Objective To profile ubiquitination in cysteinyl aspartate-specific proteinase-2(CASP2)deficient cells under heat shock and investigate the role of CASP2 in stress response.Methods Ubiquitination levels in subcellular fractions of control and C ASP2 knockout(KO)cells were detected via Western blotting.After 2 hours of heat shock treatment,Soluble Ⅱ and Pellet fractions were collected from both control and CASP2 KO cells for ubiquitinome analysis.Anti-di-glycine remnant(K-ε-GG)antibody-based proteomic analysis was performed to identify differentially ubiquitinated proteins and associated key signaling pathways.Proteins that displayed significantly upregulated ubiquitination in CASP2 KO cells under heat shock were subjected to His-tag pull-down assays to find out whether CASP2 regulated the ubiquitination of these proteins.Results Under heat shock,CASP2 KO cells displayed significantly higher accumulation of overloaded ubiquitinated conjugates in the Pellet fraction compared to controls.Ubiquitinomics analysis revealed substantial alterations in protein ubiquitination patterns following CASP2 KO.One hundred proteins exhibited significantly elevated ubiquitination levels while 36 proteins had their ubiquitination reduced relative to controls.Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway enrichment analysis indicated that hyper-ubiquitinated proteins were primarily associated with Huntington disease,Alzheimer disease,bile secretion,carbon metabolism and autophagy.His-tag pull-down assays combined with Western blotting revealed increased ubiquitination of nicotinamide adenine dinucleotide reduced-ubiquinone oxidoreductase 1 beta subcomplex subunit 3(NDUFB3)and autophagy-related protein 9A(ATG9A)in CASP2 KO cells under heat shock.Conclusion Overloaded ubiquitinated conjugates are accumulated due to CASP2 deficiency during heat shock.CASP2 modulates ubiquitination levels through multiple signaling pathways.

Objective To explore the characteristics and mechanism of phase separation between TAR DNA binding protein-43(TDP-43)and ubiquitin.Methods The TARDBP gene and its truncated genes were inserted into vectors to construct recombinant plasmids for expression and protein purification.The phase separation system of ubiquitin and TDP-43 was constructed in vitro.The characteristics of the droplets formed via liquid-liquid phase separation were observed by fluorescence microscopy.The plasmids of ubiquitin and TDP-43 were co-transfected into HEK293T cells to observe aggregates containing TDP-43 and ubiquitin and find out whether TDP-43 could be ubiquitinated.Results The GFP-8Ub,TDP-43 full-length(FL)and truncated proteins were purified.TDP-43 FL and C-terminal domain(CTD)proteins were able to form droplets via phase separation with ubiquitin.The droplets changed into solid-like aggregates after prolonged incubation.Insolvable aggregates containing TDP-43 and ubiquitin were formed.TDP-43 was ubiquitinated under stress conditions in HEK293T cells after being co-transfected with ubiquitin and TDP-43 recombinant plasmids.Conclusion TDP-43 undergoes co-phase separation with ubiquitin,mainly driven by the multivalent interaction between TDP-43′s CTD structural domain and ubiquitin.The droplets finally form aggregates with solid-like properties.Under stress conditions,especially when the protein homeostasis is disrupted,TDP-43 and ubiquitin form aggregates while TDP-43 is ubiquitinated.This study reveals the basic mechanism of TDP-43 co-phase separation with ubiquitin and liquid-solid transformation.

Background: The incidence of inflammatory bowel disease (IBD) is increasing in recent years, and the etiology and pathogenesis of IBD remain unclear.Studies showed that disorder of bile acid metabolism plays an important role in the pathological process of experimental colitis.However, serum bile acid profile in IBD patients has not been reported.Aims: To investigate the changes of serum bile acid profile in patients with IBD.Methods: Seven healthy controls, 15 patients with ulcerative colitis (UC) and 16 patients with Crohn's disease (CD) at Shanghai Xin Hua Hospital were enrolled.High-performance liquid chromatography-mass spectrometry was used to determine serum bile acid profile.Results: No significant differences in serum concentrations of primary bile acid cholic acid (CA), chenodeoxycholic acid (CDCA), glycocholic acid (GCA), taurocholic acid (TCA), glycochenodeoxycholic acid (GCDCA) were found between UC or CD and controls (P>0.05).Compared with controls, serum concentration of secondary bile acid deoxycholic acid (DCA) in UC patients was significantly decreased (P<0.05), glycodeoxycholic acid (GDCA), taurodeoxycholic acid (TDCA) in CD patients were significantly decreased (P<0.05), serum concentration of lithocholic acid (LCA) in UC and CD patients was significantly decreased (P<0.05).Conclusions: The serum bile acid profile in IBD patients is significantly changed, which suggests that it may be involved in the pathological process of IBD.

Objective To evaluate the diagnostic value of computed tomography enteroclysis/enterography (CTE),magnetic resorance enteroclysis/enterography (MRE) and ultrasonography (US) in Crohn's disease (CD) by Meta-analysis.Methods Segmental intestinal wall thickening and strengthening was taken as image diagnostic indicators in CD.Endoscopic and histopathologic findings and follow-up results were set as diagnostic standard.Retrieve and literature inclusion criteria were developed.The papers of CD diagnosed by CTE,MRE and US which met the criteria were searched and screened.The quality of the papers was evaluated by quality assessment of diagnostic accuracy studies (QUADAS).The diagnostic efficacy of CTE,MRE and US was compared,which included sensitivity,specificity,positive likelihood ratio and negative likelihood ratio,positive predictive value (PPV) and negative predictive value (NPV).Results Among the 2197 retrieved literature,a total of 12 papers met the criteria and nine of which were blinded study.The papers were grouped by three different imaging techniques and underwent QUADAS scale scoring.One paper was about CTE and MRE comparative study,so there were six papers in CTE group,the scores of all the six papers were over 10 and all of them were blinded study.There were four papers in MRE group,the scores of three papers were over 10 and all of them were blinded study.There were three papers in US group,the score of one paper was over 10 and two of these three papers were blinded study and one with unclear evaluation.The sensitivity of CTE group was the highest (89 ％),while the specificity (90 ％) was lower than that of US group (95％).The PPV (93％),NPV (95％),positive likelihood ratio (15.16) and negative likelihood ratio (0.17) of US group were the highest,those of CTE were secondary (PPV 91％,NPV 82％,positive likelihood ratio 6.25 and negative likelihood ratio 0.15).The sensitivity (74％),specificity (84％),PPV (80％),NPV (80％),positive likelihood ratio (4.18) and negative likelihood ratio (0.33) of MRE group were lower than both US group and CTE group.Conclusions The sensitivity of CTE in CD diagnosis is better than that of MRE and US,while other diagnostic indicators are poorer than US.Even though US has high diagnostic value in CD,more well designed large sample study were still needed for further evaluation.