Tracing Baculovirus AcMNPV Infection Using a Real-Time Method Based on ANCHORTM DNA Labeling Technology

Many steps in the baculovirus life cycle, from initial ingestion to the subsequent infection of all larval cells, remain largely unknown; primarily because it has hitherto not been possible to follow individual genomes and their lineages. Use of ANCHORTM technology allows a high intensity fluorescent labelling of DNA. When applied to a virus genome, it is possible to follow individual particles, and the overall course of infection. This technology has been adapted to enable labelling of the baculovirus Autographa californica Multiple NucleoPolyhedroVirus genome, as a first step to its application to other baculoviruses. AcMNPV was modified by inserting the two components of ANCHORTM: a specific DNA-binding protein fused to a fluorescent reporter, and the corresponding DNA recognition sequence. The resulting modified virus was stable, infectious, and replicated correctly in Spodoptera frugiperda 9 (Sf9) cells and in vivo. Both budded viruses and occlusion bodies were clearly distinguishable, and infecting cells or larvae allowed the infection process to be monitored in living cells or tissues. The level of fluorescence in the culture medium of infected cells in vitro showed a good correlation with the number of infectious budded viruses. A cassette that can be used in other baculoviruses has been designed. Altogether our results introduce for the first time the generation of autofluorescent baculovirus and their application to follow infection dynamics directly in living cells or tissues.

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A Fluorescent Split Aptamer for Visualizing RNA-RNA Assembly In Vivo

10.1101/109306 ◽

2017 ◽

Author(s):

Khalid K. Alam ◽

Kwaku D. Tawiah ◽

Matthew F. Lichte ◽

David Porciani ◽

Donald H. Burke

Keyword(s):

Self Assembly ◽

Living Cells ◽

Rna Aptamers ◽

Genetic Circuits ◽

Fluorescent Reporter ◽

Indirect Measures ◽

Reporter Proteins ◽

Circuit Function

AbstractRNA-RNA assembly governs key biological processes and is a powerful tool for engineering synthetic genetic circuits. Characterizing RNA assembly in living cells often involves monitoring fluorescent reporter proteins, which are at best indirect measures of underlying RNA-RNA hybridization events and are subject to additional temporal and load constraints associated with translation and activation of reporter proteins. In contrast, RNA aptamers that sequester small molecule dyes and activate their fluorescence are increasingly utilized in genetically-encoded strategies to report on RNA-level events. Split-aptamer systems have been rationally designed to generate signal upon hybridization of two or more discrete RNA transcripts, but none directly function when expressed in vivo. We reasoned that the improved physiological properties of the Broccoli aptamer enable construction of a split-aptamer system that could function in living cells. Here we present the Split-Broccoli system, in which self-assembly is nucleated by a thermostable, three-way junction RNA architecture and fluorescence activation requires both strands. Functional assembly of the system approximately follows second order kinetics in vitro and improves when cotranscribed, rather than when assembled from purified components. Split-Broccoli fluorescence is digital in vivo and retains functional modularity when fused to RNAs that regulate circuit function through RNA-RNA hybridization, as demonstrated with an RNA Toehold switch. Split-Broccoli represents the first functional split-aptamer system to operate in vivo. It offers a genetically-encoded and nondestructive platform to monitor and exploit RNA-RNA hybridization, whether as an all-RNA, stand-alone AND gate or as a tool for monitoring assembly of RNA-RNA hybrids.

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Carbon dots derived from Fusobacterium nucleatum for intracellular determination of Fe3+ and bioimaging both in vitro and in vivo

Analytical Methods ◽

10.1039/d1ay00020a ◽

2021 ◽

Author(s):

Lijuan Liu ◽

Shengting Zhang ◽

Xiaodan Zheng ◽

Hongmei Li ◽

Qi Chen ◽

...

Keyword(s):

Carbon Dots ◽

Fusobacterium Nucleatum ◽

Living Cells ◽

First Time ◽

Fluorescent Carbon Dots ◽

Fluorescent Carbon

Fusobacterium nucleatum has been employed for the first time to synthesize fluorescent carbon dots which could be applied for the determination of Fe3+ ions in living cells and bioimaging in vitro and in vivo with excellent biocompatibility.

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SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO. II

The Journal of General Physiology ◽

10.1085/jgp.16.2.233 ◽

1932 ◽

Vol 16 (2) ◽

pp. 233-242 ◽

Cited By ~ 27

Author(s):

B. G. Wilkes ◽

Elizabeth T. Palmer

Keyword(s):

Physiological Activity ◽

Outer Region ◽

Living Cells ◽

Yeast Cells ◽

Live Cells ◽

Intracellular Enzyme ◽

Relationship Of ◽

Kinetics Of

1. The pH-activity relationship of invertase has been studied in vivo and in vitro under identical external environmental conditions. 2. The effect of changing (H+) upon the sucroclastic activity of living cells of S. cerevisiae and of invertase solutions obtained therefrom has been found, within experimental error, to be identical. 3. The region of living yeast cells in which invertase exerts its physiological activity changes its pH freely and to the same extent as that of the suspending medium. It is suggested that this may indicate that this intracellular enzyme may perform its work somewhere in the outer region of the cell. 4. In using live cells containing maltase, no evidence of increased sucroclastic activity around pH 6.9, due to the action of Weidenhagen's α-glucosidase (maltase), was found.

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Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3841-3849.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3841-3849

Author(s):

B Zenzie-Gregory ◽

A Khachi ◽

I P Garraway ◽

S T Smale

Keyword(s):

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Upstream Region ◽

Promoter Strength ◽

Recognition Sequence ◽

Specific Recognition ◽

Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

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In Vitro and in Vivo Imaging of Nitroxyl with Copper Fluorescent Probe in Living Cells and Zebrafish

Molecules ◽

10.3390/molecules23102551 ◽

2018 ◽

Vol 23 (10) ◽

pp. 2551 ◽

Cited By ~ 3

Author(s):

Sathyadevi Palanisamy ◽

Yu-Liang Wang ◽

Yu-Jen Chen ◽

Chiao-Yun Chen ◽

Fu-Te Tsai ◽

...

Keyword(s):

Critical Role ◽

Model Organism ◽

Living Cells ◽

Biological Species ◽

Zebrafish Model ◽

Physiological Processes ◽

Collective Data ◽

Angeli's Salt

Nitroxyl (HNO) plays a critical role in many physiological processes which includes vasorelaxation in heart failure, neuroregulation, and myocardial contractility. Powerful imaging tools are required to obtain information for understanding the mechanisms involved in these in vivo processes. In order to develop a rapid and high sensitive probe for HNO detection in living cells and the zebrafish model organism, 2-((2-(benzothiazole-2yl)benzylidene) amino)benzoic acid (AbTCA) as a ligand, and its corresponding copper(II) complex Cu(II)-AbTCA were synthesized. The reaction results of Cu(II)-AbTCA with Angeli’s salt showed that Cu(II)-AbTCA could detect HNO quantitatively in a range of 40–360 µM with a detection limit of 9.05 µM. Furthermore, Cu(II)-AbTCA is more selective towards HNO over other biological species including thiols, reactive nitrogen, and reactive oxygen species. Importantly, Cu(II)-AbTCA was successfully applied to detect HNO in living cells and zebrafish. The collective data reveals that Cu(II)-AbTCA could be used as a potential probe for HNO detection in living systems.

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Real time imaging reveals a peroxisomal reticulum in living cells

Journal of Cell Science ◽

10.1242/jcs.113.20.3663 ◽

2000 ◽

Vol 113 (20) ◽

pp. 3663-3671 ◽

Cited By ~ 1

Author(s):

M. Schrader ◽

S.J. King ◽

T.A. Stroh ◽

T.A. Schroer

Keyword(s):

Real Time ◽

Cytoplasmic Dynein ◽

Living Cells ◽

Real Time Imaging ◽

Peroxisomal Targeting ◽

Microtubule Motor ◽

Targeting Signal ◽

Peroxisomal Targeting Signal

We have directly imaged the dynamic behavior of a variety of morphologically different peroxisomal structures in HepG2 and COS-7 cells transfected with a construct encoding GFP bearing the C-terminal peroxisomal targeting signal 1. Real time imaging revealed that moving peroxisomes interacted with each other and were engaged in transient contacts, and at higher magnification, tubular peroxisomes appeared to form a peroxisomal reticulum. Local remodeling of these structures could be observed involving the formation and detachment of tubular processes that interconnected adjacent organelles. Inhibition of cytoplasmic dynein based motility by overexpression of the dynactin subunit, dynamitin (p50), inhibited the movement of peroxisomes in vivo and interfered with the reestablishment of a uniform distribution of peroxisomes after recovery from nocodazole treatment. Isolated peroxisomes moved in vitro along microtubules in the presence of a microtubule motor fraction. Our data reveal that peroxisomal behavior in vivo is significantly more dynamic and interactive than previously thought and suggest a role for the dynein/dynactin motor in peroxisome motility.

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Involvement of proliferating cell nuclear antigen (cyclin) in DNA replication in living cells

Molecular and Cellular Biology ◽

10.1128/mcb.9.1.57-66.1989 ◽

1989 ◽

Vol 9 (1) ◽

pp. 57-66

Author(s):

M Zuber ◽

E M Tan ◽

M Ryoji

Keyword(s):

Dna Polymerase ◽

Proliferating Cell Nuclear Antigen ◽

Living Cells ◽

Nuclear Antigen ◽

Plasmid Replication ◽

Dna Polymerase Delta ◽

Dna Polymerase Alpha ◽

Proliferating Cell

Proliferating cell nuclear antigen (PCNA) (also called cyclin) is known to stimulate the activity of DNA polymerase delta but not the other DNA polymerases in vitro. We injected a human autoimmune antibody against PCNA into unfertilized eggs of Xenopus laevis and examined the effects of this antibody on the replication of injected plasmid DNA as well as egg chromosomes. The anti-PCNA antibody inhibited plasmid replication by up to 67%, demonstrating that PCNA is involved in plasmid replication in living cells. This result further implies that DNA polymerase delta is necessary for plasmid replication in vivo. Anti-PCNA antibody alone did not block plasmid replication completely, but the residual replication was abolished by coinjection of a monoclonal antibody against DNA polymerase alpha. Anti-DNA polymerase alpha alone inhibited plasmid replication by 63%. Thus, DNA polymerase alpha is also required for plasmid replication in this system. In similar studies on the replication of egg chromosomes, the inhibition by anti-PCNA antibody was only 30%, while anti-DNA polymerase alpha antibody blocked 73% of replication. We concluded that the replication machineries of chromosomes and plasmid differ in their relative content of DNA polymerase delta. In addition, we obtained evidence through the use of phenylbutyl deoxyguanosine, an inhibitor of DNA polymerase alpha, that the structure of DNA polymerase alpha holoenzyme for chromosome replication is significantly different from that for plasmid replication.

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The development of a hemicyanine-based ratiometric CO fluorescent probe with a long emission wavelength and its applications for imaging CO in vitro and in vivo

New Journal of Chemistry ◽

10.1039/d0nj00677g ◽

2020 ◽

Vol 44 (28) ◽

pp. 12107-12112 ◽

Cited By ~ 1

Author(s):

Yunyan Zhang ◽

Xiuqi Kong ◽

Yonghe Tang ◽

Min Li ◽

Yaguang Yin ◽

...

Keyword(s):

Fluorescent Probe ◽

Living Cells ◽

Emission Wavelength ◽

Long Wavelength ◽

Wavelength Emission ◽

Ratiometric Fluorescent Probe

A novel ratiometric fluorescent probe, Hcy-CO, with long-wavelength emission was developed for visualizing CO in living cells and zebrafish.

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Effect of Pyrisoxazole on Colletotrichum scovillei Infection and Anthracnose on Chili

Plant Disease ◽

10.1094/pdis-06-19-1291-re ◽

2020 ◽

Vol 104 (2) ◽

pp. 551-559

Author(s):

Y. Y. Gao ◽

X. X. Li ◽

L. F. He ◽

B. X. Li ◽

W. Mu ◽

...

Keyword(s):

Inhibitory Activity ◽

Infection Process ◽

Appressorium Formation ◽

Baseline Sensitivity ◽

Sensitivity Curves ◽

Half Maximal Effective Concentration ◽

Colletotrichum Scovillei ◽

Feasible Alternative

Anthracnose caused by Colletotrichum scovillei is one of the most destructive diseases affecting chili production. Disease control mainly relies on conventional fungicides, and repeated exposure to single-site mode-of-action fungicides may pose a risk for the development of resistant isolates within the population. Our previous study suggested that pyrisoxazole has strong inhibitory activity against C. scovillei in vitro. However, the effects of pyrisoxazole on the C. scovillei infection process and the performance of pyrisoxazole in the field remain unclear. In this study, pyrisoxazole exhibited strong inhibitory activity against the mycelial growth, appressorium formation, and appressorium diameter of C. scovillei, with half maximal effective concentration values of 0.1986, 0.0147, and 0.0269 μg/ml, respectively, but had no effect on sporulation, even at the highest concentration of 1.6 μg/ml. The baseline sensitivity curves were unimodal with a long right-hand tail. The in vivo data showed that pyrisoxazole provided both preventive and curative activity against anthracnose on chili. Pyrisoxazole decreased the incidence of anthracnose and reduced disease progress. The results of electron microscopy showed that pyrisoxazole can affect the C. scovillei infection process by altering mycelial morphology, degrading conidia and germ tubes, suppressing conidial germination and appressorium formation, and enhancing conidiophore production. Pyrisoxazole can be used to effectively control anthracnose under field conditions and increase chili yield; moreover, no phytotoxicity symptoms were observed after treatment. These results provide new insight into the mechanisms by which pyrisoxazole controls disease and suggest that pyrisoxazole is a feasible alternative for the management of anthracnose in chili.

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Force-dependent vinculin binding to talin in live cells: a crucial step in anchoring the actin cytoskeleton to focal adhesions

AJP Cell Physiology ◽

10.1152/ajpcell.00122.2013 ◽

2014 ◽

Vol 306 (6) ◽

pp. C607-C620 ◽

Cited By ~ 57

Author(s):

Hiroaki Hirata ◽

Hitoshi Tatsumi ◽

Chwee Teck Lim ◽

Masahiro Sokabe

Keyword(s):

Actin Cytoskeleton ◽

Focal Adhesions ◽

Living Cells ◽

Live Cells ◽

Mechanical Forces ◽

Actin Network ◽

Crucial Step

Mechanical forces play a pivotal role in the regulation of focal adhesions (FAs) where the actin cytoskeleton is anchored to the extracellular matrix through integrin and a variety of linker proteins including talin and vinculin. The localization of vinculin at FAs depends on mechanical forces. While in vitro studies have demonstrated the force-induced increase in vinculin binding to talin, it remains unclear whether such a mechanism exists at FAs in vivo. In this study, using fibroblasts cultured on elastic silicone substrata, we have examined the role of forces in modulating talin-vinculin binding at FAs. Stretching the substrata caused vinculin accumulation at talin-containing FAs, and this accumulation was abrogated by expressing the talin-binding domain of vinculin (domain D1, which inhibits endogenous vinculin from binding to talin). These results indicate that mechanical forces loaded to FAs facilitate vinculin binding to talin at FAs. In cell-protruding regions, the actin network moved backward over talin-containing FAs in domain D1-expressing cells while it was anchored to FAs in control cells, suggesting that the force-dependent vinculin binding to talin is crucial for anchoring the actin cytoskeleton to FAs in living cells.

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