Characterizing left atrial appendage functions in sinus rhythm and atrial fibrillation using computational models

Clinical studies show that the left atrial appendage, a blind-ended structure that is attached to the left atrium, may be the cause of 90% of atrial thrombi in atrial fibrillation (abnormal heart rhythm), and it is much reduced in sinus (normal) rhythm. In this paper, the effects of blood flows in left atrium and left atrial appendage are studied to help characterize the atrial appendage functions in sinus rhythm and atrial fibrillation using mathematical models. Our results show that the left atrial appendage is not functional in sinus rhythm because the atrial transmitral velocities remained almost identical for atria with and without appendage, which agrees with the current clinical observations. However, in atrial fibrillation, a proper atrial contraction is absent, which causes the second emptying velocity (A-wave) to be missing in both transmitral velocity and appendage filling/emptying velocity. Without the proper emptying of the blood, vortices generated in the chamber remain high strengths and with longer durations. They induce ineffective emptying of the blood in the atrium and appendage, which then lead to blood stagnation and subsequent thrombus formation.     

Ghrelin induces proliferation in human aortic endothelial cells via ERK1/2 and PI3K/Akt activation

The direct ghrelin (Ghr) involvement in cardiovascular (CV) system homeostasis has been suggested by the expression of its receptor in CV tissues and by evidence that ghrelin mediates CV activities in animals and in humans. Moreover, low Ghr plasma levels have been reported in pathological conditions characterized by high cardiovascular risk. In the present study, we investigated Ghr effect on proliferation of human aortic endothelial cell (HAEC) and involved transduction pathways. Our results indicate that ghrelin elicited proliferation in a dose-dependent manner (EC(50) about of 5nmol/L) in cultured HAEC, and that this effect was inhibited by the receptor antagonist (D-Lys3)-GHRP-6. Western blot experiments documented an activation of external receptor activated kinases (ERK1/2) and Akt in a dose-dependent fashion, as well as involvement of the cAMP pathway in ERK1/2 phosphorylation. Experiments conducted with appropriate pharmacological inhibitors to investigate Ghr-induced HAEC proliferation confirmed the involvement of ERK1/2 and I3P/Akt pathways, as well as the role of AMP cyclase/PKA pathway in ERK1/2 phosphorylation. Our results indicate that Ghr promotes HAEC proliferation, and thus may be a protective factor against vascular damage. The low ghrelin serum levels reported in insulin-resistant states may not be able to effectively counteract endothelial cell injury.     

Chitosan influence on glucose and calcium availability from yogurt: In vitro comparative study with plants fibre

Since chitosan complies with the definition of dietary fibre is necessary to study the interaction of this biopolymer with nutrients. Yogurt with fortified chitosan and different types of plants fibres like wheat, bamboo, apple, psyllium and inulin was used as a food model. The availabilities of glucose and calcium in this model were studied by an in vitro gastrointestinal tract simulation. Results showed that the different fibres decreased both glucose and calcium availabilities whereas the effect of chitosan was more pronounced. (17.7 ± 2.1% and 21.0 ± 2.5% depress respectively). This work demonstrated that the addition of chitosan to yogurts influences the availability of nutrients.     

Towards a new attenuating compound: a potentiometric, spectrophotometric and NMR equilibrium study on Fe(III), Al(III) and a new tetradentate mixed bisphosphonate-hydroxypyridinonate ligand

Coordination properties toward Fe(III) and Al(III) of a mixed bisphosphonate-hydroxypyridinonate ligand are presented. Potentiometric, spectrophotometric and NMR results allowed to conclude that Fe(III) and Al(III) coordination takes place on the pyridinone moiety. The high steric hindrance prevents the possibility of simultaneous coordination of both groups to the same metal ion. Quantum mechanical calculations confirm this finding allowing to determine the minimal length of the linker necessary for a stable conformation of complexes in which Fe(III) is coordinated both by pyridinone and bisphosphonate groups.     

Chromosomal gfp labelling of Pseudomonas aeruginosa using a mini-Tn7 transposon: application for studies of bacteria-host interactions

Analysis of bacterial interactions with host cells using multiple techniques is essential for studies on microbial pathogenesis and for the development of new antimicrobial therapies. Pseudomonas aeruginosa is an important opportunistic pathogen that can cause severe, often life-threatening pulmonary infections in individuals with impaired host defense mechanisms. Using a mini-Tn7 transposon delivery system, we have chromosomally labelled the strain P. aeruginosa PAK with a green fluorescent protein gene (gfp) and tested PAKgfp as a research tool for studies of bacteria-host interactions. We were able to reliably and rapidly measure the interactions of PAKgfp with A549 human lung epithelial cells by using flow cytometry, a fluorometric microplate reader-based assay, and fluorescence microscopy. With these analytical tools, we have demonstrated the adhesion of PAKgfp to the extracellular matrix protein fibronectin and the involvement of fibronectin in PAKgfp-A549 cell interactions. PAKgfp can be successfully used to explore the effects of various pharmacological compounds on P. aeruginosa - host cell interactions in both in vitro and in vivo systems, with potentially important medical applications.     

Unscrambling thermal stability and temperature adaptation in evolved variants of a cold-active lipase

Directed evolution by error-prone PCR was applied to stabilize the cold-active lipase from Pseudomonas fragi (PFL). PFL displays high activity at 10 degrees C, but it is highly unstable even at moderate temperatures. After two rounds of evolution, a variant was generated with a 5-fold increase in half-life at 42 degrees C and a shift of 10 degrees C in the temperature optimum, nevertheless retaining cold-activity. The evolved lipase displayed specific activity higher than the wild type enzyme in the temperature range 29-42 degrees C. Biophysical measurements did not indicate any obvious difference between the improved variant and the wild type enzyme in terms of loss of secondary structure upon heat treatment, nor a shift in the apparent melting temperature.     

A novel family of sequence-specific endoribonucleases associated with the clustered regularly interspaced short palindromic repeats

Clustered regularly interspaced short palindromic repeats (CRISPRs) together with the associated CAS proteins protect microbial cells from invasion by foreign genetic elements using presently unknown molecular mechanisms. All CRISPR systems contain proteins of the CAS2 family, suggesting that these uncharacterized proteins play a central role in this process. Here we show that the CAS2 proteins represent a novel family of endoribonucleases. Six purified CAS2 proteins from diverse organisms cleaved single-stranded RNAs preferentially within U-rich regions. A representative CAS2 enzyme, SSO1404 from Sulfolobus solfataricus, cleaved the phosphodiester linkage on the 3'-side and generated 5'-phosphate- and 3'-hydroxyl-terminated oligonucleotides. The crystal structure of SSO1404 was solved at 1.6A resolution revealing the first ribonuclease with a ferredoxin-like fold. Mutagenesis of SSO1404 identified six residues (Tyr-9, Asp-10, Arg-17, Arg-19, Arg-31, and Phe-37) that are important for enzymatic activity and suggested that Asp-10 might be the principal catalytic residue. Thus, CAS2 proteins are sequence-specific endoribonucleases, and we propose that their role in the CRISPR-mediated anti-phage defense might involve degradation of phage or cellular mRNAs.     

Salts enhance both protein stability and amyloid formation of an immunoglobulin light chain

Amyloid fibrils are associated with sulfated glycosaminoglycans in the extracellular matrix. The presence of sulfated glycosaminoglycans is known to promote amyloid formation in vitro and in vivo, with the sulfate groups playing a role in this process. In order to understand the role that sulfate plays in amyloid formation, we have studied the effect of salts from the Hofmeister series on the protein structure, stability and amyloid formation of an amyloidogenic light chain protein, AL-12. We have been able to show for the first time a direct correlation between protein stability and amyloid formation enhancement by salts from the Hofmeister series, where SO₄²⁻ conferred the most protein stability and enhancement of amyloid formation. Our study emphasizes the importance of the effect of ions in the protein bound water properties and downplays the role of specific interactions between the protein and ions.     

Inhibition of myoblast differentiation by Sfrp1 and Sfrp2

Secreted Frizzled-related proteins (Sfrps) are extracellular regulators of Wnt signalling and play important roles in developmental and oncogenic processes. They are known to be upregulated in regenerating muscle and in myoblast cultures but their function is unknown. Here, we show that the addition of recombinant Sfrp1 or Sfrp2 to C2C12 cell line cultures or to primary cultures of satellite cells results in the inhibition of myotube formation with no significant effect on the cell cycle or apoptosis. Even though at confluence, treated and untreated cultures are identical in appearance, analyses have shown that, for maximum effect, the cells have to be treated while they are proliferating. Furthermore, removal of Sfrp from the culture medium during differentiation restores normal myotube formation. We conclude that Sfrp1 and Sfrp2 act to prevent myoblasts from entering the terminal differentiation process. S. Descamps and J. Levin contributed equally to this work.