Airway epithelium controls lung inflammation and injury through the NF-kappa B pathway

Although airway epithelial cells provide important barrier and host defense functions, a crucial role for these cells in development of acute lung inflammation and injury has not been elucidated. We investigated whether NF-kappaB pathway signaling in airway epithelium could decisively impact inflammatory phenotypes in the lungs by using a tetracycline-inducible system to achieve selective NF-kappaB activation or inhibition in vivo. In transgenic mice that express a constitutively active form of IkappaB kinase 2 under control of the epithelial-specific CC10 promoter, treatment with doxycycline induced NF-kappaB activation with consequent production of a variety of proinflammatory cytokines, high-protein pulmonary edema, and neutrophilic lung inflammation. Continued treatment with doxycycline caused progressive lung injury and hypoxemia with a high mortality rate. In contrast, inducible expression of a dominant inhibitor of NF-kappaB in airway epithelium prevented lung inflammation and injury resulting from expression of constitutively active form of IkappaB kinase 2 or Escherichia coli LPS delivered directly to the airways or systemically via an osmotic pump implanted in the peritoneal cavity. Our findings indicate that the NF-kappaB pathway in airway epithelial cells is critical for generation of lung inflammation and injury in response to local and systemic stimuli; therefore, targeting inflammatory pathways in airway epithelium could prove to be an effective therapeutic strategy for inflammatory lung diseases.     

Genomic prediction of genetic merit using LD-based haplotypes in the Nordic Holstein population

Background

A haplotype approach to genomic prediction using high density data in dairy cattle as an alternative to single-marker methods is presented. With the assumption that haplotypes are in stronger linkage disequilibrium (LD) with quantitative trait loci (QTL) than single markers, this study focuses on the use of haplotype blocks (haploblocks) as explanatory variables for genomic prediction. Haploblocks were built based on the LD between markers, which allowed variable reduction. The haploblocks were then used to predict three economically important traits (milk protein, fertility and mastitis) in the Nordic Holstein population.

Results

The haploblock approach improved prediction accuracy compared with the commonly used individual single nucleotide polymorphism (SNP) approach. Furthermore, using an average LD threshold to define the haploblocks (LD≥0.45 between any two markers) increased the prediction accuracies for all three traits, although the improvement was most significant for milk protein (up to 3.1 % improvement in prediction accuracy, compared with the individual SNP approach). Hotelling’s t-tests were performed, confirming the improvement in prediction accuracy for milk protein. Because the phenotypic values were in the form of de-regressed proofs, the improved accuracy for milk protein may be due to higher reliability of the data for this trait compared with the reliability of the mastitis and fertility data. Comparisons between best linear unbiased prediction (BLUP) and Bayesian mixture models also indicated that the Bayesian model produced the most accurate predictions in every scenario for the milk protein trait, and in some scenarios for fertility.

Conclusions

The haploblock approach to genomic prediction is a promising method for genomic selection in animal breeding. Building haploblocks based on LD reduced the number of variables without the loss of information. This method may play an important role in the future genomic prediction involving while genome sequences.     

Comprehensive evaluation of canonical versus Dicer-substrate siRNA in vitro and in vivo

Since the discovery of RNA interference (RNAi), researchers have identified a variety of small interfering RNA (siRNA) structures that demonstrate the ability to silence gene expression through the classical RISC-mediated mechanism. One such structure, termed "Dicer-substrate siRNA" (dsiRNA), was proposed to have enhanced potency via RISC-mediated gene silencing, although a comprehensive comparison of canonical siRNAs and dsiRNAs remains to be described. The present study evaluates the in vitro and in vivo activities of siRNAs and dsiRNAs targeting Phosphatase and Tensin Homolog (PTEN) and Factor VII (FVII). More than 250 compounds representing both siRNA and dsiRNA structures were evaluated for silencing efficacy. Lead compounds were assessed for duration of silencing and other key parameters such as cytokine induction. We identified highly active compounds from both canonical siRNAs and 25/27 dsiRNAs. Lead compounds were comparable in potency both in vitro and in vivo as well as duration of silencing in vivo. Duplexes from both structural classes tolerated 2'-OMe chemical modifications well with respect to target silencing, although some modified dsiRNAs demonstrated reduced activity. On the other hand, dsiRNAs were more immunostimulatory as compared with the shorter siRNAs, both in vitro and in vivo. Because the dsiRNA structure does not confer any appreciable benefits in vitro or in vivo while demonstrating specific liabilities, further studies are required to support their applications in RNAi therapeutics.     

Desensitization of herpesvirus-encoded G protein-coupled receptors

Members of the herpesvirus family, including human cytomegalovirus (HCMV) and Kaposi's sarcoma-associated herpesvirus (KSHV/HHV-8), encode G protein-coupled receptor (GPCR) homologs, which strongly activate classical G protein signal transduction networks within the cell. In animal models of herpesvirus infection, the viral GPCRs appear to play physiologically important roles by enabling viral replication within tropic tissues and by promoting reactivation from latency. While a number of studies have defined intracellular signaling pathways activated by herpesviral GPCRs, it remains unclear if their physiological function is subjected to the process of desensitization as observed for cellular GPCRs. G protein-coupled receptor kinases (GRK) and arrestin proteins have been recently implicated in regulating viral GPCR signaling; however, the role that these desensitization proteins play in viral GPCR function in vivo remains unknown. Here, we review what is currently known regarding viral GPCR desensitization and discuss potential biological ramifications of viral GPCR regulation by the host cell desensitization machinery.     

定向进化人α型干扰素基因家族的研究

应用DNA改组(DNAshuffling)技术重组了12种人α型干扰素基因,并结合噬菌体表面呈现(phagedisplay)技术构建了噬菌体干扰素改组文库,采用简便的、功能性的定向竞争筛选方法,获得了比活达1 0×109IU/mg的新型α型基因工程复合干扰素,是目前国际上已投产的α2型干扰素的5倍,具有生产应用前景,上述策略也为其它细胞因子或酶的改造提供了借鉴方法。     

Microcosm and Experimental Pond Evaluation of Microbial Community Response to Synthetic Oil Contamination in Freshwater Sediments

A multivariate approach was used to evaluate the significance of synthetic oil-induced perturbations in the functional activity of sediment microbial communities. Total viable cell densities, ATP-biomass, alkaline phosphatase and dehydrogenase activity, and mineralization rates of glucose, protein, oleic acid, starch, naphthalene, and phenanthrene were monitored on a periodic basis in microcosms and experimental ponds for 11 months, both before and after exposure to synthetic oil. All variables contributed to significant discrimination between sediment microbial responses in control communities and communities exposed to a gradient of synthetic oil contamination. At high synthetic oil concentrations (4,000 ml/12 m³), a transient reduction in sediment ATP concentrations and increased rates of oleic acid mineralization were demonstrated within 1 week of exposure. These transient effects were followed within 1 month by a significant increase in rates of naphthalene and phenanthrene mineralization. After initial construction, both control and synthetic oil-exposed microbial communities demonstrated wide variability in community activity. All experimental microbial communities approached equilibrium and demonstrated good replication. However, synthetic oil perturbation was demonstrated by wide transient variability in community activity. This variability was primarily the result of the stimulation of polyaromatic hydrocarbon mineralization rates. In general, microcosms and pond communities demonstrated sufficient resiliency to recover from the effects of synthetic oil exposure within 3 months, although polyaromatic hydrocarbon mineralization rates remained significantly elevated.     

Import and Metabolism of Glutathione by Streptococcus mutans

Glutathione (γ-GluCysGly, GSH) is not found in most gram-positive bacteria, but some appear to synthesize it and others, including Streptococcus mutans ATCC 33402, import it from their growth medium. Import of oxidized glutathione (GSSG) by S. mutans 33402 in 7H9 medium was shown to require glucose and to occur with an apparent K_m of 18 ± 5 μM. GSSG, GSH, S-methylglutathione, and homocysteine-glutathione mixed disulfide (hCySSG) were imported at comparable rates (measured by depletion of substrate in the medium), as was the disulfide of γ-GluCys. In contrast, the disulfide of CysGly was not taken up at a measurable rate, indicating that the γ-Glu residue is important for efficient transport. During incubation with GSSG, little GSSG was detected in cells but GSH and γ-GluCys accumulated during the first 30 min and then declined. No significant intracellular accumulation of Cys or sulfide was found. Transient intracellular accumulation of d/l-homocysteine, as well as GSH and γ-GluCys, was observed during import of hCySSG. Although substantial levels of GSH were found in cells when S. mutans was grown on media containing glutathione, such GSH accumulation had no effect on the growth rate. However, the presence of cellular GSH did protect against growth inhibition by the thiol-oxidizing agent diamide. Import of glutathione by S. mutans ATCC 25175, which like strain 33402 does not synthesize glutathione, occurred at a rate comparable to that of strain 33402, but three species which appear to synthesize glutathione (S. agalactiae ATCC 12927, S. pyogenes ATCC 8668, and Enterococcus faecalis ATCC 29212) imported glutathione at negligible or markedly lower rates.Bacteria import peptides composed of two to eight residues by means of a number of different multiprotein uptake systems or permeases (14). Of the bacterial permeases, those of Escherichia coli, Lactococcus lactis, and Salmonella typhimurium are the best studied (6, 7). In these organisms, there are individual permeases that have high affinity for dipeptides, tripeptides, dipeptides and tripeptides, or oligopeptides. Among the bacterial peptide permeases (14), there seems to be no discrimination of the specific amino acids of the transported peptides. However, switching the stereochemistry of C_α from l to d or modifying the C-terminal carboxylate or N-terminal amine of transported peptides significantly reduces the rate of transport. One transport system which does seem to recognize peptide residue side chains has been reported to exist in Enterococcus faecalis; this system transports only peptides that possess an N-terminal Asp or Glu (13).In 1978, we reported that glutathione (γ-GluCysGly, GSH) is not synthesized by most gram-positive bacteria (4), apparent exceptions being Streptococcus agalactiae and L. lactis (previously Streptococcus lactis). However, some of the gram-positive bacteria appeared to acquire GSH by import of another form of GSH from the growth medium. Uptake of glutathione by Streptococcus mutans was later studied by Thomas (16), who found that total cellular thiol content, and radioactivity from labeled GSH or oxidized GSH (GSSG), increased with the same kinetics. A careful study of L. lactis subsp. cremoris by Wiederholt and Steele (17) established that strain Z8 efficiently accumulates GSH when grown in medium supplemented with GSH but is unable to synthesize it, whereas strain C2 can neither import nor synthesize GSH. Species of Peptostreptococcus and Fusobacterium have been shown to markedly increase their production of H₂S, apparently derived by import of glutathione from the growth medium (2). Finally, cellular accumulation of radioactivity from radiolabeled GSH or GSSG added to the incubation medium has been demonstrated in Streptococcus pneumoniae, and a mutant in which the apparent transport of glutathione is blocked has been found (9).In a recent report (10), we provided evidence for accumulation of GSH through transport and synthesis of GSH by streptococci and enterococci, but the occurrence of these processes appeared to be species dependent and even, for some species, strain dependent. Such strain dependence appears most variable for L. lactis, where different strains can synthesize GSH, accumulate GSH by import, or do neither (4, 17). In the present research, we expand on our studies of streptococci in order to gain insight into the nature of the glutathione species transported, the fate of the glutathione once it enters the cell, and the function of glutathione in the cell.