Pangenome analysis reveals transposon-driven genome evolution in cotton.

BACKGROUND: Transposable elements (TEs) have a profound influence on the trajectory of plant evolution, driving genome expansion and catalyzing phenotypic diversification. The pangenome, a comprehensive genetic pool encompassing all variations within a species, serves as an invaluable tool, unaffected by the confounding factors of intraspecific diversity. This allows for a more nuanced exploration of plant TE evolution. RESULTS: Here, we constructed a pangenome for diploid A-genome cotton using 344 accessions from representative geographical regions, including 223 from China as the main component. We found 511 Mb of non-reference sequences (NRSs) and revealed the presence of 5479 previously undiscovered protein-coding genes. Our comprehensive approach enabled us to decipher the genetic underpinnings of the distinct geographic distributions of cotton. Notably, we identified 3301 presence-absence variations (PAVs) that are closely tied to gene expression patterns within the pangenome, among which 2342 novel expression quantitative trait loci (eQTLs) were found residing in NRSs. Our investigation also unveiled contrasting patterns of transposon proliferation between diploid and tetraploid cotton, with long terminal repeat (LTR) retrotransposons exhibiting a synchronized surge in polyploids. Furthermore, the invasion of LTR retrotransposons from the A subgenome to the D subgenome triggered a substantial expansion of the latter following polyploidization. In addition, we found that TE insertions were responsible for the loss of 36.2% of species-specific genes, as well as the generation of entirely new species-specific genes. CONCLUSIONS: Our pangenome analyses provide new insights into cotton genomics and subgenome dynamics after polyploidization and demonstrate the power of pangenome approaches for elucidating transposon impacts and genome evolution.

Multiplex signal amplification for ultrasensitive CRP assay via integrated electrochemical biosensor array using MOF-derived carbon material and aptamers.

Accurate and precise detection of disease-associated proteins, such as C-reactive protein (CRP), remains a challenge in biosensor development. Herein, we present a novel approach－an integrated disposable aptasensor array－designed for precise, ultra-sensitive, and parallel detection of CRP in plasma samples. This integrated biosensing array platform enables multiplex parallel testing, ensuring the accuracy and reliability in sample analysis. The ultra-sensitivity of this biosensor is achieved through multiplex signal amplification. Leveraging the superior conductivity and extensive surface area of MOF-derived nanoporous carbon material (CMOF), the biosensor enhances recognition elements (aptamers) by catalyzing the horseradish peroxidase (HRP) label enzyme reaction to multiply the number of probe molecules. Optimized conditions yielded exceptional performance, exhibiting high accuracy (relative standard deviation, RSD≤10.0 %), a low detection limit (0.3 pg/mL, S/N = 3), ultra-sensitivity (0.16 µA/ng mL-1 mm-2), and a rapid response (seven parallel tests within 60 min). Importantly, this multi-unit integrated disposable aptasensor array accurately quantified CRP in human serum, demonstrating comparable results to commercial enzyme-linked immunosorbent assay (ELISA). This technology showcases promise for detecting various biomarkers using a unified approach, presenting an appealing strategy for early disease diagnosis and biological analysis.

Ultraspecific One-Pot CRISPR-Based "Green-Yellow-Red" Multiplex Detection Strategy Integrated with Portable Cartridge for Point-of-Care Diagnosis.

Versatile, informative, sensitive, and specific nucleic acid detection plays a crucial role in point-of-care pathogen testing, genotyping, and disease monitoring. In this study, we present a novel one-pot Cas12b-based method coupled with the "Green-Yellow-Red" strategy for multiplex detection. By integrating RT-LAMP amplification and Cas12b cleavage in a single tube, the entire detection process can be completed within 1 h. Our proposed method exhibits high specificity, enabling the discrimination of single-base mutations with detection sensitivity approaching single molecule levels. Additionally, the fluorescent results can be directly observed by the naked eye or automatically analyzed using our custom-designed software Result Analyzer. To realize point-of-care detection, we developed a portable cartridge capable of both heating and fluorescence excitation. In a clinical evaluation involving 20 potentially SARS-CoV-2-infected samples, our method achieved a 100% positive detection rate when compared to standard RT-PCR. Furthermore, the identification of SARS-CoV-2 variants using our method yielded results that were consistent with the sequencing results. Notably, our proposed method demonstrates excellent transferability, allowing for the simultaneous detection of various pathogens and the identification of mutations as low as 0.5% amidst a high background interference. These findings highlight the tremendous potential of our developed method for molecular diagnostics.

High-quality Gossypium hirsutum and Gossypium barbadense genome assemblies reveal the landscape and evolution of centromeres.

Centromere positioning and organization are crucial for genome evolution; however, research on centromere biology is largely influenced by the quality of available genome assemblies. Here, we combined Oxford Nanopore and Pacific Biosciences technologies to de novo assemble two high-quality reference genomes for Gossypium hirsutum (TM-1) and Gossypium barbadense (3-79). Compared with previously published reference genomes, our assemblies show substantial improvements, with the contig N50 improved by 4.6-fold and 5.6-fold, respectively, and thus represent the most complete cotton genomes to date. These high-quality reference genomes enable us to characterize 14 and 5 complete centromeric regions for G. hirsutum and G. barbadense, respectively. Our data revealed that the centromeres of allotetraploid cotton are occupied by members of the centromeric repeat for maize (CRM) and Tekay long terminal repeat families, and the CRM family reshapes the centromere structure of the At subgenome after polyploidization. These two intertwined families have driven the convergent evolution of centromeres between the two subgenomes, ensuring centromere function and genome stability. In addition, the repositioning and high sequence divergence of centromeres between G. hirsutum and G. barbadense have contributed to speciation and centromere diversity. This study sheds light on centromere evolution in a significant crop and provides an alternative approach for exploring the evolution of polyploid plants.

Sequence-Guided Localization of DNA Hybridization Enables Highly Selective and Robust Genotyping.

Genetic variations are always related to human diseases or susceptibility to therapies. Nucleic acid probes that precisely distinguish closely related sequences become an indispensable requisite both in research and clinical applications. Here, a Sequence-guided DNA LOCalization for leaKless DNA detection (SeqLOCK) is introduced as a technique for DNA hybridization, where the intended targets carrying distinct "guiding sequences" act selectively on the probes. In silicon modeling, experimental results reveal considerable agreement (R2  = 0.9228) that SeqLOCK is capable of preserving high discrimination capacity at an extraordinarily wide range of target concentrations. Furthermore, SeqLOCK reveals high robustness to various solution conditions and can be directly adapted to nucleic acid amplification techniques (e.g., polymerase chain reaction) without the need for laborious pre-treatments. Benefiting from the low hybridization leakage of SeqLOCK, three distinct variations with a clinically relevant mutation frequency under the background of genomic DNA can be discriminated simultaneously. This work establishes a reliable nucleic acid hybridization strategy that offers great potential for constructing robust and programmable systems for molecular sensing and computing.

Regulatory controls of duplicated gene expression during fiber development in allotetraploid cotton.

Polyploidy complicates transcriptional regulation and increases phenotypic diversity in organisms. The dynamics of genetic regulation of gene expression between coresident subgenomes in polyploids remains to be understood. Here we document the genetic regulation of fiber development in allotetraploid cotton Gossypium hirsutum by sequencing 376 genomes and 2,215 time-series transcriptomes. We characterize 1,258 genes comprising 36 genetic modules that control staged fiber development and uncover genetic components governing their partitioned expression relative to subgenomic duplicated genes (homoeologs). Only about 30% of fiber quality-related homoeologs show phenotypically favorable allele aggregation in cultivars, highlighting the potential for subgenome additivity in fiber improvement. We envision a genome-enabled breeding strategy, with particular attention to 48 favorable alleles related to fiber phenotypes that have been subjected to purifying selection during domestication. Our work delineates the dynamics of gene regulation during fiber development and highlights the potential of subgenomic coordination underpinning phenotypes in polyploid plants.

Tcbf: a novel user-friendly tool for pan-3D genome analysis of topologically associating domain in eukaryotic organisms.

SUMMARY: TAD boundaries are essential for organizing the chromatin spatial structure and regulating gene expression in eukaryotes. However, for large-scale pan-3D genome research, identifying conserved and specific TAD boundaries across different species or individuals is computationally challenging. Here, we present Tcbf, a rapid and powerful Python/R tool that integrates gene synteny blocks and homologous sequences to automatically detect conserved and specific TAD boundaries among multiple species, which can efficiently analyze huge genome datasets, greatly reduce the computational burden and enable pan-3D genome research. AVAILABILITY AND IMPLEMENTATION: Tcbf is implemented by Python/R and is available at https://github.com/TcbfGroup/Tcbf under the MIT license.

Replaceable Dielectric Film for Low-Voltage and High-Performance Electrowetting-Based Digital Microfluidics.

Electrowetting-on-dielectric (EWOD) technology has been considered as a promising candidate for digital microfluidic (DMF) applications due to its outstanding flexibility and integrability. The dielectric layer with a hydrophobic surface is the key element of an EWOD device, determining its driving voltage, reliability, and lifetime. Hereby, inspired by the ionic-liquid-filled structuring polymer with high capacitance independent on thickness, namely ion gel (IG), we develop a polymer (P)-ion gel-amorphous fluoropolymer, namely, PIGAF, composite film as a replaceable hydrophobic dielectric layer for fabrication of a high-efficiency and stable EWOD-DMF device at relatively low voltage. The results show that the proposed EWOD devices using the PIGAF-based dielectric layer can achieve a large contact angle (θ) change of ∼50° and excellent reversibility with a contact angle hysteresis of ≤5° at a relatively low voltage of 30 Vrms. More importantly, the EWOD actuation voltage did not change obviously with the PIGAF film thickness in the range of several to tens of microns, enabling the thickness of the film to be adjusted according to the demand within a certain range while keeping the actuation voltage low. An EWOD-DMF device can be prepared by simply stacking a PIGAF film onto a PCB board, demonstrating stable droplet actuation (motion) at 30 Vrms and 1 kHz as well as a maximum moving velocity of 69 mm/s at 140 Vrms and 1 kHz. The PIGAF film was highly stable and reliable, maintaining excellent EWOD performance after multiple droplet manipulations (≥50 cycles) or long-term storage of 1 year. The proposed EWOD-DMF device has been demonstrated for digital chemical reactions and biomedical sensing applications.

Sensitive electrochemical assay of acetaminophen based on 3D-hierarchical mesoporous carbon nanosheets.

Acetaminophen plays a key role in first-line Covid-19 cure as a supportive therapy of fever and pain. However, overdose of acetaminophen may give rise to severe adverse events such as acute liver failure in individual. In this work, 3D-hierarchical mesoporous carbon nanosheet (hMCNS) microspheres with superior properties were fabricated using simple and quick strategy and applied for sensitive quantification of acetaminophen in pharmaceutical formulation and rat plasmas after administration. The hMCNS microspheres are prepared via chemical etching of zinc oxide (ZnO) nanoparticles from a zinc-gallic acid precursor composite (Zn-GA) synthesized by high-temperature anaerobic pyrolysis. The obtained hMCNS could enhance analytes accessibility and accelerate proton transfer in the interface, hence increasing the electrochemical performance. Under optimized experimental conditions, the proposed electrochemical sensor achieves a detection limit of 3.5 nM for acetaminophen. The prepared electrochemical sensor has been successfully applied for quantification of acetaminophen in pharmaceutical formulations and the rat plasma samples before and after administration. Meanwhile, this sensor is compared with high-performance liquid chromatography (HPLC) as a reference technology, showing an excellent accuracy. Such an electrochemical sensor has great potential and economic benefits for applications in the fields of pharmaceutical assay and therapeutic drug monitoring (TDM).

Genomic innovation and regulatory rewiring during evolution of the cotton genus Gossypium.

Phenotypic diversity and evolutionary innovation ultimately trace to variation in genomic sequence and rewiring of regulatory networks. Here, we constructed a pan-genome of the Gossypium genus using ten representative diploid genomes. We document the genomic evolutionary history and the impact of lineage-specific transposon amplification on differential genome composition. The pan-3D genome reveals evolutionary connections between transposon-driven genome size variation and both higher-order chromatin structure reorganization and the rewiring of chromatin interactome. We linked changes in chromatin structures to phenotypic differences in cotton fiber and identified regulatory variations that decode the genetic basis of fiber length, the latter enabled by sequencing 1,005 transcriptomes during fiber development. We showcase how pan-genomic, pan-3D genomic and genetic regulatory data serve as a resource for delineating the evolutionary basis of spinnable cotton fiber. Our work provides insights into the evolution of genome organization and regulation and will inform cotton improvement by enabling regulome-based approaches.

The role of the immune microenvironment in bone, cartilage, and soft tissue regeneration: from mechanism to therapeutic opportunity.

Bone, cartilage, and soft tissue regeneration is a complex spatiotemporal process recruiting a variety of cell types, whose activity and interplay must be precisely mediated for effective healing post-injury. Although extensive strides have been made in the understanding of the immune microenvironment processes governing bone, cartilage, and soft tissue regeneration, effective clinical translation of these mechanisms remains a challenge. Regulation of the immune microenvironment is increasingly becoming a favorable target for bone, cartilage, and soft tissue regeneration; therefore, an in-depth understanding of the communication between immune cells and functional tissue cells would be valuable. Herein, we review the regulatory role of the immune microenvironment in the promotion and maintenance of stem cell states in the context of bone, cartilage, and soft tissue repair and regeneration. We discuss the roles of various immune cell subsets in bone, cartilage, and soft tissue repair and regeneration processes and introduce novel strategies, for example, biomaterial-targeting of immune cell activity, aimed at regulating healing. Understanding the mechanisms of the crosstalk between the immune microenvironment and regeneration pathways may shed light on new therapeutic opportunities for enhancing bone, cartilage, and soft tissue regeneration through regulation of the immune microenvironment.

Single-cell RNA-seq reveals fate determination control of an individual fibre cell initiation in cotton (Gossypium hirsutum).

Cotton fibre is a unicellular seed trichome, and lint fibre initials per seed as a factor determines fibre yield. However, the mechanisms controlling fibre initiation from ovule epidermis are not understood well enough. Here, with single-cell RNA sequencing (scRNA-seq), a total of 14 535 cells were identified from cotton ovule outer integument of Xu142_LF line at four developmental stages (1.5, 1, 0.5 days before anthesis and the day of anthesis). Three major cell types, fibre, non-fibre epidermis and outer pigment layer were identified and then verified by RNA in situ hybridization. A comparative analysis on scRNA-seq data between Xu142 and its fibreless mutant Xu142 fl further confirmed fibre cluster definition. The developmental trajectory of fibre cell was reconstructed, and fibre cell was identified differentiated at 1 day before anthesis. Gene regulatory networks at four stages revealed the spatiotemporal pattern of core transcription factors, and MYB25-like and HOX3 were demonstrated played key roles as commanders in fibre differentiation and tip-biased diffuse growth respectively. A model for early development of a single fibre cell was proposed here, which sheds light on further deciphering mechanism of plant trichome and the improvement of cotton fibre yield.

A liquid crystal-based biosensor for detection of insulin driven by conformational change of an aptamer at aqueous-liquid crystal interface.

Insulin is a critical predictor for the function of pancreatic islet beta cells, which plays a crucial role in diagnosing diabetes and diabetes-related disorders. Herein, we propose and validate a label-free and cost-effective aptamer-based optical LC biosensor for detection of insulin based on the directional recognition of biomolecular binding events at a responsive aqueous-liquid crystal (LC) interface. The binding of insulin and aptamer adsorbed on CTAB triggers a conformational change of the aptamer from G-quadruplex to stretched structure, inducing homeotropic to planar alignment and correspondingly dark to bright optical image change of the LC films. The molecular dynamic (MD) simulation validates that the orientational transition is associated with the interaction energy changes at the interface, which is in coordination with the optical observation. This LC biosensor takes advantages of simple preparation, easy operation, rapid sensing, high specificity for insulin determination in the range of 0.1-1.0 nM within 5 min. This sensor is also applicable for insulin detection in diluted human urine and serum. Additionally, the optical cell arrays allow to detection multiple samples of the same/different biomarkers at the same time. Such a strategy offers a potential basis for monitoring other clinical biomarkers, and for point-of-care testing (POCT) as well.

[Corrigendum] RON and c­Met facilitate metastasis through the ERK signaling pathway in prostate cancer cells.

Subsequently to the publication of the above article, the authors have discovered that the version of Fig. 5 included in the paper was an incorrect version, and that two pairs of data panels were inadvertently included in Fig. 6D (the data panels for the NC+migration and NC+HGF+U0126+invasion experiments for the PC3 cells, and the data panels for the NC+invasion and NC+HGF+U0126+invasion experiments for the DU145 cells) that contained overlapping data derived from the same source. These data were intended to represent the results obtained under different experimental conditions. Furthermore, the GAPDH control bands in Fig. 4A (DU145 cells) and the p­ERK1/2 bands in Fig. 6A (PC3 cells) were incorrectly chosen for these figures. After having consulted the original data, the authors discovered that unintended errors were made in assembling the data for these graphs. In uploading the corrected version of Fig. 5, Fig. 3C and D and Fig. 4C and D were adjusted accordingly. The corrected versions of Figs. 3, 4, 5, and 6 are shown on the subsequent pages. The authors regret the errors that were made during the preparation of the published figures, and confirm that these errors did not affect the conclusions reported in the study. The authors are grateful to the Editor of Oncology Reports for allowing them the opportunity to publish a Corrigendum, and all the authors agree to this Corrigendum. Furthermore, they apologize to the readership for any inconvenience caused. [Oncology Reports 37: 3209­3218, 2017; DOI: 10.3892/or.2017.5585].

Triple signal-enhancing electrochemical aptasensor based on rhomboid dodecahedra carbonized-ZIF67 for ultrasensitive CRP detection.

C-reactive protein (CRP) is one of the most sensitive acute-phase reactants, which is an early stage indicator of cardiovascular disease and infectious inflammation in clinic. However, it is still challenging to accurately quantification the trace content of CRP molecules in plasma samples. In this work, we propose an ultrasensitive electrochemical CRP aptasensor based on rhomboid dodecahedra carbonized-ZIF67 loaded with gold nanoparticle modified by aptamer. Aptamer biomolecules are binded to AuNPs via Au-thiol bonds for selectively capturing CRPs. The ultrasensitivity is achieved based on triple signal enhancing strategy: enhancing the specific surface area via the rhomboid dodecahedra structure of ZIF67, increasing the conductivity via carbonization of ZIF67, and multiplying the number of probe molecules via an enzyme catalyzed reaction. Experimental parameters, including the volume of C-ZIF67 dispersion, electrodeposition time of AuNPs, incubation time of aptamer-CRP and aptamer-CRP concentration, are systemically investigated and optimized. Under optimal conditions, the proposed biosensor shows excellent sensing performance with the limit of detection (LOD) of 0.44 pg mL-1 (S/N = 3), and a broad linear dynamic range of 10 pg mL-1 ‒ 10 µg mL-1 within the total readout time of 5 min. This work provides an effective electrochemical biosensor for CRP assay in plasma, being highly potential for applications in bioanalysis and point-of-care (POC) clinical diagnosis.

Dynamic 3D genome architecture of cotton fiber reveals subgenome-coordinated chromatin topology for 4-staged single-cell differentiation.

BACKGROUND: Despite remarkable advances in our knowledge of epigenetically mediated transcriptional programming of cell differentiation in plants, little is known about chromatin topology and its functional implications in this process. RESULTS: To interrogate its significance, we establish the dynamic three-dimensional (3D) genome architecture of the allotetraploid cotton fiber, representing a typical single cell undergoing staged development in plants. We show that the subgenome-relayed switching of the chromatin compartment from active to inactive is coupled with the silencing of developmentally repressed genes, pinpointing subgenome-coordinated contribution to fiber development. We identify 10,571 topologically associating domain-like (TAD-like) structures, of which 25.6% are specifically organized in different stages and 75.23% are subject to partition or fusion between two subgenomes. Notably, dissolution of intricate TAD-like structure cliques showing long-range interactions represents a prominent characteristic at the later developmental stage. Dynamic chromatin loops are found to mediate the rewiring of gene regulatory networks that exhibit a significant difference between the two subgenomes, implicating expression bias of homologous genes. CONCLUSIONS: This study sheds light on the spatial-temporal asymmetric chromatin structures of two subgenomes in the cotton fiber and offers a new insight into the regulatory orchestration of cell differentiation in plants.

A paper-based visualization chip based on nitrogen-doped carbon quantum dots nanoprobe for Hg(â ¡) detection.

Hg(II) is one of the most toxic heavy metal ions. The bioconcentration and degradation-resistant of Hg(II) bring about serious harm to the ecosystem and humans. Therefore, the establishment of an accurate and effective method for detecting mercury ions is of great significance to environmental protection, food safety and human health. In this work, a new fluorescent nanoprobe was presented using nitrogen-doped carbon quantum dots (N-CQDs) for Hg(II) sensing with high stability and selectivity. On this basis, a paper-based chip was innovatively developed for visualization detection of Hg(II). The N-CQDs were prepared through a one-step hydrothermal reaction using catechol and ethylenediamine as carbon and nitrogen sources, respectively. As-prepared N-CQDs exhibit the strong green fluorescence at the excitation/emission wavelength of 370/511 nm. In aqueous solution, a rapid and highly sensitive detection method of Hg(II) was established by the joint of dynamic and static quenching effect of Hg(II) on N-CQDs fluorescence. Under the optimized conditions, there was a stable correlation between the fluorescence intensity change of N-CQDs and the concentrations of Hg(II) in the range of 15 âˆ¼ 104 nM, and the detection limit was down to 8 nM (S/N = 3). The recoveries of water, sorghum and rice were 91.60 to 102.46%, which was consistent with ICP-MS. More importantly, the N-CQDs nanoprobe was further integrated in nitrocellulose membrane to develop paper-based chip for Hg(II) visualization detection, and the detection performance was also excellent. This strategy had significant implications for achieving low-cost, on-site real-time monitoring of mercury (II) in the environment and food.

Serum oncostatin M is a potential biomarker of disease activity and infliximab response in inflammatory bowel disease measured by chemiluminescence immunoassay.

BACKGROUND: Although endoscopy is the gold standard to assess disease activity and infliximab efficacy in inflammatory bowel disease (IBD), the invasive, costly, and time-consuming procedure limits its routine applications. We aimed to investigate the clinical value of serum oncostatin M (OSM) as a surrogate biomarker. METHODS: Fifty healthy controls, 34 non-IBD patients, and 189 IBD patients who were pre-infliximab treatment (n = 122) or in infliximab maintenance (n = 67) were enrolled. A chemiluminescence immunoassay (CLIA) was constructed to quantify serum OSM concentrations. Receiver operator characteristic (ROC) curve analysis was used to evaluate the performance of blood biomarkers for IBD management. RESULTS: The methodology of CLIA exhibited great analytical performance with a wide linear range of 31.25-25000 pg/mL, a low detection limit of 23.2 pg/mL, acceptable precision, and applicable accuracy. Patients with IBD (121.5 [43.3-249.4] pg/mL, p < 0.001) and non-IBD (72.4 [51.4-129.6] pg/mL, p = 0.005) had higher serum OSM levels than healthy controls (35.8 [23.2-56.4] pg/mL). In the analysis of clinical and endoscopic activity, serum OSM levels were elevated in moderate and severe patients compared to those in remission. IBD patients without mucosal healing had higher serum OSM levels than those with mucosal healing (AUC = 0.843). Besides, serum OSM levels were increased in clinical non-responders (287.3 [127.9-438] pg/mL) compared to responders (24.1 [23.2-53.4] pg/mL, p < 0.001), and showed great recognition ability with an AUC of 0.898. CONCLUSIONS: The newly developed methodology of CLIA had great potential for use in the clinic. Elevated serum OSM expression was a promising biomarker of severe disease and infliximab non-response in IBD patients.

A Multivariate Diagnostic Model Based on Urinary EpCAM-CD9-Positive Extracellular Vesicles for Prostate Cancer Diagnosis.

INTRODUCTION: Prostate cancer (PCa) is one of the most frequently diagnosed cancers and the leading cause of cancer death in males worldwide. Although prostate-specific antigen (PSA) screening has considerably improved the detection of PCa, it has also led to a dramatic increase in overdiagnosing indolent disease due to its low specificity. This study aimed to develop and validate a multivariate diagnostic model based on the urinary epithelial cell adhesion molecule (EpCAM)-CD9-positive extracellular vesicles (EVs) (uEVEpCAM-CD9) to improve the diagnosis of PCa. METHODS: We investigated the performance of uEVEpCAM-CD9 from urine samples of 193 participants (112 PCa patients, 55 benign prostatic hyperplasia patients, and 26 healthy donors) to diagnose PCa using our laboratory-developed chemiluminescent immunoassay. We applied machine learning to training sets and subsequently evaluated the multivariate diagnostic model based on uEVEpCAM-CD9 in validation sets. RESULTS: Results showed that uEVEpCAM-CD9 was able to distinguish PCa from controls, and a significant decrease of uEVEpCAM-CD9 was observed after prostatectomy. We further used a training set (N = 116) and constructed an exclusive multivariate diagnostic model based on uEVEpCAM-CD9, PSA, and other clinical parameters, which showed an enhanced diagnostic sensitivity and specificity and performed excellently to diagnose PCa [area under the curve (AUC) = 0.952, P < 0.0001]. When applied to a validation test (N = 77), the model achieved an AUC of 0.947 (P < 0.0001). Moreover, this diagnostic model also exhibited a superior diagnostic performance (AUC = 0.917, P < 0.0001) over PSA (AUC = 0.712, P = 0.0018) at the PSA gray zone. CONCLUSIONS: The multivariate model based on uEVEpCAM-CD9 achieved a notable diagnostic performance to diagnose PCa. In the future, this model may potentially be used to better select patients for prostate transrectal ultrasound (TRUS) biopsy.