scholarly journals Tracking of quiescence inLeishmaniaby quantifying the expression of GFP in the ribosomal DNA locus

2019 ◽  
Author(s):  
Marlene Jara ◽  
Ilse Maes ◽  
Hideo Imamura ◽  
Malgorzata A. Domagalska ◽  
Jean Claude Dujardin ◽  
...  
Keyword(s):  
Ribosomal Dna ◽  
Rdna Locus ◽  
Live Cells ◽  
Brdu Incorporation ◽  
Reference Laboratory ◽  
Latent Infections ◽  
The Individual ◽  
First Time

ABSTRACTUnder stressful conditions some microorganisms adopt a reversible non or slow proliferative quiescent stage that allows their survival. Although quiescence has been described broadly in bacteria, this phenotype has been only recently discovered inLeishmania. In the present work we developed a biosensor of quiescence that allows to monitor the physiological stage of the parasite at population and single cell levels. We inserted a GFP gene into the ribosomal DNA locus and followed the expression of this reporter gene, driven by the ribosomal promotor (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during thein vitrotransition from extracellular promastigotes to intracellular amastigotes ofL. mexicanaanL. braziliensisand that the decrease in rGFP expression was coupledin vitrowith a decrease in replication as measured by BrdU incorporation. Quiescence was not only observed in reference laboratory strains, but also among clinical isolates. We found that quiescence was reversible as the parasites could rapidly resume their metabolically active and proliferative stage when they were put back in an optimal environment for growth. We demonstrated for the first time in live cells that amastigotes are a heterogeneous population in which shallow and deep quiescent stages may coexist. Finally, we showed that rGFP expression could be monitoredin vivoand that quiescent amastigotes could reside in tissues of animals with latent infections ofL. braziliensisorL. mexicana. We propose rGFP expression as a simple parameter to define quiescent cells and further characterize them.IMPORTANCEQuiescence is a physiological diversification that allows pathogens to overcome chemotherapy without the development of drug resistance and to be invisible to the immune system of their host. Quiescent pathogens can cause latent infections and (re-) emerge in an unpredictable time during the lifetime of the individual. The phenomenon was recently described inLeishmaniain which it could explain several clinical and sub-clinical features, like therapeutic failure, reactivation of the disease and asymptomatic infections. However, a simple biosensor of quiescence forLeishmaniais not yet available. We show for the first time that the integration of GFP within the rDNA locus and the subsequent quantification of its expression can be used as a biosensor to distinguish quiescent subpopulations among live amastigotes. Moreover, we show quiescence is quickly reversible bothin vivoandin vitro. We offer a tool that will allow the further molecular characterization of quiescent parasites.

scholarly journals Tracking of quiescence in Leishmania by quantifying the expression of GFP in the ribosomal DNA locus

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marlene Jara ◽  
Ilse Maes ◽  
Hideo Imamura ◽  
Malgorzata A. Domagalska ◽  
Jean Claude Dujardin ◽  
...  
Keyword(s):  
Molecular Level ◽  
Rdna Locus ◽  
Live Cells ◽  
Brdu Incorporation ◽  
Gfp Reporter ◽  
Gfp Reporter Gene ◽  
First Time ◽  
Quiescent Stage

AbstractUnder stressful conditions some microorganisms adopt a quiescent stage characterized by a reversible non or slow proliferative condition that allows their survival. This adaptation was only recently discovered in Leishmania. We developed an in vitro model and a biosensor to track quiescence at population and single cell levels. The biosensor is a GFP reporter gene integrated within the 18S rDNA locus, which allows monitoring the expression of 18S rRNA (rGFP expression). We showed that rGFP expression decreased significantly and rapidly during the transition from extracellular promastigotes to intracellular amastigotes and that it was coupled in vitro with a decrease in replication as measured by BrdU incorporation. rGFP expression was useful to track the reversibility of quiescence in live cells and showed for the first time the heterogeneity of physiological stages among the population of amastigotes in which shallow and deep quiescent stages may coexist. We also validated the use of rGFP expression as a biosensor in animal models of latent infection. Our models and biosensor should allow further characterization of quiescence at metabolic and molecular level.

scholarly journals Interaction between Acetylated MyoD and the Bromodomain of CBP and/or p300

2001 ◽  
Vol 21 (16) ◽  
pp. 5312-5320 ◽  
Author(s):  
Anna Polesskaya ◽  
Irina Naguibneva ◽  
Arnaud Duquet ◽  
Eyal Bengal ◽  
Philippe Robin ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Protein Function ◽  
Live Cells ◽  
Regulation Of Transcription ◽  
Muscle Creatine Kinase ◽  
Nonhistone Protein ◽  
Myogenic Factor ◽  
First Time

ABSTRACT Acetylation is emerging as a posttranslational modification of nuclear proteins that is essential to the regulation of transcription and that modifies transcription factor affinity for binding sites on DNA, stability, and/or nuclear localization. Here, we present both in vitro and in vivo evidence that acetylation increases the affinity of myogenic factor MyoD for acetyltransferases CBP and p300. In myogenic cells, the fraction of endogenous MyoD that is acetylated was found associated with CBP or p300. In vitro, the interaction between MyoD and CBP was more resistant to high salt concentrations and was detected with lower doses of MyoD when MyoD was acetylated. Interestingly, an analysis of CBP mutants revealed that the interaction with acetylated MyoD involves the bromodomain of CBP. In live cells, MyoD mutants that cannot be acetylated did not associate with CBP or p300 and were strongly impaired in their ability to cooperate with CBP for transcriptional activation of a muscle creatine kinase-luciferase construct. Taken together, our data suggest a new mechanism for activation of protein function by acetylation and demonstrate for the first time an acetylation-dependent interaction between the bromodomain of CBP and a nonhistone protein.

scholarly journals Thermodynamics of protein destabilization in live cells

2015 ◽  
Vol 112 (40) ◽  
pp. 12402-12407 ◽  
Author(s):  
Jens Danielsson ◽  
Xin Mu ◽  
Lisa Lang ◽  
Huabing Wang ◽  
Andres Binolfi ◽  
...  
Keyword(s):  
Self Organization ◽  
Live Cells ◽  
Heat Capacity Change ◽  
Bacterial Cells ◽  
Crowding Effects ◽  
Capacity Change ◽  
The Individual ◽  
Cellular Components

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a β-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein’s interplay with the functionally optimized “interaction landscape” of the cellular interior.

Multi-mode light microscopy of microtubule assembly dynamics and chromosome movement in vivo and In vitro

1996 ◽  
Vol 54 ◽  
pp. 730-731
Author(s):  
E. D. Salmon ◽  
J. C. Waters ◽  
C. Waterman-Storer
Keyword(s):  
Imaging System ◽  
Ccd Camera ◽  
Transmitted Light ◽  
Microtubule Assembly ◽  
Live Cells ◽  
Optical Efficiency ◽  
Multiple Band ◽  
Multi Mode

We have developed a multi-mode digital imaging system which acquires images with a cooled CCD camera (Figure 1). A multiple band pass dichromatic mirror and robotically controlled filter wheels provide wavelength selection for epi-fluorescence. Shutters select illumination either by epi-fluorescence or by transmitted light for phase contrast or DIC. Many of our experiments involve investigations of spindle assembly dynamics and chromosome movements in live cells or unfixed reconstituted preparations in vitro in which photodamage and phototoxicity are major concerns. As a consequence, a major factor in the design was optical efficiency: achieving the highest image quality with the least number of illumination photons. This principle applies to both epi-fluorescence and transmitted light imaging modes. In living cells and extracts, microtubules are visualized using X-rhodamine labeled tubulin. Photoactivation of C2CF-fluorescein labeled tubulin is used to locally mark microtubules in studies of microtubule dynamics and translocation. Chromosomes are labeled with DAPI or Hoechst DNA intercalating dyes.

scholarly journals Hexapod Assassins’ Potion: Venom Composition and Bioactivity from the Eurasian Assassin Bug Rhynocoris iracundus

Biomedicines ◽  
2021 ◽  
Vol 9 (7) ◽  
pp. 819
Author(s):  
Nicolai Rügen ◽  
Timothy P. Jenkins ◽  
Natalie Wielsch ◽  
Heiko Vogel ◽  
Benjamin-Florian Hempel ◽  
...  
Keyword(s):  
Biomedical Applications ◽  
Galleria Mellonella ◽  
Systemic Reactions ◽  
Venom Composition ◽  
Assassin Bug ◽  
Molecular Components ◽  
First Time ◽  
Tissue Digestion

Assassin bug venoms are potent and exert diverse biological functions, making them potential biomedical goldmines. Besides feeding functions on arthropods, assassin bugs also use their venom for defense purposes causing localized and systemic reactions in vertebrates. However, assassin bug venoms remain poorly characterized. We collected the venom from the assassin bug Rhynocoris iracundus and investigated its composition and bioactivity in vitro and in vivo. It caused lysis of murine neuroblastoma, hepatoma cells, and healthy murine myoblasts. We demonstrated, for the first time, that assassin bug venom induces neurolysis and suggest that it counteracts paralysis locally via the destruction of neural networks, contributing to tissue digestion. Furthermore, the venom caused paralysis and melanization of Galleria mellonella larvae and pupae, whilst also possessing specific antibacterial activity against Escherichia coli, but not Listeria grayi and Pseudomonas aeruginosa. A combinatorial proteo-transcriptomic approach was performed to identify potential toxins responsible for the observed effects. We identified neurotoxic Ptu1, an inhibitory cystin knot (ICK) toxin homologous to ω-conotoxins from cone snails, cytolytic redulysins homologous to trialysins from hematophagous kissing bugs, and pore-forming hemolysins. Additionally, chitinases and kininogens were found and may be responsible for insecticidal and cytolytic activities. We demonstrate the multifunctionality and complexity of assassin bug venom, which renders its molecular components interesting for potential biomedical applications.

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

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.

scholarly journals Fe3O4@Pt nanoparticles to enable combinational electrodynamic/chemodynamic therapy

2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Tong Chen ◽  
Qiang Chu ◽  
Mengyang Li ◽  
Gaorong Han ◽  
Xiang Li
Keyword(s):  
Fenton Reaction ◽  
Pt Nanoparticles ◽  
Therapeutic Modality ◽  
Ros Generation ◽  
Tumor Treatment ◽  
Superior Efficacy ◽  
Platinum Nanocrystals ◽  
First Time

AbstractElectrodynamic therapy (EDT) has recently emerged as a potential external field responsive approach for tumor treatment. While it presents a number of clear superiorities, EDT inherits the intrinsic challenges of current reactive oxygen species (ROS) based therapeutic treatments owing to the complex tumor microenvironment, including glutathione (GSH) overexpression, acidity and others. Herein for the first time, iron oxide nanoparticles are decorated using platinum nanocrystals (Fe3O4@Pt NPs) to integrate the current EDT with chemodynamic phenomenon and GSH depletion. Fe3O4@Pt NPs can effectively induce ROS generation based on the catalytic reaction on the surface of Pt nanoparticles triggered by electric field (E), and meanwhile it may catalyze intracellular H2O2 into ROS via Fenton reaction. In addition, Fe3+ ions released from Fe3O4@Pt NPs under the acidic condition in tumor cells consume GSH in a rapid fashion, inhibiting ROS clearance to enhance its antitumor efficacy. As a result, considerable in vitro and in vivo tumor inhibition phenomena are observed. This study has demonstrated an alternative concept of combinational therapeutic modality with superior efficacy.

scholarly journals Cathepsin L plays a key role in SARS-CoV-2 infection in humans and humanized mice and is a promising target for new drug development

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Miao-Miao Zhao ◽  
Wei-Li Yang ◽  
Fang-Yuan Yang ◽  
Li Zhang ◽  
Wei-Jin Huang ◽  
...  
Keyword(s):  
Drug Development ◽  
Cathepsin L ◽  
Human Cells ◽  
New Drugs ◽  
Disease Course ◽  
Promising Target ◽  
First Time

AbstractTo discover new drugs to combat COVID-19, an understanding of the molecular basis of SARS-CoV-2 infection is urgently needed. Here, for the first time, we report the crucial role of cathepsin L (CTSL) in patients with COVID-19. The circulating level of CTSL was elevated after SARS-CoV-2 infection and was positively correlated with disease course and severity. Correspondingly, SARS-CoV-2 pseudovirus infection increased CTSL expression in human cells in vitro and human ACE2 transgenic mice in vivo, while CTSL overexpression, in turn, enhanced pseudovirus infection in human cells. CTSL functionally cleaved the SARS-CoV-2 spike protein and enhanced virus entry, as evidenced by CTSL overexpression and knockdown in vitro and application of CTSL inhibitor drugs in vivo. Furthermore, amantadine, a licensed anti-influenza drug, significantly inhibited CTSL activity after SARS-CoV-2 pseudovirus infection and prevented infection both in vitro and in vivo. Therefore, CTSL is a promising target for new anti-COVID-19 drug development.

scholarly journals Schistosoma mansoni eggs induce Wnt/β-catenin signaling and activate the protooncogene c-Jun in human and hamster colon

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Jakob Weglage ◽  
Friederike Wolters ◽  
Laura Hehr ◽  
Jakob Lichtenberger ◽  
Celina Wulz ◽  
...  
Keyword(s):  
Signaling Pathways ◽  
Colorectal Carcinogenesis ◽  
Sub Saharan Africa ◽  
Interleukin 4 ◽  
Health Burden ◽  
Sub Saharan ◽  
Considerable Morbidity ◽  
First Time

AbstractSchistosomiasis (bilharzia) is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, with considerable morbidity in parts of the Middle East, South America, Southeast Asia, in sub-Saharan Africa, and particularly also in Europe. The WHO describes an increasing global health burden with more than 290 million people threatened by the disease and a potential to spread into regions with temperate climates like Corsica, France. The aim of our study was to investigate the influence of S. mansoni infection on colorectal carcinogenic signaling pathways in vivo and in vitro. S. mansoni infection, soluble egg antigens (SEA) and the Interleukin-4-inducing principle from S. mansoni eggs induce Wnt/β-catenin signaling and the protooncogene c-Jun as well as downstream factor Cyclin D1 and markers for DNA-damage, such as Parp1 and γH2a.x in enterocytes. The presence of these characteristic hallmarks of colorectal carcinogenesis was confirmed in colon biopsies from S. mansoni-infected patients demonstrating the clinical relevance of our findings. For the first time it was shown that S. mansoni SEA may be involved in the induction of colorectal carcinoma-associated signaling pathways.

scholarly journals Preclinical Testing of Radiopharmaceuticals for the Detection and Characterization of Osteomyelitis: Experiences from a Porcine Model

Molecules ◽  
2021 ◽  
Vol 26 (14) ◽  
pp. 4221
Author(s):  
Aage Kristian Olsen Alstrup ◽  
Svend Borup Jensen ◽  
Ole Lerberg Nielsen ◽  
Lars Jødal ◽  
Pia Afzelius
Keyword(s):  
Animal Model ◽  
Animal Models ◽  
Porcine Model ◽  
Animal Experiments ◽  
Radioactive Tracers ◽  
The Individual ◽  
Selection Of

The development of new and better radioactive tracers capable of detecting and characterizing osteomyelitis is an ongoing process, mainly because available tracers lack selectivity towards osteomyelitis. An integrated part of developing new tracers is the performance of in vivo tests using appropriate animal models. The available animal models for osteomyelitis are also far from ideal. Therefore, developing improved animal osteomyelitis models is as important as developing new radioactive tracers. We recently published a review on radioactive tracers. In this review, we only present and discuss osteomyelitis models. Three ethical aspects (3R) are essential when exposing experimental animals to infections. Thus, we should perform experiments in vitro rather than in vivo (Replacement), use as few animals as possible (Reduction), and impose as little pain on the animal as possible (Refinement). The gain for humans should by far exceed the disadvantages for the individual experimental animal. To this end, the translational value of animal experiments is crucial. We therefore need a robust and well-characterized animal model to evaluate new osteomyelitis tracers to be sure that unpredicted variation in the animal model does not lead to a misinterpretation of the tracer behavior. In this review, we focus on how the development of radioactive tracers relies heavily on the selection of a reliable animal model, and we base the discussions on our own experience with a porcine model.

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