Association between rainfall and Escherichia coli in live bivalve molluscs harvested in Sardinia, Italy.

Rainfall is generally accepted as one of the most important factors associated with an increased level of E. coli in bivalve molluscs. Performing microbiological risk assessment is relevant to official control authorities to determine the sanitary status of harvesting areas and, therefore, develop monitoring strategies and identify management practices that could be used to improve the quality and safety of the final product. The present study aimed to investigate the impact of rainfall on the content of E. coli in bivalve molluscs farmed in Sardinia (Italy). Enumeration of E. coli was performed according to the Most Probable Number (MPN) method (ISO 16649-3) on 1,920 bivalve samples collected from 7 regional counties between 2018 and 2020. Bivalve molluscs samples included 955 mussels (Mytilus galloprovincialis), 500 oysters (Crassostrea gigas), 325 clams (Ruditapes decussatus), 94 warty venus (Venus verrucosa), and 46 lagoon cockles (Cerastoderma glaucum). Rainfall data were obtained by the Department of Meteorology of the ARPA Sardegna. For each sampling site, GPS coordinates were used to identify gauge stations within catchment areas. Cumulative rain (mm) was recorded 1, 3, 5, 7, and 15 days before sampling, among which the 7-day cumulative rain was the strongest predictor of E. coli counts. Several thresholds of 7-day cumulative rain (from <10 mm up to >300 mm) before sampling were used to estimate the chances of a non-compliant sample (E. coli levels above the limit for sanitary class A; 230 MPN/100 g). The 7-day cumulative rain was positively associated with the chances of non-compliance. When the 7-day cumulative rain before sampling was >300 mm, 80.5 % of the samples were non-compliant, and the odds of a non-compliant sample were 23.6 times higher, as compared to samples harvested when the 7-day cumulative rainfall was <10 mm. Precipitation data could be a useful tool for interpreting anomalous results from official control authorities and reduce the costs that originate from closure of production areas.

CPOP: An open source C++ cell POPulation modeler for radiation biology applications.

PURPOSE: Multicellular tumor spheroids are realistic in-vitro systems used in radiation biology research to study the effect of anticancer drugs or to evaluate the resistance of cancer cells under specific conditions. When combining the modeling of spheroids together with the simulation of radiation using Monte Carlo methods, one could estimate cell and DNA damage to be compared with experimental data. We developed a Cell Population (CPOP) modeler combined to Geant4 simulations in order to tackle how energy depositions are allocated to cells, especially when enhancing radiation outcomes using high-Z nanoparticles. CPOP manages to model large three-dimensional cell populations with independent deformable cells described with their nucleus, cytoplasm and membranes together with force law systems to manage cell-cell interactions. METHODS: CPOP is an opensource platform written in C++. It is divided into two main libraries: a "Modeler" library, for cell geometry modeling using meshes, and a Multi Agent System (MAS) library, simulating all agent (cell) interactions among the population. CPOP is fully interfaced with the Geant4 Monte Carlo toolkit and is able to directly launch Geant4 simulations after compilation. We modeled a full and realistic 3D cell population from SK-MEL28 melanoma cell population cultured experimentally. The spheroid diameter of 550 ± 40 µm corresponds to a population of approximately 1000 cells having a diameter of 17.2 ± 2.5 µm and a nucleus diameter of 11.2 ± 2.0 µm. We decided to reproduce cell irradiations performed with a X-RAD 320 Biological Irradiator (Precision XRay Inc., North Branford, CT). RESULTS: We simulated the energy spectrum of secondary particles generated in the vicinity of the spheroid and plotted the different energy spectra recovered internally to the spheroid. We evaluated also the impact of AGuIX (Gadolinium) nanoparticles modeled into the spheroid with their corresponding secondary energy spectra. CONCLUSIONS: We succeeded into modeling cell populations and combined them with Geant4 simulations. The next step will be to integrate DNA geometrical models into cell nuclei and to use the Geant4-DNA physics and radiolysis modeling capabilities in order to evaluate early strand breaks induced on DNA.

TRPV4 inhibition attenuates stretch-induced inflammatory cellular responses and lung barrier dysfunction during mechanical ventilation.

Mechanical ventilation is an important tool for supporting critically ill patients but may also exert pathological forces on lung cells leading to Ventilator-Induced Lung Injury (VILI). We hypothesised that inhibition of the force-sensitive transient receptor potential vanilloid (TRPV4) ion channel may attenuate the negative effects of mechanical ventilation. Mechanical stretch increased intracellular Ca2+ influx and induced release of pro-inflammatory cytokines in lung epithelial cells that was partially blocked by about 30% with the selective TRPV4 inhibitor GSK2193874, but nearly completely blocked with the pan-calcium channel blocker ruthenium red, suggesting the involvement of more than one calcium channel in the response to mechanical stress. Mechanical stretch also induced the release of pro-inflammatory cytokines from M1 macrophages, but in contrast this was entirely dependent upon TRPV4. In a murine ventilation model, TRPV4 inhibition attenuated both pulmonary barrier permeability increase and pro-inflammatory cytokines release due to high tidal volume ventilation. Taken together, these data suggest TRPV4 inhibitors may have utility as a prophylactic pharmacological treatment to improve the negative pathological stretch-response of lung cells during ventilation and potentially support patients receiving mechanical ventilation.

The novel cannabinoid antagonist SM-11 reduces hedonic aspect of food intake through a dopamine-dependent mechanism.

Cannabinoids, endogenous and exogenously administered, are known to positively regulate food intake and energy balance. Since CB1 receptor antagonists reduce food intake and antagonize overweight, we developed a new CB1 receptor antagonist in an attempt to identify a compound with potential application in overeating disorders. The newly developed SM-11 compound dose-dependently decreases food intake in rats by 15-20%. Moreover, SM-11 reduces self-administration of palatable food in both food restricted and ad libitum fed rats, suggesting an action on the hedonic component of food intake. Thus, we next tested the effect of SM-11 on the stimulating properties of the CB1 receptor agonist WIN55,212-2 (WIN) on the electrophysiological activity of Nucleus Accumbens-projecting dopaminergic neurons of the ventral tegmental area (VTA). SM-11 fully and readily antagonized the WIN-induced increments in single spiking and burst firing of antidromically-identified dopamine neurons. When administered to naïve (no WIN-pretreated) rats, SM-11 did not alter basal neuronal activity, thereby suggesting a pure antagonistic profile. SM-11 thus appears as a promising candidate in the search of potential anti-obesity medications.

Dosimetric evaluation of new approaches in GRID therapy using nonconventional radiation sources.

PURPOSE: Spatial fractionation of the dose has proven to be a promising approach to increase the tolerance of healthy tissue, which is the main limitation of radiotherapy. A good example of that is GRID therapy, which has been successfully used in the management of large tumors with low toxicity. The aim of this work is to explore new avenues using nonconventional sources: GRID therapy by using kilovoltage (synchrotron) x-rays, the use of very high-energy electrons, and proton GRID therapy. They share in common the use of the smallest possible grid sizes in order to exploit the dose-volume effects. METHODS: Monte Carlo simulations (penelope/peneasy and geant4/GATE codes) were used as a method to study dose distributions resulting from irradiations in different configurations of the three proposed techniques. As figure of merit, percentage (peak and valley) depth dose curves, penumbras, and central peak-to-valley dose ratios (PVDR) were evaluated. As shown in previous biological experiments, high PVDR values are requested for healthy tissue sparing. A superior tumor control may benefit from a lower PVDR. RESULTS: High PVDR values were obtained in the healthy tissue for the three cases studied. When low energy photons are used, the treatment of deep-seated tumors can still be performed with submillimetric grid sizes. Superior PVDR values were reached with the other two approaches in the first centimeters along the beam path. The use of protons has the advantage of delivering a uniform dose distribution in the tumor, while healthy tissue benefits from the spatial fractionation of the dose. In the three evaluated techniques, there is a net reduction in penumbra with respect to radiosurgery. CONCLUSIONS: The high PVDR values in the healthy tissue and the use of small grid sizes in the three presented approaches might constitute a promising alternative to treat tumors with such spatially fractionated radiotherapy techniques. The dosimetric results presented here support the interest of performing radiobiology experiments in order to evaluate these new avenues.

Proton-minibeam radiation therapy: a proof of concept.

PURPOSE: This Monte Carlo simulation work aims at studying a new radiotherapy approach called proton-minibeam radiation therapy (pMBRT). The main objective of this proof of concept was the evaluation of the possible gain in tissue sparing, thanks to the spatial fractionation of the dose, which could be used to deposit higher and potentially curative doses in clinical cases where tissue tolerances are a limit for conventional methods. METHODS: Monte Carlo simulations (GATE v.6) have been used as a method to calculate the ratio of the peak-to-valley doses (PVDR) for arrays of proton minibeams of 0.7 mm width and several center-to-center distances, at different depths in a water phantom. The beam penumbras were also evaluated as an important parameter for tissue sparing, for example, in the treatment of non-cancer diseases like epilepsy. Two proton energies were considered in this study: a clinically relevant energy (105 MeV) and a very high energy (1 GeV), to benefit from a reduced lateral scattering. For the latter case, an interlaced geometry was also evaluated. RESULTS: Higher or similar PVDR than the ones obtained in x-rays minibeam radiation therapy were achieved in several pMBRT configurations. In addition, for the two energies studied, the beam penumbras are smaller than in the case of Gamma Knife radiosurgery. CONCLUSIONS: The high PVDR obtained for some configurations and the small penumbras in comparison with existing radiosurgery techniques, suggest a potential gain in healthy tissue sparing in this new technique. Biological studies are warranted to assess the effects of pMBRT on both normal and tumoral tissues.

Nanofriction of neon films on superconducting lead.

With a quartz crystal microbalance technique we have studied the nanofriction of neon monolayers deposited on a lead surface at a temperature around 7 K. Unlike heavier adsorbates, Ne is found to systematically slide at such low temperatures without any evidence of pinning. The crossing of the Pb superconducting-metal transition is not accompanied by any change in dissipation, suggesting that the electronic contribution to friction is negligible for this system.

Gadolinium dose enhancement studies in microbeam radiation therapy.

Microbeam radiation therapy (MRT) is an innovative technique to treat brain tumors. The synchrotron generated x-ray beam, used for the treatment, is collimated and delivered in an array of narrow micrometer-sized planar rectangular fields. Several preclinical experiments performed at the Brookhaven National Laboratory (BNL) and at the European Synchrotron Radiation Facility (ESRF) have shown the sparing effect of the healthy tissue and the ablation of tumors in several animal models. It has also been determined that MRT yields a higher therapeutic index than nonsegmented beams of the same energy. This therapeutic index could be greatly improved by loading the tumor with high atomic number (Z) contrast agents. In this work, the dose enhancement factors and the peak to valley dose ratios (PVDRs) are assessed for different gadolinium (Z = 64) concentrations in the tumor and different microbeam energies by using Monte Carlo simulations (PENELOPE 2006 code). A significant decrease in the PVDR values in the tumor, and therefore a relevant increase in the dose deposition, is found in the presence of gadolinium. The optimum energy for the dose deposition in the tumor while keeping a high PVDR in the healthy tissues, which guaranties their sparing, has been investigated.

Biological equivalent dose studies for dose escalation in the stereotactic synchrotron radiation therapy clinical trials.

Synchrotron radiation is an innovative tool for the treatment of brain tumors. In the stereotactic synchrotron radiation therapy (SSRT) technique a radiation dose enhancement specific to the tumor is obtained. The tumor is loaded with a high atomic number (Z) element and it is irradiated in stereotactic conditions from several entrance angles. The aim of this work was to assess dosimetric properties of the SSRT for preparing clinical trials at the European Synchrotron Radiation Facility (ESRF). To estimate the possible risks, the doses received by the tumor and healthy tissues in the future clinical conditions have been calculated by using Monte Carlo simulations (PENELOPE code). The dose enhancement factors have been determined for different iodine concentrations in the tumor, several tumor positions, tumor sizes, and different beam sizes. A scheme for the dose escalation in the various phases of the clinical trials has been proposed. The biological equivalent doses and the normalized total doses received by the skull have been calculated in order to assure that the tolerance values are not reached.

Structural depinning of Ne monolayers on Pb at T < 6/5 K.

We have studied the nanofriction of Ne monolayers with a quartz-crystal microbalance technique at temperatures below 6.5 K and in ultrahigh-vacuum conditions. Very homogeneous and smooth lead electrodes have been physically deposited on a quartz blank at 150 K and then annealed at room temperatures. With such a Pb-plated quartz-crystal microbalance, we have observed a pronounced depinning transition separating a low-coverage region, where the film is nearly locked to the oscillating electrode, from a high-coverage region characterized by slippage at the solid-fluid boundary. Such a behavior has been found to be very reproducible. These data are suggestive of a structural depinning of the solid Ne film when it becomes incommensurate with the lead substrate, in agreement with the results of an extensive molecular-dynamics study.

Trypsin-activatable inactive renin in rat plasma.

1. Activation of inactive renin in rat plasma has been studied with different trypsin concentrations and incubation times at pH 6.2 and 4 degrees C. 2. Trypsin concentrations below 2 mg/ml, lower than endogenous rat plasma anti-trypsin activity, do not activate inactive renin, whereas maximal activation is obtained with trypsin at 6 mg/ml for 1 min at 4 degrees C, pH 6.2. 3. Under these conditions trypsin can cleave dialysable fragments from renin substrate. ANG I can be generated at 37 degrees C with a pH optimum of 5.3. Nevertheless, the ANG I formation at pH 6.2 was totally unaffected. 4. Incubations longer than 2 min with trypsin at 6 mg/ml can induce a direct cleavage of dialysable ANG I-containing fragments strongly interfering with the measurements of renin activity at pH 6.2. 5. On average 40% of the total renin measured in plasma of normotensive WK rats is in the inactive form, although a wide range of variation is observed.

Active and inactive renin after a single dose of captopril in hypertensive patients.

In 25 hypertensive patients (15 with renal artery stenosis and 10 with essential hypertension), captopril, in a single 12.5 mg dose, caused a prompt decrease in arterial pressure without changing the heart rate. Plasma active and trypsin-activated renin significantly increases, whereas inactive renin and plasma aldosterone decreased. The plasma active/inactive renin ratio was also increased, suggesting that captopril, together with a release of active renin, may induce an in vivo activation of inactive renin. No correlations were found between blood pressure changes and both pretreatment and captopril-induced variations of active, inactive and trypsin-activated renin or the active/inactive ratio. However, the percent decrease in mean arterial pressure was significantly related to the increase in the active/inactive renin ratio in a group of patients whose blood pressure was brought to normal (r = -0.78; p less than 0.001). This finding suggests the possibility that vasodilating substances, in addition to inhibiting angiotensin II formation, might play some role both in exerting a full effect of captopril on blood pressure and in triggering the in vivo mechanisms of inactive renin activation.

Studies on trypsin activation of inactive renin in rat plasma.

The experimental conditions of trypsin activation of inactive renin in rat plasma have been studied. Our results showed that the pH optimum of trypsin-activated renin is 6.2, the same as that of rat plasma active renin. Trypsin concentration and incubation time might also interfere with the activation process. Trypsin concentration below 2 mg/ml cannot activate inactive renin. Trypsin at a concentration of 6 mg/ml for 1 min. can cleave dialyzable fragments from renin substrate which can generate Angiotensin I at 37 degrees C with a pH optimum of 5.3 but which do not affect Angiotensin generation at pH 6.2. Longer incubations (more than 2 min.), on the contrary, can produce a cleavage at 4 degrees C of Angiotensin I containing fragments directly from renin substrate strongly interfering with measurements of renin activity at pH 6.2. Trypsin concentrations lower than endogenous rat plasma anti-trypsin activity do not activate inactive renin. Much higher concentrations of trypsin, however, are needed to obtain optimum activation of inactive renin. In average, 40% of the total circulating renin in rat plasma can be activated by trypsin.

Trypsin activation of inactive renin in human plasma. An assessment of some methodological aspects.

The following methodological aspects of the use of trypsin as activator of inactive renin in human plasma have been studied: a) the effect of SBTI on renin activity and angiotensin; b) the reaction velocity of trypsin on inactive renin; c) the optimum trypsin concentration; d) the ability of human plasma to neutralize exogenous trypsin. Our results show that: 1) Some commercially available SBTI may exert an angiotensinase-like effect which can be abolished by PMSF. 2) At 4 degrees C activation of inactive renin reached a maximum within the first two minutes then no further activation could be demonstrated. 3) Trypsin 2 mg/ml yielded more inactive renin than trypsin 1 or 0.5 mg/ml. A higher concentration (3 mg/ml) gave substantially equivalent activation as (with) trypsin 2 mg/ml whereas when using a still higher concentration (4 mg/ml) a degradation of the renin system components could be noted. 4) Endogenous trypsin inhibitors can eventually inactivate exogenous trypsin up to 3 mg/ml. About 20% of renin is destroyed by trypsin 4 mg/ml within 2 min at 4 degrees C while an additional 40% is lost during the incubation at 37 degrees C if no SBTI is added.

[Methodology of renal vein renin study: comparison between simultaneous and sequential sampling (author's transl)]. / Metodologia del cateterismo delle vene renali per la determinazione della secrezione reninica separata. Confronto tra prelievi simultanei e sequenziali.

Purpose of this study has been to compare the results obtained using two different procedures in blood sampling from the renal veins for measuring renal venous renin. The first is the classical procedure which employs three catheters for simultaneous sampling from both renal veins and from the inferior vena cava, or from an artery. The other one is a simplified procedure which employs a single catheter that allows blood to be collected in the following rapid sequential manner: right renal vein, inferior vena cava, left renal vein, inferior vena cava. We have studied 13 patients (8 with essential hypertension, 5 with unilateral renal artery stenosis). Two catheters were introduced through a femoral vein and inserted into both renal veins; a third catheter was inserted into the femoral artery; then the blood sampling was performed strictly simultaneously. Soon after, the blood sampling was repeated according to the above mentioned sequential single catheter procedure. PRA was measured by Angiotensin I radioimmunoassay, then the Renal Vein Ratios (RVRR) were calculated. Even though as average of less than 20 seconds elapsed between the blood sampling in a renal vein and that in inferior vena cava, our results demonstrate that the release of renin can vary so quickly that erroneous informations may be obtained unless a strictly simultaneous sampling of blood is performed. In conclusion, our study demonstrates that the only reliable renal vein renin sampling procedure must employ the simultaneous renal venous and arterial (or inferior vena cava) blood collection.