A lightweight path consistency verification based on INT in SDN.

The existing path consistency verification solutions in software-defined networking (SDN) were implemented by proactive injecting large number of probing packets or by embedding linear-scale tags as the path lengthens, which incurred significant bandwidth and communication overhead. A lightweight path consistency validation mechanism based on in-band network telemetry (INT) in SDN is proposed. Based on INT, in the scheme, the ingress switch inserts a telemetry instruction header with probability, each subsequent switch updates the telemetry data using a uniform sampling algorithm and only carries partial path information in INT packet to keep the head space size constant, the egress switch reports the final sampled telemetry data to the controller to verify the path compliance according to aggregated telemetry data. A heuristic flow selection algorithm is proposed to implement network-level path consistency validation. The proposed scheme was implemented and evaluated. The analyses and experiments demonstrate the proposed mechanism effectively limits the packet head overhead and introduces less than 7% of additional forwarding delays and 6% of throughput degradation at most.

Ionomic Combined with Transcriptomic and Metabolomic Analyses to Explore the Mechanism Underlying the Effect of Melatonin in Relieving Nutrient Stress in Apple.

Nutrient stress harms plant growth and yield. Melatonin is a biologically active, multifunctional hormone that relieves abiotic stress in plants. Although previous studies have shown that melatonin plays an important role in improving nutrient-use efficiency, the mechanism of its regulation of nutrient stress remains unclear. In this study, melatonin was applied to apple plants under nutrient stress, and morphological indices, physiological and biochemical indices, and stomatal morphology were evaluated. The response of apple plants to nutrient deficiency and the melatonin mechanism to alleviate nutrient stress were analyzed by combining ionome, transcriptome, and metabolome. The results showed that exogenous melatonin significantly alleviated the inhibitory effect of nutritional stress on the growth of apple plants by regulating stomatal morphology, improving antioxidant enzyme activity, promoting ion absorption, and utilizing and changing the absorption and distribution of minerals throughout the plant. The transcriptome results showed that melatonin alleviated nutrient stress and promoted nutrient absorption and utilization by regulating glutathione metabolism and upregulating some metal ion transport genes. The metabolome results indicated that levels of oxalic acid, L-ascorbic acid, anthocyanins (cyanidin-3-O-galactoside), lignans (lirioresinol A and syringaresinol), and melatonin significantly increased after exogenous melatonin was applied to plants under nutrient stress. These differentially expressed genes and the increase in beneficial metabolites may explain how melatonin alleviates nutrient stress in plants.

MicroRNA­378d inhibits Glut4 by targeting Rsbn1 in vitamin D deficient ovarian granulosa cells.

Vitamin D (VD) is not only associated with bone growth and development, but is also closely associated with numerous other pathological conditions. The present study aimed to investigate the effect of microRNA (miRNA/miR)­378d on ovarian granulosa cells by regulating the round spermatid basic protein 1 (Rsbn1) in the absence of VD. The abnormal expression of miRNAs in ovarian tissues of the VD­deficient mouse was analyzed using transcriptome sequencing. miR­378d, glucose transporter 4 (Glut4) and aromatase (Cyp19a) expression levels were examined via reverse transcription­quantitative (RT­q)PCR and western blotting. The expression levels of Rsbn1, Glut4 and Cyp19a were detected in transfected mouse ovarian granulosa cells. The targeting regulation between miR­378d and Rsbn1 was verified using double reporter gene assay and functional rescue experiments. Among the 672 miRNAs that were differentially expressed, cluster analysis revealed that 17 were significantly upregulated and 16 were significantly downregulated. Moreover, miR­378d showed significant upregulation, which was further verified via RT­qPCR. It was identified that the protein expression level of Rsbn1 was significantly downregulated. Furthermore, Glut4 mRNA expression was significantly decreased in the mimic group but markedly increased in the inhibitor group. By contrast, the mRNA expression levels of Rsbn1 and Cyp19a did not demonstrate any significant difference. The western blotting results indicated that the protein expression levels of Rsbn1 and Glut4 were decreased and increased, respectively, while Cyp19a did not show any significant change. In addition, the double reporter gene experiments confirmed that Rsbn1 was the target gene of miR­378d. Collectively, the present results demonstrated that miR­378d was abnormally overexpressed in the ovarian tissues of the VD­deficient mice, and that miR­378d could inhibit Glut4 production by targeting Rsbn1, which may lead to insulin resistance.

Vitamin D deficiency inhibits microRNA-196b-5p which regulates ovarian granulosa cell hormone synthesis, proliferation, and apoptosis by targeting RDX and LRRC17.

BACKGROUND: In polycystic ovary syndrome (PCOS), ovarian physiology is tightly linked to the metabolic disturbances observed in this disease. Vitamin D (VD) plays an important role in the regulation of ovulatory dysfunction and can influence genes involved in steroidogenesis in granulosa cells (GCs). However, its role in the proliferation and apoptosis of ovarian GCs is unclear. The present study aimed to investigate the role of microRNA-196-5p (miR-196b-5p) in the hormone synthesis, proliferation, and apoptosis of ovarian GCs. METHODS: The abnormal expression of miRNAs in ovarian tissues of VD-deficient mice was analyzed using transcriptome sequencing. The direct target of miR-196b-5p was predict and confirmed by bioinformatics analysis and the dual-luciferase reporter assay. Reverse transcription-quantitative PCR (RT-qPCR) was used to detect the levels of miR-196b-5p, cell proliferation was detected via the CCK8 assay, and cell apoptosis and reactive oxygen species (ROS) were measured via flow cytometry. The levels of radixin (RDX), leucine rich repeat containing 17 (LRRC17), aromatase (CYP19A1), and glucose transporter 4 (GLUT4) were detected by performing RT-qPCR or western blot. RESULTS: We found that miR-196b-5p was significantly downregulated among the 672 miRNAs that were differentially expressed (DE) in VD-deficient mice. In addition, the results demonstrated that downregulated expression of miR-196b-5p significantly increased the level of RDX and LRRC17, and reduced expression of miR-196b-5p significantly promoted ovarian GC apoptosis and inhibited cell proliferation. Downregulated expression of miR-196b-5p promoted cellular ROS production and inhibited sex hormone production and glucose uptake. Transfection with miR-196b-5p mimics significantly increased the expression of CYP19A1 and GLUT4 and decreased the RDX and LRRC17 levels in ovarian GCs. CONCLUSIONS: This study shows that miR-196b-5p can regulate the oxidative stress (OS), glucose uptake, and steroid production pathway of GCs, thus promoting follicular development and maturation. This is a step towards a feasible treatment for PCOS.

[Prenatal diagnosis of two cases with 2p15-p16.1 microdeletion syndrome].

OBJECTIVE: To detect chromosomal aberrations in two fetuses with multiple malformation. METHODS: The two fetuses were subjected to chromosomal microarray analysis (CMA) by using Affymetrix CytoScan 750K arrays. The results were analyzed by bioinformatic software. RESULTS: CMA analysis suggested that both fetuses harbored pathogenic copy number variations (CNVs) in the 2p15-16.1 region, which ranged from 255 kb to 257 kb and encompassed the XPO1 and USP34 genes. CONCLUSION: Deletion of the chr2 (61 659 957-61 733 075, hg19) encompassing the XPO1 and USP34 genes may underlie the multiple malformations in the two fetuses.

Cationic Copolymerization of Isobutylene with 4-Vinylbenzenecyclobutylene: Characteristics and Mechanisms.

A random copolymer of isobutylene (IB) and 4-vinylbenzenecyclobutylene (4-VBCB) was synthesized by cationic polymerization at -80 °C using 2-chloro-2,4,4-trimethylpentane (TMPCl) as initiator. The laws of copolymerization were investigated by changing the feed quantities of 4-VBCB. The molecular weight of the copolymer decreased, and its molecular weight distribution (MWD) increased with increasing 4-VBCB content. We proposed a possible copolymerization mechanism behind the increase in the chain transfer reaction to 4-VBCB with increasing of feed quantities of 4-VBCB. The thermal properties of the copolymers were studied by solid-phase heating and crosslinking. After crosslinking, the decomposition and glass transition temperatures (Tg) of the copolymer increased, the network structure that formed did not break when reheated, and the mechanical properties remarkably improved.

Synthesis and characterization of hydroxyl-terminated butadiene-end-capped polyisobutylene and its use as a diol for polyurethane preparation.

Hydroxyl-terminated telechelic polyisobutylene (PIB) was prepared through living cationic polymerization. A living PIB chain was formed using the t-Bu-m-DiCuOMe/TiCl4 initiating system and then capped with 1,3-butadiene (BD) to prepare chlorine-terminated telechelic PIB. The chlorine-terminated telechelic PIB was then hydrolysed with tetrabutylammonium hydroxide to form hydroxyl-terminated PIB. Nuclear magnetic resonance spectroscopy confirmed hydrolysis completion. The hydroxyl-terminated PIB was subsequently used as a diol to react with 4,4-methylenebis(phenylisocyanate) (MDI) and produce a PIB-based polyurethane, which showed stronger acid resistance, hydrolysis stability and thermal oxidation stability than a commercial polyurethane.

Investigation of the interactions between 1-butyl-3-methylimidazolium-based ionic liquids and isobutylene using density functional theory.

To identify ionic liquids (ILs) that could be used as solvents in isobutylene (IB) polymerization, the interactions between IB and eight different ILs based on the 1-butyl-3-methylimidazolium cation ([Bmim]+) were investigated using density functional theory (DFT). The anions in the ILs were chloride, hexafluorophosphate, tetrafluoroborate, bis[(trifluoromethyl)sulfonyl]imide, tetrachloroaluminate ([AlCl4]-), tetrachloroferrate, acetate, and trifluoroacetate. The interaction geometries were explained by changes in the total energy, intermolecular distances, Hirshfeld charges, and the electrostatic potential surface. The IL solvents were screened by comparing their interaction intensities with IB to the interaction intensities of reference ILs ([AlCl4]--based ILs) with IB. The microscopic mechanism for IB dissolution was rationalized by invoking a previously reported microscopic mechanism for the dissolution of gases in ILs. Computation results revealed that hydrogen (H) bonding between C2-H on the imidazolium ring and the anions plays a key role in ion pair (IP) formation. The addition of IB leads to slight changes in the dominant interactions of the IP. IB molecules occupied cavities created by small angular rearrangements of the anions, just as CO2 does when it is dissolved in an IL. The limited total free space in the ILs and the much larger size of IB than CO2 were found to be responsible for the poor solubility of IB compared with that of CO2 in the ILs.

Insights into Campylobacter jejuni colonization and enteritis using a novel infant rabbit model.

A lack of relevant disease models for Campylobacter jejuni has long been an obstacle to research into this common enteric pathogen. Here we used an infant rabbit to study C. jejuni infection, which enables us to define several previously unknown but key features of the organism. C. jejuni is capable of systemic invasion in the rabbit, and developed a diarrhea symptom that mimicked that observed in many human campylobacteriosis. The large intestine was the most consistently colonized site and produced intestinal inflammation, where specific cytokines were induced. Genes preferentially expressed during C. jejuni infection were screened, and acs, cj1385, cj0259 seem to be responsible for C. jejuni invasion. Our results demonstrates that the infant rabbit can be used as an alternative experimental model for the study of diarrheagenic Campylobacter species and will be useful in exploring the pathogenesis of other related pathogens.

[Preparation and characterization of monoclonal antibodies against cytolethal distending toxin protein of Campylobacter jejuni].

OBJECTIVE: To express Campylobacter jejuni cytolethal distending toxin B protein (CdtB) in a prokaryote to prepare monoclonal antibodies (mAbs) against the protein, and to study their antitoxic effects. METHODS: The C. jejuni cdtB gene was amplified and inserted into the expression plasmids pET-30a( + ) and pGEX-6p-1. The purified rGST-CdtB protein was used as the immunogen to screen hybridoma cells for mAbs against the protein. The mAb titers were determined with an indirect enzyme-linked immunosorbent assay (ELISA), and their specificity with a Dot-ELISA and western blotting analysis. We determined the antitoxic properties of the mAbs in CaCo-2 and HD-11 cells. RESULTS: Recombinant expression plasmids pET-30a (+)-cdtB and pGEX-6p-l-cdtB were successfully constructed, and fusion proteins rHis-CdtB and rGST-CdtB expressed, respectively. Five hybridoma cell lines, designated 1F3, IF5, 2E4, 2E11, and 2F2, were screened for the stable secretion of mAbs against CdtB. The immunoglobulin subclass of 2E11 was IgG2b and that of the other mAbs was IgG1. The mAb titers in the ascites fluids were 1:1 x 10(8) on indirect ELISA. Dot-ELISA demonstrated that the five mAbs reacted specifically with C. jejuni. Western blotting analysis confirmed that the five mAbs reacted well with the rGST-CdtB fusion protein. The mAbs significantly reduced the adhesion and invasion capacities of the bacterium in CaCo-2 cells (P < 0.01). CONCLUSION: The successful preparation of five mAbs specific for the CdtB protein will allow further study of the biological characteristics of CdtB and the pathogenesis of C. jejuni.

Use of in vivo-induced antigen technology to identify in vivo-expressed genes of Campylobacter jejuni during human infection.

Campylobacter jejuni is a prevalent foodborne pathogen worldwide. Human infection by C. jejuni primarily arises from contaminated poultry meats. Genes expressed in vivo may play an important role in the pathogenicity of C. jejuni. We applied an immunoscreening method, in vivo-induced antigen technology (IVIAT), to identify in vivo-induced genes during human infection by C. jejuni. An inducible expression library of genomic proteins was constructed from sequenced C. jejuni NCTC 11168 and was then screened using adsorbed, pooled human sera obtained from clinical patients. We successfully identified 24 unique genes expressed in vivo. These genes were implicated in metabolism, molecular biosynthesis, genetic information processing, transport, and other processes. We selected six genes with different functions to compare their expression levels in vivo and in vitro using real-time RT-PCR. The results showed that the selected six genes were significantly upregulated in vivo but not in vitro. In short, these identified in vivo-induced genes may contribute to human infection of C. jejuni, some of which may be meaningful vaccine candidate antigens or diagnosis serologic markers for campylobacteriosis. IVIAT may present a significant and efficient method for understanding the pathogenicity mechanism of Campylobacter and for finding targets for its prevention and control.

[Preparation and characterization of monoclonal antibodies against FlhF protein of Campylobacter jejuni].

OBJECTIVE: To develop a prokaryotic expression system for Campylobacrer jejuni FlhF protein and prepare monoclonal antibodies(mAb) against this protein. METHODS: The C. jejuni flhF gene was amplified and inserted into the expression plasmids, pET-30a(+) and pGEX-6p-1. Then the rHis-FlhF protein and rGST-FlhF protein were expressed and purified by affinity column chromatography. They were subsequently used as immunogen and detecting antigen to screen antibodies produced by hybridoma cells against these proteins. The titers of the mAbs were measured by indirect ELISA, and the specificities of the mAbs was evaluated by Dot-ELISA and Western blot analysis. RESULTS: The recombinant expression plasmids pET-30a(+)-flhF and pGEX-6p-1-flhF were successfully constructed, and the fusion proteins rHis-FlhF and rGST-FlhF were produced at sufficient quantities. Two hybridoma cell lines were screened, designated 2C12 and 7A9, which secrete mAbs against FlhF stably. Their immunoglobulin subclasses were both IgG1. The titers of the ascites fluid were 1×10(9); and 2×10(7); respectively. Western blot analyses confirmed that the two mAbs both reacted with the rHis-FlhF fusion protein with good sensitivity. The Dot-ELISA results demonstrated that the two mAbs reacted specifically with Campylobacrer jejuni. CONCLUSION: The mAbs against Campylobacrer jejuni FlhF protein with high specificity were successfully prepared.

A novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens using pre-adsorbed sera from infected patients.

BACKGROUND: Campylobacter jejuni is an important food-borne and zoonotic pathogen with a worldwide distribution. Humans and chickens are hosts of this pathogen. At present, there is no ideal vaccine for controlling human campylobacteriosis or the carriage of C. jejuni by chickens. Bacterial in vivo-induced antigens are useful as potential vaccine candidates and biomarkers of virulence. METHODS: In this study, we developed a novel systematic immunoproteomics approach to identify in vivo-induced antigens among the total cell proteins of C. jejuni using pre-adsorbed sera from patients infected with C. jejuni. RESULTS: Overall, 14 immunoreactive spots were probed on a PVDF membrane using pre-adsorbed human sera against C. jejuni. Then, we excised these protein spots from a duplicate gel and identified using MALDI-TOF MS. In total, 14 in vivo-induced antigens were identified using PMF and BLAST analysis. The identified proteins include CadF (CadF-1 and CadF-2), CheW, TufB, DnaK, MetK, LpxB, HslU, DmsA, PorA, ProS, CJBH_0976, CSU_0396 and hypothetical protein cje135_05017. Real-time RT-PCR was performed on 9 genes to compare their expression levels in vivo and in vitro. The data showed that 8 of the 9 analyzed genes were significantly upregulated in vivo relative to in vitro. CONCLUSION: We successfully developed a novel immunoproteomics method for identifying in vivo-induced Campylobacter jejuni antigens by using pre-adsorbed sera from infected patients. GENERAL SIGNIFICANCE: This new analysis method may prove to be useful for identifying in vivo-induced antigens within any host infected by bacteria and will contribute to the development of new subunit vaccines.