The secondary structure of nucleic acids containing quartets of guanines, termed G-quadruplexes, is known to regulate the transcription of many genes. Several G-quadruplexes can be formed in the HIV-1 long terminal repeat promoter region and their stabilization results in the inhibition of HIV-1 replication. Here, we identified helquat-based compounds as a new class of anti-HIV-1 inhibitors that inhibit HIV-1 replication at the stage of reverse transcription and provirus expression. Using Taq polymerase stop and FRET melting assays, we have demonstrated their ability to stabilize G-quadruplexes in the HIV-1 long-terminal repeat sequence. Moreover, these compounds were not binding to the general G-rich region, but rather to G-quadruplex-forming regions. Finally, docking and molecular dynamics calculations indicate that the structure of the helquat core greatly affects the binding mode to the individual G-quadruplexes. Our findings can provide useful information for the further rational design of inhibitors targeting G-quadruplexes in HIV-1.
Use of blood RNA sequencing (RNA-seq) as a splicing analysis tool for clinical interpretation of variants of uncertain significance (VUSs) found via whole-genome and exome sequencing can be difficult for genes that have low expression in the blood due to insufficient read count coverage aligned to specific genes of interest. Here, we present a short amplicon reverse transcription-polymerase chain reaction(RT-PCR) for the detection of genes with low blood expression. Short amplicon RT-PCR, is designed to span three exons where an exon harboring a variant is flanked by one upstream and one downstream exon. We tested short amplicon RT-PCRs for genes that have median transcripts per million (TPM) values less than one according to the genotype-tissue expression database. Median TPM values of genes analyzed in this study are SYN1 = 0.8549, COL1A1 = 0.6275, TCF4 = 0.4009, DSP = .2894, TTN = 0.2851, COL5A2 = 0.1036, TERT = 0.04452, NTRK2 = 0.0344, ABCA4 = 0.00744, PRPH = 0, and WT1 = 0. All these genes show insufficient exon-spanning read coverage in our RNA-seq data to allow splicing analysis. We successfully detected all genes tested except PRPH and WT1. Aberrant splicing was detected in SYN1, TCF4, NTRK2, TTN, and TERT VUSs. Therefore, our results show short amplicon RT-PCR is a useful alternative for the analysis of splicing events in genes with low TPM in blood RNA for clinical diagnostics.
- Klíčová slova
- RNA-seq, RT-PCR, VUS, aberrant splicing, blood RNA,
- MeSH
- ABC transportéry genetika MeSH
- alternativní sestřih * MeSH
- lidé MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- reverzní transkripce MeSH
- RNA * genetika MeSH
- sestřih RNA genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- ABC transportéry MeSH
- ABCA4 protein, human MeSH Prohlížeč
- RNA * MeSH
Fullerene derivatives with hydrophilic substituents have been shown to exhibit a range of biological activities, including antiviral ones. For a long time, the anti-HIV activity of fullerene derivatives was believed to be due to their binding into the hydrophobic pocket of HIV-1 protease, thereby blocking its activity. Recent work, however, brought new evidence of a novel, protease-independent mechanism of fullerene derivatives' action. We studied in more detail the mechanism of the anti-HIV-1 activity of N,N-dimethyl[70]fulleropyrrolidinium iodide fullerene derivatives. By using a combination of in vitro and cell-based approaches, we showed that these C70 derivatives inhibited neither HIV-1 protease nor HIV-1 maturation. Instead, our data indicate effects of fullerene C70 derivatives on viral genomic RNA packaging and HIV-1 cDNA synthesis during reverse transcription-without impairing reverse transcriptase activity though. Molecularly, this could be explained by a strong binding affinity of these fullerene derivatives to HIV-1 nucleocapsid domain, preventing its proper interaction with viral genomic RNA, thereby blocking reverse transcription and HIV-1 infectivity. Moreover, the fullerene derivatives' oxidative activity and fluorescence quenching, which could be one of the reasons for the inconsistency among reported anti-HIV-1 mechanisms, are discussed herein.
- Klíčová slova
- HIV-1, RNA packaging, fullerene, inhibition, nucleocapsid,
- MeSH
- fullereny metabolismus farmakologie MeSH
- genom virový účinky léků MeSH
- genové produkty gag - virus lidské imunodeficience metabolismus MeSH
- HEK293 buňky MeSH
- HIV-1 účinky léků genetika metabolismus fyziologie MeSH
- látky proti HIV metabolismus farmakologie MeSH
- lidé MeSH
- nukleokapsida - proteiny metabolismus MeSH
- reverzní transkripce MeSH
- RNA virová metabolismus MeSH
- svlékání virového obalu účinky léků MeSH
- vazba proteinů MeSH
- virion metabolismus MeSH
- zabalení virového genomu účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- fullereny MeSH
- genové produkty gag - virus lidské imunodeficience MeSH
- látky proti HIV MeSH
- nukleokapsida - proteiny MeSH
- RNA virová MeSH
Reverse transcription quantitative PCR (RT-qPCR) has delivered significant insights in understanding the gene expression landscape. Thanks to its precision, sensitivity, flexibility, and cost effectiveness, RT-qPCR has also found utility in advanced single-cell analysis. Single-cell RT-qPCR now represents a well-established method, suitable for an efficient screening prior to single-cell RNA sequencing (scRNA-Seq) experiments, or, oppositely, for validation of hypotheses formulated from high-throughput approaches. Here, we aim to provide a comprehensive summary of the scRT-qPCR method by discussing the limitations of single-cell collection methods, describing the importance of reverse transcription, providing recommendations for the preamplification and primer design, and summarizing essential data processing steps. With the detailed protocol attached in the appendix, this tutorial provides a set of guidelines that allow any researcher to perform scRT-qPCR measurements of the highest standard.
- Klíčová slova
- RT-qPCR, gene expression, preamplification, quantitative PCR, reverse transcription, sample collection, single cell,
- MeSH
- analýza jednotlivých buněk metody normy MeSH
- kvantitativní polymerázová řetězová reakce metody normy MeSH
- lidé MeSH
- reverzní transkripce genetika MeSH
- senzitivita a specificita MeSH
- stanovení celkové genové exprese metody normy MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- přehledy MeSH
RNA triplexes are significant tertiary structure motifs that are found in many functional RNAs. Hence, small molecules capable of recognition, binding, and stabilization of the triple-helical RNA structures are emerging as attractive potential molecular biology tools and therapeutic agents. Here, we utilize methods of molecular biology and biophysics to study the interactions of a series of antitumor substitution-inert polynuclear platinum complexes (SI-PPCs) with triple-helical RNA structures. We show that SI-PPCs recognize and stabilize RNA triplexes and inhibit reverse transcription preferentially in the RNA template prone to the triplex formation. These so far unexplored properties of SI-PPCs suggest that the targeting of triple-stranded regions in RNA might contribute to the biological effects of SI-PPCs.
- MeSH
- komplexní sloučeniny chemie MeSH
- konformace nukleové kyseliny MeSH
- platina chemie MeSH
- protinádorové látky chemie MeSH
- reverzní transkripce účinky léků MeSH
- RNA chemie MeSH
- sekvence nukleotidů MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- komplexní sloučeniny MeSH
- platina MeSH
- protinádorové látky MeSH
- RNA MeSH
The single-celled parasite Giardia intestinalis (Diplomonadida) has two equally sized nuclei in one cell. The nuclei have been considered identical. We have previously shown that they contain different chromosomal sets and proceed through the cell cycle with some asynchrony. Here, we demonstrate by fluorescence in situ hybridization that several genes from chromosome 5 are lost in one of the two nuclei of the WBc6 Giardia line. The missing segment stretches over at least 50 kb near the 5' chromosome end. In both WB and WBc6 Giardia cell lines, chromosome 5 is trisomic in one nucleus and monosomic in the other nucleus. The described chromosomal deletion has always been observed at the monosomic chromosome in WBc6; however, the deletion was not detected in the parent line WB. The chromosomal segment was thus initially lost after biological cloning of WB, which gave rise to clone WBc6. We show that Giardia is capable of carrying out gene expression from only one nucleus. The two nuclei display a certain level of diversity, making each of them irreplaceable. The doubled karyomastigonts of diplomonads likely have separate functions both in the mastigont/flagellar organization and in chromosomal and gene content. To our knowledge, our results offer the first methodical approach to differentiating the two, so far indistinguishable nuclei.
- Klíčová slova
- Deletion, Diplomonadida, Gene content, Gene expression, Giardia intestinalis, Karyotype, Nuclei,
- MeSH
- buněčné jádro genetika MeSH
- časové faktory MeSH
- chromozomální delece MeSH
- delece genu MeSH
- Giardia lamblia genetika ultrastruktura MeSH
- hybridizace in situ fluorescenční normy MeSH
- komplementární DNA genetika MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- mitóza MeSH
- monozomie * genetika MeSH
- polymerázová řetězová reakce s reverzní transkripcí MeSH
- regulace genové exprese fyziologie MeSH
- reverzní transkripce MeSH
- RNA protozoální genetika izolace a purifikace MeSH
- signální transdukce MeSH
- trizomie * genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- komplementární DNA MeSH
- RNA protozoální MeSH
In addition to specific RNA-binding zinc finger domains, the retroviral Gag polyprotein contains clusters of basic amino acid residues that are thought to support Gag-viral genomic RNA (gRNA) interactions. One of these clusters is the basic K16NK18EK20 region, located upstream of the first zinc finger of the Mason-Pfizer monkey virus (M-PMV) nucleocapsid (NC) protein. To investigate the role of this basic region in the M-PMV life cycle, we used a combination of in vivo and in vitro methods to study a series of mutants in which the overall charge of this region was more positive (RNRER), more negative (AEAEA), or neutral (AAAAA). The mutations markedly affected gRNA incorporation and the onset of reverse transcription. The introduction of a more negative charge (AEAEA) significantly reduced the incorporation of M-PMV gRNA into nascent particles. Moreover, the assembly of immature particles of the AEAEA Gag mutant was relocated from the perinuclear region to the plasma membrane. In contrast, an enhancement of the basicity of this region of M-PMV NC (RNRER) caused a substantially more efficient incorporation of gRNA, subsequently resulting in an increase in M-PMV RNRER infectivity. Nevertheless, despite the larger amount of gRNA packaged by the RNRER mutant, the onset of reverse transcription was delayed in comparison to that of the wild type. Our data clearly show the requirement for certain positively charged amino acid residues upstream of the first zinc finger for proper gRNA incorporation, assembly of immature particles, and proceeding of reverse transcription.IMPORTANCE We identified a short sequence within the Gag polyprotein that, together with the zinc finger domains and the previously identified RKK motif, contributes to the packaging of genomic RNA (gRNA) of Mason-Pfizer monkey virus (M-PMV). Importantly, in addition to gRNA incorporation, this basic region (KNKEK) at the N terminus of the nucleocapsid protein is crucial for the onset of reverse transcription. Mutations that change the positive charge of the region to a negative one significantly reduced specific gRNA packaging. The assembly of immature particles of this mutant was reoriented from the perinuclear region to the plasma membrane. On the contrary, an enhancement of the basic character of this region increased both the efficiency of gRNA packaging and the infectivity of the virus. However, the onset of reverse transcription was delayed even in this mutant. In summary, the basic region in M-PMV Gag plays a key role in the packaging of genomic RNA and, consequently, in assembly and reverse transcription.
- Klíčová slova
- M-PMV, RNA packaging, assembly, basic residues, human immunodeficiency virus, infectivity, nucleocapsid, retroviruses, reverse transcription,
- MeSH
- buněčné linie MeSH
- genové produkty gag genetika MeSH
- HEK293 buňky MeSH
- lidé MeSH
- Masonův-Pfizerův opičí virus genetika fyziologie MeSH
- mutace genetika MeSH
- nukleokapsida - proteiny genetika MeSH
- reverzní transkripce genetika MeSH
- RNA virová genetika MeSH
- sekvence aminokyselin genetika MeSH
- sestavení viru genetika MeSH
- zinkové prsty genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- genové produkty gag MeSH
- nukleokapsida - proteiny MeSH
- RNA virová MeSH
Annual epidemics of influenza cause death of hundreds of thousands people and they also have a significant economic impact. Hence, a need for fast and cheap influenza diagnostic method is arising. The conventional methods for an isolation of the viruses are time-consuming and require expensive instrumentation as well as trained personnel. In this study, we modified the surface of nanomaghemite (γ-Fe2 O3 ) paramagnetic core with tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane and the resulting particles were utilized for the isolation of H7N7 influenza virions. Consequently, we designed γ-Fe2 O3 paramagnetic core modified with calcium tripolyphosphate which was employed for the isolation of viral nucleic acid after virion's lysis. Both of these procedures can be performed rapidly in less than 10 min and, in combination with the RT-PCR, the whole influenza detection can be shortened to few hours. Moreover, the whole protocol could be easily automated and/or miniaturized, and thus can serve as a basis for use in a lab-on-a-chip device. We assume that magnetic isolation is an exceptional procedure which can significantly accelerate the diagnostic possibilities of a broad spectrum of diseases.
- Klíčová slova
- Influenza, Magnetic particles, PCR, Virions,
- MeSH
- chromatografie iontoměničová MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- kuřecí embryo MeSH
- polymerázová řetězová reakce metody MeSH
- reverzní transkripce MeSH
- virion izolace a purifikace MeSH
- virus chřipky A, podtyp H7N7 izolace a purifikace MeSH
- zvířata MeSH
- Check Tag
- kuřecí embryo MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Although it is generally accepted that signal transduction in plant mitogen-activated protein kinase signaling cascades is regulated via rapid posttranslational modifications, there are also several compelling examples of swift stress induced transcriptional activation of plant MAP kinase genes. A possible function of these fast and transient events is to compensate for protein losses caused by degradation of phosphorylated MAP kinases within stimulated pathways. Nevertheless, there is still need for additional evidence to precisely describe the regulatory role of plant MAP kinase transcriptional dynamics, especially in the context of whole stress stimulated pathways including also other signaling molecules and transcription factors. During the last two decades a reverse transcription quantitative real-time PCR became a golden choice for the accurate and fast quantification of the gene expression and gene expression dynamic. In here, we provide a robust, cost-effective SYBR Green-based RT-qPCR protocol that is suitable for the quantification of stress induced plant MAP kinase transcriptional dynamics in various plant species.
- MeSH
- Arabidopsis účinky léků genetika růst a vývoj fyziologie MeSH
- chlorid sodný farmakologie MeSH
- DNA primery genetika MeSH
- fyziologický stres genetika MeSH
- klonování DNA MeSH
- komplementární DNA biosyntéza genetika MeSH
- kvantitativní polymerázová řetězová reakce metody MeSH
- mitogenem aktivované proteinkinasy genetika MeSH
- osmotický tlak účinky léků MeSH
- regulace genové exprese u rostlin * účinky léků MeSH
- reverzní transkripce * účinky léků MeSH
- RNA rostlin genetika izolace a purifikace MeSH
- semenáček růst a vývoj MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- chlorid sodný MeSH
- DNA primery MeSH
- komplementární DNA MeSH
- mitogenem aktivované proteinkinasy MeSH
- RNA rostlin MeSH
Human immunodeficiency virus-1 (HIV-1) successfully escapes from host immune surveillance, vaccines and antiretroviral agents. The available antiretroviral compounds can only control viremia, but it is impossible to eliminate the virus from the organism, namely because HIV-1 provirus persists in the reservoir cells from which the virus repeatedly disseminates into new cells. Current therapeutic approaches, however, do not specifically address the stage of virus reactivation. Heme has been demonstrated as very efficient in inhibiting HIV-1 reverse transcription, while its derivative hemin ameliorated HIV-1 infection via induction of heme oxygenase-1. Normosang (heme arginate; HA) is a human hemin-containing compound used to treat acute porphyria. In this work, we studied the effects of HA in HIV-1-acutely infected T-cell lines, and in cell lines harboring either a complete HIV-1 provirus (ACH-2 cells) or an HIV-1 "mini-virus" (Jurkat clones expressing EGFP under control of HIV LTR). We demonstrate that HA inhibited HIV-1 replication during the acute infection, which was accompanied by the inhibition of reverse transcription. On the other hand, HA alone stimulated the reactivation of HIV-1 "mini-virus" and synergized with phorbol ester or TNF-α in the reactivation of HIV-1 provirus. The stimulatory effects of HA were inhibited by N-acetyl cysteine, suggesting an increased redox stress and activation of NF-κB. Further, HA induced expression of heme oxygenase-1 (HO-1) in ACH-2 cells, while HO-1 was found expressed in untreated Jurkat clones. Inhibitor of HO-1 activity, tin protoporphyrin IX, further increased HA-mediated reactivation of HIV-1 "mini-virus" in Jurkat clones, and this effect was also inhibited by N-acetyl cysteine. The stimulatory effects of HA on HIV-1 reactivation thus seem to involve HO-1 and generation of free radicals. Additionally, the effective concentrations of HA did neither affect normal T-cell activation with PMA nor induce activation of the unstimulated cells. In conclusion, HA appears to possess a combination of unique properties that could help to decrease the pool of latently infected reservoir cells, while simultaneously inhibiting HIV-1 replication in newly infected cells. Our results thus suggest a new direction to explore in treatment of HIV/AIDS disease.
- MeSH
- acetylcystein farmakologie MeSH
- aktivace viru účinky léků MeSH
- arginin farmakologie MeSH
- buněčné linie MeSH
- CD antigeny metabolismus MeSH
- diferenciační antigeny T-lymfocytů metabolismus MeSH
- hem farmakologie MeSH
- hemoxygenasa-1 antagonisté a inhibitory metabolismus MeSH
- HIV-1 účinky léků fyziologie MeSH
- Jurkat buňky MeSH
- latence viru účinky léků MeSH
- látky proti HIV farmakologie MeSH
- lektiny typu C metabolismus MeSH
- lidé MeSH
- metaloporfyriny farmakologie MeSH
- protoporfyriny farmakologie MeSH
- proviry účinky léků genetika MeSH
- replikace viru účinky léků MeSH
- reverzní transkripce účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- acetylcystein MeSH
- arginin MeSH
- CD antigeny MeSH
- CD69 antigen MeSH Prohlížeč
- diferenciační antigeny T-lymfocytů MeSH
- hem MeSH
- heme arginate MeSH Prohlížeč
- hemoxygenasa-1 MeSH
- látky proti HIV MeSH
- lektiny typu C MeSH
- metaloporfyriny MeSH
- protoporfyriny MeSH
- tin protoporphyrin IX MeSH Prohlížeč
