Multiplex cytokine analysis of dermal interstitial blister fluid defines local disease mechanisms in systemic sclerosis

IntroductionClinical diversity in systemic sclerosis (SSc) reflects multifaceted pathogenesis and the effect of key growth factors or cytokines operating within a disease-specific microenvironment. Dermal interstitial fluid sampling offers the potential to examine local mechanisms and identify proteins expressed within lesional tissue. We used multiplex cytokine analysis to profile the inflammatory and immune activity in the lesions of SSc patients.MethodsDermal interstitial fluid sample from the involved forearm skin, and synchronous plasma samples were collected from SSc patients (n = 26, diffuse cutaneous SSc (DcSSc) n = 20, limited cutaneous SSc (LcSSc) n = 6), and healthy controls (HC) (n = 10) and profiled by Luminex® array for inflammatory cytokines, chemokines, and growth factors.ResultsLuminex® profiling of the dermal blister fluid showed increased inflammatory cytokines (median interleukin ( IL)-6 in SSc 39.78 pg/ml, HC 5.51 pg/ml, p = 0.01, median IL-15 in SSc 6.27 pg/ml, HC 4.38 pg/ml, p = 0.03), chemokines (monocyte chemotactic protein (MCP)-3 9.81 pg/ml in SSc, 7.18 pg/ml HC, p = 0.04), and profibrotic growth factors (platelet derived growth factor (PDGF)-AA 10.38 pg/ml versus 6.94 pg/ml in HC, p = 0.03). In general dermal fluid and plasma cytokine levels did not correlate, consistent with predominantly local production of these factors within the dermal lesions, rather than leakage from the serum. In hierarchical clustering and network analysis IL-6 emerged as a key central mediator.ConclusionsOur data confirm that an immuno-inflammatory environment and aberrant vascular repair are intimately linked to fibroblast activation in lesional skin in SSc. This non-invasive method could be used to profile disease activity in the clinic, and identifies key inflammatory or pro-fibrotic proteins that might be targeted therapeutically. Distinct subgroups of SSc may be defined that show innate or adaptive immune cytokine signatures.     

曲酸生产菌的复合诱变选育*

以黄曲霉(Aspergillus flavus.)为出发菌株,经3次紫外线、1次^60Co、3次亚硝基胍多重复合诱变处理,选育获得曲酸生产菌UCN7—17,配以最佳培养条件,发酵7d,曲酸产量由原来的0．926％,提高到6．3％。实验证明采用多因子复合诱变,能有效改变菌株对诱变因素敏感性,提高变异率,逐步提高突变株的产酸水平。     

Ribosomal, telomeric and heterochromatin sequences localization in the karyotype of Anemone hortensis

The karyotype of the Mediterranean species Anemone hortensis L. (Ranunculaceae) was characterized with emphasis on heterochromatin distribution and localization of ribosomal (18S−5.8S−26S and 5S rDNA) and telomeric repeats (TTTAGGG). Diploid chromosome complement, 2 n  = 2 x  = 16, common to all investigated populations, consisted of three acrocentric, one meta-submetacentric and four metacentric chromosomes ranging in size from 6.34 to 10.47 µm. Fluorescence in situ hybridization (FISH) with 18S and 5S rDNA probes revealed two 18S−5.8S−26S rDNA loci on a satellite and secondary constriction of acrocentric chromosome pair 2 and terminally on acrocentric chromosome pair 3, and two 5S rDNA loci in the pericentromeric region of meta-submetacentric chromosome pair 4 and in the proximity of the 18S−5.8S−26S rDNA locus on chromosome pair 2. The only GC-rich heterochromatin, as revealed by fluorochrome Chromomycin A3 staining, was that associated with nucleolar organizer regions, whereas AT-rich heterochromatin, stained with 4,6-diamino-2-phenylindole (DAPI), was distributed intercalarly and terminally on the long arm of all three acrocentric chromosomes, and terminally on chromosomes 4 and 5. FISH with Arabidopsis -type telomeric repeats (TTTAGGG) as a probe revealed two classes of signals, small dot-like and large bands, at chromosome termini exclusively, where they corresponded to terminal DAPI-stained heterochromatin. Heteromorphism of chromosome pair 4, which refers to terminal DAPI bands and FISH signals, was observed in populations of Anemone hortensis . Chromosome pairing during meiosis was regular with formation of localized chiasmata proximal to the centromere.  © 2006 The Linnean Society of London, Botanical Journal of the Linnean Society , 2006, 150 , 177–186.     

Polymorphism Detection by Microsatellite Markers in a Magnaporthe oryzae Population From Different Geographical Areas of Brazil

This study aimed to examine Brazilian M. oryzae populations using 18 microsatellites. Fifty cultivars were sown in plastic trays for the pathotyping of 847 isolates. The DNA of 494 isolates was extracted and purified using the modified Doyle and Doyle method, the genetic structure was determined by the software Structure, and the actual number was selected from the prediction method based on the K values. Nei's genetic distance among the subpopulations was determined with the aid of the program Genetix, and the amova was performed with the program Arlequin. Out of 847 inoculated monosporic isolates, 528 infected their respective cultivars; of the 528 isolates pathotyped, there was a prevalence of group IA and pathotype IF‐1, which was the most frequent pathotype in the rice production areas of Brazil. The Bayesian clustering analysis indicated that 19 was the optimal value of K; this value was the lowest standard deviation and log (ln K) closest to zero, which predicted the 494 isolates of M. oryzae that were selected for molecular studies to be grouped into 19 subpopulations. The AMOVA detected a 37.13% variability within the 19 subpopulations and 62.87% variability among the subpopulations. The polymorphic information content (PIC) ranged from 0 to 0.756. Thirty three rare alleles were found distributed among 15 out of 19 subpopulations. The Margalef index ranged from 38.69 to 79.21 for all 18 analysed locus. The results indicated that the identification of different blast resistance genes must consider the composition of each subpopulation and that the identification is most effective when performed within a subpopulation and then between subpopulations.     

Low Doses of Ionizing Radiation Promote Tumor Growth and Metastasis by Enhancing Angiogenesis

Radiotherapy is a widely used treatment option in cancer. However, recent evidence suggests that doses of ionizing radiation (IR) delivered inside the tumor target volume, during fractionated radiotherapy, can promote tumor invasion and metastasis. Furthermore, the tissues that surround the tumor area are also exposed to low doses of IR that are lower than those delivered inside the tumor mass, because external radiotherapy is delivered to the tumor through multiple radiation beams, in order to prevent damage of organs at risk. The biological effects of these low doses of IR on the healthy tissue surrounding the tumor area, and in particular on the vasculature remain largely to be determined. We found that doses of IR lower or equal to 0.8 Gy enhance endothelial cell migration without impinging on cell proliferation or survival. Moreover, we show that low-dose IR induces a rapid phosphorylation of several endothelial cell proteins, including the Vascular Endothelial Growth Factor (VEGF) Receptor-2 and induces VEGF production in hypoxia mimicking conditions. By activating the VEGF Receptor-2, low-dose IR enhances endothelial cell migration and prevents endothelial cell death promoted by an anti-angiogenic drug, bevacizumab. In addition, we observed that low-dose IR accelerates embryonic angiogenic sprouting during zebrafish development and promotes adult angiogenesis during zebrafish fin regeneration and in the murine Matrigel assay. Using murine experimental models of leukemia and orthotopic breast cancer, we show that low-dose IR promotes tumor growth and metastasis and that these effects were prevented by the administration of a VEGF receptor-tyrosine kinase inhibitor immediately before IR exposure. These findings demonstrate a new mechanism to the understanding of the potential pro-metastatic effect of IR and may provide a new rationale basis to the improvement of current radiotherapy protocols.     

Lack of phylogenetic signal in the variation in anuran microhabitat use in southeastern Brazil

Microhabitat use is an important component of anuran behavior in both the tadpole and the adult stages. It is potentially influenced by phylogeny and extant ecological factors acting as selective pressures, such as predation, competition, or physical habitat properties. We aimed to test whether patterns of microhabitat use vary among species, habitats and sites, and how much of this variation can be explained by phylogenetic relatedness. We collected data on microhabitat use at five different sites, where we obtained a total of 4,230 records of individual tadpoles of 34 species in 15 genera and 7 families, and a total of 1,163 records of adult individuals of 39 species in 16 genera and 8 families. Mantel tests conducted to relate species dissimilarities in microhabitat use and phylogenetic relatedness indicated a weak but significant relationship for adult anurans, and no relationship for tadpoles. Our results suggest that microhabitat use is a plastic and variable trait, overcoming phylogenetic signal in tadpoles. In adult anurans, very little of the variation in microhabitat use can be explained by phylogenetic relatedness. Microhabitat use is not a good predictor of phylogeny, but it may be a very interesting subject to study natural selection and adaptation.     

Discovery and identification of a male-killing agent in the Japanese ladybird <Emphasis Type="Italic">Propylea japonica</Emphasis> (Coleoptera: Coccinellidae)

Background  

Endosymbionts that manipulate the reproduction of their hosts have been reported widely in invertebrates. One such group of endosymbionts is the male-killers. To date all male-killers reported are bacterial in nature, but comprise a diverse group. Ladybirds have been described as a model system for the study of male-killing, which has been reported in multiple species from widespread geographic locations. Whilst criteria of low egg hatch-rate and female-biased progenic sex ratio have been used to identify female hosts of male-killers, variation in vertical transmission efficiency and host genetic factors may result in variation in these phenotypic indicators of male-killer presence. Molecular identification of bacteria and screening for bacterial presence provide us with a more accurate method than breeding data alone to link the presence of the bacteria to the male-killing phenotype. In addition, by identifying the bacteria responsible we may find evidence for horizontal transfer between endosymbiont hosts and can gain insight into the evolutionary origins of male-killing. Phylogenetic placement of male-killing bacteria will allow us to address the question of whether male-killing is a potential strategy for only some, or all, maternally inherited bacteria. Together, phenotypic and molecular characterisation of male-killers will allow a deeper insight into the interactions between host and endosymbiont, which ultimately may lead to an understanding of how male-killers identify and kill male-hosts.     

Listeria monocytogenes Biofilm-Associated Protein (BapL) May Contribute to Surface Attachment of L. monocytogenes but Is Absent from Many Field Isolates

Listeria monocytogenes is a food-borne pathogen capable of adhering to a range of surfaces utilized within the food industry, including stainless steel. The factors required for the attachment of this ubiquitous organism to abiotic surfaces are still relatively unknown. In silico analysis of the L. monocytogenes EGD genome identified a putative cell wall-anchored protein (Lmo0435 [BapL]), which had similarity to proteins involved in biofilm formation by staphylococci. An insertion mutation was constructed in L. monocytogenes to determine the influence of this protein on attachment to abiotic surfaces. The results show that the protein may contribute to the surface adherence of strains that possess BapL, but it is not an essential requirement for all L. monocytogenes strains. Several BapL-negative field isolates demonstrated an ability to adhere to abiotic surfaces equivalent to that of BapL-positive strains. BapL is not required for the virulence of L. monocytogenes in mice.     

The effect of pair bonding in Cabrera vole’s scent marking

The Cabrera vole (Microtus cabrerae) is a rare rodent living in patchy grassy areas of the Iberian Peninsula where unpaired individuals of both sexes use scent marking primarily to increase their mate-finding likelihood. Cabrera voles establish long-term pair bonds with opposite-sex conspecifics constituting a breeding pair, which is expected to reduce the efforts in searching for a new mate. Under such circumstances, scent marking as a strategy to increase mate-finding likelihood became useless. Accordingly, we hypothesise that pair bonded Cabrera voles suppress mate-finding scent marking to reduce energetic costs and predation risk. To test this hypothesis, we compared scent-marking behaviour towards a clean substrate, in both paired and non-paired voles. No differences were found in the scent marks’ type and the amount of marks placed by voles in both conditions. We also analysed the scent-marking behaviour of both sex pair bonded voles when exposed simultaneously to a clean substrate, a substrate pre-marked by males and a substrate pre-marked by females. We found no significant differences in scent-marks (urine-marked area and number of faecal boli) across the three types of substrate types. In accordance with our prediction, these results suggest that pair bonded Cabrera voles did not use scent marking for mate finding, thus providing further support to the existence of a monogamous mating strategy. Furthermore, our results fail to support the use of scent marking for territorial defence purposes.     

基于psbA trnH序列的十种棘豆属植物分子系统学研究

选择内蒙古28个样地采集的10种棘豆属植物56个单株,提取样品的基因组DNA,对其叶绿体psbA trnH序列进行扩增、测序,所得序列利用ClustalX软件进行对位排列,并用MEGA50软件采用最大似然法构建系统发育树。结果显示：（1）10种棘豆属psbA trnH序列的变异位点50个,信息位点36个,种间碱基差异百分率为34%,GC含量变化范围在2318%~2572%之间。（2）棘豆属与黄芪属各为一支,自展支持率达99%,这10种棘豆属植物可能为单系起源。（3）系统树中小叶小花棘豆与小花棘豆的样本独立成一支,支持将小叶小花棘豆作为小花棘豆的变种来处理。（4）多叶棘豆、砂珍棘豆和黄毛棘豆的样本相互混杂,表明亲缘关系很近,支持《内蒙古植物志》将三者归入真棘豆亚属轮叶棘豆组的观点。（5）缘毛棘豆与薄叶棘豆的样本聚成一支,支持将二者归入矮生棘豆组。研究表明,psbA trnH序列可为棘豆属下种间系统发育关系研究提供分子证据。