Correction: Luedemann et al. Prostate Cancer-Associated miRNAs in Saliva: First Steps to an Easily Accessible and Reliable Screening Tool. Biomolecules 2022, 12, 1366.

The authors wish to make the following corrections to their paper [...].

Prostate Cancer-Associated miRNAs in Saliva: First Steps to an Easily Accessible and Reliable Screening Tool.

BACKGROUND: Common diagnostic tools for prostate cancer-prostate-specific antigen and transrectal biopsy-show only low predictive value and poor sensitivity. This study examines circulating miRNA in saliva to explore the possibility of a non-invasive and easy-to-execute diagnostic tool for prostate cancer screenings. METHODS: 16 miRNAs were extracted from salivary exosomes and analyzed via the delta-CT method. The presented method enables an application of the test in any health institution and even outpatient sector. Recruited participants were suspected to suffer from prostate cancer due to elevated PSA serum levels. Of these participants, 43 were diagnosed with prostate cancer, while 31 suffered from benign diseases and served as control group. RESULTS: hsa-mir-331-3p and hsa-mir-200b were significantly reduced in prostate cancer patients compared to the control group. ROC curve analysis revealed a reliable differentiation strength (AUC > 0.6) for both miRNAs with positive predictive values of 71% indicating prostate cancer. Differentiation of both groups based on PSA serum measurements was insufficient. The other 14 examined miRNAs showed no significant group differences. CONCLUSIONS: The presented method and miRNA are promising non-invasive tools to augment the current prostate cancer screening, thereby improving screening sensitivity and reducing numbers of false positive cancer suspects admitted to further invasive diagnostic and therapeutic steps.

Small RNAs as biomarkers to differentiate benign and malign prostate diseases: An alternative for transrectal punch biopsy of the prostate?

Prostate cancer (PCa) is the most common cancer and the third most frequent cause of male cancer death in Germany. MicroRNAs (miRNA) appear to be involved in the development and progression of PCa. A diagnostic differentiation from benign prostate hyperplasia (BPH) is often only possible through transrectal punch biopsy. This procedure is described as painful and carries risks. It was investigated whether urinary miRNAs can be used as biomarkers to differentiate the prostate diseases above. Therefore urine samples from urological patients with BPH (25) or PCa (28) were analysed using Next-Generation Sequencing to detect the expression profile of total and exosomal miRNA/piRNA. 79 miRNAs and 5 piwi-interacting RNAs (piRNAs) were significantly differentially expressed (adjusted p-value < 0.05 and log2-Fc > 1 or < -1). Of these, 6 miRNAs and 2 piRNAs could be statistically validated (AUC on test cohort > = 0.7). In addition, machine-learning algorithms were used to identify a panel of 22 additional miRNAs, whose interaction makes it possible to differentiate the groups as well. There are promising individual candidates for potential use as biomarkers in prostate cancer. The innovative approach of applying machine learning methods to this kind of data could lead to further small RNAs coming into scientific focus, which have so far been neglected.

Binary symbolic dynamics analysis to detect stress-associated changes of nonstationary heart rate variability.

Psychological stress may have harmful physiological effects and result in deteriorating health. Acute psychological stress acts also on cardiac autonomic regulation and may lead to nonstationarities in the interbeat interval series. We address the requirement of stationary RR interval series to calculate frequency domain parameters of heart rate variability (HRV) and use binary symbolic dynamics derived from RR interval differences to overcome this obstacle. 24 healthy subjects (12 female, 20-35 years) completed the following procedure: waiting period, Trier Social Stress Test to induce acute psychological stress, recovery period. An electrocardiogram was recorded throughout the procedure and HRV parameters were calculated for nine 5-min periods. Nonstationarities in RR interval series were present in all periods. During acute stress the average RR interval and SDNN decreased compared to rest before and after the stress test. Neither low frequency oscillations (LF), high frequency oscillations (HF) nor LF/HF could unambiguously reflect changes during acute stress in comparison to rest. Pattern categories derived from binary symbolic dynamics clearly identified acute stress and accompanying alterations of cardiac autonomic regulation. Methods based on RR interval differences like binary symbolic dynamics should be preferred to overcome issues related to nonstationarities.

Stress-associated changes in salivary microRNAs can be detected in response to the Trier Social Stress Test: An exploratory study.

Stress is an important co-factor for the genesis and maintenance of many diseases and is known to have an effect on gene expression via epigenetic regulation. MicroRNAs (miRNAs) appear to function as one of the key factors of this regulation. This is the first study to investigate the response of 11 stress-associated miRNAs in human saliva - as a non-invasive source - in an experimental condition of acute psychological stress, and also their correlation with established psychological (subjective stress perception), physiological (heart rate and heart rate variability) and biochemical stress parameters (salivary cortisol and alpha-amylase). 24 healthy participants between 20 and 35 years of age were investigated, using the Trier Social Stress Test (TSST) to induce acute psychological stress. Stress-associated changes were significant for miR-20b, -21 and 26b, and changes in miR-16 and -134 were close to significance, recommending further research on these miRNAs in the context of stress reactions. Significant correlations with alpha-amylase suggest their integration in sympathetic stress regulation processes. Additionally, our results demonstrate the TSST as a reliable tool for studying salivary miRNAs as non-invasive indicators of epigenetic processes in acute psychological stress reactions.

MicroRNAs in Psychological Stress Reactions and Their Use as Stress-Associated Biomarkers, Especially in Human Saliva.

MicroRNAs (miRNAs) play a central role in the regulation of many cellular processes including physiological and psychological stress reaction pathways. Psychological stress is an important factor for the genesis and maintenance of many diseases. Several miRNAs have already been described to be involved in its regulation. The presence of miRNAs in all body fluids implies a widespread role in communication throughout the whole organism and together with their stability makes them formidable candidates as biomarkers. Alterations of stress-associated miRNA expression levels have been found in the brain and whole blood of humans and animals. In this paper, we review the participation of miRNAs in stress-reactive processes as well as their usability as salivary biomarkers of such processes. In conclusion, we suggest that salivary miRNAs may be useful as noninvasive biomarkers to assess epigenetic regulation processes of chronic or acute psychological stress reactions.

Orthoformimycin, a selective inhibitor of bacterial translation elongation from Streptomyces containing an unusual orthoformate.

Upon high throughput screening of 6700 microbial fermentation extracts, we discovered a compound, designated orthoformimycin, capable of inhibiting protein synthesis in vitro with high efficiency. The molecule, whose structure was elucidated by chemical, spectrometric, and spectroscopic methods, contains an unusual orthoformate moiety (hence the name) and belongs to a novel class of translation inhibitors. This antibiotic does not affect any function of the 30S ribosomal subunit but binds to the 50S subunit causing inhibition of translation elongation and yielding polypeptide products of reduced length. Analysis by fluorescence stopped flow kinetics revealed that EF-G-dependent mRNA translocation is inhibited by orthoformimycin, whereas, surprisingly, translocation of the aminoacyl-tRNA seems to be unaffected.

Ribosome clearance by FusB-type proteins mediates resistance to the antibiotic fusidic acid.

Resistance to the antibiotic fusidic acid (FA) in the human pathogen Staphylococcus aureus usually results from expression of FusB-type proteins (FusB or FusC). These proteins bind to elongation factor G (EF-G), the target of FA, and rescue translation from FA-mediated inhibition by an unknown mechanism. Here we show that the FusB family are two-domain metalloproteins, the C-terminal domain of which contains a four-cysteine zinc finger with a unique structural fold. This domain mediates a high-affinity interaction with the C-terminal domains of EF-G. By binding to EF-G on the ribosome, FusB-type proteins promote the dissociation of stalled ribosome⋅EF-G⋅GDP complexes that form in the presence of FA, thereby allowing the ribosomes to resume translation. Ribosome clearance by these proteins represents a highly unusual antibiotic resistance mechanism, which appears to be fine-tuned by the relative abundance of FusB-type protein, ribosomes, and EF-G.

Distinct functions of elongation factor G in ribosome recycling and translocation.

Elongation factor G (EF-G) promotes the translocation step in bacterial protein synthesis and, together with ribosome recycling factor (RRF), the disassembly of the post-termination ribosome. Unlike translocation, ribosome disassembly strictly requires GTP hydrolysis by EF-G. Here we report that ribosome disassembly is strongly inhibited by vanadate, an analog of inorganic phosphate (Pi), indicating that Pi release is required for ribosome disassembly. In contrast, the function of EF-G in single-round translocation is not affected by vanadate, while the turnover reaction is strongly inhibited. We also show that the antibiotic fusidic acid blocks ribosome disassembly by EF-G/RRF at a 1000-fold lower concentration than required for the inhibition of EF-G turnover in vitro and close to the effective inhibitory concentration in vivo, suggesting that the antimicrobial activity of fusidic acid is primarily due to the direct inhibition of ribosome recycling. Our results indicate that conformational coupling between EF-G and the ribosome is principally different in translocation and ribosome disassembly. Pi release is not required for the mechanochemical function of EF-G in translocation, whereas the interactions between RRF and EF-G introduce tight coupling between the conformational change of EF-G induced by Pi release and ribosome disassembly.

Colicin E3 cleavage of 16S rRNA impairs decoding and accelerates tRNA translocation on Escherichia coli ribosomes.

The cytotoxin colicin E3 targets the 30S subunit of bacterial ribosomes and specifically cleaves 16S rRNA at the decoding centre, thereby inhibiting translation. Although the cleavage site is well known, it is not clear which step of translation is inhibited. We studied the effects of colicin E3 cleavage on ribosome functions by analysing individual steps of protein synthesis. We find that the cleavage affects predominantly the elongation step. The inhibitory effect of colicin E3 cleavage originates from the accumulation of sequential impaired decoding events, each of which results in low occupancy of the A site and, consequently, decreasing yield of elongating peptide. The accumulation leads to an almost complete halt of translation after reading of a few codons. The cleavage of 16S rRNA does not impair monitoring of codon-anticodon complexes or GTPase activation during elongation-factor Tu-dependent binding of aminoacyl-tRNA, but decreases the stability of the codon-recognition complex and slows down aminoacyl-tRNA accommodation in the A site. The tRNA-mRNA translocation is faster on colicin E3-cleaved than on intact ribosomes and is less sensitive to inhibition by the antibiotic viomycin.

Role and timing of GTP binding and hydrolysis during EF-G-dependent tRNA translocation on the ribosome.

The translocation of tRNA and mRNA through the ribosome is promoted by elongation factor G (EF-G), a GTPase that hydrolyzes GTP during the reaction. Recently, it was reported that, in contrast to previous observations, the affinity of EF-G was much weaker for GTP than for GDP and that ribosome-catalyzed GDP-GTP exchange would be required for translocation [Zavialov AV, Hauryliuk VV, Ehrenberg M (2005) J Biol 4:9]. We have reinvestigated GTP/GDP binding and show that EF-G binds GTP and GDP with affinities in the 20 to 40 microM range (37 degrees C), in accordance with earlier reports. Furthermore, GDP exchange, which is extremely rapid on unbound EF-G, is retarded, rather than accelerated, on the ribosome, which, therefore, is not a nucleotide-exchange factor for EF-G. The EF-G.GDPNP complex, which is very labile, is stabilized 30,000-fold by binding to the ribosome. These findings, together with earlier kinetic results, reveal that EF-G enters the pretranslocation ribosome in the GTP-bound form and indicate that, upon ribosome-complex formation, the nucleotide-binding pocket of EF-G is closed, presumably in conjunction with GTPase activation. GTP hydrolysis is required for rapid tRNA-mRNA movement, and P(i) release induces further rearrangements of both EF-G and the ribosome that are required for EF-G turnover.

Control of phosphate release from elongation factor G by ribosomal protein L7/12.

Ribosomal protein L7/12 is crucial for the function of elongation factor G (EF-G) on the ribosome. Here, we report the localization of a site in the C-terminal domain (CTD) of L7/12 that is critical for the interaction with EF-G. Single conserved surface amino acids were replaced in the CTD of L7/12. Whereas mutations in helices 5 and 6 had no effect, replacements of V66, I69, K70, and R73 in helix 4 increased the Michaelis constant (KM) of EF-G.GTP for the ribosome, suggesting an involvement of these residues in EF-G binding. The mutations did not appreciably affect rapid single-round GTP hydrolysis and had no effect on tRNA translocation on the ribosome. In contrast, the release of inorganic phosphate (Pi) from ribosome-bound EF-G.GDP.Pi was strongly inhibited and became rate-limiting for the turnover of EF-G. The control of Pi release by interactions between EF-G and L7/12 appears to be important for maintaining the conformational coupling between EF-G and the ribosome for translocation and for timing the dissociation of the factor from the ribosome.

Conformational changes of the small ribosomal subunit during elongation factor G-dependent tRNA-mRNA translocation.

Translocation, a coordinated movement of two tRNAs together with mRNA on the ribosome, is catalyzed by elongation factor G (EF-G). The reaction is accompanied by conformational rearrangements of the ribosome that are, as yet, not well characterized. Here, we analyze those rearrangements by restricting the conformational flexibility of the ribosome by antibiotics binding to specific sites of the ribosome. Paromomycin (Par), viomycin (Vio), spectinomycin (Spc), and hygromycin B (HygB) inhibited the tRNA-mRNA movement, while the other partial reactions of translocation, including the unlocking rearrangement of the ribosome that precedes tRNA-mRNA movement, were not affected. The functional cycle of EF-G, i.e. binding of EF-G.GTP to the ribosome, GTP hydrolysis, Pi release, and dissociation of EF-G.GDP from the ribosome, was not affected either, indicating that EF-G turnover is not coupled directly to tRNA-mRNA movement. The inhibition of translocation by Par and Vio is attributed to the stabilization of tRNA binding in the A site, whereas Spc and HygB had a direct inhibitory effect on tRNA-mRNA movement. Streptomycin (Str) had essentially no effect on translocation, although it caused a large increase in tRNA affinity to the A site. These results suggest that conformational changes in the vicinity of the decoding region at the binding sites of Spc and HygB are important for tRNA-mRNA movement, whereas Str seems to stabilize a conformation of the ribosome that is prone to rapid translocation, thereby compensating the effect on tRNA affinity.

An elongation factor G-induced ribosome rearrangement precedes tRNA-mRNA translocation.

The elongation cycle of protein synthesis is completed by translocation, a rearrangement during which two tRNAs bound to the mRNA move on the ribosome. The reaction is promoted by elongation factor G (EF-G) and accelerated by GTP hydrolysis. Here we report a pre-steady-state kinetic analysis of translocation. The kinetic model suggests that GTP hydrolysis drives a conformational rearrangement of the ribosome that precedes and limits the rates of tRNA-mRNA translocation and Pi release from EF-G.GDP.Pi. The latter two steps are intrinsically rapid and take place at random. These results indicate that the energy of GTP hydrolysis is utilized to promote the ribosome rearrangement and to bias spontaneous fluctuations within the ribosome-EF-G complex toward unidirectional movement of mRNA and tRNA.

Coupling of GTP hydrolysis by elongation factor G to translocation and factor recycling on the ribosome.

The translocation step of elongation entails the coordinated movement of tRNA and mRNA on the ribosome. Translocation is promoted by elongation factor G (EF-G) and accompanied by GTP hydrolysis, which affects both translocation and turnover of EF-G. Both reactions are much slower (50-100-fold) when GTP is replaced with non-hydrolyzable GTP analogues or GDP, indicating that the reaction rates are determined by conformational transitions induced by GTP hydrolysis. Compared to the rate of uncatalyzed, spontaneous translocation, ribosome binding of EF-G with any guanine nucleotide reduces the free energy of activation by about 18 kJ/mol, whereas GTP hydrolysis contributes another 10 kJ/mol. The acceleration by GTP hydrolysis is due to large decrease in activation enthalpy by about 30 kJ/mol, compared to the reaction with GTP analogues or GDP, whereas the activation entropy becomes unfavorable and is lowered by about 20 kJ/mol (37 degrees C). The data suggest that GTP hydrolysis induces, by a conformational change of EF-G, a rapid conformational rearrangement of the ribosome ("unlocking") which determines the rates of both tRNA-mRNA translocation and recycling of the factor.