Conditional generative adversarial network-assisted system for radiation-free evaluation of scoliosis using a single smartphone photograph: a model development and validation study.

Background: Adolescent idiopathic scoliosis (AIS) is the most common spinal disorder in children, characterized by insidious onset and rapid progression, which can lead to severe consequences if not detected in a timely manner. Currently, the diagnosis of AIS primarily relies on X-ray imaging. However, due to limitations in healthcare access and concerns over radiation exposure, this diagnostic method cannot be widely adopted. Therefore, we have developed and validated a screening system using deep learning technology, capable of generating virtual X-ray images (VXI) from two-dimensional Red Green Blue (2D-RGB) images captured by a smartphone or camera to assist spine surgeons in the rapid, accurate, and non-invasive assessment of AIS. Methods: We included 2397 patients with AIS and 48 potential patients with AIS who visited four medical institutions in mainland China from June 11th 2014 to November 28th 2023. Participants data included standing full-spine X-ray images captured by radiology technicians and 2D-RGB images taken by spine surgeons using a camera. We developed a deep learning model based on conditional generative adversarial networks (cGAN) called Swin-pix2pix to generate VXI on retrospective training (n = 1842) and validation (n = 100) dataset, then validated the performance of VXI in quantifying the curve type and severity of AIS on retrospective internal (n = 100), external (n = 135), and prospective test datasets (n = 268). The prospective test dataset included 268 participants treated in Nanjing, China, from April 19th, 2023, to November 28th, 2023, comprising 220 patients with AIS and 48 potential patients with AIS. Their data underwent strict quality control to ensure optimal data quality and consistency. Findings: Our Swin-pix2pix model generated realistic VXI, with the mean absolute error (MAE) for predicting the main and secondary Cobb angles of AIS significantly lower than other baseline cGAN models, at 3.2° and 3.1° on prospective test dataset. The diagnostic accuracy for scoliosis severity grading exceeded that of two spine surgery experts, with accuracy of 0.93 (95% CI [0.91, 0.95]) in main curve and 0.89 (95% CI [0.87, 0.91]) in secondary curve. For main curve position and curve classification, the predictive accuracy of the Swin-pix2pix model also surpassed that of the baseline cGAN models, with accuracy of 0.93 (95% CI [0.90, 0.95]) for thoracic curve and 0.97 (95% CI [0.96, 0.98]), achieving satisfactory results on three external datasets as well. Interpretation: Our developed Swin-pix2pix model holds promise for using a single photo taken with a smartphone or camera to rapidly assess AIS curve type and severity without radiation, enabling large-scale screening. However, limited data quality and quantity, a homogeneous participant population, and rotational errors during imaging may affect the applicability and accuracy of the system, requiring further improvement in the future. Funding: National Key R&D Program of China, Natural Science Foundation of Jiangsu Province, China Postdoctoral Science Foundation, Nanjing Medical Science and Technology Development Foundation, Jiangsu Provincial Key Research and Development Program, and Jiangsu Provincial Medical Innovation Centre of Orthopedic Surgery.

Human hypofunctional NCF1 variants promote pulmonary fibrosis in the bleomycin-induced mouse model and patients with systemic sclerosis via expansion of SPP1+ monocytes-derived macrophages.

OBJECTIVE: We assessed the role of a systemic lupus erythematosus causal hypofunctional variant, neutrophil cytosolic factor 1 (NCF1)-p.Arg90His (p.R90H) substitution, in systemic sclerosis (SSc). METHODS: Association of NCF1-H90 with SSc was performed in case-control cohorts, bleomycin (BLM)-treated Ncf1-R90 C57BL/6 wildtype and Ncf1-H90 knock-in (KI) littermates. Peripheral blood mononuclear cell (PBMC) subsets were analysed by cytometry by time-of-flight. RESULTS: The NCF1-H90 allele is associated with risk for diffuse cutaneous SSc (dcSSc) in Chinese and European Americans, and lung fibrosis in Chinese patients with SSc (OR=2.09, p=7.96E-10). Low copy number of NCF1 associated with lung fibrosis in European Americans (OR=4.33, p=2.60E-2). BLM-treated KI mice demonstrated increased pulmonary fibrosis, exhibiting activated type I interferon signature, elevated Spp1, Ccl2, Arg1, Timp1 and Il6 expression, enriched macrophage scores in lung tissues. In a longitudinal observation cohort, homozygous H90 patients with SSc at baseline had increased anti-nuclear antibody titres, anti-topoisomerase antibody seropositivity and anti-centromere antibody seronegativity, increased incidence of lung fibrosis and Gender-Age-lung Physiology index, elevated modified Rodnan Skin Score (mRSS) and elevated plasma osteopontin (OPN, SPP1), CCL2, ARG1, TIMP-1 and IL-6. These H90 patients with SSc sustained elevated mRSS during follow-up years with decreased survival. The 0, 1 and 2 copies of H90 carriage in SSc PBMCs exhibited dose-dependent increases in profibrotic CD14+CD68+CD11b+Tim3+monocytes. Elevated OPN, CCL2 and ARG1 in CD68+CD11b+monocyte-derived macrophages from H90 patients were decreased after co-culturing with anti-CCL2 antibody. CONCLUSION: Low NCF1 activity increases the risk for the development of dcSSc and lung fibrosis via expanding profibrotic SPP1+MoMs in a CCL2-dependent manner, contributing to the severity of lung fibrosis in both BLM-treated mice and patients with SSc.

Predicting postoperative mechanical complications with the ethnicity-adjusted global alignment and proportion score in degenerative scoliosis: does paraspinal muscle degeneration matter?

BACKGROUND: The global alignment and proportion (GAP) score was developed to predict mechanical complications (MCs) after adult spinal deformity surgery but showed limited sensitivity in the Asian population. Considering variations in sagittal parameters among different ethnic groups, our team developed the ethnicity-adjusted GAP score according to the spinopelvic parameters of 566 asymptomatic Chinese volunteers (C-GAP score). Notably, degenerative scoliosis (DS) patients with MCs following corrective surgery have more severe paraspinal muscle degeneration. For DS patients with various sagittal alignments, the unevenly distributed degeneration of paraspinal muscle may exert different influences on MC occurrence and largely affect the accuracy of the C-GAP score in clinical assessment. Therefore, incorporating paraspinal muscle degeneration indices within the C-GAP score may improve its accuracy in predicting MC occurrence. PURPOSE: We aimed to clarify the influence of paraspinal muscle degeneration on the C-GAP score predicting MC occurrence following DS surgery and modify the C-GAP score with paraspinal muscle degeneration parameters. STUDY DESIGN: A retrospective case-control study. SAMPLE SIZE: A total of 107 adult degenerative scoliosis patients. OUTCOME MEASURES: Demographic information, postoperative sagittal spinopelvic parameters, the GAP score, the C-GAP score, and paraspinal muscle degeneration parameters. METHODS: A total of 107 DS patients undergoing posterior spinal fusion surgery (≥4 vertebrae) with a minimum of 2 years follow-up (or experiencing MCs within 2 years) were retrospectively reviewed. Their C-GAP score was calculated based on our previous study and patients were divided into 3 C-GAP categories, "proportioned" (P), "moderately disproportioned" (MD), and "severely disproportioned" (SD). Relative cross-sectional area (cross-sectional area of muscle-disc ratio×100, rCSA) and fat infiltration rate, FI% at L1/2, L2/3, L3/4, and L4/5 discs were quantitatively evaluated using magnetic resonance imaging (MRI). In each C-GAP category, patients were additionally divided into the MC group and the non-MC group to analyze their paraspinal muscle degeneration. A multivariable logistic regression model consisting of the CSA-weighted average FI% (total FI%) and the C-GAP score, C-GAPM was constructed. The area under the curve (AUC) of the receiver operating characteristic (ROC) curves was used to evaluate the predictability of the GAP score, the C-GAP score, FI%, and C-GAPM. This project was supported by the National Natural Science Foundation of China (No.82272545) and Special Fund of Science and Technology Plan of Jiangsu Province (No.BE2023658). RESULTS: For all 107 patients, FI% at L1/2, L2/3, L3/4, and L4/5 discs and the total FI% of the MC group (n=32) were significantly higher than those of the non-MC group (n=75). The MC rates of 3 original GAP categories, P, MD, and SD categories were 25.00% (6/24), 27.03%(10/37), and 34.78% (16/46) (χ2=0.944, p=.624). Based on the C-GAP score, the MC rates of the P, MD, and SD categories were 11.90% (5/42), 34.69% (17/49), and 62.50% (10/16), showing significant differences (χ2=15.137, p=.001). In the C-GAP MD category, compared with the non-MC group (n=32), the MC group (n=17) has a higher total FI% (26.16(22.95, 34.00) vs. 22.67(16.39, 27.37)), p=.029). A similar trend was identified in the C-GAP SD category (34.79±11.56 vs. 19.00±5.17, p=.007), but not in the C-GAP P category (25.09(22.82, 32.66) vs. 24.66(17.36, 28.63), p=.361). The AUC of the GAP score, the C-GAP score, the total FI%, and C-GAPM were respectively 0.601, 0.722, 0.716, and 0.772. CONCLUSIONS: Paraspinal muscle degeneration exerts a significant effect on the occurrence of MC in the C-GAP MD, SD instead of P category. The integration of paraspinal muscle FI% with the C-GAP score (C-GAPM) enables a more accurate prediction of MCs following DS surgery. Surgeons should pay adequate attention to paraspinal muscle degeneration during surgical planning and postoperative management for patients in the C-GAP MD and SD categories.

Unfolded proteins in the mitochondria activate HRI and inhibit mitochondrial protein translation.

The mitochondrial unfolded protein response (UPRmt) is triggered through eIF2α phosphorylation in mammals. However, the mechanisms of UPRmt activation and the influence of eIF2α phosphorylation on mitochondrial protein translation remain unclear. In this study, we confirmed that the UPRmt is a rapid and specific stress response that occurs through pharmacological induction of eIF2α phosphorylation, along with the phosphorylation of eIF2α, ATF4, and CHOP. Moreover, with the upregulation of the expression of some chaperones, cytochrome P450 enzymes, and DDIT4, as determined by RNA-Seq and ribosome profiling, eIF2α phosphorylation was found to be essential for the expression of ATF4 and CHOP, after which ATF4 trafficked into the nucleus and initiated CHOP expression. In addition, the generation of ROS and mitochondrial morphology were not affected by the GTPP-induced UPRmt. Furthermore, we investigated the mechanism by which HRI kinase-mediated UPRmt is induced by mitochondrial unfolded proteins via CRISPR-Cas9 technology, mitochondrial recruitment of HRI and interaction with other proteins. Moreover, we confirmed that mitochondrial protein translation and mitochondrial protein import were inhibited through eIF2α phosphorylation with the accumulation of unfolded mitochondrial proteins. These findings reveal the molecular mechanism of the UPRmt and its impact on cellular protein translation, which will offer novel insights into the functions of the UPRmt, including its implications for human disease and pathobiology.

Picornavirus VP3 protein induces autophagy through the TP53-BAD-BAX axis to promote viral replication.

Macroautophagy/autophagy and apoptosis are pivotal interconnected host cell responses to viral infection, including picornaviruses. Here, the VP3 proteins of picornaviruses were determined to trigger autophagy, with the autophagic flux being triggered by the TP53-BAD-BAX axis. Using foot-and-mouth disease virus (FMDV) as a model system, we unraveled a novel mechanism of how picornavirus hijacks autophagy to bolster viral replication and enhance pathogenesis. FMDV infection induced both autophagy and apoptosis in vivo and in vitro. FMDV VP3 protein facilitated the phosphorylation and translocation of TP53 from the nucleus into the mitochondria, resulting in BAD-mediated apoptosis and BECN1-mediated autophagy. The amino acid Gly129 in VP3 is essential for its interaction with TP53, and crucial for induction of autophagy and apoptosis. VP3-induced autophagy and apoptosis are both essential for FMDV replication, while, autophagy plays a more important role in VP3-mediated pathogenesis. Mutation of Gly129 to Ala129 in VP3 abrogated the autophagic regulatory function of VP3, which significantly decreased the viral replication and pathogenesis of FMDV. This suggested that VP3-induced autophagy benefits viral replication and pathogenesis. Importantly, this Gly is conserved and showed a common function in various picornaviruses. This study provides insight for developing broad-spectrum antivirals and genetic engineering attenuated vaccines against picornaviruses.Abbreviations: 3-MA, 3-methyladenine; ATG, autophagy related; BAD, BCL2 associated agonist of cell death; BAK1, BCL2 antagonist/killer 1; BAX, BCL2 associated X, apoptosis regulator; BBC3/PUMA, BCL2 binding component 3; BCL2, BCL2 apoptosis regulator; BID, BH3 interacting domain death agonist; BIP-V5, BAX inhibitor peptide V5; CFLAR/FLIP, CASP8 and FADD like apoptosis regulator; CPE, cytopathic effects; CQ, chloroquine; CV, coxsackievirus; DAPK, death associated protein kinase; DRAM, DNA damage regulated autophagy modulator; EV71, enterovirus 71; FMDV, foot-and-mouth disease virus; HAV, hepatitis A virus; KD, knockdown; MAP1LC3/LC3, microtubule associated protein 1 light chain 3; MOI, multiplicity of infection; MTOR, mechanistic target of rapamycin kinase; PML, promyelocytic leukemia; PV, poliovirus; SVA, Seneca Valley virus; TCID50, 50% tissue culture infectious doses; TOR, target of rapamycin. TP53/p53, tumor protein p53; WCL, whole-cell lysate.

Hypothyroidism is a causal determinant of age-related cataract risk in European population: a Mendelian randomization study.

Objective: To determine whether there is a causal relationship between thyroid dysfunction and the risk of age-related cataract (ARC) in the European population. Design: A two-sample Mendelian randomization (MR) study. Methods: Hypothyroidism, hyperthyroidism, free thyroxine (fT4), and thyrotropin (TSH) were selected as exposures. The single nucleotide polymorphisms (SNP) of hypothyroidism and hyperthyroidism were obtained from the genome-wide association studies (GWAS) of the IEU database, including 337,159 subjects. Data for fT4 and TSH (72,167 subjects) were extracted from the ThyroidOmics Consortium. ARC was used as the outcome. The SNPs associated with ARC were selected from a GWAS of 216,362 individuals in the FinnGen database. The main method used was the inverse variance-weighted method, together with four complementary methods. Sensitivity analyses were performed using Cochran's Q test, MR-PRESSO, MR-Egger regression and leave-one-out test. MR pleiotropy was used to test for pleiotropy. MR Steiger test was used to test for the directionality. Results: Two-sample MR analysis revealed a positive association between genetically predicted hypothyroidism and risk of ARC (OR = 2.501, 95% CI: 1.325-4.720; P = 0.004). Hyperthyroidism, circulating fT4 and TSH levels did not have a significant causal effect on ARC (P > 0.05). The results were robust and reliable, and no horizontal pleiotropy was found after sensitivity analyses. In the MR Steiger test, we found no reverse causal effects of hypothyroidism on the ARC (P <0.001). Conclusions: Our study provides strong evidence that hypothyroidism is a causal determinant of ARC risk.

[Bovine viral diarrhea virus Erns protein expressed in Chinese hamster ovary cells and its immunogenicity analysis].

The aim of this study was to produce Erns protein of bovine viral diarrhea virus (BVDV) by using suspensively cultured CHO cells expression system and to analyze the immunogenicity of the purified Erns protein. In this study, the recombinant eukaryotic expression plasmid pcDNA3.1-BVDV-Erns was constructed based on the gene sequence of BVDV-1 NADL strain. The Erns protein was secreted and expressed in cells supernatant after transfecting the recombinant expression plasmid pcDNA3.1-BVDV-Erns into CHO cells. The expression and purification of the Erns protein was analyzed by SDS-PAGE, the reactivity was determined with anti-His monoclonal antibodies and BVDV positive serum with Western blotting. Immunogenicity analysis of the Erns protein was determined after immunizing New Zealand white rabbits, and the serum antibodies were tested by indirect ELISA (iELISA) and indirect immunofluorescence (IFA). The serum neutralizing titer of the immunized rabbits was determined by virus neutralization test. The concentration of the purified Erns protein was up to 0.886 mg/mL by BCA protein quantification kit. The results showed that the Erns protein could be detected with anti-His monoclonal antibodies and anti-BVDV sera. Serum antibodies could be detected by iELISA on the 7th day post-prime immunization, and the antibody level was maintained at a high titer until the 28th day post-immunization. The antibody titer was 1:128 000. Furthermore, the expression of the Erns protein in BVDV-infected MDBK cells could be detected with immunized rabbits sera by IFA. Moreover, antigen-specific neutralizing antibodies of 2.71 log10 was induced in rabbits. In this study, purified BVDV Erns protein was successfully produced using CHO suspension culture system, and the recombinant protein was proved to have a good immunogenicity, which may facilitate the development of BVD diagnosis method and novel subunit vaccine.

The treatment of a malunited posterior pilon fracture with talar dislocation: A staged surgical treatment protocol.

BACKGROUND: Malunited posterior pilon fractures with talus dislocation (mPPFtd) are rare and there are no appropriate treatment strategies. The purpose of this study was to introduce a stepped strategy featuring preliminary soft tissue management according to the Ilizarov principle and delayed open reduction and internal fixation (ORIF) through a modified posteromedial approach to overcome rigid soft tissue contracture. METHOD: From February 2015 to August 2021, 12 selected patients with mPPFtd who were treated with the staged protocol (Group A) were retrospectively analysed. The clinical and radiographic outcomes were evaluated using the American Orthopaedic Foot and Ankle Society (AOFAS) ankle-hindfoot score, the visual analogue scale (VAS) score, and the Burwell-Charnley score. Moreover, this case series was compared with some cases of fresh fracture (Group B) in patients that had the same baseline data from our previous study. RESULTS: In Group A, the average length of time between the date of injury and the date of surgery was 4.8 ± 3.3 months. The average time to external fixator distraction, as the first-stage treatment, was 13.4 ± 1.0 days. In the second stage of ORIF, the posterosuperior dislocation of the talus was corrected with osteotomy and leverage manoeuvres. According to the Burwell-Charnley score system, the reduction quality was excellent in 9 cases and good in 3 cases. After a mean follow-up of 3.8 ± 2.1 years, there were no infections, wound healing problems, or nerve injuries in our cohort, and union was observed in all fractures without a loss of reduction. The baseline data of the two groups were not significantly different (p>0.05). The mean AOFAS score in Group A was 85.0 ± 10.5 and that in Group B was 95.4 ± 6.1 (p<0.05). The mean VAS score in Group A was 1.7 ± 1.4 and that in Group B was 0.7 ± 0.9 (p<0.05). CONCLUSION: A staged surgical treatment strategy characterized by soft tissue management will improve the treatment of mPPFtd and produce satisfactory clinical outcomes. LEVEL OF EVIDENCE: Level IV, retrospective case series.

XBP1s gene of endoplasmic reticulum stress enhances proliferation and osteogenesis of human periodontal ligament cells.

BACKGROUND: The endoplasmic reticulum stress (ERS) pathway, inositol-requiring enzyme-1 alpha-X-box binding protein-1 (IRE1α-XBP1), has been considered as a critical factor of human periodontal ligament cells (hPDLCs) in proliferation and osteogenesis. This study aimed to explore the effect and mechanism of XBP1s, which was cleaved by IRE1α on the proliferation and osteogenesis of hPDLCs. METHODS: ERS model was induced by tunicamycin (TM); cell proliferation was assessed by CCK-8 assay; pLVX-XBP1s-hPDLCs cell line was established by lentivirus infaction; expression of ERS-related protein including eIF2α, GRP78, ATF4 and XBP1s, autophagy-related P62 and LC3, and apoptosis-related Bcl-2 and Caspase-3 were detected by Western Blot; expression of osteogenic genes was detected by RT-qPCR, and senescence of hPDLCs was explored by ß-galactosidase staining. Furthermore, the interaction between XBP1s and human bone morphogenetic protein 2 (BMP2) was examined by immunofluorescence antibody test (IFAT). RESULTS: The results showed an increase in proliferation of hPDLCs from 0 to 24 h when ERS was induced by TM treatment (P < 0.05). XBP1s overexpression induced hPDLCs proliferation, upgraded autophagy and degraded apoptosis significantly (P < 0.05). In pLVX-XBP1s-hPDLCs, the ratio of senescent cells was markedly decreased after several passages (P < 0.05); After infection with pLVX-BMP2 lentiviral supernatant, IFAT result showed that XBP1s and BMP2 well co-located in the cytoplasm of pLVX-XBP1s-hPDLCs and PERK-ATF4 ERS branch was activated, meanwhile, there were obviously more mineralized nodules and mRNA expression of osteogenesis-related genes was continually up-regulated (P < 0.05). CONCLUSIONS: XBP1s promotes the proliferation via regulating the autophagy and apoptosis, and enhances expression of osteogenic genes in hPDLCs. The mechanisms in this regard need exploring further for periodontal tissue regeneration, functionalization and clinical applications.

Regulation of proliferation and apoptosis of aging periodontal ligament cells by autophagy-related gene 7.

BACKGROUND: Human periodontal ligament cells (hPDLCs) can be applied in periodontal regeneration engineering to repair the tissue defects related to periodontitis. Theoretically, it can affect the vitality of hPDLCs that cell aging increases apoptosis and decreases autophagy. Autophagy is a highly conserved degradation mechanism, which degrades the aging and damaged intracellular organelles through autophagy lysosomes to maintain normal intracellular homeostasis. Meanwhile, autophagy-related gene 7 (ATG7) is a key gene that regulates the level of cellular autophagy. OBJECTIVE: This study was to explore the effects of autophagic regulation of aging hPDLCs on cell proliferation and cell apoptosis. METHODS: A cell model of aging hPDLCs overexpressing and silencing ATG7 were respectively constructed by lentiviral vectors in vitro. A series of experiments was performed to confirm relevant senescence phenotype on aging hPDLCs, and to detect the effects of changes in autophagy on their proliferation and apoptosis-related factors in aging hPDLCs. RESULTS: The results showed that overexpression of ATG7 could motivate autophagy, promoting proliferation of aging hPDLCs and inhibiting apoptosis synchronously (P < 0.05). On the contrary, suppressing autophagy levels by silencing ATG7 would inhibit cell proliferation and accelerate cell senescence (P < 0.05). CONCLUSION: ATG7 regulates the proliferation and apoptosis of aging hPDLCs. Hence, autophagy may act as a target to delay senescence of hPDLCs, which can be helpful in the future in-depth study on regeneration and functionalization of periodontal supporting tissues.

A geospatial platform for the tectonic interpretation of low-temperature thermochronology Big Data.

Low-temperature thermochronology is a powerful tool for constraining the thermal evolution of rocks and minerals in relation to a breadth of tectonic, geodynamic, landscape evolution, and natural resource formation processes through deep time. However, complexities inherent to these analytical techniques can make interpreting the significance of results challenging, requiring them to be placed in their geological context in 4-dimensions (3D + time). We present a novel tool for the geospatial archival, analysis and dissemination of fission-track and (U-Th)/He data, built as an extension to the open-access AusGeochem platform ( https://ausgeochem.auscope.org.au ) and freely accessible to scientists from around the world. To demonstrate the power of the platform, three regional datasets from Kenya, Australia and the Red Sea are placed in their 4D geological, geochemical, and geographic contexts, revealing insights into the tectono-thermal evolutions of these areas. Beyond facilitating data interpretation, the archival of fission track and (U-Th)/He (meta-)data in relational schemas unlocks future potential for greater integration of thermochronology and numerical geoscience techniques. The power of formatting data to interface with external tools is demonstrated through the integration of GPlates Web Service with AusGeochem, enabling thermochronology data to be readily viewed in their paleogeographic context through deep time from within the platform.

Combinational Deletions of MGF110-9L and MGF505-7R Genes from the African Swine Fever Virus Inhibit TBK1 Degradation by an Autophagy Activator PIK3C2B To Promote Type I Interferon Production.

African swine fever (ASF), caused by the African swine fever virus (ASFV), is a transboundary infectious disease of domestic pigs and wild boars, resulting in significant swine production losses. Currently, no effective commercial ASF vaccines or therapeutic options are available. A previous study has shown that deletions of ASFV MGF110-9L and MGF505-7R genes (ASFV-Δ110-9L/505-7R) attenuated virulence in pigs and provided complete protection against parental lethal ASFV CN/GS/2018 (wild-type ASFV [ASFV-WT]) challenge, but the underlying mechanism is unclear. This study found that ASFV-Δ110-9L/505-7R weakened TBK1 degradation compared with ASFV-WT through RNA sequencing (RNA-seq) and Western blotting analyses. Furthermore, we confirmed that ASFV-Δ110-9L/505-7R blocked the degradation of TBK1 through the autophagy pathway. We also identified that the downregulation of an autophagy-related protein PIK3C2B was involved in the inhibition of TBK1 degradation induced by ASFV-Δ110-9L/505-7R. Additionally, we also confirmed that PIK3C2B promoted ASFV-Δ110-9L/505-7R replication in vitro. Together, this study elucidated a novel mechanism of virulence change of ASFV-Δ110-9L/505-7R, revealing a new mechanism of ASF live attenuated vaccines (LAVs) and providing theoretical guidance for the development of ASF vaccines. IMPORTANCE African swine fever (ASF) is a contagious and lethal hemorrhagic disease of pigs caused by the African swine fever virus (ASFV), leading to significant economic consequences for the global pig industry. The development of an effective and safe ASF vaccine has been unsuccessful. Previous studies have shown that live attenuated vaccines (LAVs) of ASFV are the most effective vaccine candidates to prevent ASF. Understanding the host responses caused by LAVs of ASFV is important in optimizing vaccine design and diversifying the resources available to control ASF. Recently, our laboratory found that the live attenuated ASFV-Δ110-9L/505-7R provided complete protection against parental ASFV-WT challenge. This study further demonstrated that ASFV-Δ110-9L/505-7R inhibits TBK1 degradation mediated by an autophagy activator PIK3C2B to increase type I interferon production. These results revealed an important mechanism for candidate vaccine ASFV-Δ110-9L/505-7R, providing strategies for exploring the virulence of multigene-deleted live attenuated ASFV strains and the development of vaccines.

Deletion of MGF-110-9L gene from African swine fever virus weakens autophagic degradation of TBK1 as a mechanism for enhancing type I interferon production.

African swine fever (ASF) caused by African swine fever virus (ASFV) is a devastating disease for the global pig industry and economic benefit. The limited knowledge on the pathogenesis and infection mechanisms of ASF restricts progress toward vaccine development and ASF control. Previously, we illustrated that deletion of the MGF-110-9L gene from highly virulent ASFV CN/GS/2018 strains (ASFV∆9L) results in attenuated virulence in swine, but the underlying mechanism remains unclear. In this study, we found that the difference in virulence between wild-type ASFV (wt-ASFV) and ASFV∆9L strains was mainly caused by the difference in TANK Binding Kinase 1 (TBK1) reduction. TBK1 reduction was further identified to be mediated by the autophagy pathway and this degradative process requires the up-regulation of a positive autophagy regulation molecule- Phosphatidylinositol-4-Phosphate 3-Kinase Catalytic Subunit Type 2 Beta (PIK3C2B). Moreover, TBK1 over-expression was confirmed to inhibit ASFV replication in vitro. In summary, these results indicate that wt-ASFV counteracts type I interferon (IFN) production by degrading TBK1, while ASFVΔ9L enhanced type I IFN production by weakening TBK1 reduction, clarifying the mechanism that ASFVΔ9L present the attenuated virulence in vitro.

Antiviral Effect of Manganese against Foot-and-Mouth Disease Virus Both in PK15 Cells and Mice.

Foot-and-mouth disease (FMD) is an acute contagious disease of cloven-hoofed animals such as cattle, pigs, and sheep. Current emergency FMD vaccines are of limited use for early protection because their protective effect starts 7 days after vaccination. Therefore, antiviral drugs or additives are used to rapidly stop the spread of the virus during FMD outbreaks. Manganese (Mn2+) was recently found to be an important substance necessary for the host to protect against DNA viruses. However, its antiviral effect against RNA viruses remains unknown. In this study, we found that Mn2+ has antiviral effects on the FMD virus (FMDV) both in PK15 cells and mice. The inhibitory effect of Mn2+ on FMDV involves NF-κB activation and up-regulation of interferon-stimulated genes. Animal experiments showed that Mn2+ can be highly effective in protecting C57BL/6N mice from being infected with FMDV. Overall, we suggest Mn2+ as an effective antiviral additive for controlling FMDV infection.

Development of an Effective Double Antigen Sandwich ELISA Based on p30 Protein to Detect Antibodies against African Swine Fever Virus.

African swine fever (ASF), the highly lethal swine infectious disease caused by the African swine fever virus (ASFV), is a great threat to the swine industry. There is no effective vaccine or diagnostic method to prevent and control this disease currently. The p30 protein of ASFV is an important target for serological diagnosis, expressed in the early stage of viral replication and has high immunogenicity and sequence conservatism. Here, the CP204L gene was cloned into the expression vector pET-30a (+), and the soluble p30 protein was successfully expressed in the E. coli prokaryotic expression system and then labeled with horseradish peroxidase (HRP) to be the enzyme-labeled antigen. Using the purified recombinant p30 protein, a double-antigen sandwich ELISA for ASFV antibody detection was developed. This method exhibits excellent specificity, sensitivity and reproducibility in clinical sample detection with lower cost and shorter production cycles. Taken together, this study provides technical support for antibody detection for ASFV.

Sequential Deletions of Interferon Inhibitors MGF110-9L and MGF505-7R Result in Sterile Immunity against the Eurasia Strain of Africa Swine Fever.

African swine fever virus (ASFV) causes significant morbidity and mortality in pigs worldwide. The lack of vaccines or therapeutic options warrants urgent further investigation. To this aim, we developed a rationally designed live attenuated ASFV-Δ110-9L/505-7R mutant based on the highly pathogenic Genotype II ASFV CN/GS/2018 backbone by deleting 2 well-characterized interferon inhibitors MGF110-9L and MGF505-7R. The mutant was slightly attenuated in vitro compared to parental ASFV but highly tolerant to genetic modifications even after 30 successive passages in vitro. Groups of 5 pigs were intramuscularly inoculated with increasing doses of the mutant, ranging from 103 to 106 hemadsorption units (HAD50). Thirty-five days later, all groups were challenged with 102 HAD50 of virulent parental ASFV. All the animals were clinically normal and devoid of clinical signs consistent with ASFV at the period of inoculation. In the virulent challenge, 2 animals from 103 HAD50-inoculated group and 1 animal from 104 HAD50-inoculated group were unprotected with severe postmortem and histological lesions. The rest of animals survived and manifested with relatively normal clinical appearance accompanied by tangible histological improvements in the extent of tissue damage. Meanwhile, antibody response, as represented by p30-specific antibody titers was positively correlated to protective efficacy, potentializing its usage as an indicator of protection. Moreover, compared to 1 dose, 2 doses provided additional protection, proving that 2 doses were better than 1 dose. The sufficiency in effectiveness supports the claim that our attenuated mutant may be a viable vaccine option with which to fight ASF. IMPORTANCE African swine fever virus (ASFV) is a causative agent of acute viral hemorrhagic disease of domestic swine which is associated with significant economic losses in the pig industry. The lack of vaccines or treatment options requires urgent further investigation. ASFV MGF110-9L and MGF505-7R, 2 well-characterized interferon inhibitors, were associated with viral virulence, host range, and immune modulation. In this study, a recombinant two-gene deletion ASFV mutant with deletion of MGF110-9L and MGF505-7R was constructed. The result showed that the mutant was safe, and also highly resistant to genetic modification even after 30 successive passages. High doses of our mutant (105 and 106 HAD50) provided sterile immunity and complete protection in a virulent challenge. Two doses were superior to 1 dose and provided additional protection. This study develops a new ASFV-specific live attenuated vaccine and may be a viable vaccine option against ASF.

Surgical Treatment for Severe Cervical Hyperlordosis and Thoracolumar Kyphoscoliosis with Emery-Dreifuss Muscular Dystrophy: A Case Report and Literature Review.

BACKGROUND: Emery-Dreifuss muscular dystrophy (EDMD) is an uncommon, gradually progressive X-linked myopathy, and it could result in rigid spinal deformity. Only a few case reports have described surgical treatment of cervical hyperlordosis and thoracolumbar kyphoscoliosis secondary to EDMD. We report a rare case of EDMD to present the surgical strategies of severe cervical hyperlordosis and thoracolumbar kyphoscoliosis. CASE PRESENTATION: The patient was a 22-year-old man with EDMD who had severe cervical hyperlordosis and thoracolumbar kyphoscoliosis. A posterior spinal fusion from T9-S2 was performed to correct the thoracolumbar kyphoscoliosis at the age of 21 years. Six months later, with an anterior C7-T1 closing wedge bone-disc-bone osteotomy and a posterior-anterior-posterior cervicothoracic fusion from C4-T4, the cervical deformity was corrected, thus achieving a horizontal gaze. During 1.5-year follow-up, no loss of correction was observed. CONCLUSION: Cervical posterior-anterior-posterior closing-wedge osteotomy combined with long fusion at thoracolumbar spine can be a reliable surgical technique to correct severe spine deformity in EDMD. This two-stage revision surgical strategy can help restore a horizontal gaze on the basis of a balanced trunk. Cervical deformity in such patients should be corrected in the first stage considering its role as a "driver" of the global spine deformity.

A high-precision prediction for spatiotemporal distribution and risk assessment of antibiotics in an urban watershed using a hydrodynamic model.

A methodology for the high-precision prediction and risk assessment of antibiotics at the watershed scale was established. Antibiotic emission inventory and attenuation processes were integrated into the MIKE 11 model to predict the spatiotemporal distribution of norfloxacin, ofloxacin, enrofloxacin, erythromycin, roxithromycin, and sulfamethoxazole in the Nanfei River watershed, China. Considering the variations in antibiotic removal in sewage treatment plants, manure composting, and lagoon systems, the high, medium, and low removal efficiencies of selected antibiotics across China were obtained and used as the best, expected, and worst scenarios, respectively, to evaluate the uncertainty of antibiotic emissions. The predicted concentrations were comparable to antibiotic measurements after flow calibration. The prediction results showed that the highest concentration exposures were mainly concentrated in urban areas with a dense population. Flow variations controlled the temporal distribution characteristics of antibiotics via the dilution effect, and the concentrations of antibiotics in the dry season were 3.1 times higher than those in the wet season. The median concentrations of norfloxacin and erythromycin ranged from 111.36 ng/L to 592.33 ng/L and 106.63 ng/L to 563.01 ng/L, respectively, which both posed a high risk to cyanobacteria and a medium risk to spreading antibiotic resistance. Scenario analysis further demonstrated that high removal efficiencies of these antibiotics can considerably reduce the potential ecotoxicity risks and bacterial resistance selection. The developed methodology for predicting the distribution and risk of antibiotics was suitable for the risk assessment and control strategy of human- and livestock-sourced pollutants.

Research progress on the proteins involved in African swine fever virus infection and replication.

African swine fever (ASF) is an acute, hemorrhagic and highly contagious infectious disease caused by African swine fever virus (ASFV), which infects domestic pigs or wild boars. It is characterized by short course of disease, high fever and hemorrhagic lesions, with mortality of up to 100% from acute infection. Up to now, the lack of commercial vaccines and effective drugs has seriously threatened the healthy economic development of the global pig industry. ASFV is a double-stranded DNA virus and genome varies between about 170-194 kb, which encodes 150-200 viral proteins, including 68 structural proteins and more than 100 non-structural proteins. In recent years, although the research on structure and function of ASFV-encoded proteins has been deepened, the structure and infection process of ASFV are still not clear. This review summarizes the main process of ASFV infection, replication and functions of related viral proteins to provide scientific basis and theoretical basis for ASFV research and vaccine development.

Effect of lentivirus-mediated BMP2 from autologous tooth on the proliferative and osteogenic capacity of human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: Periodontitis is a chronic progressive inflammation that invades periodontal supporting tissues, in which periodontal tissue regeneration engineering offers new hope for prevention and treatment, including seed cells, scaffolds, and growth factors. In recent years, scholars have shown that autologous teeth can be used as new bone tissue repair materials for periodontal regeneration and bone tissue repair. The aim of this study was to establish a human periodontal ligament cell line that expresses the human bone morphogenetic protein 2 gene (BMP2) in a stable manner using lentiviral mediation in order to explore the effect of BMP2 from autologous tooth on the proliferative and osteogenic capacity of human periodontal ligament cells (hPDLCs). MATERIALS AND METHODS: Human periodontal ligament cells were cultured, subcultured, and identified, and then homologous recombinant lentivirus plasmid plv-BMP2 was constructed and transfected into the third passage (P3 ) hPDLCs. After that, the effect of BMP2 on its proliferation was detected by CCK-8, at the same time, the osteogenic induction of hPDLCs was carried out at 7, 14, and 21 days, and then the effect of BMP2 on its osteogenic ability was detected by alizarin red staining, alkaline phosphatase activity determination, and the mRNA expression levels of osteogenic-related genes using real-time fluorescence quantitative PCR, including alkaline phosphatase, runt-related transcription factor 2, bone sialoprotein, osteocalcin, osteopontin, and collagen I. Finally, spss26.0 software was used for statistical processing. RESULTS: The results showed that cells transfected with the homologous recombinant lentiviral plasmid pLV-BMP2 had a similar morphology to normal hPDLCs, showing a typical radial arrangement; the cell proliferative capacity of the pLV-BMP2 group as measured by CCK-8 was enhanced compared with the control group and the pLV-puro group (p < .05); alizarin red staining and alkaline phosphatase activity assay showed that the osteogenic ability of pLV-BMP2 was significantly enhanced compared with the control and pLV-puro groups (p < .01), and the findings of real-time fluorescence-based quantitative PCR showed high expression of osteogenic-related genes in pLV-BMP2 group (p < .01). CONCLUSION: In conclusion, a stable periodontal ligament cell line overexpressing BMP2 was successfully established by a lentivirus-mediated method, which proved that BMP2 has a strong ability to promote the proliferation and osteogenesis of hPDLCs, thereby providing an opportunity for the study of periodontal tissue regeneration as well as providing an experimental basis for the application of autologous teeth as a new type of bone repair material for periodontal therapy and even for maxillofacial bone tissue repair.