Halofuginone improves muscle-cell survival in muscular dystrophies

Halofuginone has been shown to prevent fibrosis via the transforming growth factor-β/Smad3 pathway in muscular dystrophies. We hypothesized that halofuginone would reduce apoptosis—the presumed cause of satellite-cell depletion during muscle degradation—in the mdx mouse model of Duchenne muscular dystrophy. Six-week-old mdx mouse diaphragm exhibited fourfold higher numbers of apoptotic nuclei compared with wild-type mice as determined by a TUNEL assay. Apoptotic nuclei were found in macrophages and in Pax7-expressing cells; some were located in centrally-nucleated regenerating myofibers. Halofuginone treatment of mdx mice reduced the apoptotic nuclei number in the diaphragm, together with reduction in Bax and induction in Bcl2 levels in myofibers isolated from these mice. A similar effect was observed when halofuginone was added to cultured myofibers. No apparent effect of halofuginone was observed in wild-type mice. Inhibition of apoptosis or staurosporine-induced apoptosis by halofuginone in mdx primary myoblasts and C2 myogenic cell line, respectively, was reflected by less pyknotic/apoptotic cells and reduced Bax expression. This reduction was reversed by a phosphinositide-3-kinase and mitogen-activated protein kinase/extracellular signal-regulated protein kinase inhibitors, suggesting involvement of these pathways in mediating halofuginone's effects on apoptosis. Halofuginone increased apoptosis in α smooth muscle actin- and prolyl 4-hydroxylase β-expressing cells in mdx diaphragm and in myofibroblasts, the major source of extracellular matrix. The data suggest an additional mechanism by which halofuginone improves muscle pathology and function in muscular dystrophies.     

The FtsZ-Like Protein FtsZm of Magnetospirillum gryphiswaldense Likely Interacts with Its Generic Homolog and Is Required for Biomineralization under Nitrate Deprivation

Midcell selection, septum formation, and cytokinesis in most bacteria are orchestrated by the eukaryotic tubulin homolog FtsZ. The alphaproteobacterium Magnetospirillum gryphiswaldense (MSR-1) septates asymmetrically, and cytokinesis is linked to splitting and segregation of an intracellular chain of membrane-enveloped magnetite crystals (magnetosomes). In addition to a generic, full-length ftsZ gene, MSR-1 contains a truncated ftsZ homolog (ftsZm) which is located adjacent to genes controlling biomineralization and magnetosome chain formation. We analyzed the role of FtsZm in cell division and biomineralization together with the full-length MSR-1 FtsZ protein. Our results indicate that loss of FtsZm has a strong effect on microoxic magnetite biomineralization which, however, could be rescued by the presence of nitrate in the medium. Fluorescence microscopy revealed that FtsZm-mCherry does not colocalize with the magnetosome-related proteins MamC and MamK but is confined to asymmetric spots at midcell and at the cell pole, coinciding with the FtsZ protein position. In Escherichia coli, both FtsZ homologs form distinct structures but colocalize when coexpressed, suggesting an FtsZ-dependent recruitment of FtsZm. In vitro analyses indicate that FtsZm is able to interact with the FtsZ protein. Together, our data suggest that FtsZm shares key features with its full-length homolog but is involved in redox control for magnetite crystallization.     

Diversity and abundance of glycosyl hydrolase family 5 in the North Atlantic Ocean

The diversity and abundance of glycosyl hydrolase family 5 (GH5) were studied in the North Atlantic Ocean. This family was chosen because of the large number of available sequences from cultured bacteria, the variety of substrates it targets, and the high number of similar sequences in the Sargasso Sea environmental genome database. Three clone libraries of a GH5 subcluster were constructed from the Mid-Atlantic Bight and the eastern and western North Atlantic Ocean. The two North Atlantic Ocean libraries did not differ from each other but both were significantly less diverse than the Mid-Atlantic Bight library. The abundance of GH5 genes estimated by quantitative PCR was positively correlated with chlorophyll concentrations in the eastern part of a transect from Fort Pierce, Florida, to the Azores and in a depth profile, suggesting that the supply of labile organic material selects for GH5-bearing bacteria in these waters. However, the data suggest that only <1% of all bacteria harbor the GH5 subcluster. These and other data suggest that the hydrolysis of polysaccharides requires complicated multi-enzyme systems.     

Tissue development and RNA control: "HOW" is it coordinated?

The regulation of developmental processes at the RNA level enables selective and rapid modulation of gene expression. Studies in model organisms revealed the essential contribution of the signal transduction and activation of RNA (STAR) family of RNA binding proteins to developmental processes. STAR proteins coordinate the proper timing of developmental events by delaying expression or altering the mRNA or protein levels of essential genes. Recent functional analysis of the Drosophila melanogaster STAR protein, Held Out Wing (HOW), in the context of embryonic development, provided insight into its mode of activity. Here, we describe HOW's activity in the temporal repression or elevation of gene expression that is essential for coordinating the correct timing of instructive signals.     

Proteogenomic Monitoring of Geobacter Physiology during Stimulated Uranium Bioremediation

Implementation of uranium bioremediation requires methods for monitoring the membership and activities of the subsurface microbial communities that are responsible for reduction of soluble U(VI) to insoluble U(IV). Here, we report a proteomics-based approach for simultaneously documenting the strain membership and microbial physiology of the dominant Geobacter community members during in situ acetate amendment of the U-contaminated Rifle, CO, aquifer. Three planktonic Geobacter-dominated samples were obtained from two wells down-gradient of acetate addition. Over 2,500 proteins from each of these samples were identified by matching liquid chromatography-tandem mass spectrometry spectra to peptides predicted from seven isolate Geobacter genomes. Genome-specific peptides indicate early proliferation of multiple M21 and Geobacter bemidjiensis-like strains and later possible emergence of M21 and G. bemidjiensis-like strains more closely related to Geobacter lovleyi. Throughout biostimulation, the proteome is dominated by enzymes that convert acetate to acetyl-coenzyme A and pyruvate for central metabolism, while abundant peptides matching tricarboxylic acid cycle proteins and ATP synthase subunits were also detected, indicating the importance of energy generation during the period of rapid growth following the start of biostimulation. Evolving Geobacter strain composition may be linked to changes in protein abundance over the course of biostimulation and may reflect changes in metabolic functioning. Thus, metagenomics-independent community proteogenomics can be used to diagnose the status of the subsurface consortia upon which remediation biotechnology relies.Enzymatic reduction of U(VI) to insoluble U(IV) by dissimilatory Fe(III)-reducing bacteria (DIRB) can limit subsurface U(VI) migration (2, 19, 20). This observation provided the basis for an important new biotechnology that involves remediation of contaminated groundwater by organic amendment-based stimulation of the activities of DIRB. Despite the obvious high potential value, there are significant technological challenges that must be overcome before this approach can be deployed as a functional biotechnology. In situ U(VI) reduction rates are directly coupled with microbial physiology and community composition, both of which change as bioremediation progresses (22, 24, 28). Thus, a key need is the ability to monitor changes in microbial community membership and function as they occur, so that optimal management strategies can be implemented to achieve the desired geochemical outcomes. This challenge is significant because metal reduction occurs within pore spaces deep in the aquifer and the process is associated with vast numbers of microbial cells distributed in the subsurface.Changes in the complements of DIRB proteins should reflect shifts in microbial physiology and could be used to monitor in situ bioremediation technologies if microbial proteomes could be tracked during organic amendment. Proteomic methods have previously been used to detect physiological responses of microorganisms growing in pure culture (8, 16, 17) and in genomically characterized natural microbial communities (7, 18, 26, 34). Strain-resolved proteomic approaches (18) have the potential to also track changes in microbial community composition. Previously, the use of proteomic analysis techniques to monitor subsurface bioremediation has been precluded by the lack of metagenomic data. Here, we used seven isolate Geobacter genomes to generate a reference database against which peptide data measured using two-dimensional (2D) liquid chromatography (LC)-based high-resolution tandem mass spectrometry (MS-MS) were compared for protein identification. Although differences between environmental and isolate peptide sequences may preclude peptide identification, peptides common to multiple Geobacter types and isolates enable protein identification, and peptides unique to one isolate constrain environmental genotypes. Simultaneous analysis of community structure and function in natural microbial communities that relies upon proteomics-derived genomic insights is referred to as proteogenomics (Fig. ​(Fig.1).1). In the current study, a proteogenomic approach was applied to monitor the progress of an in situ U bioremediation project carried out at the Department of Energy (DOE) Integrated Field Research Challenge site in Rifle, CO. Despite strain complexity in the recovered samples, proteomic data provided insights into the community structure and physiology of planktonic Geobacter isolates in aquifer solutions as groundwater U(VI) concentrations decreased.Open in a separate windowFIG. 1.Strain-resolved proteogenomic techniques allow identification of unique peptides and constrain the genotypes present in environmental samples. (1) Proteins were predicted from the seven isolate Geobacter genomes. (2) Proteins were aligned. In the example shown, they share a conserved region (similar colors in all seven regions) and have a variable region (indicated by the variety of colors for each protein). (3) Tryptic digest patterns are used to predict peptide sequences. Proteins were extracted from the experimental sample (4) and digested into peptides by using trypsin (5). These peptides were separated using LC (6) before high-resolution mass spectrometry on a fraction of peptides from an elution peak (7) to determine the mass of the parent ions. (8) The peptides are fragmented and mass spectrometry measurements made on fragment ions. (9) Mass spectral data are matched to peptide sequences predicted in step 3 to allow peptide identification. Peptide sequences differing by only one amino acid can be identified, as shown in the spectral diagram. (10) Detected peptides are mapped onto aligned protein sequences to identify peptides shared or unique to each of the isolate species. Spectral counts can be mapped together with peptides. Where coexisting unique peptides are detected, ratios of unique spectral counts can be used to infer relative strain abundance. As shown, the identification of a unique peptide indicates that this protein is most closely related to that of G. bemidjiensis.     

Cell-autonomous and non-cell-autonomous functions of caspase-8

Cells in vivo do not act in isolation. Therefore, when attempting to predict the results of pharmaceutical modulation of the function of a protein, we must also take into account the non-cell-autonomous consequences of such modulation. Studies of caspase-8 initially indicated that it serves as the proximal enzyme in cellular self-destruction dictated through the extrinsic cell-death pathway. Later studies revealed that it also participates in mechanisms affecting cell growth and survival. This essay presents a brief account of a study indicating that, apart from functional changes that are cell autonomous, tissue-specific deletion of caspase-8 in mice also has non-cell-autonomous effects with consequences that might even be the opposite of the cell-autonomous ones.     

The Interrelations among Stochastic Pacing,Stability, and Memory in the Heart

Low pacing variabilty in the heart has been clinically reported as a risk factor for lethal cardiac arrhythmias and arrhythmic death. In a previous simulation study, we demonstrated that stochastic pacing sustains an antiarrhythmic effect by moderating the slope of the action potential duration (APD) restitution curve, by reducing the propensity of APD alternans, converting discordant to concordant alternans, and ultimately preventing wavebreaks. However, the dynamic mechanisms relating pacing stochasticity to tissue stability are not yet known. In this work, we develop a mathematical framework to describe the APD signal using an autoregressive stochastic model, and we establish the interrelations between stochastic pacing, cardiac memory, and cardiac stability, as manifested by the degree of APD alternans. Employing stability analysis tools, we show that increased stochasticity in the ventricular tissue activation sequence works to lower the maximal absolute eigenvalues of the stochastic model, thereby contributing to increased stability. We also show that the memory coefficients of the autoregressive model are modulated by pacing stochasticity in a nonlinear, biphasic way, so that for exceedingly high levels of pacing stochasticity, the antiarrhythmic effect is hampered by increasing APD variance. This work may contribute to establishment of an optimal antiarrhythmic pacing protocol in a future study.