Chemical biology tools to study Deubiquitinases and Ubl proteases.

The reversible attachment of ubiquitin (Ub) and ubiquitin like modifiers (Ubls) to proteins are crucial post-translational modifications (PTMs) for many cellular processes. Not only do cells possess hundreds of ligases to mediate substrate specific modification with Ub and Ubls, but they also have a repertoire of more than 100 dedicated enzymes for the specific removal of ubiquitin (Deubiquitinases or DUBs) and Ubl modifications (Ubl-specific proteases or ULPs). Over the past two decades, there has been significant progress in our understanding of how DUBs and ULPs function at a molecular level and many novel DUBs and ULPs, including several new DUB classes, have been identified. Here, the development of chemical tools that can bind and trap active DUBs has played a key role. Since the introduction of the first activity-based probe for DUBs in 1986, several innovations have led to the development of more sophisticated tools to study DUBs and ULPs. In this review we discuss how chemical biology has led to the development of activity-based probes and substrates that have been invaluable to the study of DUBs and ULPs. We summarise our currently available toolbox, highlight the main achievements and give an outlook of how these tools may be applied to gain a better understanding of the regulatory mechanisms of DUBs and ULPs.

New Modifiable Risk Factors Influencing Coronary Artery Disease Severity.

Cardiovascular diseases (CVDs) remain the leading cause of death worldwide with coronary artery disease (CAD) being the first culprit in this group. In terms of CAD, not only its presence but also its severity plays a role in the patient's treatment and prognosis. CAD complexity can be assessed with the indicator named the SYNTAX score (SS). A higher SS is associated with major adverse cardiovascular event (MACE) occurrence in short- and long-term observations. Hence, the risk factors affecting CAD severity based on SS results may help lower the risk among patients with already developed CAD to reduce their impact on coronary atherosclerosis progression. The well-established risk factors of CAD are consistent with those associated with the coronary plaque burden. However, recently, it was shown that new indicators exist, which we present in this paper, that significantly contribute to CAD complexity such as inflammatory parameters, C-reactive protein (CRP), ratios based on blood smear results, and uric acid. Moreover, microbiota alteration, vitamin D deficiency, and obstructive sleep apnea (OSA) also predicted CAD severity. However, sometimes, certain indicators were revealed as significant only in terms of chronic coronary syndromes (CCSs) or specific acute coronary syndromes (ACSs). Importantly, there is a need to apply the interdisciplinary and translational approach to the novel CAD severity risk assessment to maximize the impact of secondary prevention among patients at risk of coronary atherosclerosis progression.

Variation in radiation sensitivity and repair kinetics in different parts of the spinal cord.

BACKGROUND: The spinal cord, known for its strongly serial character and high sensitivity to radiation even when a small segment is irradiated, is one of the most critical organs at risk to be spared during radiation therapy. To compare the sensitivity of different parts of the spinal cord, data for radiation myelopathy have been used. MATERIAL AND METHODS: In the present study, the relative seriality model was fitted to two different datasets of clinical radiation myelitis concerning cervical spinal cord after treating 248 patients for head and neck cancer and thoracic spinal cord after treating 43 patients with lung carcinoma. The maximum likelihood method was applied to fit the clinical data. The model parameters and their 68% confidence intervals were calculated for each dataset. The alpha/beta ratio for the thoracic cord was also was also found to be 0.9 (0-3.0) Gy. RESULTS: The dose-response curve for the more sensitive cervical myelopathy is well described by the parameters D(50)=55.9 (54.8-57.1) Gy, gamma=6.9 (5.0-9.2), s=0.13 (0.07-0.24), whereas the thoracic myelopathy is described by the parameters D(50)=75.5 (70.5-80.8) Gy, gamma=1.1 (0.6-1.6), s=36 (3.3-infinity). DISCUSSION AND CONCLUSIONS: Large differences in radiation response between the cervical and thoracic region of spinal cord are thus observed: cervical myelopathy seems to be characterized by medium seriality, while thoracic spinal cord is characterized by a highly serial dose-response. The much steeper dose-response curve for cervical spinal cord myelopathy can be interpreted as a higher number of functional subunits consistent with a higher amount of white matter close to the brain.

The dose--response relation for rat spinal cord paralysis analyzed in terms of the effective size of the functional subunit.

Radiobiological models for estimating normal tissue complication probability (NTCP) are increasingly used in order to quantify or optimize the clinical outcome of radiation therapy. A good NTCP model should fulfill at least the following two requirements: (a) it should predict the sigmoid shape of the corresponding dose-response curve and (b) it should accurately describe the probability of a specified response for arbitrary non-uniform dose delivery for a given endpoint as accurately as possible, i.e. predict the volume dependence. In recent studies of the volume effect of a rat spinal cord after irradiation with narrow and broad proton beams the authors claim that none of the existing NTCP models is able to describe their results. Published experimental data have been used here to try to quantify the change in the effective dose (D(50)) causing 50% response for different field sizes. The present study was initiated to describe the induction of white matter necrosis in a rat spinal cord after irradiation with narrow proton beams in terms of the mean dose to the effective volume of the functional subunit (FSU). The physically delivered dose distribution was convolved with a function describing the effective size or, more accurately, the sensitivity distribution of the FSU to obtain the effective mean dose deposited in it. This procedure allows the determination of the mean D(50) value of the FSUs of a certain size which is of interest for example if the cell nucleus of the oligodendrocyte is the sensitive target. Using the least-squares method to compare the effective doses for different sizes of the functional subunits with the experimental data the best fit was obtained with a length of about 9 mm. For the non-uniform dose distributions an effective FSU length of 8 mm gave the optimal fit with the probit dose-response model. The method could also be used to interpret the so-called bath and shower experiments where the heterogeneous dose delivery was used in the convolution process. The assumption of an effective FSU size is consistent with most of the effects seen when different portions of the rat spinal cord are irradiated to different doses. The effective FSU length from these experiments is about 8.5 +/- 0.5 mm. This length could be interpreted as an effective size of the functional subunits in a rat spinal cord, where multiple myelin sheaths are connected by a single oligodendrocyte and repair is limited by the range of oligodendrocyte progenitor cell diffusion. It was even possible to suggest a more likely than uniform effective FSU sensitivity distribution from the experimental data.

The impact of different dose-response parameters on biologically optimized IMRT in breast cancer.

The full potential of biologically optimized radiation therapy can only be maximized with the prediction of individual patient radiosensitivity prior to treatment. Unfortunately, the available biological parameters, derived from clinical trials, reflect an average radiosensitivity of the examined populations. In the present study, a breast cancer patient of stage I-II with positive lymph nodes was chosen in order to analyse the effect of the variation of individual radiosensitivity on the optimal dose distribution. Thus, deviations from the average biological parameters, describing tumour, heart and lung response, were introduced covering the range of patient radiosensitivity reported in the literature. Two treatment configurations of three and seven biologically optimized intensity-modulated beams were employed. The different dose distributions were analysed using biological and physical parameters such as the complication-free tumour control probability (P(+)), the biologically effective uniform dose (D), dose volume histograms, mean doses, standard deviations, maximum and minimum doses. In the three-beam plan, the difference in P(+) between the optimal dose distribution (when the individual patient radiosensitivity is known) and the reference dose distribution, which is optimal for the average patient biology, ranges up to 13.9% when varying the radiosensitivity of the target volume, up to 0.9% when varying the radiosensitivity of the heart and up to 1.3% when varying the radiosensitivity of the lung. Similarly, in the seven-beam plan, the differences in P(+) are up to 13.1% for the target, up to 1.6% for the heart and up to 0.9% for the left lung. When the radiosensitivity of the most important tissues in breast cancer radiation therapy was simultaneously changed, the maximum gain in outcome was as high as 7.7%. The impact of the dose-response uncertainties on the treatment outcome was clinically insignificant for the majority of the simulated patients. However, the jump from generalized to individualized radiation therapy may significantly increase the therapeutic window for patients with extreme radio sensitivity or radioresistance, provided that these are identified. Even for radiosensitive patients a simple treatment technique is sufficient to maximize the outcome, since no significant benefits were obtained with a more complex technique using seven intensity-modulated beams portals.

Comments on 'Reconsidering the definition of a dose-volume histogram'--dose-mass histogram (DMH) versus dose-volume histogram (DVH) for predicting radiation-induced pneumonitis.

In a recently published paper (Nioutsikou et al 2005 Phys. Med. Biol. 50 L17) the authors showed that the use of the dose-mass histogram (DMH) concept is a more accurate descriptor of the dose delivered to lung than the traditionally used dose-volume histogram (DVH) concept. Furthermore, they state that if a functional imaging modality could also be registered to the anatomical imaging modality providing a functional weighting across the organ (functional mass) then the more general and realistic concept of the dose-functioning mass histogram (D[F]MH) could be an even more appropriate descriptor. The comments of the present letter to the editor are in line with the basic arguments of that work since their general conclusions appear to be supported by the comparison of the DMH and DVH concepts using radiobiological measures. In this study, it is examined whether the dose-mass histogram (DMH) concept deviated significantly from the widely used dose-volume histogram (DVH) concept regarding the expected lung complications and if there are clinical indications supporting these results. The problem was investigated theoretically by applying two hypothetical dose distributions (Gaussian and semi-Gaussian shaped) on two lungs of uniform and varying densities. The influence of the deviation between DVHs and DMHs on the treatment outcome was estimated by using the relative seriality and LKB models using the Gagliardi et al (2000 Int. J. Radiat. Oncol. Biol. Phys. 46 373) and Seppenwoolde et al (2003 Int. J. Radiat. Oncol. Biol. Phys. 55 724) parameter sets for radiation pneumonitis, respectively. Furthermore, the biological equivalent of their difference was estimated by the biologically effective uniform dose (D) and equivalent uniform dose (EUD) concepts, respectively. It is shown that the relation between the DVHs and DMHs varies depending on the underlying cell density distribution and the applied dose distribution. However, the range of their deviation in terms of the expected clinical outcome was proven to be very large. Concluding, the effectiveness of the dose distribution delivered to the patients seems to be more closely related to the radiation effects when using the DMH concept.

Comparison of dose response models for predicting normal tissue complications from cancer radiotherapy: application in rat spinal cord.

Seven different radiobiological dose-response models have been compared with regard to their ability to describe experimental data. The first four models, namely the critical volume, the relative seriality, the inverse tumor and the critical element models are mainly based on cell survival biology. The other three models: the Lyman (Gaussian distribution), the parallel architecture and the Weibull distribution models are semi-empirical and rather based on statistical distributions. The maximum likelihood estimation was used to fit the models to experimental data and the χ2-distribution, AIC criterion and F-test were applied to compare the goodness-of-fit of the models. The comparison was performed using experimental data for rat spinal cord injury. Both the shape of the dose-response curve and the ability of handling the volume dependence were separately compared for each model. All the models were found to be acceptable in describing the present experimental dataset (p > 0.05). For the white matter necrosis dataset, the Weibull and Lyman models were clearly superior to the other models, whereas for the vascular damage case, the Relative Seriality model seems to have the best performance although the Critical volume, Inverse tumor, Critical element and Parallel architecture models gave similar results. Although the differences between many of the investigated models are rather small, they still may be of importance in indicating the advantages and limitations of each particular model. It appears that most of the models have favorable properties for describing dose-response data, which indicates that they may be suitable to be used in biologically optimized intensity modulated radiation therapy planning, provided a proper estimation of their radiobiological parameters had been performed for every tissue and clinical endpoint.

Autophagy is the dominant type of programmed cell death in breast cancer MCF-7 cells exposed to AGS 115 and EFDAC, new sesquiterpene analogs of paclitaxel.

The molecular mechanism of cell death induced by AGS 115 and EFDAC, sesquiterpene analogs of paclitaxel, was investigated in human breast cancer MCF-7 cells. The study was carried out using laser scanning cytometry, homeostatic confocal microscopy, atomic force microscopy and electron microscopy. AGS 115 and EFDAC exhibited a microtubule-stabilizing effect as confirmed by a significant increase in alpha-tubulin aggregation. Both paclitaxel analogs also induced death in MCF-7 cells. Evaluation of biochemical and morphological features suggested that the major form of programmed cell death induced by AGS 115 and EFDAC was autophagy. This was confirmed by MAP I LC3 expression and the ultrastructural pattern revealed by electron microscopy. Surface images of cells undergoing autophagy showed that, unlike during apoptosis, the dimensions remained unchanged, but the surface of the cell was deformed. The occurrence of apoptosis was confirmed by the efflux of Smac/DIABLO from mitochondria, caspase-7 activation and DNA loss, and did not exceed 9.7%. Therefore, AGS 115 and EFDAC appear to be promising candidates for further investigation in anti-cancer therapy.

Kinetics of Smac/DIABLO release from mitochondria during apoptosis of MCF-7 breast cancer cells.

Smac/DIABLO, a pro-apoptotic protein released from mitochondrial intermembrane space during apoptosis, promotes caspase activation by IAPs neutralization. The kinetics and molecular mechanism of Smac/DIABLO release from mitochondria has remained obscure. Homeostatic confocal microscopy, for the first time, showed the precise kinetics of Smac/DIABLO release from mitochondria during CPT-induced apoptosis in living MCF-7 cells. The time pattern of Smac/DIABLO escape from mitochondria comprised two phases: the initial phase of gradual protein release, followed by the second phase of plateau, appearing after 24 min of cell exposure to the drug. A similar pattern was observed during oxidative stress. The dynamics of Smac/DIABLO redistribution was confirmed by different methods: traditional confocal microscopy, immunoelectron microscopy and laser scanning cytometry. The inhibition of m-calpain prevented Smac/DIABLO release from mitochondria, which confirmed the involvement of Bax in the process. Acquired results indicate that CPT treatment triggers Bax-dependent release of Smac/DIABLO from mitochondria simultaneously with the efflux of cytochrome c.

Minute kinetics of proapoptotic proteins: BAX and Smac/DIABLO in living tumor cells revealed by homeostatic confocal microscopy.

Traditional methods of visualization and analysis based on fixed cell populations treated with the drug for a different time give the limited possibility of time-sequence analysis. In time-lapse microscopy where the whole cell is observed regardless to intracellular structure, precise localization of events and differentiation between colocalization and overlapping of the fluorescence is impossible. Furthermore prolonged experiments with living cells increased the influence of improper environmental conditions. Homeostatic confocal microscopy gives an exceptional insight into minute pattern of changes occurring in the same living cell maintained in stable conditions during whole experimental period. It is built on a confocal system equipped with the homeostatic chamber providing constant, monitored heating and moisturized, CO(2)-enriched atmosphere during long period observations. In the present study 2D/time and 4D homeostatic confocal microscopy were applied for analysis of minute pattern of changes occurring at the mitochondria. The release of Smac/DIABLO from mitochondria in tumor cells under the apoptogenic stimulus, consist of two phases: the first immediately after drug administration, and the major second one after 15 min. Furthermore the time-pattern of BAX translocation to the mitochondria and Smac/DIABLO release coincide, suggesting that the release of Smac/DIABLO is correlated with BAX translocation to the mitochondria.