Zc3h13/Flacc is required for adenosine methylation by bridging the mRNA-binding factor Rbm15/Spenito to the m6A machinery component Wtap/Fl(2)d.

N6-methyladenosine (m6A) is the most abundant mRNA modification in eukaryotes, playing crucial roles in multiple biological processes. m6A is catalyzed by the activity of methyltransferase-like 3 (Mettl3), which depends on additional proteins whose precise functions remain poorly understood. Here we identified Zc3h13 (zinc finger CCCH domain-containing protein 13)/Flacc [Fl(2)d-associated complex component] as a novel interactor of m6A methyltransferase complex components in Drosophila and mice. Like other components of this complex, Flacc controls m6A levels and is involved in sex determination in Drosophila We demonstrate that Flacc promotes m6A deposition by bridging Fl(2)d to the mRNA-binding factor Nito. Altogether, our work advances the molecular understanding of conservation and regulation of the m6A machinery.

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.

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.

Changes in genotoxic stress response, ribogenesis and PAP (3'-phosphoadenosine 5'-phosphate) levels are associated with loss of desiccation tolerance in overprimed Medicago truncatula seeds.

Re-establishment of desiccation tolerance is essential for the survival of germinated seeds facing water deficit in the soil. The molecular and ultrastructural features of desiccation tolerance maintenance and loss within the nuclear compartment are not fully resolved. In the present study, the impact of desiccation-induced genotoxic stress on nucleolar ultrastructure and ribogenesis was explored along the rehydration-dehydration cycle applied in standard seed vigorization protocols. Primed and overprimed Medicago truncatula seeds, obtained through hydropriming followed by desiccation (dry-back), were analysed. In contrast to desiccation-tolerant primed seeds, overprimed seeds enter irreversible germination and do not survive dry-back. Reactive oxygen species, DNA damage and expression profiles of antioxidant/DNA Damage Response genes were measured, as main hallmarks of the seed response to desiccation stress. Nuclear ultrastructural features were also investigated. Overprimed seeds subjected to dry-back revealed altered rRNA accumulation profiles and up-regulation of genes involved in ribogenesis control. The signal molecule PAP (3'-phosphoadenosine 5'-phosphate) accumulated during dry-back only in primed seeds, as a distinctive feature of desiccation tolerance. The presented results show the molecular and ultrastructural landscapes of the seed desiccation response, including substantial changes in nuclear organization.

Physiological and molecular aspects of seed longevity: exploring intra-species variation in eight Pisum sativum L. accessions.

Conservation of plant genetic diversity is fundamental for crop improvement, increasing agricultural production and sustainability, especially in the face of climatic changes. Although seed longevity is essential for the management of seed banks, few studies have, so far, addressed differences in this trait among the accessions of a single species. Eight Pisum sativum L. (pea) accessions were investigated to study the impact of long-term (approximately 20 years) storage, aiming to reveal contrasting seed longevity and clarify the causes for these differences. The outstanding seed longevity observed in the G4 accession provided a unique experimental system. To characterize the biochemical and physical status of stored seeds, reactive oxygen species, lipid peroxidation, tocopherols, free proline and reducing sugars were measured. Thermoanalytical measurements (thermogravimetry and differential scanning calorimetry) and transmission electron microscopy combined with immunohistochemical analysis were performed. The long-lived G4 seeds neither consumed tocopherols during storage nor showed free proline accumulation, as a deterioration hallmark, whereas reducing sugars were not affected. Thermal decomposition suggested a biomass composition compatible with the presence of low molecular weight molecules. Expansion of heterochromatic areas and reduced occurrence of γH2AX foci were highlighted in the nucleus of G4 seeds. The longevity of G4 seeds correlates with the occurrence of a reducing cellular environment and a nuclear ultrastructure favourable to genome stability. This work brings novelty to the study of within-species variations in seed longevity, underlining the relevance of multidisciplinary approaches in seed longevity research.

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.

[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.

Plasma Exosome Profile in ST-Elevation Myocardial Infarction Patients with and without Out-of-Hospital Cardiac Arrest.

The identification of new biomarkers allowing an early and more accurate characterization of patients with ST-segment elevation myocardial infarction (STEMI) is still needed, and exosomes represent an attractive diagnostic tool in this context. However, the characterization of their protein cargo in relation to cardiovascular clinical manifestation is still lacking. To this end, 35 STEMI patients (17 experiencing resuscitated out-of-hospital cardiac arrest (OHCA-STEMI) and 18 uncomplicated) and 32 patients with chronic coronary syndrome (CCS) were enrolled. Plasma exosomes were characterized by the nanoparticle tracking analysis and Western blotting. Exosomes from STEMI patients displayed a higher concentration and size and a greater expression of platelet (GPIIb) and vascular endothelial (VE-cadherin) markers, but a similar amount of cardiac troponin compared to CCS. In addition, a difference in exosome expression of acute-phase proteins (ceruloplasmin, transthyretin and fibronectin) between STEMI and CCS patients was found. GPIIb and brain-associated marker PLP1 accurately discriminated between OHCA and uncomplicated STEMI. In conclusion, the exosome profile of STEMI patients has peculiar features that differentiate it from that of CCS patients, reflecting the pathophysiological mechanisms involved in STEMI. Additionally, the exosome expression of brain- and platelet-specific markers might allow the identification of patients experiencing ischemic brain injury in STEMI.

Heat Shock Affects Mitotic Segregation of Human Chromosomes Bound to Stress-Induced Satellite III RNAs.

Heat shock activates the transcription of arrays of Satellite III (SatIII) DNA repeats in the pericentromeric heterochromatic domains of specific human chromosomes, the longest of which is on chromosome 9. Long non-coding SatIII RNAs remain associated with transcription sites where they form nuclear stress bodies or nSBs. The biology of SatIII RNAs is still poorly understood. Here, we show that SatIII RNAs and nSBs are detectable up to four days after thermal stress and are linked to defects in chromosome behavior during mitosis. Heat shock perturbs the execution of mitosis. Cells reaching mitosis during the first 3 h of recovery accumulate in pro-metaphase. During the ensuing 48 h, this block is no longer detectable; however, a significant fraction of mitoses shows chromosome segregation defects. Notably, most of lagging chromosomes and chromosomal bridges are bound to nSBs and contain arrays of SatIII DNA. Disappearance of mitotic defects at the end of day 2 coincides with the processing of long non-coding SatIII RNAs into a ladder of small RNAs associated with chromatin and ranging in size from 25 to 75 nt. The production of these molecules does not rely on DICER and Argonaute 2 components of the RNA interference apparatus. Thus, massive transcription of SatIII DNA may contribute to chromosomal instability.

The chaperone activity of 4PBA ameliorates the skeletal phenotype of Chihuahua, a zebrafish model for dominant osteogenesis imperfecta.

Classical osteogenesis imperfecta (OI) is a bone disease caused by type I collagen mutations and characterized by bone fragility, frequent fractures in absence of trauma and growth deficiency. No definitive cure is available for OI and to develop novel drug therapies, taking advantage of a repositioning strategy, the small teleost zebrafish (Danio rerio) is a particularly appealing model. Its small size, high proliferative rate, embryo transparency and small amount of drug required make zebrafish the model of choice for drug screening studies, when a valid disease model is available. We performed a deep characterization of the zebrafish mutant Chihuahua, that carries a G574D (p.G736D) substitution in the α1 chain of type I collagen. We successfully validated it as a model for classical OI. Growth of mutants was delayed compared with WT. X-ray, µCT, alizarin red/alcian blue and calcein staining revealed severe skeletal deformity, presence of fractures and delayed mineralization. Type I collagen extracted from different tissues showed abnormal electrophoretic migration and low melting temperature. The presence of endoplasmic reticulum (ER) enlargement due to mutant collagen retention in osteoblasts and fibroblasts of mutant fish was shown by electron and confocal microscopy. Two chemical chaperones, 4PBA and TUDCA, were used to ameliorate the cellular stress and indeed 4PBA ameliorated bone mineralization in larvae and skeletal deformities in adult, mainly acting on reducing ER cisternae size and favoring collagen secretion. In conclusion, our data demonstrated that ER stress is a novel target to ameliorate OI phenotype; chemical chaperones such as 4PBA may be, alone or in combination, a new class of molecules to be further investigated for OI treatment.

Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ.

To streamline the elucidation of antibacterial compounds' mechanism of action, comprehensive high-throughput assays interrogating multiple putative targets are necessary. However, current chemogenomic approaches for antibiotic target identification have not fully utilized the multiplexing potential of next-generation sequencing. Here, we used Illumina sequencing of transposon insertions to track the competitive fitness of a Burkholderia cenocepacia library containing essential gene knockdowns. Using this method, we characterized a novel benzothiadiazole derivative, 10126109 (C109), with antibacterial activity against B. cenocepacia, for which whole-genome sequencing of low-frequency spontaneous drug-resistant mutants had failed to identify the drug target. By combining the identification of hypersusceptible mutants and morphology screening, we show that C109 targets cell division. Furthermore, fluorescence microscopy of bacteria harboring green fluorescent protein (GFP) cell division protein fusions revealed that C109 prevents divisome formation by altering the localization of the essential cell division protein FtsZ. In agreement with this, C109 inhibited both the GTPase and polymerization activities of purified B. cenocepacia FtsZ. C109 displayed antibacterial activity against Gram-positive and Gram-negative cystic fibrosis pathogens, including Mycobacterium abscessus C109 effectively cleared B. cenocepacia infection in the Caenorhabditis elegans model and exhibited additive interactions with clinically relevant antibiotics. Hence, C109 is an enticing candidate for further drug development.

4-PBA ameliorates cellular homeostasis in fibroblasts from osteogenesis imperfecta patients by enhancing autophagy and stimulating protein secretion.

The clinical phenotype in osteogenesis imperfecta (OI) is attributed to the dominant negative function of mutant type I collagen molecules in the extracellular matrix, by altering its structure and function. Intracellular retention of mutant collagen has also been reported, but its effect on cellular homeostasis is less characterized. Using OI patient fibroblasts carrying mutations in the α1(I) and α2(I) chains we demonstrate that retained collagen molecules are responsible for endoplasmic reticulum (ER) enlargement and activation of the unfolded protein response (UPR) mainly through the eukaryotic translation initiation factor 2 alpha kinase 3 (PERK) branch. Cells carrying α1(I) mutations upregulate autophagy, while cells with α2(I) mutations only occasionally activate the autodegradative response. Despite the autophagy activation to face stress conditions, apoptosis occurs in all mutant fibroblasts. To reduce cellular stress, mutant fibroblasts were treated with the FDA-approved chemical chaperone 4-phenylbutyric acid. The drug rescues cell death by modulating UPR activation thanks to both its chaperone and histone deacetylase inhibitor abilities. As chaperone it increases general cellular protein secretion in all patients' cells as well as collagen secretion in cells with the most C-terminal mutation. As histone deacetylase inhibitor it enhances the expression of the autophagic gene Atg5 with a consequent stimulation of autophagy. These results demonstrate that the cellular response to ER stress can be a relevant target to ameliorate OI cell homeostasis.

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.

Ultrastructural localization of 5-methylcytosine on DNA and RNA.

DNA methylation is the major epigenetic modification and it is involved in the negative regulation of gene expression. Its alteration can lead to neoplastic transformation. Several biomolecular approaches are nowadays used to study this modification on DNA, but also on RNA molecules, which are known to play a role in different biological processes. RNA methylation is one of the most common RNA modifications and 5-methylcytosine presence has recently been suggested in mRNA. However, an analysis of nucleic acid methylation at electron microscope is still lacking. Therefore, we visualized DNA methylation status and RNA methylation sites in the interphase nucleus of HeLa cells and rat hepatocytes by ultrastructural immunocytochemistry and cytochemical staining. This approach represents an efficient alternative to study nucleic acid methylation. In particular, this ultrastructural method makes the visualization of this epigenetic modification on a single RNA molecule possible, thus overcoming the technical limitations for a (pre-)mRNA methylation analysis.

The In Vitro Effects of Enzymatic Digested Gliadin on the Functionality of the Autophagy Process.

Gliadin, the alcohol-soluble protein fraction of wheat, contains the factor toxic for celiac disease (CD), and its toxicity is not reduced by digestion with gastro-pancreatic enzymes. Importantly, it is proved that an innate immunity to gliadin plays a key role in the development of CD. The immune response induces epithelial stress and reprograms intraepithelial lymphocytes into natural killer (NK)-like cells, leading to enterocyte apoptosis and an increase in epithelium permeability. In this contribution, we have reported that in Caco-2 cells the administration of enzymatically digested gliadin (PT-gliadin) reduced significantly the expression of the autophagy-related marker LC3-II. Furthermore, electron and fluorescent microscope analysis suggested a compromised functionality of the autophagosome apparatus. The rescue of the dysregulated autophagy process, along with a reduction of PT-gliadin toxicity, was obtained with a starvation induction protocol and by 3-methyladenine administration, while rapamycin, a well-known autophagy inducer, did not produce a significant improvement in the clearance of extra- and intra-cellular fluorescent PT-gliadin amount. Altogether, our results highlighted the possible contribution of the autophagy process in the degradation and in the reduction of extra-cellular release of gliadin peptides and suggest novel molecular targets to counteract gliadin-induced toxicity in CD.

Identification of Autophagy-Related Genes and Their Regulatory miRNAs Associated with Celiac Disease in Children.

Celiac disease (CD) is a severe genetic autoimmune disorder, affecting about one in 100 people, where the ingestion of gluten leads to damage in the small intestine. Diagnosing CD is quite complex and requires blood tests and intestinal biopsy examinations. Controversy exists regarding making the diagnosis without biopsy, due to the large spectrum of manifesting symptoms; furthermore, small-intestinal gastroscopy examinations have a relatively complex management in the pediatric population. To identify novel molecular markers useful to increase the sensitivity and specificity in the diagnosis of pediatric CD patients, the expression levels of two key autophagy executor genes (ATG7 and BECN1) and their regulatory validated miRNAs (miR-17 and miR-30a, respectively) were analyzed by relative quantitative real-time-PCR on a cohort of confirmed CD patients compared to age-related controls. Among the investigated targets, the non-parametric Mann-Whitney U test and ROC analysis indicated the highest significant association of BECN1 with CD status in the blood, while in intestinal biopsies, all of the investigated sequences were positively associated with CD diagnosis. Nomogram-based analysis showed nearly opposite expression trends in blood compared to intestine tissue, while hierarchical clustering dendrograms enabled identifying CD and control subgroups based on specific genes and miRNA expression signatures. Next, using an established in vitro approach, through digested gliadin administration in Caco-2 cells, we also highlighted that the modulation of miR-17 endogenous levels using enriched exosomes increased the intracellular autophagosome content, thereby altering the autophagic status. Altogether, these results highlighted novel molecular markers that might be useful to increase the accuracy in CD diagnosis and in molecular-based stratification of the patients, further reinforcing the functional involvement of the regulation of the autophagy process within a digestive and autoimmune-related disorder as CD.

Endoplasmic Reticulum Ca(2+) Handling and Apoptotic Resistance in Tumor-Derived Endothelial Colony Forming Cells.

Truly endothelial progenitor cells (EPCs) can be mobilized from bone marrow to support the vascular network of growing tumors, thereby sustaining the metastatic switch. Endothelial colony forming cells (ECFCs) are the only EPC subtype belonging to the endothelial phenotype and capable of incorporating within neovessels. The intracellular Ca(2+) machinery plays a key role in ECFC activation and is remodeled in renal cellular carcinoma-derived ECFCs (RCC-ECFCs). Particularly, RCC-ECFCs seems to undergo a drop in endoplasmic reticulum (ER) Ca(2+) concentration ([Ca(2+) ]ER ). This feature is remarkable when considering that inositol-1,4,5-trisphosphate (InsP3 )-dependent ER-to-mitochondria Ca(2+) transfer regulates the intrinsic apoptosis pathway. Herein, we sought to assess whether: (1) the [Ca(2+) ]ER and the InsP3 -induced ER-mitochondria Ca(2+) shuttle are reduced in RCC-ECFCs; and (2) the dysregulation of ER Ca(2+) handling leads to apoptosis resistance in tumor-derived cells. RCC-ECFCs displayed a reduction both in [Ca(2+) ]ER and in the InsP3 -dependent mitochondrial Ca(2+) uptake, while they expressed normal levels of Bcl-2 and Bak. The decrease in [Ca(2+) ]ER was associated to a remarkable ER expansion in RCC-ECFCs, which is a hallmark of ER stress, and did not depend on the remodeling of the Ca(2+) -transporting and the ER Ca(2+) -storing systems. As expected, RCC-ECFCs were less sensitive to rapamycin- and thapsigargin-induced apoptosis; however, buffering intracellular Ca(2+) levels with BAPTA dampened apoptosis in both cell types. Finally, store-operated Ca(2+) entry was seemingly uncoupled from the apoptotic machinery in RCC-ECFCs. Thus, the chronic underfilling of the ER Ca(2+) pool could confer a survival advantage to RCC-ECFCs and underpin RCC resistance to pharmacological treatment. J. Cell. Biochem. 117: 2260-2271, 2016. © 2016 Wiley Periodicals, Inc.

Antifungal activity of Myriocin on clinically relevant Aspergillus fumigatus strains producing biofilm.

BACKGROUND: The human pathogenic mold Aspergillus fumigatus is able to form a complex biofilm embedded in extracellular matrix. Biofilms confer antimicrobial resistance and it is well known that aspergillosis is often refractory to the conventional antifungal therapy. The treatment of biofilm-related infections poses a significant clinical challenge on a daily basis, promoting the search for new therapeutic agents. Our aim was to exploit the modulation of sphingolipid mediators as new therapeutic target to overcome antifungal resistance in biofilm-related infections. RESULTS: Antifungal susceptibility testing was performed on 20 clinical isolates of Aspergillus fumigatus and one reference strain (A. fumigatus Af293) according the EUCAST protocol. Sessile MICs were assessed on 24-h preformed-biofilm by means of XTT-reduction assay. Myriocin (0.25-64 mg/L), a commercial sphingolipid synthesis inhibitor, was used. The MEC50 value (mg/L) of Myriocin was 8 (range 4-16) for both planktonic and sessile cells. Drug-induced morphological alterations were analyzed by optical and electron microscopy (TEM) on 24h preformed A. fumigatus Af293 biofilms. An evident hyphal damage, resulting in short, stubby, and highly branched hyphae was observed by optical microscopy. At 24h, TEM studies showed important morphological alterations, such as invaginations of the cell membrane, modification in the vacuolar system and presence of multilamellar bodies, in some cases within vacuoles. CONCLUSIONS: The direct antifungal activity, observed on both planktonic and sessile fungi, suggests that inhibition of sphingolipid synthesis could represent a new target to fight biofilm-related A. fumigatus resistance.

Autophagy is modulated in human neuroblastoma cells through direct exposition to low frequency electromagnetic fields.

In neurogenerative diseases, comprising Alzheimer's (AD), functional alteration in autophagy is considered one of the pathological hallmarks and a promising therapeutic target. Epidemiological investigations on the possible causes undergoing these diseases have suggested that electromagnetic fields (EMF) exposition can contribute to their etiology. On the other hand, EMF have therapeutic implications in reactivating neuronal functionality. To partly clarify this dualism, the effect of low-frequency EMF (LF-EMF) on the modulation of autophagy was investigated in human neuroblastoma SH-SY5Y cells, which were also subsequently exposed to Aß peptides, key players in AD. The results primarily point that LF-EMF induce a significant reduction of microRNA 30a (miR-30a) expression with a concomitant increase of Beclin1 transcript (BECN1) and its corresponding protein. Furthermore, LF-EMF counteract the induced miR-30a up-regulation in the same cells transfected with miR-30a mimic precursor molecules and, on the other side, rescue Beclin1 expression after BECN1 siRNA treatment. The expression of autophagy-related markers (ATG7 and LC3B-II) as well as the dynamics of autophagosome formation were also visualized after LF-EMF exposition. Finally, different protocols of repeated LF-EMF treatments were assayed to contrast the effects of Aß peptides in vitro administration. Overall, this research demonstrates, for the first time, that specific LF-EMF treatments can modulate in vitro the expression of a microRNA sequence, which in turn affects autophagy via Beclin1 expression. Taking into account the pivotal role of autophagy in the clearance of protein aggregates within the cells, our results indicate a potential cytoprotective effect exerted by LF-EMF in neurodegenerative diseases such as AD. J. Cell. Physiol. 229: 1776-1786, 2014. © 2014 Wiley Periodicals, Inc.