CD8+ T lymphocytes in lung tissue from patients with idiopathic pulmonary fibrosis

Objective

To study the prevalence of respiratory and atopic symptoms in (young) adults born prematurely, differences between those who did and did not develop Bronchopulmonary Disease (BPD) at neonatal age and differences in respiratory health between males and females.

Methods

Design: Prospective cohort study. Setting: Nation wide follow-up study, the Netherlands. Participants: 690 adults (19 year old) born with a gestational age below 32 completed weeks and/or with a birth weight less than 1500 g. Controls were Dutch participants of the European Community Respiratory Health Survey (ECRHS). Main outcome measures: Presence of wheeze, shortness of breath, asthma, hay fever and eczema using the ECRHS-questionnaire

Results

The prevalence of doctor-diagnosed asthma was significantly higher in the ex-preterms than in the general population, whereas eczema and hay fever were significant lower. Women reported more symptoms than men. Preterm women vs controls: asthma 13% vs 5% (p < 0.001); hay fever 8% vs 20% (p < 0.001); eczema 10% vs 42% (p < 0.001). Preterm men vs controls: asthma 9% vs 4% (p = 0.007); hay fever 8% vs 17% (p = 0.005); eczema 9% vs 31% (p < 0.001) Preterm women reported more wheeze and shortness of breath during exercise (sob) than controls: wheeze 30% vs 22% (p = 0.009); sob 27% vs 16% (p < 0.001); 19-year-old women with BPD reported a higher prevalence of doctor diagnosed asthma compared to controls (24% vs 5% p < 0.001) and shortness of breath during exercise (43% vs 16% p = 0.008). The prevalence of reported symptoms by men with BPD were comparable with the controls.

Conclusion

Our large follow-up study shows a higher prevalence of asthma, wheeze and shortness of breath in the prematurely born young adults. 19-year-old women reported more respiratory symptoms than men. Compared to the general population atopic diseases as hay fever and eczema were reported less often.     

COLD-PCR Amplification of Bisulfite-Converted DNA Allows the Enrichment and Sequencing of Rare Un-Methylated Genomic Regions

Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes.     

Comparative substrate recognition by bacterial and fungal purine transporters of the NAT/NCS2 family

We compared the interactions of purines and purine analogues with representative fungal and bacterial members of the widespread Nucleobase-Ascorbate Transporter (NAT) family. These are: UapA, a well-studied xanthine-uric acid transporter of A. nidulans, Xut1, a novel transporter from C. albicans, described for the first time in this work, and YgfO, a recently characterized xanthine transporter from E. coli. Using transport inhibition experiments with 64 different purines and purine-related analogues, we describe a kinetic approach to build models on how NAT proteins interact with their substrates. UapA, Xut1 and YgfO appear to bind several substrates via interactions with both the pyrimidine and imidazol rings. Fungal homologues interact with the pyrimidine ring of xanthine and xanthine analogues via H-bonds, principally with N1-H and =O6, and to a lower extent with =O2. The E. coli homologue interacts principally with N3-H and =O2, and less strongly with N1-H and =O6. The basic interaction with the imidazol ring appears to be via a H-bond with N9. Interestingly, while all three homologues recognize xanthines with similar high affinities, interaction with uric acid or/and oxypurinol is transporter-specific. UapA recognizes uric acid with high affinity, principally via three H-bonds with =O2, =O6 and =O8. Xut1 has a 13-fold reduced affinity for uric acid, based on a different set of interactions involving =O8, and probably H atoms from positions N1, N3, N7 or N9. YgfO does not recognize uric acid at all. Both Xut1 and UapA recognize oxypurinol, but use different interactions reflected in a nearly 26-fold difference in their affinities for this drug, while YgfO interacts with this analogue very inefficiently.     

SaMW: a probabilistic meta-heuristic algorithm for job scheduling in heterogeneous distributed systems powered by microservices

Cluster Computing - Applications are evolving in ways that demand geographically distributed resources to co-operate in order to give users better Quality of Service (QoS). There is a plethora of...     

Cloning and functional characterization of two bacterial members of the NAT/NCS2 family in Escherichia coli

The coding potential of the genome of E. coli K-12 includes YgfO and YicE, two members of the evolutionarily conserved NAT/NCS2 transporter family that are highly homologous to each other (45% residue identity) and closely related to UapA of Aspergillus nidulans, a most extensively studied microbial member of this family. YgfO and yicE were cloned from the genome, over-expressed extrachromosomally and assayed for uptake of [³H]xanthine and other nucleobases, in E. coli K-12, under conditions of negligible activity of the corresponding endogenous systems. Alternative, essentially equivalent functional versions of YgfO and YicE were engineered by C-terminal tagging with an epitope from the E. coli lactose permease and a biotin-acceptor domain from Klebsiella pneumoniae. Both YgfO and YicE were shown to be present in the plasma membrane of E. coli and function as specific, high-affinity transporters for xanthine (K_m 4.2–4.6 µM for YgfO, or 2.9–3.8 µM for YicE), in a proton motive force-dependent manner; they display no detectable transport of uracil, hypoxanthine, or uric acid at external concentrations of up to 0.1 mM. Both YgfO and YicE are inefficient in recognizing uric acid or xanthine analogues modified at position 8 of the purine ring (8-methylxanthine, 8-azaxanthine, oxypurinol, allopurinol), which distinguishes them from their fungal homologues UapA and Xut1.     

B-cell depletion therapy in patients with diffuse systemic sclerosis associates with a significant decrease in PDGFR expression and activation in spindle-like cells in the skin

Introduction

Recently, several studies assessing the clinical efficacy of rituximab (RTX) in systemic sclerosis (SSc) have reported encouraging results. We aimed at exploring whether RTX exerts its beneficial effects on fibrosis through attenuation of platelet-derived growth factor receptor (PDGFR) pathway activation.

Methods

We immunohistochemically assessed skin biopsies obtained from eight patients with SSc prior to and 6 months following RTX treatment, three control SSc patients (at the same time points) and three healthy subjects. We assessed the expression of platelet-derived growth factor, PDGFR and phosphorylated (activated) PDGFR.

Results

We found a strong correlation of PDGFRα and PDGFRβ expression on spindle-like cells and collagen deposition in SSc biopsies (r = 0.97 and r = 0.96 for PDGFRα and PDGFRβ, respectively; P < 0.0001 for both), indicating a strong link between PDGFR expression and fibrosis. Expression of PDGFRα and PDGFRβ in the papillary dermis significantly decreased following RTX administration (mean ± standard error of the mean at baseline vs. 6 months, respectively: PDGFRα, 42.05 ± 5.03 vs. 26.85 ± 3.00, P = 0.004; and PDGFRβ, 37.14 ± 4.94 vs. 24.01 ± 3.27, P = 0.012). Similarly, expression of phosphorylated PDGFRα and PDGFRβ in the papillary dermis significantly decreased following RTX administration (P = 0.006 and P = 0.013 for phospho-PDGFRα and phospho-PDGFRβ, respectively). No changes in platelet-derived growth factor tissue expression or serum levels were found following RTX treatment.

Conclusion

RTX may favorably affect skin fibrosis through attenuation of PDGFR expression and activation, a finding that supports a disease-modifying role of RTX in SSc. Large-scale, multicenter studies are needed to further explore the efficacy of RTX in SSc.     

Dispersed information diffusion with level and schema-based coordination in mobile peer to peer networks

Several open ended issues for high resource availability in mobile peer to peer networks have been examined in the recent past. Different approaches were conducted for supporting information distribution and availability, through guided or unguided packet diffusion. The majority of the recently proposed approaches try to benefit from the spatial characteristics of the dynamically varying topologies. In this work a directed information diffusion scheme is examined using a level and schema-based coordination, applied in mobile peer to peer networks. The prioritization degree of any requested advert is modeled and enables directed prioritized diffusions to end mobile users that are traversing a certain geographic region (location based advertisements). The proposed method is robust in disseminating redundant messages to users while maintaining connectivity through Gradual Energy Tree-based (GET) configuration. Simulation is performed for the examination and performance evaluation of the proposed scheme, taking into account the modeled prioritization as well as the diffusion accuracy by using the Hierarchical and Non-hierarchical GET configuration.

Cysteine-scanning analysis of the nucleobase-ascorbate transporter signature motif in YgfO permease of Escherichia coli: Gln-324 and Asn-325 are essential, and Ile-329-Val-339 form an alpha-helix

The nucleobase-ascorbate transporter (NAT) signature motif is a conserved sequence motif of the ubiquitous NAT/NCS2 family implicated in defining the function and selectivity of purine translocation pathway in the major fungal homolog UapA. To analyze the role of NAT motif more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence (315)GSIPITTFAQNNGVIQMTGVASRYVG(340) (motif underlined) was replaced individually with Cys. Of the 26 single-Cys mutants, 16 accumulate xanthine to > or =50% of the steady state observed with C-less YgfO, 4 accumulate to low levels (10-25% of C-less), F322C, N325C, and N326C accumulate marginally (5-8% of C-less), and P318C, Q324C, and G340C are inactive. When transferred to wild type, F322C(wt) and N326C(wt) are highly active, but P318G(wt), Q324C(wt), N325C(wt), and G340C(wt) are inactive, and G340A(wt) displays low activity. Immunoblot analysis shows that replacements at Pro-318 or Gly-340 are associated with low or negligible expression in the membrane. More extensive mutagenesis reveals that Gln-324 is critical for high affinity uptake and ligand recognition, and Asn-325 is irreplaceable for active xanthine transport, whereas Thr-332 and Gly-333 are important determinants of ligand specificity. All single-Cys mutants react with N-ethylmaleimide, but regarding sensitivity to inactivation, they fall to three regions; positions 315-322 are insensitive to N-ethylmaleimide, with IC(50) values > or =0.4 mM, positions 323-329 are highly sensitive, with IC(50) values of 15-80 microM, and sensitivity of positions 330-340 follows a periodicity, with mutants sensitive to inactivation clustering on one face of an alpha-helix.