Glycodelin protein and mRNA is downregulated in human first trimester abortion and partially upregulated in mole pregnancy.

Glycodelin (Gd) is a major reproductive glycoprotein and a mediator for immunomodulatory effects directed to cellular, humoral, and innate immunity. Human pregnancy depends on a diversity of physiological processes including modulation of the maternal immunosystem. We evaluated the expression of Gd protein and mRNA in first trimester decidual tissue of normal pregnancies and spontaneous abortion and hydatidiform moles. Furthermore, in vitro experiments on endometrial cancer cells to analyze the effect of human chorionic gonadotropin (hCG) on Gd regulation were performed. In decidual tissue of abortion patients, Gd expression was significantly decreased compared with normal gestation, which was confirmed by in situ hybridization. In mole pregnancy, an upregulation of Gd in the first 8 weeks of pregnancy was present. Gd is a main product of decidual tissue in the first trimester of human pregnancy. Reduced Gd expression in abortive pregnancy could lead to an increased activation of the maternal immunosystem, thus causing rejection of the developing fetus. Moreover, Gd expression in endometrial cancer cells in vitro could be stimulated by addition of hCG. Therefore, we speculate that hCG could be one of the factors regulating Gd expression because hCG is downregulated in women with abortion and upregulated in mole pregnancy. In addition, we found a positive feedback loop in Gd and hCG expression in human pregnancy.     

Wild-Type Measles Viruses with Non-Standard Genome Lengths

The length of the single stranded, negative sense RNA genome of measles virus (MeV) is highly conserved at 15,894 nucleotides (nt). MeVs can be grouped into 24 genotypes based on the highly variable 450 nucleotides coding for the carboxyl-terminus of the nucleocapsid protein (N-450). Here, we report the genomic sequences of 2 wild-type viral isolates of genotype D4 with genome lengths of 15,900 nt. Both genomes had a 7 nt insertion in the 3′ untranslated region (UTR) of the matrix (M) gene and a 1 nt deletion in the 5′ UTR of the fusion (F) gene. The net gain of 6 nt complies with the rule-of-six required for replication competency of the genomes of morbilliviruses. The insertions and deletion (indels) were confirmed in a patient sample that was the source of one of the viral isolates. The positions of the indels were identical in both viral isolates, even though epidemiological data and the 3 nt differences in N-450 between the two genomes suggested that the viruses represented separate chains of transmission. Identical indels were found in the M-F intergenic regions of 14 additional genotype D4 viral isolates that were imported into the US during 2007–2010. Viral isolates with and without indels produced plaques of similar size and replicated efficiently in A549/hSLAM and Vero/hSLAM cells. This is the first report of wild-type MeVs with genome lengths other than 15,894 nt and demonstrates that the length of the M-F UTR of wild-type MeVs is flexible.     

Patterns of Microsatellite Variability in the Drosophila Melanogaster Complex

Forty-seven microsatellite loci were amplified in Drosophila melanogaster, Drosophila simulans, Drosophila mauritiana and Drosophila sechellia. The two cosmopolitan species D. melanogaster and D. simulans were found to be the most variable ones, followed by D. mauritiana and D. sechellia. A model based clustering algorithm was applied to the population samples of D. melanogaster, D. simulans and D. sechellia. No evidence for population substructure was detected within species--most likely due to insufficient power. A Markov chain Monte Carlo method developed for demographic inference based on microsatellites provided unambiguous evidence for population contraction in D. melanogaster, D. simulans and D. sechellia, despite that the D. melanogaster and D. simulans population samples were of non-African origin and represented recently expanded populations.     

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.     

Ethylene Glycol Metabolism by Pseudomonas putida

In this study, we investigated the metabolism of ethylene glycol in the Pseudomonas putida strains KT2440 and JM37 by employing growth and bioconversion experiments, directed mutagenesis, and proteome analysis. We found that strain JM37 grew rapidly with ethylene glycol as a sole source of carbon and energy, while strain KT2440 did not grow within 2 days of incubation under the same conditions. However, bioconversion experiments revealed metabolism of ethylene glycol by both strains, with the temporal accumulation of glycolic acid and glyoxylic acid for strain KT2440. This accumulation was further increased by targeted mutagenesis. The key enzymes and specific differences between the two strains were identified by comparative proteomics. In P. putida JM37, tartronate semialdehyde synthase (Gcl), malate synthase (GlcB), and isocitrate lyase (AceA) were found to be induced in the presence of ethylene glycol or glyoxylic acid. Under the same conditions, strain KT2440 showed induction of AceA only. Despite this difference, the two strains were found to use similar periplasmic dehydrogenases for the initial oxidation step of ethylene glycol, namely, the two redundant pyrroloquinoline quinone (PQQ)-dependent enzymes PedE and PedH. From these results we constructed a new pathway for the metabolism of ethylene glycol in P. putida. Furthermore, we conclude that Pseudomonas putida might serve as a useful platform from which to establish a whole-cell biocatalyst for the production of glyoxylic acid from ethylene glycol.     

Additional haplogroups of Toxoplasma gondii out of Africa: population structure and mouse-virulence of strains from Gabon

Background

Toxoplasma gondii is found worldwide, but distribution of its genotypes as well as clinical expression of human toxoplasmosis varies across the continents. Several studies in Europe, North America and South America argued for a role of genotypes in the clinical expression of human toxoplasmosis. Genetic data concerning T. gondii isolates from Africa are scarce and not sufficient to investigate the population structure, a fundamental analysis for a better understanding of distribution, circulation, and transmission.

Methodology/Principal Findings

Seropositive animals originating from urban and rural areas in Gabon were analyzed for T. gondii isolation and genotyping. Sixty-eight isolates, including one mixed infection (69 strains), were obtained by bioassay in mice. Genotyping was performed using length polymorphism of 13 microsatellite markers located on 10 different chromosomes. Results were analyzed in terms of population structure by Bayesian statistical modeling, Neighbor-joining trees reconstruction based on genetic distances, F _ST and linkage disequilibrium. A moderate genetic diversity was detected. Three haplogroups and one single genotype clustered 27 genotypes. The majority of strains belonged to one haplogroup corresponding to the worldwide Type III. The remaining strains were distributed into two haplogroups (Africa 1 and 3) and one single genotype. Mouse virulence at isolation was significantly different between haplogroups. Africa 1 haplogroup was the most virulent.

Conclusion

Africa 1 and 3 haplogroups were proposed as being new major haplogroups of T. gondii circulating in Africa. A possible link with strains circulating in South and Central America is discussed. Analysis of population structure demonstrated a local spread within a rural area and strain circulation between the main cities of the country. This circulation, favored by human activity could lead to genetic exchanges. For the first time, key epidemiological questions were addressed for the West African T. gondii population, using the high discriminatory power of microsatellite markers, thus creating a basis for further epidemiological and clinical investigations.     

RhoA regulates peroxisome association to microtubules and the actin cytoskeleton

The current view of peroxisome inheritance provides for the formation of new peroxisomes by both budding from the endoplasmic reticulum and autonomous division. Here we investigate peroxisome-cytoskeleton interactions and show by proteomics, biochemical and immunofluorescence analyses that actin, non-muscle myosin IIA (NMM IIA), RhoA, Rho kinase II (ROCKII) and Rab8 associate with peroxisomes. Our data provide evidence that (i) RhoA in its inactive state, maintained for example by C. botulinum toxin exoenzyme C3, dissociates from peroxisomes enabling microtubule-based peroxisomal movements and (ii) dominant-active RhoA targets to peroxisomes, uncouples the organelles from microtubules and favors Rho kinase recruitment to peroxisomes. We suggest that ROCKII activates NMM IIA mediating local peroxisomal constrictions. Although our understanding of peroxisome-cytoskeleton interactions is still incomplete, a picture is emerging demonstrating alternate RhoA-dependent association of peroxisomes to the microtubular and actin cytoskeleton. Whereas association of peroxisomes to microtubules clearly serves bidirectional, long-range saltatory movements, peroxisome-acto-myosin interactions may support biogenetic functions balancing peroxisome size, shape, number, and clustering.     

Macromolecular isoforms of Daphnia magna haemoglobin

The haemoglobin (Hb) of Daphnia magna acclimated to different oxygen conditions was sampled, and in its natively assembled state it was separated by chromatofocusing. The Hb isoforms were analysed for their subunit composition under denaturating conditions by two-dimensional gel electrophoresis. The Hb system is suggested to consist of three predominant Hb aggregates, which are characterised by a specific subunit composition and synthesised in response to different ambient oxygen conditions. In normoxia, a dominant Hb aggregate (DmHbI) with a pI of 4.4-4.6 was composed of subunits B, C, E, F and G. In severe hypoxia, a different dominant Hb isoform (DmHbIII) with a pI of 5.7-5.9 was composed of subunits A, B, C, D, E and F. Further analyses in moderate hypoxia provided evidence for a third Hb isoform (DmHbII) composed of subunits B, C, D, E and F. Sequence alignment and homology modelling of the tertiary structure of the D. magna Hb domains 1 and 2 revealed functionally relevant substitutions of amino acid residues at positions B10, E7 and E11, which determine the functional properties of D. magna haemoglobin in terms of haem contact, oxygen binding and affinity. Both domains are predicted to possess the common haemoglobin fold, but helices C and D are not properly formed, and helix G is interrupted by a short coil.