OBJECTIVE: To establish and validate a novel diabetic retinopathy (DR) risk-prediction model using a whole-exome sequencing (WES)-based machine learning (ML) method. METHODS: WES was performed to identify potential single nucleotide polymorphism (SNP) or mutation sites in a DR pedigree comprising 10 members. A prediction model was established and validated in a cohort of 420 type 2 diabetic patients based on both genetic and demographic features. The contribution of each feature was assessed using Shapley Additive explanation analysis. The efficacies of the models with and without SNP were compared. RESULTS: WES revealed that seven SNPs/mutations ( rs116911833 in TRIM7, 1997T>C in LRBA, 1643T>C in PRMT10, rs117858678 in C9orf152, rs201922794 in CLDN25, rs146694895 in SH3GLB2, and rs201407189 in FANCC) were associated with DR. Notably, the model including rs146694895 and rs201407189 achieved better performance in predicting DR (accuracy: 80.2%; sensitivity: 83.3%; specificity: 76.7%; area under the receiver operating characteristic curve [AUC]: 80.0%) than the model without these SNPs (accuracy: 79.4%; sensitivity: 80.3%; specificity: 78.3%; AUC: 79.3%). CONCLUSION Novel SNP sites associated with DR were identified in the DR pedigree. Inclusion of rs146694895 and rs201407189 significantly enhanced the performance of the ML-based DR prediction model.
Diabetic Retinopathy/diagnosis* ; Humans ; Machine Learning ; Male ; Female ; Polymorphism, Single Nucleotide ; Middle Aged ; Exome Sequencing ; Aged ; Adult ; Pedigree ; Diabetes Mellitus, Type 2/complications* ; Genetic Predisposition to Disease ; Mutation

ObjectiveTranscription factor NFE2 was observed abnormal expression in myeloproliferative neoplasm (MPN) patients. However, how NFE2 is transcriptionally regulated remains ambiguous. This study aims to explore the elements and molecular mechanisms involved in the transcriptional regulation of NFE2. MethodsActive enhancers were predicted by public NGS data and conformed experimentally via dual luciferase reporter assay. After that, PRO-seq and GRO-seq data was used to detect enhancer RNAs transcribed from these enhancers. RACE was utilized to clone the full length enhancer RNA (eRNA) transcripts, and RT-qPCR was used to measure their expression in different leukemia cell lines as well as the transcript levels during induced differentiation. Finally, to investigate the molecular function of the eRNA, overexpression and knockdown of the eRNA via lentivirus system was performed in K562 cells. ResultsWe identified three enhancers regulating NFE2 transcription, which located at -3.6k, -6.2k and +6.3k from NFE2 transcription start site (TSS) respectively. At the -3.6k enhancer, we cloned an eRNA transcript and characterized that as a lncRNA which was expressed and located in the nucleus in three types of leukemia cell lines. When this lncRNA was overexpressed, expression of NFE2 was upregulated and decreases of K562 cell proliferation and migration ability were observed. While knocking down of this lncRNA, the level of NFE2 decreases correspondingly and the proliferation ability of K562 cells increases accordingly. ConclusionWe identified an enhancer lncRNA that regulates NFE2 transcription positively and suppresses K562 cell proliferation.

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.

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.

Objective:To evaluate the preoperative localization value of endoscopic ultrasound guided fine needle tattooing (EUS-FNT) for laparoscopic distal pancreatectomy in pancreatic lesions with a maximum diameter ≤3 cm.Methods:From November 2017 to October 2022, at the Second Affiliated Hospital of Soochow University, the data of patients with pancreatic lesions ≤3 cm who underwent laparoscopic distal pancreatectomy were retrospectively analyzed. Eight patients who underwent EUS-FNT assisted laparoscopic distal pancreatectomy were included in the fine needle tattooing (FNT) combined laparoscopic group. And 14 patients who underwent simple laparoscopic distal pancreatectomy were taken as the simple laparoscopic group. The success rate and complications of EUS-FNT were observed. The differences in operation time, surgery-related complications and complete resection rate of lesions between the two groups were compared. Mann-Whitney U test and descriptive analysis were used for statistical analysis. Results:In the FNT combined laparoscopic group, the lesions of 4 cases were located in the pancreatic body and 4 cases in the pancreatic tail. In the simple laparoscopic group, the lesions of 4 cases were located in the pancreatic body and 10 cases in the pancreatic tail. There was a significant difference in lesion size between the two groups (14.5 mm (10.8 mm, 16.5 mm) vs. 27.0 mm (23.5 mm, 30.0 mm), Z=-3.09, P=0.001). In the FNT combined laparoscopic group, EUS-FNT was successfully performed in all 8 patients. The average time of laparoscopy after EUS-FNT was (98.4±8.8) min. The marks were clearly visible under the laparoscopic field of view, and no complications such as abdominal hemorrhage and hematoma were observed. Laparoscopic pancreaticocaudectomy was performed in 5 cases and pancreaticocaudectomy plus splenectomy in 3 cases. The median operation time was 192.5 min (176.3 min, 203.8 min). The amount of intraoperative bleeding was large in 2 patients and blood transfusion was needed. The lesions were one-time completely resected in all 8 patients. The postoperative pathology were 6 cases of pancreatic neuroendocrine neoplasm, 1 case of intraductal papillary mucinous neoplasm (IPMN), and 1 case of solid pseudopapilloma. In the simple laparoscopic group, laparoscopic pancreaticocaudectomy was performed in 2 cases and pancreaticocaudectomy plus splenectomy in 12 cases. The median operation time was 202.5 min (192.8 min, 235.0 min), which was longer than that of FNT combined laparoscopic group, but the difference was not statistically significant ( P>0.05). The amount of intraoperative bleeding was large in 2 patients and blood transfusion was needed. In 1 patient with pancreatic body lesions, no lesion was found in the specimen examination after the first pancreatectomy, and the lesions were completely resected after the second partial pancreatectomy. Active abdominal hemorrhage occurred in 1 patient on the second day after operation, and underwent interventional embolization for hemostasis. Two weeks after surgery, 1 patient was found to have a encapsulated fluid with a long diameter of 6 cm around the pancreas by computed tomography re-examination 2 weeks after surgery. The postoperative pathology were 5 cases of pancreatic neuroendocrine neoplasm, 2 cases of IPMN, 1 case of solid pseudopapilloma, 1 case of pancreatic cyst with glandular low-grade intraepithelial neoplasia, 1 case of ectopic spleen, and 4 cases of pancreatic ductal adenocarcinoma. Conclusion:EUS-FNT can effectively localize small pancreatic lesions before laparoscopic distal pancreatectomy, shorten the operation time and improve the complete resection rate under laparoscopy.

Humans ; Heart Failure ; Stroke Volume

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

Humans ; Heart Failure ; Stroke Volume

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

Dihydroflavonol 4-reductase (DFR) plays an essential role in the biosynthesis of anthocyanin and regulation of plant flower color. Based on the transcriptome data of Cistanche tubulosa (Schenk) Wight, a full-length cDNA sequence of CtDFR gene was cloned by reverse transcription-polymerase chain reaction (RT-PCR). CtDFR contains an open reading frame (ORF) of 1 263 bp which encodes 420 amino acids with a predicted molecular weight of 47.5 kDa. The sequence analysis showed that CtDFR contains a nicotinamide adenine dinucleotide phosphate (NADPH) binding domain and a specific substrate binding domain. The expression analysis indicated that CtDFR was highly expressed in red and purple flowers, and the relative expression levels were 4.04 and 19.37 times higher than those of white flowers, respectively. The recombinant CtDFR protein was expressed in E.coli BL21 (DE3) using vector pET-28a-CtDFR and was purified. In vitro enzyme activity analysis, CtDFR could reduce three types of dihydroflavonols including dihydrokaempferol, dihydroquercetin, and dihydromyricetin to leucopelargonidin, leucocyanidin and leucodelphinidin. Subcellular localization analysis showed that CtDFR was mainly localized in the cytoplasm. These results demonstrate that CtDFR plays an important role in regulation of flower color in C. tubulosa and make a valuable contribution for the further investigation on the regulation mechanism of C. tubulosa (Schenk) Wight flower color.