Dna Ploidy Studies of Benign and Malignant Tumours: Comparison of Flow Cytometry and Image Analysis Techniques Using Two Types of Cytological Specimen

DNA ploidy studies were carried out on Feulgen stained smears and cytocentrifuge preparations from 35 malignant tumours and four benign neoplasms using the CAS image analyser. The smears were prepared from scrapings from fresh tumour tissue whereas the cytocentrifuge preparations were prepared from single nuclear suspensions from paraffin-embedded cell blocks from the same tumour. Histograms obtained by image analysis of the tumour scrapes were compared with those obtained on the cytocentrifuge preparations. Concordant results were obtained in four benign tumours (100%) and 32 malignant tumours (91%). The results obtained by image analysis were also compared with results obtained by flow cytometry of the tumour tissue. Discordant results were obtained for three malignant tumours. Possible reasons for the discrepancy include sampling error, tumour heterogeneity and selective loss of cell populations during processing.     

Taxonomic implications of genome size for all species of the genus Gasteria Duval (Aloaceae)

Nuclear DNA content (2C) is used as a new criterion to investigate all species of the genus Gasteria Duval including the three recently described species Gasteria polita van Jaarsv., G. pendulifolia van Jaarsv. and G. glauca van Jaarsv.. The 122 accessions investigated have the same chromosome number (2n=2x=14), with exception of three tetraploid plants found. The nuclear DNA content of the diploids, as measured by flow cytometry with Propidium Iodide, is demonstrated to range from 32.8–43.2 pg. This implies that the largest genome contains roughly 10¹⁰ more base pairs than the smallest. Based on DNA content the species could be divided in five groups: G. rawlinsonii Oberm. with 32.8 pg, 13 mostly inland species with 34.3–36.0 pg, five coastal species with 36.5–39.0 pg and Gasteria batesiana Rowley with 43.2 pg. The thirteen species with 34.3–36.0 pg could be divided further, in a group of eight species occupying mainly very restricted areas with 34.3–35.1 pg and a second group of five species with 35.2–36.0 pg mainly occupying large areas. These five groups did not coincide very well with the two sections and four series of Gasteria based on a cladistic analysis by van Jaarsveld et al. (1994). Based on its long leafy branches, location in the centre of Gasteria species distribution and its by far lowest DNA content, G. rawlinsonii might be the most primitive member of the genus. Nuclear DNA content as measured by flow cytometry is shown to be relevant to provide additional information on the relationships between Gasteria species.     

A Flow Cytometric Analysis of the Nuclear 2C DNA Content in 17 Phaseolus Species (53 Genotypes)

The genus Phaseolus is characterized by a highly stable karyotype of 2n = 22. Despite this constancy, the size of the chromosomes varies, and crossing of species is possible only in a few cases. We determined the 2C nuclear DNA content of a number of Phaseolus species, cultivars and genotypes by flow cytometry, in order to realize the interspecific and intraspecific variation of the 2C value. The data range from 1.03 pg to 2.18 pg without any clear correlation to systematic relationships. The mean DNA values of wild and cultivated forms, as well as those of Andean and Mesoamerican genotypes, do not differ significantly. The variation is interpreted in terms of some nucleotypic adaptations. The data may be useful for molecular biological analyses, as well as for biotechnological and classical breeding programmes.     

Pollen morphology of Justicia L. (Acanthaceae) from Thailand and its taxonomic value

The acetolysed pollen of 33 species of the genus Justicia in Thailand is investigated using both light and scanning electron microscopy. The pollen of the genus is characterised as being isopolar, bilaterally or radially symmetrical, with mostly prolate or prolate spheroidal shape. Based on characters with high factor loading in the cluster analysis and principal components analysis, the studied species are divided into two major groups; one with 3-colporate with six pseudocolpi and the other with 2-porate or 2–3-colporate with aperture areas. The pollen morphology of each group is described and illustrated. Pollen characters are useful for taxonomic delimitation and relationships among Thai species.     

On the ecology of Bonnemaisonia hamifera in its preferred habitats on the Western coast of Brittany (France)

Sexual reproduction of Bonnemaisonia hamifera Hariot is the rule on the western coast of Brittany. Each spring the alga is luxuriant in the Molène Islands, indicating that optimum conditions for growth occur there. These conditions, which include a sandy substrate and frequently renewed clear water, seem to be essential for the maturation of the male and female organs of Bonnemaisonia hamifera on the coasts of Brittany.     

Ploidy variation of progenies from intra- and inter-ploidy crosses with regard to trisomic production in asparagus (<Emphasis Type=Italic>Asparagus officinalis</Emphasis> L.)

Ploidy variation within progenies from intra- and inter-ploidy crosses of asparagus were determined in this study. The DNA content of most reduced microspores was half that of somatic cells in diploid, triploid and tetraploid plants as determined by flow cytometry. Many viable seeds were obtained from various 2x × 2x and 2x × 3x intra- and inter-ploidy crosses. Nuclear DNA contents of these progenies from reciprocal crosses between diploids and triploids were similar to those expected of diploids, but with a wider range: hyper- and hypo-diploids including trisomics seem to be involved in the progenies. Indeed, the number of chromosomes in the progenies of 2x × 3x crosses ranged from 18 to 23. Based on frequencies of viable seeds and trisomic phenotypic appearance, it was concluded that the 2x × 3x cross was most efficient for obtaining trisomic plants in Asparagus officinalis.     

流式细胞仪检测中细胞DNA样品制备的探讨

为了获得适合流式细胞仪检测的样品,通过碘化同啶标记和流式细胞仪对食管癌细胞内DNA含量分布进行分析,在不同的时间内对血液中淋巴细胞内DNA含量的测定结果进行研究。结果显示：结构完整的细胞DNA的荧光点图形成二倍体,四倍体的区域。组A各样品具有良好一致性,1～3天内差异小;组B各样品差异较大,因此细胞的完整性是DNA含量检测的基本条件,细胞经PI染色后若不能及时检测,可避光、4℃放置1～3天仍可获得较理想的实验结果。     

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.     

Hormonal control of endoreduplication in sugar beet (Beta vulgaris L.) seedlings growing in vitro

The effect on endoreduplication in sugar beet (Beta vulgaris L.) seedlings of five plant hormones in MS medium, ethylene, 24-epibrassinolide (EBR), gibberellic acid (GA(3) ), kinetin and 1-naphthaleneacetic acid (NAA), as well as a combination of kinetin and NAA at two different concentrations, was studied using flow cytometry. Analyses of DNA content in nuclei of the root, hypocotyl and cotyledons of seedlings growing in vitro were performed during their early development, starting from when the root was 0.5-1.0 cm long until expansion of the first pair of leaves. The proportions of nuclei with different DNA contents were established and the mean C-value calculated. The presence of exogenous plant hormones changed endoreduplication intensity, although to different extents, depending on the organ and developmental stage. Ethylene and NAA stimulated the process, while EBR and kinetin suppressed it and GA did not clearly affect it.     

Flow cytometric measurements do not reveal different ploidy levels in Minthostachys (Lamiaceae)

Ten of the 17 species of the taxonomically difficult Andean mint genus Minthostachys (Lamiaceae) were submitted to flow cytometric measurements of nuclear DNA content to test the hypothesis of the occurrence of different ploidy levels within the genus. Nuclear DNA content was found to vary from 1.643 to 1.775 pg, i.e by only ca. 8% between individual accessions, thus providing no evidence for polyploidy in Minthostachys. While these results do not preclude the possibility that the genus contains polyploid species nor the occurrence of heteroploidy with nearly identical nuclear DNA contents, they suggest that polyploidy did not play a major role in its diversification.     

Flow cytometric measurement of nuclear DNA content inCapsicum (Solanaceae)

Nuclei were isolated from leaf tissue of differentCapsicum species and the relative fluorescence intensity was measured by flow cytometry after propidium iodide staining.Pisum sativum nuclei with known nuclear genome size (9.07 pg) were used as internal standard to determine nuclear DNA content of the samples in absolute units. The 2C DNA contents ranged between 7.65 pg inC. annuum and 9.72 pg inC. pubescens, and the general mean of the genus was 8.42 pg. These values correspond, respectively, to 1C genome size of 3.691 (C. annuum), 4.690 (C. pubescens) and 4.063 (general mean) Mbp. In general, white-flowered species proved to have less DNA, with the exception ofC. praetermissum, which displayed a 2C DNA content of 9.23 pg. It was possible to divide the studied species into three main groups according to their DNA content, and demonstrate differences in DNA content within two of the three species complexes established on the basis of morphological traits.     

Ploidy levels among species in the 'Oxalis tuberosa alliance' as inferred by flow cytometry

The 'Oxalis tuberosa alliance' is a group of Andean Oxalis species allied to the Andean tuber crop O. tuberosa Molina (Oxalidaceae), commonly known as 'oca'. As part of a larger project studying the origins of polyploidy and domestication of cultivated oca, flow cytometry was used to survey DNA ploidy levels among Bolivian and Peruvian accessions of alliance members. In addition, this study provided a first assessment of C-values in the alliance by estimating nuclear DNA contents of these accessions using chicken erythrocytes as internal standard. Ten Bolivian accessions of cultivated O. tuberosa were confirmed to be octoploid, with a mean nuclear DNA content of approx. 3.6 pg/2C. Two Peruvian wild Oxalis species, O. phaeotricha and O. picchensis, were inferred to be tetraploid (both with approx. 1.67 pg/2C), the latter being one of the putative progenitors of O. tuberosa identified by chloroplast-expressed glutamine synthetase data in prior work. The remaining accessions (from 78 populations provisionally identified as 35 species) were DNA diploid, with nuclear DNA contents varying from 0.79 to 1.34 pg/2C.     

Flow cytometry to assess biochemical pathways in heat-stressed Cronobacter spp. (formerly Enterobacter sakazakii)

Aims: Using a flow cytometry (FC)‐based approach in combination with four selected fluorescent probes, the biochemical pathway activated following the adaptation of Cronobacter spp. to lethal heat stress was investigated. This approach assessed the physiological changes induced in four strains of Cronobacter spp. Methods and Results: Using the commercially available live/dead viability assessment fluorescence probes, live, injured or dead bacterial cells were studied. Cellular respiration and membrane potential were evaluated using the dye‐labelled probe 3,3′‐dihexylocarbocyanine iodide, metabolic activity was evaluated using a fluorescein diacetate (FDA) probe, intracellular pH changes were measured using a carboxy‐fluorescein diacetate succinimidyl ester probe, and reactive oxygen species were measured using a hydroethidine fluorescent probe. Adaptation to lethal heat stress induced physiological changes that potentially improve the survival of Cronobacter spp. Conclusions: These data showed that in situ assessment of physiological behaviour of lethally stressed cells using multiparameter FC is a useful, rapid and sensitive tool to study and assess the viability and physiological state of Cronobacter cells. Significance and Impact of the Study:  This study shows that FC is a valuable tool in the study of physiological aspects of increased survival because of sublethal adaptation to heat.     

凤仙花属（Impatiens L.）10种植物花粉形态的扫描电镜观察

利用扫描电镜观察了10种凤仙花属（Impatiens L.）植物的花粉形态。结果表明：本属花粉为单粒花粉,呈长圆形至长矩圆形,大小为20.3～46.7 μm,具角萌发孔,网状纹饰,网眼明显;根据花粉网状纹饰中网眼内是否具颗粒状突起可将其分为2类：（1）网眼内无或近无颗粒状突起,黄金凤（I. siculifer）和婺源凤仙花（I. wuyuanensis）的花粉纹饰属于这一类型;（2）网眼内有明显颗粒状突起,其余8个种的花粉纹饰均属于该类型。研究表明,花粉特征,特别是花粉粒网状纹饰中网眼内有无颗粒状突起及颗粒状突起的形态等特征,在凤仙花属内常具种水平上的可见变异,因而可作为种类划分的依据,它们在分类学上的价值应予以关注。     

DNA barcoding of Gaultheria L.in China (Ericaceae: Vaccinioideae)

Four DNA barcoding loci,chloroplast loci rbcL,matK,trnH-psbA,and nuclear locus internal transcribed spacer (ITS),were tested for the accurate discrimination of the Chinese species of Gaultheria by using intraspecific and interspecific pairwise P-distance,Wilcoxon signed rank test,and tree-based analyses.This study included 186 individuals from 89 populations representing 30 species.For all individuals,single locus markers showed high levels of sequencing universality but were ineffective for species resolvability.Polymerase chain reaction amplification and sequencing were successful for all four loci.Both ITS and matK showed significantly higher levels of interspecific species delimitation than rbcL and trnH-psbA.A combination ofmatK and ITS was the most efficient DNA barcode among all studied regions,however,they do not represent an appropriate candidate barcode for Chinese Gaultheria,by which only 11 out of 30 species can be separated.Loci rbcL,matK,and trnH-psbA,which were recently proposed as universal plant barcodes,have a very poor capacity for species separation for Chinese Gaultheria.DNA barcodes may be reliable tools to identify the evolutionary units of this group,so further studies are needed to develop more efficient DNA barcodes for Gaultheria and other genera with complicated evolutionary histories.     

Flow cytometry confirms reticulate evolution and reveals triploidy in Central European Diphasiastrum taxa (Lycopodiaceae, Lycophyta)

Background and Aims

Interspecific Diphasiastrum hybrids have been assumed to be homoploid and to produce well-formed spores serving sexual reproduction. If this were the case, forms intermediate between hybrids and parents or hybrid swarms should be expected. The purpose of this study was: (1) to check whether homoploidy consistently applies to the three hybrids throughout their Central European range; (2) to examine whether their genome sizes confirm their parentage as assumed by morphology; and (3) to perform a screening for detection of ploidy levels other than diploid and variation in DNA content due to backcrossing.

Methods

Flow cytometry was used first to measure the relative DNA values [with 4′,6-diamidino-2-phenylindole (DAPI) staining] and ploidy level as a general screening, and secondly to determine the absolute DNA 2C values [with propidium iodide (PI) staining] in a number of selected samples with the main focus on the hybrids.

Key Results

A considerable variation of DNA 2C values (5·26–7·52 pg) was detected between the three European Diphasiastrum species. The values of the diploid hybrids are highly constant without significant variation between regions. They are also intermediate between their assumed parents and agree closely with those calculated from their putative parents. This confirms their hybrid origin, assumed parentage and homoploid status. Considerably higher DNA amounts (9·48–10·30 pg) were obtained for three populations, suggesting that these represent triploid hybrids, an interpretation that is strongly supported by their morphology.

Conclusions

Diploid hybrids have retained their genetic and morphological identites throughout their Central European range, and thus no indications for diploid backcrossing were found. The triploid hybrids have probably originated from backcrossing between a diploid gametophyte of a hybrid (derived from a diplospore) and a haploid gametophyte of a diploid parental species. By repeated crossing events, reticulate evolution patterns arise that are similar to those known for a number of ferns.     

Morphological Variability and Levels of Nuclear Ploidy in Neurons of the Central Nervous System of Bivalves Relative to the Problem of Somatic Polyploidy

Ploidy of neurons was assessed by the method of nuclear DNA cytophotometry in ganglia of the central nervous system (CNS) in 28 species of Bivalvia belonging to 5 existing subclasses and having various bio-ecological characteristics. A significant variation in the nucleus size and chromatin structure was revealed on crush preparations and slides; however, almost all nuclei were diploid. Some species had nuclei with tetraploid DNA value (from 0.2 up to 1%) and with condensed chromosomes, the probable mitotic fraction of cells. It was concluded that variation in the sizes of neurons and of their nuclei in the given instance was of a functional character, and polyploidy was untypical for the nervous system of bivalves. Apparently, the polyploidy, as an evolutionary strategy of neuron growth, had no anatomical prerequisites in bivalves, which is similar to CNS oligomerization in higher gastropods, as they are devoid of any functional adaptive sense.     

甘薯属耐盐植物马鞍藤基因组大小及特征分析

马鞍藤(Ipomoea pes-caprae(L.)R. Br.)是甘薯的近缘野生种之一,为热带、亚热带海滩生长的多年生藤蔓植物,作为沿海地区的园林绿化植物之一,具有较强的耐盐性。马鞍藤基因组学的研究可为耐盐甘薯种质创制提供信息参考。本试验调查了马鞍藤基因组大小和特征概况,为后续全基因组精细图谱绘制打下基础。研究以已知基因组大小的三裂叶薯(Ipomoea triloba L.)为对照,运用流式细胞术对马鞍藤基因组大小进行初步预估;使用二代高通量测序技术(Illumina Hiseq2500)对马鞍藤基因组进行survey评估,测序深度30×,利用生物信息学方法估算马鞍藤GC含量(即鸟嘌呤和胞嘧啶所占比例)、杂合率、重复序列等基因组概况。结果表明:流式细胞术估算马鞍藤基因组大小为1012.704±17.37 Mb;经全基因组Survey测定获得马鞍藤有效数据为21.71 Gb,基因组大小经修正后估算为1041.65 Mbp;通过K-mer分布曲线估算马鞍藤基因组中重复序列所占比率为74.52%,杂合率为0.99%;经初步组装后,GC平均深度及含量分布存在异常,出现分层的现象,这可能与马鞍藤基因组杂合率较高有关。本试验首次报导甘薯属耐盐植物马鞍藤基因组大小及特征信息,为马鞍藤进一步的全基因组深度测序和甘薯耐盐基因挖掘打下基础。