Statistical Model of Shape Moments with Active Contour Evolution for Shape Detection and Segmentation

This paper describes a novel method for shape representation and robust image segmentation. The proposed method combines two well known methodologies, namely, statistical shape models and active contours implemented in level set framework. The shape detection is achieved by maximizing a posterior function that consists of a prior shape probability model and image likelihood function conditioned on shapes. The statistical shape model is built as a result of a learning process based on nonparametric probability estimation in a PCA reduced feature space formed by the Legendre moments of training silhouette images. A greedy strategy is applied to optimize the proposed cost function by iteratively evolving an implicit active contour in the image space and subsequent constrained optimization of the evolved shape in the reduced shape feature space. Experimental results presented in the paper demonstrate that the proposed method, contrary to many other active contour segmentation methods, is highly resilient to severe random and structural noise that could be present in the data

Human microRNA hsa-miR-125a-5p interferes with expression of hepatitis B virus surface antigen

MicroRNAs are small non-coding RNAs that modulate gene expression at post-transcriptional level, playing a crucial role in cell differentiation and development. Recently, some reports have shown that a limited number of mammalian microRNAs are also involved in anti-viral defense. In this study, the analysis of the hepatitis B virus (HBV) genome by the computer program MiRanda led to the identification of seven sites that are potential targets for human liver microRNAs. These sites were found to be clustered in a 995-bp segment within the viral polymerase ORF and the overlapping surface antigen ORF, and conserved among the most common HBV subtypes. The HBV genomic targets were then subjected to a validation test based on cultured hepatic cells (HepG2, HuH-7 and PLC/PRF/5) and luciferase reporter genes. In this test, one of the selected microRNAs, hsa-miR-125a-5p, was found to interact with the viral sequence and to suppress the reporter activity markedly. The microRNA was then shown to interfere with the viral translation, down-regulating the expression of the surface antigen. Overall, these results support the emerging concept that some mammalian microRNAs play a role in virus-host interaction. Furthermore, they provide the basis for the development of new strategies for anti-HBV intervention

Wood Utilization Is Dependent on Catalase Activities in the Filamentous Fungus Podospora anserina

Catalases are enzymes that play critical roles in protecting cells against the toxic effects of hydrogen peroxide. They are implicated in various physiological and pathological conditions but some of their functions remain unclear. In order to decipher the role(s) of catalases during the life cycle of Podospora anserina, we analyzed the role of the four monofunctional catalases and one bifunctional catalase-peroxidase genes present in its genome. The five genes were deleted and the phenotypes of each single and all multiple mutants were investigated. Intriguingly, although the genes are differently expressed during the life cycle, catalase activity is dispensable during both vegetative growth and sexual reproduction in laboratory conditions. Catalases are also not essential for cellulose or fatty acid assimilation. In contrast, they are strictly required for efficient utilization of more complex biomass like wood shavings by allowing growth in the presence of lignin. The secreted CATB and cytosolic CAT2 are the major catalases implicated in peroxide resistance, while CAT2 is the major player during complex biomass assimilation. Our results suggest that P. anserina produces external H2O2 to assimilate complex biomass and that catalases are necessary to protect the cells during this process. In addition, the phenotypes of strains lacking only one catalase gene suggest that a decrease of catalase activity improves the capacity of the fungus to degrade complex biomass

Systematic Deletion of Homeobox Genes in Podospora anserina Uncovers Their Roles in Shaping the Fruiting Body

Higher fungi, which comprise ascomycetes and basidiomycetes, play major roles in the biosphere. Their evolutionary success may be due to the extended dikaryotic stage of their life cycle, which is the basis for their scientific name: the Dikarya. Dikaryosis is maintained by similar structures, the clamp in basidiomycetes and the crozier in ascomycetes. Homeodomain transcription factors are required for clamp formation in all basidiomycetes studied. We identified all the homeobox genes in the filamentous ascomycete fungus Podospora anserina and constructed deletion mutants for each of these genes and for a number of gene combinations. Croziers developed normally in these mutants, including those with up to six deleted homeogenes. However, some mutants had defects in maturation of the fruiting body, an effect that could be rescued by providing wild-type maternal hyphae. Analysis of mutants deficient in multiple homeogenes revealed interactions between the genes, suggesting that they operate as a complex network. Similar to their role in animals and plants, homeodomain transcription factors in ascomycetes are involved in shaping multicellular structures

Unc45b Forms a Cytosolic Complex with Hsp90 and Targets the Unfolded Myosin Motor Domain

Myosin folding and assembly in striated muscle is mediated by the general chaperones Hsc70 and Hsp90 and a myosin specific co-chaperone, UNC45. Two UNC45 genes are found in vertebrates, including a striated muscle specific form, Unc45b. We have investigated the role of Unc45b in myosin folding. Epitope tagged murine Unc45b (Unc45bFlag) was expressed in muscle and non-muscle cells and bacteria, isolated and characterized. The protein is a soluble monomer in solution with a compact folded rod-shaped structure of ∼19 nm length by electron microscopy. When over-expressed in striated muscle cells, Unc45bFlag fractionates as a cytosolic protein and isolates as a stable complex with Hsp90. Purified Unc45bFlag re-binds Hsp90 and forms a stable complex in solution. The endogenous Unc45b in muscle cell lysates is also found associated with Hsp90. The Unc45bFlag/Hsp90 complex binds the partially folded myosin motor domain when incubated with myosin subfragments synthesized in a reticulocyte lysate. This binding is independent of the myosin rod or light chains. Unc45bFlag does not bind native myosin subfragments consistent with a chaperone function. More importantly, Unc45bFlag enhances myosin motor domain folding during de novo motor domain synthesis indicating that it has a direct role in myosin maturation. Thus, mammalian Unc45b is a cytosolic protein that forms a stable complex with Hsp90, selectively binds the unfolded conformation of the myosin motor domain, and promotes motor domain folding

Biological Roles of the Podospora anserina Mitochondrial Lon Protease and the Importance of Its N-Domain

Mitochondria have their own ATP-dependent proteases that maintain the functional state of the organelle. All multicellular eukaryotes, including filamentous fungi, possess the same set of mitochondrial proteases, unlike in unicellular yeasts, where ClpXP, one of the two matricial proteases, is absent. Despite the presence of ClpXP in the filamentous fungus Podospora anserina, deletion of the gene encoding the other matricial protease, PaLon1, leads to lethality at high and low temperatures, indicating that PaLON1 plays a main role in protein quality control. Under normal physiological conditions, the PaLon1 deletion is viable but decreases life span. PaLon1 deletion also leads to defects in two steps during development, ascospore germination and sexual reproduction, which suggests that PaLON1 ensures important regulatory functions during fungal development. Mitochondrial Lon proteases are composed of a central ATPase domain flanked by a large non-catalytic N-domain and a C-terminal protease domain. We found that three mutations in the N-domain of PaLON1 affected fungal life cycle, PaLON1 protein expression and mitochondrial proteolytic activity, which reveals the functional importance of the N-domain of the mitochondrial Lon protease. All PaLon1 mutations affected the C-terminal part of the N-domain. Considering that the C-terminal part is predicted to have an α helical arrangement in which the number, length and position of the helices are conserved with the solved structure of its bacterial homologs, we propose that this all-helical structure participates in Lon substrate interaction

A Re-Examination of Global Suppression of RNA Interference by HIV-1

The nature of the interaction between replicating HIV-1 and the cellular RNAi pathway has been controversial, but it is clear that it can be complex and multifaceted. It has been proposed that the interaction is bi-directional, whereby cellular silencing pathways can restrict HIV-1 replication, and in turn, HIV-1 can suppress silencing pathways. Overall suppression of RNAi has been suggested to occur via direct binding and inhibition of Dicer by the HIV-1 Tat protein or through sequestration of TRBP, a Dicer co-factor, by the structured TAR element of HIV-1 transcripts. The role of Tat as an inhibitor of Dicer has been questioned and our results support and extend the conclusion that Tat does not inhibit RNAi that is mediated by either exogenous or endogenous miRNAs. Similarly, we find no suppression of silencing pathways in cells with replicating virus, suggesting that viral products such as the TAR RNA elements also do not reduce the efficacy of cellular RNA silencing. However, knockdown of Dicer does allow increased viral replication and this occurs at a post-transcriptional level. These results support the idea that although individual miRNAs can act to restrict HIV-1 replication, the virus does not counter these effects through a global suppression of RNAi synthesis or processing

Differentially expressed microRNAs in experimental cerebral malaria and their involvement in endocytosis, adherens junctions, FoxO and TGF-β signalling pathways

Cerebral malaria (CM) is the most severe manifestation of infection with Plasmodium, however its pathogenesis is still not completely understood. microRNA (miRNA) have been an area of focus in infectious disease research, due to their ability to affect normal biological processes, and have been shown to play roles in various viral, bacterial and parasitic infections, including malaria. The expression of miRNA was studied following infection of CBA mice with either Plasmodium berghei ANKA (causing CM), or Plasmodium yoelii (causing severe but non-cerebral malaria (NCM)). Using microarray analysis, miRNA expression was compared in the brains of non-infected (NI), NCM and CM mice. Six miRNA were significantly dysregulated between NCM and CM mice, and four of these, miR-19a-3p, miR-19b-3p, miR-142-3p and miR-223-3p, were further validated by qPCR assays. These miRNA are significantly involved in several pathways relevant to CM, including the TGF-β and endocytosis pathways. Dysregulation of these miRNA during CM specifically compared with NCM suggests that these miRNA, through their regulation of downstream targets, may be vitally involved in the neurological syndrome. Our data implies that, at least in the mouse model, miRNA may play a regulatory role in CM pathogenesis.This work was funded by the National Health and Medical Research Council (#1099920 for GEG). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.S

Alternative Oxidase Dependent Respiration Leads to an Increased Mitochondrial Content in Two Long-Lived Mutants of the Ageing Model Podospora anserina

The retrograde response constitutes an important signalling pathway from mitochondria to the nucleus which induces several genes to allow compensation of mitochondrial impairments. In the filamentous ascomycete Podospora anserina, an example for such a response is the induction of a nuclear-encoded and iron-dependent alternative oxidase (AOX) occurring when cytochrome-c oxidase (COX) dependent respiration is affected. Several long-lived mutants are known which predominantly or exclusively respire via AOX. Here we show that two AOX-utilising mutants, grisea and PaCox17::ble, are able to compensate partially for lowered OXPHOS efficiency resulting from AOX-dependent respiration by increasing mitochondrial content. At the physiological level this is demonstrated by an elevated oxygen consumption and increased heat production. However, in the two mutants, ATP levels do not reach WT levels. Interestingly, mutant PaCox17::ble is characterized by a highly increased release of the reactive oxygen species (ROS) hydrogen peroxide. Both grisea and PaCox17::ble contain elevated levels of mitochondrial proteins involved in quality control, i. e. LON protease and the molecular chaperone HSP60. Taken together, our work demonstrates that AOX-dependent respiration in two mutants of the ageing model P. anserina is linked to a novel mechanism involved in the retrograde response pathway, mitochondrial biogenesis, which might also play an important role for cellular maintenance in other organisms