Large amplicon droplet digital PCR for DNA‐based monitoring of pediatric chronic myeloid leukaemia

Quantification of tumour‐specific molecular markers at the RNA and DNA level for treatment response monitoring is crucial for risk‐adapted stratification and guidance of individualized therapy in leukaemia and other malignancies. Most pediatric leukaemias and solid tumours of mesenchymal origin are characterized by a relatively low mutation burden at the single nucleotide level and the presence of recurrent chromosomal translocations. The genomic fusion sites resulting from translocations are stable molecular tumour markers; however, repeat‐rich DNA sequences flanking intronic breakpoints limit the design of high sensitivity PCR assays for minimal residual disease (MRD) monitoring. Here, we quantitatively evaluated the impact of repeat elements on assay selection and the feasibility of using extended amplicons (≤1330 bp) amplified by droplet digital PCR to monitor pediatric chronic myeloid leukaemia (CML). Molecular characterization of 178 genomic BCR‐ABL1 fusion sites showed that 64% were located within sequence repeat elements, impeding optimal primer/probe design. Comparative quantification of DNA and RNA BCR‐ABL1 copy numbers in 687 specimens from 55 pediatric patients revealed that their levels were highly correlated. The combination of droplet digital PCR, double quenched probes and extended amplicons represents a valuable tool for sensitive MRD assessment in CML and may be adapted to other translocation‐positive tumours.     

Introduction of transformed chloroplasts from tobacco into petunia by asymmetric cell fusion

Plastid engineering technique has been established only in Nicotiana tabacum, and the widespread application is severely limited so far. In order to exploit a method to transfer the genetically transformed plastomes already obtained in tobacco into other plant species, somatic cell fusion was conducted between a plastome transformant of tobacco and a cultivar of petunia (Petunia hybrida). A tobacco strain whose plastids had been transformed with aadA (a streptomycin/spectinomycin adenylyltransferase gene) and mdar [a gene for monodehydroascorbate reductase (MDAR)] and a petunia variety, ‘Telstar’, were used as cell fusion partners. An efficient regeneration system from the protoplasts of both the parents, and effectiveness of selection for the aadA gene with spectinomycin were established before the cell fusion. In addition, the influence of UV irradiation on the callus development from the protoplasts and shoot regeneration of tobacco was investigated. Protoplasts were cultured after cell fusion treatment with polyethylene glycol, and asymmetric somatic cybrids were selected using the aadA gene as a marker. Although many shoots of tobacco that had escaped the UV irradiation regenerated, several shoots possessing the morphology of petunia and the resistance to spectinomycin were obtained. Molecular analyses of the petunia type regenerants demonstrated that they had the nuclear and mitochondrial genomes derived from petunia besides the chloroplasts of tobacco transformed with aadA and mdar. Furthermore, it was ascertained that mdar was transcribed in the somatic cybrids. The results indicate the success in intergeneric transfer of transformed plastids of tobacco into petunia.     

Interspecies protoplast fusion inLarix: Comparison of electric and chemical methods

Summary Larix was chosen for the study on interspecies protoplast fusion due to its ability to regenerate plants from protoplasts derived from embryogenic cultures.L. laricina line L2 was used in fusion experiments with eitherL. × eurolepis line L6 orL. × leptoeuropaea line L5. A method of unambiguous labeling of parental protoplasts prior to fusion was developed using vital fluorescent dyes. Of a number of dyes tested, only rhodamine B hexyl ester chloride (R6) and 3,3′-dihexylox-carbocyanine iodide (DiOC₆) stained the protoplasts in a consistent and uniform fashion. The fusion of mixed parental protoplasts that were internally labeled was carried out either in the presence of a 20% polyethylene glycol (PEG) solution or in an electric field. The progress of fusion was readily observed, taking only minutes under the experimental conditions. The fusion products could be identified by dual fluorescence several h after the onset of fusion. Heterofusion frequencies of approximately 18% and 6% in the presence of PEG and an electric field, respectively, were attained. Postfusion cultures betweenL. × laricina protoplasts and protoplasts ofL. × leptoeuropaea gave rise to cell colonies and betweenL. laricina andL. × eurolepis, to mature somatic embryos.     

Characterization of the wound-inducible protein ipomoelin from sweet potato

The ipomoelin (IPO) gene, a wound- and methyl jasmonate-inducible gene, was isolated from sweet potato (Ipomoea batatas cv. Tainung 57), and previously demonstrated to be regulated by dephosphorylated proteins and calcium ion (Chen Y.-C. et al. Plant Cell and Environment 26, 1373–1383, 2003). In this report, the function of the IPO protein was further studied. The IPO gene was characterized as having one intron and presenting two copies within the genome of sweet potato. The IPO protein appeared 1 d after the leaves of sweet potato were wounded. Surprisingly, the accumulation of the IPO protein remained for 7 d after wounding. Additionally, after the IPO protein was fused to a histidine tag , the His-IPO fusion protein produced from Escherichia coli BL21DE3 was then used to perform the haemagglutination test, which demonstrated that His-IPO fusion protein agglutinated human blood cells. Furthermore, several carbohydrates, including methyl α- d -glucopyranoside, methyl α- d -mannopyranoside, maltose, mannose, glucose, galactose, and lactose, reduced the efficiency of the His-IPO fusion protein in agglutinating human blood cells. These experimental results may indicate that the IPO protein is a lectin , a carbohydrate-binding protein. Notably, the IPO protein retarded the growth and development of silkworm, and thus reduced silkworm survival rates. Therefore, these findings indicate that the function of the IPO protein is to protect plants from insect attack.     

ULTRASTRUCTURAL ASPECTS OF SEXUAL REPRODUCTION IN THE RED TIDE DINOFLAGELLATE GONYAULAX TAMARENSIS1

The marine dinoflagellate Gonyaulax tamarensis Lebour is best known for its propensity to form blooms known as red tides in coastal waters worldwide. This paper examines the sexual cycle of this organism using light and electron microscopy. Sexual reproduction begins with contact between thecate gametes which subsequently shed their thecae to fuse along their pellicular layers. Nuclear fusion occurs well after cytoplasmic fusion and is characterized by several distinctive features: a highly vesiculate nucleoplasm without microtubules; nucleoli and V-shaped chromosomes abut the nuclear envelope distal to the region of nuclear contact; and each chromosome possesses a longitudinal line, the central chromosomal axis. Fusion results in a planozygote with numerous cytoplasmic storage products and a slightly thickened layer beneath the pellicle. Subsequent loss of thecal plates and a thickening of the sub-pellicular layer results in a non-motile hypnozygote. A newly-formed hypnozygote possesses numerous minute papillae along its outer surface, formed by the up-folding of the accumulating wall layer. Maturation of the hypnozygote wall results in a smooth three-layered wall, the outermost layer of which is the pellicular layer. Hypnozygote germination produces a large quadriflagellate plan-omeiocyte with a single nucleus and thecal plates identical to vegetative cells. Two subsequent divisions, presumably meiotic, result in Jour cells morphologically identical to vegetative cells.     

Reprogramming of somatic cells induced by fusion of embryonic stem cells using hemagglutinating virus of Japan envelope (HVJ-E)

In this research, hemagglutinating virus of Japan envelope (HVJ-E) was used to reprogram somatic cells by fusion with mouse embryonic stem (ES) cells. Neomycin-resistant mouse embryonic fibroblasts (MEFs) were used as somatic cells. Nanog-overexpressing puromycin-resistant EB3 cells were used as mouse ES cells. These two cells were fused by exposing to HVJ-E and the generated fusion cells were selected by puromycin and G418 to get the stable fusion cell line. The fusion cells form colonies in feeder-free culture system. Microsatellite analysis of the fusion cells showed that they possessed genes from both ES cells and fibroblasts. The fusion cells were tetraploid, had alkali phosphatase activity, and expressed stem cell marker genes such as Pou5f1, Nanog, and Sox2, but not the fibroblast cell marker genes such as Col1a1 and Col1a2. The pluripotency of fusion cells was confirmed by their expression of marker genes for all the three germ layers after differentiation induction, and by their ability to form teratoma which contained all the three primary layers. Our results show that HVJ-E can be used as a fusion reagent for reprogramming of somatic cells.     

High-level expression of an antimicrobial peptide histonin as a natural form by multimerization and furin-mediated cleavage

Direct expression of an antimicrobial peptide (AMP) in Escherichia coli causes several problems such as the toxicity of AMP to the host cell, its susceptibility to proteolytic degradation, and decreased antimicrobial activity due to the additional residue(s) introduced after cleavage of AMPs from fusion partners. To overcome these problems and produce a large quantity of a potent AMP histonin (RAGLQFPVGKLLKKLLKRLKR) in E. coli, an efficient expression system was developed, in which the toxicity of histonin was neutralized by a fusion partner F4 (a truncated fragment of PurF protein) and the productivity was increased by a multimeric expression of a histonin gene. The expression level of the fusion proteins reached a maximum with a 12-mer of a histonin gene. In addition, because of the RLKR residues present at the C terminus of histonin, furin cleavage of the multimeric histonin expressed produces an intact, natural histonin. The AMP activity of the histonin produced in E. coli was identical to that of a synthetic histonin. With our expression system, 167 mg of histonin was obtained from 1 l of E. coli culture. These results may lead to a cost-effective solution for the mass production of AMPs that are toxic to a host.     

How do groups of red imported fire ants (Hymenoptera: Formicidae) feed on a droplet of sugar water?

Many previous studies have focused on the foraging behaviors and strategies of the red imported fire ants, Solenopsis invicta Buren on solid food or granular bait; little attention has been paid to how liquid sugar is fed upon. In the present study, behavioral responses of S. invicta to 25% sucrose water droplets were observed. Five foraging patterns were identified in S. invicta colonies under laboratory conditions: (i) no feeding, no sucrose water feeding was observed; (ii) surround feeding, ants surrounded and fed along the edge of the sucrose droplet; (iii) stacked feeding, ants stacked and fed along the edge of the sucrose droplet; (iv) droplet‐break feeding, ants broke the liquid droplet and sucked sucrose water that spread on surface of the substance or soil particles previously transported by ants; and (v) cover feeding, whole surface of the sucrose droplet was covered by layers of feeding ants. This is the first time cover feeding in S. invicta has been reported, which obviously requires more ants compared to the other patterns. In addition, individual ants were tracked in videos under laboratory conditions, and behavioral repertoires that led to stacking, covering and droplet‐breaking were identified and described. The field investigation showed that surround feeding was most frequently performed by S. invicta foragers; however, cover feeding was not observed under field conditions during this study. Both laboratory and field studies showed colony‐level variations in sugar‐water feeding.     

A theory of self-organization of an excitable membrane formed on the protoplasmic droplet isolated from Nitella

The process of self-organization of an excitable membrane of protoplasmic droplet of Nitella is studied theoretically by taking the interaction of the local electric current caused by the spatially non-uniform distribution of active domains into account. The theoretical model employed is that the surface membrane forms a mosaic structure composed of lipids and protein molecules, and that each element of the mosaic structure (domain) on the membrane has two distinct conformations corresponding to excited and resting states. The molecules constituting the membrane are derived from the inside of the protoplasm by diffusion. The excitability of the surface membrane appears suddenly after a morphogenetic structure of the membrane is formed with time on the surface of droplet. Time courses of the variation in membrane potential and in membrane resistance are calculated, and the results are compared with experimental data obtained with the protoplasmic droplet of Nitella.     

Peg-mediated fusion of Rubus idaeus (raspberry) and R. fruticosus (blackberry) protoplasts,selection and characterisation of callus lines

ABSTRACT

Cell suspension-derived protoplasts of two cultivated Rubus species, Rubus idaeus-raspberry (subgenus Idaeobatus 2n=2x=14) and R. fruticosus-blackberry (a complex species aggregate within the subgenus Eubatus, 2n=4x=28) were fused using different polyethylene glycol (PEG) fusion treatments. Duration of PEG treatment and choice of culture media influenced the rate of cell divisions and plating efficiency. Colony formation was initiated on solid media for the production of several callus lines. Cytological analyses were performed on selected callus lines with hexaploid chromosome number. Two hexaploid fusion callus lines, selected for their homogeneity in growth and ploidy level, were examined by molecular cytogenetic techniques of fluorescent in situ hybridisation (FISH) and genomic in situ hybridisation (GISH). GISH revealed the presence of the heterokaryon within the fusion callus lines. FISH probed with ribosomal DNA (rDNA) showed variable numbers and sizes of loci. Aberrant distribution and condensation of rDNA were common in interphase cells. FISH results suggest that large karyotype rearrangements occurred, including variation in chromosome number and rDNA loci translocations. Attempts to regenerate plants from the hexaploid callus lines following several applications of plant growth regulator combinations were unsuccessful. This may be attributed to the genomic reorganisation and instability of these long-term fusion callus cultures.     

In Vitro Characterization and Mosquito (Aedes aegypti) Repellent Activity of Essential-Oils-Loaded Nanoemulsions

The nanoemulsions composed of citronella oil, hairy basil oil, and vetiver oil with mean droplet sizes ranging from 150 to 220 nm were prepared and investigated both in vitro and in vivo. Larger emulsion droplets (195–220 nm) shifted toward a smaller size (150–160 nm) after high-pressure homogenization and resulted in higher release rate. We proposed that thin films obtained from the nanoemulsions with smaller droplet size would have higher integrity, thus increasing the vaporization of essential oils and subsequently prolonging the mosquito repellant activity. The release rates were fitted with Avrami’s equations and n values were in the same range of 0.6 to 1.0, implying that the release of encapsulated limonene was controlled by the diffusion mechanism from the emulsion droplet. By using high-pressure homogenization together with optimum concentrations of 5% (w/w) hairy basil oil, 5% (w/w) vetiver oil (5%), and 10% (w/w) citronella oil could improve physical stability and prolong mosquito protection time to 4.7 h due to the combination of these three essential oils as well as small droplet size of nanoemulsion.     

Interspecific somatic hybrids between Solanum bulbocastanum and S. tuberosum and their haploidization for potato breeding

Protoplast fusion between incongruent Solanum bulbocastanum and S. tuberosum haploids was accomplished to produce hybrids combining elite traits from both parents. We identified 11 somatic hybrids out of 42 regenerants analyzed through ISSR markers. Some hybrids had loss or gain of fragments compared to the parents, likely due to rearrangements and deletions of chromosome segments after fusion, and/or to somaclonal variation during hybrid regeneration. Increased heterotic vigor for some traits as well as high diversity was observed as the effect of both ploidy and fusion combination. Microsporogenesis analysis indicated the occurrence of multivalent configurations and several meiotic abnormalities, such as chromosomes bridges and various spindle orientations. Since all hybrids were sterile, in vitro anther culture was employed for haploidization as a possible strategy to overcome barriers to hybridizations. Haploids were obtained from all the tetraploid S. bulbocastanum (+) S. tuberosum somatic hybrids tested, although with differences in both the number of embryos per 100 anthers cultured and the number of differentiated green plantlets. This is the first report on the successful production of haploid plants from S. bulbocastanum (+) S. tuberosum hybrids.     

Biogenic and taphonomic processes affecting the development of shell assemblages: an actuopaleontological case study from mussel banks on North Sea tidal flats

Summary From spring 1997 to winter 2000, the composition and structure of epibenthic bivalve clusters and patches on a backbarrier tidal flat of the East Frisian Wadden Sea were continoously analyzed. The studied bivalve aggregates developed from intense and approximately simultaneous spatfalls of Mytilus edulis and Cerastoderma edule in 1996, after a severe winter (1995/96) with 64 days of driftice coverage. Spatfall occurred on a bare sandy tidal flat, whereas adjacent shell layers situated at the southern margin of the spatfall site were not colonized. Usually, such shell layers are the most common secondary hard substrates in the backbarrier area favorable for initial settlement of Mytilus larvae. The shell layers predominantly originate from erosion of former Mya arenaria and C. edule populations. So far, the studied bivalve aggregates are composed of approximately equal proportions of living specimens of both species, M. edulis and C. edule. The incorporated cockles were prevented from burrowing into the rediment by the coherent byssal mesh. In contrast to the results of previous studies, the great bulk of epibenthic living C. edule did survive for several years within the clusters. Therefore, the common terms ‘Mytilus banks’, ‘Mytilus patches’ or ‘Mytilus clumps’ may be too general and may erroneously imply a dominance of (or mono-specific composition by) M. edulis. In the fossil record, i.e. in diagenetically consolidated shell layers, the enforced epibenthic life-mode of C. edule and the remarkable species composition as well as the temporal development of the aggregates would not be recognizable.     

Model of droplet dynamics in the Argentine ant Linepithema humile (Mayr)

The formation of droplets of ants Linepithema humile (Mayr) is observed under certain experimental conditions: a fluctuating aggregate forms at the end of a rod and a droplet containing up to 40 ants eventually falls down. When the flux of incoming ants is sufficient, this process can continue for several hours, leading to the formation and fall of tens of droplets. Previous work indicates that the time series of drop-to-drop intervals may result from a nonlinear low-dimensional dynamics, and the interdrop increments exhibit long-range anticorrelations. A model of aggregation and droplet formation, based on experimental observations, is introduced and shown to reproduce these properties.     

Multiplication and cryopreservation of vanilla (<Emphasis Type="Italic">Vanilla planifolia</Emphasis> ‘Andrews’)

A simple and efficient method for multiplication of vanilla (Vanilla planifolia) was developed using in vitro fragmented explants (IFEs) as propagules. IFEs were obtained after dissecting apices from in vitro propagated clusters of plantlets, by cutting the remaining base of these plant clusters into segments of about 1 cm in length. After 4 months of culture on multiplication medium, 100% of IFEs produced up to 15 new shoots per explant, providing an efficient additional method for in vitro propagation of vanilla that maximizes the use of available material. Cryopreservation of apices from in vitro grown plants was achieved using the droplet vitrification protocol. Maximum survival (30%) and further regeneration (10%) of new shoots were obtained for apices derived from clusters of in vitro plantlets produced from microcuttings through a three-step droplet vitrification protocol: 1-d preculture of apices on solid MS medium with 0.3 M sucrose; loading with a 0.4 M sucrose + 2 M glycerol solution for 20–30 min; and exposure to plant vitrification solution PVS3 for 30 min at room temperature. Even though the cryogenic protocol needs to be optimized to improve results, this work represents the first successful report of cryopreservation of vanilla apices.     

Cellular dynamics of conjugation in the ciliate euplotes aediculatus. I. Cytoskeletal elements

The fate and possible roles of the cytoskeleton in the process of conjugation in the hyptrich ciliate Euplotes aediculatus were investigated. Following the coalescence of the plasma membranes of the conjugant cells, a fusion zone or bridge of cytoplasm contributed by both partners is constructed. The sub-alveolar microtubule layers of the vegetative cell cortex remain in place to define the fusion zone boundaries after cell union. The initial fusion zone consists primarily of featureless ground cytoplasm; soon the ground plasm becomes crowded with microtubules and anastomosing smooth endoplasmic reticulum, which become displaced only late in conjugation as the migratory pronuclei are exchanged between partners. Fusion zone microtubules, functioning in some undetermined way, may be involved in the nuclear migration. Resorption of the posterior portion of each partner's buccal apparatus results in the degradation of the component cilia within acid phosphatase-positive autophagic bodies. Silver staining for light microscopy shows that the late fusion zone contracts forward from the posterior border, then constricts to separate the conjugants. In some separating pairs remnants of a microfilamentous assembly are seen at the posterior edge of the fusion zone; the full extent of this system may be masked by partial degradation due to osmium tetroxide fixation. Treatment of conjugants for 6 hours with cytochalasin B prevents separation, possibly through inhibition of the actin-like microfilament assembly in the fusion zone. The observations and experiments favor a model of cell separation following conjugation in which the fusion zone is resorbed by motile or contractile processes occurring within or around the fusion bridge itself.     

Influence of ants (Hymenoptera: Formicidae) on honeydew excretion and escape behaviors in a myrmecophile,dalbulus quinquenotatus (Homoptera: Cicadellidae), and its congeners

Few leafhopper species are known to be ant-attended. Evidence is presented that unequivocally demonstrates that Dalbulus quinquenotatusis a myrmecophile. In a greenhouse study, the behavior of D. quinquenotatusand four Dalbulusspecies not associated with ants was observed in the absence and presence of the pavement ant, Tetramorium caespitum. D. quinquenotatusis readily contacted by tending ants and responds to stroking on the abdomen from ants' antennae by excreting and holding honeydew droplets until droplets are removed by ants. Nonattended Dalbulusspecies avoid contact with ants by walking,jumping, or flying away when approached. D. quinquenotatusexcretes three to six times the volume of honeydew as do two nonattended species, D. maidisand D. gelbus.Droplets of D. quinquenotatusare about 23% larger in diameter and excreted two to four times more frequently than for the other species. D. quinquenotatustakes about 0.15 s to form honeydew droplets on the anal tube, then holds the droplet an average of 0.28 s before expelling it when ants are absent. In the presence of ants, D. quinquenotatusholds the droplet an average of 1.31 s, which gives ants time to harvest the droplet. Nonattended species, however, immediately expel droplets from the anal tube after droplet formation. This ant-leafhopper mutualism apparently has evolved due to the ability of D. quinquenotatusto have extensive physical contact with ants and excrete large amounts of honeydew. D. chiapensis,a possible descendent of D. quinquenotatus,may secondarily have lost its mutualistic relationship with ants.     

Phagocytized Intracellular Microsporidian Blocks Phagosome Acidification and Phagosome-Lysosome Fusion

This study examines the relationship between phagosome acidification and phagosome-lysosome fusion events using phagocytized Glugea hertwigi spores. The incidence of lysosome fusion with Glugea spores in phagosomes of mouse peritoneal macrophages and of Tetrahymena was monitored using colloidal gold and acridine orange as labels for secondary lysosomes. Over 80% of the Glugea phagosomes remained segregated from the labeled compartments in macrophages after 60 min; this inhibition of fusion was still evident after 4 h. In Tetrahymena, Glugea spores also showed a high capacity to block fusion with secondary lysosomes (67%); however, spores coated with cationized ferritin showed an 80% fusion rate with labeled acidic compartments (i.e. lysosomes) after 60 min with both Tetrahymena and macrophages. The pH of phagosome compartments was monitored by measuring the emissions of fluorescein isothiocyanate (FITQ-labeled Glugea ingested by Tetrahymena. Tetrahymena phagosomes with FITC-Glugea did not acidify within the first hour after phagocytosis; however, phagosomes with cationized ferritin-labeled Glugea underwent acidification during this time period. This acidification took place although the capability of the host cells' lysosomes to fuse was blocked by pretreatment with poly-D-glutamic acid. The cationized ferritin bound to Glugea spores was uncoupled from the spore wall prior to fusion with colloidal gold-labeled compartments. In vitro testing showed that ferritin dissociation requires an acid pH, indicating that phagosomes acidify prior to lysosome fusion.