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Translation initiation is the rate-limiting and most complexly regulated step of protein synthesis in prokaryotes and eukaryotes. In the last few years, cryo-electron microscopy has provided several novel insights into the universal process of translation initiation. Structures of prokaryotic 30S and 70S ribosomal initiation complexes with initiator transfer RNA (tRNA), messenger RNA (mRNA), and initiation factors have recently revealed the mechanism of initiator tRNA recruitment to the assembling ribosomal machinery, involving molecular rearrangements of the ribosome and associated factors. First three-dimensional pictures of the particularly complex eukaryotic translation initiation machinery have been obtained, revealing how initiation factors tune the ribosome for recruiting the mRNA. A comparison of the available prokaryotic and eukaryotic structures shows that--besides significant differences--some key ribosomal features are universally conserved.

During the past few years, our knowledge on RNA-based regulation in many organisms has tremendously increased. In bacteria, although transcriptional regulatory proteins remain key players in gene regulation, a wide variety of post-transcriptional regulatory mechanisms discovered highlights the importance of the mRNA structure in the regulation of gene expression. RNA-dependent regulation largely contributes to rapidly adapt the bacterial metabolism in response to environmental changes, stress and in establishment of virulence. Bacteria exploit the extraordinary ability of mRNA to fold into different structures in response to various signals (environmental cues, ligand binding). Induced mRNA conformational rearrangements can potentially regulate transcription, translation and mRNA stability. The present review focuses on the structures of regulatory regions of mRNA that have evolved to permit productive interactions with trans-acting regulators, such as protein or non-coding RNAs. Finally, we describe how particular properties of these regulatory complexes regulate translation initiation.

The translation factor IF6 is shared by the Archaea and the Eukarya, but is not found in Bacteria. The properties of eukaryal IF6 (eIF6) have been extensively studied, but remain somewhat elusive. eIF6 behaves as a ribosome-anti-association factor and is involved in miRNA-mediated gene silencing; however, it also seems to participate in ribosome synthesis and export. Here we have determined the function and ribosomal localization of the archaeal (Sulfolobus solfataricus) IF6 homologue (aIF6). We find that aIF6 binds specifically to the 50S ribosomal subunits, hindering the formation of 70S ribosomes and strongly inhibiting translation. aIF6 is uniformly expressed along the cell cycle, but it is upregulated following both cold- and heat shock. The aIF6 ribosomal binding site lies in the middle of the 30-S interacting surface of the 50S subunit, including a number of critical RNA and protein determinants involved in subunit association. The data suggest that the IF6 protein evolved in the archaeal-eukaryal lineage to modulate translational efficiency under unfavourable environmental conditions, perhaps acquiring additional functions during eukaryotic evolution.

Toeprinting was developed to study the formation of ribosomal initiation complexes in bacteria. This approach, based on the inhibition of reverse transcriptase elongation, was used to monitor the effect of ribosomal components and translational factors on the formation of the active ribosomal initiation complex. Moreover, this method offers an easy way to study in vitro how mRNA conformational changes alter ribosome binding at the initiation site. These changes can be induced either by environmental cues (temperature, ion concentration), or by the binding of metabolites, regulatory proteins, and trans-acting RNAs. An experimental guide is given to follow the different steps of the formation of ribosomal initiation complexes in Escherichia coli and Staphylococcus aureus, and to monitor the mechanism of action of several regulators on translation initiation in vitro. Protocols to prepare the ribosome and the subunits are also given for Thermus thermophilus, Staphylococcus aureus, and Escherichia coli.

Translation initiation, the rate-limiting step of the universal process of protein synthesis, proceeds through sequential, tightly regulated steps. In bacteria, the correct messenger RNA start site and the reading frame are selected when, with the help of initiation factors IF1, IF2 and IF3, the initiation codon is decoded in the peptidyl site of the 30S ribosomal subunit by the fMet-tRNA(fMet) anticodon. This yields a 30S initiation complex (30SIC) that is an intermediate in the formation of the 70S initiation complex (70SIC) that occurs on joining of the 50S ribosomal subunit to the 30SIC and release of the initiation factors. The localization of IF2 in the 30SIC has proved to be difficult so far using biochemical approaches, but could now be addressed using cryo-electron microscopy and advanced particle separation techniques on the basis of three-dimensional statistical analysis. Here we report the direct visualization of a 30SIC containing mRNA, fMet-tRNA(fMet) and initiation factors IF1 and GTP-bound IF2. We demonstrate that the fMet-tRNA(fMet) is held in a characteristic and precise position and conformation by two interactions that contribute to the formation of a stable complex: one involves the transfer RNA decoding stem which is buried in the 30S peptidyl site, and the other occurs between the carboxy-terminal domain of IF2 and the tRNA acceptor end. The structure provides insights into the mechanism of 70SIC assembly and rationalizes the rapid activation of GTP hydrolysis triggered on 30SIC-50S joining by showing that the GTP-binding domain of IF2 would directly face the GTPase-activated centre of the 50S subunit.

Purpose: Recent studies have suggested an alpha/beta ratio in prostate cancer of 1.5-3 Gy, which is lower than that assumed for late-responsive normal tissues. Therefore the administration of a single, intraoperative dose of irradiation should represent a convenient irradiation modality in prostate cancer., Materials and Methods: Between February 2002 and June 2004, 34 patients with localized prostate cancer with only one risk factor (Gleason score > or =7, Clinical Stage [cT] > or =2c, or prostate-specific antigen [PSA] of 11-20 ng/mL) and without clinical evidence of lymph node metastases were treated with radical prostatectomy (RP) and intraoperative radiotherapy on the tumor bed. A dose-finding procedure based on the Fibonacci method was employed. Dose levels of 16, 18, and 20 Gy were selected, which are biologically equivalent to total doses of about 60-80 Gy administered with conventional fractionation, using an alpha/beta ratio value of 3., Results: At a median follow-up of 41 months, 24 (71%) patients were alive with an undetectable PSA value. No patients died from disease, whereas 2 patients died from other malignancies. Locoregional failures were detected in 3 (9%) patients, 2 in the prostate bed and 1 in the common iliac node chain outside the radiation field. A PSA rise without local or distant disease was observed in 7 (21%) cases. The overall 3-year biochemical progression-free survival rate was 77.3%., Conclusions: Our dose-finding study demonstrated the feasibility of intraoperative radiotherapy in prostate cancer also at the highest administered dose.

A large variety of RNA-based mechanisms have been uncovered in all living organisms to regulate gene expression in response to internal and external changes, and to rapidly adapt cell growth in response to these signals. In bacteria, structural elements in the 5' leader regions of mRNAs have direct effects on translation initiation of the downstream coding sequences. The docking and unfolding of these mRNAs on the 30S subunit are critical steps in the initiation process directly modulating and timing translation. Structural elements can also undergo conformational changes in response to environmental cues (i.e., temperature sensors) or upon binding of a variety of trans-acting factors, such as metabolites, non-coding RNAs or regulatory proteins. These RNA switches can temporally regulate translation, leading either to repression or to activation of protein synthesis.

Purpose: The aim of this work is to report a retrospective study of radiobiological indicators based on Dose-Volume Histograms analysis obtained by stereotactic radiotherapy treatments., Methods and Materials: Fifty-five patients for a total of sixty-seven brain metastases with a mean target volume of 8.49 cc were treated by Dynamic Conformal Arc Therapy (DCAT) and Intensity-Modulated Stereotactic Radiotherapy (IMRST). The Delivered prescription dose was chosen on the basis of tumor size and location so as to ensure a 100% isodose coverage to the target volume., Results: The treatment plans reported a mean value of 10% and 2.19% for the inhomogeneity and conformal index, respectively. The F factor showed we overdosed sixty-three patients delivering an additional 7% dose more than calculated values. The radiobiological parameters: TCP and NTCP showed a complete tumor control limiting the organs at risk damage., Conclusion: One goal of stereotactic radiotherapy is to design a treatment plan in which the steep dose gradient achievable minimizes the amount of radiation delivered outside the tumor region. This technique allows to deliver a much larger dose to the target without exceeding the radiation-related tolerance of normal tissues and improving patients' quality of life.

The purpose of this work is to introduce a new treatment approach and technique for partial breast irradiation in only one session to patients in prone position by using a dedicated positioning device. The patients were treated on a home-made treatment table top that allows the breast to hang down. A particular immobilization system was introduced in order to assure the reproducibility of patient positioning between the CT acquisition session and the treatment session. The clinical target volume (CTV) was outlined according to surgical clips position and/or tumor location on preoperative mammography. Because of negligible movement due to respiration, only an additional margin of 3 mm was added to obtain the planning target volume (PTV). Based on radiobiological calculations, a dose of 21 Gy was prescribed to PTV. The tumor bed was treated with 3D-CRT technique by using 5 fields and rotating the table while the gantry was approximately 90 or 270 degrees. Thirty patients were enrolled for this study chosen in conformity to an approved clinical protocol. The average percentage of PTV volume enclosed in the 90% and 95% of prescribed dose were 99.9 and 98.6% respectively, while only 3.4% of PTV volume received more than 105% of prescribed dose. Dose to 3% of skin volume was, on average, 15.2 Gy. In 97% of patients, less than 50% of the ipsilateral breast received a dose greater than half the prescribed dose. Mean doses to lungs, heart and contralateral breast were negligible. With a median follow-up of 9 months, no important early toxicity was observed both for skin and breast tissue. The treatment of breast tumor bed in prone position in only one session by using the 3D-CRT is technically feasible and seems to be a promising alternative to other accelerated partial breast irradiation techniques.

IF3 has a fidelity function in the initiation of translation, inducing the dissociation of fMet-tRNA(fMet) from the 30 S initiation complexes (30SIC) containing a non-canonical initiation triplet (e.g. AUU) in place of a canonical initiation triplet (e.g., AUG). IF2 has a complementary role, selectively promoting initiator tRNA binding to the ribosome. Here, we used parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light-scattering, and changes in intensities of fluorophore-labeled IF2 and fMet-tRNA(fMet) to determine the effects on both 30SIC formation and 30SIC conversion to 70 S initiation complexes (70SIC) of (a) substituting AUG with AUU, and/or (b) omitting IF3, and/or (c) replacing GTP with the non-hydrolyzable analog GDPCP. We demonstrate that the presence or absence of IF3 has, at most, minor effects on the rate of 30SIC formation using either AUG or AUU as the initiation codon, and conclude that the high affinity of IF2 for both 30 S subunit and initiator tRNA overrides any perturbation of the codon-anticodon interaction resulting from AUU for AUG substitution. In contrast, replacement of AUG by AUU leads to a dramatic reduction in the rate of 70SIC formation from 30SIC upon addition of 50 S subunits. Interpreting our results in the framework of a quantitative kinetic scheme leads to the conclusion that, within the overall process of 70SIC formation, the step most affected by substituting AUU for AUG involves the conversion of an initially labile 70 S ribosome into a more stable complex. In the absence of IF3, the difference between AUG and AUU largely disappears, with each initiation codon affording rapid 70SIC formation, leading to the hypothesis that it is the rate of IF3 dissociation from the 70 S ribosome during IC70S formation that is critical to its fidelity function.

Association of the 30 S initiation complex (30SIC) and the 50 S ribosomal subunit, leading to formation of the 70 S initiation complex (70SIC), is a critical step of the translation initiation pathway. The 70SIC contains initiator tRNA, fMet-tRNA(fMet), bound in the P (peptidyl)-site in response to the AUG start codon. We have formulated a quantitative kinetic scheme for the formation of an active 70SIC from 30SIC and 50 S subunits on the basis of parallel rapid kinetics measurements of GTP hydrolysis, Pi release, light-scattering, and changes in fluorescence intensities of fluorophore-labeled IF2 and fMet-tRNA(f)(Met). According to this scheme, an initially formed labile 70 S complex, which promotes rapid IF2-dependent GTP hydrolysis, either dissociates reversibly into 30 S and 50 S subunits or is converted to a more stable form, leading to 70SIC formation. The latter process takes place with intervening conformational changes of ribosome-bound IF2 and fMet-tRNA(fMet), which are monitored by spectral changes of fluorescent derivatives of IF2 and fMet-tRNA(fMet). The availability of such a scheme provides a useful framework for precisely elucidating the mechanisms by which substituting the non-hydrolyzable analog GDPCP for GTP or adding thiostrepton inhibit formation of a productive 70SIC. GDPCP does not affect stable 70 S formation, but perturbs fMet-tRNA(fMet) positioning in the P-site. In contrast, thiostrepton severely retards stable 70 S formation, but allows normal binding of fMet-tRNA(fMet)(prf20) to the P-site.

Gene expression can be regulated at the level of initiation of protein biosynthesis via structural elements present at the 5' untranslated region of mRNAs. These folded mRNA segments may bind to the ribosome, thus blocking translation until the mRNA unfolds. Here, we report a series of cryo-electron microscopy snapshots of ribosomal complexes directly visualizing either the mRNA structure blocked by repressor protein S15 or the unfolded, active mRNA. In the stalled state, the folded mRNA prevents the start codon from reaching the peptidyl-tRNA (P) site inside the ribosome. Upon repressor release, the mRNA unfolds and moves into the mRNA channel allowing translation initiation. A comparative structure and sequence analysis suggests the existence of a universal stand-by site on the ribosome (the 30S platform) dedicated for binding regulatory 5' mRNA elements. Different types of mRNA structures may be accommodated during translation preinitiation and regulate gene expression by transiently stalling the ribosome.

Background and Purpose: To report the treatment-related morbidity in patients with prostate cancer treated with an optimized pelvic intensity-modulated radiation therapy (IMRT) and simultaneous integrated dose escalation to prostate/prostate bed., Materials and Methods: Between November 2003 and May 2006, 55 patients with localized prostate cancer and >15% risk of lymph node involvement were treated with pelvic IMRT and simultaneous dose escalation to prostate area. Twenty-four patients received a radical radiation therapy program, and the remaining thirty-one patients received a postoperative irradiation as adjuvant treatment or after biochemical or macroscopic local/regional relapse. After a customized immobilization all patients underwent contrast-enhanced CT. On the CT slices CTV1 and CTV2 were delineated. CTV(1) included the prostate and seminal vesicles or prostate bed. CTV(2) consisted of CTV(1) plus pelvic nodes. CTV(1) and CTV(2) were then expanded by 0.5 and 1cm, respectively, to generate the planning target volumes. IMRT treatment plans were generated using commercial inverse planning software. Total doses of 66-80 Gy and 50-59 Gy in 33-40 fractions were prescribed to the prostate area and pelvis, respectively. The worst acute and late rectal, intestinal and GU toxicities during and after treatment were scored according to the EORTC/RTOG scales., Results: The IMRT dose distribution provided excellent PTV coverage and satisfying sparing of all the organs at risk, with no patient experiencing >grade 2 acute or late toxicities. Patients without acute grade 2 intestinal, rectal, and GU toxicity were 91%, 71%, and 63%, respectively. After a median follow-up of 19 months (interquartile range of 9 to 28 months), late grade 2 toxicity was detected only for rectum, with an actuarial 2-year rate of freedom from G2 rectal bleeding of 92%. (CI 95% 0.83-0.99.), Conclusions: Pelvic IMRT and simultaneous dose escalation to prostate area is a well-tolerated technique in patients with prostate cancer requiring treatment of pelvic lymph nodes, and seems to be associated with a lower frequency and severity of side effects when compared with conventional techniques reported in other series.

The protein IF2/eIF5B is one of the few translation initiation factors shared by all three primary domains of life (bacteria, archaea, eukarya). Despite its phylogenetic conservation, the factor is known to present marked functional divergences in the bacteria and the eukarya. In this work, the function in translation of the archaeal homologue (aIF2/5B) has been analysed in detail for the first time using a variety of in vitro assays. The results revealed that the protein is a ribosome-dependent GTPase which strongly stimulates the binding of initiator tRNA to the ribosomes even in the absence of other factors. In agreement with this finding, aIF2/5B enhances the translation of both leadered and leaderless mRNAs when expressed in a cell-free protein-synthesizing system. Moreover, the degree of functional conservation of the IF2-like factors in the archaeal and bacterial lineages was investigated by analysing the behaviour of 'chimeric' proteins produced by swapping domains between the Sulfolobus solfataricus aIF2/5B factor and the IF2 protein of the thermophilic bacterium Bacillus stearothermophilus. Beside evidencing similarities and differences between the archaeal and bacterial factors, these experiments have provided insight into the common role played by the IF2/5B proteins in all extant cells.

The purpose of this study was to check the setup and dose delivered to the patients during intraoperative electron beam radiation therapy (IORT) for prostate cancer. Twenty eight patients underwent IORT after radical prostatectomy for prostate cancer by means of a dedicated mobile accelerator, Novac7 (by Hitesys, SpA, Italy). A 9 MeV electron beam at high dose per pulse was used. Eighteen patients received IORT at escalating doses of 16, 18, and 20 Gy at 85% isodose, six patients for each dose level. Further, ten patients received 20 Gy at 85% isodose. The electron applicator position was checked in all cases by means of two orthogonal images obtained with brilliance intensifier. Target and organ at risk doses were measured in vivo by a MOSFETs dosimetry system. MOSFETs and microMOSFET dosimeters were inserted into sterile catheters and directly positioned into the rectal lumen, for ten patients, and into the bladder to urethra anastomosis, in the last 14 cases. Verification at 0 degree led to very few adjustments of setup while verifications at 90 degrees often suggested to bring the applicator closer to the target. In vivo dosimetry showed an absorbed dose into the rectum wall < or =1% of the total dose. The average dose value inside the anastomosis, for the 12 patients analyzed, was 23.7 Gy with a standard deviation of +/-7.6%, when the prescription was 20 Gy at 85% isodose. Using a C-arm mobile image intensifier, it is possible to assess if the positioning is correct and safe. Radio-opaque clips and liquid were necessary to obtain good visible images. In vivo MOSFETs dosimetry is feasible and reliable. A satisfactory agreement between measured and expected doses was found.

Purpose: The purpose of this article was to investigate how exceeding specified rectal wall dose-volume constraints impacts on the risk of late rectal bleeding by using radiobiologic calculations., Methods and Materials: Dose-volume histograms (DVH) of the rectal wall of 250 patients with prostate cancer were analyzed. All patients were treated by three-dimensional conformal radiation therapy, receiving mean target doses of 80 Gy. To study the main features of the patient population, the average and the standard deviation of the distribution of DVHs were generated. The mean dose , generalized equivalent uniform dose formulation (gEUD), modified equivalent uniform dose formulation (mEUD)(0), and normal tissue complication probability (NTCP) distributions were also produced. The DVHs set was then binned into eight classes on the basis of the exceeding or the fulfilling of three dose-volume constraints: V(40) = 60%, V(50) = 50%, and V(70) = 25%. Comparisons were made between them by , gEUD, mEUD(0), and NTCP., Results: The radiobiologic calculations suggest that late rectal toxicity is mostly influenced by V(70). The gEUD and mEUD(0) are risk factors of toxicity always concordant with NTCP, inside each DVH class. The mean dose, although a reliable index, may be misleading in critical situations., Conclusions: Both in three-dimensional conformal radiation therapy and particularly in intensity-modulated radiation therapy, it should be known what the relative importance of each specified dose-volume constraint is for each organ at risk. This requires a greater awareness of radiobiologic properties of tissues and radiobiologic indices may help to gradually become aware of this issue.

Intensity-modulated radiotherapy (IMRT) (1) is an advanced form of 3-D conformal radiotherapy. It uses non uniform spatial modifications in the intensity of the beams across the irradiated field. Consequently, it is necessary to develop sophisticated tools to compare measured and calculated dose distributions in order to verify the accuracy of the results of the planned dose distribution. Different methods have been developed to evaluate the accordance between measured and calculated doses, such as the point-to-point dose difference or the evaluation of the distance between two closed points having the same dose value (2-4). The verification method proposed by Low (5-7) seems to be more complete since it takes into account both the dose difference (DD) and the distance to agreement (DTA), allowing to define a "score", the gamma value, at each point of interest. A software tool (DDE: Dose Distribution Evaluator), based on Low's method, to evaluate the agreement between dose distribution matrices has been implemented. In particular, the proposed gamma curve, as a function of the isodose levels, gives real-time information useful for decision making about the treatment plan. The paper describes the software, and reports the obtained results in a simple geometry and in several clinical cases (head-neck and prostate). Comparison between measured data (film and MapCheck) and calculated data (CadPlan) using DDE has shown very good agreements. Thanks to its higher resolution, film dosimetry showed better accuracy than the MapCheck technique. Similar results can be obtained also with the MapCheck technique when proper measurement methods are used.

Purpose: To assess the influence of setup errors and organ motion in terms of the probability of tumor control and normal-tissue complications by tumor control probability and normal-tissue complication probability., Methods and Materials: Twelve patients were treated for prostate cancer with intensity-modulated radiation therapy. Two orthogonal portal images were taken daily. All patients underwent three computed tomography scans during the 8-week treatment time (i.e., baseline, intermediate, and final). The original treatment plans were re-evaluated, taking into account setup errors and organ motion., Results: The mean shifts +/- standard deviation of the whole patient population in the lateral, anterior-posterior, and craniocaudal direction were 1.0 +/- 1.5 mm, 0.9 +/- 2.1 mm, and 1.9 +/- 2.1 mm, respectively. In most of the recalculated dose-volume histograms, the coverage of clinical target volume was granted despite organ motion, whereas the rectal wall histograms were often very different from the planned ones., Conclusion: We have studied the impact of prostate and rectum motion, as well as setup errors, on dose-volume histograms. The estimate of these effects may have implications for predictive indications when planning intensity-modulated radiation therapy treatments on prostate.

Initiation of protein synthesis is a universally conserved event that requires initiation factors IF1, IF2 and IF3 in prokaryotes. IF2 is a GTPase essential for binding initiator transfer RNA to the 30S ribosomal subunit and recruiting the 50S subunit into the 70S initiation complex. We present two cryo-EM structures of the assembled 70S initiation complex comprising mRNA, fMet-tRNA(fMet) and IF2 with either a non-hydrolyzable GTP analog or GDP. Transition from the GTP-bound to the GDP-bound state involves substantial conformational changes of IF2 and of the entire ribosome. In the GTP analog-bound state, IF2 interacts mostly with the 30S subunit and extends to the initiator tRNA in the peptidyl (P) site, whereas in the GDP-bound state IF2 steps back and adopts a 'ready-to-leave' conformation. Our data also provide insights into the molecular mechanism guiding release of IF1 and IF3.

Purpose: To investigate the use of metal oxide silicon field effect transistors (MOSFETs) as in vivo dosimetry detectors during electron beams at high dose-per-pulse intraoperative radiotherapy., Methods and Materials: The MOSFET system response in terms of reproducibility, energy, dose rate and temperature dependence, dose-linearity from 1 to 25 Gy, angular response, and dose perturbation was analyzed in the 6-9-MeV electron beam energy range produced by an intraoperative radiotherapy-dedicated mobile accelerator. We compared these with the 6- and 9-MeV electron beams produced by a conventional accelerator. MOSFETs were also used in clinical dosimetry., Results: In experimental conditions, the overall uncertainty of the MOSFET response was within 3.5% (+/-SD). The investigated electron energies and the dose rate did not significantly influence the MOSFET calibration factors. The dose perturbation was negligible. In vivo dosimetry results were in accordance with the predicted values within +/-5%. A discordance occurred either for an incorrect position of the dosimeter on the patient or when a great difference existed between the clinical and calibration setup, particularly when performing exit dose measurements., Conclusion: Metal oxide silicon field effect transistors are suitable for in vivo dosimetry during intraoperative radiotherapy because their overall uncertainty is comparable to the accuracy required in target dose delivery.

Bacterial translation initiation factor IF2 was localized on the ribosome by rRNA cleavage using free Cu(II):1,10-orthophenanthroline. The results indicated proximity of IF2 to helix 89, to the sarcin-ricin loop and to helices 43 and 44, which constitute the "L11/thiostrepton" stem-loops of 23S rRNA. These findings prompted an investigation of the L11 contribution to IF2 activity and a re-examination of the controversial issue of the effect on IF2 functions of thiostrepton, a peptide antibiotic known primarily as a powerful inhibitor of translocation. Ribosomes lacking L11 were found to have wild-type capacity to bind IF2 but a strongly reduced ability to elicit its GTPase activity. We found that thiostrepton caused a faster recycling of this factor on and off the 70S ribosomes and 50S subunits, which in turn resulted in an increased rate of the multiple turnover IF2-dependent GTPase. Although thiostrepton did not inhibit the P-site binding of fMet-tRNA, the A-site binding of the EF-Tu-GTP-Phe-tRNA or the activity of the ribosomal peptidyl transferase center (as measured by the formation of fMet-puromycin), it severely inhibited IF2-dependent initiation dipeptide formation. This inhibition can probably be traced back to a thiostrepton-induced distortion of the ribosomal-binding site of IF2, which leads to a non-productive interaction between the ribosome and the aminoacyl-tRNA substrates of the peptidyl transferase reaction. Overall, our data indicate that the translation initiation function of IF2 is as sensitive as the translocation function of EF-G to thiostrepton inhibition.

Bacterial translation initiation factor IF2 is a GTP-binding protein that catalyzes binding of initiator fMet-tRNA in the ribosomal P site. The topographical localization of IF2 on the ribosomal subunits, a prerequisite for understanding the mechanism of initiation complex formation, has remained elusive. Here, we present a model for the positioning of IF2 in the 70S initiation complex as determined by cleavage of rRNA by the chemical nucleases Cu(II):1,10-orthophenanthroline and Fe(II):EDTA tethered to cysteine residues introduced into IF2. Two specific amino acids in the GII domain of IF2 are in proximity to helices H3, H4, H17, and H18 of 16S rRNA. Furthermore, the junction of the C-1 and C-2 domains is in proximity to H89 and the thiostrepton region of 23S rRNA. The docking is further constrained by the requisite proximity of the C-2 domain with P-site-bound tRNA and by the conserved GI domain of the IF2 with the large subunit's factor-binding center. Comparison of our present findings with previous data further suggests that the IF2 orientation on the 30S subunit changes during the transition from the 30S to 70S initiation complex.

Dihydropyridines (DHPs), synthetic molecules used as antihypertensive agents, bind to plasma membrane receptors following diffusion through the hydrophobic phase. In this study, MRS technique has been used to clarify the interactions of the dihydrophyridines Nifedipine and Lacidipine within the lipid bilayer. 1D and 2D 1H MRS at high field have been employed to examine the behavior of unilamellar dimyristoyl-phosphatidylcholine liposomes when the two drugs have been inserted in the bilayer. In particular, the study represents an innovative application of 2D 1H NOESY technique to clarify different mechanisms of interactions of small molecules inside model membranes. On the other hand, 31P measurements have been performed in multilamellar dimyristoyl-phosphatidylcholine lipsomes to detect alterations of lipid polymorphic phases. The experiments show that the two dihydropyridines interact with the lipids by different modalities. Lacidipine undergoes a very strong interaction with lipids, possibly inducing a phase segregation of lipid molecules into the bilayer, while self-association seems to be the prevalent interaction of Nifedipine inside the bilayer.

Intensity modulated radiation therapy (IMRT) is one of the most innovative techniques in oncological radiotherapy, allowing to conform the dose delivery to the tumoral target, preserving the normal tissue. The high number of parameters involved in the IMRT treatment planning requires an automated approach to the beam modulation. Such optimization process consists in the search of the global minimum of a cost function representing a quality index for the treatment. The complexity of this task, has been analyzed with a statistical approach for three clinical cases of particular interest in IMRT. Our main result is that a cost function based on dose-volume constraints entails lower complexity of the optimization process, in terms of the choice of the parameters defining the cost function and in a smaller sensitivity to the initial conditions for the optimization algorithm.