Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression

BACKGROUND: In the search for generic expression strategies for mammalian protein families several bacterial expression vectors were examined for their ability to promote high yields of soluble protein. Proteins studied included cell surface receptors (Ephrins and Eph receptors, CD44), kinases (EGFR-cytoplasmic domain, CDK2 and 4), proteases (MMP1, CASP2), signal transduction proteins (GRB2, RAF1, HRAS) and transcription factors (GATA2, Fli1, Trp53, Mdm2, JUN, FOS, MAD, MAX). Over 400 experiments were performed where expression of 30 full-length proteins and protein domains were evaluated with 6 different N-terminal and 8 C-terminal fusion partners. Expression of an additional set of 95 mammalian proteins was also performed to test the conclusions of this study. RESULTS: Several protein features correlated with soluble protein expression yield including molecular weight and the number of contiguous hydrophobic residues and low complexity regions. There was no relationship between successful expression and protein pI, grand average of hydropathicity (GRAVY), or sub-cellular location. Only small globular cytoplasmic proteins with an average molecular weight of 23 kDa did not require a solubility enhancing tag for high level soluble expression. Thioredoxin (Trx) and maltose binding protein (MBP) were the best N-terminal protein fusions to promote soluble expression, but MBP was most effective as a C-terminal fusion. 63 of 95 mammalian proteins expressed at soluble levels of greater than 1 mg/l as N-terminal H10-MBP fusions and those that failed possessed, on average, a higher molecular weight and greater number of contiguous hydrophobic amino acids and low complexity regions. CONCLUSIONS: By analysis of the protein features identified here, this study will help predict which mammalian proteins and domains can be successfully expressed in E. coli as soluble product and also which are best targeted for a eukaryotic expression system. In some cases proteins may be truncated to minimise molecular weight and the numbers of contiguous hydrophobic amino acids and low complexity regions to aid soluble expression in E. coli

The influence of neighborhood characteristics on police officers\u27 encounters with persons suspected to have a serious mental illness

Objective: Police officers\u27 decisions and behaviors are impacted by the neighborhood context in which police encounters occur. For example, officers may use greater force and be more likely to make arrests in disadvantaged neighborhoods. We examined whether neighborhood characteristics influence police encounters with individuals suspected to have a serious mental illness, addictive disorder, or developmental disability. Method: We obtained data on 916 encounters from 166 officers in six jurisdictions in Georgia, USA and abstracted geographical data pertaining to the location of these encounters from United States Decennial Census data. Encounters were nested within 163 census tracts. Officer-reported data covered general encounter characteristics, the officer\u27s perception of the subject\u27s condition, subject demographics, use of force, and disposition of the encounter (e.g., arrest v. referral or transport to treatment services). Geographical data included 17 variables representing population and housing characteristics of the census tracts, from which three indices pertaining to neighborhood income, stability, and immigration status were derived using factor-analytic techniques. We then examined associations of these indices with various encounter-related variables using multi-level analysis. Results: Encounters taking place in higher-income and higher-stability census tracts were more likely to be dispatch-initiated and take place in a private home compared to those in lower-income and lower-stability neighborhoods. In higher-income neighborhoods, encounters were more likely to involve a subject suspected to have a mental illness (as opposed to an addictive disorder or developmental disability) and less likely to involve a subject suspected to have alcohol problems. The officer\u27s level of force used was not associated with neighborhood factors. Regarding disposition, although the likelihood of arrest was unrelated to neighborhood characteristics, encounters taking place in higher-immigrant neighborhoods were more likely to result in referral or transport to services than those in lower-immigrant neighborhoods. Conclusion: Neighborhood characteristics are important to consider in research on police interactions with individuals with serious mental illnesses, addictive disorders, or developmental disabilities. Such research could inform departmental training policies and procedures based on the needs of the jurisdictions served. (C) 2014 Elsevier Ltd. All rights reserved

Bubble collisions and measures of the multiverse

To compute the spectrum of bubble collisions seen by an observer in an eternally-inflating multiverse, one must choose a measure over the diverging spacetime volume, including choosing an "initial" hypersurface below which there are no bubble nucleations. Previous calculations focused on the case where the initial hypersurface is pushed arbitrarily deep into the past. Interestingly, the observed spectrum depends on the orientation of the initial hypersurface, however one's ability observe the effect rapidly decreases with the ratio of inflationary Hubble rates inside and outside one's bubble. We investigate whether this conclusion might be avoided under more general circumstances, in particular placing the observer's bubble near the initial hypersurface. We find that it is not. As a point of reference, a substantial appendix reviews relevant aspects of the measure problem of eternal inflation.Comment: 24 pages, two figures, plus 16-page appendix with one figure; v2: minor improvements and clarifications, conclusions unchanged (version to appear in JCAP

Hypothermia protects brain mitochondrial function from hypoxemia in a murine model of sepsis

Sepsis is commonly associated with brain dysfunction, but the underlying mechanisms remain unclear, although mitochondrial dysfunction and microvascular abnormalities have been implicated. We therefore assessed whether cerebral mitochondrial dysfunction during systemic endotoxemia in mice increased mitochondrial sensitivity to a further bioenergetic insult (hyoxemia), and whether hypothermia could improve outcome. Mice (C57bl/6) were injected intraperitoneally with lipopolysaccharide (LPS) (5 mg/kg; n = 85) or saline (0.01 ml/g; n = 47). Six, 24 and 48 h later, we used confocal imaging in vivo to assess cerebral mitochondrial redox potential and cortical oxygenation in response to changes in inspired oxygen. The fraction of inspired oxygen (FiO2) at which the cortical redox potential changed was compared between groups. In a subset of animals, spontaneous hypothermia was maintained or controlled hypothermia induced during imaging. Decreasing FiO2 resulted in a more reduced cerebral redox state around veins, but preserved oxidation around arteries. This pattern appeared at a higher FiO2 in LPS-injected animals, suggesting an increased sensitivity of cortical mitochondria to hypoxemia. This increased sensitivity was accompanied by a decrease in cortical oxygenation, but was attenuated by hypothermia. These results suggest that systemic endotoxemia influences cortical oxygenation and mitochondrial function, and that therapeutic hypothermia can be protective

Fluctuations of an Atomic Ledge Bordering a Crystalline Facet

When a high symmetry facet joins the rounded part of a crystal, the step line density vanishes as sqrt(r) with r denoting the distance from the facet edge. This means that the ledge bordering the facet has a lot of space to meander as caused by thermal activation. We investigate the statistical properties of the border ledge fluctuations. In the scaling regime they turn out to be non-Gaussian and related to the edge statistics of GUE multi-matrix models.Comment: Version with major revisions -- RevTeX, 4 pages, 2 figure

The CMB and the measure of the multiverse

In the context of eternal inflation, cosmological predictions depend on the choice of measure to regulate the diverging spacetime volume. The spectrum of inflationary perturbations is no exception, as we demonstrate by comparing the predictions of the fat geodesic and causal patch measures. To highlight the effect of the measure---as opposed to any effects related to a possible landscape of vacua---we take the cosmological model, including the model of inflation, to be fixed. We also condition on the average CMB temperature accompanying the measurement. Both measures predict a 1-point expectation value for the gauge-invariant Newtonian potential, which takes the form of a (scale-dependent) monopole, in addition to a related contribution to the 3-point correlation function, with the detailed form of these quantities differing between the measures. However, for both measures both effects are well within cosmic variance. Our results make clear the theoretical relevance of the measure, and at the same time validate the standard inflationary predictions in the context of eternal inflation.Comment: 28 pages; v2: reference added, some clarification

A novel retinoblastoma therapy from genomic and epigenetic analyses.

Retinoblastoma is an aggressive childhood cancer of the developing retina that is initiated by the biallelic loss of RB1. Tumours progress very quickly following RB1 inactivation but the underlying mechanism is not known. Here we show that the retinoblastoma genome is stable, but that multiple cancer pathways can be epigenetically deregulated. To identify the mutations that cooperate with RB1 loss, we performed whole-genome sequencing of retinoblastomas. The overall mutational rate was very low; RB1 was the only known cancer gene mutated. We then evaluated the role of RB1 in genome stability and considered non-genetic mechanisms of cancer pathway deregulation. For example, the proto-oncogene SYK is upregulated in retinoblastoma and is required for tumour cell survival. Targeting SYK with a small-molecule inhibitor induced retinoblastoma tumour cell death in vitro and in vivo. Thus, retinoblastomas may develop quickly as a result of the epigenetic deregulation of key cancer pathways as a direct or indirect result of RB1 loss

Quantization of Nonstandard Hamiltonian Systems

The quantization of classical theories that admit more than one Hamiltonian description is considered. This is done from a geometrical viewpoint, both at the quantization level (geometric quantization) and at the level of the dynamics of the quantum theory. A spin-1/2 system is taken as an example in which all the steps can be completed. It is shown that the geometry of the quantum theory imposes restrictions on the physically allowed nonstandard quantum theories.Comment: Revtex file, 23 pages, no figure

Monotonicity of quantum ground state energies: Bosonic atoms and stars

The N-dependence of the non-relativistic bosonic ground state energy is studied for quantum N-body systems with either Coulomb or Newton interactions. The Coulomb systems are "bosonic atoms," with their nucleus fixed, and the Newton systems are "bosonic stars". In either case there exists some third order polynomial in N such that the ratio of the ground state energy to the respective polynomial grows monotonically in N. Some applications of these new monotonicity results are discussed

Of Bounces, Branes and Bounds

Some recent studies have considered a Randall-Sundrum-like brane world evolving in the background of an anti-de Sitter Reissner-Nordstrom black hole. For this scenario, it has been shown that, when the bulk charge is non-vanishing, a singularity-free ``bounce'' universe will always be obtained. However, for the physically relevant case of a de Sitter brane world, we have recently argued that, from a holographic (c-theorem) perspective, such brane worlds may not be physically viable. In the current paper, we reconsider the validity of such models by appealing to the so-called ``causal entropy bound''. In this framework, a paradoxical outcome is obtained: these brane worlds are indeed holographically viable, provided that the bulk charge is not too small. We go on to argue that this new finding is likely the more reliable one.Comment: 15 pages, Revtex; references added and very minor change