ObjectiveTo investigate the effect of laminin subunit α3 （LAMA3） on the epithelial-mesenchymal transition （EMT）， invasion， and metastasis abilities of pancreatic cancer （PC）. MethodsA comprehensive analysis was performed for tumor- and EMT-related databases to identify the EMT genes associated with PC， especially LAMA3. The methods of qRT-PCR and Western blot were used to measure the expression level of LAMA3 in PC tissue and cell lines； immunofluorescence assay was used to determine the localization of LAMA3 in PANC-1 cells； Transwell assay was used to investigate the effect of LAMA3 on the invasion and migration abilities of PC cells. The t-test was used for comparison of continuous data between groups. ResultsThe analysis of the TCGA database identified 3 EMT-related oncogenes for PC， i.e.， LAMA3， AREG， and SDC1. The LASSO-Cox regression model showed that LAMA3 had the most significant impact on the prognosis of PC （risk score=0.256 1×LAMA3+0.043 1×SDC1+0.071 4×AREG）. The Cox model and nomogram showed that the high expression of LAMA3 was an independent risk factor for the poor prognosis of PC （hazard ratio=1.32， 95% confidence interval： 1.07‍ ‍—‍ 1.62， P<0.01）. Experimental results showed that there was a significant increase in the expression of LAMA3 in pancreatic cancer tissue compared with the normal pancreatic tissue. Compared with the HPDE cell line， there were varying degrees of increase in the expression of LAMA3 in pancreatic cancer AsPC-1， BxPC-3， PANC-1， MIA PaCa-2， and SW1990 cell lines， with the highest expression level in PANC-1 cells. The enrichment analysis showed that LAMA3 was associated with the biological processes and signaling pathways such as EMT， collagen metabolism， extracellular matrix degradation， the TGF-β pathway， and the PI3K pathway. After the knockdown of LAMA3， there were significant reductions in the expression levels of N-Cadherin， Vimentin， and Snail， while there was a significant increase in the expression level of E-Cadherin. Transwell assay showed that there were significant reductions in the invasion and migration abilities of PANC-1 cells after the knockdown of LAMA3. ConclusionLAMA3 is highly expressed in PC and can promote the EMT， invasion， and migration of PC cells， and therefore， LAMA3 may be used as a novel diagnostic marker and a new therapeutic target for PC.

Background/Aims: Metabolic dysfunction–associated steatotic liver disease (MASLD) is a chronic liver disease characterized by hepatic steatosis. Ubiquitin-specific protease 29 (USP29) plays pivotal roles in hepatic ischemiareperfusion injury and hepatocellular carcinoma, but its role in MASLD remains unexplored. Therefore, the aim of this study was to reveal the effects and underlying mechanisms of USP29 in MASLD progression. Methods: USP29 expression was assessed in liver samples from MASLD patients and mice. The role and molecular mechanism of USP29 in MASLD were assessed in high-fat diet-fed and high-fat/high-cholesterol diet-fed mice and palmitic acid and oleic acid treated hepatocytes. Results: USP29 protein levels were significantly reduced in mice and humans with MASLD. Hepatic steatosis, inflammation and fibrosis were significantly exacerbated by USP29 deletion and relieved by USP29 overexpression. Mechanistically, USP29 significantly activated the expression of genes related to fatty acid β-oxidation (FAO) under metabolic stimulation, directly interacted with long-chain acyl-CoA synthase 5 (ACSL5) and repressed ACSL5 degradation by increasing ACSL5 K48-linked deubiquitination. Moreover, the effect of USP29 on hepatocyte lipid accumulation and MASLD was dependent on ACSL5. Conclusions USP29 functions as a novel negative regulator of MASLD by stabilizing ACSL5 to promote FAO. The activation of the USP29-ACSL5 axis may represent a potential therapeutic strategy for MASLD.

Background/Aims: Metabolic dysfunction–associated steatotic liver disease (MASLD) is a chronic liver disease characterized by hepatic steatosis. Ubiquitin-specific protease 29 (USP29) plays pivotal roles in hepatic ischemiareperfusion injury and hepatocellular carcinoma, but its role in MASLD remains unexplored. Therefore, the aim of this study was to reveal the effects and underlying mechanisms of USP29 in MASLD progression. Methods: USP29 expression was assessed in liver samples from MASLD patients and mice. The role and molecular mechanism of USP29 in MASLD were assessed in high-fat diet-fed and high-fat/high-cholesterol diet-fed mice and palmitic acid and oleic acid treated hepatocytes. Results: USP29 protein levels were significantly reduced in mice and humans with MASLD. Hepatic steatosis, inflammation and fibrosis were significantly exacerbated by USP29 deletion and relieved by USP29 overexpression. Mechanistically, USP29 significantly activated the expression of genes related to fatty acid β-oxidation (FAO) under metabolic stimulation, directly interacted with long-chain acyl-CoA synthase 5 (ACSL5) and repressed ACSL5 degradation by increasing ACSL5 K48-linked deubiquitination. Moreover, the effect of USP29 on hepatocyte lipid accumulation and MASLD was dependent on ACSL5. Conclusions USP29 functions as a novel negative regulator of MASLD by stabilizing ACSL5 to promote FAO. The activation of the USP29-ACSL5 axis may represent a potential therapeutic strategy for MASLD.

Background/Aims: Metabolic dysfunction–associated steatotic liver disease (MASLD) is a chronic liver disease characterized by hepatic steatosis. Ubiquitin-specific protease 29 (USP29) plays pivotal roles in hepatic ischemiareperfusion injury and hepatocellular carcinoma, but its role in MASLD remains unexplored. Therefore, the aim of this study was to reveal the effects and underlying mechanisms of USP29 in MASLD progression. Methods: USP29 expression was assessed in liver samples from MASLD patients and mice. The role and molecular mechanism of USP29 in MASLD were assessed in high-fat diet-fed and high-fat/high-cholesterol diet-fed mice and palmitic acid and oleic acid treated hepatocytes. Results: USP29 protein levels were significantly reduced in mice and humans with MASLD. Hepatic steatosis, inflammation and fibrosis were significantly exacerbated by USP29 deletion and relieved by USP29 overexpression. Mechanistically, USP29 significantly activated the expression of genes related to fatty acid β-oxidation (FAO) under metabolic stimulation, directly interacted with long-chain acyl-CoA synthase 5 (ACSL5) and repressed ACSL5 degradation by increasing ACSL5 K48-linked deubiquitination. Moreover, the effect of USP29 on hepatocyte lipid accumulation and MASLD was dependent on ACSL5. Conclusions USP29 functions as a novel negative regulator of MASLD by stabilizing ACSL5 to promote FAO. The activation of the USP29-ACSL5 axis may represent a potential therapeutic strategy for MASLD.

Objective:To analyze the normal expression levels of different lung cancer tumor markers(TM)in the cerebrospinal fluid and to explore the influence of serum TM levels and brain parenchymal metastasis,to more accurately determine whether the cerebrospinal fluid TM levels of patients with suspected meningeal metastasis is elevated.Methods:The clinical data of 80 patients diagnosed with non-lepto-meningeal metastasis at Tianjin Medical University Cancer Hospital between January 2015 and February 2024 were collected,including 16 patients without lung cancer and 64 patients with lung cancer.Normal TM levels in the cerebrospinal fluid of patients without lung cancer and the difference in TM levels between the cerebrospinal fluid and serum samples were analyzed.The correlation between serum and cerebrospinal fluid TM levels was also analyzed.We then compared the differences in TM levels in the cerebrospinal fluid between groups with brain parenchymal metastasis and without brain parenchymal metastasis.Results:Normal levels of TPSA,CA19-9,CEA,Cyfra21-1,and SCC in the cerebrospinal fluid were lower than those in the serum(P<0.05);however,the levels of ProGRP and NSE in the cerebrospinal fluid were higher than those in the serum(P<0.05).The levels of TPSA,SCC,ProGRP,NSE,CEA,CA19-9,and Cyfra21-1 in the cerebrospinal fluid did not correlate with those in the serum(all P>0.05).The cerebrospinal fluid levels of TPSA,SCC,ProGRP,and CA19-9 were not significantly increased in patients with brain parenchymal metastasis compared to those in patients without brain parenchymal metastasis(P>0.05).Al-though CEA and Cyfra21-1 levels increased(P<0.05),their median values increased by less than 2 times and were all within the reference range;whereas,the level of NSE in the group with brain parenchymal metastasis was lower than that in the control group.Conclusions:The basal levels of ProGRP and NSE in normal cerebrospinal fluid were significantly higher than those in the serum;whereas,the expression levels of other TM in the cerebrospinal fluid were significantly lower than those in the serum.Whether the levels of TM in the serum were elevated and whether brain parenchymal metastasis was present,did not have a clinically significant impact on the TM levels in the cerebrospinal fluid.

Sucrose synthase plays a crucial role in the plant sugar metabolism pathway by catalyzing the production of uridine diphosphate (UDP)-glucose, which serves as a bioactive glycosyl donor for various metabolic processes. In this study, a sucrose synthase gene named CtSus was cloned from Cistanche tubulosa, a traditional Chinese medicine known for its rich content of diverse glycosides, based on transcriptome analysis results. The open reading frame of CtSus was 2 418 bp long encoding 805 amino acids. Sequence analysis revealed conserved domains associated with the plant sucrose synthase family at both the N-terminal and C-terminal regions of CtSus protein. Phylogenetic analysis demonstrated that CtSus shares a close evolutionary relationship with sucrose synthases from other plants within the same order. Functional identification of CtSus was performed through whole-cell catalysis coupling with UGT71BD1, a characterized glycosyltransferase enzyme. The results showed that the introduction of CtSus significantly enhanced the conversion rate of glycosylation catalyzed by UGT71BD1. The soluble expression of CtSus in Escherichia coli was further achieved using the pCold^TM TF expression vector. In vitro enzymatic assay indicated the activity of CtSus to catalyze the formation of UDP-glucose in the presence of sucrose and UDP. Real-time quantitative-polymerase chain reaction results showed that the expression patterns of CtSus gene in different parts of C. tubulosa and the suspension cell cultures of C. tubulosa under drought stress correlated with the accumulation patterns observed for phenylethanol glycosides, respectively. Furthermore, key amino acids and their interactions between enzyme and substrate were explored based on protein structure prediction and molecular docking results. Overall, our findings identify a sucrose synthase CtSus responsible for the supply of the active glycosyl donor UDP-glucose during the biosynthesis of glycoside products in C. tubulosa and have also provided a gene element that can be utilized in engineering strain construction for glycoside products production.

To develop antimicrobials against Staphylococcus aureus by high throughput screening of drug library. The type of this study is experimental research. The clinical isolates of S. aureus were collected from the sputum samples of respiratory inpatient department of the Third Xiangya Hospital of Central South University. The anti-planktonic cells growth inhibition activity of FDA-approved drugs library (including 1 573 molecules) was assessed by building a planktonic cells screening platform; The biofilm inhibitory effect of the FDA-approved drugs was detected by building a biofilm screening platform combined with crystal violet staining; Minimal inhibitory concentrations of the selected hits were determined by broth microdilution assay. Finally, the cytotoxicity of the selected hits was detected by CCK-8 assay. The results showed that 218 hits were exhibited effective growth inhibitory effects against S. aureus by setting the concentrations of the molecules in the FDA-approved library to 100 μmol/L. These selected molecules are mainly anti-infective drugs, accounting for 118 hits; Followed by anti-cancer drugs, anti-inflammatory/-immune drugs, neurological drugs, cardiovascular drugs, endocrine drugs, and metabolic disease drugs, which accounts for 40, 19, 12, 9, 8, and 3 hits; Other unclassified drugs accounts for 9 hits. The top 10 hits exhibiting anti-planktonic cells activity against S. aureus were mainly including antitumor drugs, followed by neurological drugs and unclassified drugs like vitamin K3 with the inhibition rate of 99.65%-100%. Similarly, the top 10 hits showing biofilm inhibitory effects against S. aureus were also mainly including antitumor drugs, followed by neurological drugs and anti-inflammatory/-immune drugs with the inhibition rate of 50.22%-92.95%. The minimal inhibitory concentration (MIC) of the 51 hits by second round screening was determined by micro-dilution assay, which mainly include the antitumor drugs, cardiovascular drugs, endocrine drugs, anti-inflammatory/-immune drugs, metabolic disease drugs, neurological drugs and other unclassified drugs accounted for 22, 5, 3, 9, 2, 5 and 5 hits, respectively, with the MICs of 1.56-50 μmol/L, 6.25-25 μmol/L, 6.25-25 μmol/L, 0.2-50 μmol/L, 25-50 μmol/L, 1.56-50 μmol/L and 0.1-12.5 μmol/L, respectively. In conclusion, the minimum inhibitory concentrations of small molecules screened through high-throughput assay are at the level of micromolar with strong drug development potential and high modifiability. The high effective anti-planktonic cells and anti-biofilm activity by these molecules are expected to provide new ideas for the development of new antimicrobials against S. aureus.
Humans ; Staphylococcus aureus ; Anti-Bacterial Agents/pharmacology* ; High-Throughput Screening Assays ; Staphylococcal Infections ; Anti-Infective Agents/pharmacology* ; Microbial Sensitivity Tests ; Biofilms ; Antineoplastic Agents/pharmacology* ; Anti-Inflammatory Agents/pharmacology* ; Cardiovascular Agents/pharmacology* ; Metabolic Diseases

To develop antimicrobials against Staphylococcus aureus by high throughput screening of drug library. The type of this study is experimental research. The clinical isolates of S. aureus were collected from the sputum samples of respiratory inpatient department of the Third Xiangya Hospital of Central South University. The anti-planktonic cells growth inhibition activity of FDA-approved drugs library (including 1 573 molecules) was assessed by building a planktonic cells screening platform; The biofilm inhibitory effect of the FDA-approved drugs was detected by building a biofilm screening platform combined with crystal violet staining; Minimal inhibitory concentrations of the selected hits were determined by broth microdilution assay. Finally, the cytotoxicity of the selected hits was detected by CCK-8 assay. The results showed that 218 hits were exhibited effective growth inhibitory effects against S. aureus by setting the concentrations of the molecules in the FDA-approved library to 100 μmol/L. These selected molecules are mainly anti-infective drugs, accounting for 118 hits; Followed by anti-cancer drugs, anti-inflammatory/-immune drugs, neurological drugs, cardiovascular drugs, endocrine drugs, and metabolic disease drugs, which accounts for 40, 19, 12, 9, 8, and 3 hits; Other unclassified drugs accounts for 9 hits. The top 10 hits exhibiting anti-planktonic cells activity against S. aureus were mainly including antitumor drugs, followed by neurological drugs and unclassified drugs like vitamin K3 with the inhibition rate of 99.65%-100%. Similarly, the top 10 hits showing biofilm inhibitory effects against S. aureus were also mainly including antitumor drugs, followed by neurological drugs and anti-inflammatory/-immune drugs with the inhibition rate of 50.22%-92.95%. The minimal inhibitory concentration (MIC) of the 51 hits by second round screening was determined by micro-dilution assay, which mainly include the antitumor drugs, cardiovascular drugs, endocrine drugs, anti-inflammatory/-immune drugs, metabolic disease drugs, neurological drugs and other unclassified drugs accounted for 22, 5, 3, 9, 2, 5 and 5 hits, respectively, with the MICs of 1.56-50 μmol/L, 6.25-25 μmol/L, 6.25-25 μmol/L, 0.2-50 μmol/L, 25-50 μmol/L, 1.56-50 μmol/L and 0.1-12.5 μmol/L, respectively. In conclusion, the minimum inhibitory concentrations of small molecules screened through high-throughput assay are at the level of micromolar with strong drug development potential and high modifiability. The high effective anti-planktonic cells and anti-biofilm activity by these molecules are expected to provide new ideas for the development of new antimicrobials against S. aureus.
Humans ; Staphylococcus aureus ; Anti-Bacterial Agents/pharmacology* ; High-Throughput Screening Assays ; Staphylococcal Infections ; Anti-Infective Agents/pharmacology* ; Microbial Sensitivity Tests ; Biofilms ; Antineoplastic Agents/pharmacology* ; Anti-Inflammatory Agents/pharmacology* ; Cardiovascular Agents/pharmacology* ; Metabolic Diseases

Objective To explore the in vitro and in vivo antimicrobial activity of antipsychotic agent pimozide against Staphylococcus aureus(S.aureu).Methods The minimal inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of pimozide were determined by micro-dilution assay.Biofilm was cultured in 96-well cell culture plate,and the anti-biofilm activity of pimozide was detected by turbidimetry.The effect of pimozide on biofilm was further observed through laser confocal microscopy and SYTO9/PI staining.Combined antimicrobial effect of pimozide and other antimicrobial agents was detected by chessboard dilution method,and cytotoxicity of pimozide was detected by CCK-8 assay kit.A model of skin abscess was constructed,in vivo antimicrobial activity and toxicity of pimozide was tested.Results Pimozide showed significant dose-dependent antimicrobial activity against S.aureu,with a MIC of 8-16 μg/mL.It could significantly inhibit the formation of S.aureu biofilm and disperse the formed biofilm.The combination of pimozide and doxycycline has a synergistic antimicrobial effect in vitro,with a synergistic antimicrobial index of 0.5.It can significantly reduce the bacterial load in mouse abscess tissue in vivo,and reduce the live bacterial count from(8.25±0.13)lgarithmic value of CFU/abscess to(3.31± 0.81)logarithmic value of CFU/abscess(q=3.74,P＜0.05).The cytotoxicity of pimozide was extremely low,with a half inhibitory concentration of 64 μg/mL on cells.Conclusion Pimozide exhibits significant antimicrobial activity in vitro and in vivo with extremely low toxicity,thus is promising for the treatment of S.aureu-related local infection in psychiatric patients.

Heat stroke(HS)is a serious life-threatening disease caused by heat injury and characterized by a core body temperature>40℃with central nervous system dysfunction and multi-organ failure.The main pathophysiological manifestations of HS are the thermal acute phase response and thermoregulatory imbalance.Proteins are particularly sensitive to heat,and the thermal environment can cause massive protein denaturation,resulting in the deposition of unfolded and misfolded proteins in the cytoplasm,causing cellular dysfunction and even death.The unfolded protein response(UPR),mainly divided into the endoplasmic reticulum UPR and the mitochondrial UPR,is an important physiological process that helps proteins to fold correctly or degrade irretrievably denatured proteins.This paper summarizes the regulatory mechanisms of UPR,the relationship between UPR and severe diseases,as well as the relationship between HS and UPR to provide new ideas for the treatment of HS.