15,259 research outputs found

    Prototype Foamy Virus Capsid – Nucleic Acid Interactions: Mechanistic Insights & Application for Efficient RNA Transfer

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    Foamy viruses (FV) represent a distinct genus in the retrovirus family and separate themselves from the large group of orthoretroviruses by various distinct features in their replication cycle (reviewed in Lindemann & Rethwilm, 2011). In gene therapy retroviruses are commonly used as vectors to deliver genetic information into target cells and also FV has been successfully used for example in a canine genetic disease model (Trobridge et al., 2009). Here we investigated the interactions between the FV capsid-forming protein ‘Gag’ and nucleic acids. We found that prototype FV (PFV) Gag binds various cellular mRNAs, incorporates them into the nascent particle and thereby enables their transfer into the cytosol of target cells. There these mRNAs can serve as template for protein translation. This feature seems uniquely efficient for PFV and we developed it further into a “RNA transfer vector system” allowing efficient transgene mRNA transfer into target cells, as showed in proof-of-principle experiments in vitro and in vivo (Hamann et al., 2014a). In parallel we started investigating the specificity in viral RNA genome packaging (Hamann et al., 2014b). To date little is known how PFV selects its RNA genome over the vast excess of cellular RNAs present in the cytosol. Elevated fundamental knowledge of this mechanism could help to make the “RNA transfer vector system” even more efficient since it would allow enrichment of certain specific “designer-RNAs” in virus particles

    Multiple functions and regulatory network of miR-150 in B lymphocyte-related diseases

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    MicroRNAs (miRNAs) play vital roles in the post-transcriptional regulation of gene expression. Previous studies have shown that miR-150 is a crucial regulator of B cell proliferation, differentiation, metabolism, and apoptosis. miR-150 regulates the immune homeostasis during the development of obesity and is aberrantly expressed in multiple B-cell-related malignant tumors. Additionally, the altered expression of MIR-150 is a diagnostic biomarker of various autoimmune diseases. Furthermore, exosome-derived miR-150 is considered as prognostic tool in B cell lymphoma, autoimmune diseases and immune-mediated disorders, suggesting miR-150 plays a vital role in disease onset and progression. In this review, we summarized the miR-150-dependent regulation of B cell function in B cell-related immune diseases

    OLIG2 neural progenitor cell development and fate in Down syndrome

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    Down syndrome (DS) is caused by triplication of human chromosome 21 (HSA21) and is the most common genetic form of intellectual disability. It is unknown precisely how triplication of HSA21 results in the intellectual disability, but it is thought that the global transcriptional dysregulation caused by trisomy 21 perturbs multiple aspects of neurodevelopment that cumulatively contribute to its etiology. While the characteristics associated with DS can arise from any of the genes triplicated on HSA21, in this work we focus on oligodendrocyte transcription factor 2 (OLIG2). The progeny of neural progenitor cells (NPCs) expressing OLIG2 are likely to be involved in many of the cellular changes underlying the intellectual disability in DS. To explore the fate of OLIG2+ neural progenitors, we took advantage of two distinct models of DS, the Ts65Dn mouse model and induced pluripotent stem cells (iPSCs) derived from individuals with DS. Our results from these two systems identified multiple perturbations in development in the cellular progeny of OLIG2+ NPCs. In Ts65Dn, we identified alterations in neurons and glia derived from the OLIG2 expressing progenitor domain in the ventral spinal cord. There were significant differences in the number of motor neurons and interneurons present in the trisomic lumbar spinal cord depending on age of the animal pointing both to a neurodevelopment and a neurodegeneration phenotype in the Ts65Dn mice. Of particular note, we identified changes in oligodendrocyte (OL) maturation in the trisomic mice that are dependent on spatial location and developmental origin. In the dorsal corticospinal tract, there were significantly fewer mature OLs in the trisomic mice, and in the lateral funiculus we observed the opposite phenotype with more mature OLs being present in the trisomic animals. We then transitioned our studies into iPSCs where we were able to pattern OLIG2+ NPCs to either a spinal cord-like or a brain-like identity and study the OL lineage that differentiated from each progenitor pool. Similar to the region-specific dysregulation found in the Ts65Dn spinal cord, we identified perturbations in trisomic OLs that were dependent on whether the NPCs had been patterned to a brain-like or spinal cord-like fate. In the spinal cord-like NPCs, there was no difference in the proportion of cells expressing either OLIG2 or NKX2.2, the two transcription factors whose co-expression is essential for OL differentiation. Conversely, in the brain-like NPCs, there was a significant increase in OLIG2+ cells in the trisomic culture and a decrease in NKX2.2 mRNA expression. We identified a sonic hedgehog (SHH) signaling based mechanism underlying these changes in OLIG2 and NKX2.2 expression in the brain-like NPCs and normalized the proportion of trisomic cells expressing the transcription factors to euploid levels by modulating the activity of the SHH pathway. Finally, we continued the differentiation of the brain-like and spinal cord-like NPCs to committed OL precursor cells (OPCs) and allowed them to mature. We identified an increase in OPC production in the spinal cord-like trisomic culture which was not present in the brain-like OPCs. Conversely, we identified a maturation deficit in the brain-like trisomic OLs that was not present in the spinal cord-like OPCs. These results underscore the importance of regional patterning in characterizing changes in cell differentiation and fate in DS. Together, the findings presented in this work contribute to the understanding of the cellular and molecular etiology of the intellectual disability in DS and in particular the contribution of cells differentiated from OLIG2+ progenitors

    Extracellular Vesicles as Biomarkers and Therapeutic Targets in Cancers

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    Extracellular vesicles refer to exosomes, apoptotic bodies, microvesicles and large oncosomes, which are membrane bound structures secreted by cells including cancer cells. The pathological role and translational potential of extracellular vesicles (EVs) in cancers are receiving research attention recently. The cargoes of cancer-derived EVs retain the molecular properties of their sources and cancer cells actively release EVs into body fluids that are easy to access. EVs released from cancer cells not only promote cancer progression through the delivery of cancer-associated molecules but also reflect alterations in the state of cancers during therapy. They are considered promising biomarkers for therapeutic response evaluation, especially resistance to therapy and diagnostics. This chapter discusses the various roles of extracellular vesicles in cancers and their potential as therapeutic targets

    Deciphering Regulation in Escherichia coli: From Genes to Genomes

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    Advances in DNA sequencing have revolutionized our ability to read genomes. However, even in the most well-studied of organisms, the bacterium Escherichia coli, for ≈ 65% of promoters we remain ignorant of their regulation. Until we crack this regulatory Rosetta Stone, efforts to read and write genomes will remain haphazard. We introduce a new method, Reg-Seq, that links massively-parallel reporter assays with mass spectrometry to produce a base pair resolution dissection of more than 100 E. coli promoters in 12 growth conditions. We demonstrate that the method recapitulates known regulatory information. Then, we examine regulatory architectures for more than 80 promoters which previously had no known regulatory information. In many cases, we also identify which transcription factors mediate their regulation. This method clears a path for highly multiplexed investigations of the regulatory genome of model organisms, with the potential of moving to an array of microbes of ecological and medical relevance.</p

    Cellular Senescence in Health, Disease and Aging: Blessing or Curse?

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    Dear Colleagues, When Hayflick and Moorhead coined the term “cellular senescence” (CS) almost 60 years ago, this phenomenon was understood as a mechanism, usually induced by activation of the DNA-repair machinery, to prevent uncontrolled proliferation. Meanwhile, additional beneficial roles for CS have been identified, such as embryonic development and wound healing. The senescence associated secretory phenotype (SASP) activated in most senescent cells (SC) signals to the immune system “come here and remove me”. In organisms with young and functional immune systems, occurring SC are usually detected and removed. If SC remain in the tissue expressing the SASP, this will cause not just a damaging local inflammation but can also induce remodeling and regeneration of the surrounding tissue as well as spreading of senescence. Old organisms show reduced regenerative potential and immune function which leads to accumulation of SC. Accordingly, accumulation of SC was observed in tissues of aged individuals, but importantly also in the context of age-related disorders, neurodegenerative, or cardiovascular diseases and others. Because of its detrimental effect of the surrounding tissue, accumulation of SC is not just a consequence, but can rather been understood as a major driver of aging. In line with this, recent studies described that removal of SC showed beneficial effects on healthspan and lifespan. This exciting research led to the discovery of “senolytics”, drugs which can kill SC. Given the heterogeneity of cell types that show senescence-like phenotypes, including heart muscle and post-mitotic neuronal cells, further research is required to unravel the molecular background that renders a cell type vulnerable to senesce. Additionally, it will be important to understand how senescence is cell type-specifically induced and which molecules serve as drug targets to prevent senescence and its spreading, or actively kill SC. This special issue will shed light on the molecular pathways of CS and inflammaging and on possible strategies to interfere with these processes. Dr. Markus Riessland Guest Edito

    Structure, Activity, and Function of Protein Methyltransferases

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    This collection of review articles describes the structure, function and mechanism of individual protein methyltransferase enzymes including protein lysine methyltransferases, protein arginine methyltransferases, and also the less abundant protein histidine methyltransferases and protein N-terminal end methyltransferases. The topics covered in the individual reviews include structural aspects (domain architecture, homologs and paralogs, and structure), biochemical properties (mechanism, sequence specificity, product specificity, regulation, and histone and non-histone substrates), cellular features (subcellular localization, expression patterns, cellular roles and function, biological effects of substrate protein methylation, connection to cell signaling pathways, and connection to chromatin regulation) and their role in diseases. This review book is a useful resource for scientists working on protein methylation and protein methyltransferases and those interested in joining this emerging research field

    Molecular Mechanisms of Sensorineural Hearing Loss and Development of Inner Ear Therapeutics

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    The sense of hearing is vulnerable to environmental challenges, such as exposure to noise. More than 1.5 billion people experience some decline in hearing ability during their lifetime, of whom at least 430 million will be affected by disabling hearing loss. If not identified and addressed in a timely way, hearing loss can severely reduce the quality of life at various stages. Some causes of hearing loss can be prevented, for example from occupational or leisure noise. The World Health Organization estimates that more than 1 billion young people put themselves at risk of permanent hearing loss by listening to loud music over long periods of time. Mitigating such risks through public health action is essential to reduce the impact of hearing loss in the community. The etiology of sensorineural hearing loss is complex and multifactorial, arising from congenital and acquired causes. This book highlights the diverse range of approaches to sensorineural hearing loss, from designing new animal models of age-related hearing loss, to the use of microRNAs as biomarkers of cochlear injury and drug repurposing for the therapy of age-related and noise-induced hearing loss. Further investigation into the underlying molecular mechanisms of sensorineural hearing loss and the integration of the novel drug, cell, and gene therapy strategies into controlled clinical studies will permit significant advances in a field where there are currently many unmet needs

    INSIGHTS INTO DINOFLAGELLATE NATURAL PRODUCT SYNTHESIS VIA CATALYTIC DOMAIN INTERACTIONS

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    Dinoflagellates are protists that can be split into two evolutionary groups, the parasitic syndinians and the largely photosynthetic “core” dinoflagellates. They represent a major portion of aquatic biomass which means that they are responsible for large portions of carbon that are both fixed and released. Other than biomass, the fixed carbon can be made into natural products such as polyunsaturated fatty acids that support the biota of many ecosystems or toxins that are harmful to aquatic life and humans. DNA and RNA analyses have been used to discover the putative genes that may make these compounds, but their non-colinear arrangement in the genome is very different from model organisms and their gene copy number is very high, making it nearly impossible to determine the exact biosynthetic pathways. The goal of my studies was to develop methods to differentiate biosynthetic pathways such as lipid and toxin synthesis by comparing the ability of domains to interact with each other with the assumption that domains that preferentially interact are more likely to participate in the same pathway. Initially, a survey was performed on available dinoflagellate transcriptomes to enumerate domains potentially involved in natural product synthesis and bin them based on sequence similarity to identify genes that could be used in biochemical assays. An interesting integration of analogous genes involved in lipid synthesis with those involved in natural product synthesis was observed as well as trends in domain expansion and contraction during core dinoflagellate evolution. Ultimately, the domain that scaffolds natural product synthesis, the thiolation domain, was chosen for further study because it exhibited two clear functional bins and is acted on directly by another enzyme, a phosphopantetheinyl transferase (PPTase). The PPTase activates the thiolation domain by transferring the phosphopantetheinate group from Coenzyme A to the thiolation domain, creating a free thiol group upon which the natural products are synthesized. These PPTases were then enumerated in dinoflagellates and characterized by looking for sequence motifs and observing expression patterns over a diel cycle as well as during growth in the model species Amphidinium carterae, a basal toxic dinoflagellate. Amphidinium carterae appears to have three PPTases, two of which (PPTase 1 and 2) are very similar, except that PPTase 2 does not appear to have a stop codon and has never been observed as a full-size protein. The remaining two PPTases (PPTase 1 and 3) had alternating expression patterns that did not appear to directly correlate to the acyl carrier protein, the thiolation domain required specifically for lipid biosynthesis. This carrier protein, like other enzymes for natural product synthesis in dinoflagellates, had a chloroplast targeting sequence while the three PPTases did not. To investigate the ability of these three PPTases to activate various thiolation domains, a total of 8 domains from A. carterae were substituted into the blue pigment synthesizing gene BpsA from Streptomyces lavendulae. These recombinant constructs were used for coexpression in E. coli as well as in vitro to reduce as many artifacts as possible and assess the interactions of each PPTase with the thiolation domains. Some of the recombinant BpsA genes were able to make blue dye with all three PPTases, while others never made blue dye both in E. coli as well as in vitro. In vitro quantification of free thiol added by the PPTase showed that all the thiolation domains, as well as the acyl carrier protein could be phosphopantetheinated by all the PPTases. This generalist substrate recognition, along with the alternating expression patterns and lack of chloroplast signaling peptide, indicate that the two active PPTases are performing the same function on all available thiolation domains, probably before export to the chloroplast. This lack of pathway segregation by PPTases is a completely novel way of synthesizing natural products compared to bacteria and fungi, likely due to the acquisition of both photosynthesis and natural product/lipid biosynthesis during dinoflagellate evolution that was not present in the common ancestor. Additionally, the techniques to identify genes of interest and perform biochemical characterization developed here are useful for future experiments annotating the function of dinoflagellate genes

    Epigenetics : a catalyst of plant immunity against pathogens

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    The plant immune system protects against pests and diseases. The recognition of stress-related molecular patterns triggers localised immune responses, which are often followed by longer-lasting systemic priming and/or up-regulation of defences. In some cases, this induced resistance (IR) can be transmitted to following generations. Such transgenerational IR is gradually reversed in the absence of stress at a rate that is proportional to the severity of disease experienced in previous generations. This review outlines the mechanisms by which epigenetic responses to pathogen infection shape the plant immune system across expanding time scales. We review the cis- and trans-acting mechanisms by which stress-inducible epigenetic changes at transposable elements (TEs) regulate genome-wide defence gene expression and draw particular attention to one regulatory model that is supported by recent evidence about the function of AGO1 and H2A.Z in transcriptional control of defence genes. Additionally, we explore how stress-induced mobilisation of epigenetically controlled TEs acts as a catalyst of Darwinian evolution by generating (epi)genetic diversity at environmentally responsive genes. This raises questions about the long-term evolutionary consequences of stress-induced diversification of the plant immune system in relation to the long-held dichotomy between Darwinian and Lamarckian evolution
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