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The image analysis tool FRIC (Fluorescence Ratiometric Imaging of Chromatin) quantitatively monitors dynamic spatiotemporal distribution of euchromatin and total chromatin in live cells. A vector (pTandemH) assures stoichiometrically constant expression of the histone variants Histone 3.3 and Histone 2B, fused to EGFP and mCherry, respectively. Quantitative ratiometric (H3.3/H2B) imaging displayed a concentrated distribution of heterochromatin in the periphery of U2OS cell nuclei. As a proof of concept, peripheral heterochromatin responded to experimental manipulation of histone acetylation as well as expression of the mutant lamin A protein “progerin,” which causes Hutchinson-Gilford Progeria Syndrome. In summary FRIC is versatile, unbiased, robust, requires a minimum of experimental steps and is suitable for screening purposes.

Cell-penetrating peptides (CPPs) are a promising non-viral vector for gene and drug delivery. CPPs exhibit high cell transfection, and are biocompatible. They can be also conjugated with organic and inorganic nanomaterials, such as magnetic nanoparticles (MNPs), graphene oxide (GO), metal-organic frameworks (MOFs), and chitosan. Nanomaterials offered a high specific surface area and provided relatively straightforward methods to be modified with biomolecules including CPPs and oligonucleotides (ONs). Novel nanomaterials conjugates with CPP/ONs complexes are therefore of interest for cell transfection with high efficiency. In this chapter, we described a summary of the non-viral vectors consisting of CPPs and nanomaterials. The book chapter also included a protocol to generate hybrid biomaterials consisting of CPPs and nanoparticles (NPs) for the delivery of oligonucleotides. The conjugation of NPs with CPPs serves as an effective platform for gene therapy with high cell transfection efficiency. The protocol is simple, offers high cell transfection compared to the CPPs-ONs complexes, and can be used for further improvements using external stimuli.

Mitochondria represent an important drug target for many phatology, including neurodegeneration, metabolic disease, heart failure, ischemia-reperfusion injury, and cancer. Mitochondrial dysfunctions are caused by mutation in mitochondrial DNA or in nuclear genes encoding mitochondrial proteins. Cell-penetrating peptides (CPPs) have been employed to overcome biological barriers, target this organelle, and therapeuticaly restore mitochondrial functions. Here, we describe recent methods used to deliver oligonucleotides targeting mitochondrial protein by using mitochondrial penetrating peptides. In particular, we highlight recent advances of formulated peptides/oligonucleotides nanocomplexes as a proof-of-principle for pharmaceutical form of peptide-based therapeutics., QC 20220602

Hyssopus officinalisL. is a well-known aromatic plant used in traditional medicine and the foodand cosmetics industry. The aim of this study is to assess the antioxidant, genotoxic, antigenotoxic andcytotoxic properties of characterized hyssop essential oils and methanol extracts. Chemical compositionwas analyzed by gas chromatography - mass spectrometry (GC-MS) and liquid chromatography with diodearray detection and mass spectrometry (LC-DAD-MS), respectively. Antioxidant activity was examinedby 2,2-diphenyl-1-picrylhydrazyl (DPPH) and ferric reducing/antioxidant power (FRAP) tests; genotoxicand antigenotoxic activity were examined by the comet assay, while cytotoxicity was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide dye (MTT) test against tumor cell lines (SW480,MDA-MB 231, HeLa) and non-transformed human lung fibroblast cell lines (MRC-5). The essential oilswere rich in monoterpene hydrocarbons (e.g., limonene; 7.99–23.81%), oxygenated monoterpenes (1,8-cineole; 38.19–67.1%) and phenylpropanoids (methyl eugenol; 0.00–28.33%). In methanol extracts, the mostabundant phenolics were chlorogenic and rosmarinic acid (23.35–33.46 and 3.53–17.98 mg/g, respectively).Methanol extracts expressed moderate to weak antioxidant activity (DPPH IC50= 56.04–199.89μg/mL,FRAP = 0.667–0.959 mmol Fe2+/g). Hyssop preparations significantly reduced DNA damage in humanwhole blood cells, induced by pretreatment with hydrogen peroxide. Methanol extracts exhibited selectiveand potent dose- and time-dependent activity against the HeLa cell line. Results of the current studydemonstrated notableH.officinalismedicinal potential, which calls for further investigation.

Cx43 hemichannels (HCs) are electrically and chemically gated transmembrane pores with low open probability and multiple conductance states, which makes kinetic studies of channel gating in large datasets challenging. Here, we developed open access software, named HemiGUI, to analyze HC gating transitions and investigated voltage-induced HC opening based on up to ≈4000 events recorded in HeLa-Cx43-overexpressing cells. We performed a detailed characterization of Cx43 HC gating profiles and specifically focused on the role of the C-terminal tail (CT) domain by recording the impact of adding an EGFP tag to the Cx43 CT end (Cx43-EGFP) or by supplying the Cx43 HC-inhibiting peptide Gap19 that interferes with CT interaction with the cytoplasmic loop (CL). We found that Gap19 not only decreased HC opening activity to the open state (≈217 pS) but also increased the propensity of subconductance (≈80 pS) transitions that additionally became slower as compared to the control. The work demonstrates that large sample transition analysis allows detailed investigations on Cx43 HC gating and shows that Gap19 acts as a HC gating modifier by interacting with the CT that forms a crucial gating element. © 2020 by the authors. Licensee MDPI, Basel, Switzerland.

Lung cancer is the leading cause of cancer mortality worldwide, resulting in 88% deaths of all diagnosed patients. Hence, novel therapeutic modalities are urgently needed. Single-stranded oligonucleotide-based aptamers (APTs) are excellent ligands for tumor cell targeting. However, the molecular mechanisms underlying their internalization into living cells have been poorly studied. Towards the application of APTs for active drug targeting to cancer cells, we herein studied the mechanism underlying S15-APT internalization into human non-small cell lung cancer A549 cells. We thus delineated the mode of entry of a model nanomedical system based on quantum dots (QDs) decorated with S15-APTs as a selective targeting moiety for uptake by A549 cells. These APT-decorated QDs displayed selective binding to, and internalization by target A549 cells, but not by normal human bronchial epithelial BEAS2B, cervical carcinoma (HeLa) and colon adenocarcinoma CaCo-2 cells, hence demonstrating high specificity. Flow cytometric analysis revealed a remarkably low dissociation constant of S15-APTs-decorated QDs to A549 cells (Kd = 13.1 ± 1.6 nM). Through the systematic application of a series of established inhibitors of known mechanisms of endocytosis, we show that the uptake of S15-APTs proceeds via a classical clathrin-dependent receptor-mediated endocytosis. This cancer cell-selective mode of entry could possibly be used in the future to evade plasma membranelocalized multidrug resistance efflux pumps, thereby overcoming an important mechanism of cancer multidrug resistance. © Engelberg et al.

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by membrane association and GTP loading of SAR1, followed by the recruitment of the SEC23-SEC24 and SEC13-SEC31 subcomplexes. Both of these two subcomplexes stimulate GTP hydrolysis and coat disassembly. This inherent disassembly capacity of COPII complexes needs to be regulated to allow sufficient time for cargo sorting and transport carrier formation. By performing fluorescence recovery after photobleaching (FRAP) and mathematical modeling, we show that p150glued (also known as DCTN1), a component of the dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. Concentration of the secretory marker ts-O45-G at ERES is reduced in the presence of a C-terminal p150glued fragment that prevents binding of endogenous p150glued to SEC23. A similar cargo reduction is observed upon p150glued knockdown. Taken together, our data suggest that cargo concentration at ERES is regulated by p150glued to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules. © 2015. Published by The Company of Biologists Ltd.

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by membrane association and GTP loading of SAR1, followed by the recruitment of the SEC23-SEC24 and SEC13-SEC31 subcomplexes. Both of these two subcomplexes stimulate GTP hydrolysis and coat disassembly. This inherent disassembly capacity of COPII complexes needs to be regulated to allow sufficient time for cargo sorting and transport carrier formation. By performing fluorescence recovery after photobleaching (FRAP) and mathematical modeling, we show that p150glued (also known as DCTN1), a component of the dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. Concentration of the secretory marker ts-O45-G at ERES is reduced in the presence of a C-terminal p150glued fragment that prevents binding of endogenous p150glued to SEC23. A similar cargo reduction is observed upon p150glued knockdown. Taken together, our data suggest that cargo concentration at ERES is regulated by p150glued to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules. © 2015. Published by The Company of Biologists Ltd.

Newly synthesized proteins are sorted into COPII-coated transport carriers at the endoplasmic reticulum (ER). Assembly of the COPII coat complex, which occurs at ER exit sites (ERES), is initiated by membrane association and GTP loading of SAR1, followed by the recruitment of the SEC23-SEC24 and SEC13-SEC31 subcomplexes. Both of these two subcomplexes stimulate GTP hydrolysis and coat disassembly. This inherent disassembly capacity of COPII complexes needs to be regulated to allow sufficient time for cargo sorting and transport carrier formation. By performing fluorescence recovery after photobleaching (FRAP) and mathematical modeling, we show that p150glued (also known as DCTN1), a component of the dynactin complex, stabilizes the COPII pre-budding complex on ER membranes in a microtubule-independent manner. Concentration of the secretory marker ts-O45-G at ERES is reduced in the presence of a C-terminal p150glued fragment that prevents binding of endogenous p150glued to SEC23. A similar cargo reduction is observed upon p150glued knockdown. Taken together, our data suggest that cargo concentration at ERES is regulated by p150glued to coordinate protein sorting and transport carrier formation with the subsequent long-range transport towards the Golgi complex along microtubules. © 2015. Published by The Company of Biologists Ltd.

Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMPa proteins exhibit dynamic synthesis and subcellular localization during cellular differentiation, including during spermatogenesis, little is known of how this affects cell fate. To investigate how IMPas control cellular development, we conducted a yeast two-hybrid screen for IMPalpha2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPalpha2 expression coincident with germ-line masculization. We identified paraspeckle protein 1(PSPC1), the original defining component of nuclear paraspeckles, as an IMPalpha2-binding partner. PSPC1-IMPalpha2 binding in testis was confirmed in immunoprecipitations and pull downs, and an enzyme-linked immunosorbent assay-based assay demonstrated direct, high-affinity PSPC1 binding to either IMPalpha2/IMPbeta1 or IMPalpha6/IMPbeta1. Coexpression of full-length PSPC1 and IMPalpha2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High-throughput image analysis of >3500 cells indicated IMPalpha2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMPalpha2 isoform or small interfering RNA knockdown of IMPalpha2 each reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPalpha2 nuclear import cargo in fetal testis provides novel evidence that PSPC1 delivery to paraspeckles, and consequently paraspeckle function, may be controlled by modulated synthesis of specific IMPs.Copyright © 2015 Major et al.

Importin (IMP) superfamily members mediate regulated nucleocytoplasmic transport, which is central to key cellular processes. Although individual IMPa proteins exhibit dynamic synthesis and subcellular localization during cellular differentiation, including during spermatogenesis, little is known of how this affects cell fate. To investigate how IMPas control cellular development, we conducted a yeast two-hybrid screen for IMPalpha2 cargoes in embryonic day 12.5 mouse testis, a site of peak IMPalpha2 expression coincident with germ-line masculization. We identified paraspeckle protein 1(PSPC1), the original defining component of nuclear paraspeckles, as an IMPalpha2-binding partner. PSPC1-IMPalpha2 binding in testis was confirmed in immunoprecipitations and pull downs, and an enzyme-linked immunosorbent assay-based assay demonstrated direct, high-affinity PSPC1 binding to either IMPalpha2/IMPbeta1 or IMPalpha6/IMPbeta1. Coexpression of full-length PSPC1 and IMPalpha2 in HeLa cells yielded increased PSPC1 localization in nuclear paraspeckles. High-throughput image analysis of >3500 cells indicated IMPalpha2 levels can directly determine PSPC1-positive nuclear speckle numbers and size; a transport-deficient IMPalpha2 isoform or small interfering RNA knockdown of IMPalpha2 each reduced endogenous PSPC1 accumulation in speckles. This first validation of an IMPalpha2 nuclear import cargo in fetal testis provides novel evidence that PSPC1 delivery to paraspeckles, and consequently paraspeckle function, may be controlled by modulated synthesis of specific IMPs.Copyright © 2015 Major et al.

The aim of this study was the preparation of novel polyester nanoparticles based on folic acid (FA)–functionalized poly(ethylene glycol)–poly(propylene succinate) (PEG–PPSu) copolymer and loaded with the new anticancer drug ixabepilone (IXA). These nanoparticles may serve as a more selective (targeted) treatment of breast cancer tumors overexpressing the folate receptor. The synthesized materials were characterized by 1H-NMR, FTIR, XRD and DSC. The nanoparticles were prepared by a double emulsification and solvent evaporation method and characterized with regard to their morphology by scanning electron microscopy, drug loading with HPLC–UV and size by dynamic light scattering. An average size of 195 nm and satisfactory drug loading efficiency (3.5 %) were observed. XRD data indicated that IXA was incorporated into nanoparticles in amorphous form. The nanoparticles exhibited sustained drug release properties in vitro. Based on in vitro cytotoxicity studies, the blank FA–PEG–PPSu nanoparticles were found to be non-toxic to the cells. Fluorescent nanoparticles were prepared by conjugating Rhodanine B to PEG–PPSu, and live cell, fluorescence, confocal microscopy was applied in order to demonstrate the ability of FA–PEG–PPSu nanoparticles to enter into human breast cancer cells expressing the folate receptor. Graphical Abstract: [Figure not available: see fulltext.] © 2015, Springer Science+Business Media New York.

Long interspersed element 1 (LINE-1 or L1) retrotransposons compose 17% of the human genome. Active L1 elements are capable of replicative transposition (mobilization) and can act as drivers of genetic diversity. However, this mobilization is mutagenic and may be detrimental to the host, and therefore it is under strict control. Somatic cells usually silence L1 activity by DNA methylation of the L1 promoter. In hypomethylated cells, such as cancer cells and induced pluripotent stem cells (iPSCs), a window of opportunity for L1 reactivation emerges, and with it comes an increased risk of genomic instability and tumorigenesis. Here we show that miR-128 represses new retrotransposition events in human cancer cells and iPSCs by binding directly to L1 RNA. Thus, we have identified and characterized a new function of microRNAs: mediating genomic stability by suppressing the mobility of endogenous retrotransposons. © 2015 Nature America, Inc. All rights reserved.

Long interspersed element 1 (LINE-1 or L1) retrotransposons compose 17% of the human genome. Active L1 elements are capable of replicative transposition (mobilization) and can act as drivers of genetic diversity. However, this mobilization is mutagenic and may be detrimental to the host, and therefore it is under strict control. Somatic cells usually silence L1 activity by DNA methylation of the L1 promoter. In hypomethylated cells, such as cancer cells and induced pluripotent stem cells (iPSCs), a window of opportunity for L1 reactivation emerges, and with it comes an increased risk of genomic instability and tumorigenesis. Here we show that miR-128 represses new retrotransposition events in human cancer cells and iPSCs by binding directly to L1 RNA. Thus, we have identified and characterized a new function of microRNAs: mediating genomic stability by suppressing the mobility of endogenous retrotransposons. © 2015 Nature America, Inc. All rights reserved.

The aim of this study was the preparation of novel polyester nanoparticles based on folic acid (FA)–functionalized poly(ethylene glycol)–poly(propylene succinate) (PEG–PPSu) copolymer and loaded with the new anticancer drug ixabepilone (IXA). These nanoparticles may serve as a more selective (targeted) treatment of breast cancer tumors overexpressing the folate receptor. The synthesized materials were characterized by 1H-NMR, FTIR, XRD and DSC. The nanoparticles were prepared by a double emulsification and solvent evaporation method and characterized with regard to their morphology by scanning electron microscopy, drug loading with HPLC–UV and size by dynamic light scattering. An average size of 195 nm and satisfactory drug loading efficiency (3.5 %) were observed. XRD data indicated that IXA was incorporated into nanoparticles in amorphous form. The nanoparticles exhibited sustained drug release properties in vitro. Based on in vitro cytotoxicity studies, the blank FA–PEG–PPSu nanoparticles were found to be non-toxic to the cells. Fluorescent nanoparticles were prepared by conjugating Rhodanine B to PEG–PPSu, and live cell, fluorescence, confocal microscopy was applied in order to demonstrate the ability of FA–PEG–PPSu nanoparticles to enter into human breast cancer cells expressing the folate receptor. Graphical Abstract: [Figure not available: see fulltext.] © 2015, Springer Science+Business Media New York.

We have performed a series of in vitro tests proposed for the reliable assessment of safety associated with nanoparticles-cell interaction. A thorough analysis of toxicity of three different coating iron oxide nanoparticles on HeLa cells has been carried out including, methyl thiazol tetrazolium bromide (MTT) and Trypan blue exclusion tests, cell morphology observation by optical and Scanning Electron Microscopy (SEM), study of cytoskeletal components, analysis of cell cycle and the presence of reactive oxygen species (ROS). We have quantified magnetic nanoparticle internalization, determined possible indirect cell damages and related it to the nanoparticle coating. The results confirm a very low toxicity of the analyzed iron oxide nanoparticles into HeLa cells by multiple assays and pave the way for a more successful cancer diagnostic and treatment without secondary effects. From the Clinical Editor: In this paper, three different iron oxide nanoparticles are studied and compared from the standpoint of safety and toxicity in HeLa cells, demonstrating low toxicity for each preparation, and paving the way to potential future clinical applications. © 2014 Elsevier Inc.

Cilj ovog rada bio je uspostavljanje odgovarajućeg in vitro model sistema za proučavanje transkripcione regulacije ekspresije humanog SOX18 gena. U ovom radu prikazana je ekspresiona analiza SOX18 gena u permanentnim ćelijskim linijama kao i prva karakterizacija uzvodnog regulatornog regiona ovog gena. Primenom RT-PCR, Northern i Western blot eseja, ustanovljeno je da je SOX18 geneks­primiranu HeLa ćelijskoj liniji i da ove ćelije predstavljaju adekvatan model sistem za proučavanje transkripcione regulacije ekspresije ovog gena. Takođe, u ovom radu je prikazano i kloniranje i prva karakterizacija uzvodnog regulatornog regiona SOX18 gena. Pokazano je da region 892 bp uzvodno od ATG kodona sadrži regulatorne elemente neophodne za transkripciju i da ovaj region sadrži promotor SOX18 gena., The aim of this study was to establish an adequate in vitro model system for studying transcriptional regulation of the human SOX18 gene. The paper presents an analysis of expression of this gene in cultured cell lines and characterization of its 5' flanking region. Using RT-PCR, Northern and Western blot analysis, we demonstrated SOX18 expression in HeLa cells, indicating that this cell line provides a suitable model system for studying transcriptional regulation of the given gene. We also cloned, sequenced and for the first time characterized the human SOX18 5’ flanking region. It is shown that the region 892 bp in size immediately upstream from the start codone harbors regulatory elements sufficient for transcription and represents an SOX18 promoter region.