Autoreactive B cells discriminate CpG-rich and CpG-poor DNA and this response is modulated by IFN-alpha

Autoreactive B cells are activated by DNA, chromatin, or chromatin-containing immune complexes (ICs) through a mechanism dependent on dual engagement of the BCR and TLR9. We examined the contribution of endogenous DNA sequence elements to this process. DNA sequence can determine both recognition by the BCR and by TLR9. DNA fragments containing CpG islands, a natural source of unmethylated CpG dinucleotides, promote the activation of DNA-reactive B cells derived from BCR transgenic mice as well as DNA-reactive B cells present in the normal repertoire. ICs containing these CpG island fragments are potent ligands for AM14 IgG2a-reactive B cells. In contrast, ICs containing total mammalian DNA, or DNA fragments lacking immunostimulatory motifs, fail to induce B cell proliferation, indicating that BCR crosslinking alone is insufficient to activate low-affinity autoreactive B cells. Importantly, priming B cells with IFN-alpha lowers the BCR activation threshold and relaxes the selectivity for CpG-containing DNA. Taken together, our findings underscore the importance of endogenous CpG-containing DNAs in the TLR9-dependent activation of autoreactive B cells and further identify an important mechanism through which IFN-alpha can contribute to the pathogenesis of systemic lupus erythematosus.     

Integration of biological networks and gene expression data using Cytoscape

Cytoscape is a free software package for visualizing, modeling and analyzing molecular and genetic interaction networks. This protocol explains how to use Cytoscape to analyze the results of mRNA expression profiling, and other functional genomics and proteomics experiments, in the context of an interaction network obtained for genes of interest. Five major steps are described: (i) obtaining a gene or protein network, (ii) displaying the network using layout algorithms, (iii) integrating with gene expression and other functional attributes, (iv) identifying putative complexes and functional modules and (v) identifying enriched Gene Ontology annotations in the network. These steps provide a broad sample of the types of analyses performed by Cytoscape.     

Left-sided infective endocarditis caused by Pseudomonas aeruginosa treated medically

Infective endocarditis due to Pseudomonas aeruginosa is a rare clinical condition, difficult to diagnose and associated with high mortality. Herein we present a case of a 51 years old male without history of intravenous drug use or valvular disease, with past medical history of cholecystectomy in the previous month, who presented to the emergency department with fever, gastrointestinal symptoms, and subsequent signs of distant embolization, positive blood cultures for P. aeruginosa and development of multiple complications of the disease. The clinical presentation of infective endocarditis is nonspecific, leading to delayed diagnosis, and preventing early and effective treatment. Valvular replacement is indicated in fungal or P. aeruginosa endocarditis. This case is notable because of the resolution with amikacin combined with meropenem, in a patient with several complications that contraindicated surgery.     

Ultrasound imaging in an experimental model of fatty liver disease and cirrhosis in rats

Background

Atypical scrapie was first identified in Norwegian sheep in 1998 and has subsequently been identified in many countries. Retrospective studies have identified cases predating the initial identification of this form of scrapie, and epidemiological studies have indicated that it does not conform to the behaviour of an infectious disease, giving rise to the hypothesis that it represents spontaneous disease. However, atypical scrapie isolates have been shown to be infectious experimentally, through intracerebral inoculation in transgenic mice and sheep. The first successful challenge of a sheep with 'field' atypical scrapie from an homologous donor sheep was reported in 2007.

Results

This study demonstrates that atypical scrapie has distinct clinical, pathological and biochemical characteristics which are maintained on transmission and sub-passage, and which are distinct from other strains of transmissible spongiform encephalopathies in the same host genotype.

Conclusions

Atypical scrapie is consistently transmissible within AHQ homozygous sheep, and the disease phenotype is preserved on sub-passage.     

RNA Binding Activity of Heterodimeric Splicing Factor U2AF: at Least One RS Domain Is Required for High-Affinity Binding

The pre-mRNA splicing factor U2AF (U2 small nuclear ribonucleoprotein particle [snRNP] auxiliary factor) plays a critical role in 3′ splice site selection. U2AF binds site specifically to the intron pyrimidine tract between the branchpoint and the 3′ splice site and targets U2 snRNP to the branch site at an early step in spliceosome assembly. Human U2AF is a heterodimer composed of large (hU2AF⁶⁵) and small (hU2AF³⁵) subunits. hU2AF⁶⁵ contains an arginine-serine-rich (RS) domain and three RNA recognition motifs (RRMs). hU2AF³⁵ has a degenerate RRM and a carboxyl-terminal RS domain. Genetic studies have recently shown that the RS domains on the Drosophila U2AF subunit homologs are each inessential and might have redundant functions in vivo. The site-specific pyrimidine tract binding activity of the U2AF heterodimer has previously been assigned to hU2AF⁶⁵. While the requirement for the three RRMs on hU2AF⁶⁵ is firmly established, a role for the large-subunit RS domain in RNA binding remains unresolved. We have analyzed the RNA binding activity of the U2AF heterodimer in vitro. When the Drosophila small-subunit homolog (dU2AF³⁸) was complexed with the large-subunit (dU2AF⁵⁰) pyrimidine tract, RNA binding activity increased 20-fold over that of free dU2AF⁵⁰. We detected a similar increase in RNA binding activity when we compared the human U2AF heterodimer and hU2AF⁶⁵. Surprisingly, the RS domain on dU2AF³⁸ was necessary for the increased binding activity of the dU2AF heterodimer. In addition, removal of the RS domain from the Drosophila large-subunit monomer (dU2AF⁵⁰ΔRS) severely impaired its binding activity. However, if the dU2AF³⁸ RS domain was supplied in a complex with dU2AF⁵⁰ΔRS, high-affinity binding was restored. These results suggest that the presence of one RS domain of U2AF, on either the large or small subunit, promotes high-affinity pyrimidine tract RNA binding activity, consistent with redundant roles for the U2AF RS domains in vivo.     

A new FISH assay to simultaneously detect structural and numerical chromosomal abnormalities in mouse sperm

De novo aberrations in chromosome structure represent important categories of paternally transmitted genetic damage. Unlike numerical abnormalities, the majority of de novo structural aberrations among human offspring are of paternal origin. We report the development of a three-color fluorescence in situ hybridization (FISH) assay (CT8) to detect mouse sperm carrying structural and numerical chromosomal abnormalities. The CT8 assay uses DNA probes for the centromeric and telomeric regions of chromosome 2, and a probe for the subcentromeric region of chromosome 8. The CT8 assay was used to measure the frequencies of sperm carrying certain structural aberrations involving chromosome 2 (del2ter, dup2ter, del2cen, dup2cen), disomy 2, disomy 8, and sperm diploidy. Analysis of approximately 80,000 sperm from eight B6C3F1 mice revealed an average baseline frequency of 2.5 per 10,000 sperm carrying partial duplications and deletions of chromosome 2. Extrapolated to the entire haploid genome, approximately 0.4% of mouse sperm are estimated to carry structural chromosomal aberrations, which is more than fivefold lower than the spontaneous frequencies of sperm with chromosome structural aberrations in man. We validated the CT8 assay by comparing the frequencies of abnormal segregants in sperm of T(2;14) translocation carriers detected by this assay against those detected by chromosome painting cytogenetic analysis of meiosis II spermatocytes. The CT8 sperm FISH assay is a promising method for detecting structural chromosome aberrations in mouse sperm with widespread applications in genetics, physiology, and genetic toxicology.     

Effect of P144? (Anti-TGF-β) in an “In Vivo” Human Hypertrophic Scar Model in Nude Mice

Background

Hypertrophic scars are one of the most important complications in surgery due to their cosmetic and functional impairments. Previous studies in tissue fibrotic disorders have shown promising results by inhibiting the biological activity effect of Transforming Growth Factor-beta 1 (TGF-β1). The aim of the current study was to determine the clinical effect of the inhibition of TGF-β1 signaling in human hypertrophic scars implanted in nude mice by topical application of an inhibitor of TGF-β1 (P144®).

Material and Methods

A total of 30 human hypertrophic scars were implanted in 60 nude mice. The animals were divided in two groups, group A (placebo) and group B (treatment). Group C (basal) was considered as the preimplanted scar samples and they were not implanted in the nude mice. After the shedding period, topical application of a lipogel containing placebo (group A) or P144 (group B) was daily administered during two weeks. The animals were sacrificed upon completion of the study. Total area, thickness and collagen fibers area were measure and compared across all groups. Immunohistochemistry was also performed in order to quantify collagen type I and type III and elastic fiber expressions present in the dermis.

Results

Successful shedding was achieved in 83,3% of the xenografts. The mean time for shedding was 35±5.4 days. Statistically significant differences were found in the total area, collagen fibers area and thickness between the groups. Increased elastic fibers and decreased collagen I were found in the P144-treated group compared to the basal group.

Conclusion

Topical application of an inhibitor of TGF-β1 may promote scar maturation and clinical improvement of hypertrophic scar morphology features in an “in vivo” model in nude mice after two weeks of treatment.     

Detection and identification of microorganisms in wine: a review of molecular techniques

The microbial ecology of wine is complex. Microbes can play both positive and negative roles in the quality of the final product. Due to this impact, the microbial ecology of wine has been well studied. Traditional indirect methods, such as plating, have largely been replaced by a number of molecular methods. These methods are typically either indirect methods used for identification of cultured organisms, or direct methods used to profile whole populations or identify specific microbes in a mixed population. These molecular methods offer a number of advantages over traditional methods including speed and precision. This review will examine both direct and indirect molecular methods, provide examples of their impact on the study of the microbial ecology of wine, and also discuss their strengths and limitations.     

Short-term effects of a summer wildfire on a desert grassland arthropod community in New Mexico

Surface-active arthropods were sampled after a lightning-caused wildfire in desert grassland habitat on the Sevilleta National Wildlife Refuge, Socorro County, NM. Pitfall traps (n = 32 per treatment) were used to evaluate species-specific "activity-density" indices after the June wildfire in both burned and unburned areas. In total, 5,302 individuals were collected from 69 taxa. Herbivore activity-densities generally decreased, whereas predators often increased in the burned area; pitfall trap bias likely contributed to this latter observation. Fire caused the virtual extirpation of scaly crickets (Mogoplistidae), field crickets (Gryllidae), and camel crickets (Raphidophoridae), but recolonization began during the first postfire growing season. Several grasshoppers (Acrididae) also exhibited significant postfire declines [Ageneotettix deorum (Scudder), Eritettix simplex (Scudder), Melanoplus bowditchi Scudder, and Amphitornus coloradus (Thomas)]. Some beetles showed lower activity-density, including Pasimachus obsoletus LeConte (Carabidae) and Eleodes extricatus (Say) (Tenebrionidae). Taxa exhibiting significant postfire increases in activity-density included acridid grasshoppers (Aulocara femoratum (Scudder), Hesperotettix viridis (Thomas), Trimerotropis pallidipennis (Burmeis.), and Xanthippus corallipes Haldeman); carabid beetles (Amblycheila picolominii Reiche, Cicindela punctulata Olivier), tenebrionid beetles (Eleodes longicollis LeConte, Edrotes rotundus (Say), Glyptasida sordida (LeConte), Stenomorpha consors (Casey); the centipedes Taiyubius harrietae Chamberlin (Lithobiidae) and Scolopendra polymorpha Wood (Scolopendridae); scorpions (Vaejovis spp.; Vaejovidae); and sun spiders (Eremobates spp.; Eremobatidae). Native sand roaches (Arenivaga erratica Rehn, Eremoblata subdiaphana (Scudder); Polyphagidae) displayed no significant fire response. Overall, arthropod responses to fire in this desert grassland (with comparatively low and patchy fuel loads) were comparable to those in mesic grasslands with much higher and more continuous fuel loads.     

Myelin Gene Expression in Immortalized Schwann Cells: Relationship to Cell Density and Proliferation

Abstract: Myelin gene expression was investigated in the immortalized S16 Schwann cell line grown in the presence and absence of serum and at different densities. Protein expression was monitored by western blotting, and message levels were determined by RNase protection assays. To study cell proliferation rates at different cell densities and serum conditions. [³H]thymidine uptake assays and cell counts were performed. Although serum deprivation decreased cell proliferation as expected, the proliferation of S16 cells was unchanged or slightly increased at high density under the conditions of our experiments in either serum-containing or serum-free medium. This increased cell division at high density appeared to be due to greater release of an autocrine growth factor to the medium by dense cell populations. For both sparse and dense cells, substantially more P₀ glycoprotein (P₀) and myelin-associated glycoprotein (MAG) per milligram of total cellular protein were expressed when the cells were proliferating slowly in defined medium in comparison with more rapidly proliferating cells in serum-containing medium. Furthermore, in both serum-containing and defined media, dense cell populations expressed more MAG and P₀ than sparse ones. P₀ mRNA and MAG mRNA levels generally paralleled protein levels. The level of mRNA for peripheral myelin protein-22 (PMP-22) was also increased at high cell density but did not change much when proliferation was decreased by serum deprivation. PMP-22 protein was not detected under any of the growth conditions. The changes in expression of these genes with growth conditions may be specific for myelin proteins, because the expression of a nonmyelin glycoprotein, L1, remained constant. The level of cyclic AMP in the cells did not change with the different growth conditions tested. The results indicate that the S16 Schwann cell line mimics primary or secondary Schwann cells by down-regulating myelin gene expression when it proliferates more rapidly in the presence of serum. Furthermore, in both the presence and absence of serum, there was greater expression of myelin genes at high cell density that was not associated with a decreased proliferative rate. Because evidence for a role of secretory factors in affecting myelin gene expression was not obtained by treating sparse S16 cells with medium conditioned by dense S16 cells, the results suggest that the higher expression of myelin genes at high density may be mediated by cell-to-cell contact.