Structure, Physicochemical Properties and Biological Activity of Lipopolysaccharide from the Rhizospheric Bacterium Ochrobactrum quorumnocens T1Kr02, Containing d-Fucose Residues.

Lipopolysaccharides (LPSs) are major components of the outer membranes of Gram-negative bacteria. In this work, the structure of the O-polysaccharide of Ochrobactrum quorumnocens T1Kr02 was identified by nuclear magnetic resonance (NMR), and the physical-chemical properties and biological activity of LPS were also investigated. The NMR analysis showed that the O-polysaccharide has the following structure: →2)-ß-d-Fucf-(1→3)-ß-d-Fucp-(1→. The structure of the periplasmic glucan coextracted with LPS was established by NMR spectroscopy and chemical methods: →2)-ß-d-Glcp-(1→. Non-stoichiometric modifications were identified in both polysaccharides: 50% of d-fucofuranose residues at position 3 were O-acetylated, and 15% of d-Glcp residues at position 6 were linked with succinate. This is the first report of a polysaccharide containing both d-fucopyranose and d-fucofuranose residues. The fatty acid analysis of the LPS showed the prevalence of 3-hydroxytetradecanoic, hexadecenoic, octadecenoic, lactobacillic, and 27-hydroxyoctacosanoic acids. The dynamic light scattering demonstrated that LPS (in an aqueous solution) formed supramolecular particles with a size of 72.2 nm and a zeta-potential of -21.5 mV. The LPS solution (10 mkg/mL) promoted the growth of potato microplants under in vitro conditions. Thus, LPS of O. quorumnocens T1Kr02 can be recommended as a promoter for plants and as a source of biotechnological production of d-fucose.

Interleukin-15 Signaling in HIF-1α Regulation in Natural Killer Cells, Insights Through Mathematical Models.

Natural killer (NK) cells belong to the first line of host defense against infection and cancer. Cytokines, including interleukin-15 (IL-15), critically regulate NK cell activity, resulting in recognition and direct killing of transformed and infected target cells. NK cells have to adapt and respond in inflamed and often hypoxic areas. Cellular stabilization and accumulation of the transcription factor hypoxia-inducible factor-1α (HIF-1α) is a key mechanism of the cellular hypoxia response. At the same time, HIF-1α plays a critical role in both innate and adaptive immunity. While the HIF-1α hydroxylation and degradation pathway has been recently described with the help of mathematical methods, less is known concerning the mechanistic mathematical description of processes regulating the levels of HIF-1α mRNA and protein. In this work we combine mathematical modeling with experimental laboratory analysis and examine the dynamic relationship between HIF-1α mRNA, HIF-1α protein, and IL-15-mediated upstream signaling events in NK cells from human blood. We propose a system of non-linear ordinary differential equations with positive and negative feedback loops for describing the complex interplay of HIF-1α regulators. The experimental design is optimized with the help of mathematical methods, and numerical optimization techniques yield reliable parameter estimates. The mathematical model allows for the investigation and prediction of HIF-1α stabilization under different inflammatory conditions and provides a better understanding of mechanisms mediating cellular enrichment of HIF-1α. Thanks to the combination of in vitro experimental data and in silico predictions we identified the mammalian target of rapamycin (mTOR), the nuclear factor-κB (NF-κB), and the signal transducer and activator of transcription 3 (STAT3) as central regulators of HIF-1α accumulation. We hypothesize that the regulatory pathway proposed here for NK cells can be extended to other types of immune cells. Understanding the molecular mechanisms involved in the dynamic regulation of the HIF-1α pathway in immune cells is of central importance to the immune cell function and could be a promising strategy in the design of treatments for human inflammatory diseases and cancer.

Photo-sensitive degron variants for tuning protein stability by light.

BACKGROUND: Regulated proteolysis by the proteasome is one of the fundamental mechanisms used in eukaryotic cells to control cellular behavior. Efficient tools to regulate protein stability offer synthetic influence on molecular level on a selected biological process. Optogenetic control of protein stability has been achieved with the photo-sensitive degron (psd) module. This engineered tool consists of the photoreceptor domain light oxygen voltage 2 (LOV2) from Arabidopsis thaliana phototropin1 fused to a sequence that induces direct proteasomal degradation, which was derived from the carboxy-terminal degron of murine ornithine decarboxylase. The abundance of target proteins tagged with the psd module can be regulated by blue light if the degradation tag is exposed to the cytoplasm or the nucleus. RESULTS: We used the model organism Saccharomyces cerevisiae to generate psd module variants with increased and decreased stabilities in darkness or when exposed to blue light using site-specific and random mutagenesis. The variants were characterized as fusions to fluorescent reporter proteins and showed half-lives between 6 and 75 minutes in cells exposed to blue light and 14 to 187 minutes in darkness. In blue light, ten variants showed accelerated degradation and four variants increased stability compared to the original psd module. Measuring the dark/light ratio of selected constructs in yeast cells showed that two variants were obtained with ratios twice as high as in the wild type psd module. In silico modeling of photoreceptor variant characteristics suggested that for most cases alterations in behavior were induced by changes in the light-response of the LOV2 domain. CONCLUSIONS: In total, the mutational analysis resulted in psd module variants, which provide tuning of protein stability over a broad range by blue light. Two variants showed characteristics that are profoundly improved compared to the original construct. The modular usage of the LOV2 domain in optogenetic tools allows the usage of the mutants in the context of other applications in synthetic and systems biology as well.

A strategy for identifying fluorescence intensity profiles of single rod-shaped cells.

The extraction of fluorescence intensity profiles of single cells from image data is a common challenge in cell biology. The manual segmentation of cells, the extraction of cell orientation and finally the extraction of intensity profiles are time-consuming tasks. This article proposes a routine for the segmentation of single rod-shaped cells (i.e. without neighboring cells in a distance of the cell length) from image data combined with an extraction of intensity distributions along the longitudinal cell axis under the aggravated conditions of (i) a low spatial resolution and (ii) lacking information on the imaging system i.e. the point spread function and signal-to-noise ratio. The algorithm named cipsa transfers a new approach from particle streak velocimetry to cell classification interpreting the rod-shaped as streak-like structures. An automatic reduction of systematic errors such as photobleaching and defocusing is included to guarantee robustness of the proposed approach under the described conditions and to the convenience of end-users unfamiliar with image processing. Performance of the algorithm has been tested on image sequences with high noise level produced by an overlay of different error sources. The developed algorithm provides a user-friendly, stand-alone procedure.