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TGFβ counteracts LYVE-1-mediated induction of lymphangiogenesis by small hyaluronan oligosaccharides.
During tissue injury, inflammation, and tumor growth, enhanced production and degradation of the extracellular matrix glycosaminoglycan hyaluronan (HA) can lead to the accumulation of small HA (sHA) oligosaccharides. We have previously reported that accumulation of sHA in colorectal tumors correlates with lymphatic invasion and lymph node metastasis, and therefore, investigated here are the effects of sHA on the lymphatic endothelium. Using cultured primary lymphatic endothelial cells (LECs) and ex vivo and in vivo lymphangiogenesis assays, we found that in contrast to high-molecular-weight HA (HMW-HA), sHA of 4-25 disaccharides in length can promote the proliferation of LECs and lymphangiogenesis in a manner that is dependent on their size and concentration. At pathophysiologically relevant concentrations found in tumor interstitial fluid, sHA is pro-proliferative, acts synergistically with VEGF-C and FGF-2, and stimulates the outgrowth of lymphatic capillaries in ex vivo lymphangiogenesis assays. In vivo, intradermally injected sHA acts together with VEGF-C to increase lymphatic vessel density. Higher concentrations of sHA were found to induce expression of the anti-lymphangiogenic cytokine TGFβ in LECs, which serves to counter-regulate sHA-induced LEC proliferation and lymphangiogenesis. Using appropriate knockout mice and blocking antibodies, we found that the effects of sHA are mediated by the sialylated form of the lymphatic HA receptor LYVE-1, but not by CD44 or TLR-4. These data are consistent with the notion that accumulation of sHA in tumors may contribute to tumor-induced lymphangiogenesis, leading to increased dissemination to regional lymph nodes. KEY MESSAGES : sHA promotes lymphangiogenesis primarily through increased LEC proliferation sHA induces proliferation in a narrow concentration window due to upregulated TGFβ Smaller HA oligosaccharides more potently induce proliferation than larger ones VEGF-C and FGF-2-induced LEC proliferation and lymphangiogenesis is augmented by sHA Sialylated LYVE-1, but not CD44 or TLR-4, mediate the effects of sHA on LEC.
Boron deficiency affects rhizobia cell surface polysaccharides important for suppression of plant defense mechanisms during legume recognition and for development of nitrogen-fixing symbiosis
Background and aims: Boron (B) deficiency negatively affects legume-rhizobia symbiotic interactions and the development of N2-fixing nodules. Many described alterations are related to plant-derived carbohydrates involved in plant-microbe interactions; however, the effects of B on the bacterial polysaccharides that are crucial for correct symbiosis are unknown. Methods: Exopolysaccharide (EPS) production in several rhizobial strains grown in B-free media was analyzed following acetone extraction and silver-stained electrophoretic profiles of lipopolysaccharide (LPS). Moreover, the effects of B deficiency and mutations of the pathogenesis-related ABR17 protein on rhizobia cell surface polysaccharides on legume root colonization, nodulation, nitrogen fixation, and induction in pea nodules were investigated. Results: B-deficiency led to a 65-80 % reduction in the amount of EPS and to modifications of LPS in all strains tested. B-deficient rhizobia were not affected in the degree of adsorption to roots. However, nodulation and nitrogen fixation were reduced or inhibited by B starvation or in plants inoculated with EPS or LPS defective mutants, and ABR17 was induced. Conclusion: The results provide evidence that B is important for production of rhizobia cell surface polysaccharides essential to establish a symbiotic rather than a pathogenic-like interaction, and for development of the N2-fixing legume root nodule. © 2012 Springer Science+Business Media B.V.
Functional Characterization of the Co2+ Transporter AitP in Sinorhizobium meliloti: A New Player in Fe2+ Homeostasis.
Co2+ induces the increase of the labile-Fe pool (LIP) by Fe-S cluster damage, heme synthesis inhibition, and "free" iron import, which affects cell viability. The N2-fixing bacteria, Sinorhizobium meliloti, is a suitable model to determine the roles of Co2+-transporting cation diffusion facilitator exporters (Co-eCDF) in Fe2+ homeostasis because it has a putative member of this subfamily, AitP, and two specific Fe2+-export systems. An insertional mutant of AitP showed Co2+ sensitivity and accumulation, Fe accumulation and hydrogen peroxide sensitivity, but not Fe2+ sensitivity, despite AitP being a bona fide low affinity Fe2+ exporter as demonstrated by the kinetic analyses of Fe2+ uptake into everted membrane vesicles. Suggesting concomitant Fe2+-dependent induced stress, Co2+ sensitivity was increased in strains carrying mutations in AitP and Fe2+ exporters which did not correlate with the Co2+ accumulation. Growth in the presence of sublethal Fe2+ and Co2+ concentrations suggested that free Fe-import might contribute to Co2+ toxicity. Supporting this, Co2+ induced transcription of Fe-import system and genes associated with Fe homeostasis. Analyses of total protoporphyrin content indicates Fe-S cluster attack as the major source for LIP. AitP-mediated Fe2+-export is likely counterbalanced via a nonfutile Fe2+-import pathway. Two lines of evidence support this: (i) an increased hemin uptake in the presence of Co2+ was observed in wild-type (WT) versus AitP mutant, and (ii) hemin reversed the Co2+ sensitivity in the AitP mutant. Thus, the simultaneous detoxification mediated by AitP aids cells to orchestrate an Fe-S cluster salvage response, avoiding the increase in the LIP caused by the disassembly of Fe-S clusters or free iron uptake. IMPORTANCE Cross-talk between iron and cobalt has been long recognized in biological systems. This is due to the capacity of cobalt to interfere with proper iron utilization. Cells can detoxify cobalt by exporting mechanisms involving membrane proteins known as exporters. Highlighting the cross-talk, the capacity of several cobalt exporters to also export iron is emerging. Although biologically less important than Fe2+, Co2+ induces toxicity by promoting intracellular Fe release, which ultimately causes additional toxic effects. In this work, we describe how the rhizobia cells solve this perturbation by clearing Fe through a Co2+ exporter, in order to reestablish intracellular Fe levels by importing nonfree Fe, heme. This piggyback-ride type of transport may aid bacterial cells to survive in free-living conditions where high anthropogenic Co2+ content may be encountered.
Boron nutrition affects growth, adaptation to stressful environments, and exopolysaccharide synthesis of Ensifer meliloti
Boron (B) is a micronutrient required for the development of symbiotic legume nodules but is apparently not essential for rhizobia, although it has been related to bacteria cell surface formation and adaptation to stress responses. In this paper, we explore whether B nutrition can influence the growth of several strains of Ensifer meliloti (formerly Sinorhizobium meliloti), including mutants defective in exopolysaccharide (EPS) production. All the strains were able to grow in B-deficient (-B) media, except for the non-nodulating Sm11605 mutant, which was unable to produce EPS. The addition of boric acid before the exponential phase of growth (in all strains, including Sm11605) showed that 50 μM H3BO3 (50 B) resulted in the highest growth rate. Moreover, the addition of B increased the tolerance of E. meliloti to salinity (up to 0.5 mM NaCl), to the cell surface stressor SDS (up to 100 μg mL−1 SDS), and to 0.5 mM H2O2. Furthermore, 50 B increased survival rate after 90 min of exposure to 1 mM H2O2, which can be crucial to overcoming transient oxidative burst due to ROS production by root legumes during the initial phases of infection. Given that rhizobia exopolysaccharides (EPSs) are involved in the responses to stress and in the adaptation to and endophytic environment, we hypothesized that B affects EPS production and it can be crucial for growth and survival under stressful environments. Indeed, the amount of EPS extracted from –B cultures was 4–5 times reduced. Gene expression analysis by using qRT-PCR revealed that the genes mucR (a positive regulator of EPS-I but negative regulator of EPS-II synthesis), and emmA (a negative regulator of EPS-I), were overexpressed in –B cells. Meanwhile, expG (a positive regulator of EPS-II), and the genes exoY and expA1 (involved in the synthesis of EPS-I and EPS-II, respectively) were downregulated. These results are consistent with a reduction of both EPS-I and EPS-II synthesis under B deficiency supporting our hypothesis.
Metal transport in Medicago truncatula nodule rhizobia-infected cells
Symbiotic nitrogen fixation requires relatively large quantities of essential transition metals (iron, copper, zinc, etc.) as essential cofactors of many of the enzymes involved in this process. These nutrients are delivered by the host plant, in a complex process that requires soluble metal‐binding molecules and dedicated metal transporters. In the last years, the metal transporters responsible for iron, copper, zinc, and molybdate uptake by rhizobia‐infected cells in Medicago truncatula nodules have been identified, and the role that metal binding molecules such as citrate and nicotianamine play in symbiotic nitrogen fixation is gaining more attention. In this chapter, we will present the recent advances in this field and discuss the aspects of nodule transition metal homeostasis that still need addressing.
Sparse haplotype-based fine-scale local ancestry inference at scale reveals recent selection on immune responses.
Increasingly efficient methods for inferring the ancestral origin of genome regions are needed to gain insights into genetic function and history as biobanks grow in scale. Here we describe two near-linear time algorithms to learn ancestry harnessing the strengths of a Positional Burrows-Wheeler Transform. SparsePainter is a faster, sparse replacement of previous model-based 'chromosome painting' algorithms to identify recently shared haplotypes, whilst PBWTpaint uses further approximations to obtain lightning-fast estimation optimized for genome-wide relatedness estimation. The computational efficiency gains of these tools for fine-scale local ancestry inference offer the possibility to analyse large-scale genomic datasets using different approaches. Application to the UK Biobank shows that haplotypes better represent ancestries than principal components, whilst linkage-disequilibrium of ancestry identifies signals of recent changes to population-specific selection for many genomic regions associated with immune responses, suggesting avenues for understanding the pathogen-immune system interplay on a historical timescale.
Bacteriophage
The present invention discloses an engineered bacteriophage comprising a polynucleotide encoding a heterologous protein under the control of a repressible promoter. Also disclosed are processes for producing the engineered bacteriophage, pharmaceutical compositions comprising the engineered bacteriophage and methods of treatment using the engineered bacteriophage.
Lentiviral vector packaging and producer cell lines yield titers equivalent to the industry-standard four-plasmid process.
Lentiviral vector (LVV)-mediated cell and gene therapies have the potential to cure diseases that currently require lifelong intervention. However, the requirement for plasmid transfection hinders large-scale LVV manufacture. Moreover, large-scale plasmid production, testing, and transfection contribute to operational risk and the high cost associated with this therapeutic modality. Thus, we developed LVV packaging and producer cell lines, which reduce or eliminate the need for plasmid transfection during LVV manufacture. To develop a packaging cell line, lentiviral packaging genes were stably integrated by random integration of linearized plasmid DNA. Then, to develop EGFP- and anti-CD19 chimeric antigen receptor-encoding producer cell lines, transfer plasmids were integrated by transposase-mediated integration. Single-cell isolation and testing were performed to isolate the top-performing clonal packaging and producer cell lines. Production of LVVs that encode various cargo genes revealed consistency in the production performance of the packaging and producer cell lines compared to the industry-standard four-plasmid transfection method. By reducing or eliminating the requirement for plasmid transfection, while achieving production performance consistent with the current industry standard, the packaging and producer cell lines developed here can reduce costs and operational risks of LVV manufacture, thus increasing patient access to LVV-mediated cell and gene therapies.
Computing exponentially faster: implementing a non-deterministic universal Turing machine using DNA
The theory of computer science is based around universal Turing machines (UTMs): abstract machines able to execute all possible algorithms. Modern digital computers are physical embodiments of classical UTMs. For the most important class of problem in computer science, non-deterministic polynomial complete problems, non-deterministic UTMs (NUTMs) are theoretically exponentially faster than both classical UTMs and quantum mechanical UTMs (QUTMs). However, no attempt has previously been made to build an NUTM, and their construction has been regarded as impossible. Here, we demonstrate the first physical design of an NUTM. This design is based on Thue string rewriting systems, and thereby avoids the limitations of most previous DNA computing schemes: all the computation is local (simple edits to strings) so there is no need for communication, and there is no need to order operations. The design exploits DNA's ability to replicate to execute an exponential number of computational paths in P time. Each Thue rewriting step is embodied in a DNA edit implemented using a novel combination of polymerase chain reactions and site-directed mutagenesis. We demonstrate that the design works using both computational modelling and in vitro molecular biology experimentation: the design is thermodynamically favourable, microprogramming can be used to encode arbitrary Thue rules, all classes of Thue rule can be implemented, and non-deterministic rule implementation. In an NUTM, the resource limitation is space, which contrasts with classical UTMs and QUTMs where it is time. This fundamental difference enables an NUTM to trade space for time, which is significant for both theoretical computer science and physics. It is also of practical importance, for to quote Richard Feynman ‘there's plenty of room at the bottom’. This means that a desktop DNA NUTM could potentially utilize more processors than all the electronic computers in the world combined, and thereby outperform the world's current fastest supercomputer, while consuming a tiny fraction of its energy.