The curvHDR method for gating flow cytometry samples

Background  

High-throughput flow cytometry experiments produce hundreds of large multivariate samples of cellular characteristics. These samples require specialized processing to obtain clinically meaningful measurements. A major component of this processing is a form of cell subsetting known as gating. Manual gating is time-consuming and subjective. Good automatic and semi-automatic gating algorithms are very beneficial to high-throughput flow cytometry.     

濒危植物大别山五针松母树林花粉传播规律

大别山五针松是典型的风媒传粉植物,花粉流是种群基因流的重要形式。用重力玻片法研究大别山五针松母树林的花粉飞散特征,分析了时间和空间对花粉飞散的影响,结合林分密度和气象因子探讨花粉飞散的特点。结果表明:大别山五针松从5月中旬开始传粉,散粉期为14d,盛粉期为3d,种植密度低的下坡花粉密度比密度大的上坡的要高。日散粉最大值出现在10:00～14:00,植株中上部花粉密度较高,距花粉源50m处花粉密度最高。建议对种植密度大的区域移栽以促进花芽生成,以保证结实率与提高种子品质。     

Optimization of Two Immunofluorescent Antibodies for the Detection of <Emphasis Type="Italic">Escherichia coli</Emphasis> Using Immunofluorescent Microscopy and Flow Cytometry

Two commercially available fluorescein isothiocyanate (FITC) -conjugated anti-Escherichia coli antibodies, tested for immunofluorescence were assessed for their suitability in screening E. coli using flow cytometry. Staining efficacy was initially tested using immunofluorescent microscopy; and further optimization was carried out using flow cytometry. Initially, an acetone fixation step was utilized; however, it was determined statistically that the step could be omitted without impacting the assay and thus reduce the time involved. There was no statistical difference between the staining proficiency of the two antibodies employed. The percentage staining was quite low, approximately 10% for the two antibodies, which indicated that both were equally sensitive but ultimately, more specific antibodies are required for the detection of E. coli. Known proportions of target-E. coli (10⁵, 10⁶, and 10⁷ cells/ml) were mixed with large quantities of non-target bacteria; there was a significant correlation among all the antibodies at the different bacterial cell concentrations. Therefore, despite the low staining percentage achieved on the bacterial cultures, there is a representative and comparative level of staining occurring, between samples and between bacterial strains.     

Density dynamics of diverse Spiroplasma strains naturally infecting different species of Drosophila

Facultative heritable bacterial endosymbionts can have dramatic effects on their hosts, ranging from mutualistic to parasitic. Within-host bacterial endosymbiont density plays a critical role in maintenance of a symbiotic relationship, as it can affect levels of vertical transmission and expression of phenotypic effects, both of which influence the infection prevalence in host populations. Species of genus Drosophila are infected with Spiroplasma, whose characterized phenotypic effects range from that of a male-killing reproductive parasite to beneficial defensive endosymbiont. For many strains of Spiroplasma infecting at least 17 species of Drosophila, however, the phenotypic effects are obscure. The infection prevalence of these Spiroplasma vary within and among Drosophila species, and little is known about the within-host density dynamics of these diverse strains. To characterize the patterns of Spiroplasma density variation among Drosophila we used quantitative PCR to assess bacterial titer at various life stages of three species of Drosophila naturally-infected with two different types of Spiroplasma. For naturally infected Drosophila species we found that non-male-killing infections had consistently lower densities than the male-killing infection. The patterns of Spiroplasma titer change during aging varied among Drosophila species infected with different Spiroplasma strains. Bacterial density varied within and among populations of Drosophila, with individuals from the population with the highest prevalence of infection having the highest density. This density variation underscores the complex interaction of Spiroplasma strain and host genetic background in determining endosymbiont density.     

Quantifying CD4 receptor protein in two human CD4+ lymphocyte preparations for quantitative flow cytometry

Background

In our previous study that characterized different human CD4+ lymphocyte preparations, it was found that both commercially available cryopreserved peripheral blood mononuclear cells (PBMC) and a commercially available lyophilized PBMC (Cyto-Trol™) preparation fulfilled a set of criteria for serving as biological calibrators for quantitative flow cytometry. However, the biomarker CD4 protein expression level measured for T helper cells from Cyto-Trol was about 16% lower than those for cryopreserved PBMC and fresh whole blood using flow cytometry and mass cytometry. A primary reason was hypothesized to be due to steric interference in anti- CD4 antibody binding to the smaller sized lyophilized control cells.

Method

Targeted multiple reaction monitoring (MRM) mass spectrometry (MS) is used to quantify the copy number of CD4 receptor protein per CD4+ lymphocyte. Scanning electron microscopy (SEM) is utilized to assist searching the underlying reasons for the observed difference in CD4 receptor copy number per cell determined by MRM MS and CD4 expression measured previously by flow cytometry.

Results

The copy number of CD4 receptor proteins on the surface of the CD4+ lymphocyte in cryopreserved PBMCs and in lyophilized control cells is determined to be (1.45 ± 0.09) × 10⁵ and (0.85 ± 0.11) × 10⁵, respectively, averaged over four signature peptides using MRM MS. In comparison with cryopreserved PBMCs, there are more variations in the CD4 copy number in lyophilized control cells determined based on each signature peptide. SEM images of CD4+ lymphocytes from lyophilized control cells are very different when compared to the CD4+ T cells from whole blood and cryopreserved PBMC.

Conclusion

Because of the lyophilization process applied to Cyto-Trol control cells, a lower CD4 density value, defined as the copy number of CD4 receptors per CD4+ lymphocyte, averaged over three different production lots is most likely explained by the loss of the CD4 receptors on damaged and/or broken microvilli where CD4 receptors reside. Steric hindrance of antibody binding and the association of CD4 receptors with other biomolecules likely contribute significantly to the nearly 50% lower CD4 receptor density value for cryopreserved PBMC determined from flow cytometry compared to the value obtained from MRM MS.

Electronic supplementary material

The online version of this article (doi:10.1186/1559-0275-11-43) contains supplementary material, which is available to authorized users.     

Flow Cytometry Pulse Width Data Enables Rapid and Sensitive Estimation of Biomass Dry Weight in the Microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD₇₅₀) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD₇₅₀ measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth.     

Temporal and spatial structure in the suprabenthic community of a shallow estuary (western Portugal: Mondego river estuary)

The suprabenthic fauna of the Mondego river estuary (western Portugal) was sampled monthly between June 1996 and June 1997. Quantitative samples were taken, with a suprabenthic 500-μm mesh size net, at regularly spaced stations covering the entire south arm of the estuary. The diversity of the samples and the distribution of the species were assessed. Suprabenthic communities were identified using a divisive multivariate statistical technique and species composition, density and biomass of the dominant species of each community were compared among communities. Diversity was highest in the mouth of the estuary where density and biomass were lowest. Diversity decreased upstream and was lowest in the mid and inner estuary where density and biomass reached maximal values. Suprabenthic animals, mainly the mysid Mesopodopsis slabberi, reached high densities in inner and upstream stations, whereas the more seaward stations had lower densities but a higher number of species. The spatial patterns dominated over the temporal patterns.     

疏伐对黄土丘陵区刺槐林蒸腾的影响

黄土高原地区植被建设已达到土壤水分承载力的阈值,需要对现有林分进行结构优化并提升其生态功能。不合理的林分密度是导致黄土丘陵区刺槐林土壤干化、生长衰退的主要原因之一。疏伐可以优化林分结构,并能够通过控制蒸腾耗水来调控土壤水分,是促进刺槐林可持续生长的有效手段。疏伐对黄土丘陵区刺槐林蒸腾有何影响,目前并不清楚。研究基于树干液流法估算了4个不同疏伐强度（样地1：52%、样地2：48%、样地3：35%、样地4：未疏伐）下刺槐单株尺度的液流速率与林分尺度的日平均蒸腾量,并分析了不同时间尺度下液流速率与环境因子的关系,以阐明疏伐对黄土丘陵区刺槐林蒸腾的影响。结果表明：（1） 单株尺度刺槐蒸腾速率（即液流速率）随疏伐强度减小（林分密度增大）呈现下降趋势（样地1：0.53 kg cm^-2 d^-1、样地2：0.41 kg cm^-2 d^-1、样地3：0.31 kg cm^-2 d^-1、样地4：0.33 kg cm^-2 d^-1）;（2） 观测期林分尺度日平均蒸腾量随疏伐强度减小呈现上升趋势（样地1：0.90 mm/d、样地2：1.18 mm/d、样地3：1.04 mm/d、样地4：1.44 mm/d）;（3） 在半小时尺度与日尺度上,各样地液流速率与环境因子的关系没有显著差异,半小时尺度单株液流速率均与太阳辐射相关性最高（相关系数0.883-0.908）,液流速率日变化过程与环境因子日变化过程存在时滞现象;日尺度单株液流速率与饱和水汽压亏缺相关性最高（相关系数0.843-0.913）,样地间日尺度单株液流速率的差异性随着饱和水汽压亏缺增大而增大。研究结果初步反映了疏伐导致的林分密度变化对刺槐蒸腾的影响,将为黄土丘陵区刺槐林的结构改造、功能提升和土壤水分调控提供理论支持。     

Predicting the current distribution and potential spread of the exotic grass Eragrostis plana Nees in South America and identifying a bioclimatic niche shift during invasion

Eragrostis plana (Poaceae) is a perennial grass introduced from South Africa to the state of Rio Grande do Sul in southern Brazil. Currently, it is considered an invasive grass in several regions of the world, including South America, where it has caused negative ecological and socio‐economic impacts. Ecological niche models, using bioclimatic variables, are often used to predict the potential distribution of invasive species. In this study we prepared two bioclimatic models for E. plana using the Genetic Algorithm for Rule‐set Production, the first based on data from its native region (South Africa) and the second on data from both the native and invaded (South America) regions. We then projected each model onto South America to identify regions vulnerable to invasion by the species, and compared our results with available records of the species in South America. Finally, we explored the model's predictions for the existence of a bioclimatic niche shift during the invasion process of E. plana in South America, using multivariate statistical analysis. The model created with native distribution data was only able to predict (with highly suitable habitat) the region of introduction of E. plana in South America. However, the current distribution, as well as the region of introduction of the species, was reliably predicted by the model created with data from both native and invaded regions. Our multivariate analysis supports a hypothesis of bioclimatic niche shift during the invasion process of E. plana in South America.     

Fine‐scale geographic patterns of gene flow and reproductive character displacement in Drosophila subquinaria and Drosophila recens

When two species are incompletely isolated, strengthening premating isolation barriers in response to the production of low fitness hybrids may complete the speciation process. Here, we use the sister species Drosophila subquinaria and Drosophila recens to study the conditions under which this reinforcement of species boundaries occurs in natural populations. We first extend the region of known sympatry between these species, and then we conduct a fine‐scale geographic survey of mate discrimination coupled with estimates of gene flow within and admixture between species. Within D. subquinaria, reinforcement is extremely effective: we find variation in mate discrimination both against D. recens males and against conspecific allopatric males on the scale of a few kilometres and in the face of gene flow both from conspecific populations and introgression from D. recens. In D. recens, we do not find evidence for increased mate discrimination in sympatry, even where D. recens is rare, consistent with substantial gene flow throughout the species’ range. Finally, we find that introgression between species is asymmetric, with more from D. recens into D. subquinaria than vice versa. Within each species, admixture is highest in the geographic region where it is rare relative to the other species, suggesting that when hybrids are produced they are of low fitness. In sum, reinforcement within D. subquinaria is effective at maintaining species boundaries, but even when reinforcing selection is strong it may not always result in a pattern of strong reproductive character displacement due to variation in the frequency of hybridization and gene flow from neighbouring populations.     

Ecology of wildlife rabies in Europe

• 1 The number of wildlife rabies cases has increased in Europe in recent years. We review the epizootiology of wildlife rabies in Europe, paying special attention to recent changes to the situation of two important vector species: the red fox and the raccoon dog. Red fox Vulpes vulpes has been the main vector of rabies since 1945, but the number and proportion of raccoon dog Nyctereutes procyonoides cases has rapidly increased during the past few years, particularly in north‐eastern Europe.
• 2 The transmission rate (average number of susceptible animals infected by each rabid animal) is critical for rabies spread and is partly determined by population density. Both raccoon dogs and foxes live in pairs. Foxes also live in family groups. Pairs and groups share their territories. Home range size usually correlates negatively with population density. Fox home ranges are 50–1500 ha, those of raccoon dogs 150–700 ha. The threshold value for rabies spread among foxes is estimated to be 0.63 individuals/km². Although fox density in eastern and northern Europe may be lower than this, the pooled density of foxes and raccoon dogs exceeds the threshold density.
• 3 Animal movements, especially dispersal of young, pose a risk for rabies spread. Although the likelihood of an epizootic is highest where fox and raccoon dog densities are highest, rabies may spread fastest where population densities are lower, because dispersal distances tend to correlate negatively with population density.
• 4 Oral vaccinations have been more effective in rabies control than culling foxes. Where two vector species exist, vaccination should be conducted twice a year, because most raccoon dogs disperse in autumn but some foxes do not disperse before mid‐ or late winter.
• 5 New rabies models, based on two vector species and their interaction, and which take into account the hibernation period of raccoon dogs, are needed for north‐eastern Europe.

     

Wet and dry density of Bacillus anthracis and other Bacillus species

Aims: To determine the wet and dry density of spores of Bacillus anthracis and compare these values with the densities of other Bacillus species grown and sporulated under similar conditions. Methods and Results: We prepared and studied spores from several Bacillus species, including four virulent and three attenuated strains of B. anthracis, two Bacillus species commonly used to simulate B. anthracis (Bacillus atrophaeus and Bacillus subtilis) and four close neighbours (Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis and Bacillus stearothermophilus), using identical media, protocols and instruments. We determined the wet densities of all spores by measuring their buoyant density in gradients of Percoll and their dry density in gradients of two organic solvents, one of high and the other of low chemical density. The wet density of different strains of B. anthracis fell into two different groups. One group comprised strains of B. anthracis producing spores with densities between 1·162 and 1·165 g ml^?1 and the other group included strains whose spores showed higher density values between 1·174 and 1·186 g ml^?1. Both Bacillus atrophaeus and B. subtilis were denser than all the B. anthracis spores studied. Interestingly and in spite of the significant differences in wet density, the dry densities of all spore species and strains were similar. In addition, we correlated the spore density with spore volume derived from measurements made by electron microscopy analysis. There was a strong correlation (R² = 0·95) between density and volume for the spores of all strains and species studied. Conclusions: The data presented here indicate that the two commonly used simulants of B. anthracis, B. atrophaeus and B. subtilis were considerably denser and smaller than all B. anthracis spores studied and hence, these simulants could behave aerodynamically different than B. anthracis. Bacillus thuringiensis had spore density and volume within the range observed for the various strains of B. anthracis. The clear correlation between wet density and volume of the B. anthracis spores suggest that mass differences among spore strains may be because of different amounts of water contained within wet dormant spores. Significance and Impact of the Study: Spores of nonvirulent Bacillus species are often used as simulants in the development and testing of countermeasures for biodefense against B. anthracis. The similarities and difference in density and volume that we found should assist in the selection of simulants that better resemble properties of B. anthracis and, thus more accurately represent the performance of countermeasures against this threat agent where spore density, size, volume, mass or related properties are relevant.     

Optimization of a Saccharomyces cerevisiae fermentation process for production of a therapeutic recombinant protein using a multivariate bayesian approach

Various approaches have been applied to optimize biological product fermentation processes and define design space. In this article, we present a stepwise approach to optimize a Saccharomyces cerevisiae fermentation process through risk assessment analysis, statistical design of experiments (DoE), and multivariate Bayesian predictive approach. The critical process parameters (CPPs) were first identified through a risk assessment. The response surface for each attribute was modeled using the results from the DoE study with consideration given to interactions between CPPs. A multivariate Bayesian predictive approach was then used to identify the region of process operating conditions where all attributes met their specifications simultaneously. The model prediction was verified by twelve consistency runs where all batches achieved broth titer more than 1.53 g/L of broth and quality attributes within the expected ranges. The calculated probability was used to define the reliable operating region. To our knowledge, this is the first case study to implement the multivariate Bayesian predictive approach to the process optimization for the industrial application and its corresponding verification at two different production scales. This approach can be extended to other fermentation process optimizations and reliable operating region quantitation. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1095–1105, 2012     

Population systematics of the snake genus Naja (Reptilia: Serpentes: Elapidae) in Indochina: Multivariate morphometrics and comparative mitochondrial DNA sequencing (cytochrome oxidase I)

We analyze the population systematics of Asiatic cobras in Indochina, China and the Andaman Islands by means of comparative sequencing of the cytochrome oxidase subunit I gene of the mitochondrial DNA molecule and multivariate analysis of morphological characters. Canonical variate analysis and mtDNA sequence information reveal that the cobras of this region comprise four distinct species: Naja atra from China and northern Vietnam, Naja kaouthia from Burma, central Thailand, Cambodia and southern Vietnam, Naja siamensis from Thailand, Cambodia and southern Vietnam, and Naja sagittifera from the Andaman Islands. The subspecies N. kaouthia suphanensis Nutaphand 1986 shows no mtDNA sequence difference from typical N. kaouthia from central Thailand, and multivariate analysis does not reveal differences in general phenotypic profile; the subspecies is therefore synonymised with Naja kaouthia. The cytochrome oxidase subunit I gene, little used in molecular taxonomy, is shown to be well suited for studies at the species level, as it shows taxonomically useful levels of interspecific divergence but low levels of intraspecific variation; this is particularly relevant for studies of rare species, where sample size is a problem. The combination of multivariate morphometrics and molecular systematics can be particularly powerful in resolving systematic problems in such cases.     

Outdoor stocking density in free-range laying hens: effects on behaviour and welfare

Free-range laying hen systems are increasing within Australia and research is needed to determine optimal outdoor stocking densities. Six small (n=150 hens) experimental flocks of ISA Brown laying hens were housed with access to ranges simulating one of three outdoor stocking densities with two pen replicates per density: 2000 hens/ha, 10 000 hens/ha or 20 000 hens/ha. Birds were provided daily range access from 21 to 36 weeks of age and the range usage of 50% of hens was tracked using radio-frequency identification technology. Throughout the study, basic external health assessments following a modified version of the Welfare Quality® protocol showed most birds were in visibly good condition (although keel damage was increasingly present with age) with few differences between stocking densities. Toenail length at 36 weeks of age was negatively correlated with hours spent ranging for all pens of birds (all r⩾−0.23, P⩽0.04). At 23 weeks of age, there were no differences between outdoor stocking densities in albumen corticosterone concentrations (P=0.44). At 35 weeks of age, density effects were significant (P<0.001) where the eggs from hens in the highest outdoor stocking density showed the highest albumen corticosterone concentrations, although eggs from hens in the 10 000 hens/ha density showed the lowest concentrations (P<0.017). Behavioural observations of hens both on the range and indoors showed more dust bathing and foraging (scratching followed by ground-pecking) was performed outdoors, but more resting indoors (all P<0.001). Hens from the 2000 hens/ha densities showed the least foraging on the range but the most resting outdoors, with hens from the 20 000 hens/ha densities showing the least amount of resting outdoors (all P<0.017). Proportions of dust bathing outdoors tended to differ between the stocking densities (P=0.08). For each of the health and behavioural measures there were differences between pen replicates within stocking densities. These data show outdoor stocking density has some effects on hen welfare, and it appears that consideration of both individual and group-level behaviour is necessary when developing optimal stocking density guidelines and free-range system management practices.     

Ongoing hybridization obscures phylogenetic relationships in the Drosophila subquinaria species complex

Inferring evolutionary relationships among recently diverged lineages is necessary to understand how isolating barriers produce independent lineages. Here, we investigate the phylogenetic relationships between three incompletely isolated and closely related mushroom‐feeding Drosophila species. These species form the Drosophila subquinaria species complex and consist of one Eurasian species (D. transversa) and two widespread North American species (D. subquinaria and D. recens) that are sympatric in central Canada. Although patterns of pre‐ and post‐mating isolation among these species are well characterized, previous work on their phylogenetic relationships is limited and conflicting. In this study, we generated a multi‐locus data set of 29 loci from across the genome sequenced in a population sample from each species, and then, we inferred species relationships and patterns of introgression. We find strong statistical support that D. subquinaria is paraphyletic, showing that samples from the geographic region sympatric with D. recens are most closely related to D. recens, whereas samples from the geographic region allopatric with D. recens are most closely related to D. transversa. We present several lines of evidence that both incomplete lineage sorting and gene flow are causing phylogenetic discordance. We suggest that ongoing gene flow primarily from D. recens into D. subquinaria in the sympatric part of their ranges causes phylogenetic uncertainty in the evolutionary history of these species. Our results highlight how population genetic data can be used to disentangle the sources of phylogenetic discordance among closely related species.     

Organic carbon content and distribution in soils under different land uses in tropical and subtropical China

Tropical and subtropical China comprises a land area of about 215 Mha, but reports on its soil C storage and contents are limited. The objective of this study was to investigate the C density, stocks and distribution in soils of this region under different land uses by using soil species data from the Second National Soil Survey and the Vegetation Map of the People's Republic of China (1:4 M). It was estimated that there is a total of about 28.7±8.2 Pg organic C stored in the upper 1 m of soils of the entire region. Changes of C content (C) with depth (D) were observed following a relationship of C = (c + D)/ (a + bD), or C = 1/(a + bD). Of the various patterns of land uses in the region, soil C density was generally higher in the west than in the east, and while small differences were found in croplands, there were large variations in natural soils. In the west, the C density of meadow and herbaceous swamp soil was the highest (about 40 kg C/m²), followed by coniferous and broad-leaf forest soils (19.6 and 19.2 kg C/m², respectively). The C density of paddy, bush and coppice forest soils showed a density of 12.6 and 14.6 kg C/m², respectively. Upland and grass-savanah soils ranked the lowest (9.4 and 10.5 kg C/m², respectively). In the east, meadow and herbaceous swamp soil had the highest C density (25.2 kg C/m²), but differences in C density among soils under coniferous forest, broad-leaf forest, bush and coppice forest, and rice were small, varying from 10.2 to 11.4 kg C/m². The C density of upland soil (7.2 kg C/m²), appeared a little higher than that of grass-savanah soil (6.3 kg C/m²). For the various land uses in the region, the C density estimation is accompanied by relatively large variations.     

Sperm number assessed by flow cytometry in species of Daphnia (Crustacea,Cladocera)

Daphnia magna and Daphnia pulex are two important model species in ecotoxicology. In daphniids, studies of the effects of contaminants have mostly focused on female life history traits, yet it would also be important to examine male reproductive traits, particularly in relation to endocrine disruptors. In this study, we developed a protocol that uses flow cytometry to measure sperm number in individual males of different species of Daphnia. We tested our protocol on 114 males from several clones of three common species of Daphnia. Sperm count varied widely among individuals and reached high numbers (up to 1.45 × 10⁵). Positive relationships between male length and sperm number were observed in D. pulex and Daphnia pulicaria, but not in D. magna. Important inter‐clonal differences in sperm production were observed in all species, with some clones producing very little sperm. Duplicated sperm samples showed on average only 6% difference in sperm counts. Sperm counts were stable at least over a 2‐hr period and up to 5 hr for most samples. This sperm isolation protocol and flow cytometric enumeration approach will be of major interest to ecotoxicologists.     

Identification of a Murine Erythroblast Subpopulation Enriched in Enucleating Events by Multi-spectral Imaging Flow Cytometry

Erythropoiesis in mammals concludes with the dramatic process of enucleation that results in reticulocyte formation. The mechanism of enucleation has not yet been fully elucidated. A common problem encountered when studying the localization of key proteins and structures within enucleating erythroblasts by microscopy is the difficulty to observe a sufficient number of cells undergoing enucleation. We have developed a novel analysis protocol using multiparameter high-speed cell imaging in flow (Multi-Spectral Imaging Flow Cytometry), a method that combines immunofluorescent microscopy with flow cytometry, in order to identify efficiently a significant number of enucleating events, that allows to obtain measurements and perform statistical analysis.We first describe here two in vitro erythropoiesis culture methods used in order to synchronize murine erythroblasts and increase the probability of capturing enucleation at the time of evaluation. Then, we describe in detail the staining of erythroblasts after fixation and permeabilization in order to study the localization of intracellular proteins or lipid rafts during enucleation by multi-spectral imaging flow cytometry. Along with size and DNA/Ter119 staining which are used to identify the orthochromatic erythroblasts, we utilize the parameters “aspect ratio” of a cell in the bright-field channel that aids in the recognition of elongated cells and “delta centroid XY Ter119/Draq5” that allows the identification of cellular events in which the center of Ter119 staining (nascent reticulocyte) is far apart from the center of Draq5 staining (nucleus undergoing extrusion), thus indicating a cell about to enucleate. The subset of the orthochromatic erythroblast population with high delta centroid and low aspect ratio is highly enriched in enucleating cells.     

皖南凹耳臭蛙地理分布格局与生境选择

凹耳臭蛙(Odorrana tormota)是一种小型山溪蛙类,由于叫声独特,种内具有超声通讯功能而引起国内外相关专家的广泛关注。首次针对皖南凹耳臭蛙地理分布、生境选择以及人为干扰强度对种群密度的影响进行了研究。共调查了9县1区32条不同的山溪,发现其中的16个有凹耳臭蛙分布,除黄山区的香溪和浮溪具文献记录外,其余14个分布点均为首次报道。基于现存凹耳臭蛙分布点的水系关系,推测该物种在皖南的分布呈现出以青弋江、新安江和水阳江三大水系为纽带、间断分布的格局。对其中10个分布点222只凹耳臭蛙生境选择的统计结果表明,在夜晚活动周期内,该物种对乔木、灌木丛、草本植物、岩石、沙滩和倒伏朽木等不同生境的选择具有显著差异(F5,54=25.75,P0.001),栖息在草本植物和灌丛中蛙的数量明显多于栖息在其它几种生境类型中的蛙类。暗示溪流沿岸的草本植物和灌丛对该物种的生存和繁衍具有重要意义,乔木对其非活动周期的隐蔽可能具有不可替代的作用。根据人类活动对其栖息地破坏程度,将人为干扰程度划分为强、中和弱3种不同强度,对不同干扰强度下种群相对密度进行单因素方差分析。结果表明,中等干扰强度下的种群密度显著高于其它两种强度下的种群密度(F2,13=8.155,P=0.005)。推测适度的人为干扰可能增加了栖息地内昆虫的种类和数量,对蛙类的生存和繁衍更为有利。但随着人为干扰强度的增强,对溪流沿岸植被的破坏更为剧烈,种群密度显著下降。