Control of intracellular pH and growth by fibronectin in capillary endothelial cells

The aim of this work was to analyze the mechanism by which fibronectin (FN) regulates capillary endothelial cell proliferation. Endothelial cell growth can be controlled in chemically-defined medium by varying the density of FN coated on the substratum (Ingber, D. E., and J. Folkman. J. Cell Biol. 1989. 109:317-330). In this system, DNA synthetic rates are stimulated by FN in direct proportion to its effect on cell extension (projected cell areas) both in the presence and absence of saturating amounts of basic FGF. To investigate direct growth signaling by FN, we carried out microfluorometric measurements of intracellular pH (pHi), a cytoplasmic signal that is commonly influenced by soluble mitogens. pHi increased 0.18 pH units as FN coating densities were raised and cells progressed from round to spread. Intracellular alkalinization induced by attachment to FN was rapid and followed the time course of cell spreading. When measured in the presence and absence of FGF, the effects of FN and FGF on pHi were found to be independent and additive. Furthermore, DNA synthesis correlated with pHi for all combinations of FGF and FN. Ethylisopropylamiloride, a specific inhibitor of the plasma membrane Na+/H+ antiporter, completely suppressed the effects of FN on both pHi and DNA synthesis. However, cytoplasmic pH per se did not appear to be a critical determinant of growth since DNA synthesis was not significantly inhibited when pHi was lowered over the physiological range by varying the pH of the medium. We conclude that FN and FGF exert their growth-modulating effects in part through activation of the Na+/H+ exchanger, although they appear to trigger this system via separate pathways.     

Numerical investigations of the mechanical properties of a braided non-vascular stent design using finite element method

This paper discusses various issues relating to the mechanical properties of a braided non-vascular stent made of a Ni–Ti alloy. The design of the stent is a major factor which determines its reliability after implantation into a stenosed non-vascular cavity. This paper presents the effect of the main structural parameters on the mechanical properties of braided stents. A parametric analysis of a commercial stent model is developed using the commercial finite element code ANSYS. As a consequence of the analytical results that the pitch of wire has a greater effect than other structural parameters, a new design of a variable pitch stent is presented to improve mechanical properties of these braided stents. The effect of structural parameters on mechanical properties is compared for both stent models: constant and variable pitches. When the pitches of the left and right quarters of the stent are 50% larger and 100% larger than that of the central portion, respectively, the radial stiffness in the central portion increases by 10% and 38.8%, while the radial stiffness at the end portions decreases by 128% and 164.7%, the axial elongation by 25.6% and 56.6% and the bending deflection by 3.96% and 10.15%. It has been demonstrated by finite element analysis that the variable pitch stent can better meet the clinical requirements.     

Reactivation of Chromosomally Integrated Human Herpesvirus-6 by Telomeric Circle Formation

More than 95% of the human population is infected with human herpesvirus-6 (HHV-6) during early childhood and maintains latent HHV-6 genomes either in an extra-chromosomal form or as a chromosomally integrated HHV-6 (ciHHV-6). In addition, approximately 1% of humans are born with an inheritable form of ciHHV-6 integrated into the telomeres of chromosomes. Immunosuppression and stress conditions can reactivate latent HHV-6 replication, which is associated with clinical complications and even death. We have previously shown that Chlamydia trachomatis infection reactivates ciHHV-6 and induces the formation of extra-chromosomal viral DNA in ciHHV-6 cells. Here, we propose a model and provide experimental evidence for the mechanism of ciHHV-6 reactivation. Infection with Chlamydia induced a transient shortening of telomeric ends, which subsequently led to increased telomeric circle (t-circle) formation and incomplete reconstitution of circular viral genomes containing single viral direct repeat (DR). Correspondingly, short t-circles containing parts of the HHV-6 DR were detected in cells from individuals with genetically inherited ciHHV-6. Furthermore, telomere shortening induced in the absence of Chlamydia infection also caused circularization of ciHHV-6, supporting a t-circle based mechanism for ciHHV-6 reactivation.     

Chlamydia trachomatis impairs host base excision repair by downregulating polymerase β

Chlamydia trachomatis infections have been associated with ovarian cancer by several epidemiological studies. Here, we show that C. trachomatis‐infected primary human ovarian epithelial cells display elevated oxidative DNA damage. Base excision repair, an important cellular mechanism to repair oxidative DNA lesions, was impaired in infected primary ovarian and in several other types of cells. Polymerase β was downregulated in infected cells associated with upregulation of microRNA‐499a (miR‐499a). Stabilising polymerase β by inhibiting miR‐499a significantly improved repair. Moreover, downregulation of tumour suppressor p53 also resulted in attenuated repair in these cells. Thus, our data show that downregulation of polymerase β by direct inhibition through miR‐499a and downregulation of p53 debilitate the host‐cell base excision repair during C. trachomatis infection.     

Alkali and alkaline earth metals in decomposing macrophytes in a wetland system

The foraging habitat selections of brown-eared pheasant (Crossoptilon mantchuricum) and the common pheasant (Phasianus colchicus) were studied in Huanglongshan Nature Reserve Shaanxi, China. Foraging habitat characteristics were measured on the basis of expected differences between species at 183 sites from November to December 2006 and January 2007. The results showed that both species selected foraging habitats with altitude (<1200 m), conifer forest, half sunny and half shady slope, sunny slope, density of trees (<5 individuals/100 m²), cover of shrub (>50%), visibility class (<10%) and distance to water source (<300 m). However, the brown-eared pheasant selected habitats with cover of trees (30–50%), middle or lower slope location, distance to edge of woods (<300 m) and human disturbance (<500 m), and the selection on density of shrub was not observed, compared to the selections on cover of trees (<30%), lower slope location, distance to edge of woods (<500 m) and human disturbance (<300 m), and density of shrub (>500 individuals/100 m²) for common pheasant. We also found that the common pheasant avoid predators by concealment whereas brown-eared pheasant evade predations by running away strategy.     

A Pathogenesis Assay Using Saccharomyces cerevisiae and Caenorhabditis elegans Reveals Novel Roles for Yeast AP-1, Yap1, and Host Dual Oxidase BLI-3 in Fungal Pathogenesis

Treatment of systemic fungal infections is difficult because of the limited number of antimycotic drugs available. Thus, there is an immediate need for simple and innovative systems to assay the contribution of individual genes to fungal pathogenesis. We have developed a pathogenesis assay using Caenorhabditis elegans, an established model host, with Saccharomyces cerevisiae as the invading fungus. We have found that yeast infects nematodes, causing disease and death. Our data indicate that the host produces reactive oxygen species (ROS) in response to fungal infection. Yeast mutants sod1Δ and yap1Δ, which cannot withstand ROS, fail to cause disease, except in bli-3 worms, which carry a mutation in a dual oxidase gene. Chemical inhibition of the NADPH oxidase activity abolishes ROS production in worms exposed to yeast. This pathogenesis assay is useful for conducting systematic, whole-genome screens to identify fungal virulence factors as alternative targets for drug development and exploration of host responses to fungal infections.Nosocomial microbial infections are a growing health problem. Among these, fungal infections are especially threatening, with an estimated mortality rate of 40% (47). The key reason for this alarming mortality rate is the limited range of antifungal agents. Identification of new drug targets requires high-throughput infection assays that are complicated by the very fact that they involve two organisms: a host and a pathogen.We have taken a reductionistic approach to studying host-pathogen interactions and have developed a Saccharomyces cerevisiae-based assay to understand the genetic and molecular mechanisms of fungal pathogenesis. Using Caenorhabditis elegans as a model host, we have found that S. cerevisiae infects the worm, producing visible disease phenotypes. The two organisms used in our study are specifically suited for host-pathogen infection studies because both genomic sequences have been completely determined and mutants are readily available. A complete genome knockout collection is available for S. cerevisiae, a resource that does not exist for any fungal pathogen. Likewise an RNA interference (RNAi)-mediated knockdown genomic library is available for C. elegans. These unique tools are key in the context of a genetic screen and allow us to systematically scan the entire genomes to identify fungal virulence factors and modulators of host immunity that combat a fungal pathogen.The budding yeast S. cerevisiae has recently been described as an emerging pathogen and has been isolated from human patients (34, 35). It is routinely used as a model for pathogenic fungi because a large proportion of its genes are conserved in pathogenic fungi (for a review, see reference 32). Homologs of genes and pathways identified in S. cerevisiae have been shown to be important in bona fide pathogens. It has also been used for the identification of gene products important for fungal survival in the mammalian host environment (21, 46). For example, the SSD1 allele type affects pathogenicity of yeast, indicating that allelic variation at the SSD1 locus may be important for survival under various conditions (46). This has allowed investigators to use reverse genetic approaches to study contributions of genes whose importance has been established in S. cerevisiae.Caenorhabditis elegans has emerged as a valuable model host in which to study pathogenesis and innate immunity (for a review, see reference 22). Microbial genes essential for virulence in mammalian models have been shown to be required for pathogenicity in nematodes (43). These studies have primarily explored bacterial species and have tested only a few fungi, such as Cryptococcus neoformans and Candida albicans, to explore virulence strategies. These studies focus on a killing assay using C. elegans and have identified several virulence factors with homologs in S. cerevisiae (4, 37), suggesting that genes and pathways we have identified in S. cerevisiae are likely to be found in pathogens. Moreover, other pathogenic fungi tested are limited in the repertoire of laboratory tools available for their study, making them recalcitrant to genetic manipulation and inappropriate for whole-genome high-throughput approaches to studying fungal virulence. Recently, Breger et al. described the application of a C. elegans-based infection assay as a tool to screen a chemical library for candidate antifungal compounds (9). Our investigation complements these studies in two significant ways. First, it allows us to identify genes that exacerbate as well as attenuate the pathogenic process, because we use an intermediate disease phenotype, while most other studies have used death as an end point phenotype. This aspect, taken together with the fact that S. cerevisiae shares significant genetic identity with pathogenic fungi, suggests that our study will yield a basic understanding of fungal pathogenesis. Second, it allows us to conduct a systematic, unbiased, whole-genome screen, which is currently not available for pathogenic fungi. Furthermore, genes and pathways identified may be targeted for antimycotic drug development.Facets of innate immunity are evolutionarily conserved from nematodes to mammals. For example, a common defense strategy of mammals (phagocytes), (14), plants (3), and insects (23) is to produce reactive oxygen species (ROS), which directly damage pathogens. In human phagocytes, an NADPH oxidase enzyme complex produces ROS in host defense (19, 41). In Drosophila melanogaster, ROS are generated in the intestine by a NADPH oxidase to combat ingested bacteria (23). Loss of NADPH oxidase activity makes the fly susceptible to the bacterial infection (23, 24). Likewise, C. elegans has also been shown to produce ROS, such as superoxide and/or hydrogen peroxide, when it ingests bacterial pathogens (12). In each case, pathogen death can be abrogated by the addition of enzymes such as catalase that break down ROS (8, 27, 36), suggesting that ROS production plays a key role in a variety of pathogenic interactions.We have found that S. cerevisiae can cause infection and death in C. elegans. Our data indicate that the nematode host produces ROS in response to fungal infection. We demonstrate that mutant yeast carrying deletions of genes that mediate oxidative stress responses fail to induce the Dar disease phenotype except in mutant worms with an altered dual oxidase gene, suggesting that the generation of ROS is a part of the defense strategy for the host and the neutralization of ROS is needed for persistent fungal infection.     

Nutrient utilisation,growth performance and blood metabolites in Murrah buffalo calves (Bubalus bubalis) divergently selected for residual feed intake

The aim of this study was to evaluate differences in efficiency of feed utilisation between buffalo calves with low and high residual feed intake (RFI) by comparing feed intake, nutrient digestibility, growth traits and blood metabolites. Eighteen male Murrah buffalo calves (aged 4–6 months; 70 ± 1.0 kg body weight) were fed ad libitum with a total mixed ration for 120 d. Based on linear regression models involving dry matter intake (DMI), average daily gain (ADG) and mid-test metabolic body size, calves were assigned into low and high RFI groups. The RFI varied from ?0.33 to +0.28 kg DM/d with an average RFI of ?0.14 and 0.14 kg DM/d in low and high RFI calves, respectively. Calves had a mean DMI of 1.9 and 2.4 kg/d and an ADG of 0.5 and 0.6 kg/d in low and high RFI groups, respectively. Low RFI calves ate 19.0% less DM each day and required significantly less metabolisable energy for maintenance compared with high RFI calves (12.5 vs. 16.7 MJ/d). Nutrient digestibility and nitrogen balance did not differ among low and high RFI calves. In more efficient animals (low RFI calves) higher (p < 0.05) plasma level of growth hormone, insulin-like growth factor-1 (IGF-1), triiodothyronine (T₃) and lower concentration of thyroxin hormone were detected. No significant differences in levels of insulin, hydroxyproline, plasma and urine creatinine, total protein and albumin between high and low RFI groups were found. Blood metabolites showed significant (p < 0.05) differences at initial and final stages of study in both groups. At final stage of study, RFI showed negative correlations with growth hormone, IGF-1, T₃, urine creatinine and albumin. Low RFI buffalo calves are more efficient in feed utilisation and the differences in blood metabolites are probably due to differences in feed intake and body metabolism.     

Chlamydia trachomatis Infection Induces Replication of Latent HHV-6

Human herpesvirus-6 (HHV-6) exists in latent form either as a nuclear episome or integrated into human chromosomes in more than 90% of healthy individuals without causing clinical symptoms. Immunosuppression and stress conditions can reactivate HHV-6 replication, associated with clinical complications and even death. We have previously shown that co-infection of Chlamydia trachomatis and HHV-6 promotes chlamydial persistence and increases viral uptake in an in vitro cell culture model. Here we investigated C. trachomatis-induced HHV-6 activation in cell lines and fresh blood samples from patients having Chromosomally integrated HHV-6 (CiHHV-6). We observed activation of latent HHV-6 DNA replication in CiHHV-6 cell lines and fresh blood cells without formation of viral particles. Interestingly, we detected HHV-6 DNA in blood as well as cervical swabs from C. trachomatis-infected women. Low virus titers correlated with high C. trachomatis load and vice versa, demonstrating a potentially significant interaction of these pathogens in blood cells and in the cervix of infected patients. Our data suggest a thus far underestimated interference of HHV-6 and C. trachomatis with a likely impact on the disease outcome as consequence of co-infection.