Astaxanthin from Haematococcus pluvialis Prevents Oxidative Stress on Human Endothelial Cells without Toxicity.

Astaxanthin, a powerful antioxidant, is a good candidate for the prevention of intracellular oxidative stress. The aim of the study was to compare the antioxidant activity of astaxanthin present in two natural extracts from Haematococcus pluvialis, a microalgae strain, with that of synthetic astaxanthin. Natural extracts were obtained either by solvent or supercritical extraction methods. UV, HPLC-DAD and (HPLC-(atmospheric pressure chemical ionization (APCI)+)/ion trap-MS) characterizations of both natural extracts showed similar compositions of carotenoids, but different percentages in free astaxanthin and its ester derivatives. The Trolox equivalent antioxidant capacity (TEAC) assay showed that natural extracts containing esters displayed stronger antioxidant activities than free astaxanthin. Their antioxidant capacities to inhibit intracellular oxidative stress were then evaluated on HUVEC cells. The intracellular antioxidant activity in natural extracts was approximately 90-times higher than synthetic astaxanthin (5 µM). No modification, neither in the morphology nor in the viability, of vascular human cells was observed by in vitro biocompatibility study up to 10 µM astaxanthin concentrations. Therefore, these results revealed the therapeutic potential of the natural extracts in vascular human cell protection against oxidative stress without toxicity, which could be exploited in prevention and/or treatment of cardiovascular diseases.

Search for poly(A) polymerase targets in E. coli reveals its implication in surveillance of Glu tRNA processing and degradation of stable RNAs.

Polyadenylation is a universal post-transcriptional modification involved in degradation and quality control of bacterial RNAs. In Escherichia coli, it is admitted that any accessible RNA 3' end can be tagged by a poly(A) tail for decay. However, we do not have yet an overall view of the population of polyadenylated molecules. The sampling of polyadenylated RNAs presented here demonstrates that rRNA fragments and tRNA precursors originating from the internal spacer regions of the rrn operons, in particular, rrnB are abundant poly(A) polymerase targets. Focused analysis showed that Glu tRNA precursors originating from the rrnB and rrnG transcripts exhibit long 3' trailers that are primarily removed by PNPase and to a lesser extent by RNase II and poly(A) polymerase. Moreover, 3' trimming by exoribonucleases precedes 5' end maturation by RNase P. Interestingly, characterization of RNA fragments that accumulate in a PNPase deficient strain showed that Glu tRNA precursors still harbouring the 5' leader can be degraded by a 3' to 5' quality control pathway involving poly(A) polymerase. This demonstrates that the surveillance of tRNA maturation described for a defective tRNA also applies to a wild-type tRNA.

The interplay of Hfq, poly(A) polymerase I and exoribonucleases at the 3' ends of RNAs resulting from Rho-independent termination: A tentative model.

Discovered in eukaryotes as a modification essential for mRNA function, polyadenylation was then identified as a means used by all cells to destabilize RNA. In Escherichia coli, most accessible 3' RNA extremities are believed to be potential targets of poly(A) polymerase I. However, some RNAs might be preferentially adenylated. After a short statement of the current knowledge of poly(A) metabolism, we discuss how Hfq could affect recognition and polyadenylation of RNA terminated by Rho-independent terminators. Comparison of RNA terminus leads to the proposal that RNAs harboring 3' terminal features required for Hfq binding are not polyadenylated, whereas those lacking these structural elements can gain the oligo(A) tails that initiate exonucleolytic degradation. We also speculate that Hfq stimulates the synthesis of longer tails that could be used as Hfq-binding sites involved in non-characterized functions of Hfq-dependent sRNAs.

The poly(A)-dependent degradation pathway of rpsO mRNA is primarily mediated by RNase R.

Polyadenylation is an important factor controlling RNA degradation and RNA quality control mechanisms. In this report we demonstrate for the first time that RNase R has in vivo affinity for polyadenylated RNA and can be a key enzyme involved in poly(A) metabolism. RNase II and PNPase, two major RNA exonucleases present in Escherichia coli, could not account for all the poly(A)-dependent degradation of the rpsO mRNA. RNase II can remove the poly(A) tails but fails to degrade the mRNA as it cannot overcome the RNA termination hairpin, while PNPase plays only a modest role in this degradation. We now demonstrate that in the absence of RNase E, RNase R is the relevant factor in the poly(A)-dependent degradation of the rpsO mRNA. Moreover, we have found that the RNase R inactivation counteracts the extended degradation of this transcript observed in RNase II-deficient cells. Elongated rpsO transcripts harboring increasing poly(A) tails are specifically recognized by RNase R and strongly accumulate in the absence of this exonuclease. The 3' oligo(A) extension may stimulate the binding of RNase R, allowing the complete degradation of the mRNA, as RNase R is not susceptible to RNA secondary structures. Moreover, this regulation is shown to occur despite the presence of PNPase. Similar results were observed with the rpsT mRNA. This report shows that polyadenylation favors in vivo the RNase R-mediated pathways of RNA degradation.

Hfq affects mRNA levels independently of degradation.

BACKGROUND: The bacterial Lsm protein, Hfq, is an RNA chaperone involved in many reactions related to RNA metabolism, such as replication and stability, control of small RNA activity and polyadenylation. Despite this wide spectrum of known functions, the global role of Hfq is almost certainly undervalued; its capacity to bind DNA and to interact with many other proteins are only now beginning to be taken into account. RESULTS: The role of Hfq in the maturation and degradation of the rpsO mRNA of E. coli was investigated in vivo. The data revealed a decrease in rpsO mRNA abundance concomitant to an increase in its stability when Hfq is absent. This indicates that the change in mRNA levels in hfq mutants does not result from its modification of RNA stability. Moreover, a series of independent experiments have revealed that the decrease in mRNA level is not a consequence of a reduction of translation efficiency and that Hfq is not directly implicated in translational control of rpsO expression. Reduced steady-state mRNA levels in the absence of Hfq were also shown for rpsT, rpsB and rpsB-tsf, but not for lpp, pnp or tRNA transcripts. The abundance of chimeric transcripts rpsO-lacZ and rpsB-lacZ, whose expression was driven by rpsO and rpsB promoters, respectively, was also lower in the hfq null-mutants, while the beta-galactosidase yield remained about the same as in the parent wild-type strain. CONCLUSIONS: The data obtained suggest that alteration of rpsO, rpsT and rpsB-tsf transcript levels observed under conditions of Hfq deficiency is not caused by the post-transcriptional events, such as mRNA destabilization or changes in translation control, and may rather result from changes in transcriptional activity. So far, how Hfq affects transcription remains unclear. We propose that one of the likely mechanisms of Hfq-mediated modulation of transcription might operate early in the elongation step, when interaction of Hfq with a nascent transcript would help to overcome transcription pauses and to prevent preliminary transcript release.

Auto-assembly of E. coli DsrA small noncoding RNA: Molecular characteristics and functional consequences.

RNA molecules are important factors involved in different cellular processes and have a multitude of roles in the cell. These roles include serving as a temporary copy of genes used for protein synthesis or functions in translational machinery. Interestingly, RNA is so far the only biological molecule that serves both as a catalyst (like proteins) and as information storage (like DNA). However, in contrast to proteins well known to be able to self-associate in order to maintain the architecture of the cell, such RNA polymers are not prevalent in cells and are usually not favored by the flexibility of this molecule. In this work, we present evidence that such a polymer of a natural RNA, the DsrA RNA, exists in the bacterial cell. DsrA is a small noncoding RNA (87 nucleotides) of Escherichia coli that acts by base-pairing to mRNA in order to control the translation and the turnover of some mRNA, including rpoS mRNA, which encodes the sigma(s) RNA polymerase subunit involved in bacterial stress response. A putative model is proposed for the structure of this RNA polymer. Although the function of this polymerization is not known completely, we propose that the formation of such a structure could be involved in the regulation of DsrA ncRNA concentration in vivo or in a quality control mechanism used by the cell to eliminate misfolded RNAs.

Spectroscopic observation of RNA chaperone activities of Hfq in post-transcriptional regulation by a small non-coding RNA.

Hfq protein is vital for the function of many non-coding small (s)RNAs in bacteria but the mechanism by which Hfq facilitates the function of sRNA is still debated. We developed a fluorescence resonance energy transfer assay to probe how Hfq modulates the interaction between a sRNA, DsrA, and its regulatory target mRNA, rpoS. The relevant RNA fragments were labelled so that changes in intra- and intermolecular RNA structures can be monitored in real time. Our data show that Hfq promotes the strand exchange reaction in which the internal structure of rpoS is replaced by pairing with DsrA such that the Shine-Dalgarno sequence of the mRNA becomes exposed. Hfq appears to carry out strand exchange by inducing rapid association of DsrA and a premelted rpoS and by aiding in the slow disruption of the rpoS secondary structure. Unexpectedly, Hfq also disrupts a preformed complex between rpoS and DsrA. While it may not be a frequent event in vivo, this melting activity may have implications in the reversal of sRNA-based regulation. Overall, our data suggests that Hfq not only promotes strand exchange by binding rapidly to both DsrA and rpoS but also possesses RNA chaperoning properties that facilitates dynamic RNA-RNA interactions.

Polyadenylation of a functional mRNA controls gene expression in Escherichia coli.

Although usually implicated in the stabilization of mRNAs in eukaryotes, polyadenylation was initially shown to destabilize RNA in bacteria. All the data are consistent with polyadenylation being part of a quality control process targeting folded RNA fragments and non-functional RNA molecules to degradation. We report here an example in Escherichia coli, where polyadenylation directly controls the level of expression of a gene by modulating the stability of a functional transcript. Inactivation of poly(A)polymerase I causes overexpression of glucosamine-6-phosphate synthase (GlmS) and both the accumulation and stabilization of the glmS transcript. Moreover, we show that the glmS mRNA results from the processing of the glmU-glmS cotranscript by RNase E. Interestingly, the glmU-glmS cotranscript and the mRNA fragment encoding GlmU only slightly accumulated in the absence of poly(A)polymerase, suggesting that the endonucleolytically generated glmS mRNA harbouring a 5' monophosphate and a 3' stable hairpin is highly susceptible to poly(A)-dependent degradation.

Deep Learning for Automated Segmentation of Liver Lesions at CT in Patients with Colorectal Cancer Liver Metastases.

PURPOSE: To evaluate the performance, agreement, and efficiency of a fully convolutional network (FCN) for liver lesion detection and segmentation at CT examinations in patients with colorectal liver metastases (CLMs). MATERIALS AND METHODS: This retrospective study evaluated an automated method using an FCN that was trained, validated, and tested with 115, 15, and 26 contrast material-enhanced CT examinations containing 261, 22, and 105 lesions, respectively. Manual detection and segmentation by a radiologist was the reference standard. Performance of fully automated and user-corrected segmentations was compared with that of manual segmentations. The interuser agreement and interaction time of manual and user-corrected segmentations were assessed. Analyses included sensitivity and positive predictive value of detection, segmentation accuracy, Cohen κ, Bland-Altman analyses, and analysis of variance. RESULTS: In the test cohort, for lesion size smaller than 10 mm (n = 30), 10-20 mm (n = 35), and larger than 20 mm (n = 40), the detection sensitivity of the automated method was 10%, 71%, and 85%; positive predictive value was 25%, 83%, and 94%; Dice similarity coefficient was 0.14, 0.53, and 0.68; maximum symmetric surface distance was 5.2, 6.0, and 10.4 mm; and average symmetric surface distance was 2.7, 1.7, and 2.8 mm, respectively. For manual and user-corrected segmentation, κ values were 0.42 (95% confidence interval: 0.24, 0.63) and 0.52 (95% confidence interval: 0.36, 0.72); normalized interreader agreement for lesion volume was -0.10 ± 0.07 (95% confidence interval) and -0.10 ± 0.08; and mean interaction time was 7.7 minutes ± 2.4 (standard deviation) and 4.8 minutes ± 2.1 (P < .001), respectively. CONCLUSION: Automated detection and segmentation of CLM by using deep learning with convolutional neural networks, when manually corrected, improved efficiency but did not substantially change agreement on volumetric measurements.© RSNA, 2019Supplemental material is available for this article.

Hfq variant with altered RNA binding functions.

The interaction between Hfq and RNA is central to multiple regulatory processes. Using site-directed mutagenesis, we have found a missense mutation in Hfq (V43R) which strongly affects2 the RNA binding capacity of the Hfq protein and its ability to stimulate poly(A) tail elongation by poly(A)-polymerase in vitro. In vivo, overexpression of this Hfq variant fails to stimulate rpoS-lacZ expression and does not restore a normal growth rate in hfq null mutant. Cells in which the wild-type gene has been replaced by the hfqV43R allele exhibit a phenotype intermediate between those of the wild-type and of the hfq minus or null strains. This missense mutation derepresses Hfq synthesis. However, not all Hfq functions are affected by this mutation. For example, HfqV43R represses OppA synthesis as strongly as the wild-type protein. The dominant negative effect of the V43R mutation over the wild-type allele suggests that hexamers containing variant and genuine subunits are presumably not functional. Finally, molecular dynamics studies indicate that the V43R substitution mainly changes the position of the K56 and Y55 side chains involved in the Hfq-RNA interaction but has probably no effect on the folding and the oligomerization of the protein.

Absolute Regulatory Small Noncoding RNA Concentration and Decay Rates Measurements in Escherichia coli.

Regulation of RNA turnover is of utmost importance for controlling the concentration of transcripts and consequently cellular protein levels. Among the processes controlling RNA decay, small noncoding regulatory RNAs (sRNAs) have recently emerged as major new players. In this chapter, we describe and discuss protocols that can be used to measure sRNA concentration in vivo and to assess sRNA decay rates in Gram-negative bacteria. Precisely, we focus our analyses on the Escherichia coli Gram-negative bacterium as a model. The information described in this chapter provides a guideline to help develop a protocol in order to assess these important parameters and to identify RNA-processing enzymes involved in sRNA degradation processes.

Three-dimensional structures of fibrillar Sm proteins: Hfq and other Sm-like proteins.

Hfq is a nucleic acid-binding protein that functions as a global regulator of gene expression by virtue of its interactions with several small, non-coding RNA species. Originally identified as an Escherichia coli host factor required for RNA phage Qbeta replication, Hfq is now known to post-transcriptionally regulate bacterial gene expression by modulating both mRNA stability and translational activity. Recently shown to be a member of the diverse Sm protein family, Hfq adopts the OB-like fold typical of other Sm and Sm-like (Lsm) proteins, and also assembles into toroidal homo-oligomers that bind single-stranded RNA. Similarities between the structures, functions, and evolution of Sm/Lsm proteins and Hfq are continually being discovered, and we now report an additional, unexpected biophysical property that is shared by Hfq and other Sm proteins: E.coli Hfq polymerizes into well-ordered fibres whose morphologies closely resemble those found for Sm-like archaeal proteins (SmAPs). However, the hierarchical assembly of these fibres is dissimilar: whereas SmAPs polymerize into polar tubes (and striated bundles of such tubes) by head-to-tail stacking of individual homo-heptamers, helical Hfq fibres are formed by cylindrical slab-like layers that consist of 36 subunits arranged as a hexamer of Hfq homo-hexamers (i.e. protofilaments in a 6 x 6 arrangement). The different fibrillar ultrastructures formed by Hfq and SmAP are presented and examined herein, with the overall goal of elucidating another similarity amongst the diverse members of the Sm protein family.

The poly(A) binding protein Hfq protects RNA from RNase E and exoribonucleolytic degradation.

The Hfq protein, which shares sequence and structural homology with the Sm and Lsm proteins, binds to various RNAs, primarily recognizing AU-rich single-stranded regions. In this paper, we study the ability of the Escherichia coli Hfq protein to bind to a polyadenylated fragment of rpsO mRNA. Hfq exhibits a high specificity for a 100-nucleotide RNA harboring 18 3'-terminal A-residues. Structural analysis of the adenylated RNA-Hfq complex and gel shift assays revealed the presence of two Hfq binding sites. Hfq binds primarily to the poly(A) tail, and to a lesser extent a U-rich sequence in a single-stranded region located between two hairpin structures. The oligo(A) tail and the interhelical region are sensitive to 3'-5' exoribonucleases and RNase E hydrolysis, respectively, in vivo. In vitro assays demonstrate that Hfq protects poly(A) tails from exonucleolytic degradation by both PNPase and RNase II. In addition, RNase E processing, which occurred close to the U-rich sequence, is impaired by the presence of Hfq. These data suggest that Hfq modulates the sensitivity of RNA to ribonucleases in the cell.

Hfq affects the length and the frequency of short oligo(A) tails at the 3' end of Escherichia coli rpsO mRNAs.

Polyadenylation plays an important role in RNA degradation in bacterial cells. In Escherichia coli, exoribonucleases, mostly RNase II and polynucleotide phosphorylase, antagonize the synthesis of poly(A) tails by poly(A) polymerase I (PAP I). In accordance with earlier observations showing that only a small fraction of bacterial RNA is polyadenylated, we demonstrate here that approximately 10% of rpsO mRNA harbors short oligo(A) tails ranging from one to five A residues in wild-type cells. We also compared the length, frequency and distribution of poly(A) tails at the 3'-end of rpsO transcripts in vivo in the presence and absence of Hfq, a host factor that in vitro stimulates the activity of PAP I, and found that Hfq affects all three parameters. In the hfq(+) strain the average length of oligo(A) tails and frequency of polyadenylated transcripts was higher than in the hfq(-) strain and a smaller proportion of tails was found at the 3' end of transcripts terminated at the Rho- independent terminator. Our data led us to the conclusion that Hfq is involved in the recognition of 3' RNA extremities by PAP I.

Stimulation of poly(A) synthesis by Escherichia coli poly(A)polymerase I is correlated with Hfq binding to poly(A) tails.

The bacterial Lsm protein, host factor I (Hfq), is an RNA chaperone involved in many types of RNA transactions such as replication and stability, control of small RNA activity and polyadenylation. In this latter case, Hfq stimulates poly(A) synthesis and binds poly(A) tails that it protects from exonucleolytic degradation. We show here, that there is a correlation between Hfq binding to the 3' end of an RNA molecule and its ability to stimulate RNA elongation catalyzed by poly(A)polymerase I. In contrast, formation of the Hfq-RNA complex inhibits elongation of the RNA by polynucleotide phosphorylase. We demonstrate also that Hfq binding is not affected by the phosphorylation status of the RNA molecule and occurs equally well at terminal or internal stretches of poly(A).

Fate of mRNA extremities generated by intrinsic termination: detailed analysis of reactions catalyzed by ribonuclease II and poly(A) polymerase.

In all living cells 3' ends of RNA are posttranscriptionally elongated or shortened by nucleotidyl transferases and ribonucleases. The detailed analysis of the rpsO mRNA of Escherichia coli presented here demonstrates that transcription terminates in vivo at two sites located seven and eight nucleotides downstream from the GC-rich hairpin of the intrinsic terminator and that primary transcripts can be shortened by RNase II. The shortest RNA identified in the cell result from nibbling of primary transcripts. Primary transcripts and nibbled molecules can also be adenylated by poly(A) polymerase I (PAP I). In addition, kinetics of decay performed in vitro demonstrate that RNase II rapidly degrades poly(A) tails longer than 7-8 As processively while it slowly nibbles shorter tails and non adenylated RNAs distributively. Comparison of the kinetics of nibbling of oligoadenylated rpsO mRNA in vivo and in vitro lead us to conclude that the rates of shortening and elongation of the oligo(A) tails detected in vivo are very slow: about 0.5-7 nucleotides per min. We finally speculate that the slowness of oligo(A) synthesis may explain why polyadenylation does not affect the stability of mRNAs whose degradation is controlled by RNase E.

Structural Modelling of the Sm-like Protein Hfq from Escherichia coli.

The Hfq polypeptide of Escherichia coli is a nucleic acid-binding protein involved in the expression of many proteins. Derivation of its three-dimensional structure is important for our understanding of its role in gene regulation at the molecular level. In this study, we combined computational and biophysical analysis to derive a possible structure for Hfq. As a first step towards determining the structure, we searched for possible sequence-structure compatibility, using secondary structure prediction and protein domain and fold-recognition methods available on the WEB. One fold, essentially beta sheet in character, the Sm motif of small nuclear ribonucleoproteins, even though it initially fell well below the confidence thresholds, was proposed and further validated by a series of biophysical and biochemical studies. The Hfq hexamer structure was modelled on the human Sm D3B structure using optimised sequence alignments and molecular mechanics methods. This structure accounts for the physico-chemical properties of Hfq and highlights amino acid residues that could interact with RNA.

RNase E and polyadenyl polymerase I are involved in maturation of CI RNA, the P4 phage immunity factor.

Bacteriophage P4 immunity is controlled by a small stable RNA (CI RNA) that derives from the processing of primary transcripts. In previous works, we observed that the endonuclease RNase P is required for the maturation of CI RNA 5'-end; moreover, we found that polynucleotide phosphorylase (PNPase), a 3' to 5' RNA-degrading enzyme, is required for efficient 5'-end processing of CI RNA, suggesting that 3'-end degradation of the primary transcript might be involved in the production of proper RNase P substrates. Here, we demonstrate that another Escherichia coli nuclease, RNase E, would appear to be involved in this process. We found that transcripts of the P4 immunity region are modified by the post-transcriptional addition of short poly(A) tails and heteropolymeric tails with prevalence of A residues. Most oligoadenylated transcripts encompass the whole cI locus and are thus compatible as intermediates in the CI RNA maturation pathway. On the contrary, in a polynucleotide phosphorylase (PNPase)-defective host, adenylation occurred most frequently within cI, implying that such transcripts are targeted for degradation. We did not find polyadenylation in a pcnB mutant, suggesting that the pcnB-encoded polyadenyl polymerase I (PAP I) is the only enzyme responsible for modification of P4 immunity transcripts. Maturation of CI RNA 5'-end in such a mutant was impaired, further supporting the idea that processing of the 3'-end of primary transcripts is an important step for efficient maturation of CI RNA by RNase P.

Pretest probability of a normal echocardiography: validation of a simple and practical algorithm for routine use.

BACKGROUND: Management of increased referrals for transthoracic echocardiography (TTE) examinations is a challenge. Patients with normal TTE examinations take less time to explore than those with heart abnormalities. A reliable method for assessing pretest probability of a normal TTE may optimize management of requests. AIM: To establish and validate, based on requests for examinations, a simple algorithm for defining pretest probability of a normal TTE. METHODS: In a retrospective phase, factors associated with normality were investigated and an algorithm was designed. In a prospective phase, patients were classified in accordance with the algorithm as being at high or low probability of having a normal TTE. RESULTS: In the retrospective phase, 42% of 618 examinations were normal. In multivariable analysis, age and absence of cardiac history were associated to normality. Low pretest probability of normal TTE was defined by known cardiac history or, in case of doubt about cardiac history, by age>70 years. In the prospective phase, the prevalences of normality were 72% and 25% in high (n=167) and low (n=241) pretest probability of normality groups, respectively. The mean duration of normal examinations was significantly shorter than abnormal examinations (13.8 ± 9.2 min vs 17.6 ± 11.1 min; P=0.0003). CONCLUSION: A simple algorithm can classify patients referred for TTE as being at high or low pretest probability of having a normal examination. This algorithm might help to optimize management of requests in routine practice.

Is the secondary putative RNA-RNA interaction site relevant to GcvB mediated regulation of oppA mRNA in Escherichia coli?

GcvB is a non-coding RNA that regulates oppA mRNA in different bacterial species by binding a GcvB GU-rich region named R1 to oppA mRNA. A secondary putative interaction site (PS1) was identified in this study that is able to form a second nearly perfect 10 base-pair duplex between these two RNAs in Escherichia coli. In this work, we have studied whether the formation of a second interaction site could help stabilize the previously reported GcvB/oppA complex. Several mutations and the full deletion of PS1 were engineered. None of these modifications affected the ability of GcvB to control OppA expression. Therefore the second, putative, interaction site appears to be unnecessary for the regulatory function of GcvB with regard to its oppA target mRNA.