Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification

Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.     

11-(pyridinylphenyl)steroids--a new class of mixed-profile progesterone agonists/antagonists

Recently, a new class (often referred to as SPRMs: selective progesterone receptor modulators) of progesterone receptor ligands with mixed agonist/antagonist properties has been described. Such compounds are envisaged, for example, as treatment for endometriosis, uterine fibroids, and leiomyomas. Existing SPRMs include Asoprisnil 1 and Uliprisnil acetate 2. In our hands, however, these compounds proved to have a predominantly or exclusively antagonistic in vitro profile, which may make this type of compound less attractive, for example, as contraceptives. We therefore aimed at a class of mixed-profile compounds that would show a more evenly balanced agonist/antagonist profile. A novel class of 11beta-[4-(heteroaryl)phenyl]-substituted pregnanes was identified that displayed the desired balance. Contrary to known SPRMs, this novel class of MPP (mixed-profile progestagen) was found to have a truly mixed activity, including a sizeable agonist component.     

Activation of Cu,Zn-Superoxide Dismutase in the Absence of Oxygen and the Copper Chaperone CCS

Eukaryotic Cu,Zn-superoxide dismutases (SOD1s) are generally thought to acquire the essential copper cofactor and intramolecular disulfide bond through the action of the CCS copper chaperone. However, several metazoan SOD1s have been shown to acquire activity in vivo in the absence of CCS, and the Cu,Zn-SOD from Caenorhabditis elegans has evolved complete independence from CCS. To investigate SOD1 activation in the absence of CCS, we compared and contrasted the CCS-independent activation of C. elegans and human SOD1 to the strict CCS-dependent activation of Saccharomyces cerevisiae SOD1. Using a yeast expression system, both pathways were seen to acquire copper derived from cell surface transporters and compete for the same intracellular pool of copper. Like CCS, CCS-independent activation occurs rapidly with a preexisting pool of apo-SOD1 without the need for new protein synthesis. The two pathways, however, strongly diverge when assayed for the SOD1 disulfide. SOD1 molecules that are activated without CCS exhibit disulfide oxidation in vivo without oxygen and under copper-depleted conditions. The strict requirement for copper, oxygen, and CCS in disulfide bond oxidation appears exclusive to yeast SOD1, and we find that a unique proline at position 144 in yeast SOD1 is responsible for this disulfide effect. CCS-dependent and -independent pathways also exhibit differential requirements for molecular oxygen. CCS activation of SOD1 requires oxygen, whereas the CCS-independent pathway is able to activate SOD1s even under anaerobic conditions. In this manner, Cu,Zn-SOD from metazoans may retain activity over a wide range of physiological oxygen tensions.Oxygen is essential for aerobic respiration, but reactive byproducts of oxygen metabolism, such as the superoxide anion, can damage cellular molecules, including proteins, DNA, and lipids (1–3). SOD1s (copper- and zinc-containing superoxide dismutases) provide the primary defense against superoxide damage by catalytically removing it through a disproportionation reaction (4). This reaction involves redox cycling at the copper active site (5). SOD1s require several post-translational modifications to form an active molecule. Copper and zinc are bound by the enzyme, and an intramolecular disulfide bond is formed between two conserved cysteine residues. Although the zinc ion and disulfide bond are not directly involved in the disproportionation reaction, these modifications are required for proper stability and formation of the active site (6–10). The presence of an intramolecular disulfide bond is intriguing, given the fact that the cytosol favors reduced thiols.The activity of SOD1s in vivo is largely controlled through the aforementioned post-translational modifications. Most of what is currently known about activation of SOD1 in vivo has emerged through studies of the bakers'' yeast Saccharomyces cerevisiae SOD1. Here insertion of the catalytic copper requires the action of the copper chaperone for SOD³ (CCS) (11). CCS physically interacts with SOD1 to deliver the copper ion and catalyze the disulfide bond formation in an oxygen-dependent manner (12–15). In fact, S. cerevisiae SOD1 (ySOD1) is completely dependent on CCS for insertion of the catalytic copper and oxidation of the disulfide bond (11, 15, 16).Although ySOD1 is dependent on CCS for activity, other eukaryotic SOD1s are not. Mouse and human SOD1 (hSOD1), when expressed in CCS^−/− mouse fibroblasts and in ccs1Δ yeast, still retain some SOD1 activity (17–19). Moreover, the genome for the nematode Caenorhabditis elegans does not contain a CCS-like gene, yet harbors several Cu,Zn-SODs. Previous studies with C. elegans SOD-1 (wSOD-1) have shown that this SOD is activated completely independently of CCS (20). Together, these studies present a strong case for a second SOD1 activation mechanism independent of CCS.There must be inherent differences in SOD1 sequences that dictate whether the enzyme uses CCS or the CCS-independent pathway or both. Through targeted mutagenesis, sequences near the C terminus have been previously identified as being important (19). Yeast SOD1 contains dual prolines at positions 142 and 144, which when mutated in combination allow for CCS-independent activation. Conversely, hSOD1 and wSOD-1 contain non-proline residues at these positions, and if dual prolines are introduced, then CSS-independent activation is blocked (19, 20). How this pair of prolines influences SOD1 activation is not understood.It is interesting that nature has developed two activation mechanisms for such a key enzyme in oxidative stress protection, and these are not likely to be redundant. It was previously predicted that the two pathways draw upon distinct sources of copper (19), since the addition of the catalytic copper ion is limiting for enzyme activation. However, since disulfide oxidation is also limiting for enzyme activity, it is possible that the two pathways diverge at this level. In the current study, we investigate the requirements and regulation of the CCS-dependent and -independent SOD1 activation pathways. Our results strongly indicate that the two pathways do not diverge at the level of upstream copper transporter sources or the kinetics of copper incorporation into SOD1 but rather at the level of disulfide bond formation. Copper is required for CCS-mediated disulfide bond oxidation in yeast SOD1, whereas SOD1s that can be activated without CCS show no such requirement for copper in disulfide oxidation. Moreover, oxygen is required for enzyme activation through CCS, but the CCS-independent pathway is able to bypass the need for molecular oxygen. This allows for significant SOD1 activity to be found at a variety of oxygen concentrations by utilizing two activation pathways.     

Probing the role of interfacial residues in a dimerization inhibitor of HIV-1 protease.

The importance of each side chain of a cross-linked interfacial peptide inhibitor of HIV-1 protease was evaluated using an alanine scanning approach. Whereas the parent inhibitor has an IC50 value of 350 nM, values for the mutations reported here range from 280-9200 nM. The relative importance or each residue was thus assigned and correlated to the solvent accessible surface area (SASA) exposed upon mutation.     

Modulation of antibody galactosylation through feeding of uridine, manganese chloride, and galactose

Through process transfer and optimization for increased antibody production to 3 g/L for a GS-CHO cell line, an undesirable drop in antibody Fc galactosylation was observed. Uridine (U), manganese chloride (M), and galactose (G), constituents involved in the intracellular galactosylation process, were evaluated in 2-L bioreactors for their potential to specifically increase antibody galactosylation. These components were placed in the feed medium at proportionally increasing concentrations from 0 to 20 × UMG, where a 1× concentration of U was 1 mM, a 1× concentration of M was 0.002 mM, and a 1× concentration of G was 5 mM. Antibody galactosylation increased rapidly from 3% at 0× UMG up to 21% at 8× UMG and then more slowly to 23% at 20× UMG. The increase was primarily due to a shift from G0F to G1F, with minimal impact on other glycoforms or product quality attributes. Cell culture performance was largely not impacted by addition of up to 20× UMG except for suppression of glucose consumption and lactate production at 16 and 20× UMG and a slight drop in antibody concentration at 20× UMG. Higher accumulation of free galactose in the medium was observed at 8× UMG and above, coincident with achieving the plateau of maximal galactosylation. A concentration of 4× UMG resulted in achieving the target of 18% galactosylation at 2-L scale, a result that was reproduced in a 1,000-L run. Follow-up studies to evaluate the addition of each component individually up to 12× concentration revealed that the effect was synergistic; the combination of all three components gave a higher level of galactosylation than addition of the each effect independently. The approach was found generally useful since a second cell line responded similarly, with an increase in galactosylation from 5% to 29% from 0 to 8× UMG and no further increase or impact on culture performance up to 12× UMG. These results demonstrate a useful approach to provide exact and specific control of antibody galactosylation through manipulation of the concentrations of uridine, manganese chloride, and galactose in the cell culture medium.     

Unidirectional cross-activation of GRPR by MOR1D uncouples itch and analgesia induced by opioids

Spinal opioid-induced itch, a prevalent side effect of pain management, has been proposed to result from pain inhibition. We now report that the μ-opioid receptor (MOR) isoform MOR1D is essential for morphine-induced scratching (MIS), whereas the isoform MOR1 is required only for morphine-induced analgesia (MIA). MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, relaying itch information. We show that morphine triggers internalization of both GRPR and MOR1D, whereas GRP specifically triggers GRPR internalization and morphine-independent scratching. Providing potential insight into opioid-induced itch prevention, we demonstrate that molecular and pharmacologic inhibition of PLCβ3 and IP3R3, downstream effectors of GRPR, specifically block MIS but not MIA. In addition, blocking MOR1D-GRPR association attenuates MIS but not MIA. Together, these data suggest that opioid-induced itch is an active process concomitant with but independent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D heterodimerization.     

Elaborate visual and acoustic signals evolve independently in a large,phenotypically diverse radiation of songbirds

The concept of a macroevolutionary trade-off among sexual signals has a storied history in evolutionary biology. Theory predicts that if multiple sexual signals are costly for males to produce or maintain and females prefer a single, sexually selected trait, then an inverse correlation between sexual signal elaborations is expected among species. However, empirical evidence for what has been termed the ‘transfer hypothesis’ is mixed, which may reflect different selective pressures among lineages, evolutionary covariates or methodological differences among studies. Here, we examine interspecific correlations between song and plumage elaboration in a phenotypically diverse, widespread radiation of songbirds, the tanagers. The tanagers (Thraupidae) are the largest family of songbirds, representing nearly 10% of all songbirds. We assess variation in song and plumage elaboration across 301 species, representing the largest scale comparative study of multimodal sexual signalling to date. We consider whether evolutionary covariates, including habitat, structural and carotenoid-based coloration, and subfamily groupings influence the relationship between song and plumage elaboration. We find that song and plumage elaboration are uncorrelated when considering all tanagers, although the relationship between song and plumage complexity varies among subfamilies. Taken together, we find that elaborate visual and vocal sexual signals evolve independently among tanagers.     

Computing Exact p-values for a Cross-correlation Shotgun Proteomics Score Function

The core of every protein mass spectrometry analysis pipeline is a function that assesses the quality of a match between an observed spectrum and a candidate peptide. We describe a procedure for computing exact p-values for the oldest and still widely used score function, SEQUEST XCorr. The procedure uses dynamic programming to enumerate efficiently the full distribution of scores for all possible peptides whose masses are close to that of the spectrum precursor mass. Ranking identified spectra by p-value rather than XCorr significantly reduces variance because of spectrum-specific effects on the score. In combination with the Percolator postprocessor, the XCorr p-value yields more spectrum and peptide identifications at a fixed false discovery rate than Mascot, X!Tandem, Comet, and MS-GF+ across a variety of data sets.A high-throughput proteomics experiment generates many thousands of candidate hypotheses, only a fraction of which are true and an even smaller fraction of which are of significant biological interest. Consequently, the accurate and efficient assignment of statistical confidence estimates to identified fragmentation spectra is critical to making efficient use of shotgun proteomics data sets.Historically, the field has shifted from focusing on discrimination—the ability of a search engine to distinguish between correct and incorrect spectrum identifications—to calibration. If a score function is well-calibrated, then the score of x assigned to one spectrum is directly comparable to a score of x assigned to a different spectrum. A well-known example of poor calibration is the distribution of SEQUEST XCorr scores produced on spectra of varying charges (Fig. 1A), such that a score of 1.8 for a doubly charged (2+) spectrum indicates a good quality identification, whereas the same score assigned to a 3+ spectrum corresponds to a much poorer match. Improving the calibration of a given score function across spectra can lead to large improvements in the number of identified spectra at a fixed statistical confidence threshold. Score calibration can be carried out using empirical curve fitting procedures to estimate p-values (1, 2, 3) or, more recently, using dynamic programming to calculate an exact p-value for each observed score (4). Machine learning postprocessors such as PeptideProphet (5) and Percolator (6) simultaneously calibrate scores and incorporate additional information, leading to even larger improvements in identification rates.Open in a separate windowFig. 1.Distribution of scores for charge 2+ and charge 3+ spectra from yeast data set. A, Standard XCorr scores. B, XCorr Šidák-corrected p-values.In this work, we describe a dynamic programming method for computing exact p-values for the oldest and one of the most widely used score functions, SEQUEST XCorr (7, 8). We demonstrate analytically and empirically that the resulting p-values are valid relative to a widely accepted null model. Furthermore, we show that, across a variety of data sets, our XCorr p-value yields significantly improved statistical power relative to competing, state-of-the-art methods, including SEQUEST, Mascot (9), X!Tandem (10), and Comet (3), and is competitive with other dynamic programming-based calibration methods like MS-GF+ (11). Strikingly, the improved calibration from our scoring scheme is complementary to that provided by Percolator, so that the combination of the two methods yields even better results, evaluated both at the spectrum and peptide levels.