Autophagy serves as a crucial defense mechanism against Mycobacterium tuberculosis (Mtb) survival within infected macrophages. Transcription factor EB (TFEB) and upstream stimulatory factor 2 (USF2) belong to the bHLH-Zip family and regulate the transcription of autophagy-related genes, thereby modulating host-pathogen interactions. However, the mechanisms by which Mtb regulates these transcriptional regulatory factors to inhibit infection remain largely unexplored. This study demonstrated that PE_PGRS23 protein of Mtb impairs macrophage autophagy by inhibiting the transcription of the autophagy gene, thereby enhancing Mtb intracellular survival. Importantly, PE_PGRS23 facilitates the nuclear translocation of TFEB through PI3K-AKT-mTOR-mediated dephosphorylation. Concurrently, PE_PGRS23 promotes the nuclear translocation of USF2, which competes with TFEB for binding to the MAPLC3 promoter, ultimately suppressing MAPLC3 transcription and inhibiting autophagy. Furthermore, murine infection models demonstrated that PE_PGRS23 enhances Mtb survival and exacerbates Mtb-induced lung tissue damage. These findings underscore the critical role of the Mtb PE_PGRS23 protein in inhibiting autophagy by competitively binding of TFEB and USF2 at the MAPLC3 promoter. This mechanism facilitates the intracellular persistence of Mtb, providing theoretical insights into how the pathogen evades innate immune responses.

Mycobacterium tuberculosis (MTB), the causative agent of tuberculosis, is currently resistant to antibiotics and has undergone transcriptional adaptation to them, yet only a few transcription factors regulating mycobacterial drug resistance have been identified. In this study, the transcriptional regulatory protein Rv2250c of the TetR family was found to be a regulatory molecule of INH. Compared with wild-type strains, strains lacking the rv2250c gene showed higher minimum inhibitory concentrations (MICs) and greater intracellular survival in macrophages under INH stress. Furthermore, the INH competition with Rv2250c weakens its interaction with the efflux pump gene rv3728, thereby reducing its negative regulatory effect. This enhances the function of the MTB cell wall efflux pump, reducing the accumulation of ethidium bromide (EtBr) within the bacteria. In summary, we found that Rv2250c can regulate INH susceptibility by modulating the expression of efflux pump-encoding genes.

Tumor heterogeneity, immunosuppression, and frequent adverse effects present major challenges in colorectal cancer therapy. Although the combination of oxaliplatin (OXA, chemotherapy) and fruquintinib (FRU, antiangiogenic) shows clinical promise, their divergent physicochemical properties and inadequate tumor selectivity lead to suboptimal efficacy and systemic toxicity. To overcome the challenge, we developed a tumor-targeted nanosystem based on chitosan and fucoidan for co-delivery of hydrophilic oxaliplatin and hydrophobic fruquintinib (CS-Arg/Fuc-Bio@OF). The nanoparticle leverages the inherent P-selectin affinity of fucoidan and active targeting given by biotin modification to achieve precise tumor accumulation and microenvironment-responsive drug release. In vitro studies demonstrated that CS-Arg/Fuc-Bio@OF effectively eliminated HCT116 and HT29 cancer cells by inducing robust immunogenic cell death (ICD) and exerted potent anti-angiogenic effects. The combination of OXA-induced ICD with FRU-mediated angiogenesis suppression and polysaccharide-promoted immunomodulation synergistically reprogrammed the immunosuppressive tumor microenvironment, facilitating an effective anti-tumor immune response. In vivo, the nanoparticles significantly inhibited tumor growth and demonstrated good biosafety, with a hemolysis rate < 5% and no appreciable organ toxicity observed. This work not only validates CS-Arg/Fuc-Bio@OF as an efficient and safe combination therapy carrier but also provides a novel strategy for developing drug delivery systems based on natural polysaccharides.

[Objective]This study aimed to develop an indirect ELISA detection method for antibodies against Mycobacterium avium subsp.paratuberculosis(MAP)in sheep,for providing an efficient and reliable technique for the epidemiological surveillance and serological testing of Johne's disease(JD)in sheep.[Method]In this study,the culture supernatant of the strain(MAP-XJB13)was isolated in the laboratory during earlier stage,which was selected as the MAP-coated antigen.The optimal reaction conditions and critical values for indirect ELISA were determined through the systematic screening and optimization of various parameters,including the coating solution and conditions,blocking solution and conditions,antigen coating concentration,serum dilution ratio,antibody incubation and color development time,brand of color development solution,sample dilution solution and enzyme-labeled secondary antibody protective solution.The efficacy of the developed indirect ELISA antibody detection method for sheep MAP was assessed in terms of sensitivity,specificity,repeatability,preservation period,and coincidence rate.Finally,the initially assembled reagent kits were utilized for the clinical detection of samples from in Heilongjiang and Inner Mongolia.[Result]The optimal conditions for the ELISA were determined as follows:the coating solution utilized as CBS buffer,with the coating condition process conducted at 37℃for 4 hours.The blocking solution comprised 5%fish gelatin,5%trehalose,and 12%PEG4000,with the blocking procedure also performed at 37℃for 2 hours.The antigen coating concentration was set at 80 µg·mL-1,the serum dilution ratio was 1:40,and the dilution ratio for the enzyme-labeled secondary antibody was 1:30 000.The incubation parameters included primary antibody incubation at 25℃for 30 minutes,followed by a 30-minute incubation of the enzyme-labeled secondary antibody,and a 15-minute color development phase.The color development solution employed was Biodragon,while the sample dilution solution consisted of 1%ovalbumin and 0.5%trehalose.Additionally,the protective solution for the enzyme-labeled secondary antibody contained 0.1%ovalbumin.The critical threshold for the developed indirect ELISA method for detecting antibodies against sheep MAP was determined to be 0.460,with a sensitivity of 95.89%and a specificity of 96.12%.The cross-reactivity analysis demonstrated that,based on the premise that the positive and negative results were valid,there was no cross-reactivity with the following:positive serum for Brucella in sheep,positive serum for Mycoplasma mycoides in sheep,positive serum for Corynebacterium pseudomycosis in goats,positive serum for tuberculosis in sheep,positive serum for peste des petits ruminants virus in goats,positive serum for peste des petits ruminants in sheep,and positive serum for poxvirus in sheep.The intra-batch and inter-batch coefficients of variation ranged from 0.754%to 7.812%and 1.252%to 7.277%,respectively,and the stability of the results was maintained for up to 8 months.The sheep MAP indirect ELISA antibody detection kit exhibited a positive concordance rate of 95.89%and a negative concordance rate of 95.55%when compared to the ID.vet MAP ELISA antibody detection kit,resulting in an overall concordance rate of 98.56%.The prevalence of MAP antibodies in sheep from Heilongjiang and Inner Mongolia was found to be 10.81%.[Conclusion]This study successfully developed an indirect ELISA method for the detection of antibodies MAP in sheep.The method exhibited exceptional specificity,high sensitivity and a long shelf life,thereby offering robust technical support for the prevention and management of JD.

The mycobacterial cell wall is a complex structure that plays a critical role in resisting external environmental stress and contributing to pathogenesis, with mycolic acid being a key component in maintaining cell wall integrity. Moreover, the enzymes involved in cell wall synthesis are frequently targeted in antimycobacterial drug research. In this study, we demonstrate that the conserved extracellular DNase XthA from mycobacteria exhibits phospholipase A1 (PLA1) activity, whereas this activity is absent in non-pathogenic mycobacteria. Moreover, XthA PLA1 activity facilitates the formation of cord-like structures, promotes the proliferation of mycobacteria, and enhances their resistance to environmental stressors. Results from ethidium bromide and minimum inhibitory concentration assays suggest that XthA PLA1 activity reduces the permeability of the mycobacterial cell wall. Furthermore, analyses using scanning electron microscopy and mass spectrometry demonstrated that PLA1 activity contributes to cell wall integrity and enhances the synthesis of mycolic acids. Additionally, qPCR analysis indicated that XthA PLA1 activity upregulates the transcript of key genes involved in the MAs synthesis pathway. Collectively, these findings suggest that pathogenic mycobacteria utilize XthA PLA1 to facilitate cell wall functionality by regulating mycolic acid synthesis, thereby underscoring its potential as a drug target for disrupting the cell wall of pathogenic mycobacteria.

The potential links between psychiatric disorders and cancer risk have attracted significant attention in recent years. However, the causal relationships between these conditions remain unclear. To assess causality, a Mendelian randomization analysis was conducted using single nucleotide polymorphisms from large-scale European genome-wide association studies (GWAS). The inverse variance weighted (IVW) method was applied as the primary method for causal estimation, with additional sensitivity analyses performed to evaluate potential pleiotropic effects. IVW estimates revealed that bipolar disorder was associated with an increased risk of prostate cancer (odds ratio (OR) = 1.07; 95% confidence interval (CI): 1.003-1.15; P = .04). Genetically predicted depression was causally linked to a significantly higher risk of breast cancer (OR = 2.49; 95% CI: 1.26-4.9; P = .008), particularly in estrogen receptor-positive (ER+) breast cancer (OR = 2.82; 95% CI: 1.17-6.81; P = .02). Schizophrenia was associated with a significantly higher risk of multiple cancer types, including lung cancer (OR = 1.16; 95% CI: 1.03-1.3; P = .01), breast cancer (OR = 1.05; 95% CI: 1.02-1.09; P = .002), ER+ breast cancer (OR = 1.06; 95% CI: 1.02-1.09; P = .002), endometrial cancer (OR = 1.05; 95% CI: 1.002-1.1; P = .04), and ovarian cancer (OR = 1.08; 95% CI: 1.03-1.13; P = .0007). No significant pleiotropy of the instrumental variables was observed. This psycho-oncology study, characterized by minimal pleiotropic effects, provides genetic evidence supporting an increased incidence of various cancers associated with psychiatric disorders. These findings suggest that underlying psychiatric processes may contribute to cancer development.

Innate immunity is dominant in protecting the host's defense against intracellular bacterial infections. The secretion of IL-1β and activation of NLRP3 inflammasome in macrophages play a critical role in combating Mycobacterium tuberculosis (M.tb) infections. M.tb is an extremely successful intracellular pathogen that evades host innate immunity by interfering with a wide range of macrophage functions. However, the precise infection mechanism remains unclear. This study demonstrates that the mycobacterial serine protease Rv2569c interacts with RhoG in macrophages, effectively blocking the NF-κB signaling pathway's initiation and suppressing NLRP3 inflammasome activation, ultimately leading to a decrease in IL-1β secretion and promoting mycobacterial survival within macrophages. To investigate the role of Rv2569c in M.tb infection, an Rv2569c-deficient strain (H37RvΔRv2569c) was used to demonstrate a weakened suppression of the inflammatory response and lower intracellular survival compared to the wild-type (H37Rv) and complemented strain (H37RvΔRv2569c + Rv2569c) through in vitro and in vivo experiments. The findings provide the first proof that RhoG serves as an endogenous host sensor for pathogens and that Rv2569c-RhoG-mediated inflammatory response plays a crucial role in mycobacterial immune evasion.

Epithelial cells function as the primary line of defense against invading pathogens. However, bacterial pathogens possess the ability to compromise this barrier and facilitate the transmigration of bacteria. Nonetheless, the specific molecular mechanism employed by Mycobacterium tuberculosis (M.tb) in this process is not fully understood. Here, we investigated the role of Rv2569c in M.tb translocation by assessing its ability to cleave E-cadherin, a crucial component of cell-cell adhesion junctions that are disrupted during bacterial invasion. By utilizing recombinant Rv2569c expressed in Escherichia coli and subsequently purified through affinity chromatography, we demonstrated that Rv2569c exhibited cell wall-associated serine protease activity. Furthermore, Rv2569c was capable of degrading a range of protein substrates, including casein, fibrinogen, fibronectin, and E-cadherin. We also determined that the optimal conditions for the protease activity of Rv2569c occurred at a temperature of 37°C and a pH of 9.0, in the presence of MgCl2. To investigate the function of Rv2569c in M.tb, a deletion mutant of Rv2569c and its complemented strains were generated and used to infect A549 cells and mice. The results of the A549-cell infection experiments revealed that Rv2569c had the ability to cleave E-cadherin and facilitate the transmigration of M.tb through polarized A549 epithelial cell layers. Furthermore, in vivo infection assays demonstrated that Rv2569c could disrupt E-cadherin, enhance the colonization of M.tb, and induce pathological damage in the lungs of C57BL/6 mice. Collectively, these results strongly suggest that M.tb employs the serine protease Rv2569c to disrupt epithelial defenses and facilitate its systemic dissemination by crossing the epithelial barrier.

Macrophages serve as the primary immune cells responsible for the innate immune defense against Mycobacterium tuberculosis (MTB) infection within the host. Specifically, NLRP3, a member of the NLRs family, plays a significant role in conferring resistance against MTB infection. Conversely, MTB evades innate immune killing by impeding the activation of the NLRP3 inflammasome, although the precise mechanism remains uncertain. In this study, we have identified PE12 (Rv1172c), a member of the PE/PPE family proteins, as an extracellular protein of MTB. PE12 interacts with Toll like receptor 4 (TLR4) in macrophages, forming the PE12-TLR4 complex which subsequently inhibits the transcription and expression of NLRP3. As a result, the transcription and secretion of IL-1β are reduced through the PE12-TLR4-NLRP3-IL-1β immune pathway. In vitro and in vivo experiments using a PE12-deficient strain (H37RvΔPE12) demonstrate a weakening of the suppression of the inflammatory response to MTB infection. Our findings highlight the role of the PE12 protein in not only inhibiting the transcription and release of inflammatory cytokines but also mediating the killing of MTB escape macrophages through TLR4 and inducing lung injury in MTB-infected mice. These results provide evidence that PE12 plays a significant role in the inhibition of the host immune response by MTB.

BackgroundIntervertebral disc degeneration (IDD) is one of the most common health problems in the elderly and a major causative factor in low back pain (LBP). An increasing number of studies have shown that IDD is closely associated with autophagy and immune dysregulation. Therefore, the aim of this study was to identify autophagy-related biomarkers and gene regulatory networks in IDD and potential therapeutic targets.MethodsWe obtained the gene expression profiles of IDD by downloading the datasets GSE176205 and GSE167931 from the Gene Expression Omnibus (GEO) public database. Subsequently, differentially expressed genes (DEGs) analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, gene ontology (GO), and gene set enrichment analysis (GSEA) were performed to explore the biological functions of DEGs. Differentially expressed autophagy-related genes (DE-ARGs) were then crossed with the autophagy gene database. The hub genes were screened using the DE-ARGs protein–protein interaction (PPI) network. The correlation between the hub genes and immune infiltration and the construction of the gene regulatory network of the hub genes were confirmed. Finally, quantitative PCR (qPCR) was used to validate the correlation of hub genes in a rat IDD model.ResultsWe obtained 636 DEGs enriched in the autophagy pathway. Our analysis revealed 30 DE-ARGs, of which six hub genes (MAPK8, CTSB, PRKCD, SNCA, CAPN1, and EGFR) were identified using the MCODE plugin. Immune cell infiltration analysis revealed that there was an increased proportion of CD8+ T cells and M0 macrophages in IDD, whereas CD4+ memory T cells, neutrophils, resting dendritic cells, follicular helper T cells, and monocytes were much less abundant. Subsequently, the competitive endogenous RNA (ceRNA) network was constructed using 15 long non-coding RNAs (lncRNAs) and 21 microRNAs (miRNAs). In quantitative PCR (qPCR) validation, two hub genes, MAPK8 and CAPN1, were shown to be consistent with the bioinformatic analysis results.ConclusionOur study identified MAPK8 and CAPN1 as key biomarkers of IDD. These key hub genes may be potential therapeutic targets for IDD.

Hand, foot, and mouth disease (HFMD) is a common children infectious disease caused by human enteroviruses. Most of the cases have minimal symptoms, however, some patients may develop serious neurological, cardiac complications, or even death. The pathological mechanism leading to severe HFMD is not clearly understood, and the immunological status of the individual patient may play an important role. Transcriptomes of peripheral blood mononuclear cells from EV71-infected patients (n = 45) and healthy controls (n = 36) were examined. Immune pathways were up-regulated in patients with mild disease symptoms (n = 11, M) compared to the healthy controls (n = 36, H), demonstrating an effective anti-viral response upon EV71 infection. However, in patients with severe symptoms (n = 23, S) as well as severe patients following treatment (n = 11, A), their innate and acquired immune pathways were down-regulated, indicating a global immunity suppression. Such immune suppression characteristics could thus provide an opportunity for early EV-71 infection prognosis prediction. Based on our cohort, an SVM model using RNA-seq expression levels of five genes (MCL1, ZBTB37, PLEKHM1P, IFNAR2 and YEATS2) was developed and achieved a high ROC-AUC (91.3%) in predicting severe HFMD. Meanwhile, qPCR fold-changes method was performed based three genes (MCL1, IFNAR2 and YEATS2) on additional cohort. This qPCR method achieved a ROC-AUC of 78.6% in predicting severe HFMD, which the patients could be distinguished in 2-3 h. Therefore, our models demonstrate the possibility of HFMD severity prediction based on the selected biomarkers that predict severe HFMD effectively.

Mycobacterium tuberculosis (Mtb) is an intracellular bacterium that causes a highly contagious and potentially lethal tuberculosis (TB) in humans. It can maintain a dormant TB infection within the host. DosR (dormancy survival regulator) (Rv3133c) has been recognized as one of the key transcriptional proteins regulating bacterial dormancy and participating in various metabolic processes. In this study, we extensively investigate the still not well-comprehended role and mechanism of DosR in Mycobacterium bovis (M. bovis) Bacillus Calmette-Guérin (BCG) through a combined omics analysis. Our study finds that deleting DosR significantly affects the transcriptional levels of 104 genes and 179 proteins. Targeted metabolomics data for amino acids indicate that DosR knockout significantly upregulates L-Aspartic acid and serine synthesis, while downregulating seven other amino acids, including L-histidine and lysine. This suggests that DosR regulates amino acid synthesis and metabolism. Taken together, these findings provide molecular and metabolic bases for DosR effects, suggesting that DosR may be a novel regulatory target.

l-Arginine serves as a carbon and nitrogen source and is critical for Mycobacterium tuberculosis (Mtb) survival in the host. Generally, ArgR acts as a repressor regulating arginine biosynthesis by binding to the promoter of the argCJBDFGH gene cluster. In this study, we report that the dormancy regulator DosR is a novel arginine regulator binding to the promoter region of argC (rv1652), which regulates arginine synthesis. Phosphorylation modification promoted DosR binding to a region upstream of the promoter. Cofactors, including arginine and metal ions, had an inhibitory effect on this association. Furthermore, DosR regulatory function relies on the interaction of the 167, 181, 182, and 197 amino acid residues with an inverse complementary sequence. Arginine also binds to DosR and directly affects its DNA-binding ability. Together, the results demonstrate that DosR acts as a novel transcriptional regulator of arginine synthesis in Mycobacterium bovis bacille Calmette-Guerin.

Abstract Mutations in AT‐rich interactive domain‐containing protein 1A (ARID1A) cause Coffin‐Siris syndrome (CSS), a rare genetic disorder that results in mild to severe intellectual disabilities. However, the biological role of ARID1A in the brain remains unclear. In this study, we report that the haploinsufficiency of ARID1A in excitatory neurons causes cognitive impairment and defects in hippocampal synaptic transmission and dendritic morphology in mice. Similarly, human embryonic stem cell‐derived excitatory neurons with deleted ARID1A exhibit fewer dendritic branches and spines, and abnormal electrophysiological activity. Importantly, supplementation of acetate, an epigenetic metabolite, can ameliorate the morphological and electrophysiological deficits observed in mice with Arid1a haploinsufficiency, as well as in ARID1A‐null human excitatory neurons. Mechanistically, transcriptomic and ChIP‐seq analyses demonstrate that acetate supplementation can increase the levels of H3K27 acetylation at the promoters of key regulatory genes associated with neural development and synaptic transmission. Collectively, these findings support the essential roles of ARID1A in the excitatory neurons and cognition and suggest that acetate supplementation could be a potential therapeutic intervention for CSS.

Introduction: Non-small cell lung carcinoma (NSCLC) constitutes most lung cancers and has a poor prognosis. LncRNAs are a potential repository for the discovery of cancer prognostic markers. This study explored the role of LINC01929 in NSCLC, both the clinical prognostic significance and the mechanism of its influence on cells.Materials and Methods: LINC01929 levels in 143 pairs of NSCLC tissues and non-cancerous tissues were detected by RT-qPCR. Kaplan-Meier curves and multivariate Cox regression assays were generated for evaluating the prognostic values of LINC01929. To evaluate the cellular function, an XTT assay and transwell invasion assays were performed.Results: LINC01929 was up-regulated in NSCLC tissues compared with healthy tissues. A positive correlation was observed between LINC01929 expression level and tumor T (p = 0.002) or N stage (p = 0.010). Patients with higher LINC01929 levels had shorter overall survival (p = 0.009). Compared with other factors, high LINC01929 expression was significantly associated with poor survival in univariate Cox analysis (HR: 2.485, 95%CI: 1.220-5.060, p = 0.012). After multivariate Cox regression assays, LINC01929 was a independent prognostic factor (HR: 3.021, 95%CI: 1.377-6.628, p = 0.006). miR-1179 was a target miRNA of LINC01929. Inhibited expression of LINC01929 significantly reduced the proliferation, migration, and invasion of NSCLC cells by targeting miR-1179.Discussion: This study revealed the upregulation of LINC01929 in NSCLC. This study supports previous studies showing LINC01929 as a potential prognostic factor for NSCLC.

Inflammaging refers to low-grade, chronically activated innate immunity that has deleterious effects on healthy lifespan. However, little is known about the intrinsic signaling pathway that elicits innate immune genes during aging. Here, using Drosophila melanogaster, we profile the microRNA targetomes in young and aged animals, and reveal Dawdle, an activin-like ligand of the TGF-β pathway, as a physiological target of microRNA-252. We show that microRNA-252 cooperates with Forkhead box O, a conserved transcriptional factor implicated in aging, to repress Dawdle. Unopposed Dawdle triggers hyperactivation of innate immune genes coupled with a decline in organismal survival. Using adult muscle tissues, single-cell sequencing analysis describes that Dawdle and its downstream innate immune genes are expressed in distinct cell types, suggesting a cell nonautonomous mode of regulation. We further determine the genetic cascade by which Dawdle signaling leads to increased Kenny/IKKγ protein, which in turn activates Relish/NF-κB protein and consequentially innate immune genes. Finally, transgenic increase of microRNA-252 and Forkhead box O pathway factors in wild-type Drosophila extends lifespan and mitigates the induction of innate immune genes in aging. Together, we propose that microRNA-252 and Forkhead box O promote healthy longevity by cooperative inhibition on Dawdle-mediated inflammaging.

l-Arginine serves as a carbon and nitrogen source and is critical for Mycobacterium tuberculosis (Mtb) survival in the host. Generally, ArgR acts as a repressor regulating arginine biosynthesis by binding to the promoter of the argCJBDFGH gene cluster. In this study, we report that the dormancy regulator DosR is a novel arginine regulator binding to the promoter region of argC (rv1652), which regulates arginine synthesis. Phosphorylation modification promoted DosR binding to a region upstream of the promoter. Cofactors, including arginine and metal ions, had an inhibitory effect on this association. Furthermore, DosR regulatory function relies on the interaction of the 167, 181, 182, and 197 amino acid residues with an inverse complementary sequence. Arginine also binds to DosR and directly affects its DNA-binding ability. Together, the results demonstrate that DosR acts as a novel transcriptional regulator of arginine synthesis in Mycobacterium bovis bacille Calmette-Guerin.

Mutations in the embryonic ectoderm development (EED) cause Weaver syndrome, but whether and how EED affects embryonic brain development remains elusive. Here, we generated a mouse model in which Eed was deleted in the forebrain to investigate the role of EED. We found that deletion of Eed decreased the number of upper-layer neurons but not deeper-layer neurons starting at E16.5. Transcriptomic and genomic occupancy analyses revealed that the epigenetic states of a group of cortical neurogenesis-related genes were altered in Eed knockout forebrains, followed by a decrease of H3K27me3 and an increase of H3K27ac marks within the promoter regions. The switching of H3K27me3 to H3K27ac modification promoted the recruitment of RNA-Pol2, thereby enhancing its expression level. The small molecule activator SAG or Ptch1 knockout for activating Hedgehog signaling can partially rescue aberrant cortical neurogenesis. Taken together, we proposed a novel EED-Gli3-Gli1 regulatory axis that is critical for embryonic brain development.

BACKGROUND:Fecal immunochemical test (FIT), DNA mutation, DNA methylation, and microbial dysbiosis all showed promising in colorectal cancer (CRC) non-invasive detection. We assessed CRC detection with an assay combining all these strategies and investigated the effect of clinical features on the performance of this comprehensive test.METHODS:We performed a multidimensional analysis study using stool samples collected from 108 patients with CRC, 18 patients with colorectal adenoma, and 36 individuals with no evidence of colorectal disease. The multidimensional analysis of stool samples including FIT, stool DNA (sDNA) tests for three methylated genes (Septin9, NDRG4, BMP3) and three mutated genes (KRAS, BRAF, PI3KCA) using next generation sequencing as well as detection of stool bacteria level of Fusobacterium nucleatum and Parvimonas micra using qPCR method. We used a linear support vector classification model to analyze the data.RESULTS:The sensitivity of FIT alone was 69.4% for CRC and 11.1% for adenoma. Separately, the sensitivity of the detection of intestinal bacteria, DNA mutation, and DNA methylation for CRC was 58.3, 50.0, and 51.9%, respectively. The combination of FIT and sDNA tests had a sensitivity of 81.5% for CRC (AUC: 0.93, better than FIT alone, P = 0.017) and 27.8% for adenoma with 94.4% specificity. Sensitivity of the multidimensional test to detect CRC with stage II (84.6%) and III (91.9%) CRC was relatively higher (88.2%) than that of patients with stage I (60.0%) and stage IV (75.0%) (P = 0.024). The rate of CRC detection increased with tumor size (P = 0.008) and age (P = 0.04). Interestingly, the rate of CRC detection was higher in smoking persons than non-smokers with marginal significance (P = 0.08).CONCLUSIONS:The multidimensional assay of stool samples combining FIT and stool DNA tests further improved the diagnostic sensitivity for CRC. This could provide new approach for improvement of CRC screening and further demonstrations are warranted.

Colorectal cancer (CRC) is one of the most common and deadliest cancers worldwide. Patient survival can be greatly improved if cancerous lesions are detected early.1 Despite a strong recommendation of colonoscopy for CRC screening, patients prefer noninvasive tests, and 83% favor blood-based tests.2 Blood tests that rely on a single DNA methylation biomarker have been approved3; however, they are not recommended due to the low sensitivity for detecting early-stage CRC.4