Pyroptosis,a form of programmed cell death primarily mediated by gasdermin D(GSD-MD),plays a crucial role in infections and inflammatory responses.Although the OTU deubiquiti-nase 4(OTUD4)has shown its importance in various cellular processes,its specific function in py-roptosis remains unclear,which necessitates a deeper understanding of its role.This study aims to explore the role of OTUD4 in regulating GSDMD-mediated pyroptosis.Through experiments inclu-ding cell transfection,lactate dehydrogenase(LDH)release assays,co-immunoprecipitation(Co-IP),and Western blot(WB)analysis,we investigated the interaction between OTUD4 and GSD-MD and their influence on GSDMD-mediated pyroptosis.Our results indicated that OTUD4 could bind to GSDMD,significantly reduce Caspase-1-mediated cleavage of GSDMD,and lower protein levels of GSDMD-p30,thereby inhibiting pyroptosis.Conversely,the enzymatically inactive mutant of OTUD4(C45A)exhibited a markedly diminished inhibitory effect on pyroptosis.Furthermore,OTUD4 also demonstrated a significant inhibitory effect on pyroptosis in porcine cells.In conclu-sion,this study reveals that OTUD4 can inhibit GSDMD-p30-mediated pyroptosis through its deu-biquitinating activity,highlighting the critical function of OTUD4 in the regulation of pyroptosis and suggesting a potential target for the development of new therapeutic strategies.

The study aims to investigate the effects of adding different proportions of Codonopsis radix compound crude extracts to the rabbit diet on growth performance,immune status,intesti-nal enzyme activity,structure,and microbial composition.A total of 96 5-week-old New Zealand White rabbits were randomly divided into 4 groups,with 6 replicates per group.The control group(BC)was fed a basal diet,while the experimental groups(CM-H and CM-L)were fed a basal diet supplemented with 1 000 mg/kg and 500 mg/kg of Codonopsis radix compound crude extracts,re-spectively.The antibiotic group(CK)was fed a basal diet supplemented with 300 mg/kg of keto-tifen.The experimental period was 42 days.Blood samples were collected at days 21 and 42,and se-rum biochemical and immune markers were determined.Intestinal segments and contents were col-lected at day 42 for analysis of intestinal health.The results showed that compared with the BC group,the average daily gain,feed-to-gain ratio,and diarrhea rate were significantly higher(P＜0.05)in the CM-H and CM-L groups.The total cholesterol(Tchol)content in the serum was sig-nificantly lower in the CM-H group at day 21 and the CM-L group at day 42(P＜0.05).The high-density lipoprotein(HDL)was significantly higher in the CM-H and CM-L groups than in the CK group at day 42(P＜0.05),and the total protein(TP)in the serum was significantly higher in the CM-H and CM-L groups than in the BC group(P＜0.05).The IgG and IgM levels in the serum were significantly higher in the CM-H and CM-L groups than in the BC group(P＜0.05).In the CM-H and CM-L groups,the content of acetic acid in the colon was significantly higher than that in the BC group(P＜0.05).The content of propionic acid in the colon of the CM-L group was also significantly higher than that in the BC group(P＜0.05).The content of α-amylase in the duode-num,the content of trypsin in the duodenum,the pancreas,and the ileum of the CM-H group were significantly higher than those in the BC group(P＜0.05),and the content of trypsin in the duode-num of the CM-H group was significantly higher than those in the BC group and the CM-L group(P＜0.05).Compared with the BC group,the content of GPX1 in the ileum and jejunum of the CM-L group and the ileum of the CM-H group was significantly increased(P＜0.05),and the length of the villi in the duodenum of the CM-H group was significantly increased(P＜0.05).Compared with the BC group,the expression level of ZO-1 in the ileum of the CM-H group was significantly upregulated(P＜0.05),and the expression level of Claudin in the jejunum of the CM-H group and the CM-L group was significantly higher than that in the CK group(P＜0.05).The high-throughput sequencing results showed that the Sob index was significantly higher in the CM-L group compared to the BC group(P＜0.05).At the phylum level,the Firmicutes and Bacteroid-ota phyla were the main phyla.At the genus level,Akkermansia and Ruminococcus were the main genera.The relative abundance of Papillibacter and Eubacterium_ruminantium_group in the CM-L group was significantly higher than that in the CK group(P＜0.05).In summary,adding a Codonopsis radix compound crude extract to the diet can improve the growth performance,immu-nity,antioxidant capacity,integrity of intestinal mucosal structure,enzyme activity in the intestine,and increase the diversity of microorganisms in the blind intestine when the diet is supplemented with 500 mg/kg of Codonopsis radix compound crude extract.

Pyroptosis,a form of programmed cell death primarily mediated by gasdermin D(GSD-MD),plays a crucial role in infections and inflammatory responses.Although the OTU deubiquiti-nase 4(OTUD4)has shown its importance in various cellular processes,its specific function in py-roptosis remains unclear,which necessitates a deeper understanding of its role.This study aims to explore the role of OTUD4 in regulating GSDMD-mediated pyroptosis.Through experiments inclu-ding cell transfection,lactate dehydrogenase(LDH)release assays,co-immunoprecipitation(Co-IP),and Western blot(WB)analysis,we investigated the interaction between OTUD4 and GSD-MD and their influence on GSDMD-mediated pyroptosis.Our results indicated that OTUD4 could bind to GSDMD,significantly reduce Caspase-1-mediated cleavage of GSDMD,and lower protein levels of GSDMD-p30,thereby inhibiting pyroptosis.Conversely,the enzymatically inactive mutant of OTUD4(C45A)exhibited a markedly diminished inhibitory effect on pyroptosis.Furthermore,OTUD4 also demonstrated a significant inhibitory effect on pyroptosis in porcine cells.In conclu-sion,this study reveals that OTUD4 can inhibit GSDMD-p30-mediated pyroptosis through its deu-biquitinating activity,highlighting the critical function of OTUD4 in the regulation of pyroptosis and suggesting a potential target for the development of new therapeutic strategies.

The study aims to investigate the effects of adding different proportions of Codonopsis radix compound crude extracts to the rabbit diet on growth performance,immune status,intesti-nal enzyme activity,structure,and microbial composition.A total of 96 5-week-old New Zealand White rabbits were randomly divided into 4 groups,with 6 replicates per group.The control group(BC)was fed a basal diet,while the experimental groups(CM-H and CM-L)were fed a basal diet supplemented with 1 000 mg/kg and 500 mg/kg of Codonopsis radix compound crude extracts,re-spectively.The antibiotic group(CK)was fed a basal diet supplemented with 300 mg/kg of keto-tifen.The experimental period was 42 days.Blood samples were collected at days 21 and 42,and se-rum biochemical and immune markers were determined.Intestinal segments and contents were col-lected at day 42 for analysis of intestinal health.The results showed that compared with the BC group,the average daily gain,feed-to-gain ratio,and diarrhea rate were significantly higher(P＜0.05)in the CM-H and CM-L groups.The total cholesterol(Tchol)content in the serum was sig-nificantly lower in the CM-H group at day 21 and the CM-L group at day 42(P＜0.05).The high-density lipoprotein(HDL)was significantly higher in the CM-H and CM-L groups than in the CK group at day 42(P＜0.05),and the total protein(TP)in the serum was significantly higher in the CM-H and CM-L groups than in the BC group(P＜0.05).The IgG and IgM levels in the serum were significantly higher in the CM-H and CM-L groups than in the BC group(P＜0.05).In the CM-H and CM-L groups,the content of acetic acid in the colon was significantly higher than that in the BC group(P＜0.05).The content of propionic acid in the colon of the CM-L group was also significantly higher than that in the BC group(P＜0.05).The content of α-amylase in the duode-num,the content of trypsin in the duodenum,the pancreas,and the ileum of the CM-H group were significantly higher than those in the BC group(P＜0.05),and the content of trypsin in the duode-num of the CM-H group was significantly higher than those in the BC group and the CM-L group(P＜0.05).Compared with the BC group,the content of GPX1 in the ileum and jejunum of the CM-L group and the ileum of the CM-H group was significantly increased(P＜0.05),and the length of the villi in the duodenum of the CM-H group was significantly increased(P＜0.05).Compared with the BC group,the expression level of ZO-1 in the ileum of the CM-H group was significantly upregulated(P＜0.05),and the expression level of Claudin in the jejunum of the CM-H group and the CM-L group was significantly higher than that in the CK group(P＜0.05).The high-throughput sequencing results showed that the Sob index was significantly higher in the CM-L group compared to the BC group(P＜0.05).At the phylum level,the Firmicutes and Bacteroid-ota phyla were the main phyla.At the genus level,Akkermansia and Ruminococcus were the main genera.The relative abundance of Papillibacter and Eubacterium_ruminantium_group in the CM-L group was significantly higher than that in the CK group(P＜0.05).In summary,adding a Codonopsis radix compound crude extract to the diet can improve the growth performance,immu-nity,antioxidant capacity,integrity of intestinal mucosal structure,enzyme activity in the intestine,and increase the diversity of microorganisms in the blind intestine when the diet is supplemented with 500 mg/kg of Codonopsis radix compound crude extract.

The aim of this experiment is to investigate the mechanism of liver cell damage induced by nonylphenol(NP).After establishing an in vitro model of NP induced BRL-3A liver cell injury,changes of oxidative damage markers were evaluated,Fe2+content was determined,the ultrastruc-ture of BRL-3A was observed,and the expression level of iron death marker proteins were deter-mined.The results showed that NP could significantly increase ROS and MDA in BRL-3A cells(P＜0.05);the GSH content,GSH Px activity,and SOD activity were significantly reduced(P＜0.05);the Fe2+content significantly increased(P＜0.05)and showed a dose-dependent effect;the mitochondrial volume of BRL-3A cells significantly decreases,the mitochondrial cristae break or e-ven disappear,and some mitochondria show vacuolization;the expression levels of iron homeostasis related proteins TFR1 and FTH1 were significantly reduced(P＜0.05).The results indicate that ferroptosis is involved in the mechanism of NP induced liver cell damage,which bene-fits for further exploration of NP pathogenesis.

Cadmium is a non-essential,non-degradable heavy metal element,the accumulation of cadmium can cause kidney damage.The purpose of this study was to reveal the ferroptosis mecha-nism induced by cadmium exposure in porcine kidney PK-15 cells.First of all,cell viability of gradi-ent concentration of cadmium chloride(CdCl2)on PK-15 cells was detected by CCK-8 method to screen suitable work concentration of CdCl2.Secondly,PK-15 cells were treated with 10 μmol/L CdCl2 for 3,6 and 12 h.The contents of lactate dehydrogenase(LDH)malondialdehyde(MDA)were detected by colorimetry,the expression of ferroptosis related protein were detected by West-ern blot,and the content of ferrous ion(Fe2+)was detected by fluorescence probe.Finally,PK-15 cells were pretreated with 10 μmol/L HO-1 inhibitor zinc protoporphyrin(ZnPP)for 6 h,and then treated with 10 μmol/L CdCl2 for 12 h.The morphology of PK-15 cells was observed by phase contrast microscope and the expression of ferroptosis related proteins was detected by West-ern blot.The results showed that CdCl2 treatment caused a significant decrease in cell viability.The levels of LDH,MDA and Fe2+,the protein levels of Nrf2,HO-1 and ALOX5,and the expression level of FTH1 protein were significantly decreased in CdCl2 treatment cells(P＜0.01).ZnPP could significantly improve the ferroptosis of PK-15 cells induced by CdCl2 treatment.Compared with CdCl2 treatment cells,the cell morphology was significantly improved,the protein levels of Nrf2,HO-1 and ALOX5 protein were significantly decreased,and the protein levels of FTH1 protein were significantly increased in ZnPP pretreatment group(P＜0.01).The results showed that cad-mium induced iron overload and lipid peroxidation,which result in ferroptosis in PK-15 cells through Nrf2/HO-1 pathway.

Pyroptosis is a programmed death of inflammatory cells triggered by pathogen invasion,dependent on caspase activation,through both classical and non-classical pyroptosis pathways.Cell pyroptosis is related to the occurrence and development of a variety of animal infectious diseases caused by microbial infection.After microorganisms invading,cells are stimulated by pathology-re-lated molecular patterns,causing strong immune response,stimulating inflammatory signaling pathways,and then activating inflammasome,leading to pyroptosis.The immune system has e-volved multiple mechanisms to fight microbial infections and regulate inflammatory responses.The innate immune system,by recognizing microbial molecules in pathogens and responding quickly by producing inflammasome and activating pyroptosis,helps clear pathogens to prevent infection and maintain the normal functioning of the body.A thorough study of the pathogenesis and immune es-cape mechanism of cell pyroptosis in animal infectious diseases will provide a new direction for the treatment of animal infectious diseases.

Porcine kidney cell line(PK-15)with HO-1 gene knockout was constructed by CRISPR/Cas9 system to provide experimental materials for exploring the role and molecular mechanism of HO-1 in cadmium-induced ferroptosis in PK-15 cells.Three sgRNA targeting HO-1 gene were de-signed,synthesized and ligated to lentiviral vector Lenti CRISPRv2.The constructed lentiviral plas-mid was transfected into human embryonic cells(HEK293FT)to obtain recombinant lentivirus,which was used to infect PK-15 cells.The monoclonal cell line with HO-1 knockout was obtained by puromycin screening and limited dilution method.The knockout effect was analyzed by sequencing and Western blot detection.The HO-1 gene knockout cells were treated with 10 μmol/L cadmium chloride(CdCl2).The cell viability was detected by CCK-8 method,the cell morpholo-gy was observed by phase contrast microscope,and the content of ferrous ion(Fe2+)was detected by fluorescence probe.The results showed that sgRNA2 possessed the highest editing efficiency.The base insertion or deletion of HO-1 gene and the frameshift of the target gene occurred in all five knockout cells.Western blot results showed that no expression of HO-1 protein was detected,indicating that the PK-15 cell line with HO-1 gene knockout was successfully constructed.After CdCl2 treatment,compared with the control cells,the cell viability was significantly increased and the Fe2+content was significantly decreased in PK-15 cells with HO-1 gene deletion,indicating that HO-1 gene knockout could significantly alleviate the abnormal iron metabolism caused by cadmium treatment.In conclusion,this study successfully constructed a PK-15 cell line knocking out HO-1 gene by using CRISPR/Cas9 gene editing technique,and confirmed that HO-1 plays an important role in iron metabolism abnormality in PK-15 cells induced by cadmium treatment,which lays a foundation for further study on the regulatory mechanism of HO-1 in ferroptosis in PK-15 cells induced by cadmium exposure.

We aimed to obtain the recombinant aminopeptidase encoded by Listeria monocytogenes (L. monocytogenes) gene lmo1711, and characterized the enzyme. First, the amino acid sequences of Lmo1711 from L. monocytogenes EGD-e and its homologues in other microbial species were aligned and the putative active sites were analyzed. The putative model of Lmo1711 was constructed through the SWISS-MODEL Workspace. Then, the plasmid pET30a-Lmo1711 was constructed and transformed into E. coli for expression of the recombinant Lmo1711. The his-tagged soluble protein was purified using the nickel-chelated affinity column chromatography. With the amino acid-p-nitroaniline as the substrate, Lmo1711 hydrolyzed the substrate to free p-nitroaniline monomers, whose absorbance measured at 405 nm reflected the aminopeptidase activity. The specificity of Lmo1711 to substrates was then examined by changing various substrates, and the effect of metal ions on the catalytic efficiency of this enzyme was further determined. Based on the bioinformatics data, Lmo1711 is a member of the M29 family aminopeptidases, containing a highly conserved catalytic motif (Glu-Glu-His-Tyr-His-Asp) with typical structure arrangements of the peptidase family. The recombinant Lmo1711 with a size of about 49.3 kDa exhibited aminopeptidase activity and had a selectivity to the substrates, with the highest degree of affinity for leucine-p-nitroaniline. Interestingly, the enzymatic activity of Lmo1711 can be activated by Cd²⁺, Zn²⁺, and is strongly stimulated by Co²⁺. We here, for the first time demonstrate that L. monocytogenes lmo1711 encodes a cobalt-activated aminopeptidase of M29 family.

Fumonisin B1 (FB1) is a carcinogenic mycotoxin found in commodities such as corn and corn-originated products. An aptamer-based method for detection of FB1 was developed using the fluorescent dye PicoGreen, which can recognize and bind double-stranded DNA. A peak fluorescence of PicoGreen was obtained in 15 min in the presence of FB1 aptamer, which formed a double-stranded hybridizer DNA with its complementary strand. The excitation and emission wavelengths for PicoGreen detection were 480 nm and 520 nm, respectively. The sensitivity of this aptamer/PicoGreen-based method was 0.1 μg/L. This method showed a good linearity for FB1 concentration ranging from 0.1 to 1 μg/L. The entire detection procedure for FB1 could be completed within 40 min. No cross reactions were observed with any other mycotoxins against aflatoxin B1, ochratoxin A, citrinin and zearalenone, demonstrating high specificity towards FB1 aptamer. Agreement between commercial, antibody-based enzyme-linked immunosorbent assay (ELISA) kit and aptamer method was excellent with a kappa value of 0.857. Taken together, this aptamer/PicoGreen-based method is more cost-effective, time-saving and useful than ELISA for detection of FB1.
Aflatoxin B1 ; Enzyme-Linked Immunosorbent Assay ; Fluorescence ; Fluorescent Dyes ; chemistry ; Fumonisins ; analysis ; Mycotoxins ; analysis ; Ochratoxins ; Organic Chemicals ; chemistry ; Staining and Labeling ; Zea mays