Spiropiperidine CCR5 antagonists

A novel series of CCR5 antagonists has been identified, utilizing leads from high-throughput screening which were further modified based on insights from competitor molecules. Lead optimization was pursued by balancing opposing trends of metabolic stability and potency. Selective and potent analogs with good pharmacokinetic properties were successfully developed.     

Helix α4 of the Bacillus thuringiensis Cry1Aa Toxin Plays a Critical Role in the Postbinding Steps of Pore Formation

Helix α4 of Bacillus thuringiensis Cry toxins is thought to play a critical role in the toxins'' mode of action. Accordingly, single-site substitutions of many Cry1Aa helix α4 amino acid residues have previously been shown to cause substantial reductions in the protein''s pore-forming activity. Changes in protein structure and formation of intermolecular disulfide bonds were investigated as possible factors responsible for the inactivity of these mutants. Incubation of each mutant with trypsin and chymotrypsin for 12 h did not reveal overt structural differences with Cry1Aa, although circular dichroism was slightly decreased in the 190- to 210-nm region for the I132C, S139C, and V150C mutants. The addition of dithiothreitol stimulated pore formation by the E128C, I132C, S139C, T142C, I145C, P146C, and V150C mutants. However, in the presence of these mutants, the membrane permeability never reached that measured for Cry1Aa, indicating that the formation of disulfide bridges could only partially explain their loss of activity. The ability of a number of inactive mutants to compete with wild-type Cry1Aa for pore formation in brush border membrane vesicles isolated from Manduca sexta was also investigated with an osmotic swelling assay. With the exception of the L147C mutant, all mutants tested could inhibit the formation of pores by Cry1Aa, indicating that they retained receptor binding ability. These results strongly suggest that helix α4 is involved mainly in the postbinding steps of pore formation.During the last few decades, the insecticidal toxins produced by Bacillus thuringiensis have been used increasingly in the forms of formulated sprays and transgenic plants for the highly focused biological control of insect pests (29). At the same time, the mechanism by which these proteins form pores in the apical membrane of midgut epithelial cells of targeted insects has been studied extensively (7, 29). In the case of the three-domain Cry toxins, specificity is mostly attributable to their capacity to bind to certain proteins located on the surface of the intestinal membrane through specific segments of domains II and III, composed mainly of β sheets (16, 27). On the other hand, membrane insertion and pore formation are thought to occur through elements of domain I, composed of a bundle of six amphipathic α-helices surrounding the highly hydrophobic helix α5 (17, 20).Several lines of evidence indicate that helices α4 and α5 play a particularly important role in these processes (3). Spectroscopic studies with synthetic peptides corresponding to domain I helices revealed that α4 and α5 have the greatest propensity for insertion into artificial membranes, although insertion and pore formation were most efficient when α4 and α5 were connected by a segment corresponding to the α4-α5 loop of the toxin (13, 14). A particularly large number of single-site mutations with altered amino acids from these helices, which lead to a strong reduction in the toxicity and pore-forming ability of the toxin, have been characterized (2, 9, 10, 15, 18, 23, 25, 30, 31, 33). Finally, a site-directed chemical modification study has provided strong evidence that α4 lines the lumens of the pores formed by the toxin (23).Recent studies have established that toxin activity is especially sensitive to modifications not only in the charged residues of α4 (31) but in most of its hydrophilic residues (15). Furthermore, the loss of activity of most of these mutants did not result from an altered selectivity or size of the pores but from a reduced pore-forming capacity of the toxin (15, 31). In order to better understand the role of α4 in the mechanism of pore formation, the present study was carried out with a series of previously characterized Cry1Aa mutants in which most of the residues from this helix were replaced by cysteines (15). By subjecting these mutants to circular dichroism (CD), protease sensitivity, pore formation inhibition, and electrophoretic mobility analyses, our data suggest that the mutations in α4 which alter the pore-forming ability of Cry1Aa do so mainly by preventing the proper oligomerization or membrane insertion of the toxin.     

COMMD1 forms oligomeric complexes targeted to the endocytic membranes via specific interactions with phosphatidylinositol 4,5-bisphosphate

Copper metabolism Murr1 domain 1 (COMMD1) is a 21-kDa protein involved in copper export from the liver, NF-kappaB signaling, HIV infection, and sodium transport. The precise function of COMMD and the mechanism through which COMMD1 performs its multiple roles are not understood. Recombinant COMMD1 is a soluble protein, yet in cells COMMD1 is largely seen as targeted to cellular membranes. Using co-localization with organelle markers and cell fractionation, we determined that COMMD1 is located in the vesicles of the endocytic pathway, whereas little COMMD1 is detected in either the trans-Golgi network or lysosomes. The mechanism of COMMD1 recruitment to cell membranes was investigated using lipid-spotted arrays and liposomes. COMMD1 specifically binds phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in the absence of other proteins and does not bind structural lipids; the phosphorylation of PtdIns at position 4 is essential for COMMD1 binding. Proteolytic sensitivity and molecular modeling experiments identified two distinct domains in the structure of COMMD1. The C-terminal domain appears sufficient for lipid binding, because both the full-length and C-terminal domain proteins bind to PtdIns(4,5)P2. In native conditions, endogenous COMMD1 forms large oligomeric complexes both in the cytosol and at the membrane; interaction with PtdIns(4,5)P2 increases the stability of oligomers. Altogether, our results suggest that COMMD1 is a scaffold protein in a distinct sub-compartment of endocytic pathway and offer first clues to its role as a regulator of structurally unrelated membrane transporters.     

Variation in exotic and native seed arrival and recruitment of bird dispersed species in subtropical forest restoration and regrowth

Invasive bird-dispersed plants often share the same suite of dispersers as co-occurring native species, resulting in a complex management issue. Integrated management strategies could incorporate manipulation of dispersal or establishment processes. To improve our understanding of these processes, we quantified seed rain, recruit and seed bank density, and species richness for bird-dispersed invasive and native species in three early successional subtropical habitats in eastern Australia: tree regrowth, shrub regrowth and native restoration plantings. We investigated the effects of environmental factors (leaf area index (LAI), distance to edge, herbaceous ground cover and distance to nearest neighbour) on seed rain, seed bank and recruit abundance. Propagule availability was not always a good predictor of recruitment. For instance, although native tree seed rain density was similar, and species richness was higher, in native plantings, compared with tree regrowth, recruit density and species richness were lower. Native plantings also received lower densities of invasive tree seed rain than did tree regrowth habitats, but supported a similar density of invasive tree recruits. Invasive shrub seed rain was recorded in highest densities in shrub regrowth sites, but recruit density was similar between habitats. We discuss the role of microsite characteristics in influencing post-dispersal processes and recruit composition, and suggest ways of manipulating these processes as part of an integrated management strategy for bird-dispersed weeds in natural areas.     

High-throughput quantification of splicing isoforms

Most human messenger RNAs (mRNAs) are alternatively spliced and many exhibit disease-specific splicing patterns. However, the contribution of most splicing events to the development and maintenance of human diseases remains unclear. As the contribution of alternative splicing events to diagnosis and prognosis is becoming increasingly recognized, it becomes important to develop precise methods to quantify the abundance of these isoforms in clinical samples. Here we present a pipeline for real-time PCR annotation of splicing events (RASE) that allows accurate identification of a large number of splicing isoforms in human tissues. The RASE automatically designed specific primer pairs for 81% of all alternative splicing events in the NCBI build 36 database. Experimentally, the majority of the RASE designed primers resulted in isoform-specific amplification suitable for quantification in human cell lines or in formalin-fixed, paraffin-embedded (FFPE) RNA extract. Using this pipeline it is now possible to rapidly identify splicing isoform signatures in different types of human tissues or to validate complete sets of data generated by microarray expression profiling and deep sequencing techniques.     

Exploiting protein structure data to explore the evolution of protein function and biological complexity

New directions in biology are being driven by the complete sequencing of genomes, which has given us the protein repertoires of diverse organisms from all kingdoms of life. In tandem with this accumulation of sequence data, worldwide structural genomics initiatives, advanced by the development of improved technologies in X-ray crystallography and NMR, are expanding our knowledge of structural families and increasing our fold libraries. Methods for detecting remote sequence similarities have also been made more sensitive and this means that we can map domains from these structural families onto genome sequences to understand how these families are distributed throughout the genomes and reveal how they might influence the functional repertoires and biological complexities of the organisms. We have used robust protocols to assign sequences from completed genomes to domain structures in the CATH database, allowing up to 60% of domain sequences in these genomes, depending on the organism, to be assigned to a domain family of known structure. Analysis of the distribution of these families throughout bacterial genomes identified more than 300 universal families, some of which had expanded significantly in proportion to genome size. These highly expanded families are primarily involved in metabolism and regulation and appear to make major contributions to the functional repertoire and complexity of bacterial organisms. When comparisons are made across all kingdoms of life, we find a smaller set of universal domain families (approx. 140), of which families involved in protein biosynthesis are the largest conserved component. Analysis of the behaviour of other families reveals that some (e.g. those involved in metabolism, regulation) have remained highly innovative during evolution, making it harder to trace their evolutionary ancestry. Structural analyses of metabolic families provide some insights into the mechanisms of functional innovation, which include changes in domain partnerships and significant structural embellishments leading to modulation of active sites and protein interactions.     

The immune inhibitory receptor LAIR-1 is highly expressed by plasmacytoid dendritic cells and acts complementary with NKp44 to control IFNα production

Plasmacytoid dendritic cells (pDCs) are a subset of dendritic cells endowed with the capacity of producing large amounts of IFNα. Here we show that the Leukocyte-Associated Ig-like Receptor-1 (LAIR-1) is abundantly expressed on pDCs (the highest expression among all leukocytes) and its cross-linking inhibits IFNα production in response to Toll-like receptor ligands. Remarkably, LAIR-1 expression in pDCs is down-regulated in the presence of interleukin (IL)-3, thus indicating coordinated functions with NKp44, another pDC inhibitory receptor, which is conversely induced by IL-3. Nevertheless, the expression of NKp44 in pDCs isolated from secondary lymphoid organs, which is thought to be influenced by IL-3, is not coupled to a decreased expression of LAIR-1. Interestingly, pDCs isolated from peripheral blood of systemic lupus erithematosus (SLE) patients express lower levels of LAIR-1 while displaying slight but consistent expression of NKp44, usually undetectable on pDCs derived from healthy donors. Using sera derived from SLE patients, we show that LAIR-1 and NKp44 display synergistic inhibitory effects on IFNα production by interleukin IL-3 cultured pDCs stimulated with DNA immunocomplexes. In conclusion, our results indicate that the inhibitory function of LAIR-1 may play a relevant role in the mechanisms controlling IFNα production by pDCs both in normal and pathological innate immune responses.     

Managed Wildfire Effects on Forest Resilience and Water in the Sierra Nevada

Fire suppression in many dry forest types has left a legacy of dense, homogeneous forests. Such landscapes have high water demands and fuel loads, and when burned can result in catastrophically large fires. These characteristics are undesirable in the face of projected warming and drying in the western US. Alternative forest and fire treatments based on managed wildfire—a regime in which fires are allowed to burn naturally and only suppressed under defined management conditions—offer a potential strategy to ameliorate the effects of fire suppression. Understanding the long-term effects of this strategy on vegetation, water, and forest resilience is increasingly important as the use of managed wildfire becomes more widely accepted. The Illilouette Creek Basin in Yosemite National Park has experienced 40 years of managed wildfire, reducing forest cover by 22%, and increasing meadow areas by 200% and shrublands by 24%. Statistical upscaling of 3300 soil moisture observations made since 2013 suggests that large increases in wetness occurred in sites where fire caused transitions from forests to dense meadows. The runoff ratio (ratio of annual runoff to precipitation) from the basin appears to be increasing or stable since 1973, compared to declines in runoff ratio for nearby, unburned watersheds. Managed wildfire appears to increase landscape heterogeneity, and likely improves resilience to disturbances, such as fire and drought, although more detailed analysis of fire effects on basin-scale hydrology is needed.     

Mitochondrial thioredoxin reductase 2 is elevated in long‐lived primate as well as rodent species and extends fly mean lifespan

In a survey of enzymes related to protein oxidation and cellular redox state, we found activity of the redox enzyme thioredoxin reductase (TXNRD) to be elevated in cells from long‐lived species of rodents, primates, and birds. Elevated TXNRD activity in long‐lived species reflected increases in the mitochondrial form, TXNRD2, rather than the cytosolic forms TXNRD1 and TXNRD3. Analysis of published RNA‐Seq data showed elevated TXNRD2 mRNA in multiple organs of longer‐lived primates, suggesting that the phenomenon is not limited to skin‐derived fibroblasts. Elevation of TXNRD2 activity and protein levels was also noted in liver of three different long‐lived mutant mice, and in normal male mice treated with a drug that extends lifespan in males. Overexpression of mitochondrial TXNRD2 in Drosophila melanogaster extended median (but not maximum) lifespan in female flies with a small lifespan extension in males; in contrast, overexpression of the cytosolic form, TXNRD1, did not produce a lifespan extension.