967 research outputs found
Reversal of aging-induced increases in aortic stiffness by targeting cytoskeletal protein-protein interfaces
BACKGROUND: The proximal aorta normally functions as a critical shock absorber that protects small downstream vessels from damage by pressure and flow pulsatility generated by the heart during systole. This shock absorber function is impaired with age because of aortic stiffening.
METHODS AND RESULTS: We examined the contribution of common genetic variation to aortic stiffness in humans by interrogating results from the AortaGen Consortium genomeâwide association study of carotidâfemoral pulse wave velocity. Common genetic variation in the NâWASP (WASL) locus is associated with carotidâfemoral pulse wave velocity (rs600420, P=0.0051). Thus, we tested the hypothesis that decoy proteins designed to disrupt the interaction of cytoskeletal proteins such as NâWASP with its binding partners in the vascular smooth muscle cytoskeleton could decrease ex vivo stiffness of aortas from a mouse model of aging. A synthetic decoy peptide construct of NâWASP significantly reduced activated stiffness in ex vivo aortas of aged mice. Two other cytoskeletal constructs targeted to VASP and talinâvinculin interfaces similarly decreased agingâinduced ex vivo active stiffness by onâtarget specific actions. Furthermore, packaging these decoy peptides into microbubbles enables the peptides to be ultrasoundâtargeted to the wall of the proximal aorta to attenuate ex vivo active stiffness.
CONCLUSIONS: We conclude that decoy peptides targeted to vascular smooth muscle cytoskeletal proteinâprotein interfaces and microbubble packaged can decrease aortic stiffness ex vivo. Our results provide proof of concept at the ex vivo level that decoy peptides targeted to cytoskeletal proteinâprotein interfaces may lead to substantive dynamic modulation of aortic stiffness.Published versio
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Phosphorylation at in the ATP-Binding Site of /Calmodulin-Dependent Kinase II as a Mechanism for Switching off the Kinase Activity
CaMKII /calmodulin-dependent kinase II) is a serine/threonine phosphotransferase that is capable of long-term retention of activity due to autophosphorylation at a specific threonine residue within each subunit of its oligomeric structure. The isoform of CaMKII is a significant regulator of vascular contractility. Here, we show that phosphorylation of CaMKII at , a residue located within the ATP-binding site, terminates the sustained activity of the enzyme. To test the physiological importance of phosphorylation at , we generated a phosphospecific antibody and demonstrated an increase in phosphorylation upon depolarization and contraction of blood vessels. To determine if the phosphorylation of affects the kinase activity, we mutated to alanine or aspartic acid. The S26D mutation mimicking the phosphorylated state of CaMKII causes a dramatic decrease in autophosphorylation levels and greatly reduces the catalytic activity towards an exogenous substrate (autocamtide-3), whereas the S26A mutation has no effect. These data combined with molecular modelling indicate that a negative charge at of CaMKII inhibits the catalytic activity of the enzyme towards its autophosphorylation site at most probably by blocking ATP binding. We propose that phosphorylation constitutes an important mechanism for switching off CaMKII activity
Reversal of aging-induced increases in aortic stiffness by targeting cytoskeletal protein-protein interfaces
Background: The proximal aorta normally functions as a critical shock absorber that protects small downstream vessels from damage by pressure and flow pulsatility generated by the heart during systole. This shock absorber function is impaired with age because of aortic stiffening.
Methods and Results: We examined the contribution of common genetic variation to aortic stiffness in humans by interrogating results from the AortaGen Consortium genome-wide association study of carotid-femoral pulse wave velocity. Common genetic variation in the N-WASP (WASL) locus is associated with carotid-femoral pulse wave velocity (rs600420, P=0.0051). Thus, we tested the hypothesis that decoy proteins designed to disrupt the interaction of cytoskeletal proteins such as N-WASP with its binding partners in the vascular smooth muscle cytoskeleton could decrease ex vivo stiffness of aortas from a mouse model of aging. A synthetic decoy peptide construct of N-WASP significantly reduced activated stiffness in ex vivo aortas of aged mice. Two other cytoskeletal constructs targeted to VASP and talin-vinculin interfaces similarly decreased aging-induced ex vivo active stiffness by on-target specific actions. Furthermore, packaging these decoy peptides into microbubbles enables the peptides to be ultrasound-targeted to the wall of the proximal aorta to attenuate ex vivo active stiffness.
Conclusions: We conclude that decoy peptides targeted to vascular smooth muscle cytoskeletal protein-protein interfaces and microbubble packaged can decrease aortic stiffness ex vivo. Our results provide proof of concept at the ex vivo level that decoy peptides targeted to cytoskeletal protein-protein interfaces may lead to substantive dynamic modulation of aortic stiffness
ccrABEnt serine recombinase genes are widely distributed in the Enterococcus faecium and Enterococcus casseliflavus species groups and are expressed in E. faecium
The presence, distribution and expression of cassette chromosome recombinase (ccr) genes, which are homologous to the staphylococcal ccrAB genes and are designated ccrABEnt genes, were examined in enterococcal isolates (n=421) representing 13 different species. A total of 118 (28â%) isolates were positive for ccrABEnt genes by PCR, and a number of these were confirmed by Southern hybridization with a ccrAEnt probe (n=76) and partial DNA sequencing of ccrAEnt and ccrBEnt genes (n=38). ccrABEnt genes were present in Enterococcus faecium (58/216, 27â%), Enterococcus durans (31/38, 82â%), Enterococcus hirae (27/52, 50â%), Enterococcus casseliflavus (1/4, 25â%) and Enterococcus gallinarum (1/2, 50â%). In the eight other species tested, including Enterococcus faecalis (n=94), ccrABEnt genes were not found. Thirty-eight sequenced ccrABEnt genes from five different enterococcal species showed 94â100â% nucleotide sequence identity and linkage PCRs showed heterogeneity in the ccrABEnt flanking chromosomal genes. Expression analysis of ccrABEnt genes from the E. faecium DO strain showed constitutive expression as a bicistronic mRNA. The ccrABEnt mRNA levels were lower during log phase than stationary phase in relation to total mRNA. Multilocus sequence typing was performed on 39 isolates. ccrABEnt genes were detected in both hospital-related (10/29, 34â%) and non-hospital (4/10, 40â%) strains of E. faecium. Various sequence types were represented by both ccrABEnt positive and negative isolates, suggesting acquisition or loss of ccrABEnt in E. faecium. In summary, ccrABEnt genes, potentially involved in genome plasticity, are expressed in E. faecium and are widely distributed in the E. faecium and E. casseliflavus species groups
Characterization of a Vancomycin-resistant Enterococcus faecium Outbreak Caused by 2 Genetically Different Clones at a Neonatal Intensive Care Unit
In July 2010, we identified an outbreak of vancomycin-resistant enterococci (VRE) in our 26-bed neonatal intensive care unit. We performed an epidemiological investigation after clinical cultures of 2 neonates were positive for VRE. Identification, susceptibility testing, and molecular characterization were performed. Cultures of 3 surveillance stool samples of inpatients and 5 environmental samples were positive for VRE. All isolates were identified as Enterococcus faecium containing the vanA gene. Two distinct clones were identified by performing pulsed-field gel electrophoresis. The 2 clones exhibited different pulsotypes, but they represented identical Tn1546 types. Two sequence types, ST18 and ST192, were identified among all of the isolates with multilocus sequence typing. Our investigation determined that the outbreak in the neonatal intensive care unit was caused by 2 genetically different clones. The outbreak may have occurred through clonal spread and horizontal transfer of the van gene
The Work of the Course: validity and reliability in assessing English Literature
© 2017 National Association for the Teaching of English This article reflects on the values and practices of a revolutionary UK A level (senior secondary) course that achieved a high degree of validity and reliability in assessing the study of English literature. John Hodgson and Bill Greenwell were involved in its teaching and assessment from an early stage, and Greenwell's comments on an early draft of the article have been incorporated. The practice of literary response enshrined in the course was based on a striking application of âpersonal responseâ to literature, gave students opportunities to show capability in studying and writing a range of literary styles and genres, and engaged teachers regionally and nationally in a developed professional community of practice. It remains a touchstone of quality as well as of innovation in English curriculum and assessment
Immune monitoring and treatment in immune-mediated inflammatory diseases
Immune monitoring assists in the diagnosis and clinical management of immune-mediated inflammatory diseases
In patients with primary Sjögrenâs syndrome innate-like MAIT cells display upregulated IL-7R, IFN-Îł, and IL-21 expression and have increased proportions of CCR9 and CXCR5-expressing cells
Introduction: Mucosal-associated invariant T (MAIT) cells might play a role in B cell hyperactivity and local inflammation in primary Sjögrenâs syndrome (pSS), just like previously studied mucosa-associated CCR9+ and CXCR5+ T helper cells. Here, we investigated expression of CCR9, CXCR5, IL-18R and IL-7R on MAIT cells in pSS, and assessed the capacity of DMARDs to inhibit the activity of MAIT cells. Methods: Circulating CD161+ and IL-18Rα+ TCRVα7.2+ MAIT cells from pSS patients and healthy controls (HC) were assessed using flow cytometry, and expression of CCR9, CXCR5, and IL-7R on MAIT cells was studied. Production of IFN-Îł and IL-21 by MAIT cells was measured upon IL-7 stimulation in the presence of leflunomide (LEF) and hydroxychloroquine (HCQ). Results: The numbers of CD161+ and IL-18Rα+ MAIT cells were decreased in pSS patients compared to HC. Relative increased percentages of CD4 MAIT cells in pSS patients caused significantly higher CD4/CD8 ratios in MAIT cells. The numbers of CCR9 and CXCR5-expressing MAIT cells were significantly higher in pSS patients. IL-7R expression was higher in CD8 MAIT cells as compared to all CD8 T cells, and changes in IL-7R expression correlated to several clinical parameters. The elevated production of IL-21 by MAIT cells was significantly inhibited by LEF/HCQ treatment. Conclusion: Circulating CD161+ and IL-18Rα+ MAIT cell numbers are decreased in pSS patients. Given their enriched CCR9/CXCR5 expression this may facilitate migration to inflamed salivary glands known to overexpress CCL25/CXCL13. Given the pivotal role of IL-7 and IL-21 in inflammation in pSS this indicates a potential role for MAIT cells in driving pSS immunopathology
Evolutionary origins of the emergent ST796 clone of vancomycin resistant Enterococcus faecium
From early 2012, a novel clone of vancomycin resistant Enterococcus faecium (assigned
the multi locus sequence type ST796) was simultaneously isolated from geographically
separate hospitals in south eastern Australia and New Zealand. Here we describe the
complete genome sequence of Ef_aus0233, a representative ST796 E. faecium isolate.
We used PacBio single molecule real-time sequencing to establish a high quality,
fully assembled genome comprising a circular chromosome of 2,888,087 bp and five
plasmids. Comparison of Ef_aus0233 to other E. faecium genomes shows Ef_aus0233
is a member of the epidemic hospital-adapted lineage and has evolved from an ST555-
like ancestral progenitor by the accumulation or modification of five mosaic plasmids
and five putative prophage, acquisition of two cryptic genomic islands, accrued
chromosomal single nucleotide polymorphisms and a 80 kb region of recombination,
also gaining Tn1549 and Tn916, transposons conferring resistance to vancomycin and
tetracycline respectively. The genomic dissection of this new clone presented here
underscores the propensity of the hospital E. faecium lineage to change, presumably
in response to the specific conditions of hospital and healthcare environments
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