Highly Sensitive Weaving Sensor of Hybrid Graphene Nanoribbons and Carbon Nanotubes for Enhanced Pressure Sensing Function.

Carbon nanotubes (CNTs) hold great promise in next-generation sensors because of their remarkable physical properties. Yet, maintaining precise stacking configurations of CNTs to make full use of their remarkable properties is challenging because of their susceptibility to spontaneous reconstruction. Inspired by the weaving technology, we propose a CNT-graphene nanoribbon hybrid woven model that can maintain the specific structure of CNTs to achieve their elaborately designed function. In this study, comprehensive molecular dynamics simulations are carried out to investigate the thermal stability of the CNT-graphene hybrid woven model, as well as their potential for pressure sensing applications by utilizing the unique response of thermal transport to mechanical deformation at heterojunctions. The thermal stability is sensitive to the size of the graphene nanoribbon, and the woven structure remains stable from 200-500 K when its width is greater than 2.0 nm. Moreover, it is exciting that the sensors are effective at predicting the shapes of externally loaded objects through the analysis of the thermal conductivity distribution, which can be derived from the relationship between the thermal conduction and the pressure. Our findings shed light on the bottom-up functional design of nanomaterials and expand wider applications of high-performance nanosensors in other related fields.

Bioinformatics analysis identifies potential biomarkers involved in the metastasis of locoregionally advanced nasopharyngeal carcinoma.

Nasopharyngeal carcinoma (NPC) is one of the malignant epithelial tumors with a high metastasis rate. This study aimed to screen potential novel biomarkers involved in NPC metastasis. Microarray data of locoregionally advanced NPC (LA-NPC; GSE103611) were obtained from the database of Gene Expression Omnibus. The differentially expressed genes (DEGs) between LA-NPC tissues with and without distant metastasis after radical treatment were screened. Functional analysis was performed and the protein-protein interaction and submodule were analyzed. The univariate Cox regression analysis was performed to identify prognostic genes in NPC in the validation microarray dataset GSE102349. The drug-gene interactions and key genes were identified. Totally, 107 DEGs were identified. The upregulated DEGs and the key nodes in the protein-protein interaction network were associated with pathways or biological processes related to the cell cycle. Four genes including CD44, B2M, PTPN11, and TRIM74 were associated with disease-free survival in NPC. The drug-gene interaction analysis revealed that upregulated genes CXCL10, CD44, B2M, XRCC5, and RPL11 might be potential druggable genes for patients with LA-NPC metastasis by regulating cell cycle, autophagy, and drug resistance. Upregulated CXCL10, CD44, B2M, XRCC5, and RPL11 might play important roles in LA-NPC metastasis by regulating cell cycle-related pathways.

Water flow inside various geometric nano-confinement channels.

In nano-confined systems, the properties of a fluid are different from those of macroscopic systems, and the properties of a nanotube can significantly affect water transport. However, our knowledge of the effects of nanotube shape is far from adequate. In the present work, we study the properties of a fluid transporting in different nano-confined configurations by molecular dynamics simulations. This study is aimed at gaining insight into the transport of water molecules in carbon nanotubes with different configurations. We find that the closer of channel shape to the circular nanotube (more sides of the channel), the lower friction coefficient of the solid-liquid interface has and the friction coefficient of nanochannels increases with R when R < 1.0 nm. The friction coefficient converges to a stable value (close to the friction coefficient of graphene/water) when R > 1.0 nm. A variety of configurations leads to the variation of the fluid properties in nanotubes. Our results can be applied to the nanofluid properties of a complex channel structure and water nanochannel microscopic design.

High levels of plasma selenium are associated with metabolic syndrome and elevated fasting plasma glucose in a Chinese population: A case-control study.

BACKGROUND: Selenium is important for human health and involved in various metabolic processes. Deficiency of selenium associates with increased risk for cancer and cardiovascular diseases. There has been an increase use of selenium supplements for the treatment of autoimmune thyroid conditions. However, the potential biological effects of selenium overload arouse the public concern. The aim of this study was to investigate the associations of plasma selenium concentrations of adults with metabolic syndrome (MS) in Chinese population. METHODS: A matched case-control study including 204 metabolic syndrome patients and 204 healthy controls was conducted in 2012. The MS cases were defined according to the criteria of Chinese Diabetes Society (CDS). Healthy controls without abnormality of metabolic components were matched with cases in age, gender and region. Plasma concentrations of selenium were determined by graphite furnace atomic absorption spectrometry (GFAAS). Fasting plasma glucose (FPG), total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (HDL), and low density lipoprotein cholesterol (LDL) were detected by automatic biochemical analyzer. RESULTS: The median levels of plasma selenium in MS group were 146.3 (107.3-199.4)µg/L, which were significantly higher than that in the control group (127.4: 95.7-176.0)µg/L; Plasma levels of selenium were related to the risk of MS in dose-response manner. Risk of MS was significantly higher in subjects with plasma selenium in the highest tertile (T3: ≥176.0µg/L) compared to those in the lowest tertile (T1: <95.7µg/L) [odds ratio (OR)=2.416 (95% CI: 1.289-4.526)]. The plasma levels of selenium were positively correlated with fasting plasma glucose (FPG) (rs=0.268, P<0.001). Plasma selenium at the median (T2: 95.7-176.0µg/L) or upper tertile (T3: ≥176.0µg/L) was associated with increased risk of elevated FPG (defined by FPG≥6.1mmol/L) as compared with the lowest tertile (T1: ≤95.7µg/L) [T2 vs. T1, OR=3.487 (1.738-6.996); T3 vs. T1, OR=6.245 (3.005-12.981)]. CONCLUSIONS: Higher levels of plasma selenium might increase the risk of metabolic syndrome and elevated fasting plasma glucose. Selenium supplements should be used with prudence for CVD and cancer prevention.

Establishment of a rapid and scalable gene expression system in livestock by site-specific integration.

Somatic cell-mediated transgenesis is routinely used to transfer exogenous genes to livestock genomes. However, transgene insertion events are essentially random which may lead to transgene silencing or alter animal phenotype because of insertional mutagenesis. To overcome these problems, we established a gene manipulation system in goat somatic cells based on homologous recombination and flp recombinase-mediated site-specific integration. First, we performed gene targeting to introduce an frt-docking site into the α1 (I) procollagen (ColA1) locus in goat somatic cells. Second, the targeted cell clones were rejuvenated by embryo cloning, and the vigorous cells with targeted frt were reestablished. Third, a gene-replacement system was used to introduce an EGFP reporter gene into the targeted ColA1 locus via flp mediated recombination. As a result, the transgenic somatic cell exhibited faithful expression of EGFP gene under control of the CMV promoter. Similarly, other expression vectors can be introduced into the defined site to evaluate gene functions or express valuable proteins. The gene manipulation system described here will be applicable in other livestock somatic cells, and would allow for the rapid generation of livestock with transgene targeted to the defined site.

Large-scale production of functional human lysozyme in transgenic cloned goats.

Human lysozyme (hLZ), an essential protein against many types of microorganisms, has been expressed in transgenic livestock to improve their health status and milk quality. However, the large-scale production of hLZ in transgenic livestock is currently unavailable. Here we describe the generation of transgenic goats, by somatic cell-mediated transgenic cloning, that express large amounts of recombinant human lysozyme (rhLZ) in milk. Specifically, two optimized lysozyme expression cassettes (ß-casein/hLZ and ß-lactoglobulin/hLZ) were designed and introduced into goat somatic cells by cell transfection. Using transgenic cell colonies, which were screened by 0.8 mg/mL G418, as a nuclear donor, we obtained 10 transgenic cloned goats containing one copy of hLZ hybrid gene. An ELISA assay indicated that the transgenic goats secreted up to 6.2 g/L of rhLZ in their milk during the natural lactation period, which is approximately 5-10 times higher than human milk. The average rhLZ expression levels in ß-casein/hLZ and ß-lactoglobulin/hLZ transgenic goats were 2.3 g/L and 3.6 g/L, respectively. Therefore, both rhLZ expression cassettes could induce high levels of expression of the rhLZ in goat mammary glands. In addition, the rhLZ purified from goat milk has similar physicochemical properties as the natural human lysozyme, including the molecular mass, N-terminal sequence, lytic activity, and thermal and pH stability. An antibacterial analysis revealed that rhLZ and hLZ were equally effective in two bacterial inhibition experiments using Staphylococcus aureus and Escherichia coli. Taken together, our experiments not only underlined that the large-scale production of biologically active rhLZ in animal mammary gland is realistic, but also demonstrated that rhLZ purified from goat milk will be potentially useful in biopharmaceuticals.

[Deletion of marker gene in transgenic goat by Cre/LoxP system].

In producing transgenic livestock, selectable marker genes (SMGs) are usually used to screen transgenic cells from numerous normal cells. That results in SMGs integrating into the genome and transmitting to offspring. In fact, SMGs could dramatically affect gene regulation at integration sites and also make the safety evaluation of transgenic animals complicated. In order to determine the deletion time and methods in the process of producing transgenic goats, the feasibility of deleting SMGs was explored by Cre/LoxP before or after somatic cell cloning. In addition, we compared the efficiency of protein transduction with plasmids co-transduction. We could delete 43.9% SMGs after screening out the transgenic cell clones, but these cells could not be applied to somatic cells cloning because of serious aging after two gene modifications. The SMG-free cells suitable for nuclear transfer were accessible by using the cells of transgenic goats, but this approach was more time consuming. Finally, we found that the Cre plasmid could delete SMGs with an efficiency of 7.81%, but about 30% in SMG-free cells had sequences of Cre plasmid. Compared with Cre plasmid, the integration of new exogenous gene could be avoided by TAT-CRE protein transduction, and the deletion rate of TAT-CRE transduction was between 43.9 and 72.8%. Therefore, TAT-Cre transduction could be an effective method for deleting selectable marker genes.

The dominant expression of functional human lactoferrin in transgenic cloned goats using a hybrid lactoferrin expression construct.

Human Lactoferrin (hLF) is an iron-binding protein with multiple physiological functions. As the availability of natural hLF is limited, alternative means of producing this biopharmaceutical protein have been extensively studied. Here we report on the dominant expression of recombinant human lactoferrin (rhLF) in transgenic cloned goats using a novel optimised construct made by fusing a 3.3 kb hLF minigene to the regulatory elements of the ß-casein gene. The transgenic goat produced more than 30 mg/ml rhLF in its milk, and rhLF expression was stable during the entire lactation cycle. The rhLF purification efficiency from whole goat milk is approximately 70%, and its purity is above 98%. Compared with natural hLF, the rhLF from transgenic goats has similar biological characteristics including molecular mass, N-terminal sequence, isoelectric point, immunoreactivity and digestive stability. More importantly, the purified rhLF showed specific anti-tumour activity in the mouse model of melanoma experimental metastasis. Therefore, our study shows that the large-scale production of functional rhLF in transgenic goat milk could be an economical and promising source of human therapeutic use in the future.

Production of Prnp-/- goats by gene targeting in adult fibroblasts.

Homozygous mice devoid of functional Prnp are resistant to scrapie and prion propagation, but heterozygous mice for Prnp disruption still suffer from prion disease and prion deposition. We have previously generated heterozygous cloned goats with one allele of Prnp functional disruption. To obtain goats with both alleles of Prnp be disrupted which would be resistant to scrapie completely, a second-round gene targeting was applied to disrupt the wild type allele of Prnp in the heterozygous goats. By second-round gene targeting, we successfully disrupted the wild type allele of Prnp in primary Prnp (+/-) goat skin fibroblasts and obtained a Prnp (-/-) cell line without Prnp expression. This is the first report on successful targeting modification in primary adult somatic cells of animals. These cells were used as nuclear donors for somatic cell cloning to produce Prnp (-/-) goats. A total of 57 morulae or blastocytes developed from the reconstructed embryos were transferred to 31 recipients, which produced 7 pregnancies at day 35. At 73 days of gestation, we obtained one cloned fetus with Prnp (-/-) genotype. Our research not only indicated that multiple genetic modifications could be accomplished by multi-round gene targeting in primary somatic cells, but also provided strong evidence that gene targeting in adult cells other than fetal cells could be applied to introduce precise genetic modifications in animals without destroying the embryos.

Goat MII ooplasts support preimplantation development of embryos cloned from other species.

The preimplantation development competences of somatic cell nuclear transfer (SCNT) embryos reconstructed with enuleated goat (Capra hircus) Metaphase II (MII) oocytes matured in vivo and whole cells derived from adult fibroblasts of several mammalian species (goat, boer goat, bovine, tahr, panda) and human patient were evaluated. Results obtained from our experiments revealed that these reconstructed SCNT embryos could complete preimplantation development to form blastocysts. The fusion rate and blastocyst rate of intra-species SCNT embryos (Capra hircus as control) was 78.67 (557/708); 56.29% (264/469), that of sub-species or inter-species SCNT embryos were: boer goat 78.18% (541/692); 33.90% (40/118), bovine 70.53% (146/207); 22.52% (25/111), tahr 53.51% (61/114); 5.26% (3/570), panda 79.82% (1159/1452); 8.35% (75/898) and human 68.76% (317/461); 5.41% (16/296), respectively. It is concluded that (1) there are no relationships between fusion rate and relativeness of the recipient cytoplasm to nucleus donor cells, (2) cytoplast of the goat MII oocyte can support the preimplantation development of SCNT embryos reconstructed with nucleus from other species, (3) the blastocyst rate of close relative inter-species SCNT embryos is higher than that of distant relative inter-species SCNT embryos.

Excision of selectable genes from transgenic goat cells by a protein transducible TAT-Cre recombinase.

The Cre/loxP site-specific recombination system is a widely used tool for genetic engineering of mammalian genomes. Recombination of loxP-modified alleles is often induced by introduction of foreign DNA vector expressing Cre into the cells. But the introduced DNA vector has the potential to integrate into the genome of the cells and continuous expression of Cre recombinase from the foreign vector has the potential to yield cytotoxicity and genotoxicity in various cells. In this study, we investigate the possibility of overcoming this limitation by using a cell-permeable TAT-Cre recombinase. We found that TAT-Cre treatment of transgenic goat fibroblast cells did not compromise the development competency of reconstructed embryos by using these TAT-Cre-treated cells as nuclear donor in nuclear transfer. Finally, we obtained two live cloned goats where a selectable gene cassette was removed. Our work not only provided an efficient protein transduction-based system for removing selectable genes from transgenic goats, but also presented strong evidence that no severe damage was made to the host cells during the process of protein transduction.

Expression of active recombinant human lactoferrin in the milk of transgenic goats.

This report details the establishment of a transgenic goat model in order to produce human lactoferrin (hLf) in the mammary gland for large-scale application and research. Two transgenic male goats were generated by microinjecting sequence encoding hLf cDNA to the pronuclear. In the two lines, derived from the two founders, eight lactating female goats could secrete recombinant human lactoferrin (rhLf) at concentrations of up to 0.765 mg/ml. The method of purifying the rhLf from the milk was achieved using ion-exchange chromatography and resulted in 97% purity. Biochemical and physicochemical characteristics of rhLf were similar to native lactoferrin (nhLf); this included N-terminal sequence, isoelectric point, molecular mass, glycosylation, iron-binding/releasing ability, thermal stability, and proteolysis. The rhLf showed broad spectrum antibacterial activity inhibiting the growth of several pathogenic bacterial strains. Also investigated, although to a lesser degree, was a practicable pasteurization method for the downstream processing of rhLf and, further, a method for the oral administration of rhLf. On the basis of these results, our studies show an optimistic and promising approach for the large-scale production and therapeutic application of rhLf expressed in transgenic goats.

Factors derived from mouse embryonic stem cells promote self-renewal of goat embryonic stem-like cells.

Goat embryonic stem (ES)-like cells could be isolated from primary materials-inner cell masses (ICMs) and remain undifferentiated for eight passages in a new culture system containing mouse ES cell conditioned medium (ESCCM) and on a feeder layer of mouse embryo fibroblasts (MEFs). However, when cultured in medium without mouse ESCCM, goat ES-like cells could not survive for more than three passages. In addition, no ES-like cells could be obtained when ICMs were cultured on goat embryo fibroblasts or the primary materials-whole goat blastocysts were cultured on MEFs. Goat ES-like cells isolated from ICMs had a normal karyotype and highly expressed alkaline phosphatase. Multiple differentiation potency of the ES-like cells was confirmed by differentiation into neural cells and fibroblast-like cells in vitro. These results suggest that mouse ES cells might secrete factors playing important roles in promoting goat ES-like cells' self-renewal, moreover, the feeder layers and primary materials could also influence the successful isolation of goat ES-like cells.

Functional disruption of the prion protein gene in cloned goats.

The cellular prion protein (PrPC), a membrane glycoprotein anchored to the outer surface of neurons, lymphocytes and other cells, is associated directly with the pathogenesis of the transmissible spongiform encephalopathies (TSEs) occurring mainly in humans, cattle, sheep and goats. Although mice lacking PrPC develop and reproduce normally and are resistant to scrapie infection, large animals lacking PrPC, especially those species in which TSE occurs naturally, are currently not available. Here, five live PRNP+/- goats cloned by gene targeting are reported. Detailed RNA-transcription and protein-expression analysis of one PRNP+/- goat showed that one allele of the caprine PRNP gene had been disrupted functionally. No gross abnormal development or behaviour could be seen in these PRNP+/- goats up to at least 3 months of age. These heterozygous PRNP+/- goats are ready to be used in producing homozygous PRNP-/- goats in which no PrPC should be expressed.