Ab Initio Structural Energetics of Beta-Si3N4 Surfaces

Motivated by recent electron microscopy studies on the Si3N4/rare-earth oxide interfaces, the atomic and electronic structures of bare beta-Si3N4 surfaces are investigated from first principles. The equilibrium shape of a Si3N4 crystal is found to have a hexagonal cross section and a faceted dome-like base in agreement with experimental observations. The large atomic relaxations on the prismatic planes are driven by the tendency of Si to saturate its dangling bonds, which gives rise to resonant-bond configurations or planar sp^2-type bonding. We predict three bare surfaces with lower energies than the open-ring (10-10) surface observed at the interface, which indicate that non-stoichiometry and the presence of the rare-earth oxide play crucial roles in determining the termination of the Si3N4 matrix grains.Comment: 4 Pages, 4 Figures, 1 tabl

An integrated multi-omics approach identifies the landscape of interferon-α-mediated responses of human pancreatic beta cells

Interferon-α (IFNα), a type I interferon, is expressed in the islets of type 1 diabetic individuals, and its expression and signaling are regulated by T1D genetic risk variants and viral infections associated with T1D. We presently characterize human beta cell responses to IFNα by combining ATAC-seq, RNA-seq and proteomics assays. The initial response to IFNα is characterized by chromatin remodeling, followed by changes in transcriptional and translational regulation. IFNα induces changes in alternative splicing (AS) and first exon usage, increasing the diversity of transcripts expressed by the beta cells. This, combined with changes observed on protein modification/degradation, ER stress and MHC class I, may expand antigens presented by beta cells to the immune system. Beta cells also up-regulate the checkpoint proteins PDL1 and HLA-E that may exert a protective role against the autoimmune assault. Data mining of the present multi-omics analysis identifies two compound classes that antagonize IFNα effects on human beta cells

PhosTryp: a phosphorylation site predictor specific for parasitic protozoa of the family trypanosomatidae

<p>Abstract</p> <p>Background</p> <p>Protein phosphorylation modulates protein function in organisms at all levels of complexity. Parasites of the <it>Leishmania </it>genus undergo various developmental transitions in their life cycle triggered by changes in the environment. The molecular mechanisms that these organisms use to process and integrate these external cues are largely unknown. However <it>Leishmania </it>lacks transcription factors, therefore most regulatory processes may occur at a post-translational level and phosphorylation has recently been demonstrated to be an important player in this process. Experimental identification of phosphorylation sites is a time-consuming task. Moreover some sites could be missed due to the highly dynamic nature of this process or to difficulties in phospho-peptide enrichment.</p> <p>Results</p> <p>Here we present PhosTryp, a phosphorylation site predictor specific for trypansomatids. This method uses an SVM-based approach and has been trained with recent <it>Leishmania </it>phosphosproteomics data. PhosTryp achieved a 17% improvement in prediction performance compared with Netphos, a non organism-specific predictor. The analysis of the peptides correctly predicted by our method but missed by Netphos demonstrates that PhosTryp captures <it>Leishmania</it>-specific phosphorylation features. More specifically our results show that <it>Leishmania </it>kinases have sequence specificities which are different from their counterparts in higher eukaryotes. Consequently we were able to propose two possible <it>Leishmania</it>-specific phosphorylation motifs.</p> <p>We further demonstrate that this improvement in performance extends to the related trypanosomatids <it>Trypanosoma brucei </it>and <it>Trypanosoma cruzi</it>. Finally, in order to maximize the usefulness of PhosTryp, we trained a predictor combining all the peptides from <it>L. infantum, T. brucei and T. cruzi</it>.</p> <p>Conclusions</p> <p>Our work demonstrates that training on organism-specific data results in an improvement that extends to related species. PhosTryp is freely available at <url>http://phostryp.bio.uniroma2.it</url></p

Akt1-associated actomyosin remodelling is required for nuclear lamina dispersal and nuclear shrinkage in epidermal terminal differentiation

Keratinocyte cornification and epidermal barrier formation are tightly controlled processes, which require complete degradation of intracellular organelles, including removal of keratinocyte nuclei. Keratinocyte nuclear destruction requires Akt1-dependent phosphorylation and degradation of the nuclear lamina protein, Lamin A/C, essential for nuclear integrity. However, the molecular mechanisms that result in complete nuclear removal and their regulation are not well defined. Post-confluent cultures of rat epidermal keratinocytes (REKs) undergo spontaneous and complete differentiation, allowing visualisation and perturbation of the differentiation process in vitro. We demonstrate that there is dispersal of phosphorylated Lamin A/C to structures throughout the cytoplasm in differentiating keratinocytes. We show that the dispersal of phosphorylated Lamin A/C is Akt1-dependent and these structures are specific for the removal of Lamin A/C from the nuclear lamina; nuclear contents and Lamin B were not present in these structures. Immunoprecipitation identified a group of functionally related Akt1 target proteins involved in Lamin A/C dispersal, including actin, which forms cytoskeletal microfilaments, Arp3, required for actin filament nucleation, and Myh9, a component of myosin IIa, a molecular motor that can translocate along actin filaments. Disruption of actin filament polymerisation, nucleation or myosin IIa activity prevented formation and dispersal of cytoplasmic Lamin A/C structures. Live imaging of keratinocytes expressing fluorescently tagged nuclear proteins showed a nuclear volume reduction step taking less than 40 min precedes final nuclear destruction. Preventing Akt1-dependent Lamin A/C phosphorylation and disrupting cytoskeletal Akt1-associated proteins prevented nuclear volume reduction. We propose keratinocyte nuclear destruction and differentiation requires myosin II activity and the actin cytoskeleton for two intermediate processes: Lamin A/C dispersal and rapid nuclear volume reduction

Transcript Expression Analysis of Putative Trypanosoma brucei GPI-Anchored Surface Proteins during Development in the Tsetse and Mammalian Hosts

Human African Trypanosomiasis is a devastating disease caused by the parasite Trypanosoma brucei. Trypanosomes live extracellularly in both the tsetse fly and the mammal. Trypanosome surface proteins can directly interact with the host environment, allowing parasites to effectively establish and maintain infections. Glycosylphosphatidylinositol (GPI) anchoring is a common posttranslational modification associated with eukaryotic surface proteins. In T. brucei, three GPI-anchored major surface proteins have been identified: variant surface glycoproteins (VSGs), procyclic acidic repetitive protein (PARP or procyclins), and brucei alanine rich proteins (BARP). The objective of this study was to select genes encoding predicted GPI-anchored proteins with unknown function(s) from the T. brucei genome and characterize the expression profile of a subset during cyclical development in the tsetse and mammalian hosts. An initial in silico screen of putative T. brucei proteins by Big PI algorithm identified 163 predicted GPI-anchored proteins, 106 of which had no known functions. Application of a second GPI-anchor prediction algorithm (FragAnchor), signal peptide and trans-membrane domain prediction software resulted in the identification of 25 putative hypothetical proteins. Eighty-one gene products with hypothetical functions were analyzed for stage-regulated expression using semi-quantitative RT-PCR. The expression of most of these genes were found to be upregulated in trypanosomes infecting tsetse salivary gland and proventriculus tissues, and 38% were specifically expressed only by parasites infecting salivary gland tissues. Transcripts for all of the genes specifically expressed in salivary glands were also detected in mammalian infective metacyclic trypomastigotes, suggesting a possible role for these putative proteins in invasion and/or establishment processes in the mammalian host. These results represent the first large-scale report of the differential expression of unknown genes encoding predicted T. brucei surface proteins during the complete developmental cycle. This knowledge may form the foundation for the development of future novel transmission blocking strategies against metacyclic parasites

DNA replication and the GINS complex: localization on extended chromatin fibers

<p>Abstract</p> <p>Background</p> <p>The GINS complex is thought to be essential for the processes of initiation and elongation of DNA replication. This complex contains four subunits, one of which (Psf1) is proposed to bind to both chromatin and DNA replication-associated proteins. To date there have been no microscopic analyses to evaluate the chromatin distribution of this complex. Here, we show the organization of GINS complexes on extended chromatin fibers in relation to sites of DNA replication and replication-associated proteins.</p> <p>Results</p> <p>Using immunofluorescence microscopy we were able to visualize ORC1, ORC2, PCNA, and GINS complex proteins Psf1 and Psf2 bound to extended chromatin fibers. We were also able to detect these proteins concurrently with the visualization of tracks of recently replicated DNA where EdU, a thymidine analog, was incorporated. This allowed us to assess the chromatin association of proteins of interest in relation to the process of DNA replication. ORC and GINS proteins were found on chromatin fibers before replication could be detected. These proteins were also associated with newly replicated DNA in bead-like structures. Additionally, GINS proteins co-localized with PCNA at sites of active replication.</p> <p>Conclusion</p> <p>In agreement with its proposed role in the initiation of DNA replication, GINS proteins associated with chromatin near sites of ORC binding that were devoid of EdU (absence of DNA replication). The association of GINS proteins with PCNA was consistent with a role in the process of elongation. Additionally, the large size of our chromatin fibers (up to approximately 7 Mb) allowed for a more expansive analysis of the distance between active replicons than previously reported.</p

Lipidomic Analysis of Extracellular Vesicles from the Pathogenic Phase of Paracoccidioides brasiliensis

Background: Fungal extracellular vesicles are able to cross the cell wall and transport molecules that help in nutrient acquisition, cell defense, and modulation of the host defense machinery.Methodology/Principal Findings: Here we present a detailed lipidomic analysis of extracellular vesicles released by Paracoccidioides brasiliensis at the yeast pathogenic phase. We compared data of two representative isolates, Pb3 and Pb18, which have distinct virulence profiles and phylogenetic background. Vesicle lipids were fractionated into different classes and analyzed by either electrospray ionization- or gas chromatography-mass spectrometry. We found two species of monohexosylceramide and 33 phospholipid species, including phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, phosphatidylserine, phosphatidylinositol, and phosphatidylglycerol. Among the phospholipid-bound fatty acids in extracellular vesicles, C181 predominated in Pb3, whereas C18:2 prevailed in Pb18. the prevalent sterol in Pb3 and Pb18 vesicles was brassicasterol, followed by ergosterol and lanosterol. Inter-isolate differences in sterol composition were observed, and also between extracellular vesicles and whole cells.Conclusions/Significance: the extensive lipidomic analysis of extracellular vesicles from two P. brasiliensis isolates will help to understand the composition of these fungal components/organelles and will hopefully be useful to study their biogenesis and role in host-pathogen interactions.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)National Institutes of Health (NIH)Universidade Federal de São Paulo, UNIFESP, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilUniv Texas El Paso, Dept Biol Sci, Border Biomed Res Ctr, El Paso, TX 79968 USAUniversidade Federal de São Paulo, UNIFESP, Dept Microbiol Imunol & Parasitol, São Paulo, BrazilFAPESP: 06/05095-6FAPESP: 07/04757-8FAPESP: 07/59768-4CNPq: 301666/2010-5National Institutes of Health (NIH): 5G12RR008124-16A1National Institutes of Health (NIH): 5G12RR008124-16A1S1National Institutes of Health (NIH): G12MD007592Web of Scienc