Optimization of an HPLC-MS/MS method to analyze chlordecone in bovine serum and correlations with levels in liver, muscle and fat.

Chlordecone is an organochlorine pesticide used from 1972 to 1993 in the French West Indies. Its extensive use and high persistence in soils induced massive contamination of the environment and of the food chain, especially in cattle through contaminated soil ingestion. To ensure suitability for consumption of bovine meat, monitoring plans are set up based on perirenal fat concentrations after slaughtering. In the present study, we have investigated an in-vivo monitoring approach by measuring chlordecone levels in serum samples. For this purpose, a sensitive high-performance liquid-chromatography-tandem mass spectrometry (HPLC-MS/MS) method following a QuEChERS extraction method was successfully optimized and validated, reaching a limit of quantification of 0.05 ng g-1 fresh weight. This method was applied to 121 serum samples collected from bovines originating from contaminated areas of Martinique and Guadeloupe. Chlordecone was detected in 88% of the samples, and quantified in 77% of the samples, with concentrations ranging from 0.05 to 22 ng g-1. Perirenal fat, liver, and muscle were also sampled on the same animals and the measured concentrations of chlordecone were statistically correlated to the levels determined in serum. Mean concentration ratios of 6.5 for fat/serum, 27.5 for liver/serum, and 3.3 for muscle/serum were calculated, meaning that chlordecone was not only distribute in fat (as expected), muscle and liver, but also in serum. Good correlations were found to allow prediction of chlordecone concentrations in muscle based on concentrations measured in serum. This study opens the door to possible pre-control of bovines before slaughter. In cases of probable non-compliance with maximum residue levels (MRLs), farm management could proceed to allow for depuration under controlled conditions. This would have a strong impact on both economic and food safety management measures.

The Ultraviolet Irradiation of Keratinocytes Induces Ectopic Expression of LINE-1 Retrotransposon Machinery and Leads to Cellular Senescence.

Retrotransposons have played an important role in evolution through their transposable activity. The largest and the only currently active human group of mobile DNAs are the LINE-1 retrotransposons. The ectopic expression of LINE-1 has been correlated with genomic instability. Narrow-band ultraviolet B (NB-UVB) and broad-band ultraviolet B (BB-UVB) phototherapy is commonly used for the treatment of dermatological diseases. UVB exposure is carcinogenic and can lead, in keratinocytes, to genomic instability. We hypothesize that LINE-1 reactivation occurs at a high rate in response to UVB exposure on the skin, which significantly contributes to genomic instability and DNA damage leading to cellular senescence and photoaging. Immortalized N/TERT1 and HaCaT human keratinocyte cell lines were irradiated in vitro with either NB-UVB or BB-UVB. Using immunofluorescence and Western blotting, we confirmed UVB-induced protein expression of LINE-1. Using RT-qPCR, we measured the mRNA expression of LINE-1 and senescence markers that were upregulated after several NB-UVB exposures. Selected miRNAs that are known to bind LINE-1 mRNA were measured using RT-qPCR, and the expression of miR-16 was downregulated with UVB exposure. Our findings demonstrate that UVB irradiation induces LINE-1 reactivation and DNA damage in normal keratinocytes along with the associated upregulation of cellular senescence markers and change in miR-16 expression.

Ectopically Expressed Meiosis-Specific Cancer Testis Antigen HORMAD1 Promotes Genomic Instability in Squamous Cell Carcinomas.

Genomic instability is a prominent hallmark of cancer, however the mechanisms that drive and sustain this process remain elusive. Research demonstrates that numerous cancers with increased levels of genomic instability ectopically express meiosis-specific genes and undergo meiomitosis, the clash of mitotic and meiotic processes. These meiotic genes may represent novel therapeutic targets for the treatment of cancer. We studied the relationship between the expression of the meiosis protein HORMAD1 and genomic instability in squamous cell carcinomas (SCCs). First, we assessed markers of DNA damage and genomic instability following knockdown and overexpression of HORMAD1 in different cell lines representing SCCs and epithelial cancers. shRNA-mediated depletion of HORMAD1 expression resulted in increased genomic instability, DNA damage, increased sensitivity to etoposide, and decreased expression of DNA damage response/repair genes. Conversely, overexpression of HORMAD1 exhibited protective effects leading to decreased DNA damage, enhanced survival and decreased sensitivity to etoposide. Furthermore, we identified a meiotic molecular pathway that regulates HORMAD1 expression by targeting the upstream meiosis transcription factor STRA8. Our results highlight a specific relationship between HORMAD1 and genomic instability in SCCs, suggesting that selectively inhibiting HORMAD1, possibly, through STRA8 signaling, may provide a new paradigm of treatment options for HORMAD1-expressing SCCs.

A tRNA-derived fragment present in E. coli OMVs regulates host cell gene expression and proliferation.

RNA-sequencing has led to a spectacular increase in the repertoire of bacterial sRNAs and improved our understanding of their biological functions. Bacterial sRNAs have also been found in outer membrane vesicles (OMVs), raising questions about their potential involvement in bacteria-host relationship, but few studies have documented this issue. Recent RNA-Sequencing analyses of bacterial RNA unveiled the existence of abundant very small RNAs (vsRNAs) shorter than 16 nt. These especially include tRNA fragments (tRFs) that are selectively loaded in OMVs and are predicted to target host mRNAs. Here, in Escherichia coli (E. coli), we report the existence of an abundant vsRNA, Ile-tRF-5X, which is selectively modulated by environmental stress, while remaining unaffected by inhibition of transcription or translation. Ile-tRF-5X is released through OMVs and can be transferred to human HCT116 cells, where it promoted MAP3K4 expression. Our findings provide a novel perspective and paradigm on the existing symbiosis between bacteria and human cells.

The Contributions of Cancer-Testis and Developmental Genes to the Pathogenesis of Keratinocyte Carcinomas.

Keratinocyte carcinomas are among the most prevalent malignancies worldwide. Basal cell carcinoma (BCC) and cutaneous squamous cell carcinoma (cSCC) are the two cancers recognized as keratinocyte carcinomas. The standard of care for treating these cancers includes surgery and ablative therapies. However, in recent years, targeted therapies (e.g., cetuximab for cSCC and vismodegib/sonidegib for BCC) have been used to treat advanced disease as well as immunotherapy (e.g., cemiplimab). These treatments are expensive and have significant toxicities with objective response rates approaching ~50-65%. Hence, there is a need to dissect the molecular pathogenesis of these cancers to identify novel biomarkers and therapeutic targets to improve disease management. Several cancer-testis antigens (CTA) and developmental genes (including embryonic stem cell factors and fetal genes) are ectopically expressed in BCC and cSCC. When ectopically expressed in malignant tissues, functions of these genes may be recaptured to promote tumorigenesis. CTAs and developmental genes are emerging as important players in the pathogenesis of BCC and cSCC, positioning themselves as attractive candidate biomarkers and therapeutic targets requiring rigorous testing. Herein, we review the current research and offer perspectives on the contributions of CTAs and developmental genes to the pathogenesis of keratinocyte carcinomas.

An Expanded Landscape of Unusually Short RNAs in 11 Samples from Six Eukaryotic Organisms.

Small RNA sequencing (sRNA-Seq) approaches unveiled sequences derived from longer non-coding RNAs, such as transfer RNA (tRNA) and ribosomal RNA (rRNA) fragments, known as tRFs and rRFs, respectively. However, rRNAs and RNAs shorter than 16 nt are often depleted from library preparations/sequencing analyses, although they may be functional. Here, we sought to obtain a complete repertoire of small RNAs by sequencing the total RNA from 11 samples of 6 different eukaryotic organisms, from yeasts to human, in an extended 8- to 30-nt window of RNA length. The 8- to 15-nt window essentially contained fragments of longer non-coding RNAs, such as microRNAs, PIWI-associated RNAs (piRNAs), small nucleolar RNAs (snoRNAs), tRNAs and rRNAs. Notably, unusually short RNAs < 16 nt were more abundant than those >16 nt in bilaterian organisms. A new RT-qPCR method confirmed that two unusually short rRFs of 12 and 13 nt were more overly abundant (~3-log difference) than two microRNAs. We propose to not deplete rRNA and to reduce the lower threshold of RNA length to include unusually short RNAs in sRNA-Seq analyses and datasets, as their abundance and diversity support their potential role and importance as biomarkers of disease and/or mediators of cellular function.

Elucidating the Color of Rosé Wines Using Polyphenol-Targeted Metabolomics.

The color of rosé wines is extremely diverse and a key element in their marketing. It is due to the presence of anthocyanins and of additional pigments derived from them and from other wine constituents. To explore the pigment composition and determine its links with color, 268 commercial rosé wines were analysed. The concentration of 125 polyphenolic compounds was determined by a targeted metabolomics approach using ultra high-performance liquid chromatography coupled to triple quadrupole mass spectrometry (UHPLC-QqQ-MS) analysis in the Multiple Reaction Monitoring (MRM) mode and the color characterised by spectrophotometry and CieLab parameters. Chemometrics analysis of the composition and color data showed that although color intensity is primarily determined by polyphenol extraction (especially anthocyanins and flavanols) from the grapes, different color styles correspond to different pigment compositions. The salmon shade of light rosé wines is mostly due to pyranoanthocyanin pigments, resulting from reactions of anthocyanins with phenolic acids and pyruvic acid, a yeast metabolite. Redness of intermediate color wines is related to anthocyanins and carboxypoyranoanthocyanins and that of dark rosé wines to products of anthocyanin reactions with flavanols while yellowness of these wines is associated to oxidation.

Identification of Abundant and Functional dodecaRNAs (doRNAs) Derived from Ribosomal RNA.

Using a modified RNA-sequencing (RNA-seq) approach, we discovered a new family of unusually short RNAs mapping to ribosomal RNA 5.8S, which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. Using a new quantitative detection method that we developed, we confirmed our RNA-seq data and determined that the minimal core doRNA sequence and its 13-nt variant C-doRNA (doRNA with a 5' Cytosine) are the two most abundant doRNAs, which, together, may outnumber microRNAs. The C-doRNA/doRNA ratio is stable within species but differed between species. doRNA and C-doRNA are mainly cytoplasmic and interact with heterogeneous nuclear ribonucleoproteins (hnRNP) A0, A1 and A2B1, but not Argonaute 2. Reporter gene activity assays suggest that C-doRNA may function as a regulator of Annexin II receptor (AXIIR) expression. doRNAs are differentially expressed in prostate cancer cells/tissues and may control cell migration. These findings suggest that unusually short RNAs may be more abundant and important than previously thought.

A New Specific and Sensitive RT-qPCR Method Based on Splinted 5' Ligation for the Quantitative Detection of RNA Species Shorter than microRNAs.

Recently, we discovered a new family of unusually short RNAs mapping to 5.8S ribosomal RNA (rRNA) and which we named dodecaRNAs (doRNAs), according to the number of core nucleotides (12 nt) their members contain. To confirm these small RNA-sequencing (RNA-Seq) data, validate the existence of the two overly abundant doRNAs-the minimal core 12-nt doRNA sequence and its + 1-nt variant bearing a 5' Cytosine, C-doRNA-and streamline their analysis, we developed a new specific and sensitive splinted 5' ligation reverse transcription (RT)-quantitative polymerase chain reaction (qPCR) method. This method is based on a splint-assisted ligation of an adapter to the 5' end of doRNAs, followed by RT-qPCR amplification and quantitation. Our optimized protocol, which may discriminate between doRNA, C-doRNA, mutated and precursor sequences, can accurately detect as low as 240 copies and is quantitatively linear over a range of 7 logs. This method provides a unique tool to expand and facilitate studies exploring the molecular and cellular biology of RNA species shorter than microRNAs.

Correlation between endemic chlordecone concentrations in three bovine tissues determined by isotopic dilution liquid chromatography-tandem mass spectrometry.

Chlordecone (CLD) is an organochlorine pesticide widely used from the 1970s to the 1990s in the French West Indies that induced long-term pollution of the ecosystem. Due to involuntary soil ingestion, some species bred in open-air areas can be contaminated. As CLD is distributed in various tissues depending on the breeding species, this study focuses on the distribution of CLD in bovines. For this purpose, three tissues, i.e. fat, muscle, and liver, from 200 bovines originating from Martinique and Guadeloupe were sampled in 2016 to determine their endemic contamination levels. Analyses were performed with the official method for veterinary controls, isotopic dilution liquid chromatography-tandem mass spectrometry, which has been fully validated and which reaches a limit of quantification of 3 µg.kg-1 fresh weight (fw). Irrespective of the matrices, CLD was detected in 68% of samples (404 samples above the LOD) and quantified in 59% of samples (332 samples above the LOQ). Regarding contamination levels, the liver had a broader range of concentrations (LOQ up to 420.6 µg.kg-1 fw) than fat (LOQ up to 124.6 µg.kg-1 fw) and muscle (LOQ up to 67.6 µg.kg-1 fw). This confirms the atypical behaviour of CLD compared to other persistent organochlorine pollutants. Statistical processing demonstrated a correlation between CLD concentrations among the three studied tissues. The CLD concentration ratios were 0.54 for muscle/fat, 3.75 for liver/fat, and 0.14 for muscle/liver.

Altered microRNA Transcriptome in Cultured Human Liver Cells upon Infection with Ebola Virus.

Ebola virus (EBOV) is a virulent pathogen, notorious for inducing life-threatening hemorrhagic fever, that has been responsible for several outbreaks in Africa and remains a public health threat. Yet, its pathogenesis is still not completely understood. Although there have been numerous studies on host transcriptional response to EBOV, with an emphasis on the clinical features, the impact of EBOV infection on post-transcriptional regulatory elements, such as microRNAs (miRNAs), remains largely unexplored. MiRNAs are involved in inflammation and immunity and are believed to be important modulators of the host response to viral infection. Here, we have used small RNA sequencing (sRNA-Seq), qPCR and functional analyses to obtain the first comparative miRNA transcriptome (miRNome) of a human liver cell line (Huh7) infected with one of the following three EBOV strains: Mayinga (responsible for the first Zaire outbreak in 1976), Makona (responsible for the West Africa outbreak in 2013-2016) and the epizootic Reston (presumably innocuous to humans). Our results highlight specific miRNA-based immunity pathways and substantial differences between the strains beyond their clinical manifestation and pathogenicity. These analyses shed new light into the molecular signature of liver cells upon EBOV infection and reveal new insights into miRNA-based virus attack and host defense strategy.

Complexity of the microRNA transcriptome of cow milk and milk-derived extracellular vesicles isolated via differential ultracentrifugation.

MicroRNAs (miRNAs) are small gene-regulatory noncoding RNA that are highly enriched in cow milk. They are encapsulated in different extracellular vesicle (EV) subsets that protect them from the extracellular milieu and the harsh conditions of the gastrointestinal tract during digestion. Here, we isolated pellets enriched in 4 different EV subsets, via differential ultracentrifugation of commercial cow milk: 12,000 × g (P12K), 35,000 × g (P35K), 70,000 × g (P70K), and 100,000 × g (P100K). Small RNA sequencing (sRNA-Seq) analyses revealed an unprecedented level of diversity in the complete miRNA repertoire and features of unfractionated cow milk and derived EV subsets. Although 5 miRNA sequences represented more than 50% of all miRNAs, milk EV exhibited heterogeneous content of miRNAs and isomeric variants (termed isomiR): P100K EV were enriched in reference miRNA sequences, and P12K and P35K EV in related isomiR. Incubation of milk EV with human cultured HeLa cells led to cellular enrichment in miRNA miR-223, which was concomitant with decreased expression of a reporter gene placed under the control of miR-223, thereby demonstrating the functionality of miR-223. These results suggest that cow milk EV may transfer their miRNAs to human cells and regulate recipient cell gene expression programming in a manner as complex as that of their miRNA transcriptome. The biological activity and relevance of the different milk EV subsets and bioactive mediators, including small noncoding RNA, in health and disease, warrants further investigation.

French infant total diet study: Dietary exposure to heat-induced compounds (acrylamide, furan and polycyclic aromatic hydrocarbons) and associated health risks.

A total diet study (TDS) was conducted between 2010 and 2016 to assess the risk associated with chemicals in food of non-breast-fed children from 1 to 36 months living in France. Food samples were collected, prepared "as consumed", and analyzed for chemicals of public health interest. Acrylamide, furan and polycyclic aromatic hydrocarbons (PAHs) were analyzed as heat-induced compounds produced mainly during thermal processing of foods. Dietary exposure was assessed for 705 representative children using food consumptions recorded through a 3-consecutive-days record. As all calculated margins of exposure (MOE) for PAHs exceeded 10 000, dietary exposure of the infant and toddler population was deemed tolerable with regard to the carcinogenic risk. Conversely, the exposure levels to acrylamide and furan were considered as of concern, requiring management measures to reduce the exposure essentially by reducing the formation of heat-induced compounds during food production or preparation processes. Efforts should mainly focus on major contributors to the exposure, i.e. sweet and savoury biscuits and bars, and potatoes and potato products for acrylamide, baby jars of vegetables, with or without meat or fish for acrylamide and furan.

Small Non-Coding RNAs Derived From Eukaryotic Ribosomal RNA.

The advent of RNA-sequencing (RNA-Seq) technologies has markedly improved our knowledge and expanded the compendium of small non-coding RNAs, most of which derive from the processing of longer RNA precursors. In this review article, we will present a nonexhaustive list of referenced small non-coding RNAs (ncRNAs) derived from eukaryotic ribosomal RNA (rRNA), called rRNA fragments (rRFs). We will focus on the rRFs that are experimentally verified, and discuss their origin, length, structure, biogenesis, association with known regulatory proteins, and potential role(s) as regulator of gene expression. This relatively new class of ncRNAs remained poorly investigated and underappreciated until recently, due mainly to the a priori exclusion of rRNA sequences-because of their overabundance-from RNA-Seq datasets. The situation surrounding rRFs resembles that of microRNAs (miRNAs), which used to be readily discarded from further analyses, for more than five decades, because no one could believe that RNA of such a short length could bear biological significance. As if we had not yet learned our lesson not to restrain our investigative, scientific mind from challenging widely accepted beliefs or dogmas, and from looking for the hidden treasures in the most unexpected places.

Levels of furan in foods from the first French Total Diet Study on infants and toddlers.

This study describes an optimisation and validation process on a method using gas chromatography coupled with mass spectrometry to quantify furan in foods consumed mainly by infants and toddlers. The method that we developed allowed for low limits of quantification for liquid (1 µg kg-1) and solid (2 µg kg-1) samples. Our method was then applied to 134 food samples from the first French Total Diet Study on infants and toddlers. Furan was detected in 84% and quantified in 61% of the samples, at average lower and upper bound (LB/UB) concentrations ranging from 0 to 44 µg kg-1. The sugar and sugar derivatives, milk, growth milk, infant formulae and "other hot beverages categories contained the lowest average content (LB/UB ≤ 1 µg kg-1) and breakfast cereals contained the highest (LB/UB = 44 µg kg-1).

Levels of acrylamide in foods included in 'the first French total diet study on infants and toddlers'.

This study describes an optimisation and validation process using liquid chromatography coupled with tandem mass spectrometry to quantify acrylamide in foods mainly consumed by infants and toddlers. A limit of quantification of 5µg.kg-1 for both solid and liquid samples was achieved, except for unprepared infant cereals (LOQ of 18µg.kg-1). The method was then applied to 141 food samples from the first French total diet study on infants and toddlers. Acrylamide was detected in most samples at mean LB/UB concentrations ranging from 0.14 to 102µg.kg-1. The "Follow-on formula" and "Infant formula" products contained the lowest average content (LB/UB of 0.14/2.2µg.kg-1 and 0.60/2.9µg.kg-1 respectively) and the "Sweet and savoury biscuits and bars" (102µg.kg-1; n=1 represented by a plain dry biscuit) contained the highest.

TWEAK Negatively Regulates Human Dicer.

The ribonuclease Dicer plays a central role in the microRNA pathway by processing microRNA precursors (pre-microRNAs) into microRNAs, a class of 19- to 24-nucleotide non-coding RNAs that regulate expression of ≈60% of the genes in humans. To gain further insights into the function and regulation of Dicer in human cells, we performed a yeast two-hybrid (Y2HB) screen using human Dicer double-stranded RNA-binding domain (dsRBD) as bait. This approach identified tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) as a Dicer-interacting protein candidate. Confocal immunofluorescence microscopy revealed the colocalization of Dicer and TWEAK proteins at the perinuclear region of HeLa cells. The Dicer-TWEAK protein interaction was confirmed by coimmunoprecipitation and found not likely to be mediated by RNA. TWEAK dose-dependently reduced pre-microRNA conversion into mature microRNA in Dicer activity assays using extracts of transfected human HEK 293 cells. TWEAK expression also impaired microRNA-guided RNA silencing of a reporter gene induced by a pre-microRNA. These findings suggest a role for TWEAK-a pro-inflammatory cytokine-in regulating Dicer function and microRNA biogenesis, and its possible involvement in regulating gene expression during inflammatory processes and diseases.

A High-Throughput UHPLC-QqQ-MS Method for Polyphenol Profiling in Rosé Wines.

A rapid, sensitive and selective analysis method using Ultra High Performance Liquid Chromatography coupled to triple-quadrupole Mass Spectrometry (UHPLC-QqQ-MS) has been developed for the quantification of polyphenols in rosé wines. The compound detection being based on specific MS transitions in Multiple Reaction Monitoring (MRM) mode, the present method allows the selective quantification of up to 152 phenolic and two additional non-phenolic wine compounds in 30 min without sample purification or pre-concentration, even at low concentration levels. This method was repeatably applied to a set of 12 rosé wines and thus proved to be suitable for high-throughput and large-scale metabolomics studies.

Overexpression of active Aurora-C kinase results in cell transformation and tumour formation.

Aurora kinases belong to a conserved family of serine/threonine kinases key regulators of cell cycle progression. Aurora-A and Aurora-B are expressed in somatic cells and involved mainly in mitosis while Aurora-C is expressed during spermatogenesis and oogenesis and is involved in meiosis. Aurora-C is hardly detectable in normal somatic cells. However all three kinases are overexpressed in many cancer lines. Aurora-A possesses an oncogenic activity while Aurora-B does not. Here we investigated whether Aurora-C possesses such an oncogenic activity. We report that overexpression of Aurora-C induces abnormal cell division resulting in centrosome amplification and multinucleation in both transiently transfected cells and in stable cell lines. Only stable NIH3T3 cell clones overexpressing active Aurora-C formed foci of colonies when grown on soft agar, indicating that a gain of Aurora-C activity is sufficient to transform cells. Furthermore, we reported that NIH-3T3 stable cell lines overexpressing Aurora-C induced tumour formation when injected into nude mice, demonstrating the oncogenic activity of enzymatically active Aurora kinase C. Interestingly enough tumor aggressiveness was positively correlated with the quantity of active kinase, making Aurora-C a potential anti-cancer therapeutic target.