Combining Flow Cytometry and Real-Time PCR Methodology to Demonstrate Consumption by Prymnesium parvum1

Bowers, Holly A., Andreas Brutemark, Wanderson F. Carvalho, and Edna Granéli, 2010. Combining Flow Cytometry and Real-Time PCR Methodology to Demonstrate Consumption by Prymnesium parvum. Journal of the American Water Resources Association (JAWRA) 46(1):133-143. DOI: 10.1111/j.1752-1688.2009.00397.x Abstract: Harmful algal bloom species can persist in the environment, impacting aquatic life and human health. One of the mechanisms by which some harmful algal bloom species are able to persist is by consumption of organic particles. Methods to demonstrate and measure consumption can yield insight into how populations thrive. Here, we combine flow cytometry and real-time PCR to demonstrate consumption of a cryptophyte species (Rhodomonas sp.) by a toxic mixotrophic haptophyte (Prymnesium parvum). Using flow cytometry, the feeding frequency of a population of P. parvum cells was calculated using the phycoerythrin (PE) fluorescence signal from Rhodomonas sp. and the fluorescence of an acidotropic probe labeling the food vacuoles. Feeding frequency increased in the beginning of the experiment and then began to decline, reaching a maximum of 47.5% of the whole P. parvum population after 212 min. The maximum number of consumed Rhodomonas sp. cells was 0.8 per P. parvum cell, and occurred after 114 min corresponding to an ingestion rate of 0.4 Rhodomonas sp. cells/P. parvum/h. Cells from the feeding P. parvum population were sorted, washed, and subjected to a real-time PCR assay targeting the cryptophyte 18S locus. There was a correlation between cycle threshold (Ct) values and number of consumed prey cells calculated by fluorescence. Overall, this study shows that flow cytometric analysis, of the acidotropic probe and prey pigments, is an efficient and rapid tool in enumerating food vacuoles and the number of prey cells consumed. Furthermore, we suggest that real-time PCR can be applied to cells sorted by flow cytometry, thus allowing for the detection and potential quantification of the targeted prey cells.     

TFM实时成像技术在缺陷检测中的应用

在压力容器的超声检测中,为解决传统相控阵（PA）二维成像存在缺陷图像畸变,难以准确定性等问题,采用1种基于全聚焦法（TFM）的实时超声成像技术,使用一维线阵和二维面阵分别对孔等典型实际缺陷进行扫查,获得缺陷的二维和三维图像,从定量角度对比分析2者的准确度。结果表明:该方法获得的三维图像测量误差在8%以内,具有更高的精确度和检出率,对于孔类缺陷的还原度更高,这对于缺陷检测与评估以及和特种设备的安全生产具有重要意义。     

武汉冬季大气PM2.5小时分辨率源贡献识别及潜在影响域分析

冬季是我国大气细颗粒物(PM_2.5)污染较为严重的时段,武汉市PM_2.5受到明显的区域传输影响.本研究基于小时分辨率PM_2.5组分观测数据,采用受体模型,解析武汉冬季大气PM_2.5各类源的实时贡献.结合轨迹聚类和浓度权重,识别影响各类源的传输路径和潜在源区.武汉冬季大气平均ρ(PM_2.5)为（75.1±29.2）μg·m^-3.观测期间共有两次污染过程,第一次污染过程主要受西北方向气团影响,水溶性离子升高是PM_2.5呈现高值的主要原因,ρ（NH⁺₄）、ρ（NO^-₃）和ρ（SO■）分别是清洁时段的1.6、 1.7和2.1倍;第二次污染过程则以正东方向气团为主,二次有机组分有明显的生成.对武汉冬季大气PM_2.5贡献最大的是二次源（34.1%）,其次是机动车尾气（23.7%）、燃煤（11.5%）、道路尘（10.9%）、钢铁冶炼（8.7...     

Wildlife identified as major source of Escherichia coli in agriculturally dominated watersheds by BOX A1R-derived genetic fingerprints

The presence of Escherichia coli in recreational and potable waters is a major concern to the general public as elevated levels of E. coli suggest the presence of pathogenic bacteria and viruses. Unfortunately, traditional microbial techniques do not allow specific identification of the source of E. coli. This reduces the ability to target management practices that reduce bacterial contamination. In the Finger Lakes region of western New York, USA, wildlife resides in relatively high densities on watersheds dominated by people and dairy farms, and as a result, the sources of fecal degradation of potable and recreational waters are often unknown. In the Conesus Lake watershed, the sources of microbial contamination were assessed using Rep-PCR molecular tools, a method of amplifying repetitive DNA sequences found throughout the E. coli genome to produce distinct fingerprints for a given ecotype. Molecular fingerprints of E. coli isolated from regional populations of cattle, humans, geese and deer were compared to E. coli isolated from stream water samples. Canonical discriminant function analysis indicated that the DNA fingerprints of the original source group isolates were correctly predicted 90.2% of the time. Since land use in the sub-watersheds was dominated by dairy and cash crop farms, it was expected that the majority of E. coli isolated would be identified as cows; however, an unexpectedly high percentage of isolates were identified as wildlife (geese and deer). Geese were the dominant source of E. coli (44.7-73.7% of the total sources) in four sub-watersheds followed by cows (10.5-21.1%), deer (10.5-18.4%), humans (5.3-12.9%) and unidentifiable sources (0.0-11.8%). Management practices intended to decrease the number of cattle or the amount of manure spread in a sub-watershed were reflected in a decrease of E. coli ecotypes associated with dairy cows.     

Case report: birth after preimplantation genetic diagnosis of a subtle mutation in SMN1 gene

Spinal muscular atrophy (SMA) preimplantation genetic diagnosis (PGD) has been available since 1998. Protocols are based on the detection of the homozygous deletion of exon 7, which are present in 90–98% of SMA patients. A couple where the woman was a heterozygous carrier of the usual SMN1 Del7 mutation and the man was a heterozygous carrier of pMet263Arg substitution in exon 6 of SMN1 gene was referred for PGD. The usual PGD test being unsuitable for this couple, we developed a novel duplex polymerase chain reaction (PCR)-based PGD test for the detection of the mutation pMet263Arg by allele specific amplification, combined with the amplification of D5S641 extragenic polymorphic marker. PCR conditions were established using single control lymphoblasts and lymphocytes from the pMet263Arg substitution carrier. Amplification was obtained in 100% of the 86 single cells tested, amplification refractory mutation system (ARMS) PCR was specific in 100% of single cells tested and a complete genotype (mutation plus D5S641) was achieved in 88% of them. A PGD cycle was performed successfully and a pregnancy was obtained. An unaffected girl was born and postnatal diagnosis confirmed PGD results. This is the first PGD described for SMA because of another mutation than the major homozygous exon 7 deletion of SMN1. In the future, a similar strategy could be adopted for other subtle mutations of this gene. Copyright © 2006 John Wiley & Sons, Ltd.