Impact of heat and cold shock on epigenetics and chromatin structure.

Cells are continuously exposed to various sources of insults, among which temperature variations are extremely common. Epigenetic mechanisms, critical players in gene expression regulation, undergo alterations due to these stressors, potentially leading to health issues. Despite the significance of DNA methylation and histone modifications in gene expression regulation, their changes following heat and cold shock in human cells remain poorly understood. In this study, we investigated the epigenetic profiles of human cells subjected to hyperthermia and hypothermia, revealing significant variations. Heat shock primarily led to DNA methylation increments and epigenetic modifications associated with gene expression silencing. In contrast, cold shock presented a complex scenario, with both methylation and demethylation levels increasing, indicating different epigenetic responses to the opposite thermal stresses. These temperature-induced alterations in the epigenome, particularly their impact on chromatin structural organization, represent an understudied area that could offer important insights into genome function and potential prospects for therapeutic targets.

Histochemistry for nucleic acid research: 60 years in the European Journal of Histochemistry.

Since the discovery of DNA structure in 1953, the deoxyribonucleic acid has always been playing a central role in biological research. As physical and ordered nucleotides sequence, it stands at the base of genes existence. Furthermore, beside this 2-dimensional sequence, DNA is characterized by a 3D structural and functional organization, which is of interest for the scientific community due to multiple levels of expression regulation, of interaction with other biomolecules, and much more. Analogously, the nucleic acid counterpart of DNA, RNA, represents a central issue in research, because of its fundamental role in gene expression and regulation, and for the DNA-RNA interplay. Because of their importance, DNA and RNA have always been mentioned and studied in several publications, and the European Journal of Histochemistry is no exception. Here, we review and discuss the papers published in the last 60 years of this Journal, focusing on its contribution in deepening the knowledge about this topic and analysing papers that reflect the interest this Journal always granted to the world of DNA and RNA.

The impact of storage on extracellular vesicles: A systematic study.

Mounting evidence suggests that storage has an impact on extracellular vesicles (EVs) properties. While -80°C storage is a widespread approach, some authors proposed improved storage strategies with conflicting results. Here, we designed a systematic study to assess the impact of -80°C storage and freeze-thaw cycles on EVs. We tested the differences among eight storage strategies and investigated the possible fusion phenomena occurring during storage. EVs were collected from human plasma and murine microglia culture by size exclusion chromatography and ultracentrifugation, respectively. The analysis included: concentration, size and zeta potential (tunable resistive pulse sensing), contaminant protein assessment; flow cytometry for the analysis of two single fluorescent-tagged EVs populations (GFP and mCherry), mixed before preservation. We found that -80°C storage reduces EVs concentration and sample purity in a time-dependent manner. Furthermore, it increases the particle size and size variability and modifies EVs zeta potential, with a shift of EVs in size-charge plots. None of the tested conditions prevented the observed effects. Freeze-thaw cycles lead to an EVs reduction after the first cycle and to a cycle-dependent increase in particle size. With flow cytometry, after storage, we observed a significant population of double-positive EVs (GFP+ -mCherry+ ). This observation may suggest the occurrence of fusion phenomena during storage. Our findings show a significant impact of storage on EVs samples in terms of particle loss, purity reduction and fusion phenomena leading to artefactual particles. Depending on downstream analyses and experimental settings, EVs should probably be processed from fresh, non-archival, samples in majority of cases.

Oxidative Stress and Nuclear Reprogramming: A Pilot Study of the Effects of Reactive Oxygen Species on Architectural and Epigenetic Landscapes.

Cell genome integrity is continuously threatened by various sources, both endogenous and exogenous. Oxidative stress causes a multitude of damages, severely affecting cell viability, fidelity of genetic information inheritance, and determining profound alterations in gene expression. Epigenetics represents a major form of gene expression modulation, influencing DNA accessibility to transcription factors and the overall nuclear architecture. When assessing the stress-induced epigenome reprogramming, widely diffused biochemical and molecular approaches commonly fail to incorporate analyses such as architectural chromatin alterations and target molecules precise spatial localization. Unveiling the significance of the nuclear response to the oxidative stress, as well as the functional effects over the chromatin organization, may reveal targets and strategies for approaches aiming at limiting the impact on cellular stability. For these reasons, we utilized potassium bromate treatment, a stressor able to induce DNA damages without altering the cellular microenvironment, hence purely modeling nuclear oxidative stress. By means of high-resolution techniques, we described profound alterations in DNA and histone epigenetic modifications and in chromatin organization in response to the reactive oxygen species.

Targeting cellular stress in vitro improves osteoblast homeostasis, matrix collagen content and mineralization in two murine models of osteogenesis imperfecta.

Most cases of dominantly inherited osteogenesis imperfecta (OI) are caused by glycine substitutions in the triple helical domain of type I collagen α chains, which delay collagen folding, and cause the synthesis of collagen triple helical molecules with abnormal structure and post-translational modification. A variable extent of mutant collagen ER retention and other secondary mutation effects perturb osteoblast homeostasis and impair bone matrix quality. Amelioration of OI osteoblast homeostasis could be beneficial both to osteoblast anabolic activity and to the content of the extracellular matrix they deposit. Therefore, the effect of the chemical chaperone 4-phenylbutyrate (4-PBA) on cell homeostasis, collagen trafficking, matrix production and mineralization was investigated in primary osteoblasts from two murine models of moderate OI, Col1a1+/G349C and Col1a2+/G610C. At the cellular level, 4-PBA prevented intracellular accumulation of collagen and increased protein secretion, reducing aggregates within the mutant cells and normalizing ER morphology. At the extracellular level, increased collagen incorporation into matrix, associated with more mature collagen fibrils, was observed in osteoblasts from both models. 4-PBA also promoted OI osteoblast mineral deposition by increasing alkaline phosphatase expression and activity. Targeting osteoblast stress with 4-PBA improved both cellular and matrix abnormalities in culture, supporting further in vivo studies of its effect on bone tissue composition, strength and mineralization as a potential treatment for classical OI.

[Pt(O,O'-acac)(γ-acac)(DMS)]: Alternative Strategies to Overcome Cisplatin-Induced Side Effects and Resistance in T98G Glioma Cells.

Cisplatin (CDDP) is one of the most effective chemotherapeutic agents, used for the treatment of diverse tumors, including neuroblastoma and glioblastoma. CDDP induces cell death through different apoptotic pathways. Despite its clinical benefits, CDDP causes several side effects and drug resistance.[Pt(O,O'-acac)(γ-acac)(DMS)], namely PtAcacDMS, a new platinum(II) complex containing two acetylacetonate (acac) and a dimethylsulphide (DMS) in the coordination sphere of metal, has been recently synthesized and showed 100 times higher cytotoxicity than CDDP. Additionally, PtAcacDMS was associated to a decreased neurotoxicity in developing rat central nervous system, also displaying great antitumor and antiangiogenic activity both in vivo and in vitro. Thus, based on the knowledge that several chemotherapeutics induce cancer cell death through an aberrant increase in [Ca2+]i, in the present in vitro study we compared CDDP and PtAcacDMS effects on apoptosis and intracellular Ca2+ dynamics in human glioblastoma T98G cells, applying a battery of complementary techniques, i.e., flow cytometry, immunocytochemistry, electron microscopy, Western blotting, qRT-PCR, and epifluorescent Ca2+ imaging. The results confirmed that (i) platinum compounds may induce cell death through an aberrant increase in [Ca2+]i and (ii) PtAcacDMS exerted stronger cytotoxic effect than CDDP, associated to a larger increase in resting [Ca2+]i. These findings corroborate the use of PtAcacDMS as a promising approach to improve Pt-based chemotherapy against gliomas, either by inducing a chemosensitization or reducing chemoresistance in cell lineages resilient to CDDP treatment.

The dynamics of the nuclear environment and their impact on gene function.

In the last decades, it has become increasingly clear how the modulation of spatial organization of chromatin over time and through the cell cycle is closely connected to gene function regulation. Different physicochemical stimuli contribute to the realization of specific transcriptional programs and finally to a specific cellular phenotype. In this review, we aim to describe the current knowledge about the dynamics regulating the movements and the interactions of molecules within the nucleus and their impact on gene functions. In particular, taking into account that these forces exert their effect in a nuclear environment characterized by a high concentration of molecules, we will discuss the role of proteins and structures that regulate these movements and transduce physicochemical signals acting on the cell to the nucleus.

Timing of Cytosine Methylation on Newly Synthesized RNA by Electron Microscopy.

Increasing evidence demonstrates that RNA nucleotides undergo epigenetic modifications, such as methylation on cytosine. Although the presence of modified bases on mRNA has been proven, their molecular significance is largely undefined. We describe here a methodology to dissect the timing of modification of cytosine to 5-methylcytosine (5mC or m5C) in relation to RNA elongation and processing. To do this we use chlorouridine and iodouridine, two synthetically modified nucleotide bases which can be recognized by RNA polymerase II and incorporated into nascent RNA. These modified bases are added to a cell culture for defined intervals of time, and then immunocytochemical staining using antibodies against the modified nucleotides is carried out. This procedure allows us to identify the range of time in which 5mC is produced in nascent mRNA. This method provides the ultra-resolution of electron microscopy and allows following nascent RNA molecules during their elongation.

Novel Medicinal Mushroom Blend as a Promising Supplement in Integrative Oncology: A Multi-Tiered Study using 4T1 Triple-Negative Mouse Breast Cancer Model.

Although medicinal mushroom extracts have been proposed as promising anti-cancer agents, their precise impacts on metastatic breast cancer are still to be clarified. For this purpose, the present study exploited the effect of a novel medicinal mushroom blend, namely Micotherapy U-care, in a 4T1 triple-negative mouse breast cancer model. Mice were orally administered with Micotherapy U-care, consisting of a mixture of Agaricus blazei, Ophiocordyceps sinensis, Ganoderma lucidum, Grifola frondosa, and Lentinula edodes. The syngeneic tumor-bearing mice were generated by injecting 4T1 cells in both supplemented and non-supplemented mice. After sacrifice 25 days later, specific endpoints and pathological outcomes of the murine pulmonary tissue were evaluated. (i) Histopathological and ultrastructural analysis and (ii) immunohistochemical assessment of TGF-ß1, IL-6 and NOS2, COX2, SOD1 as markers of inflammation and oxidative stress were performed. The QoL was comparatively evaluated. Micotherapy U-care supplementation, starting before 4T1 injection and lasting until the end of the experiment, dramatically reduced the pulmonary metastases density, also triggering a decrease of fibrotic response, and reducing IL-6, NOS, and COX2 expression. SOD1 and TGF-ß1 results were also discussed. These findings support the valuable potential of Micotherapy U-care as adjuvant therapy in the critical management of triple-negative breast cancer.

Extra-Small Gold Nanospheres Decorated With a Thiol Functionalized Biodegradable and Biocompatible Linear Polyamidoamine as Nanovectors of Anticancer Molecules.

Gold nanoparticles are elective candidate for cancer therapy. Current efforts are devoted to developing innovative methods for their synthesis. Besides, understanding their interaction with cells have become increasingly important for their clinical application. This work aims to describe a simple approach for the synthesis of extra-small gold nanoparticles for breast cancer therapy. In brief, a biocompatible and biodegradable polyamidoamine (named AGMA1-SH), bearing 20%, on a molar basis, thiol-functionalized repeat units, is employed to stabilize and coat extra-small gold nanospheres of different sizes (2.5, 3.5, and 5 nm in gold core), and to generate a nanoplatform for the link with Trastuzumab monoclonal antibody for HER2-positive breast cancer targeting. Dynamic light scattering, transmission electron microscopy, ultraviolet visible spectroscopy, X-ray powder diffraction, circular dichroism, protein quantification assays are used for the characterization. The targeting properties of the nanosystems are explored to achieve enhanced and selective uptake of AGMA1-SH-gold nanoparticles by in vitro studies against HER-2 overexpressing cells, SKBR-3 and compared to HER-2 low expressing cells, MCF-7, and normal fibroblast cell line, NIH-3T3. In vitro physicochemical characterization demonstrates that gold nanoparticles modified with AGMA1-SH are more stable in aqueous solution than the unmodified ones. Additionally, the greater gold nanoparticles size (5-nm) is associated with a higher stability and conjugation efficiency with Trastuzumab, which retains its folding and anticancer activity after the conjugation. In particular, the larger Trastuzumab functionalized nanoparticles displays the highest efficacy (via the pro-apoptotic protein increase, anti-apoptotic components decrease, survival-proliferation pathways downregulation) and internalization (via the activation of the classical clathrin-mediated endocytosis) in HER-2 overexpressing SKBR-3 cells, without eliciting significant effects on the other cell lines. The use of biocompatible AGMA1-SH for producing covalently stabilized gold nanoparticles to achieve selective targeting, cytotoxicity and uptake is completely novel, offering an important advancement for developing new anticancer conjugated-gold nanoparticles.

Squaring the Circle: A New Study of Inward and Outward-Rectifying Potassium Currents in U251 GBM Cells.

In the present study, the functional role of the inwardly rectifying K+ channel, Kir4.1, and large-conductance Ca2+-activated K+ (BK) channel during cell migration in U251 cell line was investigated. We focused on polarised cells which are positive for the active-Cdc42 migration marker. The perforated patch technique was used to avoid intracellular dialysis and to maintain physiological changes in intracellular calcium. Wound healing was employed to assay migration after 24 h. Polarised cells recorded displayed different hallmarks of undifferentiated glial cells: depolarised resting membrane potential and high membrane resistance. Cells recorded outside wounded area did not display either constitutive inward or outward rectification. After migration, U251 cells were characterised by a constitutively smaller Kir4.1 and larger BK currents with a linearly related amplitude. Menthol modulation increased both currents in a linearly dependent manner, indicating a common mechanism triggered by activation of transient receptor potential melastatin 8 (TRPM8), a Ca2+-permeable non-selective cation channel. We hypothesised that both migration and menthol modulation would share an increase of intracellular calcium triggering the increase in Kir4.1 and BK channels. Immunocytochemistry demonstrated the cytoplasmic expression of both Kir4.1 and BK channels and a mislocation in the nucleus under basal conditions. Before and after migration, polarised cells increased the expression of Kir4.1 and BK channels both in the cytoplasm and nucleus. TEM ultrastructural analysis displayed a different nuclear distribution of Kir4.1 and BK channels. In the present study, the physiological role of Kir4.1 and BK currents at membrane potential, their involvement in migration, and the functional role of nuclear channels were discussed.

Perichromatin region: a moveable feast.

The perichromatin region is an elusive zone of the cell nucleus located at the periphery of the condensed chromatin areas. This region is visible at the electron microscope level under special staining treatments, otherwise it is merged with the border of condensed chromatin. In this 200 nm-thick area, several fundamental cell processes take place, such as replication, DNA repair and transcription. In addition, RNA processing occurs in the perichromatin region, including 5'-capping and 3'-polyadenylation as well as splicing. Recently, it has become clear that also some epigenetics modifications take place there, such as methylation of DNA and RNA on cytosine and adenosine. In summary, this thin interface between chromatin and the interchromatinic space represents the zone where the majority of the functions of DNA in interphase occur, in a place where there is no steric hindrance of condensed chromatin, the products can easily move away toward their target and the enzymes can freely dock.