| 1. |
- Beal, Jacob, et al.
(författare)
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Robust estimation of bacterial cell count from optical density
- 2020
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Ingår i: Communications Biology. - : Springer Science and Business Media LLC. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data.
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| 2. |
- Chowdhury, Sounak, et al.
(författare)
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Streptococcus pyogenes Forms Serotype- and Local Environment-Dependent Interspecies Protein Complexes
- 2021
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Ingår i: mSystems. - 2379-5077. ; 6:5, s. 1-19
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Tidskriftsartikel (refereegranskat)abstract
- Streptococcus pyogenes is known to cause both mucosal and systemic infections in humans. In this study, we used a combination of quantitative and structural mass spectrometry techniques to determine the composition and structure of the interaction network formed between human plasma proteins and the surfaces of different S. pyogenes serotypes. Quantitative network analysis revealed that S. pyogenes forms serotype-specific interaction networks that are highly dependent on the domain arrangement of the surface-attached M protein. Subsequent structural mass spectrometry analysis and computational modeling of one of the M proteins, M28, revealed that the network structure changes across different host microenvironments. We report that M28 binds secretory IgA via two separate binding sites with high affinity in saliva. During vascular leakage mimicked by increasing plasma concentrations in saliva, the binding of secretory IgA was replaced by the binding of monomeric IgA and C4b-binding protein (C4BP). This indicates that an upsurge of C4BP in the local microenvironment due to damage to the mucosal membrane drives the binding of C4BP and monomeric IgA to M28. These results suggest that S. pyogenes has evolved to form microenvironment-dependent host-pathogen protein complexes to combat human immune surveillance during both mucosal and systemic infections. IMPORTANCE Streptococcus pyogenes (group A Streptococcus [GAS]), is a human-specific Gram-positive bacterium. Each year, the bacterium affects 700 million people globally, leading to 160,000 deaths. The clinical manifestations of S. pyogenes are diverse, ranging from mild and common infections like tonsillitis and impetigo to life-threatening systemic conditions such as sepsis and necrotizing fasciitis. S. pyogenes expresses multiple virulence factors on its surface to localize and initiate infections in humans. Among all these expressed virulence factors, the M protein is the most important antigen. In this study, we perform an in-depth characterization of the human protein interactions formed around one of the foremost human pathogens. This strategy allowed us to decipher the protein interaction networks around different S. pyogenes strains on a global scale and to compare and visualize how such interactions are mediated by M proteins.
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| 3. |
- Eisenberg, Tobias, et al.
(författare)
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Cardioprotection and lifespan extension by the natural polyamine spermidine
- 2016
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Ingår i: Nature Medicine. - : Springer Science and Business Media LLC. - 1078-8956 .- 1546-170X. ; 22:12, s. 1428-1438
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Tidskriftsartikel (refereegranskat)abstract
- Aging is associated with an increased risk of cardiovascular disease and death. Here we show that oral supplementation of the natural polyamine spermidine extends the lifespan of mice and exerts cardioprotective effects, reducing cardiac hypertrophy and preserving diastolic function in old mice. Spermidine feeding enhanced cardiac autophagy, mitophagy and mitochondrial respiration, and it also improved the mechano-elastical properties of cardiomyocytes in vivo, coinciding with increased titin phosphorylation and suppressed subclinical inflammation. Spermidine feeding failed to provide cardioprotection in mice that lack the autophagy-related protein Atg5 in cardiomyocytes. In Dahl salt-sensitive rats that were fed a high-salt diet, a model for hypertension-induced congestive heart failure, spermidine feeding reduced systemic blood pressure, increased titin phosphorylation and prevented cardiac hypertrophy and a decline in diastolic function, thus delaying the progression to heart failure. In humans, high levels of dietary spermidine, as assessed from food questionnaires, correlated with reduced blood pressure and a lower incidence of cardiovascular disease. Our results suggest a new and feasible strategy for protection against cardiovascular disease.
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| 4. |
- Gurung, Jyoti Mohan, 1984-
(författare)
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Coordinating type III secretion system biogenesis in Yersinia pseudotuberculosis
- 2020
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Various Gram-negative bacteria utilize type III secretion system (T3SS) to deliver effectors into eukaryotic host cells and establish mutualistic or pathogenic interactions. An example is the Ysc-Yop T3SS of pathogenic Yersinia species. The T3SS resembles a molecular syringe with a wide cylindrical membrane-spanning basal body that scaffolds a hollow extracellular needle with a pore-forming translocon complex crowned at the needle tip. Together they form a continuous conduit between bacteria and host cells that allow delivery of effector proteins. Dedicated actions of cytoplasmic chaperones, regulators and components of the cytoplasmic complex orchestrates hierarchical assembly of T3SS. On the basis of secretion hierarchy, proteins can be categorized as ‘early’ needle complex proteins, ‘middle’ translocators and ‘late’ Yop effectors. However, how the system recognizes, prepares and mediates temporal delivery of T3S substrates is not fully understood. Herein, we have investigated the roles of YscX and YscY (present specifically in the Ysc family of T3SS), as well as YopN-TyeA (broadly distributed among T3SS families) to provide a better understanding of some of the molecular mechanisms governing spatiotemporal control of T3SS assembly.Despite reciprocal YscX-YscY binary and YscX-YscY-SctV ternary interactions between the member proteins, functional interchangeability in Yersinia was not successful. This revealed YscX and YscY must perform functions unique to Yersinia T3SS. Defined domain swapping and site-directed mutagenesis identified two highly conserved cysteine residues important for YscX function. Moreover, the N-terminal region of YscX harboured an independent T3S signal. Manipulating the YscX N-terminus by exchanging it with equivalent secretion signals from different T3S substrates abrogated T3S activity. This was explained by the need for the YscX N-terminus to correctly localize and/or assemble the ‘early’ SctI inner adapter and SctF needle protein. Therefore, N-terminal YscX performs dual functions; one as a secretion signal and the other as a structural signal to control early stage assembly of T3SS.In Ysc-Yop T3SS, YopN-TyeA complex is involved in the later stage of T3SS assembly, inhibiting Yops secretion until host cell contact is achieved. Analysis of the YopN C-terminus identified a specific domain stretching 279-287 critical for regulating Ysc-Yop T3SS activity. The regulation was mediated by specific hydrophobic contacts between W279 of YopN and F8 of TyeA.In conclusion, this work has provided novel molecular mechanisms regarding the spatiotemporal assembly of T3SS. While the N-terminal region of YscX contributes to the early stage of T3SS assembly, the C-terminal region of YopN is critical for regulating Ysc-Yop activity at a later stage of T3SS assembly.
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| 5. |
- Heil, Jan, et al.
(författare)
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Sarcopenia predicts reduced liver growth and reduced resectability in patients undergoing portal vein embolization before liver resection-A DRAGON collaborative analysis of 306 patients
- 2022
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Ingår i: HPB. - : ELSEVIER SCI LTD. - 1365-182X .- 1477-2574. ; 24:3, s. 413-421
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Tidskriftsartikel (refereegranskat)abstract
- Background: After portal vein embolization (PVE) 30% fail to achieve liver resection. Malnutrition is a modifiable risk factor and can be assessed by radiological indices. This study investigates, if sarcopenia affects resectability and kinetic growth rate (KGR) after PVE. Methods: A retrospective study was performed of the outcome of PVE at 8 centres of the DRAGON collaborative from 2010 to 2019. All malignant tumour types were included. Sarcopenia was defined using gender, body mass and skeletal muscle index. First imaging after PVE was used for liver volumetry. Primary and secondary endpoints were resectability and KGR. Risk factors impacting liver growth were assessed in a multivariable analysis. Results: Eight centres identified 368 patients undergoing PVE. 62 patients (17%) had to be excluded due to unavailability of data. Among the 306 included patients, 112 (37%) were non-sarcopenic and 194 (63%) were sarcopenic. Sarcopenic patients had a 21% lower resectability rate (87% vs. 66%, p < 0.001) and a 23% reduced KGR (p = 0.02) after PVE. In a multivariable model dichotomized for KGR >2.3% standardized FLR (sFLR)/week, only sarcopenia and sFLR before embolization correlated with KGR. Conclusion: In this largest study of risk factors, sarcopenia was associated with reduced resectability and KGR in patients undergoing PVE.
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| 6. |
- Höpken, Wolfram, et al.
(författare)
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Context-based Adaptation of Ubiquitous Web Applications in Tourism
- 2008
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Ingår i: Information and Communication Technologies in Tourism 2008 Proceedings of the International Conference in Innsbruck, Austria, 2008. - Vienna & New York : Springer. - 9783211772799 ; , s. 533-544
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Konferensbidrag (refereegranskat)abstract
- The customer nowadays expects ubiquitous access to information exactly relevant within the current context during all trip phases. I.e. information has to be provided in a highly personalised way and has to be accessible anytime and anywhere by any possible devices. Consequently, information systems have to adapt themselves to the current usage context. This paper presents an approach to dynamically adapt ubiquitous web applications in the dimensions content, design and behaviour to the complete usage context (i.e. user, current situation and used device), making use of an XML-based neutral representation of the web application’s user interface. The paper discusses existing approaches for adaptive systems, presents a general-purpose adaptation model and a corresponding IT-framework and introduces the application of the proposed approach within the Innsbruck.mobile web application
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| 7. |
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| 8. |
- Mühlenkamp, Melanie C., et al.
(författare)
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Vitronectin Binds to a Specific Stretch within the Head Region of Yersinia Adhesin A and Thereby Modulates Yersinia enterocolitica Host Interaction
- 2017
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Ingår i: Journal of Innate Immunity. - : S. Karger AG. - 1662-811X .- 1662-8128. ; 9:1, s. 33-51
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Tidskriftsartikel (refereegranskat)abstract
- Complement resistance is an important virulence trait of Yersinia enterocolitica (Ye). The predominant virulence factor expressed by Ye is Yersinia adhesin A (YadA), which enables bacterial attachment to host cells and extracellular matrix and additionally allows the acquisition of soluble serum factors. The serum glycoprotein vitronectin (Vn) acts as an inhibitory regulator of the terminal complement complex by inhibiting the lytic pore formation. Here, we show YadA-mediated direct interaction of Ye with Vn and investigated the role of this Vn binding during mouse infection in vivo. Using different Yersinia strains, we identified a short stretch in the YadA head domain of Ye O:9 E40, similar to the ‘uptake region' of Y. pseudotuberculosis YPIII YadA, as crucial for efficient Vn binding. Using recombinant fragments of Vn, we found the C-terminal part of Vn, including heparin-binding domain 3, to be responsible for binding to YadA. Moreover, we found that Vn bound to the bacterial surface is still functionally active and thus inhibits C5b-9 formation. In a mouse infection model, we demonstrate that Vn reduces complement-mediated killing of Ye O:9 E40 and, thus, improved bacterial survival. Taken together, these findings show that YadA-mediated Vn binding influences Ye pathogenesis.
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| 9. |
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| 10. |
- Shen, Xiantao, et al.
(författare)
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Bacterial Imprinting at Pickering Emulsion Interfaces.
- 2014
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Ingår i: Angewandte Chemie (International edition). - : Wiley. - 1521-3773. ; 53:40, s. 10687-10690
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Tidskriftsartikel (refereegranskat)abstract
- The tendency of bacteria to assemble at oil-water interfaces can be utilized to create microbial recognition sites on the surface of polymer beads. In this work, two different groups of bacteria were first treated with acryloyl-functionalized chitosan and then used to stabilize an oil-in-water emulsion composed of cross-linking monomers that were dispersed in aqueous buffer. Polymerization of the oil phase followed by removal of the bacterial template resulted in well-defined polymer beads bearing bacterial imprints. Chemical passivation of chitosan and cell displacement assays indicate that the bacterial recognition on the polymer beads was dependent on the nature of the pre-polymer and the target bacteria. The functional materials for microbial recognition show great potential for constructing cell-cell communication networks, biosensors, and new platforms for testing antibiotic drugs.
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