Immunophenotypic Profiling of Erythroid Progenitor-Derived Extracellular Vesicles in Diamond-Blackfan Anaemia: A New Diagnostic Strategy

Diamond-Blackfan Anaemia (DBA) is a rare inherited anaemia caused by heterozygous mutations in one of 13 ribosomal protein genes. Erythroid progenitors (BFU-E and CFU-E) in bone marrow (BM) show a proapoptotic phenotype. Suspicion of DBA is reached after exclusion of other forms of BM failure syndromes. To improve DBA diagnosis, which is confirmed by mutation analysis, we tested a new approach based on the study of extracellular vesicles (EVs) isolated from plasma by differential centrifugations and analysed by flow cytometry. We chose CD34, CD71 and CD235a markers to study erythroid EVs. We characterised the EVs immunophentoypic profiles of 13 DBA patients, 22 healthy controls and 16 patients with other haematological diseases. Among the three EVs clusters we found, only the CD34+/CD71_low population showed statistically significant differences between DBA patients and controls (p< 0.05). The absence of this cluster is in agreement with the low levels of BFU-E found in DBA patients. The assessment of ROC curves demonstrated the potential diagnostic value of this population. We suggest that this assay may be useful to improve DBA diagnosis as a quicker and less invasive alternative to BM BFU-E culture analysis.     

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.     

Serum amyloid P component controls chromatin degradation and prevents antinuclear autoimmunity.

Serum amyloid P component (SAP), a highly conserved plasma protein named for its universal presence in amyloid deposits, is the single normal circulating protein that shows specific calcium-dependent binding to DNA and chromatin in physiological conditions. The avid binding of SAP displaces H1-type histones and thereby solubilizes native long chromatin, which is otherwise profoundly insoluble at the physiological ionic strength of extracellular fluids. Furthermore, SAP binds in vivo both to apoptotic cells, the surface blebs of which bear chromatin fragments, and to nuclear debris released by necrosis. SAP may therefore participate in handling of chromatin exposed by cell death. Here we show that mice with targeted deletion of the SAP gene spontaneously develop antinuclear autoimmunity and severe glomerulonephritis, a phenotype resembling human systemic lupus erythematosus, a serious autoimmune disease. The SAP-/- mice also have enhanced anti-DNA responses to immunization with extrinsic chromatin, and we demonstrate that degradation of long chromatin is retarded in the presence of SAP both in vitro and in vivo. These findings indicate that SAP has an important physiological role, inhibiting the formation of pathogenic autoantibodies against chromatin and DNA, probably by binding to chromatin and regulating its degradation.     

Stable isotope profiles of partially migratory salmonid populations in Atlantic rivers of Patagonia

In the present study, profiles of stable isotope composition were characterized for two species with partially migratory populations in rivers along the latitudinal gradient of Patagonia, brown trout Salmo trutta and rainbow trout Oncorhynchus mykiss . The effects of factors ( e.g. ontogeny of fishes, location, species and fasting) that may influence the stable isotope analysis (SIA) were evaluated, as was SIA evaluated as a tool to assign individual fish to their corresponding ecotype. Anadromous fishes exhibited enriched δ¹⁵N (15·2 ± 1·0‰; mean ± s . d .) and δ¹³C (−19·2 ± 1·3‰) relative to resident fishes'δ¹⁵N (8·8 ± 1·1‰) and δ¹³C (−23·2 ± 2·5‰). For both species, the difference in δ¹⁵N was larger between resident (range 6·8–10·7‰) and anadromous (range 14·3–17·8‰) fishes than that in δ¹³C. Values of δ¹³C, while not as dramatically contrasting in rainbow trout, provided a powerful anadromy marker for brown trout in the region. Increases were found in both δ¹⁵N and δ¹³C during the spawning migration of anadromous rainbow trout, most likely due to fasting. Differences in stable isotopes between location, size and species were found, suggesting different stable isotopes base levels in freshwater environments and different trophic levels and feeding location of anadromous populations. The SIA was demonstrated as a powerful tool for ecotype discrimination in Patagonian Rivers, overriding any effect of sampling location, size or species.     

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.     

Potential for controlling codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) in Argentina using the sterile insect technique and egg parasitoids

Codling moth is the main pest affecting apples and pears worldwide. Most pest control strategies used against this insect have relied on the use of broad‐spectrum insecticides which have led to non‐desirable effects like pesticide resistance, residues in the environment, human health concerns and the reduction of access to international markets. Therefore, alternative pest control strategies that would result in sustainable fruit production systems while taking care of the environment are strongly promoted. The use of the sterile insect technique has proven to be a valuable pest control tactic within area‐wide integrated pest management strategies, and its synergistic effect for Lepidoptera pests when combined with other biological control tactics such as parasitoids has been documented. The purposes of this research were to evaluate the response of an Argentinean codling moth strain to a sub‐sterilizing radiation dose of 100 Gy and to assess the acceptability and suitability of sterile codling moth eggs by the egg parasitoids, Trichogramma cacoeciae (Marchal) and Trichogramma nerudai (Pintureau and Gerding). Irradiated female moths survived better than irradiated male moths and non‐irradiated male and female moths. Also, the fecundity of irradiated female moths was reduced by more than 30% as compared to non‐irradiated ones whereas their fertility was close to zero. The F₁ generation was male biased with a lower fertility (inherited sterility) than the parental generation. Trichogramma cacoeciae and T. nerudai parasitized both fertile and sterile eggs. However, there was a significant reduction in acceptability for sterile eggs. Trichogramma nerudai parasitized more eggs than T. cacoeciae, but egg acceptability for this species was proportionally lower than for T. cacoeciae especially on eggs oviposited by irradiated females. Development to adult of both parasitoids species was not substantially affected by the origin of the eggs and the wasps had acceptable levels of adult emergence, survival and fecundity. These results provided useful information on the potential for controlling the codling moth using egg parasitoids and the sterile insect technique in Argentina.     

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.     

Biological studies on Trichogrammatoidea bactrae Nagaraja (Hymenoptera: Trichogrammatidae), egg parasitoid of Tuta absoluta Meyrick (Lepidoptera: Gelechiidae)

The tomato moth, Tuta absoluta Meyrick, is one of the most important tomato pests in South America. In Argentina, management strategies include only chemical control. In this work, the parasitoid wasp Trichogrammatoidea bactrae Nagaraja was evaluated as a potential natural enemy against this pest. Biological and population parameters were estimated by developing a life table under laboratory conditions at 25 ± 1oC, 14:10 photoperiod and 60 ± 10% RH. Three cohorts of 26-30 T. bactrae females each were placed with one of the three following treatments: 1 - Sitotroga cerealella (Olivier) eggs on a piece of cardboard; 2 - S. cerealella eggs on a piece of tomato leaf, and 3- T. absoluta eggs on a piece on tomato leaf. The following parameters were estimated for each cohort: survival (egg to adult), longevity, fecundity and oviposition period of females, sex proportion of the F1, net rate of reproduction (Ro), mean generation time (T) and intrinsic rate of population increase (r m). Survival of the T. bactrae immatures was higher than 90% on both, S. cerealella and T. absoluta eggs. The female survival curves corresponded to type III and showed no significant differences among treatments. The three cohorts did not show significant differences between sex ratio, female longevity, oviposition period, fecundity and the population parameters studied. These results indicate that T. bactrae would be a potential biological control agent of T. absoluta.