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Major Technical Challenge (major + technical_challenge)
Selected AbstractsCommunity heterogeneity and single-cell digestive activity of estuarine heterotrophic nanoflagellates assessed using lysotracker and flow cytometryENVIRONMENTAL MICROBIOLOGY, Issue 7 2010Eva Sintes Summary Heterotrophic nanoflagellates (HNFs) are an essential component of all aquatic microbial food webs, and yet the exploration of the numerical and single-cell responses of these organisms in mixed assemblages still represents a major technical challenge. LysoTracker Green staining combined with flow cytometry was recently proposed for the enumeration of aquatic HNFs. Here we show that LysoTracker Green not only allows the enumeration of HNFs in estuarine samples with a wide range of HNF abundances, but also allows the discrimination of distinct HNF populations in mixed assemblages. In addition, the resulting cytometric parameters can be used to characterize cell size and the level of activity of the cells in the different populations that are detected. LysoTracker Green accumulates preferentially in lysosomes, and we demonstrate that the green fluorescence emission from HNF cells stained with LysoTracker strongly correlates with cell-specific ,-glucosaminidase (,-Gam) activity, a key digestive enzyme of lysosomal origin in eukaryotic cells. Our results further show that different populations that develop in estuarine regrowth cultures are characterized by different intrinsic ranges of size and of feeding activity, and that there is a wide range of single-cell responses within these HNF populations. We found a large degree of uncoupling between cell size and feeding activity, both between and within HNF populations, and there appears to be no clear allometric scaling of feeding activity. We were able to reconstruct the succession of distinct HNF populations that developed during the regrowth experiments, and explore the complex interactions that occurred between numerical (change in abundance of the cytometric populations) and single-cell HNF responses. [source] Determination of haplotypes from single DNA molecules: a method for single-molecule barcoding,,HUMAN MUTATION, Issue 9 2007Ming Xiao Abstract Determining the haplotypes in a diploid individual is a major technical challenge in genetic studies of human complex traits. Here we report a method of molecular haplotyping by directly imaging multiple polymorphic sites on individual DNA molecules simultaneously. DNA fragments amplified by long-range PCR were labeled with fluorescent dyes at each polymorphic site using a modified gap-filled padlock probe ligation approach. The labeled DNA molecules were then stretched into linear form on a functionalized glass surface and imaged with multicolor total internal reflection fluorescence (TIRF) microscopy. By determining the colors and positions of the fluorescent labels with respect to the backbone at polymorphic sites, the haplotype can be inferred accurately, in a manner similar to reading a barcode, even when the DNA fragments are not fully labeled. The feasibility of this technology is demonstrated by the determination of the haplotype of a 9.3-kbp DNA fragment containing four SNPs. Hum Mutat 28(9), 913,921, 2007. Published 2007 Wiley-Liss, Inc. [source] Atomic force microscopy study of living diatoms in ambient conditionsJOURNAL OF MICROSCOPY, Issue 3 2003I. C. Gebeshuber Summary We present the first in vivo study of diatoms using atomic force microscopy (AFM). Three chain-forming, benthic freshwater species ,Eunotia sudetica, Navicula seminulum and a yet unidentified species , are directly imaged while growing on glass slides. Using the AFM, we imaged the topography of the diatom frustules at the nanometre range scale and we determined the thickness of the organic case enveloping the siliceous skeleton of the cell (10 nm). Imaging proved to be stable for several hours, thereby offering the possibility to study long-term dynamic changes, such as biomineralization or cell movement, as they occur. We also focused on the natural adhesives produced by these unicellular organisms to adhere to other cells or the substratum. Most man-made adhesives fail in wet conditions, owing to chemical modification of the adhesive or its substrate. Diatoms produce adhesives that are extremely strong and robust both in fresh- and in seawater environments. Our phase-imaging and force-pulling experiments reveal the characteristics of these natural adhesives that might be of use in designing man-made analogues that function in wet environments. Engineering stable underwater adhesives currently poses a major technical challenge. [source] Proteomic analysis of mouse growth plate cartilagePROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 24 2006Daniele Belluoccio Abstract Cartilage is a highly specialized load-bearing tissue with a small number of cells and a high proportion of extracellular matrix (ECM). The abundance of heavily sulfated proteoglycans and a poorly soluble collagenous ECM presents a major technical challenge to 2-DE. Here we report proteomic analysis of mouse growth plate cartilage using novel methodology for tissue dissection and sample prefractionation. We have successfully resolved cartilage tissue extracts by 2-DE for the first time and identified cartilage ECM proteins by Western blotting and MS/MS. 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