Homologous recombination is required for AAV-mediated gene targeting

High frequencies of gene targeting can be achieved by infection of mammalian cells with recombinant adeno-associated virus (rAAV) vectors [D. W. Russell and R. K. Hirata (1998) Nature Genet., 18, 325–330; D. W. Russell and R. K. Hirata (2000) J. Virol., 74, 4612–4620; R. Hirata et al. (2002) Nat. Biotechnol., 20, 735–738], but the mechanism of targeting is unclear and random integration often occurs in parallel. We assessed the role of specific DNA repair and recombination pathways in rAAV gene targeting by measuring correction of a mutated enhanced green fluorescent protein (EGFP) gene in cells where homologous recombination (HR) or non-homologous end-joining (NHEJ) had been suppressed by RNAi. EGFP-negative cells were transduced with rAAV vectors carrying a different inactivating deletion in the EGFP, and in parallel with rAAV vectors carrying red fluorescent protein (RFP). Expression of RFP accounted for viral transduction efficiency and long-term random integration. Approximately 0.02% of the infected GFP-negative cells were stably converted to GFP positive cells. Silencing of the essential NHEJ component DNA-PK had no significant effect on the frequency of targeting at any time point examined. Silencing of the SNF2/SWI2 family members RAD54L or RAD54B, which are important for HR, reduced the rate of stable rAAV gene targeting ~5-fold. Further, partial silencing of the Rad51 paralogue XRCC3 completely abolished stable long-term EGFP expression. These results show that rAAV gene targeting requires the Rad51/Rad54 pathway of HR.     

Does host plant richness explain diversity of ectomycorrhizal fungi? Re‐evaluation of Gao et al. (2013) data sets reveals sampling effects

The generally positive relationship between biodiversity of groups of directly or indirectly interacting organisms is one of the most important ecological concepts (Gaston, 2000 Nature, 405 , 220–227; Scherber C, Eisenhauer N, Weisser WW et al., 2010 Nature, 468 , 553–556). In a recent issue of Molecular Ecology, Gao C, Shi N‐N, Liu Y‐X et al. (2013: 22 , 3403–3414) reported that the richness of plants and ectomycorrhizal fungi is positively correlated both at local and at global scales. Here, we challenge these findings by re‐analysis of data and ascribe the reported results to sampling effect and poor data compilation.     

Chromium (III) enhanced diamine silver staining of proteins and DNA in gels

Chromium (III) enhanced the sensitivities of diamine silver staining of four proteins between 6- and 50-fold over that of the Coomassie Brilliant Blue (CBB)-chromium modified thiosulfate-silver staining method (Zhou et al. Biotechnology Letters, 2002, 24: 1561–1567). Using six dsDNA fragments, the detection limits of this new method was 10 to 30 pg per band, being 10- to 25-fold more sensitive than previous methods.     

Cambrian Acritarchs from the Col di Foglia (Agordo) southalpine metamorphic basement, Italian Eastern Alps: the oldest biostratigraphic record in the alps

Abstract  The Lower Palaeozoic biostratigraphic records in the Alps are briefly reviewed and the result of a new study of the acritarch assemblage found by Sassi et al. (1984) in the greenschist facies black metapelites of the Southalpine metamorphic basement at Col di Foglia, and studied by Kalvacheva et al. (1986), is presented. The new  taxonomic and biostratigraphic study indicates a late Cambrian age, which is the oldest unquestionable, recently assessed, biostratigraphic dating of the entire Alps, as well as of the Italian peninsular. Keywords Alps, Southalpine metamorphic basement, Eastern Alps, Agordo, Acritarchs, Cambrian Subject codes: G17002     

On taxonomy and distribution of fossil Cerophytidae (Coleoptera: Elateriformia) with description of a new Mesozoic species of Necromera Martynov 1926

In this paper the data on fossils of the family Cerophytidae are reviewed. New synonymy on generic names Necromera Martynov 1926 (proposed in composition of the family Oedemeridae); Idiomerus Dolin in Dolin et al. 1980, n. syn. (proposed in composition of Elateridae) and Leptocnemus Hong & Wang 1990, n. syn. (proposed without any family attribution) is established. New materials on this family from Mesozoic deposits of Asia are cited. As a result, it was established that this spreads in deposits of both Upper Jurassic and Lower Cretaceous. Mercata festira Lin 1986 described from Lower Jurassic as member of Silphidae and Abrotus reconditus Dolin in Dolin et al. 1980 described from Upper Jurassic as a member of Elateridae are transferred to Cerophytidae. Diagnoses of the genus Necromera and family Cerophytidae in compression fossils are elaborated. Necromera admiranda n. sp. is described from the Upper Jurassic-Lower Cretaceous in Liaoning, China. The historical development of the family from the Lower Jurassic is discussed.     

腺相关病毒载体工程毒株精细化纯化

[背景] 大量制备高纯度的重组腺相关病毒（Recombinant Adeno-Associated Virus,rAAV）,是以腺相关病毒（AAV）为投递载体的基因靶向治疗或基因工程药物研发过程中需要解决的重要问题。[目的] 利用层析柱法大量纯化质粒和rAAV细胞培养液,以获得高纯度的rAAV颗粒。[方法] 对组装rAAV的3种质粒用HiPrep^TM10 Sepharose 6FF和HiTrap PlasmidSelect Xtra凝胶层析柱纯化,目的是得到组装AAV的超螺旋质粒DNA,然后对组装rAAV过程中的AAV-293细胞培养方法进行优化,组装成功病毒后,再对收集到的细胞提取液用HiLoad^TM10Q和HiLoad^TM10SP阴阳离子交换层析柱纯化,最后用纯化浓缩后的rAAV感染HT1080细胞,通过流式细胞仪检测荧光表达细胞数,计算浓缩后活病毒感染滴度。[结果] HiPrep和HiTrap层析柱纯化后得到大量高纯度的超螺旋质粒DNA,组装病毒后,用HiLoad柱纯化获得大量高纯度的rAAV颗粒,通过测定,rAAV基因组滴度达到（2.46±0.37）×10¹² TCID₅₀/mL （MOI=1.0×10⁴）。[结论] 通过一系列精细化组装纯化制备出高纯度、高滴度rAAV病毒颗粒。     

An interfacial equilibria model for the electrokinetic properties of a fat emulsion. Part III: A predictive computer model

Abstract

The semi-empirical thermodynamic model for the binding of metal ions to an emulsion (intralipid 20%) reported previously (Hall et al., 1991 ; Gaskin et al., 1993) is incorporated into a thermodynamic computer model. This permits the zeta potential and emulsion stability together with precipitate formation to be estimated for any intravenous nutrition regimen. A regimen frequently used in the intravenous nutrition of patients is considered in this modeling study. The effect of solution pH and calcium on the zeta potential of the emulsion is predicted. Calcium and magnesium are the only metal cations which are predicted to be of importance when considering stability of this emulsion.     

Evaluation of Antibody-Dependent Enhancement of SARS-CoV Infection in Rhesus Macaques Immunized with an Inactivated SARS-CoV Vaccine

正Dear Editor,In 2003,severe acute respiratory syndrome coronavirus(SARS-CoV)emerged in Guangdong Province,China,infected more than 8000 individuals,and resulted in a 10%mortality rate(Rota et al.2003).Later,in 2012,a novel CoV,Middle East respiratory syndrome coronavirus(MERS-CoV),was isolated from the sputum of a man in Saudi Arabia(Perl et al.2014).Notably,MERS-CoV     

Proximity probing assays for simultaneous visualization of protein complexes in situ

Evaluation of: Leuchowius KJ, Clausson CM, Grannas K et al. Parallel visualization of multiple protein complexes in individual cells in tumor tissue. Mol. Cell Proteomics doi:10.1074/mcp.O112.023374 (2013) (Epub ahead of print).

Techniques for in situ detection and quantification of proteins in fixed tissue remain an important element of both basic biological analyses and clinical biomarker research. The practical importance of such techniques can be exemplified by the everyday clinical use of immunohistochemical detection of the estrogen receptor and HER2 in tissues from breast cancer patients. Several techniques are currently available for detection of single proteins and post-translational modifications, but very few are suitable for detection of protein complexes. Methods that enable simultaneous detection and quantification of protein complexes provide novel possibilities for understanding the biological role(s) of protein complexes and may open new opportunities to improve clinical biomarker research. Leuchowius et al. describe an improved proximity ligation assay for in situ detection of protein complexes, which is able to detect and quantify several protein complexes simultaneously in the same tissue specimen.     

Het up mould unleashes a sporekiller prion

Conclusion het-s/het-S incompatibility is one of only a handful of fungal heterokaryon incompatibility systems that have begun to be molecularly analysed (for a review see Saupe 2000). The spore killer phenotype also is one of only a few segregation ratio distortions that has been studied in any detail. Finally the [HET-s] prion is one of only three examples known in non-mammalian systems (for a review see Wickneret al 1999). Clearly thehet-s/het-S story intersects with several exciting areas of research that offer many opportunities for young scientists.     

Search for the Cellular Functions of Plant Hsp100/Clp Family Proteins

Referee: Dr. Peter Csermely, Department of Medical Chemistry, Semmeliweis Univ. School of Medicine, P.O. Box 260, H-1444 Budapest 8, Hungary Hsp100/Clp family of proteins is ubiquitously distributed in living systems. Detailed work carried out in bacterial and yeast cells has shown that regulatory members of the Clp family (mainly ClpA, ClpB, and ClpC), together with the catalytic subunit (mainly ClpP), comprise an ATP-dependent two-component proteolytic system. Members of the Hsp100/Clp protein family are not only involved in the regulation of energy-dependent protein hydrolysis but also function as molecular chaperones. However, the biochemical/physiological role(s) of the Hsp100/Clp protein family in higher plants has yet to be elucidated. Recently, this protein family has been implicated in plant stress responses: the hot1 mutant of Arabidopsis thaliana, which has mutation in hsp101 gene, and is defective in tolerance to high temperature (S.-W. Hong and E. Vierling, 2000, Proc Natl Acad Sci USA, 97 (8), 4392-4397) and the transgenic Arabidopsis thaliana plants overexpressing AtHsp101 gene exhibit high temperature tolerance (C. Quietsch et al., 2000, Plant Cell, 12, 479–492). Furthermore, the Hsp101 protein is involved in the translational regulation of cellular mRNAs and one such candidate has been identified as the photosynthetic electron transport gene Ferredoxin 1 mRNA (J. Ling et al., 2000, Plant Cell, 12, 1213–1227). We present what is known about the bacterial, yeast, and plant Hsp100/Clp proteins, discuss their possible relationship, and, more importantly, examine the cellular roles that this important family of proteins plays in plants.     

Purification of Stearidonic Acid (18:4(n-3)) and Hexadecatetraenoic Acid (16:4(n-3)) from Algal Fatty Acid with Lipase and Medium Pressure Liquid Chromatography

Stearidonic acid (18:4(n-3)) and hexadecatetraenoic acid (16:4(n-3)) are included in some edible marine algae such as Undaria pinnatifida and Ulva pertusa with relatively high compositions (up to 40%) of total fatty acids. In order to prepare 16:4(n-3) and 18:4(n-3) enriched fatty acid concentrates, we screened for a suitable lipase which concentrates these acids by the removal of other fatty acids in the selective esterification reaction reported by Shimada et al. (Shimada et al. (1997), J. Am. Oil Chem. Soc., 74, 1465-1470). In combination with the lipase reaction and reversed-phase medium pressure liquid chromatography, we purified 18:4(n-3) and 16:4(n-3) to more than 95% purity.     

Why some parasites are widespread and abundant while others are local and rare?

Abundances and distributions of species are usually associated. This implies that as a species declines in abundance so does the number of sites it occupies. Conversely, when there is an increase in a species' range size, it is usually followed by an increase in population size (Gaston et al. 2000 ). This ecological phenomenon, also known as the abundance–occupancy relationship (AOR), is well documented in several species of animals and plants (Gaston et al. 2000 ) but has been little investigated in parasites. In this issue of Molecular Ecology, Drovetski et al. ( 2014 ) investigated the AOR in avian haemosporidians (vector‐borne blood parasites) using data from four well‐sampled bird communities. In support of the AOR, the research group found that the abundance of parasite cytochrome b lineages (a commonly used proxy for species identification within this group of parasites) was positively linked with the abundance of susceptible avian host species and that the most abundant haemospordian lineages were those with larger ranges. Drovetski et al. ( 2014 ) also found evidence for both hypotheses proposed to explain the AOR in parasites: the trade‐off hypothesis (TOH) and the niche‐breadth hypothesis (NBH). Interestingly, the main predictor of the AOR was the number of susceptible hosts (i.e. number of infected birds) and not the number of host species the parasites were able to exploit.     

Abnormality in the fissioning control of a strain of asexual Dugesia dorotocephala found near Fort Collins,Colorado

Asexual Dugesia dorotocephala, collected from a new site several miles northwest of Fort Collins, Colorado, appeared similar, at the time of collection, to ones collected from other sites in the same general locale. However, these exhibited marked differences in the more crowded conditions of laboratory culture: abnormally long, 30–50 mm specimens, seemingly unable to fission, developed. Since previous studies of asexual D. dorotocephala had revealed an inhibitory effect of cohorts on fissioning (Best et al., 1969, 1974, 1975; Pigon et al., 1974), experiments were conducted with this new strain to ascertain the effect of population density (group size) on fissioning incidence. Results are described which show that this inhibitory effect of cohorts on fissioning is exaggerated in this new strain.     

Reconsideration of anopheline mosquito phylogeny (Diptera: Culicidae: Anophelinae) based on morphological data

The phylogeny of anopheline mosquitoes (Culicidae: Anophelinae) is re‐examined using morphological data derived from adults, fourth‐instar larvae and pupae. Based on the data set of Sallum et al. (2000), we add some previously missing data and simplify and recode characters to eliminate ambiguities and more accurately reflect homologies, with special emphasis on characters of the male genitalia that provide the main criteria for the subgeneric classification of genus Anopheles. The principal aim of the study is to assess objectively the phylogenetic relationships and classification of two taxa not included by Sallum et al. (2000): Anopheles corethroides, a representative of the Australasian Stigmaticus Group, and An. kyondawensis, an unusual Oriental species whose adult and pupal stages were only recently discovered. The revised data set consists of 167 characters for 66 species representing the three traditionally recognised genera of Anophelinae, the six traditionally accepted subgenera of genus Anopheles and all informal series and most species groups of subgenera Anopheles, Cellia and Nyssorhynchus. The data are analysed using equal weighting (EW) and implied weighting (IW). Analysis under EW generates a strict consensus tree with principal lineages consistent with those reported by Sallum et al. (2000). Analysis under IW supports the monophyly of Anophelinae, the basal position of Chagasia, the monophyly of subgenera Cellia, Kerteszia and Nyssorhynchus, and the sister relationship of Kerteszia + Nyssorhynchus, but otherwise yields relationships that differ significantly in one respect or another from those obtained in all previous analyses of both morphological and molecular data. Subgenus Anopheles is arrayed as a polyphyletic lineage basal to a monophyletic clade comprising the Neotropical Kerteszia + Nyssorhynchus and the Old World Cellia in a sister‐group relationship. Bironella, Lophopodomyia and Stethomyia are firmly nested within subgenus Anopheles, which would nevertheless still be paraphyletic if these taxa were subsumed within it. Anopheles kyondawensis is well supported as the sister group of Bironella + all other Anopheles. Bironella, Stethomyia, An. corethroides and several other Anopheles clades are each strongly supported in a pectinate series of relationships, terminating in the clade comprising subgenera Cellia, Kerteszia and Nyssorhynchus. These relationships and other aspects of the phylogeny are discussed in relation to the formal and informal classification of genus Anopheles.     

Ectopic Localization of Mitochondrial ATP Synthase: A Target for Anti-Angiogenesis Intervention?

A receptor for angiostatin was identified on the surface of endothelial cells as F₁–F₀ ATP synthase (Moser et al., 1999). Proc. Natl. Acad. Sci. U.S.A. 96, 2811–2816. This ectopic ATP synthase catalyzes ATP synthesis and is inhibited by angiostatin over a wide pH range. Endothelial cells grown at normal pH suffer no ill effects from this angiostatin-mediated inhibition of ATP synthase, whereas endothelial cells grown at low, tumor-like extracellular pH cannot maintain a normal intracellular pH and die. Angiostatin inhibits both ATP synthesis and ATP hydrolysis (Moser et al., 2001) and interferes with intracellular pH regulation (Wahl and Grant, 2002; Wahl et al., 2002). Although angiostatin administered intravenously is cleared from the circulation in a matter of minutes, angiostatin-mimetics that are more stable have potential for clinical application. An angiostatin-mimetic activity has recently been observed using a polyclonal antibody against the β catalytic subunit of ATP synthase. In order to explore the mechanism of action of angiostatin and its mimetics, further work needs to be done to evaluate clinical applicability, specificity, and contraindications for this class of therapeutics.     

Botulinum Neurotoxin Types A,B, and E: Fragmentations by Autoproteolysis and Other Mechanisms Including by <Emphasis Type="Italic">O</Emphasis>-Phenanthroline–Dithiothreitol,and Association of the Dinucleotides NAD<Superscript>+</Superscript>/NADH with the Heavy Chain of the Three Neurotoxins

The first evidence of autoproteolytic activity of the ~50-kDa light chain of the clostridial neurotoxins (NT) is traceable to the observations that the light chains of botulinum NT serotypes A and E, separated from their ~100-kDa heavy chain conjugate, were found cleaved at the amino side of Tyr250 and Arg244, respectively [DasGupta and Foley (1989). Biochimie 71: 1183–1200]. Specific cleavages of the recombinant light chain of NT type A, including at Tyr249–Tyr250, firmly established that the cleavages reported earlier were due to autoproteolysis [Ahmed et al. (2001). J. Protein Chem. 20: 221–231; Ahmed et al. (2003). Biochemistry 42:12539–12549] and not by contaminating proteases or non-enzymatic. We now report many cleavages in the NT types A, B and E and also in their separated light and heavy chains, and identification of several of the peptide bonds cleaved. None of the identified cleaved bonds (–P₁–P₁′ –) in one serotype (except Asp–Pro) was found common in other serotypes or cleaved within itself at a second site. After separation from the heavy chain self-cleavages of the light chains of type A, B and E at Tyr249–Tyr250, Gln258–Ser259 and Ile243–Arg244, respectively indicate an intriguing feature (in the aligned sequences these bonds of type A and B are 2 and type A and E are 4 peptide bonds apart) that may have some role in the NT’s structure–function relationship yet to be understood. We point out that autoproteolysis of a single peptide bond (Phe418–Thr419 or Phe422–Glu423) in NT type A reported by Ahmed et al. (2001) can potentially generate proteolytically active light chain freed of the heavy chain; this is an efficient pathway, that by-passes nicking by a trypsin-like protease(s) inside the intrachain disulfide bridge and its reductive cleavage. We offer probable explanations for the observed cleavages such as acid- and metal-mediated (non-catalytic and non-stoichiometric) reactions in addition to autoproteolysis but cannot predict which mechanism(s) of cleavage occur or prevail following NT’s entry in the body as poison or therapeutic agent. The metal chelator O-phenanthroline (above critical miceller concentration) in the presence of dithiothreitol cleaved type E NT at limited sites generating discrete 114-, 87-, 49-, 42-, and 31-kDa fragments but degraded NTs type A and B extensively. The limited cleavage of type E NT was dependent on the presence of metal ion(s) bound to the protein and its native (urea sensitive) conformation. The self-cleavage of the NTs at specific sites prompted us to search for specific binding sites on the NTs analogous to SNARE-motifs—the 9-residuelong motifs present on the NT’s natural substrates (SNAP-25, syntaxin, VAMP/synaptobrevin); such putative binding motifs (sites) noted on all clostridial NTs are reported here. Their relationship to the observed autoproteolysis remains to be determined experimentally. The dinucleotide NAD⁺/NADH associated with the NTs type A, B and E (2–3 NADH per protein molecule) via their H-chains, and a portion of the H-chain (toward the C-terminus) appears to exhibit limited amino acid sequence homology with lactate dehydrogenase—a representative NAD⁺/NADH binding protein.     

Seeing the forest beyond the trees

In a recent paper (Mitchard et al. 2014, Global Ecology and Biogeography, 23 , 935–946) a new map of forest biomass based on a geostatistical model of field data for the Amazon (and surrounding forests) was presented and contrasted with two earlier maps based on remote‐sensing data Saatchi et al. (2011; RS1) and Baccini et al. (2012; RS2). Mitchard et al. concluded that both the earlier remote‐sensing based maps were incorrect because they did not conform to Mitchard et al. interpretation of the field‐based results. In making their case, however, they misrepresented the fundamental nature of primary field and remote‐sensing data and committed critical errors in their assumptions about the accuracy of research plots, the interpolation methodology and the statistical analysis. By ignoring the large uncertainty associated with ground estimates of biomass and the significant under‐sampling and spatial bias of research plots, Mitchard et al. reported erroneous trends and artificial patterns of biomass over Amazonia. Because of these misrepresentations and methodological flaws, we find their critique of the satellite‐derived maps to be invalid.