8,994 research outputs found

    Discovery of selective saccharide receptors via Dynamic Combinatorial Chemistry

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    The diagnosis of various diseases and pathological conditions can be accomplished by screening and detecting glycans in cells. Certain glycans serve as excellent biomarkers, being related to cell malfunctioning, while other structurally similar glycans perform completely different functions and are naturally present in healthy cells. Despite the theoretical feasibility of using glycans as biomarkers for early disease detection, our current inability to discriminate between them limits their use. One promising approach to distinguishing between glycans is targeting their dissimilarities in saccharide chains. However, designing selective receptors for saccharides is challenging due to the complexity of these molecules. Their vast diversity, the fact that they exist in many interconvertible forms, their lack of recognisable functional groups, or the fact that they are normally heavily solvated in aqueous environments have made the design of receptors for saccharides a challenge that has kept the scientific community busy for the last 35 years. Although there have been ground-breaking discoveries in the field, improvements are needed to enhance our disease detection and risk stratification tools. To address this challenge, we employed a technique known as Dynamic Combinatorial Chemistry (DCC). DCC enables the self-formation and self-selection of the best possible receptor for a given target from a pool or library of potentially good ligands. DCC has been effective for creating receptors for biomolecules such as DNA, RNA, and proteins, but its use for discovering sugar receptors is less explored. In this work, we filled this gap by implementing DCC for screening common saccharides (glucose, galactose, mannose, and fructose) using small, simple, and inexpensive building blocks. Our results indicated that molecule 2DD, which consists of a benzene ring with 2 units of amino acid aspartic acid derivatives connected in positions 1 and 3, is the best receptor in a library of very similar structures for the saccharides glucose, galactose, and mannose. For fructose, molecule 1P, a benzene ring linked to just one unit of the amino acid phenylaldehyde, was appointed as the best receptor. The differential behaviour of fructose can provide insight into the relatively unknown processes behind molecular recognition of sugars. Molecules 2DD and 1P, as well as some other library members as negative controls, were then synthesised for further testing and DCC results were then validated by Isothermal Titration Calorimetry (ITC) and NMR techniques, proving the effectiveness of DCC as a molecular recognition tool for the creation of receptors for saccharides. Moreover, molecule 1P was found to have a high binding constant (Ka_{a} = 1762 M1^{-1}) and selectivity (50-100 times over other sugars) for fructose, which is surprisingly good considering the simplicity of the receptor. A much more challenging approach was attempted by employing short peptides as scaffolds in DCC experiments. The benefits of using peptides are numerous but can be summarised in three bullet points: customisability, flexibility, and easiness in their synthesis. Unfortunately, we encountered many difficulties for the complete functionalisation of the peptides within the Dynamic Combinatorial Library (DCL) and this approach did not yield the desired results before the research project came to an end. However, we believe in its potential and the knowledge that we gained on the topic helped to stablish the foundations on which new research will be carried out in the near future within the research group. In summary, this thesis reports the development of a rapid methodology for the discovery of selective receptors for monosaccharides, employing a library of simple and inexpensive starting building blocks. While this was a proof-of-concept study, it can be scalable to larger library sizes and to target more complex biomolecules, becoming a useful tool that could accelerate the discovery of new molecules with biomedical applications

    Molecular characterization of the PhiKo endolysin from Thermus thermophilus HB27 bacteriophage phiKo and its cryptic lytic peptide RAP-29

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    IntroductionIn the era of increasing bacterial resistance to antibiotics, new bactericidal substances are sought, and lysins derived from extremophilic organisms have the undoubted advantage of being stable under harsh environmental conditions. The PhiKo endolysin is derived from the phiKo bacteriophage infecting Gram-negative extremophilic bacterium Thermus thermophilus HB27. This enzyme shows similarity to two previously investigated thermostable type-2 amidases, the Ts2631 and Ph2119 from Thermus scotoductus bacteriophages, that revealed high lytic activity not only against thermophiles but also against Gram-negative mesophilic bacteria. Therefore, antibacterial potential of the PhiKo endolysin was investigated in the study presented here.MethodsEnzyme activity was assessed using turbidity reduction assays (TRAs) and antibacterial tests. Differential scanning calorimetry was applied to evaluate protein stability. The Collection of Anti-Microbial Peptides (CAMP) and Antimicrobial Peptide Calculator and Predictor (APD3) were used to predict regions with antimicrobial potential in the PhiKo primary sequence. The minimum inhibitory concentration (MIC) of the RAP-29 synthetic peptide was determined against Gram-positive and Gram-negative selected strains, and mechanism of action was investigated with use of membrane potential sensitive fluorescent dye 3,3′-Dipropylthiacarbocyanine iodide (DiSC3(5)).Results and discussionThe PhiKo endolysin is highly thermostable with melting temperature of 91.70°C. However, despite its lytic effect against such extremophiles as: T. thermophilus, Thermus flavus, Thermus parvatiensis, Thermus scotoductus, and Deinococcus radiodurans, PhiKo showed moderate antibacterial activity against mesophiles. Consequently, its protein sequence was searched for regions with potential antibacterial activity. A highly positively charged region was identified and synthetized (PhiKo105-133). The novel RAP-29 peptide lysed mesophilic strains of staphylococci and Gram-negative bacteria, reducing the number of cells by 3.7–7.1 log units and reaching the minimum inhibitory concentration values in the range of 2–31 μM. This peptide is unstructured in an aqueous solution but forms an α-helix in the presence of detergents. Moreover, it binds lipoteichoic acid and lipopolysaccharide, and causes depolarization of bacterial membranes. The RAP-29 peptide is a promising candidate for combating bacterial pathogens. The existence of this cryptic peptide testifies to a much wider panel of antimicrobial peptides than thought previously

    Human norovirus emergence and circulation in humans and animals

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    Structural analysis of PLD3 reveals insights into the mechanism of lysosomal 5′ exonuclease-mediated nucleic acid degradation

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    The phospholipase D (PLD) family is comprised of enzymes bearing phospholipase activity towards lipids or endo- and exonuclease activity towards nucleic acids. PLD3 is synthesized as a type II transmembrane protein and proteolytically cleaved in lysosomes, yielding a soluble active form. The deficiency of PLD3 leads to the slowed degradation of nucleic acids in lysosomes and chronic activation of nucleic acid-specific intracellular toll-like receptors. While the mechanism of PLD phospholipase activity has been extensively characterized, not much is known about how PLDs bind and hydrolyze nucleic acids. Here, we determined the high-resolution crystal structure of the luminal N-glycosylated domain of human PLD3 in its apo- and single-stranded DNA-bound forms. PLD3 has a typical phospholipase fold and forms homodimers with two independent catalytic centers via a newly identified dimerization interface. The structure of PLD3 in complex with an ssDNA-derived thymidine product in the catalytic center provides insights into the substrate binding mode of nucleic acids in the PLD family. Our structural data suggest a mechanism for substrate binding and nuclease activity in the PLD family and provide the structural basis to design immunomodulatory drugs targeting PLD3

    Investigating the role of versican in immune exclusion in triple negative breast cancer

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    Triple negative breast cancer has the highest T cell infiltrate in comparison to other subtypes of breast cancer. To try to improve the anti-tumour response of these T cells, immunotherapy has been trialled, however clinical trials showed poor results. The response to immunotherapy in solid tumours is limited and this has been attributed to the presence of the extracellular matrix (ECM). The ECM can interact with T cells biochemically or physically, affecting their trafficking in the tumour. This can cause the restriction of T cells in the stroma limiting their contact with the tumour epithelial cells, leading to an immune excluded phenotype. Identifying key components of the ECM that are associated with the restriction of immune cells can provide potential targets that could be degraded to improve anti-tumour immunity. From previous work in the lab a signature of molecules were identified which were associated with immunosuppression. In the initial analysis of these molecules in a subset of TNBC tissues, versican (VCAN) was identified as an ECM component that associates with immune cell infiltration into the tumour epithelium. VCAN is a proteoglycan which has the glycosaminoglycan chondroitin sulphate (CS) attached to the peptide backbone. Through its multiple domains and glycan post-translational modifications, VCAN has been shown to have a role in inflammation and cancer progression. To study how VCAN may affect the trafficking of T cells, I first looked at how VCAN expression associated with immune excluded tissues. It was observed that VCAN levels were higher in the epithelial zone of excluded tissues compared to inflamed tissues. CS levels were then explored within the tissues where the sulphation patterns on CS in the stroma led to the discovery of CS-C being higher in excluded tissues and CS-A being higher in inflamed tissues. To observe this effect in-vitro, VCAN was enriched from TNBC and fibroblast cell line secretions. The effect of CS was tested through chondroitinase (CSase) treatment of VCAN enriched protein in a transwell model. An increase in invasion was observed following CSase treatment of protein with high levels of CS-C. To conclude, from the study I identified that within TNBC tissues the excluded immune phenotype associates with epithelial zone expressed VCAN which has a different CS sulphation pattern compared to inflamed tissues, and this difference in sulphation inhibits T-cell trafficking in in vitro models, which can be overcome through enzymatic digestion of the CS. Therefore, targeting VCAN by degrading CS may provide a way to drive excluded tumours into an inflamed and therapy responsive phenotype. Such an approach could be coupled with immunotherapy such as cell-based T-cell therapies

    Otitis media: recent advances in otitis media vaccine development and model systems

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    Otitis media is an inflammatory disorder of the middle ear caused by airways-associated bacterial or viral infections. It is one of the most common childhood infections as globally more than 80% of children are diagnosed with acute otitis media by 3 years of age and it is a common reason for doctor’s visits, antibiotics prescriptions, and surgery among children. Otitis media is a multifactorial disease with various genetic, immunologic, infectious, and environmental factors predisposing children to develop ear infections. Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the most common culprits responsible for acute otitis media. Despite the massive global disease burden, the pathogenesis of otitis media is still unclear and requires extensive future research. Antibiotics are the preferred treatment to cure middle ear infections, however, the antimicrobial resistance rate of common middle ear pathogens has increased considerably over the years. At present, pneumococcal and influenza vaccines are administered as a preventive measure against otitis media, nevertheless, these vaccines are only beneficial in preventing carriage and/or disease caused by vaccine serotypes. Otitis media caused by non-vaccine serotype pneumococci, non-typeable H. influenza, and M. catarrhalis remain an important healthcare burden. The development of multi-species vaccines is an arduous process but is required to reduce the global burden of this disease. Many novel vaccines against S. pneumoniae, non-typeable H. influenza, and M. catarrhalis are in preclinical trials. It is anticipated that these vaccines will lower the disease burden and provide better protection against otitis media. To study disease pathology the rat, mouse, and chinchilla are commonly used to induce experimental acute otitis media to test new therapeutics, including antibiotics and vaccines. Each of these models has its advantages and disadvantages, yet there is still a need to develop an improved animal model providing a better correlated mechanistic understanding of human middle ear infections, thereby underpinning the development of more effective otitis media therapeutics. This review provides an updated summary of current vaccines against otitis media, various animal models of otitis media, their limitations, and some future insights in this field providing a springboard in the development of new animal models and novel vaccines for otitis media

    Human gut microbes’ transmission, persistence, and contribution to lactose tolerance

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    Human genotypes and their environment interact to produce selectable phenotypes. How microbes of the human gut microbiome interact with their host genotype to shape phenotype is not fully understood. Microbiota that inhabit the human body are environmentally acquired, yet many are passed intergenerationally between related family members, raising the possibility that they could act like genes. Here, I present three studies aimed at better understanding how certain gut microbiota contribute to host phenotypes. In a first study, I assessed mother to child transmission in understudied populations. I collected stool samples from 386 mother-infant pairs in Gabon and Vietnam, which are relatively under-studied for microbiome dynamics, and in Germany. Using metagenomic sequencing I characterized microbial strain diversity. I found that 25-50% of strains detected in mother-infant pairs were shared, and that strain-sharing between unrelated individuals was rare overall. These observations indicate that vertical transmission of microbes is widespread in human populations. Second, to test whether strains acquired during infancy persist into adulthood (similar to human genes), I collected stool from an adolescent previously surveyed for microbiome diversity as an infant. This dataset represents the longest follow-up to date for the persistence of strains seeded in infancy. I observed two strains that had persisted in the gut despite over 10 years passing, as well as 5 additional strains shared between the subject and his parents. Taken together, the results of these first two studies suggest that gut microbial strains persist throughout life and transmit between host-generations, dynamics more similar to those of the host’s own genome than of their environment. Third, I tested whether gut microbes could confer a phenotype (lactose tolerance) to individuals lacking the necessary genotypes (lactase persistence). I studied 784 women in Gabon, Vietnam and Germany for lactase persistence (genotype), lactose tolerance (phenotype), and characterized their gut microbiomes through metagenomic sequencing. Despite the genotype, I observed that 13% of participants were lactose tolerant by clinical criteria; I termed this novel phenotype microbially-acquired lactose tolerance (MALT). Those with MALT harbored microbiomes enriched for Bifidobacteria, a known lactose degrader. These results indicate that Bifidobacteria - which is passed intergenerationally - can confer a phenotype previously thought to be under only host genetic control. Taken together, my thesis work lends weight to the concept that specific microbes inhabiting the human gut have the potential to behave as epigenetic factors in evolution

    Antibody glycosylation in COVID-19

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    Proteomic

    An extracellular receptor tyrosine kinase motif orchestrating intracellular STAT activation

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    The ErbB4 receptor isoforms JM-a and JM-b differ within their extracellular juxtamembrane (eJM) domains. Here, ErbB4 isoforms are used as a model to address the effect of structural variation in the eJM domain of receptor tyrosine kinases (RTK) on downstream signaling. A specific JM-a-like sequence motif is discovered, and its presence or absence (in JM-b-like RTKs) in the eJM domains of several RTKs is demonstrated to dictate selective STAT activation. STAT5a activation by RTKs including the JM-a like motif is shown to involve interaction with oligosaccharides of N-glycosylated cell surface proteins such as β1 integrin, whereas STAT5b activation by JM-b is dependent on TYK2. ErbB4 JM-a- and JM-b-like RTKs are shown to associate with specific signaling complexes at different cell surface compartments using analyses of RTK interactomes and super-resolution imaging. These findings provide evidence for a conserved mechanism linking a ubiquitous extracellular motif in RTKs with selective intracellular STAT signaling

    Endoplasmic reticulum localization of phosphoinositide specific phospholipase C enzymes in U73122 cultured human osteoblasts

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    Different signal transduction pathways contribute to the differentiation and metabolic activities of osteoblasts, with special regard to the calcium-related pathway of phosphoinositide specific phospholipase C (PLC) enzyme family. PLC enzymes were demonstrated to be involved in the differentiation of osteoblasts and differently localize in the nucleus, cytoplasm or both depending on the isoform. The amino-steroid molecule U-73122 inhibits the enzymes belonging to the PLC family. In addition to the temporary block of the enzymatic activity, U-73122 promotes off-target effects, including modulation of the expression of selected PLC genes and different localization of PLC enzymes, depending on the cell line, in different cell lines. In order to evaluate possible off-target effects of the molecule in human osteoblasts, we investigated the expression of PLC genes and the localization of PLC enzymes in cultured human osteoblasts (hOBs) in the presence of low dose U-73122. Our results confirm that all PLC genes are transcribed in hOBs, that probably splicing variants of selected PLC genes are expressed and that all PLC enzymes are present in hOBs, except for PLC δ3 in quiescent hOBs at seeding. Our results confirm literature data excluding toxicity of U-73122 on cell survival. Our results indicate that U-73122 did not significantly affect the transcription of PLC genes. It acts upon the localization of PLC enzymes, as PLC enzymes are detected in cell protrusions or pseudopodia-like structures, at the nuclear or the plasma membrane, in membrane ruffles and/or in the endoplasmic reticulum
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