Establishment and application of a TaqMan-based multiplex real-time PCR for simultaneous detection of three porcine diarrhea viruses.

Introduction: Porcine viral diarrhea is a common clinical disease, which results in high mortality and economic losses in the pig industry. Porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PoRV), and porcine deltacoronavirus (PDCoV) are important diarrhea viruses in pig herds. The similarities of their clinical symptoms and pathological changes make it difficult to distinguish these three viruses clinically. Therefore, there is a need for a highly sensitive and specific method to simultaneously detect and differentiate these viruses. Methods: A multiplex real-time PCR assay using TaqMan probes was developed to simultaneously detect PEDV, PoRV, and PDCoV. To assess the efficacy of the established assay, 30 clinical samples with diarrhea symptoms were used to compare the results obtained from the multiplex real-time PCR assay with those obtained from commercial singleplex real-time PCR kit. Importantly, a total of 4,800 diarrhea samples were tested and analyzed to validate the utility of the assay. Results: This multiplex real-time PCR assay showed high sensitivity, specificity, and excellent repeatability with a detection limit of 1 × 102 copies/µL. Comparing the results of the commercial singleplex real-time PCR kit and the multiplex real-time PCR method for detecting PEDV, PoRV, and PDCoV, there was complete agreement between the two approaches. Clinical data revealed single infection rates of 6.56% for PEDV, 21.69% for PoRV, and 6.65% for PDCoV. The co-infection rates were 11.83% for PEDV + PoRV, 0.29% for PEDV + PDCoV, 5.71% for PoRV + PDCoV, and 1.29% for PEDV + PDCoV + PoRV, respectively. Discussion: The multiplex real-time PCR method established in this study is a valuable diagnostic tool for simultaneously differentiating PEDV, PoRV, and PDCoV. This method is expected to significantly contribute to prevent and control the spread of infectious diseases, as well as aid in conducting epidemiological investigations.

UFM1 inhibits hypoxia-induced angiogenesis via promoting proteasome degradation of HIF-1α.

Angiogenesis is crucial for blood flow recovery and ischemic tissue repair of peripheral artery disease (PAD). Exploration of new mechanisms underlying angiogenesis will shed light on the treatment of PAD. Ubiquitin-fold modifier 1 (UFM1), a newly identified ubiquitin-like molecule, has been discovered to be involved in various pathophysiological processes. However, the role of UFM1 in the pathogenesis of PAD, especially in endothelial angiogenesis remains obscure, and we aimed to clarify this issue in this study. We initially found UFM1 was significantly upregulated in gastrocnemius muscles of PAD patients and hind limb ischemia mice. And UFM1 was mainly colocalized with endothelial cells in ischemic muscle tissues. Further, elevated expression of UFM1 was observed in hypoxic endothelial cells. Subsequent genetic inhibition of UFM1 dramatically enhanced migration, invasion, adhesion, and tube formation of endothelial cells under hypoxia. Mechanistically, UFM1 reduced the stability of hypoxia-inducible factor-1α (HIF-1α) and promoted the von Hippel-Lindau-mediated K48-linked ubiquitin-proteasome degradation of HIF-1α, which in turn decreased angiogenic factor VEGFA expression and suppressed VEGFA related signaling pathway. Consistently, overexpression of UFM1 inhibited the angiogenesis of endothelial cells under hypoxic conditions, whereas overexpression of HIF-1α reversed this effect. Collectively, our data reveal that UFM1 inhibits the angiogenesis of endothelial cells under hypoxia through promoting ubiquitin-proteasome degradation of HIF-1α, suggesting UFM1 might serve as a potential therapeutic target for PAD.

Advances in the study of S100A9 in cardiovascular diseases.

Cardiovascular disease (CVD) is a group of diseases that primarily affect the heart or blood vessels, with high disability and mortality rates, posing a serious threat to human health. The causative factors, pathogenesis, and characteristics of common CVD differ, but they all involve common pathological processes such as inflammation, oxidative stress, and fibrosis. S100A9 belongs to the S100 family of calcium-binding proteins, which are mainly secreted by myeloid cells and bind to the Toll-like receptor 4 and receptor for advanced glycation end products and is involved in regulating pathological processes such as inflammatory response, fibrosis, vascular calcification, and endothelial barrier function in CVD. The latest research has found that S100A9 is a key biomarker for diagnosing and predicting various CVD. Therefore, this article reviews the latest research progress on the diagnostic and predictive, and therapeutic value of S100A9 in inflammatory-related CVD such as atherosclerosis, myocardial infarction, and arterial aneurysm and summarizes its molecular mechanisms in the progression of CVD, aiming to explore new predictive methods and to identify potential intervention targets for CVD in clinical practice.

Recent progress in plasma modification of 2D metal chalcogenides for electronic devices and optoelectronic devices.

Two-dimensional metal chalcogenides (2D MCs) present a great opportunity for overcoming the size limitation of traditional silicon-based complementary metal-oxide-semiconductor (CMOS) devices. Controllable modulation compatible with CMOS processes is essential for the improvement of performance and the large-scale applications of 2D MCs. In this review, we summarize the recent progress in plasma modification of 2D MCs, including substitutional doping, defect engineering, surface charge transfer, interlayer coupling modulation, thickness control, and nano-array pattern etching in the fields of electronic devices and optoelectronic devices. Finally, challenges and outlooks for plasma modulation of 2D MCs are presented to offer valuable references for future studies.

Clinical evaluation of a new COVID-19 antigen rapid test kit for detection of SARS-CoV-2.

The antigen rapid diagnostic test (Ag-RDT) is an assay kit for detecting the SARS-COV-2 nucleocapsid proteins, based on the colloidal gold method.Accurate diagnosis has an important role in limiting the transmission of SARS-COV-2, and also helps patients to receive earlier treatment .The object of this study was to perform the clinical evaluation of a novel Ag-RDTs with samples collected from two different swabs.DEEPBLUE®COVID-19 antigen detection kit used for the examination of the subjects in the experiment.For antigen testing on samples collected with nasal swabs, sensitivity was 91.7 % (95 % CI 83.6-96.6 %) and specificity was 100 %(95 %CI 98.1-100 %).For nasopharyngeal swabs, the sensitivity was 96.8 % (95 % CI 93.6-98.7 %) and the specificity was 100 % (95 % CI 98.2-100 %).Fisher Precision test showed a significant correlation between nasopharyngeal swab Ag-RDTs and nasal swab Ag-RDTs and RT-qPCR test (p-value <0.001).The results showed that the patients use the kit for testing were comparable to the RT-qPCR.

Multifaceted SOX2-chromatin interaction underpins pluripotency progression in early embryos.

Pioneer transcription factors (TFs), such as OCT4 and SOX2, play crucial roles in pluripotency regulation. However, the master TF-governed pluripotency regulatory circuitry was largely inferred from cultured cells. In this work, we investigated SOX2 binding from embryonic day 3.5 (E3.5) to E7.5 in the mouse. In E3.5 inner cell mass (ICM), SOX2 regulates the ICM-trophectoderm program but is dispensable for opening global enhancers. Instead, SOX2 occupies preaccessible enhancers in part opened by early-stage expressing TFs TFAP2C and NR5A2. SOX2 then widely redistributes when cells adopt naive and formative pluripotency by opening enhancers or poising them for rapid future activation. Hence, multifaceted pioneer TF-enhancer interaction underpins pluripotency progression in embryos, including a distinctive state in E3.5 ICM that bridges totipotency and pluripotency.

SPIRAL: integrating and aligning spatially resolved transcriptomics data across different experiments, conditions, and technologies.

Properly integrating spatially resolved transcriptomics (SRT) generated from different batches into a unified gene-spatial coordinate system could enable the construction of a comprehensive spatial transcriptome atlas. Here, we propose SPIRAL, consisting of two consecutive modules: SPIRAL-integration, with graph domain adaptation-based data integration, and SPIRAL-alignment, with cluster-aware optimal transport-based coordination alignment. We verify SPIRAL with both synthetic and real SRT datasets. By encoding spatial correlations to gene expressions, SPIRAL-integration surpasses state-of-the-art methods in both batch effect removal and joint spatial domain identification. By aligning spots cluster-wise, SPIRAL-alignment achieves more accurate coordinate alignments than existing methods.

Changes of Gut Microbiota in Maintenance Hemodialysis Patients and Their Impact on Patient's Microinflammation Status.

This study was to investigate the changes in gut microbiota in maintenance hemodialysis patients and analyze their impact on patient's microinflammation status. For this purpose, thirty-nine chronic kidney disease (CKD) maintenance hemodialysis patients admitted to our hospital from March 2019 to March 2022 were selected as the experimental group, and 40 healthy individuals with examination results during the same period were selected as the control group. The levels of gut microbiota (Lactobacillus, Bifidobacterium, Escherichia coli, and Enterococcus faecalis) and microinflammation indicators [interleukin-6 (IL-6), tumor necrosis factor α (TNF-α), and high-sensitivity C-reactive protein (hs-CRP)] were measured in both groups. The relationship between changes in gut microbiota and microinflammation in maintenance hemodialysis CKD patients was analyzed. Results showed that the levels of Lactobacillus and Bifidobacterium in the experimental group were significantly lower than those in the control group (all, P<0.05), while the levels of Escherichia coli and Enterococcus faecalis in the experimental group were significantly higher than those in the control group (all, P<0.05). The IL-6, TNF-α, and hs-CRP levels in the experimental group were significantly higher than those in the control group (all, P<0.05). Using microinflammation indicators as dependent variables and microbiota indicators as independent variables for stepwise regression analysis, the results showed that the levels of Lactobacillus were negatively correlated with IL-6 and TNF-α levels in patients (r=-0.358, -0.942, P<0.05); the levels of Bifidobacterium were negatively correlated with IL-6, TNF-α, and hs-CRP levels in patients (r=-0.394, -0.211, -0.547, P<0.05); the levels of Escherichia coli were positively correlated with IL-6 and TNF-α levels in patients (r=0.221, 0.268, P<0.05); the levels of Enterococcus faecalis were positively correlated with IL-6 and hs-CRP levels in patients (r=0.253, 0.378, P<0.05). In conclusion, patients with maintenance hemodialysis for CKD commonly exhibit gut microbiota dysbiosis and varying degrees of low-grade inflammation. Compared to healthy individuals, maintenance hemodialysis patients with CKD have lower levels of Bifidobacterium and Lactobacillus and higher levels of Escherichia coli and Enterococcus in their gut. Bifidobacterium, Lactobacillus, Escherichia coli, and Enterococcus all have a certain impact on the low-grade inflammation status of patients with maintenance hemodialysis for CKD.

Ufmylation on UFBP1 alleviates non-alcoholic fatty liver disease by modulating hepatic endoplasmic reticulum stress.

Non-alcoholic fatty liver disease (NAFLD) is the most common liver disease characterized by lipid accumulation and endoplasmic reticulum (ER) stress, while effective therapies targeting the specific characteristics of NAFLD are limited. Ufmylation is a newly found post-translational modification process that involves the attachment of the Ubiquitin-fold modifier 1 (UFM1) protein to its substrates via ufmylation modification system. Ufmylation regulates ER stress via modifying UFM1 binding protein 1 (UFBP1), suggesting a potential role for ufmylation in NAFLD pathogenesis. However, the precise role of ufmylation in NAFLD remains unclear. Herein, we aim to elucidate the impact of ufmylation on UFBP1 in NAFLD and explore the underlying mechanisms involved. We observed increased expression of UFM1-conjugated proteins and ufmylation modification system components in livers with steatosis derived from NAFLD patients and NAFLD models. Upregulation of ufmylation on hepatic proteins appeared to be an adaptive response to hepatic ER stress in NAFLD. In vitro, knocking down UFBP1 resulted in increased lipid accumulation and lipogenesis in hepatocytes treated with free fatty acids (FFA), which could be rescued by wild-type UFBP1 (WT UFBP1) but not by a mutant form of UFBP1 lacking the main ufmylation site lys267 (UFBP1 K267R). In vivo, ufmylation on UFBP1 ameliorated obesity, hepatic steatosis, hepatic lipogenesis, dyslipidemia, insulin resistance and liver damage in mice with NAFLD induced by a high fat diet (HFD). We also demonstrated that the downregulation of UFBP1 induced ER stress, whereas the reintroduction or overexpression of UFBP1 alleviated ER stress in a manner dependent on ufmylation in NAFLD. This mechanism could be responsible for the amelioration of aberrant hepatic lipogenesis and insulin resistance in NAFLD. Our data reveal a protective role of ufmylation on UFBP1 against NAFLD and offer a specific target for NAFLD treatment.

OBOX regulates mouse zygotic genome activation and early development.

Zygotic genome activation (ZGA) activates the quiescent genome to enable the maternal-to-zygotic transition1,2. However, the identity of transcription factors that underlie mammalian ZGA in vivo remains elusive. Here we show that OBOX, a PRD-like homeobox domain transcription factor family (OBOX1-OBOX8)3-5, are key regulators of mouse ZGA. Mice deficient for maternally transcribed Obox1/2/5/7 and zygotically expressed Obox3/4 had a two-cell to four-cell arrest, accompanied by impaired ZGA. The Obox knockout defects could be rescued by restoring either maternal and zygotic OBOX, which suggests that maternal and zygotic OBOX redundantly support embryonic development. Chromatin-binding analysis showed that Obox knockout preferentially affected OBOX-binding targets. Mechanistically, OBOX facilitated the 'preconfiguration' of RNA polymerase II, as the polymerase relocated from the initial one-cell binding targets to ZGA gene promoters and distal enhancers. Impaired polymerase II preconfiguration in Obox mutants was accompanied by defective ZGA and chromatin accessibility transition, as well as aberrant activation of one-cell polymerase II targets. Finally, ectopic expression of OBOX activated ZGA genes and MERVL repeats in mouse embryonic stem cells. These data thus demonstrate that OBOX regulates mouse ZGA and early embryogenesis.

TRAIP serves as a potential prognostic biomarker and correlates with immune infiltrates in lung adenocarcinoma.

BACKGROUND: Lung adenocarcinoma (LUAD) is one of the major types of lung cancer with high morbidity and mortality. The TRAF-interacting protein (TRAIP) is a ring-type E3 ubiquitin ligase which has been recently identified to play pivotal roles in various cancers. However, the expression and function of TRAIP in LUAD remain elusive. METHODS: In this study, we used bioinformatic tools as well as molecular experiments to explore the exact role of TRAIP and the underlying mechanism. RESULTS: Data mining across the UALCAN, GEPIA and GTEx, GEO and HPA databases revealed that TRAIP was significantly overexpressed in LUAD tissues than that in adjacent normal tissues. Kaplan-Meier curve showed that high TRAIP expression was associated with poor overall survival (OS) and relapse-free survival (RFS). Univariate and multivariate cox regression analysis revealed that TRAIP was an independent risk factor in LUAD. And the TRAIP-based nomogram further supported the prognostic role of TRAIP in LUAD. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis demonstrated that TRAIP-associated genes were mainly involved in DNA replication, cell cycle and other processes. The immune infiltration analysis indicated that TRAIP expression was tightly correlated with the infiltration of diverse immune cell types, including B cell, CD8 + T cell, neutrophil and dendritic cell. Moreover, TRAIP expression was observed to be significantly associated with tumor infiltrating lymphocytes (TILs) and immune checkpoint molecules. In vitro experiments further confirmed knockdown of TRAIP inhibited cell migration and invasion, as well as decreasing chemokine production and inhibiting M2-like macrophage recruitment. Lastly, CMap analysis identified 10 small molecule compounds that may target TRAIP, providing potential therapies for LUAD. CONCLUSIONS: Collectively, our study found that TRAIP is an oncogenic gene in LUAD, which may be a potential prognostic biomarker and promising therapeutic target for LUAD.

Maternal TDP-43 interacts with RNA Pol II and regulates zygotic genome activation.

Zygotic genome activation (ZGA) is essential for early embryonic development. However, the regulation of ZGA remains elusive in mammals. Here we report that a maternal factor TDP-43, a nuclear transactive response DNA-binding protein, regulates ZGA through RNA Pol II and is essential for mouse early embryogenesis. Maternal TDP-43 translocates from the cytoplasm into the nucleus at the early two-cell stage when minor to major ZGA transition occurs. Genetic deletion of maternal TDP-43 results in mouse early embryos arrested at the two-cell stage. TDP-43 co-occupies with RNA Pol II as large foci in the nucleus and also at the promoters of ZGA genes at the late two-cell stage. Biochemical evidence indicates that TDP-43 binds Polr2a and Cyclin T1. Depletion of maternal TDP-43 caused the loss of Pol II foci and reduced Pol II binding on chromatin at major ZGA genes, accompanied by defective ZGA. Collectively, our results suggest that maternal TDP-43 is critical for mouse early embryonic development, in part through facilitating the correct RNA Pol II configuration and zygotic genome activation.

Interrelationships between sarcopenia, bone turnover markers and low bone mineral density in patients on hemodialysis.

BACKGROUND: Hemodialysis (HD) patients are at risk for sarcopenia (SP) and bone loss, which may impact falls and bone fragility and lead to poor prognosis. Patients with HD and those with osteoporosis (OP) are still underdiagnosed and untreated. The aims of the present study were to evaluate the factors that affect bone mineral density (BMD) loss in HD patients, and explore traditional and novel approaches to manage chronic kidney disease-mineral-bone disorder (CKD-MBD). METHODS: Patients who underwent regular HD at the First Affiliated Hospital of Soochow University were retrospectively evaluated. According to the WHO osteoporosis criteria, patients were categorized into three groups: normal BMD, osteopenia, and osteoporosis. Demographic and clinical data, skeletal muscle mass, and bone turnover markers(BTM) were compared between the three groups. The correlation between bone density and muscle mass was calculated, and related risk factors were analyzed. RESULTS: This study enrolled 130 HD patients, 36 patients were diagnosed with sarcopenia (27.7%), 44 patients were diagnosed with osteopenia (33.8%), 19 patients were diagnosed with osteoporosis (14.6%), and 23 patients were diagnosed with osteosarcopenia (17.7%). The SMI was positively correlated with the BMD of the lumbar spine (r = 0.23, p < 0.01) and femoral neck (r = 0.22, p < 0.05). In ordinal logistic regression analysis, the odds ratio (OR) for low BMD was high for patients with sarcopenia (OR = 5.894, 95% CI 1.592-21.830, p < 0.01), older age (OR = 1.095, 95% CI 1.041-1.153, p < 0.001), higher TRACP-5b levels (OR = 1.597, 95% CI 1.230-2.072, p < 0.01), and lower 25-OH vitamin D levels (OR = 0.631, 95% CI 0.544-0.733, p < 0.001). CONCLUSION: The preservation of skeletal muscle mass could be important to prevent a BMD decrease in HD patients. Adequate intake of vitamin D and control of TRACP-5b levels will help reduce the occurrence and progression of osteopenia/sarcopenia in HD patients.

Inhibition of Th17 cells by donepezil ameliorates experimental lung fibrosis and pulmonary hypertension.

Rationale: Pulmonary hypertension (PH) secondary to lung fibrosis belongs to WHO Group III, one of the most common subgroups of PH; however, it lacks effective treatment options. Cholinesterase inhibitor donepezil (DON) has been shown to effectively improve Group I PH. However, its effects on Group III PH are unknown. Methods: A lung fibrosis-induced PH mouse model was constructed using a single intratracheal instillation of bleomycin (BLM), after which DON was administered daily. Pulmonary artery and right ventricle (RV) remodeling were evaluated at the end of the study. Lung tissue in each group was analyzed using RNA sequencing, and the results were further verified with datasets from patients with PH. The mechanisms underlying DON-induced effects on PH were verified both in vivo and in vitro. Results: DON effectively improved pulmonary artery and RV remodeling in the BLM-induced mouse model. Transcriptomic profiles of lung tissue indicated that the expression of inflammatory and fibrotic genes was significantly changed in this process. In the animal model and patients with PH, T helper 17 lymphocytes (Th17) were the most common inflammatory cells infiltrating the lung tissue. DON significantly inhibited lung fibroblast activation; thus, preventing lung fibrosis and reducing the inflammatory response and Th17 cell infiltration in the BLM-induced lung tissue. In addition, Th17 cells could activate lung fibroblasts by secreting IL17A, and DON-mediated inhibition of Th17 cell differentiation was found to depend on the α7nAchR-JAK2-STAT3 pathway. Conclusion: DON can alleviate lung fibrosis and PH in an experimental mouse model. It inhibited pro-inflammatory Th17 cell differentiation, which is dependent on a cholinergic receptor pathway, thereby regulating fibroblast activation.

A cross-sectional study on gut microbiota in patients with chronic kidney disease undergoing kidney transplant or hemodialysis.

OBJECTIVE: To investigate whether the gut microbiota differs in patients with chronic kidney disease receiving kidney transplant or hemodialysis, and to explore the relationship between the gut microbiota and clinical indicators. METHODS: A total of 72 patients with kidney transplantation (RT) and 78 patients with hemodialysis (HD) admitted to the First Affiliated Hospital of Soochow University from January 2019 to October 2022 were selected as the research subjects. The V3-V4 region sequences of 16S rRNA were used for high-throughput sequencing to analyze the differences in gut microbiome between the two groups and its relationship with clinical indicators. RESULTS: Gut microbial α diversity (Chao1, Ace, Shannon, Simpson) was significantly decreased in RT patients compared with that in HD patients (P<0.05). The relative abundance of Bacteroides, Megamonas, and Prevotella was significantly higher in HD patients than that in RT patients (P<0.05). There was a negative association between the Bacteroides and ß2-microglobulin (ß2-MG) (P<0.05). Lactobacillus was negatively correlated with uric acid (UA) (P<0.05). CONCLUSION: This study elucidates the composition and changes of the gut microbiota in RT and HD patients and its association with clinical indicators, providing a scientific basis for the regulatory mechanism of gut microbiota in the treatment of chronic kidney disease.

Macular Vascular Complexity Analysis of Diabetes Mellitus by Swept-Source Optical Coherence Tomographic Angiography.

PURPOSE: This study was designed to evaluate the associations between retinal vascular complexity features, including fractal dimension (FD) and blood vessel tortuosity (BVT), and the severity of diabetic retinopathy (DR) by using optical coherence tomographic angiography (OCTA). METHODS: In this prospective cross-sectional study, 1,282 ocular-treatment-naive patients with type 2 diabetes mellitus (DM) (1,059 without DR and 223 with DR) registered in the community of Guangzhou, China, were enrolled. OCTA was used to measure FD and BVT in the superficial capillary plexus (SCP) and the deep capillary plexus (DCP). Univariate and multivariate linear regression analyses were performed to analyze the correlation of FD and BVT in different layers with DR severity. RESULTS: In this study, 1,282 patients with DM (1,282 eyes), with a mean age of 64.2 ± 7.8 years, were included. FD in the DCP decreased and BVT in the DCP increased in patients with DR compared with those in patients without DR, even after adjusting for confounding factors (p < 0.05). Trend analysis showed a significant decrease in the FD values as the DR progressed, whereas the BVT progressively increased with worsening DR severity (p < 0.01). The FD in DCP had a statistically significant positive correlation with FD in SCP and a negative correlation with BVT in SCP and BVT in DCP in all of the participants, including the non-DR group, moderate DR group, and severe DR group (p < 0.01). CONCLUSIONS: FD and BVT determined using OCTA might be useful parameters for objectively distinguishing DR from non-DR and indicating DR progression.

UFMylation System: An Emerging Player in Tumorigenesis.

Ubiquitin-fold modifier 1 (UFM1), a newly identified ubiquitin-like molecule (UBLs), is evolutionarily expressed in multiple species except yeast. Similarly to ubiquitin, UFM1 is covalently attached to its substrates through a well-orchestrated three-step enzymatic reaction involving E1, the UFM1-activating enzyme (ubiquitin-like modifier-activating enzyme 5, UBA5); E2, the UFM1-conjugating enzyme 1 (UFC1); and E3, the UFM1-specific ligase 1 (UFL1). To date, numerous studies have shown that UFM1 modification is implicated in various cellular processes, including endoplasmic reticulum (ER) stress, DNA damage response and erythroid development. An abnormal UFM1 cascade is closely related to a variety of diseases, especially tumors. Herein, we summarize the process and functions of UFM1 modification, illustrating the relationship and mechanisms between aberrant UFMylation and diversified tumors, aiming to provide novel diagnostic biomarkers or therapeutic targets for cancer treatments.

P4HB UFMylation regulates mitochondrial function and oxidative stress.

UFMylation is a ubiquitin-like modification which attaches the ubiquitin-fold modifier 1 to target proteins. To date, only a few UFMylation targets have been identified. In the current study, we demonstrated that P4HB is a new target protein for UFMylation and it can be UFMylated at three lysine residues in the form of mono-UFMylation. P4HB has oxidoreductase, chaperone and isomerase effects. It presents in the endoplasmic reticulum, mitochondria and cytosol. Next, we generated a stable HepG2 cell line, the hepatocellular cells, with defective P4HB UFMylation. Our data show that P4HB UFMylation defect promotes P4HB protein degradation via the ubiquitin-proteasome pathway. Defective P4HB UFMylation causes mitochondrial function damage, oxidative stress, and endoplasmic reticulum stress in HepG2 cells. These effects are more obvious when treating HepG2 cells with palmitic acid, which is frequently used as one of the cell models of non-alcoholic fatty liver disease (NAFLD). Our results identify UFMylation as a key post-translational modification for the maintenance of P4HB stability and biological functions in HepG2 cells, and point to P4HB UFMylation as a potential direction in the study of NAFLD.