Acute podocyte vascular endothelial growth factor (VEGF-A) knockdown disrupts alphaVbeta3 integrin signaling in the glomerulus

Podocyte or endothelial cell VEGF-A knockout causes thrombotic microangiopathy in adult mice. To study the mechanism involved in acute and local injury caused by low podocyte VEGF-A we developed an inducible, podocyte-specific VEGF-A knockdown mouse, and we generated an immortalized podocyte cell line (VEGF(KD)) that downregulates VEGF-A upon doxycycline exposure. Tet-O-siVEGF:podocin-rtTA mice express VEGF shRNA in podocytes in a doxycycline-regulated manner, decreasing VEGF-A mRNA and VEGF-A protein levels in isolated glomeruli to ~20% of non-induced controls and urine VEGF-A to ~30% of control values a week after doxycycline induction. Induced tet-O-siVEGF:podocin-rtTA mice developed acute renal failure and proteinuria, associated with mesangiolysis and microaneurisms. Glomerular ultrastructure revealed endothelial cell swelling, GBM lamination and podocyte effacement. VEGF knockdown decreased podocyte fibronectin and glomerular endothelial alpha(V)beta(3) integrin in vivo. VEGF receptor-2 (VEGFR2) interacts with beta(3) integrin and neuropilin-1 in the kidney in vivo and in VEGF(KD) podocytes. Podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activation in glomeruli, detected by WOW1-Fab. VEGF silencing in cultured VEGF(KD) podocytes downregulates fibronectin and disrupts alpha(V)beta(3) integrin activation cell-autonomously. Collectively, these studies indicate that podocyte VEGF-A regulates alpha(V)beta(3) integrin signaling in the glomerulus, and that podocyte VEGF knockdown disrupts alpha(V)beta(3) integrin activity via decreased VEGFR2 signaling, thereby damaging the three layers of the glomerular filtration barrier, causing proteinuria and acute renal failure.     

Uncovering the origin of the black stains in Lascaux Cave in France

Lascaux Cave in France was discovered in 1940. Since being opened to visitors the cave has suffered three major microbial outbreaks. The current problem is the fast dissemination of black stains which are threatening the Palaeolithic paintings. Previous data pointed to the involvement of new fungal species in the formation of black stains on the rock walls and ceiling. However, it appears that there could be other reasons for the formation of different and extensive black stains coating the surface of the clayey sediments. Our analyses reveal that black stains on clayey sediments are mainly produced by Acremonium nepalense, a manganese oxide‐depositing fungus, widely distributed in the cave. Thus, in Lascaux Cave, the black stains have a dual origin: on limestone rocks they are mainly produced by the accumulation of fungal melanins, and on clayey sediments by the biogenic deposition of black manganese oxides.     

Molecular and Electrophysiological Characterization of a Novel Cation Channel of Trypanosoma cruzi

We report the identification, functional expression, purification, reconstitution and electrophysiological characterization of a novel cation channel (TcCat) from Trypanosoma cruzi, the etiologic agent of Chagas disease. This channel is potassium permeable and shows inward rectification in the presence of magnesium. Western blot analyses with specific antibodies indicated that the protein is expressed in the three main life cycle stages of the parasite. Surprisingly, the parasites have the unprecedented ability to rapidly change the localization of the channel when they are exposed to different environmental stresses. TcCat rapidly translocates to the tip of the flagellum when trypomastigotes are submitted to acidic pH, to the plasma membrane when epimastigotes are submitted to hyperosmotic stress, and to the cell surface when amastigotes are released to the extracellular medium. Pharmacological block of TcCat activity also resulted in alterations in the trypomastigotes ability to respond to hyperosmotic stress. We also demonstrate the feasibility of purifying and reconstituting a functional ion channel from T. cruzi after recombinant expression in bacteria. The peculiar characteristics of TcCat could be important for the development of specific inhibitors with therapeutic potential against trypanosomes.     

Wall stress and flow dynamics in abdominal aortic aneurysms: finite element analysis vs. fluid-structure interaction

Abdominal aortic aneurysm (AAA) rupture is the clinical manifestation of an induced force exceeding the resistance provided by the strength of the arterial wall. This force is most frequently assumed to be the product of a uniform luminal pressure acting along the diseased wall. However fluid dynamics is a known contributor to the pathogenesis of AAAs, and the dynamic interaction of blood flow and the arterial wall represents the in vivo environment at the macro-scale. The primary objective of this investigation is to assess the significance of assuming an arbitrary estimated peak fluid pressure inside the aneurysm sac for the evaluation of AAA wall mechanics, as compared with the non-uniform pressure resulting from a coupled fluid-structure interaction (FSI) analysis. In addition, a finite element approach is utilised to estimate the effects of asymmetry and wall thickness on the wall stress and fluid dynamics of ten idealised AAA models and one non-aneurysmal control. Five degrees of asymmetry with uniform and variable wall thickness are used. Each was modelled under a static pressure-deformation analysis, as well as a transient FSI. The results show that the inclusion of fluid flow yields a maximum AAA wall stress up to 20% higher compared to that obtained with a static wall stress analysis with an assumed peak luminal pressure of 117 mmHg. The variable wall models have a maximum wall stress nearly four times that of a uniform wall thickness, and also increasing with asymmetry in both instances. The inclusion of an axial stretch and external pressure to the computational domain decreases the wall stress by 17%.     

Dynamics of initial colonization of nonconserved perennial ryegrass by anaerobic fungi in the bovine rumen

Anaerobic fungi (Neocallimastigales) are active degraders of fibrous plant material in the rumen. However, only limited information is available relating to how quickly they colonize ingested feed particles. The aim of this study was to determine the dynamics of initial colonization of forage by anaerobic fungi in the rumen and the impact of different postsampling wash procedures used to remove loosely associated microorganisms. Neocallimastigales-specific molecular techniques were optimized to ensure maximal coverage before application to assess the population size (quantitative PCR) and composition (automated ribosomal intergenic spacer analysis) of the colonizing anaerobic fungi. Colonization of perennial ryegrass (PRG) was evident within 5 min, with no consistent effect of time or wash procedure on fungal population composition. Wash procedure had no effect on population size unlike time, which had a significant effect. Colonizing fungal population size continued to increase over the incubation period after an initial lag of c. 4 min. This dynamic differs from that reported previously for rumen bacteria, where substantial colonization of PRG occurred within 5 min. The observed delay in colonization of plant material by anaerobic fungi is suggested to be primarily mediated by the time taken for fungal zoospores to locate, attach and encyst on plant material.     

Expression of uncoupling protein-3 in subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its modulation by fasting.

Uncoupling protein-3 (UCP3) is a mitochondrial inner-membrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differentially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully validated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxidative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mitochondria. Taken together, these results suggest that oxidative muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24-h fast, which greatly increases UCP3 mRNA (16.4-fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5-fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes.     

A new role for the p85-phosphatidylinositol 3-kinase regulatory subunit linking FRAP to p70 S6 kinase activation.

The serine/threonine kinase p70 S6 kinase (p70S6K) phosphorylates the 40 S ribosomal protein S6, modulating the translation of an mRNA subset that encodes ribosomal proteins and translation elongation factors. p70S6K is activated in response to mitogenic stimuli and is required for progression through the G(1) phase of the cell cycle and for cell growth. Activation of p70S6K is regulated by phosphorylation of seven different residues distributed throughout the protein, a subset of which depends on the activity of p85/p110 phosphatidylinositol 3-kinase (PI3K); in fact, the phosphorylation status of Thr(229) and Thr(389) is intimately linked to PI3K activity. In the full-length enzyme, however, these sites are also acutely sensitive to the action of FKBP 12-rapamycin-associated protein (FRAP). The mechanism by which PI3K and FRAP cooperate to induce p70S6K activation remains unclear. Here we show that the p85 regulatory subunit of PI3K also controls p70S6K activation by mediating formation of a ternary complex with p70S6K and FRAP. The p85 C-terminal SH2 domain is responsible for p85 coupling to p70S6K and FRAP, because deletion of the C-terminal SH2 domain inhibits complex formation and impairs p70S6K activation by PI3K. Formation of this complex is not required for activation of a FRAP-independent form of p70S6K, however, underscoring the role of p85 in regulating FRAP-dependent p70S6K activation. These studies thus show that, in addition to the contribution of PI3K activity, the p85 regulatory subunit plays a critical role in p70S6K activation.