To investigate the anchoring of plasmid DNA/anti-DNA antibody/cationic lipid tri-complex (DAC micelles) onto bisphosphonate-modified 316 L coronary stents for cardiovascular site-specific gene delivery.

The objective of this experiment was to evaluate the effect of dietary lysine concentration on meat quality and carcass traits in 2 chicken lines, SD02 and SD03, and their crosses, both originating from a Chinese native breed, the Erlang Mountainous chicken. The lines were selected for 4 generations by Sichuan Agricultural University (Ya'an, China); for the present study, chickens from the 2 lines and their crosses were randomly assigned at hatch to 1 of 2 dietary groups. One group was offered diets containing 1, 0.85, and 0.70% total lysine, whereas the other was offered diets with 1.15, 1, and 0.85% total lysine from d 1 to 28, d 29 to 49, and d 50 to 70, respectively. In total, 252 chickens were commercially processed at 70 d old. Traits measured included live BW, subcutaneous fat thickness, weight of carcass, eviscerated carcass, semi-eviscerated carcass, breast muscle (left pectoralis major and minor), leg muscle (boneless left drum plus thigh), heart, gizzard, proventriculus, spleen, liver, comb, and abdominal fat, color parameters lightness, redness, or yellowness (L*, a*, and b*), pH, and breast muscle intramuscular fat content. The results indicated that, although dietary lysine concentration did not affect subcutaneous fat thickness, color parameters, pH, intramuscular fat content, and organ weights, there were effects on feed conversion and muscle and BW (P < 0.05). Males and females displayed major differences in feed conversion, BW, muscle growth, and organ weight. The Line SD02 chickens grew faster and displayed less fat deposition and superior feed conversion compared with Line SD03 and the reciprocal crosses. In conclusion, performance of the chicken stocks evaluated in this study differs substantially in muscle weight and carcass weight.

Bone tissue engineering is a new approach for the repair of orbital defects. The aim of the present study was to evaluate prefabricated beta-tricalcium phosphate (β-TCP) combined with autologous bone marrow stromal cells (BMSCs) to repair orbital wall defect in canine models. Defects measuring 10 mm in diameter were created in the orbital medial walls of 12 dogs. The orbits were randomly divided into five groups: group 1, repaired with osteogenesis-induced BMSCs/TCP constructs; group 2, repaired with noninduced BMSCs/TCP constructs; group 3, repaired with β-TCP scaffolds only; group 4, normal group; group 5, negative control (bone defect without treatment). Computed tomography (CT) scanning, gross observation, bone density measurements, micro-CT, and histological observations were performed. In group 1, new bone was observed with only a small amount of residual material, and bony union was achieved 3 months after surgery. In contrast, the constructs showed slow degradation with minimal bone formation in groups 2 and 3. Furthermore, the appearance and bone density of the constructs in group 1 were similar to that of normal bone: the constructs were covered with complete mucosa, and new alveolate plate grew into the ethmoidal sinuses. A large bone defect remained in group 5. This study demonstrated that biologic scaffolds composed of β-TCP and osteogenesis-induced BMSCs have been successfully used to restore bone functionality in animal models, which may provide a potential clinical approach for orbital wall repair and bone regeneration in humans. © 2013 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 2013.

Manure fertilizers are widely used in agriculture and highly impacted the soil microbial communities such as ammonia oxidizers. However, the knowledge on the communities of archaeal versus bacterial ammonia oxidizers in paddy soil affected by manure fertilization remains largely unknown, especially for a long-term influence. In present work, the impact of manure fertilization on the population of ammonia oxidizers, related potential nitrification rates (PNRs) and the key factors manipulating the impact were investigated through studying two composite soil cores (long-term fed with manure fertilization versus undisturbed). Moreover, soil incubated with NH4 (+) for 5 weeks was designed to verify the field research. The results showed that the copy numbers of bacterial amoA gene in the manure fed soil were significant higher than those in the unfed soil (p < 0.05), suggesting a clear stimulating effect of long-term manure fertilization on the population of ammonia-oxidizing bacteria (AOB). The detected PNRs in the manure fed soil core (14-218 nmol L(-1)  N g(-1)  h(-1) ) were significant higher than those in the unfed soil core (5-72 nmol L(-1)  N g(-1)  h(-1) ; p < 0.05). Highly correlations between the PNRs and the bacterial amoA gene copies rather than archaeal amoA gene were observed, indicating strong nitrification capacity related to bacterial ammonia oxidizers. The NH4 (+) -N significantly correlated to the abundance of AOB (p < 0.01) and explained 96.1% of the environmental variation, showing the NH4 (+) -N was the main factor impacting the population of AOB. The incubation experiment demonstrated a clear increase of the bacterial amoA gene abundance (2.0 × 10(6) to 8.4 × 10(6)  g(-1) d.w.s. and 1.6 × 10(4) to 4.8 × 10(5)  g(-1) d.w.s.) in both soil but not for the archaeal amoA gene, in agreement with the field observation. Overall, our results suggested that manure fertilization promoted the population size of bacterial ammonia oxidizers rather than their archaeal counterparts whether in long-term or short-term usage and the NH4 (+) -N was the key impact factor.

OBJECTIVE: To develop a reliable and specific method for rapid prenatal diagnosis of Trisomy 21 (Down syndrome). METHODS: We established a dual color competitive fluorescent Polymerase Chain Reaction (PCR) to measure the gene dosage of Down syndrome critical region (DSCR), a single copy sequence in chromosome 21. Another unique single copy sequence located on chromosome 2 (USC2) but not glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was chose as reference gene. RESULTS: The DSCR3/USC2 ratio of peripheral blood in trisomy 21 syndrome patients to normal subjects was 1.41∼1.74 to 0.93∼1.15, respectively (p < 0.01). Dual color competitive fluorescent PCR technique effectively differentiates the normal subjects from the Down syndrome patients. Next, according to the dual color competitive fluorescence quantitative PCR, among the 46 pregnant women, 3 cases were Down syndrome and 43 cases were normal, and these were confirmed by cytogenetic karyotype analysis. CONCLUSION: This indicated that the new technique may be a reliable and specific method for the rapid prenatal diagnosis of Trisomy 21.

Type 2 diabetes mellitus is the most common endocrine disease all over the world, while existing therapies can only ameliorate hyperglycemia or temporarily improve the response to insulin in target tissues, they cannot retard or improve the progressive β-cell dysfunction persistently. Combined therapy of stem cells and sitagliptin might resolve this problem, we verified this hypothesis in a diabetic rat model. Except ten Wistar rats in normal control group, diabetic rats were divided into diabetic control group, WJ-MSCs group, sitagliptin group and WJ-MSCs + sitagliptin group and received homologous therapy. Ten weeks after therapy, diabetic symptoms, FPG and GHbA1c in WJ-MSCs group, sitagliptin group and WJ-MSCs + sitagliptin group were significantly less than those in diabetic control group (P < 0.05), while fasting C-peptide and number of β cells in WJ-MSCs group and WJ-MSCs + sitagliptin group was significantly higher than those in diabetic control and sitagliptin group (P < 0.01). Glucagon and number of α cells in sitagliptin group and WJ-MSCs + sitagliptin group were significantly lower than those in WJ-MSCs group and diabetic control group (P < 0.01). No symptoms of rejection and toxic effect were observed. Combined therapy of WJ-MSCs and sitagliptin can effectively ameliorate hyperglycemia, promote regeneration of islet β cells and suppress generation of islet α cells in diabetic rats, presenting a new therapy for type 2 diabetes although the exact mechanisms are unclear.

Lipopolysaccharides (LPS) from the outer membrane of Gram-negative bacteria serve as endotoxin to exert potent immune responses. However, the effect of LPS on adipogenesis has not been elucidated. The present study was designed to examine the effect of LPS on adipogenesis in 3T3-L1 preadipocytes and possible mechanism(s) of action involved. Our results revealed that LPS challenge significantly suppressed adipogenesis in 3T3-L1 preadipocytes mainly through downregulated expression of the late adipogenic markers PPARγ and aP2 as well as AMP-activated protein kinase (AMPK) expression and activity. As an inflammatory factor, LPS was found to lead to an overt reduction in IκBα levels compared with the time-matched controls, consolidating its pro-inflammatory property in 3T3-L1 preadipocytes. Our data also revealed that LPS retarded adipogenesis, the effect of which was partially reversed by the selective inhibitor of IKKβ. IκBα was found to be involved in the anti-adipogenic effect of LPS. In conclusion, LPS is capable of inhibiting adipogenesis in 3T3-L1 adipocytes possibly through activation of NF-κB and inhibition of AMPK. With the activation of NF-κB pathway and inhibition of AMPK, LPS suppresses C/EBP α DNA-binding activity and the expression of late adipogenic markers PPARγ and aP2.

To explore whether experiencing inflammatory pain has an impact upon intracortical synaptic organization, the planar multi-electrode array (MEA) technique and 2-dimensional current source density (2D-CSD) imaging were used in slice preparations of the anterior cingulate cortex (ACC) from rats. Synaptic activity across different layers of the ACC was evoked by deep layer stimulation through one electrode. The layer-localization of both local field potentials (LFPs) and the spread of current sink calculated by 2D-CSD analysis was characterized pharmacologically. Moreover, the induction of long-term potentiation (LTP) and changes in LTP magnitude were also evaluated. We found that under naïve conditions, the current sink was initially generated in layer VI, then spread to layer V and finally confined to layers II-III. This spatial pattern of current sink movement typically reflected changes in depolarized sites from deep layers (V-VI) to superficial layers (II-III) where intra- and extracortical inputs terminate. In the ACC slices from rats in an inflamed state (for 2 h) caused by intraplantar bee-venom injection, the spatial profile of intra-ACC synaptic organization was significantly changed, showing an enlarged current sink distribution and a leftward shift of the stimulus-response curves relative to the naïve and saline controls. The change was more distinct in the superficial layers (II-III) than in the deep site. In terms of temporal properties, the rate of LTP induction was significantly increased in layers II-III by inflammatory pain. However, the magnitude of LTP was not significantly enhanced by this treatment. Taken together, these results show that inflammatory pain results in distinct spatial and temporal plasticity of synaptic organization in the ACC, which may lead to altered synaptic transmission and modulation.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) secreting cellular tumor vaccines contribute to the induction of potent antitumor immune responses in murine models and patients suffering from cancers. Hepatocellular carcinoma (HCC) is one of the most frequent and malignant cancers in China. We describe, for the first time, a GM-CSF releasing vaccine strategy that represents a step toward combating this type of cancer. In this study, a bystander cell-based GM-CSF secreting vaccine against murine HCC, Hepa1-6/B78H1-GM-CSF, was co-administered with a low dose of cyclophosphamide (CY). After challenging with tumor and vaccination, immunological assays demonstrated that the cellular antitumor immune responses were efficiently activated and that tumor development was significantly retarded, which was dependent on synergy with CY. The promising outcome of the anti-HCC vaccine in the murine model demonstrates the feasibility of a future clinical application for this treatment in HCC patients.Cellular & Molecular Immunology advance online publication, 20 May 2013; doi:10.1038/cmi.2013.20.

Aim:Vasodilator-stimulated phosphoprotein (VASP) expression is upregulated in human cancers and correlates with more invasive advanced tumor stages. The aim of this study was to elucidate the mechanisms by which matrine, an alkaloid derived from Sophora species plants, acted on the VASP protein in human gastric cancer cells in vitro.Methods:VASP was expressed and purified. Intrinsic fluorescence spectroscopy was used to study the binding of matrine to VASP. CD spectroscopy was used to examine the changes in the VASP protein secondary structure. Human gastric carcinoma cell line BGC823 was tested. Scratch wound and cell adhesion assays were used to detect the cell migration and adhesion, respectively. Real-time PCR and Western blot assays were used to measure mRNA and protein expression of VASP.Results:In the fluorescence assay, the dissociation constant for binding of matrine to VASP protein was 0.86 mmol/L, thus the direct binding between the two molecules was weak. However, matrine (50 μg/mL) caused obvious change in the secondary structure of VASP protein shown in CD spectrum. Treatments of BGC823 cells with matrine (50 μg/mL) significantly inhibited the cell migration and adhesion. The alkaloid changed the subcellular distribution of VASP and formation of actin stress fibers in BGC823 cells. The alkaloid caused small but statistically significant decreases in VASP protein expression and phosphorylation, but had no significant effect on VASP mRNA expression.Conclusion:Matrine modulates the structure, subcellular distribution, expression and phosphorylation of VASP in human gastric cancer cells, thus inhibiting the cancer cell adhesion and migration.

The reactivity of sp(2) carbon materials is studied using the adsorption and dissociation of O2 on graphene and graphene oxide as model systems. The reactions on the basal plane, zigzag and armchair edges of graphene and graphene oxide with different oxygen-containing groups are calculated using first principles calculations. Two Brønsted-Evans-Polanyi relationships are identified and an electron delocalization model is suggested to understand the general trend of reactivity for sp(2) carbon materials.

The aim of the present study was to determine the efficacy of ulinastatin (UTI) for the treatment of sepsis and to investigate the associated molecular mechanisms. Twenty‑four male rabbits were randomly divided into 4 groups, the normal, sham, sepsis model and UTI groups, each containing 6 rabbits. Serum levels of interleukin (IL)‑10 and tumor necrosis factor‑α (TNF‑α) were measured by enzyme‑linked immunosorbent assay (ELISA). Liver, kidney and lung tissues were stained with hematoxylin and eosin (H&E) 36 h after sacrifice and morphological changes were observed under an optical microscope. The expression levels of IL‑10 and TNF‑α proteins in rabbit kidney tissue in each group were determined by immunohistochemical detection and western blot analysis. ELISA results indicated that, compared with the sepsis model, IL‑10 levels were significantly higher in the UTI treatment group (183.91±11.521 pg/ml) at 36 h (P=0.000), while serum TNF‑α concentration decreased significantly in the UTI treatment group (31.637±2.770 pg/ml; P=0.000). Results of western blot analysis were consistent with the immunohistochemistry, indicating that UTI upregulates IL‑10 and downregulates TNF‑α levels. In the current study, UTI was demonstrated to effectively treat urinary sepsis and alleviate the inflammatory response in tissues. These effects were mediated by the upregulation of IL‑10 and downregulation of TNF‑α levels.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus associated with the development of both lymphoid and epithelial tumors. EBNA1 is the only viral protein expressed in all EBV-associated malignancies and plays important roles in EBV latency. Thus, EBNA1 is thought to be a promising antigen for immunotherapy of all EBV-associated malignancies. This study was undertaken to produce recombinant EBNA1 protein in Pichia pastoris and evaluate its immunogenicity. The truncated EBNA1 (E1ΔGA, codons 390-641) was expressed as a secretory protein with an N-terminal histidine tag in the methylotrophic yeast P. pastoris and purified by Ni-NTA affinity chromatography. The purified proteins were then used as antigens to immunize BALB/c mice for production of polyclonal antibodies. Western blot analysis showed that the polyclonal antibodies specifically recognized the EBNA1 protein in B95-8 cell lysates. The recombinant E1ΔGA also induced strong lymphoproliferative and Th1 cytokine responses in mice. Furthermore, mice immunized with E1ΔGA developed CD4(+) and CD8(+) T cell responses. These findings showed that the yeast-expressed E1ΔGA retained good immunogenicity and might be a promising vaccine candidate against EBV-associated malignancies.

Parallel imaging and compressed sensing have been arguably the most successful and widely used techniques for fast magnetic resonance imaging (MRI). Recent studies have shown that the combination of these two techniques is useful for solving the inverse problem of recovering the image from highly under-sampled k-space data. In sparsity-enforced sensitivity encoding (SENSE) reconstruction, the optimization problem involves data fidelity (L2-norm) constraint and a number of L1-norm regularization terms (i.e. total variation or TV, and L1 norm). This makes the optimization problem difficult to solve due to the non-smooth nature of the regularization terms. In this paper, to effectively solve the sparsity-regularized SENSE reconstruction, we utilize a new optimization method, called fast composite splitting algorithm (FCSA), which was developed for compressed sensing MRI. By using a combination of variable splitting and operator splitting techniques, the FCSA algorithm decouples the large optimization problem into TV and L1 sub-problems, which are then, solved efficiently using existing fast methods. The operator splitting separates the smooth terms from the non-smooth terms, so that both terms are treated in an efficient manner. The final solution to the SENSE reconstruction is obtained by weighted solutions to the sub-problems through an iterative optimization procedure. The FCSA-based parallel MRI technique is tested on MR brain image reconstructions at various acceleration rates and with different sampling trajectories. The results indicate that, for sparsity-regularized SENSE reconstruction, the FCSA-based method is capable of achieving significant improvements in reconstruction accuracy when compared with the state-of-the-art reconstruction method.

Complement system is known as highly sophisticated immune defense mechanism forantigen recognition as well as effector functions. Activation of the terminal pathway of the complementsystem leads to the assembly of terminal complement complexes (C5b-9), which induces thecharacteristic complement-mediated cytolysis. The lytic activity of shark complement involvesfunctional analogues of mammalian C8 and C9. In this article, a full-length cDNA of C9 (CpC9) isidentified from cartilaginous species, the whitespotted bambooshark, Chiloscyllium plagiosum byRACE. The CpC9 cDNA is 2263 bp in length, encoding a protein of 603 amino acids, which shares 42%and 43% identity with human and Xenopus C9 respectively. Through sequence alignment andcomparative analysis, the CpC9 protein was found well conserved, with the typical modulararchitecture in TCCs and nearly unanimous cysteine composition from fish to mammal. Phylogeneticanalysis places it in a clade with C9 orthologs in higher veterbrate and as a sister taxa to the Xenopus.Expression analysis revealed that CpC9 is constitutively highly expressed in shark liver, with much lessor even undetectable expression in other tissues; demonstrating liver is the primary tissue for C9synthesis. To sum up, the structural conservation and distinctive phylogenetics might indicate thepotentially vital role of CpC9 in shark immune response, though it remains to be confirmed by furtherstudy.

Full-length sequences were determined and analyzed for two human (MO and W3) and one swine (W2-5) hepatitis E virus (HEV) isolates from Beijing, China. The genomes of the three strains were composed of 7242, 7239, 7239 nucleotides, respectively, excluding the poly (A) tails, and were 84% identical to each other. All were classified into genotype 4. Sequence analysis shows that the 2 human isolates have up to 91-94% nucleotide identity in full length genome with swine strains isolated in China, while the swine isolate share 92% identity with the human strain T1 from Beijing. At the amino acid level, the three strains share 94%, 97% and 89-92% identity in the ORF1, ORF2 and ORF3 proteins respectively. The human strains MO and W3 have the highest identity, 97%, 98%-99% and 96%-98% in ORFs 1-3, respectively, to swine strains CHN-XJ-SW13 and CHN-XJ-SW33 from Xinjiang, China, while swine strain W2-5 has highest identity with the human strain HE-JA2, 96%, 99% and 91% in ORFs 1-3, respectively. Genotype specific amino acid substitutions were found at a single site in all three ORFs by sequences alignment, and genotype specific short sequences (5-10 aa in length) were found in ORF1 and the C-terminus of ORF3. However, no difference was found at any amino acid position that discriminates between human and swine HEVs within genotype 4 for any of the three ORFs. These results indicated that the genotype 4 HEV strains from humans and pigs in China may evolve from the common ancestor.

OBJECTIVE: The arteriovenous fistula (AVF) still suffers from a high number of failures caused by insufficient outward remodeling and intimal hyperplasia (IH) formation from which the exact mechanism is largely unknown. A suitable animal model is of vital importance in the unraveling of the underlying pathophysiology. However, current murine models of AVF failure do not incorporate the surgical configuration that is commonly used in humans. Because the hemodynamic profile is one of the key determinants that play a role in vascular remodeling in the AVF, it is preferable to use this same configuration in an animal model. Here we describe a novel murine model of AVF failure in which the configuration (end-to-side) is similar to what is most frequently performed in humans. METHODS: An AVF was created in 45 C57BL/6 mice by anastomosing the end of a branch of the external jugular vein to the side of the common carotid artery with interrupted sutures. The AVFs were harvested and analyzed histologically at days 7, 14, and 28. Identical veins of unoperated-on mice served as controls. Intravenous near-infrared fluorescent fluorophores were used to assess the patency of the fistula. RESULTS: The patency rates at days 7, 14, and 28 days were 88%, 90%, and 50%, respectively. The mean circumference increased up to day 14, with a maximum 1.4-fold increase at day 7 compared with the control group (1.82 ± 0.7 vs 1.33 ± 0.3 mm; P = .443). Between days 14 and 28, the circumference remained constant (2.36 ± 0.2 vs 2.45 ± 0.2 mm; P = .996). At 7 days after surgery, the intimal area consisted mainly of an acellular layer that was structurally analogous to a focal adherent thrombus. Starting at 14 days after surgery, venous IH increased significantly compared with the unoperated-on group (14 days: 115,090 ± 22,594 μm(2), 28 days: 234,619 ± 47,828 μm(2), unoperated group: 2368 ± 1056 μm(2); P = .001 and P < .001, respectively) and was mainly composed of cells positive for α-smooth muscle actin. We observed leukocytes in the adventitial side of the vein at all time points. CONCLUSIONS: Our novel murine AVF model, which incorporates a clinically relevant configuration of the anastomosis, displays similar features that are characteristic of failing human AVFs. Moreover, our findings suggest that coagulation and inflammation could both potentially play an important role in the formation of IH and subsequent AVF failure. Near-infrared fluoroscopy was a suitable alternative for conventional imaging techniques. This murine AVF-model is a valuable addition to the AVF animal model arsenal.

The present research described the preparation, purification, and identification of antioxidant peptides from goat milk casein (GMC). Goat milk casein was hydrolyzed by using a combination of neutral and alkaline proteases to obtain goat milk casein hydrolysates (GMCH) with high antioxidant activity. After desalting by nonpolar macroporous absorption resin, GMCH was isolated and purified by gel filtration chromatography and reversed-phase HPLC, respectively, and further identified by nanoliter electrospray ionization-tandem mass spectrometry. Antioxidant activities of GMC, GMCH, and pure peptides were evaluated and compared using free radical scavenging activity, metal ion chelating ability, and anti-lipid peroxidation ability. Compared with GMC, the free radical-scavenging ability and ferrous ion-chelating ability of GMCH increased significantly. The inhibition effect of lipid peroxidation of GMCH was much stronger than that of tert-butylhydroquinone and phytogermine and a little lower than that of ascorbic acid. The antioxidant activity of GMCH could be attributed to the high antioxidant activity of oligopeptides, especially 5 novel oligopeptides: Val-Tyr-Pro-Phe, Phe-Gly-Gly-Met-Ala-His, Phe-Pro-Tyr-Cys-Ala-Pro, Tyr-Val-Pro-Glu-Pro-Phe, and Tyr-Pro-Pro-Tyr-Glu-Thr-Tyr, which were first observed in GMCH. The antioxidant activity of these 5 novel oligopeptides and GMCH increased 3.59 to 380 times compared with GMC, combining anti-lipid peroxidation ability of GMCH, which indicated that GMCH and its purified fractions in different stages could be used as functional food ingredients, food additives, and pharmaceutical agents in the future.