Expanded Cocirculation of Stable Subtypes,Emerging Lineages,and New Sporadic Reassortants of Porcine Influenza Viruses in Swine Populations in Northwest Germany

The emergence of the human 2009 pandemic H1N1 (H1N1pdm) virus from swine populations refocused public and scientific attention on swine as an important source of influenza A viruses bearing zoonotic potential. Widespread and year-round circulation of at least four stable lineages of porcine influenza viruses between 2009 and 2012 in a region of Germany with a high-density swine population is documented here. European avian influenza virus-derived H1N1 (H1N1av) viruses dominated the epidemiology, followed by human-derived subtypes H1N2 and H3N2. H1N1pdm viruses and, in particular, recently emerging reassortants between H1N1pdm and porcine HxN2 viruses (H1pdmN2) were detected in about 8% of cases. Further reassortants between these main lineages were diagnosed sporadically. Ongoing diversification both at the phylogenetic and at the antigenic level was evident for the H1N1av lineage and for some of its reassortants. The H1avN2 reassortant R1931/11 displayed conspicuously distinct genetic and antigenic features and was easily transmitted from pig to pig in an experimental infection. Continuing diverging evolution was also observed in the H1pdmN2 lineage. These viruses carry seven genome segments of the H1N1pdm virus, including a hemagglutinin gene that encodes a markedly antigenically altered protein. The zoonotic potential of this lineage remains to be determined. The results highlight the relevance of surveillance and control of porcine influenza virus infections. This is important for the health status of swine herds. In addition, a more exhaustive tracing of the formation, transmission, and spread of new reassortant influenza A viruses with unknown zoonotic potential is urgently required.     

Microsatellite markers from a microdissected swine chromosome 6 genomic library

To develop additional microsatellite (MS) markers in the region of the porcine skeletal muscle ryanodine receptor gene (RYR1), a microdissected genomic library was generated from the proximal half of the q arm of swine chromosome 6. Purified DNA was restriction enzyme-digested, ligated to oligonucleotide adaptors and amplified by PCR using primers complementary to the adaptor sequences. The purity of the amplified products and boundaries of the microdissected chromosomal region were verified by fluorescence in situ hybridization. (CA)n-containing sequences were then identified in a small insert genomic library generated from the PCR-amplified microdissected DNA. Oligonucleotide primers were developed for the PCR amplification of 30 of the 46 (CA)n repeat-containing clones, which were subsequently used to amplify DNA isolated from unrelated pigs of different breeds to determine the informativeness of these MS markers. Twenty-two of these MS markers were genotyped on the University of Illinois Yorkshire x Meishan swine reference population. These 22 markers were all assigned within a 50.7-CM region of the swine chromosome 6 linkage map, indicating the specificity of the microdissected library.     

Human genome diversity data reveal that L564P is the predominant TPC2 variant and a prerequisite for the blond hair associated M484L gain-of-function effect

The endo-lysosomal two-pore channel (TPC2) has been established as an intracellular cation channel of significant physiological and pathophysiological relevance in recent years. For example, TPC2^-/- mice show defects in cholesterol degradation, leading to hypercholesterinemia; TPC2 absence also results in mature-onset obesity, and a role in glucagon secretion and diabetes has been proposed. Infections with bacterial toxins or viruses e.g., cholera toxin or Ebola virus result in reduced infectivity rates in the absence of TPC2 or after pharmacological blockage, and TPC2^-/- cancer cells lose their ability to migrate and metastasize efficiently. Finally, melanin production is affected by changes in hTPC2 activity, resulting in pigmentation defects and hair color variation. Here, we analyzed several publicly available genome variation data sets and identified multiple variations in the TPC2 protein in distinct human populations. Surprisingly, one variation, L564P, was found to be the predominant TPC2 isoform on a global scale. By applying endo-lysosomal patch-clamp electrophysiology, we found that L564P is a prerequisite for the previously described M484L gain-of-function effect that is associated with blond hair. Additionally, other gain-of-function variants with distinct geographical and ethnic distribution were discovered and functionally characterized. A meta-analysis of genome-wide association studies was performed, finding the polymorphisms to be associated with both distinct and overlapping traits. In sum, we present the first systematic analysis of variations in TPC2. We functionally characterized the most common variations and assessed their association with various disease traits. With TPC2 emerging as a novel drug target for the treatment of various diseases, this study provides valuable insights into ethnic and geographical distribution of TPC2 polymorphisms and their effects on channel activity.     

Ribosomal DNA shows extremely low genetic divergence in a world-wide distributed,but disjunct and highly adapted marine protozoan (Virgulinella fragilis,Foraminiferida)

Virgulinella fragilis can be mainly observed in different, separated, oxygen-depleted and sulfide-enriched environments around the world and seems to be well adapted to such extreme habitats. Dispersal mechanisms behind this geographical distribution pattern are not yet understood. To analyze the genetic differentiation of geographically isolated populations, we conducted molecular phylogenetic analyses of the small subunit (SSU) ribosomal DNA (rDNA) and internal transcribed spacers (ITS) of rDNA nucleotide sequences in populations of V. fragilis collected in the South Atlantic (upwelling area off Namibia) and in the Pacific (Wellington Harbor, New Zealand, and Namako-ike, Japan). Our molecular analyses revealed SSU rDNA and ITS sequences strikingly similar or identical among these three disjunct populations. Such a low molecular genetic differentiation, a fixation rate converging to zero, could either arise from rapid dispersal, ultraslow mutation rates due to a strictly asexual mode of reproduction, unlimited genetic exchange between populations or the existence of a resting stage for survival under unfavorable conditions. We discuss each explanation and conclude that V. fragilis might possibly represent a protozoan trapped in evolutionary stasis.     

The Arabidopsis apyrase AtAPY1 is localized in the Golgi instead of the extracellular space

ABSTRACT: BACKGROUND: The two highly similar Arabidopsis apyrases AtAPY1 and AtAPY2 were previously shown to be involved in plant growth and development, evidently by regulating extracellular ATP signals. The subcellular localization of AtAPY1 was investigated to corroborate an extracellular function. RESULTS: Transgenic Arabidopsis lines expressing AtAPY1 fused to the SNAP-(O6-alkylguanine-DNA alkyltransferase)-tag were used for indirect immunofluorescence and AtAPY1 was detected in punctate structures within the cell. The same signal pattern was found in seedlings stably overexpressing AtAPY1-GFP by indirect immunofluorescence and live imaging. In order to identify the nature of the AtAPY1-positive structures, AtAPY1-GFP expressing seedlings were treated with the endocytic marker stain FM4-64 (N-(3-triethylammoniumpropyl)-4-(p-diethylamino-phenyl-hexatrienyl)-pyridinium dibromide) and crossed with a transgenic line expressing the trans-Golgi marker Rab E1d. Neither FM4-64 nor Rab E1d co-localized with AtAPY1. However, live imaging of transgenic Arabidopsis lines expressing AtAPY1-GFP and either the fluorescent protein-tagged Golgi marker Membrin 12, Syntaxin of plants 32 or Golgi transport 1 protein homolog showed co-localization. The Golgi localization was confirmed by immunogold labeling of AtAPY1-GFP. There was no indication of extracellular AtAPY1 by indirect immunofluorescence using antibodies against SNAP and GFP, live imaging of AtAPY1-GFP and immunogold labeling of AtAPY1-GFP. Activity assays with AtAPY1-GFP revealed GDP, UDP and IDP as substrates, but neither ATP nor ADP. To determine if AtAPY1 is a soluble or membrane protein, microsomal membranes were isolated and treated with various solubilizing agents. Only SDS and urea (not alkaline or high salt conditions) were able to release the AtAPY1 protein from microsomal membranes. CONCLUSIONS: AtAPY1 is an integral Golgi protein with the substrate specificity typical for Golgi apyrases. It is therefore not likely to regulate extracellular nucleotide signals as previously thought. We propose instead that AtAPY1 exerts its growth and developmental effects by possibly regulating glycosylation reactions in the Golgi.     

Intermembrane Space Proteome of Yeast Mitochondria

The intermembrane space (IMS) represents the smallest subcompartment of mitochondria. Nevertheless, it plays important roles in the transport and modification of proteins, lipids, and metal ions and in the regulation and assembly of the respiratory chain complexes. Moreover, it is involved in many redox processes and coordinates key steps in programmed cell death. A comprehensive profiling of IMS proteins has not been performed so far. We have established a method that uses the proapoptotic protein Bax to release IMS proteins from isolated mitochondria, and we profiled the protein composition of this compartment. Using stable isotope-labeled mitochondria from Saccharomyces cerevisiae, we were able to measure specific Bax-dependent protein release and distinguish between quantitatively released IMS proteins and the background efflux of matrix proteins. From the known 31 soluble IMS proteins, 29 proteins were reproducibly identified, corresponding to a coverage of >90%. In addition, we found 20 novel intermembrane space proteins, out of which 10 had not been localized to mitochondria before. Many of these novel IMS proteins have unknown functions or have been reported to play a role in redox regulation. We confirmed IMS localization for 15 proteins using in organello import, protease accessibility upon osmotic swelling, and Bax-release assays. Moreover, we identified two novel mitochondrial proteins, Ymr244c-a (Coa6) and Ybl107c (Mic23), as substrates of the MIA import pathway that have unusual cysteine motifs and found the protein phosphatase Ptc5 to be a novel substrate of the inner membrane protease (IMP). For Coa6 we discovered a role as a novel assembly factor of the cytochrome c oxidase complex. We present here the first and comprehensive proteome of IMS proteins of yeast mitochondria with 51 proteins in total. The IMS proteome will serve as a valuable source for further studies on the role of the IMS in cell life and death.Mitochondria are double-membrane-bound organelles that fulfill a multitude of important cellular functions. Proteomic analysis of purified mitochondria revealed that they contain approximately 1000 (yeast) to 1500 (human) different proteins (1–3). However, the distribution of these proteins among the four mitochondrial subcompartments (outer membrane, inner membrane, matrix, and intermembrane space) has been only marginally studied through global approaches. This is attributed to the high complexity of purifying submitochondrial fractions to a grade suitable for proteomic analysis. The best-studied submitochondrial proteomes comprise the outer membranes of S. cerevisae, N. crassa, and A. thaliana (4–6). The mitochondrial intermembrane space (IMS)¹ represents a highly interesting compartment for several reasons: it provides a redox active space that promotes oxidation of cysteine residues similar to the endoplasmic reticulum and the bacterial periplasm, but unlike cytosol, nucleus, or the mitochondrial matrix where the presence of thioredoxins or glutaredoxins prevents the risk of unwanted cysteine oxidation (7, 8). Furthermore in higher eukaryotes IMS proteins are released into the cytosol upon apoptotic induction, which triggers the activation of a cell-killing protease activation cascade (9, 10). The IMS can also exchange proteins, lipids, metal ions, and various metabolites with other cellular compartments, allowing mitochondrial metabolism to adapt to cellular homeostasis. In particular, the biogenesis and activity of the respiratory chain were shown to be controlled by various proteins of the IMS (11–13). Most of the currently known IMS proteins are soluble proteins; however, some inner membrane proteins have been annotated as IMS proteins as well, such as proteins that are peripherally attached to the inner membrane or membrane proteins that expose enzyme activity toward the IMS (8).All IMS proteins are encoded in the nuclear DNA and have to be imported after translation in the cytosol (14–19). Two main pathways are known to mediate the import and sorting of proteins into the IMS. One class of proteins contains bipartite presequences that consist of a matrix targeting signal and a hydrophobic sorting signal. These signals arrest the incoming preprotein at the inner membrane translocase TIM23. After insertion into the inner membrane, the soluble, mature protein can be released into the IMS by the inner membrane protease (IMP) (20–22). The second class of IMS proteins possesses characteristic cysteine motifs that typically are either twin CX₉C or twin CX₃C motifs (23, 24). Upon translocation across the outer membrane via the TOM complex, disulfide bonds are formed within the preproteins, which traps them in the IMS. Disulfide bond formation is mediated by the MIA machinery, which consists of the inner-membrane-anchored Mia40 and the soluble IMS protein Erv1 (25–28).The release of cytochrome c from the IMS upon binding and insertion of Bax at the outer membrane is a hallmark of programmed cell death. Although Bax is found only in higher eukaryotes, it was shown that recombinant mammalian Bax induces the release of cytochrome c upon incubation with isolated yeast mitochondria (29, 30). Furthermore, we found that not only cytochrome c but also other soluble IMS proteins are released from Bax-treated yeast mitochondria, whereas soluble matrix proteins largely remain within the organelle (30).We used this apparently conserved mechanism to systematically profile the protein composition of the yeast mitochondrial IMS by employing an experimental approach based on stable isotope labeling, which allowed for the specific identification of Bax-dependent protein release. Almost the entire set of known soluble IMS proteins was identified, and 20 additional, novel soluble IMS proteins were found. We confirmed IMS localization for 15 proteins through biochemical assays. Among these proteins, we identified novel proteins that fall into several classes: (i) those that are involved in maintaining protein redox homeostasis (thioredoxins, thioredoxin reductases, or thiol peroxidases), (ii) those that undergo proteolytic processing by IMP (Ptc5), (iii) those that utilize the MIA pathway for their import (Mic23 and Coa6), and (iv) those that play a role in the assembly of cytochrome c oxidase (Coa6).