scholarly journals Improved Methodology for Identification of Protists and Microalgae from Plankton Samples Preserved in Lugol's Iodine Solution: Combining Microscopic Analysis with Single-Cell PCR

2008 ◽  
Vol 74 (8) ◽  
pp. 2505-2510 ◽  
Author(s):  
Barbara M. Auinger ◽  
Karin Pfandl ◽  
Jens Boenigk
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Sodium Thiosulfate ◽  
Proteinase K ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Iodine Solution ◽  
Lugol’S Iodine ◽  
Single Cell Pcr ◽  
Field Samples

ABSTRACT Here we introduce a method for quantitative analysis of planktonic protists and microalgae from preserved field samples combining morphological and small-subunit (SSU) rRNA gene sequence analysis. We linked a microscopic screening with PCR of single cells using field samples preserved with Lugol's iodine solution. Cells possessing a rigid cell wall were incubated with Viscozyme and subsequently with proteinase K for cell disruption; this was unnecessary for fragile cells. The addition of sodium thiosulfate to the PCR tube considerably decreased the inhibiting effect of the fixative (iodine) on the PCR and thus allowed for successful single-cell PCR even of long DNA fragments (up to as many as 3,000 base pairs). We further applied the protocol to investigate the dominant SSU rRNA genotypes in distinct flagellate morphospecies originating from different samples. We hypothesized that despite the morphological similarity, protist morphospecies in different habitats or sampled during different seasons are represented by different genotypes. Our results indicate species-specific differences: the two species Ochromonas sp. and Dinobryon divergens were represented by several different genotypes each, and for the latter species, the dominating genotype differed with habitat. In contrast, Dinobryon pediforme, Dinobryon bavaricum, and Synura sphagnicola were exclusively represented by a single genotype each, and the respective genotype was the same in different samples. In summary, our results highlight the significance of molecular variation within protist morphospecies.

scholarly journals RNA splicing programs define tissue compartments and cell types at single cell resolution

2021 ◽  
Author(s):  
Julia Eve Olivieri ◽  
Roozbeh Dehghannasiri ◽  
Peter Wang ◽  
SoRi Jang ◽  
Antoine de Morree ◽  
...  
Keyword(s):  
Gene Expression ◽  
Single Cell ◽  
High Throughput ◽  
Rna Splicing ◽  
Single Cells ◽  
Cell Types ◽  
Mouse Lemur ◽  
Cell Type ◽  
Multiple Organs ◽  
Single Cell Pcr

More than 95% of human genes are alternatively spliced. Yet, the extent splicing is regulated at single-cell resolution has remained controversial due to both available data and methods to interpret it. We apply the SpliZ, a new statistical approach that is agnostic to transcript annotation, to detect cell-type-specific regulated splicing in > 110K carefully annotated single cells from 12 human tissues. Using 10x data for discovery, 9.1% of genes with computable SpliZ scores are cell-type specifically spliced. These results are validated with RNA FISH, single cell PCR, and in high throughput with Smart-seq2. Regulated splicing is found in ubiquitously expressed genes such as actin light chain subunit MYL6 and ribosomal protein RPS24, which has an epithelial-specific microexon. 13% of the statistically most variable splice sites in cell-type specifically regulated genes are also most variable in mouse lemur or mouse. SpliZ analysis further reveals 170 genes with regulated splicing during sperm development using, 10 of which are conserved in mouse and mouse lemur. The statistical properties of the SpliZ allow model-based identification of subpopulations within otherwise indistinguishable cells based on gene expression, illustrated by subpopulations of classical monocytes with stereotyped splicing, including an un-annotated exon, in SAT1, a Diamine acetyltransferase. Together, this unsupervised and annotation-free analysis of differential splicing in ultra high throughput droplet-based sequencing of human cells across multiple organs establishes splicing is regulated cell-type-specifically independent of gene expression.

scholarly journals Comparative Single-Cell Genomics of Chloroflexi from the Okinawa Trough Deep-Subsurface Biosphere

2016 ◽  
Vol 82 (10) ◽  
pp. 3000-3008 ◽  
Author(s):  
Heather Fullerton ◽  
Craig L. Moyer
Keyword(s):  
Single Cell ◽  
Okinawa Trough ◽  
Reductive Dehalogenation ◽  
Rrna Gene ◽  
Ssu Rrna ◽  
Metabolic Potential ◽  
Deep Subsurface ◽  
Content Type ◽  
Reductive Dehalogenase ◽  
The Okinawa Trough

ABSTRACTChloroflexismall-subunit (SSU) rRNA gene sequences are frequently recovered from subseafloor environments, but the metabolic potential of the phylum is poorly understood. The phylumChloroflexiis represented by isolates with diverse metabolic strategies, including anoxic phototrophy, fermentation, and reductive dehalogenation; therefore, function cannot be attributed to these organisms based solely on phylogeny. Single-cell genomics can provide metabolic insights into uncultured organisms, like the deep-subsurfaceChloroflexi. Nine SSU rRNA gene sequences were identified from single-cell sorts of whole-round core material collected from the Okinawa Trough at Iheya North hydrothermal field as part of Integrated Ocean Drilling Program (IODP) expedition 331 (Deep Hot Biosphere). Previous studies of subsurfaceChloroflexisingle amplified genomes (SAGs) suggested heterotrophic or lithotrophic metabolisms and provided no evidence for growth by reductive dehalogenation. Our nineChloroflexiSAGs (seven of which are from the orderAnaerolineales) indicate that, in addition to genes for the Wood-Ljungdahl pathway, exogenous carbon sources can be actively transported into cells. At least one subunit for pyruvate ferredoxin oxidoreductase was found in four of theChloroflexiSAGs. This protein can provide a link between the Wood-Ljungdahl pathway and other carbon anabolic pathways. Finally, one of the sevenAnaerolinealesSAGs contains a distinct reductive dehalogenase homologous (rdhA) gene.IMPORTANCEThrough the use of single amplified genomes (SAGs), we have extended the metabolic potential of an understudied group of subsurface microbes, theChloroflexi. These microbes are frequently detected in the subsurface biosphere, though their metabolic capabilities have remained elusive. In contrast to previously examinedChloroflexiSAGs, our genomes (several are from the orderAnaerolineales) were recovered from a hydrothermally driven system and therefore provide a unique window into the metabolic potential of this type of habitat. In addition, a reductive dehalogenase gene (rdhA) has been directly linked to marine subsurfaceChloroflexi, suggesting that reductive dehalogenation is not limited to the classDehalococcoidia. This discovery expands the nutrient-cycling and metabolic potential present within the deep subsurface and provides functional gene information relating to this enigmatic group.

PCR Amplification of Microsatellites from Single Cells of Karenia brevis Preserved in Lugol’s Iodine Solution

2007 ◽  
Vol 10 (2) ◽  
pp. 122-127 ◽  
Author(s):  
D. W. Henrichs ◽  
M. A. Renshaw ◽  
C. A. Santamaria ◽  
B. Richardson ◽  
J. R. Gold ◽  
...  
Keyword(s):  
Single Cells ◽  
Pcr Amplification ◽  
Karenia Brevis ◽  
Iodine Solution ◽  
Lugol’S Iodine

scholarly journals Homoacetogenesis in Deep-SeaChloroflexi, as Inferred by Single-Cell Genomics, Provides a Link to Reductive Dehalogenation in TerrestrialDehalococcoidetes

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Holly L. Sewell ◽  
Anne-Kristin Kaster ◽  
Alfred M. Spormann
Keyword(s):  
Single Cell ◽  
Deep Sea ◽  
Formate Dehydrogenase ◽  
Single Cells ◽  
Reductive Dehalogenation ◽  
Evolutionary Significance ◽  
Rrna Gene ◽  
Deep Sea Sediment ◽  
Deep Sea Sediments ◽  
Anaerobic Benzoate Oxidation

ABSTRACTThe deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylumChloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments of the Peruvian Margin, which are enriched in suchChloroflexi. 16S rRNA gene sequence analysis placed two of these single-cell-derived genomes (DscP3 and Dsc4) in a clade of subphylum IChloroflexiwhich were previously recovered from deep-sea sediment in the Okinawa Trough and a third (DscP2-2) as a member of the previously reported DscP2 population from Peruvian Margin site 1230. The presence of genes encoding enzymes of a complete Wood-Ljungdahl pathway, glycolysis/gluconeogenesis, aRhodobacternitrogen fixation (Rnf) complex, glyosyltransferases, and formate dehydrogenases in the single-cell genomes of DscP3 and Dsc4 and the presence of an NADH-dependent reduced ferredoxin:NADP oxidoreductase (Nfn) and Rnf in the genome of DscP2-2 imply a homoacetogenic lifestyle of these abundant marineChloroflexi. We also report here the first complete pathway for anaerobic benzoate oxidation to acetyl coenzyme A (CoA) in the phylumChloroflexi(DscP3 and Dsc4), including a class I benzoyl-CoA reductase. Of remarkable evolutionary significance, we discovered a gene encoding a formate dehydrogenase (FdnI) with reciprocal closest identity to the formate dehydrogenase-like protein (complex iron-sulfur molybdoenzyme [CISM], DET0187) of terrestrialDehalococcoides/Dehalogenimonasspp. This formate dehydrogenase-like protein has been shown to lack formate dehydrogenase activity inDehalococcoides/Dehalogenimonasspp. and is instead hypothesized to couple HupL hydrogenase to a reductive dehalogenase in the catabolic reductive dehalogenation pathway. This finding of a close functional homologue provides an important missing link for understanding the origin and the metabolic core of terrestrialDehalococcoides/Dehalogenimonasspp. and of reductive dehalogenation, as well as the biology of abundant deep-seaChloroflexi.IMPORTANCEThe deep marine subsurface is one of the largest unexplored biospheres on Earth and is widely inhabited by members of the phylumChloroflexi. In this report, we investigated genomes of single cells obtained from deep-sea sediments and provide evidence for a homacetogenic lifestyle of these abundant marineChloroflexi. Moreover, genome signature and key metabolic genes indicate an evolutionary relationship between these deep-sea sediment microbes and terrestrial, reductively dehalogenatingDehalococcoides.

In-Situ Dual Beam (FIBSEM) Techniques for Probe Pad Deposition and Dielectric Integrity Inspection in 0.2 μm Technology DRAM Single Cells

1999 ◽  
Author(s):  
Gunnar Zimmermann ◽  
Richard Chapman
Keyword(s):  
Electron Beam ◽  
Single Cell ◽  
Single Cells ◽  
Ion Beam ◽  
Gate Oxide ◽  
Ion Milling ◽  
Dual Beam ◽  
Sem Imaging ◽  
Innovative Techniques

Abstract Dual beam FIBSEM systems invite the use of innovative techniques to localize IC fails both electrically and physically. For electrical localization, we present a quick and reliable in-situ FIBSEM technique to deposit probe pads with very low parasitic leakage (Ipara < 4E-11A at 3V). The probe pads were Pt, deposited with ion beam assistance, on top of highly insulating SiOx, deposited with electron beam assistance. The buried plate (n-Band), p-well, wordline and bitline of a failing and a good 0.2 μm technology DRAM single cell were contacted. Both cells shared the same wordline for direct comparison of cell characteristics. Through this technique we electrically isolated the fail to a single cell by detecting leakage between the polysilicon wordline gate and the cell diffusion. For physical localization, we present a completely in-situ FIBSEM technique that combines ion milling, XeF2 staining and SEM imaging. With this technique, the electrically isolated fail was found to be a hole in the gate oxide at the bad cell.

scholarly journals Visually precise, low-damage, single-cell spatial manipulation with single-pixel resolution

2021 ◽  
Vol 12 (11) ◽  
pp. 4111-4118
Author(s):  
Qi Zhang ◽  
Yunlong Shao ◽  
Boye Li ◽  
Yuanyuan Wu ◽  
Jingying Dong ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Pixel Resolution ◽  
Single Pixel

We achieved the low-damage spatial puncture of single cells at specific visual points with an accuracy of <65 nm.

scholarly journals Single cell transcriptome atlas of mouse mammary epithelial cells across development

2021 ◽  
Vol 23 (1) ◽  
Author(s):  
Bhupinder Pal ◽  
Yunshun Chen ◽  
Michael J. G. Milevskiy ◽  
François Vaillant ◽  
Lexie Prokopuk ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Single Cell ◽  
Developmental Stages ◽  
Mammary Epithelial Cells ◽  
Expression Profiles ◽  
Single Cells ◽  
Chromatin Accessibility ◽  
Mammary Epithelial ◽  
Cell Transcriptome ◽  
Single Cell Transcriptome

Abstract Background Heterogeneity within the mouse mammary epithelium and potential lineage relationships have been recently explored by single-cell RNA profiling. To further understand how cellular diversity changes during mammary ontogeny, we profiled single cells from nine different developmental stages spanning late embryogenesis, early postnatal, prepuberty, adult, mid-pregnancy, late-pregnancy, and post-involution, as well as the transcriptomes of micro-dissected terminal end buds (TEBs) and subtending ducts during puberty. Methods The single cell transcriptomes of 132,599 mammary epithelial cells from 9 different developmental stages were determined on the 10x Genomics Chromium platform, and integrative analyses were performed to compare specific time points. Results The mammary rudiment at E18.5 closely aligned with the basal lineage, while prepubertal epithelial cells exhibited lineage segregation but to a less differentiated state than their adult counterparts. Comparison of micro-dissected TEBs versus ducts showed that luminal cells within TEBs harbored intermediate expression profiles. Ductal basal cells exhibited increased chromatin accessibility of luminal genes compared to their TEB counterparts suggesting that lineage-specific chromatin is established within the subtending ducts during puberty. An integrative analysis of five stages spanning the pregnancy cycle revealed distinct stage-specific profiles and the presence of cycling basal, mixed-lineage, and 'late' alveolar intermediates in pregnancy. Moreover, a number of intermediates were uncovered along the basal-luminal progenitor cell axis, suggesting a continuum of alveolar-restricted progenitor states. Conclusions This extended single cell transcriptome atlas of mouse mammary epithelial cells provides the most complete coverage for mammary epithelial cells during morphogenesis to date. Together with chromatin accessibility analysis of TEB structures, it represents a valuable framework for understanding developmental decisions within the mouse mammary gland.

Sign in / Sign up

Export Citation Format

Share Document