A brief history of the Feulgen reaction.

One hundred years ago, Robert Feulgen published a landmark paper in which he described the first method to stain DNA in cells and tissues. Although a century has passed since the discovery by Feulgen and Rossenbeck, the chemical reaction still exerts an important influence in current histochemical studies. Its contribution in diverse fields, spanning from biomedicine to plant biology, has paved the way for the most significant studies that constitute our current knowledge. The possibility to specifically explore the DNA in cell nuclei while quantifying its content makes it a contemporary and timeless method. Indeed, many histocytochemical studies following the 1924 paper have led to a deep understanding of genome organization in general as well as several specific mechanisms (e.g. DNA duplication or tumour pathology) that, nowadays, constitute some of the most fundamental pillars in biological investigations. In this review, we discuss the chemistry and application of the Feulgen reaction to both light and electron microscopy.

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.

Effects of Ferrocene and Ferrocenium on MCF-7 Breast Cancer Cells and Interconnection with Regulated Cell Death Pathways.

The effects of ferrocene (Fc) and ferrocenium (Fc+) induced in triple negative human breast cancer MCF-7 cells were explored by immunofluorescence, flow cytometry, and transmission electron microscopy analysis. The different abilities of Fc and Fc+ to produce reactive oxygen species and induce oxidative stress were clearly observed by activating apoptosis and morphological changes after treatment, but also after tests performed on the model organism D. discoideum, particularly in the case of Fc+. The induction of ferroptosis, an iron-dependent form of regulated cell death driven by an overload of lipid peroxides in cellular membranes, occurred after 2 h of treatment with Fc+ but not Fc. However, the more stable Fc showed its effects by activating necroptosis after a longer-lasting treatment. The differences observed in terms of cell death mechanisms and timing may be due to rapid interconversion between the two oxidative forms of internalized iron species (from Fe2+ to Fe3+ and vice versa). Potential limitations include the fact that iron metabolism and mitophagy have not been investigated. However, the ability of both Fc and Fc+ to trigger different and interregulated types of cell death makes them suitable to potentially overcome the shortcomings of traditional apoptosis-mediated anticancer therapies.

Carbonic anhydrase IV in lizard chemical signals.

The evolution of chemical signals is subject to environmental constraints. A multicomponent signal may combine semiochemical molecules with supporting compounds able to enhance communication efficacy. Carbonic anhydrases (CAs) are ubiquitous enzymes catalysing the reversible hydration of carbon dioxide, a reaction involved in a variety of physiological processes as it controls the chemical environment of the different tissues or cellular compartments, thus contributing to the overall system homeostasis. CA-IV isoform has been recently identified by mass spectrometry in the femoral gland secretions (FG) of the marine iguana, where it has been hypothesized to contribute to the chemical stability of the signal, by regulating blend pH. Lizards, indeed, use FG to communicate by delivering the waxy secretion on bare substrate, where it is exposed to environmental stressors. Therefore, we expect that some molecules in the mixture may play supporting functions, enhancing the stability of the chemical environment, or even conferring homeostatic properties to the blend. CA-IV may well represent an important candidate to this hypothesized supporting/homeostatic function, and, therefore, we can expect it to be common in FG secretions of other lizard species. To evaluate this prediction and definitely validate CA identity, we analysed FG secretions of eight species of wall lizards (genus Podarcis), combining mass spectrometry, immunoblotting, immunocytochemistry, and transmission electron microscopy. We demonstrate CA-IV to actually occur in the FG of seven out of the eight considered species, providing an immunochemistry validation of mass-spectrometry identifications, and localizing the enzyme within the secretion mass. The predicted structure of the identified CA is compatible with the known enzymatic activity of CA-IV, supporting the hypothesis that CA play a signal homeostasis function and opening to new perspective about the role of proteins in vertebrate chemical communication.

Cytological, molecular, cytogenetic, and physiological characterization of a novel immortalized human enteric glial cell line.

Enteric glial cells (EGCs), the major components of the enteric nervous system (ENS), are implicated in the maintenance of gut homeostasis, thereby leading to severe pathological conditions when impaired. However, due to technical difficulties associated with EGCs isolation and cell culture maintenance that results in a lack of valuable in vitro models, their roles in physiological and pathological contexts have been poorly investigated so far. To this aim, we developed for the first time, a human immortalized EGC line (referred as ClK clone) through a validated lentiviral transgene protocol. As a result, ClK phenotypic glial features were confirmed by morphological and molecular evaluations, also providing the consensus karyotype and finely mapping the chromosomal rearrangements as well as HLA-related genotypes. Lastly, we investigated the ATP- and acetylcholine, serotonin and glutamate neurotransmitters mediated intracellular Ca2+ signaling activation and the response of EGCs markers (GFAP, SOX10, S100ß, PLP1, and CCL2) upon inflammatory stimuli, further confirming the glial nature of the analyzed cells. Overall, this contribution provided a novel potential in vitro tool to finely characterize the EGCs behavior under physiological and pathological conditions in humans.

Study on the activation of cell death mechanisms: in search of new therapeutic targets in glioblastoma multiforme.

Malignant primary brain tumors remain among the most difficult cancers to treat, in particular, Glioblastoma Multiforme (GBM) is the deadliest brain tumor. The standard therapies currently used are not efficient enough in improving patients' survival and quality of life. Cisplatin (CDDP), a platinum-based drug, has shown efficacy against different solid neoplasms, but it is also associated to different forms of off-target toxicity. To overcome the limitation in the use of CDDP in the treatment of GBM patients, fourth generation platinum compounds are been synthesized, one of them is the Pt(IV)Ac-POA, a prodrug with a medium-chain fatty acid as axial ligand, which acts as a histone 3 deacetylase inhibitor. Moreover, recently, the antioxidant effects of medicinal mushrooms have been shown to induce a lowering of the toxicity of chemotherapy drugs, inducing greater therapeutic efficiency, thus the combined therapy of chemotherapy and micotherapy could be helpful in the treatment of GBM reducing the adverse effects of the former thanks to phytotherapy's antioxidant, anti-inflammatory, immunomodulatory and antitumoral activities. Here, through immunoblotting, ultrastructural and immunofluorescence analysis, we evaluated the contribution in the activation of different cell death pathway of Micotherapy U-Care, a medicinal blend supplement, used together with platinum-based compounds on human glioblastoma U251 cells.

Rediscover Potassium Permanganate as a Stain for Basic Proteins on Ultrathin Sections at Transmission Electron Microscopy.

Potassium permanganate solution has been used both as a fixative and as a staining for ultrathin sections at transmission electron microscopy, due to its ability to provide good contrast of different tissue components. Subsequently, it has been forgotten due to disadvantages such as conspicuous formation of precipitates and fragility of the tissue sections treated with this dye when placed under the electron beam. Here we demonstrate that the observed granularity of the sections is not related to the formation of non-specific precipitates, but rather to basic proteins such as chromatin proteins closely associated with DNA and ribosomal particles which are intensely stained. This results in a marked contrast of the nuclei, in particular of the heterochromatin areas, the granular component of the nucleoli, and the rough endoplasmic reticulum, that are rich in these protein complexes. We also show how the embedding in LR white acrylic resin can preserve a good morphology and be less sensitive to the treatment with potassium permanganate than the epoxy resin sections, also allowing to perform immunocytochemistry. The fragility of the epoxy resin sections can be partially improved by using formvar-coated grids.

Specific RNA Visualization at Electron Microscopy via Terbium Citrate Vapors.

Terbium citrate staining represents the method of choice for RNA visualization at transmission electron microscopy. Because of its affinity for guanosines in RNA rather than in DNA, terbium citrate gives precise results being a selective staining. However, difficulties often arise when performing this technique, especially in crucial and delicate steps such as rinses, when it is not uncommon to excessively reduce the already feeble contrast. For these reasons, we developed a straightforward and secure approach to overcome such complications. Here we report a new method for RNA single molecule localization by means of terbium citrate vapors, viable for every type of fixation and embedding.

Mercury chloride alters heterochromatin domain organization and nucleolar activity in mouse liver.

Mercury is a highly toxic element that induces severe alterations and a broad range of adverse effects on health. Its exposure is a global concern because it is widespread in the environment due to its multiple industrial, domestic, agricultural and medical usages. Among its various chemical forms, both humans and animals are mainly exposed to mercury chloride (HgCl2), methylmercury and elemental mercury. HgCl2 is metabolized primarily in the liver. We analysed the effects on the nuclear architecture of an increasing dosage of HgCl2 in mouse hepatocytes cell culture and in mouse liver, focusing specifically on the organization, on some epigenetic features of the heterochromatin domains and on the nucleolar morphology and activity. Through the combination of molecular and imaging approaches both at optical and electron microscopy, we show that mercury chloride induces modifications of the heterochromatin domains and a decrease of some histones post-translational modifications associated to heterochromatin. This is accompanied by an increase in nucleolar activity which is reflected by bigger nucleoli. We hypothesized that heterochromatin decondensation and nucleolar activation following mercury chloride exposure could be functional to express proteins necessary to counteract the harmful stimulus and reach a new equilibrium.

Enhanced Delivery of Rose Bengal by Amino Acids Starvation and Exosomes Inhibition in Human Astrocytoma Cells to Potentiate Anticancer Photodynamic Therapy Effects.

Photodynamic therapy (PDT) is a promising anticancer strategy based on the light energy stimulation of photosensitizers (PS) molecules within a malignant cell. Among a multitude of recently challenged PS, Rose bengal (RB) has been already reported as an inducer of cytotoxicity in different tumor cells. However, RB displays a low penetration capability across cell membranes. We have therefore developed a short-term amino acids starvation protocol that significantly increases RB uptake in human astrocytoma cells compared to normal rat astrocytes. Following induced starvation uptake, RB is released outside cells by the exocytosis of extracellular vesicles (EVs). Thus, we have introduced a specific pharmacological treatment, based on the GW4869 exosomes inhibitor, to interfere with RB extracellular release. These combined treatments allow significantly reduced nanomolar amounts of administered RB and a decrease in the time interval required for PDT stimulation. The overall conditions affected astrocytoma viability through the activation of apoptotic pathways. In conclusion, we have developed for the first time a combined scheme to simultaneously increase the RB uptake in human astrocytoma cells, reduce the extracellular release of the drug by EVs, and improve the effectiveness of PDT-based treatments. Importantly, this strategy might be a valuable approach to efficiently deliver other PS or chemotherapeutic drugs in tumor cells.

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.

Nicotinic Acid Adenine Dinucleotide Phosphate Induces Intracellular Ca2+ Signalling and Stimulates Proliferation in Human Cardiac Mesenchymal Stromal Cells.

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a newly discovered second messenger that gates two pore channels 1 (TPC1) and 2 (TPC2) to elicit endo-lysosomal (EL) Ca2+ release. NAADP-induced lysosomal Ca2+ release may be amplified by the endoplasmic reticulum (ER) through the Ca2+-induced Ca2+ release (CICR) mechanism. NAADP-induced intracellular Ca2+ signals were shown to modulate a growing number of functions in the cardiovascular system, but their occurrence and role in cardiac mesenchymal stromal cells (C-MSCs) is still unknown. Herein, we found that exogenous delivery of NAADP-AM induced a robust Ca2+ signal that was abolished by disrupting the lysosomal Ca2+ store with Gly-Phe ß-naphthylamide, nigericin, and bafilomycin A1, and blocking TPC1 and TPC2, that are both expressed at protein level in C-MSCs. Furthermore, NAADP-induced EL Ca2+ release resulted in the Ca2+-dependent recruitment of ER-embedded InsP3Rs and SOCE activation. Transmission electron microscopy revealed clearly visible membrane contact sites between lysosome and ER membranes, which are predicted to provide the sub-cellular framework for lysosomal Ca2+ to recruit ER-embedded InsP3Rs through CICR. NAADP-induced EL Ca2+ mobilization via EL TPC was found to trigger the intracellular Ca2+ signals whereby Fetal Bovine Serum (FBS) induces C-MSC proliferation. Furthermore, NAADP-evoked Ca2+ release was required to mediate FBS-induced extracellular signal-regulated kinase (ERK), but not Akt, phosphorylation in C-MSCs. These finding support the notion that NAADP-induced TPC activation could be targeted to boost proliferation in C-MSCs and pave the way for future studies assessing whether aberrant NAADP signaling in C-MSCs could be involved in cardiac disorders.

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.

Berberine Photo-Activation Potentiates Cytotoxicity in Human Astrocytoma Cells through Apoptosis Induction.

Photodynamic therapy (PDT) has recently attracted interest as an innovative and adjuvant treatment for different cancers including malignant gliomas. Among these, Glioblastoma (GBM) is the most prevalent neoplasm in the central nervous system. Despite conventional therapeutic approaches that include surgical removal, radiation, and chemotherapy, GBM is characterized by an extremely poor prognosis and a high rate of recurrence. PDT is a physical process that induces tumor cell death through the genesis and accumulation of reactive oxygen species (ROS) produced by light energy interaction with a photosensitizing agent. In this contribution, we explored the potentiality of the plant alkaloid berberine (BBR) as a photosensitizing and cytotoxic agent coupled with a PDT scheme using a blue light source in human established astrocytoma cell lines. Our data mainly indicated for the combined BBR-PDT scheme a potent activation of the apoptosis pathway, through a massive ROS production, a great extent of mitochondria depolarization, and the sub-sequent activation of caspases. Altogether, these results demonstrated that BBR is an efficient photosensitizer agent and that its association with PDT may be a potential anticancer strategy for high malignant gliomas.

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.