Identification and validation of metastasis-related gene ZG16 in the prognosis and progression in colorectal cancer.

Background: Metastasis remains the leading cause of mortality among colorectal cancer (CRC) patients. Identification of new metastasis-related genes are critical to improve colorectal cancer prognosis. Methods: Data on mRNA expression in metastatic and primary CRC was obtained from the Gene Expression Omnibus (GEO) database, including GSE81986, GSE41568, GSE71222, GSE21510, and GSE14333. Additionally, data concerning mRNA expression in colon cancer (COAD) and adjacent normal tissues were acquired from The Cancer Genome Atlas (TCGA) database. Hub genes were identified by weighted gene co-expression network analysis (WGCNA) and differential gene expression analysis. Moreover, we assessed the impact of hub gene expression on both overall survival (OS) and disease-free survival (DFS) in patients and identified ZG16 as a potential target. We generated CRC cell lines transfected with lentivirus OE-ZG16 to investigate proliferation, invasion, and migration in vitro. To further elucidate the involvement of ZG16, we utilized gene set enrichment analysis (GSEA) to identify enriched pathways, which were subsequently validated via Western blot analysis. Results: Five datasets containing primary and metastatic CRC samples from GEO database and CRC samples from TCGA database were included in this study and 29 hub genes were identified by WGCNA and differentially expressed gene (DEG) analysis. Low expression of the hub genes (CLCA1 and ZG16) was associated with poor DFS and OS. We confirmed the low expression of ZG16 in CRC using external database and IHC analysis at both transcriptional and protein levels. In addition, the expression of ZG16 was notably elevated in NCM460 cells in comparison to CRC cell lines. The overexpression of ZG16 in CRC cells has been shown to inhibit the proliferation, invasion, and migration of CRC cells. Furthermore, the overexpression of ZG16 has been found to suppress the activation of the epithelial-mesenchymal transition (EMT) and Wnt/ß-catenin signaling pathways in CRC. Conclusion: ZG16 may serve as a promising therapeutic target for metastatic CRC treatment.

Acid-activated α-MnO2 for photothermal co-catalytic oxidative degradation of propane: Activity and reaction mechanism.

In order to further reduce the energy consumption of the conventional thermal catalytic oxidation system and improve the degradation efficiency of pollutants, photothermal synergistic catalytic oxidation (PTSCO) system was constructed in this paper with propane as simulated pollutant representing VOCs, and then the modified α-MnO2 catalysts were prepared by using the acid activation method, which were used for the catalytic oxidation of propane in PTSCO. The α-MnO2 with appropriate acid concentration possessed excellent low-temperature reducibility, abundant active oxygen species, fast oxygen migration rate and a large number of acid sites. The optimal catalyst, H0.05-MnO2, had a T90 of 204 °C in the PTSCO system, which reduced by more than 30 °C relative to the α-MnO2 (T90 of 235 °C). Moreover, H0.05-MnO2 demonstrated excellent water resistance and long-term stability (T = 45 h). It was shown that the combination of photocatalysis and thermocatalysis can improve propane degradation by examining the kinetics of propane degradation in the PTSCO system and the conformational relationship of propane degradation by catalysts. Furthermore, a multi-pathway synergistic mechanism between photocatalysis and thermocatalysis in the PTSCO system was proposed. This work provided a theoretical basis for the preparation of high-performance catalysts and the catalytic degradation of propane.

A σC-protein-based indirect enzyme-linked immunosorbent assay for clinical detection of antiavian reovirus antibodies.

Avian reovirus (ARV) is the causative agent of avian viral arthritis and causes significant economic losses to the global poultry industry. For clinical diagnosis, detecting ARV-specific antibodies is crucial. We successfully expressed the ARV-σC protein in insect cells using the baculovirus expression vector system, achieving an expression level of approximately 200 mg/L. We developed an indirect enzyme-linked immunosorbent assay (iELISA) using the ARV-σC protein as a coating antigen to detect antibodies against it. The inter-batch and intrabatch coefficients of iELISA variation were less than 10%. Its sensitivity (1:12,800 diluted in serum) was 4 times higher than that of the indirect immunofluorescence assay (IFA; 1:3200 diluted in serum), and it showed no cross-reactivity with antibodies against other common avian viruses (such as Infectious bursal disease virus, Newcastle disease virus). The practicality of the iELISA was further evaluated using clinical samples. 300 clinical sera from chickens vaccinated with the ARV attenuated vaccine and 20 SPF sera were tested using both the iELISA and the IFA, demonstrating a 100% conformity rate. In conclusion, these results suggest that the iELISA developed in this study is a rapid, sensitive, and specific method that could serve as an effective diagnostic tool for monitoring and controlling avian viral arthritis.

Mangosteen extract reduces the bacterial load of eggshell and improves egg quality.

The increasing emergence and spread of antibiotic resistance accelerate the desire for antibiotic alternatives. Plant extracts have emerged as a promising and relatively unexplored area of research as potential substitutes. Herein, we investigated the prevalence and distribution patterns of bacteria on egg surfaces and evaluated the inhibitory effects of mangosteen extract on these surface bacteria. In addition, we examined the antioxidant activity and egg quality in improving the ability of mangosteen extract. The results showed that the predominant bacteria isolated from eggshells were Gram-positive, with Staphylococcus and Micrococcus as the dominant genera. Notably, mangosteen extract exhibited significant bactericidal activity, effectively inhibiting Gram-positive bacteria on the surface of chicken eggshells. Moreover, the supplementation of mangosteen extract in the feed of laying hens yielded a noteworthy improvement in egg quality, accompanied by positively shaped structure and function of microbial communities on the egg surface and in the feces. Collectively, our findings suggested that mangosteen extract was an effective alternative to traditional antibiotics, offering valuable insights for animal husbandry development.

Hematological indicator-based machine learning models for preoperative prediction of lymph node metastasis in cervical cancer.

Background: Lymph node metastasis (LNM) is an important prognostic factor for cervical cancer (CC) and determines the treatment strategy. Hematological indicators have been reported as being useful biomarkers for the prognosis of a variety of cancers. This study aimed to evaluate the feasibility of machine learning models characterized by preoperative hematological indicators to predict the LNM status of CC patients before surgery. Methods: The clinical data of 236 patients with pathologically confirmed CC were retrospectively analyzed at the Gynecology Oncology Department of the First Affiliated Hospital of Bengbu Medical University from November 2020 to August 2022. The least absolute shrinkage and selection operator (LASSO) was used to select 21 features from 35 hematological indicators and for the construction of 6 machine learning predictive models, including Adaptive Boosting (AdaBoost), Gaussian Naive Bayes (GNB), and Logistic Regression (LR), as well as Random Forest (RF), Support Vector Machines (SVM), and Extreme Gradient Boosting (XGBoost). Evaluation metrics of predictive models included the area under the receiver operating characteristic curve (AUC), accuracy, specificity, sensitivity, and F1-score. Results: RF has the best overall predictive performance for ten-fold cross-validation in the training set. The specific performance indicators of RF were AUC (0.910, 95% confidence interval [CI]: 0.820-1.000), accuracy (0.831, 95% CI: 0.702-0.960), specificity (0.835, 95% CI: 0.708-0.962), sensitivity (0.831, 95% CI: 0.702-0.960), and F1-score (0.829, 95% CI: 0.696-0.962). RF had the highest AUC in the testing set (AUC = 0.854). Conclusion: RF based on preoperative hematological indicators that are easily available in clinical practice showed superior performance in the preoperative prediction of CC LNM. However, investigations on larger external cohorts of patients are required for further validation of our findings.

Fast and High-Performance Learned Image Compression With Improved Checkerboard Context Model, Deformable Residual Module, and Knowledge Distillation.

Deep learning-based image compression has made great progresses recently. However, some leading schemes use serial context-adaptive entropy model to improve the rate-distortion (R-D) performance, which is very slow. In addition, the complexities of the encoding and decoding networks are quite high and not suitable for many practical applications. In this paper, we propose four techniques to balance the trade-off between the complexity and performance. We first introduce the deformable residual module to remove more redundancies in the input image, thereby enhancing compression performance. Second, we design an improved checkerboard context model with two separate distribution parameter estimation networks and different probability models, which enables parallel decoding without sacrificing the performance compared to the sequential context-adaptive model. Third, we develop a three-pass knowledge distillation scheme to retrain the decoder and entropy coding, and reduce the complexity of the core decoder network, which transfers both the final and intermediate results of the teacher network to the student network to improve its performance. Fourth, we introduce L1 regularization to make the numerical values of the latent representation more sparse, and we only encode non-zero channels in the encoding and decoding process to reduce the bit rate. This also reduces the encoding and decoding time. Experiments show that compared to the state-of-the-art learned image coding scheme, our method can be about 20 times faster in encoding and 70-90 times faster in decoding, and our R-D performance is also 2.3% higher. Our method achieves better rate-distortion performance than classical image codecs including H.266/VVC-intra (4:4:4) and some recent learned methods, as measured by both PSNR and MS-SSIM metrics on the Kodak and Tecnick-40 datasets.

Synergistic intravesical instillation for bladder cancer: CRISPR-Cas13a and fenbendazole combination therapy.

BACKGROUND: CRISPR-Cas13a is renowned for its precise and potent RNA editing capabilities in cancer therapy. While various material systems have demonstrated efficacy in supporting CRISPR-Cas13a to execute cellular functions in vitro efficiently and specifically, the development of CRISPR-Cas13a-based therapeutic agents for intravesical instillation in bladder cancer (BCa) remains unexplored. METHODS: In this study, we introduce a CRISPR-Cas13a nanoplatform, which effectively inhibits PDL1 expression following intravesical instillation. This system utilizes a fusion protein CAST, created through the genetic fusion of CRISPR-Cas13 and the transmembrane peptide TAT. CAST acts as a potent transmembrane RNA editor and is assembled with the transepithelial delivery carrier fluorinated chitosan (FCS). Upon intravesical administration into the bladder, the CAST-crRNAa/FCS nanoparticles (NPs) exhibit remarkable transepithelial capabilities, significantly suppressing PDL1 expression in tumor tissues.To augment immune activation within the tumor microenvironment, we integrated a fenbendazole (FBZ) intravesical system (FBZ@BSA/FCS NPs). This system is formulated through BSA encapsulation followed by FCS coating, positioning FBZ as a powerful chemo-immunological agent. RESULTS: In an orthotropic BCa model, the FBZ@BSA/FCS NPs demonstrated pronounced tumor cell apoptosis, synergistically reduced PDL1 expression, and restructured the immune microenvironment. This culminated in an enhanced synergistic intravesical instillation approach for BCa. Consequently, our study unveils a novel RNA editor nanoagent formulation and proposes a potential synergistic therapeutic strategy. This approach significantly bolsters therapeutic efficacy, holding promise for the clinical translation of CRISPR-Cas13-based cancer perfusion treatments.

Light footprint of microalgae cultivation system addressed by modified Cornet model.

The cultivation of microalgae is significantly influenced by light intensity and utilization efficiency. This study developed a modified Cornet (M-Cornet) model to assess the distribution of light intensity and flux in microalgae cultivation systems. Algal biofilm cultivation represents a more concentrated approach of algal suspension cultivation. Both follow the M-Cornet model and exhibit the same growth rates when cultured under identical conditions. Algal pigments and morphologies greatly impact the light absorption and scattering, resulting in light attenuation in intensity, penetration distance and light flux distribution. Algae varieties exhibit diverse light flux characteristics. 37% - 90% of the incident light is absorbed, of which, 80% to 90% is dissipated as heat. 10% to 63% of the incident light is scattered off the photobioreactor. The overall light utilization efficiency ranges 6% to 13%. The light footprint using the M-Cornet model offers valuable insights for photobioreactors designing and cultivation operating.

Genome-wide variation study and inter-tissue communication analysis unveil regulatory mechanisms of egg-laying performance in chickens.

Egg-laying performance is of great economic importance in poultry, but the underlying genetic mechanisms are still elusive. In this work, we conduct a multi-omics and multi-tissue integrative study in hens with distinct egg production, to detect the hub candidate genes and construct hub molecular networks contributing to egg-laying phenotypic differences. We identifiy three hub candidate genes as egg-laying facilitators: TFPI2, which promotes the GnRH secretion in hypothalamic neuron cells; CAMK2D, which promotes the FSHß and LHß secretion in pituitary cells; and OSTN, which promotes granulosa cell proliferation and the synthesis of sex steroid hormones. We reveal key endocrine factors involving egg production by inter-tissue crosstalk analysis, and demonstrate that both a hepatokine, APOA4, and an adipokine, ANGPTL2, could increase egg production by inter-tissue communication with hypothalamic-pituitary-ovarian axis. Together, These results reveal the molecular mechanisms of multi-tissue coordinative regulation of chicken egg-laying performance and provide key insights to avian reproductive regulation.

Biomechanical analysis of spinal cord injury during scoliosis correction surgery.

Introduction: Surgical correction is a common treatment for severe scoliosis. Due to the significant spinal deformation that occurs with this condition, spinal cord injuries during corrective surgery can occur, sometimes leading to paralysis. Methods: Such events are associated with biomechanical changes in the spinal cord during surgery, however, their underlying mechanisms are not well understood. Six patient-specific cases of scoliosis either with or without spinal complications were examined. Finite element analyses (FEA) were performed to assess the dynamic changes and stress distribution of spinal cords after surgical correction. The FEA method is a numerical technique that simplifies problem solving by replacing complex problem solving with simplified numerical computations. Results: In four patients with poor prognosis, there was a concentration of stress in the spinal cord. The predicted spinal cord injury areas in this study were consistent with the clinical manifestations of the patients. In two patients with good prognosis, the stress distribution in the spinal cord models was uniform, and they showed no abnormal clinical manifestations postoperatively. Discussion: This study identified a potential biomechanical mechanism of spinal cord injury caused by surgical correction of scoliosis. Numerical prediction of postoperative spinal cord stress distribution might improve surgical planning and avoid complications.

Combined Inhibition of PI3K and STAT3 signaling effectively inhibits bladder cancer growth.

Bladder cancer is characterized by aberrant activation of the phosphatidylinositol-3-OH kinase (PI3K) signaling, underscoring the significance of directing therapeutic efforts toward the PI3K pathway as a promising strategy. In this study, we discovered that PI3K serves as a potent therapeutic target for bladder cancer through a high-throughput screening of inhibitory molecules. The PI3K inhibitor demonstrated a robust anti-tumor efficacy, validated both in vitro and in vivo settings. Nevertheless, the feedback activation of JAK1-STAT3 signaling reinstated cell and organoid survival, leading to resistance against the PI3K inhibitor. Mechanistically, the PI3K inhibitor suppresses PTPN11 expression, a negative regulator of the JAK-STAT pathway, thereby activating STAT3. Conversely, restoration of PTPN11 enhances the sensitivity of cancer cells to the PI3K inhibitor. Simultaneous inhibition of both PI3K and STAT3 with small-molecule inhibitors resulted in sustained tumor regression in patient-derived bladder cancer xenografts. These findings advocate for a combinational therapeutic approach targeting both PI3K and STAT3 pathways to achieve enduring cancer eradication in vitro and in vivo, underscoring their promising therapeutic efficacy for treating bladder cancer.

Extracellular vesicle surface display of αPD-L1 and αCD3 antibodies via engineered late domain-based scaffold to activate T-cell anti-tumor immunity.

Extracellular vesicles (EVs) are emerging as promising carriers for the delivery of therapeutic biologics. Genetic engineering represents a robust strategy for loading proteins of interest into EVs. Identification of EV-enriched proteins facilitates protein cargo loading efficiency. Many EV-enriched proteins are sorted into EVs via an endosomal sorting complex required for transport (ESCRT)-dependent pathway. In parallel, viruses hijack this EV biosynthesis machinery via conserved late domain motifs to promote egress from host cells. Inspired by the similarity of biogenesis between EVs and viruses, we developed a synthetic, Late domain-based EV scaffold protein that enables the display of a set of single chain variable fragments (scFvs) on the EV surface. We named this scaffold the Late domain-based exosomal antibody surface display platform (LEAP). We applied the LEAP scaffold to reprogramme HEK293T cell-derived EVs to elicit T-cell anti-tumor immunity by simultaneously displaying αPD-L1 and αCD3 scFvs on the EV surface (denoted as αPD-L1×αCD3 bispecific T-cell engaging exosomes, BiTExos). We demonstrated that αPD-L1×αCD3 BiTExos actively redirected T cells to bind to PD-L1+ tumor cells, promoting T-cell activation, proliferation and tumoricidal cytokine production. Furthermore, the αPD-L1×αCD3 BiTExos promoted T-cell infiltration into the tumor microenvironment to mitigate the tumor burden in vivo. Our study suggested that the LEAP scaffold may serve as a platform for EV surface display and could be applied for a broad range of EV-based biomedical applications.

Treatment of Leakage Following Sleeve Gastrectomy by Laparo-Endoscopic Gastrostomy (LEG).

Sleeve gastrectomy (SG) is widely recognized as the leading bariatric procedure worldwide. However, leakage, its major complication, remains a significant concern. This study focuses on the challenges of managing leakage, especially when conventional endoscopic treatments are ineffective. Although a novel one-step approach as reported by Pulimuttil James Zachariah from Wei-Jei Lee's team has demonstrated promise, further investigations and reports on its efficacy are currently insufficient. Between January 2021 and November 2023, we analyzed five patients treated at our center for SG leakage. Patient data include demographics, comorbidities, surgical details, and outcomes. The study details Laparo-Endoscopic Gastrostomy procedures performed post-SG leakage diagnosis, highlighting differences between acute and chronic instances. The study effectively implemented Zachariah's one-step approach, achieving favorable results in all five cases. Patient characteristics, presentation, postoperative progression, and additional treatments were documented. The outcome supports Zachariah's assertion that the one-step approach is a simple, safe, and cost-effective approach for SG leakage, avoiding digestive tract reconstruction. Despite potential limitations, including challenges in closing large defects and extended healing times, the procedure's effectiveness in decompression, drainage, and nutritional support significantly contributes to its elevated healing rate. The study emphasizes the importance of timely abdominal drain removal based on clinical conditions, challenging traditional practices for better clinical outcomes.

Genome editing in plants using the TnpB transposase system.

The widely used clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease (Cas) system is thought to have evolved from IS200/IS605 transposons. TnpB proteins, encoded by one type of IS200/IS605 transposon, are considered to be the evolutionary ancestors of Cas12 nucleases, which have been engineered to function as RNA-guided DNA endonucleases for genome editing in bacteria and human cells. TnpB nucleases, which are smaller than Cas nucleases, have been engineered for use in genome editing in animal systems, but the feasibility of this approach in plants remained unknown. Here, we obtained stably transformed genome-edited mutants in rice (Oryza sativa) by adapting three recently identified TnpB genome editing vectors, encoding distinct TnpB nucleases (ISAam1, ISDra2, and ISYmu1), for use in plants, demonstrating that the hypercompact TnpB proteins can effectively edit plant genomes. ISDra2 and ISYmu1 precisely edited their target sequences, with no off-target mutations detected, showing that TnpB transposon nucleases are suitable for development into a new genome editing tool for plants. Future modifications improving the genome-editing efficiency of the TnpB system will facilitate plant functional studies and breeding programs. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-024-00172-6.

Enhanced performance of thermally activated delayed fluorescent light emitting diodes by optimized host polarity.

The interaction between the intrinsic polarity of the host material and the TADF guest material affects charge injection and transport, exciton formation, charge recombination, and emission mechanisms. Therefore, understanding and controlling the interaction between the intrinsic polarity of the host material and the TADF guest material is very important to realize efficient TADF-OLED devices. This study investigated the molecular interaction between different polar host materials and a thermally activated delayed fluorescence material (DMAc-PPM). It has been found that interaction between the host and guest (π-π stacking interaction, multiple CH/π contacts) greatly influence the molecular transition dipole moment orientation of the guest. And the OLED devices based on the strong polar host (DPEPO) exhibited the highest EQEmax and lowest luminescence intensity, while devices using the weaker polar hosts mCP and CBP achieved higher luminance and lower EQEmax. Then, the strong polar host DPEPO was mixed with the weaker polar hosts CBP and mCP, respectively. The devices prepared based on the mixed-host DPEPO: mCP showed a 2.2 times improvement in EQEmax from 6.3% to 20.1% compared to the single-host mCP. The devices prepared based on the mixed-host DPEPO: CBP showed a 3.1 times improvement in luminance intensity from 1023 cd/m2 to 4236 cd/m2 compared to the single host of DPEPO. This suggests that optimizing the polarity of host materials has the potential to enhance the performance of solution prepared OLED devices.

Gga-miR-200a-3p suppresses avian reovirus-induced apoptosis and viral replication via targeting GRB2.

Avian reovirus (ARV) is a significant pathogen that causes various clinical diseases in chickens, including viral arthritis, chronic respiratory diseases, retarded growth, and malabsorption syndrome. These conditions result in substantial economic losses for the global poultry industry. MicroRNAs (miRNAs), a type of small noncoding RNAs that regulate gene expression post transcriptionally by silencing or degrading their RNA targets, play crucial roles in response to pathogenic infections. In this study, transfection of DF-1 cells with gga-miR-200a-3p, an upregulated miRNA observed in ARV-infected cells, significantly suppressed ARV-induced apoptosis by directly targeting GRB2 and impeded ARV replication. Conversely, knockdown of endogenous gga-miR-200a-3p in DF-1 cells using a specific miRNA inhibitor enhanced ARV-induced apoptosis and promoted GRB2 expression, thereby facilitating viral growth within cells. Consistently, inhibition of GRB2 activity through siRNA-mediated knockdown reduced viral titers. Therefore, gga-miR-200a-3p plays a vital antiviral role in the host response to ARV infection by suppressing apoptosis via direct targeting of GRB2 protein. This information enhances our understanding of the mechanisms by which host cells combat against ARV infection through self-encoded small RNA molecules and expands our knowledge regarding the involvement of microRNAs in the host response to pathogenic infections.

Modified pedicle subtraction osteotomy for osteoporotic vertebral compression fractures: a retrospective study of 104 patients.

BACKGROUND: Osteoporotic vertebral compression fractures (OVCF) caused by osteoporosis is a common clinical fracture type. There are many surgical treatment options for OVCF, but there is a lack of comparison among different options. Therefore, we counted a total of 104 cases of OVCF operations with different surgical plans, followed up the patients, and compared the surgical outcome indications before, after and during the follow-up. METHOD: 104 patients who underwent posterior osteotomy (Modified PSO, SPO, PSO, VCR) and kyphosis correction surgery at our hospital between April 2006 and August 2021 with a minimum follow-up period of 24 months were included. All cases were injuries induced by a fall incurred while standing or lifting heavy objects without high-energy trauma. The mean CT value was 71 HU, which was below 110 HU, indicating severe osteoporosis. The indications for surgery included gait disturbance due to severe pain with pseudarthrosis, increased kyphotic angle, and progressive neurological symptoms. Pre- and postoperative CL, TLK, TK, PrTK, TKmax, GK, LL, PI, SS, PT, SVA, TPA, were investigated radiologically. Additionally, We evaluated estimated blood loss, surgical time and perioperative symptom. RESULT: The results show, after operation, TLK (37.32 ± 10.61° vs. 11.01 ± 8.06°, P < 0.001), TK (35.42 ± 17.64° vs. 25.62 ± 12.24°, P < 0.001), TKmax (49.71 ± 16.32° vs. 24.12 ± 13.34°, P < 0.001), SVA (44.91 ± 48.67 vs. 23.52 ± 30.21, P = 0.013), CL (20.23 ± 13.21° vs. 11.45 ± 9.85°, P = 0.024) and TPA (27.44 ± 12.76° vs. 13.91 ± 9.24°, P = 0.009) were improved significantly in modified Pedicle subtraction osteotomy (mPSO) after operation. During follow-up, TLK (37.32 ± 10.61° vs. 13.88 ± 10.02°, P < 0.001) and TKmax (49.71 ± 16.32° vs. 24.12 ± 13.34°, P < 0.001) were improved significantly in Modified PSO group. In additon, estimated blood loss (790.0 ± 552.2 ml vs. 987.0 ± 638.5 ml, P = 0.038), time of operation (244.1 ± 63.0 min vs. 292.4 ± 87.6 min, P = 0.025) were favorable in Modified PSO group compared to control group. CONCLUSION: To conclude, mPSO could acquire a favorable degree of kyphosis correction as well as fewer follow-up complications. Compared with other surgical methods, it also has the advantages of less surgical trauma and shorter operation time. It can be an effective solution for the treatment of OVCF.

Examination of Pyroptosis by Flow Cytometry.

Pyroptosis is an inflammatory type of programmed cell death predominantly driven by the formation of plasma membrane pores by the N-terminus generated from the cleaved Gasdermin (GSDM) family proteins. Examination of membrane-attached GSDM-NT by Western Blot is the most commonly used method for evaluating pyroptosis. However, it is difficult to differentiate cells with pyroptosis from other forms of cell death using this method. In this study, Infectious Bursal Disease Virus (IBDV)-infected DF-1 cells were employed as a model to quantify the proportion of cells undergoing pyroptosis by flow cytometry, utilizing specific antibodies against the N-terminal fragment of chicken GSDME (chGSDME-NT) and propidium iodide (PI) staining. The chGSDME-NT-positive cells were readily detectable by flow cytometry using Alexa Fluor 647-labeled anti-chGSDME-NT antibodies. Moreover, the proportion of chGSDME-NT/PI double-positive cells in IBDV-infected cells (around 33%) was significantly greater than in mock-infected controls (P < 0.001). These findings indicate that examination of membrane-bound chGSDME-NT by flow cytometry is an effective approach for determining pyroptotic cells among cells undergoing cell death.

Generation of a novel attenuated IBDV vaccine strain by mutation of critical amino acids in IBDV VP5.

Infectious bursal disease virus (IBDV) is an acute and highly infectious RNA virus known for its immunosuppressive capabilities, chiefly inflicting rapid damage to the bursa of Fabricius (BF) of chickens. Current clinical control of IBDV infection relies on vaccination. However, the emergence of novel variant IBDV (nVarIBDV) has posed a threat to the poultry industry across the globe, underscoring the great demand for innovative and effective vaccines. Our previous studies have highlighted the critical role of IBDV VP5 as an apoptosis-inducer in host cells. In this study, we engineered IBDV mutants via a reverse genetic system to introduce amino acid mutations in VP5. We found that the mutant IBDV-VP5/3m strain caused reduced host cell mortality, and that strategic mutations in VP5 reduced IBDV replication early after infection, thereby delaying cell death. Furthermore, inoculation of chickens with IBDV-VP5/3m effectively reduced damage to BF and induced neutralizing antibody production comparable to that of parental IBDV WT strain. Importantly, vaccination with IBDV-VP5/3m protected chickens against challenges with nVarIBDV, an emerging IBDV variant strain in China, reducing nVarIBDV loads in BF while alleviating bursal atrophy and splenomegaly, suggesting that IBDV-VP5/3m might serve as a novel vaccine candidate that could be further developed as an effective vaccine for clinical control of IBD. This study provides a new clue to the development of novel and effective vaccines.

TRPV4 mediates IL-1-induced Ca2+ signaling, ERK activation and MMP expression.

Ca2+ permeation through TRPV4 in fibroblasts is associated with pathological matrix degradation. In human gingival fibroblasts, IL-1ß binding to its signaling receptor (IL-1R1) induces activation of extracellular regulated kinase (ERK) and MMP1 expression, processes that require Ca2+ flux across the plasma membrane. It is not known how IL-1R1, which does not conduct Ca2+, generates Ca2+ signals in response to IL-1. We examined whether TRPV4 mediates the Ca2+ fluxes required for ERK signaling in IL-1 stimulated gingival fibroblasts. TRPV4 was immunostained in fibroblasts of human gingival connective tissue and in focal adhesions of cultured mouse gingival fibroblasts. Human gingival fibroblasts treated with IL-1ß showed no change of TRPV4 expression but there was increased MMP1 expression. In mouse, gingival fibroblasts expressing TRPV4, IL-1 strongly increased [Ca2+]i. Pre-incubation of cells with IL-1 Receptor Antagonist blocked Ca2+ entry induced by IL-1 or the TRPV4 agonist GSK101. Knockout of TRPV4 or expression of a non-Ca2+-conducting TRPV4 pore-mutant or pre-incubation with the TRPV4 inhibitor RN1734, blocked IL-1-induced Ca2+ transients and expression of the mouse interstitial collagenase, MMP13. Treatment of mouse gingival fibroblasts with GSK101 phenocopied Ca2+ and ERK responses induced by IL-1; these responses were absent in TRPV4-null cells or cells expressing a non-conducting TRPV4 pore-mutant. Immunostained IL-1R1 localized with TRPV4 in adhesions within cell extensions. While TRPV4 immunoprecipitates analyzed by mass spectrometry showed no association with IL-1R1, TRPV4 associated with Src-related proteins and Src co-immunoprecipitated with TRPV4. Src inhibition reduced IL-1-induced Ca2+ responses. The functional linkage of TRPV4 with IL-1R1 expands its repertoire of innate immune signaling processes by mediating IL-1-driven Ca2+ responses that drive matrix remodeling in fibroblasts. Thus, inhibiting TRPV4 activity may provide a new pharmacological approach for blunting matrix degradation in inflammatory diseases.