Biomarker discovery in asthma‐related inflammation and remodeling

Asthma is a complex inflammatory disease of airways. A network of reciprocal interactions between inflammatory cells, peptidic mediators, extracellular matrix components, and proteases is thought to be involved in the installation and maintenance of asthma‐related airway inflammation and remodeling. To date, new proteic mediators displaying significant activity in the pathophysiology of asthma are still to be unveiled. The main objective of this study was to uncover potential target proteins by using surface‐enhanced laser desorption/ionization‐time of flight‐mass spectrometry (SELDI‐TOF‐MS) on lung samples from mouse models of allergen‐induced airway inflammation and remodeling. In this model, we pointed out several protein or peptide peaks that were preferentially expressed in diseased mice as compared to controls. We report the identification of different five proteins: found inflammatory zone 1 or RELMα (FIZZ‐1), calcyclin (S100A6), clara cell secretory protein 10 (CC10), Ubiquitin, and Histone H4.     

The role of flavonoids in the establishment of plant roots endosymbioses with arbuscular mycorrhiza fungi,rhizobia and Frankia bacteria

Flavonoids are a group of secondary metabolites derived from the phenylpropanoid pathway. They are ubiquitous in the plant kingdom and have many diverse functions including key roles at different levels of root endosymbioses. While there is a lot of information on the role of particular flavonoids in the Rhizobium-legume symbiosis, yet their exact role during the establishment of arbuscular mycorrhiza and actinorhizal symbioses still remains unclear. Within the context of the latest data suggesting a common symbiotic signaling pathway for both plant-fungal and plant bacterial endosymbioses between legumes and actinorhiza-forming fagales, this mini-review highlights some of the recent studies on the three major types of root endosymbioses. Implication of the molecular knowledge of endosymbioses signaling and genetic manipulation of flavonoid biosynthetic pathway on the development of strategies for the transfer and optimization of nodulation are also discussed.     

苏云金芽胞杆菌LM1212菌株和突变株的全基因组序列比较分析

【背景】LM1212菌株是昆虫病原菌苏云金芽胞杆菌(Bacillus thuringiensis,Bt)中的一员,其芽胞和晶体分别产生于芽胞形成细胞和晶体产生细胞中,具有独特的细胞分化表型。与野生株LM1212相比,突变株LM1212-DB芽胞细胞比例明显降低并产生更高比例的晶体产生细胞,这使得LM1212-DB菌株成为研究晶体产生细胞形成机制和提高菌株杀虫活性的绝佳实验材料。【目的】比较LM1212菌株和LM1212-DB菌株的基因组差异,以便于揭示导致这两个菌株表型差异的原因。【方法】利用单分子测序技术(single molecular real-time,SMRT)和Pacbio RS II测序平台对两个菌株进行全基因组测序,对染色体和质粒、双组分信号系统和插入序列等进行差异分析,并构建表型特性相关基因的系统发育树。【结果】基因组分析发现,LM1212和LM1212-DB菌株均含有丰富的插入序列和双组分信号系统,暗示两个菌株极易发生基因重排且具有较强的环境适应性。与LM1212菌株相比,突变株LM1212-DB中发生了染色体和质粒片段缺失、质粒重排、质粒拷贝数变异。进一步分析缺失基因的功能发现,一些环境胁迫响应基因(如sigB)和芽胞形成相关基因(如abrB)等缺失;通过分析质粒拷贝数变异发现,具有增加晶体细胞比例功能的转录因子CpcR所在质粒的拷贝数增加1个,同时对CpcR的进化分析发现,与其亲缘关系最近的基因的从属菌株也产生与LM1212菌株相似的细胞分化表型。这些重要功能基因的缺失和拷贝数变异可能是导致两个菌株表型差异的原因。此外,突变株LM1212-DB缺失I型限制-修饰系统,这使得突变株LM1212-DB与野生菌株LM1212相比具有更好的外源DNA兼容性。【结论】突变株LM1212-DB染色体和质粒的结构变异可能是导致与野生株LM1212表型差异的潜在原因,这将为研究LM1212菌株的晶体细胞分化机制提供指导方向。     

Regulation of NADPH Oxidase Activity in Phagocytes: RELATIONSHIP BETWEEN FAD/NADPH BINDING AND OXIDASE COMPLEX ASSEMBLY*

The X⁺-linked chronic granulomatous disease (X⁺-CGD) variants are natural mutants characterized by defective NADPH oxidase activity but with normal Nox2 expression. According to the three-dimensional model of the cytosolic Nox2 domain, most of the X⁺-CGD mutations are located in/or close to the FAD/NADPH binding regions. A structure/function study of this domain was conducted in X⁺-CGD PLB-985 cells exactly mimicking 10 human variants: T341K, C369R, G408E, G408R, P415H, P415L, Δ507QKT509-HIWAinsert, C537R, L546P, and E568K. Diaphorase activity is defective in all these mutants. NADPH oxidase assembly is normal for P415H/P415L and T341K mutants where mutation occurs in the consensus sequences of NADPH- and FAD-binding sites, respectively. This is in accordance with their buried position in the three-dimensional model of the cytosolic Nox2 domain. FAD incorporation is abolished only in the T341K mutant explaining its absence of diaphorase activity. This demonstrates that NADPH oxidase assembly can occur without FAD incorporation. In addition, a defect of NADPH binding is a plausible explanation for the diaphorase activity inhibition in the P415H, P415L, and C537R mutants. In contrast, Cys-369, Gly-408, Leu-546, and Glu-568 are essential for NADPH oxidase complex assembly. However, according to their position in the three-dimensional model of the cytosolic domain of Nox2, only Cys-369 could be in direct contact with cytosolic factors during oxidase assembly. In addition, the defect in oxidase assembly observed in the C369R, G408E, G408R, and E568K mutants correlates with the lack of FAD incorporation. Thus, the NADPH oxidase assembly process and FAD incorporation are closely related events essential for the diaphorase activity of Nox2.     

PrPSc binding antibodies are potent inhibitors of prion replication in cell lines

Conversion of the cellular alpha-helical prion protein (PrP(C)) into a disease-associated isoform (PrP(Sc)) is central to the pathogenesis of prion diseases. Molecules targeting either normal or disease-associated isoforms may be of therapeutic interest, and the antibodies binding PrP(C) have been shown to inhibit prion accumulation in vitro. Here we investigate whether antibodies that additionally target disease-associated isoforms such as PrP(Sc) inhibit prion replication in ovine PrP-inducible scrapie-infected Rov cells. We conclude from these experiments that antibodies exclusively binding PrP(C) were relatively inefficient inhibitors of ScRov cell PrP(Sc) accumulation compared with antibodies that additionally targeted disease-associated PrP isoforms. Although the mechanism by which these monoclonal antibodies inhibit prion replication is unclear, some of the data suggest that antibodies might actively increase PrP(Sc) turnover. Thus antibodies that bind to both normal and disease-associated isoforms represent very promising anti-prion agents.     

Genome-wide association mapping of sexual incompatibility genes in cacao (<Emphasis Type="Italic">Theobroma cacao</Emphasis> L.)

Sexual compatibility limits the production of cacao plantations, being an important selection criterion in breeding programs. However, the current method for characterizing compatibility, based on the frequency of flower setting after controlled pollination, is time consuming, requiring a long time to identify self-compatible individuals. The identification of molecular markers in genomic regions can be an alternative to allow early selection of self-compatible plants. The present study aimed to identify SNP markers associated with sexual compatibility in cacao, by utilizing genome-wide association (GWAS) mapping. A population of 295 individuals mostly from third-generation breeding populations, but also founder clones, was used. This population was phenotypically characterized by hand pollinating 8199 flowers and evaluating the flower retention 15 days after pollination. In addition, leaf samples of each individual were collected and DNA extracted for genotyping by sequencing, generating 5301 SNP markers after cleaning. Genome-wide association mapping analysis was performed using Synbreed, GCTA, and TASSEL softwares. Significant markers associated to incompatibility, likely in strong linkage disequilibrium, were found within a region of 196 kb, in the proximal end of chromosome 4, suggesting the existence of a major gene in that region. However, this result should be validated in a larger population, considering that only 295 trees were used here. When the SNP effects were treated as random in the estimation process, many other regions in the genome appears to be involved with sexual incompatibility in cacao. Candidate genes were found not only in the proximal end of chromosome 4 but also spread in several other regions of the genome.     

Mouse Model of Respiratory Tract Infection Induced by Waddlia chondrophila

Waddlia chondrophila, an obligate intracellular bacterium belonging to the Chlamydiales order, is considered as an emerging pathogen. Some clinical studies highlighted a possible role of W. chondrophila in bronchiolitis, pneumonia and miscarriage. This pathogenic potential is further supported by the ability of W. chondrophila to infect and replicate within human pneumocytes, macrophages and endometrial cells. Considering that W. chondrophila might be a causative agent of respiratory tract infection, we developed a mouse model of respiratory tract infection to get insight into the pathogenesis of W. chondrophila. Following intranasal inoculation of 2 x 10⁸W. chondrophila, mice lost up to 40% of their body weight, and succumbed rapidly from infection with a death rate reaching 50% at day 4 post-inoculation. Bacterial loads, estimated by qPCR, increased from day 0 to day 3 post-infection and decreased thereafter in surviving mice. Bacterial growth was confirmed by detecting dividing bacteria using electron microscopy, and living bacteria were isolated from lungs 14 days post-infection. Immunohistochemistry and histopathology of infected lungs revealed the presence of bacteria associated with pneumonia characterized by an important multifocal inflammation. The high inflammatory score in the lungs was associated with the presence of pro-inflammatory cytokines in both serum and lungs at day 3 post-infection. This animal model supports the role of W. chondrophila as an agent of respiratory tract infection, and will help understanding the pathogenesis of this strict intracellular bacterium.     

Molecular phylogeny and phylogeography of the Cuban cave-fishes of the genus Lucifuga: evidence for cryptic allopatric diversity

Underground environments are increasingly recognized as reservoirs of faunal diversity. Extreme environmental conditions and limited dispersal ability of underground organisms have been acknowledged as important factors promoting divergence between species and conspecific populations. However, in many instances, there is no correlation between genetic divergence and morphological differentiation. Lucifuga Poey is a stygobiotic fish genus that lives in Cuban and Bahamian caves. In Cuba, it offers a unique opportunity to study the influence of habitat fragmentation on the genetic divergence of stygobiotic species and populations. The genus includes four species and one morphological variant that have contrasting geographical distributions. In this study, we first performed a molecular phylogenetic analysis of the Lucifuga Cuban species using mitochondrial and nuclear markers. The mitochondrial phylogeny revealed three deeply divergent clades that were supported by nuclear and morphological characters. Within two of these main clades, we identified five lineages that are candidate cryptic species and a taxonomical synonymy between Lucifuga subterranea and Lucifuga teresinarum. Secondly, phylogeographic analysis using a fragment of the cytochrome b gene was performed for Lucifuga dentata, the most widely distributed species. We found strong geographical organization of the haplotype clades at different geographic scales that can be explained by episodes of dispersal and population expansion followed by population fragmentation and restricted gene flow. At a larger temporal scale, these processes could also explain the diversification and the distribution of the different species.     

The moss genes <Emphasis Type="Italic">PpSKI1</Emphasis> and <Emphasis Type="Italic">PpSKI2</Emphasis> encode nuclear SnRK1 interacting proteins with homologues in vascular plants

The yeast Snf1, animal AMPK, and plant SnRK1 protein kinases constitute a family of related proteins that have been proposed to serve as metabolic sensors of the eukaryotic cell. We have previously reported the characterization of two redundant SnRK1 encoding genes (PpSNF1a and PpSNF1b) in the moss Physcomitrella patens. Phenotypic analysis of the snf1a snf1b double knockout mutant suggested that SnRK1 is important for the plant’s ability to recognize and adapt to conditions of limited energy supply, and also suggested a possible role of SnRK1 in the control of plant development. We have now used a yeast two-hybrid system to screen for PpSnf1a interacting proteins. Two new moss genes were found, PpSKI1 and PpSKI2, which encode highly similar proteins with homologues in vascular plants. Fusions of the two encoded proteins to the green fluorescent protein localize to the nucleus. Knockout mutants for either gene have an excess of gametophores under low light conditions, and exhibit reduced gametophore stem lengths. Possible functions of the new proteins and their connection to the SnRK1 kinase are discussed.