TLR9 acts as a first-line host defense against pathogens recognizing DNA comprising unmethylated CpG motifs present in bacteria and viruses. Species- and sequence-specific recognition differences ...were demonstrated for TLR9 receptors. Activation of human (h)TLR9 requires a pair of closely positioned CpG motifs within oligodeoxyribonucleotides (ODNs), whereas mouse TLR9 is effectively activated by an ODN with a single CpG motif. Molecular model-directed mutagenesis identified two regions, site A and site B, as important for receptor activation. Amino acid residues Gln
and Arg
within site A contribute to the sequence-specific recognition by hTLR9 in determining the bias for two appropriately spaced CpG motifs within immunostimulatory ODNs. Mutation of Gln
at site B, in combination with Gln
and Arg
mutations of mouse counterparts, increased activation of hTLR9 by mouse-specific ODN, mammalian genomic DNA, and bacterial DNA. We propose that the double CpG motif sequence-specificity of hTLR9 results in decreased activation by ODNs with a lower frequency of CpG motifs, such as from mammalian genomic DNA, which increases hTLR9 selectivity for pathogen versus host DNA.
Toll-like receptors encounter a diversity of degradation products in endosomes. TLR7 and TLR8 have been shown to be activated by RNA degradation products. Here we show that although TLR9 requires ...single-stranded DNA longer than 20 nucleotides for a robust response, TLR9 activation is augmented by CpG-containing oligodeoxyribonucleotides (sODNs) as short as 2 nucleotides, which, by themselves, do not induce activation in cell cultures, as well as in mice. sODNs also activate human TLR9 in combination with ODNs containing a single CpG motif that by themselves do not activate human TLR9. The specific sequence motif of sODN and colocalization of ODN and sODN suggest that the mechanism of activation involves binding of both ODN and sODN to TLR9. sODNs augment TLR9 activation by mammalian genomic DNA indicating the role of short DNA degradation products in the endosomes in response to infection or in autoimmune disease, particularly at limiting concentrations of ODNs.
Alginate is the dominant structural polysaccharide found in brown seaweed. The industrial process systems for alginic acid isolation use formalin to insolubilize unwanted molecules like polyphenols ...so they do not get extracted along with the desired polysaccharide. In our present study, we tested six formalin alternatives that would not only allow us to isolate alginate but also retrieve the polyphenol-rich fraction which has uses in the food, cosmetic, and pharmaceutical industries. Sodium alginate was isolated from the marine macroalgae species
Laminaria hyperborea
with the aid of a common 0.2 wt% formalin solution and compared against water and polar organic fluids, i.e., pure- or a 50 wt% aqueous phase mixture of methanol, ethanol, or acetone. Beside solute molecular dissolution, several commercial mechanical pre-treatment steps were successfully tested, which showed a positive effect correlation with the yield of the produced alginate-rich fraction. Identification of alginate in the samples was confirmed using FT-IR analysis, where some possible impurities were indicated. Evaluated methodologies mostly contributed to higher biopolymer fractions, which were obtained. Furthermore, it was possible to extract up to 128 mg GAE (100 g)
−1
of dry weight of polyphenol-rich fraction in resource side streams, impacting the valorization of feedstock.
Toll-like receptor 9 (TLR9) stimulatory CpG-containing oligodeoxynucleotides (ODNs) with phosphorothioate backbones have successfully replaced the naturally occurring agonists of TLR9 in drug ...development due to their increased stability. Replacing the nonbridging oxygen with a sulfur atom in the phosphate linkage of ODNs has been accepted as having a minor impact on the chemical and physical properties of the agonists. Here, we report that the TLR9 binding site exhibits a strong bias in favor of a phosphodiester backbone over the phosphorothioate backbone of the CpG motif. Furthermore, we show that while single point mutations of W47, W96 and K690 within the TLR9 binding site retains full TLR9 activation by phosphodiester-based ODNs, activation by phosphorothioate-based ODNs is strongly impaired. The substitution of a phosphorothioate linkage for a phosphodiester linkage of just the CpG motif considerably improves the activation potency of a phosphorothioate-based oligonucleotide for human B-cells and plasmacytoid dendritic cells, as well as for mouse bone marrow-derived dendritic cells and macrophages. Our results highlight the functional significance of the phosphodiester linkage of a CpG dinucleotide for binding, which is important in designing improved immunostimulatory TLR9 agonists.
The paper begins with a brief introduction to case and case marking and a description of the Hittite corpus and the corpus of the research. The main part is devoted to the case marking of ...Sumerograms, an as yet unresearched and undescribed topic in Hittitology. The paper examines different possibilities for the case marking of Sumerograms on the level of the sentence and the functional value of phonetic complements. It also attempts to discern whether competition between formally case-marked and case-unmarked Sumerograms in the grammatical roles of subject and direct object was driven by grammatical factors, economy of writing or by other reasons.
Listeria monocytogenes is a food and soil-borne pathogen that secretes a pore-forming toxin listeriolysin O (LLO) as its major virulence factor. We tested the effects of LLO on an intestinal ...epithelial cell line Caco-2 and compared them to an unrelated pore-forming toxin equinatoxin II (EqtII). Results showed that apical application of both toxins causes a significant drop in transepithelial electrical resistance (TEER), with higher LLO concentrations or prolonged exposure time needed to achieve the same magnitude of response than with EqtII. The drop in TEER was due to pore formation and coincided with rearrangement of claudin-1 within tight junctions and associated actin cytoskeleton; however, no significant increase in permeability to fluorescein or 3 kDa FITC-dextran was observed. Influx of calcium after pore formation affected the magnitude of the drop in TEER. Both toxins exhibit similar effects on epithelium morphology and physiology. Importantly, LLO action upon the membrane is much slower and results in compromised epithelium on a longer time scale at lower concentrations than EqtII. This could favor listerial invasion in hosts resistant to E-cadherin related infection.
Predstavljeni odlomek se začne na točki, ko se Krevza skupaj s starcem in služabnicami napoti k Apolonovemu templju, da bi izvedela prerokbo. V njem smo priča Krevzini bolečini, ko izve, da ne bo ...dobila otroka in da jo je Ksut izdal. Temu se pridruži še njena zgodba o Apolonovem posilstvu in izpostavitvi otroka. Starec ji predlaga maščevanje nad Ksutom in Ionom, Krevza pa se odloči zgolj za to, da bo poskusila odstraniti Iona. Njena spletka, da bi Iona zastrupila s kapljico Gorgonine krvi, je razkrita in Krevza je obsojena na smrt s kamenjanjem. Odlomek se zaključi s tožbo zbora ob smrtni obsodbi.
•Lignin valorization is aggravated by its complex and heterogeneous structure.•Fungal enzymes are a greener and a more specific alternative for lignin conversion.•Oxidoreductase enzymes use a ...radical-based mechanism.•β-etherase system targets the β-O-4 bond.•Enzyme activity, kinetics and product yields are influenced by many factors.
Lignin is a wasted renewable source of biomass-derived value-added chemicals. However, due to its material resistance to degradation, it remains highly underutilized. In order to develop new, catalysed and more environment friendly reaction processes for lignin valorization, science has turned a selective concentrated attention to microbial enzymes. This present work looks at the enzymes involved with the main reference focus on the different elementary mechanisms of action/conversion rate kinetics. Pathways, like with laccases/peroxidases, employ radicals, which more readily result in polymerization than de-polymerization. The β-etherase system interaction of proteins targets β-O-4 ether covalent bond, which targets lower molecular weight product species. Enzymatic activity is influenced by a wide variety of different factors which need to be considered in order to obtain the best functionality and synthesis yields.
Listeriolysin O (LLO) is a cytolysin capable of forming pores in cholesterol-rich lipid membranes of host cells. It is conveniently suited for engineering a pH-governed responsiveness, due to a pH ...sensor identified in its structure that was shown before to affect its stability. Here we introduced a new level of control of its hemolytic activity by making a variant with hemolytic activity that was pH-dependent. Based on detailed structural analysis coupled with molecular dynamics and mutational analysis, we found that the bulky side chain of Tyr406 allosterically affects the pH sensor. Molecular dynamics simulation further suggested which other amino acid residues may also allosterically influence the pH-sensor. LLO was engineered to the point where it can, in a pH-regulated manner, perforate artificial and cellular membranes. The single mutant Tyr406Ala bound to membranes and oligomerized similarly to the wild-type LLO, however, the final membrane insertion step was pH-affected by the introduced mutation. We show that the mutant toxin can be activated at the surface of artificial membranes or living cells by a single wash with slightly acidic pH buffer. Y406A mutant has a high potential in development of novel nanobiotechnological applications such as controlled release of substances or as a sensor of environmental pH.
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