A Database of Accurate Electrophoretic Migration Patterns for Human Proteins

Native molecular weight (MW) is one of the defining features of proteins. Denaturing gel electrophoresis (SDS-PAGE) is a very popular technique for separating proteins and determining their MW. Coupled with antibody-based detection, SDS-PAGE is widely applied for protein identification and quantitation. Yet, electrophoresis is poorly reproducible and the MWs obtained are often inaccurate. This hampers antibody validation and negatively impacts the reliability of western blot data, resulting worldwide in a considerable waste of reagents and labour. We argue that, to alleviate these problems there is a need to establish a database of reference MWs measured by SDS-PAGE. Using mass spectrometry as an orthogonal detection method, we acquired electrophoretic migration patterns for approximately 10′000 human proteins in five commonly used cell lines. We applied a robust internal calibration of migration to determine accurate and reproducible molecular weights. This in turn allows merging replicates to increase accuracy, but also enables comparing different cell lines. Mining of the data obtained highlights structural factors that affect migration of distinct classes of proteins. When combined with peptide coverage, the data produced recapitulates known post-translational modifications and differential splicing and can be used to formulate hypotheses on new or poorly known processing events. The full information is freely accessible as a web resource through a user friendly graphical interface (https://pumba.dcsr.unil.ch/). We anticipate that this database will be useful to investigators worldwide for troubleshooting western blot experiments, but could also contribute to the characterization of human proteoforms.     

Rearranged Genomic RNA Segments Offer a New Approach to the Reverse Genetics of Rotaviruses

Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The RV genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. We report here a reverse genetics system for RV based on the preferential packaging of rearranged RNA segments. Using this system, wild-type or in vitro-engineered forms of rearranged segment 7 from a human rotavirus (encoding the NSP3 protein), derived from cloned cDNAs and transcribed in the cytoplasm of COS-7 cells with the help of T7 RNA polymerase, replaced the wild-type segment 7 of a bovine helper virus (strain RF). Recombinant RF viruses (i.e., engineered monoreassortant RF viruses) containing an exogenous rearranged RNA were recovered by propagating the viral progeny in MA-104 cells, with no need for additional selective pressure. Our findings offer the possibility to extend RV reverse genetics to segments encoding nonstructural or structural proteins for which no potent selective tools, such as neutralizing antibodies, are available. In addition, the system described here is the first to enable the introduction of a mutated gene expressing a modified nonstructural protein into an infectious RV. This reverse genetics system offers new perspectives for investigating RV protein functions and developing recombinant live RV vaccines containing specific changes targeted for attenuation.Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile viral gastroenteritis and are responsible for up to 700,000 deaths each year (6, 24). The RV genome consists of 11 segments of double-stranded RNA (dsRNA) which can be separated by polyacrylamide gel electrophoresis (PAGE), resulting in typical dsRNA profiles exhibiting four well-defined size classes of segments. However, some RV show unusual dsRNA profiles in which standard-sized segments are replaced by larger, rearranged forms (for a review, see reference 5). Gene rearrangements were first reported for RV isolated from immunodeficient children with chronic infection (11, 26) and can be obtained experimentally by serial passages at a high multiplicity of infection (MOI) in cell culture (10, 14, 21). We recently showed that rearrangements also occur during acute RV infection (36). Gene rearrangement usually consists of a partial head-to-tail duplication of a segment sequence. In most cases, sequence duplication occurs after the stop codon, leaving the open reading frame (ORF) untouched and the encoded protein unchanged (5, 6). Less frequently, the duplication occurs within the ORF, which thus encodes a modified protein (8, 38).Manipulation at the cDNA level of most positive- and negative-strand RNA viral genomes, followed by rescue of infectious virus, is now well established and has provided a better understanding of RNA virus replication and pathogenesis. In the case of Reoviridae, the development of reverse genetics systems has been hampered by the nature of the genome, which carries 10 to 12 dsRNA segments that are densely packed within the viral particle and are transcribed and replicated within a subviral structure. Recent major advances were obtained in the development of reverse genetics systems for some Reoviridae. For mammalian orthoreo- and orbiviruses, reverse genetics systems based on the transfection of plasmid cDNAs (13) or cDNA-derived mRNAs (2) corresponding to a complete set of 10 RNA segments have been established, allowing the rescue of infectious viral progeny. However, for RV, there is no report indicating that transfection of a complete set of viral mRNAs can result in the rescue of infectious viruses, and it is still unknown whether the RV genome can be infectious. In 2006, Komoto et al. (16) described the first reverse genetics system for RV, based on a model originally developed for influenza viruses by Palese and colleagues (17). In this system, an exogenous RV mRNA synthesized in the cell cytoplasm by the T7 RNA polymerase (T7pol), expressed by a recombinant vaccinia virus, is packaged in place of its homologous counterpart into a helper RV. This reverse genetics system has allowed the recovery of engineered monoreassortant infectious RV (designated recombinant RV) with an incorporated exogenous segment 4 encoding the spike protein VP4. An in vitro-modified cDNA-derived segment 4 was also successfully introduced into a recombinant RV to obtain a virus carrying a VP4 antigenic chimera (15). However, this system is restricted to segments encoding antigenically distinct viral surface proteins (like VP4) because the selection of recombinant viruses requires the use of specific and potent neutralizing antibodies to eliminate wild-type (WT) helper viruses.Results obtained from a preliminary study suggested that rearranged RNA segments might overcome this limitation. Indeed, we first characterized human RV clones containing rearranged segment 7, 11, or both (8) and then compared the fitness levels of rearranged versus WT viruses. We found that viruses with rearranged segment 7 or 11 replicated less well than or equally to WT viruses, as judged by viral growth curve experiments. Surprisingly, in competition growth experiments, rearranged segment 7 or 11 was always selected into the viral progenies, even when mixed infections were performed with a ratio of 1 rearranged to 1,000 WT viruses (4). The absence of a growth advantage conferred on the virus by rearranged segments, combined with their preferential segregation into the viral progenies, suggested that rearranged RNA segments might be packaged preferentially. These observations are in agreement with results of earlier studies showing that rearranged segment 5 or 11 segregated preferentially into viral progenies issued from mixed infections with WT virus (10, 19).We developed a reverse genetics system for RV on the basis of the preferential packaging of rearranged RNAs. We report here the rescue of recombinant viruses carrying cDNA-derived rearranged segment 7 (either unmodified or containing silent mutations introduced by site-directed mutagenesis to generate restriction enzyme sites as markers), with no selection pressure other than serial passage in cell culture. We also report the rescue of a recombinant virus expressing a double-sized recombinant NSP3 protein encoded by an in vitro-modified cDNA-derived rearranged segment 7, showing for the first time that an in vitro-engineered gene encoding a modified nonstructural protein can be introduced into an infectious RV.     

Heat Shock Protein 27 Plays Two Distinct Roles in Controlling Human Breast Cancer Cell Migration on Laminin-5

It has recently been reported that phosphorylation of the small heat shock protein 27 (hsp27) enhances p38 MAP kinase dependent migration of bovine and human vascular endothelial cells. We have examined the role of hsp27 in controlling the constitutive migration of human breast cancer cells on the extracellular matrix molecule laminin-5. In a haptotaxis assay, anisomycin- or heat shock-induced phosphorylation of hsp27 enhances migration of MDA-MB-231 breast cancer cells constitutively overexpressing hsp27. Under these conditions, hsp27 redistributes to the nucleus. Unphosphorylated hsp27, which remains in the cytosol, induces resistance to a subset of drugs that inhibit haptotactic migration of these cells. We conclude that hsp27 plays two distinct roles in controlling migration of breast cancer cells: phosphorylated hsp27 enhances migration, while unphosphorylated hsp27 can sustain migration in the presence of inhibitory drugs.     

The Viral KSHV Chemokine vMIP-II Inhibits the Migration of Naive and Activated Human NK Cells by Antagonizing Two Distinct Chemokine Receptors

Natural killer (NK) cells are innate immune cells able to rapidly kill virus-infected and tumor cells. Two NK cell populations are found in the blood; the majority (90%) expresses the CD16 receptor and also express the CD56 protein in intermediate levels (CD56^Dim CD16^Pos) while the remaining 10% are CD16 negative and express CD56 in high levels (CD56^Bright CD16^Neg). NK cells also reside in some tissues and traffic to various infected organs through the usage of different chemokines and chemokine receptors. Kaposi''s sarcoma-associated herpesvirus (KSHV) is a human virus that has developed numerous sophisticated and versatile strategies to escape the attack of immune cells such as NK cells. Here, we investigate whether the KSHV derived cytokine (vIL-6) and chemokines (vMIP-I, vMIP-II, vMIP-III) affect NK cell activity. Using transwell migration assays, KSHV infected cells, as well as fusion and recombinant proteins, we show that out of the four cytokine/chemokines encoded by KSHV, vMIP-II is the only one that binds to the majority of NK cells, affecting their migration. We demonstrate that vMIP-II binds to two different receptors, CX3CR1 and CCR5, expressed by naïve CD56^Dim CD16^Pos NK cells and activated NK cells, respectively. Furthermore, we show that the binding of vMIP-II to CX3CR1 and CCR5 blocks the binding of the natural ligands of these receptors, Fractalkine (Fck) and RANTES, respectively. Finally, we show that vMIP-II inhibits the migration of naïve and activated NK cells towards Fck and RANTES. Thus, we present here a novel mechanism in which KSHV uses a unique protein that antagonizes the activity of two distinct chemokine receptors to inhibit the migration of naïve and activated NK cells.     

On Two Measures of Unbalancedness in a One-Way Model and Their Relation to Efficiency

This paper discusses two measures of unbalancedness in a one-way model and shows that for a given statistical procedure they may serve as measures of efficiency of a design. They also allow to compare for example estimation methods for variance components in designs with a fixed level of unbalancedness.     

Patterns of Sperm Transfer Behavior in a Pholcid Spider with Two Distinct Copulatory Phases

Sexual selection is the responsible force for the evolution and maintenance of genital diversity and function. This is the case for example, of genital movements performed by males during mating and copulation duration. Spiders perform ritualized copulations whereby males carry out different types of movements using their pedipalps with varying duration. The function and duration of these pedipalp movements is unclear. In the pholcid spider, Holocnemus pluchei males that copulate with virgin females perform two copulatory phases: phase I in which the pedipalps move and phase II in which pedipalps remain motionless. Using H. pluchei as a study species, our study aims were: 1) to assess if sperm transfer occurs when pedipalps move or are still and quantify the number of sperm in male bulbs and in the female uterus externus after copulation; and, 2) to determine if amount of sperm transferred to females is associated with duration of each copulatory phase. Two experimental groups (i. e. complete copulation and interrupted copulation) were established in which the amount of sperm remaining in the male bulbs and the amount of sperm stored by females were determined. Our results show that sperm transfer occurs during phase I, that males transfer almost all sperm from their bulbs while the females store only 20% of that male amount. There was no relation between the amount of sperm transferred or stored and the duration of the copulatory phases. These results support the hypothesis that while both phases may serve a copulatory courtship, only phase I (when pedipalps move) serves for sperm transfer.     

Convergent Evolution of Argonaute-2 Slicer Antagonism in Two Distinct Insect RNA Viruses

RNA interference (RNAi) is a major antiviral pathway that shapes evolution of RNA viruses. We show here that Nora virus, a natural Drosophila pathogen, is both a target and suppressor of RNAi. We detected viral small RNAs with a signature of Dicer-2 dependent small interfering RNAs in Nora virus infected Drosophila. Furthermore, we demonstrate that the Nora virus VP1 protein contains RNAi suppressive activity in vitro and in vivo that enhances pathogenicity of recombinant Sindbis virus in an RNAi dependent manner. Nora virus VP1 and the viral suppressor of RNAi of Cricket paralysis virus (1A) antagonized Argonaute-2 (AGO2) Slicer activity of RNA induced silencing complexes pre-loaded with a methylated single-stranded guide strand. The convergent evolution of AGO2 suppression in two unrelated insect RNA viruses highlights the importance of AGO2 in antiviral defense.     

两种生境条件下西南桦的生殖构件及其空间格局

在广西靖西土石山生态交错区调查了西南桦(Betula alnoides)生殖植株的生长状况,并研究土山和石山两种生境条件下西南桦的生殖构件及其在冠层的分布格局.结果表明:石山西南桦开始开花结实的林木胸径明显小于土山;两种立地上西南桦植株的花芽、花序数以及雄花序比率与其胸径、树高均不显著相关(P>0.05);土山和石山的西南桦雄花序数随着总花序数的增加均呈对数曲线增长趋势,在生殖投资较小的情况下植株趋向于优先发育雄花序,而在生殖投资较大时主要增加雌花序的数量;西南桦生殖构件在两种生境间存在显著差异,石山植株的雄花芽数、每个雄发芽发育出的雄花序数均显著低于土山(P<0.05),雄花序少于雌花序的植株比例远高于土山,生殖枝比率亦显著低于土山(P<0.05);尽管石山雄花序、雌花序和总花序数量以及雄花序比率低于土山,其雌花序比率高于土山,但是由于两种立地上单株间变异幅度大,独立t检验表明其差异均未达到显著水平;两种立地上西南桦花序大多着生于树冠上、中层,且90%以上的生殖枝居于树冠外层.     

Cladoceran Periodicity Patterns in Relation to Selected Environmental Factors in Two Cascading Warm-Water Reservoirs Over a Decade

Hydrobiologia - Temporal changes in occurrence, population density and reproductive intensity (% of breeding individuals, clutch size) of species of the planktonic cladoceran assemblage in two...     

Two Distinct Varieties of Giardia in a Mixed Infection from a Single Human Patient

ABSTRACT. Twenty-five in vitro cultures of Giardia duodenalis derived from a Brisbane patient were established to assess the genetic heterogeneity of a population. Each of the established lines carried a predominance of one of two distinct varieties of Giardia . The two varieties were heterogeneous by four unambiguous criteria that were representative of the whole genome. These included restriction enzyme polymorphisms, hybridization with the cloned rDNA repeat and with a gene encoding a cysteine-rich surface protein, electrophoretic karyotyping and DNA fingerprinting. Differences between parasites derived from this patient were greater than have been seen between all other established G. duodenalis in vitro cultures from both human and animals. The culture were heavily selected such that a single Giardia line carried a predominance of one genotype and was not representative of the entire original population.     

Alternative Responses to Predation in Two Headwater Stream Minnows Is Reflected in Their Contrasting Diel Activity Patterns

Animals exhibit diel periodicity in their activity in part to meet energy requirements whilst evading predation. A competing hypothesis suggests that partitioning of diel activities is less important because animals capitalise on opportunity. To test these hypotheses we examined the diel activity patterns for two cyprinid minnows, chubbyhead barb Barbus anoplus and the Eastern Cape redfin minnow Pseudobarbus afer that both occur within headwater streams in the Eastern Cape, South Africa. Chubbyhead barbs exhibited consistent nocturnal activity based on both field and laboratory observations. Due to the absence of fish predators within its habitat, its nocturnal behaviour suggests a response to the cost associated with diurnal activity, such as predation risk by diving and wading birds. In contrast, redfin minnows showed high diurnal activity and a shoaling behaviour in the wild, whereas, in the laboratory, they showed high refuge use during the diel cycle. Despite their preference for refuge in the laboratory, they were diurnally active, a behaviour that was consistent with observations in the wild. The diurnal activity of this species suggests a response to the cost associated with nocturnal activity. Such a cost could be inferred from the presence of the longfin eel, a native predator that was active at night, whereas the daytime shoaling behaviour suggests an anti-predator mechanism to diurnal visual predators. The implications of these findings relate to the impacts associated with the potential invasions by non-native piscivores that occur in the mainstem sections. Diurnal activity patterns for redfin minnows, that are IUCN-listed as endangered, may, in part, explain their susceptibility to high predation by visual non-native piscivores, such as bass and trout. In contrast, the nocturnal habits of chubbyhead barbs suggest a probable pre-adaptation to visual predation. The likelihood of invasion by nocturnally-active sharptooth catfish Clarias gariepinus, however, may compromise this prior advantage.     

Automated Learning of Subcellular Variation among Punctate Protein Patterns and a Generative Model of Their Relation to Microtubules

Characterizing the spatial distribution of proteins directly from microscopy images is a difficult problem with numerous applications in cell biology (e.g. identifying motor-related proteins) and clinical research (e.g. identification of cancer biomarkers). Here we describe the design of a system that provides automated analysis of punctate protein patterns in microscope images, including quantification of their relationships to microtubules. We constructed the system using confocal immunofluorescence microscopy images from the Human Protein Atlas project for 11 punctate proteins in three cultured cell lines. These proteins have previously been characterized as being primarily located in punctate structures, but their images had all been annotated by visual examination as being simply “vesicular”. We were able to show that these patterns could be distinguished from each other with high accuracy, and we were able to assign to one of these subclasses hundreds of proteins whose subcellular localization had not previously been well defined. In addition to providing these novel annotations, we built a generative approach to modeling of punctate distributions that captures the essential characteristics of the distinct patterns. Such models are expected to be valuable for representing and summarizing each pattern and for constructing systems biology simulations of cell behaviors.     

CCR2 Defines a Distinct Population of NK Cells and Mediates Their Migration during Influenza Virus Infection in Mice

Natural killer (NK) cells are innate lymphocytes that play an important role in control of viral infections. We recently showed that intranasal infection of mice with influenza virus induced the accumulation of NK cells in the airways. NK cells however did not proliferate in the airways or in the draining lymph node, but in the bone marrow mainly. As also monocyte-precursors undergo vigorous proliferation in the bone marrow (BM) during infections and then egress CCR2-dependently, we decided to determine the role of CCR2 in NK cell migration during intranasal influenza virus infection. We show that a unique population of NK cells in the BM expressed CCR2 and that monocyte chemotactic protein-1 (MCP-1), one of the CCR2 ligands, was produced in the airways of influenza virus infected mice. Analysis of BM chimeric mice reconstituted with a mix of wild-type (wt) and CCR2-deficient BM cells showed that upon influenza virus infection, a significantly lower proportion of CCR2-deficient than wt NK cells was recovered from the bronchoalveolar lavage (BAL). Taken together, our data demonstrate that during influenza virus infection a proportion of NK cells migrate in a CCR2-dependent fashion.