Sudden sensorineural hearing loss (SSNHL) can be a prodromal symptom of ischemic stroke, especially posterior circulation strokes in the anterior inferior cerebellar artery (AICA) area. Early diagnosis and optimal treatment for vascular SSNHL provide an opportunity to prevent more extensive area infarction. The objective of our research was to find clues that suggest stroke at the stage of isolated sudden hearing loss.We retrospectively investigated the medical records of patients who received an initial diagnosis of sudden sensorineural hearing loss upon admission from January 2017 to December 2022 at Capital Medical University Affiliated Beijing Tiantan Hospital. Among these patients, 30 individuals who developed acute ischemic stroke during their hospital stay were enrolled as the case group. To create a control group, we matched individuals from the nonstroke idiopathic SSNHL patients to the case group in terms of age (±3 years old) at a ratio of 1:4. We collected the clinical characteristics, pure tone hearing threshold test results, and imaging information for all patients included in the study.Three models were constructed to simulate different clinical situations and to identify vascular sudden sensorineural hearing loss (SSNHL). The results revealed that patients with SSNHL who had three or more stroke risk factors, bilateral hearing loss, moderately severe to total hearing loss, and any intracranial large artery stenosis and occlusion (≥50%) were at a higher risk of developing ischemic stroke during hospitalization. Consistent with previous studies, the presence of vertigo at onset also played a significant role in the early detection of upcoming stroke.Clinicians should be alert to SSNHL patients with bilateral hearing loss, moderately severe to total hearing loss and other aforementioned features. Early pure tone audiometric hearing assessment and vascular assessment are necessary for high-risk patients with SSNHL. Sudden sensorineural hearing loss (SSNHL), a subset of sensorineural hearing loss (SHL) , is widePrevious studies indicated that 1.8%−4.2% of SSNHL patients were diagnosed with ischemic stroke, which was a higher percentage than that of people who were never diagnosed with SSNHL . SeveralConsidering the time window for thrombolytic or interventional treatment, it is crucial to identify vascular SSNHL early before more devastating cerebral infarction. However, to our knowledge, there is still a lack of research on risk factors of stroke in isolated SSNHL patients. This case–control study aims to investigate the clinical characteristics, laboratory tests, auditory, and neuroimaging findings to facilitate the early identification of SSNHL related to stroke.Retrospectively, we investigated the medical records of patients who received an initial diagnosis of sudden sensorineural hearing loss upon admission from January 2017 to December 2022 at Capital Medical University Affiliated Beijing Tiantan Hospital. According to the Guidelines for Diagnosis and Treatment of Sudden Deafness 2015, China , the diaThe controls were nonstroke idiopathic SSNHL inpatients in the same period (January 2017 to December 2022) as the case group. Patients with identifiable causes of hearing loss, such as noise-induced hearing loss, traumatic hearing loss, ototoxic drug poisoning, and Meniere's disease, were excluded. Given that atherosclerosis is commonly associated with advancing age , age-relWe edited a case report form (CRF) and digitized it using Epidata 3.1 software developed by EpiData Association in Copenhagen, Denmark. The study encompassed the collection of demographic and medical records from all 150 patients, including information on sex, age, hypertension, diabetes, hyperlipidaemia, history of stroke, and drinking and smoking history.The detailed definitions of the six risk factors for stroke mentioned above are as follows: hypertension, indicated by a history of high blood pressure with a measurement of ≥140/90 mmHg as reported by the individual or diagnosed by a physician ; dyslipiThe auditory assessment was ascertained by measuring pure tone averages (PTAs) from 500 Hz to 4,000 Hz for both bone and air conduction (ISO/EC17025), and the auditory assessments were performed by audiologists. According to the World Report on Hearing 2021, the degree of hearing loss was determined using the average hearing thresholds at 500 Hz, 1,000 Hz, 2,000 Hz, and 4,000 Hz. Hearing loss was classified as either mild to moderately severe (20–50 dB) or moderately severe to total hearing loss (≥50 dB) . MoreoveBrain MRI and magnetic resonance angiography (MRA) imaging were performed on all but one patient who had previously undergone coronary artery bypass graft surgery. However, one patient had typical focal neurological deficits (dysarthria and diplopia) after SSNHL, and subsequent CT (11 days later) showed hypodensity in the right brachium pontis. The interpretation of neuroimaging was independently achieved by a neurologist and a neuroradiologist to ensure accuracy and reliability. The vascular territories of infarct lesions in the case group were determined using MRI anatomical templates that have been previously validated for diagnosing arterial territories , 24. TheIn addition, we evaluated major intracranial large arteries in all 150 patients, including the internal carotid arteries , anterior cerebral arteries , middle cerebral arteries , posterior cerebral arteries , vertebral arteries , and basilar artery (BA). We used magnetic resonance angiography (MRA) with the WASID  criteria to measure the degree of intracranial artery stenosis (ICAS). This was graded as normal to mild stenosis (<50%) and moderate stenosis to occlusion (≥50%) .t test was applied to compare data between the groups. The chi-square test was applied to analyse comparisons for dichotomous data; p < 0.05 was regarded as statistically significant. Three multivariate logistic regression models were constructed based on the different situations in clinical practice. Due to the limited sample size, we combined stroke risk factors such as “smoking history” from p values (p < 0.05) were assumed to be statistically significant. A receiver operating characteristic curve (ROC) was plotted, and the area under the ROC curve (AUC) was calculated.SPSS version 26.0  was used for statistical analysis. We expressed dichotomous data as the numbers (percentages) and expressed continuous data as the median  or mean standard deviation. For continuous data, the Mann–Whitney U test or Student's p < 0.05) in terms of sex, dyslipidaemia, hypertension, stroke history, smoking, and drinking history. In the case group, 14 patients (46.67%) had three or more stroke risk factors, while in the control group, there were 12 patients (10%) with the same condition (p < 0.001). The incidence of vertigo in the case group was significantly higher (p = 0.011) than that in the control group among the accompanying symptoms. No significant differences were found between the two groups in terms of dizziness, headache, tinnitus, or other accompanying symptoms.The case group consisted of twenty five males and five females, with an average age of 57.13 ± 10.64 years. The nonstroke control group consisted of 120 patients, comprised of 58 females and 62 males, with an average age of 56.67 ± 10.82 years. p = 0.003). With respect to the degree of SSNHL, a significant distinction was observed in the range of moderately severe to total hearing loss (p = 0.017). Therefore, individuals with SSNHL displaying a degree of moderately severe to total HL have an increased likelihood of developing ischemic stroke. For lab tests, statistically significant differences (p < 0.001) were observed in serum creatinine (Scr). No significant difference was observed in the other indicators (p > 0.05).As depicted in Among the individuals in the case group, the infarcts involved the cerebellum in seventeen cases, pons in nine cases, corona radiata in five cases, occipital lobe in four cases, frontotemporal lobe in three cases, corpus callosum in three cases, medulla oblongata in two cases, midbrain in one case, and thalamus in two cases. For the assessment of intracranial arteries, 22 of 30 (73.3%) patients in the case group had intracranial large artery stenosis or occlusion, among whom 20 (66.6%) patients had ≥50% stenosis or occlusion, including 18 (60.0%) cases presenting with ≥50% stenosis or occlusion in the vertebral arteries and/or basilar artery. Additionally, out of the 120 patients in the nonstroke control group, 24 individuals (20%) were found to have moderate to severe stenosis or occlusion in their large arteries, with a statistically significant difference (p <0.001).To improve clinical applicability, three distinct multivariate logistic regression models were designed to determine the risk factors associated with ischemic stroke in patients with sudden sensorineural hearing loss (SSNHL).p = 0.004). Vertigo, when present at onset, was a significant risk factor of stroke-related SSNHL . Similarly, patients with higher serum creatinine levels on admission were at a greater risk of stroke , as shown in For this primary model, the hypothesized scenarios were that patients did not undergo pure tone audiometry and MRI during their clinic visits. Patients with three or more stroke risk factors showed a markedly higher risk of ischemic stroke  and moderate to severe hearing loss  were associated with an increased risk of stroke.For cases where complete audiometry was carried out, findings related to the side and degree of hearing loss were integrated into Model-1. Audiometric data indicated that both bilateral hearing loss .Building on Model-2, the presence of intracranial artery stenosis or occlusion (≥50%) was introduced to develop Model-3. Factoring in intracranial artery issues became a strong risk factor for evolving into cerebral infarction during hospitalization . MRI of the brain revealed acute infarction in the bilateral cerebellum, pons, and left occipital lobe . MagnetiRegardless of the inclusion or exclusion of audiometric and neuroimaging variables in model-1 to model-3, the presence of accompanying vertigo consistently serves as a dependable risk factor for stroke. Similarly, Tzu-Pu Chang et al. found that individuals who presented with both sudden hearing loss and vertigo had a higher likelihood of stroke than those with either vertigo or sudden hearing loss alone. Furthermore, there seemed to be an increasing risk of stroke as the interval between episodes of these two symptoms diminished . This maBilateral SSNHL is rare, accounting for approximately 5% of all SSNHL cases, and is often associated with poor prognosis . Our stuApart from which side hearing loss occurs, the multivariate logistic model-2 and model-3 showed that the severity of hearingloss was a risk indicator for stroke. A previous study showed that individuals with more severe hearing loss tend to have a higher CHADS2 score, suggesting that the severity of hearing loss was positively related to the degree of atherosclerosis . AnotherIn contrast to previous studies that exclusively examined patients with anterior inferior cerebellar artery infarction –42, our This study represents the initial endeavor to explore the risk of stroke among patients with sudden sensorineural hearing loss (SSNHL) during their hospitalization. In cases where there is no noticeable neurological deficit or it appears too late, thrombolytic or antithrombotic treatment may not be feasible. This study provides clues to this apparent conundrum. We conducted an exploration to build three models simulating different clinical situations, incorporating clinical characteristics, laboratory tests, pure tone audiometry, and neuroimaging. These models aim to assist clinicians and serve as a reference for future research. The innovative findings reveal that bilateral sudden sensorineural hearing loss and moderately severe to total hearing loss are independent risk factors for subsequent stroke. Additionally, our study supports earlier findings indicating that individuals with SSNHL accompanied by vertigo at onset are at an elevated risk of stroke , 44.Despite our study's implications, we acknowledge its limitations. First, this study is a retrospective case–control study with a limited sample size and thus is at risk of information bias. Second, pure tone audiometry was measured as the average of 500, 1,000, 2,000, and 4,000 Hz. Quartering is a common method of hearing calculation and may not be sufficient to assess hearing loss at low or high frequencies alone; therefore, additional studies in patients with high-tone or low-tone hearing loss are needed. Third, due to the retrospective nature of this study, detailed time information between SSNHL and neurological deficits was not fully collected. As a result, the study was unable to determine the ideal time point for magnetic resonance imaging. There is still a need for further sample size expansion and multicentre validation in the future.We cautiously consider that SSNHL may not be the direct cause of stroke but rather a potential indicator or warning sign, particularly for posterior circulation stroke in the middle territory, during the progression of the disease. According to our research, clinicians should be alert to patients with three or more stroke risk factors, bilateral SSNHL, and moderately severe to total hearing loss, effectively screen high-risk vascular SSNHL groups and complete brain structure and vascular imaging evaluations.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.Ethical approval was not required for the study involving humans in accordance with the local legislation and institutional requirements. Written informed consent to participate in this study was not required from the participants or the participants' legal guardians/next of kin in accordance with the national legislation and the institutional requirements.YZ: Investigation, Writing—original draft, Data curation, Methodology. HL: Writing—review & editing, Investigation. GL: Data curation, Writing—review & editing. JL: Formal analysis, Writing—review & editing. J-JM: Methodology, Writing—review & editing. XZ: Conceptualization, Funding acquisition, Writing—review & editing. YJ: Conceptualization, Funding acquisition, Methodology, Supervision, Writing—review & editing.


SOD2 and MGST3 are proposed as mechanistically associated with cisplatin-induced hearing loss. Further, the models in this study focus on individual patient benefit and incorporation of quality of life measures to identify hearing loss impact. To study short- and long-term effects of chemotherapy, testicular cancer is ideal as a model disease for other cancers, as patients are young with long life-expectancy and without significant comorbidity. With small adjustments, the model can likely be applied in the treatment of other cancers where cisplatin is used, thus helping with choice of treatment without risking a trade-off in efficacy, standing to influence clinical practice.To our knowledge, this is the first study that presents a machine learning setup incorporating genetics and clinical factors to predict hearing loss in a large and fairly unique cohort of testicular cancer patients, with follow-up data examining long-range side effects of chemotherapy. Genetic variants in Testicular cancer is predominantly curable, but the long-term side effects of chemotherapy have a severe impact on life quality. In this research study, we focus on hearing loss as a part of overall chemotherapy-induced ototoxicity. This is a unique approach where we combine clinical data from the acclaimed nationwide Danish Testicular Cancer (DaTeCa)-Late database. Clinical and genetic data on 433 patients were collected from hospital files in October 2014. Hearing loss was classified according to the FACT/GOG-Ntx-11 version 4 self-reported Ntx6. Machine learning models combining a genome-wide association study within a nested cross-validated logistic regression were applied to identify patients at high risk of hearing loss. The model comprising clinical and genetic data identified 67% of the patients with hearing loss; however, this was with a false discovery rate of 49%. For the non-affected patients, the model identified 66% of the patients with a false omission rate of 19%. An area under the receiver operating characteristic (ROC-AUC) curve of 0.73  was obtained, and the model suggests genes SOD2 and MGST3 as important in improving prediction over the clinical-only model with a ROC-AUC of 0.66 . Such prediction models may be used to allow earlier detection and prevention of hearing loss. We suggest a possible biological mechanism for cisplatin-induced hearing loss development. On confirmation in larger studies, such models can help balance treatment in clinical practice. Testicular cancer is the most common cancer in men below 40 years of age in developed countries with a continuously rising incidence in many countries . It is a2 increase in cisplatin dose, a 3.2 dB decrease in age-adjusted hearing threshold has been identified [Prevalence of ototoxicity has been associated with cumulative cisplatin doses and age at diagnosis. For example, with every 100 mg/mentified . In a laentified . In anotentified . There ientified ,10,11.Platinum ototoxicity has been associated with death of hair cells in the organ of Corti, which reside within a blood–labyrinth barrier ,12, whicn = 2572). Clinical features were identified in hospital files [Long-term TCS were identified in the DaTeCa-Late cohort  with patal files . In Octoal files .Patients gave informed consent to participate in this study, and the study was approved by the regional ethical committee  and the National Board of Data Protection .2 and etoposide 100 mg/m2 for five days each cycle, while 43 (10%) received double-dose cisplatin (40 mg/m2) and etoposide (200 mg/m2) as part of a research protocol. Bleomycin was administered at a dose of 15.000 IE/m2 with a cumulative maximum dose of 150.000/m2.All patients received bleomycin–etoposide–cisplatin (BEP) for disseminated testicular cancer, for three cycles or more, as previously described . The majClinical information consisted of age at diagnosis and at time of completion of the questionnaire, body mass index (BMI), glomerular filtration rate before treatment, cumulative cisplatin dose per square meter of body surface area (BSA), number of BEP cycles, histology (seminoma vs. non-seminoma), prognostic classification as per International Germ Cell Cancer Collaborative Group (IGCCCG) , alcoholSelf-perceived hearing loss was assessed with the Ntx subscale of the FACT/GOG-Ntx-11, version 4, which evaluates the severity and impact of neuropathy . The queHere, we aim at predicting hearing loss; thus, only FACT/GOG-Ntx6 is further explored. FACT/GOG-Ntx6 and FACT/GOG-Ntx7 were not strongly correlated, which may indicate different biological etiologies. For FACT/GOG-Ntx6, to ensure clinical relevance, the outcome was dichotomized. Low-risk (score from 0 to 1) and high-risk groups (score from 2 to 4) were considered.It is important to point out that the FACT/GOG-Ntx questionnaire was completed in 2014, and patients had answered FACT/GOG-Ntx6 according to their current experience of hearing levels. However, at that time, the patients were also asked if they recalled experiencing worse hearing during treatment , and whether it returned to normal afterwards . Even though HC Q1 and HC Q2 are not validated at the same level as FACT/GOG-Ntx , we used® HumanOmniExpressExome-8-v1-2-B-b37 chip (approximately 1 million markers).DNA samples were prepared at DTU Multi-Assay Core  and genotyped at AROS Applied Biotechnology A/S company  using Illumina® (v2011.1) with PLINK Input Report Plug-in (v2.1.3). Variants with genotyping call rate < 0.98, not in Hardy–Weinberg equilibrium , or with a minor allele frequency < 0.01 were excluded. Quality control for both single nucleotide polymorphisms (SNPs) and patient samples is described in detail in Genotyping data were converted into pedigree format using GenomeStudioCYP2J2 rs11572279, MGST3 rs9333378, ABCA12 rs10498027, ABCC5 rs939336, WFS1 rs1801206, SLC44A4 rs494620, NOX3 rs12195525, CEP78 rs17787781, CYP2C9 rs4917639, CYP2C8 rs2071426, SYCE1 rs2149616, ABCC8 rs2074308, DUSP6 rs808820, DMXL2 rs2414105, ABCA10 rs10491178, ABCA7 rs3752229, CYP2B6 rs2279345, ERCC1 rs3212986, MCM8 rs3761873. Additionally, seven SNPs reported to be associated with cisplatin-induced hearing loss in a recent systematic review [LRP2 rs2075252, LRP2 rs4668123, TPMT rs1800460, SOD2 rs4880, GSTP1 rs1695, COMT rs4646316, COMT rs9332377.SNPs were selected via a genome-wide association study (GWAS) within a nested cross-validated logistic regression  and a systematic review search. Genes linked with cisplatin metabolism or ototoxicity were obtained  from datAll patients included after quality control had complete hearing loss outcome data. In patients with missing values in predictors (for BMI and smoking), a multiple imputation method ,27 with A nested five-outer, five-inner cross-validation logistic regression was implemented using SciKit-learn  ) training . Then, fThe dataset was randomly split 30 different times in training, validation, and test sets to ensure model reproducibility and robustness. More information on model hyperparameters, encoding of variables, and feature normalization is included in For the model with clinical data only, permutation tests were applied to ensure the model was not fitting random noise. For the model with clinical and genetic data, this was achieved by adding randomly selected SNPs.n = 278) patients had a median age (IQR) of 29 (26–36) years. Demographic features are presented in Out of 478 patients from the Danish Testicular Cancer (DaTeCa)-Late cohort , 45 patiAfter genotype quality control, 393 patients with data on 611,129 SNPs were available for analysis .In this study, we used the FACT/GOG-Ntx-11 version 4, which provides a targeted assessment of peripheral neuropathy such as auditory neuropathy. Auditory neuropathy comprises two different questions, where FACT/GOG-Ntx6 measures difficulty hearing, and FACT/GOG-Ntx7 measures tinnitus . A moderFirst, the prediction ability of the routinely available clinical information was assessed. Nine features were incrementally included in the model through exploring exhaustive permutations with each single feature addition. The two most informative clinical features , SOD2 rs4880, MGST3 rs9333378, intergenic rs4389005, ABCA10 rs10491178, ABCA12 rs10498027, MCM8 rs3761873 .Prediction performance, assessed as ROC-AUC, reached a plateau when six genetic features were added to the model , with a mean ROC-AUC of 0.73  A. The mos3761873 . Out of s3761873 . Only th metrics  were SODs9333378 . HomozygFor each sample, prediction scores ranged between 0 and 1, where a value closer to 1 indicated a higher probability of hearing loss. Using a default cut-off of 0.50, a sensitivity of 67% was reached and a positive predictive value of 51%. Correspondingly, this resulted in a specificity of 66% and a negative predictive value of 80% B. The moFor most patients (320 out of 393), adding genetic data improved hearing loss prediction; however, for 42 out of 320, this was still not enough to correctly classify these patients. In 7 out of 393 patients, the addition of genetic data led to misclassification. For 55 out of 393 patients, neither clinical nor genetic data helped on prediction and/or classification .Overall, we were able to improve prediction performance when adding genetic features to clinical data (ROC-AUC 0.73) compared to the models with only clinical data (ROC-AUC 0.66).To test robustness and non-randomness of the selected models, in the models with only clinical data, all variables were permutated, which led to a mean ROC-AUC close to 0.50 throughout the forward feature selection . The meaIn an additional test, random genetic variants were added to the model with the informative clinical features (age at diagnosis and number of treatment cycles). Mean ROC-AUC was 0.67  for the model with six random genetic variants and two informative clinical features , which wIn this study, we present a model for the prediction of hearing loss after cisplatin-containing chemotherapy based on a combination of clinical and genetic features, achieving a classification performance of ROC-AUC 0.73. We observed an improved prediction after the inclusion of genetic data compared to clinical data only. Age at diagnosis and number of treatment cycles were the most important clinical predictors, matching what has previously been reported ,31.We have focused on hearing loss as part of ototoxicity, as we did not observe a strong correlation between hearing loss (FACT/GOG-Ntx6), and tinnitus (FACT/GOG-Ntx7), which may indicate independent biological mechanisms. Indeed, not all people who suffer from hearing loss have tinnitus, and vice versa, and studies on the genetics behind tinnitus are still at an early stage ,33.SOD2 that codes for the superoxide dismutase 2 (SOD2), a mitochondrial protein [SOD2 SNP [SOD2 rs4880:GG individuals compared with SOD2 rs4880:AA, or SOD2 rs4880:GA [The first SNP selected in the model, the functional rs4880 SNP, is located on codon 16 exon 2 of  protein . SNP rs4SOD2 SNP ; howeverSOD2 SNP . Some stSOD2 SNP . A folloSOD2 SNP . Howevers4880:GA .MGST3, that codes for the microsomal glutathione S-transferase 3 (MGST3) [The second SNP selected in the model, SNP rs9333378, is located in  (MGST3) . Among t (MGST3) .SOD2 rs4880 and MGST3 rs9333378 on cisplatin-induced hearing loss.Here, we hypothesized a combined effect of When platinum enters the cells, it is metabolized by the mitochondria, which will lead to the production of reactive oxygen species (ROS) such as superoxide. SOD2 will then degrade superoxide into hydrogen peroxide until complete superoxide anion degradation. If SOD2 is prevented from entering the mitochondria due to partial arrest of beta-helix, this may lead to an accumulation of ROS. ROS cause lipid peroxidation, activation of pro-inflammatory factors, and cell death by apoptosis, including hair cells ,42. IndeGPR12 , a G protein-coupled receptor (GPCR). GPCRs have been seen to be involved in several physiological and pathological functions [ABCA10 rs10491178 and ABCA12 rs10498027, both leading to stop-gains within the ABCA genes which encode ATP-binding cassette (ABC) transporters. Overexpression of ABC transporters have been associated with multidrug resistance, including cisplatin, in multiple tumors [ABCA10 rs10491178:GG has been associated with lower expression of ABCA6 [MCM8 rs3761873 was the last SNP selected by the model, which leads to a stop-gain. MCM8 encodes the mini-chromosome maintenance 8 homologous recombinant repair factor protein (MCM8), and in a recent mouse study, inhibition of MCM8 (and MCM9) hypersensitized cells to cisplatin [Additionally, potential novel variants associated with cisplatin-induced hearing loss were selected on the logistic regression model. SNP rs4389005 located in an intergenic region was found in the cross-validated GWAS. The closest gene is unctions . The sube tumors . ABCA10 of ABCA6 . MCM8 rsisplatin .2 and three treatment cycles). This points to other relevant features that led this patient to develop hearing loss, either clinical or genetic predispositions that might be underrepresented in this dataset and, hence, may not have been detected.While we observed a false discovery rate of 49% using a 0.50 cut-off, it is promising to see that only four of the twenty-three patients with the highest score (FACT/GOG-Ntx6 = 4) were misclassified. Three of them had a prediction score very close to 0.50 (two patients with 0.48 and one with 0.49 prediction scores). The last misclassified patient had a prediction score of 0.31 and was also the youngest of the 23. Furthermore, he received one of the lowest amounts of cisplatin , or low risk (66% specificity) of hearing loss after cisplatin-based treatment.SOD2 rs4880 and MGST3 rs9333378 on cisplatin-induced hearing loss development. In our study, these SNPs have not yielded significant results when single associations between SNPs and outcome have been performed; thus, a combination of cross-validated GWAS and systemic review search is suggested as a feature selection approach for machine learning.We also propose a combined effect involving Following confirmation in a prospective clinical setting and replication in larger independent studies, such a model could be used as a complement to support clinical decision-making and help in reducing hearing loss cases by adjusting treatment for patients in the high-risk group, especially with treatment of other cancers where cisplatin is used.


The microbiota inhabiting soil plays a significant role in essential life-supporting element cycles. Here, we investigated the occurrence of horizontal gene transfer (HGT) and established the HGT network of carbon metabolic genes in 764 soil-borne microbiota genomes. Our study sheds light on the crucial role of HGT components in microbiological diversification that could have far-reaching implications in understanding how these microbial communities adapt to changing environments, ultimately impacting agricultural practices. In the overall HGT network of carbon metabolic genes in soil-borne microbiota, a total of 6,770 nodes and 3,812 edges are present. Among these nodes, phyla Proteobacteria, Actinobacteriota, Bacteroidota, and Firmicutes are predominant. Regarding specific classes, Actinobacteria, Gammaproteobacteria, Alphaproteobacteria, Bacteroidia, Actinomycetia, Betaproteobacteria, and Clostridia are dominant. The Kyoto Encyclopedia of Genes and Genomes (KEGG) functional assignments of glycosyltransferase (18.5%), glycolysis/gluconeogenesis (8.8%), carbohydrate-related transporter (7.9%), fatty acid biosynthesis (6.5%), benzoate degradation (3.1%) and butanoate metabolism (3.0%) are primarily identified. Glycosyltransferase involved in cell wall biosynthesis, glycosylation, and primary/secondary metabolism (with 363 HGT entries), ranks first overwhelmingly in the list of most frequently identified carbon metabolic HGT enzymes, followed by pimeloyl-ACP methyl ester carboxylesterase, alcohol dehydrogenase, and 3-oxoacyl-ACP reductase. Such HGT events mainly occur in the peripheral functions of the carbon metabolic pathway instead of the core section. The inter-microbe HGT genetic traits in soil-borne microbiota genetic sequences that we recognized, as well as their involvement in the metabolism and regulation processes of carbon organic, suggest a pervasive and substantial effect of HGT on the evolution of microbes. Employi via HGT , and in  via HGT .via MGEs  has been highlighted as a public concern  for downstream analyses. Putative microbial horizontally transferred genes (HTGs) in these genomes were identified employing the BLASTP-based IMGAP pipeline under default mode  with the keyword “soil” within the “biosample” or “habitat” regions. We then chose and downloaded from the public database (Genbank/IMG) the out-coming high-quality genomes of soil microbial isolates  to Streptomyces yangpuensis fd2-tb (Actinobacteriota) having the highest HGT frequency (n = 14) (see Supplementary Table S3 at https://doi.org/10.6084/m9.figshare.22154828.v1). The chimeric nature and high externalization rate  of Haloarchaea have been highlighted previously  functional assignments, glycosyltransferases (18.5%), glycolysis/gluconeogenesis (8.8%), carbohydrate-related transporters (7.9%), fatty acid biosynthesis (6.5%), benzoate degradation (3.1%), and butanoate metabolism (3.0%) are most frequently identified . These rhttps://doi.org/10.6084/m9.figshare.22154828.v1). The most commonly used centrality measurement, degree, counts the edges that link a specific node. In a directed graph, the arrows are directional, pointing from one node (HGT donor) to another (HGT acceptor), so the number of arrows pointing to each node is its entry degree. In contrast, the number of arrows pointing away from this node is its outgoing degree. Of the top 30 nodes having the highest degree values (>25), class Actinomycetia occupies the most (43.2%) of these nodes (see Supplementary Table S4 at https://doi.org/10.6084/m9.figshare.22154828.v1), followed by classes Deltaproteobacteria (13.3%) and Betaproteobacteria (13.3%). In comparison, most other nodes (92.5%) in the overall HGT network of carbon metabolic genes in soil-borne microbiota display a degree value <5. In particular, the highest degree values in correspondent classes were seen in the genomes of Kribbella soli VKM Ac-2,540 , Anaeromyxobacter sp. Red232 , Paenibacillus piri MS74 , Pseudoduganella violaceinigra DSM 15887, and Pyrinomonas methylaliphatogenes K22 .We then used two classical network centralities, degree and betweenness, to examine what taxonomic groups are holding and shaping the HGT network , class Actinomycetia also occupies most (60.0%) of these nodes, followed by class Deltaproteobacteria (10.0%). Primarily exhibiting the highest betweenness centrality values in correspondent classes are genomes of Jiangella anatolica GTF31 , Pyrinomonas methylaliphatogenes K22 , Saccharicrinis fermentans DSM 9555 , Methylovulum miyakonense HT12 , and Nitrosospira multiformis ATCC 25196 . In comparison, most other nodes (98.2%) in the overall HGT network of carbon metabolic genes in soil-borne microbiota display a betweenness centrality value <5. On the other hand, nodes that display lower betweenness centralities either occupy peripheral positions in the network or form their linked constituents.Another centrality measurement, betweenness, measures how frequently a specific node is located on the shortest path possible between any pair of nodes within the network. Nodes with large betweenness centralities act as essential reservoirs or communicators for the common-good genetic codes. Of the top 30 nodes having the highest betweenness centrality values (>20) in the overall HGT network of carbon metabolic genes in soil-borne microbiota  degree, as well as average path length were higher in the “acceptor” network than in the “donor” one in the corresponding networks of Proteobacteria and Actinobacteria, respectively. While the connector component (number of subgraphs in which each pair of nodes is connected) shows the opposite trend (higher in the “acceptor”). Average eigenvector centralities (that measure the transitive influence of nodes) decline from the Proteobacteria network to that of Actinobacteria, and, finally, Bacteroidota. Other network properties like modularity  show only slight variations among groups .2.2.We further created sub-networks that extracted HGT events (edges) corresponding to specific carbon metabolic functions  and nodes acting as HGT donors and acceptors, respectively, as well as extracting their direct edges .In the sub-network reflecting HGT episodes of core carbon metabolism , genes attributed to pathways of glycolysis/gluconeogenesis (10.14%), butanoate metabolism (7.29%), pyruvate metabolism (7.29%), and galactose metabolism (7.00%) are predominant. Microbial classes Actinobacteria (27.66%), Gammaproteobacteria (13.83%), and Alphaproteobacteria (12.59%) are mostly seen .https://doi.org/10.6084/m9.figshare.22154828.v1). Alcohol dehydrogenase exhibits a wide range of substrate specificity additionally, oxidizing mainly primary and secondary aliphatic alcohols when utilizingNAD+ as a co-substrate. It is also able to reduce aldehydes and ketones  is dominant in the current sub-network of core carbon metabolism . In the sub-network, HGT genes encoding acetolactate synthase (28.3%), succinate-semialdehyde dehydrogenase (22.64%), and 3-hydroxybutyryl-CoA dehydrogenase (9.43%) are predominant. Microbial classes Actinobacteria (26.14%), Actinomycetia (21.59%), and Alphaproteobacteria (13.64%) are mostly seen .Likewise, in the sub-network reflecting HGT episodes of lipid metabolism , genes attributed to fatty acid biosynthesis (28.88%), primary bile acid biosynthesis (2.94%), and fatty acid degradation (2.67%) are predominant. Microbial classes Actinobacteria (29.65%), Alphaproteobacteria (15.47%), Actinomycetia (14.0%), Gammaproteobacteria (9.02%), and Betaproteobacteria (7.73%) are mostly seen at the same time .https://doi.org/10.6084/m9.figshare.22154828.v1). Pimeloyl-ACP methyl ester carboxylesterase catalyzes the hydrolysis of the ester bonds of pimeloyl-ACP esters that allow the synthesis of pimeloyl-ACP via the fatty acid synthetic pathway (Polaromonas spp. isolated from Arctic and Antarctic glaciers (https://doi.org/10.6084/m9.figshare.22154828.v1). FabG is the key enzyme directly responsible for the synthesis of 3-hydroxyacyl-ACPs in the fatty acid synthesis elongation cycle  is dominant in the current sub-network of lipid metabolism , genes attributed to benzoate degradation (47.71%), chloroalkane/chloroalkene degradation (11.93%), steroid degradation (9.17%), fluorobenzoate degradation (7.34%), dioxin degradation (6.42%), and toluene degradation (6.42%) are predominant. At the same time, microbial classes Actinomycetia (19.07%), Actinobacteria (18.56%), Gammaproteobacteria (15.98%), Alphaproteobacteria (14.43%), and Betaproteobacteria (10.31%) are mostly observed .https://doi.org/10.6084/m9.figshare.22154828.v1). While microbial classes Gammaproteobacteria (14.37%), Actinobacteria (9.58%), Alphaproteobacteria (9.58%), Actinomycetia (8.54%), Bacteroidia, (8.12%), Betaproteobacteria (6.04%), and Bacilli (5.42%) are mostly observed to encode glycosyltransferase  ranks first overwhelmingly in the list of most frequently identified carbon-metabolism-related HGT enzymes from the soil-borne microbiota  of organic carbon compounds were also visualized by sub-network creation . In the https://doi.org/10.6084/m9.figshare.22154828.v1), such as beta-lactamase family enzymes (159 entries) that degrade multiple metabolites  for the use of the substrates as nutrients or carbon sources (https://doi.org/10.6084/m9.figshare.22154828.v1).Also, there are other specific carbon metabolic enzymes enriched in the HGT network  . We thouA variety of soil-borne carbon-cycle HGT events of cross-class and even cross-phylum levels were discovered with considerably high transfer frequency . The conPsudomonas putida KT2440, Escherichia coli MG1655, and Kluyvera sp. can be transferred to a wide range of bacterial phyla from agricultural soils . Similar findings were also reported previously. For example, according to research on the horizontally gained genes within the E. coli metabolic pathways  v.5.0  with the keyword “soil” within the “biosample” or “habitat” regions. We have then chosen and downloaded from the public database (Genbank/IMG) the resultant high-quality 764 genomes of soil microbial isolates as listed in Supplementary Table S1 at P) v.5.0  under deP) v.5.0 , 2022. IP) v.5.0 . This reThe original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.LL and ZL conceived and designed the research. YL, QX, and ZX provided the computing sources. LL, DM, ZY, and HY analyzed the data. LL wrote the manuscript. All authors contributed to the article and approved the submitted version.This research was supported by the key project of Science and Technology of Hunan Branch of China National Tobacco Corporation , Natural Science Foundation of Changsha (No. kq2202089), Fundamental Research Funds for the Central Universities of Central South University (No. 2022ZZTS0420), Hunan International Scientific and Technological Cooperation Base of Environmental Microbiome and Application (No. 2018WK4019), and key project of Science and Technology of China National Tobacco Corporation (No. 110202201004) (JY-04).YL, QX, and ZX were employed by Zhangjiajie Tobacco Company of Hunan Province.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


We compared the real-time PCR-determined expression levels of the target miRNAs between the two groups. We calculated the air conduction pure-tone-averaged (PTA) audiometric thresholds in affected ears at the initial and final follow-up visits. We undertook inter-group comparisons of hearing outcome status and initial and final PTA audiometric thresholds. There was no significant inter-group difference in miRNA expression level, hearing recovery status and initial and final affected ear PTA audiometric thresholds.Sudden sensorineural hearing loss (SSNHL) is an acquired idiopathic hearing loss. Serum levels of small, non-coding RNAs and microRNAs (miRNAs) miR-195-5p/-132-3p/-30a-3p/-128-3p/-140-3p/-186-5p/-375-3p/-590-5p are differentially expressed in SSNHL patients within 28 days of hearing loss onset. This study determines if these changes persist by comparing the serum miRNA expression profile of SSNHL patients within 1 month of hearing loss onset with that of patients 3–12 months after hearing loss onset. We collected serum from consenting adult SSNHL patients at presentation or during clinic follow-up. We matched patient samples drawn 3–12 months after hearing loss onset (delayed group,  MicroRNAs (miRNAs) are short (approximately 23 nucleotides) non-coding RNA strands that regulate gene expression. RNA polymerase II transcribes these nucleotides to create initial miRNAs in the nucleus (pre-miRNAs). Pre-miRNAs are precursor miRNAs with a hairpin structure that are derived by RNA polymerase III transcription and DGCR8 protein cleavage before transfer to the cytoplasm by exportin-5 (RNA binding protein). The RNA-induced silencing complex (RISC) is formed in the cytoplasm from loading one arm of the miRNA duplex resulting from the cleavage of pre-miRNAs by Dicer into Argonaute protein. The RISC binds to complementary sequences on target messenger RNA (mRNA), to achieve mRNA translation inhibition and mRNA degradation. MiRNAs thus control target gene expression at the post-transcriptional stage  based onMiRNAs have been identified in a wide range of bodily fluids, such as serum, plasma, urine, saliva, and other bodily fluids ,6,7; theChanges in the circulating levels of miRNAs have been identified in acquired sensorineural hearing loss . A betteSudden sensorineural hearing loss (SSNHL) is a type of acquired idiopathic hearing loss that develops within 72 h . SSNHL hThere is early evidence of miRNA involvement in SSNHL ,27. Li eThe reversibility of the identified DEM findings in SSNHL patients has not been studied. Hence, this study determines and compares the serum expression level of previously identified DEMs in SSNHL patients within 1 month of hearing loss onset with that of patients 3–12 months post-hearing loss onset.Nine SSNHL patients whose serum samples were classified, based on the time of collection , as beinp = 0.11). The initial median PTA audiometric threshold in the affected ears of immediate group patients, 66.9  dB, was not statistically different from the 76.3  dB median PTA audiometric threshold recorded in the delayed group . There was no significant inter-group difference in the proportion of patients experiencing hearing recovery, six of fourteen (42.9%) in the immediate group and five of nine (55.6%) in the delayed group. The final median PTA-affected ear audiometric threshold in immediate and delayed group patients, 63.3  dB and 67.5  dB, respectively, were also not significantly different. Both groups were thus similarly matched for presenting hearing loss severity, the proportion of patients with dizziness, and the rate of hearing recovery (The mean ages ± standard deviation (SD) of the patients in the immediate and delayed groups, 58.07 ± 16.95 and 59.56 ± 15.86 years, respectively, were similar. The male:female ratios in the immediate and delayed sample patient groups, 7:7 and 5:4, respectively, were similar . Overallrecovery .p > 0.005 Bonferroni-corrected Mann–Whitney U-test) (The median expression levels ∆Ct (IQR) of miRs -128-3p/-132-3p/-375-3p/-590-5p/-30a-3p/-140-3p/-186-5p and -195-5p were 2.68 (1.12), 3.29 (1.86), −1.66 (1.54), 4.34 (2.85), 4.88 (1.87), 2.37 (2), 0.70 (1.07), 3.38 (0.97) in immediate group patients and 2.92 (1.48), 3.80 (1.38), −0.59 (1.06), 5.96 (3.05), 4.96 (1.65), 2.96 (2.08), 0.68 (2.63), 4.35 (2.77) in delayed group patients, respectively, were not statistically different ( U-test) .p > 0.05, Mann–Whitney U-test). Furthermore, hearing recovery as well as final PTA thresholds of the affected ears were not statistically significantly different between immediate and delayed SSNHL patient groups. The stability of these miRNA findings over time suggests that these miRNAs have translational potential as SSNHL disease biomarkers. The expression levels of eight mature miRNAs found to be differentially expressed in the serum of SSNHL patients within 28 days of the onset of hearing loss compared to normal hearing healthy control individuals  were selThe spiral modiolar artery (SMA) is, to the best of our knowledge, the only artery that supplies blood to the cochlea. This artery ascends through the modiolus and is primarily divided into two parallel capillary networks of the stria vascularis and the spiral ligament in the cochlear lateral wall . The strMiRNAs that are abundantly found in the brain play critical roles in the development and function of neuronal networks, including neurogenesis, synaptogenesis, and morphogenesis regulation ,40. Of tHIF-1α) is a target of miR-140-3p [MiR-140-3p, another brain-enriched miRNA, has been demonstrated to orchestrate neuroinflammation and neuron apoptosis . MiR-140R-140-3p ,54. SimiR-140-3p . AdditioR-140-3p ,57. ThisR-140-3p  persistsSlc26a5) and antiapoptotic B-cell lymphoma 2 (BCL2) genes [The nervous system has been shown to contain large amounts of miR-195 and miR-132 . MiRs -12) genes ,64. Ther2) genes . Both miAcHE), an important TLR regulator particularly relevant in neuroinflammation, which results in the inhibition of TLR-induced cytokine production [IFNβ) and IFN-stimulated gene of 15 kDa (IsG15), and interleukin -1β (IL-1β) and -6 (IL-6), allowing the virus to propagate [Additionally, miR-132 expression is upregulated by Toll-like receptors (TLR) in inflammation, which in turn exerts an inhibitory effect on inflammatory processes . It downoduction . The dowropagate . In addiropagate . Zhou etropagate  have shoropagate . IL-6 reropagate . These pCirculating miRNAs have been shown to have high stability in the extracellular environment . HoweverAdult patients, 18 years and older, presenting with SSNHL as defined by the AAOHNS criteria , were rePatients who were unable to provide any blood samples, complete diagnostic physical and hearing tests, or with any known cause for hearing loss, major intercurrent medical illness or coexisting ear pathologies were excluded from the study. A full clinical examination of the participants’ ears was performed by the College of Physicians and Surgeons of British Columbia registered Otolaryngologist Head and Neck Surgeons. The position and shape of the external ears were inspected. Pain upon palpation over the tragus, pinna, or mastoid process was recorded if present. Otoscopic examination of the ear canals and tympanic membranes was undertaken to identify potential causes of conductive hearing loss resulting from middle ear fluid, otitis media, foreign bodies, cerumen impaction, otitis externa, or trauma. Webber’s tests were undertaken with 256 or 512Hz tuning forks to categorize hearing losses as sensorineural or conductive . Patients with serum samples drawn 3-12 months after the onset of SSNHL (delayed group) were matched by age and sex to patients with samples drawn within 28 days of SSNHL onset (immediate group) . This stAll participants underwent pure tone audiometric assessment by the College of Speech and Hearing Health Professionals of British Columbia registered Audiologists or Hearing Instrument Practitioners. The assessments were undertaken in sound-proofed environments using insert or supra-aural earphones and bone vibrators according to accepted practice guidelines . Copies A sensorineural hearing loss was defined for study purposes as a loss of >25 dB at any frequency with an air-bone gap of <10 dB. A conductive hearing loss was defined as an air-bone gap of >25 dB at any frequency. A mixed hearing loss was defined as the occurrence of AC and BC losses of >25 dB, with an air-bone gap >10 dB ,75. Durig for 15 min at 4 °C. The aqueous phase containing RNA was transferred to a new tube, and isopropanol  was added. The samples were incubated at room temperature for 10 min followed by centrifugation at 12,000× g for 15 min at 4 °C. In total, 1.5 volumes of 100% ethanol were added and mixed thoroughly by pipetting. Up to 700 μL of the sample, including any precipitate, was pipetted into an RNeasy MinElute spin column in a 2 mL collection tube and centrifuge at ≥8000× g for 15 s at room temperature. Then, 700 μL Buffer RWT was added to the RNeasy MinElute spin column and centrifuge for 15 s at ≥8000× g. Then, 500 μL Buffer RPE was added onto the RNeasy MinElute spin column and centrifuge for 15 s at ≥8000× g. Then, 500 μL of 80% ethanol was added to the RNeasy MinElute spin column and centrifuged for 2 min at ≥8000× g. RNeasy MinElute spin column was placed in a new 2 mL collection tube with an open lid and centrifuged at full speed for 5 min to dry the membrane. After discarding the flow-through, 14 μL RNase-free water was added directly to the center of the spin column membrane.Blood samples were collected for serum separation, and aliquots were stored at −80 °C for subsequent RNA extraction. Total RNA was extracted from 200 microliters of serum using miRNeasy Mini Kit  according to the manufacturer’s instructions. Briefly, 1 mL of Qiazol Lysis reagent was added to 200 μL of serum, followed by incubation at room temperature for 5 min. Then, 200 μL Chloroform  was added, and the samples were vortexed and incubated for 5 min at room temperature followed by centrifugation at 12,000× Reverse transcription (RT) was undertaken with a TaqMan Advanced cDNA synthesis kit  utilizing a preamplification step followed by TaqMan™ advanced miRNA Assay . In brief, a total of 30 μL and 2 μL of microRNAs, 3 μL of poly(A) reaction mix, 10 μL of ligation reaction mix, and 15 μL of reverse transcriptase reagents were incubated at 42 °C for 15 min and deactivated at 85 °C for 5 min. Then, 5 μL of the RT reaction product was incubated with 45 μL of the miR-Amp Reaction Mix before extracting and diluting cDNA 1:10 with nuclease-free water for microRNA real-time PCR analysis. ® Advanced miRNA Assays and Taqman Fast Advanced master mix on a QuantStudio Real-time PCR System . Individual qPCR assays were performed in triplicate with a total reaction volume of 20 μL using hsa-miR-30a-3p/-128-3p/-132-3p/-140-3p/-186-5p/-195-5p -375-3p/-590-5p/primers according to the manufacturer’s instructions. The PCR thermocycler was programmed for denaturation at 95C for 20 s, followed by 40 annealing cycles of 95°C for 1 s and 60°C for 20 s. The normalization of microRNA expression levels was performed based on reference microRNA, hsa-miR-191-5p. The miRNA expression level was calculated using the delta Ct method [Real-time PCR was performed using Taqmant method .Pure tone audiometric testing was used to confirm the diagnosis of SSNHL, categorize the degree of hearing loss at presentation, and determine patients’ hearing recovery status. Air conduction pure-tone average thresholds at 4 low  or 3 high  frequencies were calculated for affected ears . The heat-test or Welch’s t-test if the standard deviations in both groups were similar or different, respectively, and the inter-group proportion of females were compared with Pearson’s chi-square test. The groups’ patients’ clinical SSNHL profiles were compared statistically: initial and final PTA thresholds with Mann–Whitney U tests proportions of patients who recovered hearing and presented with associated dizziness were compared with Pearson’s chi-squared tests. A Bonferroni-corrected p-value of < 0.005 was adopted as significant to adjust for multiple t and u-tests. Box and Whisker plots were generated using GraphPad Prism version 9.0.The expression level of the test miRNAs in the serum of SSNHL patients in the immediate and delayed groups was compared with Mann–Whitney U-tests using SPSS version 26 . To determine if the groups were otherwise equally matched biologically inter-group, averaged age was statistically compared with independent samples. Student’s


Morus alba is used as a traditional Chinese medicine due to its various biological activities. Phenylpropanoid metabolism is one of the most important pathways in Morus alba to produce secondary metabolites and response to stress. From the general phenylpropanoid pathway, there are two metabolic branches in M. alba, including flavonoid and lignin biosynthesis, which also play roles in response to stress. However, the dynamic changes between flavonoid and lignin biosynthesis under Botrytis cinerea infection and UV-B stress in M. alba were unclear. To explore the different regulation mode of flavonoid and lignin biosynthesis in M. alba leaves’ response to biotic and abiotic stress, a combined proteomic and metabolomic study of M. alba leaves under UV-B stress and B. cinerea infection was performed. The results showed that most of the proteins involved in the lignin and flavonoid biosynthesis pathway were increased under either UV-B stress or B. cinerea infection in M. alba. This was also confirmed by enzyme assays and metabolomics analysis. Additionally, the abundance of proteins involved in the biosynthesis of jasmonic acid was increased after B. cinerea infection. This suggests that both flavonoid and lignin biosynthesis participate in the responses to abiotic and biotic stress in M. alba, but they might be regulated by different hormone signaling. Arabidopsis thaliana [Arabidopsis [Phenylpropanoid metabolism is one of the most important metabolisms in plants, yielding more than 8000 metabolites which contribute to plant development and plant-environment interplay . Flavonothaliana . There wbidopsis ,7.Populus ussuriensis resulted in enhanced tolerance to drought stress by means of anti-oxidation and mechanical supporting [Lignin biosynthesis is another important branch of phenylpropanoid metabolism. Lignin is a complex biomacromolecular polymer that usually surrounds the polysaccharide components of plant cell walls and provides structural support, giving rigidity and strength to stems . The actpporting . In addipporting . Moreovepporting .Botrytis allii [Lignin biosynthesis and flavonoid biosynthesis share the same precursor and early steps. Both of them could contribute to the tolerance of biotic and abiotic stress. However, a preference for lignin biosynthesis and the suppression of flavonoid biosynthesis has been reported in the studies of some species under biotic stress. The accumulation of flavonols induced by UV-B was attenuated by the concurrent application of the bacterial elicitor flg22 (simulating biotic stress), which correlated with the strong suppression of the flavonol biosynthesis genes . The supis allii . Since tis allii . AlthougMorus alba is widely cultivated because its leaves can be used to feed silkworms [M. alba is also used as an herb in traditional Chinese medicine. Secondary metabolites of M. alba have been proved to have multiple physiological activities, including antiviral, antibacterial, and anti-inflammatory activities [M. alba leaves under different stresses, we treated M. alba leaves using two kinds of stress: B. cinerea infection, which is biotic stress, and high-level UV-B radiation, which is abiotic stress. Proteomic analysis was performed, and the results showed that flavonoid biosynthesis and lignin biosynthesis were significantly up-regulated by BC stress and UV-B stress. Although B. cinerea infection stress is biotic stress, no suppression of flavonoid biosynthesis has been observed compared with UV-B stress. This was confirmed by enzyme activity and metabolite analysis. After comparing the changes in lignin biosynthesis and flavonoid biosynthesis in the B. cinerea infection group (BC) and UV-B radiation group (UVB), the results suggested that resistance to UV-B and BC stress may be achieved through different pathways, but both of them could finally activate the biosynthesis of flavonoid and lignin.ilkworms . M. albativities ,17,18,19M. alba leaves. In the BC group, 408 differentially expressed proteins (DEPs) were identified, including 165 decreased and 243 increased DEPs. In the UVB group, 393 DEPs were identified. Among them, 189 DEPs were decreased and 204 were increased , jasmonic acid (JA), and abscisic acid (ABA), were analyzed based on the proteomics data. The results showed that only proteins related to JA biosynthesis were significantly altered in the BC group, which was different from the UVB group B. Two liGlehnia littoralis cells, Arabidopsis seedings, Brassica napus seedings, etc. [M. alba leaves under BC stress. The abundance of DEPs involved in flavonoid and lignin biosynthesis was increased, and the contents of flavonoids in the BC and UVB groups were also increased. The up-regulation of flavonoid biosynthesis under BC stress was also exhibited on a transcript level [B. cinerea infection and UV-B stress were two different types of stress, and the distinct change in JA biosynthesis showed that there was a difference in the initial steps of the response under these two types of stress. The similar changes observed in flavonoid and lignin biosynthesis suggested that there might be a same node in the responses to B. cinerea infection and UV-B stress, which could up-regulate the proteins involved in flavonoid and lignin biosynthesis simultaneously.Through a Venn diagram, more than half of the DEPs were common in the UVB and BC groups. It seems that many of the changes in response to the UVB and BC treatments were similar, although they were two completely different types of stress. Moreover, the similarity was more obvious when we considered the changes in the pathways related to phenylpropanoid biosynthesis. The common DEPs were not only identified in the lignin biosynthesis pathway and flavonoid biosynthesis pathway, but were also systematically increased. It has been reported that plants suppress the accumulation of flavonoids and up-regulate lignin and phytotoxin biosynthesis when facing biotic stress, especially the down-regulation of the CHS . This kigs, etc. ,20,21. Hpt level   exhibited an impaired accumulation of flavonoids and were more sensitive to osmotic and drought stresses, which indicated that the suppressed tolerance caused by the deficiency of flavonoid biosynthesis could not be complemented by other processes [Phytophthora citrophthora attack in fruit. In addition, the fruits treated with 6-benzylaminopurine could increase the level of polymethoxyflavone and consequently show greater resistance to an attack by P. citrophthora [In the UVB and BC groups, phenylpropanoid biosynthesis and flavonoids biosynthesis were in the several top enriched GO and KEGG terms. The results of pathway mapping showed that many of the key enzymes involved in these two pathways were increased after treatments. Moreover, diverse flavonoids were also identified to be differentially accumulated through non-target metabolomic analysis. Although there were DEPs identified in other secondary metabolite biosynthesis pathways, none of them exhibited such a systematic change like phenylpropanoid biosynthesis, especially flavonoid biosynthesis. The systematic activation of flavonoid biosynthesis was observed on a protein level as well as a metabolite level. It has been reported that plants enhance stress tolerance by promoting flavonoid accumulation. The production of Flavonol Glycosides 3 (PFG3), an R2R3-MYB TF, has been reported to regulate flavonoid biosynthesis in rocesses . The polophthora . So, theM. alba leaves, including kuwanon H, kuwanon J, kuwanol E, guangsangon E, chalcomoracin, etc. These complex compounds showed different activities, including antibacterial and photoprotection. Kuwanon G is an inhibitor of protein tyrosine phosphatases A and B, and showed an inhibition of Mycobacterium tuberculosis growth by 61% [2O2-induced cellular ROS and the enhanced repair of DNA damage induced by UV-A [B. cinerea stress. A previous study of Liu et al. showed that the content of chalcomoracin was increased after UVB treatment and BC treatment, which further emphasized its role in stress resistance [Besides the benefit of flavonoid itself, the accumulated flavonoids could serve as a precursor for the production of complex secondary metabolites. Methylcyclohexene motifs, which are accessible via Diels-Alder reactions, are common in Moraceae natural products . Flavonoh by 61% . Kuwanonh by 61% . Kuwanon by UV-A . Chalcom by UV-A . While tsistance .B. cinerea infection and UV-B stress. Lignin is an indispensable polymer in plants which provides the strength for plants to grow tall in gravitropic environments and a hydrophobic lining to facilitate water and nutrient transport in plant vasculature [B. cinerea require the penetration of the host tissues by the hyphae. Under BC stress, activated lignin biosynthesis can benefit M. alba leaves through the strengthened cell wall.Proteomic data, enzyme assays, and qRT-PCR showed that lignin biosynthesis was activated after culature . Apart fculature . Lignin culature . The infArabidopsis mutants defective in the ability to synthesize sinapate esters in ferulic acid hydroxylase 1 (fah1) are more sensitive to UV-B than the wild-type. And despite its ability to accumulate flavonoids, the ferulic acid hydroxylase mutant fah1 exhibits more physiological injury than either the wild-type or mutants defective in the synthesis of flavonoids in transparent testa 5 (tt5) [A. thaliana plants expressing R2R3-MYB TF, ZmMYB31, exhibited a significant decrease in lignin content and a reduction in sinapoyl malate biosynthesis relative to the control plants, but increased the flavonoid biosynthesis relative to the controls. Despite the increase in flavonoids, transgenic A. thaliana plants were still highly sensitive to UV radiation compared with the controls, displaying an upward leaf curling phenotype resembling those observed in mutant plants deficient in UV-protectant compounds [It was commonly reported that flavonoids and lignin had excellent UV absorption ability owing to their aromatic structure and the presence of numerous phenolic, ketone, and intramolecular hydrogen bonds . It has  5 (tt5) . A. thalompounds . These sM. alba were washed and sown in soil for gemination. The seedlings were then grown at 25 ± 1 °C with a 16 h light/8 h dark cycle  for 5–6 weeks. The treatments of UVB and BC were conducted according to Liu et al. [−2 d−1 [B. cinerea grown on potato dextrose agar medium and placed in a covered Petri dish and incubated for 36 h with a 16 h light/8 h dark cycle. After that, the leaves were frozen in liquid nitrogen, then stored at −80 °C for future use. Three biological replications were performed.The seeds of u et al. , with mi [−2 d−1 . The UV g, 4 °C for 20 min. The supernatant was discarded, and the pellet was washed twice with 0.07% 2-mercaptoethanol in acetone. The pellet was dried using a Speed-Vacconcentrator and resuspended in lysis solution  by vortex at room temperature for 1 h. The supernatant was collected after centrifugation at 13,000× g for 20 min. Protein concentration was determined using a Bradford assay with bovine serum albumin as the standard. The proteins (100 μg) were purified with methanol and chloroform to remove impurities from the samples. After that, the protein was reduced with 50 mM dithiothreitol at 56 °C for 1 h and alkylated with 50 mM iodoacetamide in the dark at 37 °C for 1 h. Then, the protein was digested with trypsin at 1:100 enzyme/protein ratio (w/w) at 37 °C overnight. The obtained peptides were desalted with ZipTip , lyophilized, and then dissolved in 0.1% formic acid for nanoflow liquid chromatography-mass spectrometry.Protein extraction, purification, and digestion were performed according to Zhu et al. . Briefly−1 for 120 min.The instrument method was carried out according to Liu et al. . In briep-value of less than 0.05 were regarded as differentially expressed proteins (DEPs).The raw data were analyzed using Proteome Discoverer software , and tandem mass spectra were searched against Morus notabilis proteins in the UniProt database . The oxidation of methionine was set as a variable modification, and the carbamidomethylation of cysteine was set as a fixed modification. The mass tolerance values of MS and MS/MS were set to 10 ppm and 0.2 Da, respectively. The enzyme digestion method was set as trypsinization. Proteins with unique peptides of less than 2 were excluded. Protein abundances in the UVB and BC groups were compared with those in the control group, and proteins with an adjust The identified proteins were annotated by the eggNOG database . Gene OnM. alba leaf powder was extracted with 0.5 mL 70% methanol for three times. The supernatants were combined, lyophilized, and reconstituted in 50 µL of distilled water. Untargeted metabolomics was conducted using a Vanquish Horizon UPLC system  coupled with a Thermo Scientific Orbitrap Exploris 480 mass spectrometer. A reverse-phase C18 column  was used for metabolite separation with 0.1% formic acid in water as solvent A and 0.1% formic acid in acetonitrile as solvent B. The flow rate was 0.4 mL min−1. The LC gradient started at 0.1% solvent B for 0.5 min, then a linear gradient from 0.1% B to 40% B over 21 min, then a linear gradient from 40% B to 90% B over 2 min, held at 90% B for 2 min, and then return to 0.1% B.Metabolites were extracted according to the method of Zhong et al. . In briep-value of less than 0.05 were regarded as differential abundance metabolites (DAM).The raw data of metabolomic analysis were processed using Compound Discoverer 3.2 . Metabolite annotation was performed by searching the raw data against the mzCloud, mzVault, KEGG, AraCyc, BioCyc, Plant Cyc, and ChemSpider databases with a mass tolerance of 10 ppm. The identified metabolites were assigned as “level 2-identified compounds” because of MS/MS database matching . The con−ΔΔCt method.RNA extraction and qRT-PCR analysis were performed according to Liu et al. . The priThe enzyme activities of Cinnamyl-alcohol dehydrogenase (CAD) and L-phenylalanine ammonialyase (PAL) were measured using the CAD assay kit  and PAL assay kit  according to the manuals. The contents of total flavonoids were measured using the total flavonoids content assay kit .B. cinerea stress and UV-B stress in M. alba leaves. Proteomic analysis showed that flavonoid biosynthesis and lignin biosynthesis were both activated under two types of stress. The activation was further confirmed by enzyme activity assays and metabolite analysis. Phenylalanine and tyrosine biosynthesis, which is the upstream of flavonoid biosynthesis and lignin biosynthesis, were also up-regulated. In addition, the biosynthesis of different phytohormones was also evaluated, and a distinction was found in the JA biosynthesis between B. cinerea infection and UV-B stress. These results indicated that although B. cinerea infection and UV-B radiation are two different types of stress and may activate different signaling pathways, both of them could finally up-regulate flavonoid biosynthesis and lignin biosynthesis. Moreover, these two branches of phenylpropanoid metabolism contributed to the resistance to both types of stress in M. alba.In this study, we compared the response to


Neuroendocrine neoplasms (NENs), which are characterized by neuroendocrine differentiation, can arise in various organs. NENs have been divided into well-differentiated neuroendocrine tumors (NETs) and poorly differentiated neuroendocrine carcinomas (NECs) based on morphological differentiation, each of which has a distinct etiology, molecular profile, and clinicopathological features. While the majority of NECs originate in the pulmonary organs, extrapulmonary NECs occur most predominantly in the gastro-entero-pancreatic (GEP) system. Although platinum-based chemotherapy is the main therapeutic option for recurrent or metastatic GEP-NEC patients, the clinical benefits are limited and associated with a poor prognosis, indicating the clinically urgent need for effective therapeutic agents. The clinical development of molecular-targeted therapies has been hampered due to the rarity of GEP-NECs and the paucity of knowledge on their biology. In this review, we summarize the biology, current treatments, and molecular profiles of GEP-NECs based on the findings of pivotal comprehensive molecular analyses; we also highlight potent therapeutic targets for future precision medicine based on the most recent results of clinical trials. Neuroendocrine neoplasms (NENs), which are characterized by neuroendocrine differentiation, can arise in various organs. NENs have generally been divided into two types based on morphological differentiation: neuroendocrine tumors (NETs) and neuroendocrine carcinomas (NECs) , each ofThe existing strategy for treating GEP-NEC has been extrapolated from methods directed toward small-cell lung cancer (SCLC) that is the most predominant histology among NECs, because of the rarity of GEP-NEC and their biological similarities with SCLC –8. HowevIn this review, we summarize the biology and current treatments of GEP-NEC, as well as the state-of-the-art knowledge of its molecular landscape that has emerged from existing comprehensive analyses. Site-specific genetic alterations are also addressed according to the organ of origin. In addition, the similarities and differences between GEP-NEC and SCLC are highlighted, where appropriate, in terms of clinicopathological and molecular features. Finally, we discuss potential therapeutic targets from both basic and clinical viewpoints.NETs and NECs are distinct entities with widely differing etiologies, clinicopathologies, and genomic profiles.While GEP-NENs share a neuroendocrine phenotype, they are heterogenous malignancies that can originate from different anatomical sites. Based on grading of the Ki-67 proliferation index of the World Health Organization (WHO) classification in 2010, GEP-NENs can be categorized as low grade (G1), intermediate grade (G2), or high grade (G3), with Ki-67 values of < 3%, 3–20%, and > 20%, respectively . FurtherThe clinicopathological features of NECs and NETs are shown in Table Approximately 10% of all NECs originate from the extrapulmonary organs, among which one-third develop in the GEP system , 8, 17. TP53 and RB transcriptional corepressor 1 (RB1) [TP53 and RB1 have also been observed in GEP-NEC at frequencies ranging from 57 to 89% and 9 to 46%, respectively [TP53 mutation in GEP-NEC [RB1 mutations emerge at a much lower rate in GEP-NEC compared to SCLC, the RB1 pathway appears to be suppressed by other mechanisms, such as copy number alterations affecting RB1, silencing of p16 via promoter methylation of cyclin-dependent kinase inhibitor 2A (CDKN2A), and amplifications of MYC or cyclin E1 (CCNE1) as an RB1 antagonist [Developments in high-throughput genomic technologies have led to a better understanding of the molecular profiles of GEP-NEC compared with SCLC or GEP-NET Table . SCLC is 1 (RB1) –46. NEC  1 (RB1) –50. Gene 1 (RB1) , 51. Genectively , 52–57, ectively . Chromot GEP-NEC . Althougtagonist , 58, 59.tagonist , 60–63. tagonist , 64, 65.tagonist .Table 3DTP53 and RB1, other frequently mutated genes in GEP-NECs are KRAS, BRAF, adenomatosis polyposis coli (APC), CCNE1, CDKN2A, Notch receptor 1 (NOTCH1), F-box and WD repeat domain containing-7 (FBXW7), catenin beta 1 (CTNNB1), and phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha (PIK3CA)/phosphatase and tensine homolog (PTEN) [ATM) gene was subject to frequent copy number losses, whereas the MYC gene was frequently amplified [In addition to the mutations of g (PTEN) , 55, 57 mplified . In a symplified . Notablymplified .SOX2 gene is recurrently amplified in SCLC [SCLC has typically been classified into four molecular subtypes based on the expression status of distinct neuroendocrine-lineage-specific transcription factors, including achaete-scute family bHLH transcription factor 1 (ASCL1), neuronal differentiation 1 (NEUROD1), POU class 2 homeobox 3 (POU2F3), and yes1-associated transcriptional regulator (YAP1) . These s in SCLC  and has  in SCLC . In GEP- in SCLC . Thus, sKMT2D), lysine methyltransferase 2C (KMT2C), CREB-binding protein (CREBBP), and E1A-binding protein p300 (EP300), have been shown to be frequently altered in SCLC [ARID1A), are more common in GEP-NEC and GEP-NET, but rare in SCLC [Epigenetic aberrations are among the most oncogenic processes in SCLC  and GEP- in SCLC , and mut in SCLC , 56, 58  in SCLC , 42, 52.BRAF and APC mutations in colorectal NECs [KRAS mutation in colorectal and pancreatic NECs [NOTCH1 mutation in esophageal NECs [ELF3) mutation in ampullary NECs [Although GEP-NECs share some genomic alterations characterized by neuroendocrine lineage regardless of different primary organ sites, they also have organ-specific mutational signatures . A pivottal NECs , 84–88, tic NECs , 55, 57,eal NECs , 12, 57,ary NECs , 89  and death-domain-associated protein (DAXX) play a role in chromatin remodeling at telomeres and other genomic sites , and the in NECs , 68, 100 in NECs , 52, 101and KRAS , 62, 102and KRAS , 55.Collectively, GEP-NECs adopt a subset of genomic and epigenomic characteristics of SCLCs, but some key molecular alterations are organ specific, even in the GEP system. In addition, distinct molecular profiles between NECs and NETs support the notion that NECs are not derived directly from NETs , 9.GEP-NEC patients are often diagnosed at advanced stages and are not eligible for curative treatment. For such patients, systemic treatment is the main therapeutic option for prolonging survival and improving their symptoms and quality of life. The current therapeutic strategies for GEP-NENs differ according to NET and NEC subtypes Fig. . SystemiTherapeutic strategies for patients with GEP-NECs originated from those designed for SCLCs because of their close tumoral entity and the rarity of GEP-NEC , 31, 103Unfortunately, there is no standard chemotherapy for a second-line setting. In a systematic review and meta-analysis of second-line treatment in 582 patients with advanced extrapulmonary NEC, the ORR and median PFS were 18% and 2.5 months, respectively . SimilarAlthough many molecular-targeted agents are currently approved for various solid tumors, no targeted therapies have been established for the clinical management of NECs. Therefore, novel therapies tailored to their molecular composition are urgently required to improve prognosis. A growing number of comprehensive molecular analyses have provided potential targets for GEP-NEC , 55, 57,KRAS mutational activation leads to downstream signaling of the MAPK and PI3K/v-akt murine thymoma viral oncogene homolog (Akt) pathways, which play crucial roles in tumorigenesis, proliferation, survival, angiogenesis, and metastasis. In GEP-NECs, KRAS mutations are reported at a similar frequency as in conventional cancers arising at the same sites: KRAS genes are frequently mutated in colorectal and pancreatic NECs, while they are less common in esophageal NECs [KRAS G12C allele-specific covalent inhibitors demonstrated a profound clinical impact in KRAS G12C-mutated non-small-cell lung cancer (NSCLC) [KRAS G12C mutations have been observed in NENs [KRAS G12C mutation may represent a potential target even for GEP-NEC, the efficacy is likely to be contingent upon RTK dependency and signaling rebound kinetics [KRAS G12C-mutated GEP-NEC, further preclinical and clinical studies are needed for GEP-NEC. In addition, the success of targeting KRAS G12C will provide hope that a range of mutant RAS allele-specific targeted therapies could become therapeutically tractable [Aberrant activation of receptor tyrosine kinase (RTK) pathways is common in non-neuroendocrine epithelial cancers, and eal NECs , 12 (Tab (NSCLC) , 144. Th in NENs , 146. Alkinetics , 148. Asractable .V600E) of the BRAF gene has been identified in various tumor types, and BRAF inhibitors have yielded clinical benefits for patients with BRAF V600E-mutated cancers, especially melanoma and NSCLC [BRAF V600E mutation [An activating missense mutation in codon 600 of exon 15  in response to cellular DNA damage . Preclinand TP53 . Thus, tn cancer , chemothtant CRC , AZD1775tant CRC , or dualMetabolic rewiring evokes cellular mechanisms that reduce therapeutic mightiness. Aberration of MYC also leads to reprogramming of cellular metabolism, which creates reliance on arginine biosynthetic pathways, including polyamine biosynthesis and mTOR pathway activation , 184. ArWhile MYC-targeted therapies have emerged as a promising approach for GEP-NEC treatment, they have been hampered due to the lack of available clinical data, highlighting the need for further clinical trials for GEP-NEC.BRCA-mutated breast, ovarian, prostate, and pancreatic cancers has provided proof-of-concept for synthetic lethality as a novel therapeutic strategy. DDR pathways are also sometimes deficient in SCLC and extrapulmonary NEC [Recently, targeting components of DNA repair pathways has emerged as a therapeutic strategy , 186. DNnary NEC , 189. Innary NEC . In a ranary NEC . A phasenary NEC .In addition to PARP, DDR kinases, such as ataxia telangiectasia and RAD3-related (ATR), ATM, CHK1, and WEE1, have emerged as attractive targeted molecules because of their central roles in DNA repair , 193. ATMost pancreatic NETs have an extraordinary tumor vascularization due to overexpression of pro-angiogenic factors, including vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) . In factAlthough there are no approved anti-angiogenetic agents for NECs, a preclinical study has demonstrated potent antitumor activity for two anti-VEGF antibodies, bevacizumab and aflibercept, in xenograft models of SCLC and colon NEC cell lines . ClinicaDelta-like canonical Notch ligand 3 (DLL3) is an inhibitory ligand of the Notch receptor pathway and is highly expressed in most pulmonary NECs , 209, whDLL3 is an attracted molecule that delivers cytotoxic compounds selectively and directly to NEC cells. Rovalpituzumab tesirine is an ADC comprising the cytotoxic payload pyrrolobenzodiazepine, which is conjugated by a linker to a monoclonal DLL3 antibody. Despite promising preclinical and early-phase clinical antitumor activity , 210, phTarlatamab AMG 757) is a half-life extended BiTE designed to specifically bind DLL3 on cancer cells and CD3 on T cells, resulting in T-cell-dependent killing of cancer cells with DLL3 expression in the SCLC patient-derived xenograft models . A phase57 is a hEpigenetic alternations, including DNA methylation, histone acetylation, and histone methylation, regulate gene expression and interact with numerous transcription factors that have fundamental functions in cancer progression . The enhBromodomain and extra-terminal (BET) proteins bind acetylated histones and recruit protein complexes to promote transcription, among which BRD4 serves as a transcriptional regulator of MYC , 227. BE177Lu) or β-emitting 90Yttrium (90Y), in order to kill neoplastic cells with lethal radiation [Somatostatin receptors (SSTRs) are a family of G protein-coupled receptors that are implicated in the regulation of hormone secretion and tumor proliferation in NET . Since Sadiation . A phaseadiation Lu labeleadiation  octreotaadiation . Based oAlthough NECs have generally absent or reduced SSTR expression , 30, 31,SSTR2 is one of the most frequently expressed subtypes of SSTRs in GEP-NENs. PEN-221 is a small peptide drug conjugate that selectively targets SSTR2, with a cleavable linker to a cytotoxic payload DM1. Treatment with PEN-221 was found to enable efficient drug delivery to SSTR2-positive cells, resulting in complete and durable regressions in SSTR2-positive SCLC xenograft mouse models . A phaseOther potential cellular targets in GEP-NECs have been identified, such as exportin-1 (XPO1), the BCL2 apoptosis regulator (BCL2), and lurbinectedin  and its ligand PD-L1, promote antitumor immune escape during the cancer–immunity cycle process , 257. ClARID1A is a more common altered gene in GEP-NEC [ARID1A mutations, while only one of 27 non-responders harbored mutations [Based on the clinical benefit of anti-PD-1 Ab pembrolizumab for patients with microsatellite instability-high (MSI-H)/deficient mismatch repair (dMMR) tumors in pivotal clinical trials , the FDA GEP-NEC –12, 52.  GEP-NEC . ARID1A  GEP-NEC . Preclin GEP-NEC . Clinicautations . Thus, autations .Several treatment strategies have been examined to turn immunologically “cold” tumors with poor immune activation into “hot” tumors with strong immune infiltration in clinical trials combining the anti-PD-1/PD-L1 antibody with other immune-modulating treatments, including other ICIs, chemotherapy, angiogenetic inhibitors, and molecular-targeted agents Table . CurrentThe PD-1/PD-L1 interaction is not the only immune checkpoint pathway that regulates T-cell activation in the tumor microenvironment (TME). Lymphocyte activation gene 3 protein (LAG3), T-cell immunoglobulin mucin receptor 3 (TIM3), and T-cell immunoreceptor with Ig and ITIM domains (TIGIT) are overexpressed on effector CD4 + and CD8 + T cells, regulatory T cells (Tregs), and natural killer cells, which act as inhibitory immune checkpoint modulators . In factChemotherapy can promote immune responses by increasing the immunogenicity of cancer cells or inhibiting immunosuppressive circuitries . The cliAs the VEGF/VEGFR signaling pathway induces immunosuppressive effects via the downregulation of MHC expression, the activation of inhibitory immune checkpoint molecules, and the inhibition of TILs and dendritic cell differentiation in addition to angiogenesis , the comHDAC inhibitors have been found to enhance tumor immunogenicity through not only increased MHC presentation  but alsoThe DDR pathway is sometimes deficient in NECs , 189, whNEC is a rare histological subtype among cancers in the GEP system, and the unsatisfying prognosis highlights the clinically urgent need for effective therapeutic compounds. NEC partially shares molecular features specific to SCLC across primary organ sites, whereas NEC also has key genetic aberrations similar to the non-neuroendocrine conventional cancer arising in the same organ sites. Thus, the molecular landscape of GEP-NECs is composed of both neuroendocrine-specific and site-specific alterations, indicating that there is potential in the extrapolation of effective treatment strategies, not only from SCLC but also from conventional cancers at the same site of origin. A better understanding of GEP-NEC biology could reveal a population vulnerable to specific molecular inhibition, which may pave the way for the establishment of personalized medicine. In addition, the assessment of ctDNA will guide the selection of patients who may benefit from molecular-targeted agents by identifying clonally altered genes in GEP-NECs with intratumoral heterogeneity.