Synthesis of α-glucan in mycobacteria involves a hetero-octameric complex of trehalose synthase TreS and Maltokinase Pep2

Recent evidence established that the cell envelope of Mycobacterium tuberculosis, the bacillus causing tuberculosis (TB), is coated by an α-glucan-containing capsule that has been implicated in persistence in a mouse infection model. As one of three known metabolic routes to α-glucan in mycobacteria, the cytoplasmic GlgE-pathway converts trehalose to α(1 → 4),α(1 → 6)-linked glucan in 4 steps. Whether individual reaction steps, catalyzed by trehalose synthase TreS, maltokinase Pep2, and glycosyltransferases GlgE and GlgB, occur independently or in a coordinated fashion is not known. Here, we report the crystal structure of M. tuberculosis TreS, and show by small-angle X-ray scattering and analytical ultracentrifugation that TreS forms tetramers in solution. Together with Pep2, TreS forms a hetero-octameric complex, and we demonstrate that complex formation markedly accelerates maltokinase activity of Pep2. Thus, complex formation may act as part of a regulatory mechanism of the GlgE pathway, which overall must avoid accumulation of toxic pathway intermediates, such as maltose-1-phosphate, and optimize the use of scarce nutrients

Synthesis and cytotoxicity of a biotinylated CC-1065 analogue

BACKGROUND: The use of pretargeting technology for cancer imaging and treatment has made significant progress in the last few years. This approach takes advantage of the fact that biotin binds strongly to proteins avidin and streptavidin. Thus, a non-toxic tumor cell specific antibody is conjugated with avidin/streptavidin, and is administered to patients. After the antibody binds to tumor cells (usually 24–48 h); a clearing agent is given to remove the residual circulating antibodies in blood. Lastly, a toxic biotin-radioisotope conjugate is administered. Due to the small size of the biotin-radioisotope molecule and tight binding between biotin and avidin/streptavidin, the biotin-radioisotope rapidly binds to tumor cells with high specificity. CC-1065 (1) is one of a few classes of extremely potent antitumor agents, and a biotinalyted CBI-bearing CC-1065 analogue is a promising candidate to be used in the pretargeting technology to treat cancer. RESULTS: A biotinalyted CBI-bearing CC-1065 analogue, 6, was synthesized. The IC(50) of 6 was 0.7 nM against U937 cells. Compound 6 caused apototsis of U937 cells. CONCLUSIONS: For the first time, a biotinalyted CBI-bearing CC-1065 analogue, 6, was synthesized. The biotinylated 6 can serve as a model compound to explore the usefulness of non-radioactive small molecule anticancer drugs in the pretargeting strategy for cancer imaging and therapy

The structures of E. coli NfsA bound to the antibiotic nitrofurantoin; to 1,4-benzoquinone and to FMN

NfsA is a dimeric flavoprotein that catalyses the reduction in nitroaromatics and quinones by NADPH. This reduction is required for the activity of nitrofuran antibiotics. The crystal structure of free Escherichia coli NfsA and several homologues have been determined previously, but there is no structure of the enzyme with ligands. We present here crystal structures of oxidised E. coli NfsA in the presence of several ligands, including the antibiotic nitrofurantoin. Nitrofurantoin binds with the furan ring, rather than the nitro group that is reduced, near the N5 of the FMN. Molecular dynamics simulations show that this orientation is only favourable in the oxidised enzyme, while potentiometry suggests that little semiquinone is formed in the free protein. This suggests that the reduction occurs by direct hydride transfer from FMNH(−) to nitrofurantoin bound in the reverse orientation to that in the crystal structure. We present a model of nitrofurantoin bound to reduced NfsA in a viable hydride transfer orientation. The substrate 1,4-benzoquinone and the product hydroquinone are positioned close to the FMN N5 in the respective crystal structures with NfsA, suitable for reaction, but are mobile within the active site. The structure with a second FMN, bound as a ligand, shows that a mobile loop in the free protein forms a phosphate-binding pocket. NfsA is specific for NADPH and a similar conformational change, forming a phosphate-binding pocket, is likely to also occur with the natural cofactor

¹H, ¹³C and ¹⁵N NMR assignments of self-incompatibility protein homologue 15 from <i>Arabidopsis thaliana</i>

The SPH proteins are a large family of small, disulphide-bonded, secreted proteins, originally found to be involved in the self-incompatibility response in the field poppy (Papaver rhoeas). They are now known to be widely distributed in plants, many containing multiple members of this protein family. Apart from the PrsS proteins in Papaver the function of these proteins is unknown but they are thought to be involved in plant development and cell signalling. There has been no structural study of SPH proteins to date. Using the Origami strain of E. coli, we cloned and expressed one member of this family, SPH15 from Arabidopsis thaliana, as a folded thioredoxin-fusion protein, purified it from the cytosol, and cleaved it to obtain the secreted protein. We here report the assignment of the NMR spectra of SPH15, which contains 112 residues plus three N-terminal amino acids from the vector. The secondary structure propensity from TALOS+ shows that it contains eight beta strands and connecting loops. This is largely in agreement with predictions from the amino acid sequence, which show an additional C-terminal strand

Flexibility of KorA, a plasmid-encoded, global transcription regulator, in the presence and the absence of its operator

The IncP (Incompatibility group P) plasmids are important carriers in the spread of antibiotic resistance across Gram-negative bacteria. Gene expression in the IncP-1 plasmids is stringently controlled by a network of four global repressors, KorA, KorB, TrbA and KorC interacting cooperatively. Intriguingly, KorA and KorB can act as co-repressors at varying distances between their operators, even when they are moved to be on opposite sides of the DNA. KorA is a homodimer with the 101-amino acid subunits, folding into an N-terminal DNA-binding domain and a C-terminal dimerization domain. In this study, we have determined the structures of the free KorA repressor and two complexes each bound to a 20-bp palindromic DNA duplex containing its consensus operator sequence. Using a combination of X-ray crystallography, nuclear magnetic resonance spectroscopy, SAXS and molecular dynamics calculations, we show that the linker between the two domains is very flexible and the protein remains highly mobile in the presence of DNA. This flexibility allows the DNA-binding domains of the dimer to straddle the operator DNA on binding and is likely to be important in cooperative binding to KorB. Unexpectedly, the C-terminal domain of KorA is structurally similar to the dimerization domain of the tumour suppressor p53

The Seventh Data Release of the Sloan Digital Sky Survey

This paper describes the Seventh Data Release of the Sloan Digital Sky Survey (SDSS), marking the completion of the original goals of the SDSS and the end of the phase known as SDSS-II. It includes 11663 deg^2 of imaging data, with most of the roughly 2000 deg^2 increment over the previous data release lying in regions of low Galactic latitude. The catalog contains five-band photometry for 357 million distinct objects. The survey also includes repeat photometry over 250 deg^2 along the Celestial Equator in the Southern Galactic Cap. A coaddition of these data goes roughly two magnitudes fainter than the main survey. The spectroscopy is now complete over a contiguous area of 7500 deg^2 in the Northern Galactic Cap, closing the gap that was present in previous data releases. There are over 1.6 million spectra in total, including 930,000 galaxies, 120,000 quasars, and 460,000 stars. The data release includes improved stellar photometry at low Galactic latitude. The astrometry has all been recalibrated with the second version of the USNO CCD Astrograph Catalog (UCAC-2), reducing the rms statistical errors at the bright end to 45 milli-arcseconds per coordinate. A systematic error in bright galaxy photometr is less severe than previously reported for the majority of galaxies. Finally, we describe a series of improvements to the spectroscopic reductions, including better flat-fielding and improved wavelength calibration at the blue end, better processing of objects with extremely strong narrow emission lines, and an improved determination of stellar metallicities. (Abridged)Comment: 20 pages, 10 embedded figures. Accepted to ApJS after minor correction

Life values as predictors of pain, disability and sick leave among Swedish registered nurses: a longitudinal study

<p>Abstract</p> <p>Background</p> <p>Prospective studies on high-risk populations, such as subgroups of health care staff, are limited, especially prospective studies among staff not on sick-leave. This paper is a report of a longitudinal study conducted to describe and compare the importance and consistency of life domains among registered nurses (RNs) working in a Swedish hospital and evaluate a model based on the consistency of valued life domains for prediction of pain, disability and sick leave.</p> <p>Method</p> <p>Importance and consistency ratings of life values, in 9 domains, were collected during 2003 and 2006 from 196 RNs using the Valued Living Questionnaire (VLQ). Logistic regression analyses were used for prediction of pain, disability and sick leave at the three-year follow-up. The predictors family relations, marriage couples/intimate relations, parenting, friends/social life, work, education, leisure time, psychological well-being, and physical self-care were used at baseline.</p> <p>Results</p> <p>RNs rated life values regarding parenting as most important and with the highest consistency both at baseline and at follow-up. No significant differences were found between RNs' ratings of importance and consistency over the three-year period, except for friends/social relations that revealed a significant decrease in importance at follow-up. The explanatory models for pain, disability and sick leave significantly predicted pain and disability at follow-up. The odds of having pain were significantly increased by one consistency rating (psychological well-being), while the odds were significantly decreased by physical self-care. In the model predicting disability, consistency in psychological well-being and education significantly increased the odds of being disabled, while consistency in physical self-care significantly decreased the odds.</p> <p>Conclusion</p> <p>The results suggest that there might be a link between intra-individual factors reflecting different aspects of appraised life values and musculoskeletal pain (MSP).</p

Quantitative characterization of metabolism and metabolic shifts during growth of the new human cell line AGE1.HN using time resolved metabolic flux analysis

For the improved production of vaccines and therapeutic proteins, a detailed understanding of the metabolic dynamics during batch or fed-batch production is requested. To study the new human cell line AGE1.HN, a flexible metabolic flux analysis method was developed that is considering dynamic changes in growth and metabolism during cultivation. This method comprises analysis of formation of cellular components as well as conversion of major substrates and products, spline fitting of dynamic data and flux estimation using metabolite balancing. During batch cultivation of AGE1.HN three distinct phases were observed, an initial one with consumption of pyruvate and high glycolytic activity, a second characterized by a highly efficient metabolism with very little energy spilling waste production and a third with glutamine limitation and decreasing viability. Main events triggering changes in cellular metabolism were depletion of pyruvate and glutamine. Potential targets for the improvement identified from the analysis are (i) reduction of overflow metabolism in the beginning of cultivation, e.g. accomplished by reduction of pyruvate content in the medium and (ii) prolongation of phase 2 with its highly efficient energy metabolism applying e.g. specific feeding strategies. The method presented allows fast and reliable metabolic flux analysis during the development of producer cells and production processes from microtiter plate to large scale reactors with moderate analytical and computational effort. It seems well suited to guide media optimization and genetic engineering of producing cell lines

The LifeCycle Project-EU Child Cohort Network : a federated analysis infrastructure and harmonized data of more than 250,000 children and parents

Early life is an important window of opportunity to improve health across the full lifecycle. An accumulating body of evidence suggests that exposure to adverse stressors during early life leads to developmental adaptations, which subsequently affect disease risk in later life. Also, geographical, socio-economic, and ethnic differences are related to health inequalities from early life onwards. To address these important public health challenges, many European pregnancy and childhood cohorts have been established over the last 30 years. The enormous wealth of data of these cohorts has led to important new biological insights and important impact for health from early life onwards. The impact of these cohorts and their data could be further increased by combining data from different cohorts. Combining data will lead to the possibility of identifying smaller effect estimates, and the opportunity to better identify risk groups and risk factors leading to disease across the lifecycle across countries. Also, it enables research on better causal understanding and modelling of life course health trajectories. The EU Child Cohort Network, established by the Horizon2020-funded LifeCycle Project, brings together nineteen pregnancy and childhood cohorts, together including more than 250,000 children and their parents. A large set of variables has been harmonised and standardized across these cohorts. The harmonized data are kept within each institution and can be accessed by external researchers through a shared federated data analysis platform using the R-based platform DataSHIELD, which takes relevant national and international data regulations into account. The EU Child Cohort Network has an open character. All protocols for data harmonization and setting up the data analysis platform are available online. The EU Child Cohort Network creates great opportunities for researchers to use data from different cohorts, during and beyond the LifeCycle Project duration. It also provides a novel model for collaborative research in large research infrastructures with individual-level data. The LifeCycle Project will translate results from research using the EU Child Cohort Network into recommendations for targeted prevention strategies to improve health trajectories for current and future generations by optimizing their earliest phases of life.Peer reviewe