Loop-mediated isothermal amplification (LAMP) continuously generates strand-displaced single-stranded DNA intermediates, providing the possibility of assembling DNA fragments. Here, we developed a novel two-DNA-fragment fusion technique, termed fusion LAMP, which is an isothermal DNA-fusion strategy that enables the fusion of two independent DNA fragments within a single amplification reaction. By combining fusion LAMP with CRISPR/Cas13a, we further established an "AND-gate" nucleic acid detection platform, termed Fusion LAMP-Coupled CRISPR/Cas13a (FLCC), which enables concurrent detection of two targets by reading the fusion product-triggered fluorescence signals. This platform generates signals only when two targets are present simultaneously. To prove this concept, we then employed FLCC to identify the methicillin-resistant (MRSA). This method achieved a limit of detection of 10 copies/mu L of MRSA genomic DNA and showed no cross-reactivity with closely related bacterial strains. Furthermore, we validated its feasibility by detecting 19 clinical isolates, demonstrating a simple and accurate approach for MRSA detection. Collectively, the FLCC platform ensures identifying pathogens accurately and provides a promising diagnostic approach for detecting complex genetic targets.

Loop-mediated isothermal amplification (LAMP) continuously generates strand-displaced single-stranded DNA intermediates, providing the possibility of assembling DNA fragments. Here, we developed a novel two-DNA-fragment fusion technique, termed fusion LAMP, which is an isothermal DNA-fusion strategy that enables the fusion of two independent DNA fragments within a single amplification reaction. By combining fusion LAMP with CRISPR/Cas13a, we further established an "AND-gate" nucleic acid detection platform, termed Fusion LAMP-Coupled CRISPR/Cas13a (FLCC), which enables concurrent detection of two targets by reading the fusion product-triggered fluorescence signals. This platform generates signals only when two targets are present simultaneously. To prove this concept, we then employed FLCC to identify the methicillin-resistant Staphylococcus aureus (MRSA). This method achieved a limit of detection of 10 copies/μL of MRSA genomic DNA and showed no cross-reactivity with closely related bacterial strains. Furthermore, we validated its feasibility by detecting 19 clinical isolates, demonstrating a simple and accurate approach for MRSA detection. Collectively, the FLCC platform ensures identifying pathogens accurately and provides a promising diagnostic approach for detecting complex genetic targets.

The correlation between persistent human cytomegalovirus (HCMV) infection and poor prognosis in colorectal cancer (CRC) patients has garnered increasing attention. UL82 is a tegument protein of HCMV, and our previous research indicated that the presence of UL82 is significantly associated with reduced overall survival in CRC patients. However, the mechanism by which UL82 affects the prognosis of CRC patients remains unclear. In this study, we investigated the role of UL82 in CRC progression through both in vitro and in vivo experiments, and revealed its downstream regulatory pathways by integrating transcriptomics, metabolomics, and proteomics. Our findings first revealed that UL82 significantly promoted CRC cell proliferation by increasing the proportion of cells in the S phase of the cell cycle. Additionally, UL82 enhanced the expression of the oncogene AGR2, while knockdown of AGR2 abolished the proliferative effect of UL82. Interestingly, UL82 interacted with the transcription factor DDX5, which transcriptionally inhibited AGR2 expression. Furthermore, this UL82-AGR2 axis promoted nucleotide metabolism in CRC cells by enhancing the levels of nucleotide synthesis enzymes DTYMK, RRM2, and TYMS. In conclusion, our study suggests that the UL82/DDX5 complex may promote nucleotide metabolism and cell cycle progression of CRC by upregulating AGR2 and UL82 may serve as a potential prognostic biomarker for CRC patients.

Human cytomegalovirus (HCMV) infection and its association with tumorigenesis and tumor progression have garnered increasing attention. Previous research results have indicated that the detection rate of HCMV UL82 is higher in colorectal cancer (CRC) tissues and is correlated with poor prognosis in patients, yet the underlying mechanisms remain unclear. In this study, a cell model transfected with UL82 was established. Through in vitro and in vivo experiments, it was found that UL82 promoted the proliferation of CRC cells. Transcriptomic and metabolomic analyses revealed that UL82 could influence CRC glucose metabolism and cell proliferation by upregulating the key TCA cycle enzyme OGDH. Additionally, UL82 also affected CRC cell proliferation by upregulating the expression of ANGPT2, and silencing ANGPT2 resulted in a reduction in OGDH protein levels. Finally, through the investigation of the ubiquitin-mediated degradation pathway of OGDH, it was demonstrated that ANGPT2 inhibited the protein degradation of OGDH via deubiquitination, thereby maintaining its stability. In summary, UL82 promotes CRC cell proliferation by upregulating ANGPT2, which inhibits the ubiquitin-mediated degradation of OGDH, suggesting that targeting the UL82/ANGPT2/OGDH axis may offer a potential clinical strategy for the diagnosis of CRC.

Introduction:Central nervous system (CNS) infections represent a significant global public health concern and are characterized by high morbidity and mortality rates. In this study, we developed an integrated diagnostic approach for CNS infections by combining high-throughput nanopore sequencing with multiplex PCR amplification, designated targeted nanopore sequencing (tNPS). Methods:The tNPS assay employed a dual detection strategy incorporating pathogen-specific primers targeting 17 prevalent CNS pathogens (seven bacteria, one fungus and nine DNA viruses), with universal primers for the comprehensive amplification of full-length 16S ribosomal RNA (16S rRNA) and internal transcribed spacer (ITS) regions. Results:Analytical validation of tNPS was successfully carried out using the 12 positive reference strains (seven bacteria, one fungus, and four DNA viruses) individually, the ZymoBIOMICS microbial community (eight bacteria and two fungi), the laboratory synthetic community of bacteria and fungi (seven bacteria and one fungus), and the laboratory synthetic community of viruses (five DNA viruses). With accelerated turnaround time within 8 h, the tNPS also assayed 11 clinical cerebrospinal fluid (CSF) samples, which further confirmed the feasibility of precise identification of CNS pathogens compared to CSF culture and metagenomic next-generation sequencing. Discussion:Our tNPS as a culture-independent diagnostic assay offered enhanced efficiency, high-throughput capability, and an expanded pathogen detection spectrum, facilitating potential implementation in molecular diagnosis of CNS infection.

Since the 20th century, global health security related biosafety risks have progressively intensified. Major public health crises, primarily caused by infectious diseases, have become more frequent. Additional concerns, such as population aging, the prevalence of chronic non-communicable diseases like hypertension and diabetes, the growing problem of antimicrobial resistance, and the ongoing effects of global environmental changes and worsening air pollution, have increased health risks for humanity. One Health, a multidisciplinary and cross-regional collaboration strategy that encompasses human, animal, and environmental health care, has become a key internationally recognized strategy for addressing significant and complex human health issues. On April 2, 2024, the One Health Innovation and Development Forum was held at Wenzhou Medical University. At this forum, experts from around the world gathered to discuss topics such as infectious disease prevention and control, vaccine development, antibiotic resistance, food safety, and environmental health. They reached a consensus and took action to jointly promote the realization of a global community of health for all.

Tuberculosis (TB) remains the leading cause of human mortality in infectious diseases. Drug-resistant TB, particularly multidrug-resistant TB and extensively drug-resistant TB, poses a pressing clinical and public health challenge. The main causative agents of TB are known as Mycobacterium tuberculosis (MTB), which exhibits a highly complex drug resistance profile. Traditional culture-based phenotypic drug susceptibility testing is time-consuming, and PCR-based assays are restricted to detecting known mutational hotspots. In this study, we present a protocol leveraging high-throughput nanopore sequencing technology in conjunction with multiplex PCR, termed targeted nanopore sequencing, for the identification of MTB and analysis of its drug resistance. Our method for MTB drug resistance assessment offers the benefits of being culture-free, efficient, high-throughput, and highly accurate, which could significantly aid in clinical patient management and the control of TB infections.

Bladder cancer is the most common type of malignant tumor in the urinary system, which occurs in the bladder mucosa. Due to the lack of precise diagnostic tools, bladder cancer has a high recurrence rate. Fluorescence cystoscopy is recognized as a promising tool for improving the detection rate of bladder cancer, but its clinical application is hindered due to the scarcity of fluorescent agents. To address this issue, an aminopeptidase N (APN)-activated fluorescent agent with specific recognition and labeling abilities for bladder cancer has been developed. The fluorescent agent, namely, Flu-FAPN, exhibited high sensitivity and specificity toward APN under physiological conditions. The fluorescent intensity increased more than 37-fold after reacting with APN, and the detection limit is 0.10 ng/mL for APN. In addition, the hydrolysis of alanine catalyzed by APN could initiate the intramolecular electron rearrangement process, which subsequently contributed to the formation of protein-fluorescein adduct and thereby achieved covalent labeling of bladder cancer cells. Cell experiments conducted in cancer and normal cells demonstrate that Flu-FAPN has low cytotoxicity and high specificity for bladder cancer cells with an obvious fluorescence signal, which could be retained over 80% even in fixed cells. Finally, Flu-FAPN was successfully applied for the fluorescence-image-guided resection of tumor tissues in mice with orthotopic bladder cancer. We hope this covalent labeling-based agent can provide a promising tool for surgery navigation and accelerate the clinical application of fluorescence cystoscopy, thereby improving the cure rate of bladder cancer.

Highly sensitive airborne virus monitoring is critical for preventing and containing epidemics. However, the detection of airborne viruses at ultra-low concentrations remains challenging due to the lack of ultra-sensitive methods and easy-to-deployment equipment. Here, we present an integrated microfluidic cartridge that can accurately detect SARS-COV-2, Influenza A, B, and respiratory syncytial virus with a sensitivity of 10 copies/mL. When integrated with a high-flow aerosol sampler, our microdevice can achieve a sub-single-copy spatial resolution of 0.83 copies/m3 for airborne virus surveillance with an air flow rate of 400 L/min and a sampling time of 30 minutes. We then designed a series of virus-in-aerosols monitoring systems (RIAMs), including versions of a multi-site sampling RIAMs (M-RIAMs), a stationary real-time RIAMs (S-RIAMs), and a roaming real-time RIAMs (R-RIAMs) for different application scenarios. Using M-RIAMs, we performed a comprehensive evaluation of 210 environmental samples from COVID-19 patient wards, including 30 aerosol samples. The highest positive detection rate of aerosol samples (60%) proved the aerosol-based SARS-CoV-2 monitoring represents an effective method for spatial risk assessment. The detection of 78 aerosol samples in real-world settings via S-RIAMs confirmed its reliability for ultra-sensitive and continuous airborne virus monitoring. Therefore, RIAMs shows the potential as an effective solution for mitigating the risk of airborne virus transmission. Effective surveillance of respiratory virus particles in air requires highly sensitive tools that can produce timely and accurate results. Here, the authors develop a device for detection of four respiratory viruses in air with high spatial viral resolution and test it in different settings.

Hemicyanine dyes are known as widely used fluorophores with good biocompatibility and excellent photophysical property, but poor ability to produce singlet oxygen has limited their application in photodynamic therapy (PDT). Introduction of heavy atoms seems to be a feasible way to enhance the PDT efficiency, but undesired toxicity is inevitable. Herein, a radical enhanced intersystem crossing-based hemicyanine dye Cy-OT is synthesized and successfully applied in PDT process. The singlet oxygen quantum yield of Cy-OT increases by 4.2 times through the introduction of 4-hydroxyl-2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) unit. Cell experiments carried out in MCF-7 cells demonstrates that Cy-OT is localized in mitochondria and can produce single oxygen under the irradiation of 660 nm light which subsequently results in mitochondria membrane potential (MMP) loss and induces cell apoptosis. The therapeutic experiment of 4T1 tumor bearing mice further indicates that Cy-OT can serve as an ideal photosensitizer with good antitumor effect and negligible side effect to normal tissues.

Drug-resistant tuberculosis (TB), especially multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), is one of the urgent clinical problems and public health challenges. Culture-based phenotypic drug susceptibility testing (pDST) is time-consuming, and PCR-based assays are limited to hotspot mutations. In this study, we developed and validated a convenient and efficient approach based on high-throughput nanopore sequencing technology combined with multiplex PCR, namely nanopore targeted sequencing (NTS), to simultaneously sequence 18 genes associated with antibiotic resistance in Mycobacterium tuberculosis (MTB). The analytical performance of NTS was evaluated, and 99 clinical samples were collected to assess its clinical performance. The NTS results showed that MTB and its drug resistance were successfully identified in approximately 7.5 h. Furthermore, compared to the pDST and Xpert MTB/RIF assays, NTS provided much more drug resistance information, covering 14 anti-TB drugs, and it identified 20 clinical cases of drug-resistant MTB. The mutations underlying these drug-resistant cases were all verified using Sanger sequencing. Our approach for this TB drug resistance assay offers several advantages, including being culture-free, efficient, high-throughput, and highly accurate, which would be very helpful for clinical patient management and TB infection control.

The aim of this study was to investigate the underlying molecular mechanism behind the promotion of cell survival under conditions of glucose deprivation by l-lactate. To accomplish this, we performed tissue microarray and immunohistochemistry staining to analyze the correlation between the abundance of pan-Lysine lactylation and prognosis. In vivo evaluations of tumor growth were conducted using the KPC and nude mice xenograft tumor model. For mechanistic studies, multi-omics analysis, RNA interference, and site-directed mutagenesis techniques were utilized. Our findings robustly confirmed that l-lactate promotes cell survival under glucose deprivation conditions, primarily by relying on GLS1-mediated glutaminolysis to support mitochondrial respiration. Mechanistically, we discovered that l-lactate enhances the NMNAT1-mediated NAD+ salvage pathway while concurrently inactivating p-38 MAPK signaling and suppressing DDIT3 transcription. Notably, Pan-Kla abundance was significantly upregulated in patients with Pancreatic adenocarcinoma (PAAD) and associated with poor prognosis. We identified the 128th Lysine residue of NMNAT1 as a critical site for lactylation and revealed EP300 as a key lactyltransferase responsible for catalyzing lactylation. Importantly, we elucidated that lactylation of NMNAT1 enhances its nuclear localization and maintains enzymatic activity, thereby supporting the nuclear NAD+ salvage pathway and facilitating cancer growth. Finally, we demonstrated that the NMNAT1-dependent NAD+ salvage pathway promotes cell survival under glucose deprivation conditions and is reliant on the activity of Sirt1. Collectively, our study has unraveled a novel molecular mechanism by which l-lactate promotes cell survival under glucose deprivation conditions, presenting a promising strategy for targeting lactate and NAD+ metabolism in the treatment of PAAD.

Circulating tumor cells (CTCs) are tumor cells that exist in human peripheral blood, which could spread to other tissues or organs via the blood circulation system and develop into metastatic foci, leading to tumor recurrence or metastasis in postoperative patients and thereby increasing the mortality of malignant tumor patients. Evaluation of CTC levels can be used for tumor metastasis prediction, prognosis evaluation, drug exploitation, individualized treatment, liquid biopsy, etc., which exhibit outstanding clinical application prospects. In recent years, accurately capturing and analyzing CTCs has become a research hotspot in the early diagnosis and precise treatment of tumors. This review summarized various enrichment and isolation technologies for evaluating CTCs based on the design principle and discussed the challenges and perspectives in this field.

BACKGROUND:In the era of tyrosine kinase inhibitor (TKI) treatment, the progression of chronic myeloid leukemia (CML) remains a significant clinical challenge, and genetic biomarkers for the early identification of CML patients at risk for progression are limited. This study explored whether essential circular RNAs (circRNAs) can be used as biomarkers for diagnosing and monitoring CML disease progression and assessing CML prognosis. METHODS:Peripheral blood (PB) samples were collected from 173 CML patients (138 patients with chronic phase CML [CML-CP] and 35 patients with accelerated phase/blast phase CML [CML-AP/BP]) and 63 healthy controls (HCs). High-throughput RNA sequencing (RNA-Seq) was used to screen dysregulated candidate circRNAs for a circRNA signature associated with CML disease progression. Quantitative real-time PCR (qRT-PCR) was used for preliminary verification and screening of candidate dysregulated genes, as well as subsequent exploration of clinical applications. Receiver operating characteristic (ROC) curve analysis, Spearman's rho correlation test, and the Kaplan-Meier method were used for statistical analysis. RESULTS:The aberrant expression of hsa_circ_0006010 and hsa_circ_0002903 during CML progression could serve as valuable biomarkers for differentiating CML-AP/BP patients from CMP-CP patients or HCs. In addition, the expression levels of hsa_circ_0006010 and hsa_circ_0002903 were significantly associated with the clinical features of CML patients but were not directly related to the four scoring systems. Furthermore, survival analysis revealed that high hsa_circ_0006010 expression and low hsa_circ_0002903 expression indicated poor progression-free survival (PFS) in CML patients. Finally, PB hsa_circ_0006010 and hsa_circ_0002903 expression at diagnosis may also serve as disease progression surveillance markers for CML patients but were not correlated with PB BCR-ABL1/ABL1IS. CONCLUSIONS:Our study demonstrated that PB levels of hsa_circ_0006010 and hsa_circ_0002903 may serve as novel diagnostic, surveillance, and prognostic biomarkers for CML disease progression and may contribute to assisting in the diagnosis of CML patients at risk for progression and accurate management of advanced CML patients.

Abstract Background Microsatellite instability-high (MSI-H) has emerged as a significant biological characteristic of colorectal cancer (CRC). Studies reported that MSI-H CRC generally had a better prognosis than microsatellite stable (MSS)/microsatellite instability-low (MSI-L) CRC, but some MSI-H CRC patients exhibited distinctive molecular characteristics and experienced a less favorable prognosis. In this study, our objective was to explore the metabolic transcript-related subtypes of MSI-H CRC and identify a biomarker for predicting survival outcomes. Methods Single-cell RNA sequencing (scRNA-seq) data of MSI-H CRC patients were obtained from the Gene Expression Omnibus (GEO) database. By utilizing the copy number variation (CNV) score, a malignant cell subpopulation was identified at the single-cell level. The metabolic landscape of various cell types was examined using metabolic pathway gene sets. Subsequently, functional experiments were conducted to investigate the biological significance of the hub gene in MSI-H CRC. Finally, the predictive potential of the hub gene was assessed using a nomogram. Results This study revealed a malignant tumor cell subpopulation from the single-cell RNA sequencing (scRNA-seq) data. MSI-H CRC was clustered into two subtypes based on the expression profiles of metabolism-related genes, and ENO2 was identified as a hub gene. Functional experiments with ENO2 knockdown and overexpression demonstrated its role in promoting CRC cell migration, invasion, glycolysis, and epithelial-mesenchymal transition (EMT) in vitro. High expression of ENO2 in MSI-H CRC patients was associated with worse clinical outcomes, including increased tumor invasion depth (p = 0.007) and greater likelihood of perineural invasion (p = 0.015). Furthermore, the nomogram and calibration curves based on ENO2 showed potential prognosis predictive performance. Conclusion Our findings suggest that ENO2 serves as a novel prognostic biomarker and is associated with the progression of MSI-H CRC.

Objectives Constitutive activation of Janus kinase 2 (JAK2)/signal transducer and activator of transcription (STAT) signaling pathway is central to the pathogenesis of myeloproliferative neoplasms (MPNs). Long noncoding RNAs (lncRNAs) regulate diverse biological processes. However, the role of lncRNAs in MPN pathogenesis is not well studied.Methods The expression of lnc-AC004893 in MPN patients was measured by quantitative real-time PCR (qRT-PCR). Gene-specific short hairpin RNAs (shRNAs) were designed to inhibit the expression of lnc-AC004893, and western blot was performed to explore the role of lnc-AC004893 via regulating the JAK2/STAT5 signaling pathway. Furthermore, co-IP was performed to determine the binding ability of lnc-AC004893 and STAT5 protein. Finally, the BaF3-JAK2V617F-transplanted mouse model was used to assess the biological role of lnc-ac004893 in vivo.Results We report that lnc-AC004893, a poorly conserved pseudogene-209, is substantially upregulated in MPN cells compared with normal controls (NCs). Knockdown of lnc-AC004893 by specific shRNAs suppressed cell proliferation and decreased colony formation. Furthermore, the knockdown of lnc-AC004893 reduced the expression of p-STAT5 but not total STAT5 in HEL and murine IL-3-dependent Ba/F3 cells, which present constitutive and inducible activation of JAK2/STAT5 signaling. In addition, inhibition of murine lnc-ac004893 attenuated BaF3-JAK2V617F-transplanted phenotypes and extended the overall survival. Mechanistically, knockdown of lnc-AC004893 enhanced the binding ability of STAT5 and protein tyrosine phosphatase SHP1. Furthermore, knockdown of lnc-AC004893 decreased STAT5-lnc-AC004893 interaction but not SHP1-lnc-AC004893 interaction.Conclusion Lnc-AC004893 regulates STAT5 phosphorylation by affecting the interaction of STAT5 and SHP1. Lnc-AC004893 might be a potential therapeutic target for MPN patients.

Purpose: To determine the effect of formononetin (FMNT) in the proliferation, drug resistance, and DNA damage of myeloproliferative neoplasm (MPN), and to evaluate the potential of FMNT as a therapeutic target. Methods: Cell viability curves and colony formation assays were used to characterize the proliferation of HEL or HEL/R cells. Flow cytometry was conducted to assess the apoptosis of HEL or HEL/R cells, while western blotting was performed to evaluate the expressions of DNA H2AX, DNA-PK, and Rad51, which are indicative of DNA damage, and the phosphorylation levels of JAK2 and STAT3 were determined. Results: Formononetin (FMNT) suppressed cell proliferation of HEL cells in a dose-dependent manner. It significantly reduced colony formation and promoted apoptosis of HEL cells (p < 0.05). For HEL/R cells, FMNT treatment significantly reduced cell viability, and resistance, and promoted apoptosis. Moreover, FMNT elevated H2AX levels and significantly reduced the expressions of DNA-PK and Rad51 in TG101209 (TG)-induced JAK-TKI-resistant cells (p < 0.05). Furthermore, FMNT decreased JAK2 and STAT3 phosphorylation in JAK- TKI-resistant cells. Conclusion: Treatment with FMNT represses proliferation, promotes apoptosis, weakens JAK-TKI resistance, and strengthens DNA damage in MPN cells by suppressing JAK/STAT3 pathway. This suggests that FMNT is a potential therapeutic candidate for the treatment of MPN.

Background The prognostic value of cytokeratin 19 fragment (CYFRA 21 − 1) and Ki67 in advanced non-small cell lung cancer (NSCLC) patients with wild-type epidermal growth factor receptor (EGFR) remains to be explored. Methods In this study, 983 primary NSCLC patients from January 2016 to December 2019 were retrospectively reviewed. Finally, 117 advanced NSCLC patients with wild-type EGFR and 37 patients with EGFR mutation were included and prognostic value of CYFRA 21 − 1 and Ki67 were also identified. Results The patients age, smoking history and the Eastern Corporative Oncology Group (ECOG) performance scores were significantly different between CYFRA21-1 positive and negative groups ( p < 0.05), while no significant differences were found in Ki67 high and low groups. The results of over survival (OS) demonstrated that patients with CYFRA21-1 positive had markedly shorter survival time than CYFRA21-1 negative ( p < 0.001, For whole cohorts; p = 0.002, For wild-type EGFR). Besides, patients with wild-type EGFR also had shorter survival times than Ki67 high group. Moreover, In CYFRA 21 − 1 positive group, patients with Ki67 high had obviously shorter survival time compared to patients with Ki67 low (median: 24vs23.5 months; p = 0.048). However, Ki67 could not be used as an adverse risk factor for patients with EGFR mutation. Multivariate cox analysis showed that age (HR, 1.031; 95%CI, 1.003 ~ 1.006; p = 0.028), Histopathology (HR, 1.760; 95%CI,1.152 ~ 2.690; p = 0.009), CYFRA 21 − 1 (HR, 2.304; 95%CI,1.224 ~ 4.335; p = 0.01) and Ki67 (HR, 2.130; 95%CI,1.242 ~ 3.652; p = 0.006) served as independent prognostic risk factor for advanced NSCLC patients. Conclusions Our finding indicated that CYFRA 21 − 1 was an independent prognostic factor for advanced NSCLC patients and Ki67 status could be a risk stratification marker for CYFRA 21 − 1 positive NSCLC patients with wild-type EGFR.

One of the most potent anti-human cytomegalovirus (HCMV) immune mechanisms possessed by host cells is type I interferon (IFN1), which induces the expression of IFN-stimulated genes (ISGs). During this process, mitochondria play an important role in the IFN1 response, and mitofusin 1 (MFN1) is a key regulator of mitochondrial fusion located on the outer mitochondrial membrane. However, the underlying mechanism of MFN1's promotion of IFN1 during HCMV infection still remains unknown. In this study, HCMV infection promoted IFN1 production and enhanced ISG expression. Meanwhile, it promoted the increase of mitochondrial fusion in THP-1 cells and peripheral blood mononuclear cells (PBMCs), especially the expression of MFN1. Phosphorylation of tank binding kinase 1 (p-TBK1), interferon regulatory factor 3 (p-IRF3), and ISGs was significantly decreased in MFN1 or mitochondrial antiviral signaling protein (MAVS)-knockdown THP-1 cells, and MFN1 was constitutively associated with MAVS, positively regulated mitochondrial fusion, and IFN1 production. Knockdown of MFN1 inhibited the MAVS redistribution without affecting the MAVS expression, whereas the HCMV-induced IFN1 production decreased. Conversely, leflunomide could induce the expression of MFN1, thereby producing IFN1 and stimulating the expression of ISG in leflunomide-treated THP-1 cells. These observations reveal that HCMV infection leads to MFN1-mediated redistribution of MAVS and then induces an antiviral response of IFN1 and that the MFN-agonist leflunomide promotes IFN1 responses and may serve as a potential anti-HCMV therapy. IMPORTANCE Human cytomegalovirus (HCMV) infection is ubiquitous and is often asymptomatic in healthy individuals, but it can cause great damage to newborns, AIDS patients, and other immune deficiency patients. In this study, we found that HCMV infection caused mitochondrial fusion, and expression of mitofusin 1 (MFN1), which is a protein associated with mitochondrial antiviral signaling protein (MAVS), positively regulates mitochondrial fusion and HCMV-induced IFN1 response. Knockdown of MFN1 or MAVS can inhibit the HCMV-induced IFN1 production. What is more, confocal laser-scanning microscope showed that knockdown of MFN1 inhibits the HCMV-induced redistribution of MAVS. Conversely, MFN1 agonist leflunomide could induce IFN1 production. In conclusion, we provide new insight into the relationship between MFN1 and IFN1 during HCMV infection and show that MFN1 may serve as a potential strategy against HCMV infection.

Background and AimSevere acute pancreatitis (SAP) in patients progresses rapidly and can cause multiple organ failures associated with high mortality. We aimed to train a machine learning (ML) model and establish a nomogram that could identify SAP, early in the course of acute pancreatitis (AP). MethodsIn this retrospective study, 631 patients with AP were enrolled in the training cohort. For predicting SAP early, five supervised ML models were employed, such as random forest (RF), K-nearest neighbors (KNN), and naive Bayes (NB), which were evaluated by accuracy (ACC) and the areas under the receiver operating characteristic curve (AUC). The nomogram was established, and the predictive ability was assessed by the calibration curve and AUC. They were externally validated by an independent cohort of 109 patients with AP. ResultsIn the training cohort, the AUC of RF, KNN, and NB models were 0.969, 0.954, and 0.951, respectively, while the AUC of the Bedside Index for Severity in Acute Pancreatitis (BISAP), Ranson and Glasgow scores were only 0.796, 0.847, and 0.837, respectively. In the validation cohort, the RF model also showed the highest AUC, which was 0.961. The AUC for the nomogram was 0.888 and 0.955 in the training and validation cohort, respectively. ConclusionsOur findings suggested that the RF model exhibited the best predictive performance, and the nomogram provided a visual scoring model for clinical practice. Our models may serve as practical tools for facilitating personalized treatment options and improving clinical outcomes through pre-treatment stratification of patients with AP.