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Various in vivo experimental works carried out on different animals and organs have shown that it is possible to reduce the damage caused to healthy tissue still preserving the therapeutic efficacy on the tumor tissue, by drastically reducing the total time of dose delivery (in vivo experiments. Moreover, the work discusses the saturation problems of the most common radiotherapy dosimeters when used in the very high dose-per-pulse FLASH conditions and provides some preliminary experimental data on their behavior.

Since Favaudon’s paper of 2014, there has been an increasing interest in FLASH radiotherapy. The FLASH modality could represent a breakthrough in radiation oncology; nevertheless, it brings new scientific and technological challenges. Currently, one of the main limits the scientific community has to cope with is the lack of a common technological platform to experiment with. Considering this framework, the possibility of readapting existing linac platforms to produce a FLASH beam is particularly attractive and different attempts have been already made. The purpose of this article is to illustrate how it is possible to transform a dedicated Intra Operative Radio Therapy (IORT) mobile linac into a FLASH research machine. Compared to the modification required by a standard medical linac, such transformation is easier, does not affect the machine settings and can be rapidly performed by the final user. NOVAC 7 is an IORT linac which can reach a maximum dose-per-pulse up to 13 cGy/pulse (average dose rate 39 Gy/min); such dose rate can be significantly increased by modifying the collimation system. Four different Source Surface Distance (SSD) can be obtained: -Clinical reference configuration; -Upper applicator only (SSD 50 cm); -Monitor chambers housing only (SSD 7 cm); -Dismounted monitor chambers (SSD 1.6 cm). The fourth configuration allows reaching values of dose-per-pulse up to around 18 Gy/pulse and dose rates up to around 500 Gy/s, at a Pulse Repetition Frequency (PRF) of 30 Hz. The other three configurations can be obtained without using any tool and without changing NOVAC settings, until reaching a FLASH dose rate in the third configuration. For FLASH configurations, relative and absolute dosimetric characterization of the beam were performed using radiochromic films EBT3. NOVAC7 transformed in FLASH mode can be used both for dosimetric testing and characterization of detectors and for radiobiological studies on cells and organoids, offering a wide range of dose-per-pulse, from 3 cGy/pulse up to 18 Gy/pulse; dose rates correspondingly change from 3 cGy/s up to 540 Gy/s.

The spatial and temporal changes of morphological and mechanical properties of living cells reflect complex functionally-associated processes. Monitoring these modifications could provide a direct information on the cellular functional state. Here we present an integrated biophysical approach to the quantification of the morphological and mechanical phenotype of single cells along a maturation pathway. Specifically, quantitative phase microscopy and single cell biomechanical testing were applied to the characterization of the maturation of human foetal osteoblasts, demonstrating the ability to identify effective label-free biomarkers along this fundamental biological process.

A novel real-time charged particle detector for medical accelerators is proposed in this paper. The system, by means of a passive resonant cavity, performs a non destructive measurement of the beam current and provides a real time monitoring of the absorbed dose delivered to the patient. The system complies with requirements of the relevant International Standards for being used as a Radiation Detector for Charged Particles. The formulated theory allows the system design versus the accelerator features by providing analytical formulas and a development strategy. A prototype has been realized and tested in order to check the compliance with the expected behavior in the operating conditions of the Medical Accelerator. The system has been proved to be equivalent to the standard ionization based monitor chambers, furthermore offering several advantages: the absence of bias high voltage, more compactness, small size and the fact that this system measures the physical observable quantity directly related with the dose, the beam current.

ClC-5 is a 2Cl−/1H+ antiporter highly expressed in endosomes of proximal tubule cells. It is essential for endocytosis and mutations in ClC-5 cause Dent's disease, potentially leading to renal failure. However, the physiological role of ClC-5 is still unclear. One of the main issues is whether the strong rectification of ClC-5 currents observed in heterologous systems, with currents elicited only at positive voltages, is preserved in vivo and what is the origin of this rectification. In this work we identified a ClC-5 mutation, D76H, which, besides the typical outward currents of the wild-type (WT), shows inward tail currents at negative potentials that allow the estimation of the reversal of ClC-5 currents for the first time. A detailed analysis of the dependence of these inward tail currents on internal and external pH and [Cl−] shows that they are generated by a coupled transport of Cl− and H+ with a 2 : 1 stoichiometry. From this result we conclude that the inward tail currents are caused by a gating mechanism that regulates ClC-5 transport activity and not by a major alteration of the transport mechanism itself. This implies that the strong rectification of the currents of WT ClC-5 is at least in part caused by a gating mechanism that activates the transporter at positive potentials. These results elucidate the biophysical properties of ClC-5 and contribute to the understanding of its physiological role.

ClC-5 is a Cl-/H+ antiporter that functions in endosomes and is important for endocytosis in the proximal tubule. The mechanism of transport coupling and voltage dependence in ClC-5 is unclear. Recently, a transport-deficient ClC-5 mutant (E268A) was shown to exhibit transient capacitive currents. Here, we studied the external and internal Cl- and pH dependence of the currents of E268A. Transient currents were almost completely independent of the intracellular pH. Even though the transient currents are modulated by extracellular pH, we could exclude that they are generated by proton-binding/unbinding reactions. In contrast, the charge movement showed a nontrivial dependence on external chloride, strongly supporting a model in which the movement of an intrinsic gating charge is followed by the voltage-dependent low-affinity binding of extracellular chloride ions. Mutation of the external Glu-211 (a residue implicated in the coupling of Cl- and proton transport) to aspartate abolished steady-state transport, but revealed transient currents that were shifted by similar to 150 mV to negative voltages compared to E268A. This identifies Glu(ext) as a major component of the gating charge underlying the transient currents of the electrogenic ClC-5 transporter. The molecular events underlying the transient currents of ClC-5 emerging from these results can be explained by an inward movement of the side chain of Glu(ext), followed by the binding of extracellular Cl- ions.

Human ClC-5 is a Cl−/H+ antiporter belonging to the CLC family, which includes both Cl− channels and Cl−/H+ antiporters. Small organic molecules have been useful tools for studying ion channels and many commercial drugs target specific ion channel proteins. Some blockers have been found to inhibit the plasma membrane localized CLC channels ClC-0, ClC-1 and ClC-Ka. However, high-affinity ligands for most CLCs are missing.Here, we tested the benzofuran derivative RT-931,2 on ClC-5 and ClC-4. Interestingly RT-93 inhibits ClC-5 (EC50 ∼ 200 μM), but has only a small effect on the highly homologous antiporter ClC-4. Towards our goal to identify the binding site of RT-93 on ClC-5, we constructed 12 chimeras between ClC-4 and ClC-5. Guided by the crystal structure of a bacterial CLC protein and using the alignment between ClC-4 and ClC-5, we identified the sequence stretches with the main differences between these two proteins and substituted in ClC-5 the portions with the corresponding sequence of ClC-4. We expect to remove the RT-93 sensitivity at least in one of these. So far we tested 8 chimeras, none of which, however, showed a significant difference in comparison with ClC-5. If none of the chimeras eliminates the binding site we will proceed with constructing larger chimeras.Discovering the binding site of this blocker might help in creating a more efficient ligand that, apart from being a useful biophysical tool, may provide a way to modulate protein function in vivo.(Supported by PRIN, Telethon (GGP08064; GGP12008) and IIT).1. Liantonio et al. 2008. PNAS2. Liantonio et al. 2012. J Hypertens.

Osteoporosis, characterized by excessive osteoclast mediated bone resorbtion, affects millions of people. ClC-7 is a chloride-proton exchanger member of the CLC protein family localized in lysosomes and in the ruffled border of osteoclasts. Loss of function of ClC-7 leads to osteopetrosis, neurodegeneration and lysosomal storage disease. The osteopetrotic phenotype is explained by the fact that the ion transport activity of ClC-7 is essential for the osteoclast mediated bone resorption. Thus, blocking ClC-7 can be expected to provide an effective treatment of osteoporosis. Here, we describe a purely optical assay of ClC-7 function employing the E2GFP-dsRed Cl−/pH sensor1 fused to the C-terminus of ClC-7, carrying in addition a mutation of a leucine motif at the N-terminus resulting in a partial redirection of ClC-7 to the plasma membrane2-3. For functional activity, ClC-7 associates with the beta-subunit Ostm14. To ensure the simultaneous and equivalent expression of ClC-7 and Ostm1, we constructed a plasmid in which the two cDNAs are linked by a self-cleavable 2AP peptide4. In addition, we introduced a mutation of the gating glutamate (E245A) which renders the transporter voltage-independent and abolishes proton transport3. The assay consists in lowering [Cl]ext and monitoring the resulting decrease in ClC-7 mediated decrease of [Cl]int by measuring the E2GFP/DSRed fluorescence ratio. This simple assay can be applied in HTS and may lead to the design of specific drugs modulating ClC-7 activity.Supported by IIT, Seed project1. Arosio D et al (2010) Nature Methods7, 516.2. Stauber T, Jentsch TJ (2010). J Biol Chem 285: 34537.3. Leisle L et al. (2011 EMBO J. 30, 2140.4. Trishas G et al (2008). BMC Biol. Sep 15;6.

Background: The mechanism of anion selectivity of Cl− transporting CLC proteins is not fully understood. Results: Mutational analysis of the extracellularly exposed CLC-5 residue Lys210 indicates its essential role in anion discrimination. Conclusion: A positive charge at position 210 is crucial to confer NO3− over Cl− preference in coupled anion/proton transport. Significance: These findings suggest a novel mechanism responsible for anion discrimination in CLC proteins., Mammalian CLC proteins comprise both Cl− channels and Cl−/H+ antiporters that carry out fundamental physiological tasks by transporting Cl− across plasma membrane and intracellular compartments. The NO3− over Cl− preference of a plant CLC transporter has been pinpointed to a conserved serine residue located at Scen and it is generally assumed that the other two binding sites of CLCs, Sext and Sin, do not substantially contribute to anion selectivity. Here we show for the Cl−/H+ antiporter CLC-5 that the conserved and extracellularly exposed Lys210 residue is critical to determine the anion specificity for transport activity. In particular, mutations that neutralize or invert the charge at this position reverse the NO3− over Cl− preference of WT CLC-5 at a concentration of 100 mm, but do not modify the coupling stoichiometry with H+. The importance of the electrical charge is shown by chemical modification of K210C with positively charged cysteine-reactive compounds that reintroduce the WT preference for Cl−. At saturating extracellular anion concentrations, neutralization of Lys210 is of little impact on the anion preference, suggesting an important role of Lys210 on the association rate of extracellular anions to Sext.

The tissue specificity of fibrillar deposition in dialysis-related amyloidosis is most likely associated with the peculiar interaction of beta2-microglobulin (beta2-m) with collagen fibers. However, other co-factors such as glycosaminoglycans might facilitate amyloid formation. In this study we have investigated the role of heparin in the process of collagen-driven amyloidogenesis. In fact, heparin is a well known positive effector of fibrillogenesis, and the elucidation of its potential effect in this type of amyloidosis is particularly relevant because heparin is regularly given to patients subject to hemodialysis to prevent blood clotting. We have monitored by atomic force microscopy the formation of beta2-m amyloid fibrils in the presence of collagen fibers, and we have discovered that heparin strongly accelerates amyloid deposition. The mechanism of this effect is still largely unexplained. Using dynamic light scattering, we have found that heparin promotes beta2-m aggregation in solution at pH 6.4. Morphology and structure of fibrils obtained in the presence of collagen and heparin are highly similar to those of natural fibrils. The fibril surface topology, investigated by limited proteolysis, suggests that the general assembly of amyloid fibrils grown under these conditions and in vitro at low pH is similar. The exposure of these fibrils to trypsin generates a cleavage at the C-terminal of lysine 6 and creates the 7-99 truncated form of beta2-m (DeltaN6beta2-m) that is a ubiquitous constituent of the natural beta2-m fibrils. The formation of this beta2-m species, which has a strong propensity to aggregate, might play an important role in the acceleration of local amyloid deposition.

Dialysis-related amyloidosis is characterized by the deposition of insoluble fibrils of beta(2)-microglobulin (beta(2)-m) in the musculoskeletal system. Atomic force microscopy inspection of ex vivo amyloid material reveals the presence of bundles of fibrils often associated to collagen fibrils. Aggregation experiments were undertaken in vitro with the aim of reproducing the physiopathological fibrillation process. To this purpose, atomic force microscopy, fluorescence techniques, and NMR were employed. We found that in temperature and pH conditions similar to those occurring in periarticular tissues in the presence of flogistic processes, beta(2)-m fibrillogenesis takes place in the presence of fibrillar collagen, whereas no fibrils are obtained without collagen. Moreover, the morphology of beta(2)-m fibrils obtained in vitro in the presence of collagen is extremely similar to that observed in the ex vivo sample. This result indicates that collagen plays a crucial role in beta(2)-m amyloid deposition under physiopathological conditions and suggests an explanation for the strict specificity of dialysis-related amyloidosis for the tissues of the skeletal system. We hypothesize that positively charged regions along the collagen fiber could play a direct role in beta(2)-m fibrillogenesis. This hypothesis is sustained by aggregation experiments performed by replacing collagen with a poly-L-lysine-coated mica surface. As shown by NMR measurements, no similar process occurs when poly-L-lysine is dissolved in solution with beta(2)-m. Overall, the findings are consistent with the estimates resulting from a simplified collagen model whereby electrostatic effects can lead to high local concentrations of oppositely charged species, such as beta(2)-m, that decay on moving away from the fiber surface.

ClC-5 is a 2 Cl- / 1 H+ antiporter highly expressed in endosomes of proximal tubule cells. It is essential for endocytosis and mutations in ClC-5 cause Dent's disease potentially leading to renal failure. However, the physiological role of ClC-5 is still unclear. One of the main issues is whether the strong rectification of ClC-5 currents observed in heterologous systems, with currents elicited only at positive voltages, is preserved in vivo and which is the origin of this rectification. We identified a ClC-5 mutation, D76H, that besides the typical outward currents of the WT, shows inward tail currents at negative potentials that allow estimation of the reversal of ClC-5 currents for the first time. A detailed analysis of the dependence of these inward tail currents on internal and external pH and [Cl-] shows that they are generated by a coupled transport of Cl- and H+ with a 2 : 1 stoichiometry. From this result we conclude that the inward tail currents are due to a gating mechanism that regulates ClC-5 transport activity and not to a major alteration of the transport mechanism itself. This implies that the strong rectification of the currents of WT ClC-5 is at least in part due to a gating mechanism that activates the transporter at positive potentials.

Structural and functional studies identified two critical residues for the transport mechanism of CLC transporters; the so-called gating glutamate (E211 in ClC-5) that controls the access of the anions to the extracellular space and is critical for anion/proton coupling and the proton glutamate (E268 in ClC-5), likely the intracellular entry/exit point for protons. However, the mechanism of voltage-sensitivity of CLC transporters is still poorly understood.Interestingly, it has been recently reported that the E268A mutant of the endosomal Cl-/H+ antiporter ClC-5, beside inhibiting steady-state transports, exhibits transient currents upon voltage steps to large positive voltages. These transient currents may offer the possibility to glean information on the molecular details of transport coupling and voltage-sensitivity. Here we studied the dependence of the transient currents on the extracellular and intracellular pH and Cl- concentration. We conclude that the transient currents represent the movement of an intrinsic gating charge followed by the voltage dependent binding of extracellular Cl- ions. In addition, we find that the gating glutamate mutation E211D abolishes stationary transport but displays transient currents which are shifted by ∼ 150 mV compared to the proton glutamate mutation, identifying E211 as a major component of the voltage sensing mechanism of ClC-5.