scholarly journals Single-cell Multiplexed Fluorescence Imaging to Visualize Viral Nucleic Acids and Proteins and Monitor HIV, HTLV, HBV, HCV, Zika Virus, and Influenza Infection

10.3791/61843 ◽  
2020 ◽  
Author(s):  
Raven Shah ◽  
Shuiyun Lan ◽  
Maritza N. Puray-Chavez ◽  
Dandan Liu ◽  
Philip R. Tedbury ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Single Cell ◽  
Fluorescence Imaging ◽  
Zika Virus ◽  
Influenza Infection ◽  
Multiplexed Fluorescence Imaging

scholarly journals Multiplexed fluorescence imaging of GPCR downstream signaling dynamics at the single-cell level

2021 ◽  
Author(s):  
Ryosuke Tany ◽  
Yuhei Goto ◽  
Yohei Kondo ◽  
Kazuhiro Aoki
Keyword(s):  
Single Cell ◽  
G Protein ◽  
Fluorescence Imaging ◽  
Imaging System ◽  
Single Cell Level ◽  
Cell Level ◽  
Gpcr Signaling ◽  
Downstream Signaling ◽  
Signaling Dynamics ◽  
Multiplexed Fluorescence Imaging

AbstractG-protein-coupled receptors (GPCRs) play an important role in sensing various extracellular stimuli, such as neurotransmitters, hormones, and tastants, and transducing the input information into the cell. While the human genome encodes more than 800 GPCR genes, only four Gα-proteins (Gαs, Gαi/o, Gαq/11, and Gα12/13) are known to couple with GPCRs. It remains unclear how such divergent GPCR information is translated into the downstream G-protein signaling dynamics. To answer this question, we report a multiplexed fluorescence imaging system for monitoring GPCR downstream signaling dynamics at the single-cell level. Genetically encoded biosensors for cAMP, Ca2+, RhoA, and ERK were selected as markers for GPCR downstream signaling, and were stably expressed in HeLa cells. GPCR was further transiently overexpressed in the cells. As a proof-of-concept, we visualized GPCR signaling dynamics of 5 dopamine receptors and 12 serotonin receptors, and found heterogeneity between GPCRs and between cells. Even when the same Gα proteins were known to be coupled, the patterns of dynamics in GPCR downstream signaling, including the signal strength and duration, were substantially distinct among GPCRs. These results suggest the importance of dynamical encoding in GPCR signaling.

scholarly journals Improved Synthesis Strategy for Peptide Nucleic Acids (PNA) appropriate for Cell-specific Fluorescence Imaging

2012 ◽  
Vol 9 (1) ◽  
pp. 1-10 ◽  
Author(s):  
Rüdiger Pipkorn ◽  
Manfred Wiessler ◽  
Waldemar Waldeck ◽  
Ute Hennrich ◽  
Kiyoshi Nokihara ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Fluorescence Imaging ◽  
Peptide Nucleic Acids ◽  
Specific Fluorescence ◽  
Synthesis Strategy

scholarly journals Integrative single-cell and cell-free plasma RNA transcriptomics elucidates placental cellular dynamics

2017 ◽  
Vol 114 (37) ◽  
pp. E7786-E7795 ◽  
Author(s):  
Jason C. H. Tsang ◽  
Joaquim S. L. Vong ◽  
Lu Ji ◽  
Liona C. Y. Poon ◽  
Peiyong Jiang ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Single Cell ◽  
Human Placenta ◽  
Single Cells ◽  
Specific Gene ◽  
Hypertensive Disorder ◽  
Cellular Dynamics ◽  
Maternal Cell ◽  
Plasma Rna ◽  
Free Plasma

The human placenta is a dynamic and heterogeneous organ critical in the establishment of the fetomaternal interface and the maintenance of gestational well-being. It is also the major source of cell-free fetal nucleic acids in the maternal circulation. Placental dysfunction contributes to significant complications, such as preeclampsia, a potentially lethal hypertensive disorder during pregnancy. Previous studies have identified significant changes in the expression profiles of preeclamptic placentas using whole-tissue analysis. Moreover, studies have shown increased levels of targeted RNA transcripts, overall and placental contributions in maternal cell-free nucleic acids during pregnancy progression and gestational complications, but it remains infeasible to noninvasively delineate placental cellular dynamics and dysfunction at the cellular level using maternal cell-free nucleic acid analysis. In this study, we addressed this issue by first dissecting the cellular heterogeneity of the human placenta and defined individual cell-type–specific gene signatures by analyzing more than 24,000 nonmarker selected cells from full-term and early preeclamptic placentas using large-scale microfluidic single-cell transcriptomic technology. Our dataset identified diverse cellular subtypes in the human placenta and enabled reconstruction of the trophoblast differentiation trajectory. Through integrative analysis with maternal plasma cell-free RNA, we resolved the longitudinal cellular dynamics of hematopoietic and placental cells in pregnancy progression. Furthermore, we were able to noninvasively uncover the cellular dysfunction of extravillous trophoblasts in early preeclamptic placentas. Our work showed the potential of integrating transcriptomic information derived from single cells into the interpretation of cell-free plasma RNA, enabling the noninvasive elucidation of cellular dynamics in complex pathological conditions.

Fluorescence imaging-based methods for single-cell protein analysis

2019 ◽  
Vol 411 (19) ◽  
pp. 4339-4347
Author(s):  
Siwen Wang ◽  
Fei Ji ◽  
Zhonghan Li ◽  
Min Xue
Keyword(s):  
Single Cell ◽  
Fluorescence Imaging ◽  
Protein Analysis ◽  
Single Cell Protein ◽  
Cell Protein

scholarly journals 973. Single-cell RNA Sequencing Analysis of Zika Virus Infection in Human Stem Cell-Derived Cerebral Organoids

2019 ◽  
Vol 6 (Supplement_2) ◽  
pp. S33-S34
Author(s):  
Karen Ocwieja ◽  
Alexandra Stanton ◽  
Alexsia Richards ◽  
Jenna Antonucci ◽  
Travis Hughes ◽  
...  
Keyword(s):  
Stem Cell ◽  
Human Brain ◽  
Single Cell ◽  
Rna Sequencing ◽  
Zika Virus ◽  
Model Systems ◽  
Sequencing Analysis ◽  
Human Stem Cell ◽  
Zikv Infection ◽  
Single Cell Rna Sequencing

Abstract Background The molecular mechanisms underpinning the neurologic and congenital pathologies caused by Zika virus (ZIKV) infection remain poorly understood. One barrier has been the lack of relevant model systems for the developing human brain; however, thanks to advances in the stem cell field, we can now evaluate ZIKV central nervous system infections in human stem cell-derived cerebral organoids which recapitulate complex 3-dimensional neural architecture. Methods We apply Seq-Well—a simple, portable platform for massively parallel single-cell RNA sequencing—to characterize cerebral organoids infected with ZIKV. Using this sequencing method, and published transcriptional profiles, we identify multiple cellular populations in our organoids, including neuroprogenitor cells, intermediate progenitor cells, and terminally differentiated neurons. We detect and quantify host mRNA transcripts and viral RNA with single-cell resolution, defining transcriptional features of uninfected cells and infected cells. Results In this model of the developing brain, we identify preferred tropisms of ZIKV infection and pronounced effects on cell division, differentiation, and death. Our data additionally reveal differences in cellular populations and gene expression within organoids infected by historic and contemporary ZIKV strains from a variety of geographic locations. This finding might help explain phenotypic differences attributed to the viruses, including variable propensity to cause microcephaly. Conclusion Overall, our work provides insight into normal and diseased human brain development, and suggests that both virus replication and host response mechanisms underlie the neuropathology of ZIKV infection. Disclosures All Authors: No reported Disclosures.

scholarly journals Combining fluorescence imaging with Hi-C to study 3D genome architecture of the same single cell

2018 ◽  
Vol 13 (5) ◽  
pp. 1034-1061 ◽  
Author(s):  
David Lando ◽  
Srinjan Basu ◽  
Tim J Stevens ◽  
Andy Riddell ◽  
Kai J Wohlfahrt ◽  
...  
Keyword(s):  
Single Cell ◽  
Fluorescence Imaging ◽  
Genome Architecture ◽  
3D Genome

Abstract 3880: Synthesis of complex oligonucleotide libraries enables flexible and precise high-resolution fluorescence imaging of cellular nucleic acids

2011 ◽  
Author(s):  
Robert A. Ach ◽  
N. Alice Yamada ◽  
Peter Tsang ◽  
Alicia Scheffer-Wong ◽  
Laurakay Bruhn
Keyword(s):  
High Resolution ◽  
Nucleic Acids ◽  
Fluorescence Imaging

scholarly journals Uncultured Microbial Phyla Suggest Mechanisms for Multi-Thousand-Year Subsistence in Baltic Sea Sediments

mBio ◽  
2019 ◽  
Vol 10 (2) ◽  
Author(s):  
Jordan T. Bird ◽  
Eric D. Tague ◽  
Laura Zinke ◽  
Jenna M. Schmidt ◽  
Andrew D. Steen ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Baltic Sea ◽  
Single Cell ◽  
Marine Sediments ◽  
Niche Differentiation ◽  
Enzyme Assays ◽  
Sodium Pumps ◽  
Cellular Life ◽  
Zero Growth ◽  
Life On Earth

ABSTRACTEnergy-starved microbes in deep marine sediments subsist at near-zero growth for thousands of years, yet the mechanisms for their subsistence are unknown because no model strains have been cultivated from most of these groups. We investigated Baltic Sea sediments with single-cell genomics, metabolomics, metatranscriptomics, and enzyme assays to identify possible subsistence mechanisms employed by unculturedAtribacteria,Aminicenantes,Actinobacteriagroup OPB41,Aerophobetes,Chloroflexi,Deltaproteobacteria,Desulfatiglans,Bathyarchaeota, andEuryarchaeotamarine group II lineages. Some functions appeared to be shared by multiple lineages, such as trehalose production and NAD+-consuming deacetylation, both of which have been shown to increase cellular life spans in other organisms by stabilizing proteins and nucleic acids, respectively. Other possible subsistence mechanisms differed between lineages, possibly providing them different physiological niches. Enzyme assays and transcripts suggested thatAtribacteriaandActinobacteriagroup OPB41 catabolized sugars, whereasAminicenantesandAtribacteriacatabolized peptides. Metabolite and transcript data suggested thatAtribacteriautilized allantoin, possibly as an energetic substrate or chemical protectant, and also possessed energy-efficient sodium pumps.Atribacteriasingle-cell amplified genomes (SAGs) recruited transcripts for full pathways for the production of all 20 canonical amino acids, and the gene for amino acid exporter YddG was one of their most highly transcribed genes, suggesting that they may benefit from metabolic interdependence with other cells. Subsistence of uncultured phyla in deep subsurface sediments may occur through shared strategies of using chemical protectants for biomolecular stabilization, but also by differentiating into physiological niches and metabolic interdependencies.IMPORTANCEMuch of life on Earth exists in a very slow-growing state, with microbes from deeply buried marine sediments representing an extreme example. These environments are like natural laboratories that have run multi-thousand-year experiments that are impossible to perform in a laboratory. We borrowed some techniques that are commonly used in laboratory experiments and applied them to these natural samples to make hypotheses about how these microbes subsist for so long at low activity. We found that some methods for stabilizing proteins and nucleic acids might be used by many members of the community. We also found evidence for niche differentiation strategies, and possibly cross-feeding, suggesting that even though they are barely growing, complex ecological interactions continue to occur over ultralong timescales.

scholarly journals Single cell fluorescence imaging of glycan uptake by intestinal bacteria

2019 ◽  
Vol 13 (7) ◽  
pp. 1883-1889 ◽  
Author(s):  
Jan-Hendrik Hehemann ◽  
Greta Reintjes ◽  
Leeann Klassen ◽  
Adam D. Smith ◽  
Didier Ndeh ◽  
...  
Keyword(s):  
Single Cell ◽  
Fluorescence Imaging ◽  
Intestinal Bacteria
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