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Accurate assessment of material and energy exchange between land and atmosphere is essential for water resources management and sustainable development of agriculture. To understand the characteristics of energy distribution and the dynamic change process of water and heat fluxes within the maize farmland ecosystem in the old course of Yellow River and their response to meteorological factors, we utilized the eddy covariance measurements and the full-element automatic weather station to continuously observe energy fluxes and conventional meteorological elements of summer maize farmland in the old course of Yellow River during 2019-2020. We analyzed the variation of energy fluxes and the effects of environmental factors, such as temperature, precipitation, and wind speed. Additionally, we calculated the energy closure rate and the proportion of energy distribution during the growth stage. The results showed that the peaks of net radiation, sensible heat flux, and latent heat flux occurred between 11:00 and 14:00, and the peak of soil heat flux occurred between 14:00 and 15:00. In terms of energy distribution, energy consumption of summer maize farmland during the whole growth period was dominated by latent heat flux and sensible heat flux. Energy was mainly consumed by sensible heat flux at sowing-emergence stage, accounting for 37.1% of net radiation, respectively. Energy in the rest of growth stages was dominated by latent heat flux. The energy closure rate during the whole growth period was better, with a coefficient of determination of 0.83, and the closure rate was higher in day and lower at night. Precipitation affected latent heat flux and sensible heat flux, and latent heat flux was more sensitive to precipitation. The increase of latent heat flux after rainfall was lower in late growth stage than in early growth stage. During the whole growth period of summer maize, solar radiation was the most significant meteorological factor affecting both sensible heat flux and latent heat flux, followed by vapor pressure deficit. The contribution of temperature and vapor pressure deficit to latent heat flux was significantly higher than sensible heat flux, while the relative contribution of wind speed, relative humidity, and solar radiation to latent heat flux was lower than sensible heat flux. Leaf area index and fractional vegetation cover had a significant positive correlation with latent heat flux and a significant negative correlation with sensible heat flux. Our results could deepen the understanding of water and heat transfer law of summer maize farmland in the old course of Yellow River, providing a theoretical basis for efficient water use of crops.

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Non-alcoholic fatty liver disease (NAFLD) and myocardial infarction (MI) are two major health burdens with significant prevalence and mortality. This study aimed to explore the co-expressed genes to understand the relationship between NAFLD and MI and identify potential crucial biomarkers of NAFLD-related MI using bioinformatics and machine learning. Functional enrichment analysis was conducted, a co-protein-protein interaction (PPI) network diagram was constructed, and support vector machine-recursive feature elimination (SVM-RFE) and least absolute shrinkage and selection operator (LASSO) techniques were employed to identify one differentially expressed gene (DEG), Thrombospondin 1 (THBS1). THBS1 demonstrated strong performance in distinguishing NAFLD patients (AUC = 0.981) and MI patients (AUC = 0.900). Immuno-infiltration analysis revealed significantly lower CD8+ T cells and higher neutrophil levels in patients with NAFLD and MI. CD8+ T cells and neutrophils were effective in distinguishing NAFLD/MI from healthy controls. Correlation analysis showed that THBS1 was positively correlated with CCR (chemokine receptor), MHC class (major histocompatibility complex class), neutrophils, parainflammation, and Tfh (follicular helper T cells), and negatively correlated with CD8+ T cells, cytolytic activity, and TIL (tumor-infiltrating lymphocytes) in NAFLD and MI patients. THBS1 emerged as a novel biomarker for diagnosing NAFLD/MI in comparison to healthy controls. The results indicate that CD8+ T cells and neutrophils could serve as inflammatory immune features for differentiating patients with NAFLD/MI from healthy individuals.

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Background: Long-term outcomes for knee osteoarthritis patients undergoing unicompartmental knee arthroplasty (UKA) and total knee arthroplasty (TKA) remain inconclusive. Objectives: This study aims to evaluate the long-term outcomes over five years, including Knee Society Pain Scores (KSPS), Knee Society Scores (KSS), Knee Society Function Scores (KSFS), range of motion (ROM), and survival rates-of UKA vs. TKA in knee osteoarthritis patients. Design: Systematic review using data from randomized controlled and cohort trials, and world databases. Data sources: Researchers searched Medline, Embase, Cochrane Controlled Register of Trials, and ClinicalTrials.gov from January 1990 to March 2024. Eligibility criteria for selecting studies: The researchers selected studies based on adult participants with knee osteoarthritis. Eligible studies compare UKA and TKA reports on clinical or surgical outcomes, including KSPS, KSS, KSFS, ROM and survival rates, over 5 years. The researchers excluded the studies fewer than five years, or if English text was unavailable. Results: Researchers categorized twenty-nine eligible studies into three groups: five randomized controlled trials, 11 registries and database studies, and 13 cohort studies. The analysis revealed that neither TKA nor UKA definitively outperformed the other in terms of pain (SMD (95% CI): -0.06 [-0.41, 0.28], I 2 = 90%) and KSS scores (SMD (95% CI): -0.07 [-0.23, 0.008], I 2 = 81%) over a period of five years. However, KSFS (SMD (95% CI): -0.30 [-0.43, -0.17], I 2 = 74%) and ROM (SMD (95% CI): -0.78 [-1.11, -0.46], I 2 = 92%) tended to favor UKA, and survival rate favor TKA at 5 or over 5-year follow-up periods. Conclusions: UKA shows a trend towards better outcomes in KSFS and ROM, alongside a more favorable survival rate in TKA at the five-year and beyond follow-up periods. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?RecordID=517835, PROSPERO (CRD42024517835).

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High-strength, lightweight, ultrathin, and flexible electromagnetic interference (EMI) shielding materials with a high shielding effectiveness (SE) are essential for modern integrated electronics. Herein, cellulose nanofibrils (CNFs) are employed to homogeneously disperse graphene nanoplates (GNPs) into an aramid nanofiber (ANF) network and silver nanowire (AgNW) network, respectively, producing high-performance nanopapers. These nanopapers, featuring nacre-mimetic microstructures and layered architectures, exhibited high tensile strength (601.11 MPa) and good toughness (103.56 MJ m-3) with a thickness of only 24.58 µm. Their specific tensile strength reaches 447.59 MPa·g-1·cm3, which is 1.74 times that of titanium alloys (257 MPa·g-1·cm3). The AgNW/GNP composite conductive layers exhibit an electrical conductivity of 12010.00 S cm-1, providing the nanopapers with great EMI shielding performance, achieving an EMI SE of 63.87 dB and an EMI SE/t of 25978.80 dB cm-1. The nanopapers also show reliable durability, retaining a tensile strength of 500.96 MPa and an EMI SE of 57.59 dB after 120,000 folding cycles. Additionally, they have a good electrical heating performance with a fast response time, low driving voltage, effective deicing capability, and reliable heating capacity in water. This work presents a strategy to develop a high-performance nanopaper, showing great potential for applications in electromagnetic compatibility, national defense, smart electronics, and human health.

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In this study, the synergistic effect and weak gel mechanism of XG and Gleditsia sinensis polysaccharide (GSP) in different ratios were studied through the rheological properties, microstructure and molecular simulation based on density functional theory (DFT). The results of rheological properties showed that the mixtures formed a weak gel at the concentration of 0.5 % (w/v), with the synergistic impact peaking at a XG/GSP ratio of 3:7. Weak gels produced by XG and GSP had the intersection of G' and G" within the temperature sweep range, and the largest change in the G' slope at a XG/GSP ratio of 3:7. By calculating the interaction energy, it was found that the backbone of XG was more likely to interact with the backbone of GSP. Furthermore, the XG mainchain intersected with the backbone of GSP in a cross shape ("X" shape). As a result, this paper proposed a possible mechanism for the formation of the XG/GSP weak gel, with XG as the main chain and GSP as the grid point, and the main interaction type being hydrogen bonding, with the van der Waals force also involved. The results provide new insight for designing and producing physical gels with specific interactions in food industry.

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As the first identified multidrug efflux pump in Mycobacterium tuberculosis (Mtb), EfpA is an essential protein and promising drug target. However, the functional and inhibitory mechanisms of EfpA are poorly understood. Here we report cryo-EM structures of EfpA in outward-open conformation, either bound to three endogenous lipids or the inhibitor BRD-8000.3. Three lipids inside EfpA span from the inner leaflet to the outer leaflet of the membrane. BRD-8000.3 occupies one lipid site at the level of inner membrane leaflet, competitively inhibiting lipid binding. EfpA resembles the related lysophospholipid transporter MFSD2A in both overall structure and lipid binding sites and may function as a lipid flippase. Combining AlphaFold-predicted EfpA structure, which is inward-open, we propose a complete conformational transition cycle for EfpA. Together, our results provide a structural and mechanistic foundation to comprehend EfpA function and develop EfpA-targeting anti-TB drugs.

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BACKGROUND: High blood pressure (HBP) and diabetes mellitus (DM) are two of the most prevalent cardiometabolic disorders globally, especially among individuals with lower socio-economic status (SES). Studies have linked residential greenness to decreased risks of HBP and DM. However, there has been limited evidence on whether SES may modify the associations of residential greenness with HBP and DM. METHODS: Based on a national representative cross-sectional study among 44,876 adults, we generated the normalized difference vegetation index (NDVI) at 1 km spatial resolution to characterize individuals' residential greenness level. Administrative classification (urban/rural), nighttime light index (NLI), individual income, and educational levels were used to characterize regional urbanicity and individual SES levels. RESULTS: We observed weaker inverse associations of NDVI with HBP and DM in rural regions compared to urban regions. For instance, along with per interquartile range (IQR, 0.26) increment in residential NDVI at 0∼5 year moving averages, the ORs of HBP were 1.04 (95%CI: 0.94, 1.15) in rural regions and 0.85 (95%CI: 0.79, 0.93) in urban regions (P = 0.003). Along with the decrease in NLI levels, there were continuously decreasing inverse associations of NDVI with DM prevalence (P for interaction <0.001). In addition, weaker inverse associations of residential NDVI with HBP and DM prevalence were found among individuals with lower income and lower education levels compared to their counterparts. CONCLUSIONS: Lower regional urbanicity and individual SES could attenuate the associations of residential greenness with odds of HBP and DM prevalence.

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Polycyclic aromatic hydrocarbons (PAHs) constitute a class of persistent organic pollutants with strong lipophilicity, which readily accumulate within organisms and have the effect to induce disorders in lipid metabolism. The present study aimed to investigate the accumulation localization and pattern of PAHs in Ruditapes philippinarum, and to reveal the association between PAHs and lipids metabolism. The 21-day exposure experiment was conducted using a mixture of phenanthrene, chrysene, and benzo[a]pyrene (the proportion is 1:1:1) at concentrations of 0.4 µg/L, 2 µg/L, and 10 µg/L. The tissue distribution of PAHs indicated that the digestive gland was the primary site of PAHs accumulation. Meanwhile, fluorescence colocalization suggested that PAHs primarily accumulated within the lipid droplets of digestive gland cells. This study further determined the transcriptomic and lipidomic profiles of the digestive gland to analyze the key genes involved in disrupted lipid metabolism and the major lipids affected. Lipidomic analysis identified the key differential metabolites as triglycerides (TGs). Furthermore, TGs were upregulated in the digestive gland had a total carbon atom number of 50-64 and a total number of 3-9 double bonds in the acyl side chains. Biochemical analysis experiments and oil red O stained frozen sections confirmed that the content of TGs steadily increased in various tissues during the experiment, leading to an elevated digestive gland index. Changes of lipid metabolism associated genes expression level also indicated that the synthesis of lipid in digestive gland were up-regulated while the decomposition was down-regulated. This study is the first to demonstrate the cellular localization of PAHs accumulation in bivalves and confirms the pattern of variation in TGs, providing new insights into the mechanisms of PAHs bioaccumulation and lipid metabolism disruption.

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In neurological diseases, the regulation of autophagy plays a crucial role in their pathology, particularly the relationship between autophagy and hepatic encephalopathy (HE) which merits detailed investigation. Glycosphingolipids are abundant and broadly functional in the nervous system and are closely associated with autophagy. However, the specific link and mechanisms between glycosphingolipids and autophagy in HE remain unclear. This study aims to explore the impact of glycosphingolipid changes on the autophagy in HE and its potential mechanisms. Utilizing lectin microarrays, we observed elevated expression levels of α2-3 sialylated glycosphingolipid in the brain tissue of HBV transgenic mice and ammonia-induced astrocyte models, suggesting that the increase in α2-3 sialylated glycosphingolipid is related to HE. Further research revealed that the increased expression of α2-3 sialylated glycosphingolipid, mediated by ST3GAL2, affects autophagy by regulating the autophagy initiation complex Vps34-Beclin-1. In summary, our research not only comprehensively reveals the changes in brain glycosphingolipid during HBV-related HE but also elucidates the interactions and regulatory mechanisms between α2-3 sialylated glycosphingolipid and autophagy. This study provides a new perspective on understanding the pathogenesis of HE and offers novel theories and targets for future research and treatment strategies.

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A remote steric hindrance ligand (m-tBu)2C6H3PCy2 (L1) was synthesized to promote Ni-catalyzed C-O bond activation. The reaction achieved high yields for secondary benzylic C(sp3)-O borylation in non-π-extended systems under mild conditions. Mechanistic studies indicate that the nickel complex containing 1 equiv of L1 serves as the active catalyst, while increased loading of L1 gives the inactive bisligated Ni species. Acetanilide is crucial for the cross-coupling reaction, which facilitates generation of the monoligated nickel species.

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The sensitive detection of trace biomarkers in exhaled breath for lung cancer diagnosis represents a critical area of research in life analytical chemistry, with profound implications for early disease detection, therapeutic intervention, and prognosis monitoring. Despite its potential, the analytical process faces significant challenges due to the ultratrace levels of disease biomarkers present and the complex, high-humidity composition of exhaled breath. This study introduces a highly sensitive method for detecting aldehyde biomarkers in exhaled breath by integrating the use of amino-functionalized microporous organic networks (NH2-MON) as a solid-phase microextraction (SPME) fiber coating with gas chromatography-triple quadrupole mass spectrometry (GC-MS/MS) analysis. The method innovatively combines sample collection and extraction, achieving a dual-step enrichment process that significantly enhances both the enrichment efficiency and reproducibility of biomarker detection while effectively mitigating the interference caused by water vapor in exhaled breath. The NH2-MON, utilized as an SPME fiber coating, demonstrates exceptional enrichment capacity for five key aldehyde biomarkers, facilitating the development of a highly sensitive detection approach for these biomarkers in exhaled breath. Compared to previously reported methods, the proposed technique exhibits significantly lower limits of quantification, ranging from 0.77 to 11.89 pg mL-1, and achieves substantially higher enrichment factors, ranging from 9156- to 35723-fold. The practicality and feasibility of the method were validated through the analysis of exhaled breath samples from lung cancer patients, underscoring its potential application in the early diagnosis and monitoring of lung cancer.

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Whether maternal exposure to dust-sourced particulate matter (hereafter, dust PM2.5) is associated with stillbirth remains unknown. We adopted a sibling-matched case-control design to analyze 9332 stillbirths and 17,421 live births. We associated the risk of stillbirth simultaneously with dust and nondust components of PM2.5 and developed a nonlinear joint exposure-response function. Next, we estimated the burden of stillbirths attributable to the PM2.5 mixture. The concentration index was used to evaluate whether the burden of PM2.5-related stillbirths was disproportionally distributed among pregnancies exposed to dust-rich particles. Each 10 µg/m3 increase in dust PM2.5 was associated with a 14.5% (95% confidence interval: 5.5, 24.2%) increase in the odds of stillbirth. Based on the risk assessment across 137 countries, sand dust contributed to about 15% of the PM2.5 exposure but to about 45% of the PM2.5-related stillbirths during 2003-2019. In 2015, 30% of the PM2.5-related stillbirths were concentrated within 15% of pregnancies exposed to the dust-richest PM2.5. The index increased in subregions, such as South Asia, suggesting the growth of health inequality due to exposure to dust PM2.5. Based on our findings, land management, such as halting desertification, will help prevent stillbirths and reduce global maternal health inequality.

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Xinjiang is a major province of sheep breeding in China, which plays an important role in meeting people's needs for meat products, increasing farmers' income and sustainable development of animal husbandry. However, the genetic differentiation relationship between breeds was not clear, and most sheep had low fecundity, which seriously restricted the efficient development of sheep industry. Therefore, this study used the whole genome resequencing to detect the genetic variation of Dexin mutton and fine-wool sheep, explored the selected regions and important genes of the litter size traits, analyzed the genetic mechanism of reproductive traits, and provided new insights for the high fecundity breeding of sheep. A total of 5,236.338 G genome data and 35,884,037 SNPs were obtained. Furthermore, we identified 39 selection signals spanning candidate genes, 99 genes were significantly associated related to growth, reproduction and immunity, among which, BRIP1, BMPR1B, BMP4, NGF, etc. genes, and MAKP signaling pathway, Fanconi anemia pathway and Thyroid hormone signaling pathway and other signaling pathways were significantly correlated with litter size trait. Among them, we identified NGF, TrKA and BRIP1 genes was the important genes for sheep litter size traits and the mutation frequencies of 9 SNPs in BRIP1 gene were significantly different in domestic sheep in the world. The research provided new insights for the breeding of self-cultivated meat fine-wool sheep.

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Titanium dioxide nanoparticles (TiO2 NPs) can cause mitochondrial apoptosis of TM4 cells associated with reactive oxygen species (ROS) accumulation and Ca2+ overload, but the relations among these processes remain unclear. This study aimed to evaluate whether the accumulation of ROS caused by TiO2 NPs inhibits MCUb expression, leading to mitochondrial calcium overload and subsequent cell apoptosis through the mitochondrial pathway. TM4 cells were exposed to different concentrations of TiO2 NPs (0, 25, 50, 75, 100 µg/mL) for 24 h. We assessed cell viability, ROS level, MCUb and VDAC1 expression, mitochondrial and cytoplasmic Ca2+ levels, mitochondrial membrane potential (MMP), apoptosis rate, and key proteins related to mitochondrial apoptosis (Bcl-2, Bax, Caspase 3, Caspase 9, p53 and Cyt c). Additionally, the effect of N-acetylcysteine (NAC) on MCUb expression, calcium homeostasis, and cell apoptosis was evaluated. Compared to control group, TiO2 NPs significantly increased ROS level, downregulated MCUb expression, elevated Ca2+ levels in mitochondria and cytoplasm, and enhanced mitochondria-regulated apoptosis, starting from the 50 µg/mL TiO2 NPs group. However, NAC significantly increased MCUb expression, attenuated Ca2+ levels in mitochondria and cytoplasm, and reduced mitochondria-related apoptosis. In conclusion, TiO2 NPs induced ROS accumulation, which inhibited the expression of MCUb. The decreased MCUb level led to Ca2+ overload in mitochondria, causing TM4 cell apoptosis via the mitochondrial pathway. This research elucidates, for the first time, the role of MCUb and its relation with ROS in apoptosis of TM4 cells induced by TiO2 NPs, which supplementing the molecular mechanism of cell apoptosis caused by TiO2 NPs.

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Banana Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4), is a major disease of banana plants worldwide. Effector proteins play critical roles in banana-Foc TR4 interaction. Our previous studies highlighted a ribonuclease protein belonging to the T2 family (named as FocRnt2) in the Foc TR4 secretome, which was predicted to be an effector. However, its biological function in Foc TR4 infection is still unclear. Herein, we observed significant expression of FocRnt2 during the early stage of fungal infection in planta. A yeast signal sequence trap assay showed that FocRnt2 contained a functional signal peptide for secretion. FocRnt2 possessed ribonuclease activity that could degrade the banana total RNA in vitro. Subcellular localization showed that FocRnt2 was localized in the nucleus and cytoplasm of Nicotiana benthamiana leaves. Transient expression of FocRnt2 suppressed the expression of salicylic acid- and jasmonic acid-signalling marker genes, reactive oxygen species accumulation, and BAX-mediated cell death in N. benthamiana. FocRnt2 deletion limited fungal penetration, reduced fusaric acid biosynthesis in Foc TR4, and attenuated fungal virulence against banana plants, but had little effect on Foc TR4 growth and sensitivity to various stresses. Furthermore, FocRnt2 deletion mutants induced higher expression of the defence-related genes in banana plants. These results suggest that FocRnt2 plays an important role in full virulence of Foc TR4, further improving our understanding of effector-mediated Foc TR4 pathogenesis.

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As one of rare high-value ocotillol (OCT)-type ginsenosides, pseudoginsenoside Rt5 has been identified with significant pharmacological activities. UDP-glycosyltransferases (UGTs) play pivotal roles in catalyzing the transfer of a glycosyl moiety from a donor to an acceptor. In this study, the novel UGT, PjUGT10, was screened from the transcriptome database of Panax japonicus and identified with the enzymatic activity of transferring a glucosyl group on OCT to produce Rt5. The catalytic efficiency of PjUGT10 was further enhanced by employing site-directed mutation. Notably, the variant M7 exhibited a remarkable 6.16 × 103-fold increase in kcat/Km towards 20S,24R-ocotillol and a significant 2.02 × 103-fold increase to UDP-glucose, respectively. Moreover, molecular dynamics simulations illustrated a reduced distance between 20S,24R-ocotillol and the catalytic residue His15 or UDP-glucose, favoring conformation interactions between the enzyme and substrates. Subsequently, Rt5 was synthesized in an engineered Escherichia coli strain M7 coupled with a UDP-glucose synthetic system. This study not only shed light on the protein engineering that can enhance the catalytic activity of PjUGT10, but also established a whole-cell approach for the production of Rt5.

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AgNW networks show high promise as a conductive material due to excellent flexibility, low resistance, high transparency, and ease of large-scale preparation. However, the application of AgNW networks has been hindered by their inherent characteristics, such as easy oxidation degradation, chemical corrosion, and structural instability at high temperatures. In this study, a dense SiOx protective layer derived from perhydropolysilazane was introduced to fabricate a robust SiOx/AgNW nanocomposite coating through an all-solution process at room temperature. The achieved nanocomposite coating shows outstanding thermal stability up to 450 °C, resistance to ultraviolet radiation, and excellent mechanical performance by maintaining stability after 10,000 cycles of bending at a radius of 2.5 mm, 1000 cycles of peeling, and 1200 cycles of wearing. Meanwhile, the nanocomposite coating demonstrates exceptional chemical tolerance against HCl, Na2S, and organic solvents. A transparent heater based on the nanocomposite coating achieves a remarkable benchmark with a maximum temperature of 400 °C at 20 V. These features highlight the potential of the nanocomposite coating in flexible electronics, optoelectronics, touch screens, and high-performance heaters.

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Identifying the antibacterial mechanisms of elemental silver at the nanoscale remains a significant challenge due to the intertwining behaviors between the particles and their released ions. The open question is which of the above factor dominate the antibacterial behaviors when silver nanoparticles (Ag NPs) with different sizes. Considering the high reactivity of Ag NPs, prior research has primarily concentrated on coated particles, which inevitably hinder the release of Ag+ ions due to additional chemical agents. In this study, we synthesized various Ag NPs, both coated and uncoated, using the laser ablation in liquids (LAL) technique. By analyzing both the changes in particle size and Ag+ ions release, the impacts of various Ag NPs on the cellular activity and morphological changes of gram-negative (E. coil) and gram-positive (S. aureus) bacteria were evaluated. Our findings revealed that for uncoated Ag NPs, smaller particles exhibited greater ions release efficiency and enhanced antibacterial efficacy. Specifically, particles approximately 1.5 nm in size released up to 55 % of their Ag+ ions within 4 h, significantly inhibiting bacterial growth. Additionally, larger particles tended to aggregate on the bacterial cell membrane surface, whereas smaller particles were more likely to be internalized by the bacteria. Notably, treatment with smaller Ag NPs led to more pronounced bacterial morphological changes and elevated levels of intracellular reactive oxygen species (ROS). We proposed that the bactericidal activity of Ag NPs stems from the synergistic effect between particle-cell interaction and the ionic silver, which is dependent on the crucial parameter of particle size.

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Betacoronaviruses encode a conserved accessory gene within the +1 open reading frame (ORF) of nucleocapsid called the internal N gene. This gene is referred to as "I" for mouse hepatitis virus (MHV), ORF9b for severe acute respiratory CoV (SARS-CoV) and SARS-CoV-2, and ORF8b for Middle East respiratory syndrome CoV (MERS-CoV). Previous studies have shown ORF8b and ORF9b have immunoevasive properties, while the only known information for MHV I is its localization within the virion of the hepatotropic/neurotropic A59 strain of MHV. Whether MHV I is an innate immune antagonist or has other functions has not been evaluated. In this report, we show that the I protein of the neurotropic JHM strain of MHV (JHMV) lacks a N terminal domain present in other MHV strains, has immunoevasive properties, and is a component of the virion. Genetic deletion of JHMV I (rJHMVIΔ57-137) resulted in a highly attenuated virus both in vitro and in vivo that displayed a post RNA replication/transcription defect that ultimately resulted in fewer infectious virions packaged compared with wild-type virus. This phenotype was only seen for rJHMVIΔ57-137, suggesting the structural changes predicted for A59 I altered its function, as genetic deletion of A59 I did not change viral replication or pathogenicity. Together, these data show that JHMV I both acts as a mild innate immune antagonist and aids in viral assembly and infectious virus production, and suggest that the internal N proteins from different betacoronaviruses have both common and virus strain-specific properties.IMPORTANCECoV accessory genes are largely studied in overexpression assays and have been identified as innate immune antagonists. However, functions identified after overexpression are often not confirmed in the infected animal host. Furthermore, some accessory proteins are components of the CoV virion, but their role in viral replication and release remains unclear. Here, we utilized reverse genetics to abrogate expression of a conserved CoV accessory gene, the internal N ("I") gene, of the neurotropic JHMV strain of MHV and found that loss of the I gene resulted in a post replication defect that reduced virion assembly and ultimately infectious virus production, while also increasing some inflammatory molecule expression. Thus, the JHMV I protein has roles in virion assembly that were previously underappreciated and in immunoevasion.

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Extracellular matrix (ECM) metabolism disorders in the inflammatory microenvironment play a key role in the pathogenesis of intervertebral disc degeneration (IDD). Interleukin-32 (IL-32) has been reported to be involved in the progression of various inflammatory diseases; however, it remains unclear whether it participates in the matrix metabolism of nucleus pulposus (NP) cells. Therefore, this study aimed to investigate the mechanism of IL-32 on regulating the ECM metabolism in the inflammatory microenvironment. RNA-seq was used to identify aberrantly expressed genes in NP cells in the inflammatory microenvironment. Western blotting, real-time quantitative PCR, immunohistochemistry and immunofluorescence analysis were performed to measure the expression of IL-32 and metabolic markers in human NP tissues or NP cells treated with or without tumor necrosis factor-α (TNF-α). In vivo, an adeno-associated virus overexpressing IL-32 was injected into the caudal intervertebral discs of rats to assess its effect on IDD. Proteins interacting with IL-32 were identified via immunoprecipitation and mass spectrometry. Lentivirus overexpressing IL-32 or knocking down Fat atypical cadherin 4 (FAT4), yes-associated protein (YAP) inhibitor-Verteporfin (VP) were used to treat human NP cells, to explore the pathogenesis of IL-32. Hippo/YAP signaling activity was verified in human NP tissues. IL-32 expression was significantly upregulated in degenerative NP tissues, as indicated in the clinical samples. Furthermore, IL-32 was remarkably overexpressed in TNF-α-induced degenerative NP cells. IL-32 overexpression induced IDD progression in the rat model. Mechanistically, the elevation of IL-32 in the inflammatory microenvironment enhanced its interactions with FAT4 and mammalian sterile 20-like kinase1/2 (MST1/2) proteins, prompting MST1/2 phosphorylation, and activating the Hippo/YAP signaling pathway, causing matrix metabolism disorder in NP cells. Our results suggest that IL-32 mediates matrix metabolism disorders in NP cells in the inflammatory micro-environment via the FAT4/MST/YAP axis, providing a theoretical basis for the precise treatment of IDD.

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