Vibrio vulnificus, a foodborne pathogen, has a high mortality rate. Despite its relevance to public health, the identification of virulence genes associated with the pathogenicity of currently known clinical isolates of V. vulnificus is incomplete and its synergistic pathogenesis remains unclear. Here, we integrate whole genome sequencing (WGS), genome-wide association studies (GWAS), and genome-wide epistasis studies (GWES), along with phenotype characterization to investigate the pathogenesis and survival strategies of V. vulnificus. GWAS and GWES identified a total of six genes  associated with the pathogenicity of clinical isolates related to nucleotide/amino acid transport and metabolism, cell membrane biogenesis, signal transduction mechanisms, and protein turnover. Of these, five were newly discovered potential specific virulence genes of V. vulnificus in this study. Furthermore, GWES combined with phenotype experiments indicated that V. vulnificus isolates were clustered into two ecological groups (EGs) that shared distinct biotic and abiotic factors, and ecological strategies. Our study reveals pathogenic mechanisms and their evolution in V. vulnificus to provide a solid foundation for designing new vaccines and therapeutic targets. Primary septicemia is the most lethal consequence of V. vulnificus foodborne infection, with a mortality rate of more than 50% in immunocompromised patients, while wound infection has a mortality rate of 17%  analysis of variations in the virulence-correlated gene (vcg) . Thus, expanding the V. vulnificus genome dataset is necessary for a comprehensive reconstruction of the phylogenetic structure of the species.Given its potential as an emerging infectious disease, an understanding of the variation in and distribution of ne (vcg) , 16S rRNne (vcg) , multi-lne (vcg) , multi-lne (vcg) , and thene (vcg) . Traditilineages . HoweverV. vulnificus pathogenicity (V. vulnificus remain unknown. Genome-wide association studies (GWAS) aim to capture associations of genotypes with phenotypes by testing hundreds of thousands of genetic variants across many genomes. GWAS have been successfully used in bacterial research to uncover antibiotic resistance, virulence, host specificity, and prognosis and can potentially be applied to any heritable bacterial traits , to detect co-selection signals in a group , and no . vulnificus isolates and their isolation sources by GWAS. Then, we systematically examine co-adaptation patterns among V. vulnificus genetic variants, including core and accessory genome variants using GWES. We identify six genes linked to the pathogenicity of V. vulnificus clinical isolates, of which five were newly discovered in this study. We also uncover complicated gene interactions indicating that core and accessory genomes have co-evolved to produce coadapted gene complexes that encode distinct ecological strategies. Our findings indicate that these isolates and the genetic variants encoding them can be characterized by hierarchical clustering into groups that reflect patterns in the evolution of V. vulnificus. Finally, we explore the conditions that likely shaped V. vulnificus evolution by characterizing the phenotypes of isolates under different environmental conditions. Our results suggest that V. vulnificus has gradually altered its fitness landscape through co-adaptation.In this study, we first analyze the whole genome sequences of 518 V22.1V. vulnificus isolates used in this study is listed in https://www.ncbi.nlm.nih.gov/genome/browse#!/prokaryotes/189/).In total, 518 isolates  were used in this study, including 325 newly sequenced isolates  and 193 publicly available isolates . Detailed sampling information for the 2.2All isolates were taken from the freezer (-80°C), streaked on Columbia blood agar (CBA) plates, and grown at 37°C for 24 hours. A single colony of a strain was transferred to 5 mL Luria-Bertani (LB) medium containing 2% NaCl and cultured at 37°C to the exponential phase with shaking.2.3DNA was extracted from samples using the QIAGEN UltraClean® Microbial DNA Isolation kit, Catalog no. 12224-50, as per the manufacturer’s instructions.2.4De novo assembly of the filtered reads was performed using Shovill version 1.0.4  . De novoon 1.0.4  with sta2.5SNPs identified from aligning the assemblies to the reference genome YJ016 with the NUCmer module in the software MUMmer 3.0  were useAll genomic sequences were annotated using Prokka . We used2.6. vulnificus isolates, including 87 clinical and 420 environmental isolates, were analyzed using the software Pyseer , and k-mers-based GWAS can reflect diverse genetic events, including SNPs, insertion and deletion of accessory genes, and cover the noncoding regions, including those related to transcriptional and translational regulation, overcoming limitations of analysis at the level of SNPs and non-core gene insertions/deletions , this method groups cDBG nodes pinpointed by the association model into subgraphs defined by their neighborhood in the initial cDBG. DBGWAS does not require prior annotation or reference genomes. Importantly, it is also computationally efficient.DBGWAS is a 2.7. vulnificus isolates using the GWES tool SpydrPick . Bacteria were cultured as described above (“Culture Conditions”).2.8.2V. vulnificus were incubated in 2% NaCl-LB medium at 37°C to reach an A600 (absorbance) of 0.8. We estimated the growth of each culture every hour for 24 hours at different temperatures , different osmotic conditions , and varied pH levels  using A600. All experiments were carried out in triplicate and repeated three times.Isolates of 2.8.3A600 of 0.8. Swimming ability was recorded by measuring the diameter of the colony at 12, 24, 36, 48, and 72 h after inoculation onto a swimming plate at 37°C. Swarming ability was recorded after 72 h on a swarming plate at 37°C.Isolates were inoculated on a swimming plate (LB media containing 0.3% agar) and a swarming plate (LB agar with 3% NaCl) after being cultivated at 37°C overnight to an 2.8.4A590 >0.06), low-grade positive (0.03< A590 ≤0.06), or negative (A590 ≤0.03).The overnight bacterium liquid was added to a 24-well cell culture plate at 37°C for 24 h without shaking; 2% NaCl-LB medium served as a negative control. Each well was rinsed three times with 1× phosphate-buffered saline (PBS) after the supernatant was removed. Adherent bacteria were fixed with methanol for 15 min and then methane was aspirated from the culture wells, leaving them to air dry naturally. Plate cells were then stained with 0.1% crystal violet for 5 min, followed by washing with 1× PBS three times. The bound dye was dissolved with 33% glacial acetic acid. Finally, the optical density of the solution was measured using a Multiskan Spectrum at 590 nm . The exp2.8.52, 5% CO2, and 5% H2 was used to create anaerobic conditions. The isolates were inoculated on a Columbia blood agar plate and cultivated at 37°C for 5 days under anaerobic conditions.An anaerobic chamber (Gene Science) filled with a gas mixture comprised of 90% N2.8.6The VITEK-2 automated microbial identification and drug sensitivity analyzer (BioMérieux) was employed for measuring biochemical parameters and antimicrobial susceptibility.33.1V. vulnificus isolates from 14 countries between 1964 and 2018. Isolates included 193 isolates from the NCBI database – 58 clinical, 124 environmental, and 11 unspecified; and 325 isolates from newly collected samples – 29 clinical and 296 environmental (V. vulnificus isolates.We performed whole genome variation detection of 518 V. vulnificus YJ016 as the reference sequence divided the species into six well-defined lineages (A maximum likelihood (ML) tree of the 518 isolates based on core-genome SNPs with lineages Figure 13.2V. vulnificus, we analyzed GWAS results of the 518 V. vulnificus isolates. We used Pyseer to identify variants in the whole genome sequences of V. vulnificus associated with clinical and environmental phenotypes based on SNPs, insertion and deletion of accessory genes, and k-mers, respectively. To reduce the false positive rate, we used a strict threshold to judge the analysis results in combination with prior knowledge  >8.76; V. vulnificus, located on 567 coding genes at the k-mers level. Twenty-eight genetic variants were identified at both the SNPs level and k-mers level, and eleven genetic variants were identified at both the insertion and deletion of accessory genes level and k-mers level. These variants involved genes that encode structural proteins, transport proteins, metabolic proteins, and signal proteins; thus, they can directly or indirectly affect the pathogenicity of V. vulnificus through their roles in energy production and conversion, cell cycle control, translation, ribosomal structure and biogenesis, replication, recombination, and repair, cell wall/membrane/envelope biogenesis, cell motility, intracellular transport, nucleotide/amino acid/carbohydrate/coenzyme/lipid transport and metabolism, inorganic ion transport and metabolism, post-translational modification, protein turnover, chaperone functions, and signal transduction mechanisms. Moreover, the purH  (pldA (encoding phospholipase A1)  , and pldpase A1)  genes idk-mers and clinical vs. environmental phenotypes. Overall, we found 100 nodes related to the clinical phenotypes of V. vulnificus, located on 20 coding genes [P-value = 2.10 x 10-7, -log10 >6.68; purH  were newly discovered in this study , 28,508 co-adaptation groups were identified that could be further subdivided into 166 co-adaptation networks containing 464 core-genome SNPs  on 34 core genes and 750 accessory genes (We used SpydrPick on the whole genome sequences of 518 V. vulnificus virulence genes. The high-frequency genes in the largest co-adaptation network (N1) were flgK, flgE, and flgL . The purH gene has previously been shown (by deletion mutants) to alter the lethality and cytotoxic activity of V. vulnificus in mice and is a virulence gene of V. vulnificus  regulates the enzyme for synthesis of cyclic di-GMP. Cyclic di-GMP has emerged as one of the most common and essential bacterial second messengers, with the ability to influence biofilm formation, motility, virulence, the cell cycle, differentiation, and other processes, hence influencing V. vulnificus pathogenicity (yiaV (encodes inner membrane protein yiaV precursor) (dsbD (encodes disulfide interchange protein dsbD precursor) affect antimicrobial drug tolerance of Escherichia coli (ramA (encodes R-stereoselective amidase) affects antimicrobial drug tolerance of Klebsiella pneumoniae   and dsbDia coli . The geneumoniae . The genlnificus .3.4The largest co-adaptation network (N1) included most of the interacting SNPs, which challenged interpretation due to its abundance of pairwise interactions. We thus used hierarchical clustering based on N1 variants to classify variants into two ecological groups (EGs), EG1 and EG2  identified by SpydrPick.Given the complex structure of the co-adaptation network N1, we also did a cluster analysis of the coadaptation loci, which separated the coadapted complex loci into two tiers. Tier 1 loci variants were stable in EG1 but highly variable in EG2, containing genes We detected 4 co-adaptation networks  involving SNPs that were interactions of the type “incompatibility”, meaning that when one or more genes exist, at least other genes are absent. We detected a further 161 co-adaptation networks  involving accessory genes that were “genome island-like”.3.5V. vulnificus grew best at 2% NaCl concentration, but EG2 isolates were more tolerant to high salt environments than EG1 isolates  favor the growth and reproduction of V. vulnificus. Alkaline conditions (pH 8-9) not only impede the growth and reproduction of V. vulnificus but also inhibit its activity  to measure relevant phenotypes: growth rate under temperature challenges , osmotic challenges , and pH challenges ; motility assay, biofilm formation ability, survival under anaerobic conditions, biochemical parameters, and antimicrobial susceptibility. Neither EG1 nor EG2 isolates grew at 4°C or 45°C. EG2 isolates had a faster growth rate than EG1 isolates at 37°C. All isolates Figure 6efazolin .4V. vulnificus for the first time. In this study, we used a robust mix of WGS, GWAS, and GWES, as well as phenotype description, to analyze 518 genomes of V. vulnificus isolates collected worldwide from a variety of sources to investigate genetic diversity, virulence genes, the drivers of genotypic and phenotypic evolution, and the emergence of coadapted gene complexes. Our phylogenomic analysis indicates that V. vulnificus has diverged into six distinct lineages, L1 through L6. This result improves our resolution of V. vulnificus population structure, identifying one more lineage than a previous study  were newly discovered in this study. Our findings can be applied to find candidate targets for vaccine development against V. vulnificus and provide an important foundation for further insight into the pathogenic mechanism of this species. Future studies should consider gene knockout and in vivo infection experiments on these targets.We were successful in examining potential epistatic interactions using GWES to identify co-evolved proteins and thus hypothesize potential networks of functionally linked genes. Our GWES results combined with GWAS results showed that a total of six genes correlate with the pathogenicity of V. vulnificus isolates could be separated into two EGs based on core-genome SNPs by GWES. To further elucidate the evolutionary drivers behind the divergence of the two EGs, we conducted phenotypic experiments. The two EGs exhibit distinct ecologies based on our investigation of V. vulnificus phenotypes, genomic evolution, and distribution. EG1 members appear to be more adapted to low-nutrient, lower-salinity, and brackish-like water environments, whereas EG2 members were more likely to come from rich-nutrient, higher-salinity, ocean-like environments. Our phenotypic experiments indicate EG2 isolates can withstand a wider range of stressors and have a competitive advantage in colonizing and growing in diverse hosts, which might explain why most isolates were obtained from this cluster. It was noteworthy that the proportion of clinical samples in EG1 isolates (1/59) was lower than that in EG2 isolates (86/459), indicating a lower virulence potential of this ecological group in humans. Interesting findings here include the clear presence of clinical isolates in both EG1 and EG2 that confirm work that “clinical isolates” are not phylogenetically grouped. We speculate that these phylogenetically distant clusters occupy distinct niches that may differ in their natural hosts or habitats, and this physical isolation may lead to obvious evolutionary pressures that reduce the likelihood of encounters and recombination, leading to genetic isolation and the emergence of distinct ecotypes, with potentially devastating consequences for aquaculture and human health. Despite unresolved issues, our findings emphasize the central role of lateral motility in constructing ecologically significant variation within the species. We also found that the co-adaptation networks went through progressive stages, consistent with the previous research results in Vibrio parahaemolyticus  with distinct patterns of bacterial behavior in our investigation. Our study can be utilized to better understand the pathogenic mechanism and evolution of V. vulnificus.We combined WGS, GWAS, and GWES, as well as detailed phenotypic analysis of The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/J-XZ: Conceptualization, Data curation, Formal Analysis, Investigation, Methodology, Software, Validation, Visualization, Writing - original draft, Writing - review & editing. YY: Conceptualization, Writing - review & editing. Q-HH: Resources, Writing - review & editing. D-ZJ: Resources, Writing - review & editing. YB: Resources, Writing - review & editing. W-WX: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing - review & editing. LK: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing - review & editing. J-LW: Conceptualization, Funding acquisition, Project administration, Resources, Supervision, Writing - review & editing.


Antibiotic resistance has become a serious threat to global public health and economic development. Rapid and accurate identification of a patient status for antimicrobial resistance (AMR) are urgently needed in clinical diagnosis. Here we describe the development of an assay method for activity fingerprinting of AMR β-lactamases using panels of 7 β-lactam antibiotics in 35 min. New Deli Metallo β-lactamase-1 (NDM-1) and penicillinase were demonstrated as two different classes of β-lactamases. The panel consisted of three classes of antibiotics, including: penicillins , cephalosporins  and carbapenems (meropenem and imipenem). The assay employed a scheme combines the catalytic reaction of AMR β-lactamases on antibiotic substrates with a flow-injected thermometric biosensor that allows the direct detection of the heat generated from the enzymatic catalysis, and eliminates the need for custom substrates and multiple detection schemes. In order to differentiate classes of β-lactamases, characterization of the enzyme activity under different catalytic condition, such as, buffer composition, ion strength and pH were investigated. This assay could provide a tool for fast diagnosis of patient AMR status which makes possible for the future accurate treatment with selected antibiotics. Since the discovery of penicillin, a broad range of antibiotics have been discovered which have enhanced our ability to treat bacterial infections. Antibiotics have played a critical role in medicine, as well as in other fields . HoweverPhysicians are on the front lines in the effort to treat patients infected with multi-resistant pathogens. However, in order to determine the most effective course of treatment, physicians require AMR diagnostics capable of rapidly and specifically determining the patient’s AMR profile. Although there are multiple mechanisms by which pathogens achieve resistance, the most common mechanism is the production of enzymes that catalytically inactivate the antibiotics. Numerous physical and activity based methods have been developed to detect these AMR enzymes ranging from traditional methods, such as the disk method to new techniques, such as multiplex PCR, DNA microarrays, colorimetric assays, immune-chromatographic technique and thermometric analysis. Each technique has advantages and disadvantages. However, none of these techniques is capable of providing results/diagnostics of multi-resistance in a clinically relevant timeframe. Efforts must be renewed to address this issue . Rapid, There are several methods that have been conventionally used to detect antibiotic resistance in recent decades, such as the double dilution method and susceptibility testing . HoweverHere we describe the development of a novel AMR strategy that profiles AMR enzymes by testing a panel of antibiotics. The diagnostic strategy specifically and efficiently identifies the AMR enzymatic activity profile using a flow injected thermal biosensor device. For these studies, we have chosen to use a panel of antibiotics that are widely used in clinical settings. The panel consisted of three different classes of antibiotics: Penicillins , Cephalosprins  and Carbapenems . In order to verify the profile based characterization, two classes of AMR β-lactamases: New Deli Metallo β-lactamase-1 (NDM-1) and penicillinase were chosen as examples in the study. The effects of the reaction conditions, such as: ion strength, buffer composition, and pH on the activity profiling of the AMR β-lactamases catalyzing this panel of antibiotics were investigated.Penicillin G, Piperacillin, Ceftriaxone, Cefepime, Cefazolin, Meropenem were purchased from Solarbio Co., Ltd. . Imipenem was bought from Fresenius Kabi . HEPES was obtained from Sangon Biotech Co., Ltd. . All other reagents were purchased from Sigma-Aldrich Co . The controlled pore glass (CPG) beads (125–140 μm particle diameter and 50 nm pore size) were originally purchased from VEB Trisola, Steinach, Germany. The construction of plasmid encoding NDM-1, expression and purification of recombinant NDM-1(rNDM-1) were performed as previously described . The purPhosphate buffer (PB) and HEPES buffer in different ionic strengths  and pHs  were prepared as running buffers and for sample preparation. All water used for preparing the buffers and samples was from a Milli-Q system .Stock solutions of each of the antibiotics: Penicillin G, Piperacillin, Ceftriaxone, Cefepime, Cefazolin, Meropenem, and Imipenem, were prepared in PB and HEPES at the various ionic strengths and pHs, which were then serially diluted: 10 mM, 5 mM, 2.5 mM, 1.25 mM, and 0.625 mM.The Enzyme Thermal biosensor (ET) is a flow-injected analytical device  has been employed in this study. The enzyme, e.g. β-lactamase, was immobilized on spherical porous glass beads (CPG), loaded in an enzyme column and substrate samples were injected (e.g. antibiotics) with running buffers at various concentrations. Meanwhile the simultaneously generated heat can be precisely and continuously detected with high resolution thermistor sensors . The then is the number of moles of product; ΔH is the enthalpy change of the biochemical reaction; ΔT is the temperature change of the reaction system; SC is the heat capacity of the reaction system. This formula shows that there is a linear relationship between the temperature change and the substrate concentration. Thus, the peak height or area can be used for determination of an analyte concentration  and high resolution thermistors in coupled with signal amplification.-1 and sample volumes of 30 and 75 μL were used. However, for the comparative study, the response signals were adjusted to 10 μL/analyte. Each sample measurement was performed in triplicate.The enzyme column was loaded with ca. 50 IU immobilized β-lactamase (e.g. penicillinase or NDM-1) onto CPG. The working temperature was set at 25°C. A flow rate of 0.6 mL minst, 3rd and 4th generation cephalosporins, respectively.We first evaluated effects of different buffers on degradation of 7 β-lactam antibiotics: penicillin G, piperacillin, ceftriaxone, cefepime, cefazolin, meropenem and imipenem. Among these 7 antibiotics, penicillin G and piperacillin belong to penicillin class; ceftriaxone, cefepime, cefazolin belong to cephalosporin class; and meropenem and imipenem belong to carbapenem class. Cefazolin, ceftriaxone and cefepime are representative of the 1Studies were performed to determine the effect that buffer had on assay sensitivity 3. In thAs penicillinase can only efficiently catalyze penicillin class, only penicillin G and piperacillin response curves were presented in buffers of PB , cephalosporins  and Carbapenems , were investigated using PB Figure 4In contrast to Penicillinase in The pH effects on the catalytic reactions were evaluated in PB and HEPES buffers at pHs 6.8, 7.2, 7.6 and 8.0 Figure 6As enzymatic degradation of antibiotics is the most common mechanism of AMR, we have developed a thermometric biosensor strategy for the rapid identification of AMR enzymes in 35 min using a panel of antibiotics to profile their substrate specificity. In this study, a substrate specificity profile for NDM-1 and penicillinase has been created using a panel of 7 antibiotics including penicillin G, piperacillin, cefepime, ceftriaxone, cefazolin, meropenem, and imipenem. The results show that the strategy is both feasible and reliable.The concentration dependent calibration curves were prepared using PB and HEPES. The results show that buffer conditions can be employed to further increase the informational content of the assay scheme should the antibiotic profile alone be inadequate to identify the AMR enzyme. The results of the buffer and pH effects on the profiles provided the possibility for differentiation of classes of antibiotics, e.g. carbapenem and peniciilin or cephalosporin in which the carbapenem antibiotics showed highest sensitivity vs cepalosprin which showed the smallest changes in buffer concentration of 100 mM. This conclusion is also supported by the pH studies. Furthermore, the results also indicate that it is feasible to differentiate β-lactamases, e.g. penicillinase and NDM-1 by varying the buffer concentrations. The lowest buffer concentration (10 mM) gave the highest sensitivity for penicillinase reactions vs the lowest sensitivity for NDM-1 reactions but the highest sensitivity in 100 mM buffer.As expected, penicillinase was most active against the penicillin class of antibiotics, penicillin and Piperacillin. In contrast, NDM-1 could hydrolize all three classes of antibiotics in particular carbapenems. Compared to other methods (see The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.CS: Investigation, comparative study, data curation, formal analysis, visualization, writing - review & editing, XS: Data curation, formal analysis, investigation, methodology, validation, visualization, writing - original draft, writing - review & editing. YW, Investigation, data curation, formal analysis, LB: Funding acquisition, review & editing, supervision, resources. MM: Review &editing, English proofreading. CW: funding acquisition, supervision QM: project administration, formal analysis, writing - review & editing, funding acquisition, resources. BX: Conceptualization, project administration, data curation, formal analysis, methodology, writing - review & editing, funding acquisition, resources, supervision. All authors contributed to the article and approved the submitted version.


Background: Men diagnosed with Klinefelter syndrome (KS) commonly exhibit non-obstructive azoospermia or rarely having sperm in their ejaculate, rendering them traditionally considered sterile prior to the introduction of intracytoplasmic sperm injection (ICSI). The presence of mosaic KS may mask the classical phenotype, resulting in underdiagnosis throughout their lifetime. Surgical sperm retrieval through Microdissection Testicular Sperm Extraction (Micro-TESE) combined with ICSI has become the gold standard approach, maximizing reproductive outcomes in these individuals. However, it is noteworthy that approximately 7% of men with KS may exhibit sperm in their ejaculate, providing an opportunity for them to achieve biological parenthood through ICSI.Case Presentation: In this report, we present an exceptional case of a 45-year-old man with Mosaic KS and severe oligozoospermia who successfully achieved pregnancy utilizing ICSI with freshly ejaculated sperm. Remarkably, this case represents the oldest recorded instance of a man with Klinefelter syndrome fathering his own biological child using sperm derived from fresh ejaculate.Conclusion: Although this case is exceedingly rare, it underscores the critical importance of exhausting all possibilities to facilitate biological parenthood in men with KS before considering alternative options such as sperm donation or adoption. By recognizing the potential for successful conception using ejaculated sperm in this population, we can provide individuals with mosaic KS the opportunity to fulfill their desire for biological offspring. Klinefelter syndrome (KS), initially described by Dr. Harry Klinefelter in 1942, is characterized by bilateral gynecomastia, small testes, aspermatogenesis, reduced Leydig cell function, and elevated follicle-stimulating hormone (FSH) levels . In 1959Hypogonadism is a common symptom in KS regardless of genotype . Non-mosAzoospermia, is a consistent finding in KS, with only approximately 7% of KS males exhibiting detectable spermatozoa in their semen . Prior tThe wide phenotypic spectrum of KS contributes to underdiagnosis or delayed diagnosis Notably, KS is frequently diagnosed when individuals seek medical attention for infertility investigations. Consequently, the average age of diagnosis is in the mid-30s, and it is estimated that only 25%–50% of individuals with KS receive a diagnosis .The case presented herein focuses on a unique scenario involving a 45-year-old man with mosaic KS and severe oligozoospermia who achieved successful pregnancy through intracytoplasmic sperm injection (ICSI) using ejaculated fresh sperm. This is the first report of the oldest man with mosaic KS to father his offspring using ejaculated spermatozoa.By highlighting this exceptional case, we aim to increase awareness among healthcare professionals about the potential for successful assisted reproduction in older men with KS. It is crucial to recognize that advanced age in KS individuals should not be a deterrent when considering assisted reproductive technology as an option for achieving biological parenthood.Approval to publish this case study was granted from the local research committee and the local ethical board . Permission to publish was also granted and written consent was obtained from the family.A 45-year-old male and his 32-year-old female partner presented with a 3-year history of primary infertility. Semen analysis of the male partner revealed severe oligozoospermia on two occasions. He had a history of smoking ten cigarettes per day and was undergoing treatment with Sertraline for depression. The patient had no significant family, surgical, or social history.Physical examination of the male partner showed no abnormalities, with a height of 173 cm and a weight of 74 kg. He exhibited appropriate virilization and had no signs of gynecomastia. Local genital examination revealed normal consistency and small-volume testes, each measuring 6 mL using an orchidometer. The epididymis appeared normal bilaterally, and examination of the spermatic cord revealed intact vas deferens without evidence of clinical varicocele.6/ml, 42% forward progressive motility, and 0% normal morphology.Given the severity of oligozoospermia, the patient was advised to cryopreserve his sperm as a precautionary measure for future intracytoplasmic sperm injection (ICSI) procedures. Subsequent semen analyses were performed according to the To investigate the cause of severe oligozoospermia, the patient underwent chromosomal analysis, Y chromosome microdeletion testing, and hormonal profiling; Karyotyping by G- and C-banding was performed and revealed Mosaic Klinefelter Syndrome, the resolution of G-banding was no less than 550 bands  and 47, XXY (91 cells). No Y chromosome microdeletion was detected. Hormonal profiling revealed a morning serum total testosterone level of 14.6 nmol/L, sex hormone-binding globulin (SHBG) level of 47 nmol/L, estradiol (E2) level of 89 pmol/L, follicle-stimulating hormone (FSH) level of 12.5 U/L, and luteinizing hormone (LH) level of 9.9 IU/L.The female partner had a regular menstrual cycle and reported mild dysmenorrhea but no dyspareunia. She had no history of pelvic inflammatory disease or sexually transmitted infections. Pelvic ultrasound showed no uterine pathology, and the ovaries displayed no cysts, with a total antral follicle count of 13 (8 in the right ovary and 5 in the left ovary). Her Anti-Mullerian Hormone (AMH) level was 8.1 pmol/L (1.13 ng/mL).Genetic counselling was offered to the couple, but they declined termination of pregnancy if KS was detected. Instead, they opted for prenatal non-invasive genetic screening.In September 2020, a standard antagonist protocol was initiated, starting from cycle day 3, by administering a daily dosage of 225 IU of recombinant FSH  for 9 days. On cycle day 8, a gonadotropin-releasing hormone (GnRH) antagonist  was introduced to prevent premature ovulation. Follicle development was monitored by transvaginal ultrasound. Once three follicles measuring ≥17 mm in diameter were detected, hCG  was administered to trigger ovulation and induce final oocyte maturation. Oocyte retrieval was planned 37 h after trigger using a 17-gauge/35 cm single-lumen needle  through transvaginal follicle aspiration under ultrasound guidance. Four mature oocytes were retrieved. Simultaneously, a fresh semen sample was obtained from the male partner. A single motile sperm was selected and injected into each of the four metaphase II oocytes.Results: Three oocytes were fertilized normally, while one embryo arrested. The remaining two embryos developed into blastocysts by day 5 and 6 post-fertilization. The first blastocyst, graded as 3BB according to the Gardner scoring system , was suiThe patient’s β-hCG level was positive (159 mIU/mL) 9 days after embryo transfer, confirming clinical pregnancy. At 6 weeks and 4 days of gestation, a single intrauterine gestational sac with fetal heartbeat was documented via transvaginal ultrasound, confirming a viable pregnancy. Prenatal genetic screening showed no aneuploidy. The patient successfully delivered a live female offspring weighing 3050 g via caesarean section (due to failure to progress) at 40 weeks and 3 days of gestation in June 2021. The newborn showed no apparent abnormalities.This case highlights the successful utilization of ICSI in a 45-year-old male with mosaic KS to achieve a viable pregnancy using his own ejaculated spermatozoa. The advanced age of the patient further adds to the uniqueness of this case, positioning him as the oldest man reported to father his own biological child in the setting of KS. Natural conception in men with KS is exceedingly rare, however the first reported case claimed paternity was through blood-group matching . Since tIn our case, the diagnosis of KS was initially made during infertility investigations, consistent with previous reports indicating that approximately half of KS cases are diagnosed in adulthood due to fertility-related issues . The patThe intricate relationship between genotype and phenotype in Klinefelter syndrome (KS) remains an ongoing challenge to elucidate comprehensively. While the impact of a specific gene, SHOX, has been identified in contributing to certain phenotypic aspects of KS, the broader genotype-phenotype correlation remains enigmatic. Available literature underscores the notion that a singular genetic mechanism does not solely account for the wide-ranging phenotypic spectrum and variability seen in KS. Rather, it appears that a confluence of factors, including genetic, such as number of CAG repeats in the androgen receptor, parent of origin of the extra X chromosome or skewed inactivation of one X chromosome, additionally epigenetic, hormonal, environmental, and individual influences, likely interact to shape the observed variations .The presence of an additional X chromosome and the associated gene dosage effect, along with hypogonadism, can partially explain certain phenotypic features observed in KS .The cytogenetic analysis of our case demonstrated clearly a 9% mosaicism. However, it is crucial to recognize that the karyotype observed in lymphocytes may not directly correlate with sperm production or accurately represent the karyotype of the testicular germ cell line. Therefore, caution must be exercised when using karyotype as a predictive factor for sperm retrieval in cases of non-mosaic KS .Furthermore, men with KS may exhibit tissue-specific mosaicism, indicating that relying solely on peripheral karyotyping may fail to capture the mosaic status of the condition . While pDifferent predictive markers have been explored for successful sperm retrieval in KS, including mosaic state, testosterone level, and age. While some studies suggest that age has no effect on predicting sperm retrieval in KS men , others To date, a total of 10 couples with KS male partners who have sperm in their ejaculate have achieved live births, resulting in 15 children (including 5 singletons and 5 sets of twins). The male partners’ ages ranged from 29 to 35 years old . In all Although NIPT has limitations in detecting sex-chromosome aneuploidies, it has become more accessible to patients. A large study evaluating the performance of NIPT in detecting sex chromosome aneuploidy reported a positive predictive value of 66.67% for 47, XXY . The lowThis was a rare case of a man with mosaic Klinefelter syndrome who presented with severe oligospermia and a successful live birth was achieved using assisted reproductive technology, the classical presentation of KS is azoospermia and surgical sperm retrieval using MicroTESE is required with variable success rate. We hope that this case sheds light on the awareness of the phenotypic variation of KS with respect to age and contributes to the existing literature.


The C60 layer is assembled on the well-defined molecular network, allowing appropriate molecular orbital hybridization. Upon thermal activation, covalent coupling perpendicular to the surface via [4 + 2] cycloaddition between C60 and the phenyl ring of the molecule is realized; the resultant adduct shows frozen orientation and distinct sub-molecular feature at room temperature and further enables lateral covalent bonding via [2 + 2] cycloaddition. This work unlocks an unconventional route for bottom-up precise synthesis of three-dimensional covalently-bonded organic architectures/devices on surfaces.As an efficient molecular engineering approach, on-surface synthesis (OSS) defines a special opportunity to investigate intermolecular coupling at the sub-molecular level and has delivered many appealing polymers. So far, all OSS is based on the lateral covalent bonding of molecular precursors within a single molecular layer; extending OSS from two to three dimensions is yet to be realized. Herein, we address this challenge by cycloaddition between C 60 and an aromatic compound allowing covalent coupling perpendicular to the surface and multiple non-planar covalent coupling of C60.On-surface synthesis (OSS) defines a special opportunity to investigate intermolecular coupling at. sub-molecular level and has delivered many appealing polymers but so-far OSS is limited to the. lateral covalent bonding of molecular precursors. Here, the authors demonstrate cycloaddition. between C Although it is straightforward to pile up molecular layers by van der Waals (vdW) interaction or hydrogen bonding5, assemblies based on noncovalent bonding often have lower thermal stability, conductivity, and mechanical properties compared with the covalently-bonded products8. As an efficient bottom-up approach, on-surface synthesis (OSS) defines a special opportunity to investigate intermolecular coupling at the sub-molecular level and has successfully delivered various covalently-bonded two-dimensional (2D) polymers15. So far, these systems have been all based on the lateral coupling of molecular precursors within a single molecular layer. Extending OSS from two to three dimensions requires not only the lateral coupling parallel to the substrate but also the covalent bonding perpendicular to the substrate, e.g., interlayer coupling between molecular layers, which is yet to be realized.Constructing heterostructured molecular layers with atomic/molecular level precision and extending molecular engineering from two to three dimensions hold great importance for both fundamental science and application purposes in the fields of nanoelectronic devices, photonics, and quantum science60 and its derivatives have shown magnificent physical properties/potentials ; the chemical reactions of C60 with organic compounds have also sparked considerable interests22. Especially, the unique 3D conjugated electronic structure and the geometric combination of pentagons and hexagons of C60 molecules make their reactions significantly distinct from those of graphene or planar polyaromatic molecules24. The electron-deficient curved π-conjunction allows C60 to form adducts with various dienes  as a dienophile;25 due to the multiple non-planar reactive sites , C60 is expected to act as a steric spherical “multi-plug” reactant27. However, the close-packed arrangement of C60 molecules32 and their strong interaction with the substrates35 make the on-surface reaction of C60 with other molecules rather difficult. Moreover, the coupling between C60 and the targeting functional group(s) requires a specific spatial geometry37. There is no precedent for the on-surface reaction of C60 with aromatic compounds yet.As the most representative fullerene, C60 and 3,5-bis(carboxylic acid)-phenyl-3-maleimide  and their adducts on Au(111) surface  of C60 and the highest occupied molecular orbital (HOMO) of BCPM, 2D ordered BCPM layer is constructed on Au(111) beneath C60. As demonstrated by scanning tunneling microscopy (STM) imaging and density functional theory (DFT) calculations, interlayer [4 + 2] cycloaddition between C60 and BCPM perpendicular to the substrate is triggered upon thermal activation. The resultant adduct, i.e., C60-BCPM, shows obvious differences from pristine C60, including the frozen orientation hence triangular sub-molecular feature observed at room temperature (RT), increased adsorption height, and distinct domain structure and reactivity. Especially, forming macrocycles with the hexamer rings with C6v symmetry dominated, lateral intermolecular [2 + 2] cycloaddition among C60-BCPMs parallel to the substrate is evidenced. Using C60 and BCPM as the first example, this work presents an unconventional strategy for bottom-up synthesis of 3D covalently-bonded organic architectures, extending OSS from 2D to 3D by cycloaddition reactions with fullerenes.Herein, we report the cycloaddition of Ce BCPM, C2H7NO6 ana | = | b | = 27.2 ± 0.2 Å and an angle of 60° between the two vectors. Although the lateral molecular arrangement is similar to that formed on graphene epitaxially grown on Cu(111) (G-Cu)38, close inspection -phenyl group adsorbs in a flat geometry at a height of 3.4 Å above Au(111), while maleimide adopts a tilted configuration with the ring rotated by 20.5° from the horizon Fig. . The net60 molecules were then deposited onto the BCPM layer-covered Au(111) at room temperature (RT). At low dosages, C60 adorbs preferentially in the circular pores of the BCPM network and is imaged as a bright circular protrusion  leads to a coating of the C60 layer on the BCPM layer. These C60 molecules follow the same symmetry and periodicity as the underlying BCPM network . The coverage increases with the C60 dosage until the BCPM network is fully covered  surface 29, linescans across the two domains show that C60 in D1 is 3.5 Å higher than in D2 28, D1 is smooth, showing a uniform contrast; iii) surprisingly, even upon scanning at RT, distinct triangular sub-molecular feature of C60 is observed from D1, suggesting that the C60 are firmly frozen, adopting the same orientation  was only observed when quenching the molecules under cryogenic conditions;29 at RT, pristine C60 molecules can only be imaged as featureless spheres on Au(111) due to their high rotational mobility at this temperature41.After annealing the Cved Fig. . The bri D2 Fig. ; ii) whiion Fig. . This is60 on top of one BCPM, thermo-activated coupling between C60 and BCPM is rationalized. Confirmed by the frontier molecular orbital analysis, the orbital matching and the appropriate steric arrangement can enable [4 + 2] cycloaddition between bond at the bottom of C60 and the C atoms in para-positions of BCPM phenyl ring, forming a covalently-bonded adduct, i.e., C60-BCPM , consistent with the experiments. Upon [4 + 2] cycloaddition, hydrogen bonding between C60-BCPM is largely weakened compared with that between pristine BCPMs  to self-assemble into D2. Meanwhile, the cycloaddition confines the rotation of C60, allowing the sub-molecular resolution at RT. The molecular feature  the greater height of molecules in D1 than that in D2, ii) the frozen morphology showing intramolecular features at RT, iii) the varied lateral arrangement of molecules compared with that before annealing Fig., iv) andCPM Fig. . Top of n enable  + 2 cyclure Fig.  and lateage Fig.  match th60-BCPMs, the sample   are dominant . Linescans show that the HR’s diameter is 16.8 Å  is based on the dissociation/formation of organic precursors’ σ bonds at the terminal of molecular precursors, which can be achieved by intermolecular lateral coupling on surfaces15, thus allowing 2D synthesis only; the present case forms covalent bonding between adjacent molecular layers based on the rehybridization of π orbitals of the reactants. Such cycloaddition is also different from that of BCPM with graphene, where cycloaddition occurs between C = C of maleimide of BCPM and graphene, involving both [2 + 2] and [4 + 2] cycloaddition with different pairs of carbon atoms of graphene hexagons38. In the present system, cycloaddition between C60 and BCPM maleimide is not observed. Although the energy barrier of their [2 + 2] cycloaddition in the gas phase (1.3 eV) is only 0.1 eV higher than that of [4 + 2] cycloaddition between C60 and BCPM phenyl ring   are not formed synchronously: C–C bond with Carbon A on the BCPM’s phenyl ring forms first , and then the second C–C bond with Carbon B forms ; this is different from cycloaddition of C60 with benzene where the two bonds of [4 + 2] cycloaddition form simultaneously. In TS, Carbon A and B locate at 3.6 Å and 4.3 Å above the Au(111), respectively, i.e., being lifted by 0.2 Å and 0.9 Å compared with the geometry of pristine BCPM on Au(111). The phenyl ring after cycloaddition becomes non-flat and distorted. We compared the energy barrier for cycloaddition of C60 with benzene, benzene-1,3-dicarboxylic acid, N-phenylmaleimide, and BCPM  calculations to understand the cycloaddition pathways. For [4 + 2] cycloaddition between BCPM and C60 on Au1  for C60-BCPM is also 0.16 eV lower than that of C60 . The carboxylic acid groups of BCPM enable the formation of a well-defined template for C60, while the out-of-plane maleimide group enables strong adsorption of the molecules on the substrate. The configuration of C60 on BCPM allows appropriate molecular orbital hybridization between them meanwhile the tilted maleimide group of BCPM restrains the lateral diffusion of C60, enabling covalent bonding perpendicular to the surface via interlayer [4 + 2] cycloaddition upon thermal activation. The cycloaddition is demonstrated by the increased adsorption height, the distinct domain structure, and the frozen orientation hence the triangular sub-molecular feature at RT, distinct from pristine C60. Moreover, the rotation-frozen C60 in C60-BCPMs, their close-packed molecular arrangement, and the multiple non-planar reactive sites of C60 allow, upon elevated annealing, a next-step lateral [2 + 2] cycloaddition of C60-BCPMs to form macrocycles, further distinguishing the difference of C60-BCPM and pristine C60. Moreover, both C60 and BCPM are able to have a second cycloaddition along the direction perpendicular to the surface. Allowing both vertical and lateral covalent bonding, this work unlocks an efficient route for bottom-up synthesis of covalently-bonded 3D organic framework on surfaces via cycloaddition of organic compounds with fullerenes--the steric multi-plug reactants, thus extending OSS from 2D to 3D. The strategy of interlayer covalent bonding may innovate the design of robust hetero-layered materials for engineering cutting-edge devices with molecular precision.3D covalent bonding has been realized by cycloaddition of C–10 mbar. The Au(111) substrate was cleaned by repeated cycles of argon ion sputtering (1000 eV) and annealing (at 770 K). BCPM compound and C60 were sublimated from the crucibles of a low-temperature evaporator at 330 K and 470 K, respectively, onto the surface kept at RT. All STM images were collected in the constant current mode at RT.All experiments were performed in an ultrahigh vacuum (UHV) chamber equipped with a variable-temperature Aarhus scanning tunneling microscope. The typical base pressure of the UHV chamber was 1 × 1044. The interaction between electrons and ion cores was described by the projected augmented wave (PAW) method45 with a plane-wave cutoff energy of 400 eV. The exchange and correlation effects between electrons were treated by Perdew-Burke-Ernzerhof (PBE) exchange-correlation density functional46. The vdW correction was performed using the zero damping DFT-D3 method of Grimme47. The BCPM network, BCPM network with C60 hosted in the pores of that network, and the steric assembly of C60 on atop of BCPM were calculated in a 9 × 9 supercell on the Au(111) substrate. The close-packed arrangement of the C60-BCPM adduct was calculated in a 2√3 × 2√3 − R30° supercell on Au(111). The hexamer ring composed of six C60-BCPMs was calculated in a 16 × 16 supercell on Au(111). The CI-NEB calculations48 for [4 + 2] cycloaddition between C60 and BCPM and [2 + 2] cycloaddition between the C60-BCPMs were performed in a 9 × 9 supercell on Au(111), with 12 and 11 images inserted between the initial and final states, respectively. The Brillouin zone was sampled through the Monkhorst-Pack scheme with 3 × 3 × 1 mesh for the 2√3 × 2√3-R30° supercell and only Gamma point for the other cells with lattice parameters larger than 20 Å. In the structural relaxation, all structures were optimized until the residual forces were found smaller than 0.02 eV/Å. In the Cl-NEB calculations, the force criterion was 0.05 eV/Å for each image. The Tersoff-Hamman method was employed for the calculations of the STM images49.DFT calculations were performed using the Vienna ab initio simulation package (VASP)Supplementary InformationPeer Review File


Paralemnalia thyrsoides. The in vitro cytotoxic potential of the ethyl acetate extracts of the different five isolates were evaluated using MTS assay against four cancer cell lines; A549, CT-26, MDA-MB-231, and U87. Metabolomics profiling of the different extracts using LC-HR-ESI-MS, besides molecular docking studies for the dereplicated compounds were performed to unveil the chemical profile and the cytotoxic mechanism of the soft coral associated fungi.Cancer continues to be one of the biggest causes of death that affects human health. Chemical resistance is still a problem in conventional cancer treatments. Fortunately, numerous natural compounds originating from different microbes, including fungi, possess cytotoxic characteristics that are now well known. This study aims to investigate the anticancer prospects of five fungal strains that were cultivated and isolated from the Red Sea soft coral Penicillium griseofulvum (RD1), Cladosporium sphaerospermum (RD2), Cladosporium liminiforme (RD3), Penicillium chrysogenum (RD4), and Epicoccum nigrum (RD5). The in vitro study showed that the ethyl acetate extract of RD4 exhibited the strongest cytotoxic potency against three cancer cell lines A549, CT-26 and MDA-MB-231 with IC50 values of 1.45 ± 8.54, 1.58 ± 6.55 and 1.39 ± 2.0 µg/mL, respectively, also, RD3 revealed selective cytotoxic potency against A549 with IC50 value of 6.99 ± 3.47 µg/mL. Docking study of 32 compounds dereplicated from the metabolomics profiling demonstrated a promising binding conformation with EGFR tyrosine kinase that resembled its co-crystallized ligand albeit with better binding energy score.The five isolated fungal strains were identified as Our results highlight the importance of soft coral-associated fungi as a promising source for anticancer metabolites for future drug discovery.The online version contains supplementary material available at 10.1186/s12866-023-03045-y. Cancer is defined as a group of disorders that can affect different body organs and are characterized by the invasion of healthy tissues and the unchecked proliferation of aberrant cells. New tumors can be formed as a result of cancer cells spreading to different bodily regions , 2. WithNatural products play a significant role in the development of chemotherapeutic medicines, and they are also thought to be a main source of novel therapies that feed the existing clinical pipeline for treating cancer. Taxanes (e.g. Taxol), vinblastine, vincristine, and the podophyllotoxin are examples of clinically important antitumor drugs originating from higher plants and have largely contributed to the management of human diseases since their discovery early in the 20th century –9.Terrestrial fungi have long been known as a rich source of biologically active secondary metabolites and for treatment of many disorders since the discovery of penicillin by Sir Alexander Fleming 1928, which has led to a breakthrough in the treatment of bacterial infections –12. EndoParalemnalia thyrsoides (Ehrenberg 1834), which is one of the most common marine invertebrates, natively distributed throughout tropical and subtropical regions of the Indo-Pacific Ocean − 243.123 , 329.232  and 313.237 , were identified as a monoterpene Penicimonoterpene (1), polyketide derivatives penicitide B (2) and penicitide A (3), corresponding to the molecular formulas C12H20O5, C18H34O5 and C18H34O4. In addition, the mass ion peak at m/z 259.118 [M-H]− , in conformity with the predicted molecular formula C12H20O6, was characterized as penicierythritols B (4). The mass ion peak at m/z 637.155 [M-H]−  for the suggested molecular formula C32H30O14 was recognized as chrysoxanthone A (5). Moreover, the mass ion peak m/z 505.351 [M-H]− , in consonance with the predicted molecular formula C30H50O6 was identified as penicisteroid A (6). Besides, the metabolite, namely chrysogeamide E (7) with molecular formula C35H55N5O7 was dereplicated from the mass ion peak at m/z 656.401 [M-H]− . The metabolites 1–7 were found to be previously isolated from Penicillium chrysogenum [9H16O3 was dereplicated from the mass ion peak at m/z 171.102 [M-H]−  as decarestrictine L (8), it was formerly obtained from Penicillium simplicissimum [m/z [M + H] + 213.148 , corresponding to the molecular formula C12H20O3 was identified as patulolide C (9) which was earlier isolated from Penicillium urticae [m/z 213.112 [M-H]−  in accordance with the molecular formula C11H18O4, was identified as citreoviral (10) and was previously reported from Penicillium citreoviride [m/z 309.206 and 323.186 [M-H]−  corresponding to the molecular formulas C18H30O4 and C18H28O5 were characterized as cephalosporolide H (11) and hynapene A (12), which were found to be also isolated from Penicillium sp. [18H30O5 and C20H28O5 were dereplicated from the mass ion peaks at m/z 327.216 and 349.200 [M + H] +  and were identified as penisporolide A (13) and rezishanone B (14), they were reported to be isolated from Penicillium sp. −  in compliance with the molecular formulas C23H24O5 and C23H26O5. Another mass ion peak at m/z 637.155 [M-H]− , in conformity with the molecular formula C32H30O14, was identified as rugulotrosin A (17) and rugulotrosin B (18), also, previously isolated from Penicillium sp. [m/z 469.261 [M-H]−  for the predicted molecular formula C29H34N4O2 was distinguished as communesin G (19) that was formerly obtained from Penicillium rivulum [m/z 427.175 [M-H]−  for the molecular formula C24H28O7 was dereplicated as paraherquonin (20), which has been reported from Penicillium paraherquei + .The annotated compounds . These metabolites were identified as (+)- epicoccone C (22) −  in agreement with the molecular formula C10H8O6, it was reported to be isolated from the fungus Cladosporium herbarum as herbaric acid (25) −  in consonance with the molecular formula C14H26O5S. Three cyclohexene derivatives, cladoscyclitol A (32), cladoscyclitol C (27), and cladoscyclitol D (28) were reported to be isolated from Cladosporium sp. JJM22 −  in compliance with the molecular formulas with molecular formula C12H20O5, C12H22O4, and C12H22O5, respectively. Furthermore, two tetramic acids, cladosporiumin B (29) and (Z)-cladosin K (31) were dereplicated from the mass ion peaks at m/z 350.202 [M + H] +  and at m/z 418.208 [M-H]−  corresponding to the molecular formulas C19H27NO5 and C25H29N3O3. They were isolated from Cladosporium sp. SCSIO z0025 [Cladosporium sphaerospermum [m/z 227.128 [M-H]−  in match with the molecular formula C12H20O4, it was previously isolated from Cladosporium sp. TZP29 [(30).In addition to the above mentioned metabolites, the metabolomics analysis revealed the presence of three metabolites, were reported in e C (22) , (±)-5 h2) [(23) , and amp3) [(24) , matchedcid (25) . One macIO z0025  and Cladospermum , respectin silico evaluated against the Epidermal Growth Factor Receptor tyrosine kinase (EGFR). The molecular docking results for the 32 dereplicated compounds were presented in (Table S4) and the 2D as well as the 3D conformations of the best fitted derivatives were illustrated in Figs. To better understand the pharmacological mechanism of the fungal extracts in inhibiting the tested cancerous cell lines, their dereplicated compounds were n Table S and the (5), chrysogeamide E (7), rugulotrosin A (17) and (Z)-cladosin K (31) revealing − 10.33, -11.66, -10.51 and − 9.79 Kcal/mol, respectively that surpassed the co-crystallized ligand AQ4 . Moreover, the carbonyl of chrysoxanthone A (5) managed to form an ionic bond with Lys721 with a distance of 3.22 Å and additional H-bond with Gln767 that resembled AQ4  exhibited the best binding energy to EGFR and was capable to interact with several amino acid residues through hydrogen bonds such as Lys721, Met769, Cys773, Arg817 and Asp831 in addition to two H-pi interaction with Phe699  bound to the crucial Met769 as well as extensive H-bonds formation with Ala719, Cys751, Thr766, Leu821, Asp831 and Thr830  formed hydrophobic interactions with both Leu694 and Leu820 that supported its tight binding with EGFR. In a similar way, the hydroxyl group in compound (31) formed H-bond with Met769 of 3.27 Å distance and hydrophobic interactions with the crucial Lys721 and Cys773 by its 6-membered and 5-membered rings, respectively , penicisteroid A (6), rugulotrosin B (18) and citrinadin A (21) that showed − 9.16, -9.11, -9.66 and − 9.53 Kcal/mol, respectively. Their 2D interactions with EGFR binding site revealed multiple interactions through hydrogen and ionic bonding with the bind site residues  bound to Asp776 and Glu780 with an average distance of 3.05 Å as well as other H- bond formation with Leu694 and Cys773 , Cladosporium sphaerospermum (RD2), Cladosporium liminiforme (RD3), Penicillium chrysogenum (RD4), and Epicoccum nigrum (RD5). The in vitro cytotoxic assay of the ethyl acetate extracts of the five different fungal strains revealed a significant effect in killing cancer cells, as the RD4 fungal extract showed the strongest potency with IC50 values of 1.45 ± 8.54, 1.58 ± 6.55 and 1.39 ± 2.0 µg/mL against the three cancer cell lines A549, CT-26 and MDA-MB-231. Likewise, RD3 revealed selective cytotoxic potency against A549 with IC50 value of 6.99 ± 3.47 µg/mL and moderate activity against MDA-MB-231 cell line (IC50 37.50 ± 7.73 µg/mL). Interestingly, in an attempt to unveil the chemical profile of the soft coral associated fungi, the extracts were subjected to LC-HR-ESI-MS based metabolomics analysis, leading to the characterization of 32 metabolites predominantly alkaloids, terpenoids, phenolic derivatives and benzenoids. Additionally, Molecular docking analysis of the dereplicated secondary metabolites was accomplished to explain the mechanism of the fungal extracts in inhibiting the tested cancerous cell lines. The study suggested that EGFR tyrosine kinase inhibitory potential is a plausible mechanism for their cytotoxic activities, in particular chrysoxanthone A (5), chrysogeamide E (7), rugulotrosin A (17) and (Z)-cladosin K (31), which displayed exceptional binding abilities with EGFR tyrosine kinase. Our study highlighted the importance of Paralemnalia thyrsoides-associated fungi as a promising source of antitumor compounds. Marine fungal endophytes have drawn numerous researchers’ attention in basic and applied research as a distinguished reservoir of bioactive metabolites for the development of diverse natural cytotoxic medications. Nevertheless, further investigations could be dedicated for in-depth understanding of the cytotoxic activity of the extracts and metabolites isolated from these microorganisms.The current work revealed the identification and purification of five fungal isolates from Red Sea soft coral, Below is the link to the electronic supplementary material.Supplementary Material 1


Vibrio vulnificus is a bacterium that inhabits warm seawater or brackish water environments and causes foodborne diseases and wound infections. In severe cases, V. vulnificus invades the skeletal muscle tissue, where bacterial proliferation leads to septicemia and necrotizing fasciitis with high mortality. Despite this characteristic, information on metabolic changes in tissue infected with V. vulnificus is not available. Here, we elucidated the metabolic changes in V. vulnificus-infected mouse skeletal muscle using capillary electrophoresis time-of-flight mass spectrometry (CE-TOFMS). Metabolome analysis revealed changes in muscle catabolites and energy metabolites during V. vulnificus infection. In particular, succinic acid accumulated but fumaric acid decreased in the infected muscle. However, the virulence factor deletion mutant revealed that changes in metabolites and bacterial proliferation were abolished in skeletal muscle infected with a multifunctional-autoprocessing repeats-in-toxin (MARTX) mutant. On the other hand, mice that were immunosuppressed via cyclophosphamide (CPA) treatment exhibited a similar level of bacterial counts and metabolites between the wild type and MARTX mutant. Therefore, our data indicate that V. vulnificus induces metabolic changes in mouse skeletal muscle and proliferates by using the MARTX toxin to evade the host immune system. This study indicates a new correlation between V. vulnificus infections and metabolic changes that lead to severe reactions or damage to host skeletal muscle.IMPORTANCEV. vulnificus causes necrotizing skin and soft tissue infections (NSSTIs) in severe cases, with high mortality and sign of rapid deterioration. Despite the severity of the infection, the dysfunction of the host metabolism in skeletal muscle triggered by V. vulnificus is poorly understood. In this study, by using a mouse wound infection model, we revealed characteristic changes in muscle catabolism and energy metabolism in skeletal muscle associated with bacterial proliferation in the infected tissues. Understanding such metabolic changes in V. vulnificus-infected tissue may provide crucial information to identify the mechanism via which V. vulnificus induces severe infections. Moreover, our metabolite data may be useful for the recognition, identification, or detection of V. vulnificus infections in clinical studies. Vibrio vulnificus is a halophilic Gram-negative bacterium that inhabits warm seawater or brackish water environments. V. vulnificus causes foodborne disease after the ingestion of raw or undercooked seafood, characterized by several gastrointestinal symptoms, such as abdominal pain, vomiting, fever, chills, and nausea, which resolve within a few days in healthy people , and more than 95% of cases of VNSSTIs are associated with the subtropical western Pacific and Atlantic coastal regions of the Northern Hemisphere, such as Taiwan, South Korea, Japan, or the Gulf of Mexico  and the multifunctional-autoprocessing repeats-in-toxin (MARTX) are regarded as significant toxins of V. vulnificus. Researchers have identified several functions for each toxin. VVH is a pore-forming toxin that promotes bacterial translocation from the intestine to the bloodstream by causing intestinal tissue damage with cell death  have been used to investigate the mechanism of necrotizing fasciitis in bacterial infection. In particular, a metabolic analysis indicated differences in metabolites during the acute phase of infection in patients. Moreover, previous studies have indicated that bacterial growth and viability were closely associated and resulted in host metabolic changes. In fact, a variety of group A streptococci (GAS) respond to specific metabolites . AccordiV. vulnificus-infected skeletal muscles in a mouse wound infection model using capillary electrophoresis–time-of-flight mass spectrometry (CE-TOFMS). We found that muscle catabolites changed upon infection by Vibrio spp., whereas V. vulnificus alone significantly affected the succinate-fumarate pathway of the tricarboxylic acid (TCA) cycle in skeletal muscle. The V. vulnificus toxin MARTX facilitated the proliferation of bacteria in skeletal muscle and was indirectly important for triggering metabolic changes in mouse skeletal muscle. Even during short infection, these metabolic changes were detected in skeletal muscle. This study is the first report showing metabolic changes in V. vulnificus-infected muscle tissue.In this study, we attempted to estimate the metabolic changes in V. vulnificus wound infection model, we checked that V. vulnificus induces inflammation of skeletal muscle  Hematoxylin and eosin staining showing the effect of PBS treatment or V. vulnificus infection on mouse skeletal muscles. (B) The expression levels of the MIP-2, TNF-α, and IL-6 mRNAs were estimated by RT-qPCR. The expression of each mRNA was normalized to that of the GAPDH mRNA. These samples were divided into four groups as follows: PBS-treated leg (PBS), V. vulnificus-injected leg (Vv), the leg opposite the PBS-treated leg (OP), and the leg opposite the V. vulnificus-injected leg (OV) (9 or 10/group). (C) Concentrations of arginine, creatine, and creatinine in mouse blood were extracted from the CE-TOFMS data at 9 h after infection. The samples were divided into four groups as described above (3/group). *, P < 0.05, and **, P < 0.01, versus the control group (t test). P values were determined by one-way ANOVA followed by Dunnett’s test. †, FDR < 0.05, versus the Vv group. Download FIG S1, TIF file, 8.2 MB.Copyright © 2023 Ishida et al.2023Ishida et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the V. vulnificus infection, we analyzed the metabolic conversion of ornithine to creatinine in host skeletal muscle after 9 h of V. vulnificus infection. Mouse skeletal muscles were treated by subcutaneous injection into the right femoral region of phosphate-buffered saline  or V. vulnificus , and each opposite leg was defined as OP (leg opposite the PBS leg) or OV (leg opposite the V. vulnificus leg). These skeletal muscles were collected after 9 h of infection, and the metabolites were analyzed by CE-TOFMS. We found that creatine and creatinine were decreased in the Vv group . Those data indicated changes in metabolite associated with infection damage , using CE-TOFMS . These dd muscle . The datn damage . Upon anon model . In conton model , the heaon model , or the on model . Furthered group . These d10.1128/msystems.00682-22.2FIG S2V. vulnificus. Metabolites in mouse whole blood were measured by CE-TOFMS after 9 h of infection with V. vulnificus. (A) PCA plots showing configure to different cluster between PBS- and Vv-treated tissues. The red and blue dots indicate PBS and V. vulnificus (Vv)-infected tissues (3 to 5/group). (B) A heat map analysis reported the metabolic changes of each metabolite via the autoscaling method; the results are expressed using a red and blue color scheme, indicating high and low concentrations of metabolites, respectively. x axis, 25.67%; y axis, 16.99%; z axis, 18.06% (3 to 5/group). (C) The relative changes in the metabolites in the Vv group are indicated by the volcano plot, with red denoting significantly changed metabolites (by more than 2-fold), blue indicates metabolites that changed by more than 2-fold but without significant differences detected, and orange indicates metabolites with significant differences but showing changes of less than 2-fold (3 to 5/group). (D) The relative changes of the metabolites in the Vv group were indicated by the volcano plot in Vv-infected mice were categorized into six metabolic pathways  (3 to 5/group). Download FIG S2, TIF file, 0.6 MB.Profiling of the mouse blood metabolites triggered by infection with Copyright © 2023 Ishida et al.2023Ishida et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/msystems.00682-22.5TABLE S1Table S1, PDF file, 0.3 MB.Calculated concentrations of metabolites in this study. Download Copyright © 2023 Ishida et al.2023Ishida et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the P. aeruginosa modulates the host TCA cycle in a variety of tissues    and B. IV. vulnificus-infected skeletal muscle. These data imply that the muscle catabolism and TCA cycle metabolites are associated with bacterial growth.In order to reveal the metabolic change in the early infection phase, we investigated the bacterial cell counts between the different infection time points in the host skeletal muscle and measured metabolites in the host skeletal muscle after 3 h of infection  to E. ThvvhA and ΔrtxA1 mutants and the double knockout [DKO] mutant) in the V. vulnificus wound infection model. First, we confirmed the inflammation of the skeletal muscle in the presence of V. vulnificus toxins  showed a decrease in metabolic changes in the creatine to creatinine pathway in skeletal muscle. As shown in vvhA deletion mutant exhibited metabolic changes in the TCA cycle metabolites in host skeletal muscle that were similar to those observed under wild-type conditions. However, these metabolic changes were abolished in the rtxA1 deletion mutant. These data indicate that MARTX is associated with the modulation of the host TCA cycle and skeletal muscle catabolism.Previous studies revealed that VVH and MARTX induced apoptosis and mitochondrial damage , 21. To s toxins  and B. Tl muscle  and B. T10.1128/msystems.00682-22.3FIG S3rtxA1 deletion mutant. Mice were treated with or without CPA and treated with PBS or infected with V. vulnificus (WT and ΔrtxA1) strains. The samples were divided into six groups as follows: PBS treatment, PBS plus CPA treatment, V. vulnificus infection (Vv), V. vulnificus infection and cyclophosphamide treatment (Vv+CPA), MARTX (ΔrtxA1) mutant infection, and MARTX mutant infection and CPA treatment (ΔrtxA1+CPA). (A) Hematoxylin and eosin staining showed the effect of PBS treatment or infection with the V. vulnificus  strains. (B) The changes in the expression levels of the MIP-2, TNF-α, and IL-6 mRNAs triggered by infection with V. vulnificus WT or mutant  strains were estimated by RT-qPCR. The expression of each mRNA was normalized to that of the GAPDH mRNA (3 to 5/group). (C) The number of bacteria in skeletal muscle tissues was estimated using a CFU assay. These skeletal muscles were infected with V. vulnificus  strains for 1, 3, and 9 h. The asterisks denote significant differences versus the wild-type group (3 to 5/group). (D) The expression levels of the 16S rRNA transcript were estimated by RT-qPCR (3 to 5/group). (E) White blood cells were counted in mouse whole blood (n = 4/group). (F) The expression levels of the 16S rRNA transcript were estimated by RT-qPCR (4/group). (G) The number of bacteria in whole blood was estimated using a CFU assay. Skeletal muscles were infected for 6 h (4/group). (H) Hematoxylin and eosin staining showed the effects of PBS treatment or infection with V. vulnificus (WT and ΔrtxA1) strains on mouse skeletal muscles treated with or without CPA. (I) A Western blot analysis of myeloperoxidase in mouse skeletal muscles was conducted at 6 h of infection. (J) Metabolites of creatine and creatinine in skeletal muscles were extracted from the CE-TOFMS data at 6 h of infection (4/group). *, P < 0.05, and **, P < 0.01, versus the control group (t test). P values were determined by one-way ANOVA followed by Tukey’s test. Different letters indicate significant difference. A P value of <0.05 is considered significant. Download FIG S3, TIF file, 2.6 MB.Immunodeficient mice exhibited changes in skeletal muscle catabolism metabolites after infection with the Copyright © 2023 Ishida et al.2023Ishida et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the vvhA deletion mutant strains yielded a gradual increase in bacterial cell counts in mouse skeletal muscle, whereas the rtxA1 deletion mutant strain did not . We examined the number of white blood cells  staining  for 9 h, and the resulting metabolic changes were analyzed , V. parahaemolyticus (Vp), V. mimicus (Vm), or V. cholerae (Vc) for 9 h. (A) H&E staining showed the effects of PBS treatment or V. vulnificus, V. parahaemolyticus, V. mimicus, or V. cholerae infection on mouse skeletal muscles. (B) The expression levels of the MIP-2, TNF-α, and IL-6 mRNAs were estimated by RT-qPCR. The expression of each mRNA was normalized to that of the GAPDH mRNA (4/group). (C) Metabolites of creatine and creatinine in blood were extracted from the CE-TOFMS data after treatment with PBS or infection with the Vibrio spp. for 9 h (4/group). *, P < 0.05, and **, P < 0.01, versus the control group (t test). P values were determined by one-way ANOVA followed by Tukey’s test. Different letters indicate significant differences. A P value of <0.05 is considered significant. Download FIG S4, TIF file, 8.2 MB.Other Copyright © 2023 Ishida et al.2023Ishida et al.https://creativecommons.org/licenses/by/4.0/Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the V. parahaemolyticus- and V. mimicus-infected muscle, similar to V. vulnificus infection. Despite the similar catabolic pattern, citric acid, isocitric acid, and succinic acid were not changed in V. parahaemolyticus and V. mimicus infection. These results indicated that the increase in TCA cycle metabolites was specific to V. vulnificus-infected skeletal muscle.Next, the mouse skeletal muscle was isolated, and its metabolites were analyzed by CE-TOFMS. The metabolites associated with muscle catabolites, the creatine-to-creatinine pathway, were changed in V. vulnificus infection , affected the changes in metabolism simultaneously with the increase in the bacterial counts in skeletal muscle . These data supported the contention that V. vulnificus promotes succinate accumulation via the downregulation of host succinate dehydrogenase. Taken together, these findings suggest that the inhibition of the succinate-fumarate metabolic pathway provides succinate more effectively to V. vulnificus, with consequent development of dramatic inflammation in infected skeletal muscles.To elucidate the wound infection caused by CA cycle . In partCA cycle . In addilusively . It is pogenesis , 34. Pernfection . Thus, wV. parahaemolyticus and V. mimicus also secrete pore-forming toxins (such as thermostable direct hemolysin [TDH]), which induce permeability, cytotoxicity, and apoptosis in host cells  derived from CMCP6 were provided by Takashige Kashimoto at Kitasato University , Osaka University. The bacterial strains used in this experiment are listed in V. vulnificus strains were cultured in 2% NaCl–LB broth , V. parahaemolyticus was cultured in 3% NaCl–LB broth, and V. mimicus and V. cholerae were cultured in 1% NaCl–LB broth overnight at 37°C with shaking (170 rpm). Bacterial culture media were centrifuged at 12,000 rpm for 2 min and concentrated, and organisms were grown on thiosulfate-citrate-bile salts-sucrose (TCBS) agar overnight. A single colony was picked from the TCBS agar and cultured in LB broth for 8 h. The bacterial suspension was mixed with a glycerol solution  and stored at −80°C. For experiments, bacterial strains were picked from the stock and were cultured in 1%, 2%, or 3% LB broth overnight. Subsequently, the culture broth was recultured in new LB broth for 2 h and then concentrated to an optical density at 600 nm (OD600) of 1.0 with PBS.iversity , 47, andWe purchased 5-week-old male Crl:CD1 mice . The mice were housed in a controlled environment with a 12-h/12-h light/dark cycle and maintained at 23°C. One week later, the mice were utilized in the animal experiments.V. vulnificus mouse wound infection model  at 23°C after anesthesia using isoflurane. The mice were sacrificed 9 h after the injections. The mouse muscles were dissected and placed in a 1.5-mL tube. Mouse blood samples were collected in 1.5-mL tubes and EDTA dipotassium (EDTA-2K) at 1 mg/mL was added, to prevent blood coagulation.Previously, Yamazaki et al. established a on model . Mice weNeutropenic mice, 6 weeks old, were prepared via a 2-dose intraperitoneal administration of cyclophosphamide monohydrate at 150 mg/kg of body weight at day −4 and 100 mg/kg at day −1 from infection. Neutropenic mice were infected with bacteria using the method used in another experiment. The mice were sacrificed 6 h after the injections. The mouse muscles were dissected and placed in 1.5-mL tubes. Mouse blood samples were collected in 1.5-mL tubes, and EDTA-2K at 1 mg/mL was added to prevent blood coagulation.g for 5 min at 4°C, and the supernatant was filtered using ultracentrifugal 5-kDa-cutoff filters (Millipore) at 9,100 × g for 4 h at 4°C. The filtrates were concentrated in a vacuum evaporator and dissolved with ultrapure water containing internal controls .The tissues were frozen rapidly in liquid nitrogen and stored at −80°C. The skeletal muscles were cut on ice, and 50-mg samples were collected in 1.5-mL tubes. The skeletal muscles were mixed with 500 μL of methanol containing 10 μM methionine sulfone and camphor-10-sulfonic acid, as an internal control, in each tube. The muscles were homogenized using a beads cell disrupter  and the lysates were removed into a new tube. The blood (50 μL) was placed in 2-mL tubes and mixed with 500 μL of methanol containing 10 μM methionine sulfone and camphor-10-sulfonic acid, as an internal control, in each tube. These samples were mixed with 200 μL of ultrapure water and 500 μL of chloroform. After vortex mixing, the samples were centrifuged at 9,100 × m/z range.The samples were analyzed using Agilent 7100 capillary electrophoresis (CE) system coupled with an Agilent 6230 TOF LC-MS  by Human Metabolome Technologies, Inc. . CE-TOFMS analysis of cationic and anionic metabolites was performed as described previously –51. Cati–m/z, migration time, and peak area to compare with the standard . Each peak area was normalized based on internal standard levels, and then the resultant relative area values were normalized by sample amounts to obtain a relative level for each metabolite. Methionine sulfone was used for the density correction of cationic metabolites, and camphor-10-sulfonic acid was used for the density correction of anionic metabolites.Analyzed raw data were processed using Agilent MassHunter qualitative analysis (version B.07.00). Each metabolite was identified and quantified based on the reference peak information, including −1 to 10−5). Mouse blood was collected in 100-μL portions in 1.5-mL tubes and EDTA-2K at 1 mg/mL was added to prevent blood coagulation. These skeletal muscles and blood samples with infection with V. vulnificus strains were plated at 100 μL on 2% LB agar containing 50 μg/mL rifampicin, followed by incubation for 12 h at 37°C. V. parahaemolyticus was plated at 100 μL on 3% LB agar and incubated for 12 h at 37°C. V. mimicus and V. cholerae were plated at 100 μL on 1% LB agar and incubated for 12 h at 37°C. Live bacterial cells were counted by calculating the number of CFU/50 mg of skeletal muscle or CFU/100 μL of blood.The homogenized Mouse skeletal muscles were weighed to 100 mg, added to 900 μL of PBS, homogenized using a homogenizer pestle, and diluted gradually . cDNA was synthesized using the Primescript RT reagent kit . Gene expression levels were measured using quantitative real-time PCR with TB Green premix Ex Taq . The mouse-specific primer pairs used in this experiment are listed in Muscle tissues were fixed in 4% paraformaldehyde for 24 h, washed in PBS, and embedded in paraffin. Two-micrometer sections were stained with H&E according to standard procedures.Mouse whole blood was collected in 10-μL portions in 1.5-mL tubes, and the number of white blood cells was measured using an automatic blood cell counter for animals .Mouse skeletal muscles were added to 500 μL of radio immunoprecipitation assay (RIPA) buffer  per 10 mg of muscle tissue and homogenized sufficiently using a homogenizer . The mixture was centrifuged  and the supernatant was collected. The supernatant was then mixed with 5× sample buffer  and boiled for 5 min at 95°C. Equal amounts of protein were loaded on SDS polyacrylamide gels, separated by electrophoresis, and transferred to polyvinylidene difluoride membranes, which were then blocked with Tris-buffered saline containing Tween 20  for 1 h at 23°C and incubated with the primary antibodies overnight at 4°C. Subsequently, the membranes were washed 3 times with TBS-T, incubated with immunoglobulin G (IgG)–horseradish peroxidase-conjugated secondary antibodies for 2 h at 23°C, washed 3 times with TBS-T, visualized using the enhanced chemiluminescence (ECL) Western blotting kit , and imaged using LASS-2000. Antibodies against myeloperoxidase (MPO) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were purchased from Santa Cruz Biotechnology, Inc. . Horseradish peroxidase-conjugated goat anti-rabbit IgG was from Medical & Biological Laboratories Co., Ltd. .P values were calculated using the data presented as the means and SEM for all experiments. Hierarchical clustering by heat map and PCA of metabolite data sets were computed using MassHunter Mass Profiler Professional . P values were calculated using Student's t test with the threshold for significance set at a P value of <0.05. For metabolic analysis, comparisons between groups were estimated by Dunnett’s test with Benjamini-Hochberg normalization using IBM SPSS Statistics 25 . A Benjamini-Hochberg false discovery rate  of <0.05 was considered statistically significant. Multiple data analysis was performed based on one-way analysis of variance (ANOVA) and Tukey’s honestly significant difference test using IBM SPSS Statistics 25 . A P value of <0.05 was considered significant.All studies were conducted in triplicate and on 3 separate days. The data are presented as means and standard errors of the means (SEM) for all experiments. https://www.ebi.ac.uk/metabolights) with the data set identifier MTBLS6837; these data are summarized in The data from metabolic analysis using CE-MS are publicly available at EMBL-EBI’s MetaboLights repository (


Vf) for the lowest Vp.This study focuses on the retting effect on the mechanical properties of flax biobased materials. For the technical fiber, a direct link was established between the biochemical alteration of technical flax and their mechanical properties. In function of the retting level, technical fibers appeared smoother and more individualized; nevertheless, a decrease in the ultimate modulus and maximum stress was recorded. A biochemical alteration was observed as the retting increased (a decrease in the soluble fraction from 10.4 ± 0.2 to 4.5 ± 1.2% and an increase in the holocellulose fractions). Regarding the mechanical behavior of biocomposites manufactured by thermocompression, a non-elastic behavior was observed for the tested samples. Young moduli (E1 and E2) gradually increased with retting. The retting effect was more pronounced when a normalization was performed (according to the fiber volume and porosity). A 40% increase in elastic modulus could be observed between under-retting (−) and over-retting (+). Moreover, the porosity content (Vp) increased overall with fiber content. Setup 3, with optimized processing parameters, was the most desirable processing protocol because it allowed the highest fiber fraction ( For the development of ecofriendly and more efficient materials, research and industrial focus on the use of natural resources as an alternative to the petrol-based materials is highly favored. In this context, flax fiber-reinforced composites have attracted much attention from industrial and researchers. Compared to conventional fiber, flax fibers possess a lower price, relatively high specific strength and modulus, good sound absorption and heat insulation performance, recyclability and availability ,2. DespifV) of 50%, the authors of [Considering their different botanical origins, varieties, and variability, many studies ,12,13 hathors of ,18,19 reThis highlights the importance of understanding the flax behavior during retting in order to promote the integration of flax in biobased composites. In this context, this study aims to focus on the way in which retting could affect the mechanical performance of flax-based biocomposites. An advanced analysis of technical flax fibers associated to three different retting levels: textile-optimized (0), under-retting (−) and over-retting (+) with the goal to analyze the impact of retting time on the mechanical properties of technical fibers and the derived technical flax biobased composites. The originality of this work lies in the idea of studying the effect of the technical flax fibers, not of the single fiber.Epoxy XB3513 resin and Aradur 5021 polyimide crosslinker were used as the matrix polymer and provided by Vitech Composites . For the plant fiber, flax of the Bolchoi variety was used. It was grown in Romilly La Puthenay, Normandy, France. The seeding, harvesting and retting conditions are summarized in −2. The water-diluted epoxy was then sprayed on the flax bands and finally pre-cured at 120 °C. The resulting flax–epoxy prepregs presented a fiber content of Wf = 50% ± 3. After that, flax–epoxy prepregs were cut into 30 cm squares. Then, they were stacked in a UD pattern and placed between two metal plates. Two sheets of PTFE were added to facilitate the demolding step. At the end, we obtained biobased composite plates with an average thickness of 493 ± 31 µm and average density of 1.262 ± 0.024 g·cm−3. The thermocompression molding process was tested according to parameters summarized in −2.For the composite preparation, flax veils were impregnated with the epoxy resin to form prepregs sheets. The impregnation step was conducted on non-woven UD flax bands with a 110 g·mfV) and the porosity content (Vp) of our biobased composites was achieved by the combined use of TGA and pycnometer.The determination of the fiber content . The displacement rate was set at 1 mm·minw method  to deter−1 and carried out at 23 °C and 65% HR. Specimens prepared according to ISO 527 were cut by a laser beam with a cutting speed of 35 mm·s−1. Flax–epoxy plates were glued at the area pinched by the jaws to avoid early breaks. For each material, five samples were tested.Tensile tests were carried out on a Shimadzu traction machine with a 50 kN capacity load and self-tightening jaws. The displacement rate was fixed at 2 mm·min−1. During this analysis, the oscillation was strain-controlled, with a dynamic amplitude up to 30 µm and a force range of 4 ± 4 N. Measurements were conducted from 0.1 to 1 Hz frequencies.Dynamic mechanical analysis (DMA) was carried out in 3-point bending in a Netzsch DMA 242 system. Sample dimensions were 40 × 10 × 0.5 mm. The heating setup consists of a temperature slope from 30 to 130 °C at 3 °C·min2SO4 (%) (w/w) insoluble residue.The chemical composition  on the flax samples was performed according to the AFNOR standard (XPU44-162), derived from the Van Soest method . Tests w−3 for the theoretical elementary fiber and decreased from 47.6 to 29.8 GPa/g·cm−3 for the 100 mm technical fiber. The specific tensile strength varied from 631 to 553 MPa/g·cm−3 and from 477 to 224 MPa/g·cm−3 for the theoretical elementary fiber and the 100 mm technical fiber, respectively. Similar results have been reported by Zhu et al. [Moreover, the elastic modulus and tensile strength decreased when the retting level was more pronounced . The ultu et al. . Whateve(i)Area 1 (0–0.1% deformation): an elastic transition. This area is used to calculate the E1 modulus .(ii)Area 2 (0.1–0.3% deformation): a plastic transition and attributed to quick rearrangement of crystalline cellulose microfibrils between them and leading to a reduction in the stiffness between area 1 and area 3.(iii)Area 3 (over 0.3%): the second elastic transition. The E2 modulus (for large deformations) was calculated between 0.3 and 0.5% of deformation.The variation in Young moduli  and tensile strength according to the influence of retting and setup is illustrated in fV) in cE is composite Young’s modulus; fE is longitudinal Young’s modulus of the fiber; mE is matrix Young’s modulus; mV is volumic matrix content; fV is volumic fiber content; η0 is fiber orientation efficiency (=1); η1 is fiber length efficiency (=1).For better understanding, we present the standardized mechanical properties of the tested materials  from setup 1 to setup 3. Furthermore, E’ increased by 15%  from lowest retted (−) to highest retted (+) level. This double positive impact is even more pronounced in the rubbery state, where a 45% increase in E’ was observed between retting (−) and (+) at 130 °C. Yang et al. also noted a similar trend using kenaf fibers mixed with PBAT-PHBV copolymer matrix composite [Regardless of temperature and frequency, a double positive effect of both retting and processing parameters on the storage modulus was determined. At mild conditions , E′ increased by 13%  gradually increased with retting. The latter had a positive impact on the mechanical effective performance  to (+). The specific tensile strength decreased by −13% and −53% for the theoretical elementary fiber and the technical fiber, respectively. The degradation of the middle lamella, mostly made of pectin, explains the loss of elasticity properties due to retting. The middle lamella degradation decreased the internal cohesion between the fibrils and consequently affected the elasticity and rigidity performances of technical flax fibers. From a mechanical perspective, advanced retting coupled with setup 3 leads to optimal and highly specific mechanical performance, synonymous with good correlation between high cellulose fraction, high crystallinity and mechanical properties. Performing a normalization of E1 modulus according to the fiber volume, the retting effect was particularly pronounced. Switching from retting (−) to (+) improved the elasticity moduli by 40%. Moreover, the higher the fiber content, the higher the porosity rates (Vp). The same trend was also deduced for viscoelastic behavior. With optimal conditions (retting (+) and improved processing setup 3), the storage modulus was also significantly improved, confirming the efficiency of retting (+)/setup 3 conditions at different scales of mechanical performances. Processing parameters strongly affect the conversion rate of the epoxy monomers and up to 90% of monomer conversion leads to a restriction on the macromolecular chains. Further viscoelastic and wettability analysis will be necessary to better understand the matrix/reinforcement interaction and quality.