of-function variant in MG.

Although evolutionarily just as ancient as IgM, it has been thought for many years that IgD is not present in birds. Based on the recently sequenced genomes of 48 bird species as well as high-throughput transcriptome sequencing of immune-related tissues, we demonstrate in this work that the ostrich (Struthio camelus) possesses a functional δ gene that encodes a membrane-bound IgD H chain with seven CH domains. Furthermore, δ sequences were clearly identified in many other bird species, demonstrating that the δ gene is widely distributed among birds and is only absent in certain bird species. We also show that the ostrich possesses two μ genes (μ1, μ2) and two υ genes (υ1, υ2), in addition to the δ and α genes. Phylogenetic analyses suggest that subclass diversification of both the μ and υ genes occurred during the early stages of bird evolution, after their divergence from nonavian reptiles. Although the positions of the two υ genes are unknown, physical mapping showed that the remaining genes are organized in the order μ1-δ-α-μ2, with the α gene being inverted relative to the others. Together with previous studies, our data suggest that birds and nonavian reptile species most likely shared a common ancestral IgH gene locus containing a δ gene and an inverted α gene. The δ gene was then evolutionarily lost in selected birds, whereas the α gene lost in selected nonavian reptiles. The data obtained in this study provide significant insights into the understanding of IgH gene evolution in tetrapods.

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most devastating pathogens in the swine industry worldwide. Due to the lack of robust cell lines and small animal models, the pathogenesis of PRRSV infection and mechanism for protective vaccination are still not yet well understood. To obtain useful cell lines, several groups have attempted to construct different transgenic cell lines with three PRRSV receptors: CD163, CD169, and CD151. The results showed that CD163 is essential for PRRSV entry into target cells and replication, and both CD169 and CD151 play key roles during PRRSV infection. However, their interplay and combined effect remains unclear. In this study, we generated transgenic BHK-21 derived cell lines co-expressing different combinations of the three receptors, which were transfected with CD163 alone, or the combination of CD163 and CD169, or the combination of CD163 and CD151, or the combination of CD163, CD169, and CD151 using the PiggyBac transposon system. Our results showed that the synergistic interaction among the three receptors was important to improve the susceptibility of cells during PRRSV infection. Through a series of comparable analyses, we confirmed that the cell line co-expressing triple receptors sustained viral infection and replication, and was superior to the current cell platform used for the PRRSV study, MARC-145 cells. Moreover, we found that PRRSV infection of the transgenic cell lines could trigger IFN-stimulated gene responses similar to those of porcine alveolar macrophages and MARC-145 cells. In summary, we developed a stable transgenic cell line susceptible to PRRSV, which may not only provide a useful tool for virus propagation, vaccine development, and pathogenesis studies, but also establish the foundation for small animal model development.

Cloning by somatic nuclear transfer is an inefficient process in which many of the cloned animals die shortly after birth and display organ abnormalities. In an effort to determine the possible roles IGFs played in neonatal death and organ abnormalities, we have examined expression patterns of eight genes in insulin-like growth factor (IGF) systems (IGF1, IGF2, IGF1R, IGF2R, IGFBP-1, IGFBP-2, IGFBP-3, and IGFBP-4) in six organs (heart, liver, spleen, lung, kidney, and brain) of both neonatal death cloned bovines (n = 9) and normal control calves (n = 3) produced by artificial insemination (AI) using real-time quantitative RT-PCR. The effect of the age of the fibroblast donor cell on the gene expression profiles was also investigated. Aberrant expressions of six genes (IGF2, IGF1R, IGF2R, IGFBP-2, IGFBP-3, and IGFBP-4) were found in some studied tissues, but the expression of two genes (IGF1 and IGFBP-1) had similar levels with the normal controls. For the studied genes, kidney was the organ that was most affected (five genes) by gene downregulation, whereas spleen was the organ that was not affected. The two upregulation genes were in brain, but both of downregulation and upregulation were found in the heart, liver, and lung. The expression of three genes (IGF2R, IGFBP-4, and IGF2) in some tissues showed significant differences between AF cell-derived and FF cell-derived clones. Our results suggest that aberrations in gene expression within IGF systems were found in most cloned bovine tissues of neonatal death. Because IGF systems play an important role in embryo development and organogenesis, the aberrant transcription patterns detected in these clones may contribute to the defects of organs reported in neonatal death of clones.

Transgenic mice expressing a recombinant human monoclonal antibody (rHMAb) against hantavirus were generated. These mice could be used as models to explore the possibilities of producing rHMAbs for therapeutic purposes. The highest concentration of the rHMAb in the milk of the transgenic females was 6.6 mg/ml. The rHMAb was also detected in the sera of pups fed by the transgenic females. Both the rHMAbs in the milk of transgenic mice and those in the sera of suckling pups were found to be active against hantaviruses, although the light chain of the antibody absorbed by the pups was modified by N-linked glycosylation.

Cloning by somatic nuclear transfer is an inefficient process in which many of the cloned animals died shortly after birth and displayed organ abnormalities. In an effort to determine the possible genetic causes of neonatal death and organ abnormalities, we have examined expression patterns of four genes that modified chromatin (DNMT1, PCAF, MeCP2 and EED) in six organs (heart, liver, spleen, lung, kidney and brain) of both neonatal death cloned bovines (n=9) and normal control calves produced by artificial insemination (Al) using real-time quantitative RT-PCR. The effect of the age of the fibroblast donor cell on the gene expression profiles was also investigated. Aberrant expressions of DNMT1 and PCAF were found in some studied tissues, but the expression of MeCP2 and EED had similar levels to those of the normal controls. The expression of DNMT1 showed a higher level in heart, liver and brain of both cloned bovines. A higher expression level of PCAF was seen in heart and liver of both cloned bovines, but a lower level was seen only in spleen of adult fibroblast (AF) cell-derived clones. Our results suggest that aberrant expression in gene that modified chromatins were found in cloned bovine tissues of neonatal death. Because DNMT1 and PCAF play an important role in DNA methylation and histone acetylation on nuclear chromatin respectively, and normal expression of DNMT1 and PCAF is needed for precious reprogramming of donor nuclear, the aberrant transcription patterns of DNMT1 and PCAF in these clones may contribute to the defects of organs reported in neonatal death of clones.

Cloning by somatic nuclear transfer is an inefficient process in which some of the cloned animals die shortly after birth and display organ abnormalities. In an effort to determine the possible genetic causes of neonatal death and organ abnormalities, we used real-time quantitative reverse transcription-polymerase chain reaction to examine expression patterns of eight developmentally important genes (PCAF, Xist, FGFR2, PDGFRa, FGF10, BMP4, Hsp70.1, and VEGF) in six organs (heart, liver, spleen, lung, kidney, and brain) of both cloned bovines that died soon after birth (n = 9) and normal control calves produced by artificial insemination. In somatic cloning of cattle, fibroblasts have often been used for doner nuclei, and the effect of the age of the fibroblast donor cells on gene expression profiles was investigated. Aberrant expressions of seven genes were found in these clones. The majority of aberrantly expressed genes were common in clones derived from adult fibroblast (AF) and in clones derived from fetal fibroblast (FF) compared to controls, whereas some genes were dysregulated either in AF cell-derived or in FF cell-derived clones. For the studied genes, kidney was the organ least affected by gene dysregulation, and heart was the organ most affected, in which five genes were aberrant. Most dysregulations (12 of 19) were up-regulation, but PDGFRa only showed down-regulation. VEGF, BMP-4, PCAF, and Hsp70.1 were extremely dysregulated, whereas the other four genes had a low level of gene dysregulation. Our results suggest that the aberrant gene expression occurred in most tissues of cloned bovines that died soon after birth. For each specific gene, aberrant expression resulting from nuclear transfer was tissue-specific. Because these genes play important roles in embryo development and organogenesis, the aberrant transcription patterns detected in these clones may contribute to the defects of organs reported in neonatal death of clones.

Summary Effective treatment of chronic myelogenous leukemia (CML) largely depends on the eradication of CML leukemic stem cells (LSCs). We recently showed that CML LSCs depend on Tcf1 and Lef1 factors for self-renewal. Using a connectivity map, we identified prostaglandin E1 (PGE1) as a small molecule that partly elicited the gene expression changes in LSCs caused by Tcf1/Lef1 deficiency. Although it has little impact on normal hematopoiesis, we found that PGE1 treatment impaired the persistence and activity of LSCs in a pre-clinical murine CML model and a xenograft model of transplanted CML patient CD34 + stem/progenitor cells. Mechanistically, PGE1 acted on the EP4 receptor and repressed Fosb and Fos AP-1 factors in a β-catenin-independent manner. Misoprostol, an FDA-approved EP4 agonist, conferred similar protection against CML. These findings suggest that activation of this PGE1-EP4 pathway specifically targets CML LSCs and that the combination of PGE1/misoprostol with conventional tyrosine-kinase inhibitors could provide effective therapy for CML.

The development of a hematopoietic reporter is crucial for determining the fate of lineages derived from cell-based therapies. A marking system will enable safer embryonic stem and induced pluripotent stem cell-based derivation of blood lineages and facilitate the development of efficient cellular reprogramming strategies based on direct fibroblast conversion. Here we report that the protein tyrosine phosphatase CD45 is an ideal candidate gene on which to base a hematopoietic reporter. CD45 regulatory elements were discovered by analyzing transcription factor chromatin occupancy (ChIP-seq) and promoter nuclease sensitivity (DNase-seq) to identify minimally sufficient sequences required for expression. After cloning the CD45 regulatory elements into an attenuated lentiviral backbone, we found that two transcriptional initiation regions were essential for high-level expression. Expressing CD45 promoters containing these regions and tethered to green fluorescent protein (GFP) in a primary B-cell differentiation assay and a transplantation model resulted in high levels of GFP in lymphoid, myeloid, and nucleated erythroid cells in mouse and human blood cell lineages. Moreover, GFP levels remained high 5 months after secondary transplantation, indicating persistence of the reporter. No CD45-driven GFP expression is observed after fibroblast or embryonic stem cell transduction. The GFP reporter is seen only after embryonic stem cells differentiate into hematopoietic cell progenitors and lineages, suggesting that this hematopoietic reporter system could be useful in validating potential autologous blood cell therapies.

Interleukin-7 receptor α chain (IL-7Rα)-derived signals are critical for normal T cell development, mature T cell homeostasis, and longevity of memory T cells. IL-7Rα expression in T cells is dynamically regulated at different developmental and antigen-responding stages. However, the molecular mechanism underlying the dynamic regulation is not completely understood. Here we describe generation of a bacterial artificial chromosome (BAC)-based reporter transgenic mouse strain, which contains 210 kb DNA sequence flanking the Il7r locus. We used in vitro validated EGFP reporter and insulator sequences to facilitate the reporter transgene expression. Consistent with endogenous IL-7Rα expression, the BAC transgene was expressed in mature T cells, a portion of natural killer cells but not in mature B cells. In the thymus, the EGFP reporter and endogenous IL-7Rα showed synchronized silencing in CD4(+)CD8(+) double positive stage, were both upregulated in CD4(+) or CD8(+) single positive thymocytes, and both continued to be co-expressed in naïve T cells in the periphery. Upon encountering antigen, the antigen-specific effector CD8(+) T cells downregulated both endogenous IL-7Rα and the EGFP reporter, which were upregulated in synchrony in antigen-specific memory CD8 T cells. These results indicate that the BAC-EGFP transgene reports endogenous IL-7Rα regulation with high fidelity, and further suggest that the 210 kb sequence flanking the Il7r locus contains sufficient genetic information to regulate its expression changes in T lineage cells. Our approach thus represents a critical initial step towards systematic dissection of the cis regulatory elements controlling dynamic IL-7Rα regulation during T cell development and cellular immune responses.

Maintaining a steady pool of self-renewing hematopoietic stem cells (HSCs) is critical for sustained production of multiple blood lineages. Many transcription factors and molecules involved in chromatin and epigenetic modifications have been found to be critical for HSC self-renewal and differentiation; however, their interplay is less understood. The transcription factor GA binding protein (GABP), consisting of DNA-binding subunit GABPα and transactivating subunit GABPβ, is essential for lymphopoiesis as shown in our previous studies. Here we demonstrate cell-intrinsic, absolute dependence on GABPα for maintenance and differentiation of hematopoietic stem/progenitor cells. Through genome-wide mapping of GABPα binding and transcriptomic analysis of GABPα-deficient HSCs, we identified Zfx and Etv6 transcription factors and prosurvival Bcl-2 family members including Bcl-2, Bcl-XL, and Mcl-1 as direct GABP target genes, underlying its pivotal role in HSC survival. GABP also directly regulates Foxo3 and Pten and hence sustains HSC quiescence. Furthermore, GABP activates transcription of DNA methyltransferases and histone acetylases including p300, contributing to regulation of HSC self-renewal and differentiation. These systematic analyses revealed a GABP-controlled gene regulatory module that programs multiple aspects of HSC biology. Our studies thus constitute a critical first step in decoding how transcription factors are orchestrated to regulate maintenance and multipotency of HSCs.

In this paper, we consider cooperative spectrum detection based on energy detection in distributed antenna systems. Soft combination of the observed energies from different cognitive radio users is investigated. Based on the Neyman-Pearson criterion, we obtain an optimal soft combination scheme that maximizes the detection probability for a given false alarm probability. We derive closed-form expressions for the probabilities of detection and false alarm for the optimal soft combination scheme. Finally, we show through numerical results the improvement in cooperative spectrum detection with the optimal soft combination.

T cell factor 1 (TCF-1) is a transcription factor known to act downstream of the canonical Wnt pathway and is essential for normal T cell development. However, its physiological roles in mature CD8(+) T cell responses are unknown. Here we showed that TCF-1 deficiency limited proliferation of CD8(+) effector T cells and impaired their differentiation toward a central memory phenotype. Moreover, TCF-1-deficient memory CD8(+) T cells were progressively lost over time, exhibiting reduced expression of the antiapoptotic molecule Bcl-2 and interleukin-2 receptor beta chain and diminished IL-15-driven proliferation. TCF-1 was directly associated with the Eomes allele and the Wnt-TCF-1 pathway was necessary and sufficient for optimal Eomes expression in naive and memory CD8(+) T cells. Importantly, forced expression of Eomes partly protected TCF-1-deficient memory CD8(+) T cells from time-dependent attrition. Our studies thus identify TCF-1 as a critical player in a transcriptional program that regulates memory CD8 differentiation and longevity.

GA binding protein (GABP) consists of GABPalpha and GABPbeta subunits. GABPalpha is a member of Ets family transcription factors and binds DNA via its conserved Ets domain, whereas GABPbeta does not bind DNA but possesses transactivation activity. In T cells, GABP has been demonstrated to regulate the gene expression of interleukin-7 receptor alpha chain (IL-7Ralpha) and postulated to be critical in T cell development. To directly investigate its function in early thymocyte development, we used GABPalpha conditional knock-out mice where the exons encoding the Ets DNA-binding domain are flanked with LoxP sites. Ablation of GABPalpha with the Lck-Cre transgene greatly diminished thymic cellularity, blocked thymocyte development at the double negative 3 (DN3) stage, and resulted in reduced expression of T cell receptor (TCR) beta chain in DN4 thymocytes. By chromatin immunoprecipitation, we demonstrated in DN thymocytes that GABPalpha is associated with transcription initiation sites of genes encoding key molecules in TCR rearrangements. Among these GABP-associated genes, knockdown of GABPalpha expression by RNA interference diminished expression of DNA ligase IV, Artemis, and Ku80 components in DNA-dependent protein kinase complex. Interestingly, forced expression of prearranged TCR but not IL-7Ralpha can alleviate the DN3 block in GABPalpha-targeted mice. Our observations collectively indicate that in addition to regulating IL-7Ralpha expression, GABP is critically required for TCR rearrangements and hence normal T cell development.

T cell factor-1 (TCF-1) and lymphoid enhancer-binding factor 1, the effector transcription factors of the canonical Wnt pathway, are known to be critical for normal thymocyte development. However, it is largely unknown if it has a role in regulating mature T cell activation and T cell-mediated immune responses. In this study, we demonstrate that, like IL-7Rα and CD62L, TCF-1 and lymphoid enhancer-binding factor 1 exhibit dynamic expression changes during T cell responses, being highly expressed in naive T cells, downregulated in effector T cells, and upregulated again in memory T cells. Enforced expression of a p45 TCF-1 isoform limited the expansion of Ag-specific CD8 T cells in response to Listeria monocytogenes infection. However, when the p45 transgene was coupled with ectopic expression of stabilized β-catenin, more Ag-specific memory CD8 T cells were generated, with enhanced ability to produce IL-2. Moreover, these memory CD8 T cells expanded to a larger number of secondary effectors and cleared bacteria faster when the immunized mice were rechallenged with virulent L. monocytogenes. Furthermore, in response to vaccinia virus or lymphocytic choriomeningitis virus infection, more Ag-specific memory CD8 T cells were generated in the presence of p45 and stabilized β-catenin transgenes. Although activated Wnt signaling also resulted in larger numbers of Ag-specific memory CD4 T cells, their functional attributes and expansion after the secondary infection were not improved. Thus, constitutive activation of the canonical Wnt pathway favors memory CD8 T cell formation during initial immunization, resulting in enhanced immunity upon second encounter with the same pathogen.

One of the major drawbacks in Orthogonal Frequency Division Multiplexing (OFDM) is its high Peak-to-Average Power Ratio (PAR). Clipping and filtering is the simplest and most effective method to reduce PAR for OFDM. But clipping and filtering gives rise to serious distortion which seriously degrades the bit error rate (BER) performance. Mitigating the disadvantages while inheriting the advantages of clipping and filtering, a new scheme called clipping and clipped power re-allocation with filtering is proposed for PAR reduction in this paper. The proposed scheme re-allocates the clipped power to other low-power time-domain symbols in the same OFDM block to reduce the distortion. Simulation results show that the BER performance of the proposed scheme is much better than conventional clipping and filtering technique and the PAR performance is slightly better than clipping and filtering. Besides, the proposed scheme is compatible with existing OFDM standards since no changes are required in the receiver structure.

A highly efficient cloning vector was constructed for cloning PCR products by inserting an 80 bp DNA fragment into pGEM-5zf (+) vector. The Xcm I digestion of this vector gave rise to a 3′ overhanging deoxythymidine offering the possibility of cloning PCR products with 3′ adenosine overhang created by Taq DNA polymerase. Furthermore, two EcoR I sites were added to the construct for identification of recombinant plasmids using a single restriction enzyme. Taken together, the more efficient cloning performance and the lower cost of this vector as compared to the commercial T vector, suggests that it may be one of the best T vectors for cloning of PCR products.

Software-defined radio (SDR) can support multiple communication standards and provide flexible services. In this paper, a feasible radio frequency (RF) bandpass sampling architecture of single-channel SDR receiver is proposed. The tradeoff between the sampling frequency and resolution of the analog-to-digital converter (ADC) and the selectivity and tunability of the bandpass filter provides a natural way to alleviate the design challenge of RF bandpass filter. The passband of the bandpass filter can be wider than the bandwidth of the desired signal, resulting in increasing the sampling frequency and resolution. The minimum acceptable sampling frequency is based on the bandwidth of the signal and the passband of the bandpass filter. The out-of-band interferences are further attenuated in digital domain. The signal can be decimated by sample rate conversion after it has been converted to baseband to reduce the processing rate.

The neonatal Fc receptor (FcRn) protects immunoglobulin G (IgG) from catabolism and is also responsible for IgG absorption in the neonatal small intestine. However, whether it mediates the transfer of IgG from plasma to milk still remains speculative. In the present study, we have generated transgenic mice that over-express the bovine FcRn (bFcRn) in their lactating mammary glands. Significantly increased IgG levels were observed in the sera and milk from transgenic animals, suggesting that the over-expressed bFcRn could bind and protect endogenous mouse IgG and thus extend its lifespan. We also found that injected human IgG showed a significantly longer half-life (7-8 days) in the transgenic mice than in controls (2.9 days). Altogether, the data suggested that bFcRn could bind both mouse and human IgG, showing a cross-species FcRn-IgG binding activity. However, we found no selective accumulation of endogenous mouse IgG or injected bovine IgG in the milk of the transgenic females, supporting a previous hypothesis that IgG was transported from serum to milk in an inverse correlation to its binding affinity to FcRn.

Based on the known partial cDNA sequence of dragline silk protein an artificial gene monomer, a 360 bp sequence, was designed and polymerized to encode an analog of dragline silk protein. Six tandem copies of monomer were cloned into pBC1 vector and microinjected into the pronuclei of fertilized Kunming White eggs. Transgenic mice were screened by Polymerase Chain Reaction (PCR) and Southern blot which revealed that 10 mice (5 male, 5 female) among 58 mice were transgenic positive. Milk of five F0 mice and eight F1 mice was analyzed by Western blot, and two F0 mice and seven F1 mice expressed recombinant dragline silk protein. In transgenic mice milk a maximum of concentration of recombinant dragline silk protein was 11.7 mg/L by radioimmunoassay.

Only three Ig isotypes, IgM, IgX, and IgY, were previously known in amphibians. Here, we describe a heavy-chain isotype inXenopus tropicalis, IgF (encoded by Cφ), with only two constant region domains. IgF is similar to amphibian IgY in sequence, but the gene contains a hinge exon, making it the earliest example, in evolution, of an Ig isotype with a separately encoded genetic hinge. We also characterized a gene for the heavy chain of IgD, located immediately 3′ of Cμ, that shares features with the Cδgene in fish and mammals. The latter gene contains eight constant-region-encoding exons and, unlike the chimeric splicing of μCH1 onto the IgD heavy chain in teleost fish, it is expressed as a unique IgD heavy chain. The IgH locus ofX. tropicalisshows a 5′ VH-DH-JH-Cμ-Cδ-Cχ-Cυ-Cφ3′ organization, suggesting that the mammalian and amphibian Ig heavy-chain loci share a common ancestor.

Human milk lysozyme is an important protein for innate immunity, but human breast milk is a fairly poor source for commercial production of this enzyme. Research on the expression of recombinant human lysozyme (rHlys) is therefore potentially valuable to the dairy industry. In this study, 2 different kinds of transgenic mice, PBC-hLY and PBC-sighLY, were generated and used as system models to express rHlys. Six lines of PBC-hLY transgenic mice with human lysozyme genomic DNA-based constructs were generated, and a maximum expression level of rHlys approaching 0.154 mg/mL was achieved. Antibacterial activity of the whey from PBC-hLY female transgenic mice was determined by a turbidimetric assay. Results showed that antibacterial activity of the whey was strongly enhanced, and confirmed that rHlys retained full activity. For rHlys to be secreted efficiently into the milk of transgenic mice, 5 lines of mice were also generated, in which the signal peptide DNA of bovine beta-casein was substituted for that of lysozyme in PBC-hLY transgenic mice. Compared with PBC-hLY transgenic mice, both the expression levels of rHlys and the antibacterial activity of the whey were much higher in the PBC-sighLY transgenic mice. The concentration of rHlys in one of these mice amounted to 1.405 mg/mL-3 times higher than the level in human whey. The antibacterial activity of the whey was also 3 times higher than that of human whey. The rHlys from both PBC-hLY and PBC-sighLY transgenic mice had the same antibacterial activity as human milk lysozyme. The effect of the signal peptide and copy numbers of the transgene on expression of rHlys was also evaluated. This work will certainly permit a better understanding of how mammary gland bioreactor systems can be applied to produce rHlys in other mammals, such as cattle.

Myostatin (GDF8, MSTN) is a member of the transforming growth factor beta superfamily that is essential for proper regulation of skeletal muscle mass. In order to study its expression and regulatory mechanism deeply, we have presented a comparative analysis of about 170-kb pig BAC sequence containing the myostatin gene among pig, human and mouse. The genomic region is characterized by high interspersed repeats and low G+C content. As for the myostatin gene, a higher sequence similarity is found between human and pig than between these species and the mouse. One striking feature is that the structure of two TATA-boxes in the nearby downstream of CCAAT-box is identified in the promoter. Further analysis reveals that the TATA-box1 is responsible for the transcription in pig and human, but the TATA-box2 acts on the transcription in mouse. The other interesting feature is that two polyadenylation signal sequences (AATAAA) exist in 3'UTR of the pig myostatin gene. Moreover, a large number of potential transcription factor-binding sites are also identified in evolutionary conserved regions (ECRs), which may be associated with the regulation of myostatin. Many putative transcription factors play an important role in the muscle development, and the complex interaction between myostatin and these factors may be required for proper muscle development.

To the editor: Notch-derived signals are essential for specification of hematopoietic progenitors to T-cell lineage and for promotion of β-selection at the CD4–CD8– double-negative 3 (DN3) stage. However, these signals are not required for further thymocyte maturation.[1][1] Accordingly, the

Cloning by somatic nuclear transfer is an inefficient process in which some of the cloned animals die shortly after birth and display organ abnormalities. In an effort to determine the possible genetic causes of neonatal death and organ abnormalities, we used real-time quantitative reverse transcription-polymerase chain reaction to examine expression patterns of eight developmentally important genes (PCAF, Xist, FGFR2, PDGFRa, FGF10, BMP4, Hsp70.1, and VEGF) in six organs (heart, liver, spleen, lung, kidney, and brain) of both cloned bovines that died soon after birth (n=9) and normal control calves produced by artificial insemination. In somatic cloning of cattle, fibroblasts have often been used for doner nuclei, and the effect of the age of the fibroblast donor cells on gene expression profiles was investigated. Aberrant expressions of seven genes were found in these clones. The majority of aberrantly expressed genes were common in clones derived from adult fibroblast (AF) and in clones derived from fetal fibroblast (FF) compared to controls, whereas some genes were dysregulated either in AF cell-derived or in FF cell-derived clones. For the studied genes, kidney was the organ least affected by gene dysregulation, and heart was the organ most affected, in which five genes were aberrant. Most dysregulations (12 of 19) were up-regulation, but PDGFRa only showed down-regulation. VEGF, BMP-4, PCAF, and Hsp70.1 were extremely dysregulated, whereas the other four genes had a low level of gene dysregulation. Our results suggest that the aberrant gene expression occurred in most tissues of cloned bovines that died soon after birth. For each specific gene, aberrant expression resulting from nuclear transfer was tissue-specific. Because these genes play important roles in embryo development and organogenesis, the aberrant transcription patterns detected in these clones may contribute to the defects of organs reported in neonatal death of clones.

During late stages of T cell development, positively selected CD4+CD8+ (DP) thymocytes must make a lineage choice decision to become MHC-I-restricted cytotoxic CD8+ T cells or MHC-II-restricted helper CD4+ T cells. This lineage choice process is determined by extrinsic signals such as TCR and γc cytokines and by intrinsic transcriptional factors such as Myb, Gata3, ThPOK and Runx. The canonical Wnt effector transcription factors, TCF-1 and LEF-1, are essential for the transition from CD4-CD8- (DN) to the DP stage, but their requirement beyond the DP stage is unknown. Here we show that deficiency in TCF-1 resulted in a preferential loss of CD4+ T cells and reduced CD4/CD8 ratio, and this phenotype was exacerbated upon loss of both TCF-1 and LEF-1. By crossing TCF-1-/- mice to MHC-I-deficient strain or OT-2 CD4+ TCR transgenic strain, we found that DP thymocytes selected on MHC-II were re-directed to the CD8 lineage in the absence of TCF-1 and/or LEF-1. Mechanistically, we observed that Myb expression is greatly diminished in post-select DP thymocytes lacking TCF-1 and/or LEF-1, and that activation of the Wnt signaling pathway in post-select DP thymocytes induced Myb expression. Collectively, these findings revealed a novel role of Wnt effectors TCF-1 and LEF-1 in promoting CD4+ lineage choice, which is likely mediated by Wnt-dependent upregulation of Myb.

GA binding protein (GABP) consists of GABPalpha and GABPbeta subunits. GABPalpha is a member of Ets family transcription factors and binds DNA via its conserved Ets domain, whereas GABPbeta does not bind DNA but possesses transactivation activity. In T cells, GABP has been demonstrated to regulate the gene expression of interleukin-7 receptor alpha chain (IL-7Ralpha) and postulated to be critical in T cell development. To directly investigate its function in early thymocyte development, we used GABPalpha conditional knockout mice where the exons encoding the Ets DNA-binding domain are flanked with LoxP sites. Ablation of GABPalpha with the Lck-Cre transgene greatly diminished thymic cellularity, blocked thymocyte development at the double negative (DN)-3 stage, and resulted in reduced expression of T cell receptor (TCR) beta chain in DN4 thymocytes. By chromatin immunoprecipitation, we demonstrated in DN thymocytes that GABPalpha is associated with transcription initiation sites of genes encoding key molecules in TCR rearrangements. Among these GABP-associated genes, knockdown of GABPalpha expression by RNA interference diminished expression of DNA ligase IV, Artemis, and Ku80 components in DNA-dependent protein kinase complex. Interestingly, forced expression of pre-arranged TCR but not IL-7Ralpha can alleviate the DN3 block in GABPalpha-targeted mice. Our observations collectively GABP is critically required for TCR rearrangements and hence normal T cell development.

To evaluate the effects of animal milk containing fucosylated antigens on Helicobacter pylori (H. pylori) binding to Lewis b antigen.A mammary gland expression vector containing human alpha1-3/4-fucosyltransferase cDNA sequences was constructed. Transient expression of human alpha1-3/4-fucosyltransferase cDNA in goat mammary cell and establishment of transgenic mice were performed. The adhesion inhibitory properties of milk samples were analyzed by using H. pylori.Goat milk samples were found to inhibit bacterial binding to Lewis b antigen. The highest inhibition was observed 42 h after injection of the plasmid. The binding activity of H. pylori to Lewis b antigen reduced mostly, by 83%, however milk samples from transgenic mice did not inhibit H. pylori binding to Lewis b antigen.The use of "humanized" animal milk produced by the transgenic introduction of fucosylated antigen can perhaps provide an alternative therapy and preventive measure for H. pylori infection.