Combined analysis of Perca fluviatilis reproductive performance and oocyte proteomic profile

The success of reproduction depends greatly upon gamete quality, especially oocytes which carry most of the molecular material necessary for early embryogenesis. However, it remains difficult to find relevant morphologic and/or biochemical parameters to assess oocyte quality and thus have a reliable prediction of the reproduction performance. To understand which criteria are the most reliable to assess the reproductive success of the Eurasian perch (Perca fluviatilis), we measured 14 parameters characterizing female, spawn, oocyte, and embryonic or larval development on 20 independent spawn. A data analysis allowed the definition of two clusters of spawn with different larval characteristics: the first cluster was composed of spawn which led mainly to strong large larvae presenting a low deformity rate, while the second cluster rather corresponds to spawn leading to smaller and weaker larvae with a higher deformity rate. Moreover, a third cluster (unfertilized spawn) was studied. Our analysis revealed that most of the prefertilization biological traits that we studied appeared poorly relevant to predict larval features, proper embryonic development and deformity occurrences. We thus performed a large scale proteomic analysis to highlight proteins differently expressed in each spawn cluster. A 2D-DIGE study followed by an MS/MS spectrometry allowed the identification of 32 proteins involved in several biological functions and differently expressed between spawn clusters. Among them, proteins involved in cell response to the oxidative stress, as well as energetic metabolism, heat shock proteins and Vitellogenins are of particular interest. Several functions appear specific to a spawn cluster and could thus explain their corresponding reproduction performance. In the future, proteins involved in those cellular mechanisms may constitute molecular markers predictive of the reproduction performance in Perca fluviatilis.     

Dynamic regulation of HIV-1 mRNA populations analyzed by single-molecule enrichment and long-read sequencing

Alternative RNA splicing greatly expands the repertoire of proteins encoded by genomes. Next-generation sequencing (NGS) is attractive for studying alternative splicing because of the efficiency and low cost per base, but short reads typical of NGS only report mRNA fragments containing one or few splice junctions. Here, we used single-molecule amplification and long-read sequencing to study the HIV-1 provirus, which is only 9700 bp in length, but encodes nine major proteins via alternative splicing. Our data showed that the clinical isolate HIV-1_89.6 produces at least 109 different spliced RNAs, including a previously unappreciated ∼1 kb class of messages, two of which encode new proteins. HIV-1 message populations differed between cell types, longitudinally during infection, and among T cells from different human donors. These findings open a new window on a little studied aspect of HIV-1 replication, suggest therapeutic opportunities and provide advanced tools for the study of alternative splicing.     

Receptor protein tyrosine phosphatase mu expression as a marker for endothelial cell heterogeneity; analysis of RPTPmu gene expression using LacZ knock-in mice

The receptor-like protein tyrosine phosphatase mu (RPTPmu) belongs to the subfamily of meprin, A5, RPTPmu (MAM) domain-containing RPTPs, which are thought to play an important role in cell-cell adhesion mediated processes. The current study was designed to examine the expression pattern of RPTPmu in mice. We have generated RPTPmu-LacZ knock-in mice that express the beta-galactosidase (LacZ) reporter gene under the control of the RPTPmu promoter. LacZ expression patterns were analysed in embryos and adult mice by whole mount LacZ staining. Analysis of beta-galactosidase activity of heterozygous embryos and adult tissues revealed RPTPmu expression in endothelial cells of arteries and capillaries. In contrast, expression was virtually absent in endothelial cells of veins and in fenestrated endothelial cells in the adult liver and spleen. Moreover, RPTPmu expression was found in endothelial cells from the endocardium and the aorta in embryos, but not in adult mice. In addition to heterogeneous expression in endothelial cells, RPTPmu expression was found in cardiac muscle cells but not in skeletal muscle cells or smooth muscle cells. Expression was also found in Type II pneumonocytes in the lung alveoli and in Purkinje cells and other neurons in the brain. The specific expression of RPTPmu in arterial endothelial cells and in cardiac myocytes suggests that RPTPmu may play a role in the regulation of cardiovascular functions.     

Notch signaling coordinates the patterning of striatal compartments

Numerous lines of evidence suggest that Notch signaling plays a pivotal role in controlling the production of neurons from progenitor cells. However, most experiments have relied on gain-of-function approaches because perturbation of Notch signaling results in death prior to the onset of neurogenesis. Here, we examine the requirement for Notch signaling in the development of the striatum through the analysis of different single and compound Notch1 conditional and Notch3 null mutants. We find that normal development of the striatum depends on the presence of appropriate Notch signals in progenitors during a critical window of embryonic development. Early removal of Notch1 prior to neurogenesis alters early-born patch neurons but not late-born matrix neurons in the striatum. We further show that the late-born striatal neurons in these mutants are spared as a result of functional compensation by Notch3. Notably, however, the removal of Notch signaling subsequent to cells leaving the germinal zone has no obvious effect on striatal organization and patterning. These results indicate that Notch signaling is required in neural progenitor cells to control cell fate in the striatum, but is dispensable during subsequent phases of neuronal migration and differentiation.     

NF-κB pathway controls mitochondrial dynamics

The Optic atrophy 1 protein (OPA1) is a key element in the dynamics and morphology of mitochondria. We demonstrated that the absence of IκB kinase-α, which is a key element of the nonclassical NF-κB pathway, has an impact on the mitochondrial network morphology and OPA1 expression. In contrast, the absence of NF-κB essential modulator (NEMO) or IκB kinase-β, both of which are essential for the canonical NF-κB pathway, has no impact on mitochondrial dynamics. Whereas Parkin has been reported to positively regulate the expression of OPA1 through NEMO, herein we found that PARK2 overexpression did not modify the expression of OPA1. PARK2 expression reduced the levels of Bax, and it prevented stress-induced cell death only in Bak-deficient mouse embryonic fibroblast cells. Collectively, our results point out a role of the nonclassical NF-κB pathway in the regulation of mitochondrial dynamics and OPA1 expression.Mitochondria perform multiple functions that are critical to the maintenance of cellular homeostasis. Mitochondrial dysfunctions have been linked to the development of degenerative diseases and aging. Damaged mitochondria are removed by mitophagy, a process partially regulated by the PARK2-encoded E3 ubiquitin ligase (Parkin) in a PTEN-induced putative protein kinase 1 (PINK1)-dependent manner.^{1, 2, 3, 4} During mitophagy, the phosphorylation of mitofusin (Mfn) 2 by PINK1 has been suggested to induce the recruitment of Parkin to the mitochondria in cardiomyocytes.⁵ However, previous groups have shown that that Mfn 1 and 2 are dispensable for Parkin-dependent mitophagy in fibroblasts, whereas the Parkin-dependent degradation of these proteins may impair fusion of damaged mitochondria with the healthy network.^{6, 7, 8} PINK1 and Parkin thus act as a quality control machinery on the outer mitochondrial membrane (OMM) to preserve mitochondrial integrity through the ubiquitination of OMM proteins.^{9, 10} Moreover, through its E3 ubiquitin ligase activity,^{11, 12} Parkin was reported to bind to the linear ubiquitin chain assembly complex (LUBAC) and to increase the ubiquitination of NF-κB essential modulator (NEMO),¹³ a component of the classical NF-κB signaling pathway.¹⁴ Müller–Rischart et al. also proposed that Parkin positively regulates the expression of the mitochondrial guanosine triphosphatase Optic atrophy 1 protein (OPA1) through linear ubiquitination of NEMO.¹³ OPA1 is a regulator of mitochondrial inner membrane fusion and cristae remodeling.^{15, 16, 17} A defect in OPA1 expression is associated with mitochondrial network fragmentation and enhanced sensitivity of the cells to undergo apoptosis by promoting cytochrome c release from the mitochondria.^{18, 19, 20} Because NEMO-deficient mouse embryonic fibroblast (MEF) cells display a normal mitochondrial network morphology, we decided to re-examine the role of Parkin in regulating OPA1 expression through the NF-κB signaling pathway.     

Soybean ferritin: isolation, characterization, and free radical generation

The main aim of this work was to assess the multi-task role of ferritin(Ft)in the oxidative metabolism of soybean(Glycine max).Soybean seeds incubated for 24 h yielded 41 ± 5 μg Ft/g fresh weight.The rate of in vitro incorporation of iron(Fe)into Ft was tested by supplementing the reaction medium with physiological Fe chelators.The control rate,observed in the presence of 100 μM Fe,was not significantly different from the values observed in the presence of 100 μM Fe-his.However,it was significantly higher in the presence of 100 μM Fe-citrate(approximately 4.5-fold)or of 100 μM Fe-ATP(approximately 14-fold).Moreover,a substantial decrease in the Trp-dependent fluorescence of the Ft protein was determined during Fe uptake from Fe-citrate,as compared with the control.On the other hand,Ft addition to homogenates from soybean embryonic axes reduced endogenously generated ascorbyl radical,according to its capacity for Fe uptake.The data presented here suggest that Ft could be involved in the generation of free radicals,such as hydroxyl radical,by Fe-catalyzed reactions.Moreover,the scavenging of these radicals by Ft itself could then lead to protein damage.However,Ft could also prevent cellular damage by the uptake of catalytically active Fe.     

RAPD-PCR analysis of Staphylococcus aureus strains isolated from bovine and human hosts

Staphylococcus aureus is one of the most important pathogens in humans and animals. In this study eighty strains were analyzed by RAPD-PCR to assess the genetic relationship between S. aureus isolates from bovine and human hosts. Results were compared with those obtained by biotyping. Fifty-two percent of the S. aureus isolates belonged to a host specific biotype (human, bovine and poultry). Bovine and human ecovars were the most prevalent. Dendrogram obtained by RAPD results showed that all the isolates clustered into eleven groups (A-K) at a relative genetic similarity of less than 30% when analyzed with the three primers. Group A clustered 95% of the human host isolates and the remaining groups (B-K) clustered the bovine host isolates. Principal coordinate analysis also showed that the isolates could be arbitrarily divided into two groups, bovine and human, by the second coordinate. Only 9 isolates (11%) were not clustered into these groups. The genetic diversity among the S. aureus isolates from bovine hosts is relatively low compared to that of isolates from human hosts. There were no statistically significant differences among isolated from bovine and human hosts. This study shows that RAPD-PCR assayed with three primers can be successfully applied to assess the genetic relationship of S. aureus isolates from different hosts.     

The importance of modelling the spread of insecticide resistance in a heterogeneous environment: the example of adding synergists to bed nets

ABSTRACT: BACKGROUND: Insecticides are an effective and practical tool for reducing malaria transmission but the development of resistance to the insecticides can potentially compromise controls efforts. In this study a mathematical model was developed to explore the effects on mosquito populations of spatial heterogeneous deployment of insecticides. This model was used to identify important parameters in the evolution of insecticide resistance and to examine the contribution of new generation long-lasting insecticidal bed nets, that incorporate a chemical synergist on the roof panel, in delaying insecticide resistance. METHODS: A genetic model was developed to predict changes in mosquito fitness and resistance allele frequency. Parameters describing insecticide selection, fitness cost and the additional use of synergist were incorporated. Uncertainty and sensitivity analysis were performed followed by investigation of the evolution of resistance under scenarios of fully effective or ineffective synergists. RESULTS: The spread of resistance was most sensitive to selection coefficients, fitness cost and dominance coefficients while mean fitness was most affected by baseline fitness levels. Using a synergist delayed the spread of resistance but could, in specific circumstances that were thoroughly investigated, actually increase the rate of spread. Different spread dynamics were observed, with simulations leading to fixation, loss and most interestingly, equilibrium (without explicit overdominance) of the resistance allele. CONCLUSIONS: This strategy has the potential to delay the spread of resistance but note that in an heterogeneous environment it can also lead to the opposite effect, i.e., increasing the rate of spread. This clearly emphasizes that selection pressure acting inside the house cannot be treated in isolation but must be placed in context of overall insecticide use in an heterogeneous environment.     

Expression of canonical SOS genes is not under LexA repression in Bdellovibrio bacteriovorus

The here-reported identification of the LexA-binding sequence of Bdellovibrio bacteriovorus, a bacterial predator belonging to the delta-Proteobacteria, has made possible a detailed study of its LexA regulatory network. Surprisingly, only the lexA gene and a multiple gene cassette including dinP and dnaE homologues are regulated by the LexA protein in this bacterium. In vivo expression analyses have confirmed that this gene cassette indeed forms a polycistronic unit that, like the lexA gene, is DNA damage inducible in B. bacteriovorus. Conversely, genes such as recA, uvrA, ruvCAB, and ssb, which constitute the canonical core of the Proteobacteria SOS system, are not repressed by the LexA protein in this organism, hinting at a persistent selective pressure to maintain both the lexA gene and its regulation on the reported multiple gene cassette. In turn, in vitro experiments show that the B. bacteriovorus LexA-binding sequence is not recognized by other delta-Proteobacteria LexA proteins but binds to the cyanobacterial LexA repressor. This places B. bacteriovorus LexA at the base of the delta-Proteobacteria LexA family, revealing a high degree of conservation in the LexA regulatory sequence prior to the diversification and specialization seen in deeper groups of the Proteobacteria phylum.