Genetic characteristics associated with the virulence of porcine epidemic diarrhea virus (PEDV) with a naturally occurring truncated ORF3 gene.

Porcine epidemic diarrhea virus (PEDV) has emerged in American countries, and it has reemerged in Asia and Europe, causing significant economic losses to the pig industry worldwide. In the present study, the 17GXCZ-1ORF3d strain, which has a naturally large deletion at the 172-554 bp position of the ORF3 gene, together with the 17GXCZ-1ORF3c strain, was serially propagated in Vero cells for up to 120 passages. The adaptability of the two strains gradually increased through serial passages in vitro. Genetic variation analysis of the variants of the two strains from different generations revealed that the naturally truncated ORF3 gene in the 17GXCZ-1ORF3d variants was stably inherited. Furthermore, the survival, viral shedding and histopathological lesions following inoculation of piglets demonstrated that the virulence of 17GXCZ-1ORF3d-P120 was significantly attenuated. These results indicate that the naturally truncated ORF3 gene may accelerate the attenuation of virulence and is involved in PEDV virulence together with mutations in other structural genes. Importantly, immunization of sows with G2b 17GXCZ-1ORF3d-P120 increased PEDV-specific IgG and IgA antibody levels in piglets and conferred partial passive protection against heterologous G2a PEDV strains. Our findings suggest that an attenuated strain with a truncated ORF3 gene may be a promising candidate for protection against PEDV.

Enhancing representation in radiography-reports foundation model: a granular alignment algorithm using masked contrastive learning.

Recently, multi-modal vision-language foundation models have gained significant attention in the medical field. While these models offer great opportunities, they still face crucial challenges, such as the requirement for fine-grained knowledge understanding in computer-aided diagnosis and the capability of utilizing very limited or even no task-specific labeled data in real-world clinical applications. In this study, we present MaCo, a masked contrastive chest X-ray foundation model that tackles these challenges. MaCo explores masked contrastive learning to simultaneously achieve fine-grained image understanding and zero-shot learning for a variety of medical imaging tasks. It designs a correlation weighting mechanism to adjust the correlation between masked chest X-ray image patches and their corresponding reports, thereby enhancing the model's representation learning capabilities. To evaluate the performance of MaCo, we conducted extensive experiments using 6 well-known open-source X-ray datasets. The experimental results demonstrate the superiority of MaCo over 10 state-of-the-art approaches across tasks such as classification, segmentation, detection, and phrase grounding. These findings highlight the significant potential of MaCo in advancing a wide range of medical image analysis tasks.

Long-term expanded hepatic progenitor cells ameliorate D-GalN/LPS-induced acute liver failure through repolarizing M1 macrophage to M2-Like phenotype via activation of the IL-10/JAK2/STAT3 signaling pathway.

Acute liver failure (ALF) is a devastating liver disease characterized by the rapid deterioration of hepatocytes, which causes a series of clinical complications, including hepatic dysfunction, coagulopathy, encephalopathy, and multiorgan failure. Cell-based therapy is a promising alternative as it can bridge patients until their livers regenerate, releasing immunomodulatory molecules to suppress inflammation. This study reports an iPSCs-derived long-term expanded hepatic progenitor cell (LTHepPCs), which can differentiate into hepatocyte-like cells (HLCs) in vivo. When introduced into drug-induced ALF models, LTHepPCs mitigate liver damage by modulating the local immune microenvironment. This is achieved by shifting macrophages/Kupffer cells towards an anti-inflammatory state, resulting in a decrease in the expression of inflammatory cytokines such as TNF-a, IL-1ß, and IL-8, and an increase in the expression of anti-inflammatory cytokines such as IL-10 and ARG-1. In vitro co-culturing of THP-1 or mBMDMs with LTHepPCs suggested that LTHepPCs could activate the anti-inflammatory state of macrophages/Kupffer cells via the IL-10/JAK2/STAT3 signaling pathway. Therefore, LTHepPC transplantation is a promising therapy for ALF patients.

The C-terminal amino acid motifs of NS1 protein affect the replication and virulence of naturally NS-truncated H1N1 canine influenza virus.

The vast majority of data obtained from sequence analysis of influenza A viruses (IAVs) have revealed that nonstructural 1 (NS1) proteins from H1N1 swine, H3N8 equine, H3N2 avian and the correspondent subtypes from dogs have a conserved four C-terminal amino acid motif when independent cross-species transmission occurs between these species. To test the influence of the C-terminal amino acid motifs of NS1 protein on the replication and virulence of IAVs, we systematically generated 7 recombinants, which carried naturally truncated NS1 proteins, and their last four C-terminal residues were replaced with PEQK and SEQK (for H1N1), EPEV and KPEI (for H3N8) and ESEV and ESEI (for H3N2) IAVs. Another recombinant was generated by removing the C-terminal residues by reverse genetics. Remarkably, the ESEI and KPEI motifs circulating in canines largely contributed efficient replication in cultured cells and these had enhanced virulence. In contrast, the avian ESEV motif was only responsible for high pathogenicity in mice. We examined the effects of these motifs upon interferon (IFN) induction. The 7 mutant viruses replicated in vitro in an IFN-independent manner, and the canine SEQK motif was able to induced higher levels of IFN-ß in human cell lines. These findings shed further new light on the role of the four C-terminal residues in replication and virulence of IAVs and suggest that these motifs can modulate viral replication in a species-specific manner.

Comparison of acute hemodynamic effect of prioritizing ventricular resynchronization versus left ventricular filling during optimization of cardiac resynchronization therapy.

BACKGROUND: Targeting the maximal ventricular resynchronization, with the shortest QRS duration (QRSd), is commonly implemented after cardiac resynchronization therapy (CRT). OBJECTIVE: We compared optimization of ventricular resynchronization with optimization of left ventricular (LV) filling during CRT by measuring their acute hemodynamic effects. METHODS: Patients with standard CRT indications, recruited from two centers, underwent biventricular pacing (BVP) and left bundle branch pacing (LBBP). We performed a within-patient comparison of acute hemodynamic response of systolic blood pressure (SBP) at the atrioventricular delay (AVD) with the shortest QRSd against the AVD with the most efficient LV filling. In a validation sub-study, we also performed electrical assessment using QRS area (QRSa) and hemodynamic assessment with the maximum rate of LV pressure rise (dP/dtmax). RESULTS: Thirty patients (age 65 ± 10, 53% male) were recruited. The AVD producing maximal ventricular resynchronization was associated with a significantly shorter QRSd (difference 15 ± 12 ms for BVP and 18 ± 13 ms for LBBP, both P < 0.01) and a significantly smaller improvement in SBP (difference 3 ± 4 mmHg for BVP, and 2 ± 2 mmHg for LBBP, both P < 0.01) compared with the AVD that optimized filling. Similar findings were observed in the sub-study, with a significantly smaller improvement in dP/dtmax assessed with QRSd and QRSa (difference 9 ± 7% and 6 ± 4% during BVP, and 5 ± 6% and 3 ± 3% during LBBP, all P < 0.01). CONCLUSION: Targeting the maximal ventricular resynchronization results in suboptimal acute hemodynamic performance with both BVP and LBBP as CRT. These findings support prioritizing LV filling when programming AVD for CRT.

Sex differences outcomes in conduction system pacing for patients with typical left bundle branch block.

BACKGROUND: Biventricular pacing (BVP) appears to confer more pronounced advantages in women, yet the impact of conduction system pacing (CSP) remains insufficiently characterized. This investigation seeks to elucidate sex-specific disparities in clinical outcomes among patients with typical left bundle branch block (LBBB) undergoing CSP, with a particular focus on assessing contributory factors. METHODS: Consecutive patients diagnosed with nonischemic cardiomyopathy, exhibiting left ventricular ejection fraction (LVEF) ≤ 40%, and manifesting typical LBBB as Strauss criteria, underwent CSP. Subsequent longitudinal monitoring assessed improvements in LVEF and the composite endpoint of mortality or heart failure hospitalization (HFH). RESULTS: Among the included 176 patients, women (n = 84, mean age: 69.5 ± 8.8 years) displayed smaller heart size (LVEDd, 62.0 ± 8.3 mm vs. 64.8 ± 7.9 mm, P = 0.023) and shorter baseline QRSd (163.5 ± 17.7 ms vs. 169.7 ± 15.1 ms; P = 0.013) than men. Of the 171 patients who completed the follow-up, super-response was observed in 120 (70%), with a higher occurrence in women than men (78.3% vs. 62.5%, P = 0.024). The incidence of death or HFH was numerically lower in women (7.1% Vs 13%, Log-rank P = 0.216). Notably, the super-response showed a significant difference in women compared to men at the same electrocardiography and/or echocardiographic parameters value. Mediation analysis between sex and super-response revealed that LVEDd and pQRSd play an intermediary role, with the mediation proportion of 26.07% and 27.98%, respectively. CONCLUSIONS: Women may derive more benefits from CSP, and pQRSd and LVEDd partly drive this difference.

Isolation and identification of a novel porcine-related recombinant mammalian orthoreovirus type 3 strain from cattle in Guangxi Province, China.

The Mammalian orthoreovirus (MRV) infects various mammals, including humans, and is linked to gastrointestinal, respiratory, and neurological diseases. A recent outbreak in Liuzhou, Guangxi, China, led to the isolation of a new MRV strain, GXLZ2301, from fecal samples. This strain replicates in multiple cell lines and forms lattice-like structures. Infected cells exhibit single-cell death and syncytia formation. The virus's titers peaked at 107.2 TCID50/0.1 mL in PK-15 and BHK cells, with the lowest at 103.88 TCID50/0.1 mL in A549 cells. Electron microscopy showed no envelope with a diameter of about 70 nm. Genetic analysis revealed GXLZ2301 as a recombinant strain with gene segments from humans, cows, and pigs, similar to type 3 MRV strains from Italy (2015-2016). Pathogenicity tests indicated that while the bovine MRV strain did not cause clinical symptoms in mice, it caused significant damage to the gut, lungs, liver, kidneys, and brain. The emergence of this MRV strain may pose a threat to the health of animals and humans, and it is recommended that its epidemiology and recombination be closely monitored.

A Comprehensive Analysis of LYAR in Colorectal Cancer: Prognostic Marker and Therapeutic Target.

Background: Colorectal cancer (CRC) is a major global health challenge with a need for new biomarkers and therapeutic targets. This work aimed to investigate the biological mechanisms and clinical value of Ly1 antibody reactive (LYAR) in CRC. Methods: We analyzed LYAR mRNA expression across multiple public databases, including genotype-tissue expression, gene expression omnibus, Oncomine, and the cancer genome atlas, alongside in-house immunohistochemical data to evaluate LYAR protein expression in CRC and non-CRC colorectal tissues. Gene set enrichment analysis (GSEA) was used to elucidate LYAR's biological functions, and its impact on the tumor immune microenvironment was assessed using CIBERSORT, ESTIMATE, and single-cell RNA sequencing techniques. In addition, LYAR's association with clinicopathological features and patient prognosis was explored, and its influence on drug sensitivity was investigated using the Connectivity Map database. Results: LYAR was significantly upregulated in CRC tissues compared with non-CRC colorectal counterparts, associated with altered immune cell composition and enhanced RNA processing, splicing, and cell cycle regulation. High LYAR expression correlated with poor disease-free and overall survival, underscoring its prognostic value. GSEA revealed LYAR's involvement in critical cellular processes and pathways, including DNA repair, cell cycle, and mTORC1 signaling. Correlation analysis identified genes positively and negatively associated with LYAR, leading to the discovery of temsirolimus and WYE-354, mTOR inhibitors, as potential therapeutic agents for CRC. Furthermore, LYAR expression predicted increased sensitivity to cetuximab in RAS wild-type metastatic CRC, indicating its utility as a biomarker for treatment responsiveness. Conclusions: LYAR's upregulation in CRC highlights its potential as a biomarker for prognosis and therapeutic targeting, offering insights into CRC pathology and suggesting new avenues for treatment optimization.

Macrophage OTUD1-CARD9 axis drives isoproterenol-induced inflammatory heart remodelling.

BACKGROUND: Chronic inflammation contributes to the progression of isoproterenol (ISO)-induced heart failure (HF). Caspase-associated recruitment domain (CARD) families are crucial proteins for initiation of inflammation in innate immunity. Nonetheless, the relevance of CARDs in ISO-driven cardiac remodelling is little explored. METHODS: This study utilized Card9-/- mice and reconstituted C57BL/6 mice with either Card9-/- or Otud1-/- marrow-derived cells. Mechanistic studies were conducted in primary macrophages, cardiomyocytes, fibroblasts and HEK-293T cells. RESULTS: Here, we demonstrated that CARD9 was substantially upregulated in murine hearts infused with ISO. Either whole-body CARD9 knockout or myeloid-specific CARD9 deletion inhibited ISO-driven murine cardiac inflammation, remodelling and dysfunction. CARD9 deficiency in macrophages prevented ISO-induced inflammation and alleviated remodelling changes in cardiomyocytes and fibroblasts. Mechanistically, we found that ISO enhances the activity of CARD9 by upregulating ovarian tumour deubiquitinase 1 (OTUD1) in macrophages. We further demonstrated that OTUD1 directly binds to the CARD9 and then removes the K33-linked ubiquitin from CARD9 to promote the assembly of the CARD9-BCL10-MALT1 (CBM) complex, without affecting CARD9 stability. The ISO-activated CBM complex results in NF-κB activation and macrophage-based inflammatory gene overproduction, which then enhances cardiomyocyte hypertrophy and fibroblast fibrosis, respectively. Myeloid-specific OTUD1 deletion also attenuated ISO-induced murine cardiac inflammation and remodelling. CONCLUSIONS: These results suggested that the OTUD1-CARD9 axis is a new pro-inflammatory signal in ISO-challenged macrophages and targeting this axis has a protective effect against ISO-induced HF. KEY POINTS: Macrophage CARD9 was elevated in heart tissues of mice under chronic ISO administration. Either whole-body CARD9 knockout or myeloid-specific CARD9 deficiency protected mice from ISO-induced inflammatory heart remodeling. ISO promoted the assembly of CBM complex and then activated NF-κB signaling in macrophages through OTUD1-mediated deubiquitinating modification. OTUD1 deletion in myeloid cells protected hearts from ISO-induced injuries in mice.

Enhancing the vision-language foundation model with key semantic knowledge-emphasized report refinement.

Recently, vision-language representation learning has made remarkable advancements in building up medical foundation models, holding immense potential for transforming the landscape of clinical research and medical care. The underlying hypothesis is that the rich knowledge embedded in radiology reports can effectively assist and guide the learning process, reducing the need for additional labels. However, these reports tend to be complex and sometimes even consist of redundant descriptions that make the representation learning too challenging to capture the key semantic information. This paper develops a novel iterative vision-language representation learning framework by proposing a key semantic knowledge-emphasized report refinement method. Particularly, raw radiology reports are refined to highlight the key information according to a constructed clinical dictionary and two model-optimized knowledge-enhancement metrics. The iterative framework is designed to progressively learn, starting from gaining a general understanding of the patient's condition based on raw reports and gradually refines and extracts critical information essential to the fine-grained analysis tasks. The effectiveness of the proposed framework is validated on various downstream medical image analysis tasks, including disease classification, region-of-interest segmentation, and phrase grounding. Our framework surpasses seven state-of-the-art methods in both fine-tuning and zero-shot settings, demonstrating its encouraging potential for different clinical applications.

Identification and Full-Length Sequence Analysis of a Novel Recombinant Goat Astrovirus Genotype in Guangxi, China.

Astroviruses are single-stranded, positive-sense RNA viruses capable of infecting humans as well as a wide range of mammalian and avian species, with a length of approximately 6.6-7.7 kb. In this study, 139 goat fecal samples collected from the Guangxi province were used for the RT-PCR detection, and two of these were positive for goat astrovirus, with a positivity rate of 1.44% (2/139). The complete genome sequence of an astrovirus strain and the partial genome sequence of a strain astrovirus, named GX WZ 2023 and GX HC 2023, were amplified and sequenced, and their sequence lengths were 6284 nt and 6213 nt, respectively. Among them, the capsid protein of goat astrovirus GX HC 2023 showed the highest amino acid identity of 95.9% with ovine astrovirus GX, which belonged to the MAstV-2 genotype. However, the closest relative of the GX WZ 2023 strain was found to be the caprine astrovirus Sichuan, with a nucleotide sequence identity of 76.8%. The ORF1ab nonstructural protein of this strain showed the highest amino acid identities of 89.2 and 95.8% with the ovine astrovirus S5.1 and caprine astrovirus G5.1 strains, respectively. However, its ORF2 capsid protein has 68.4% amino acid identity with the bovine astrovirus (BAstV) 16 2021 CHN strain and only 21.9-64% amino acid identity with all available strains of goat astrovirus. The GX WZ 2023 strain was recombined with the Chinese (BAstV 16 2021 CHN) and Japanese bovine strains (BAstV JPN 2015) in the ORF2 region. Therefore, the goat astrovirus GX WZ 2023 is proposed as a new member of the family goat astroviridae based on the species classification criteria of the International Committee on Taxonomy of Viruses. These findings enhance our understanding of the prevalence and genetic evolution of goat astrovirus and provide a scientific basis for future studies of these viruses in other animals.

A recombinant Getah Virus expressing a GFP gene for rapid neutralization testing and antiviral drug screening assay.

Getah virus (GETV) is a re-emerging mosquito-borne RNA virus that induces fever, hind limb edema, swollen submandibular lymph nodes, and urticaria in horses. In pigs, the virus often results in stillbirths among pregnant sows, and neurological symptoms leading to death in piglets. Currently, there are no specific treatments or drugs available for GETV infection. The use of reporter viruses to monitor viral replication and spread in real-time within infected cells and animals provides a powerful tool for targeting antiviral drugs throughout the viral life cycle. Their fluorescence-tracked characteristics greatly facilitate virus neutralization tests (VNTs). In this study, we engineered two recombinant viruses by inserting different reporter protein genes at the 3' end of the structural protein gene, an unreported location that can accommodate exogenous genes. The rGEEiLOV and rGEEGFP viruses demonstrated genetic stability for at least five passages and replicated at a rate similar to that of the parental virus in BHK-21 cells. The rGEEGFP virus facilitated viral neutralization testing. Additionally, we used the reporter virus rGEEGFP to confirm ivermectin, a broad-spectrum antiparasitic agent, as a potential inhibitor of GETV in vitro. Ivermectin appears to inhibit the early replication stages of the virus and can block cell-to-cell viral transmission. In conclusion, rGEEGFP holds significant potential for antiviral screening to identify specific inhibitors against GETV and for use in viral neutralization tests.

The emergence, isolation, and phylogenetic analysis of a closely related human strain of parainfluenza virus 5 from a case of porcine reproductive and respiratory syndrome in China.

Reports of Parainfluenza virus 5 (PIV5) epidemics have been on a global upward trend, with an expanding host range across various animals. In 2020, we isolated a PIV5 strain from a PRRSV-positive serum sample. This strain was named GX2020. Genetic analysis revealed that GX2020 belongs to group A, represented by the AGS strain isolated from a human in the USA. Comparisons of amino acid identity in the coding regions showed that GX2020 had the highest amino acid identity (99.6%) with the AGS strain. The emergence of PIV5 strains genetically similar to human strains in pigs highlights its zoonotic potential and underscores the need for enhanced PIV5 surveillance in the future.

Isolation, identification, and pathogenicity of two novel goose astrovirus from goslings with severe gout in China.

Goose astroviruses (GAstVs) are important pathogens which can cause gout in goslings leading to huge economic losses for the goose farming industry in China. In 2023, an infectious disease characterized by visceral gout broke out in commercial goose farms in Guangxi and Guangdong provinces of China. In this study, two GAstV strains of GXNN and GDCS were successfully isolated from these two disease-ridden goose farms. The complete genomic lengths of these two strains were 7166 bp, and phylogenetic analysis showed that they were both GAstV-2 subtypes. The 3-dimensional structures of the capsid protein were predicted and six characteristic mutation sites at amino acid positions 60, 61, 228, 229, 456 and 523 were found within the strong antigenic regions. A recombination event occurred at 6833-7070 nt between the GAstV TZ03 and Turkey astrovirus CA/00 and this was detected in both the GXNN and GDCS strains. Another recombinant event occurred at 63-2747 nt between the GAstV XT1 and GAstV SDPY and this was detected in the GDCS strain. When 1-day-old goslings were infected with the novel GXNN and GDCS strains, they showed severe visceral gout. This was accompanied by enlarged spleens, liver hemorrhages and urate deposits in the kidneys and ureters and their blood urea nitrogen levels were significantly elevated. The mortality rates of the GXNN- and GDCS-infected groups were pathogenically high at 80 % and 60 %, respectively. These results will promote our understanding of the evolution and epidemic potential of GAstVs in China.

Percutaneous left atrial appendage closure with SeaLA device in non-valvular atrial fibrillation.

OBJECTIVES: The clinical efficacy and safety of a novel left atrial appendage (LAA) occluder of the SeaLA closure system in patients with nonvalvular atrial fibrillation (NVAF) were reported. BACKGROUND: Patients with NVAF are at a higher risk of stroke compared to healthy individuals. Left atrial appendage closure (LAAC) has emerged as a prominent strategy for reducing the risk of thrombosis in individuals with NVAF. METHODS: A prospective, multicenter study was conducted in NVAF patients with a high risk of stroke. RESULTS: The LAAC was successfully performed in 163 patients. The mean age was 66.93 ± 7.92 years, with a mean preoperative CHA2DS2-VASc score of 4.17 ± 1.48. One patient with residual flow >3 mm was observed at the 6-month follow-up, confirmed by TEE. During the follow-up, 2 severe pericardiac effusions were noted, and 2 ischemic strokes were observed. Four device-related thromboses were resolved after anticoagulation treatment. There was no device embolism. CONCLUSIONS: The LAAC with the SeaLA device demonstrates encouraging feasibility, safety, and efficacy outcomes.

Isolation and characterization of Chlamydia felis and its pathogenesis in cats.

Feline upper respiratory tract disease (URTD) is a common but complicated disease that occurs in domestic cats, worldwide. 396 cats in Guangxi Province, China were screened for URTD-associated pathogens from March 2022 to August 2023. Mycoplasma felis was found to be the most prevalent infectious agent with a positivity rate of 24.75 %, followed by feline calicivirus (FCV), Chlamydia felis, feline herpesvirus 1 (FHV-1) and feline influenza A virus (FeIAV) with rates of 15.91, 11.62, 5.56 and 1.52 %, respectively. In particular, C. felis and M. felis were found in 13 of 55 co-infected cats. Of the 46 C. felis-positive samples, one strain, named as GXNN36, was successfully isolated using chicken embryos and it was characterized both in vivo and in vitro. For the cat studies, both high- and low-dose challenged groups showed severe conjunctivitis, accompanied by transient fever and respiratory symptoms. C. felis replicated well in turbinate, trachea and lung tissues with high copy numbers and the infection subsequently spread to the livers, spleens, pancreas, kidneys, hearts and intestines. These findings will help our understanding of the role of C. felis in feline URTD and provide a valuable model to evaluate the efficacy of vaccines and therapeutic remedies in the future.

Hunnivirus structural protein VP2 inhibits beta interferon production by targeting the IRF3 essential modulator.

Water buffalo Hunnivirus (BufHuV) belongs to the family Picornaviridae and is a newly discovered member of the Hunnivirus A genus. It causes intestinal diseases in cattle, mainly lead to subclinical infections, thereby seriously threatening the health of cattle herds. In addition, it can also bring about various clinical disease syndromes which results in severe economic losses to the cattle industry. To date, there have been no reports worldwide on the study of Hunnivirus virus infecting host cells and causing innate immune responses. In this study, we found that interferon treatment effectively blocked BufHuV replication and infection with the virus weakened the host antiviral responses. Inhibiting the transcription of IFN-ß and ISGs induced by either Sendai virus (SeV) or poly(I:C) in MDBK and HCT-8 cells, were dependent on the IRF3 or NF-κB signaling pathways, and this inhibited the activation of IFN-ß promoter by TBK1 and its upstream molecules, RIGI and MDA5. By constructing and screening five BufHuV proteins, we found that VP2, 2 C, 3 C and 3D inhibited the activation of IFN-ß promoter induced by SeV. Subsequently, we showed that VP2 inhibited the activation of IRF3 induced by SeV or poly (I:C), and it inhibited IRF3 activation by inhibiting its phosphorylation and nuclear translocation. In addition, we confirmed that VP2 inhibited the activation of IFNß induced by signaling molecules, MDA5 and TBKI. In summary, these findings provide new insights into the pathogenesis of Hunnivirus and its mechanisms involved in evading host immune responses.

Macrophage-Derived GSDMD Plays an Essential Role in Atherosclerosis and Cross Talk Between Macrophages via the Mitochondria-STING-IRF3/NF-κB Axis.

BACKGROUND: Macrophages play a crucial role in atherosclerotic plaque formation, and the death of macrophages is a vital factor in determining the fate of atherosclerosis. GSDMD (gasdermin D)-mediated pyroptosis is a programmed cell death, characterized by membrane pore formation and inflammatory factor release. METHODS: ApoE-/- and Gsdmd-/- ApoE-/- mice, bone marrow transplantation, and AAV (adeno-associated virus serotype 9)-F4/80-shGSDMD (shRNA-GSDMD) were used to examine the effect of macrophage-derived GSDMD on atherosclerosis. Single-cell RNA sequencing was used to investigate the changing profile of different cellular components and the cellular localization of GSDMD during atherosclerosis. RESULTS: First, we found that GSDMD is activated in human and mouse atherosclerotic plaques and Gsdmd-/- attenuates the atherosclerotic lesion area in high-fat diet-fed ApoE-/- mice. We performed single-cell RNA sequencing of ApoE-/- and Gsdmd-/- ApoE-/- mouse aortas and showed that GSDMD is principally expressed in atherosclerotic macrophages. Using bone marrow transplantation and AAV-F4/80-shGSDMD, we identified the potential role of macrophage-derived GSDMD in aortic pyroptosis and atherosclerotic injuries in vivo. Mechanistically, GSDMD contributes to mitochondrial perforation and mitochondrial DNA leakage and subsequently activates the STING (stimulator of interferon gene)-IRF3 (interferon regulatory factor 3)/NF-κB (nuclear factor kappa B) axis. Meanwhile, GSDMD regulates the STING pathway activation and macrophage migration via cytokine secretion. Inhibition of GSDMD with GSDMD-specific inhibitor GI-Y1 (GSDMD inhibitor Y1) can effectively alleviate the progression of atherosclerosis. CONCLUSIONS: Our study has provided a novel macrophage-derived GSDMD mechanism in the promotion of atherosclerosis and demonstrated that GSDMD can be a potential therapeutic target for atherosclerosis.

Identification of B-cell epitope on the N protein of type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) using monoclonal antibody and construction of epitope-mutated virus.

The escalating epidemic of PRRSV-1 in China has prompted widespread concern regarding the evolution of strains, disparities in pathogenicity to herds, and immunological detection of emerging strains. The nucleocapsid (N) protein, as a highly conserved protein with immunogenic properties in PRRSV, is a subject of intensive study. In this research, the recombinant His-N protein was expressed based on the N gene of PRRSV-1 using a prokaryotic expression system and then administered to BALB/c mice. A cell fusion protocol was implemented between SP2/0 cells and splenocytes, resulting in the successful screening of a monoclonal antibody against the N protein, designated as mAb 2D7, by indirect ELISA. Western Blot analysis and Indirect Immunofluorescence Assay (IFA) confirmed that mAb 2D7 positively responded to PRRSV-1. By constructing and expressing a series of truncated His-fused N proteins, a B-cell epitope of N protein, 59-AAEDDIR-65, was identified. A sequence alignment of two genotypes of PRRSV revealed that this epitope is relatively conserved in PRRSV, yet more so in genotype 1. Cross-reactivity analysis by Western blot analysis demonstrated that the B-cell epitope containing D62Y mutation could not be recognized by mAb 2D7. The inability of mAb 2D7 to recognize the epitope carrying the D62Y mutation was further determined using an infectious clone of PRRSV. This research may shed light on the biological significance of the N protein of PRRSV, paving the way for the advancement of immunological detection and development of future recombinant marker vaccine.