Computational identification of phospho-tyrosine sub-networks related to acanthocyte generation in neuroacanthocytosis

Acanthocytes, abnormal thorny red blood cells (RBC), are one of the biological hallmarks of neuroacanthocytosis syndromes (NA), a group of rare hereditary neurodegenerative disorders. Since RBCs are easily accessible, the study of acanthocytes in NA may provide insights into potential mechanisms of neurodegeneration. Previous studies have shown that changes in RBC membrane protein phosphorylation state affect RBC membrane mechanical stability and morphology. Here, we coupled tyrosine-phosphoproteomic analysis to topological network analysis. We aimed to predict signaling sub-networks possibly involved in the generation of acanthocytes in patients affected by the two core NA disorders, namely McLeod syndrome (MLS, XK-related, Xk protein) and chorea-acanthocytosis (ChAc, VPS13A-related, chorein protein). The experimentally determined phosphoproteomic data-sets allowed us to relate the subsequent network analysis to the pathogenetic background. To reduce the network complexity, we combined several algorithms of topological network analysis including cluster determination by shortest path analysis, protein categorization based on centrality indexes, along with annotation-based node filtering. We first identified XK- and VPS13A-related protein-protein interaction networks by identifying all the interactomic shortest paths linking Xk and chorein to the corresponding set of proteins whose tyrosine phosphorylation was altered in patients. These networks include the most likely paths of functional influence of Xk and chorein on phosphorylated proteins. We further refined the analysis by extracting restricted sets of highly interacting signaling proteins representing a common molecular background bridging the generation of acanthocytes in MLS and ChAc. The final analysis pointed to a novel, very restricted, signaling module of 14 highly interconnected kinases, whose alteration is possibly involved in generation of acanthocytes in MLS and ChAc.     

Activation of eosinophils interacting with dermal fibroblasts by pruritogenic cytokine IL-31 and alarmin IL-33: implications in atopic dermatitis

Background

IL-31 is a pruritogenic cytokine, and IL-33 is an alarmin for damaging inflammation. They together relate to the pathogenesis of atopic dermatitis (AD). Eosinophil infiltration into the inner dermal compartment is a predominant pathological feature of AD. We herein investigated the in vitro inflammatory effects of IL-31 and IL-33 on the activation of human eosinophils and dermal fibroblasts.

Methodology/Principal Findings

Receptors, adhesion molecules and signaling molecules were assessed by Western blot or flow cytometry. Chemokines and cytokine were quantitated by multiplex assay. Functional IL-31 receptor component IL-31RA, OSMR-β and IL-33 receptor component ST2 were constitutively expressed on the surface of eosinophils. Co-culture of eosinophils and fibroblasts significantly induced pro-inflammatory cytokine IL-6 and AD-related chemokines CXCL1, CXCL10, CCL2 and CCL5. Such inductions were further enhanced with IL-31 and IL-33 stimulation. IL-31 and IL-33 could significantly provoke the release of CXCL8 from eosinophils and fibroblasts, respectively, which was further enhanced upon co-culture. In co-culture, eosinophils and fibroblasts were the main source for the release of CCL5, and IL-6, CXCL1, CXCL8, CXCL10 and CCL2, respectively. Direct interaction between eosinophils and fibroblasts was required for CXCL1, CXCL10, CXCL8 and CCL5 release. Cell surface expression of intercellular adhesion molecule-1 on eosinophils and fibroblasts was up-regulated in co-culture upon IL-31 and IL-33 stimulation. The interaction between eosinophils and fibroblasts under IL-31 and IL-33 stimulation differentially activated extracellular signal-regulated kinase, c-Jun N-terminal kinase, p38 mitogen-activated protein kinase, nuclear factor-κB and phosphatidylinositol 3-kinase–Akt pathways. Using specific signaling molecule inhibitors, the differential induction of IL-31 and IL-33-mediated release of cytokines and chemokines such as IL-6 and CXCL8 from co-culture should be related to their distinct activation profile of intracellular signaling pathways.

Conclusions/Significance

The above findings suggest a crucial immunopathological role of IL-31 and IL-33 in AD through the activation of eosinophils-fibroblasts interaction via differential intracellular signaling mechanisms.     

Genetic variation in cell death genes and risk of non-Hodgkin lymphoma

Background

Non-Hodgkin lymphomas are a heterogeneous group of solid tumours that constitute the 5^th highest cause of cancer mortality in the United States and Canada. Poor control of cell death in lymphocytes can lead to autoimmune disease or cancer, making genes involved in programmed cell death of lymphocytes logical candidate genes for lymphoma susceptibility.

Materials and Methods

We tested for genetic association with NHL and NHL subtypes, of SNPs in lymphocyte cell death genes using an established population-based study. 17 candidate genes were chosen based on biological function, with 123 SNPs tested. These included tagSNPs from HapMap and novel SNPs discovered by re-sequencing 47 cases in genes for which SNP representation was judged to be low. The main analysis, which estimated odds ratios by fitting data to an additive logistic regression model, used European ancestry samples that passed quality control measures (569 cases and 547 controls). A two-tiered approach for multiple testing correction was used: correction for number of tests within each gene by permutation-based methodology, followed by correction for the number of genes tested using the false discovery rate.

Results

Variant rs928883, near miR-155, showed an association (OR per A-allele: 2.80 [95% CI: 1.63–4.82]; p_F = 0.027) with marginal zone lymphoma that is significant after correction for multiple testing.

Conclusions

This is the first reported association between a germline polymorphism at a miRNA locus and lymphoma.     

C-terminal fluorescent labeling impairs functionality of DNA mismatch repair proteins

The human DNA mismatch repair (MMR) process is crucial to maintain the integrity of the genome and requires many different proteins which interact perfectly and coordinated. Germline mutations in MMR genes are responsible for the development of the hereditary form of colorectal cancer called Lynch syndrome. Various mutations mainly in two MMR proteins, MLH1 and MSH2, have been identified so far, whereas 55% are detected within MLH1, the essential component of the heterodimer MutLα (MLH1 and PMS2). Most of those MLH1 variants are pathogenic but the relevance of missense mutations often remains unclear. Many different recombinant systems are applied to filter out disease-associated proteins whereby fluorescent tagged proteins are frequently used. However, dye labeling might have deleterious effects on MutLα's functionality. Therefore, we analyzed the consequences of N- and C-terminal fluorescent labeling on expression level, cellular localization and MMR activity of MutLα. Besides significant influence of GFP- or Red-fusion on protein expression we detected incorrect shuttling of single expressed C-terminal GFP-tagged PMS2 into the nucleus and found that C-terminal dye labeling impaired MMR function of MutLα. In contrast, N-terminal tagged MutLαs retained correct functionality and can be recommended both for the analysis of cellular localization and MMR efficiency.     

Biosafety evaluation of recombinant protein production in goat mammary gland using adenoviral vectors: preliminary study

The production of recombinant proteins in the milk of non-transgenic goats can be achieved by transducing the mammary gland with recombinant adenoviral vectors. However, this process involves several regulatory issues. The current study evaluates the biosafety of this production system. We present a preliminary biosafety profile based on detection of adenoviral particles in different body fluids and the antibody response after adenoviral transduction of the goat mammary gland. In addition, two methods of adenoviral inactivation in milk were tested. Although adenoviral particles were detected in the milk until day 4 after transduction, they were absent in serum, saliva, urine and feces. Anti-adenovirus antibodies were detected in serum and milk. The virus inactivation methods neutralized adenoviral particles and preserved the immunological identity of the recombinant protein. These results support the idea of a safe production of recombinant proteins using adenoviral vectors.     

Oleanane glycosides from the roots of Alhagi maurorum

Three new oleanane-type triterpene glycosides (1–3), along with four known compounds (4–7) glycosides, were isolated from the roots of Alhagi maurorum. Their structures were elucidated by 1D and 2D-NMR experiments as well as ESI-MS analysis. The antiproliferative activity of the isolated compounds was evaluated against a small panel of cancer cell lines including human breast cancer (MCF-7), human lung adenocarcinoma (A549), human prostate cancer (PC-3) and human leukemia (U937) cell lines. None of the tested compounds, in a range of concentrations between 1 and 50 μM, caused a significant reduction of the cell number.     

The relationship between symptoms of depression and body weight in younger adults

A bidirectional relationship between obesity and depression may exist, though previous results are conflicting. The objectives of our study were to determine whether there is a bidirectional relationship between obesity and symptoms of depression in younger adults and whether this relationship varies with sociodemographic factors. We used data from 7,980 participants in the National Longitudinal Survey of Youth 1979 to examine whether baseline depressive symptoms (score ≥ 10 on a seven-item subscale of the CES-D) in 1992, predicted adjusted percent change in BMI between 1992 and 1994. We then examined whether obesity in 1992 predicted the development of symptoms of depression in 1994, after adjustment for confounders. We found that the presence of baseline depressive symptoms was not prospectively associated with increase in percent BMI, except in Hispanic women. Additionally, baseline obesity was not associated with higher risk of future symptoms of depression in the sample overall (adjusted risk ratio (RR) 1.20; 99% CI 0.91-1.60). However, in those of higher socioeconomic status, obesity was associated with almost double the risk of depressive symptoms compared to nonobese (highest income category: adjusted RR 1.97; 99% CI 1.14-3.40). We concluded that although obesity was not associated with risk of depression symptoms in the population overall, obesity was associated with an increased risk of developing depressive symptoms in those of higher socioeconomic status. Sociodemographic factors may be important modifiers of the relationship between obesity and depression.     

Reliability and intermethod agreement for body fat assessment among two field and two laboratory methods in adolescents

To increase knowledge about reliability and intermethods agreement for body fat (BF) is of interest for assessment, interpretation, and comparison purposes. It was aimed to examine intra- and inter-rater reliability, interday variability, and degree of agreement for BF using air-displacement plethysmography (Bod-Pod), dual-energy X-ray absorptiometry (DXA), bioelectrical impedance analysis (BIA), and skinfold measurements in European adolescents. Fifty-four adolescents (25 females) from Zaragoza and 30 (14 females) from Stockholm, aged 13-17 years participated in this study. Two trained raters in each center assessed BF with Bod-Pod, DXA, BIA, and anthropometry (DXA only in Zaragoza). Intermethod agreement and reliability were studied using a 4-way ANOVA for the same rater on the first day and two additional measurements on a second day, one each rater. Technical error of measurement (TEM) and percentage coefficient of reliability (%R) were also reported. No significant intrarater, inter-rater, or interday effect was observed for %BF for any method in either of the cities. In Zaragoza, %BF was significantly different when measured by Bod-Pod and BIA in comparison with anthropometry and DXA (all P < 0.001). The same result was observed in Stockholm (P < 0.001), except that DXA was not measured. Bod-Pod, DXA, BIA, and anthropometry are reliable for %BF repeated assessment within the same day by the same or different raters or in consecutive days by the same rater. Bod-Pod showed close agreement with BIA as did DXA with anthropometry; however, Bod-Pod and BIA presented higher values of %BF than anthropometry and DXA.     

Human erythema and matrix metalloproteinase-1 mRNA induction, in vivo, share an action spectrum which suggests common chromophores

Matrix metalloproteinase 1 (MMP-1) is widely regarded as a biomarker of photoageing. We tested the hypothesis that MMP-1 mRNA expression and erythema share a common action spectrum by comparing the effects of erythemally equivalent doses of UVB, UVA1 and solar simulated radiation (SSR) on acute MMP-1 mRNA expression in whole human skin in vivo. Our results show comparable MMP-1 expression with all three spectra, which supports our hypothesis. The sharing of an action spectrum implies common chromophores, one of which is likely to be DNA. We have previously shown that all spectra that we used readily induce cyclobutane thymine dimers (T<>T) in human epidermis in vivo but we lack quantitative data on damage to dermal DNA. This is important because we do not know if dermal MMP-1 induction occurs via direct damage to the dermis, or indirectly via damage to the epidermis. Our results show that UVB induces about 3 times more T<>T compared with erythemally equivalent doses of UVA1, which is similar to our published epidermal data. This supports previously published work that also implicates an unknown UVA1 chromophore for erythema and MMP-1 induction. However, the distribution of the dermal DNA damage varies considerably with spectrum. In the case of UVB it is primarily in the upper dermis, but with UVA1 it is evenly distributed. Thus, irrespective of chromophores, MMP-1 induction by direct dermal damage by both spectra is possible. The practical conclusions of our data are that the small (<5%) UVB content of solar UVR is likely to be the main cause of photoageing, at least in terms of MMP-1 expression. Furthermore, prevention of erythema by sunscreen use is likely to result in reduced MMP-1 expression.