The anti-inflammatory effect of lutein in broilers is mediated by regulating Toll-like receptor 4/myeloid-differentiation-factor 88 signaling pathway.

The anti-inflammatory role of lutein has been widely recognized, however, the underlying mechanism is still not fully elucidated. Hence, the effects of lutein on the intestinal health and growth performance of broilers and the action of mechanism were investigated. 288 male yellow-feathered broilers (1-day old) were randomly allocated to 3 treatment groups with 8 replicates of 12 birds each, and the control group was fed a broken rice-soybean basal diet, while the test groups were fed a basal diet added with 20 mg/kg and 40 mg/kg of lutein (LU20, LU40), respectively. The feeding trial lasted for 21 d. The results showed that 40 mg/kg lutein supplementation tended to increase ADFI (P = 0.10) and ADG (P = 0.08) of broilers. Moreover, the addition of lutein caused a decreasing trend of gene expression and concentration of proinflammatory cytokines IL-1ß (P = 0.08, P = 0.10, respectively) and IL-6 (P = 0.06, P = 0.06, respectively) and also tended to decrease the gene expression of TLR4 (P = 0.09) and MyD88 (P = 0.07) while increasing gene expression and concentration of anti-inflammatory cytokines IL-4 and IL-10 (P < 0.05) in the jejunum mucosa of broilers. Additionally, lutein supplementation increased the jejunal villi height of broilers (P < 0.05) and reduced villi damage. The experiment in vitro showed that lutein treatment reduced the gene expression of IL-1ß, IL-6, and IFN-γ in chicken intestinal epithelial cells (P < 0.05). However, this effect was diminished after knock-down of TLR4 or MyD88 genes using RNAi technology. In conclusion, lutein can inhibit the expression and secretion of proinflammatory cytokines in the jejunum mucosa and promote intestinal development of broilers, and the anti-inflammatory effect may be achieved by regulating TLR4/MyD88 signaling pathway.

Analysis on the Effect of Radiofrequency Ablation and Electrocautery in the Treatment of Vaginal Intraepithelial Neoplasia.

Objective: This research intends to investigate the clinical efficacy of radiofrequency ablation and electrocautery in treating grade I or II vaginal intraepithelial neoplasia (VaIN). Methods: This is a single-center retrospective study, which collected the clinical data of 100 patients with VaIN diagnosed by colposcopy and pathological biopsy in the Gynecology and Cervical Center of Xiangzhu Branch of the Maternal and Child Health Hospital of Guangxi Zhuang Autonomous Region between January 2020 and June 2021. Patients were divided into the study group (radiofrequency ablation treatment) and the control group (electrocautery) according to differences in treatment approaches. 6- and 12-month follow-ups were performed on all patients. Gynecological examination results, liquid-based thin-layer cytology (TCT), negative conversion of human papillomavirus (HPV), curative effects, and prognosis were recorded. Results: All patients completed regular follow-ups that lasted for 6 and 12 months. The 6- and 12-month cure rates of the study group were 76.0% and 92.0%, respectively, and the data in the control group were 70.0% and 82.0%, respectively. In terms of the 6- and 12-month negative conversion rates of HPV, the data in the study group were 68.0% and 78.0%, versus 60% and 68% in the control group, respectively. The lesion duration rate showed no statistical significance between the study group (8.0%) and the control group (P > 0.05). The analysis of postoperative follow-up complications revealed that the study group had a statistically lower overall incidence of vaginal bleeding, excessive vaginal discharge, vaginal burning sensation, and decreased vaginal elasticity than the control group (8.0% vs. 24.0% P < 0.05). Conclusion: Both radiofrequency ablation and electrocautery have obvious clinical effects in patients with grade I or II VaIN, but the former contributed to fewer operative complications and a good prognosis, which deserves clinical promotion.

[Analysis of Rainfall Runoff Pollution and Pollution Load Estimation for Urban Communities in a Highly Urbanized Region].

The Taihu Lake plain is a highly urbanized region in China with many water-related environmental problems. Although point-source pollution has been effectively controlled by government legislation, urban surface runoff pollution is still a major issue. Different types of urban communities were selected for rainfall runoff experiments. According to the monitoring data of rainfall events, multiple methods were used to analyze the characteristics of surface runoff pollution and estimate the pollution load for different types of communities. The results indicated that surface runoff from urban communities reduced the river water quality. Certain degrees of the 'first flush' effect occurred in different types of urban communities. The surface runoff pollution in the commercial residential community was weaker than that in commercial and private residential communities; however, the first flush occurred more frequently in the commercial residential community. Holding back 30% of the surface runoff could effectively improve the runoff water quality in commercial and private residential communities as well as the commercial residential community with restaurants. In the commercial residential community, 25% of surface runoff should be held to improve runoff water quality effectively. The loads of pollutants, especially nitrogen and phosphorus, in urban communities in the Taihu Lake basin were higher than those in other regions in China. This research can assist with the reduction of surface runoff pollution in highly urbanized communities.

A universal multiplex PCR strategy for 100-plex amplification using a hydrophobically patterned microarray.

Both basic research and clinical medicine have urgent demands for highly efficient strategies to simultaneously identify many different DNA sequences within a single tube. Effective and simultaneous amplification of multiple target sequences is a prerequisite for any successful multiple nucleic acid detection method. Multiplex PCR is one of the best choices for this purpose. However, due to the intrinsic interference and competition among primer pairs in the same tube, multiple rounds of highly empirical optimization procedures are usually required to establish a successful multiplex PCR reaction. To address this challenge, we report here a universal multiplex PCR strategy that is capable of over 100-plex amplification using a specially designed microarray in which hydrophilic microwells are patterned on a hydrophobic chip. On such an array, primer pairs tagged with a universal sequence are physically separated in individual hydrophilic microwells on an otherwise hydrophobic chip, enabling many unique PCR reactions to be proceeded simultaneously during the first step of the procedure. The PCR products are then isolated and further amplified from the universal sequences, producing a sufficient amount of material for analysis by conventional gel electrophoresis or DNA microarray technology. This strategy is abbreviated as "MPH&HPM" for "Multiplex PCR on a Hydrophobically and Hydrophilically Patterned Microarray". The feasibility of this method is first demonstrated by a multiplex PCR reaction for the simultaneous detection of eleven pneumonia-causing pathogens. Further, we demonstrate the power of this strategy with a highly successful 116-plex PCR reaction that required only little prior optimization. The effectiveness of the MPH&HPM strategy with clinical samples is then illustrated with the detection of deleted exons of the Duchenne Muscular Dystrophy (DMD) gene, the results are in excellent agreement with the clinical records. Because of its generality, simplicity, flexibility, specificity and capacity of more than 100-plex amplification, the MPH&HPM strategy should have broad applications in both laboratory research and clinical applications when multiplex nucleic acid analysis is required.

NF-kappaB transcription factor p50 critically regulates tissue factor in deep vein thrombosis.

NF-kappaB transcription factors regulate the expression of tissue factor (TF), a principal initiator of the coagulation cascade. Dominant among them is the p50/p65 heterodimer. Here we report that Andrographolide (Andro; a p50 inhibitor) and genetic deletion of p50 attenuated TF activity in stimulated endothelial cells and monocytes/macrophages. Results of the electrophoretic mobility "supershift" assay and chromatin immunoprecipitation demonstrated the direct interaction of the p50/p65 heterodimer with the NF-kappaB site of the human TF promoter. Andro-treated and p50 null mice both exhibited blunted TF expression and reduced venous thrombosis, which were recapitulated by an anti-murine TF antibody in vivo. Our findings thus indicate that regulation of TF by NF-kappaB transcription factor p50 is essential for the pathogenesis of deep vein thrombosis and suggest that specific inhibitors of p50, such as Andro, may be therapeutically valuable for preventing and perhaps treating venous thrombosis.

[Specific antitumor effect of adeno-associated virus vector carrying TRAIL gene under the control of hTERT promoter].

BACKGROUND & OBJECTIVE: Adeno-associated virus (AAV) has been widely used in tumor gene therapy. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a safe and potent anti-tumor gene which could induce apoptosis of many tumor cells. This study was to use tumor-specific promoter hTERT to construct AAV-hTERT-TRAIL, and explore its antitumor effect and mechanism in vitro. METHODS: Purified AAV-hTERT-TRAIL was obtained after co-transfection of HEK293 with pAAV-hTERT-TRAIL and two other help plasmids. After transfection of AAV-hTERT-TRAIL into three tumor cell lines, SW620, HepG2, A549 and two normal cell lines, NHLF and MRC5, the expression of TRAIL was detected by RT-PCR, Western blot and immunohistochemistry (IHC); the influence of AAV-hTERT-TRAIL transfection on cell proliferation was evaluated using MTT assay. Activation of caspase-3 and PARP was measured by Western blot. Cell apoptosis was assessed using ELISA and flow cytometry. RESULTS: AAV-hTERT-TRAIL was successfully packaged in HEK293 cells. After AAV-hTERT-TRAIL infection, specific expression of TRAIL was detected in three tumor cell lines, but not in two normal cell lines. Cell proliferation rates in SW620, A549, HepG2, NHLF and MRC5 cells were 41.55%, 44.29%, 49.95%, 84.59% and 87.22%, respectively after transfection of AAV-hTERT-TRAIL at a multiplicity of infection (MOI) of 100 for 96 h. AAV-hTERT-TRAIL activated caspase-3 apoptotic pathway and induced apoptosis in tumor cell lines, but not in normal cell lines. CONCLUSIONS: hTERT increases selectivity and safety of AAV vector. hTERT promoter controls the expression of anti-tumor genes to specifically induce death of tumor cells.

[Expression and identification of recombinant P-selectin and P-selectin glycoprotein ligand-1].

P-selectin, one of the membrane proteins, expresses on platelet and endothelia and interacts with P-selectin glycoprotein ligand-1 (PSGL-1) on leukocyte membrane. This interaction mediates leukocytes rolling on endothelial membrane and then induces leukocyte recruitment to the site of infection or tissue injury. In the present study, we constructed the recombinant wild type human P-selectin, its calcium-binding sites mutants and recombinant PSGL-1-globulin (PSGL-1-Rg). They expressed in Sf9 cells by using the baculovirus expression system and were purified by TalonTM metal or Protein A affinity chromatography. The results showed that the recombinant PSGL-1-Rg interacted with recombinant wild type P-selectin and two P-selectin mutants with 2 calcium-binding sites mutation respectively, but could not bind to the P-selectin mutant with all 4 calcium-binding sites mutation. Therefore, we verified the importance of P-selectin calcium-binding sites for its interaction with PSGL-1.

In vivo biocompatibility and mechanical properties of porous zein scaffolds.

In our previous study, a three-dimensional zein porous scaffold with a compressive Young's modulus of up to 86.6+/-19.9 MPa and a compressive strength of up to 11.8+/-1.7 MPa was prepared, and was suitable for culture of mesenchymal stem cells (MSCs) in vitro. In this study, we examined its tissue compatibility in a rabbit subcutaneous implantation model; histological analysis revealed a good tissue response and degradability. To improve its mechanical property (especially the brittleness), the scaffolds were prepared using the club-shaped mannitol as the porogen, and stearic acid or oleic acid was added. The scaffolds obtained had an interconnected tubular pore structure, 100-380 microm in pore size, and about 80% porosity. The maximum values of the compressive strength and modulus, the tensile strength and modulus, and the flexural strength and modulus were obtained at the lowest porosity, reaching 51.81+/-8.70 and 563.8+/-23.4 MPa; 3.91+/-0.86 and 751.63+/-58.85 MPa; and 17.71+/-3.02 and 514.39+/-19.02 MPa, respectively. Addition of 15% stearic acid or 20% oleic acid did not affect the proliferation and osteogenic differentiation of MSCs, and a successful improvement of mechanical properties, especially the brittleness of the zein scaffold could be achieved.

EMMA: an efficient massive mapping algorithm using improved approximate mapping filtering.

Efficient massive mapping algorithm (EMMA), an algorithm on efficiently mapping massive cDNAs onto genomic sequences, has recently been developed. The process of mapping massive cDNAs onto genomic sequences has been improved using more approximate mapping filtering based on an enhanced suffix array coupled with a pruned fast hash table, algorithms of block alignment extensions, and k-longest paths. When compared with the classical BLAT software in this field, the computing of EMMA ranges from two to forty-one times faster under similar prediction precisions.

Human acyl-CoA:cholesterol acyltransferase 2 gene expression in intestinal Caco-2 cells and in hepatocellular carcinoma.

Humans express two ACAT (acyl-CoA:cholesterol acyltransferase) genes, ACAT1 and ACAT2. ACAT1 is ubiquitously expressed, whereas ACAT2 is primarily expressed in intestinal mucosa and plays an important role in intestinal cholesterol absorption. To investigate the molecular mechanism(s) responsible for the tissue-specific expression of ACAT2, we identified five cis-elements within the human ACAT2 promoter, four for the intestinal-specific transcription factor CDX2 (caudal type homeobox transcription factor 2), and one for the transcription factor HNF1alpha (hepatocyte nuclear factor 1alpha). Results of luciferase reporter and electrophoretic mobility shift assays show that CDX2 and HNF1alpha exert a synergistic effect, enhancing the ACAT2 promoter activity through binding to these cis-elements. In undifferentiated Caco-2 cells, the ACAT2 expression is increased when exogenous CDX2 and/or HNF1alpha are expressed by co-transfection. In differentiated Caco-2 cells, the ACAT2 expression significantly decreases when the endogenous CDX2 or HNF1alpha expression is suppressed by using RNAi (RNA interference) technology. The expression levels of CDX2, HNF1alpha, and ACAT2 are all greatly increased when the Caco-2 cells differentiate to become intestinal-like cells. These results provide a molecular mechanism for the tissue-specific expression of ACAT2 in intestine. In normal adult human liver, CDX2 expression is not detectable and the ACAT2 expression is very low. In the hepatoma cell line HepG2 the CDX2 expression is elevated, accounting for its elevated ACAT2 expression. A high percentage (seven of fourteen) of liver samples from patients affected with hepatocellular carcinoma exhibited elevated ACAT2 expression. Thus, the elevated ACAT2 expression may serve as a new biomarker for certain form(s) of hepatocellular carcinoma.

Two human ACAT2 mRNA variants produced by alternative splicing and coding for novel isoenzymes.

Acyl coenzyme A:cholesterol acyltransferase 2 (ACAT2) plays an important role in cholesterol absorption. Human ACAT2 is highly expressed in small intestine and fetal liver, but its expression is greatly diminished in adult liver. The full-length human ACAT2 mRNA encodes a protein, designated ACAT2a, with 522 amino acids. We have previously reported the organization of the human ACAT2 gene and the differentiation-dependent promoter activity in intestinal Caco-2 cells. In the current work, two human ACAT2 mRNA variants produced by alternative splicing are cloned and predicted to encode two novel ACAT2 isoforms, named ACAT2b and ACAT2c, with 502 and 379 amino acids, respectively. These mRNA variants differ from ACAT2a mRNA by lack of the exon 4 (ACAT2b mRNA) and exons 4-5 plus 8-9-10 (ACAT2c mRNA). Significantly, comparable amounts of the alternatively spliced ACAT2 mRNA variants were detected by RT-PCR, and Western blot analysis confirmed the presence of their corresponding proteins in human liver and intestinecells. Furthermore, phosphorylation and enzymatic activity analyses demonstrated that the novel isoenzymes ACAT2b and ACAT2c lacked the phosphorylatable site SLLD, and their enzymatic activities reduced to 25%-35% of that of ACAT2a. These evidences indicate that alternative splicing produces two human ACAT2 mRNA variants that encode the novel ACAT2 isoenzymes. Our findings might help to understand the regulation of the ACAT2 gene expression under certain physiological and pathological conditions.

Variations of dominant microbial populations in groundwater in response to the leachate from Laogang Landfill.

Temporal changes of dominant microbial populations in groundwater in response to the leachate from Shanghai Laogang Landfill were investigated. Concentrations of dissolved redox-relevant species in groundwater suggested that the dominating redox process had changed from denitrification to methane-production/sulfate-reduction due to landfilling. Dominant microbial populations were determined using restriction fragment length polymorphism( RFLP) analyses of 16S rRNA gene libraries, which were further studied by sequencing and phylogenetic analyses. The results indicated that obvious shifts of dominant microbial populations had occurred in groundwater in response to the pollution of leachate. The closest relatives of some dominant clones are accordant with the dominating redox processes determined by hydrochemical analyses, based on the GenBank's indications on the ability to perform redox reactions.

Comparison of cytocompatibility of zein film with other biomaterials and its degradability in vitro.

UNLABELLED: Cytocompatibility of particle zein (Pzein) and film zein (Fzein) was evaluated and compared with polyhydroxybutyrate (PHB), its copolymer poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV), polylactic acid (PLA), and collagen, using HL-7702 cells, in terms of cell attachment rate within 3 h, and cell viabilities at 3 and 6 days determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The zein degradation test was carried out using collagenase and trypsin, and the degradation product was added to the culture medium at different concentrations in order to examine the concentration-dependent cytotoxic effect. RESULT: The adhesion rate of the HL-7702 cells on both Pzein and Fzein was higher than that on collagen film. Cell viabilities were higher on both Pzein and Fzein than on films of PLA, PHB, PHBV, and collagen from fish skin. Zein can be degraded by both trypsin and collagenase, and the degradation product can enhance cell viability within a certain range of concentrations.

Heparin-loaded zein microsphere film and hemocompatibility.

Zein was studied as a drug-eluting coating film composed of zein microspheres for cardiovascular devices (e.g. stent). In vitro 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) analysis showed that both zein film and its degraded product had better biocompatibility compared with Corning culture plate on the growth of human umbilical veins endothelial cells (HUVECs, p<0.05, n=6), and the effect of zein degraded product on HUVECs was dose-dependent. The best result was obtained at 0.3 mg/ml of the addition. The encapsulation efficiency of heparin and heparin loading varied with the amount of both zein and heparin, and the highest encapsulation efficiency (heparin 1.33 mg/ml and zein 16 mg/ml) was 22.77+/-1.33% (n=3). Scanning electron microscope (SEM) observation indicated that the zein film was made of microspheres in diameter from nano- to micrometer, which could be controlled. Sizes of heparin-loaded zein microspheres changed before and after release of heparin because of conglutination among zein microspheres. Release rate of heparin from microsphere film reached to 33.5+/-1.2% within 12 h, and began to get into subsequent "slow release" phase; about 55% of the entrapped heparin was released after 20 days. Both zein film and heparin-loaded zein microsphere film were effective in suppressing platelet adhesion, and the heparin-loaded film showed a better anticoagulation as determined with thrombin time (TT) assay. These results suggest that zein film could be used directly as a new type of coating material for its better biocompatibility with HUVECs. Moreover, the heparin-loaded zein microsphere film can significantly improve the hemocompatibility.

[Effect of methylmercury on expression of immediate early gene c-jun mRNA in rat brain].

In order to probe into the early prediction molecular index and the signal transduction molecular mechanism of methyl mercury chloride (MMC) neurotoxicity, the expression of c-jun mRNA in rat brains induced by different concentration MMC for different times were observed by using reverse transcription polymerase chain reaction (RT-PCR) methods (the control group was physiological saline of 0.9%, the concentrations of expose groups were 0.05, 0.5, 5 mg x kg(-1) respectively, the sampling times were 20, 60, 240, 1440 min). The result showed the expression of c-jun mRNA in rat brains was prior to the accumulation of mercury, and the expression of c-jun mRNA in rat brains could early predict the neurotoxicity of MMC. IEG (c-jun) participated in the toxicity process of injury by MMC.

Identification and characterization of a rat novel gene RSEP4 expressed specifically in central nervous system.

The low-abundantly expressed genes composed the majorities of the mRNAs expressed in the central nervous system (CNS), and were thought to be important for the normal brain functions. Through differential screening a low-abundance cDNA sublibrary with mRNA from neuropathic pain of chronic constriction injury (CCI) model, we have identified a novel rat gene, rat spinal-cord expression protein 4 gene (RSEP4). The total length of RSEP4 cDNA is 2006 bp, with a 501 nucleotide open reading frame (ORF) that encodes a 167 amino acid polypeptide. Northern blot revealed that RSEP4 was expressed specifically in the CNS. In situ hybridization showed that the mRNA of RSEP4 was strongly expressed in the CA1, CA2, CA3 and DG regions of hippocampus, the Purkinje cells of cerebellum, and the small sensory neurons of dorsal horn and large motor neurons of ventral horn of spinal cord. Over-expression of RSEP4-EGFP fusion protein in the human embryonic kidney 293T cells showed that RSEP4 protein was mainly localized in the cell cytoplasm. These results suggest that RSEP4 may play some roles in the CNS.

Preparation of an anti-Cdx-2 antibody for analysis of different species Cdx-2 binding to acat2 promoter.

The homeodomain protein, Cdx-2, as transcription factor has been implicated in the transcriptional regulation of genes expressed in small intestine and the process of tumorgenesis. In current work, a conserved mouse Cdx-2 domain (mCdx-2D) coded by its cDNA fragment, which was amplified and cloned into the expression vector pGEX-4T1, was expressed as a fusion protein with GST (GST-mCd x-2D) and purified by one step of affinity chromatography. A polyclonal antibody against Cdx-2 was raised by using the recombinant fusion protein GST-mCdx-2D as antigen and was fractionated from the rabbit anti-serum. Western blot and EMSA (electrophoretic mobility shift assay) demonstrate that the natural and denatured Cdx-2s from different species (mouse and human) can be detected by the prepared anti-Cdx-2 antibody. Most notably, we found that the Cdx-2 in human intestine cell line Caco-2 is expressed in a differentiation-dependent manner and can efficiently bind to the mouse and human acat2 (acyl-coenzyme A: cholesterol acyltransferase 2) promoter regions, suggesting that the transcriptional factor Cdx-2 may play a role in regulating the acat2 expression in the intestinal cells.

[Preparation and application of gel chip].

A new method for manufacturing three-dimensional gel film-coated chips was described in this paper and its advantages were evaluated by its application. A patch of polyacrylamide gel (15mm x 15mm x 20 microm) was fixed on the glass surface with Bind-Silane treatment, then activated by glutaraldehyde. The aldehyde groups in gel provided reactive sites that allowed covalent immobilization of molecules containing amino groups. Oligonucleotides were mechanically spotted by GMS 417 Arrayer. After hybridization with Cy-3 labeled probes, fluorescence signals of perfect binding can be discriminated from mismatched ones. Compared with two-dimensional glass chip, the capacity of oligonucleotides immobilized on gel film-coated chip is over 100 times. And the gel film-coated chip have lower background and shorter hybridization time. Monoclonal antibodys of cytokine IL-4, IL-5, IL-6, IL-7, ANG, I-309 and VEGF were also immobilized on the gel film-coated chips to make protein microarrays. After incubation with serum of breast cancer patients or normal persons, the microarray reacted with biotin-labeled second antibodys of cytokines and Cy-3-labeled streptavidin sequentially. Results show IL-4, IL-5, I-309 and VEGF of patients have higher expression level than normal persons. This kind of protein microarrays can be potentially helpful to clinical diagnosis. Furthermore different oligonucleotides or proteins can be performed in parallel in a single reaction with minimal amount of binding reagents. Such gel film-coated chips can be used widely in the fabrication of oligonucleotides and proteins microarrays.

A cDNA clone for beta-caryophyllene synthase from Artemisia annua.

An homology-based cloning strategy yielded a full-length cDNA from Artemisia annua that encoded a protein of 60.3 kDa which resembled a sesquiterpene synthase in sequence. Heterologous expression of the gene in Escherichia coli provided a soluble recombinant enzyme capable of catalyzing the divalent metal ion-dependent conversion of farnesyl diphosphate to beta-caryophyllene, a sesquiterpene olefin found in the essential oil of A. annua. In reaction parameters and kinetic properties, beta-caryophyllene synthase resembles other sesquiterpene synthases of angiosperms. The beta-caryophyllene synthase gene is expressed in most plant tissues during early development, and is induced in mature tissue in response to fungal elicitor thus suggesting a role for beta-caryophyllene in plant defense.