Not Every Low-Dose Is Low-Dose: Impact of Revising Low-Dose CT Protocol on Mean Effective Radiation Exposure.

Introduction: According to the American Urological Association imaging guidelines, patients presenting with renal colic should undergo low-dose (LD) rather than standard-dose (SD) noncontrast CT. The aim of the present study was to assess how often physicians ordered LD CT scans and to calculate mean effective radiation exposure (ERE) from CT scans from dose length products, and determine mean cumulative ERE over 1-year follow-up period. Methods: After obtaining ethics approval, a retrospective chart review was conducted for patients with renal colic presenting to the emergency department between August 1, 2015 and July 31, 2016 (Phase I) and between April 1, 2019 and October 1, 2019 (Phase II). All imaging studies performed within 1-year of initial presentation were cataloged. Results: In Phase I, 146 patients, with mean age of 51 years and mean body mass index (BMI) of 28.6 kg/m2, underwent 220 CT scans. In Phase II, 225 patients, with mean age of 55 years and mean BMI of 26.7 kg/m2, underwent 273 CT scans. Urologists were the only physicians ordering LD CT scans and they ordered significantly more LD than SD CT scans (71.3% vs 28.7%, p < 0.001). In Phase II, after revision of LD CT scan protocol in March 2019, the mean ERE per LD CT significantly decreased (6.5 vs 1.6 mSv, p < 0.001). In addition, there were significant differences in mean ERE from LD CT scans between two hospitals in the same health system (1.6 vs 7.8 mSv, p < 0.001). The mean cumulative ERE in Phase II over the 1-year period was 19.3 mSv, with 6.9% of patients exceeding 50 mSv. Conclusions: Although LD CT scans are being ordered, a small percentage of patients continue to exceed the 50 mSv annual threshold. It is important to keep track of mean ERE of LD CT scans and collaborate with medical physicists and the diagnostic imaging department to further refine LD CT scan protocols since not every low-dose is low-dose.

Effect of allosteric inhibition of non-muscle myosin 2 on its intracellular diffusion.

Subcellular dynamics of non-muscle myosin 2 (NM2) is crucial for a broad-array of cellular functions. To unveil mechanisms of NM2 pharmacological control, we determined how the dynamics of NM2 diffusion is affected by NM2's allosteric inhibitors, i.e. blebbistatin derivatives, as compared to Y-27632 inhibiting ROCK, NM2's upstream regulator. We found that NM2 diffusion is markedly faster in central fibers than in peripheral stress fibers. Y-27632 accelerated NM2 diffusion in both peripheral and central fibers, whereas in peripheral fibers blebbistatin derivatives slightly accelerated NM2 diffusion at low, but markedly slowed it at high inhibitor concentrations. In contrast, rapid NM2 diffusion in central fibers was unaffected by direct NM2 inhibition. Using our optopharmacological tool, Molecular Tattoo, sub-effective concentrations of a photo-crosslinkable blebbistatin derivative were increased to effective levels in a small, irradiated area of peripheral fibers. These findings suggest that direct allosteric inhibition affects the diffusion profile of NM2 in a markedly different manner compared to the disruption of the upstream control of NM2. The pharmacological action of myosin inhibitors is channeled through autonomous molecular processes and might be affected by the load acting on the NM2 proteins.

A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative.

Blebbistatin is a commonly used molecular tool for the specific inhibition of various myosin II isoforms both in vitro and in vivo. Despite its popularity, the use of blebbistatin is hindered by its poor water-solubility (below 10 micromolar in aqueous buffer) and blue-light sensitivity, resulting in the photoconversion of the molecule, causing severe cellular phototoxicity in addition to its cytotoxicity. Furthermore, blebbistatin forms insoluble aggregates in water-based media above 10 micromolar with extremely high fluorescence and also high adherence to different types of surfaces, which biases its experimental usage. Here, we report a highly soluble (440 micromolar in aqueous buffer), non-fluorescent and photostable C15 amino-substituted derivative of blebbistatin, called para-aminoblebbistatin. Importantly, it is neither photo- nor cytotoxic, as demonstrated on HeLa cells and zebrafish embryos. Additionally, para-aminoblebbistatin bears similar myosin II inhibitory properties to blebbistatin or para-nitroblebbistatin (not to be confused with the C7 substituted nitroblebbistatin), tested on rabbit skeletal muscle myosin S1 and on M2 and HeLa cells. Due to its drastically improved solubility and photochemical feature, as well as lack of photo- or cytotoxicity, para-aminoblebbistatin may become a feasible replacement for blebbistatin, especially at applications when high concentrations of the inhibitor or blue light irradiation is required.

A protocol for EBT3 radiochromic film dosimetry using reflection scanning.

PURPOSE: To evaluate the performance of the EBT3 radiochromic film dosimetry system using reflection measurements and to suggest a calibration protocol for precise and accurate reflection film dosimetry. METHODS: A set of 14 Gafchromic EBT3 film pieces were irradiated to various doses ranging from 0 to 8 Gy and subsequently scanned using both the reflection and transmission mode. Scanning resolution varied from 50 to 508 dpi (0.5-0.05 mm/pixel). Both the red and green color channels of scanned images were used to relate the film response to the dose. A sensitivity, uncertainty, and accuracy analysis was performed for all scanning modes and color channels. The total uncertainty, along with the fitting and experimental uncertainty components, was identified and analyzed. A microscope resolution target was used to evaluate possible resolution losses under reflection scanning. The calibration range was optimized for reflection scanning in the low (<2 Gy) and high (>2 Gy) dose regions based on the reported results. RESULTS: Reflection scanning using the red channel exhibited the highest sensitivity among all modes, being up to 150% higher than transmission mode in the red channel for the lowest dose level. Furthermore, there was no apparent loss in resolution between the two modes. However, higher uncertainties and reduced accuracy were observed for the red channel under reflection mode, especially at dose levels higher than 2 Gy. These uncertainties were mainly attributed to saturation effects which were translated in poor fitting results. By restricting the calibration to the 0-2 Gy dose range, the situation is reversed and the red reflection mode was superior to the transmission mode. For higher doses, the green channel in reflection mode presented comparable results to the red transmission. CONCLUSIONS: A two-color reflection scanning protocol can be suggested for EBT3 radiochromic film dosimetry using the red channel for doses less than 2 Gy and the green channel for higher doses. The precision and accuracy are significantly improved in the low dose region following such a protocol.

para-Nitroblebbistatin, the non-cytotoxic and photostable myosin†II inhibitor.

Blebbistatin, the best characterized myosin II-inhibitor, is commonly used to study the biological roles of various myosin II isoforms. Despite its popularity, the use of blebbistatin is greatly hindered by its blue-light sensitivity, resulting in phototoxicity and photoconversion of the molecule. Additionally, blebbistatin has serious cytotoxic side effects even in the absence of irradiation, which may easily lead to the misinterpretation of experimental results since the cytotoxicity-derived phenotype could be attributed to the inhibition of the myosin II function. Here we report the synthesis as well as the in vitro and in vivo characterization of a photostable, C15 nitro derivative of blebbistatin with unaffected myosin II inhibitory properties. Importantly, para-nitroblebbistatin is neither phototoxic nor cytotoxic, as shown by cellular and animal tests; therefore it can serve as an unrestricted and complete replacement of blebbistatin both in vitro and in vivo.

Azidoblebbistatin, a photoreactive myosin inhibitor.

Photoreactive compounds are important tools in life sciences that allow precisely timed covalent crosslinking of ligands and targets. Using a unique technique we have synthesized azidoblebbistatin, which is a derivative of blebbistatin, the most widely used myosin inhibitor. Without UV irradiation azidoblebbistatin exhibits identical inhibitory properties to those of blebbistatin. Using UV irradiation, azidoblebbistatin can be covalently crosslinked to myosin, which greatly enhances its in vitro and in vivo effectiveness. Photo-crosslinking also eliminates limitations associated with the relatively low myosin affinity and water solubility of blebbistatin. The wavelength used for photo-crosslinking is not toxic for cells and tissues, which confers a great advantage in in vivo tests. Because the crosslink results in an irreversible association of the inhibitor to myosin and the irradiation eliminates the residual activity of unbound inhibitor molecules, azidoblebbistatin has a great potential to become a highly effective tool in both structural studies of actomyosin contractility and the investigation of cellular and physiological functions of myosin II. We used azidoblebbistatin to identify previously unknown low-affinity targets of the inhibitor (EC(50) ≥ 50 µM) in Dictyostelium discoideum, while the strongest interactant was found to be myosin II (EC(50) = 5 µM). Our results demonstrate that azidoblebbistatin, and potentially other azidated drugs, can become highly useful tools for the identification of strong- and weak-binding cellular targets and the determination of the apparent binding affinities in in vivo conditions.

Cross-linked long-pitch actin dimer forms stoichiometric complexes with gelsolin segment 1 and/or deoxyribonuclease I that nonproductively interact with myosin subfragment 1.

Actin dimer cross-linked along the long pitch of the F-actin helix by N-(4-azido)-2-nitrophenyl (ANP) was purified by gel filtration. Purified dimers were found to polymerize on increasing the ionic strength, although at reduced rate and extent in comparison with native actin. Purified actin dimer interacts with the actin-binding proteins (ABPs) deoxyribonuclease I (DNase I) and gelsolin segment-1 (G1) as analyzed by gel filtration and native gel electrophoresis. Complex formation of the actin dimer with these ABPs inhibits its ability to polymerize. The interaction with rabbit skeletal muscle myosin subfragment 1 (S1) was analyzed for polymerized actin dimer and dimer complexed with gelsolin segment 1 or DNase I by measurement of the actin-stimulated myosin S1-ATPase and gel filtration. The data obtained indicate binding of subfragment 1 to actin dimer, albeit with considerably lower affinity than to F-actin. Polymerized actin dimer was able to stimulate the S1-ATPase activity to about 50% of the level of native F-actin. In contrast, the actin dimer complexed to DNase I or gelsolin segment 1 or to both proteins was unable to significantly stimulate the S1-ATPase. Similarly, G1:dimer complex at 20 microM stimulated the rate of release of subfragment 1 bound nucleotide (mant-ADP) only 1.6-fold in comparison to about 9-fold by native F-actin at a concentration of 0.5 microM. Using rapid kinetic techniques, a dissociation constant of 2.4 x 10 (-6) M for subfragment 1 binding to G1:dimer was determined in comparison to 3 x 10 (-8) M for native F-actin under identical conditions. Since the rate of association of subfragment 1 to G1:dimer was considerably lower than to native F-actin, we suspect that the ATP-hydrolysis by S1 was catalyzed before its association to the dimer. These data suggest an altered, nonproductive mode for the interaction of subfragment 1 with the isolated long-pitch actin dimer.

Intermonomer cross-linking of F-actin alters the dynamics of its interaction with H-meromyosin in the weak-binding state.

Previous cross-linking studies [Kim E, Bobkova E, Hegyi G, Muhlrad A & Reisler E (2002) Biochemistry 41, 86-93] have shown that site-specific cross-linking among F-actin monomers inhibits the motion and force generation of actomyosin. However, it does not change the steady-state ATPase parameters of actomyosin. These apparently contradictory findings have been attributed to the uncoupling of force generation from other processes of actomyosin interaction as a consequence of reduced flexibility at the interface between actin subdomains-1 and -2. In this study, we use EPR spectroscopy to investigate the effects of cross-linking constituent monomers upon the molecular dynamics of the F-actin complex. We show that cross-linking reduces the rotational mobility of an attached probe. It is consistent with the filaments becoming more rigid. Addition of heavy meromyosin (HMM) to the cross-linked filaments further restricts the rotational mobility of the probe. The effect of HMM on the actin filaments is highly cooperative: even a 1 : 10 molar ratio of HMM to actin strongly restricts the dynamics of the filaments. More interesting results are obtained when nucleotides are also added. In the presence of HMM and ADP, similar strongly reduced mobility of the probe was found than in a rigor state. In the presence of adenosine 5'[betagamma-imido] triphosphate (AMPPNP), a nonhydrolyzable analogue of ATP, weak binding of HMM to either cross-linked or native F-actin increases probe mobility. By contrast, weak binding by the HMM/ADP/AlF4 complex has different effects upon the two systems. This protein-nucleotide complex increases probe mobility in native actin filaments, as does HMM + AMPPNP. However, its addition to cross-linked filaments leaves probe mobility as constrained as in the rigor state. These findings suggest that the dynamic change upon weak binding by HMM/ADP/AlF4 which is inhibited by cross-linking is essential to the proper mechanical behaviour of the filaments during movement.

The crystal structure of a cross-linked actin dimer suggests a detailed molecular interface in F-actin.

The 2.5-A resolution crystal structure is reported for an actin dimer, composed of two protomers cross-linked along the longitudinal (or vertical) direction of the F-actin filament. The crystal structure provides an atomic resolution view of a molecular interface between actin protomers, which we argue represents a near-native interaction in the F-actin filament. The interaction involves subdomains 3 and 4 from distinct protomers. The atomic positions in the interface visualized differ by 5-10 A from those suggested by previous models of F-actin. Such differences fall within the range of uncertainties allowed by the fiber diffraction and electron microscopy methods on which previous models have been based. In the crystal, the translational arrangement of protomers lacks the slow twist found in native filaments. A plausible model of F-actin can be constructed by reintroducing the known filament twist, without disturbing significantly the interface observed in the actin dimer crystal.

Dosimetric properties of improved GafChromic films for seven different digitizers.

Two recently introduced GafChromic film models, HS and XR-T, have been developed as more sensitive and uniform alternatives to GafChromic MD-55-2 film. The HS model has been specifically designed for measurement of absorbed dose in high-energy photon beams (above 1 MeV), while the XR-T model has been introduced for dose measurements of low energy (0.1 MeV) photons. The goal of this study is to compare the sensitometric curves and estimated dosimetric uncertainties associated with seven different GafChromic film dosimetry systems for the two new film models. The densitometers tested are: LKB Pharmacia UltroScan XL, Molecular Dynamics Personal Densitometer, Nuclear Associates Radiochromic Densitometer Model 37-443, Photoelectron Corporation CMR-604, Laser Pro 16, Vidar VXR-16, and AGFA Arcus II document scanner. Pieces of film were exposed to different doses in a dose range from 0.5 to 50 Gy using 6 MV photon beam. Functional forms for dose vs net optical density have been determined for each of the GafChromic film-dosimetry systems used in this comparison. Two sources of uncertainties in dose measurements, governed by the experimental measurement and calibration curve fit procedure, have been compared for the densitometers used. Among the densitometers tested, it is found that for the HS film type the uncertainty caused by the experimental measurement varies from 1% to 3% while the calibration fit uncertainty ranges from 2% to 4% for doses above 5 Gy. Corresponding uncertainties for XR-T film model are somewhat higher and range from 1% to 5% for experimental and from 2% to 7% for the fit uncertainty estimates. Notwithstanding the significant variations in sensitivity, the studied densitometers exhibit very similar precision for GafChromic film based dose measurements above 5 Gy.

Comparative skin dose measurement in the treatment of anal canal cancer: conventional versus conformal therapy.

The subject of this work was to compare the effect of Conventional and Conformal techniques, used for anal canal cancer treatments, on the skin dose deposition. Skin dose was measured on a Rando phantom using XR-T GAFCHROMIC film. A skin surface dose histogram was constructed and a skin dose profile in the sagittal direction of the perineal region was measured, for both techniques. The measured skin dose in the anterior and posterior region of the skin exposed to radiation is from two to ten times higher when using a conventional technique. In the perineal region, an 85% of the prescription isodose line spreads over 25% of the perineum for conformal technique as compared to 65% with conventional techniques. In addition, conformal technique dose profiles confine better the anatomical position of the anal verge than conventional techniques. Results presented in this work confirm clinically observed improvement in the radiation-induced dermatitis when using the conformal technique.

Contribution of conformal therapy in the treatment of anal canal carcinoma with combined chemotherapy and radiotherapy: results of a phase II study.

PURPOSE: To evaluate the feasibility of delivering conformal radiotherapy (RT) and concurrent chemotherapy without a mandatory break in patients with anal canal carcinoma. METHODS AND MATERIALS: Thirty patients with T2-T4 tumors were treated with a combination of 54 Gy in 30 fractions and two cycles of 5-fluorouracil and mitomycin C. Dose-volume histograms were obtained for bone marrow, small bowel, and skin to compare the conventional technique using the Radiation Therapy Oncology Group standard with our conformal technique. RESULTS: The mean dose ratio of the conventional compared with the conformal technique for bone marrow, small bowel, and skin was, respectively, 2.1-2.7, 3.0, and 2.0, in favor of the conformal technique. All patients completed their treatment without a treatment break. An incidence of Grade 3 toxicity for bone marrow, bowel, and skin of 13.3%, 3.3%, and 20%, respectively, was observed. With a median follow-up of 33 months, a 4-year local recurrence-free survival rate of 91% was observed. CONCLUSION: The results of this study have shown that conformal RT leads to a well-tolerated treatment. The treatment time is shortened to 6 weeks. A significant decrease in the acute toxicity rate suggests that by decreasing the "volume factor," conformal RT improves the therapeutic index in patients treated with combined chemotherapy and RT.

Actin cross-linking and inhibition of the actomyosin motor.

Intrastrand cross-linking of actin filaments by ANP, N-(4-azido-2-nitrophenyl) putrescine, between Gln-41 in subdomain 2 and Cys-374 at the C-terminus, was shown to inhibit force generation with myosin in the in vitro motility assays [Kim et al. (1998) Biochemistry 37, 17801-17809]. To clarify the immobilization of which of these two sites inhibits the actomyosin motor, the properties of actins with partially overlapping cross-linked sites were examined. pPDM (N,N'-p-phenylenedimaleimide) and ABP [N-(4-azidobenzoyl) putrescine] were used to obtain actin filaments cross-linked ( approximately 50%) between Cys-374 and Lys-191 (interstrand) and Gln-41 and Lys-113 (intrastrand), respectively. ANP, ABP, and pPDM cross-linked filaments showed similar inhibition of their sliding speeds and force generation with myosin ( approximately 25%) in the in vitro motility assays. In analogy to ANP cross-linking of actin, pPDM and ABP cross-linkings did not change the strong S1 binding to actin and the V(max) and K(m) parameters of actomyosin ATPase. The similar effects of these three cross-linkings reveal the tight coupling between structural elements of the subdomain 2/subdomain 1 interface and show the importance of its dynamic flexibility to force generation with myosin. The possibility that actin cross-linkings inhibit rate-limiting steps in motion and force generation during myosin cross-bridge cycle was tested in stopped-flow experiments. Measurements of the rates of mantADP release from actoS1 and ATP-induced dissociation of actoS1 did not reveal any differences between un-cross-linked and ANP cross-linked actin in these complexes. These findings are discussed in terms of the uncoupling between force generation and other aspects of actomyosin interactions due to a constrained dynamic flexibility of the subdomain 2/subdomain 1 interface in cross-linked actin filaments.