Recover then aggregate: unified cross-modal deep clustering with global structural information for single-cell data.

Single-cell cross-modal joint clustering has been extensively utilized to investigate the tumor microenvironment. Although numerous approaches have been suggested, accurate clustering remains the main challenge. First, the gene expression matrix frequently contains numerous missing values due to measurement limitations. The majority of existing clustering methods treat it as a typical multi-modal dataset without further processing. Few methods conduct recovery before clustering and do not sufficiently engage with the underlying research, leading to suboptimal outcomes. Additionally, the existing cross-modal information fusion strategy does not ensure consistency of representations across different modes, potentially leading to the integration of conflicting information, which could degrade performance. To address these challenges, we propose the 'Recover then Aggregate' strategy and introduce the Unified Cross-Modal Deep Clustering model. Specifically, we have developed a data augmentation technique based on neighborhood similarity, iteratively imposing rank constraints on the Laplacian matrix, thus updating the similarity matrix and recovering dropout events. Concurrently, we integrate cross-modal features and employ contrastive learning to align modality-specific representations with consistent ones, enhancing the effective integration of diverse modal information. Comprehensive experiments on five real-world multi-modal datasets have demonstrated this method's superior effectiveness in single-cell clustering tasks.

Ultrasound and its coupled oak blocks treatment based on absorbed energy density for comprehensive insight and scale-up consideration of phenolic and astringency profiles in red wines.

Ultrasound and ultrasound-coupled oak blocks techniques on the phenolic and astringency profiles of Petit Verdot wines were discussed in this study. The relationship between techniques at varying absorbed energy density (AED) gradients and astringency was revealed, elucidating potential molecular mechanisms regarding compound interactions. Ultrasound was found to promote phenolics degradation and condensed tannins maturation, while oak blocks improved ellagitannins release into wines, facilitating polycondensation reactions to form larger complexes. Phenolics binding to salivary proteins decreased at low AED values (0-33.07 J/mL), enhanced at high AED values (66.14-165.34 J/mL), and reduced salivary proteins' precipitation ability. Treated samples significantly altered astringency's global terminology and sensory attributes across oral regions, with oak blocks enriching the astringency hierarchy. Multivariate analysis identified an optimal ultrasound AED of 0 to 33.07 J/mL, and an effective PLS-DA model was developed for industrial scale-up considerations, aiming to cost-effectively enhance the organoleptic quality of wines.

Achieving 1.7 GPa Considerable Ductility High-Strength Low-Alloy Steel Using Hot-Rolling and Tempering Processes.

To achieve a balanced combination of high strength and high plasticity in high-strength low-alloy (HSLA) steel through a hot-rolling process, post-heat treatment is essential. The effects of post-roll air cooling and oil quenching and subsequent tempering treatment on the microstructure and mechanical properties of HSLA steels were investigated, and the relevant strengthening and toughening mechanisms were analyzed. The microstructure after hot rolling consists of fine martensite and/or bainite with a high density of internal dislocations and lattice defects. Grain boundary strengthening and dislocation strengthening are the main strengthening mechanisms. After tempering, the specimens' microstructures are dominated by tempered martensite, with fine carbides precipitated inside. The oil-quenched and tempered specimens exhibit tempering performance, with a yield strength (YS) of 1410.5 MPa, an ultimate tensile strength (UTS) of 1758.6 MPa, and an elongation of 15.02%, which realizes the optimization of the comprehensive performance of HSLA steel.

In-vitro and in-vivo comparative studies of treatment effects on enamel demineralization during orthodontic therapy: implications for clinical early-intervention strategy.

OBJECTIVES: This study aimed to investigate if CPP-ACP / infiltrating resin was superior in treating enamel demineralization during orthodontic therapy compared with fluoride varnish, in order to provide early-intervention implications for dental professionals. MATERIALS AND METHODS: In the in-vitro study, premolars were grouped into four: remineralization with fluoride varnish / CPP-ACP, sealing with infiltrating resin, and negative control. Experimental demineralization of enamel surfaces was analyzed using techniques of QLF, SEM, EDS and micro-hardness testing. An in-vivo intervention study was conducted on patients randomly assigned into three groups. At the baseline and every-3-month follow-up, QLF parameters were compared temporally and parallelly to yield potential implications for promotion in clinical practice. RESULTS: The in-vitro study performed on 48 experimental tooth surfaces demonstrated that sealing with infiltrating resin reduced enamel surface porosity and increased surface micro-hardness significantly. In the in-vivo intervention study on 163 tooth surfaces, it was suggested that for those who meet the criteria of -10 < ΔF < -6 and - 1000 < ΔQ < -20 at the baseline, all these treatment methods could achieve acceptable outcomes; with the rising of absolute values of ΔF and ΔQ, sealing with infiltrating resin showed more evident advantages. CONCLUSION: For enamel demineralization during orthodontic therapy, all the treatment methods involved in this study showed acceptable effectiveness but had respective characteristics in treatment effects. QLF parameters could be used as indicators for clinical early-intervention strategy with regards to this clinical issue. CLINICAL RELEVANCE: With QLF parameters, clinical early-intervention strategy for enamel demineralization during orthodontic therapy could be optimized.

The study on 4D culture system of squamous cell carcinoma of tongue.

Traditional cell culture methods often fail to accurately replicate the intricate microenvironments crucial for studying specific cell growth patterns. In our study, we developed a 4D cell culture model-a precision instrument comprising an electromagnet, a force transducer, and a cantilever bracket. The experimental setup involves placing a Petri dish above the electromagnet, where gel beads encapsulating magnetic nanoparticles and tongue cancer cells are positioned. In this model, a magnetic force is generated on the magnetic nanoparticles in the culture medium to drive the gel to move and deform when the magnet is energized, thereby exerting an external force on the cells. This setup can mimic the microenvironment of tongue squamous cell carcinoma CAL-27 cells under mechanical stress induced by tongue movements. Electron microscopy and rheological analysis were performed on the hydrogels to confirm the porosity of alginate and its favorable viscoelastic properties. Additionally, Calcein-AM/PI staining was conducted to verify the biosafety of the hydrogel culture system. It mimics the microenvironment where tongue squamous cell carcinoma CAL-27 cells are stimulated by mechanical stress during tongue movement. Electron microscopy and rheological analysis experiments were conducted on hydrogels to assess the porosity of alginate and its viscoelastic properties. Calcein-AM/PI staining was performed to evaluate the biosafety of the hydrogel culture system. We confirmed that the proliferation of CAL-27 tongue squamous cells significantly increased with increased matrix stiffness after 5 d as assessed by MTT. After 15 d of incubation, the tumor spheroid diameter of the 1%-4D group was larger than that of the hydrogel-only culture. The Transwell assay demonstrated that mechanical stress stimulation and increased matrix stiffness could enhance cell aggressiveness. Flow cytometry experiments revealed a decrease in the number of cells in the resting or growth phase (G0/G1 phase), coupled with an increase in the proportion of cells in the preparation-for-division phase (G2/M phase). RT-PCR confirmed decreased expression levels of P53 and integrinß3 RNA in the 1%-4D group after 21 d of 4D culture, alongside significant increases in the expression levels of Kindlin-2 and integrinαv. Immunofluorescence assays confirmed that 4D culture enhances tissue oxygenation and diminishes nuclear aggregation of HIF-1α. This device mimics the microenvironment of tongue cancer cells under mechanical force and increased matrix hardness during tongue movement, faithfully reproducing cell growthin vivo, and offering a solid foundation for further research on the pathogenic matrix of tongue cancer and drug treatments.

Partial substitution of wheat flour with kiwi starch: Rheology, microstructure changes in dough and the quality properties of bread.

Kiwi starch (KS) is a new fruit-derived starch-based food material. In this study, wheat flour was partially replaced with 10-20% KS to make bread, and the influence of this substitution on mixed flour, dough processing performance, bread quality, and shelf life was investigated. KS substitution improved the water-binding ability of mixed flour, making it easier to gelatinize while improving viscoelasticity but reducing the integrity of the dough's gluten network structure. As the substitution rate increases, the hardness, air-cell ratio, and width-to-height ratio of bread significantly increased, while the springiness, resilience, baking loss, and specific volume reduced significantly (p < 0.05). KS enriched the bread's color and flavor by promoting the Maillard reaction during baking. Overall acceptability of 10% KS group was highest in sensory evaluation. KS substitution significantly reduced starch digestibility and expected glycemic index (GI), inhibited mold growth and reproduction during storage and prolonged the shelf life of the bread at 25 °C.

Metabolic reprogramming in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a malignancy with high incidence in China. Due to the lack of effective molecular targets, the prognosis of ESCC patients is poor. It is urgent to explore the pathogenesis of ESCC to identify promising therapeutic targets. Metabolic reprogramming is an emerging hallmark of ESCC, providing a novel perspective for revealing the biological features of ESCC. In the hypoxic and nutrient-limited tumor microenvironment, ESCC cells have to reprogram their metabolic phenotypes to fulfill the demands of bioenergetics, biosynthesis and redox homostasis of ESCC cells. In this review, we summarized the metabolic reprogramming of ESCC cells that involves glucose metabolism, lipid metabolism, and amino acid metabolism and explore how reprogrammed metabolism provokes novel opportunities for biomarkers and potential therapeutic targets of ESCC.

ACSL3 regulates breast cancer progression via lipid metabolism reprogramming and the YES1/YAP axis.

OBJECTIVE: Mitochondrial fatty acid oxidation is a metabolic pathway whose dysregulation is recognized as a critical factor in various cancers, because it sustains cancer cell survival, proliferation, and metastasis. The acyl-CoA synthetase long-chain (ACSL) family is known to activate long-chain fatty acids, yet the specific role of ACSL3 in breast cancer has not been determined. METHODS: We assessed the prognostic value of ACSL3 in breast cancer by using data from tumor samples. Gain-of-function and loss-of-function assays were also conducted to determine the roles and downstream regulatory mechanisms of ACSL3 in vitro and in vivo. RESULTS: ACSL3 expression was notably downregulated in breast cancer tissues compared with normal tissues, and this phenotype correlated with improved survival outcomes. Functional experiments revealed that ACSL3 knockdown in breast cancer cells promoted cell proliferation, migration, and epithelial-mesenchymal transition. Mechanistically, ACSL3 was found to inhibit ß-oxidation and the formation of associated byproducts, thereby suppressing malignant behavior in breast cancer. Importantly, ACSL3 was found to interact with YES proto-oncogene 1, a member of the Src family of tyrosine kinases, and to suppress its activation through phosphorylation at Tyr419. The decrease in activated YES1 consequently inhibited YAP1 nuclear colocalization and transcriptional complex formation, and the expression of its downstream genes in breast cancer cell nuclei. CONCLUSIONS: ACSL3 suppresses breast cancer progression by impeding lipid metabolism reprogramming, and inhibiting malignant behaviors through phospho-YES1 mediated inhibition of YAP1 and its downstream pathways. These findings suggest that ACSL3 may serve as a potential biomarker and target for comprehensive therapeutic strategies for breast cancer.

Geographical characterization of wines from seven regions of China by chemical composition combined with chemometrics: Quality characteristics of Chinese 'Marselan' wines.

This study investigated the basic and functional compositions, volatile compounds, intelligent sensory characteristics and antioxidant capacity of the commercial 'Marselan' wines from seven Chinese regions. The Nei Mongol wines featured high total reducing sugar, fructose, ammonia nitrogen, 17 monomeric phenolic acids contents and elevated antioxidant capacity. Malic acid was the only organic acid that significantly different in all seven regions. Malvidin-3-O-glucoside and trans-peonidin-3-O-(6-O-p-coumaryl)-glucoside showed the highest and lowest contents. A total of 102 volatiles was detected and Hebei wines had the most (91). Hexanoic acid and ß-damascenone were considered to have high potential sensory effects (OAV ≥ 1) as compounds detected in all regions. Floral, sweet, and fruity were the most important aroma series. E-eye analysis revealed the colors of the samples tended to yellowish with aging. PCA and OPLS-DA based on the basic wine composition, monomeric organic acids and anthocyanins allowed achieving a discrimination of the seven regions, respectively.

Graphene/ chitosan tubes inoculated with dental pulp stem cells promotes repair of facial nerve injury.

Introduction: Facial nerve injury significantly impacts both the physical and psychological] wellbeing of patients. Despite advancements, there are still limitations associated with autografts transplantation. Consequently, there is an urgent need for effective artificial grafts to address these limitations and repair injuries. Recent years have witnessed the recognition of the beneficial effects of chitosan (CS) and graphene in the realm of nerve repair. Dental pulp stem cells (DPSCs) hold great promise due to their high proliferative and multi-directional differentiation capabilities. Methods: In this study, Graphene/CS (G/CST) composite tubes were synthesized and their physical, chemical and biological properties were evaluated, then DPSCs were employed as seed cells and G/CST as a scaffold to investigate their combined effect on promoting facial nerve injury repair. Results and Disscussion: The experimental results indicate that G/CST possesses favorable physical and chemical properties, along with good cyto-compatibility. making it suitable for repairing facial nerve transection injuries. Furthermore, the synergistic application of G/CST and DPSCs significantly enhanced the repair process for a 10 mm facial nerve defect in rabbits, highlighting the efficacy of graphene as a reinforcement material and DPSCs as a functional material in facial nerve injury repair. This approach offers an effective treatment strategy and introduces a novel concept for clinically managing facial nerve injuries.

Transcriptomic-metabolomic analysis reveals the effect of copper toxicity on fermentation properties in Saccharomyces cerevisiae.

Copper is one of the unavoidable heavy metals in wine production. In this study, the effects on fermentation performance and physiological metabolism of Saccharomyces cerevisiae under copper stress were investigated. EC1118 was the most copper-resistant among the six strains. The ethanol accumulation of EC1118 was 26.16-20 mg/L Cu2+, which was 1.90-3.15 times higher than that of other strains. The fermentation rate was significantly reduced by copper, and the inhibition was relieved after 4-10 days of adjustment. Metabolomic-transcriptomic analysis revealed that amino acid and nucleotide had the highest number of downregulated and upregulated differentially expressed metabolites, respectively. The metabolism of fructose and mannose was quickly affected, which then triggered the metabolism of galactose in copper stress. Pathways such as oxidative and organic acid metabolic processes were significantly affected in the early time, resulting in a significant decrease in the amount of carboxylic acids. The pathways related to protein synthesis and metabolism under copper stress, such as translation and peptide biosynthetic process, was also significantly affected. In conclusion, this study analyzed the metabolite-gene interaction network and molecular response during the alcohol fermentation of S. cerevisiae under copper stress, providing theoretical basis for addressing the influence of copper stress in wine production.

Bridging-Solvent Strategy for Quasi-Single-Crystal Perovskite Films and Stable Solar Cells.

Controllable fabrication of formamidinium (FA)-based perovskite solar cells (PSCs) with both high efficiency and long-term stability is the key to their further commercialization. However, the diversity of PbI2 complexes and perovskite compositions usually leads to light sensitive PbI2 residues and phase impurities in the film, which can accelerate the device degradation. Here, the crystallization kinetics of FA-based perovskite films are studied and a bridging-solvent strategy is proposed to modulate the reaction kinetics between PbI2 and ammonium salts by prohibiting the formation of undesired intermediates. N-methylpyrrolidone (NMP) solvent is introduced into the PbI2 precursor solution to obtain stable and homogeneous PbI2-NMP complex films. The strong interaction between NMP and formamidinium iodide (FAI) molecules promotes the conversion from PbI2-NMP into (001)-oriented quasi-single-crystal perovskite films with negligible impurities, long carrier lifetime of 1.5 µs and a large grain size of 3 µm. The optimized PSCs exhibit a high power conversion efficiency of 24.1%, as well as superior shelf stability which maintains 95% initial efficiency after storage in air for 1200 h (T95 = 1200 h), and operating stability with T96 = 300 h under continuous working at the maximum power point. This work offers a simple and reproducible method for fabricating phase-pure and uniaxially oriented perovskite films.

Comparison of the safety profiles for pirfenidone and nintedanib: a disproportionality analysis of the US food and drug administration adverse event reporting system.

Background: Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive interstitial lung disease of unknown etiology. Pirfenidone (PFD) and nintedanib (NDN) were both conditionally recommended in the clinical practice guideline published in 2015. Safety and tolerability are related to the risk of treatment discontinuation. Therefore, this study evaluated and compared the adverse events (AEs) of PFD and NDN in a large real-world population by analyzing data from the FDA Adverse Event Reporting System (FAERS) to provide a reference for their rational and safe use. Methods: The AEs of PFD and NDN were extracted from the FAERS database. The pharmacovigilance online analysis tool OpenVigil 2.1 was used to retrieve data from the FAERS database from the first quarter of 2012 to the second quarter of 2022. The reporting odds ratio (ROR) and proportional reporting ratio were used to detect the risk signals. Results: The database included 26,728 and 11,720 reports for PFD and NDN, respectively. The most frequent AEs of PFD and NDN were gastrointestinal disorders. The RORs for these drugs were 5.874 and 5.899, respectively. "Cardiac disorders" was the most statistically significant system order class for NDN with an ROR of 9.382 (95% confidence interval = 8.308-10.594). Furthermore, the numbers of designated medical events of PFD and NDN were 552 and 656, respectively. Notably, liver injury was reported more frequently for NDN (11.096%) than for PFD (6.076%). Conclusion: This study revealed differences in the reporting of AEs between PFD and NDN. The findings provide reference for physicians in clinical practice. Attention should be paid to the risks of cardiac disorders and liver injury associated with NDN.

PLC-CN-NFAT1 signaling-mediated Aß and IL-1ß crosstalk synergistically promotes hippocampal neuronal damage.

Alzheimer's disease (AD) is a progressive neurodegenerative disease. Neuronal calcium overload plays an important role in Aß deposition and neuroinflammation, which are strongly associated with AD. However, the specific mechanisms by which calcium overload contributes to neuroinflammation and AD and the relationship between them have not been elucidated. Phospholipase C (PLC) is involved in regulation of calcium homeostasis, and CN-NFAT1 signaling is dependent on intracellular Ca2+ ([Ca2+]i) to regulate transcription of genes. Therefore, we hypothesized that the PLC-CN-NFAT1 signaling might mediate the interaction between Aß and inflammation to promote neuronal injury in AD. In this experiment, the results showed that the levels of Aß, IL-1ß and [Ca2+]i in the hippocampal primary neurons of APP/PS1 mice (APP neurons) were significantly increased. IL-1ß exposure also significantly increased Aß and [Ca2+]i in HT22 cells, suggesting a close association between Aß and IL-1ß in the development of AD. Furthermore, PLC activation induced significant calcium homeostasis imbalance, cell apoptosis, Aß and ROS production, and significantly increased expressions of CN and NFAT1, while PLC inhibitor significantly reversed these changes in APP neurons and IL-1ß-induced HT22 cells. Further results indicated that PLC activation significantly increased the expressions of NOX2, APP, BACE1, and NCSTN, which were inhibited by PLC inhibitor in APP neurons and IL-1ß-induced HT22 cells. All indications point to a synergistic interaction between Aß and IL-1ß by activating the PLC-CN-NFAT1 signal, ultimately causing a vicious cycle, resulting in neuronal damage in AD. The study may provide a new idea and target for treatment of AD.

Oxidized sodium alginate hydrogel-mouse nerve growth factor sustained release system promotes repair of peripheral nerve injury.

In recent years, mouse nerve growth factor (mNGF) has emerged as an important biological regulator to repair peripheral nerve injury, but its systemic application is restricted by low efficiency and large dosage requirement. These limitations prompted us to search for biomaterials that can be locally loaded. Oxidized sodium alginate hydrogel (OSA) exhibits good biocompatibility and physicochemical properties, and can be loaded with drugs to construct a sustained-release system that can act locally on nerve injury. Here, we constructed a sustained-release system of OSA-mouse nerve growth factor (mNGF), and investigated the loading and release of the drug through Fourier transform infrared spectroscopy and drug release curves. In vitro and in vivo experiments showed that OSA-mNGF significantly promoted the biological activities of RSC-96 cells and facilitated the recovery from sciatic nerve crush injury in rats. This observation may be attributed to the additive effect of OSA on promoting Schwann cell biological activities or its synergistic effect of cross-activating phosphoinositide 3-kinase (PI3K) through extracellular signal regulated kinase (ERK) signaling. Although the specific mechanism of OSA action needs to be explored in the future, the current results provide a valuable preliminary research basis for the clinical application of the OSA-mNGF sustained-release system for nerve repair.

Study on pulse current auxiliary heating process of submerged arc welding.

During the welding process, temperature filed distribution of weldment is one of the key factors which influence welding quality. In order to improve the mechanical properties of welding joint, a technology for heating process of weldment using auxiliary pulse current was proposed in this article. Firstly, through metallographic experiment, strength experiment and hardness experiment, it was proved that the auxiliary pulse current not only can refine grain size but also improves the mechanical property of the welding joint. Then the paper used ANSYS and its ANSYS Parametric Design Language parametric design language to simulate the welding process with assistant pulse current, and the temperature field, the pulse current density distribution and the time of effective pulse current in the welded joint were obtained. Quantitative analyses were undertaken on the influence laws of auxiliary pulse current parameters on weld temperature field, auxiliary pulse current density distribution and the time of effective pulse current. A new parameter was defined as time-current density (the time integral of current density) to demonstrate the change rule of auxiliary pulse current density distribution with time. Finally, analyzing the reasons for raising the welding quality. There were two reasons for such phenomenon: one was auxiliary pulse current change the state of heat distribution in the weld seam area, the other was that the impulse oscillation of the auxiliary pulse influences the nucleation process of melting region.

Characterization of in vitro viral neutralization targets of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) in alveolar macrophage and evaluation of protection potential against HP-PRRSV challenged based on combination of HP-PRRSV-structure proteins in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) poses a significant threat to the global pork industry, resulting in substantial economic losses. Current control measures rely on modified live virus (MLV) vaccines with safety concerns. However, the lack of consensus on protective PRRSV antigens is impeding the development of effective and safety subunit vaccines. In this study, we conducted in vitro virus neutralization (VN) assays in MARC-145 and CRL-2843CD163/CD169 cell lines and primary porcine alveolar macrophages (PAMs) to systemically identify PRRSV structural proteins (SPs) recognized by virus-neutralizing antibodies in hyperimmune serum collected from piglets infected with highly pathogenic PRRSV (HP-PRRSV). Additionally, piglets immunized with different combinations of recombinant PRRSV-SPs were challenged with HP-PRRSV to evaluate their in vivo protection potential. Intriguingly, different in vitro VN activities of serum antibodies elicited by each PRRSV SP were observed depending on the cell type used in the VN assay. Notably, antibodies specific for GP3, GP4, and M exhibited highest in vitro VN activities in PAMs, correlating with complete protection (100% survival) against HP-PRRSV challenge in vivo after immunization of piglets with combination of GP3, GP4, M and N (GP3/GP4/M/N). Further analysis of lung pathology, weight gain, and viremia post-challenge revealed that the combination of GP3/GP4/M/N provided superior protective efficacy against severe infection. These findings underscore the potential of this SP combination to serve as an effective PRRSV subunit vaccine, marking a significant advancement in pork industry disease management.

Uniaxial-Oriented Perovskite Films with Controllable Orientation.

Perovskite films with large crystal size, preferred orientation, and facile fabrication process, combining advantages of single-crystal and polycrystalline films, have gained considerable attention recently. However, there is little research on the facet properties of perovskite films. Here, (111)- and (001)-oriented perovskite films with bandgaps ranging from 1.53 to 1.77 eV, and systematically investigated their orientation-dependent properties are achieved. The (111)-oriented films show electron-dominated traps and the (001)-oriented films show hole-dominated traps, which are related to their atomic arrangement at the surface. Compared with the (001)-oriented films, the (111)-oriented films exhibit lower work function and superior water/oxygen robustness. For the wide-bandgap films, the lattice of the (001)-oriented film provides an unobstructed passage for ion migration. Comparably, the (111)-oriented films exhibit suppressed ion migration and excellent phase stability. The optimized unencapsulated solar cells based on both (001) and (111) orientations show a similar high efficiency of ≈23%. The (111)-oriented solar cell exhibits excellent stability, maintaining 95% of its initial efficiency after 1500 h maximum power point (MPP) tracking test, and 97% initial efficiency after 3000 h aging in ambient conditions. This work paves the way for the rational design, controllable synthesis, and targeted optimization of uniaxial-oriented perovskite films for various electronic applications.

Porcine HERC6 acts as major E3 ligase for ISGylation and is auto-ISGylated for effective ISGylation in porcine.

Interferon-stimulated gene product 15 (ISG15) can be conjugated to substrates through ISGylation. Currently, the E3 ligase for porcine ISGylation remains unclear. Here, we identified porcine HERC5 and HERC6 (pHERC5/6) as ISGylation E3 ligases with pHERC6 acting as a major one by reconstitution of porcine ISGylation system in HEK-293 T cell via co-transfecting E1, E2 and porcine ISG15(pISG15) genes. Meanwhile, our data demonstrated that co-transfection of pISG15 and pHERC5/6 was sufficient to confer ISGylation, suggesting E1 and E2 of ISGylation are interchangeable between human and porcine. Using an immunoprecipitation based ISGylation analysis, our data revealed pHERC6 was a substrate for ISGylation and confirmed that K707 and K993 of pHERC6 were auto-ISGylation sites. Mutation of these sites reduced pHERC6 half-life and inhibited ISGylation, suggesting that auto-ISGylation of pHERC6 was required for effective ISGylation. Conversely, sustained ISGylation induced by overexpression of pISG15 and pHERC6 could be inhibited by a well-defined porcine ISGylation antagonist, the ovarian tumor (OTU) protease domain of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)-nsp2 and PRRSV-nsp1ß, further indicating such method could be used for identification of virus-encoded ISG15 antagonist. In conclusion, our study contributes new insights towards porcine ISGylation system and provides a novel tool for screening viral-encoded ISG15 antagonist.