NGS-PrimerPlex: High-throughput primer design for multiplex polymerase chain reactions

Multiplex polymerase chain reaction (PCR) has multiple applications in molecular biology, including developing new targeted next-generation sequencing (NGS) panels. We present NGS-PrimerPlex, an efficient and versatile command-line application that designs primers for different refined types of amplicon-based genome target enrichment. It supports nested and anchored multiplex PCR, redistribution among multiplex reactions of primers constructed earlier, and extension of existing NGS-panels. The primer design process takes into consideration the formation of secondary structures, non-target amplicons between all primers of a pool, primers and high-frequent genome single-nucleotide polymorphisms (SNPs) overlapping. Moreover, users of NGS-PrimerPlex are free from manually defining input genome regions, because it can be done automatically from a list of genes or their parts like exon or codon numbers. Using the program, the NGS-panel for sequencing the LRRK2 gene coding regions was created, and 354 DNA samples were studied successfully with a median coverage of 97.4% of target regions by at least 30 reads. To show that NGS-PrimerPlex can also be applied for bacterial genomes, we designed primers to detect foodborne pathogens Salmonella enterica, Escherichia coli O157:H7, Listeria monocytogenes, and Staphylococcus aureus considering variable positions of the genomes.     

Occurrence of Ditylenchus weischeri and Not D. dipsaci in Field Pea Harvest Samples and Cirsium arvense in the Canadian Prairies

The stem nematode, a parasite of the herbaceous perennial weed, Cirsium arvense (L.) Scop. and identified as Ditylenchus dipsaci (Kühn) Filipjev, was reported in the Canadian prairies in 1979. Recently, D. weischeri Chizhov parasitizing Cirsium arvense was described in Russia, and it has been shown that this species is not an agricultural pest. In this study, we examined Ditylenchus species found in field pea (Pisum sativum L.) grain harvest samples in 2009 and 2010 and from C. arvense shoots in pea fields in the Saskatchewan, Alberta, and Manitoba provinces. Samples from 538 fields (mainly yellow pea) were provided by 151 growers throughout the main pea-growing area of the Canadian prairies. Of the samples collected, 2% were positive for Ditylenchus. The population density of the nematode ranged between 4 and 1,500 nematodes kg^-1 pea harvest sample and related to presence of C. arvense seeds. Positive samples occurred in 2009 but not in 2010 and were from throughout the pea-growing area of the Canadian prairies and not related to cropping history. C. arvense collected from yellow pea fields in Saskatchewan and Manitoba, but not Alberta, were infested with Ditylenchus. Morphological and molecular (ITS-PCR-RFLP) traits indicated that this species belongs to D. weischeri. The results indicated the stem nematode found in yellow pea grain is D. weischeri which resided with C. arvense seeds and debris to pea samples. Unlike D. dipsaci, D. weischeri is not a nematode pest of economic importance; therefore, its presence in the pea harvest samples was not a concern.     

A Pipeline for Determining Protein–Protein Interactions and Proximities in the Cellular Milieu

It remains extraordinarily challenging to elucidate endogenous protein-protein interactions and proximities within the cellular milieu. The dynamic nature and the large range of affinities of these interactions augment the difficulty of this undertaking. Among the most useful tools for extracting such information are those based on affinity capture of target bait proteins in combination with mass spectrometric readout of the co-isolated species. Although highly enabling, the utility of affinity-based methods is generally limited by difficulties in distinguishing specific from nonspecific interactors, preserving and isolating all unique interactions including those that are weak, transient, or rapidly exchanging, and differentiating proximal interactions from those that are more distal. Here, we have devised and optimized a set of methods to address these challenges. The resulting pipeline involves flash-freezing cells in liquid nitrogen to preserve the cellular environment at the moment of freezing; cryomilling to fracture the frozen cells into intact micron chunks to allow for rapid access of a chemical reagent and to stabilize the intact endogenous subcellular assemblies and interactors upon thawing; and utilizing the high reactivity of glutaraldehyde to achieve sufficiently rapid stabilization at low temperatures to preserve native cellular interactions. In the course of this work, we determined that relatively low molar ratios of glutaraldehyde to reactive amines within the cellular milieu were sufficient to preserve even labile and transient interactions. This mild treatment enables efficient and rapid affinity capture of the protein assemblies of interest under nondenaturing conditions, followed by bottom-up MS to identify and quantify the protein constituents. For convenience, we have termed this approach Stabilized Affinity Capture Mass Spectrometry. Here, we demonstrate that Stabilized Affinity Capture Mass Spectrometry allows us to stabilize and elucidate local, distant, and transient protein interactions within complex cellular milieux, many of which are not observed in the absence of chemical stabilization.Insights into many cellular processes require detailed information about interactions between the participating proteins. However, the analysis of such interactions can be challenging because of the often-diverse physicochemical properties and the abundances of the constituent proteins, as well as the sometimes wide range of affinities and complex dynamics of the interactions. One of the key challenges has been acquiring information concerning transient, low affinity interactions in highly complex cellular milieux (3, 4).Methods that allow elucidation of such information include co-localization microscopy (5), fluorescence protein Förster resonance energy transfer (4), immunoelectron microscopy (5), yeast two-hybrid (6), and affinity capture (7, 8). Among these, affinity capture (AC)¹ has the unique potential to detect all specific in vivo interactions simultaneously, including those that interact both directly and indirectly. In recent times, the efficacy of such affinity isolation experiments has been greatly enhanced through the use of sensitive modern mass spectrometric protein identification techniques (9). Nevertheless, AC suffers from several shortcomings. These include the problem of 1) distinguishing specific from nonspecific interactors (10, 11); 2) preserving and isolating all unique interactions including those that are weak and/or transient, as well as those that exchange rapidly (10, 12, 13); and 3) differentiating proximal from more distant interactions (14).We describe here an approach to address these issues, which makes use of chemical stabilization of protein assemblies in the complex cellular milieu prior to AC. Chemical stabilization is an emerging technique for stabilizing and elucidating protein associations both in vitro (15–20) and in vivo (3, 12, 14, 21–29), with mass spectrometric (MS) readout of the AC proteins and their connectivities. Such chemical stabilization methods are indeed well-established and are often used in electron microscopy for preserving complexes and subcellular structures both in the cellular milieu (3) and in purified complexes (30, 31), wherein the most reliable, stable, and established stabilization reagents is glutaraldehyde. Recently, glutaraldehyde has been applied in the “GraFix” protocol in which purified protein complexes are subjected to centrifugation through a density gradient that also contains a gradient of glutaraldehyde (30, 31), allowing for optimal stabilization of authentic complexes and minimization of nonspecific associations and aggregation. GraFix has also been combined with mass spectrometry on purified complexes bound to EM grids to obtain a compositional analysis of the complexes (32), thereby raising the possibility that glutaraldehyde can be successfully utilized in conjunction with AC in complex cellular milieux directly.In this work, we present a robust pipeline for determining specific protein-protein interactions and proximities from cellular milieux. The first steps of the pipeline involve the well-established techniques of flash freezing the cells of interest in liquid nitrogen and cryomilling, which have been known for over a decade (33, 34) to preserve the cellular environment, as well as having shown outstanding performance when used in analysis of macromolecular interactions in yeast (35–39), bacterial (40, 41), trypanosome (42), mouse (43), and human (44–47) systems. The resulting frozen powder, composed of intact micron chunks of cells that have great surface area and outstanding solvent accessibility, is well suited for rapid low temperature chemical stabilization using glutaraldehyde. We selected glutaraldehyde for our procedure based on the fact that it is a very reactive stabilizing reagent, even at lower temperatures, and because it has already been shown to stabilize enzymes in their functional state (48–50). We employed highly efficient, rapid, single stage affinity capture (36, 51) for isolation and bottom-up MS for analysis of the macromolecular assemblies of interest (52–54). For convenience, we have termed this approach Stabilized Affinity-Capture Mass Spectrometry (SAC-MS).     

Evolution of the gall-forming plant parasitic nematodes (Tylenchida: Anguinidae) and their relationships with hosts as inferred from Internal Transcribed Spacer sequences of nuclear ribosomal DNA

Phylogenetic relationships among gall-forming plant parasitic nematodes of the subfamily Anguininae are reconstructed by maximum parsimony and maximum likelihood analyses. Sequences of the ITS of rDNA from 53 populations and species of gall-forming nematodes and five populations of the Ditylenchus dipsaci species complex were analysed. The phylogenetic trees strongly support monophyly of the genus Anguina and show nonmonophyly for the genera Mesoanguina and Heteroanguina. Morphological and biological characters are generally congruent with the anguinid groups identified in the rDNA phylogeny. Analyses of evolution of different gall types among anguinids reveal that there are apparent evolutionary trends in gall evolution: from abnormal swelling and growth of infested plant organs toward small localised galls, and from infestation of vegetative toward generative organs. Our study demonstrates that the main anguinid groups are generally associated with host plants belonging to the same or related systematic groups. The comparison of the ITS phylogenies of anguinids parasitising Poaceae and their host grasses shows a high level of cospeciation events.     

Effect of Light Intensity on the Morphogenesis of Stevia rebaudiana under In Vitro Conditions

Russian Journal of Plant Physiology - A study of the effect of various light intensity (75, 135, 230, and 382 µmol/(m2 s)) on the morphogenesis of Stevia rebaudiana (Bertoni) Bertoni in vitro...     

A van der Pol coupled-oscillator model as a basis for developing a system for suppressing MHD instabilities in a tokamak

The main parameters of tokamak discharges are known to be limited by large-scale MHD instabilities. Sometimes, the instabilities lead to a rapid (on time scales of tens of microseconds) disruption of the discharge current and to the release of all the energy stored in the plasma column at the discharge chamber wall. This process, which is called the disruptive instability, may have irreversible catastrophic consequences for the operation of a fusion reactor. In the present paper, a study is made of the dynamics of self-oscillations in systems of two and six van der Pol coupled oscillators. A van der Pol coupled-oscillator model is used to develop a multivariable feedback controller based on the combined principle of compensating for internal cross feedbacks within the object and introducing damping feedbacks in each control channel. By using mathematical simulation methods, it is shown that the controller designed guarantees the suppression of self-oscillations in a system of van der Pol oscillators over a fairly broad range of parameters of the object under control (and thereby provides the structural stability of the object). The nonlinear control system model makes it possible to suppress coupled MHD perturbations developing in a tokamak plasma.     

Poly(T) Variation in Heteroderid Nematode Mitochondrial Genomes is Predominantly an Artefact of Amplification

We assessed the rate of in vitro polymerase errors at polythymidine [poly(T)] tracts in the mitochondrial DNA (mtDNA) of a heteroderid nematode (Heterodera cajani). The mtDNA of these nematodes contain unusually high numbers of poly(T) tracts, and have previously been suggested to contain biological poly(T) length variation. However, using a cloned molecule, we observed that poly(T) variation was generated in vitro at regions containing more than six consecutive Ts. This artefactual error rate was estimated at 7.3 × 10⁻⁵ indels/poly(T) tract >6 Ts/cycle. This rate was then compared to the rate of poly(T) variation detected after the amplification of a biological sample, in order to estimate the ‘biological + artefactual’ rate of poly(T) variation. There was no significant difference between the artefactual and the artefactual + biological rates, suggesting that the majority of poly(T) variation in the biological sample was artefactual. We then examined the generation of poly(T) variation in a range of templates with tracts up to 16 Ts long, utilizing a range of Heteroderidae species. We observed that T deletions occurred five times more frequently than insertions, and a trend towards increasing error rates with increasing poly(T) tract length. These findings have significant implications for studies involving genomes with many homopolymer tracts.