The Normal, the Radiosensitive, and the Ataxic in the Era of Precision Radiotherapy: A Narrative Review.

(1) Background: radiotherapy is a cornerstone of cancer treatment. When delivering a tumoricidal dose, the risk of severe late toxicities is usually kept below 5% using dose-volume constraints. However, individual radiation sensitivity (iRS) is responsible (with other technical factors) for unexpected toxicities after exposure to a dose that induces no toxicity in the general population. Diagnosing iRS before radiotherapy could avoid unnecessary toxicities in patients with a grossly normal phenotype. Thus, we reviewed iRS diagnostic data and their impact on decision-making processes and the RT workflow; (2) Methods: following a description of radiation toxicities, we conducted a critical review of the current state of the knowledge on individual determinants of cellular/tissue radiation; (3) Results: tremendous advances in technology now allow minimally-invasive genomic, epigenetic and functional testing and a better understanding of iRS. Ongoing large translational studies implement various tests and enriched NTCP models designed to improve the prediction of toxicities. iRS testing could better support informed radiotherapy decisions for individuals with a normal phenotype who experience unusual toxicities. Ethics of medical decisions with an accurate prediction of personalized radiotherapy's risk/benefits and its health economics impact are at stake; (4) Conclusions: iRS testing represents a critical unmet need to design personalized radiotherapy protocols relying on extended NTCP models integrating iRS.

Fatty acid desaturase genetic variations and dietary omega-3 fatty acid intake associate with arterial stiffness.

Aims: Long-chain polyunsaturated fatty acids (PUFAs) generate diverse bioactive lipid mediators, which tightly regulate vascular inflammation. The effects of omega-3 PUFA supplementation in cardiovascular prevention however remain controversial. In addition to direct dietary intake, fatty acid desaturases (FADS) determine PUFA levels. Increased arterial stiffness represents an independent predictor of mortality and cardiovascular events. The aim of the present study was to determine the association of PUFA intake, FADS1 genotype, and FADS expression with arterial stiffness. Methods and results: A cross-sectional population-based cohort study of 1464 participants without overt cardiovascular disease was conducted. Dietary intake was assessed using a food frequency questionnaire. Arterial stiffness was assessed by carotid-femoral pulse wave velocity (cfPWV), and the FADS1 locus variant was determined. Blood cell transcriptomics was performed in a subset of 410 individuals. Pulse wave velocity was significantly associated with the FADS1 locus variant. Differential associations between PWV and omega-3 PUFA intake were observed depending on the FADS1 genotype. High omega-3 PUFA intake attenuated the FADS1 genotype-dependent associations. Carriers of the minor FADS1 locus variant exhibited increased expression of FADS2, which is associated with PWV. Conclusion: Taken together, these findings point to FADS1 genotype-dependent associations of omega-3 PUFA intake on subclinical cardiovascular disease. These findings may have implications for identifying responders and non-responders to omega-3 PUFA supplementation and open up for personalized dietary counselling in cardiovascular prevention.

Impact of natriuretic peptide polymorphisms on diastolic and metabolic function in a populational cohort: insights from the STANISLAS cohort.

AIMS: Elevated brain natriuretic peptide (BNP) and the N-terminal fragment of its pro-hormone (NT-proBNP) have become established biomarkers for heart failure and are associated with cardiovascular morbidity and mortality. Investigating sources of inter-individual heterogeneity, particularly genetic factors, could help better identify patients at risk of future cardiovascular disease. The aim of this study was to estimate the heritability of circulating NT-proBNP levels, to perform a genome-wide association study (GWAS) and gene-candidate analysis focused on NPPB-NPPA genes on these levels, and to examine their association with cardiovascular or metabolic outcomes. METHODS AND RESULTS: A total of 1555 individuals from the STANISLAS study were included. The heritability of circulating NT-proBNP levels was estimated at 15%, with seven single nucleotide polymorphisms (SNPs) reaching the significant threshold in the GWAS. All above SNPs were located on the same gene cluster constituted of MTHFR, CLCN6, NPPA, NPPB, and C1orf167. NPPA gene expression was also associated with NT-proBNP levels. Moreover, six other SNPs from NPPA-NPPB genes were associated with diastolic function (lateral e' on echocardiography) and metabolic features (glycated haemoglobin). CONCLUSIONS: The heritability of natriuretic peptides appears relatively low (15%) and mainly based on the same gene cluster constituted of MTHFR, CLCN6, NPPA, NPPB, and C1orf167. Natriuretic peptide polymorphisms are associated with natriuretic peptide levels and diastolic function. These results suggest that natriuretic peptide polymorphisms may have an impact in the early stages of cardiovascular and metabolic disease.

Implication of repeat insertion domains in the trans-activity of the long non-coding RNA ANRIL.

Long non-coding RNAs have emerged as critical regulators of cell homeostasis by modulating gene expression at chromatin level for instance. Here, we report that the lncRNA ANRIL, associated with several pathologies, binds to thousands of loci dispersed throughout the mammalian genome sharing a 21-bp motif enriched in G/A residues. By combining ANRIL genomic occupancy with transcriptomic analysis, we established a list of 65 and 123 genes potentially directly activated and silenced by ANRIL in trans, respectively. We also found that Exon8 of ANRIL, mainly made of transposable elements, contributes to ANRIL genomic association and consequently to its trans-activity. Furthermore, we showed that Exon8 favors ANRIL's association with the FIRRE, TPD52L1 and IGFBP3 loci to modulate their expression through H3K27me3 deposition. We also investigated the mechanisms engaged by Exon8 to favor ANRIL's association with the genome. Our data refine ANRIL's trans-activity and highlight the functional importance of TEs on ANRIL's activity.

Non-Coding RNA Silencing in Mammalian Cells by Antisense LNA GapmeRs Transfection.

The assessment of non-coding RNAs (ncRNAs) functions highly relies on loss of function studies. However, due to their exclusive or partial nuclear localization, many small and long ncRNAs are not efficiently silenced by RNA interference. Antisense LNA GapmeRs constitute a good alternative to RNAi. They allow an effective knockdown of ncRNAs with sizes greater than 80 nucleotides, regardless of their cellular localization. This chapter focuses on the silencing of two different nuclear ncRNAs (ANRIL and SATIII RNAs) in mammalian cells using antisense LNA GapmeRs with two different transfection methods: calcium phosphate-mediated transfection and LipofectamineTM 2000.

Fluorescence In Situ Hybridization of Small Non-Coding RNAs.

The determination of the cellular localization of a noncoding RNA (ncRNA) is highly helpful to decipher its function. RNA-FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules within individual cells and tissues. Refined RNA-FISH methods have also been developed to determine RNA transcription and degradation rates. This chapter describes an RNA-FISH protocol that we developed to study the expression and localization of satellite III (SATIII) RNAs. This specific class of ncRNAs is expressed in response to various cellular stresses, including heat shock. The protocol is based on the use of a biotinylated LNA probe subsequently detected by a Streptavidin, Alexa Fluor® 488 conjugate. A protocol allowing efficient coupling of RNA-FISH and protein detection by immunofluorescence is also described as well as the bioinformatics pipeline, Substructure Analyzer, we recently developed to automate fluorescence signal analysis.

Study of Genome-Wide Occupancy of Long Non-Coding RNAs Using Chromatin Isolation by RNA Purification (ChIRP).

Long noncoding RNAs (lncRNAs) have recently emerged as masters of gene expression regulation by exerting their functions in all cell compartments through a wide repertoire of mechanisms. A high portion of lncRNAs are robustly enriched in the chromatin fraction suggesting a broad regulatory role in the nuclear compartment. Despite the advances in this field, the interaction between lncRNAs and the chromatin is still poorly understood. This led to the emergence of numerous hybridization capture assays such as the Chromatin Isolation by RNA Purification (ChIRP) which revealed at high resolution the genomic binding sites of several nuclear lncRNAs. In this chapter, we describe the ChIRP protocol that was successfully applied to the lncRNA ANRIL. We also provide a user-friendly bioinformatic pipeline for ChIRP-seq data analysis.

Microarray Analysis of Whole-Transcriptome RNAs Including Non-Coding RNAs.

Non-coding RNAs participate in most cellular processes and play a causative role in several diseases. In addition to their relevance as targets or tools for therapy, ncRNAs have been extensively detected in body fluids supporting their role as easily accessible and minimally invasive biomarkers. However, the precise measurement of circulating ncRNAs remains challenging due to their low abundance and the heterogeneity of the ncRNA population (size, polyadenylation status, circular forms). Microarrays constitute a very powerful method to analyze the expression level and the splicing pattern of circulating ncRNAs since they preserve sample integrity (no need to remove globin or rRNA) and allow precise quantification of low-abundance transcripts (no limitation by read depth). This chapter describes the protocols used in our lab to extract and purify total RNAs from PAXgene RNA Blood Tubes and to perform RNA labeling and hybridization on the Clariom™ D microarrays from Affymetrix.

Substructure Analyzer: A User-Friendly Workflow for Rapid Exploration and Accurate Analysis of Cellular Bodies in Fluorescence Microscopy Images.

The last decade has been characterized by breakthroughs in fluorescence microscopy techniques illustrated by spatial resolution improvement but also in live-cell imaging and high-throughput microscopy techniques. This led to a constant increase in the amount and complexity of the microscopy data for a single experiment. Because manual analysis of microscopy data is very time consuming, subjective, and prohibits quantitative analyses, automation of bioimage analysis is becoming almost unavoidable. We built an informatics workflow called Substructure Analyzer to fully automate signal analysis in bioimages from fluorescent microscopy. This workflow is developed on the user-friendly open-source platform Icy and is completed by functionalities from ImageJ. It includes the pre-processing of images to improve the signal to noise ratio, the individual segmentation of cells (detection of cell boundaries) and the detection/quantification of cell bodies enriched in specific cell compartments. The main advantage of this workflow is to propose complex bio-imaging functionalities to users without image analysis expertise through a user-friendly interface. Moreover, it is highly modular and adapted to several issues from the characterization of nuclear/cytoplasmic translocation to the comparative analysis of different cell bodies in different cellular sub-structures. The functionality of this workflow is illustrated through the study of the Cajal (coiled) Bodies under oxidative stress (OS) conditions. Data from fluorescence microscopy show that their integrity in human cells is impacted a few hours after the induction of OS. This effect is characterized by a decrease of coilin nucleation into characteristic Cajal Bodies, associated with a nucleoplasmic redistribution of coilin into an increased number of smaller foci. The central role of coilin in the exchange between CB components and the surrounding nucleoplasm suggests that OS induced redistribution of coilin could affect the composition and the functionality of Cajal Bodies.

Radiomics Method for the Differential Diagnosis of Radionecrosis Versus Progression after Fractionated Stereotactic Body Radiotherapy for Brain Oligometastasis.

Stereotactic radiotherapy (SRT) is recommended for treatment of brain oligometastasis (BoM) in patients with controlled primary disease. Where contrast enhancement enlargement occurs during follow-up, distinguishing between radionecrosis and progression presents a critical challenge. Without pathological confirmation, decision-making may be inappropriate and delayed. Quantitative imaging features extracted from routinely performed examinations are of interest in potentially addressing this problem. We explored the added value of the radiomics method for the differential diagnosis of these two entities. Twenty patients who received SRT for BoM, from any primary location, were included (8 radionecrosis, 12 progressions, pathologically confirmed). We assessed the clinical relevance of 1,766 radiomics features, extracted using IBEX software, from the first T1-weighted postcontrast magnetic resonance imaging (MRI) after SRT showing a lesion modification. We evaluated seven feature-selection methods and 12 classification methods in terms of respective predictive performance. The classification accuracy was measured using Cohen's kappa after leave-one-out cross-validation. In this work, the best predictive power reached was a Cohen's kappa of 0.68 (overall accuracy of 85%), expressing a strong agreement between the algorithm prediction and the histological gold standard. Prediction accuracy was 75% for radionecrosis, and 91% for progression. The area under a curve reached 0.83 using a bagging algorithm trained with the chi-square score features set. These findings indicated that the radiomics method is able to discriminate radionecrosis from progression in an accurate, early and noninvasive way. This promising study is a proof of concept, preceding a larger prospective study for defining a robust model to support decision-making in BoM. In summary, distinguishing between radionecrosis and progression is challenging without pathology. We built a classification model based on imaging data and machine learning. Using this model, we were able predict progression and radionecrosis in, respectively, 91% and 75% of cases.

Enhanced Probe-Based RT-qPCR Quantification of MicroRNAs Using Poly(A) Tailing and 5' Adaptor Ligation.

Probe-based quantitative PCR (qPCR) is a commonly used tool in the realm of real-time qPCR experiments since it is one of the most sensitive detection methods allowing an accurate and reproducible analysis. It uses real-time fluorescence from a fluorescently labeled probe that specifically targets the desired PCR product to measure DNA amplification at each cycle of the PCR. Coupled to a proper reverse transcription step, probe-based qPCR can be efficiently used for the analysis of the expression of difficult targets such as miRNAs. In this chapter, we describe the TaqMan® advanced miRNA assay in which, owing to a poly(A)-tailing step, the reverse transcription is advantageously performed at once for all the miRNAs in a given sample, and, coupled to the ligation of a 5' universal adapter, allows for a supplementary pre-qPCR amplification step increasing the sensitivity of the assay. Along this protocol, we also provide our general guidelines and advices to perform a reliable and successful quantitative analysis.

Contribution of protein Gar1 to the RNA-guided and RNA-independent rRNA:Ψ-synthase activities of the archaeal Cbf5 protein.

Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ2603 in Pyrococcus abyssi 23S rRNA and of Ψ55 in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ2607 (Ψ2603 in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.

Deep Structural Analysis of RPAP3 and PIH1D1, Two Components of the HSP90 Co-chaperone R2TP Complex.

RPAP3 and PIH1D1 are part of the HSP90 co-chaperone R2TP complex involved in the assembly process of many molecular machines. In this study, we performed a deep structural investigation of the HSP binding abilities of the two TPR domains of RPAP3. We combined 3D NMR, non-denaturing MS, and ITC techniques with Y2H, IP-LUMIER, FRET, and ATPase activity assays and explain the fundamental role played by the second TPR domain of RPAP3 in the specific recruitment of HSP90. We also established the 3D structure of an RPAP3:PIH1D1 sub-complex demonstrating the need for a 34-residue insertion, specific of RPAP3 isoform 1, for the tight binding of PIH1D1. We also confirm the existence of a complex lacking PIH1D1 in human cells (R2T), which shows differential binding to certain clients. These results highlight similarities and differences between the yeast and human R2TP complexes, and document the diversification of this family of co-chaperone complexes in human.

Specific G-quadruplex ligands modulate the alternative splicing of Bcl-X.

Sequences with the potential to form RNA G-quadruplexes (G4s) are common in mammalian introns, especially in the proximity of the 5' splice site (5'SS). However, the difficulty of demonstrating that G4s form in pre-mRNA in functional conditions has meant that little is known about their effects or mechanisms of action. We have shown previously that two G4s form in Bcl-X pre-mRNA, one close to each of the two alternative 5'SS. If these G4s affect splicing but are in competition with other RNA structures or RNA binding proteins, then ligands that stabilize them would increase the proportion of Bcl-X pre-mRNA molecules in which either or both G4s had formed, shifting Bcl-X splicing. We show here that a restricted set of G4 ligands do affect splicing, that their activity and specificity are strongly dependent on their structures and that they act independently at the two splice sites. One of the ligands, the ellipticine GQC-05, antagonizes the major 5'SS that expresses the anti-apoptotic isoform of Bcl-X and activates the alternative 5'SS that expresses a pro-apoptotic isoform. We propose mechanisms that would account for these see-saw effects and suggest that these effects contribute to the ability of GQC-05 to induce apoptosis.

MicroRNA-29b Contributes to Collagens Imbalance in Human Osteoarthritic and Dedifferentiated Articular Chondrocytes.

OBJECTIVE: Decreased expression of collagen type II in favour of collagen type I or X is one hallmark of chondrocyte phenotype changes in osteoarthritic (OA) cartilage. MicroRNA- (miR-) 29b was previously shown to target collagens in several tissues. We studied whether it could contribute to collagen imbalance in chondrocytes with an impaired phenotype. METHODS: After preliminary microarrays screening, miR-29b levels were measured by RT- quantitative PCR in in vitro models of chondrocyte phenotype changes (IL-1ß challenge or serial subculturing) and in chondrocytes from OA and non-OA patients. Potential miR-29b targets identified in silico in 3'-UTRs of collagens mRNAs were tested with luciferase reporter assays. The impact of premiR-29b overexpression in ATDC5 cells was studied on collagen mRNA levels and synthesis (Sirius red staining) during chondrogenesis. RESULTS: MiR-29b level increased significantly in IL-1ß-stimulated and weakly in subcultured chondrocytes. A 5.8-fold increase was observed in chondrocytes from OA versus non-OA patients. Reporter assays showed that miR-29b targeted COL2A1 and COL1A2 3'-UTRs although with a variable recovery upon mutation. In ATDC5 cells overexpressing premiR-29b, collagen production was reduced while mRNA levels increased. CONCLUSIONS: By acting probably as a posttranscriptional regulator with a different efficacy on COL2A1 and COL1A2 expression, miR-29b can contribute to the collagens imbalance associated with an abnormal chondrocyte phenotype.

Identification of G-quadruplexes in long functional RNAs using 7-deazaguanine RNA.

RNA G-quadruplex (G4) structures are thought to affect biological processes, including translation and pre-mRNA splicing, but it is not possible at present to demonstrate that they form naturally at specific sequences in long functional RNA molecules. We developed a new strategy, footprinting of long 7-deazaguanine-substituted RNAs (FOLDeR), that allows the formation of G4s to be confirmed in long RNAs and under functional conditions.

Enhanced SRSF5 Protein Expression Reinforces Lamin A mRNA Production in HeLa Cells and Fibroblasts of Progeria Patients.

The Hutchinson Gilford Progeria Syndrome (HGPS) is a rare genetic disease leading to accelerated aging. Three mutations of the LMNA gene leading to HGPS were identified. The more frequent ones, c.1824C>T and c.1822G>A, enhance the use of the intron 11 progerin 5'splice site (5'SS) instead of the LMNA 5'SS, leading to the production of the truncated dominant negative progerin. The less frequent c.1868C>G mutation creates a novel 5'SS (LAΔ35 5'SS), inducing the production of another truncated LMNA protein (LAΔ35). Our data show that the progerin 5'SS is used at low yield in the absence of HGPS mutation, whereas utilization of the LAΔ35 5'SS is dependent upon the presence of the c.1868C>G mutation. In the perspective to correct HGPS splicing defects, we investigated whether SR proteins can modify the relative yields of utilization of intron 11 5'SSs. By in cellulo and in vitro assays, we identified SRSF5 as a direct key regulator increasing the utilization of the LMNA 5'SS in the presence of the HGPS mutations. Enhanced SRSF5 expression in dermal fibroblasts of HGPS patients as well as PDGF-BB stimulation of these cells decreased the utilization of the progerin 5'SS, and improves nuclear morphology, opening new therapeutic perspectives for premature aging.

Fluorescence in situ hybridization of small non-coding RNAs.

RNA FISH is a powerful method to detect specific RNAs in fixed cells. It allows both localization and quantification of RNA molecules within individual cells and tissues. Refined RNA FISH methods have also been developed to determine RNA transcription and degradation rates. This chapter describes an RNA FISH protocol that we developed in order to study the expression and localization of satellite III RNAs. This specific class of non-coding RNAs is expressed in response to various cellular stresses including heat shock. This protocol is based on the use of a biotinylated LNA probe subsequently detected by a streptavidin-Alexa Fluor(®) 488 conjugate. A protocol allowing efficient coupling of RNA FISH and protein detection by immunofluorescence is also described in this chapter.

Probing small non-coding RNAs structures.

The diverse roles of RNAs depend on their ability to fold so as to form biologically functional structures. Thus, understanding the function of a given RNA molecule often requires experimental analysis of its secondary structure by in vitro RNA probing, which is more accurate than using prediction programs only. This chapter presents in vitro RNA probing protocols that we routinely use, from RNA transcript production and purification to RNA structure determination using enzymatic (RNases T1, T2, and V1) and chemical (DMS, CMCT, kethoxal, and Pb(2+)) probing performed on both unlabeled and end-labeled RNAs.

A conserved splicing mechanism of the LMNA gene controls premature aging.

Hutchinson-Gilford progeria syndrome (HGPS) is a rare genetic disorder phenotypically characterized by many features of premature aging. Most cases of HGPS are due to a heterozygous silent mutation (c.1824C>T; p.Gly608Gly) that enhances the use of an internal 5' splice site (5'SS) in exon 11 of the LMNA pre-mRNA and leads to the production of a truncated protein (progerin) with a dominant negative effect. Here we show that HGPS mutation changes the accessibility of the 5'SS of LMNA exon 11 which is sequestered in a conserved RNA structure. Our results also reveal a regulatory role of a subset of serine-arginine (SR)-rich proteins, including serine-arginine rich splicing factor 1 (SRSF1) and SRSF6, on utilization of the 5'SS leading to lamin A or progerin production and a modulation of this regulation in the presence of the c.1824C>T mutation is shown directly on HGPS patient cells. Mutant mice carrying the equivalent mutation in the LMNA gene (c.1827C>T) also accumulate progerin and phenocopy the main cellular alterations and clinical defects of HGPS patients. RNAi-induced depletion of SRSF1 in the HGPS-like mouse embryonic fibroblasts (MEFs) allowed progerin reduction and dysmorphic nuclei phenotype correction, whereas SRSF6 depletion aggravated the HGPS-like MEF's phenotype. We demonstrate that changes in the splicing ratio between lamin A and progerin are key factors for lifespan since heterozygous mice harboring the mutation lived longer than homozygous littermates but less than the wild-type. Genetic and biochemical data together favor the view that physiological progerin production is under tight control of a conserved splicing mechanism to avoid precocious aging.