Melanin is an essential component for the integrity of the cell wall of Aspergillus fumigatus conidia

Background  

Aspergillus fumigatus is the most common agent of invasive aspergillosis, a feared complication in severely immunocompromised patients. Despite the recent commercialisation of new antifungal drugs, the prognosis for this infection remains uncertain. Thus, there is a real need to discover new targets for therapy. Particular attention has been paid to the biochemical composition and organisation of the fungal cell wall, because it mediates the host-fungus interplay. Conidia, which are responsible for infections, have melanin as one of the cell wall components. Melanin has been established as an important virulence factor, protecting the fungus against the host's immune defences. We suggested that it might also have an indirect role in virulence, because it is required for correct assembly of the cell wall layers of the conidia.     

Early and long-standing rheumatoid arthritis: distinct molecular signatures identified by gene-expression profiling in synovia

Introduction

Autoantibodies against C1q correlate with lupus nephritis. We compared titers of anti-C1q and anti-dsDNA in 70 systemic lupus erythematosus patients with (n = 15) or without (n = 55) subsequent biopsy-proven lupus nephritis.

Methods

The 15 patients with subsequent lupus nephritis had anti-C1q assays during clinical flares (mean Systemic Lupus Erythematosus Disease Activity Index (SLEDAI), 10.0 ± 4.7; range, 3 to 22) before the diagnosis of lupus nephritis (median, 24 months; range 3 to 192). Among the 55 others, 33 patients had active lupus (mean SLEDAI, 10.3 ± 6.2; range, 4 to 30) without renal disease during follow-up (median 13 years; range 2 to 17 years) and 22 had inactive lupus (mean SLEDAI, 0; range, 0 to 3).

Results

Anti-C1q titers were elevated in 15/15 (100%) patients who subsequently developed nephritis (class IV, n = 14; class V, n = 1) and in 15/33 (45%) patients without renal disease (P < 0.001). The median anti-C1q titer differed significantly between the groups (P = 0.003). Anti-C1q titers were persistently positive at the time of glomerulonephritis diagnosis in 70% (7/10) of patients, with no difference in titers compared with pre-nephritis values (median, 147 U/ml; interquartile range (IQR), 69 to 213 versus 116 U/ml; 50 to 284, respectively). Titers decreased after 6 months' treatment with immunosuppressive drugs and corticosteroids (median, 76 U/ml; IQR, 33 to 106) but remained above normal in 6/8 (75%) patients. Anti-dsDNA antibodies were increased in 14/15 (93.3%) patients with subsequent nephritis and 24/33 (72.7%) patients without nephritis (P = ns). Anti-C1q did not correlate with anti-dsDNA or the SLEDAI in either group.

Conclusions

Anti-C1q elevation had 50% positive predictive value (15/30) and 100% (18/18) negative predictive value for subsequent class IV or V lupus nephritis.     

Analysis of Blood Stem Cell Activity and Cystatin Gene Expression in a Mouse Model Presenting a Chromosomal Deletion Encompassing Csta and Stfa2l1

The cystatin protein superfamily is characterized by the presence of conserved sequences that display cysteine protease inhibitory activity (e.g., towards cathepsins). Type 1 and 2 cystatins are encoded by 25 genes of which 23 are grouped in 2 clusters localized on mouse chromosomes 16 and 2. The expression and essential roles of most of these genes in mouse development and hematopoiesis remain poorly characterized. In this study, we describe a set of quantitative real-time PCR assays and a global expression profile of cystatin genes in normal mouse tissues. Benefiting from our collection of DelES embryonic stem cell clones harboring large chromosomal deletions (to be reported elsewhere), we selected a clone in which a 95-kb region of chromosome 16 is missing (Del^16qB3Δ/+). In this particular clone, 2 cystatin genes, namely Csta and Stfa2l1 are absent along with 2 other genes (Fam162a, Ccdc58) and associated intergenic regions. From this line, we established a new homozygous mutant mouse model (Del^{16qB3Δ/16qB3Δ}) to assess the in vivo biological functions of the 2 deleted cystatins. Stfa2l1 gene expression is high in wild-type fetal liver, bone marrow, and spleen, while Csta is ubiquitously expressed. Homozygous Del^{16qB3Δ/16qB3Δ} animals are phenotypically normal, fertile, and not overtly susceptible to spontaneous or irradiation-induced tumor formation. The hematopoietic stem and progenitor cell activity in these mutant mice are also normal. Interestingly, quantitative real-time PCR expression profiling reveals a marked increase in the expression levels of Stfa2l1/Csta phylogenetically-related genes (Stfa1, Stfa2, and Stfa3) in Del^{16qB3Δ/16qB3Δ} hematopoietic tissues, suggesting that these candidate genes might be contributing to compensatory mechanisms. Overall, this study presents an optimized approach to globally monitor cystatin gene expression as well as a new mouse model deficient in Stfa2l1/Csta genes, expanding the available tools to dissect cystatin roles under normal and pathological conditions.     

A Novel Lipidomic Strategy Reveals Plasma Phospholipid Signatures Associated with Respiratory Disease Severity in Cystic Fibrosis Patients

The aim of this study was to search for lipid signatures in blood plasma from cystic fibrosis (CF) patients using a novel MALDI-TOF-ClinProTools™ strategy, initially developed for protein analysis, and thin layer chromatography coupled to MALDI-TOF (TLC-MALDI). Samples from 33 CF patients and 18 healthy children were subjected to organic extraction and column chromatography separation of lipid classes. Extracts were analyzed by MALDI-TOF, ion signatures were compared by the ClinProTools™ software and by parallel statistical analyses. Relevant peaks were identified by LC-MSn. The ensemble of analyses provided 11 and 4 peaks differentially displayed in CF vs healthy and in mild vs severe patients respectively. Ten ions were significantly decreased in all patients, corresponding to 4 lysophosphatidylcholine (18∶0, 18∶2, 20∶3, and 20∶5) and 6 phosphatidylcholine (36∶5, O-38∶0, 38∶4, 38∶5, 38∶6, and P-40∶1) species. One sphingolipid, SM(d18∶0), was significantly increased in all patients. Four PC forms (36∶3, 36∶5, 38∶5, and 38∶6) were consistently downregulated in severe vs mild patients. These observations were confirmed by TLC-MALDI. These results suggest that plasma phospholipid signatures may be able to discriminate mild and severe forms of CF, and show for the first time MALDI-TOF-ClinProTools™ as a suitable methodology for the search of lipid markers in CF.     

Grb14 inhibits FGF receptor signaling through the regulation of PLCγ recruitment and activation

To decipher the mechanism involved in Grb14 binding to the activated fibroblast growth factor receptor (FGFR), we used the bioluminescence resonance energy transfer (BRET) technique and the Xenopus oocyte model. We showed that Grb14 was recruited to FGFR1 into a trimeric complex containing also phospholipase C gamma (PLCγ). The presence of Grb14 altered FGF-induced PLCγ phosphorylation and activation. Grb14-FGFR interaction involved the Grb14-SH2 domain and the FGFR pY766 residue, which is the PLCγ binding site. Our data led to a molecular model whereby Grb14 binding to the phosphorylated FGFR induces a conformational change that unmasks a PLCγ binding motif on Grb14, allowing trapping and inactivation of PLCγ.

Structured summary

MINT-8019680: Grb14 (uniprotkb:O88900) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by anti tag coimmunoprecipitation (MI:0007)MINT-8019693, MINT-8019727: Grb14 (uniprotkb:O88900) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by bioluminescence resonance energy transfer (MI:0012)MINT-8019714, MINT-8019746: PLC gamma1 (uniprotkb:P19174) physically interacts (MI:0915) with FGFR1 (uniprotkb:P11362) by bioluminescence resonance energy transfer (MI:0012)     

RasV12 induces Survivin/AuroraB pathway conferring tumor cell apoptosis resistance

Several cancers are treated by interferons α and γ in association with conventional chemotherapy due to the resistance observed with interferon treatment alone. The frequency of un-sensitive cancer depends on tumor origin and oncogenic genes. Preclinical studies have highlighted interferon resistance in many cancers such as colon carcinoma due to oncogenic Ras. However, the resistance mechanism remains elusive. Apoptosis and proliferation of Ras^wt and mutated Ras^V12 transformed colon carcinoma cells treated with several recombinant interferon combinations were analyzed by flow cytometer and immunoblot. Apoptotic pathways of resistant Ras^V12 cells were investigated using siRNA strategy to determine key proteins involved in this process. We show that interferons α and γ synergized to induce human Ras^wt colon carcinoma cell (HT29) apoptosis by caspases and PARP-1 cleavages in contrast to Ras^V12 mutated colon carcinoma cells (SW480, HT29 clone). However, Ras^V12 siRNA restored interferon sensitivity of Ras^V12-HT29 clone to apoptosis. Survivin siRNA increased interferon apoptosis in Ras^wt cells demonstrating the key role of this protein in cell survival. Ras^V12 mutation in HT29 clone neutralized the interferon effect on Survivin suppression and maintained high level of phospho-Aurora-B/Histone H3, which protected cells from apoptosis. SiRNA strategy against both Aurora-B and Survivin in Ras^V12 cells synergized to restore interferon -induced apoptosis. Ras^V12 cells are less sensitive than Ras^wt cells to interferon induced cell apoptosis due to a Survivin/Aurora-B survival alternative pathway. Taken together, these results may provide interest in siRNA-therapeutic strategy and diagnostic relevance for therapy.     

Role of the extracellular amino terminus and first membrane-spanning helix of dopamine D1 and D5 receptors in shaping ligand selectivity and efficacy

Transmembrane (TM) helices of human D1-like dopaminergic receptors (hD1R and hD5R) harbor the same residues implicated in ligand binding and activation of catecholamine G protein-coupled receptors (GPCRs). Yet, hD1R and hD5R naturally display the distinct functional properties shared by wild type and constitutively active mutant GPCRs, respectively. Interestingly, we show in the present study that a class of synthetic phenylbenzazepine agonists containing a methyl on the azepine ring exhibited lower affinity for the more constitutively activated hD5R. These results cannot be explained by the “allosteric ternary complex model” postulating a higher agonist affinity for constitutively active GPCRs. We have also explored the functional role of distinct extracellular amino terminus (NT) and TM1 regions of hD1R and hD5R using a chimerical approach. Of these two regions, our studies suggest that TM1 predominantly shapes D1-like ligand affinity and selectivity. Additionally, NT and TM1 of hD1R and hD5R play no role in receptor constitutive activity but differentially modulate dopamine-mediated responsiveness. The TM1 exchange mediated drastic changes in intrinsic efficacy and activity of phenylbenzazepine drugs displaying partial agonism at hD1R and hD5R. Phenylbenzazepines were converted into strong partial agonists or full agonists in cells expressing hD1R-TM1^D5 chimera while being switched from full agonists to partial agonists and partial agonists to antagonists in cells harboring hD5R-TM1^D1 chimera. TM1 exchange had no effect on antipsychotic-mediated inverse agonism. In summary, our study shows that NT and TM1 of D1-like receptors control ligand binding and agonist-induced activation, poising these regions as important structural determinants for catecholamine GPCR function.     

Platelet serotonin level predicts survival in amyotrophic lateral sclerosis

Background

Amyotrophic lateral sclerosis (ALS) is a life-threatening neurodegenerative disease involving upper and lower motor neurons loss. Clinical features are highly variable among patients and there are currently few known disease-modifying factors underlying this heterogeneity. Serotonin is involved in a range of functions altered in ALS, including motor neuron excitability and energy metabolism. However, whether serotoninergic activity represents a disease modifier of ALS natural history remains unknown.

Methodology

Platelet and plasma unconjugated concentrations of serotonin and plasma 5-HIAA, the major serotonin metabolite, levels were measured using HPLC with coulometric detection in a cohort of 85 patients with ALS all followed-up until death and compared to a control group of 29 subjects.

Principal Findings

Platelet serotonin levels were significantly decreased in ALS patients. Platelet serotonin levels did not correlate with disease duration but were positively correlated with survival of the patients. Univariate Cox model analysis showed a 57% decreased risk of death for patients with platelet serotonin levels in the normal range relative to patients with abnormally low platelet serotonin (p = 0.0195). This protective effect remained significant after adjustment with age, gender or site of onset in multivariate analysis. Plasma unconjugated serotonin and 5-HIAA levels were unchanged in ALS patients compared to controls and did not correlate with clinical parameters.

Conclusions/Significance

The positive correlation between platelet serotonin levels and survival strongly suggests that serotonin influences the course of ALS disease.     

JULIDE: a software tool for 3D reconstruction and statistical analysis of autoradiographic mouse brain sections

In this article we introduce JULIDE, a software toolkit developed to perform the 3D reconstruction, intensity normalization, volume standardization by 3D image registration and voxel-wise statistical analysis of autoradiographs of mouse brain sections. This software tool has been developed in the open-source ITK software framework and is freely available under a GPL license. The article presents the complete image processing chain from raw data acquisition to 3D statistical group analysis. Results of the group comparison in the context of a study on spatial learning are shown as an illustration of the data that can be obtained with this tool.