A novel histone exchange factor, protein phosphatase 2Cγ, mediates the exchange and dephosphorylation of H2A–H2B

In eukaryotic nuclei, DNA is wrapped around a protein octamer composed of the core histones H2A, H2B, H3, and H4, forming nucleosomes as the fundamental units of chromatin. The modification and deposition of specific histone variants play key roles in chromatin function. In this study, we established an in vitro system based on permeabilized cells that allows the assembly and exchange of histones in situ. H2A and H2B, each tagged with green fluorescent protein (GFP), are incorporated into euchromatin by exchange independently of DNA replication, and H3.1-GFP is assembled into replicated chromatin, as found in living cells. By purifying the cellular factors that assist in the incorporation of H2A–H2B, we identified protein phosphatase (PP) 2C γ subtype (PP2Cγ/PPM1G) as a histone chaperone that binds to and dephosphorylates H2A–H2B. The disruption of PP2Cγ in chicken DT40 cells increased the sensitivity to caffeine, a reagent that disturbs DNA replication and damage checkpoints, suggesting the involvement of PP2Cγ-mediated histone dephosphorylation and exchange in damage response or checkpoint recovery in higher eukaryotes.

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In vitro FRAP reveals the ATP-dependent nuclear mobilization of the exon junction complex protein SRm160

The Journal of Cell Biology ◽

10.1083/jcb.200307002 ◽

2004 ◽

Vol 164 (6) ◽

pp. 843-850 ◽

Cited By ~ 21

Author(s):

Stefan Wagner ◽

Simion Chiosea ◽

Maria Ivshina ◽

Jeffrey A. Nickerson

Keyword(s):

Rna Splicing ◽

Fluorescence Recovery After Photobleaching ◽

Fluorescent Protein ◽

Fluorescence Recovery ◽

Exon Junction Complex ◽

Permeabilized Cells ◽

Exon Junction ◽

Rna Export ◽

Green Fluorescent

We present a new in vitro system for characterizing the binding and mobility of enhanced green fluorescent protein (EGFP)–labeled nuclear proteins by fluorescence recovery after photobleaching in digitonin-permeabilized cells. This assay reveals that SRm160, a splicing coactivator and component of the exon junction complex (EJC) involved in RNA export, has an adenosine triphosphate (ATP)–dependent mobility. Endogenous SRm160, lacking the EGFP moiety, could also be released from sites at splicing speckled domains by an ATP-dependent mechanism. A second EJC protein, RNPS1, also has an ATP-dependent mobility, but SRm300, a protein that binds to SRm160 and participates with it in RNA splicing, remains immobile after ATP supplementation. This finding suggests that SRm160-containing RNA export, but not splicing, complexes have an ATP-dependent mobility. We propose that RNA export complexes have an ATP-regulated mechanism for release from binding sites at splicing speckled domains. In vitro fluorescence recovery after photobleaching is a powerful tool for identifying cofactors required for nuclear binding and mobility.

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Nucleocytoplasmic Shuttling Factors Including Ran and CRM1 Mediate Nuclear Export of NFAT In Vitro

The Journal of Cell Biology ◽

10.1083/jcb.141.4.863 ◽

1998 ◽

Vol 141 (4) ◽

pp. 863-874 ◽

Cited By ~ 121

Author(s):

Ralph H. Kehlenbach ◽

Achim Dickmanns ◽

Larry Gerace

Keyword(s):

Nuclear Export ◽

Fluorescent Protein ◽

Dependent Manner ◽

Nucleocytoplasmic Shuttling ◽

Permeabilized Cell ◽

Permeabilized Cells ◽

Digitonin Permeabilization ◽

Green Fluorescent ◽

Rate Limiting

We have developed a permeabilized cell assay to study the nuclear export of the shuttling transcription factor NFAT, which contains a leucine-rich export signal. The assay uses HeLa cells that are stably transfected with NFAT fused to the green fluorescent protein (GFP). Nuclear export of GFP–NFAT in digitonin-permeabilized cells occurs in a temperature- and ATP-dependent manner and can be quantified by flow cytometry. In vitro NFAT export requires the GTPase Ran, which is released from cells during the digitonin permeabilization. At least one additional rate-limiting export factor is depleted from permeabilized cells by a preincubation at 30°C in the absence of cytosol. This activity can be provided by cytosolic or nucleoplasmic extracts in a subsequent export step. Using this assay, we have purified a second major export activity from cytosol. We found that it corresponds to CRM1, a protein recently reported to be a receptor for certain leucine-rich export sequences. CRM1 appears to be imported into the nucleus by a Ran-dependent mechanism that is distinct from conventional signaling pathways. Considered together, our studies directly demonstrate by fractionation and reconstitution that nuclear export of NFAT is mediated by multiple nucleocytoplasmic shuttling factors, including Ran and CRM1.

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Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

Microorganisms ◽

10.3390/microorganisms9051005 ◽

2021 ◽

Vol 9 (5) ◽

pp. 1005

Author(s):

Olga Chervyakova ◽

Elmira Tailakova ◽

Nurlan Kozhabergenov ◽

Sandugash Sadikaliyeva ◽

Kulyaisan Sultankulova ◽

...

Keyword(s):

Fluorescent Protein ◽

Viral Vector ◽

Foot And Mouth Disease ◽

Open Reading Frames ◽

Foreign Gene ◽

Mouth Disease Virus ◽

Foreign Genes ◽

Green Fluorescent ◽

Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

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Development of a Fluorescent Tool for Studying Legionella bozemanae Intracellular Infection

Microorganisms ◽

10.3390/microorganisms9020379 ◽

2021 ◽

Vol 9 (2) ◽

pp. 379

Author(s):

Breanne M. Head ◽

Christopher I. Graham ◽

Teassa MacMartin ◽

Yoav Keynan ◽

Ann Karen C. Brassinga

Keyword(s):

Growth Kinetics ◽

Legionella Pneumophila ◽

Fluorescent Protein ◽

Disease Incidence ◽

Acanthamoeba Castellanii ◽

Infection Model ◽

Intracellular Infection ◽

Green Fluorescent

Legionnaires’ disease incidence is on the rise, with the majority of cases attributed to the intracellular pathogen, Legionella pneumophila. Nominally a parasite of protozoa, L. pneumophila can also infect alveolar macrophages when bacteria-laden aerosols enter the lungs of immunocompromised individuals. L. pneumophila pathogenesis has been well characterized; however, little is known about the >25 different Legionella spp. that can cause disease in humans. Here, we report for the first time a study demonstrating the intracellular infection of an L. bozemanae clinical isolate using approaches previously established for L. pneumophila investigations. Specifically, we report on the modification and use of a green fluorescent protein (GFP)-expressing plasmid as a tool to monitor the L. bozemanae presence in the Acanthamoeba castellanii protozoan infection model. As comparative controls, L. pneumophila strains were also transformed with the GFP-expressing plasmid. In vitro and in vivo growth kinetics of the Legionella parental and GFP-expressing strains were conducted followed by confocal microscopy. Results suggest that the metabolic burden imposed by GFP expression did not impact cell viability, as growth kinetics were similar between the GFP-expressing Legionella spp. and their parental strains. This study demonstrates that the use of a GFP-expressing plasmid can serve as a viable approach for investigating Legionella non-pneumophila spp. in real time.

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CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo

Veterinary Research ◽

10.1186/s13567-021-00964-4 ◽

2021 ◽

Vol 52 (1) ◽

Author(s):

Peng-Fei Fu ◽

Xuan Cheng ◽

Bing-Qian Su ◽

Li-Fang Duan ◽

Cong-Rong Wang ◽

...

Keyword(s):

Pseudorabies Virus ◽

Fluorescent Protein ◽

Antiviral Agents ◽

Firefly Luciferase ◽

Inhibitory Effect ◽

Economic Losses ◽

Green Fluorescent ◽

Swine Herds

AbstractPseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.

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Paracetamol modulates biofilm formation in Staphylococcus aureus clonal complex 8 strains

Scientific Reports ◽

10.1038/s41598-021-84505-1 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Andi R. Sultan ◽

Kirby R. Lattwein ◽

Nicole A. Lemmens-den Toom ◽

Susan V. Snijders ◽

Klazina Kooiman ◽

...

Keyword(s):

Staphylococcus Aureus ◽

Biofilm Formation ◽

Fluorescent Protein ◽

Clonal Complex ◽

Regulatory Pathways ◽

Immune Modulator ◽

Viability Assay ◽

Analgesic Agents ◽

Green Fluorescent

AbstractStaphylococcus aureus biofilms are a major problem in modern healthcare due to their resistance to immune system defenses and antibiotic treatments. Certain analgesic agents are able to modulate S. aureus biofilm formation, but currently no evidence exists if paracetamol, often combined with antibiotic treatment, also has this effect. Therefore, we aimed to investigate if paracetamol can modulate S. aureus biofilm formation. Considering that certain regulatory pathways for biofilm formation and virulence factor production by S. aureus are linked, we further investigated the effect of paracetamol on immune modulator production. The in vitro biofilm mass of 21 S. aureus strains from 9 genetic backgrounds was measured in the presence of paracetamol. Based on biofilm mass quantity, we further investigated paracetamol-induced biofilm alterations using a bacterial viability assay combined with N-Acetylglucosamine staining. Isothermal microcalorimetry was used to monitor the effect of paracetamol on bacterial metabolism within biofilms and green fluorescent protein (GFP) promoter fusion technology for transcription of staphylococcal complement inhibitor (SCIN). Clinically relevant concentrations of paracetamol enhanced biofilm formation particularly among strains belonging to clonal complex 8 (CC8), but had minimal effect on S. aureus planktonic growth. The increase of biofilm mass can be attributed to the marked increase of N-Acetylglucosamine containing components of the extracellular matrix, presumably polysaccharide intercellular adhesion. Biofilms of RN6390A (CC8) showed a significant increase in the immune modulator SCIN transcription during co-incubation with low concentrations of paracetamol. Our data indicate that paracetamol can enhance biofilm formation. The clinical relevance needs to be further investigated.

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Development and Characterization of a cDNA-Launch Recombinant Simian Hemorrhagic Fever Virus Expressing Enhanced Green Fluorescent Protein: ORF 2b’ Is Not Required for In Vitro Virus Replication

Viruses ◽

10.3390/v13040632 ◽

2021 ◽

Vol 13 (4) ◽

pp. 632

Author(s):

Yingyun Cai ◽

Shuiqing Yu ◽

Ying Fang ◽

Laura Bollinger ◽

Yanhua Li ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Lines ◽

Enhanced Green Fluorescent Protein ◽

Fluorescent Protein ◽

Infectious Clone ◽

Hemorrhagic Fever ◽

Simian Hemorrhagic Fever Virus ◽

Hemorrhagic Fever Virus ◽

Green Fluorescent

Simian hemorrhagic fever virus (SHFV) causes acute, lethal disease in macaques. We developed a single-plasmid cDNA-launch infectious clone of SHFV (rSHFV) and modified the clone to rescue an enhanced green fluorescent protein-expressing rSHFV-eGFP that can be used for rapid and quantitative detection of infection. SHFV has a narrow cell tropism in vitro, with only the grivet MA-104 cell line and a few other grivet cell lines being susceptible to virion entry and permissive to infection. Using rSHFV-eGFP, we demonstrate that one cricetid rodent cell line and three ape cell lines also fully support SHFV replication, whereas 55 human cell lines, 11 bat cell lines, and three rodent cells do not. Interestingly, some human and other mammalian cell lines apparently resistant to SHFV infection are permissive after transfection with the rSHFV-eGFP cDNA-launch plasmid. To further demonstrate the investigative potential of the infectious clone system, we introduced stop codons into eight viral open reading frames (ORFs). This approach suggested that at least one ORF, ORF 2b’, is dispensable for SHFV in vitro replication. Our proof-of-principle experiments indicated that rSHFV-eGFP is a useful tool for illuminating the understudied molecular biology of SHFV.

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The Great Capacity on Promoting Melanogenesis of Three Compatible Components in Vernonia anthelmintica (L.) Willd.

International Journal of Molecular Sciences ◽

10.3390/ijms22084073 ◽

2021 ◽

Vol 22 (8) ◽

pp. 4073

Author(s):

Yifan Lai ◽

Qingyuan Feng ◽

Rui Zhang ◽

Jing Shang ◽

Hui Zhong

Keyword(s):

Herbal Medicine ◽

Caffeic Acid ◽

Fluorescent Protein ◽

Traditional Chinese Herbal Medicine ◽

Expression Of Genes ◽

Proliferation And Differentiation ◽

Green Fluorescent ◽

Vernonia Anthelmintica

To investigate a possible methodology of exploiting herbal medicine and design polytherapy for the treatment of skin depigmentation disorder, we have made use of Vernonia anthelmintica (L.) Willd., a traditional Chinese herbal medicine that has been proven to be effective in treating vitiligo. Here, we report that the extract of Vernonia anthelmintica (L.) Willd. effectively enhances melanogenesis responses in B16F10. In its compound library, we found three ingredients (butin, caffeic acid and luteolin) also have the activity of promoting melanogenesis in vivo and in vitro. They can reduce the accumulation of ROS induced by hydrogen peroxide and inflammatory response induced by sublethal concentrations of copper sulfate in wild type and green fluorescent protein (GFP)-labeled leukocytes zebrafish larvae. The overall objective of the present study aims to identify which compatibility proportions of the medicines may be more effective in promoting pigmentation. We utilized the D-optimal response surface methodology to optimize the ratio among three molecules. Combining three indicators of promoting melanogenesis, anti-inflammatory and antioxidant capacities, we get the best effect of butin, caffeic acid and luteolin at the ratio (butin:caffeic acid:luteolin = 7.38:28.30:64.32) on zebrafish. Moreover, the effect of melanin content recovery in the best combination is stronger than that of the monomer, which suggests that the three compounds have a synergistic effect on inducing melanogenesis. After simply verifying the result, we performed in situ hybridization on whole-mount zebrafish embryos to further explore the effects of multi-drugs combination on the proliferation and differentiation of melanocytes and the expression of genes (tyr, mitfa, dct, kit) related to melanin synthesis. In conclusion, the above three compatible compounds can significantly enhance melanogenesis and improve depigmentation in vivo.

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Human Keratinocytes Adopt Neuronal Fates After In Utero Transplantation in the Developing Rat Brain

Cell Transplantation ◽

10.1177/0963689720978219 ◽

2021 ◽

Vol 30 ◽

pp. 096368972097821

Author(s):

Andrea Tenorio-Mina ◽

Daniel Cortés ◽

Joel Esquivel-Estudillo ◽

Adolfo López-Ornelas ◽

Alejandro Cabrera-Wrooman ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Fluorescent Protein ◽

Ectopic Expression ◽

Neural Induction ◽

Human Keratinocytes ◽

Differentiation Potential ◽

Neuronal Proteins ◽

Green Fluorescent

Human skin contains keratinocytes in the epidermis. Such cells share their ectodermal origin with the central nervous system (CNS). Recent studies have demonstrated that terminally differentiated somatic cells can adopt a pluripotent state, or can directly convert its phenotype to neurons, after ectopic expression of transcription factors. In this article we tested the hypothesis that human keratinocytes can adopt neural fates after culturing them in suspension with a neural medium. Initially, keratinocytes expressed Keratins and Vimentin. After neural induction, transcriptional upregulation of NESTIN, SOX2, VIMENTIN, SOX1, and MUSASHI1 was observed, concomitant with significant increases in NESTIN detected by immunostaining. However, in vitro differentiation did not yield the expression of neuronal or astrocytic markers. We tested the differentiation potential of control and neural-induced keratinocytes by grafting them in the developing CNS of rats, through ultrasound-guided injection. For this purpose, keratinocytes were transduced with lentivirus that contained the coding sequence of green fluorescent protein. Cell sorting was employed to select cells with high fluorescence. Unexpectedly, 4 days after grafting these cells in the ventricles, both control and neural-induced cells expressed green fluorescent protein together with the neuronal proteins βIII-Tubulin and Microtubule-Associated Protein 2. These results support the notion that in vivo environment provides appropriate signals to evaluate the neuronal differentiation potential of keratinocytes or other non-neural cell populations.

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Kiss1 Neurons Drastically Change Their Firing Activity in Accordance With the Reproductive State: Insights From a Seasonal Breeder

Endocrinology ◽

10.1210/en.2014-1472 ◽

2014 ◽

Vol 155 (12) ◽

pp. 4868-4880 ◽

Cited By ~ 8

Author(s):

Masaharu Hasebe ◽

Shinji Kanda ◽

Hiroyuki Shimada ◽

Yasuhisa Akazome ◽

Hideki Abe ◽

...

Keyword(s):

Fluorescent Protein ◽

Resting Membrane Potential ◽

Reproductive State ◽

Firing Patterns ◽

State Dependent ◽

Channel Expression ◽

Physiological Analysis ◽

Seasonal Breeder ◽

Green Fluorescent

Kisspeptin (Kiss) neurons show drastic changes in kisspeptin expression in response to the serum sex steroid concentration in various vertebrate species. Thus, according to the reproductive states, kisspeptin neurons are suggested to modulate various neuronal activities, including the regulation of GnRH neurons in mammals. However, despite their reproductive state-dependent regulation, there is no physiological analysis of kisspeptin neurons in seasonal breeders. Here we generated the first kiss1-enhanced green fluorescent protein transgenic line of a seasonal breeder, medaka, for histological and electrophysiological analyses using a whole-brain in vitro preparation in which most synaptic connections are intact. We found histologically that Kiss1 neurons in the nucleus ventralis tuberis (NVT) projected to the preoptic area, hypothalamus, pituitary, and ventral telencephalon. Therefore, NVT Kiss1 neurons may regulate various homeostatic functions and innate behaviors. Electrophysiological analyses revealed that they show various firing patterns, including bursting. Furthermore, we found that their firings are regulated by the resting membrane potential. However, bursting was not induced from the other firing patterns with a current injection, suggesting that it requires some chronic modulations of intrinsic properties such as channel expression. Finally, we found that NVT Kiss1 neurons drastically change their neuronal activities according to the reproductive state and the estradiol levels. Taken together with the previous reports, we here conclude that the breeding condition drastically alters the Kiss1 neuron activities in both gene expression and firing activities, the latter of which is strongly related to Kiss1 release, and the Kiss1 peptides regulate the activities of various neural circuits through their axonal projections.

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