Structural Features of Single-Stranded Integron Cassette attC Sites and Their Role in Strand Selection

We recently showed that cassette integration and deletion in integron platforms were occurring through unconventional site-specific recombination reactions involving only the bottom strand of attC sites. The lack of sequence conservation among attC sites led us to hypothesize that sequence-independent structural recognition determinants must exist within attC sites. The structural data obtained from a synaptic complex of the Vibrio cholerae integrase with the bottom strand of an attC site has shown the importance of extra helical bases (EHB) inside the stem-loop structure formed from the bottom strand. Here, we systematically determined the contribution of three structural elements common to all known single-stranded attC site recombination substrates (the EHBs, the unpaired central spacer (UCS), and the variable terminal structure (VTS)) to strand choice and recombination. Their roles have been evaluated in vivo in the attl x attC reaction context using the suicide conjugation assay we previously developed, but also in an attC x attC reaction using a deletion assay. Conjugation was used to deliver the attC sites in single-stranded form. Our results show that strand choice is primarily directed by the first EHB, but the presence of the two other EHBs also serves to increase this strand selection. We found that the structure of the central spacer is essential to achieve high level recombination of the bottom strand, suggesting a dual role for this structure in active site exclusion and for hindering the reverse reaction after the first strand exchange. Moreover, we have shown that the VTS has apparently no role in strand selectivity

ACE2 gene expression is up-regulated in the human failing heart

BACKGROUND: ACE2 is a novel homologue of angiotensin converting enzyme (ACE). ACE2 is highly expressed in human heart and animal data suggest that ACE2 is an essential regulator of cardiac function in vivo. Since overactivity of the renin-angiotensin system contributes to the progression of heart failure, this investigation assessed changes in gene expression of ACE2, ACE, AT(1 )receptor and renin in the human failing heart. METHODS: The sensitive technique of quantitative reverse transcriptase polymerase chain reaction was used to determine the level of mRNA expression of ACE and ACE2 in human ventricular myocardium from donors with non-diseased hearts (n = 9), idiopathic dilated cardiomyopathy (IDC, n = 11) and ischemic cardiomyopathy (ICM, n = 12). Following logarithmic transformation of the data, a one-way analysis of variance was performed for each target gene followed by a Dunnett's test to compare the two disease groups IDC and ICM versus control. RESULTS: As anticipated, ACE mRNA was found to be significantly increased in the failing heart with a 3.1 and 2.4-fold up-regulation found in IDC and ICM relative to non-diseased myocardium. Expression of ACE2 mRNA was also significantly up-regulated in IDC (2.4-fold increase) and ICM (1.8-fold increase) versus non-diseased myocardium. No change in angiotensin AT(1 )receptor mRNA expression was found in failing myocardium and renin mRNA was not detected. CONCLUSIONS: These data suggest that ACE2 is up-regulated in human IDC and ICM and are consistent with the hypothesis that differential regulation of this enzyme may have important functional consequences in heart failure. This strengthens the hypothesis that ACE2 may be a relevant target for the treatment of heart failure and will hopefully spur further studies to clarify the functional effects in human myocardium of ACE2 derived peptides

Mechanical stretch and shear flow induced reorganization and recruitment of fibronectin in fibroblasts

It was our objective to study the role of mechanical stimulation on fibronectin (FN) reorganization and recruitment by exposing fibroblasts to shear fluid flow and equibiaxial stretch. Mechanical stimulation was also combined with a Rho inhibitor to probe their coupled effects on FN. Mechanically stimulated cells revealed a localization of FN around the cell periphery as well as an increase in FN fibril formation. Mechanical stimulation coupled with chemical stimulation also revealed an increase in FN fibrils around the cell periphery. Complimentary to this, fibroblasts exposed to fluid shear stress structurally rearranged pre-coated surface FN, but unstimulated and stretched cells did not. These results show that mechanical stimulation directly affected FN reorganization and recruitment, despite perturbation by chemical stimulation. Our findings will help elucidate the mechanisms of FN biosynthesis and organization by furthering the link of the role of mechanics with FN

Angiotensin-(1–7) and the G Protein-Coupled Receptor Mas Are Key Players in Renal Inflammation

Angiotensin (Ang) II mediates pathophysiologial changes in the kidney. Ang-(1–7) by interacting with the G protein-coupled receptor Mas may also have important biological activities

Hyperglycemia and Diabetes Downregulate the Functional Expression of TRPV4 Channels in Retinal Microvascular Endothelium

Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca2+]i in RMECs and this response was largely abolished using siRNA targeted against TRPV4. These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25 mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months' streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy

A target specificity switch in IS911 transposition: the role of the OrfA protein

The role played by insertion sequence IS911 proteins, OrfA and OrfAB, in the choice of a target for insertion was studied. IS911 transposition occurs in several steps: synapsis of the two transposon ends (IRR and IRL); formation of a figure-of-eight intermediate where both ends are joined by a single-strand bridge; resolution into a circular form carrying an IRR–IRL junction; and insertion into a DNA target. In vivo, with OrfAB alone, an IS911-based transposon integrated with high probability next to an IS911 end located on the target plasmid. OrfA greatly reduced the proportion of these events. This was confirmed in vitro using a transposon with a preformed IRR–IRL junction to examine the final insertion step. Addition of OrfA resulted in a large increase in insertion frequency and greatly increased the proportion of non-targeted insertions. The intermolecular reaction leading to targeted insertion may resemble the intramolecular reaction involving figure-of-eight molecules, which leads to the formation of circles. OrfA could, therefore, be considered as a molecular switch modulating the site-specific recombination activity of OrfAB and facilitating dispersion of the insertion sequence (IS) to ‘non-homologous’ target sites

Bias between the Left and Right Inverted Repeats during IS911 Targeted Insertion ▿

IS911 is a bacterial insertion sequence composed of two consecutive overlapping open reading frames (ORFs [orfA and orfB]) encoding the transposase (OrfAB) as well as a regulatory protein (OrfA). These ORFs are bordered by terminal left and right inverted repeats (IRL and IRR, respectively) with several differences in nucleotide sequence. IS911 transposition is asymmetric: each end is cleaved on one strand to generate a free 3′-OH, which is then used as the nucleophile in attacking the opposite insertion sequence (IS) end to generate a free IS circle. This will be inserted into a new target site. We show here that the ends exhibit functional differences which, in vivo, may favor the use of one compared to the other during transposition. Electromobility shift assays showed that a truncated form of the transposase [OrfAB(1-149)] exhibits higher affinity for IRR than for IRL. While there was no detectable difference in IR activities during the early steps of transposition, IRR was more efficient during the final insertion steps. We show here that the differential activities between the two IRs correlate with the different affinities of OrfAB(1-149) for the IRs during assembly of the nucleoprotein complexes leading to transposition. We conclude that the two inverted repeats are not equivalent during IS911 transposition and that this asymmetry may intervene to determine the ordered assembly of the different protein-DNA complexes involved in the reaction

Adaptation to global change : a transposable element-epigenetics perspective

Understanding how organisms cope with global change is a major scientific challenge. The molecular pathways underlying rapid adaptive phenotypic responses to global change remain poorly understood. Here, we highlight the relevance of two environment-sensitive molecular elements: transposable elements (TEs) and epigenetic components (ECs). We first outline the sensitivity of these elements to global change stressors and review how they interact with each other. We then propose an integrative molecular engine coupling TEs and ECs and allowing organisms to fine-tune phenotypes in a real-time fashion, adjust the production of phenotypic and genetic variation, and produce heritable phenotypes with different levels of transmission fidelity. We finally discuss the implications of this molecular engine in the context of global change

Elucidating the spatio-temporal dynamics of an emerging wildlife pathogen using approximate Bayesian computation

Emerging pathogens constitute a severe threat for human health and biodiversity. Determining the status (native or non-native) of emerging pathogens, and tracing back their spatio-temporal dynamics, is crucial to understand the eco-evolutionary factors promoting their emergence, to control their spread and mitigate their impacts. However, tracing back the spatio-temporal dynamics of emerging wildlife pathogens is challenging because (i) they are often neglected until they become sufficiently abundant and pose socio-economical concerns and (ii) their geographical range is often little known. Here, we combined classical population genetics tools and approximate Bayesian computation (i.e. ABC) to retrace the dynamics of Tracheliastes polycolpus, a poorly documented pathogenic ectoparasite emerging in Western Europe that threatens several freshwater fish species. Our results strongly suggest that populations of T. polycolpus in France emerged from individuals originating from a unique genetic pool that were most likely introduced in the 1920s in central France. From this initial population, three waves of colonization occurred into peripheral watersheds within the next two decades. We further demonstrated that populations remained at low densities, and hence undetectable, during 10 years before a major demographic expansion occurred, and before its official detection in France. These findings corroborate and expand the few historical records available for this emerging pathogen. More generally, our study demonstrates how ABC can be used to determine the status, reconstruct the colonization history and infer key evolutionary parameters of emerging wildlife pathogens with low data availability, and for which samples from the putative native area are inaccessible