Induction and regulation of matrix metalloproteinase-12in human airway smooth muscle cells

BACKGROUND: The elastolytic enzyme matrix metalloproteinase (MMP)-12 has been implicated in the development of airway inflammation and remodeling. We investigated whether human airway smooth muscle cells could express and secrete MMP-12, thereby participating in the pathogenesis of airway inflammatory diseases. METHODS: Laser capture microdissection was used to collect smooth muscle cells from human bronchial biopsy sections. MMP-12 mRNA expression was analysed by quantitative real-time RT-PCR. MMP-12 protein expression and secretion from cultured primary airway smooth muscle cells was further analysed by Western blot. MMP-12 protein localization in bronchial tissue sections was detected by immunohistochemistry. MMP-12 activity was determined by zymography. The TransAM AP-1 family kit was used to measure c-Jun activation and nuclear binding. Analysis of variance was used to determine statistical significance. RESULTS: We provide evidence that MMP-12 mRNA and protein are expressed by in-situ human airway smooth muscle cells obtained from bronchial biopsies of normal volunteers, and of patients with asthma, COPD and chronic cough. The pro-inflammatory cytokine, interleukin (IL)-1β, induced a >100-fold increase in MMP-12 gene expression and a >10-fold enhancement in MMP-12 activity of primary airway smooth muscle cell cultures. Selective inhibitors of extracellular signal-regulated kinase, c-Jun N-terminal kinase and phosphatidylinositol 3-kinase reduced the activity of IL-1β on MMP-12, indicating a role for these kinases in IL-1β-induced induction and release of MMP-12. IL-1β-induced MMP-12 activity and gene expression was down-regulated by the corticosteroid dexamethasone but up-regulated by the inflammatory cytokine tumour necrosis factor (TNF)-α through enhancing activator protein-1 activation by IL-1β. Transforming growth factor-β had no significant effect on MMP-12 induction. CONCLUSION: Our findings indicate that human airway smooth muscle cells express and secrete MMP-12 that is up-regulated by IL-1β and TNF-α. Bronchial smooth muscle cells may be an important source of elastolytic activity, thereby participating in remodeling in airway diseases such as COPD and chronic asthma

A Position Effect on the Heritability of Epigenetic Silencing

In animals and yeast, position effects have been well documented. In animals, the best example of this process is Position Effect Variegation (PEV) in Drosophila melanogaster. In PEV, when genes are moved into close proximity to constitutive heterochromatin, their expression can become unstable, resulting in variegated patches of gene expression. This process is regulated by a variety of proteins implicated in both chromatin remodeling and RNAi-based silencing. A similar phenomenon is observed when transgenes are inserted into heterochromatic regions in fission yeast. In contrast, there are few examples of position effects in plants, and there are no documented examples in either plants or animals for positions that are associated with the reversal of previously established silenced states. MuDR transposons in maize can be heritably silenced by a naturally occurring rearranged version of MuDR. This element, Muk, produces a long hairpin RNA molecule that can trigger DNA methylation and heritable silencing of one or many MuDR elements. In most cases, MuDR elements remain inactive even after Muk segregates away. Thus, Muk-induced silencing involves a directed and heritable change in gene activity in the absence of changes in DNA sequence. Using classical genetic analysis, we have identified an exceptional position at which MuDR element silencing is unstable. Muk effectively silences the MuDR element at this position. However, after Muk is segregated away, element activity is restored. This restoration is accompanied by a reversal of DNA methylation. To our knowledge, this is the first documented example of a position effect that is associated with the reversal of epigenetic silencing. This observation suggests that there are cis-acting sequences that alter the propensity of an epigenetically silenced gene to remain inactive. This raises the interesting possibility that an important feature of local chromatin environments may be the capacity to erase previously established epigenetic marks

ApoB siRNA-induced Liver Steatosis is Resistant to Clearance by the Loss of Fatty Acid Transport Protein 5 (Fatp5)

The association between hypercholesterolemia and elevated serum apolipoprotein B (APOB) has generated interest in APOB as a therapeutic target for patients at risk of developing cardiovascular disease. In the clinic, mipomersen, an antisense oligonucleotide (ASO) APOB inhibitor, was associated with a trend toward increased hepatic triglycerides, and liver steatosis remains a concern. We found that siRNA-mediated knockdown of ApoB led to elevated hepatic triglycerides and liver steatosis in mice engineered to exhibit a human-like lipid profile. Many genes required for fatty acid synthesis were reduced, suggesting that the observed elevation in hepatic triglycerides is maintained by the cell through fatty acid uptake as opposed to fatty acid synthesis. Fatty acid transport protein 5 (Fatp5/Slc27a5) is required for long chain fatty acid (LCFA) uptake and bile acid reconjugation by the liver. Fatp5 knockout mice exhibited lower levels of hepatic triglycerides due to decreased fatty acid uptake, and shRNA-mediated knockdown of Fatp5 protected mice from diet-induced liver steatosis. Here, we evaluated if siRNA-mediated knockdown of Fatp5 was sufficient to alleviate ApoB knockdown-induced steatosis. We determined that, although Fatp5 siRNA treatment was sufficient to increase the proportion of unconjugated bile acids 100-fold, consistent with FATP5's role in bile acid reconjugation, Fatp5 knockdown failed to influence the degree, zonal distribution, or composition of the hepatic triglycerides that accumulated following ApoB siRNA treatment

Heritable Epigenetic Variation among Maize Inbreds

Epigenetic variation describes heritable differences that are not attributable to changes in DNA sequence. There is the potential for pure epigenetic variation that occurs in the absence of any genetic change or for more complex situations that involve both genetic and epigenetic differences. Methylation of cytosine residues provides one mechanism for the inheritance of epigenetic information. A genome-wide profiling of DNA methylation in two different genotypes of Zea mays (ssp. mays), an organism with a complex genome of interspersed genes and repetitive elements, allowed the identification and characterization of examples of natural epigenetic variation. The distribution of DNA methylation was profiled using immunoprecipitation of methylated DNA followed by hybridization to a high-density tiling microarray. The comparison of the DNA methylation levels in the two genotypes, B73 and Mo17, allowed for the identification of approximately 700 differentially methylated regions (DMRs). Several of these DMRs occur in genomic regions that are apparently identical by descent in B73 and Mo17 suggesting that they may be examples of pure epigenetic variation. The methylation levels of the DMRs were further studied in a panel of near-isogenic lines to evaluate the stable inheritance of the methylation levels and to assess the contribution of cis- and trans- acting information to natural epigenetic variation. The majority of DMRs that occur in genomic regions without genetic variation are controlled by cis-acting differences and exhibit relatively stable inheritance. This study provides evidence for naturally occurring epigenetic variation in maize, including examples of pure epigenetic variation that is not conditioned by genetic differences. The epigenetic differences are variable within maize populations and exhibit relatively stable trans-generational inheritance. The detected examples of epigenetic variation, including some without tightly linked genetic variation, may contribute to complex trait variation

Gibberellin Acts through Jasmonate to Control the Expression of MYB21, MYB24, and MYB57 to Promote Stamen Filament Growth in Arabidopsis

Precise coordination between stamen and pistil development is essential to make a fertile flower. Mutations impairing stamen filament elongation, pollen maturation, or anther dehiscence will cause male sterility. Deficiency in plant hormone gibberellin (GA) causes male sterility due to accumulation of DELLA proteins, and GA triggers DELLA degradation to promote stamen development. Deficiency in plant hormone jasmonate (JA) also causes male sterility. However, little is known about the relationship between GA and JA in controlling stamen development. Here, we show that MYB21, MYB24, and MYB57 are GA-dependent stamen-enriched genes. Loss-of-function of two DELLAs RGA and RGL2 restores the expression of these three MYB genes together with restoration of stamen filament growth in GA-deficient plants. Genetic analysis showed that the myb21-t1 myb24-t1 myb57-t1 triple mutant confers a short stamen phenotype leading to male sterility. Further genetic and molecular studies demonstrate that GA suppresses DELLAs to mobilize the expression of the key JA biosynthesis gene DAD1, and this is consistent with the observation that the JA content in the young flower buds of the GA-deficient quadruple mutant ga1-3 gai-t6 rga-t2 rgl1-1 is much lower than that in the WT. We conclude that GA promotes JA biosynthesis to control the expression of MYB21, MYB24, and MYB57. Therefore, we have established a hierarchical relationship between GA and JA in that modulation of JA pathway by GA is one of the prerequisites for GA to regulate the normal stamen development in Arabidopsis

Estimating the Number of Paediatric Fevers Associated with Malaria Infection Presenting to Africa's Public Health Sector in 2007

Peter Gething and colleagues compute the number of fevers likely to present to public health facilities in Africa and the estimated number of these fevers likely to be infected with Plasmodium falciparum malaria parasites

The relationship between reported fever and Plasmodium falciparum infection in African children

<p>Abstract</p> <p>Background</p> <p>Fever has traditionally served as the entry point for presumptive treatment of malaria in African children. However, recent changes in the epidemiology of malaria across many places in Africa would suggest that the predictive accuracy of a fever history as a marker of disease has changed prompting calls for the change to diagnosis-based treatment strategies.</p> <p>Methods</p> <p>Using data from six national malaria indicator surveys undertaken between 2007 and 2009, the relationship between childhood (6-59 months) reported fever on the day of survey and the likelihood of coincidental <it>Plasmodium falciparum </it>infection recorded using a rapid diagnostic test was evaluated across a range of endemicities characteristic of Africa today.</p> <p>Results</p> <p>Of 16,903 children surveyed, 3% were febrile and infected, 9% were febrile without infection, 12% were infected but were not febrile and 76% were uninfected and not febrile. Children with fever on the day of the survey had a 1.98 times greater chance of being infected with <it>P. falciparum </it>compared to children without a history of fever on the day of the survey after adjusting for age and location (OR 1.98; 95% CI 1.74-2.34). There was a strong linear relationship between the percentage of febrile children with infection and infection prevalence (R²= 0.9147). The prevalence of infection in reported fevers was consistently greater than would be expected solely by chance and this increased with increasing transmission intensity. The data suggest that in areas where community-based infection prevalence in childhood is above 34-37%, 50% or more of fevers are likely to be associated with infection.</p> <p>Conclusion</p> <p>The potential benefits of diagnosis will depend on the prevalence of infection among children who report fever. The study has demonstrated a predictable relationship between parasite prevalence in the community and risks of infection among febrile children suggesting that current maps of parasite prevalence could be used to guide diagnostic strategies in Africa.</p

Validation of a new test that assesses functional performance of the upper extremity and neck (FIT-HaNSA) in patients with shoulder pathology

<p>Abstract</p> <p>Background</p> <p>There is a lack of standardized tests that assess functional performance for sustained upper extremity activity. This study describes development of a new test for measuring functional performance of the upper extremity and neck and assesses reliability and concurrent validity in patients with shoulder pathology.</p> <p>Methods</p> <p>A series of developmental tests were conducted to develop a protocol for assessing upper extremity tasks that required multi-level movement and sustained elevation. Kinematics of movement were investigated to inform subtask structure. Tasks and test composition were refined to fit clinical applicability criteria and pilot tested on 5 patients awaiting surgery for shoulder impingement and age-sex matched controls. Test-retest reliability was assessed on 10 subjects. Then a cohort of patients with mild to moderate (n = 17) shoulder pathology and 19 controls (17 were age-sex matched to patients) were tested to further validate the Functional Impairment Test-Hand, and Neck/Shoulder/Arm (FIT-HaNSA) by comparing it to self-reported function and measured strength. The FIT-HaNSA, DASH and SPADI were tested on a single occasion. Impairments in isometric strength were measured using hand-held dynamometry. Discriminative validity was determined by comparing scores to those of age-sex matched controls (n = 34), using ANOVA. Pearson correlations between outcome measures (n = 41) were examined to establish criterion and convergent validity.</p> <p>Results</p> <p>A test protocol based on three five-minute subtasks, each either comprised of moving objects to waist-height shelves, eye-level shelves, or sustained manipulation of overhead nuts/bolts, was developed. Test scores for the latter 2 subtasks (or total scores) were different between controls as compared to either surgical-list patients with shoulder impingement or a variety of milder shoulder pathologies (p < 0.01). Test 1 correlated the highest with the DASH (r = -0.83), whereas Test 2 correlated highest with the SPADI (r = -0.76).</p> <p>Conclusion</p> <p>Initial data suggest the FIT-HaNSA provides valid assessment of impaired functional performance in patients with shoulder pathology. It discriminates between patients and controls, is related to self-reported function, and yet provides distinct information. Longitudinal testing is warranted.</p

Co-Orientation of Replication and Transcription Preserves Genome Integrity

In many bacteria, there is a genome-wide bias towards co-orientation of replication and transcription, with essential and/or highly-expressed genes further enriched co-directionally. We previously found that reversing this bias in the bacterium Bacillus subtilis slows replication elongation, and we proposed that this effect contributes to the evolutionary pressure selecting the transcription-replication co-orientation bias. This selection might have been based purely on selection for speedy replication; alternatively, the slowed replication might actually represent an average of individual replication-disruption events, each of which is counter-selected independently because genome integrity is selected. To differentiate these possibilities and define the precise forces driving this aspect of genome organization, we generated new strains with inversions either over ∼1/4 of the chromosome or at ribosomal RNA (rRNA) operons. Applying mathematical analysis to genomic microarray snapshots, we found that replication rates vary dramatically within the inverted genome. Replication is moderately impeded throughout the inverted region, which results in a small but significant competitive disadvantage in minimal medium. Importantly, replication is strongly obstructed at inverted rRNA loci in rich medium. This obstruction results in disruption of DNA replication, activation of DNA damage responses, loss of genome integrity, and cell death. Our results strongly suggest that preservation of genome integrity drives the evolution of co-orientation of replication and transcription, a conserved feature of genome organization