Dmrt1, Amh, Sox9, Tex14, Trim71, Slc26a8, Spam1, Tdrp, Tsga10, Boc, Cxcl14, and Hsd17b3) were identified to be specifically related to duck testicular development, and 11 genes  were specifically related to duck ovarian development. Seven genes  were screened to be specifically involved in the female sterility of Mule ducks; eight genes  were specifically involved in male sterility; and five genes  were involved in both female and male sterility. This study provides an insight into the differential development between male and female gonads of ducks and the sterility mechanism of Mule ducks through function, pathway, and protein interaction analyses. Our findings provide theoretical basis for the further research on sex determination and differentiation of birds and the sterility of Mule ducks.The key genes of avian gonadal development are of great significance for sex determination. Transcriptome sequencing analysis of Mule duck gonad as potential sterile model is expected to screen candidate genes related to avian gonad development. In this study, the embryonic gonadal tissues of Mule ducks, Jinding ducks, and Muscovy ducks were collected and identified. Six sample groups including female Mule duck (A), male Mule duck (B), female Jinding duck (C), male Jinding duck (D), female Muscovy duck (E), and male Muscovy duck (F) were subjected to RNA sequencing analysis. A total of 9,471 differential genes (DEGs) and 691 protein-protein interaction pairs were obtained. Totally, 12 genes ( Sox9) is marker gene of testicular development, and cytochrome P450 family 19 subfamily A member 1(Cyp19a1) is ovarian developmental marker genes. In mammals, the expression of Sox9 is activated by Sry, whereas the expression of Sox9 must be initiated by another male-specific gene (probably a Z-linked gene) due to the lack of Sry in birds. Aromatase encoded by Cyp19a1 is a key enzyme in the estrogen synthesis pathway (Amh), Steroidogenic factor-1 (Sf1), Wilm’s tumor1 gene (Wt1), and Gata binding protein 4 (Gata4) in birds were different from those in other species  and their chromosome structures are highly conserved .PCR amplification was performed with gonadal tissue DNA as a template, and gene-specific PCR primers were designed according to the s female . PCR wass female , 10 μM, The 500 eggs of Jinding ducks (domestic ducks), Muscovy ducks and their hybridization offspring Mule ducks were collected, respectively. When eggs were hatched to the 1/2 embryonic stage, the gonadal tissues were collected to extract DNA for sex identification. After sex identification, 10–20 paired gonads were combined into one sample. Six groups were respectively marked as A, female Mule ducks; B, male Mule ducks; C, female Jinding ducks; D, male Jinding ducks; E, female Muscovy ducks; F, male Muscovy ducks and a total of 18 samples were constructed with three biological replicates per group. The sample collection was approved by the Ethics Committee of Wuhan Academy of Agricultural Science.Total RNA was extracted from all tissues using TRIzol Reagent  according to the manufacturer’s instructions. The integrity of the obtained RNA was evaluated using an Agilent Bioanalyzer 2100 system .After quality evaluation of total RNA, high-quality total RNA samples were purified to obtain mRNA ending with Poly-A using the oligo (dT) magnetic beads, followed by fragmentation (about 300 bp) and cDNA synthesis with RNA as template. The RNA library was prepared using a total amount of 1 μg RNA per sample. The sequencing library was generated using NEB Next UltraTM RNA Library prep Kit for Illumina  according to the instruction manual. A total of 18 cDNA libraries were constructed and paired-end sequenced using Illumina high-throughput sequencing platforms.Anas platyrhynchos reference genome (ftp://ftp.ensembl.org/pub/release100/fasta/anas_platyrhynch-os/dna/) using HISAT2 software (http://ccb.jhu.edu/software/hisat2/index.shtml). The Read Count value aligned to each gene using HTSeq software was used as the original expression level of the gene. Expression levels were normalized using the FPKM method  prediction of DEGs was based on the retrieved associations from STRING  following the manufacturer’s recommended procedures, and the resultant cDNAs were stored at -20°C. The quantitative real-time PCR (qRT-PCR) was conducted on an CFX-96/384  in a 10 μl system containing 5 μl of 2 × SYBR Green qPCR Master Mix , 0.2 μl of each forward and reverse primer  (10 μM),t method .http://www.ncbi.nlm.nih.gov/sra/) under accession number PRJNA874907.A total of 18 sequencing libraries of six sample groups were constructed  with three biological replicates per group. A total of 798216036 clean reads were obtained after paired-end sequencing on the Illumina sequencing platform, and the reference genome alignment results showed that the unique alignment rate was above 87% . The rawIn order to evaluate the robustness of each group, we performed Spearman’s correlation analyses. The results showed that the average correlation coefficient of samples within the same group was above 0.9, and the average correlation coefficient between the Muscovy duck group and the other two groups was around 0.7 . In addip-value < 0.05 and absolute value of log2 FoldChange >1 , the hierarchical clustering analysis was performed with the threshold of hange >1 . A totalhange >1 . The volHsd17b3 and Amh) and one down-regulated DEG Cyp17a1 were significantly enriched in sex differentiation. The GO enrichment analysis results also showed that three up-regulated DEGs  and three down-regulated DEGs  were significantly enriched in positive regulation of cell differentiation or developmental process. In C vs. D, one up-regulated DEG (Spam1) and four down-regulated DEGs  were significantly enriched in developmental process and reproductive process. Additionally, there were four shared DEGs  among A vs. B, C vs. D, and E vs. F were significantly enriched in estrogen biosynthetic process, calcium ion transport, sex differentiation, and multicellular organism development.GO enrichment analysis of DEGs in intra-breed comparison  was performed. The top 10 shared GO terms in which DEGs were enriched mainly included ion transport or multicellular organismal process-related biological processes (BP), plasma membrane or extracellular space-related cellular component (CC), and ion channel activation related molecular function (MF) . In A vsTsga10, Stra8, Terb1, Spam1, Tex14 were up-regulated, and Plcd4, Bmp15 were down-regulated. The down-regulated DEG Plcd4 was also enriched in such biological processes as sexual reproduction and gamete generation. In A vs. C, four up-regulated DEGs  were significantly enriched in meiotic cell cycle. In inter-breed male comparison of B vs. D, three down-regulated DEGs  were enriched in biological processes of male gamete generation and spermatogenesis, and in B vs. F, five up-regulated DEGs  and two down-regulated DEGs (Plcd4 and Hsd17b3) were enriched in reproductive process  were involved in aromatic amino acid family catabolic process in B vs. F. It should be noticed that up-regulated DEG Aldh4a1 was enriched in proline catabolic process.In addition, in inter-breed female comparison (A vs. E and A vs. C), the top 10 shared GO terms in which DEGs were enriched mainly included immune response-or reproduction-related BP, telomere cap complex- or axonemal dynein complex-related CC, as well as ion channel activation- or receptor activation-related MF . In A vs process . BesidesKEGG pathway analysis of each comparison group was performed. The results showed that the DEGs in intra-breed comparison were enriched in such signaling pathways as Calcium signaling pathway, Steroid hormone biosynthesis, and TGF-beta signaling pathway. Specifically, the DEGs in A vs. B, C vs. D, and E vs. F were also significantly enriched in GnRH signaling pathway, MAPK signaling pathway, Drug metabolism-cytochrome P450, respectively . For intp-value = 7.7 E-5),and the interaction score of NOG-BMP7 was 0.989. In addition, 3 DEGs  in the subnetwork in A vs. E were annotated to the redox biological processes and lipid metabolism signaling pathway  network. The results showed that a total of 691 gene pairs were searched including 122 in A vs. E, 65 in A vs. C, 176 in B vs. F, 34 in B vs. D, 164 in A vs. B, 119 in C vs. D, and 11 in E vs. F. The PPI network contained 1893 DEGs and 164 (Score>0.95) interaction pairs in A vs. B . Five ge pathway . The res pathway . The PPIDmrt1, Amh, Sox9, Tex14, Trim71, Slc26a8, Spam1, Tdrp, Tsga10, Boc, Cxcl14, Hsd17b3, Hsd17b1, Cyp19a1, Cyp17a1, Hhipl2, Uts2r, Cdon, Axin2, Nxph1, Brinp2, and Brinp3 were identified as sex-related genes in Mule ducks, Jinding ducks, and Muscovy ducks. Most of these 22 sex-related genes were annotated to sexual differentiation- and reproduction-related biological processes, of which, seven genes  were strongly expressed in male gonads of at least two duck breeds and simultaneously lowly expressed in female gonads of all three duck breeds. The 11 genes  were highly expressed in female gonads of at least two duck breeds and meanwhile lowly expressed in male gonads of all three breeds. In addition, seven genes including Stra8, Dmc1, Terb1, Tex14, Tsga10, Spam1, and Plcd4 were involved in meiotic cell cycle, reproductive process, and gamete generation, and they were identified as the genes causing female sterility in Mule ducks. The expression of three genes  in female gonads were lower in Mule ducks than in other two breeds. Thirteen genes including Gtsf1, Nalcn, Tat, Slc26a8, Kmo, Plcd4, Aldh4a1, Hgd, Tsga10, Hsd17b3, Stra8, Terb1, and Tex14 involved in male gamete generation, spermatogenesis, sperm motility, proline catabolic process, and reproductive process were identified as the genes causing male sterility in Mule ducks. Among them, the expression of three genes  in male gonads was lower in Mule ducks than in other two breeds. and the expression of five genes  in male gonads of Mule ducks was lower than that of Muscovy ducks but higher than that of Jinding ducks.A total of 34 key candidate genes were identified as gonadal development- or infertility-related genes according to our clustering analyses, functional enrichment analyses, and the published literature , of whicCyp17a1, Hhipl2, hsd17β1, Nxph1, ENSAPLG00020014497, and ENSAPLG0002-0010883, were more highly expressed in males than in females of both three breeds. ENSAPLG000200-14475 was more highly expressed in both females and males of Mule ducks than in those of Muscovy ducks. Particularly, the expression level of topors was higher in Mule duck females than males. The expression of Nxph1 was higher in the females of Mule ducks and Muscovy ducks than their males. Eleven DEGs identified by qRT-PCR coincided with the RNA-Seq results, indicating the reliability of RNA-Seq results.Eleven DEGs were randomly selected for validation by qRT-PCR. As shown in Dmrt1 has been reported to have important effect on gonadal development in multiple species   . Our datvy ducks . Moreovein birds , implyintor Sox9 , and Dmrntiation . Based oelopment .Tex14) is a novel protein localized to intercellular bridges of germ cells  is a nuclear factor, and in Tdrp-deletion mouse model, many sperm motility indices such as average path velocity (VAP), straight line velocity (VSL), and curvilinear velocity (VCL) are significantly lowered (p < 0.05)  , implyinHsd17b3 located on the Z chromosome of ducks was more highly expressed in males than in females in Mule ducks and Jinding ducks. Besides, Hsd17b3 was significantly enriched in sex differentiation GO term. In contrast, Hsd17b1 was more highly expressed in females than in males in all three breeds, which was consistent with the observation during the gonad developmental stage in flatfish (Cyp19a1 can convert androstenedione and testosterone into estrone (E1) and estradiol (E2), respectively. Hsd17b1 can efficiently catalyze the conversion of E1 with low biological activity into E2 with high biological activity , as an important member of the cytochrome P450 subfamily, exhibits both hydroxylase and lyase activities, and it is expressed in the Leydig cells and the granulosa cells of ovarian follicles  or testosterone (T) in XX Nile tilapia  exhibited high expression in females but low expression in males in all three duck breeds, of which Uts2r and Cdon were involved in multicellular organismal process. Nxph1, Brinp2, and Brinp3 were highly expressed in females but lowly expressed in males in Mule ducks and Muscovy ducks. of which Brinp2 and Brinp3 were involved in regulation of developmental process.The final step in androgen biosynthesis in the testis is the conversion of androstenedione (Δ4) into testosterone , and this step is catalyzed by 17β-HSD-3 . The incflatfish . Aromataactivity . Overexpale mice . In thisollicles . Mutatio tilapia . ConsistStra8, Dmc1, Terb1, and Tex14 were involved in meiotic cell cycle, of which 3 genes  were more highly expressed in Jinding duck females and Muscovy duck females than in Mule duck females. Stimulated by retinoic acid gene 8 (Stra8) is considered a key regulator of meiosis in mammals , a meiosis-specific gene, is crucial for assembling DNA double strand breaks (DSBs) site in homologous recombination and for searching DNA sequences located on homologous chromatids . In mammals, meiosis is initiated in different life stages for different sexes, and female germ cells enter meiosis shortly after sex determination during embryogenesis, while male germ cells do not enter meiosis until puberty . Our int mammals . In fema meiosis . Althougrm cells . DNA meiromatids . Dmc1-kner ovary . Follicuogenesis . In addius cycle . In thisGtsf1, Nalcn, Tat, and Slc26a8) were higher in male Mule ducks than in male Jinding ducks but lower than in male Muscovy ducks. The three genes  were lowly expressed in male Mule ducks whereas highly expressed in males of the other two breeds. These results suggested that the above-mentioned seven genes were related to duck male sterility. Gametocyte-specific factor 1 (Gtsf1) is evolutionarily highly conserved. Primary spermatocytes of Gtsf1−/− male mice stopped proliferation before the zygotene of the first meiotic prophase, thus causing massive germ cell apoptosis on postnatal day 14, eventually leading to male sterility (Plcd4) gene homozygote deleted fail to initiate acrosome reaction, thereby resulting in male sterility or small number per litter in mice  (Aldh4a1 expression is significantly decreased in oocytes of postpartum mouse (increased in sperm from patients with severe disruption of semen proteins (d-Aspartate (D-Asp) and N-methyl-d-aspartate (NMDA) promote spermatogenesis by stimulating the biosynthesis of sex steroid hormones  revealed that DEGs were significantly enriched in biological processes such as male gamete generation, spermatogenesis, proline catabolic process. Of these DEGs, the expression of four genes  , whose pum mouse . Prolineum mouse . Antioxium mouse . More stproteins , that d-hormones , that D-hormones . Additiohormones . Based ohormones .Tsga10, Terb1, Stra8, Tex14, and Spam1) were predicted to be associated with sterility in male and female Mule ducks. Our data showed that the expression level of these five genes was significantly lower in Mule ducks than that in Muscovy ducks for both males and females. In this study, Tsga10 and Spam1 were found to be potentially related to the female sterility in Mule ducks, of which testis-specific gene antigen 10 (Tsga10) has been reported to be highly expressed in various species, especially in the middle and main parts of sperm tails . Furthermore, the imbalance between OXPHOS and spermatogonia glycolysis could lead to meiosis failure  were identified to be specifically related to testicular development of ducks, and 11 genes  specifically related to the female. Seven genes  were screened to be specifically responsible for the female sterility of Mule ducks, eight genes  for male sterility, and five genes  for both female and male sterility. This study lays a solid foundation for elucidating the mechanism of avian gonad development.Taken together, the transcriptome analysis of 1/2 embryonic gonads of three duck breeds was performed by RNA-seq. A total of 9471 DEGs were identified by intra- and inter-breed comparison between males and females of Mule ducks, Muscovy ducks, and Jinding ducks. Twelve genes (


Compared to previous methods, the proposed sensor exhibits good detection accuracy and excellent sensitivity. Therefore, the proposed HCOOH sensor could be used as a new detection method for HCOOH and could provide a new design path for other sensors.With several major polarity and weak optical properties, the sensitive detection of HCOOH remains a major challenge. Given the special role of HCOOH in assisting in the catalytic hydrogenation process of Ir complexes, HCOOH (as a hydrogen source) could rapidly activate Ir complexes as catalysts and further reduce the substrates. This work developed a facile and sensitive HCOOH fluorescence sensor utilizing an optimal catalytic fluorescence generation system, which consists of the phenyl-pyrazole-type Ir-complex PP-Ir-Cl and the coumarin-type fluorescence probe P-coumarin. The sensor demonstrates excellent sensitivity and specificity for HCOOH and formates; the limits of detection for HCOOH, HCOONa, and HCOOEt The concentration of HCOOH could be obtained by titrating the generated I2 with sodium thiosulfate (Na2S2O3). The analysis of trace amounts of HCOOH is currently performed using expensive instruments, such as HPLC [Currently, the main industrial analytical method of HCOOH is the potassium permanganate  is 7825 h−1 at 37 °C) when HCOONa is used as a hydrogen source [In various catalytic hydrogenation processes, HCOOH/HCOONa is often used as an important hydrogen source, which has many advantages, such as low cost, nontoxicity, stability, and good water solubility ,22. In pn source . Based o2) are also essential, besides the catalysts and substrates. As shown in 3N). Compared to NMR analytical methods, the proposed sensor exhibits good detection accuracy and higher sensitivity, which could be used as a potential sensitive detection method for trace HCOOH analysis and could provide a new design path for more sensors.Here, a novel HCOOH fluorescence sensor was developed based on highly active catalytic hydrogenation reactions, which consists of highly active and stable phenyl pyrazole Ir-complex catalysis and a sensitive quinone hydrogenation fluorescence turn-on probe. For a complete catalytic hydrogenation system, the hydrogen sources  were incubated at 60 °C for 30 min, and then, the fluorescence intensity was tested. As shown in In order to obtain the optimal conditions, we investigated the effects of various experimental parameters, including the pH of the reaction solution, the reaction solvents, temperature, the amount of the catalyst PP-Ir-Cl, and the reaction time, on the fluorescence response performance of the HCOOH sensor. As shown in methanol C. We four 30 min D. The ber 30 min E. The flHCOOH − 136.53 with a linear correlation coefficient of 0.9871, where I is the recovery intensity value of fluorescence. Compared to other proposed methods of HCOOH detection, the HCOOH fluorescence sensor exhibits a lower LOD and a satisfactory linear range  was calculated to be 1.0 µM (146 ppb) according to the 3σ/s. HCOOEt3N − 1449.2 with a linear correlation coefficient of 0.9902, where I is the recovery intensity value of fluorescence.Moreover, the Ir-complex catalytic fluorescence sensor was used to detect two important formates: HCOONa and HCOOEt2(CH2)5COOH, glucose, KCl, acetic acid HAc, NaAc, NaH2PO4, Na2HPO4, sodium citrate, and NaCl, were used as the interfering agents because they are common in the environment. As shown in As an important organic acid, the detection of HCOOH is easily disturbed by other organic acids, especially ethanoic acid and propanoic acid, due to similar physical and chemical properties. Thus, the specificity of the HCOOH sensor was also evaluated using various interfering substances (5.0 mM), instead of HCOOH or formate (1.0 mM), under the same experimental conditions. Various inorganic and organic salts, and organic acids, including methanol MeOH, ethanol EtOH, tert-Butanol, NADH, oxalic acid, sodium sulfate, 2-bromopropionic acid, 4-bromobutyric acid, mandelic acid, NH2 chemocatalysis and electrocatalysis systems. Three parallel samples were obtained and underwent the standard recovery test. The results are shown in To investigate the practicability of the developed HCOOH sensor, the sensor was applied to detect HCOOH in triethylamine solution, which is usually used as the reaction solution in CO3N, methanol, ethanol, tert-butanol, nicotinamide adenine dinucleotide disodium salt (NADH), oxalic acid, sodium sulfate, glucose, KCl, acetic acid HAc, sodium acetate NaAc, NaH2PO4, Na2HPO4, sodium citrate, and NaCl were purchased from the Shanghai Aladdin Biochemical Technology Co. . 2-Bromopropionic acid, 4-bromobutyric acid, mandelic acid, and aminocaproic acid NH2(CH2)5COOH were acquired from the MACKLIN reagent . The Ir-complex PP-Ir-Cl and the fluorescence probe P-coumarin were synthesized according to our previous work [2HPO4 and NaH2PO4. HAc-NaAc buffer solution was prepared using HAc and NaAc. Fluorescence spectra were collected using an F-4700 fluorescence spectrophotometer .HCOOH, HCOONa, HCOOEtous work ,24. The v/v% of different organic solvents, 100 uM of the fluorescence probe P-coumarin, and different amounts of the Ir-complex PP-Ir-Cl was added to different concentrations of HCOOH or formate solution and incubated for 1 h at 60 °C. The fluorescence intensity of each sample was then tested. Finally, a standard curve line was constructed between various concentrations of HCOOH or formates and the recovery values of fluorescence intensity. In addition, the fluorescence probe selectivity was studied under the optimal conditions using various interfering substances instead of HCOOH or formates.In a typical detection experiment, 990 µL of 0.2 M phosphate buffer with different pH or an HAc-NaAc buffer containing 10 −. To study the performance of the sensor in a CO2 hydrogenation reaction, the sensor was applied to detect the produced HCOOH in a triethylamine environment. Briefly, 1.0 mL of triethylamine solution containing various concentrations of HCOOH  was first removed of free triethylamine using a vacuum distillation, and then 1.0 mL of HAc-NaAc buffer  containing 10 v/v% of methanol, 100 uM of the fluorescence probe P-coumarin, and 5.0 uM of the Ir-complex PP-Ir-Cl was added and incubated for 1.0 h at 60 °C. Finally, the fluorescence spectroscopy of each sample was tested. Three experiments were performed in parallel, and the RSD was calculated.The fluorescence sensor had excellent specificity for HCOO3N, with a satisfactory linear range and a low LOD (50.6 ppb), and it can achieve an accurate determination of HCOOH samples in a CO2 catalytic hydrogenation system. Therefore, the HCOOH sensor could be used as a new detection method for HCOOH and can provide a new design path for other sensors.In conclusion, we have developed a facile and rapid fluorescence sensor for HCOOH based on highly active Ir-complexes’ catalytic transfer hydrogen fluorescence turn-on reaction and using HCOOH or formates as the hydrogen source. The excellent HCOOH-assisted catalytic fluorescence response system was obtained by optimizing various experimental conditions, including pH, reaction solvents, temperature, catalyst amount, and reaction time. The proposed HCOOH sensor shows good sensitivity and specificity for the detection of HCOOH, HCOONa, and HCOOEt


In February 2021, an explosion of cases of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia overwhelmed the only hospital in Mayotte. To report a case series of patients with acute respiratory failure (ARF) due to SARS-CoV-2 who were evacuated by air from Mayotte to Reunion Island.This retrospective observational study evaluated all consecutive patients with ARF due to SARS-CoV-2 who were evacuated by air from Mayotte Hospital to the intensive care unit (ICU) of Félix Guyon University Hospital in Reunion Island between February 2, and March 5, 2021.2/FiO2 ratio was lower on ICU admission (140 [102–192] mmHg) than on departure . Factors associated with severe hypoxemia during air transport was lack of treatment with curare (P = .012) and lack of invasive mechanical ventilation (P = .003). Nine patients (23.7%) received veno-venous extracorporeal membrane oxygenation support in our ICU. Seven deaths (18.4%) occurred in hospital.A total of 43 patients with SARS-CoV-2 pneumonia were evacuated by air, for a total flight time of 2 hours and a total travel time of 6 hours. Of these, 38 patients (88.4%) with a median age of 55 (46–65) years presented with ARF and were hospitalized in our ICU. Fifteen patients were screened for the SARS-CoV-2 501Y.V2 variant, all of whom tested positive. Thirteen patients (34.2%) developed an episode of severe hypoxemia during air transport, and the median paOEmergency air evacuation of patients with ARF due to SARS-CoV-2 was associated with severe hypoxemia but remained feasible. In cases of ARF due to SARS-CoV-2 requiring emergency air evacuation, sedated patients receiving invasive mechanical ventilation and curare should be prioritized over nonintubated patients. It is noteworthy that patients with SARS-CoV-2 pneumonia related to the 501Y.V2 variant were very severe despite their young age. In February 2021, the only hospital of the island, Mayotte Hospital, was overwhelmed by the explosion in the number of cases, as it is equipped with only 16 intensive care unit (ICU) beds for a population of 270,000 inhabitants. The medical infrastructure in Reunion Island meets European standards . When Mayotte Hospital became overwhelmed with cases of SARS-CoV-2 pneumonia, several patients were transferred to Félix Guyon University Hospital in Reunion Island, in a context of solidarity among islands of the Indian Ocean region. All patients had to be evacuated by air due to the emergency situation. There is no transplantation center (except kidney) on the French overseas territories of Reunion Island and Mayotte which are located in the Indian Ocean. As a result, patients needing heart, lung or liver transplantation must be transferred to Paris, over a distance of 10,000 km. It is to be noted that medical teams in both Reunion Island and Mayotte have experience with emergency air evacuations, including for patients receiving extracorporeal membrane oxygenation support.,6 It was necessary to urgently transfer patients with acute respiratory failure (ARF) due to SARS-CoV-2 from Mayotte to the only ICU in Indian Ocean equipped with many VV-ECMO supports. To date, few studies have examined the outcome of patients with SARS-CoV-2 pneumonia evacuated by air,–9 and only a handful of them have focused on intensive care patients.,11 Here, we report a case series of patients with ARF due to SARS-CoV-2 who were evacuated by air from Mayotte to Reunion Island.Mayotte  and Reunion Island  are 2 French overseas departments located in the Indian Ocean at a distance of 10,000 km from Paris. Until January 2021, Mayotte was relatively spared by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. On January 6, 2021, the incidence of deaths caused by SARS-CoV-2 was 20.7 per 100,000 inhabitants on the island .22.1Commission nationale de l’informatique et des libertés . This study complies with the Strengthening the Reporting of Observational studies in Epidemiology recommendations statement.This retrospective observational study evaluated all consecutive patients with ARF due to SARS-CoV-2 who were evacuated by air from Mayotte Hospital in Mamoudzou to the ICU of Félix Guyon University Hospital in Reunion Island between February 2 and March 5, 2021. All patients or their legally authorized representative were verbally informed about the study and could refuse to participate; moreover, they received a written information notice about the process of data collection. The study was approved by the Ethics Committee of the French Society of Infectious Disease and Tropical Medicine (CER-MIT), and was declared to the ,14All evaluated patients had positive confirmatory respiratory sample with SARS-CoV-2 by real time reverse transcription polymerase chain reaction using kit targeting IP2 and IP4 regions and N gene. Samples from nasopharyngeal swabs or patient's respiratory specimens were extracted using NucliSens easyMAG system (BioMérieux). The genomes were secondly obtained by sequencing using the Oxford Nanopore technology and the Artic Network's overlapping amplicon protocol.2.2rd generation cephalosporin or piperacillin-tazobactam after realization of respiratory sample; dexamethasone at a dosage of 6 mg/day for 10 days; systematic deworming with ivermectine or albendazole; enhanced systematic anticoagulation according to the guidelines of the French Society of Hemostasis and the French Society of Anesthesia and Intensive Care.In accordance with our protocol, all patients with ARF due to SARS-CoV-2 were treated with: a 3High flow nasal cannula oxygenation was initiated in patients who need standard oxygen ≥9 L/min to maintain peripheral arterial oxygenation saturation ≥92%. The decision of the timing of intubation and mechanical ventilation was not protocolized but determined by the ICU team.2.32 <0.6, PEEP <15, and norepinephrine <1 mg/h. The use of curares in patients receiving invasive mechanical ventilation was not protocolized and was left to the discretion of the medical team.The protocol of air evacuation had been established by the prehospital emergency medical system of La Reunion and Mayotte and there was no specific training for these transports. In order to be transported, patients had to meet the following criteria: FiOTotal flight time was 2 hours and total travel time between the 2 hospitals was 6 hours. Patients were evacuated on board an Embraer 135 aircraft specially equipped for medical evacuation missions (12 seats and 3 stretchers). The vast majority of flights carried 3 patients, 2 of whom received invasive mechanical ventilation and 1 received conventional oxygen therapy. The medical crew, was composed of 3 doctors and 3 nurses, experienced in inter-hospital transport. Both the cockpit and a delimited area at the bottom of the aircraft were considered “clean”; the rest of the aircraft was deemed “contaminated.” The medical team was fully dressed in protective clothing for the duration of the flight including examination gloves, filtering facepiece class 2 mask (FFP2) and goggles for eye protection. Each patient was assigned 1 doctor, 1 nurse, and 1 emergency kit composed of a vacuum mattress, an aspirator, a defibrillator, a transport ventilator, and suitcases with ventilation material and emergency medication  <88% during >15 minutes).The primary outcome was the occurrence of severe hypoxemia during air transport , duration of invasive mechanical ventilation, in-ICU length of stay and hospital mortality.Other complications during air transport were evaluated: oxygen desaturation requiring increase of oxygen requirements or introduction of curares, hypotension  requiring positive inotropic therapy or fluid challenge >500 mL of crystalloid, endotracheal tube or vascular catheter dislodgement, arrhythmia, cardiac arrest, agitation requiring modification of sedation.2.6th–75th percentiles). Categorical variables were compared using Fisher exact test. Continuous variables were compared using the nonparametric Wilcoxon test or Mann–Whitney U test, as appropriate. A P value <.05 was considered significant. Statistical analyses were conducted using SPSS 15.0 .Categorical variables were expressed as total number (percentage) and continuous variables as median  presenting with ARF were transferred directly to our ICU and included in the study cohort. During air transport, 4 patients (10.5%) received conventional oxygen therapy (between 6 L/min and 9 L/min) before departure and 34 patients (89.5%) received invasive mechanical ventilation; 3 (8.8%) of the ventilated patients also received VV-ECMO support.For all patients the indication for emergency air evacuation was to free up ICU beds in the hospital of Mayotte.2. Fifteen patients (39.5%) were screened for the SARS-CoV-2 501Y.V2 variant, all of whom tested positive.The characteristics of patients are shown in Tables The median time from onset of symptoms to air evacuation was 12 (9–14) days. For patients receiving invasive mechanical ventilation, the median time from orotracheal intubation to air evacuation was 4 (2–7) days. Eight patients (21.1%) were treated with noradrenaline during air transport.A total of 13 patients (34.2%) developed an episode of severe hypoxemia during air transport.P > .9) (Table P = .012), lack of invasive mechanical ventilation (P = .003)  Table . In univ2/FiO2 ratio was significantly lower on admission to our ICU (140 [102–192] mmHg) than on departure from Mayotte Hospital   and fluid challenge >500 mL of crystalloid during air transport.Agitation requiring modification of sedation occurred in 2 patients (5.3%) and oxygen desaturation requiring increase of fraction of inspired oxygen, sedation enhancement and introduction of neuromuscular blocking agents occurred in 8 patients (21%).No cardiac arrest, arrhythmia, endotracheal tube or vascular catheter dislodgement occurred during air transport.The median plasma lactate concentration on admission to ICU was 1.3 (1.1–1.6) mmol/L Table .3.1One patient had a fatal cardiac arrest due to pulmonary embolism in our ICU within 24 hours of departure from Mayotte Hospital.One patient who presented with acute coronary syndrome in Mayotte underwent coronary angiography in our ICU. The intervention revealed severe 3-vessel disease requiring revascularization.Of the 35 patients who were not on VV-ECMO support on departure from Mayotte, 6 (17.1%) required VV-ECMO support for refractory acute respiratory distress syndrome in our ICU. Overall, 9 (23.7%) of 38 patients received VV-ECMO support for refractory acute respiratory distress syndrome  and 19 patients (50%), respectively.P = .584) (Table The median length of stay in ICU was 21 13–46) days, with no significant difference between patients with and without an episode of severe hypoxemia during air transport  (Table After a median follow-up of 56 (49–66) days, 7 deaths (18.4%) occurred, with no significant difference between patients with and without an episode of severe hypoxemia during air transport P = .85  were able to receive VV-ECMO support in Reunion Island, where 16 machines are presently available compared to 2 machines in Mayotte. Moreover, 1 patient who presented with acute coronary syndrome in Mayotte was able to undergo coronary angiography in Reunion Island; the intervention revealed three-vessel disease, prompting treatment with coronary revascularization. From an ethical perspective; however, the benefits of emergency air evacuation existed only at the collective level .,11 The lack of ethical benefit at the personal level explains why many patients and/or their families refused the evacuation. Lastly, it should be stressed that patients in our case series were critically ill, with almost a quarter of them requiring VV-ECMO support despite their young age. This could due to the increased virulence of variant of concern, which has been highlighted in several studies.,26Studies have shown that the transfer of critically ill patients receiving mechanical ventilation can increase ventilation duration and patient mortality.The other issues to be highlighted are the numerous transfers to manage to reach the ICU at the hospital of Reunion Island  and the discomfort of the protective equipment in a tropical zone where temperature and hygrometry are very high at that season which corresponded to austral summer. examined only 13 patients, none of whom were on VV-ECMO support; moreover, travel time was shorter than in our study. Similarly, the study by Turc et al evaluated only 36 patients  who traveled for a much shorter period of time (70 minutes) than in our study.Our study has many limitations. Biases may have been introduced due to the retrospective nature of the study. In addition, the number of evaluated patients was relatively small. Yet, in their study of patients with SARS-CoV-2 pneumonia evacuated by air, Pavain et alIn conclusion, emergency air evacuation of patients with ARF due to SARS-CoV-2 501Y.V2 variant was associated with respiratory complications but remained feasible. In cases of ARF due to SARS-CoV-2 requiring emergency air evacuation, sedated patients receiving invasive mechanical ventilation and curare should be prioritized over non-intubated patients. It is noteworthy that patients with SARS-CoV-2 pneumonia related to the 501Y.V2 variant were very severe despite their young age.HB, YB, NA had full access to all the study data and take responsibility for the completeness of the data and the accuracy of the analysis.Study concept and design: HB, IL, PR, CA, RB, AL, BP, CC, CV, MC, MCJB, CC, NO, HC, JA, YB, NA, LDAcquisition of data: HB, YE, HKAnalysis and interpretation of data: HB, IL, PR, CA, RB, AL, BP, CC, CV, MC, MCJB, CC, NO, HC, JA, YB, NA, LDDrafting of the manuscript and critical revision of the manuscript for important intellectual content: HB, IL, PR, CA, RB, AL, BP, CC, CV, MC, MCJB, CC, NO, HC, JA, YB, NAStatistical analysis: NA, JAFunding: Support was provided solely by institutional and/or departmental sources.Administrative, technical and material support and study supervision: HB, IL, PR, CA, RB, AL, BP, CC, CV, MC, MCJB, CC, NO, HC, JA, YB, NAConceptualization: Hamza Berguigua, Ludovic Iche, Cyril Aubert, Renaud Blondé, Antoine Legrand, Bérénice Puech, Chloé Combe, Charles Vidal, Margot Caron, Marie-Christine Jaffar-Bandjee, Christophe Caralp, Nora Oulehri, Yvonnick Boué, Nicolas Allou.Data curation: Hamza Berguigua, Ludovic Iche, Philippe Roche, Cyril Aubert, Renaud Blondé, Chloé Combe, Charles Vidal, Nora Oulehri, Hugo Kerambrun, Yvonnick Boué.Formal analysis: Hamza Berguigua, Ludovic Iche, Cyril Aubert, Renaud Blondé, Antoine Legrand, Bérénice Puech, Chloé Combe, Charles Vidal, Christophe Caralp, Jérôme Allyn, Yvonnick Boué, Nicolas Allou.Investigation: Hamza Berguigua, Philippe Roche, Renaud Blondé, Antoine Legrand, Bérénice Puech, Charles Vidal, Margot Caron, Marie-Christine Jaffar-Bandjee, Nora Oulehri, Hugo Kerambrun, Yvonnick Boué.Methodology: Hamza Berguigua, Ludovic Iche, Renaud Blondé, Charles Vidal, Christophe Caralp, Nora Oulehri, Jérôme Allyn, Yvonnick Boué, Nicolas Allou.Project administration: Hamza Berguigua.Resources: Christophe Caralp, Nora Oulehri, Hugo Kerambrun.Supervision: Hamza Berguigua, Ludovic Iche, Philippe Roche, Margot Caron, Hugo Kerambrun, Yvonnick Boué, Nicolas Allou.Validation: Hamza Berguigua, Ludovic Iche, Jérôme Allyn, Yvonnick Boué, Nicolas Allou.


Cancer stem cells (CSCs) are a small subset of cancer cells and are thought to play a critical role in the initiation and maintenance of tumor mass. CSCs exhibit similar hallmarks to normal stem cells, such as self-renewal, differentiation, and homeostasis. In addition, CSCs are equipped with several features so as to evade anticancer mechanisms. Therefore, it is hard to eliminate CSCs by conventional anticancer therapeutics that are effective at clearing bulk cancer cells. Interferons are innate cytokines and are the key players in immune surveillance to respond to invaded pathogens. Interferons are also crucial for adaptive immunity for the killing of specific aliens including cancer cells. However, CSCs usually evolve to escape from interferon-mediated immune surveillance and to shape the niche as a “cold” tumor microenvironment (TME). These CSC characteristics are related to their unique epigenetic regulations that are different from those of normal and bulk cancer cells. In this review, we introduce the roles of epigenetic modifiers, focusing on LSD1, BMI1, G9a, and SETDB1, in contributing to CSC characteristics and discussing the interplay between CSCs and interferon response. We also discuss the emerging strategy for eradicating CSCs by targeting these epigenetic modifiers, which can elevate cytosolic nuclei acids, trigger interferon response, and reshape a “hot” TME for improving cancer immunotherapy. The key epigenetic and immune genes involved in this crosstalk can be used as biomarkers for precision oncology. Cancer stem cells (CSCs) are a small subset of cancer-initiating and/or cancer maintenance cells in the cancer mass. CSCs exhibit similar characteristics as that of normal stem cells, such as self-renewal, differentiation, and homeostasis. However, CSCs usually acquire genetic and epigenetic alterations that allow CSCs to evade tumor suppression mechanisms  1,2,3,4,3,42,3,4Immune evasion is a fundamental feature of CSCs, which is a significant obstacle in immune checkpoint blockade (ICB) therapy ,10,11. CUnlike bulk tumor cells, CSCs exhibit distinct epigenetic landscapes that are marked by altered DNA methylation and histone modifications ,17. ThesThe type I interferons (IFNs), including IFN-α and IFN-β, are innate cytokines that are equipped to every cell as the first-line immune response to combat invaded pathogens. Upon infection, the pathogenic nucleic acids can be detected by a variety of pattern recognition receptors (PRRs), which then transmit these “danger signals” through STING-TBK1 and MAVS-TBK1 axes to activate the hub transcription factors NF-kB and interferon regulatory factor 3 (IRF3), thereby allowing these transcription factors to translocate into the nucleus and turn on the gene expressions of type I interferons and pro-inflammatory cytokines. Depending on the nature of nucleic acids in the cytosol, the cells use different PRRs to detect viral DNA, such as cGAS, IFI16, DDX41, AIM2, ZBP1/DAI, RNA polymerase III, and viral RNA, such as DDX58/RIG-I, IFH1/MDA5, and Toll-like receptors (TLRs) 3, 7, 8, 9  Figure . In addiThe induced type I interferons, which are secreted from virus infected or malignant cells, stimulate the expression of type II interferon (IFN-γ) and numerous interferon-stimulated genes (ISGs) by binding to the type I interferon receptors (IFNAR1/IFNAR2) on the cell membrane. The interferon-bound IFNAR1/IFNAR2 activate the Janus kinase 1 (JAK1) and Tyrosine kinase 2 (TYK2), which phosphorylate the interferon-stimulated gene factor 3 (ISGF3) composed of interferon regulatory factor 9 (IRF9), signal transducer and activator of transcription 1 (STAT1), and STAT2 . BecauseThe dendritic cells (DCs) and natural killer (NK) cells are well-documented innate immune cells that produce pro-inflammatory cytokines and chemokines in response to stimulation by type I interferons. These pro-inflammatory cytokines, such as IFN-γ and tumor necrosis factor (TNF), and chemokines, such as CXCL10, are critical for modulating adaptive immune response to inhibit cancer cells in the TME. For example, CXCL10 can attract the cytotoxic CD8+ T cells to the TME for cancer killing , and theThe cytotoxic CD8+ T cells are the major effector cells that kill cancer cells in cancer immunotherapy. The activation of cytotoxic CD8+ T cells requires specific antigens that are processed and presented together with MHC-I on the cell surface of professional antigen-presenting cells (APCs), such as DCs. In addition to MHC-I, co-stimulatory signals provided by the B7 molecules of APCs are also required. The B7 molecules bind to the CD28 of CD8+ T cells and induce T-cell activation and proliferation. In contrast, T-cell activity is suppressed by inhibitory checkpoint molecules, such as PD-1 and cytotoxic T-lymphocyte antigen-4 (CTLA-4), to avoid over-activation, which may lead to tissue damage and autoimmune diseases. Cancer cells can express the ligands of T-cell inhibitory molecules, such as PD-L1 and PD-L2, to inhibit the activation of CD8+ T cells and escape from immune attack. In this way, antibodies (ICIs) that block the actions of PD-1, PD-L1, and CTLA-4 are developed to activate CD8+ T cells in the TME for cancer immunotherapy. However, the efficacies of ICIs are limit in many cancers due to the “cold” TME, where is an immunosuppressive territory and is short of activated CD8+ T cells and pro-inflammatory cytokines/chemokines . TherefoThe interferon response is critical for antitumor immunosurveillance. In addition, interferons and ISGs have been demonstrated to inhibit CSC characteristics, such as chemoresistance. However, CSCs may evolve various mechanisms to evade interferon-mediated immunosurveillance . The intDoherty et al. have reported that the mesenchymal breast CSCs exhibit a significantly repressed IFN/STAT gene expression signature . The treIn the 4T1 mouse model of breast cancer, the amounts of IFN-γ and CD8+ T cells decrease but the levels of ALDH+ breast CSCs increase along with tumor growth. The administration of IFN-γ can suppress the sphere formation of 4T1 tumor in vitro and decrease the number of ALDH+ breast CSCs in the 4T1 tumor-bearing mice . MoreoveBoth IFN-β and IFN-γ can induce cell cycle arrest and the dormancy of tumor-repopulating cells (TRCs) or disseminated tumor cells (DTCs), which exhibit CSC characteristics and can re-initiate tumor growth at distant organs long after therapy. For example, Liu et al. reported that IFN-β or IFN-γ treatment induces melanoma TRCs to enter dormancy via an indolamine 2,3-dioxygenase 1-kynurenine-aryl hydrocarbon receptor-p27-dependent pathway. Moreover, the high expression of IFN-β is correlated with a longer survival in melanoma patients ,49. Lan In addition to interferons, many interferon-related genes can inhibit CSCs. The interferon-stimulated gene 15 (ISG15) is an ubiquitin-like protein, which plays pleiotropic roles in the TME. Furthermore, ISG15 can inhibit the protein translation of ABCC2 and increase drug sensitivity in cisplatin-resistant ovarian cancer cells . MoreoveAnother drug resistant gene, ABCG2, can be repressed by the interferon regulatory factor 6 (IRF6) . As is kHuang et al. reported that the interferon-induced protein 44-like (IFI44L) can decrease the chemoresistance of hepatic CSCs towards doxorubicin. In contrast, depletion of IFI44L promotes cell migration, invasion, and pulmonary metastasis of hepatic CSCs. Downregulation of IFI44L is frequently observed in hepatocellular carcinoma (HCC) and is correlated with patient’s poor survival . These rHowever, the interferon response may potentiate CSC properties in pancreatic adenocarcinoma and sarcoma ,60,61. TSeveral studies have shown that CSCs are able to evade interferon-mediated tumor suppression ,10. In alow breast CSCs properties, such as tumor sphere formation, cell plasticity, and resistance to chemotherapy and radiotherapy. Notably, an interferon-responsive gene signature is downregulated in ETV7-expressing breast cancer cells. In breast cancer patients, the low expressions of ETV7-repressed interferon signature genes are associated with worse prognoses [The precise mechanisms underlying the inhibition of the interferon response in CSCs remain to be elucidated; however, some CSC-related markers may be involved in this suppression. Li et al. have reported that CD133-enriched HCC cells are resistant to IFN-γ-induced autophagy and growth suppression . Oncostarognoses , suggestGlioma CSCs is able to evade interferon response via the downregulation of STAT1, a critical transcription factor for the induction of ISGs . MechaniThe Moloney murine leukemia virus insertion site 1 (BMI1) is a CSC marker found in several cancers . FurtherThe miR-199a is enriched in mammary stem cells (MaSCs) and in breast CSCs. In addition to stimulating CSC features, miR-199a represses the interferon response by directly targeting the nuclear receptor corepressor LCOR, which is able to sensitize MaSCs and CSCs to interferon-induced differentiation and senescence. A high expression of miR-199a is associated with poor relapse-free survival in patients with breast cancer, especially in those with estrogen receptor-negative breast cancers, which exhibit enhanced CSC properties . These sThe interferon response can be induced by the de-repression of ERV elements or DNA damage-induced cytosolic nucleic acids ,35,36,37Interestingly, LSD1 removes mono- and di-methyl groups (me1/2) from lysine 4 (K4) and lysine 9 (K9) of histone H3, thus alters chromatin configuration and gene expression . In addi6-methyladenosine (m6A) modification of BMI1 mRNA, which promotes BMI1 translation in oral cancer cells [With regard to BMI1, it is an essential component of the polycomb repressive complex 1 (PRC1) that inhibits gene expression by modifying histone H2A with ubiquitin . It player cells . The BMIer cells .Moreover, BMI1 can induce EMT and metastasis of cancer cells through cooperating with Twist1, a crucial EMT regulator . In addiA high expression of BMI1 is associated with poor outcomes in the patients with NPC, glioma, HNSCC, and NSCLC ,105,106.In addition to the PRC complex, several regulators of histone H3K9 methylation, such as the SET domain-containing histone methyltransferases SETDB1 and G9a , play important roles in regulating pluripotency and cancer stemness ,110. ImpAn overexpression of G9a is found in several types of cancer, such as neuroblastoma, ovarian, breast, bladder, and lung cancers, and is usually correlated with poor prognoses ,121,122.Additionally, SETDB1 is another histone H3K9 methyltransferase and is required for early embryonic development . It reguImportantly, SETDB1 contributes to the proliferation and migration of colorectal cancer cells via inhibiting p53 and epigenetically silencing p21 expression ,128. OveBecause LSD1, BMI1, G9a, and SETDB1 are enriched in CSCs and are crucial for CSC characteristics, targeting these histone methylation modifiers can be used as a strategy to inhibit CSCs. Another rationale for targeting these epigenetic modifiers in CSCs is to re-activate ERV elements and induce DNA damage, both can trigger interferon response and sculpt a “hot” TME for ICB therapy . The expAs is known, LSD1 plays an important role in suppressing ERV elements in mouse embryonic stem cells (mESCs) and in human cancer cells ,134. TheBy performing an analysis of TCGA data sets, the expression of LSD1 is conversely correlated with CD8+ T cell infiltration in various types of cancer, and high LSD1 expression is a predictor of poor prognosis for HNSCC patients ,140. QinIn addition to augmenting antitumor immunity, LSD1 ablation can suppress the stem cell-like properties of HNSCC, HCC, glioma, small cell lung cancer, and breast cancers of luminal-B, HER2-positive, and TNBC subtypes through attenuating Wnt/β-catenin, Notch, BMI1, and SOX2-deriven stemness signaling. Inhibition of LSD1 also promotes epithelial differentiation through de-repression of fate-determining transcription factors ,146,147.Importantly, BMI1 plays a critical role in the self-renewal and chemoresistance of HNSCC CSCs ,90. The As previously mentioned, G9a contributes to CSC characteristics and cancer progression, suggesting that targeting G9a may be a potential strategy to suppress CSCs . It has As SETDB1 is required for 53BP1 repositioning and BRCA1 function during DNA homologous recombination repair, the depletion of SETDB1 increases DNA strand breaks in G2 phase of the cell cycle . The inhIn addition to LSD1, BMI1, G9a, and SETDB1, other epigenetic modifiers are also involved in the regulation of CSC properties and can be candidate therapeutic targets. For example, targeting EZH2 can de-repress ERV and induce interferon response in Ewing sarcoma . In prosAlthough targeting epigenetic modifiers is an effective strategy by which to boost the antitumor immune response for CSC eradication in many preclinical studies, its application in clinics is still challenged. Because many epigenetic modifiers are essential for the functions and homeostasis of normal stem/progenitor cells, the depletion of these epigenetic modifiers may cause severe adverse effects to the healthy tissues and organs ,126,171.The histone methylation modifiers LSD1, BMI1, G9a, and SETDB1 are crucial for CSC characteristics, such as self-renewal, differentiation, homeostasis, EMT, chemoresistance, and immune evasion. Their expressions are usually higher in CSCs than in bulk tumor cells. Therefore, these epigenetic modifiers can be used as CSC biomarkers and therapeutic targets.The inhibition of LSD1, BMI1, G9a, or SETDB1 can de-repress ERV elements and induce DNA damage, and both events elicit cytosolic nucleic acids and trigger an interferon response. The induced interferons not only inhibit cancer cells but also activate innate and adaptive immune responses. For example, interferons can activate DC and NK cells to ensure immunosurveillance and prime Th1 and CD8+ T cells for tumor killing. Thus, the activation of the interferon response can contribute to the “hot” TME and improve ICB therapy. Moreover, interferons play a critical role in CSC dormancy and, thus, prevent cancer relapse. Nevertheless, a continued hyperactivation of interferon response may cause tissue damage and autoimmune disease. Therefore, further investigations that unveil the precise regulation of interferon response are required for future clinical applications in anticancer therapy.


These metabolites were detected in the urine and (or) feces of 74% of volunteers afterconsuming resveratrol, while 26% lacked these dehydroxylase activities.The LUNU non-producer metabotype was more prevalent in females (P < 0.05) but independent of individuals’ BMIand age. A 4-styrylphenol reductase in both metabotypes convertedstilbenes to their corresponding dibenzyls, while no 4-dehydroxylationin stilbenes or dibenzyls was observed. 4-Hydroxy-trans-stilbene, pinosylvin, dihydropinosylvin, 3-hydroxydibenzyl, and3-hydroxy-trans-stilbene were not detected in vivoor in vitro. Further research on LUNU metabotypes, their associatedgut microbiota, and their impact on health is worthwhile.We describe here for the first time the consistent observationof two metabotypes associated with resveratrol metabolism by the humangut microbiota, that is, lunularin (LUNU)-producers and LUNU non-producers.In healthy volunteers ( Theterm metabotype is an extensive concept involving individuals’differential metabolic responses to nutritional or pharmacologicalinterventions.7 Specifically, in the context of(poly)phenols’ metabolism and health, metabotype refers toa differential metabolic phenotype defined by specific metabolitesderived from the gut microbiota, characteristic of the precursor (poly)phenolmetabolism. A metabotype is also characterized by the associated microbialecology in terms of composition and activity, which could notablyimpact human health. Therefore, this feature is less influenced byexternal factors and refers to a qualitative-genuine criterion concerning the specific microbial ecology that harbors each individual.7 In this context, the metabolismof EA and the isoflavone daidzein fulfills the definition of metabotype.14 In the case of EA, three urolithin-relatedmetabotypes have been defined depending on the final urolithins produced,that is, metabotype A ,metabotype B [production of urolithins A and B (3-hydroxy-urolithin)],and (or) isourolithin A  and metabotype 0(urolithin non-producers).15 In the case of isoflavones,the equol- and O-desmethylangolesin (ODMA)-producermetabotypes have been identified so far.16 In addition, a gradient of metabolite production may also occurwithin a specific metabotype, that is, high and low urolithin or equolproducers.7 Overall, the potential biologicalactivity derived from (poly)phenol intake is conditioned by the gutmicrobiome ecology of each individual.7 Therefore, the stratification,that is, grouping of individuals according to their gut microbiota(poly)phenol metabotypes, has been proposed to understand individuals’responses to dietary (poly)phenols, that is, to explain their differentmetabolisms and health effects, which could be crucial in the contextof personalized nutrition.18There is notable human interindividualvariability in responseto (poly)phenol consumption,19 Relevant amounts of RSV can be consumed only through nutraceuticalpreparations since its presence in the human diet is limited to afew foodstuffs that contain low RSV levels, including grapes, redwine, peanuts, pistachios, and some types of berries .19Resveratrol (RSV) is the most relevant dietary stilbene duetoits well-known bioactivity, including antioxidant, anti-inflammatory,immunomodulatory, anti-diabetic, cancer chemopreventive, neuroprotective,and cardiovascular protective effects.trans-stilbene (DHST), showing high interindividual variabilityin the type of metabolites produced.9 However,this variability has been reported in a few volunteers or in vitrofecal samples.20The metabolismof RSV by the gut microbiota was previously reportedto produce dihydroresveratrol (DHRSV), 3,4′-dihydroxydibenzyl, and 3,4′-dihydroxy-n = 59) after consuming RSV.21 In that study, we observed that LUNU, but notDHST, was further dehydroxylated at the 3-position to yield 4HDB.We also observed substantial variability in the metabolism of RSVby the gut microbiota. Notably, 4HDB was only detected in those volunteersthat produced LUNU.21 However, other metabolicsteps and a possible explanation for the variability observed remainedunexplored in that study.Recently, we have reportedthe presence of 4-hydroxydibenzyl (4HDB),also known as 4-(2-phenylethyl)phenol according to the IUPAC, as anovel metabolite from the human gut microbiota in the urine of healthyvolunteers (n = 195), combiningin vivo determinations with in vitro studies to confirm catabolicsteps and specific enzymatic activities.Therefore, in the present study, weaimed to (i) decipher the metabolismof RSV by the human gut microbiota and (ii) identify the possiblepresence of metabotypes consistently associated with this metabolismin a group of healthy volunteers , trans-pinosylvin , chrysin (97%), resorcinol , 3-hydroxybenzoicacid (99%), 4-hydroxybenzoic acid (99%), 3′-hydroxyphenyl aceticacid (99%), 4′-hydroxyphenyl acetic acid (98%), 3-(3′-hydroxyphenyl)propanoicacid (98%), 3-(4′-hydroxyphenyl)propanoic acid (98%), β-glucuronidase, and sulfatase from Helix pomatia. RSV 4′-O-sulfate, RSV 3-O-glucuronide, DHRSV 3-O-glucuronide, and RSV 3-O-sulfate wereobtained as described elsewhere.22 4-Hydroxy-trans-stilbene  was obtained from ThermoFisherSci. . Ultrapure Millipore water was used throughoutthe study.HPLC-grade acetonitrile, dimethyl sulfoxide(DMSO), formic acid, and methanol were obtained from JT Baker . The following chemicals were purchased from Sigma-Aldrich: 1H NMR chemical shifts are reported relative to tetramethylsilaneand referenced via residual proton resonances of the correspondingdeuterated solvent. 1H NMR spectra were recorded at 25°C on Avance 300 and 400 MHz instruments . Coupling constants (J) are expressed inHz. The abbreviations for the coupling patterns are the following:br, broad; s, singlet; d, doublet; t, triplet; q, quadruplet; andm, multiplet.2123 Datafor DHRSV: 1H NMR : δ 9.11 , 9.02 , 6.99 , 6.65 , 6.06 , 6.02 ,2.64 .DHRSV (>97%) wassynthesized as previously described.24 Data for 4HDB: 1H NMR : δ 9.12 , 7.30–7.12 , 6.99, 6.64 , 2.84–2.71 .4-Hydroxydibenzyl (or 4-(2-phenylethyl)phenol,4HDB; >97%) wassynthesized according to Camaioni and Franz.25 Datafor 3HDB: 1H NMR : δ 7.34–7.25, 7.24–7.12 , 6.77 , 6.71–6.65 , 4.71 , 2.96–2.83.3-Hydroxydibenzyl (or3-(2-phenylethyl)phenol, 3HDB; >97%) wassynthesized as described elsewhere.26 Data for LUNU: 1H NMR: δ 7.15 , 7.07–7.01 , 6.78–6.72 ,6.69–6.63 , 4.66 , 4.61 , 2.83 .3,4′-Dihydroxydibenzyl  was synthesized following the procedure described byAliet al.26 Data for DHP: 1H NMR : δ7.32–7.26 , 7.20 , 6.25 , 6.19 , 4.75 , 2.92–2.85 , 2.84–2.78 .Dihydropinosylvin (or 3-hydroxy-(5-phenethyl)phenol, DHP;>97%)was synthesized as previously reported.trans-stilbene(or 3-(4-hydroxystyryl)phenol, DHST, >97%) was synthesized as describedelsewhere.27 Data for DHST: 1H NMR : δ 9.56, 9.36 , 7.41 ,7.13 , 7.03 , 6.98–6.88 , 6.76 , 6.63  ppm.Finally,3,4′-dihydroxy-Polygonum cuspidatum in the evening for 7 days. The capsules were manufactured by LaboratoriosAdmira S.L.  following the requirementsof the European Union’s good manufacturing practices. No placebowas included since this trial was not designed to test the effectivenessof a compound or evaluate changes in specific clinical variables.Since there was no clinical variable to accurately power (minimumsample size) for this type of study, the sample size was based onprevious stratifying studies dealing with the gut microbiota-associatedmetabotyping of individuals.28This dietary interventionwas approved (reference PI-042) by IMDEA-Food  andthe Spanish National Research Council’s Bioethics Committee(Madrid) within the MetaboGut Project . The trial followed the ethical guidelines outlined in theHelsinki Declaration of 1975 and its amendments. Healthy volunteersover 18 years old were recruited. Exclusion criteria involved theintake of resveratrol-containing foodstuffs, including red wine, peanuts,and pistachios (a list of foods and derivatives was provided to thevolunteers). In addition, antibiotics (within a month before the study),pregnancy/lactation, history of smoking, diagnosed chronic illness,taking medication or food supplements (within a month before the study),previous gastrointestinal surgery, habitual consumption of more than20 g of alcohol/day, being a vegetarian, or being on a weight lossregimen were included in the exclusion criteria. The study was fullyexplained to the volunteers, who provided written informed consentbefore participating. Three days before and during the intervention,the participants consumed a low-polyphenol diet, supervised by a nutritionist,and based on grilled meat-fish, rice, pasta, low-fat cheese, and bread,with a reduced contribution (1 portion/day) of the following: fruitsand vegetables, legumes, nuts and seeds, juices, olive oil, coffee,and tea. The volunteers consumed one daily hard gelatin capsule containing150 mg of RSV (98% purity) from 29 Samples were processed underanoxic conditions in an anaerobic chamber  with an atmosphere ofN2/H2/CO2 (85:5:10) at 37 °C.Briefly, aliquots were prepared with 10 g of stool samples and dilutedwith l-cysteine hydrochloride-supplemented nutrient brothusing a stomacher for homogenization. Filtered suspensions were inoculatedinto Wilkins-Chalgren anaerobe medium  containing l-cysteine. The suspensions contained 30μM of each standard dissolved in DMSO . The compounds  were individually added to the broth and incubatedunder anoxic conditions in the anaerobic chamber described above.Fecal inocula from each volunteer plus broth with no compounds andeach compound plus medium with no fecal inocula were used as controls.Three replicates were carried out using each fecal suspension andcompound. The recovery of all metabolites assayed in vitro was >95%.Preliminary fecal incubations were carried out at different times,that is, 1, 2, 4, 7, and 10 days. Finally, all the incubations wereperformed for 7 days. After 7 days, samples were collected, processed,and further analyzed by UPLC-QTOF-MS and (or) gas chromatography–massspectrometry(GC–MS) analyses.Twelve participants  provided baseline stool samples. The volunteerswere chosen after in vivo analyses to confirm catabolic steps in vitro.Fecal suspensions and culturing experiments were conducted as previouslydescribed.30 Besides, as described elsewhere, urine samples were treatedovernight with glucuronidase and sulfatase to deconjugate phase-IIconjugated metabolites.30 Non-hydrolyzedurine samples were analyzed by UPLC-QTOF-MS and hydrolyzed samplesby UPLC-QTOF-MS and GC–MS. Stool samples were also providedbefore and after RSV consumption for 7 days and processed as previouslydescribed.31 Fecal samples from fermentationexperiments were extracted with ethyl acetate plus formic acid andprocessed as reported elsewhere.29 Bothfecal and in vitro-fermented fecal samples were analyzed by UPLC-MS-QTOFand GC–MS. In the case of GC–MS, urine, feces, and fecalfermentation samples were analyzed with and without derivatization.For derivatization, the evaporated residues of processed samples weredissolved in 30 μL of pyridine and converted to trimethylsilylderivatives by adding 30 μL of N,O-bis-(trimethylsilyl) trifluoroacetamide containing 1% trimethylchlorosilaneas previously described.21 In the non-derivatizedsamples, the residues after speed vacuum evaporation were dissolvedin acetone and injected into the GC–MS equipment.The urine samples(baseline and after 7 days of RSV consumption) were centrifuged, filteredthrough a 0.22 μm PVDF filter, and diluted with water containing0.1% formic acid. Diuresis was standardized by measuring the urinaryexcretion of creatinine, as previously reported.30 Briefly, the analyses were performed on an Agilent 1290Infinity UPLC system coupled to a 6550 accurate-mass quadrupole-time-of-flight(QTOF) mass spectrometer using an electrospray interface (Jet Stream Technology), using chrysinas an internal control of the ionization signal. Spectra were acquiredin the m/z range of 100 to 1100in a negative polarity mode and at an acquisition rate of 1.5 spectra/s.Data were processed using Mass Hunter Qualitative Analysis software, which lists and rates possible molecularformulas consistent with the accurate mass measurement and the actualisotopic pattern. A target screening strategy was applied to qualitativelyanalyze possible metabolites that could be present after RSV consumption.In addition, targeted MS/MS analysis provided additional informationto achieve a reliable compound identification. MS/MS product ion spectrawere collected at m/z 50–800range using a retention time window of 1 min, collision energy of20 V, and an acquisition rate of 4 spectra/s.A previously validated methodwas used to analyze RSV and derived metabolites.21 AnHP5-MS (30 m × 0.25 mm in inner diameter and a film thicknessof 0.25 μm) phase capillary column was used with helium as acarrier gas at a constant rate of 1 mL/min, and the temperature ofthe injector and MS source was maintained at 200 °C. The sampleswere analyzed in a splitless mode. The MS was operated in the electronionization mode with an ionization energy of 70 eV, and the mass spectrumwas acquired in a positive electron impact (70 eV). Two methods withdifferent MS parameters were performed depending on the metabolites.First, the metabolites 3HDB and 4HDB were analyzed without silylation.In this case, the selected ion monitoring acquisition type was chosenfor the 198 and 107 m/z ions, segment retention timeswere fixed between 21 and 24 min in high-resolution mode, dwell timewas set at 25 ms, and cycle time of 13.96 Hz, and finally, an electronmultiplier voltage (EMV) of 1700 was calculated. The second methodologywas used for silylated metabolites, that is, RSV, DHRSV, PINO, LUNU,DHP, DHST, and 4HST. In this case, the MS parameters were as follows:full scan mode acquisition type with a threshold of 1000, scan massrange from 50 to 800 Da at 2.0 scan/sec, a scan speed of 1.562 u/s,a cycle time of 502.60 ms, and finally, a calculated EMV of 1360.21As recently described, sampleswere analyzed using an HP 8890 gas chromatograph with an HP 5977Bmass selective detector (Agilent).trans-stilbene (3HST), which only differed in the −OHposition. Finally, the quantification of RSV and derived metaboliteswas determined by interpolation of the calibration curves obtainedwith their corresponding standards in the urine and fecal matrices.In addition, using extracted ion chromatograms (EICs) for area calculationand quantification reduced the possibility of misinterpreting overlappingpeaks.Directcomparison with available standards was used to identify differentmetabolites. In addition, their spectral properties, molecular masses,and fragmentation patterns were used to confirm the identification.These criteria were also used to identify some metabolites tentativelywhen no standards were available. For example, in the absence of standards,tentative identification was approached for LUNU and DHST conjugates,whose exact masses coincided with the expected metabolites, and furtherhydrolysis released the corresponding free forms, which were identifiedwith the available standards. In the case of tentative phase-II 4HDBconjugates [glucuronides and (or) sulfates], its presence in urinewas assumed since free 4HDB was identified using the correspondingstandard in hydrolyzed urine after enzymatic treatment. Besides, tentativeidentification was also approached in possible isomers with identicalmass to available standards, for example, 4HST and 3-hydroxy-https://www.metaboanalyst.ca), a heatmap was performed with normalized urine LUNU conjugate values(log-transformed) after consuming RSV for 7 days to group high andlow LUNU-producers. Data plots were performed using SigmaPlot 14.5. Statistical significance wasset at *P < 0.05.Quantification of metaboliteswas expressed as mean ± SD. The statistical analyses were carriedout using the SPSS software, v. 27.0.1.0 . A multinomial logistic regression model was applied to evaluatethe relationship between metabotypes and age, BMI, and sex. UsingMetaboAnalyst 5.0 (n = 195) were recruitedin two locations in Spain, namely, Madrid (n = 91)and Murcia  to addressany possible influence of geographical location on metabotype distribution.All the recruited participants were Europeans, and the mean age rangedfrom 18 to 81 years (41.5 ± 14.4), the BMI from 18.5 to 44.6,and there were more females (63.6%) than males (36.4%) ( (36.4%) 1. No volcis-RSV,DHRSV, DHST, and RSV were detected in LUNU-producers  were selected. The in vitro experimentswere not conceived to simulate the physiological conditions. Instead,the aims of the in vitro incubations were (i) to confirm the in vivoresults and (ii) to explore if the metabolic machinery of the gutmicrobiota was capable of producing some metabolites that were notdetected in vivo 4. Therefa), which was dehydroxylated at the 5-positionto produce LUNU (step b). Then, a further 3-dehydroxylationyielded the metabolite 4HDB (step c). Notably, the metabolite3HDB was not detected in any sample in vivo or in vitro, confirmingthe lack of dehydroxylase activity of the gut microbiota at the 4′-positionof LUNU (step d). Furthermore, some DHST was also detectedafter RSV incubation (step e) but only in 2 of the 6samples of LUNU-producers, showing similarities to those observedin vivo. Thus, this confirmed that the direct 5-dehydroxylation ofRSV to produce DHST was a minor pathway, probably due to the presenceof the double bond of the 4-styrylphenol core in RSV. In addition,no further dehydroxylation of DHST to yield 4HST was observed (step f), confirming the non-production of 4HST in LUNU-producers(g), which also reinforcedthe lack of dehydroxylation at the 4-position of DHST. Furthermore,the lack of 4-dehydroxylation of stilbenes and dibenzyls was alsoconfirmed in vitro since neither PINO (step h) nor DHP(step i) were detected in any sample, confirming thein vivo results.First, the incubation of RSV in stool samples fromLUNU-producersyielded DHRSV 4, step aroducers3. Finallroducers3, we coua). However, LUNU and 4HDB were not detected inthese samples (steps b and c), confirmingthat LUNU non-producers cannot dehydroxylate at the 3- or 5-positions,in agreement with in vivo results (d). Inthe same line, DHST, 4HST, or 3HST were not detected in LUNU non-producers.Finally, as in LUNU-producers, PINO and DHP were not detected, suggestingthat the gut microbiota cannot dehydroxylate these dibenzyls and stilbenesat the 4- (or 4′)-position, independently of the capabilityto dehydroxylate at the 3- and 5- positions. Overall, all these resultsconfirmed the in vivo findings.In LUNU non-producers, DHRSV was readily producedafter RSV incubation in both LUNU-producers and non-producers,confirming that the 4-styrylphenol reductase activity was common inboth metabotypes. This fast reduction could explain the absence of4HST in vivo and in vitro, despite the fact that LUNU-producers couldtheoretically dehydroxylate DHST at the 3-position to produce 4HST.Similarly, the incubation of 4HST only yielded 4HDB (step n), which was detected in both LUNU-producers and non-producers, furtherconfirming the presence of the 4-styrylphenol reductase activity inboth LUNU metabotypes.The incubation of DHRSV andLUNU with samples from LUNU-producersconfirmed steps a, b, c, and e) but not at the 4-position. This reaction is favored in dibenzyls (DHRSV and LUNU) versusstilbenes (DHST). On the other hand, individuals from the metabotypeLUNU non-producers cannot dehydroxylate dibenzyls or stilbenes andonly produce dibenzyls from stilbenes when the corresponding precursoris available, that is, RSV, DHST, or 4HST. However, this reductionrequires the 4-styrylphenol core (not present in PINO).Overall, the individuals from the metabotypeLUNU-producers candehydroxylate at the 5- and 3-positions . However, despite thelow-polyphenol diet of individuals, these compounds were consistentlypresent in all the samples, that is, urine and fecal samples beforeand after RSV consumption and fecal cultures of all compounds in controlsand after fermentation with stools from both LUNU metabotypes. Noclear differences were observed when comparing urine and feces beforeand after consuming RSV or between LUNU-producers and non-producers.In the case of fecal fermentations, there was a tendency to increasethe amount of hydroxyphenyl benzoic, acetic, and propanoic acid derivativesas well as resorcinol, especially after incubation of DHRSV (resultsnot shown). However, all these compounds were already present in thecontrols, preventing significant changes from being quantified inthe current design.n = 195).However, this distribution differed depending on the geographicallocation (P = 0.039), that is, 68.3% of LUNU-producersand 31.7% of LUNU non-producers in Murcia, while the distributionwas 81.3% of LUNU-producers and 18.7% of LUNU non-producers in Madrid.We next explored the possible association of LUNU metabotype distributionwith participants’ BMI, sex, and age, considering all the individualsand each specific location. No significant associations between metabotypesand BMI or age were observed in all individuals or by specific geographiclocation. However, there were more female LUNU non-producers thanmale non-producers in Murcia   compared to Madrid, where non-significant differencesbetween sexes were observed  . The significant differences were also observed whenall the individuals were considered in the analysis  , and the interaction between locationand sex was also significant in the metabotype distribution (P = 0.027).The distribution ofLUNU metabotypes was 74.4% of LUNU-producers and 25.6% of LUNU non-producerswhen all the individuals were considered  of LUNU-producers after consumingRSV for 7 days revealed the presence of high, medium, and low producersof the four LUNU metabolites, that is, glucuronides 1 and 2 and sulfates1 and 2 5A.In ad33 These transformations depend on the individuals’gut microbiota and critically affect (poly)phenol activity. Thereis notable interindividual variability in the metabolism and healtheffects of (poly)phenols. Thus, the stratification of individualsaccording to their metabotypes associated with (poly)phenol metabolismhas been proposed to understand this variability, which could be crucialin the context of personalized nutrition.34 The urolithin and equol-ODMA metabotypeshave been characterized in large cohorts and arise from particularbacterial species/strains with specific enzymatic machinery to catalyzecharacteristic reactions on the phenolic core.38The catabolism of each specific (poly)phenolby the gut microbiotadepends on the number, type, and position of specific functional groups,stereoisomerism, and polymerization degrees.7In the present study, weidentify two metabotypes associated withRSV metabolism by the human gut microbiota for the first time, thatis, LUNU-producers and LUNU non-producers. The gut microbial-derivedmetabolites of RSV metabolism in the LUNU-producer metabotype wereDHRSV (and conjugates), DHST (and conjugates), and 4HDB  phenols’ metabolism by the gut microbiota, reflectingthe higher or lower abundance of bacteria capable of metabolizingthe (poly)phenols and derived metabolites, and it is directly influencedby external factors phenol ingested, sample collectiontime, the sensitivity of the analytical procedure, etc.), which meansthat this gradient is not a genuine feature of each volunteer.21 The metabolite 4HDB was consistently found inall LUNU-producers, that is, all showed 3- and 5-dehydroxylase activities.However, no complete conversion from LUNU to 4HDB was observed inany case. Therefore, taking into account the relative abundance ofLUNU and the difficult determination of 4HDB,21 LUNU is a valid marker to stratify volunteers according to the abilityof their microbiota to metabolize RSV. Finally, other significantfindings of the present study are (i) the lack of dehydroxylase activityof the gut microbiota at the 4- (or 4′)-position in dibenzylsand stilbenes, and (ii) the specific 4-styrylphenol reductase activitypresent in both metabotypes. The double-bond reduction is probablythe first and most universal reaction in gut microbiota’s catabolismof (poly)phenols, including hydroxycinnamic acids, flavonoids, stilbenes,and so forth.20 However, the existence of a specific4-styrylphenol reductase activity for stilbenes, as evidenced by theinability to reduce pinosylvin lacking the 4-hydroxy group, is reportedhere for the first time to the best of our knowledge.We have confirmed herethe metabolite 4HDB, but not 3HDB, producedafter 3-dehydroxylation of LUNU ,which was recently identified in volunteers after consuming RSV.39 However,in the present study, these metabolites were consistently found inall the samples, that is, in vivo and in vitro, including the controls.This agrees with previous studies that reported these metabolitesas final degradation products in most (poly)phenols, including procyanidins,flavanones, hydroxycinnamic acid derivatives, anthocyanins, and soforth.40 Nevertheless, in the fecal fermentations,the increase of phenolic acids and resorcinol suggested the productionof these metabolites in both LUNU metabotypes, especially after DHRSVincubation. Thus, these results suggest that scission or survivalof the meta-dihydroxyphenyl ring and (or) the openingof aromatic rings might occur in DHRSV to yield these metabolites.Overall, our results suggest that producing these metabolites is nota hallmark of RSV metabolism. However, we acknowledge that the invivo study’s design was not specifically addressed to characterizethese metabolites, taking into account their promiscuous presencein all the samples. In addition, due to solubility problems, the lowconcentration of stilbenes and dibenzyls incubated in fecal fermentationscould have prevented more significant differences versus controlsin the production of these final metabolites.Some phenolicacids, including hydroxyphenyl benzoic, acetic, andpropanoic acid derivatives, have been linked specifically to RSV metabolismby the gut microbiota in mice.14 In the urolithin metabotypes, aging is the mainfactor affecting metabotype distribution.28 In contrast, BMI, sex, dietary patterns, or other characteristicshave not been conclusively associated with urolithin metabotypes.28LUNU metabotypeswere not associated with BMI or age. However,the percentage of female versus male LUNU non-producers was higherin Murcia than in Madrid, even though there were more female thanmale participants in both locations. The explanation for this apparentassociation requires further research. In the case of the equol- orODMA-producer metabotypes, no clear associations for any particularfactor have been consistently detected, including dietary patterns,age, education level, anthropometric values, and race or ethnicity.Coriobacteriia are known.33 For example, the specific dehydroxylase activityof some gut bacteriahas been reported to be crucial in the metabolism of EA to yield urolithinmetabotypes in which Gordonibacter urolithinfaciens can convert EA to urolithin C , a commonmetabolite in urolithin metabotypes.41 Incontrast, Ellagibacter isourolithinifaciens can convert EA to isourolithin A, a characteristic metabolite ofthe urolithin metabotype B.42 Notably,the abundance of these species is very low in the urolithin non-producers(metabotype 0), supporting their inability to produce urolithins.43 In addition to EA, G. urolithinfaciens and E. isourolithinifaciens can metabolizecaffeic, dihydrocaffeic, chlorogenic, and rosmarinic acids, but notother flavonoid and non-flavonoid (poly)phenols. Notably, these bacteriadid not dehydroxylate or reduce the styrene double bond of RSV andother phenolics, suggesting a high substrate specificity.44 In contrast, the coriobacteria Slackia equolifaciens, Adlercreutziaequolifaciens, and Adlercreutzia rubneri strain ResAG-91T.8 can convert RSV into DHRSV,45 butthe gut species capable of catalyzing the sequential dehydroxylationsin stilbenes and dibenzyls remains to be elucidated.The dehydroxylase activity and substrate specificity of theclass 17 it is plausible that human gut microbiotas with different compositionsand functionalities might affect the outcome of trials also for RSVsince the biological activity of the microbial-derived metabolitesmight differ from that of RSV.47 In this regard, recentstudies have described low biological activities for DHRSV and LUNUin animal models, including caloric restriction mimetics,46 and their impact on insulin sensitivity.47 In contrast, other studies claim the high anticancerand anti-inflammatory activity of these metabolites.48 RSV, DHRSV, and LUNU conjugates were previously identifiedin plasma, urine, and mammary tissues of breast cancer patients.49 However, the lack of authentic standards of LUNU conjugates preventedtheir quantification in the bloodstream, urine, and breast tissues,which should be explored in further studies. Therefore, whether thesequential dehydroxylase activities of RSV that give rise to LUNUmetabotypes significantly impact human health deserves further research.Similarly, our ongoing studies investigate the possible gut microbiotaassociated with LUNU metabotypes. Overall, we show here the most detailedmetabolic pathway of RSV by the human gut microbiota reported to dateand describe these novel metabotypes associated with RSV metabolism.This study could contribute to understanding the substantial humaninterindividual variability in the health effects of dietary (poly)phenols,including RSV.As observedfor other (poly)phenols, such as EA and isoflavones,