Luminal-Applied Flagellin Is Internalized by Polarized Intestinal Epithelial Cells and Elicits Immune Responses via the TLR5 Dependent Mechanism

Bacteria release flagellin that elicits innate responses via Toll-like receptor 5 (TLR5). Here, we investigated the fate of apically administrated full length flagellin from virulent and avirulent bacteria, along with truncated recombinant flagellin proteins in intestinal epithelial cells and cellular responses. Flagellin was internalized by intestinal epithelial cell (IEC) monolayers of IEC-18. Additionally, apically applied flagellin was internalized by polarized human Caco-2BBe and T-84 cells in a TLR5 dependent mechanism. More, flagellin exposure did not affect the integrity of intestinal monolayers. With immunofluorescent staining, internalized flagellin was detected in both early endosomes as well as lysosomes. We found that apical exposure of polarized Caco-2BBe and T-84 to flagellin from purified Salmonella, Escherichia coli O83:H1 (isolate from Crohn’s lesion) or avirulent E. coli K12 induced comparable levels of basolateral IL-8 secretion. A recombinant protein representing the conserved amino (N) and carboxyl (C) domains (D) of the flagellin protein (ND1/2ECHCD2/1) induced IL-8 secretion from IEC similar to levels elicited by full-length flagellins. However, a recombinant flagellin protein containing only the D3 hypervariable region elicited no IL-8 secretion in both cell lines compared to un-stimulated controls. Silencing or blocking TLR5 in Caco-2BBe cells resulted in a lack of flagellin internalization and decreased IL-8 secretion. Furthermore, apical exposure to flagellin stimulated transepithelial migration of neutrophils and dendritic cells. The novel findings in this study show that luminal-applied flagellin is internalized by normal IEC via TLR5 and co-localizes to endosomal and lysosomal compartments where it is likely degraded as flagellin was not detected on the basolateral side of IEC cultures

Chemical Basis of Metabolic Network Organization

Although the metabolic networks of the three domains of life consist of different constituents and metabolic pathways, they exhibit the same scale-free organization. This phenomenon has been hypothetically explained by preferential attachment principle that the new-recruited metabolites attach preferentially to those that are already well connected. However, since metabolites are usually small molecules and metabolic processes are basically chemical reactions, we speculate that the metabolic network organization may have a chemical basis. In this paper, chemoinformatic analyses on metabolic networks of Kyoto Encyclopedia of Genes and Genomes (KEGG), Escherichia coli and Saccharomyces cerevisiae were performed. It was found that there exist qualitative and quantitative correlations between network topology and chemical properties of metabolites. The metabolites with larger degrees of connectivity (hubs) are of relatively stronger polarity. This suggests that metabolic networks are chemically organized to a certain extent, which was further elucidated in terms of high concentrations required by metabolic hubs to drive a variety of reactions. This finding not only provides a chemical explanation to the preferential attachment principle for metabolic network expansion, but also has important implications for metabolic network design and metabolite concentration prediction

Broad Spectrum Pro-Quorum-Sensing Molecules as Inhibitors of Virulence in Vibrios

Quorum sensing (QS) is a bacterial cell-cell communication process that relies on the production and detection of extracellular signal molecules called autoinducers. QS allows bacteria to perform collective activities. Vibrio cholerae, a pathogen that causes an acute disease, uses QS to repress virulence factor production and biofilm formation. Thus, molecules that activate QS in V. cholerae have the potential to control pathogenicity in this globally important bacterium. Using a whole-cell high-throughput screen, we identified eleven molecules that activate V. cholerae QS: eight molecules are receptor agonists and three molecules are antagonists of LuxO, the central NtrC-type response regulator that controls the global V. cholerae QS cascade. The LuxO inhibitors act by an uncompetitive mechanism by binding to the pre-formed LuxO-ATP complex to inhibit ATP hydrolysis. Genetic analyses suggest that the inhibitors bind in close proximity to the Walker B motif. The inhibitors display broad-spectrum capability in activation of QS in Vibrio species that employ LuxO. To the best of our knowledge, these are the first molecules identified that inhibit the ATPase activity of a NtrC-type response regulator. Our discovery supports the idea that exploiting pro-QS molecules is a promising strategy for the development of novel anti-infectives

A cutting force model based on kinematics analysis for C/SiC in rotary ultrasonic face machining

Ceramic matrix composites (CMC) superior properties and are used in the harsh conditions of high temperature and pressure, in aerospace and other industries. However, due to inhomogeneous and anisotropic properties of the composites, the machining is still challenging to achieve desired efficiency and quality. For advanced materials, Rotary ultrasonic machining is considered as a process with high efficiency technology. The cutting force is a critical factor required to be effectively predicted and controlled to reduce processing defects in composites. In this research, the rotary ultrasonic machining was used for face machining of carbon reinforced silicon carbide matrix composites (C/SiC), with a conical shaped tool. The kinematics between individual diamond abrasive and the workpiece material was analyzed to illustrate the separation characteristics in the cutting area. The condition for the intermittent machining during RUFM was obtained by establishing the mathematical relation between cutting parameters and vibration parameters. The indentation fracture theory was adopted to calculate the penetration depth into the workpiece by diamond abrasives in the RUFM. The relationship of cutting force and processing parameters including spindle speed, feed rate, and cutting depth were investigated. The comparison of the experimental and simulation data of the cutting force, showed that most of the tests, the errors were below 15 %. It is therefore stipulated that the cutting force model developed in this paper can be applied to predict cutting forces and optimize the process in the RUFM of C/SiC

Large-scale and rapid synthesis of disk-shaped and nano-sized graphene

We synthesized disk-shaped and nano-sized graphene (DSNG) though a novel ion-exchange methodology. This new methodology is achieved by constructing metal ion/ion-exchange resin framework. The morphology and size of the graphene can be modulated by changing the mass ratio of the carbon-containing resin to the cobalt-containing precursor. This is the first time to show that the DSNG formed on the granular transition metal substrate. The DSNG gives a high intensity of photoluminescence at near-UV wavelength of 311 nm which may provide a new type of fluorescence for applications in laser devices, ultraviolet detector UV-shielding agent and energy technology. The emission intensity of the DSNG is thirty times higher than that of the commercial large graphene. Our approach for graphene growth is conveniently controllable, easy to scale-up and the DSNG shows superior luminescent properties as compared to conventional large graphene

Trait evaluation and trial cultivation of Dongfang No. 2, the hybrid of a male gametophyte clone of Laminaria longissima (Laminariales, Phaeophyta) and a female one of L. japonica

Direct cultivation of the first filial generation of gametophyte clones from different Laminaria species is a highly effective way of utilizing kelp heterozygous vigor (heterosis). A male gametophyte clone of L. longissima Miyabe and a female one of L. japonica Areschoug were hybridized, generating Dongfang No. 2 hybrid kelp. This hybrid kelp was used directly in trial cultivation, and its agronomical traits were evaluated. L. longissima and L. japonica are obviously different and complement each other in their morphological characteristics and ecological performances. The hybrid of their gametophyte clones, Dongfang No. 2, showed 56.8% heterozygous vigor in yield. It also showed increased yields of 41.0 and 76.4% compared to the widely used commercial kelps Variety 1 and Variety 2, respectively. In large-scale cultivation trials at different locations and in different years, Dongfang No. 2 attained significantly higher yields than Varieties 1 and 2, increasing yield by 26.4% on average over Variety 1 and by 65.0% over the other. Dongfang No. 2 has a robust holdfast and a wide, long and deep-brown uniform blade, which shows a distinct middle groove. In addition to yield, Dongfang No. 2 also demonstrates obvious heterozygous vigor in other agronomic traits. It is resistant to strong irradiance, as the two commercial varieties are, has an appropriate vegetative maturation time, and adapts well to a range of different culture conditions. The parentage analysis using AFLP of total DNA and SNP of the ITS region of ribosomal RNA transcription unit showed that Dongfang No. 2 is the real hybrid of L. japonica and L. longissima

Trends in Notifiable Infectious Diseases in China: Implications for Surveillance and Population Health Policy

This study aimed to analyse trends in notifiable infectious diseases in China, in their historical context. Both English and Chinese literature was searched and diseases were categorised according to the type of disease or transmission route. Temporal trends of morbidity and mortality rates were calculated for eight major infectious diseases types. Strong government commitment to public health responses and improvements in quality of life has led to the eradication or containment of a wide range of infectious diseases in China. The overall infectious diseases burden experienced a dramatic drop during 1975–1995, but since then, it reverted and maintained a gradual upward trend to date. Most notifiable diseases are contained at a low endemic level; however, local small-scale outbreaks remain common. Tuberculosis, as a bacterial infection, has re-emerged since the 1990s and has become prevalent in the country. Sexually transmitted infections are in a rapid, exponential growth phase, spreading from core groups to the general population. Together human immunodeficiency virus (HIV), they account for 39% of all death cases due to infectious diseases in China in 2008. Zoonotic infections, such as severe acute respiratory syndrome (SARS), rabies and influenza, pose constant threats to Chinese residents and remain the most deadly disease type among the infected individuals. Therefore, second-generation surveillance of behavioural risks or vectors associated with pathogen transmission should be scaled up. It is necessary to implement public health interventions that target HIV and relevant coinfections, address transmission associated with highly mobile populations, and reduce the risk of cross-species transmission of zoonotic pathogens

A Systematic Screen for Micro-RNAs Regulating the Canonical Wnt Pathway

MicroRNAs (miRs) and the canonical Wnt pathway are known to be dysregulated in human cancers and play key roles during cancer initiation and progression. To identify miRs that can modulate the activity of the Wnt pathway we performed a cell-based overexpression screen of 470 miRs in human HEK293 cells. We identified 38 candidate miRs that either activate or repress the canonical Wnt pathway. A literature survey of all verified candidate miRs revealed that the Wnt-repressing miRs tend to be anti-oncomiRs and down-regulated in cancers while Wnt-activating miRs tend to be oncomiRs and upregulated during tumorigenesis. Epistasis-based functional validation of three candidate miRs, miR-1, miR-25 and miR-613, confirmed their inhibitory role in repressing the Wnt pathway and suggest that while miR-25 may function at the level of â-catenin (β-cat), miR-1 and miR-613 act upstream of β-cat. Both miR-25 and miR-1 inhibit cell proliferation and viability during selection of human colon cancer cell lines that exhibit dysregulated Wnt signaling. Finally, transduction of miR-1 expressing lentiviruses into primary mammary organoids derived from Conductin-lacZ mice significantly reduced the expression of the Wnt-sensitive β-gal reporter. In summary, these findings suggest the potential use of Wnt-modulating miRs as diagnostic and therapeutic tools in Wnt-dependent diseases, such as cancer