Activated invariant NKT cells regulate osteoclast development and function

Invariant NKT (iNKT) cells modulate innate and adaptive immune responses through activation of myeloid dendritic cells and macrophages and via enhanced clonogenicity, differentiation, and egress of their shared myeloid progenitors. Because these same progenitors give rise to osteoclasts (OCs), which also mediate the egress of hematopoietic progenitors and orchestrate bone remodeling, we hypothesized that iNKT cells would extend their myeloid cell regulatory role to the development and function of OCs. In this study, we report that selective activation of iNKT cells by α-galactosylceramide causes myeloid cell egress, enhances OC progenitor and precursor development, modifies the intramedullary kinetics of mature OCs, and enhances their resorptive activity. OC progenitor activity is positively regulated by TNF-α and negatively regulated by IFN-γ, but is IL-4 and IL-17 independent. These data demonstrate a novel role of iNKT cells that couples osteoclastogenesis with myeloid cell egress in conditions of immune activation.     

Phosphatidylinositol-4-phosphate 5-kinase and GEP100/Brag2 protein mediate antiangiogenic signaling by semaphorin 3E-plexin-D1 through Arf6 protein

The semaphorins are a family of secreted or membrane-bound proteins that are known to guide axons in the developing nervous system. Genetic evidence revealed that a class III semaphorin, semaphorin 3E (Sema3E), and its receptor Plexin-D1 also control the vascular patterning during development. At the molecular level, we have recently shown that Sema3E acts on Plexin-D1 expressed in endothelial cells, thus initiating a novel antiangiogenic signaling pathway that results in the retraction of filopodia in endothelial tip cells. Sema3E induces the rapid disassembly of integrin-mediated adhesive structures, thereby inhibiting endothelial cell adhesion to the extracellular matrix. This process requires the activation of small GTPase Arf6 (ADP-ribosylation factor 6), which regulates intracellular trafficking of β1 integrin. However, the molecular mechanisms by which Sema3E-Plexin-D1 activates Arf6 remained to be identified. Here we show that GEP100 (guanine nucleotide exchange protein 100)/Brag2, a guanine nucleotide exchange factor for Arf6, mediates Sema3E-induced Arf6 activation in endothelial cells. We provide evidence that upon activation by Sema3E, Plexin-D1 recruits phosphatidylinositol-4-phosphate 5-kinase, and its enzymatic lipid product, phosphatidylinositol 4,5-bisphosphate, binds to the pleckstrin homology domain of GEP100. Phosphatidylinositol 4,5-bisphosphate binding to GEP100 enhances its guanine nucleotide exchange factor activity toward Arf6, thus resulting in the disassembly of integrin-mediated focal adhesions and endothelial cell collapse. Our present study reveals a novel phospholipid-regulated antiangiogenic signaling pathway whereby Sema3E activates Arf6 through Plexin-D1 and consequently controls integrin-mediated endothelial cell attachment to the extracellular matrix and migration.     

Successful Agrobacterium mediated transformation of Thielaviopsis basicola by optimizing multiple conditions

Thielaviopsis basicola is a hemibiotrophic root pathogen causing black root rot in a wide range of economically important crops. Our initial attempts to transform T. basicola using standard Agrobacterium tumefaciens–mediated transformation (ATMT) protocols were unsuccessful. Successful transformation required the addition of V8 juice (to induce germination of T. basicola chlamydospores) and higher concentrations of acetosyringone in the co-cultivation medium, and of chlamydospores/endoconidia, A. tumefaciens cells during co-cultivation. With these modifications, two T. basicola strains were successfully transformed with the green (egfp) or red (AsRed) fluorescent protein genes. Chlamydospores/endoconidia transformed with the egfp gene exhibited strong green fluorescence, but their fluorescence became weaker as the germ tubes emerged. Transformants harbouring the AsRed gene displayed strong red fluorescence in both chlamydospores/endoconidia and germ tubes. Fluorescent microscopic observations of an AsRed-labelled strain colonizing roots of transgenic Nicotiana benthamiana plants, which express the actin filaments labelled with EGFP, at 24 hours post inoculation showed varying levels of fungal germination and penetration. At this stage, the infection appeared to be biotrophic with the EGFP-labelled host actin filaments not being visibly degraded, even in host root cells in close contact with the hyphae. This is the first report of ATMT of T. basicola, and the use of an AsRed-labelled strain to directly observe the root infection process.     

Pseudomonas viridiflava, a multi host plant pathogen with significant genetic variation at the molecular level

The pectinolytic species Pseudomonas viridiflava has a wide host range among plants, causing foliar and stem necrotic lesions and basal stem and root rots. However, little is known about the molecular evolution of this species. In this study we investigated the intraspecies genetic variation of P. viridiflava amongst local (Cretan), as well as international isolates of the pathogen. The genetic and phenotypic variability were investigated by molecular fingerprinting (rep-PCR) and partial sequencing of three housekeeping genes (gyrB, rpoD and rpoB), and by biochemical and pathogenicity profiling. The biochemical tests and pathogenicity profiling did not reveal any variability among the isolates studied. However, the molecular fingerprinting patterns and housekeeping gene sequences clearly differentiated them. In a broader phylogenetic comparison of housekeeping gene sequences deposited in GenBank, significant genetic variability at the molecular level was found between isolates of P. viridiflava originated from different host species as well as among isolates from the same host. Our results provide a basis for more comprehensive understanding of the biology, sources and shifts in genetic diversity and evolution of P. viridiflava populations and should support the development of molecular identification tools and epidemiological studies in diseases caused by this species.     

Epigenome-wide scans identify differentially methylated regions for age and age-related phenotypes in a healthy ageing population

Age-related changes in DNA methylation have been implicated in cellular senescence and longevity, yet the causes and functional consequences of these variants remain unclear. To elucidate the role of age-related epigenetic changes in healthy ageing and potential longevity, we tested for association between whole-blood DNA methylation patterns in 172 female twins aged 32 to 80 with age and age-related phenotypes. Twin-based DNA methylation levels at 26,690 CpG-sites showed evidence for mean genome-wide heritability of 18%, which was supported by the identification of 1,537 CpG-sites with methylation QTLs in cis at FDR 5%. We performed genome-wide analyses to discover differentially methylated regions (DMRs) for sixteen age-related phenotypes (ap-DMRs) and chronological age (a-DMRs). Epigenome-wide association scans (EWAS) identified age-related phenotype DMRs (ap-DMRs) associated with LDL (STAT5A), lung function (WT1), and maternal longevity (ARL4A, TBX20). In contrast, EWAS for chronological age identified hundreds of predominantly hyper-methylated age DMRs (490 a-DMRs at FDR 5%), of which only one (TBX20) was also associated with an age-related phenotype. Therefore, the majority of age-related changes in DNA methylation are not associated with phenotypic measures of healthy ageing in later life. We replicated a large proportion of a-DMRs in a sample of 44 younger adult MZ twins aged 20 to 61, suggesting that a-DMRs may initiate at an earlier age. We next explored potential genetic and environmental mechanisms underlying a-DMRs and ap-DMRs. Genome-wide overlap across cis-meQTLs, genotype-phenotype associations, and EWAS ap-DMRs identified CpG-sites that had cis-meQTLs with evidence for genotype-phenotype association, where the CpG-site was also an ap-DMR for the same phenotype. Monozygotic twin methylation difference analyses identified one potential environmentally-mediated ap-DMR associated with total cholesterol and LDL (CSMD1). Our results suggest that in a small set of genes DNA methylation may be a candidate mechanism of mediating not only environmental, but also genetic effects on age-related phenotypes.     

Comparison of real-time quantitative PCR and culture for the diagnosis of emerging Rickettsioses

Background

Isolation of Rickettsia species from skin biopsies may be replaced by PCR. We evaluated culture sensitivity compared to PCR based on sampling delay and previous antibiotic treatment.

Methodology/Principal Findings

Skin biopsies and ticks from patients with suspected Rickettsia infection were screened for Rickettsia spp. using qPCR, and positive results were amplified and sequenced for the gltA and ompA genes. Immunofluorescence for spotted fever group rickettsial antigens was done for 79 patients. All skin biopsies and only ticks that tested positive using qPCR were cultured in human embryonic lung (HEL) fibroblasts using the centrifugation-shell vial technique. Patients and ticks were classified as definitely having rickettsioses if there was direct evidence of infection with a Rickettsia sp. using culture or molecular assays or in patients if serology was positive. Data on previous antibiotic treatments were obtained for patients with rickettsiosis. Rickettsia spp. infection was diagnosed in 47 out of 145 patients (32%), 41 by PCR and 12 by culture, whereas 3 isolates were obtained from PCR negative biopsies. For 3 of the patients serology was positive although PCR and culture were negative. Rickettsia africae was the most common detected species (n = 25, [17.2%]) and isolated bacterium (n = 5, [3.4%]). The probability of isolating Rickettsia spp. was 12 times higher in untreated patients and 5.4 times higher in patients from our hometown. Rickettsia spp. was amplified in 24 out of 95 ticks (25%) and we isolated 7 R. slovaca and 1 R. raoultii from Dermacentor marginatus.

Conclusions/Significance

We found a positive correlation between the bacteria copies and the isolation success in skin biopsies and ticks. Culture remains critical for strain analysis but is less sensitive than serology and PCR for the diagnosis of a Rickettsia infection.