Active droplets through enzyme-free, dynamic phosphorylation.

Life continuously transduces energy to perform critical functions using energy stored in reactive molecules like ATP or NADH. ATP dynamically phosphorylates active sites on proteins and thereby regulates their function. Inspired by such machinery, regulating supramolecular functions using energy stored in reactive molecules has gained traction. Enzyme-free, synthetic systems that use dynamic phosphorylation to regulate supramolecular processes have not yet been reported, to our knowledge. Here, we show an enzyme-free reaction cycle that consumes the phosphorylating agent monoamidophosphate by transiently phosphorylating histidine and histidine-containing peptides. The phosphorylated species are labile and deactivate through hydrolysis. The cycle exhibits versatility and tunability, allowing for the dynamic phosphorylation of multiple precursors with a tunable half-life. Notably, we show the resulting phosphorylated products can regulate the peptide's phase separation, leading to active droplets that require the continuous conversion of fuel to sustain. The reaction cycle will be valuable as a model for biological phosphorylation but can also offer insights into protocell formation.

Optimising Radioligand Therapy for Patients with Gastro-Entero-Pancreatic Neuroendocrine Tumours: Expert Opinion from an Italian Multidisciplinary Group.

Radioligand therapy (RLT) with lutetium (177Lu) oxodotreotide is an approved therapy in combination with somatostatin analogues (SSAs) for patients with advanced, well-differentiated G1-G2, gastro-entero-pancreatic neuroendocrine tumours (GEP-NETs) that progress on SSAs. We conducted a series of round table meetings throughout Italy to identify issues related to RLT delivery to patients with GEP-NETs. Four key issues were identified: (1) the proper definition of tumour progression prior to RLT initiation; (2) the impact of RLT in patients with bone metastases and/or high hepatic tumour burden; (3) the optimal follow-up protocol after RLT; and (4) organisational issues related to RLT use and managerial implications. This article reviews the literature relating to the aforementioned issues and makes recommendations based on available evidence and Italian NET experts' opinions. In particular, the group recommends the development of a diagnostic-therapeutic care pathway (DTCP) for patients undergoing RLT which provides systematic guidance but can still be individualised for each patient's clinical and psychosocial needs. A DTCP may clarify the diagnostic, therapeutic and post-treatment monitoring process, and improve communication and the coordination of care between hub and spoke centres. The DTCP may also contribute to changes in the care process related to the 2013/59/EURATOM Directive and to the definition of costs when planning for future or updated reimbursement of RLT in Italy.

Optimisation of protection in the medical exposure of recurrent adult patients due to computed tomography procedures: development of recurrent exposures reference levels.

OBJECTIVES: To assess the incidence (1 year) and the cumulative incidence (3 years) of the condition of patients accruing cumulative effective doses (CED) of ≥ 100 mSv and their variability among different hospitals. To establish and validate a reference level for the CED in patients with recurrent exposures (RERL) and provide a RERL value. METHODS: Data of CT exposure was collected in 9 similar hospitals. The database included 294,222 patient*years who underwent 442,278 CT exams in 3 years. The incidence proportion of patients with CED ≥ 100 mSv in a given year (I100;1) and the 3-year cumulative incidence of patients with CED ≥ 100 mSv over 3 consecutive years (I100;3) were calculated and compared among different institutions. RESULTS: I100;1 ranged from a minimum of 0.1% to a maximum of 5.1%. The percentage of recurrent patients was quite uniform among centres ranging from 23 to 38%. The I100;3 ranged from a minimum of 1.1 to 11.4%. There was a strong positive correlation between the third quartile values of yearly CED and yearly incidence (r = 0.90; R2 = 0.81; p < 0.0001). RERL value in our study was found at 34.0 mSv. CONCLUSION: The management of patients with recurrent exposures is highly variable among hospitals leading to a 50-fold variation in I100;1 and to a tenfold variation in I100;3. RERL could be established and used by taking as a RERL quantity the CED and as a RERL value the 75th percentile of the third quartiles of the distribution of the yearly CED obtained by surveying different hospitals. CLINICAL RELEVANCE STATEMENT: This is the first ever multicentre study that quantifies recurrent exposures in terms of incidence and cumulative incidence of patients with CED ≥ 100 mSv. RERL establishment and use could benefit the optimisation of radioprotection of patients with recurrent exposures. KEY POINTS: This is the first multicentre study estimating yearly incidence and 3-year cumulative incidence of patients with cumulative effective doses ≥ 100 mSv. In this study, a 50-fold inter centre variation between the maximum (5.1%) and the minimum value (0.1%) of yearly incidence of patients with cumulative effective doses ≥ 100 mSv was reported. The range of the 3-year cumulative incidence extended from 1.1 to 11.4% (a tenfold variation) The third quartile of the yearly cumulative effective doses in a centre showed a strong positive correlation with the yearly incidence of patients with cumulative effective doses ≥ 100 mSv, with a potential of being used to set reference levels for recurrent exposures.

Liquid spherical shells are a non-equilibrium steady state of active droplets.

Liquid-liquid phase separation yields spherical droplets that eventually coarsen to one large, stable droplet governed by the principle of minimal free energy. In chemically fueled phase separation, the formation of phase-separating molecules is coupled to a fuel-driven, non-equilibrium reaction cycle. It thus yields dissipative structures sustained by a continuous fuel conversion. Such dissipative structures are ubiquitous in biology but are poorly understood as they are governed by non-equilibrium thermodynamics. Here, we bridge the gap between passive, close-to-equilibrium, and active, dissipative structures with chemically fueled phase separation. We observe that spherical, active droplets can undergo a morphological transition into a liquid, spherical shell. We demonstrate that the mechanism is related to gradients of short-lived droplet material. We characterize how far out of equilibrium the spherical shell state is and the chemical power necessary to sustain it. Our work suggests alternative avenues for assembling complex stable morphologies, which might already be exploited to form membraneless organelles by cells.

Impact of High-to-Moderate Penetrance Genes on Genetic Testing: Looking over Breast Cancer.

Breast cancer (BC) is the most common cancer and the leading cause of cancer death in women worldwide. Since the discovery of the highly penetrant susceptibility genes BRCA1 and BRCA2, many other predisposition genes that confer a moderate risk of BC have been identified. Advances in multigene panel testing have allowed the simultaneous sequencing of BRCA1/2 with these genes in a cost-effective way. Germline DNA from 521 cases with BC fulfilling diagnostic criteria for hereditary BC were screened with multigene NGS testing. Pathogenic (PVs) and likely pathogenic (LPVs) variants in moderate penetrance genes were identified in 15 out of 521 patients (2.9%), including 2 missense, 7 non-sense, 1 indel, and 3 splice variants, as well as two different exon deletions, as follows: ATM (n = 4), CHEK2 (n = 5), PALB2 (n = 2), RAD51C (n = 1), and RAD51D (n = 3). Moreover, the segregation analysis of PVs and LPVs into first-degree relatives allowed the detection of CHEK2 variant carriers diagnosed with in situ melanoma and clear cell renal cell carcinoma (ccRCC), respectively. Extended testing beyond BRCA1/2 identified PVs and LPVs in a further 2.9% of BC patients. In conclusion, panel testing yields more accurate genetic information for appropriate counselling, risk management, and preventive options than assessing BRCA1/2 alone.

The Role of Chemically Innocent Polyanions in Active, Chemically Fueled Complex Coacervate Droplets.

Complex coacervation describes the liquid-liquid phase separation of oppositely charged polymers. Active coacervates are droplets in which one of the electrolyte's affinity is regulated by chemical reactions. These droplets are particularly interesting because they are tightly regulated by reaction kinetics. For example, they serve as a model for membraneless organelles that are also often regulated by biochemical transformations such as post-translational modifications. They are also a great protocell model or could be used to synthesize life-they spontaneously emerge in response to reagents, compete, and decay when all nutrients have been consumed. However, the role of the unreactive building blocks, e.g., the polymeric compounds, is poorly understood. Here, we show the important role of the chemically innocent, unreactive polyanion of our chemically fueled coacervation droplets. We show that the polyanion drastically influences the resulting droplets' life cycle without influencing the chemical reaction cycle-either they are very dynamic or have a delayed dissolution. Additionally, we derive a mechanistic understanding of our observations and show how additives and rational polymer design help to create the desired coacervate emulsion life cycles.

Silver and Gold Pillarplex Pseudorotaxanes from α,ω-Dicarboxylic Acids.

A series of pseudorotaxanes with supramolecular organometallic silver(I) and gold(I) pillarplexes acting as rings and different α,ω-dicarboxylic acids as axle components are reported. The successful formation of the host-guest complexes is shown by 1 H NMR spectroscopy and respective NMR titration. Additional evaluation with ITC titration experiments yielded dissociation constants (Kd ) ranging from 10-5 to 10-7  M. Single-crystal X-Ray diffraction analysis reveals a particularly exciting pore alignment of different examples in the solid state depending on the length of the guest. The work highlights, that dicarboxylic acids can penetrate the tight tubular pillarplex pore, paving the way to future mechanically interlocked molecules and materials.

EFOMP Malaga Declaration 2023: An updated vision on Medical Physics in Europe.

In 2006, the European Federation of Organisations for Medical Physics (EFOMP) adopted the "Malaga Declaration". The declaration asserted the fundamental role of Medical Physics professionals in the radiation protection of patients, workers, general public, carers and comforters and research participants in hospitals. However, since that time the Medical Physics profession has evolved in Europe and new regulations and documentation have been issued, such as directive 2013/59/Euratom and the "European Guidelines on Medical Physics Expert" (RP174). EFOMP has published updated core-curricula and strived towards the recognition of the profession at the European level. In view of this, an update of the original Malaga Declaration was deemed necessary, to define the future vision that will guide the actions of the Federation in the years to come. This Declaration, which has been approved by the national member organizations of EFOMP in April 2023, is much broader than the original Malaga version. This is expected considering the rapid evolution of medical device technology over the last 17 years. The Radiation Protection Expert in hospital settings should be an MPE, since the latter has the highest level of radiation protection knowledge and training. Given the passion and energy that animated the debate, which led to the updating of the Malaga Declaration, we are confident that it represents a solid basis for the development of our profession in Europe which is in consonance with the aspirations of us all.

Impact of patient's habitus on image quality and quantitative metrics in 18F-FDG PET/CT images.

PURPOSE: To study how the quantitative parameters of 18F-FDG PET imaging change with the emission scan duration (ESD) and the body-mass-index (BMI) in phantom and patients on a time-of-flight (TOF)-PET/CT system. METHODS: The image-quality phantom with (b-NEMA-IQ, BMI = 29.2 kg/m2) and without (NEMA-IEC, BMI = 21.4 kg/m2) a 'belt' of water-bags was filled with 18F-FDG activities to obtain nominal standardized uptake values (SUV) of 19, 8 and 5. Patients with BMI ≤ 25 kg/m2 (L-BMI) and BMI > 25 kg/m2 (H-BMI) were enrolled in this study. Phantom and patients underwent list-mode PET acquisition at 120 s/bed-position. Images reconstructed with clinical protocol and different ESD (120, 90, 75, 60, 45, 30 s) were analysed for comparison of maximum SUV (SUVmax), maximum standardized uptake value lean-body-mass corrected (SULmax) and noise. RESULTS: 79 oncologic patients (45 L-BMI, 44 H-BMI) were analysed. From 90 s to 30 s, an increasing variation of SUVmax and SULmax with respect to the reference 120 s time was observed, from 18% to 60% and from 16% to 37% for phantom and patients, respectively. SUVmax values were significantly higher (+50%) in b-NEMA-IQ than NEMA-IQ phantom and in H-BMI (+33%) than L-BMI patients. No significant difference was found in SULmax for the two BMI categories in both phantom and patients. CV values decreased when increasing ESD, being higher in H-BMI patients (0.13-0.25) and b-NEMA-IQ phantom (0.15-0.28) than in L-BMI patients (0.11-0.21) and NEMA-IQ phantom (0.11-0.20). CONCLUSIONS: Reduction of ESD may severely impact on the variations of SUVmax and SULmax in 18F-FDG PET/CT imaging. This study confirms recommendations of using SUL for lesion uptake quantification, being unaffected by BMI variation.

A Carbodiimide-Fueled Reaction Cycle That Forms Transient 5(4H)-Oxazolones.

In life, molecular architectures, like the cytoskeletal proteins or the nucleolus, catalyze the conversion of chemical fuels to perform their functions. For example, tubulin catalyzes the hydrolysis of GTP to form a dynamic cytoskeletal network. In contrast, myosin uses the energy obtained by catalyzing the hydrolysis of ATP to exert forces. Artificial examples of such beautiful architectures are scarce partly because synthetic chemically fueled reaction cycles are relatively rare. Here, we introduce a new chemical reaction cycle driven by the hydration of a carbodiimide. Unlike other carbodiimide-fueled reaction cycles, the proposed cycle forms a transient 5(4H)-oxazolone. The reaction cycle is efficient in forming the transient product and is robust to operate under a wide range of fuel inputs, pH, and temperatures. The versatility of the precursors is vast, and we demonstrate several molecular designs that yield chemically fueled droplets, fibers, and crystals. We anticipate that the reaction cycle can offer a range of other assemblies and, due to its versatility, can also be incorporated into molecular motors and machines.

Regulating DNA-Hybridization Using a Chemically Fueled Reaction Cycle.

Molecular machines, such as ATPases or motor proteins, couple the catalysis of a chemical reaction, most commonly hydrolysis of nucleotide triphosphates, to their conformational change. In essence, they continuously convert a chemical fuel to drive their motion. An outstanding goal of nanotechnology remains to synthesize a nanomachine with similar functions, precision, and speed. The field of DNA nanotechnology has given rise to the engineering precision required for such a device. Simultaneously, the field of systems chemistry developed fast chemical reaction cycles that convert fuel to change the function of molecules. In this work, we thus combined a chemical reaction cycle with the precision of DNA nanotechnology to yield kinetic control over the conformational state of a DNA hairpin. Future work on such systems will result in out-of-equilibrium DNA nanodevices with precise functions.

Phase Transitions in Chemically Fueled, Multiphase Complex Coacervate Droplets.

Membraneless organelles are droplets in the cytosol that are regulated by chemical reactions. Increasing studies suggest that they are internally organized. However, how these subcompartments are regulated remains elusive. Herein, we describe a complex coacervate-based model composed of two polyanions and a short peptide. With a chemical reaction cycle, we control the affinity of the peptide for the polyelectrolytes leading to distinct regimes inside the phase diagram. We study the transitions from one regime to another and identify new transitions that can only occur under kinetic control. Finally, we show that the chemical reaction cycle controls the liquidity of the droplets offering insights into how active processes inside cells play an important role in tuning the liquid state of membraneless organelles. Our work demonstrates that not only thermodynamic properties but also kinetics should be considered in the organization of multiple phases in droplets.

Photoactivatable V-Shaped Bifunctional Quinone Methide Precursors as a New Class of Selective G-quadruplex Alkylating Agents.

Combining the selectivity of G-quadruplex (G4) ligands with the spatial and temporal control of photochemistry is an emerging strategy to elucidate the biological relevance of these structures. In this work, we developed six novel V-shaped G4 ligands that can, upon irradiation, form stable covalent adducts with G4 structures via the reactive intermediate, quinone methide (QM). We thoroughly investigated the photochemical properties of the ligands and their ability to generate QMs. Subsequently, we analyzed their specificity for various topologies of G4 and discovered a preferential binding towards the human telomeric sequence. Finally, we tested the ligand ability to act as photochemical alkylating agents, identifying the covalent adducts with G4 structures. This work introduces a novel molecular tool in the chemical biology toolkit for G4s.

Tunable induced circular dichroism in gels.

The ICD phenomenon has drawn a lot of attention in recent years in applicable fields such as chiral sensing and chiroptical devices. In this work, we first gaze at the issues of thin spin-coated films not being able to deliver consistent ICD signals. A hypothesis of the underlying problem is proposed through a brief elucidation of the spin-coating process. To confirm and eliminate the uncontrollable dynamic factors with spin coating, we then dedicate our efforts to develop a new gel system based on chiral L-/D-N',N'-Dibenzoyl-cystine. Achiral dye molecules are intercalated in a DBC gel through a "one-step" preparation procedure. Compared to the former spin-coating system, significantly improved reproducibility of the new gel system is demonstrated. Besides, the ICD signals can be customized in a broad spectral range (wavelength tunability) by substituting dye molecules. Finally, we discuss the potential applications of this interesting system.

Enhancing the impact of Artificial Intelligence in Medicine: A joint AIFM-INFN Italian initiative for a dedicated cloud-based computing infrastructure.

Artificial Intelligence (AI) techniques have been implemented in the field of Medical Imaging for more than forty years. Medical Physicists, Clinicians and Computer Scientists have been collaborating since the beginning to realize software solutions to enhance the informative content of medical images, including AI-based support systems for image interpretation. Despite the recent massive progress in this field due to the current emphasis on Radiomics, Machine Learning and Deep Learning, there are still some barriers to overcome before these tools are fully integrated into the clinical workflows to finally enable a precision medicine approach to patients' care. Nowadays, as Medical Imaging has entered the Big Data era, innovative solutions to efficiently deal with huge amounts of data and to exploit large and distributed computing resources are urgently needed. In the framework of a collaboration agreement between the Italian Association of Medical Physicists (AIFM) and the National Institute for Nuclear Physics (INFN), we propose a model of an intensive computing infrastructure, especially suited for training AI models, equipped with secure storage systems, compliant with data protection regulation, which will accelerate the development and extensive validation of AI-based solutions in the Medical Imaging field of research. This solution can be developed and made operational by Physicists and Computer Scientists working on complementary fields of research in Physics, such as High Energy Physics and Medical Physics, who have all the necessary skills to tailor the AI-technology to the needs of the Medical Imaging community and to shorten the pathway towards the clinical applicability of AI-based decision support systems.

Dosimetric optimization of nuclear medicine therapy based on the Council Directive 2013/59/EURATOM and the Italian law N. 101/2020. Position paper and recommendations by the Italian National Associations of Medical Physics (AIFM) and Nuclear Medicine (AIMN).

This recommendation by the Italian Associations of Nuclear Medicine (AIMN) and Medical Physics (AIFM) focuses on the dosimetric optimization of Nuclear Medicine Therapy (NMT) as clearly requested by the article 56 of the EURATOM Directive 2013/59 and its consequent implementation in article 158 in the Italian Law n. 101/2020. However, this statement must deal with scientific and methodological limits that still exist and, above all, with the currently available limited resources. This paper addresses these specific issues. It distinguishes among many possible kinds of NMT. For each type, dosimetric optimization is recommended or considered optional, according to the general criteria adopted in any human choice, i.e. a check of technical feasibility first, followed by a cost/benefit argument. The classification of therapies as standardized or non-standardized is presented. This is based on the complexity of the type of pathology, on the variability of the treatment outcome, and on the risks involved. According to the present document, which was officially delivered to Italian Health Ministry as necessary interpretation of the law, a therapeutic team can, in science and consciousness, overcome the indications of posology, to optimize and tailoring a treatment with dosimetry, on the basis of published national or international data or guidelines, without need of an Ethics Committee approval. Data collected in this way will provide additional evidence about optimal dosimetric reference values. As conclusion, a formal appeal is made to the European and National regulatory agencies for pharmaceuticals to obtain the official acknowledgment of this principle.

The influence of basic plan parameters on calculated small field output factors - A multicenter study.

PURPOSE: The influence of basic plan parameters such as slice thickness, grid resolution, algorithm type and field size on calculated small field output factors (OFs) was evaluated in a multicentric study. METHODS AND MATERIALS: Three computational homogeneous water phantoms with slice thicknesses (ST) 1, 2 and 3 mm were shared among twenty-one centers to calculate OFs for 1x1, 2x2 and 3x3 cm2 field sizes (FSs) (normalized to 10x10 cm2 FS), with their own treatment planning system (TPS) and the energy clinically used for stereotactic body radiation therapy delivery. OFs were calculated for each combination of grid resolution (GR) (1, 2 and 3 mm) and ST and finally compared with the OFs measured for the TPS commissioning. A multivariate analysis was performed to test the effect of basic plan parameters on calculated OFs. RESULTS: A total of 509 data points were collected. Calculated OFs are slightly higher than measured ones. The multivariate analysis showed that Center, GR, algorithm type, and FS are predictive variables of the difference between calculated and measured OFs (p < 0.001). As FS decreases, the spread in the difference between calculated and measured OFs became larger when increasing the GR. Monte Carlo and Analytical Anisotropic Algorithms, presented a dependence on GR (p < 0.01), while Collapsed Cone Convolution and Acuros did not. The effect of the ST was found to be negligible. CONCLUSIONS: Modern TPSs slightly overestimate the calculated small field OFs compared with measured ones. Grid resolution, algorithm, center number and field size influence the calculation of small field OFs.

Lean Thinking to manage a national working group on physics aspects of Stereotactic Body Radiation Therapy.

PURPOSE: To report how the adoption of a Lean Thinking mindset in the management of a national working group (WG) on the physics of stereotactic body radiation therapy (SBRT) contributed to achieve SBRT standardization objectives. METHODS: Vision for the WG has been established as fragmentation reduction and process harmonization enhancement in SBRT for Italian centers. Two main research themes of the technical aspects of SBRT emerged as areas with major standardization improvement needs, small field dosimetry and SBRT planning comparisons, to be investigated through multi-institutional studies. The management of the WG leveraged on the Lean concept of fostering self-organization in a non-hierarchical environment. Four progressive involvement levels were defined for each study. No specific "scientific" pre-experience was required to propose and coordinate a project, just requiring a voluntary commitment. People engagement was measured in terms of number of published articles. The standardization goals have been conducted through a simplified "5S" (Sort, Set in Order, Shine, Standardize, and Sustain) methodology, first considering a phase of awareness (the first three "S"), then identifying and implementing standardization actions (the last two "S"). RESULTS: Since the beginning, 157 medical physicists joined the AIFM/SBRT-WG. Twenty-four papers/reviews/letters have been published in the period 2014-2019 on major radiation oncology journals, authored by >100 physicists (>50% working in small hospitals). Six over 12 first authors worked in peripheral/small hospitals, with no prior publication as first author. These studies contributed to the awareness and standardization phases for both small-field dosimetry and planning. In particular, errors in small-field measurements in 8% of centers were detected thanks to a generalized output factor curve in function of the effective field size created by averaging data available from different Linacs. Furthermore, planner's experience in SBRT was correlated with dosimetric parameters in the awareness phase; while sharing median dose volume histograms (DVHs) reduced variability among centers while keeping the same level of plan complexity. Finally, all the dosimetric parameters statistically significant to the planner experience during the awareness phase, were no longer significantly different in the standardization phase. CONCLUSIONS: The experience of our SBRT-WG has shown how a Lean Thinking mindset could foster the SBRT procedure standardization and spread the physics of SBRT knowledge, enhancing personal growth. Our expectation is to inspire other scientific societies that have to deal with fragmented contexts or pursue processes harmonization through Lean principles.

Estimating dose delivery accuracy in stereotactic body radiation therapy: A review of in-vivo measurement methods.

Stereotactic body radiation therapy (SBRT) has been recognized as a standard treatment option for many anatomical sites. Sophisticated radiation therapy techniques have been developed for carrying out these treatments and new quality assurance (QA) programs are therefore required to guarantee high geometrical and dosimetric accuracy. This paper focuses on recent advances on in-vivo measurements methods (IVM) for SBRT treatment. More specifically, all of the online QA methods for estimating the effective dose delivered to patients were compared. Determining the optimal IVM for performing SBRT treatments would reduce the risk of errors that could jeopardize treatment outcome. A total of 89 papers were included. The papers were subdivided into the following topics: point dosimeters (PD), transmission detectors (TD), log file analysis (LFA), electronic portal imaging device dosimetry (EPID), dose accumulation methods (DAM). The detectability capability of the main IVM detectors/devices were evaluated. All of the systems have some limitations: PD has no spatial data, EPID has limited sensitivity towards set-up errors and intra-fraction motion in some anatomical sites, TD is insensitive towards patient related errors, LFA is not an independent measure, DAMs are not always based on measures. In order to minimize errors in SBRT dose delivery, we recommend using synergic combinations of two or more of the systems described in our review: on-line tumor position and patient information should be combined with MLC position and linac output detection accuracy. In this way the effects of SBRT dose delivery errors will be reduced.