Abstract: Targeted enzymatic modification of DNA, RNA and proteins by addition of a methyl group is the prevalent epigenetic regulatory mechanism in higher eukaryotes. Methylation has been linked to a variety of biological processes including regulation of gene expression and cell identity. In DNA, three methyltransferases - DNMT1, DNMT3A, and DNMT3B - facilitate the transfer of a methyl group to the fifth carbon of a cytosine residue in CpG dinucleotides generating 5-methylcytosine. Aberrant genomic methylation profiles are associated with and serve as diagnostic markers for numerous human diseases. DNA methyltransferases are attractive therapeutic targets for drug design, however, the interplay and exact roles of the three DNMT enzymes in different biological contexts remains understudied. To achieve selective catalytic tracking of each individual Dnmt enzyme, we engineered the catalytic centre of mouse Dnmt1 or Dnmt3A for preferential transfer of chemical moieties containing functional azide groups from synthetic AdoMet cofactor analogs. We derived a series of mouse and human cell lineages by installing the derived codons in one or both alleles of the Dnmt1 or Dnmt3A gene using precise genome editing. To enable selective catalysis-dependent azide-tagging of the DNMT-specific targets in vivo, we elaborated an electroporation-based procedure for pulse-internalization of an azide cofactor analog into the engineered cells or, most recently, its metabolic in-cell production from a corresponding synthetic analogue of methionine using a genome-engineered methionine-adenosyl transferase. The deposited azide groups were exploited as ‘click’ handles for precise mapping of the tagged methylation sites in the genome using TOP-seq profiling or for direct nanopore sequencing. Pilot studies of mouse ESCs and 3T3-L1 fibroblasts unveil detailed roles of the individual DNMTs during cell state transitions. Altogether, we present a new experimental platform for high-resolution genome-wide tracking of individual epigenetic writers in live cells providing unprecedented inroads into deciphering and altering mammalian epigenetic mechanisms, and paving new ways for developing next-generation diagnostic and therapeutic approaches.

Related references:
Staševskij et al. Mol Cell, 2017, 65: 554–64.
Stankevičius et al. Mol Cell, 2022, 82:1053–65.
Vilkaitis et al. Acc Chem Res, 2023, 56: 3188-97.
Gasiulė et al. J Amer Chem Soc, 2024, 146: 18722–29.

Keywords: Epigenetic mechanisms, DNA methylation, biomolecular engineering, epigenomic technologies

Biography: Prof. Saulius Klimašauskas graduated Organic Chemistry at Vilnius University and then worked on first characterization of bacterial DNA cytosine-N4 methyltransferases with Prof. Arvydas Janulaitis at the Institute of Applied Enzymology Fermentas in Vilnius, Lithuania to receive his Ph.D. in Bioorganic Chemistry in 1987. He was a postdoctoral scientist in structural and molecular biology of DNA cytosine-5 methyltransferases with Dr. Sir Richard J. Roberts (NL, FRS) at Cold Spring Harbor Laboratory, where he was a major contributor to the discovery of a novel type of DNA-protein interaction – DNA base flipping. After starting his own group in 1995 at the Institute of Biotechnology in Vilnius, Lithuania he grew through the ranks of Head of Laboratory, Head of Department to become a Distinguished Research Professor at the Institute of Biotechnology, Life Sciences Center, Vilnius University in 2017. Dr. Klimašauskas was a repeat HHMI International Research Scholar (1995–2005), a JSPS invited professor at Osaka University (2002) and a recipient of a prestigious ERC advanced grant (2016-2023). He was elected and a full member of the Lithuanian Academy of Sciences, a Fellow of the Royal Society of Chemistry and an EMBO member. His long standing research interests span mechanistic studies and molecular engineering of AdoMet-dependent methyltransferases and epigenetic mechanisms involving biological modification of DNA and RNA. He co-authored over 100 research and review articles in high impact journals and obtained over 10 international patentsand commercial licences on novel technologies for DNA/RNA labeling and epigenome analysis.

Abstract: Nervous System (NS) is formed by different neural cells, such as astrocytes, neurons and oligodendroglia in the central nervous system (CNS) and swan cells in the peripheric NS. Neurons are speciality cells that communicate each other and with all body cells. Oligodendroglia and Swan cells, are productors of myeline in the CNS and in peripheric NS respectively. Astrocytes are special cells in the NS with many functions, such as fooding the neurons and myeline productor cells with glucose; elimination of glutamate in synapsis; constituent of blood brain barrio (BBB); productor of cytokines and chemokines to control inflammation; take care of the microbiota; elimination bacteria and virus no autochthon inside the NS; have a role in memory and learning producing long and short term potentiation; pass to reactive astrocytes to protect neurons, oligodendroglia and swan cells; form a plate in the CNS when is broken to evite the communication between cells to protect NS from bacteria and virus. Furthermore, astrocytes control toxic action of Aβ peptide and plates in Alzheimer’s disease; control insulin receptor and increase pAKT to decrease GSK-3 activating GS (glutamate synthase) to eliminate glucose inside the cells and, of course reduce pTAU; increase IDE (inhibitor of degrading enzyme) that decrease insulin and Aβ; because noradrenalin can unit to its receptor and, to Aβ too producing GSK-3, astrocytes diminish the increase of pTAU production. All these demonstrate than astrocytes can be the more important cells in our nervous system.
Keywords: Neurons; Astrocytes; oligodendroglia; ATP increase; IDE; GSK-3; pTAU; Aβ peptide; BBB; CNS; PNS; learning; memory.

Acknowledgements: “Fineurol group”

Biography: In 1985, Prof. Valles graduated in Biological Science at the University of Valencia and in 1996 I finished my PhD in the Cytological Research Institute (IEC) looking for astrogliogenesis during brain development. In 1997, I spent three years at Hallamshire Hospital, Sheffield, UK, working in inflammation and regulation of IL-1 and IL-1 receptor. In 2000, I returned to Spain at Department of Physiology, Faculty of Medicine in University of Valencia until now. I was working in toxicity in developmental and neurodegenerative disease (such as Alzheimer’s disease) looking for oxidative stress and inflammation mechanisms. Actually, I have my own laboratory “FINEUROL” and I am working in neurodegeneration and neurodevelopment. I have lectures in the School of Medicine as a Professor.

Biography: Prof. Nagwa A Meguid is a professor of human genetics and special needs, Former Head of Human Genetics Institute, (NRC) in Egypt. She holds a Ph.D. in Human Genetics, and she is a Senior Geneticist at the Genetics Institute, Pasadena, California; a fellow of Uppsala University, Sweden and senior geneticist at Yale University USA. She wins the outstanding L’Oreal UNESCO Award for women in Science; Africa & Middle East. Her work spans a wide area that ties together work from different disciplines; genetics, psychiatry, behavioral neuroscience and women empowerment. Awarded the National State Award of Excellence in Advanced Medical science & Technology. She was given the Distinctive Arab Female Scientist Prize in Genetics by the Arabian Gulf University. Recently Schowman Arab Award in Autism2022. She used her expertise to identify and describe several novel genetic syndromes. Had more than 100 International publications in elite Journals. Role model for postgraduate students, Supervising 80 theses. Head of the laboratory of research in DNA in behavioral disorders and Founder of autism research study group in Egypt. Jury president L’OREAL-UNESCO Egypt awards for young researchers. Head of Child Brain Research Group and Council for Nutritional and Environmental Medicine, Norway.
She built a remarkable library of ASD cases, publishing more than 70 articles in International Journals handling discovery of new genes and new Biomarkers in autistic children. She published a Chapter on Autism in Egypt, in a book by Prof. Alfred Volkmar. She is the head and Founder of autism research group study at NRC, Egypt. Had two Licensed patents for autism. Developed new strategies in Information Communication Technology application for broken neglected women having disabled children combating the stigmatization and alienation of women who bear disabled children in Egypt. Chapter in Women and ICT in Africa and the Middle East: Changing Selves, Changing Societies (2016): Published by Zed Books in collaboration with the International Development Research Centre in Canada. Jury president L’OREAL-UNESCO Egypt awards for young women researchers. Head of CONEM Egypt Child Brain Research Group and member in Council for Nutritional and Environmental Medicine, Norway. Peer Reviewer for Scientific elite International Journals. Member in Bioethics Network at NRC. She used genetics to understand the links between brain structure and cognitive abilities of neuro-developmental disorders.. Her work has a strong gene-brain-behavior research focus.

Abstract: We aim at advancing an effective setup for rapid phenotypic Antimicrobial Susceptibility Testing (AST) and discuss the analysis performed at population versus single cell levels. Electrical Impedance Spectroscopy (EIS) is commonly used for functional characterization of living cells. EIS allows direct assessment of membrane integrity and, as such, of the cellular viability status. While entire cellular population can be electrically addressed at once, single-cell EIS analysis is provided either by Impedance Cytometry or by label-free Electrically Modulated Light Microscopy. However, only the latter allows analysis of (single) cell cycle dynamics alterations due to drug exposure. For rapid capturing and concentrating microbial cells from biological samples, we have developed a proprietary immuno-magnetic cell separation assay capable of expediting analyses either merely by EIS, as well as by electrically actuated optical microscopy. Whereas EIS analysis of a cluster (population) of magnetically tagged microorganisms allowed phenotypic AST in just 15 minutes, for thorough assessment of heterogenous microbial cell response to antimicrobials, single-cell assays have to be performed. Adding AC electrical actuation to light microscopy provides label-free, high-resolution images of both optical path and electrical impedance of living cells. These maps relate to the distribution of the refractive index and conductivity, as complementary intrinsic cellular parameters and imaging contrast elements. This multimodal method provides new capabilities for determining both electrical and optical structures of (microbial) cells and their dynamic changes per se, or in response to exposure to antimicrobial drugs. Our presentation will highlight our recent developments in: (1) cell separation, (2) in-situ AST assessment based on EIS fingerprint of magnetically tagged microbial cells as well as (3) of analysis of (single) cell cycle dynamics changes, and related heterogeneities, revealed by Electrically Modulated Light Microscopy. We will emphasize the complementarity between optical (refractive index) and electrical (conductivity) maps/images especially concerning the viability status.
Keywords: EIS, electro-optical microscopy, phenotypic antimicrobial susceptibility testing, electrical and structural fingerprint

Biography: Prof. Eugen Gheorghiu graduated (Bio)Physics at the University of Bucharest, then worked on: (1) developing microscopic models on the dielectric behavior of (non)spheroidal living cells, (2) analyzing cell cycle progression using non-linear modelling, to receive his Ph.D. in theoretical physics with Prof Aureliu Sandulescu in 1994. He then established the biophysics lab within the National Institute of Biotechnology in Bucharest. He was a JSPS fellow at Kyoto University (1996-1997), then established the International Centre of Biodynamics, under UNESCO. During 2003-2009 he coordinated the Master Programme in Biodynamics, within the University of Bucharest, since 2004- he is PhD Advisor, University of Bucharest. His long-standing research interests span developments of electrically modulated optical assays supporting non-invasive analysis of living cell dynamics (per se and exposed to drugs), rapid detection of markers in blood (using portable, multichannel SPR systems), fast point-of-care methods and devices for: (a) sensitive identification & quantitation of microbes, and (b) Phenotypic Antibacterial & Antifungal Susceptibility/Resistance Testing based on immune-magnetic capture, magnetophoresis and electro-optical fingerprinting. He is first author of over 10 international and Romanian patents, and corresponding author of over 50 papers, a recent land-mark relates to high-resolution mapping of the electrical impedance and optical path at nanoscale, based on quantitative phase imaging combined with electrical actuation, reported in Nature-LSA (2021).

Abstract: Quantitation of cell dynamics is a prerequisite for demanding cell based biosensing applications related to cytotoxicity testing.
We take the challenge to advance a novel cellular sensing assay with boosted sensitivity and reproducibility for evaluating cytotoxicity of novel drugs and bio-materials. The dynamics of the: distribution of electro-optical cellular parameters (refractive index, and electrical conductivity), membrane permeability, cell-surface and cell-cell interaction were chosen as reporter processes. This work is connected with radically new support concepts recently developed associated with improved bio-sensing capability of dynamic control of electrified bio-interfaces’ response to an exogenous rhythm in cell-based platforms.
We present a multimodal, label free, high spatial resolution and low temporal noise functional imaging instrument enabling high resolution optical and electrochemical mapping - the latter beyond the limitations of electrode-based technologies (surface or scanning ones). It exploits the electrical modulation of the refractive index of a tailored (sensing) interface and of live cells, via an externally applied sinusoidal voltage to provide label free electric contrast of adhered and hydrogel matrix incorporated model eukaryotic cells. This enables high content label free assessment of cell parameters and is capable to provide electrical, morphological and structural properties of cellular structures dynamics relevant for toxicity evaluation.
We highlight the virtues and challenges of this novel opto-electrochemical method as an enabling tool for live cells analysis when undergoing toxic and cytopathic effects or in response to a model drug (a last resort antibiotic) presented as a case study.
The unique detection characteristics of this cellular platform are essential for applications in the fields of cell signalling, drug screening and hazard evaluation.
Reference support work US Patent US 11,680,939 B2; Anal. Chem, (2020), DOI: 10.1021/acs.analchem.9b03217; Light Sci Appl (2021) 10.1038/s41377-020-00461-x

Keywords: advanced cell based biosensing, label free electro-optical imaging, toxicity testing.

Acknowledgements: This research is supported by The Romanian Ministry for Education and Research, grant BioSense, PNRR-III-C9-2023-I8, contract CF129-31.07.2023.

Biography: Research Prof. Mihaela Gheorghiu is the head of the Biosensing Department of the International Centre of Biodynamics, Bucharest, Romania and PhD thesis advisor within the University of Bucharest (Biology Doctoral School). Physicist by formation, BSc Physics and MSc in Biophysics at the University of Bucharest (1994, and 1995 respectively), she obtained her PhD in 2003 and Habilitation in 2019 in Biology, University of Bucharest. She is the recipient of several fellowships such as of the Eastern Europe Research Scientists & Students Exchange & Collaboration Programme National University Singapore, at the Department of cell physiology, Catholic University Leuven, Belgium for postdoctoral stages, and of Boehringer Ingelheim, Research stage at Institute for Molecular Biotechnology, Jena, Germany, as well as of the Grigore Antipa Award of the Romanian Academy (in 2022). Her work combines engineering, modelling and biochemical tools for advancing electro-optical analytical platforms of cellular dynamics and of the effects triggered by various stimuli (as well as drug candidates) at single cell level. Prof Gheorghiu expertise covers the analysis of both homogenous and heterogenous cultures, encompassing prokaryotic (e.g., bacteria) and eukaryotic cells (including opto-genetically engineered ones).