Chronic kidney disease induced by an adenine rich diet upregulates integrin linked kinase (ILK) and its depletion prevents the disease progression

Kidney fibrosis is one of the main pathological findings of progressive chronic kidney disease (CKD) although the pathogenesis of renal scar formation remains incompletely explained. Integrin-linked kinase (ILK), a major scaffold protein between the extracellular matrix (ECM) and intracellular signaling pathways, is involved in several pathophysiological processes during renal damage. However, ILK contribution in the CKD progress remains to be fully elucidated. In the present work, we studied 1) the renal functional and structural consequences of CKD genesis and progression when ILK is depleted and 2) the potential of ILK depletion as a therapeutic approach to delay CKD progression. We induced an experimental CKD model, based on an adenine-supplemented diet on adult wild-type (WT) and ILK-depleted mice, with a tubulointerstitial damage profile resembling that is observed in human CKD. The adenine diet induced in WT mice a progressive increase in plasma creatinine and urea concentrations. In the renal cortex it was also observed tubular damage, interstitial fibrosis and progressive increased ECM components, pro-inflammatory and chemo-attractant cytokines, EMT markers and TGF-β1 expressions. These observations were highly correlated to a simultaneous increase of ILK expression and activity. In adenine-fed transgenic ILK-depleted mice, all these changes were prevented. Additionally, we evaluated the potential role of ILK depletion to be applied after the disease induction, as an effective approach to interventions in human CKD subjects. In this scenario, two weeks after the establishment of adenine-induced CKD, ILK was abrogated in WT mice and stabilized renal damage, avoiding CKD progression. We propose ILK to be a potential target to delay renal disease progression.     

N-Naphthoyl-substituted indole thio-barbituric acid analogs inhibit the helicase activity of the hepatitis C virus NS3

The hepatitis C virus (HCV) represents a substantial threat to human health worldwide. The virus expresses a dual-function protein, NS3 having both protease and RNA helicase activities that are essential for productive viral replication and sustained infections. While viral protease and polymerase inhibitors have shown great successes in treating chronic HCV infections, drugs that specifically target the helicase activity have not advanced. A robust and quantitative 96-well plate-based fluorescent DNA unwinding assay was used to screen a class of indole thio-barbituric acid (ITBA) analogs using the full-length, recombinant HCV NS3, and identified three naphthoyl-containing analogs that efficiently inhibited NS3 helicase activity in a dose-dependent manner, with observed IC₅₀ values of 21–24?µM. Standard gel electrophoresis helicase assays using radiolabeled duplex DNA and RNA NS3 substrates confirmed the inhibition of NS3 unwinding activity. Subsequent anisotropy measurements demonstrated that the candidate compounds did not disrupt NS3 binding to nucleic acids. Additionally, the rate of ATP hydrolysis and the protease activity were also not affected by the inhibitors. Thus, these results indicate that the three ITBA analogs containing N-naphthoyl moieties are the foundation of a potential series of small molecules capable of inhibiting NS3 activity via a novel interaction with the helicase domain that prevents the productive unwinding of nucleic acid substrates, and may represent the basis for a new class of therapeutic agents with the potential to aid in the treatment and eradication of hepatitis C virus.     

Spitzenkorper localization and intracellular traffic of green fluorescent protein-labeled CHS-3 and CHS-6 chitin synthases in living hyphae of Neurospora crassa

The subcellular location and traffic of two selected chitin synthases (CHS) from Neurospora crassa, CHS-3 and CHS-6, labeled with green fluorescent protein (GFP), were studied by high-resolution confocal laser scanning microscopy. While we found some differences in the overall distribution patterns and appearances of CHS-3-GFP and CHS-6-GFP, most features were similar and were observed consistently. At the hyphal apex, fluorescence congregated into a conspicuous single body corresponding to the location of the Spitzenkörper (Spk). In distal regions (beyond 40 μm from the apex), CHS-GFP revealed a network of large endomembranous compartments that was predominantly comprised of irregular tubular shapes, while some compartments were distinctly spherical. In the distal subapex (20 to 40 μm from the apex), fluorescence was observed in globular bodies that appeared to disintegrate into vesicles as they advanced forward until reaching the proximal subapex (5 to 20 μm from the apex). CHS-GFP was also conspicuously found delineating developing septa. Analysis of fluorescence recovery after photobleaching suggested that the fluorescence of the Spk originated from the advancing population of microvesicles (chitosomes) in the subapex. The inability of brefeldin A to interfere with the traffic of CHS-containing microvesicles and the lack of colocalization of CHS-GFP with the endoplasmic reticulum (ER)-Golgi body fluorescent dyes lend support to the idea that CHS proteins are delivered to the cell surface via an alternative route distinct from the classical ER-Golgi body secretory pathway.Fungal hyphae elongate and branch by a complex process based on polarized secretion. Many studies have investigated the cellular and molecular components involved in shaping fungal cells, but no detailed understanding of the mechanisms that govern and regulate polarized fungal growth has been achieved (4, 25). In the yeast Saccharomyces cerevisiae, many of the main components of the secretory pathway, including some of the enzymes involved in cell wall formation, have been extensively characterized (32). Filamentous fungi encode homologues of some key components known from the yeast secretory pathway, but despite their apparent orthology, relatively little is known about how this pathway is organized to accomplish the highly polarized growth typical of hyphae. There are some differences in cell wall synthesis between filamentous fungi and S. cerevisiae. In hyphae of septate fungi, vesicles and other components accumulate at the apex, as part of the Spitzenkörper (Spk) (14, 22-24, 28). The composition and mode of action of this pleomorphic and dynamic structure have intrigued fungal biologists for many decades.Fungal cells have at least two types of well-defined secretory vesicles (5). It has been suggested that macrovesicles, or conventional secretory vesicles, carry the components of the amorphous phase of the cell wall, in addition to the load of extracellular enzymes (5, 27). There is a large body of evidence characterizing the chitin synthase (CHS)-carrying microvesicles as chitosomes (3, 8, 13, 30). CHS are β-glycosyltransferases that catalyze the polymerization of N-acetylglucosamine from UDP N-acetylglucosamine into chitin (47), a major structural polymer of the fungal cell wall (2). Chitin synthesis occurs in highly localized fashion both at the hyphal apices (7) and at nascent septa (29). Chitosomes are the smallest vesicles with the ability to form chitin microfibrils in vitro and have been suggested to carry and transport CHS to the cell surface at the apex of hyphae for cell wall synthesis (13, 37, 48, 55, 56). In recent years, studies on fungal CHS have concentrated mainly on gene identification. Given this wealth of information, we chose CHS as candidate markers to investigate vesicle traffic in fungal hyphae.Fungi have multiple chs genes grouped into two divisions, with seven classes, primarily on the basis of similarities in the primary sequence of the predicted proteins (12, 16, 37, 50). Division I includes classes I, II, and III, which share a catalytic domain surrounded by a hydrophilic N-terminal region and a hydrophobic C-terminal region (12). Division II includes classes IV, V, and VII, all with a catalytic domain preceded by a cytochrome b₅-like domain. In addition, classes V and VII contain an N-terminal myosin motor-like domain, suggesting a direct interaction with the actin cytoskeleton (15, 20, 58). Class VI has not been assigned to either division and includes recently identified CHS of unknown function (16). Earlier studies suggest that the various CHS have specific roles in chitin cell wall synthesis that are time or space dependent (60). In contrast to most filamentous fungi, S. cerevisiae (46) and Candida albicans (40) have only three or four CHS isozymes, respectively. S. cerevisiae Chs1p, C. albicans Chs2p, and C. albicans Chs8p belong to class I; S. cerevisiae Chs2p and C. albicans Chs1p belong to class II; and S. cerevisiae Chs3p and C. albicans Chs3p belong to class IV (46). While potential roles in hyphal growth have been suggested for some of the seven CHS classes described in filamentous fungi (9, 64, 65), we lack specific information on the cellular localization and trafficking to their sites of action in regions of active cell wall growth for most of these proteins.The goal of this study was to elucidate the traffic of CHS-containing vesicles en route from their site of genesis to their site of exocytosis in living hyphae of Neurospora crassa. The availability of an almost-complete genome sequence for this fungus allowed the identification of seven open reading frames with high homology to previously described chs genes (10). We chose to trace the intracellular location and secretory paths of CHS-3 and CHS-6. Neurospora CHS-3 belongs to the previously reported class I CHS with known homologues in all fungi tested, including S. cerevisiae Chs1p. In contrast, CHS-6 is a newly identified CHS assigned to class VI, homologous to Aspergillus fumigatus ChsD (39) and Coccidioides posadasii CHS-6 (34) but with no apparent homologues in S. cerevisiae or C. albicans. To trace both proteins, we fused green fluorescent protein (GFP) to the carboxyl terminus of the CHS coding regions and analyzed the fate of the resulting CHS-3-GFP and CHS-6-GFP fusion proteins by high-resolution confocal laser scanning microscopy (CLSM) in living hyphae of N. crassa.     

Spontaneous altruism by chimpanzees and young children

People often act on behalf of others. They do so without immediate personal gain, at cost to themselves, and even toward unfamiliar individuals. Many researchers have claimed that such altruism emanates from a species-unique psychology not found in humans' closest living evolutionary relatives, such as the chimpanzee. In favor of this view, the few experimental studies on altruism in chimpanzees have produced mostly negative results. In contrast, we report experimental evidence that chimpanzees perform basic forms of helping in the absence of rewards spontaneously and repeatedly toward humans and conspecifics. In two comparative studies, semi–free ranging chimpanzees helped an unfamiliar human to the same degree as did human infants, irrespective of being rewarded (experiment 1) or whether the helping was costly (experiment 2). In a third study, chimpanzees helped an unrelated conspecific gain access to food in a novel situation that required subjects to use a newly acquired skill on behalf of another individual. These results indicate that chimpanzees share crucial aspects of altruism with humans, suggesting that the roots of human altruism may go deeper than previous experimental evidence suggested.     

Plasmodium falciparum-infected mice: more than a tour de force

Up until recently, the relevance of Plasmodium falciparum-infected humanized mice for malaria studies has been questioned because of the low percentage of mice in which the parasite develops. Advances in the generation of new immunodeficient mouse strains combined with the use of protocols that modulate the innate immune defenses of mice have facilitated the harvesting of exoerythrocytic and intraerythrocytic stages of the parasite. These results renew the hope of working with P. falciparum in a laboratory animal and indicate that the next challenge (i.e. a complete parasite cycle in the same mouse, including transmission to mosquito) could be reached in the future.     

Identification and susceptibility against fluconazole and albaconazole of 100 yeasts' strains isolated from vaginal discharge

Vulvovaginal candidiasis is a condition that affects a great number of fertile women. It is considered the second cause of genital infection after vaginosis due to GAM complex. Candida albicans is the most frequent isolated species from vaginal discharge. However, sometimes more than one yeast species could be found in the same clinical sample that are more resistant to antifungal drugs. Nowadays, it is necessary to identify properly up to species level the isolated microorganism and to determine the antifungal susceptibility profile. One hundred strains obtained from vaginal discharge of 94 patients suffering acute vulvovaginal candidiasis were studied. The identification of the isolates showed: C. albicans 86%, Candida glabrata 6%, Candida inconspicua 3%, Candida krusei 2% and Candida intermedia, Candida holmii and Trichosporon asahii one case each. Minimal inhibitory concentrations (MIC) of all the yeasts against fluconazole and albaconazole were performed. C. glabrata, C. krusei and C. inconspicua were the most resistant against fluconazole, on the other hand albicans was susceptible to this drug. All the isolates presented MIC against albaconazole much lower than fluconazole.     

Effects of hyaluronan treatment on lipopolysaccharide-challenged fibroblast-like synovial cells

Numerous investigations have reported the efficacy of exogenous hyaluronan (HA) in modulating acute and chronic inflammation. The current study was performed to determine the in vitro effects of lower and higher molecular weight HA on lipopolysaccharide (LPS)-challenged fibroblast-like synovial cells. Normal synovial fibroblasts were cultured in triplicate to one of four groups: group 1, unchallenged; group 2, LPS-challenged (20 ng/ml); group 3, LPS-challenged following preteatment and sustained treatment with lower molecular weight HA; and group 4, LPS-challenged following pretreatment and sustained treatment with higher molecular weight HA. The response to LPS challenge and the influence of HA were compared among the four groups using cellular morphology scoring, cell number, cell viability, prostaglandin E₂ (PGE₂) production, IL-6 production, matrix metalloproteinase 3 (MMP3) production, and gene expression microarray analysis. As expected, our results demonstrated that LPS challenge induced a loss of characteristic fibroblast-like synovial cell culture morphology (P < 0.05), decreased the cell number (P < 0.05), increased PGE₂ production 1,000-fold (P < 0.05), increased IL-6 production 15-fold (P < 0.05), increased MMP3 production threefold (P < 0.05), and generated a profile of gene expression changes typical of LPS (P < 0.005). Importantly, LPS exposure at this concentration did not alter the cell viability. Higher molecular weight HA decreased the morphologic change (P < 0.05) associated with LPS exposure. Both lower and higher molecular weight HA significantly altered a similar set of 21 probe sets (P < 0.005), which represented decreased expression of inflammatory genes (PGE₂, IL-6) and catabolic genes (MMP3) and represented increased expression of anti-inflammatory and anabolic genes. The molecular weight of the HA product did not affect the cell number, the cell viability or the PGE₂, IL-6, or MMP3 production. Taken together, the anti-inflammatory and anticatabolic gene expression profiles of fibroblast-like synovial cells treated with HA and subsequently challenged with LPS support the pharmacologic benefits of treatment with HA regardless of molecular weight. The higher molecular weight HA product provided a cellular protective effect not seen with the lower molecular weight HA product.     

The Expression of Functional Vpx during Pathogenic SIVmac Infections of Rhesus Macaques Suppresses SAMHD1 in CD4+ Memory T Cells

For nearly 20 years, the principal biological function of the HIV-2/SIV Vpx gene has been thought to be required for optimal virus replication in myeloid cells. Mechanistically, this Vpx activity was recently reported to involve the degradation of Sterile Alpha Motif and HD domain-containing protein 1 (SAMHD1) in this cell lineage. Here we show that when macaques were inoculated with either the T cell tropic SIVmac239 or the macrophage tropic SIVmac316 carrying a Vpx point mutation that abrogates the recruitment of DCAF1 and the ensuing degradation of endogenous SAMHD1 in cultured CD4⁺ T cells, virus acquisition, progeny virion production in memory CD4⁺ T cells during acute infection, and the maintenance of set-point viremia were greatly attenuated. Revertant viruses emerging in two animals exhibited an augmented replication phenotype in memory CD4⁺ T lymphocytes both in vitro and in vivo, which was associated with reduced levels of endogenous SAMHD1. These results indicate that a critical role of Vpx in vivo is to promote the degradation of SAMHD1 in memory CD4⁺ T lymphocytes, thereby generating high levels of plasma viremia and the induction of immunodeficiency.     

Sec14-nodulin proteins and the patterning of phosphoinositide landmarks for developmental control of membrane morphogenesis

Polarized membrane morphogenesis is a fundamental activity of eukaryotic cells. This process is essential for the biology of cells and tissues, and its execution demands exquisite temporal coordination of functionally diverse membrane signaling reactions with high spatial resolution. Moreover, mechanisms must exist to establish and preserve such organization in the face of randomizing forces that would diffuse it. Here we identify the conserved AtSfh1 Sec14-nodulin protein as a novel effector of phosphoinositide signaling in the extreme polarized membrane growth program exhibited by growing Arabidopsis root hairs. The data are consistent with Sec14-nodulin proteins controlling the lateral organization of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P₂) landmarks for polarized membrane morphogenesis in plants. This patterning activity requires both the PtdIns(4,5)P₂ binding and homo-oligomerization activities of the AtSfh1 nodulin domain and is an essential aspect of the polarity signaling program in root hairs. Finally, the data suggest a general principle for how the phosphoinositide signaling landscape is physically bit mapped so that eukaryotic cells are able to convert a membrane surface into a high-definition lipid-signaling screen.