Upregulated miR-146a expression in peripheral blood mononuclear cells from rheumatoid arthritis patients

Introduction

MicroRNAs are small noncoding RNA molecules that negatively regulate gene expression via degradation or translational repression of their targeted mRNAs. It is known that aberrant microRNA expression can play important roles in cancer, but the role of microRNAs in autoimmune diseases is only beginning to emerge. In this study, the expression of selected microRNAs is examined in rheumatoid arthritis.

Methods

Total RNA was isolated from peripheral blood mononuclear cells obtained from patients with rheumatoid arthritis, and healthy and disease control individuals, and the expression of miR-146a, miR-155, miR-132, miR-16, and microRNA let-7a was analyzed using quantitative real-time PCR.

Results

Rheumatoid arthritis peripheral blood mononuclear cells exhibited between 1.8-fold and 2.6-fold increases in miR-146a, miR-155, miR-132, and miR-16 expression, whereas let-7a expression was not significantly different compared with healthy control individuals. In addition, two targets of miR-146a, namely tumor necrosis factor receptor-associated factor 6 (TRAF6) and IL-1 receptor-associated kinase 1 (IRAK-1), were similarly expressed between rheumatoid arthritis patients and control individuals, despite increased expression of miR-146a in patients with rheumatoid arthritis. Repression of TRAF6 and/or IRAK-1 in THP-1 cells resulted in up to an 86% reduction in tumor necrosis factor-α production, implicating that normal miR-146a function is critical for the regulation of tumor necrosis factor-α production.

Conclusions

Recent studies have shown that synovial tissue and synovial fibroblasts from patients with rheumatoid arthritis exhibit increased expression of certain microRNAs. Our data thus demonstrate that microRNA expression in rheumatoid arthritis peripheral blood mononuclear cells mimics that of synovial tissue/fibroblasts. The increased microRNA expression in rheumatoid arthritis patients is potentially useful as a marker for disease diagnosis, progression, or treatment efficacy, but this will require confirmation using a large and well defined cohort. Our data also suggest a possible mechanism contributing to rheumatoid arthritis pathogenesis, whereby miR-146a expression is increased but unable to properly function, leading to prolonged tumor necrosis factor-α production in patients with rheumatoid arthritis.     

Virulence determinants, drug resistance and mobile genetic elements of Laribacter hongkongensis: a genome-wide analysis

Background

Laribacter hongkongensis is associated with community-acquired gastroenteritis and traveler's diarrhea. In this study, we performed an in-depth annotation of the genes in its genome related to the various steps in the infective process, drug resistance and mobile genetic elements.

Results

For acid and bile resistance, L. hongkongensis possessed a urease gene cassette, two arc gene clusters and bile salt efflux systems. For intestinal colonization, it possessed a putative adhesin of the autotransporter family homologous to those of diffusely adherent Escherichia coli (E. coli) and enterotoxigenic E. coli. To evade from host defense, it possessed superoxide dismutase and catalases. For lipopolysaccharide biosynthesis, it possessed the same set of genes that encode enzymes for synthesizing lipid A, two Kdo units and heptose units as E. coli, but different genes for its symmetrical acylation pattern, and nine genes for polysaccharide side chains biosynthesis. It contained a number of CDSs that encode putative cell surface acting (RTX toxin and hemolysins) and intracellular cytotoxins (patatin-like proteins) and enzymes for invasion (outer membrane phospholipase A). It contained a broad variety of antibiotic resistance-related genes, including genes related to β-lactam (n = 10) and multidrug efflux (n = 54). It also contained eight prophages, 17 other phage-related CDSs and 26 CDSs for transposases.

Conclusions

The L. hongkongensis genome possessed genes for acid and bile resistance, intestinal mucosa colonization, evasion of host defense and cytotoxicity and invasion. A broad variety of antibiotic resistance or multidrug resistance genes, a high number of prophages, other phage-related CDSs and CDSs for transposases, were also identified.     

Speciation in sexually deceptive orchids: pollinator-driven selection maintains discrete odour phenotypes in hybridizing species

Ophrys orchids mimic the female sex pheromones of their pollinator species to attract males for pollination. Reproductive isolation in Ophrys is based on the selective attraction of only a single pollinator species. A change of floral odour can result in the attraction of a new pollinator species that acts as an isolation barrier towards other sympatrically occurring Ophrys species. Ophrys lupercalis, Ophrys bilunulata, and Ophrys fabrella grow sympatrically and bloom consecutively on Majorca and are pollinated by three species of Andrena. We investigated variation of phenotypic and genotypic flower traits, aiming to study the role of the floral odour for reproductive isolation and speciation. Using chemical and electrophysiology (gas chromatography coupled with an electroantennographic detector) methods, we show that the three Ophrys species use the same odour compounds for pollinator attraction, but in different proportions. A comparison of the floral odour bouquets in a multivariate analysis revealed a clear grouping of plants from the same species, although with an overlap between species. A comparison of the same plants using molecular markers gave a contrasting result. Although O. lupercalis and O. fabrella were genetically well separated, plants of O. bilunulata did not form a distinct group but were similar to either O. lupercalis or O. fabrella. Our data indicate gene flow and hybridization to occur between O. bilunulata and O. lupercalis as well as between O. bilunulata and O. fabrella. All plants of O. bilunulata, despite having different genotypes, showed a very similar floral odour. This reflects a strong selective pressure by the pollinating males. The overlap of genotypes of O. bilunulata and O. fabrella supports our hypothesis that O. fabrella diverged from O. bilunulata by scent variation and the attraction of a new pollinator species, Andrena fabrella. © 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98 , 439–451.     

A new immunoprecipitation-real time quantitative PCR assay for anti-Th/To and anti-U3RNP antibody detection in systemic sclerosis

Introduction

Classic anti-nucleolar antibodies anti-Th/To and U3 ribonucleoprotein (-U3RNP) can help in the diagnosis, prediction of organ involvement and prognosis in systemic sclerosis (SSc); however, no validated commercial assay is available. We aimed at establishing a novel quantitative real time PCR (qPCR) method to detect these antibodies.

Methods

Standard immunoprecipitation (IP) was performed using K562 cell extract and RNA components were extracted. cDNA was reverse transcribed from RNA components and Th RNA and U3 RNA were detected by qPCR using custom primers. Cycle threshold (Ct) values were compared in a titration experiment to determine the assay efficacy. The new assay was evaluated by testing 22 anti-Th/To and 12 anti-U3RNP positive samples in addition to 88 controls, and the results were compared with IP as a gold standard.

Results

By testing serial 1:8 dilutions of cell lysate as the substrate in the IP step, RNA extracted after IP, and its derived cDNA, linear dose response curves were noted for both anti-Th/To and -U3RNP. With every dilution, Ct values changed approximately three as expected, reflecting the eight-fold difference of cDNA. The Ct difference between positive and negative samples was 8 to 13, which was similar throughout the dilutions. In the specificity analysis, the Ct values of positive samples were clearly different from the negative groups and the results by qPCR had a near perfect correlation with IP.

Conclusions

Our new method readily detects these two clinically important antibodies in SSc. Making tests for anti-Th/To and -U3RNP antibodies widely available to clinicians should be helpful in the diagnosis and follow-up of SSc patients.     

Effect of calcification on the mechanical stability of plaque based on a three-dimensional carotid bifurcation model

Background

This study characterizes the distribution and components of plaque structure by presenting a three-dimensional blood-vessel modelling with the aim of determining mechanical properties due to the effect of lipid core and calcification within a plaque. Numerical simulation has been used to answer how cap thickness and calcium distribution in lipids influence the biomechanical stress on the plaque.

Method

Modelling atherosclerotic plaque based on structural analysis confirms the rationale for plaque mechanical examination and the feasibility of our simulation model. Meaningful validation of predictions from modelled atherosclerotic plaque model typically requires examination of bona fide atherosclerotic lesions. To analyze a more accurate plaque rupture, fluid-structure interaction is applied to three-dimensional blood-vessel carotid bifurcation modelling. A patient-specific pressure variation is applied onto the plaque to influence its vulnerability.

Results

Modelling of the human atherosclerotic artery with varying degrees of lipid core elasticity, fibrous cap thickness and calcification gap, which is defined as the distance between the fibrous cap and calcification agglomerate, form the basis of our rupture analysis. Finite element analysis shows that the calcification gap should be conservatively smaller than its threshold to maintain plaque stability. The results add new mechanistic insights and methodologically sound data to investigate plaque rupture mechanics.

Conclusion

Structural analysis using a three-dimensional calcified model represents a more realistic simulation of late-stage atherosclerotic plaque. We also demonstrate that increases of calcium content that is coupled with a decrease in lipid core volume can stabilize plaque structurally.     

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

Background

To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH).

Methods

Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test.

Results

We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase.

Conclusion

Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively.     

Major morphological effects of a regulatory gene: Pgm1-t in rainbow trout

We have investigated the morphological effects of a genetic locus, Pgm1- t, that affects the expression of a phosphoglucomutase locus (Pgm1) in liver of rainbow trout (Salmo gairdneri). We have previously shown that embryos with liver Pgm1 expression hatch earlier than those without liver Pgm1 expression. We predicted that this difference in developmental rate should cause a reduction in meristic counts in the more rapidly developing fish with liver Pgm1 expression. Eight meristic (countable) characters in nine full-sib groups segregating for the presence or absence of liver Pgm1 expression are in agreement with this prediction. In eight of the nine families, there is a significant difference in the multivariate distribution of the eight meristic counts between full sibs with and without liver Pgm1 expression. This separation in multivariate space is based on a tendency for lower meristic counts in fish with liver Pgm1 expression. The magnitude of these morphological differences is similar to that between two subspecies of cutthroat trout (Salmo clarki) that show substantial genetic divergence at structural loci encoding enzymes (Nei's D = 0.34). These data support the view that small changes in the developmental process caused by genetic differences at regulatory genes can have large effects on morphology.      

Analysis of Vibrio vulnificus from Market Oysters and Septicemia Cases for Virulence Markers

Representative encapsulated strains of Vibrio vulnificus from market oysters and oyster-associated primary septicemia cases (25 isolates each) were tested in a blinded fashion for potential virulence markers that may distinguish strains from these two sources. These isolates were analyzed for plasmid content, for the presence of a 460-bp amplicon by randomly amplified polymorphic DNA PCR, and for virulence in subcutaneously (s.c.) inoculated, iron-dextran-treated mice. Similar percentages of market oyster and clinical isolates possessed detectable plasmids (24 and 36%, respectively), produced the 460-bp amplicon (45 and 50%, respectively), and were judged to be virulent in the mouse s.c. inoculation-iron-dextran model (88% for each). Therefore, it appears that nearly all V. vulnificus strains in oysters are virulent and that genetic tests for plasmids and specific PCR size amplicons cannot distinguish between fully virulent and less virulent strains or between clinical and environmental isolates. The inability of these methods to distinguish food and clinical V. vulnificus isolates demonstrates the need for alternative subtyping approaches and virulence assays.