Reduced sleep in Drosophila Shaker mutants

Most of us sleep 7-8 h per night, and if we are deprived of sleep our performance suffers greatly; however, a few do well with just 3-4 h of sleep-a trait that seems to run in families. Determining which genes underlie this phenotype could shed light on the mechanisms and functions of sleep. To do so, we performed mutagenesis in Drosophila melanogaster, because flies also sleep for many hours and, when sleep deprived, show sleep rebound and performance impairments. By screening 9,000 mutant lines, we found minisleep (mns), a line that sleeps for one-third of the wild-type amount. We show that mns flies perform normally in a number of tasks, have preserved sleep homeostasis, but are not impaired by sleep deprivation. We then show that mns flies carry a point mutation in a conserved domain of the Shaker gene. Moreover, after crossing out genetic modifiers accumulated over many generations, other Shaker alleles also become short sleepers and fail to complement the mns phenotype. Finally, we show that short-sleeping Shaker flies have a reduced lifespan. Shaker, which encodes a voltage-dependent potassium channel controlling membrane repolarization and transmitter release, may thus regulate sleep need or efficiency.     

Regional insolation forcing of late Quaternary climate change in the Southern Hemisphere

In agreement with the Milankovitch orbital forcing hypothesis it is often assumed that glacial-interglacial climate transitions occurred synchronously in the Northern and Southern hemispheres of the Earth. It is difficult to test this assumption, because of the paucity of long, continuous climate records from the Southern Hemisphere that have not been dated by tuning them to the presumed Northern Hemisphere signals. Here we present an independently dated terrestrial pollen record from a peat bog on South Island, New Zealand, to investigate global and local factors in Southern Hemisphere climate changes during the last two glacial-interglacial cycles. Our record largely corroborates the Milankovitch model of orbital forcing but also exhibits some differences: in particular, an earlier onset and longer duration of the Last Glacial Maximum. Our results suggest that Southern Hemisphere insolation may have been responsible for these differences in timing. Our findings question the validity of applying orbital tuning to Southern Hemisphere records and suggest an alternative mechanism to the bipolar seesaw for generating interhemispheric asynchrony in climate change.     

Isolation by phage display and characterization of a single-chain antibody specific for O6-methyldeoxyguanosine

New approaches of making single chain Fv antibodies against O⁶-methyl-2′-deoxyguanosine (O⁶MdG) have been demonstrated by using the phage antibody display system. Using O⁶MdG as an antigen, 21 positive clones were identified by ELISA from this library, one of which, designated H3, specifically binds to O⁶MdG with high affinity. The H3 scFv antibody has an affinity constant (K_aff) of 5.94×10O¹¹(mol/L)^-1. H3 scFv has been successfully used to detect O⁶MdG in DNA hydrolyses from yeast or E. coli cells treated with a DNA methylating agent. To our knowledge, this is the first report of the selection of a specific scFv against DNA adducts. The results demonstrate the potential applications of the phage display technology for the detection of DNA lesions caused by mutagens and carcinogens.     

A fish survey of the White River, Nevada

In spring and summer 1991 and 1992, we surveyed fishes of the White River system, Nye and White Pine Counties, Nevada, to determine the status of natives. There are 5 known native fishes to the White River: Lepidomeda albivallis (White River spinedace), Crenichthys baileyi albivallis (Preston White River springfish), Crenichthys baileyi thermophilus (Moorman White River springfish), Catostomus clarki intermedius (White River desert sucker), and Rhinichthys osculus ssp. (White River speckled dace). All 5 had declined in range. Lepidomeda albivallis had experienced the greatest decline, with less than 50 remaining, and these were restricted to a 70-m stream reach. Rhinichthys osculus spp. was most widespread, found in 18 spring systems. Cottus bairdi (mottled sculpin) was collected for the 1st time from the White River system, where it was probably native. Protective measures should be implemented to conserve all native White River fishes to include C. bairdi.     

De novo cardiomyocytes from within the activated adult heart after injury

A significant bottleneck in cardiovascular regenerative medicine is the identification of a viable source of stem/progenitor cells that could contribute new muscle after ischaemic heart disease and acute myocardial infarction. A therapeutic ideal--relative to cell transplantation--would be to stimulate a resident source, thus avoiding the caveats of limited graft survival, restricted homing to the site of injury and host immune rejection. Here we demonstrate in mice that the adult heart contains a resident stem or progenitor cell population, which has the potential to contribute bona fide terminally differentiated cardiomyocytes after myocardial infarction. We reveal a novel genetic label of the activated adult progenitors via re-expression of a key embryonic epicardial gene, Wilm's tumour 1 (Wt1), through priming by thymosin β4, a peptide previously shown to restore vascular potential to adult epicardium-derived progenitor cells with injury. Cumulative evidence indicates an epicardial origin of the progenitor population, and embryonic reprogramming results in the mobilization of this population and concomitant differentiation to give rise to de novo cardiomyocytes. Cell transplantation confirmed a progenitor source and chromosome painting of labelled donor cells revealed transdifferentiation to a myocyte fate in the absence of cell fusion. Derived cardiomyocytes are shown here to structurally and functionally integrate with resident muscle; as such, stimulation of this adult progenitor pool represents a significant step towards resident-cell-based therapy in human ischaemic heart disease.     

Endothelial and perivascular cells maintain haematopoietic stem cells

Several cell types have been proposed to create niches for haematopoietic stem cells (HSCs). However, the expression patterns of HSC maintenance factors have not been systematically studied and no such factor has been conditionally deleted from any candidate niche cell. Thus, the cellular sources of these factors are undetermined. Stem cell factor (SCF; also known as KITL) is a key niche component that maintains HSCs. Here, using Scf(gfp) knock-in mice, we found that Scf was primarily expressed by perivascular cells throughout the bone marrow. HSC frequency and function were not affected when Scf was conditionally deleted from haematopoietic cells, osteoblasts, nestin-cre- or nestin-creER-expressing cells. However, HSCs were depleted from bone marrow when Scf was deleted from endothelial cells or leptin receptor (Lepr)-expressing perivascular stromal cells. Most HSCs were lost when Scf was deleted from both endothelial and Lepr-expressing perivascular cells. Thus, HSCs reside in a perivascular niche in which multiple cell types express factors that promote HSC maintenance.     

The deubiquitinase USP9X suppresses pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDA) remains a lethal malignancy despite much progress concerning its molecular characterization. PDA tumours harbour four signature somatic mutations in addition to numerous lower frequency genetic events of uncertain significance. Here we use Sleeping Beauty (SB) transposon-mediated insertional mutagenesis in a mouse model of pancreatic ductal preneoplasia to identify genes that cooperate with oncogenic Kras(G12D) to accelerate tumorigenesis and promote progression. Our screen revealed new candidate genes for PDA and confirmed the importance of many genes and pathways previously implicated in human PDA. The most commonly mutated gene was the X-linked deubiquitinase Usp9x, which was inactivated in over 50% of the tumours. Although previous work had attributed a pro-survival role to USP9X in human neoplasia, we found instead that loss of Usp9x enhances transformation and protects pancreatic cancer cells from anoikis. Clinically, low USP9X protein and messenger RNA expression in PDA correlates with poor survival after surgery, and USP9X levels are inversely associated with metastatic burden in advanced disease. Furthermore, chromatin modulation with trichostatin A or 5-aza-2'-deoxycytidine elevates USP9X expression in human PDA cell lines, indicating a clinical approach for certain patients. The conditional deletion of Usp9x cooperated with Kras(G12D) to accelerate pancreatic tumorigenesis in mice, validating their genetic interaction. We propose that USP9X is a major tumour suppressor gene with prognostic and therapeutic relevance in PDA.     

Coral reef diversity refutes the neutral theory of biodiversity

The global decline of coral reefs highlights the need to understand the mechanisms that regulate community structure and sustain biodiversity in these systems. The neutral theory, which assumes that individuals are demographically identical regardless of species, seeks to explain ubiquitous features of community structure and biodiversity patterns. Here we present a test of neutral-theory predictions with the use of an extensive species-level data set of Indo-Pacific coral communities. We show that coral assemblages differ markedly from neutral-model predictions for patterns of community similarity and the relative abundance of species. Within local communities, neutral models do not fit relative abundance distributions as well as the classical log-normal distribution. Relative abundances of species across local communities also differ markedly from neutral-theory predictions: coral communities exhibit community similarity values that are far more variable, and lower on average, than the neutral theory can produce. Empirical community similarities deviate from the neutral model in a direction opposite to that predicted in previous critiques of the neutral theory. Instead, our results support spatio-temporal environmental stochasticity as a major driver of diversity patterns on coral reefs.     

Global landscape of protein complexes in the yeast Saccharomyces cerevisiae

Identification of protein-protein interactions often provides insight into protein function, and many cellular processes are performed by stable protein complexes. We used tandem affinity purification to process 4,562 different tagged proteins of the yeast Saccharomyces cerevisiae. Each preparation was analysed by both matrix-assisted laser desorption/ionization-time of flight mass spectrometry and liquid chromatography tandem mass spectrometry to increase coverage and accuracy. Machine learning was used to integrate the mass spectrometry scores and assign probabilities to the protein-protein interactions. Among 4,087 different proteins identified with high confidence by mass spectrometry from 2,357 successful purifications, our core data set (median precision of 0.69) comprises 7,123 protein-protein interactions involving 2,708 proteins. A Markov clustering algorithm organized these interactions into 547 protein complexes averaging 4.9 subunits per complex, about half of them absent from the MIPS database, as well as 429 additional interactions between pairs of complexes. The data (all of which are available online) will help future studies on individual proteins as well as functional genomics and systems biology.