Airway cellularity, lipid laden macrophages and microbiology of gastric juice and airways in children with reflux oesophagitis

Background and methods

Human metapneumovirus (hMPV) is a recently discovered respiratory virus associated with bronchiolitis, pneumonia, croup and exacerbations of asthma. Since respiratory viruses are frequently detected in patients with acute exacerbations of COPD (AE-COPD) it was our aim to investigate the frequency of hMPV detection in a prospective cohort of hospitalized patients with AE-COPD compared to patients with stable COPD and to smokers without by means of quantitative real-time RT-PCR.

Results

We analysed nasal lavage and induced sputum of 130 patients with AE-COPD, 65 patients with stable COPD and 34 smokers without COPD. HMPV was detected in 3/130 (2.3%) AE-COPD patients with a mean of 6.5 × 10⁵ viral copies/ml in nasal lavage and 1.88 × 10⁵ viral copies/ml in induced sputum. It was not found in patients with stable COPD or smokers without COPD.

Conclusion

HMPV is only found in a very small number of patients with AE-COPD. However it should be considered as a further possible viral trigger of AE-COPD because asymptomatic carriage is unlikely.     

Variation in Streptococcus pyogenes NAD+ Glycohydrolase Is Associated with Tissue Tropism

Streptococcus pyogenes is an important pathogen that causes a variety of diseases. The most common infections involve the throat (pharyngitis) or skin (impetigo); however, the factors that determine tissue tropism and severity are incompletely understood. The S. pyogenes NAD⁺ glycohydrolase (SPN) is a virulence factor that has been implicated in contributing to the pathogenesis of severe infections. However, the role of SPN in determining the bacterium''s tissue tropism has not been evaluated. In this report, we examine the sequences of spn and its endogenous inhibitor ifs from a worldwide collection of S. pyogenes strains. Analysis of average pairwise nucleotide diversity, average number of nucleotide differences, and ratio of nonsynonymous to synonymous substitutions revealed significant diversity in spn and ifs. Application of established models of molecular evolution shows that SPN is evolving under positive selection and diverging into NAD⁺ glycohydrolase (NADase)-active and -inactive subtypes. Additionally, the NADase-inactive SPN subtypes maintain the characteristics of a functional gene while ifs becomes a pseudogene. Thus, NADase-inactive SPN continues to evolve under functional constraint. Furthermore, NADase activity did not correlate with invasive disease in our collection but was associated with tissue tropism. The ability to cause infection at both the pharynx and the skin (“generalist” strains) is correlated with NADase-active SPN, while the preference for causing infection at either the throat or the skin (“specialist” strains) is associated with NADase-inactive SPN. These findings suggest that SPN has a NADase-independent function and prompt a reevaluation of the role of SPN in streptococcal pathogenesis.Many bacterial pathogens that are capable of causing infection at multiple tissue sites have considerable underlying genetic diversity that is reflected by the presence or absence of different subsets of virulence genes or by the presence of alternative alleles of specific virulence genes (37, 44, 48). For the latter genes, variation in sequence may arise under pressure to avoid the immune response or reflect proteins whose functions are diverging. Horizontal gene transfer (HGT) events can initially increase diversity through the reassortment of these variant virulence genes and may result in altered pathogenicity or the ability to more efficiently exploit a given ecological niche (37). Continued selection of fitter variants adapted for infection of a specific niche can then lead to a subsequent purging of genetic diversity and a reduction in the types of clinical syndromes a particular lineage can cause (8). As a consequence, genetically discrete subpopulations with strong tropisms for different tissues emerge within the existing species, and this process may represent a key step in the formation of new species (6). Understanding the changes that occur during niche specialization can provide important insights into pathogenic mechanisms required for infection of a specific tissue.Analysis of tissue-specific adaptation is emerging as an important approach for understanding the pathogenesis of the numerous diseases caused by Streptococcus pyogenes (group A streptococcus [GAS]). This Gram-positive bacterium has a worldwide distribution and is a pathogen of humans exclusively, causing important diseases, which include those that are destructive of tissue and life-threatening (cellulitis, necrotizing fasciitis) and those associated with deregulation of immunity (glomerulonephritis, rheumatic fever) (6, 12). However, most cases of S. pyogenes disease are more superficial and self-limiting and occur at either the throat (pharyngitis) or the skin (impetigo). These two tissue sites also represent the primary reservoirs responsible for dissemination of the organism to new hosts. A large body of epidemiological evidence that suggests that there are distinct subpopulations of strains more adapted for infection of either the throat or the skin has accumulated, suggesting that specific adaptations to these two tissues are driving the evolution of its pan-genome (6). However, the specific adaptations responsible for niche specialization are not well understood.A frequently used approach for uncovering a common molecular basis behind bacterial phenotype has been to group strains based on sequence variation in housekeeping genes (18). In the case of niche specialization, continued selection for variants more highly adapted to a particular tissue will purge neutral gene diversity in the adapted population relative to the population as a whole. However, a complication in deciphering trends associated with tissue adaptation in S. pyogenes has been that despite some niche separation, there are high rates of recombination relative to mutation within the species as a whole, on par with that of Streptococcus pneumoniae, a species considered to be highly recombinogenic (6, 22, 57). Frequent recombination has resulted in a random segregation of neutral housekeeping haplotypes between S. pyogenes strains from ecologically distinct subpopulations (6). Thus, standard approaches to establishing relationships between strains have been of only limited utility for understanding niche adaptation for S. pyogenes.A more productive approach for S. pyogenes has been to look for genetic variation outside neutral housekeeping genes that is strongly associated with ecological niche. In this regard, genotypes based on the gene encoding the M protein (emm) provide a significant correlation with tissue tropism (6). The M protein is a fibrillar surface molecule that plays multiple roles in promoting virulence, and serological typing based on M protein diversity has been the traditional method for classifying S. pyogenes strains (35). It is well established that strains with certain M types have a strong preference for infection at either the throat or the skin (9, 40). There are more than 200 known M types (50), which can be divided into 4 major subfamilies based on the sequence of the peptidoglycan-spanning domain at the 3′ end of emm (25). Furthermore, the emm locus can encode one gene or a combination of subfamily genes in a tandem arrangement (7). Analyses of large strain collections have revealed that in ∼99% of strains, the organization of emm genes in the locus can be assigned to one of five patterns (designated A to E) (6). Although strains with each emm pattern may colonize the same tissue types, there is a strong correlation between emm pattern and the ability of the organism to cause disease at specific tissue sites. Strains with emm patterns A to C generally cause pharyngitis; emm pattern D strains are typically the cause of skin diseases, such as impetigo; and emm pattern E strains are “generalists,” which can cause symptomatic infection at either tissue site at approximately equal fractions of the total (6). Since emm pattern is strongly associated with tissue tropism, it is likely that characteristics consistently coinherited with the emm pattern also play a role in determining the tissue tropism of the organism (6, 29).The S. pyogenes NAD⁺ glycohydrolase (SPN, also known as Nga) is a virulence factor with characteristics that merit evaluation for a possible role in tissue tropism. This secreted toxin has an enzymatic activity (NADase) that cleaves the glycosidic bond of β-NAD⁺ to produce nicotinamide and ADP-ribose. All S. pyogenes strains examined to date possess the gene that encodes SPN (spn), but some strains produce a SPN that lacks detectable NADase activity (1, 30, 36, 42). Since there is evidence that SPN′s robust NADase activity contributes to virulence (4, 43, 52, 56), the existence of NADase-deficient SPN has yet to be explained. Epidemiological studies conducted on several limited strain collections have not been informative, as these studies have both found (1, 52) and failed to find (15) an association between NADase activity and whether a lineage has the capacity to cause invasive disease. Whether or not SPN is associated with tissue tropism is not known.SPN also has multiple complex interactions with other proteins that suggest it has an important, yet incompletely understood role in disease pathogenesis. These interactions also imply that SPN is under considerable coevolutionary pressure with its partners (47). For example, the ability of S. pyogenes to produce NADase-active SPN is absolutely dependent on the presence of an endogenous inhibitor protein, immunity factor for SPN (IFS) (31, 42). IFS is a competitive inhibitor of SPN′s β-NAD⁺ substrate and apparently acts to inhibit self-toxicity resulting from any presecretory SPN molecules that adventitiously fold prior to their export from the streptococcal cell. In the absence of IFS, SPN is lethal for S. pyogenes. Interestingly, strains that produce NADase-inactive SPN also have a truncated form of IFS (42). Once secreted, both NADase-active SPN and NADase-inactive SPN are injected into the host cell cytoplasm by a process known as cytolysin-mediated translocation (CMT), which requires interaction between multiple domains of SPN and the pore-forming cytolysin streptolysin O (SLO) (11, 20, 39, 41). When in the cytoplasmic compartment, NADase-active SPN can trigger rapid cell death, which is associated with depletion of β-NAD⁺ pools (10, 11, 39). The genes for SPN (spn), IFS (ifs), and SLO (slo) are encoded in the same operon (31, 42), as is typical of coevolving virulence factor/inhibitor pairs (47). Thus, SPN has multiple complex interactions and is suspected of being important in pathogenesis; however, there is a considerable amount of genetic and functional variation that has yet to be fully defined.In the present study, we sought to clarify the role of SPN in the infectious process through analysis of the genetic diversity in spn and ifs and the relationship this diversity has with disease severity and ecologic niche. By examining a diverse, worldwide collection of S. pyogenes strains, we identify the SPN domains evolving under positive (diversifying) and negative (purifying) selection, correlate these sites with NADase activity, and demonstrate that NADase activity is associated with tissue tropism but not invasiveness of disease.     

The island rule and a research agenda for studying ecogeographical patterns

We are currently experiencing a resurgence of interest in ecogeographical rules, which describe general trends in morphology and related traits along geographical gradients. In order to develop a more comprehensive understanding of the generality and underlying causal mechanisms for these patterns, we recommend a new, more integrated research agenda. In particular, we recommend studies that simultaneously consider different clines in morphology, geographical ranges and diversity as intricately related phenomena; all being ecological, evolutionary and biogeographical responses of organisms to selection regimes that vary non-randomly over space and time, and among species with different ecological and evolutionary histories.     

Diversification in the Mexican horned lizard Phrynosoma orbiculare across a dynamic landscape

The widespread montane Mexican horned lizard Phrynosoma orbiculare (Squamata: Phrynosomatidae) represents an ideal species to investigate the relative impacts of Neogene vicariance and Quaternary climate change on lineage diversification across the Mexican highlands. We used mitochondrial DNA to examine the maternal history of P. orbiculare and estimate the timing and tempo of lineage diversification. Based on our results, we inferred 11 geographically structured, well supported mitochondrial lineages within this species, suggesting P. orbiculare represents a species complex. Six divergences between lineages likely occurred during the Late Miocene and Pliocene, and four splits probably happened during the Pleistocene. Diversification rate appeared relatively constant through time. Spatial and temporal divergences between lineages of P. orbiculare and co-distributed taxa suggest that a distinct period of uplifting of the Transvolcanic Belt around 7.5-3 million years ago broadly impacted diversification in taxa associated with this mountain range. To the north, several river drainages acting as filter barriers differentially subdivided co-distributed highland taxa through time. Diversification patterns observed in P. orbiculare provide additional insight into the mechanisms that impacted differentiation of highland taxa across the complex Mexican highlands.     

Highly polymorphic microsatellite markers developed for the social halictine bee Lasioglossum (Chilalictus) hemichalceum

Lasioglossum ( Chilalictus ) hemichalceum is a social halictine bee species for which we developed 10 polymorphic microsatellite loci in order to investigate detailed genetic structure of cooperating indvididuals. The loci are highly polymorphic with allele numbers ranging between eight and 22. A null allele was detected at one locus in the absence of pedigree information.     

植物的遗传转化及其应用(英文)

近年来,植物遗传转化研究有了长足的发展。已经达到能够通过简单的遗传控制手段研究具有新表现型的植物,甚至达到进入商业化的程度。这些手段包括植物生物学的主要研究技术以及植物组织培养和树种改良的一些实用方法。尽管采用农瘤杆菌和鸟枪法等技术的植物遗传转化系统已经得到了广泛的应用,但是在如何开发具有能够得到控制表达的转基因高产植物方面,在如何使所得到的转基因植物远离遗传危害等方面,目前的转化系统遇到了极大的技术挑战。已经提出了各种各样的方法用于将新基因稳定地导入120多种不同植物的核基因组。本文将讨论这些遗传转化系统所需的生物学要求和实际应用方面的需求、基因转化和转基因表达的研究策略、遗传转化植物的鉴定以及转基因植物与大众的认可。本文将分为七个部分加以讨论:一、导言;二 、基因转化到细胞里的方法;三、植物遗传转化策略;四、植物遗传转化的鉴定;五、植物遗传转化的实际应用;六、转基因植物与环境;七、未来植物遗传转化的需求与发展方向。