A Minimal Model of Metabolism Based Chemotaxis

Since the pioneering work by Julius Adler in the 1960's, bacterial chemotaxis has been predominantly studied as metabolism-independent. All available simulation models of bacterial chemotaxis endorse this assumption. Recent studies have shown, however, that many metabolism-dependent chemotactic patterns occur in bacteria. We hereby present the simplest artificial protocell model capable of performing metabolism-based chemotaxis. The model serves as a proof of concept to show how even the simplest metabolism can sustain chemotactic patterns of varying sophistication. It also reproduces a set of phenomena that have recently attracted attention on bacterial chemotaxis and provides insights about alternative mechanisms that could instantiate them. We conclude that relaxing the metabolism-independent assumption provides important theoretical advances, forces us to rethink some established pre-conceptions and may help us better understand unexplored and poorly understood aspects of bacterial chemotaxis

Further investigation of confirmed urinary tract infection (UTI) in children under five years: a systematic review.

Background: Further investigation of confirmed UTI in children aims to prevent renal scarring and future complications. Methods: We conducted a systematic review to determine the most effective approach to the further investigation of confirmed urinary tract infection (UTI) in children under five years of age. Results: 73 studies were included. Many studies had methodological limitations or were poorly reported. Effectiveness of further investigations: One study found that routine imaging did not lead to a reduction in recurrent UTIs or renal scarring. Diagnostic accuracy: The studies do not support the use of less invasive tests such as ultrasound as an alternative to renal scintigraphy, either to rule out infection of the upper urinary tract (LR- = 0.57, 95%CI: 0.47, 0.68) and thus to exclude patients from further investigation or to detect renal scarring (LR+ = 3.5, 95% CI: 2.5, 4.8). None of the tests investigated can accurately predict the development of renal scarring. The available evidence supports the consideration of contrast-enhanced ultrasound techniques for detecting vesico-ureteric reflux (VUR), as an alternative to micturating cystourethrography (MCUG) (LR+ = 14.1, 95% CI: 9.5, 20.8; LR- = 0.20, 95%CI: 0.13, 0.29); these techniques have the advantage of not requiring exposure to ionising radiation. Conclusion: There is no evidence to support the clinical effectiveness of routine investigation of children with confirmed UTI. Primary research on the effectiveness, in terms of improved patient outcome, of testing at all stages in the investigation of confirmed urinary tract infection is urgently required

Promoter methylation of CDKN2A and lack of p16 expression characterize patients with hepatocellular carcinoma

<p>Abstract</p> <p>Background</p> <p>The product of CDKN2A, p16 is an essential regulator of the cell cycle controlling the entry into the S-phase. Herein, we evaluated CDKN2A promoter methylation and p16 protein expression for the differentiation of hepatocellular carcinoma (HCC) from other liver tumors.</p> <p>Methods</p> <p>Tumor and corresponding non-tumor liver tissue samples were obtained from 85 patients with liver tumors. CDKN2A promoter methylation was studied using MethyLight technique and methylation-specific PCR (MSP). In the MethyLight analysis, samples with ≥ 4% of PMR (percentage of methylated reference) were regarded as hypermethylated. p16 expression was evaluated by immunohistochemistry in tissue sections (n = 148) obtained from 81 patients using an immunoreactivity score (IRS) ranging from 0 (no expression) to 6 (strong expression).</p> <p>Results</p> <p>Hypermethylation of the CDKN2A promoter was found in 23 HCCs (69.7%; mean PMR = 42.34 ± 27.8%), six (20.7%; mean PMR = 31.85 ± 18%) liver metastases and in the extralesional tissue of only one patient. Using MSP, 32% of the non-tumor (n = 85), 70% of the HCCs, 40% of the CCCs and 24% of the liver metastases were hypermethylated. Correspondingly, nuclear p16 expression was found immunohistochemically in five (10.9%, mean IRS = 0.5) HCCs, 23 (92%; mean IRS = 4.9) metastases and only occasionally in hepatocytes of non-lesional liver tissues (mean IRS = 1.2). The difference of CDKN2A-methylation and p16 protein expression between HCCs and liver metastases was statistically significant (p < 0.01, respectively).</p> <p>Conclusion</p> <p>Promoter methylation of CDKN2A gene and lack of p16 expression characterize patients with HCC.</p

Flux balance analysis of primary metabolism in Chlamydomonas reinhardtii

Background Photosynthetic organisms convert atmospheric carbon dioxide into numerous metabolites along the pathways to make new biomass. Aquatic photosynthetic organisms, which fix almost half of global inorganic carbon, have great potential: as a carbon dioxide fixation method, for the economical production of chemicals, or as a source for lipids and starch which can then be converted to biofuels. To harness this potential through metabolic engineering and to maximize production, a more thorough understanding of photosynthetic metabolism must first be achieved. A model algal species, C. reinhardtii, was chosen and the metabolic network reconstructed. Intracellular fluxes were then calculated using flux balance analysis (FBA). Results The metabolic network of primary metabolism for a green alga, C. reinhardtii, was reconstructed using genomic and biochemical information. The reconstructed network accounts for the intracellular localization of enzymes to three compartments and includes 484 metabolic reactions and 458 intracellular metabolites. Based on BLAST searches, one newly annotated enzyme (fructose-1,6-bisphosphatase) was added to the Chlamydomonas reinhardtii database. FBA was used to predict metabolic fluxes under three growth conditions, autotrophic, heterotrophic and mixotrophic growth. Biomass yields ranged from 28.9 g per mole C for autotrophic growth to 15 g per mole C for heterotrophic growth. Conclusion The flux balance analysis model of central and intermediary metabolism in C. reinhardtii is the first such model for algae and the first model to include three metabolically active compartments. In addition to providing estimates of intracellular fluxes, metabolic reconstruction and modelling efforts also provide a comprehensive method for annotation of genome databases. As a result of our reconstruction, one new enzyme was annotated in the database and several others were found to be missing; implying new pathways or non-conserved enzymes. The use of FBA to estimate intracellular fluxes also provides flux values that can be used as a starting point for rational engineering of C. reinhardtii. From these initial estimates, it is clear that aerobic heterotrophic growth on acetate has a low yield on carbon, while mixotrophically and autotrophically grown cells are significantly more carbon efficient

A critical review of the research literature on Six Sigma, Lean and StuderGroup's Hardwiring Excellence in the United States: the need to demonstrate and communicate the effectiveness of transformation strategies in healthcare

<p>Abstract</p> <p>Background</p> <p>U.S. healthcare organizations are confronted with numerous and varied transformational strategies promising improvements along all dimensions of quality and performance. This article examines the peer-reviewed literature from the U.S. for evidence of effectiveness among three current popular transformational strategies: Six Sigma, Lean/Toyota Production System, and Studer's Hardwiring Excellence.</p> <p>Methods</p> <p>The English language health, healthcare management, and organizational science literature (up to December 2007) indexed in Medline, Web of Science, ABI/Inform, Cochrane Library, CINAHL, and ERIC was reviewed for studies on the aforementioned transformation strategies in healthcare settings. Articles were included if they: appeared in a peer-reviewed journal; described a specific intervention; were not classified as a pilot study; provided quantitative data; and were not review articles. Nine references on Six Sigma, nine on Lean/Toyota Production System, and one on StuderGroup meet the study's eligibility criteria.</p> <p>Results</p> <p>The reviewed studies universally concluded the implementations of these transformation strategies were successful in improving a variety of healthcare related processes and outcomes. Additionally, the existing literature reflects a wide application of these transformation strategies in terms of both settings and problems. However, despite these positive features, the vast majority had methodological limitations that might undermine the validity of the results. Common features included: weak study designs, inappropriate analyses, and failures to rule out alternative hypotheses. Furthermore, frequently absent was any attention to changes in organizational culture or substantial evidence of lasting effects from these efforts.</p> <p>Conclusion</p> <p>Despite the current popularity of these strategies, few studies meet the inclusion criteria for this review. Furthermore, each could have been improved substantially in order to ensure the validity of the conclusions, demonstrate sustainability, investigate changes in organizational culture, or even how one strategy interfaced with other concurrent and subsequent transformation efforts. While informative results can be gleaned from less rigorous studies, improved design and analysis can more effectively guide healthcare leaders who are motivated to transform their organizations and convince others of the need to employ such strategies. Demanding more exacting evaluation of projects consultants, or partnerships with health management researchers in academic settings, can support such efforts.</p

The Hetero-Hexameric Nature of a Chloroplast AAA+ FtsH Protease Contributes to Its Thermodynamic Stability

FtsH is an evolutionary conserved membrane-bound metalloprotease complex. While in most prokaryotes FtsH is encoded by a single gene, multiple FtsH genes are found in eukaryotes. Genetic and biochemical data suggest that the Arabidopsis chloroplast FtsH is a hetero-hexamer. This raises the question why photosynthetic organisms require a heteromeric complex, whereas in most bacteria a homomeric one is sufficient. To gain structural information of the possible complexes, the Arabidopsis FtsH2 (type B) and FtsH5 (type A) were modeled. An in silico study with mixed models of FtsH2/5 suggests that heteromeric hexamer structure with ratio of 4∶2 is more likely to exists. Specifically, calculation of the buried surface area at the interfaces between neighboring subunits revealed that a hetero-complex should be thermodynamically more stable than a homo-hexamer, due to the presence of additional hydrophobic and hydrophilic interactions. To biochemically assess this model, we generated Arabidopsis transgenic plants, expressing epitope-tagged FtsH2 and immuno-purified the protein. Mass-spectrometry analysis showed that FtsH2 is associated with FtsH1, FtsH5 and FtsH8. Interestingly, we found that ‘type B’ subunits (FtsH2 and FtsH8) were 2–3 fold more abundant than ‘type A’ (FtsH1 and FtsH5). The biochemical data corroborate the in silico model and suggest that the thylakoid FtsH hexamer is composed of two ‘type A’ and four ‘type B’ subunits

Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures

DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes

Structure-Function Studies of DNA Binding Domain of Response Regulator KdpE Reveals Equal Affinity Interactions at DNA Half-Sites

Expression of KdpFABC, a K+ pump that restores osmotic balance, is controlled by binding of the response regulator KdpE to a specific DNA sequence (kdpFABCBS) via the winged helix-turn-helix type DNA binding domain (KdpEDBD). Exploration of E. coli KdpEDBD and kdpFABCBS interaction resulted in the identification of two conserved, AT-rich 6 bp direct repeats that form half-sites. Despite binding to these half-sites, KdpEDBD was incapable of promoting gene expression in vivo. Structure-function studies guided by our 2.5 Å X-ray structure of KdpEDBD revealed the importance of residues R193 and R200 in the α-8 DNA recognition helix and T215 in the wing region for DNA binding. Mutation of these residues renders KdpE incapable of inducing expression of the kdpFABC operon. Detailed biophysical analysis of interactions using analytical ultracentrifugation revealed a 2∶1 stoichiometry of protein to DNA with dissociation constants of 200±100 and 350±100 nM at half-sites. Inactivation of one half-site does not influence binding at the other, indicating that KdpEDBD binds independently to the half-sites with approximately equal affinity and no discernable cooperativity. To our knowledge, these data are the first to describe in quantitative terms the binding at half-sites under equilibrium conditions for a member of the ubiquitous OmpR/PhoB family of proteins

A Novel Zinc Finger Protein Zfp277 Mediates Transcriptional Repression of the Ink4a/Arf Locus through Polycomb Repressive Complex 1

Polycomb group (PcG) proteins play a crucial role in cellular senescence as key transcriptional regulators of the Ink4a/Arf tumor suppressor gene locus. However, how PcG complexes target and contribute to stable gene silencing of the Ink4a/Arf locus remains little understood.We examined the function of Zinc finger domain-containing protein 277 (Zfp277), a novel zinc finger protein that interacts with the PcG protein Bmi1. Zfp277 binds to the Ink4a/Arf locus in a Bmi1-independent manner and interacts with polycomb repressor complex (PRC) 1 through direct interaction with Bmi1. Loss of Zfp277 in mouse embryonic fibroblasts (MEFs) caused dissociation of PcG proteins from the Ink4a/Arf locus, resulting in premature senescence associated with derepressed p16(Ink4a) and p19(Arf) expression. Levels of both Zfp277 and PcG proteins inversely correlated with those of reactive oxygen species (ROS) in senescing MEFs, but the treatment of Zfp277(-/-) MEFs with an antioxidant restored the binding of PRC2 but not PRC1 to the Ink4a/Arf locus. Notably, forced expression of Bmi1 in Zfp277(-/-) MEFs did not restore the binding of Bmi1 to the Ink4a/Arf locus and failed to bypass cellular senescence. A Zfp277 mutant that could not bind Bmi1 did not rescue Zfp277(-/-) MEFs from premature senescence.Our findings implicate Zfp277 in the transcriptional regulation of the Ink4a/Arf locus and suggest that the interaction of Zfp277 with Bmi1 is essential for the recruitment of PRC1 to the Ink4a/Arf locus. Our findings also highlight dynamic regulation of both Zfp277 and PcG proteins by the oxidative stress pathways

Crystal Structure Analysis Reveals Functional Flexibility in the Selenocysteine-Specific tRNA from Mouse

Selenocysteine tRNAs (tRNA(Sec)) exhibit a number of unique identity elements that are recognized specifically by proteins of the selenocysteine biosynthetic pathways and decoding machineries. Presently, these identity elements and the mechanisms by which they are interpreted by tRNA(Sec)-interacting factors are incompletely understood.We applied rational mutagenesis to obtain well diffracting crystals of murine tRNA(Sec). tRNA(Sec) lacking the single-stranded 3'-acceptor end ((ΔGCCA)RNA(Sec)) yielded a crystal structure at 2.0 Å resolution. The global structure of (ΔGCCA)RNA(Sec) resembles the structure of human tRNA(Sec) determined at 3.1 Å resolution. Structural comparisons revealed flexible regions in tRNA(Sec) used for induced fit binding to selenophosphate synthetase. Water molecules located in the present structure were involved in the stabilization of two alternative conformations of the anticodon stem-loop. Modeling of a 2'-O-methylated ribose at position U34 of the anticodon loop as found in a sub-population of tRNA(Sec)in vivo showed how this modification favors an anticodon loop conformation that is functional during decoding on the ribosome. Soaking of crystals in Mn(2+)-containing buffer revealed eight potential divalent metal ion binding sites but the located metal ions did not significantly stabilize specific structural features of tRNA(Sec).We provide the most highly resolved structure of a tRNA(Sec) molecule to date and assessed the influence of water molecules and metal ions on the molecule's conformation and dynamics. Our results suggest how conformational changes of tRNA(Sec) support its interaction with proteins