Human male recombination maps for individual chromosomes

Meiotic recombination is essential for the segregation of chromosomes and the formation of normal haploid gametes, yet we know very little about the meiotic process in humans. We present the first (to our knowledge) recombination maps for every autosome in the human male obtained by new immunofluorescence techniques followed by centromere-specific multicolor fluorescence in situ hybridization in human spermatocytes. The mean frequency of autosomal recombination foci was 49.8+/-4.3, corresponding to a genetic length of 2,490 cM. All autosomal bivalents had at least one recombination focus. In contrast, the XY bivalent had a recombination focus in 73% of nuclei, suggesting that a relatively large proportion of spermatocytes may be at risk for nondisjunction of the XY bivalent or elimination by meiotic arrest. There was a very strong correlation between mean length of the synaptonemal complex (SC) and the number of recombination foci per SC. Each bivalent presented a distinct distribution of recombination foci, but in general, foci were near the distal parts of the chromosome, with repression of foci near the centromere. The position of recombination foci demonstrated positive interference, but, in rare instances, foci were very close to one another.     

Cost of resistance and tolerance under competition: the defense-stress benefit hypothesis

Defense costs provide a major explanation for why plants in nature have not evolved to be better defended against pathogens and herbivores; however, evidence for defense costs is often lacking. Plants defend by deploying resistance traits that reduce damage, and tolerance traits that reduce the fitness effects of damage. We first tested the defense-stress cost (DSC) hypothesis that costs of defenses increase and become important under competitive stress. In a greenhouse experiment, uniparental maternal families of the host plant Arabis perennans were grown in the presence and absence of the bunch grass Bouteloua gracilis and the herbivore Plutella xylostella. Costs of resistance and tolerance manifest as reduced growth in the absence of herbivory were significant when A. perennans grew alone, but not in the competitive environment, in contrast to the DSC hypothesis. We then tested the defense-stress benefit (DSB) hypothesis that plant defenses may benefit plants in competitive situations thereby reducing net costs. For example, chemical resistance agents and tolerance may also have functions in competitive interactions. To test the DSB hypothesis, we compared differentially competitive populations for defense costs, assuming that poorer competitors from less dense habitats were less likely to have evolved defenses that also function in competition. Without competitive benefits of defenses, poorer competitors were expected to have higher net costs of defenses under competition in accordance with DSB. Populations of A. perennans and A. drummondii that differed dramatically in competitiveness were compared for costs, and as the DSB hypothesis predicts, only the poor competitor population showed costs of resistance under competition. However, cost of tolerance under competition did not differ among populations, suggesting that the poor competitors might have evolved a general stress tolerance. Although the DSC hypothesis may explain cases where defense costs increase under stress, the DSB hypothesis may explain some cases where costs decrease under competitive stress.     

Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate

The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly few direct mTOR substrates have been established to date. To tackle this gap in our knowledge, we took advantage of a combined quantitative phosphoproteomic and interactomic strategy. We analyzed the insulin- and aa-responsive phosphoproteome upon inhibition of the mTOR complex 1 (mTORC1) component raptor, and investigated in parallel the interactome of endogenous mTOR. By overlaying these two datasets, we identified acinus L as a potential novel mTORC1 target. We confirmed acinus L as a direct mTORC1 substrate by co-immunoprecipitation and MS-enhanced kinase assays. Our study delineates a triple proteomics strategy of combined phosphoproteomics, interactomics, and MS-enhanced kinase assays for the de novo-identification of mTOR network components, and provides a rich source of potential novel mTOR interactors and targets for future investigation.The serine/threonine kinase mammalian target of rapamycin (mTOR)¹ is conserved in all eukaryotes from yeast to mammals (1). mTOR is a central controller of cellular growth, whole body metabolism, and aging, and is frequently deregulated in metabolic diseases and cancer (2). Consequently, mTOR as well as its upstream and downstream cues are prime candidates for targeted drug development to alleviate the causes and symptoms of age-related diseases (3, 4). The identification of novel mTOR regulators and effectors thus remains a major goal in biomedical research. A vast body of literature describes a complex signaling network around mTOR. However, our current comparatively detailed knowledge of mTOR''s upstream cues contrasts with a rather limited set of known direct mTOR substrates.mTOR exists in two structurally and functionally distinct multiprotein complexes, termed mTORC1 and mTORC2. Both complexes contain mTOR kinase as well as the proteins mLST8 (mammalian lethal with SEC thirteen 8) (5–7), and deptor (DEP domain-containing mTOR-interacting protein) (8). mTORC1 contains the specific scaffold protein raptor (regulatory-associated protein of mTOR) (9, 10), whereas mTORC2 contains the specific binding partners rictor (rapamycin-insensitive companion of mTOR) (5–7), mSIN1 (TORC2 subunit MAPKAP1) (11–13), and PRR5/L (proline rich protein 5/-like) (14–16). The small macrolide rapamycin acutely inhibits mTORC1, but can also have long-term effects on mTORC2 (17, 18). More recently, ATP-analogs (19) that block both mTOR complexes, such as Torin 1 (20), have been developed. As rapamycin has already been available for several decades, our knowledge of signaling events associated with mTORC1 as well as its metabolic inputs and outputs is much broader as compared with mTORC2. mTORC1 responds to growth factors (insulin), nutrients (amino acids, aa) and energy (ATP). In response, mTORC1 activates anabolic processes (protein, lipid, nucleotide synthesis) and blocks catabolic processes (autophagy) to ultimately allow cellular growth (21). The insulin signal is transduced to mTORC1 via the insulin receptor (IR), and the insulin receptor substrate (IRS), which associates with class I phosphoinositide 3-kinases (PI3Ks). Subsequent phosphatidylinositol 3,4,5 trisphosphate (PIP3) binding leads to relocalization of the AGC kinases phosphoinositide-dependent protein kinase 1 (PDK1) and Akt (also termed protein kinase B, PKB) to the plasma membrane, where PDK1 phosphorylates Akt at T308 (22, 23). In response, Akt phosphorylates and inhibits the heterocomplex formed by the tuberous sclerosis complex proteins 1 and 2 (TSC1-TSC2) (24, 25). TSC1-TSC2 is the inhibitory, GTPase-activating protein for the mTORC1-inducing GTPase Ras homolog enriched in brain (rheb) (26–30), which activates mTORC1 at the lysosome. mTORC1 localization depends on the presence of aa, which in a rag GTPase-dependent manner induce mTORC1 relocalization to lysosomes (31, 32). Low energy levels are sensed by the AMP-dependent kinase (AMPK), which in turn phosphorylates the TSC1-TSC2 complex (33) and raptor (34), thereby inhibiting mTORC1.mTORC1 phosphorylates its well-described downstream substrate S6-kinase (S6K) at T389, the proline-rich Akt substrate of 40 kDa (PRAS40) at S183, and the translational repressor 4E-binding protein (4E-BP) at T37/46 (35–41). Unphosphorylated 4E-BP binds and inhibits the translation initiation factor 4G (eIF4G), which within the eIF4F complex mediates the scanning process of the ribosome to reach the start codon. Phosphorylation by mTORC1 inhibits 4E-BP''s interaction with eIF4E, thus allowing for assembly of eIF4F, and translation initiation (42, 43). More recently, also the IR-activating growth factor receptor-bound protein 10 (Grb10) (44, 45), the autophagy-initiating Unc-51-like kinase ULK1 (46), and the trifunctional enzymatic complex CAD composed of carbamoyl-phosphate synthetase 2, aspartate transcarbamoylase, and dihydroorotase (47, 48), which is required for nucleotide synthesis, have been described as direct mTORC1 substrates.mTORC2 activation is mostly described to be mediated by insulin, and this is mediated by a PI3K variant that is distinct from the PI3K upstream of mTORC1 (49, 50). Furthermore, mTORC2 responds to aa (5, 51). In response, mTORC2 phosphorylates the AGC kinases Akt at S473 (52–55), and serum and glucocorticoid kinase SGK (56) and protein kinase C alpha (PKCalpha) (7) within their hydrophobic motifs (57, 58), to control cellular motility (5–7), hepatic glycolysis, and lipogenesis (59). In addition, mTOR autophosphorylation at S2481 has been established as an mTORC2 readout in several cell lines including HeLa cells (49).Given the multiplicity of effects via which mTOR controls cellular and organismal growth and metabolism, it is surprising that only relatively few direct mTOR substrates have been established to date. Proteomic studies are widely used to identify novel interactors and substrates of protein kinases. Two studies have recently shed light on the interaction of rapamycin and ATP-analog mTOR inhibitors with TSC2 inhibition in mammalian cells (44, 45), and one study has analyzed the effects of raptor and rictor knockouts in non-stimulated cells (48).In this work, we report a functional proteomics approach to study mTORC1 substrates. We used an inducible raptor knockdown to inhibit mTORC1 in HeLa cells, and analyzed the effect in combination with insulin and aa induction by quantitative phosphoproteomics using stable isotope labeling by amino acids in cell culture (SILAC) (60). In parallel, we purified endogenous mTOR complexes and studied the interactome of mTOR by SILAC-MS. Through comparative data evaluation, we identified acinus L as a potential novel aa/insulin-sensitive mTOR substrate. We further validated acinus L by co-immunoprecipitation and MS-enhanced kinase assays as a new direct mTORC1 substrate.     

Dating the arthropod tree based on large-scale transcriptome data

Molecular sequences do not only allow the reconstruction of phylogenetic relationships among species, but also provide information on the approximate divergence times. Whereas the fossil record dates the origin of most multicellular animal phyla during the Cambrian explosion less than 540 million years ago(mya), molecular clock calculations usually suggest much older dates. Here we used a large multiple sequence alignment derived from Expressed Sequence Tags and genomes comprising 129genes (37,476 amino acid positions) and 117 taxa, including 101 arthropods. We obtained consistent divergence time estimates applying relaxed Bayesian clock models with different priors and multiple calibration points. While the influence of substitution rates, missing data, and model priors were negligible, the clock model had significant effect. A log-normal autocorrelated model was selected on basis of cross-validation. We calculated that arthropods emerged ~600 mya. Onychophorans (velvet worms) and euarthropods split ~590 mya, Pancrustacea and Myriochelata ~560 mya, Myriapoda and Chelicerata ~555 mya, and 'Crustacea' and Hexapoda ~510 mya. Endopterygote insects appeared ~390 mya. These dates are considerably younger than most previous molecular clock estimates and in better agreement with the fossil record. Nevertheless, a Precambrian origin of arthropods and other metazoan phyla is still supported. Our results also demonstrate the applicability of large datasets of random nuclear sequences for approximating the timing of multicellular animal evolution.     

Distant land use affects terrestrial and aquatic habitats of high naturalness

Biotas from all ecosystems need to respond to factors that determine habitat suitability. These factors originate from different scales. Effects can be assumed to be hierarchical in the order large-scale geographic > regional > local > small-scale in-habitat factors. We aimed at the identification of general patterns by comparisons between ecosystems (forest floor snails, hololimnic stream macroinvertebrates) and across scales, and include potential seasonal effects. Sampling sites displayed signs of naturalness, such as high levels of deadwood accumulation in the forests, or a lack of artificial stream bed fixation plus a “good” to “high” score for the assemblage-derived Multimetric Index (MMI) in the streams. Terrestrial and aquatic assemblages of non-emergent taxa fluctuated independent of seasonal effects. They differed in their relative correlation with environmental matrices with quasi-concentric effects in forests, and longitudinal effects in streams. Large-scale factors, namely geographic position, strongly influenced assemblage turnover, but the effect is based on a high covariation between geographic position and environmental factors. We thus extracted variables that best explained species turnover after correcting for spatio-temporal effects. The terrestrial community assembling was habitat-based and mainly responded to soil acidification, distance to disturbances, and regional scale deforestation and deciduous/mixed forest cover. The stream assemblages were structured by regional pasture cover, organic pollution, regional deciduous forest cover and microlithal cover. Apparently, community assembly occurs along with changes in regional forest cover and the transport of nutrients and matter that can originate from a distance, irrespective of ecosystem and assumed “naturalness”.     

Predictive Mapping of Plant Species and Communities Using GIS and Landsat Data in a Southern Mongolian Mountain Range

We assessed presence/absence prediction of plant species and communities in a southern Mongolian mountain range from geospatial data using a randomized sampling approach. One hundred randomized vegetation samples (3?×?3 m) were collected within the 2?×?2 km summit region of the Dund Saykhan range, which forms part of the core zone of the Gobi Gurvan Saykhan National Park in arid southern Mongolia. Using logistic regression, habitat preference models for all abundant species (n?=?52) and communities (n?=?5) were constructed; predictors were derived from Landsat 5 imagery and a digital elevation model. Nagelkerkes r ² was used for an initial data mining, and all significant models were validated by splitting the data and using one half for accuracy assessment based on the AUC (Area Under the receiver operating characteristic Curve)-values. Significant models could be built for half of the species. Altitude proved to be the most important predictor followed by variables derived from Landsat data. The clear altitudinal distribution patterns most definitely reflect precipitation; overall biodiversity in this arid environment is widely controlled by moisture availability. The chosen approach may prove valuable for applied studies wherever spatial data on species distributions are required for conservation efforts.     

Mutations in ACY1, the gene encoding aminoacylase 1, cause a novel inborn error of metabolism

N-terminal acetylation of proteins is a widespread and highly conserved process. Aminoacylase 1 (ACY1; EC 3.5.14) is the most abundant of the aminoacylases, a class of enzymes involved in hydrolysis of N-acetylated proteins. Here, we present four children with genetic deficiency of ACY1. They were identified through organic acid analyses using gas chromatography-mass spectrometry, revealing increased urinary excretion of several N-acetylated amino acids, including the derivatives of methionine, glutamic acid, alanine, leucine, glycine, valine, and isoleucine. Nuclear magnetic resonance spectroscopy analysis of urine samples detected a distinct pattern of N-acetylated metabolites, consistent with ACY1 dysfunction. Functional analyses of patients' lymphoblasts demonstrated ACY1 deficiency. Mutation analysis uncovered recessive loss-of-function or missense ACY1 mutations in all four individuals affected. We conclude that ACY1 mutations in these children led to functional ACY1 deficiency and excretion of N-acetylated amino acids. Questions remain, however, as to the clinical significance of ACY1 deficiency. The ACY1-deficient individuals were ascertained through urine metabolic screening because of unspecific psychomotor delay (one subject), psychomotor delay with atrophy of the vermis and syringomyelia (one subject), marked muscular hypotonia (one subject), and follow-up for early treated biotinidase deficiency and normal clinical findings (one subject). Because ACY1 is evolutionarily conserved in fish, frog, mouse, and human and is expressed in the central nervous system (CNS) in human, a role in CNS function or development is conceivable but has yet to be demonstrated. Thus, at this point, we cannot state whether ACY1 deficiency has pathogenic significance with pleiotropic clinical expression or is simply a biochemical variant. Awareness of this new genetic entity may help both in delineating its clinical significance and in avoiding erroneous diagnoses.     

The heat shock protein HSP70 promotes mouse NK cell activity against tumors that express inducible NKG2D ligands

The stress-inducible heat shock protein (HSP) 70 is known to function as an endogenous danger signal that can increase the immunogenicity of tumors and induce CTL responses. We show in this study that HSP70 also activates mouse NK cells that recognize stress-inducible NKG2D ligands on tumor cells. Tumor size and the rate of metastases derived from HSP70-overexpressing human melanoma cells were found to be reduced in T and B cell-deficient SCID mice, but not in SCID/beige mice that lack additionally functional NK cells. In the SCID mice with HSP70-overexpressing tumors, NK cells were activated so that they killed ex vivo tumor cells that expressed NKG2D ligands. In the tumors, the MHC class I chain-related (MIC) A and B molecules were found to be expressed. Interestingly, a counter selection was observed against the expression of MICA/B in HSP70-overexpressing tumors compared with control tumors in SCID, but not in SCID/beige mice, suggesting a functional relevance of MICA/B expression. The melanoma cells were found to release exosomes. HSP70-positive exosomes from the HSP70-overexpressing cells, in contrast to HSP70-negative exosomes from the control cells, were able to activate mouse NK cells in vitro to kill YAC-1 cells, which express NKG2D ligands constitutively, or the human melanoma cells, in which MICA/B expression was induced. Thus, HSP70 and inducible NKG2D ligands synergistically promote the activation of mouse NK cells resulting in a reduced tumor growth and suppression of metastatic disease.     

The unfolded protein response is shaped by the NMD pathway

Endoplasmic reticulum (ER) stress induces the unfolded protein response (UPR), an essential adaptive intracellular pathway that relieves the stress. Although the UPR is an evolutionarily conserved and beneficial pathway, its chronic activation contributes to the pathogenesis of a wide variety of human disorders. The fidelity of UPR activation must thus be tightly regulated to prevent inappropriate signaling. The nonsense-mediated RNA decay (NMD) pathway has long been known to function in RNA quality control, rapidly degrading aberrant mRNAs, and has been suggested to regulate subsets of normal mRNAs. Here, we report that the NMD pathway regulates the UPR. NMD increases the threshold for triggering the UPR in vitro and in vivo, thereby preventing UPR activation in response to normally innocuous levels of ER stress. NMD also promotes the timely termination of the UPR. We demonstrate that NMD directly targets the mRNAs encoding several UPR components, including the highly conserved UPR sensor, IRE1α, whose NMD-dependent degradation partly underpins this process. Our work not only sheds light on UPR regulation, but demonstrates the physiological relevance of NMD''s ability to regulate normal mRNAs.