scholarly journals A New Generation of Functional Tagged Proteins for HIV Fluorescence Imaging

Viruses ◽  
2021 ◽  
Vol 13 (3) ◽  
pp. 386
Author(s):  
João I. Mamede ◽  
Joseph Griffin ◽  
Stéphanie Gambut ◽  
Thomas J. Hope
Keyword(s):  
Molecular Mechanisms ◽  
Fluorescent Proteins ◽  
Functional Characterization ◽  
Hiv Replication ◽  
Virus Particles ◽  
Wild Type Phenotype ◽  
Viral Particles ◽  
The Individual ◽  
Tools And Techniques ◽  
New Generation

During the last decade, there was a marked increase in the development of tools and techniques to study the molecular mechanisms of the HIV replication cycle by using fluorescence microscopy. Researchers often apply the fusion of tags and fluorophores to viral proteins, surrogate proteins, or dyes to follow individual virus particles while they progress throughout infection. The inclusion of such fusion motifs or surrogates frequently disrupts viral infectivity or results in a change of the wild-type phenotype. Here, we detail the construction and functional characterization of two new constructs where we fused fluorescent proteins to the N-terminus of HIV-1 Integrase. In the first, IN is recruited into assembling particles via a codon optimized Gag to complement other viral constructs, while the second is fused to a Gag-Pol expression vector fully capable of integration. Our data shows that N-terminal tagged IN is functional for integration by both recovery of integration of catalytically inactive IN and by the successful infectivity of viruses carrying only labeled IN. These tools will be important to study the individual behavior of viral particles and associate such behavior to infectivity.

scholarly journals Gene edited fluorescent cerebral organoids to study human brain function and disease

2020 ◽  
Author(s):  
Lisa Bachmann ◽  
Lucia Gallego Villarejo ◽  
Natalie Heinen ◽  
David Marks ◽  
Thorsten Müller
Keyword(s):  
Human Brain ◽  
Brain Function ◽  
Gene Editing ◽  
Molecular Mechanisms ◽  
Embryoid Body ◽  
Fluorescent Proteins ◽  
Genetic Disorders ◽  
Lamin B1 ◽  
The Individual ◽  
Human Genetic Disorders

AbstractCerebral organoids are a promising model to study human brain function and disease, though the high inter-organoid variability of the mini-brains is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different hiPSC lines, which enables the identification of cells from different origin within a single organoid. The method combines a gene editing workflow and subsequent organoid differentiation and offers a unique tool to study gene function in a complex human 3D tissue-like model. Using a CRISPR/Cas9 gene editing approach, different fluorescent proteins were fused to β-actin or lamin B1 in hiPSCs and subsequently used as a marker to identify each cell line. Mixtures of differently edited cells were seeded to induce embryoid body formation and cerebral organoid differentiation. As a consequence, the development of the 3D tissue was detectable by live confocal fluorescence microscopy and immunofluorescence staining in fixed samples. Analysis of mixed organoids allowed the identification and examination of specifically labeled cells in the organoid that belong to each of the two hiPSC donor lines. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, and thus the mixed organoids overcome the inter-organoid inhomogeneity limitations. The approach aims to pave the way for the reliable analysis of human genetic disorders by the use of organoids and to fundamentally understand the molecular mechanisms underlying pathological conditions.

scholarly journals Gene Edited Fluorescent Cerebral Organoids to Study Human Brain Function and Disease

2021 ◽  
Author(s):  
Thorsten Mueller ◽  
Lisa Bachmann ◽  
Lucia Gallego Villarejo ◽  
Natalie Heinen ◽  
David Marks
Keyword(s):  
Human Brain ◽  
Brain Function ◽  
Gene Editing ◽  
Molecular Mechanisms ◽  
Embryoid Body ◽  
Fluorescent Proteins ◽  
Genetic Disorders ◽  
Induced Pluripotent Stem Cell ◽  
Induced Pluripotent ◽  
The Individual

Abstract Cerebral organoids are a promising model to study human brain function and disease, though the high inter-organoid variability of the mini-brains is still challenging. To overcome this limitation, we introduce the method of labeled mixed organoids generated from two different human induced pluripotent stem cell (hiPSC) lines, which enables the identification of cells from different origin within a single organoid. The method combines a gene editing workflow and subsequent organoid differentiation and offers a unique tool to study gene function in a complex human 3D tissue-like model. Using a CRISPR/Cas9 gene editing approach, different fluorescent proteins were fused to β-actin or lamin B1 in hiPSCs and subsequently used as a marker to identify each cell line. Mixtures of differently edited cells were seeded to induce embryoid body formation and cerebral organoid differentiation. As a consequence, the development of the 3D tissue was detectable by live confocal fluorescence microscopy and immunofluorescence staining in fixed samples. Analysis of mixed organoids allowed the identification and examination of specifically labeled cells in the organoid that belong to each of the two hiPSC donor lines. We demonstrate that a direct comparison of the individual cells is possible by having the edited and the control (or the two differentially labeled) cells within the same organoid, and thus the mixed organoids overcome the inter-organoid inhomogeneity limitations. The approach aims to pave the way for the reliable analysis of human genetic disorders by the use of organoids and to fundamentally understand the molecular mechanisms underlying pathological conditions.

scholarly journals An icosahedral virus as a fluorescent calibration standard: a method for counting protein molecules in cells by fluorescence microscopy

2016 ◽  
Author(s):  
John M. Murray
Keyword(s):  
Single Molecule ◽  
Fluorescent Protein ◽  
Fluorescent Proteins ◽  
Icosahedral Symmetry ◽  
Modern Biology ◽  
Virus Particles ◽  
Native Proteins ◽  
Icosahedral Virus ◽  
The Individual

AbstractThe ability to replace genes coding for cellular proteins with DNA that codes for fluorescent protein-tagged versions opens the way to counting the number of molecules of each protein component of macromolecular assembliesin vivoby measuring fluorescence microscopically. Converting fluorescence to absolute numbers of molecules requires a fluorescent standard whose molecular composition is known precisely. In this report the construction, properties, and mode of using a set of fluorescence calibration standards are described. The standards are based on an icosahedral virus engineered to contain exactly 240 copies of one of seven different fluorescent proteins. Two applications of the fluorescent standards to counting molecules in the human parasiteToxoplasma gondiiare described. Methods for improving the preciseness of the measurements and minimizing potential inaccuracies are emphasized.Lay AbstractA broad goal of modern biology is to understand how the machines within living cells work. It is nowadays routine to identify the individual protein components of a machine, but not yet straightforward to tell how many copies of each component are needed to build a functional assembly. In many types of cells it is now possible to substitute for the native proteins within cells altered versions that are fluorescent. If one knew how much fluorescence is generated by a single molecule of the altered protein, then one could use a light microscope to count the number of copies of the protein in a cellular machine by simply measuring the total fluorescence coming from that part of the cell. This paper describes the construction and methods for using a set of fluorescent virus particles that can be used to determine how much fluorescence is contributed by one molecule of fluorescent protein. The virus particles were chosen for this role because the particular icosahedral symmetry of their structure guarantees that each particle contains exactly 240 copies of one fluorescent protein.

Flavonoids as P-gp Inhibitors: A Systematic Review of SARs

2019 ◽  
Vol 26 (25) ◽  
pp. 4799-4831 ◽  
Author(s):  
Jiahua Cui ◽  
Xiaoyang Liu ◽  
Larry M.C. Chow
Keyword(s):  
Molecular Mechanisms ◽  
Metastatic Cancer ◽  
Drug Candidates ◽  
Chemical Structures ◽  
Transporter Family ◽  
Promising Area ◽  
New Generation ◽  
Nontoxic Inhibitors

P-glycoprotein, also known as ABCB1 in the ABC transporter family, confers the simultaneous resistance of metastatic cancer cells towards various anticancer drugs with different targets and diverse chemical structures. The exploration of safe and specific inhibitors of this pump has always been the pursuit of scientists for the past four decades. Naturally occurring flavonoids as benzopyrone derivatives were recognized as a class of nontoxic inhibitors of P-gp. The recent advent of synthetic flavonoid dimer FD18, as a potent P-gp modulator in reversing multidrug resistance both in vitro and in vivo, specifically targeted the pseudodimeric structure of the drug transporter and represented a new generation of inhibitors with high transporter binding affinity and low toxicity. This review concerned the recent updates on the structure-activity relationships of flavonoids as P-gp inhibitors, the molecular mechanisms of their action and their ability to overcome P-gp-mediated MDR in preclinical studies. It had crucial implications on the discovery of new drug candidates that modulated the efflux of ABC transporters and also provided some clues for the future development in this promising area.

scholarly journals Deciphering the Mounting Complexity of the p53 Regulatory Network in Correlation to Long Non-Coding RNAs (lncRNAs) in Ovarian Cancer

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 527 ◽  
Author(s):  
Sonali Pal ◽  
Manoj Garg ◽  
Amit Kumar Pandey
Keyword(s):  
Ovarian Cancer ◽  
Molecular Mechanisms ◽  
Human Cancer ◽  
Critical Role ◽  
Functional Characterization ◽  
Great Promise ◽  
Altered Expression ◽  
Disease Biology ◽  
Non Coding Rnas ◽  
Post Transcriptional Regulation

Amongst the various gynecological malignancies affecting female health globally, ovarian cancer is one of the predominant and lethal among all. The identification and functional characterization of long non-coding RNAs (lncRNAs) are made possible with the advent of RNA-seq and the advancement of computational logarithm in understanding human disease biology. LncRNAs can interact with deoxyribonucleic acid (DNA), ribonucleic acid (RNA), proteins and their combinations. Moreover, lncRNAs regulate orchestra of diverse functions including chromatin organization and transcriptional and post-transcriptional regulation. LncRNAs have conferred their critical role in key biological processes in human cancer including tumor initiation, proliferation, cell cycle, apoptosis, necroptosis, autophagy, and metastasis. The interwoven function of tumor-suppressor protein p53-linked lncRNAs in the ovarian cancer paradigm is of paramount importance. Several lncRNAs operate as p53 regulators or effectors and modulates a diverse array of functions either by participating in various signaling cascades or via interaction with different proteins. This review highlights the recent progress made in the identification of p53 associated lncRNAs while elucidating their molecular mechanisms behind the altered expression in ovarian cancer tumorigenesis. Moreover, the development of novel clinical and therapeutic strategies for targeting lncRNAs in human cancers harbors great promise.

scholarly journals Physical Activity and Redox Balance in the Elderly: Signal Transduction Mechanisms

2021 ◽  
Vol 11 (5) ◽  
pp. 2228
Author(s):  
Daniela Galli ◽  
Cecilia Carubbi ◽  
Elena Masselli ◽  
Mauro Vaccarezza ◽  
Valentina Presta ◽  
...  
Keyword(s):  
Physical Activity ◽  
Molecular Mechanisms ◽  
Vascular Tissue ◽  
The Elderly ◽  
Redox Balance ◽  
Antioxidant Systems ◽  
Targeted Prevention ◽  
Individual Subject ◽  
Aged People ◽  
The Individual

Reactive Oxygen Species (ROS) are molecules naturally produced by cells. If their levels are too high, the cellular antioxidant machinery intervenes to bring back their quantity to physiological conditions. Since aging often induces malfunctioning in this machinery, ROS are considered an effective cause of age-associated diseases. Exercise stimulates ROS production on one side, and the antioxidant systems on the other side. The effects of exercise on oxidative stress markers have been shown in blood, vascular tissue, brain, cardiac and skeletal muscle, both in young and aged people. However, the intensity and volume of exercise and the individual subject characteristics are important to envisage future strategies to adequately personalize the balance of the oxidant/antioxidant environment. Here, we reviewed the literature that deals with the effects of physical activity on redox balance in young and aged people, with insights into the molecular mechanisms involved. Although many molecular pathways are involved, we are still far from a comprehensive view of the mechanisms that stand behind the effects of physical activity during aging. Although we believe that future precision medicine will be able to transform exercise administration from wellness to targeted prevention, as yet we admit that the topic is still in its infancy.

scholarly journals RNA and Protein Requirements for Incorporation of the Pol Protein into Foamy Virus Particles

2005 ◽  
Vol 79 (11) ◽  
pp. 7005-7013 ◽  
Author(s):  
Katrin Peters ◽  
Tatiana Wiktorowicz ◽  
Martin Heinkelein ◽  
Axel Rethwilm
Keyword(s):  
Foamy Virus ◽  
Genomic Rna ◽  
Rna Packaging ◽  
Gag Protein ◽  
Protein Precursor ◽  
Protein Requirements ◽  
Virus Particles ◽  
Foamy Viruses ◽  
Protein Incorporation ◽  
The Individual

ABSTRACT Foamy viruses (FVs) generate their Pol protein precursor molecule independently of the Gag protein from a spliced mRNA. This mode of expression raises the question of the mechanism of Pol protein incorporation into the viral particle (capsid). We previously showed that the packaging of (pre)genomic RNA is essential for Pol encapsidation (M. Heinkelein, C. Leurs, M. Rammling, K. Peters, H. Hanenberg, and A. Rethwilm, J. Virol. 76:10069-10073, 2002). Here, we demonstrate that distinct sequences in the RNA, which we termed Pol encapsidation sequences (PES), are required to incorporate Pol protein into the FV capsid. Two PES were found, which are contained in the previously identified cis-acting sequences necessary to transfer an FV vector. One PES is located in the U5 region of the 5′ long terminal repeat and one at the 3′ end of the pol gene region. Neither element has any significant effect on RNA packaging. However, deletion of either PES resulted in a significant reduction in Pol encapsidation. On the protein level, we show that only the Pol precursor, but not the individual reverse transcriptase (RT) and integrase (IN) subunits, is incorporated into FV particles. However, enzymatic activities of the protease (PR), RT, or IN are not required. Our results strengthen the view that in FVs, (pre)genomic RNA functions as a bridging molecule between Gag and Pol precursor proteins.

scholarly journals Identification and Functional Characterization of the p66/p68 Components of the MeCP1 Complex

2002 ◽  
Vol 22 (2) ◽  
pp. 536-546 ◽  
Author(s):  
Qin Feng ◽  
Ru Cao ◽  
Li Xia ◽  
Hediye Erdjument-Bromage ◽  
Paul Tempst ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Cpg Dinucleotides ◽  
Methylated Dna ◽  
Major Mechanism ◽  
Preferential Binding ◽  
Dna Silencing ◽  
The Individual ◽  
Identification And Characterization ◽  
Eukaryotic Genomes

ABSTRACT Methylation of cytosine at CpG dinucleotides is a common feature of many higher eukaryotic genomes. A major biological consequence of DNA methylation is gene silencing. Increasing evidence indicates that recruitment of histone deacetylase complexes by methyl-CpG-binding proteins is a major mechanism of methylated DNA silencing. We have previously reported that the MeCP1 protein complex represses transcription through preferential binding, remodeling, and deacetylation of methylated nucleosomes. To understand the molecular mechanism of the functioning of the MeCP1 complex, the individual components of the MeCP1 complex need to be characterized. In this paper, we report the identification and functional characterization of the p66 and p68 components of the MeCP1 complex. We provide evidence that the two components are different forms of the same zinc finger-containing protein. Analysis of the p66 homologs from different organisms revealed two highly conserved regions, CR1 and CR2. While CR1 is involved in the association of p66 with other MeCP1 components, CR2 plays an important role in targeting p66 and MBD3 to specific loci. Thus, our study not only completes the identification of the MeCP1 components but also reveals the potential function of p66 in MeCP1 complex targeting. The identification and characterization of all the MeCP1 components set the stage for reconstitution of the MeCP1 complex.

scholarly journals Tracking the Fate of Genetically Distinct Vesicular Stomatitis Virus Matrix Proteins Highlights the Role for Late Domains in Assembly

2015 ◽  
Vol 89 (23) ◽  
pp. 11750-11760 ◽  
Author(s):  
Timothy K. Soh ◽  
Sean P. J. Whelan
Keyword(s):  
Plasma Membrane ◽  
Vesicular Stomatitis Virus ◽  
Matrix Protein ◽  
Fluorescent Proteins ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Viral Particles ◽  
The Matrix ◽  
Vesicular Stomatitis ◽  
Late Domains

ABSTRACTVesicular stomatitis virus (VSV) assembly requires condensation of the viral ribonucleoprotein (RNP) core with the matrix protein (M) during budding from the plasma membrane. The RNP core comprises the negative-sense genomic RNA completely coated by the nucleocapsid protein (N) and associated by a phosphoprotein (P) with the large polymerase protein (L). To study the assembly of single viral particles, we tagged M and P with fluorescent proteins. We selected from a library of viruses with insertions in the M gene a replication-competent virus containing a fluorescent M and combined that with our previously described virus containing fluorescent P. Virus particles containing those fusions maintained the same bullet shape appearance as wild-type VSV but had a modest increase in particle length, reflecting the increased genome size. Imaging of the released particles revealed a variation in the amount of M and P assembled into the virions, consistent with a flexible packaging mechanism. We used the recombinants to further study the importance of the late domains in M, which serve to recruit the endosomal sorting complex required for transport (ESCRT) machinery during budding. Mutations in late domains resulted in the accumulation of virions that failed to pinch off from the plasma membrane. Imaging of single virions released from cells that were coinfected with M tagged with enhanced green fluorescent protein and M tagged with mCherry variants in which the late domains of one virus were inactivated by mutation showed a strong bias against the incorporation of the late-domain mutant into the released virions. In contrast, the intracellular expression and membrane association of the two variants were unaltered. These studies provide new tools for imaging particle assembly and enhance our resolution of existing models for assembly of VSV.IMPORTANCEAssembly of vesicular stomatitis virus (VSV) particles requires the separate trafficking of the viral replication machinery, a matrix protein (M) and a glycoprotein, to the plasma membrane. The matrix protein contains a motif termed a “late domain” that engages the host endosomal sorting complex required for transport (ESCRT) machinery to facilitate the release of viral particles. Inactivation of the late domains through mutation results in the accumulation of virions arrested at the point of release. In the study described here, we developed new tools to study VSV assembly by fusing fluorescent proteins to M and to a constituent of the replication machinery, the phosphoprotein (P). We used those tools to show that the late domains of M are required for efficient incorporation into viral particles and that the particles contain a variable quantity of M and P.

scholarly journals Could metabolomics drive the fate of COVID-19 pandemic? A narrative review on lights and shadows

2021 ◽  
Vol 0 (0) ◽  
Author(s):  
Michele Mussap ◽  
Vassilios Fanos
Keyword(s):  
Molecular Mechanisms ◽  
Vascular Dysfunction ◽  
Asymptomatic Infection ◽  
Multiple Organ ◽  
Therapeutic Interventions ◽  
Patient Specific ◽  
Wide Range ◽  
Life Threatening ◽  
The Individual ◽  
Host Metabolism

Abstract Human Severe Acute Respiratory Syndrome CoronaVirus 2 (SARS-CoV-2) infection activates a complex interaction host/virus, leading to the reprogramming of the host metabolism aimed at the energy supply for viral replication. Alterations of the host metabolic homeostasis strongly influence the immune response to SARS-CoV-2, forming the basis of a wide range of outcomes, from the asymptomatic infection to the onset of COVID-19 and up to life-threatening acute respiratory distress syndrome, vascular dysfunction, multiple organ failure, and death. Deciphering the molecular mechanisms associated with the individual susceptibility to SARS-CoV-2 infection calls for a system biology approach; this strategy can address multiple goals, including which patients will respond effectively to the therapeutic treatment. The power of metabolomics lies in the ability to recognize endogenous and exogenous metabolites within a biological sample, measuring their concentration, and identifying perturbations of biochemical pathways associated with qualitative and quantitative metabolic changes. Over the last year, a limited number of metabolomics- and lipidomics-based clinical studies in COVID-19 patients have been published and are discussed in this review. Remarkable alterations in the lipid and amino acid metabolism depict the molecular phenotype of subjects infected by SARS-CoV-2; notably, structural and functional data on the lipids-virus interaction may open new perspectives on targeted therapeutic interventions. Several limitations affect most metabolomics-based studies, slowing the routine application of metabolomics. However, moving metabolomics from bench to bedside cannot imply the mere determination of a given metabolite panel; rather, slotting metabolomics into clinical practice requires the conversion of metabolic patient-specific data into actionable clinical applications.

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