Abstract
Conferences
Sensing internal and external nutrient factors in Drosophila
Gérard Manière and Julie Delescluse
Université de Bourgogne, Dijon, France
The detection of nutrients and pathogens is essential for the survival of animals. Sensing of nutrients such as amino acids is fundamental because they are the building blocks for proteins and the most abundant macromolecules in cells. In the Drosophila brain, amino acids can be detected by neurons in the mushroom bodies which serve as the memory center, through an amino acid transporter, minidiscs, via a TOR pathway. Additionally, in Drosophila, the sensing of pathogens, such as certain bacteria, can involve the direct binding of whole bacteria or cell wall component like peptidoglycan to specific receptors located in brain neurons or in gustatory neurons. As a result, bacteria can modify behaviors such as egg-laying behavior and food intake via specific pathways.
Toxicity in vitro studies of plant protection products and mycotoxins
Luna Bridgemana, Raquel Penalva-Olcinaa,b, Cristina Juana, Benoit Poinssotc, Eeva-Riikka Vehniäinenb, Ana Juan-Garcíaa
a Laboratory of food Chemistry and Toxicology, Faculty of Pharmacy, Universitat de València, Spain
b Department of Biological and Environmental Science, University of Jyväskylä, Finland
c UMR Agroécologie, INRAE, Institut Agro, Université de Bourgogne, Dijon, France
The contamination of stored food by mycotoxin together with the use of toxic herbicides leads to huge economic losses and to environmental and health damages. To assess the conceivable toxicity of natural plant protection products (PPP) such as Vacciplant(R), Alginate, Chitosan and COS-OGA and one of the most common mycotoxin’s Ochratoxin A (OTA) two different approaches were studied: i) in vitro cytotoxicity assays using non-differentiated human neuronal cells SH-SY5Y, and ii) assays with in vitro model Daphnia magna: A) acute toxicity tests by exposing D. magna at serial dilutions of all four PPP during 96h; B) heartrate measurement of D. magna exposed to the highest concentrations; C) immobility assessment after 21 days of exposure to Alginate, Vacciplant(R ) and OTA D) reproducibility by measuring number of offspring during 21 days of exposure to Alginate, Vacciplant(R) and OTA, and D) growth rate measurement after 21 days of exposure. Acute toxicity tests in SH-SY5Y cells showed that Vacciplant and Alginate were the most cytotoxic; on the contrary, for D. magna the cytotoxic potential was as follows: Chitosan>Alginate>Cos-OGA >Vacciplant(R). Regarding heart rate in D. magna, Chitosan together with OTA showed a prominent decrease at the concentrations tested. On the contrary Vacciplant(R) showed to have a delayed effect at the immobility test also affecting the reproduction. From these results, all four PPP at the concentrations assayed showed to have an effect over SH-SY5Y cells and D. magna even acute effect being able to cause environmental or human damages. This work has been supported by the Spanish Ministry of Science and Innovation PID2020-115871RB-100. RPO would like to thank the European FIT-FORTHEM program for the possibility of the research stage at the University of Jyväskylä.
Aflatoxin B1 and ochratoxin damage in rat kidney: a proteomics approach
Lara Manyes and Álvaro Lázaro
Laboratory of Food Chemistry and Toxicology, Faculty of Pharmacy, Universitat de València, Spain
Proteomics is the study of the interactions, function, composition, and structures of proteins and their cellular activities. Proteomics can analyze the expression of proteins at different levels (organ, tissue, cell line) allowing the assessment of specific quantitative and qualitative cellular responses related to the conditions studied. Quantitative proteomics can also provide deep insights into disease mechanisms, cellular functions, and biomarker discovery. Toxicoproteomics implements global protein expression technologies to toxicological and clinical research. Some proteins react to exposure to toxicants and display subtle variations in different cells and tissues. Technological development in mass spectrometry (MS) instruments, bioinformatics, and protein databases increases the opportunities for mechanistic and molecular damage research. The identification of peptide and protein sequences from MS uses a variety of bioinformatics tools, which search protein and nucleotide sequence databases. The aim of this proteomics study was to evaluate the renal toxicity of ochratoxin A (OTA) and Aflatoxin B1 (AFB1) oral exposure using Wistar rats and the potential beneficial effects of fermented whey (FW) and pumpkin (P). For the experiment, 10 groups of rats composed by 5 males and 5 females were exposed to different conditions for 28 days. Here, the preliminary results for males fed with control feed, feed containing AFB1, feed with OTA, and feed including AFB1 and OTA will be explained. Results were obtained after bioinformatics analysis using DAVID and KEGG platforms. This work was supported by the Spanish Ministry of Science and Innovation (PID2022-140722OB-I00).
Current strategies to elicit secondary metabolism in antibiotic-producing Actinomycetes
Valeria Alduina and Alessia Maria Sampino
Universita degli Studi di Palermo, Italy
Actinomycetes are ubiquitous aerobic Gram-positive bacteria, industrially relevant as producers of a wide range of bioactive secondary metabolites, such as antibiotics of clinical and commercial importance. The understanding of actinomycete biology relies on extensive studies on the model organism Streptomyces coelicolor over many years and on the availability of its complete genome sequence. S. coelicolor produces three well-characterized antibiotics – the blue pigment actinorhodin, the red pigment undecylprodigiosin, and the calcium-dependent lipopeptide antibiotic. Besides, development in genomic tools has revealed a reservoir of 25 additional silent/cryptic pathways in the S. coelicolor genome. This bacterium has an unusually complex developmental cycle that includes programmed cell death phenomena that make this bacterium a multicellular prokaryotic model. Morphological differentiation in S. coelicolor is strictly related to physiological differentiation: indeed, the onset of morphological differentiation generally coincides with the production of secondary metabolites. Understanding the mechanisms governing antibiotic biosynthesis is a challenging task, considering the importance of antibiotics in human and global health. Antibiotic biosynthesis is regulated at different levels by extracellular signals, quorum sensing-related factors, multiple master regulators, and biochemical pathways, such as bald, white, and sky. Various transcriptional regulators are involved in a hierarchical way to control development and antibiotic production. If, on the one hand, many factors and molecular mechanisms have been identified in controlling both morphological and physiological differentiation in S. coelicolor, on the other hand, for most actinomycetes, mechanisms and factors governing morphological and physiological processes still need to be understood. This presentation will provide an overview of the regulatory mechanisms known to control antibiotic production in S. coelicolor and both genetic and physiological methods adopted to improve the production of well-known antibiotics and to discover novel molecules from Actinomycetes.
Small Talk – Understanding bacterial communication for biotechnological breakthroughs
Ralf Heermann & Nazzareno Dominelli
Johannes Gutenberg-Universität Mainz, Germany
In nature, bacteria are not loners but live in close association with conspecifics, competitors or higher organisms. To act as community or to perfectly adapt to their eukaryotic hosts, bacteria must “talk” to each other. To exchange information, they use small diffusible molecules, a process that microbiologists refer to as quorum sensing. These language molecules are released by the bacteria in the environment and are then sensed by their neighbors via specific receptors. Thus, the community can arrange and adapt specific phenotypes in dependence of the cell count termed quorum. Due to the different structures and modifications of the communication molecules bacteria have evolved different languages and dialects, which can in addition give information about time and venue. Moreover, bacteria have small talk with their hosts as animals, plants and yet humans, a process referred to as inter-kingdom signaling. Especially entomopathogenic bacteria have evolved a great potential in communication processes since they harbor the largest number of putative communication receptors ever found in bacteria. Since bacterial communication is prerequisite for e.g. the infection of hosts by pathogenic bacteria or for biofilm formation, the molecular components of the bacterial communication systems are promising candidates as targets for badly needed new antimicrobial drugs or non-toxic biofilm inhibitors.
Unlocking the potential of Kitasatospora purpeofusca: a promising rare Actinomycete dealing with the stress signaling exerted by Selenite
Alessandro Presentato
Universita degli Studi di Palermo, Italy
The utilization of selenium in industrial applications, alongside the improper disposal of selenium derivatives, determined a concerning accumulation of these compounds in the environment. Among the various selenium species, oxyanion selenite is of high concern due to its solubility associated with high bioavailability and toxicity levels. Furthermore, as a rare Earth element, selenium faces the imminent threat of depletion, triggering potentially significant economic and technological repercussions. Therefore, it is important to recycle and recover selenium from selenium-containing waste. In the pursuit of sustainable practices aligned with the principles of the circular economy, microbial biotransformation of selenite into less harmful selenium species emerges as the most promising strategy. While actinomycetes are recognized as invaluable players in environmental biotechnology, their involvement in selenite biotransformation remains largely unexplored, primarily due to a limited understanding of oxyanion bioprocessing. In this study, Kitasatospora purpeofusca, a rare actinomycete isolated from agricultural soil, features a series of striking cell responses when dealing with selenite oxyanions, including morphological changes, alterations and damage of the cell membrane, an outburst of oxidative stress, the participation of thiol-based chemistry in the biotic processing of selenite, and the generation of selenium nanomaterials as a byproduct of selenite transformation. Moreover, findings on a laboratory-evolved strain of Kitasatospora purpeofusca that was repeatedly cultured in the presence of high concentrations of Se oxyanions, highlight significant distinctions from its wild-type counterpart at the genome sequence level. Precisely, the genotypic variant of the Kitasatospora strain showcases attenuation of secondary metabolite production, placing paramount importance on selenite transformation. The attributes displayed by this Kitasatospora evolved strain position it as a valuable asset in biotechnology.
Biosynthesis and signalling functions of nitric oxide (NO) in plant cells
David Wendehenne and Zoé Chaudron
UMR Agroécologie, INRAE, Institut Agro, Université de Bourgogne, Dijon, France
Nitric oxide (NO) is a gaseous free radical that functions in a wide array of physiological processes in many if not all organisms. In animals, NO is mainly produced by nitric oxide synthases (NOSs). NOSs are active as homodimers, use L-arginine (L-Arg) as substrate and catalyse NO synthesis in two distinct cycles with N-hydroxy-L-Arg as an intermediate product that is processed to L-citrulline and NO. In plants, the hunt for NO sources led to the discovery of both enzymatic and non-enzymatic processes. This search also highlighted that land plants do not possess NOS. Nonetheless, NOSs were found in several algal species including Klebsormidium nitens, a filamentous terrestrial alga used as a biological model to elucidate the early transition step from aquatic algae to land plants. Algal NOSs conserve the main structural features of animal NOS but also display singularities questioning the mechanisms underlying their catalytic cycles. First studies investigating NO functions in algae have shown that S-nitrosation, a main NO-dependent post-translational protein modification in animals, is conserved in algae. Accordingly, we identified several proteins undergoing S-nitrosation in response to an abiotic stress in K. nitens including the inositol polyphosphate multi-kinase 2 (KnIPK2). Our talk will summarize our current and ongoing work on NOSs and S-nitrosated proteins in algae.
What have we learnt about SARS-CoV-2 membrane proteins?
Ismael Mingarro and Juan Ortiz-Mateu
Universitat de València, Spain
The COVID-19 outbreak, caused by SARS-CoV-2, has triggered a global health crisis and driven extensive research into the virus’s molecular mechanisms. Among the virus’s components, the envelope proteins play a vital role in viral entry, assembly, and immune evasion. SARS-CoV-2 envelope proteins are integral components of the virion membrane, contributing to its structural integrity. They also facilitate viral entry by interacting with host receptors, leading to fusion and internalization into host cells, a critical step in infection. Additionally, these proteins are involved in viral particle assembly and release, aiding in the incorporation of the viral genome and other structural proteins. Their presence during viral assembly supports the hijacking of host cell machinery for efficient viral replication and may modify host cell membrane permeability, impacting various aspects of viral replication and pathogenesis.
In this lecture we will show the progress we have made in understanding the biogenesis and membrane disposition of some of the viral membrane proteins, aiding in the development of strategies to combat future coronavirus outbreaks.
Biological membranes affect membrane-associated processes
Dirk Schneider and Veronika Osten
Johannes Gutenberg-Universität Mainz, Germany
All cells are surrounded by membranes that separate the cytoplasm from the extracellular world. In eukaryotes, cell organelles are also separated from the cytoplasm by biological membranes. Eukaryotic membranes are composed of various (phospho)lipids with different head groups and acyl chain lengths, as well as cholesterol as the main component of the membrane. In addition, biological membranes contain membrane-associated or -integral proteins. Transmembrane proteins mediate the flow of energy, molecules, and/or signals across membranes. It is known that, on the one hand, the membrane lipid environment can modulate the activity of membrane proteins and, on the other hand, membrane proteins can also influence the structure of membranes. Several transmembrane proteins form higher-ordered oligomers within membranes, the functional state of these membrane proteins. But how does the lipid environment affect the structure and activity of (oligomeric) membrane proteins? We will discuss the relationship between the structure and activity of (oligomeric) membrane proteins and the lipid bilayer properties.
Members of the large superfamily of ATP-binding cassette transporters (ABC transporters) are transmembrane proteins that are conserved across all domains of life. They import or export diverse substrates across biological membranes by using energy gained via ATP hydrolysis. BmrA, the ABC transporter of Bacillus subtilis, became a paradigm for studying ABC transporter structure, function, and multidrug resistance, not only due to its homology to the human P-glycoprotein. In order to understand how membrane lipids regulate the activity of a membrane protein, we reconstituted BmrA into liposomes with different lipid compositions and examined the resulting ATPase activity in vitro. Our results indicate that (i) membranes with a thicker hydrophobic thickness and (ii) a certain amount of negatively charged lipids are crucial for optimal BmrA activity.
Studies on a synthetized drug and a compound from natural origin: Specific kinase pathways in enterovirus infection can be blocked by a repurposed cancer drug
Varpu Marjomaki
Dept. of Biological and Environmental Science, Nanoscience center, University of Jyväskylä, Finland
Enteroviruses are one of the most abundant viruses causing mild to serious acute infections in humans and also contributing to chronic diseases like type 1 diabetes. Presently, there are no approved antiviral drugs against enteroviruses. Here, we show that vemurafenib, an FDA-approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, prevents enterovirus translation and replication. Vemurafenib shows efficacy against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect acts through cellular phosphatidylinositol 4-kinase type IIIβ (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevents infection efficiently in acute cell models, eradicates infection in a chronic cell model, and lowers virus amounts in pancreas and heart in an acute mouse model. Our findings open new possibilities to develop drugs against enteroviruses and give hope for repurposing vemurafenib as an antiviral drug against enteroviruses.
Studies on Antimicrobial Efficacy and Mechanisms of Lignin from Birch, Straw, and Pine/Spruce
Jun Liu1, Maiju Pöysti2, Lotta-Riina Sundberg1, Varpu Marjomäki1
1Dept. of Biological and Environmental Science, Nanoscience Center, University of Jyväskylä, Finland. 2Spinnova, Jyväskylä, Finland
Wood and plant materials are rich in antiviral and antibacterial substances. Lignin is a by-product of wood industry and potentially great sustainable source of antimicrobials. Here, we studied lignin from three sources in more detail for their antimicrobial efficacy: birch (CH Bioforce), straw (CH Bioforce) and pine/spruce (Stora Enso). The EC50 (effective concentration) values of water-soluble lignin was roughly 15 to 30 -fold lower, i.e. more efficient, against non-enveloped enteroviruses than kraft lignin. Efficacy against enveloped coronaviruses (SARS-CoV-2, and seasonal corona virus HCoV-OC43) was roughly 200-fold lower than against non-enveloped enteroviruses. Antibacterial experiments with Gram-positive (S. aureus), and Gram-negative (E. coli) bacteria demonstrated all lignin solutions to cause over 99% percentage reduction in bacterial growth. TEM imaging visualized the morphological changes of viruses and bacteria. Various imaging and spectroscopy techniques are being used to evaluate the antimicrobial mechanisms of these different lignin samples. Altogether, water soluble lignin from birch and straw demonstrated broadly acting antimicrobial efficacy, acting directly on the viruses and bacteria.
Bacteriophage and bacterial infections in the mucosal milieu
Lotta-Riina Sundberg
Dept. of Biological and Environmental Science, Nanoscience center, University of Jyväskylä, Finland
The vertebrate mucosal surface is a complex environment including commensal microbiota and host immune mechanisms, and serves as the first line of defence against pathogens.
Pathogenic bacteria have a strong affinity towards mucosa, as it is their way into the body. In many cases, the interaction with the mucosa directly increases bacterial virulence, as bacteria sense they are close to invasion. Symbiotic (bacterio)phages are common and major members in the mucosal microbiome, although their role is often neglected Phages are viruses that infect specifically bacteria, and by regulating bacterial communities and stimulating the immune system they are important for our health. Bacterial invasion into mucosa thus subjects bacteria for potential phage infections by phages retained in the mucosal layer. Our studies show that the mucosal environment increases bacterial susceptibility to phage infections, and that we can colonize the mucosal surfaces with phages which provides protection against bacterial disease up to 7 days. However, the increased susceptibility to phage infection has a direct impact on bacterial resistance mechanisms against phages, favouring CRISPR-Cas resistance in the mucosal environment instead of surface resistance. The presence of mucin led to a dramatic increase in CRISPR spacer acquisition, especially in low nutrient conditions where over 60% of bacterial colonies obtained at least one new spacer. Currently, we study the phage-bacterium interplay in the mucosa using a cell culture -based approach. Understanding the ecology of pathogens and their phages in the mucosa is central for our health and for the development of tools to combat antibiotic resistant bacterial infections.
DNA methylation alterations in genomic instability and cancer
Viviana Barra and Salvatore Martino
Universita degli Studi di Palermo, Italy
Epigenetics describes modifications on the DNA that do not alter the nucleotide sequence. Epigenetic modifications are reversible but also hereditable. These changes include post-translational modifications of the histones and DNA methylation. DNA methylation was the first epigenetic modification to be discovered in humans and it is the most widely studied. It consists of the addition of a methyl group to the cytosine within CpG dinucleotides, generating the 5 methyl-cytosine (5meC). Genomic regions rich in 5meC tend to be highly compacted making them hardly accessible by the transcriptional machinery. This is the reason why DNA methylation is mainly considered as a negative regulator of gene expression. However, the majority of the genome characterized by DNA methylation is made up by repetitive DNA which is usually gene-poor. Interestingly, there are many human diseases exhibiting loss of DNA methylation at repetitive sequences, including cancer. Hence, the question is: what is the role of DNA methylation at repetitive sequences? Our research is aimed to reply to this question and here we will present some data about the importance of DNA methylation for centromere function.
Ciliary Signalling: an ancient way for cells to communicate
Helen May-Simera and Jannis von Spreckelsen
Johannes Gutenberg-Universität Mainz, Germany
Recent research underscores the importance of primary cilia, microtubule-based sensory organelles, in cellular signal reception and processing. Primary cilia dysfunction underlies a large number of genetic disorders collectively called ‘ciliopathies’. Cilia have been found to release extracellular vesicles (EVs), which are known to exert various physiological functions. While previous focus has centred on large EVs (lgEVs) budding directly from the ciliary membrane, there has been little exploration of an association between cilia and small EVs (smEVs) derived from multivesicular bodies (MVBs). In our laboratory we are exploring the role of cilia in the EV biogenesis and function, in the context of development and disease. Since kidney dysfunction is a classical symptom of numerous ciliopathies, we have focused our attention on cilia EVs in renal tissues. The use of ciliary mutant renal cell models from ciliopathy mutant mice and ciliopathy patient-derived samples and cell lines has proven valuable in studying the biogenesis and bioactivity of ciliary smEVs. We could recently show that ciliary mutant mammalian cells exhibit increased secretion of smEVs and a shift in EV composition, with a differential loading of signalling molecules upon ciliary dysfunction. Moreover, these smEVs demonstrate biological activity and the ability to modulate the WNT response in recipient cells. These findings shed light on smEV-dependent ciliary signalling mechanisms that may underlie ciliopathy disease pathogenesis.
Harnessing the signaling properties of TNF receptors for cancer therapy: are we following the right TRAILs?
Olivier Micheau & Abdelmnim Radoua
INSERM, Université de Bourgogne, Dijon, France
TNF-related apoptosis-inducing ligand (TRAIL or Apo2 or TNFSF10) belongs to the TNF superfamily. When bound to its agonistic receptors, TRAIL is able to induce apoptosis in tumour cells, but not healthy cells. This tumour selectivity prompted, over the last three decades, a large number of studies aiming at evaluating the anti-tumoural potential of TRAIL or its derivatives. Yet, emerging evidence indicates that TRAIL can also trigger, on the other hand, a non-canonical signal transduction pathway that is likely to be detrimental for its use in oncology. While therapeutic approaches have failed, so far, novel formulations are still being evaluated. We will discuss why Harnessing the signaling properties of TRAIL receptors is so important to envision functional therapeutic perspectives.
Poster session
o Nuclear involvement of the Cell Division Cycle 48 protein during the plant immune response.
Ines D., Wendehenne D., Courty P.E., Rosnoblet C.
o Study of the mode of action of innovative phytosterol-based technologies in wheat and grapevine: from the lab to the field.
Lasterre L., Claverie J., Poinssot B.
o Novel RNA Editing tools to recode Premature Termination Codon in CFTR nonsense mutations.
Titoli S., Gargano S., Chiavetta R.F., Cancemi P., Melfi R., Di Leonardo A. and Barra V.
o Identification and functional characterization of S-nitrosated proteins from Klebsormidium nitens.
Chaudron Z., Nicolas-Frances V., Klinguer A., Rosnoblet C., Besson-Bard A. and Wendehenne D.
o Study of the stress-related signaling of endoplasmic reticulum in grapevine immunity associated to LysM receptor kinases (VvLYKs).
Marzari T., Gayral M., Poinssot B.
