Biomolecular Techniques Research Articles

Proteomics to Study Parchment Degradation – From Bulk to Spatial Analysis

Abstract Implementing biomolecular techniques in the study of written cultural heritage has led to a steady development of biocodicology and the study of DNA and proteins in parchment in the past years. In this context, matrix-assisted laser desorption/ionisation mass spectrometry (MALDI-MS) has gained increasing attention as a powerful tool to study the ancient writing support. With a particular focus on the main components of parchment – different types of collagens – proteomic applications of the technique are highlighted. The review summarises advances in biocodicological studies focusing on manufacturing aspects and conservation treatments. Herein we further investigated proteomic studies on animal skin to prove the potential of MALDI-MS imaging (MALD-MSI) to broaden our knowledge and take studies on damage assessment and degradation of parchment to another level.

Ancient DNA and paleoproteomic analysis on Roman Imperial-era individuals from Histria, Romania

Ancient biomolecules have become an increasingly important part of archaeological investigations interested in understanding population movements and health. Despite their ability to elucidate historically-attested contexts of human mobility and interaction between different cultural groups, biomolecular techniques are still underutilized in certain historical and archaeological contexts. One such context is the Roman Imperial limes, or border zone, along the lower reaches of the Danube, which saw more than five hundred years of migration, conflict, and accommodation among a wide range of populations, from Mediterranean settlers to steppe pastoralists. In this region, more than a century of archaeological investigation has unearthed the remains of tens of thousands of Roman-era individuals. However, only a limited number of contexts have undergone biomolecular analyses. While these deceased humans may offer an untapped reservoir of biomolecular information, many were collected during a period when the standard precautions and protocols for ancient biomolecular research were not yet established. Because contamination is a major barrier for successfully recovering ancient DNA and proteins, conducting a pilot study to assess bimolecular preservation of a small representative dataset of human remains before embarking on a more extensive research program may prevent unnecessary sampling. This study applies ancient DNA and paleoproteomic techniques to human remains from a Roman-period cemetery at Histria, a site located just south of the Danube at the edge of the Roman province of Moesia Inferior. The individuals from whom we sampled dentin and dental calculus were excavated between the 1940s and 1980s and were housed at the Francisc J. Rainer Institute since. Our results suggest that both microbial and human ancient DNA is preserved in the dental calculus and dentin samples. We also successfully recovered sex-specific amelogenin peptides in tooth enamel from three individuals, including a juvenile. In conclusion, our results are encouraging, signifying the feasibility of future aDNA and paleoproteomic research for this skeletal collection. Our analyses also showcase how sex estimation with genomic and proteomic methods may contradict traditional osteological approaches. These findings not only offer deeper insights into the lives of these individuals but also show promise for the investigation of broader anthropological questions, such as the impact of Roman annexation in this region.

Eco-Friendly Synthesis Methods of Gold Nanoparticles, Their Characterization and Applications in Diagnostic, Therapeutic and Sensors

Nanoparticles have unique traits which make them useful for different purposes. Numerous methods are used to manufacture nanoparticles at commercial scale. Gold nanoparticles (AuNPs) are one of the most utilized and preferred nanoparticles due to their traits like low resistivity, less toxicity, optical, high stability, fluorescence quenching ability, and "surface plasmon resonance". Gold nanoparticles were utilized in ancient Roman Times for staining glasses and till now their new applications are being discovered every day. Various methodologies are utilized for Gold nanoparticle synthesis including conventional chemical methods, UV rays, polymers, ultrasound, plant and microbe-mediated techniques, etc. Conventional techniques are not eco-friendly or cost-effective. Nowadays plants and microbes being cost-effective and eco-friendly are preferred for gold nanoparticle synthesis. Various extracellular, intracellular, and biomolecular techniques are being utilized to manufacture gold nanoparticles. Gold nanoparticles have a vast scope in chemical, biomedicine, food, electronic and forensic industries. AuNPs are widely utilized as sensors, also as carriers in Drug delivery, Photothermal therapy, Heavy metal ion detection etc. This review describes various synthesis techniques, applications, and characterizations of AuNPs.

Application of biomolecular techniques on tsetse fly puparia for species identification at larvipostion sites.

Puparia are commonly found in tsetse fly larviposition sites during studies on larval ecology. This chitinous shell is representative of past or ongoing exploitation of these sites by tsetse flies. The morphological characteristics of the puparium are not sufficiently distinctive to allow identification of the species. This study explores the applicability of biomolecular techniques on empty puparia for tsetse fly species identification. Five techniques were compared for DNA extraction from tsetse fly puparia, 1/Chelex® 100 Resin, 2/CTAB, 3/Livak's protocol, 4/DEB + proteinase K and 5/QIAamp® DNA Mini kit, using two homogenisation methods (manual and automated). Using a combination of two primer pairs, Chelex, CTAB, and DEB + K proved the most efficient on fresh puparia with 90, 85, and 70% samples identified, respectively. Shifting from fresh to one- to nine-month-old puparia, the Chelex method gave the best result allowing species identification on puparia up to seven months old. The subsequent testing of the Chelex extraction protocol identified 152 (60%) of 252 field-collected puparia samples at species level. The results show that reliable genetic identification of tsetse flies species can be performed from empty puparia, what can prove of great interest for future ecological studies on larviposition sites. The Chelex technique was the most efficient for DNA extraction, though the age-limit of the samples stood at seven months, beyond which DNA degradation probably compromises the genetic analysis.

Sex Determination and Isotopic Analysis of the Nivåfjord Mesolithic Burials, Zealand, Denmark

Since 1992 the prehistoric Nivåfjord in northeast Zealand, Denmark, has yielded an appreciable number of inhumation burials and cremations dating to the Mesolithic, especially the sites of Nivå 10 and Nivågård. Unfortunately, the micro-region is characterised by poor organic preservation, restricting the successful application of biomolecular techniques to human remains, including large-scale radiocarbon dating programmes as well as both stable isotope and ancient DNA analyses. Here, we apply an alternative technique, an acid etch peptide-based method, to determine the sex of eight individuals from Nivå 10 as well as the Nivågård child. Moreover, we revisit the utility of stable carbon (δ13C), nitrogen (δ15N) and sulfur (δ34S) isotope analysis of human tissues to reconstruct the life histories and diets of 10 individuals from Nivå 10 as well as the Nivågård child. To contextualise further, we sampled 14 Capreolus capreolus and three Sus scrofa from the Nivågård site for stable isotope analysis. We demonstrate that sex can successfully be determined from contexts susceptible to poor organic preservation, and show that the Nivågård child spent a proportion of its life outside a sea spray-influenced environment, and consumed significant quantities of marine protein as demonstrated by its δ13C and δ34S values.

Interdisciplinary evidence for early domestic horse exploitation in southern Patagonia

The introduction of domestic horses transformed Indigenous societies across the grasslands of Argentina, leading to the emergence of specialized horse cultures across the Southern Cone. However, the dynamics of this introduction are poorly chronicled by historic records. Here, we apply archaeozoological and biomolecular techniques to horse remains from the site of Chorrillo Grande 1 in southern Argentina. Osteological and taphonomic analyses suggest that horses were pastorally managed and used for food by Aónikenk/Tehuelche hunter-gatherers before the onset of permanent European settlement, as early as the mid-17th century. DNA-based sex identifications suggest consumption of both male and female horses, while ceramic residue also shows use of guanaco products. Sequential isotope analyses on horse dentition reveal an origin in southern Patagonia and movement of these animals between the Río Coig and Río Gallegos basins. These results reinforce emerging evidence for rapid Indigenous-mediated dispersal of horses in the Americas and demonstrate that horses catalyzed rapid economic and social transformations.

DEfiNING SPATIAL HETEROGENEITY OF A NOVEL LIVING BIOBANK OF POST-SURGICAL RESIDUAL GLIOBLASTOMA TO DEVELOP STRATEGIES FOR TARGETED THERAPY

Abstract AIMS Glioblastoma is the most common cancer arising within the brain accounting for >2,000 deaths/year in the UK. Current standard-of-care therapy consists of maximal safe surgical resection followed by radiochemotherapy which activates the cellular DNA damage response (DDR). However, disease prognosis remains poor (median OS ~12-15 months). This stagnation in the development and clinical translation of novel therapeutics is largely attributed to extensive inter- and intra-tumoral heterogeneity and resistant glioma stem cell (GSC) niches. Traditionally, primary ex vivo and in vivo models of glioblastoma have been generated from the resected tumour mass potentially omitting the post-surgical residual tumour cells that give rise to disease recurrence and morbidity. METHOD Multiple parallel primary ex vivo GSC cell models were generated from anatomically discrete regions of en-bloc partial lobectomy glioblastoma specimens to produce 3D models of resected (tumour “core”) and typically residual (distal invasive tumour “edge”) GSC niches. These models were characterised using an array of biomolecular techniques and 3D based assays. RESULTS Preliminary RNA-seq analyses revealed largely divergent transcriptional landscapes for resected vs residual GSCs and protein expression/immunofluorescence analyses also identified key differences in GSC and DDR ex- pression, and subsequent cellular responses to current standard-of-care radio-chemotherapy regimens. CONCLUSIONS These data reveal phenotypic relationships between GSC “stemness” and DDR activity, therefore highlighting the potential clinical relevance of our novel parallel resected and residual ex vivo glioblastoma models. Characterisation of further models from within our 3D GSC biobank will also aid evaluation of novel therapeutics capable of targeting post-surgical GSC subpopulations leading to improved disease outcomes.

Modern Analytical Techniques for Berry Authentication

The health-related properties attributed to berries and the subsequent interest awakened within the market of functional foods mean that these small fruits may be potential targets for food fraud. In this review, studies on berry authentication through modern analytical techniques are discussed in detail. Most of the studies reported to date are related to chemical approaches, mainly chromatographic techniques. Other chemical (NMR, NIR, and Raman spectroscopy), biomolecular, and isotopic methods have also delivered promising results in the field of berry authentication, although there is still limited information available in this respect. Despite the potential of the methods described in the present review, to date, there is no universal one. Therefore, combinations of different approaches in order to complement each other are increasingly used (e.g., HPTLC and mass spectrometry; Raman and IR spectroscopies; biomolecular and analytical techniques…). Considering that adulteration practices are increasingly evolving, continuous research in the field of food authentication is needed, especially in the case of berries, since there are still some berry species that have not yet been included in any authentication study.

Molecular mechanisms of multi-omic regulation in breast cancer.

Breast cancer is a complex disease that is influenced by the concurrent influence of multiple genetic and environmental factors. Recent advances in genomics and other high throughput biomolecular techniques (-omics) have provided numerous insights into the molecular mechanisms underlying breast cancer development and progression. A number of these mechanisms involve multiple layers of regulation. In this review, we summarize the current knowledge on the role of multiple omics in the regulation of breast cancer, including the effects of DNA methylation, non-coding RNA, and other epigenomic changes. We comment on how integrating such diverse mechanisms is envisioned as key to a more comprehensive understanding of breast carcinogenesis and cancer biology with relevance to prognostics, diagnostics and therapeutics. We also discuss the potential clinical implications of these findings and highlight areas for future research. Overall, our understanding of the molecular mechanisms of multi-omic regulation in breast cancer is rapidly increasing and has the potential to inform the development of novel therapeutic approaches for this disease.

Pollen and Molecular Biomarkers from Sedimentary Archives in the Central Po Plain (N Italy): Assessing Their Potential to Deepen Changes in Natural and Agricultural Systems

This paper proposes to improve the information provided by biological indicators from sedimentary archives by integrating biomolecular techniques and botanical skills. This study represents a first proposal for combining pollen and biomolecular markers to detect land use and improve knowledge of past environmental change drivers. The specific aim of the research is to verify the relationship between miliacin (a pentacyclic triterpene methyl ether, usually interpreted as a broomcorn millet biomarker) and Panicum pollen in three near-site stratigraphic sequences of the Terramara S. Rosa di Poviglio (Po Plain, N Italy). The three cores span the last ~15,000 years and potentially record the beginning of Panicum miliaceum cultivation attested in the area since at least the Bronze Age within the Terramare culture. Despite the fact that Panicum pollen grains were rare in the spectra and miliacin was barely detectable in most of the 31 samples selected for biomolecular analyses, their combined evidence testifies to the local presence of the plant. Panicum pollen and sedimentary miliacin suggest the adoption of millet crops during the Recent Bronze Age by the Terramare culture, when climatic instability led to the diversification of cereal crops and the shift to drought-tolerant varieties.

#2556 BIOLOGICAL EFFECTS OF A SHORT PERIOD OF NORMOTHERMIC MACHINE PERFUSION IN MARGINAL KIDNEYS: RESULTS OF A PROTEOMICS ANALYSIS

Abstract Background and Aims Renal normothermic machine perfusion (NMP) is an organ preservation method based on the circulation of a warm (35–37°C) perfusion solution through the renal vasculature to deliver oxygen and nutrients. However, the biological effects of this technique on the marginal kidneys are uncertain. We consequently used mass spectrometry to define the proteomic profile of kidney tissue and urine from eight organs, considered unsuitable for transplantation, reconditioned for 120 min using a Kidney Assist device. Method Biopsies were taken during the pre-implantation histological evaluation (T-1), at the start of back table preparation (T0), and after 60 and 120 min of perfusion (T60, T120). Urine was collected at T0, T30, T60, and T120. Multiple algorithms, support vector machine (SVM) learning and partial least squares discriminant analysis (PLS-DA) were used to select the most discriminative proteins during NMP. Results Statistical analysis revealed the upregulation of 169 proteins and the downregulation of 196 in kidney tissue during NMP. Machine learning algorithms recognized the top 50 most discriminative proteins, five of which were concomitantly upregulated (LXN, ETFB, NUDT3, CYCS and UQCRC1) and six downregulated (CFHR3, C1S, CFI, KNG1, SERPINC1, and F9) in the kidney and urine after NMP. Functional analysis showed that the most upregulated proteins were involved in the oxidative phosphorylation system and ATP synthesis, whereas the downregulated proteins were involved in the complement system and coagulation cascade. Results were, then, validated by classical and highly conservative biomolecular lab techniques. Conclusion Our proteomic analysis revealed that even brief periods of NMP induce substantial metabolic and biochemical changes in marginal organs, which supports the use of this promising technique in the clinic.

Embracing Innovation and Accessibility in Biomolecular Research Publishing.

We are thrilled to share the latest developments at the Journal of Biomolecular Techniques (JBT), your esteemed peer-reviewed publication dedicated to advancing biotechnology research. Since its inception, JBT has been committed to promoting the pivotal role that biotechnology plays in contemporary scientific endeavors, fostering knowledge exchange among biomolecular resource facilities, and communicating the groundbreaking research conducted by the Association's Research Groups, members, and other investigators.

Revised Classification and Definition of Epilepsy Syndromes with Onset in Childhood by the International League Against Epilepsy Task Force

This article presents the updated, modified classification and definitions of epileptic syndromes, proposed by the consensus of International League Against Epilepsy (ILAE) task force. The ILAE report suggests a nosological revision of the epilepsy syndromes with onset in childhood, which are subdivided into three categories: (1) self-limited focal epilepsies; (2) genetic generalized epilepsies; and (3) developmental and/or epileptic encephalopathies. The most significant revised definition in childhood epilepsy syndromes is “self-limited,” formerly known as “benign.” These changes are essential to review and incorporate into practice when caring for patients with epilepsy. More data on the connection between genetic etiologies and epilepsy syndromes should be gathered using highly advanced biomolecular techniques for precision medicine. Key words: Childhood benign focaleEpilepsy; Benign epilepsy of childhood with centrotemporal spikes; Childhood absence epilepsy; Epilepsy syndrome.

Molecular dynamics simulations reveal the impact of NUDT15 R139C and R139H variants in structural conformation and dynamics

NUDT15, also known as MTH2, is a member of the NUDIX protein family that catalyzes the hydrolysis of nucleotides and deoxynucleotides, as well as thioguanine analogues. NUDT15 has been reported as a DNA sanitizer in humans, and more recent studies have shown that some genetic variants are related to a poor prognosis in neoplastic and immunologic diseases treated with thioguanine drugs. Despite this, the role of NUDT15 in physiology and molecular biology is quite unclear, as is the mechanism of action of this enzyme. The existence of clinically relevant variants has prompted the study of these enzymes, whose capacity to bind and hydrolyze thioguanine nucleotides is still poorly understood. By using a combination of biomolecular modeling techniques and molecular dynamics, we have studied the monomeric wild type NUDT15 as well as two important variants, R139C and R139H. Our findings reveal not only how nucleotide binding stabilizes the enzyme but also how two loops are responsible for keeping the enzyme in a packed, close conformation. Mutations in α2 helix affect a network of hydrophobic and π-interactions that enclose the active site. This knowledge contributes to the understanding of NUDT15 structural dynamics and will be valuable for the design of new chemical probes and drugs targeting this protein. Communicated by Ramaswamy H. Sarma

Precision Medicine Approach Based on Molecular Alterations for Patients with Relapsed or Refractory Multiple Myeloma: Results from the MM-EP1 Study.

Despite that cytogenetic and molecular analysis of tumor cells can rapidly identify recurring molecular abnormalities, no personalized therapy is currently available in the setting of relapsed/refractory multiple myeloma (r/r MM). MM-EP1 is a retrospective study aimed at comparing a personalized molecular-oriented (MO) versus a non-molecular-oriented (no-MO) approach in r/r MM. Actionable molecular targets and their associated therapies were the BRAF V600E mutation and BRAF inhibitors; t(11;14)(q13;q32) and BCL2 inhibitors; and t(4;14)(p16;q32) with FGFR3 fusion/rearrangements and FGFR3 inhibitors. One hundred three highly pretreated r/r MM patients with a median age of 67 years (range 44-85) were included. Seventeen (17%) patients were treated using an MO approach with BRAF inhibitors (vemurafenib or dabrafenib, n = 6), BCL2 inhibitor (venetoclax, n = 9), or FGFR3 inhibitor (erdafitinib, n = 2). Eighty-six (86%) patients received non-MO therapies. Overall response rate was 65% in MO patients versus 58% in the non-MO group (p = 0.053). Median PFS and OS were 9 and 6 months (HR = 0.96; CI95 = 0.51-1.78; p = 0.88) and 26 and 28 months (HR = 0.98; CI95 = 0.46-2.12; p = 0.98), respectively, in MO and no-MO patients. Despite the low number of patients treated with an MO approach, this study highlights the strengths and weakness of a molecular-targeted approach for the treatment of multiple myeloma. Widespread biomolecular techniques and improvement of precision medicine treatment algorithms could improve selection for precision medicine in myeloma.

Prevalence of Eucoleus garfiai in Wild Boars Hunted at Different Altitudes in the Campania and Latium Regions (Italy).

Recent reports of Eucoleus garfiai in wild boars in southern Italy have highlighted the need for collecting epidemiological data on the presence of this parasite and understanding the role of possible interactions between wild boars, E. garfiai, and the environment. This study analyses, using histopathological and biomolecular techniques, the presence of E. garfiai in tongue samples of wild boars hunted in four provinces of the Campania and Latium regions (Italy), in areas located above and below 900 m above sea level (asl). Histopathological examinations revealed the presence of adults and eggs of nematodes, which were subsequently identified as E. garfiai by biomolecular analysis, in the tongue epithelium. The detection of the parasite was more frequent in samples collected from hunting areas located above 900 m asl than in those collected from areas located below 900 m asl (66.67% vs. 38.09%; p < 0.01). Some species of earthworms are intermediate hosts of E. garfiai and it is well known that earthworms are more present in high quality soils. Therefore, we can suggest that the higher prevalence of E. garfiai at higher altitudes is probably linked to a greater presence of earthworms in the soil, due to its higher quality in these areas.

Dental calculus and isotopes reveal information about ancient diet of Boccone del Povero community (1st-2nd century CE, Latium, central Italy)

Evidence about the dietary habits of the 42 individuals recovered in the necropolis of Boccone del Povero (1st-2nd century CE; Rome, Italy) was obtained by a multidisciplinary approach. Dental calculus analysis (through optical microscopy and gas chromatography-mass spectrometry) and stable isotope investigation from bone proteins were carried out on the human skeletal series, with a particular focus on the young female unearthed from the richest burial (no. CLXXXI) of the archaeological site. Overall, carbon and nitrogen stable isotope data indicated an omnivorous diet mainly based on C3-terrestrial proteins, although for some individuals C4 plant consumption cannot be excluded. In fact, evidence from calculus suggests the use of Andropogoneae (e.g., sorghum), Triticeae, together with Rosaceae seeds (e.g., almonds), milk and dairy products. The application of morphological, biochemical, and biomolecular techniques provided a reconstruction of the subsistence pattern of this ancient population living in a suburb of the Imperial Rome.

A PCR-based method for the diagnosis of Enterobius vermicularis in stool samples, specifically designed for clinical application.

Enterobius vermicularis (E. vermicularis) is a nematode that infects up to 200 million people worldwide, despite effective medications being available. Conventional diagnostic tests are hindered by low sensitivity and poor patient compliance. Furthermore, no biomolecular techniques are available for clinical application. The aim of this study was to develop a procedure specifically designed for clinical application to detect E. vermicularis by means of PCR. Two subject groups were taken into account: a group of 27 infected patients and a control group of 27 healthy subjects. A nested-PCR was performed on fecal samples to detect E. vermicularis. Due to the intrinsic difficulties of the fecal matrix, several countermeasures were adopted to ensure the efficient performance of the method: (a) a large amount of feces for the extraction process (20 g instead of 200 mg); (b) a combination of chemical and physical treatments to grind the fecal matrix; (c) an additional purification process for the negative samples after the first nested-PCR; and (d) the selection of a very specific target region for the PCR. Due to the lack of overlap with other organisms, a sequence of the 5S ribosomal DNA (rDNA) spacer region including the tract SL1 was chosen to design appropriate external and internal primers. The first nested-PCR detected E.vermicularis in 19/27 samples from infected patients. After further purification, 5/8 of the negative samples resulted positive at the second PCR. Conversely, all the samples from healthy controls resulted negative to both PCRs. Sensitivity and specificity of the method were, respectively, 88.9% and 100%. The results prove the high diagnostic accuracy of the proposed method, addressing and overcoming the challenges posed by both conventional tests and PCR-based approaches. Therefore, the method can be proposed for clinical application.

Defective Desmosomal Adhesion Causes Arrhythmogenic Cardiomyopathy by Involving an Integrin-αVβ6/TGF-β Signaling Cascade.

Arrhythmogenic cardiomyopathy (ACM) is characterized by progressive loss of cardiomyocytes with fibrofatty tissue replacement, systolic dysfunction, and life-threatening arrhythmias. A substantial proportion of ACM is caused by mutations in genes of the desmosomal cell-cell adhesion complex, but the underlying mechanisms are not well understood. In the current study, we investigated the relevance of defective desmosomal adhesion for ACM development and progression. We mutated the binding site of DSG2 (desmoglein-2), a crucial desmosomal adhesion molecule in cardiomyocytes. This DSG2-W2A mutation abrogates the tryptophan swap, a central interaction mechanism of DSG2 on the basis of structural data. Impaired adhesive function of DSG2-W2A was confirmed by cell-cell dissociation assays and force spectroscopy measurements by atomic force microscopy. The DSG2-W2A knock-in mouse model was analyzed by echocardiography, ECG, and histologic and biomolecular techniques including RNA sequencing and transmission electron and superresolution microscopy. The results were compared with ACM patient samples, and their relevance was confirmed in vivo and in cardiac slice cultures by inhibitor studies applying the small molecule EMD527040 or an inhibitory integrin-αVβ6 antibody. The DSG2-W2A mutation impaired binding on molecular level and compromised intercellular adhesive function. Mice bearing this mutation develop a severe cardiac phenotype recalling the characteristics of ACM, including cardiac fibrosis, impaired systolic function, and arrhythmia. A comparison of the transcriptome of mutant mice with ACM patient data suggested deregulated integrin-αVβ6 and subsequent transforming growth factor-β signaling as driver of cardiac fibrosis. Blocking integrin-αVβ6 led to reduced expression of profibrotic markers and reduced fibrosis formation in mutant animals in vivo. We show that disruption of desmosomal adhesion is sufficient to induce a phenotype that fulfils the clinical criteria to establish the diagnosis of ACM, confirming the dysfunctional adhesion hypothesis. Deregulation of integrin-αVβ6 and transforming growth factor-β signaling was identified as a central step toward fibrosis. A pilot in vivo drug test revealed this pathway as a promising target to ameliorate fibrosis. This highlights the value of this model to discern mechanisms of cardiac fibrosis and to identify and test novel treatment options for ACM.

Ozone at low concentration modulates microglial activity in vitro: A multimodal microscopy and biomolecular study.

Oxygen-ozone (O2 -O3 ) therapy is an adjuvant/complementary treatment based on the activation of antioxidant and cytoprotective pathways driven by the nuclear factor erythroid 2-related factor 2 (Nrf2). Many drugs, including dimethyl fumarate (DMF), that are used to reduce inflammation in oxidative-stress-related neurodegenerative diseases, act through the Nrf2-pathway. The scope of the present investigation was to get a deeper insight into the mechanisms responsible for the beneficial result of O2 -O3 treatment in some neurodegenerative diseases. To do this, we used an integrated approach of multimodal microscopy (bright-field and fluorescence microscopy, transmission and scanning electron microscopy) and biomolecular techniques to investigate the effects of the low O3 concentrations currently used in clinical practice in lipopolysaccharide (LPS)-activated microglial cells human microglial clone 3 (HMC3) and in DMF-treated LPS-activated (LPS + DMF) HMC3 cells. The results at light and electron microscopy showed that LPS-activation induced morphological modifications of HMC3 cells from elongated/branched to larger roundish shape, cytoplasmic accumulation of lipid droplets, decreased electron density of the cytoplasm and mitochondria, decreased amount of Nrf2 and increased migration rate, while biomolecular data demonstrated that Heme oxygenase 1 gene expression and the secretion of the pro-inflammatory cytokines, Interleukin-6, and tumor necrosis factor-α augmented. O3 treatment did not affect cell viability, proliferation, and morphological features of both LPS-activated and LPS + DMF cells, whereas the cell motility and the secretion of pro-inflammatory cytokines were significantly decreased. This evidence suggests that modulation of microglia activity may contribute to the beneficial effects of the O2 -O3 therapy in patients with neurodegenerative disorders characterized by chronic inflammation. HIGHLIGHTS: Low-dose ozone (O3 ) does not damage activated microglial cells in vitro Low-dose O3 decreases cell motility and pro-inflammatory cytokine secretion in activated microglial cells in vitro Low-dose O3 potentiates the effect of an anti-inflammatory drug on activated microglial cells.