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Discovered in the 1980s, small regulatory RNAs (sRNAs) are now considered key actors in virtually all aspects of bacterial physiology and virulence. Together with transcriptional and translational regulatory proteins, they integrate and often are hubs of complex regulatory networks, responsible for bacterial response/adaptation to various perceived stimuli. The recent development of powerful RNA sequencing technologies has facilitated the identification and characterization of sRNAs (length, structure and expression conditions) and their RNA targets in several bacteria. Nevertheless, it could be very difficult for non-experts to understand the advantages and drawbacks related to each offered option and, consequently, to make an informed choice. Therefore, the main goal of this review is to provide a guide to navigate through the twists and turns of high-throughput RNA sequencing technologies, with a specific focus on those applied to the study of sRNAs. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Purpose: To investigate the relationship between the Diffusion Kurtosis Imaging (DKI) parameters and conventional metrics provided by Diffusion-weighted imaging (DWI) in patients affected by Brain or Head and Neck (HN) cancer., Methods: Ten patients affected by brain tumor and nine patients with HN tumor underwent a pre-treatment MR examination at 3 T. The largest tumor section was manually contoured by two expert neuroradiologists. The apparent diffusion coefficient (D app ) and apparent diffusional kurtosis (K app ) parameters were determined at the voxel level by using the DKI model, and compared to the apparent diffusion coefficient (ADC) and the tissue diffusion coefficient (D mono ) obtained from mono-exponential fitting methods. The Akaike Information Criteria (AIC) was calculated to assess the quality of the fitting methods. Cross-correlations between all the variables were assessed using the Spearman rank test., Results: Increased K app values were found in each lesion. All parameters were strongly related, in particular an inverse relationship emerged between median values of K app and D app /D mono /ADC in both patient groups, while D app showed positive correlations with D mono and ADC. From the analysis at the voxel level, significant inverse associations were found between K app and D mono within the lesions, while a weak or moderate association emerged between K app and ADC or D app ., Conclusions: A significant association between the apparent diffusional kurtosis K app and the tissue diffusion coefficient D mono emerged for both brain and HN tumors at 3 T, suggesting that both variables may consistently reflect deeper insight into the microstructural characteristics of tumors., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Objective: To correlate intravoxel incoherent motion (IVIM) imaging and dynamic contrast-enhanced (DCE) MRI in head and neck squamous cell carcinoma (HNSCC)., Methods: Forty untreated patients with HNSCC were included retrospectively in the study. Perfusion fraction f, diffusion coefficient D and perfusion-related diffusion coefficient D* were extracted by bi-exponential fitting of IVIM data. Semi-quantitative DCE-MRI parameters, including positive enhancement integral (PEI) and maximum slope of increase (MSI), were calculated. The relationships between all variables were assessed by Spearman's test for correlation., Results: 27 primary tumors (PTs) and 23 lymph nodes (LNs) were analyzed. The residual sum of squares (RSS), used to assess the fit quality, was significantly different between PTs and LNs, with the last showing lower values. In LNs, D* and the product D*×f were positively related to both nPEI and nMSI, while no significant correlation was found in PTs., Conclusion: Evident relationships between D* and D*×f and DCE-MRI perfusion measurements were found in LNs, while no significant association emerged in PTs. This presumably is due to the poorer agreement between the experimental data and curve fitting for PTs, as compared to LNs. Additional work is warranted to improve the reliability of the IVIM parameter estimations in primary HNSCCs., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

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.