PRP@CERIC: Scientific Seminar Series

Data are the main underlying substrate of the research process. It is imperative to facilitate the collection, creation, analysis, and discussion of data to generate new knowledge and improve research.

FAIR, an acronym for Findable, Accurate, Interoperable and Reusable indicates the main features that a data ecosystem must possess to achieve this objective.

Therefore, to create a digital platform based on FAIR principles on a multicentric and heterogenous consortium of laboratories it is crucial to analyze the research process of every member to identify the differences and, more importantly, the similarities between them, creating a shared and useful data management plan that homogenize and streamline the data collection and sharing process.

To assess these processes an interview was carried out within the researchers of Pathogen Readiness Platform (PRP@CERIC) project. Three main steps were identified that are shared with all the laboratories: 1) the sample acquisition/creation entry phase, 2) the sample analysis and data elaboration phase, and 3) the research results and data sharing phase.

The FAIRification activity intervene on these steps through a combination of methodologies and digital infrastructure software: 1) the adoption and testing of forms and metadata acceptance repositories. 2) The introduction of a shared electronic notebook (adapting the opensource ELABFTW software), integrated with automatic analysis pipelines and laboratory specific data management plans. 3) The customization of an opensource platform (NOMAD OASIS) that supports direct connection with the journal publications, external databases and uses persistent and globally unique identifiers, (i.e., Digital Object Identifiers, DOI) to maintain a direct link to the real data on the platform. This approach aims at shifting the burden of implementing a FAIR data ecosystem towards automatic processes that follow the natural internal research process. It will use real cases from the different labs to test the implementation and usability.

Speaker: Marco Prenassi, RIT (Area Science Park)

Infrared nanoscopies, such as IR s-SNOM (scattering-type scanning near-field microscopy) and PTE (photo-thermal expansion microscopy), allow to overcross the diffraction limit imposed to far-field FTIR by exploiting the near-field approach.

This is done by combining the high chemical sensitivity of infrared spectroscopy in the mid-IR range with the nanoscale lateral resolution of Atomic Force Microscopy (AFM).

Structural information on fundamental biological components, including proteins, nucleic acids, and lipids, can be thus obtained together with topographic details with lateral resolution of a few tens of nanometers.

IR s-SNOM, in particular, is the ideal technique for characterizing cellular sub-components and bio-based nanocomposites with thicknesses down to a few nanometers.

Examples include protein self-assemblies, amyloid-like structures, DNA-based adducts for gene transfer, and drug delivery systems.

This seminar will cover the fundamentals of IR nanoscopy, with a focus on selected practical applications.

Speaker: Federica Piccirilli, RIT (Area Science Park)

RNA modifications have attracted increasing attention due to their crucial roles in various biological processes. These modifications involve biochemical alterations of nucleotides that can impact RNA structure and dynamics.

Despite extensive studies, the use of molecular dynamics (MD) simulations to investigate modified RNA remains limited.

MD simulations are a powerful tool for accessing the structural dynamics of RNA at the atomistic level. The accuracy of these simulations largely depends on the quality of the force-field parameters utilized.

Therefore, it is beneficial to combine simulations with experiments, such as by fitting parameters against experimental data or enforcing experimental averages with ensemble refinement methods.

In this talk the use of ensemble refinement methods are presented, to investigate the structure of a 20-bp RNA helix containing Inosines. The maximum entropy principle, along with advanced enhanced sampling techniques, was used to generate an ensemble of structures compatible with NMR and SAXS data.

The presence of Inosines increased flexibility in the helix and allowed for the observation of sugar puckering in the C2′-endo conformation, which is not expected in ds-RNA.

Speaker: Valerio Piomponi, RIT (Area Science Park)

Biomedical research relies heavily on purified recombinant proteins, playing a crucial role in a wide spectrum of applications.

Their homogeneous nature allows for detailed reconstruction of their three-dimensional structures using techniques such as X-ray, cryoEM, and NMR, facilitating a deeper understanding of protein folding, conformational changes, molecular architectures, enabling studies of their function.

Additionally, these proteins are essential in investigating various interactions, such as protein-protein, protein-ligand, or protein-nucleic acid, and serve as critical tools in the field of therapeutics and diagnostics.

To guarantee the reliability of experimental data in these studies a good quality of recombinant protein is essential.

In this presentation the process of protein production is described, giving an overview of critical steps from construct design through expression, purification and quality control.

Speaker: Marta Semrau, RIT (Area Science Park)

Structural Biology is a consolidated scientific field that is critical for understanding of the molecular bases that lie behind the function of the different proteins, the investigation on the pathological impact of protein mutations and the development of new and better drugs.

One of the most long-standing disciplines of the field is protein crystallography, a technique that exploits single crystal x-ray diffraction to obtain atomic or near-atomic resolution models of the proteins that are investigated, allowing to delve into structure-activity relationship studies with final aims that may span from understanding the functioning of a biological process at molecular level, to the development of new therapeutic agents.

In the first part of the presentation some base concepts regarding protein crystallization are described, with a particular focus on how protein crystals can be optimized and on the techniques that may be used in order to obtain crystals, and then structures, of protein-ligand complexes.

In the second part a portion of a Structure Based Drug Design project is illustrated, providing an example of the data and results that can be obtained using single crystal x-ray diffraction, also focusing on the different approaches that might be used for the development of a drug candidate.

Speaker: Andrea Dalle Vedove, RIT (Area Science Park)

When trained on millions of protein sequences, large language models have been shown to develop emergent capabilities and to generalize across a range of applications, from prediction of mutational effects to long-range contact predictions.

Similarly, unsupervised machine learning tools are able to cluster protein sequences, identifying homologous domains and improving functional and evolutionary analyses.

In order to describe structural information, van Kempen et al. (2023) have recently introduced a discretized 20-letter structural alphabet (3Di) encoding the tertiary interactions between residues.

The 3Di alphabet allows the density peak clustering of structures starting from local Foldseek alignments, thus refining protein family classification in the twilight zone.

At the same time, new protein language models can be trained using this structure-aware vocabulary, finding applications, for instance, in protein stability due to point mutations.

Speaker: Marco Celoria, RIT (Area Science Park)

Atomic Force Microscopy (AFM) has got a fast development over the last decades mainly thanks to its versatility.

Indeed, AFM has shown unprecedented potential in the field of scanning probe microscopy, being able to operate in diverse environments and specimens, as well as to map simultaneously the topography and various physico-chemical properties of the scanned sample (e.g. adhesion, friction or elasticity) on different spatio-temporal scales, from µm to nm and from minutes to ms/µs.

In parallel, the research interest on biophysical topics for diagnostic and therapeutic purposes has been growing, along with the need for high resolution tools able to operate on biological systems in physiological conditions.

Particular attention has been paid to the field of mechanobiology, given the demonstrated correlation between mechanical properties and the patho-physiological state of many biological specimens.

In this context, AFM allows for the acquisition of simultaneous topographic and mechanical maps of several biological systems, ranging from the micro- to the nanoscale.

Recently, rising attention has been dedicated to the analysis of cellular membranes models and their interaction with small Extracellular vesicles (s-EVs), given the similarities of the latter to viral particles and their well-known capability to influence the fate of recipient cells.

This presentation gives some examples of AFM-based mechanobiology on cells, ECMs and tissues, as well as a quick review of the results obtained so far in the NanoInnovation Labs in Elettra on cellular membranes/s-EVs interactions.

Some future perspectives and the potential of the high-speed AFM acquired in the context of PRP project are also illustrated.

Speaker: Luca Puricelli, RIT (Area Science Park) – Elettra Sincrotrone Trieste

High-Performance Computing (HPC) has become a cornerstone of scientific and industrial advancement, enabling breakthroughs in diverse fields such as drug discovery, material simulations, and neural network research.

However, the intricate web of components that make up an HPC infrastructure often remains obscured, hindering a comprehensive understanding of its inner workings.

This talk aims to dissect the complex tapestry of HPC infrastructure, shedding light on each component and elucidating the intricate relationships that drive computational power to new heights.

The presentation commences with a description of ORFEO, the hardware that enables its usage as an HPC facility, and the challenges that new projects pose to this facility.

Then, the discussion seamlessly moves into the software layer of HPC infrastructure, where it unravels the complexities of orchestrating computational tasks.

Notably, the role of resource managers like SLURM and container orchestration platforms like Kubernetes in efficiently allocating and managing computing resources is explored.

The talk addresses the challenges and advantages of these systems in coordinating parallel computations, optimizing job scheduling, and adapting to dynamic workloads.

Moving beyond resource management, exploring storage solutions and identity management solutions underscores the critical role they play in serving data and securing access within an HPC environment.

Finally, this talk presents VirtualOrfeo, the first iteration of a realistic virtual environment to play with the discussed technologies.

Speaker: Ruggero Lot, RIT – Area Science Park

FAIR-by-design approach is the worldwide Gold-Standard for what concerns public data collection, both in scientific research and in administrative processes.

This talk tries to contextualize the environment in which the FAIR-by-design concept was born by retracing the main stages of what we could address as the “Open Knowledge Movement”, that brought to the definition of Open Data and Open Access.

It is discussed how Open Data have a reason for being only if they satisfy the FAIR principles, and it is explained how to be fully compliant with them implies being collected through a FAIR-by-design approach.

It is also underlined that notwithstanding FAIR Data idea was introduced in tight connection with Open Data one, FAIRness is a broader concept that applies even to Data that are not Open.

Special attention is given to the reason why the only way to have Data fully compliant with FAIR principle is collecting them through a FAIR-by-design pipeline and what this implies for a scientific laboratory.

Speaker: Federica Bazzocchi, RIT – Area Science Park

Cryo-microscopy techniques are emerging as fundamental components for the future of scientific research.

Despite significant technological advancements on the instrumental front, numerous aspects in sample preparation methodologies still require refinement and standardization.

While progress has been made across all levels, from instrumentation to sample preparation, challenges persist, demanding continued attention.

The introduction of a nanofabrication approach for producing substrates used in biological applications of cryo-microscopy is crucial for enhancing the reproducibility and statistical validity of conducted analyses.

Nanofabrication can address existing gaps in this field, significantly contributing to the enhancement of sample preparation protocols. This technology plays a key role in streamlining workflows and optimizing the precision of procedures.

Furthermore, nanofabrication extends to the production of devices used in conjunction with cryo-microscopy. These devices, such as detectors and sensors, are essential for detecting and quantifying bacterial contaminations from samples of different types and for various applications.

At IOM, two primary technologies are emerging in this context, focusing on approaches based on the use of MEMS (Micro-Electro-Mechanical Systems) and purely optical readouts to improve the effectiveness of conducted analyses.

In summary, the application of nanofabrication not only addresses gaps in sample preparation protocols but also plays a crucial role in the development of advanced technologies for the detection and characterization of biological samples using cryo-microscopy.

Speaker: Simone Dal Zilio, CNR – IOM

The research interest of the main group at the Institute of Crystallography (CNR) in Trieste is the structural and functional characterization of therapeutic targets and protein involved in human diseases.

The aim is to elucidate the structural basis of human pathologies and to develop effective drugs.

Structural studies are performed by means of X-ray crystallography and Small Angle X-ray scattering and in vitro functional characterization is mainly achieved by qualitative, semiquantitative and quantitative binding analysis.

This talk addresses the present state of the art of the intermolecular interactions characterization facility @ IC-CNR Trieste, discussing the applications and the potential of the core of the platform (i.e., Isothermal Titration Calorimetry and Grating Coupled Interferometry), and its upcoming improvements that will be achieved thanks to PRP@CERIC project.

Speaker: Sonia Covaceuszach, CNR – IC

Deep Ultraviolet Resonance Raman Spectroscopy (DUVRR) is an emerging and powerful analytical tool used for detection and characterization of biological samples in a label-free and real time approach.

The excitation in the Deep UV provides specific resonance enhancement of biological moieties, especially protein, DNA and RNA structures, allowing to efficiently detect biological Raman markers in the spectra.

It is well known that DUV radiation is a genotoxic agent. Prolonged UV light exposition induces damage to the genomes of viruses, breaking bonds and forming photodimeric lesions in RNA.

These damages prevent both transcription and replication which leads to viral inactivation. Even though the effects of UV radiation on DNA of microorganisms have been well-recorded, its impact on RNA and RNA modifications is less known.

A sample that is prone to such a damage are viruses, and with the SARS-COV-2 (RNA virus) outbreak, the need of fast tools for viral characterization and classification became even more necessary.

In this work, we will show how to obtain stable, high quality and information-rich DUVRR spectra of active Vesicular Stomatitis Virus (VSV) without affecting its viability, RNA and protein integrity.

By opportunely tuning the excitation wavelength, we can detect and assign in the vibrational spectra specific markers of protein and RNA components useful to elucidate biochemical characteristics of active VSV virus.

In addition, we can study the effect of DUV irradiation in the mechanism of inactivation of VSV viruses in order to find the most damaging wavelength for the active VSV virus. This information can further be exploited to build an effective countermeasure tool for virus deactivation.

Speaker: Denis Rajnovic, ICGEB – Elettra Sincrotrone Trieste