Gene Expression, Bioinformatics and Substrate Selectivity of Phospholipase D from Acinetobacter sp.

Microbial phospholipase D (PLD) showed a more competent potential for phospholipids production due to its higher catalytic activity and broader substrate specificity. In this study, the PLD from Acinetobacter sp. DUT-2 (ADPLD) was used as the research object. Firstly, bioinformatics was used to examine the protein sequence characteristics. Then, the recombinant plasmid was generated and heterologously expressed in Escherichia coli. The enzyme protein was purified further, and the substrate selectivity of ADPLD to phosphatidylcholine (PC) with various acyl chains was investigated. Finally, the substrate recognition mechanism of ADPLD was investigated using molecular docking and molecular dynamics simulation. Multiple sequence alignment and phylogenetic tree analysis of ADPLD and other microbial-derived PLDs revealed that the sequence similarity between ADPLD and Streptomyces-derived PLDs was less than 30%, and there was only one conserved HKD motif, indicating that the catalytic mechanism of ADPLD might differ from the reaction mechanism in traditional cognition, which required two HKD motifs to complete the PLD catalytic process. ADPLD was mostly produced as a soluble protein, and a relatively uniform protein could be purified using Ni2+ affinity chromatography at a low concentration of 50 mmol/L imidazole. When soybean PC was utilized as a substrate, the specific activity of ADPLD was about 4.09 U/mg. ADPLD showed relatively high activity in neutralizing short-chain PC (C6~C14), with a specific activity of 13.2 U/mg for 8:0/8:0-PC, which was higher than that of other PC substrates with long acyl chains. The activity of ADPLD on PC reduced dramatically when the acyl chain length of PC grew from C14 to C16. Molecular dynamics simulation and molecular docking experiments revealed that the ADPLD amino acid residues Thr205, Pro209, Phe293, Ala324, Lys329, and Phe453 might form hydrophobic interactions with PC. Arg383 and Gly326 could form hydrogen bonds with PC when the distance between Arg383 (N), Gly326 (N), and PC (P) was <3 Å. These results indicate that ADPLD can form a stable enzyme substrate intermediate with phospholipid molecules. These findings set the groundwork for ADPLD molecular modification and future industrial applications

A four-microRNA panel in serum may serve as potential biomarker for renal cell carcinoma diagnosis

BackgroundRenal cell carcinoma (RCC) is one out of the most universal malignant tumors globally, and its incidence is increasing annually. MicroRNA (miRNA) in serum could be considered as a non-invasive detecting biomarker for RCC diagnosis.MethodA total of 224 participants (112 RCC patients (RCCs) and 112 normal controls (NCs)) were enrolled in the three-phrase study. Reverse transcription quantitative PCR (RT-qPCR) was applied to reveal the miRNA expression levels in RCCs and NCs. Receiver operating characteristic (ROC) curves and the area under the ROC curve (AUC) were utilized to predict the diagnostic ability of serum miRNAs for RCC. Bioinformatic analysis and survival analysis were also included in our study.ResultsCompared to NCs, the expression degree of miR-155-5p, miR-224-5p in serum was significantly upregulated in RCC patients, and miR-1-3p, miR-124-3p, miR-129-5p, and miR-200b-3p were downregulated. A four-miRNA panel was construed, and the AUC of the panel was 0.903 (95% CI: 0.847–0.944; p &lt; 0.001; sensitivity = 75.61%, specificity = 93.67%). Results from GEPIA database indicated that CHL1, MPP5, and SORT1 could be seen as promising target genes of the four-miRNA panel. Survival analysis of candidate miRNAs manifested that miR-155-5p was associated with the survival rate of RCC significantly.ConclusionsThe four-miRNA panel in serum has a great potential to be non-invasive biomarkers for RCC sift to check

Developing neural network diagnostic models and potential drugs based on novel identified immune-related biomarkers for celiac disease

Abstract Background As one of the most common intestinal inflammatory diseases, celiac disease (CD) is typically characterized by an autoimmune disorder resulting from ingesting gluten proteins. Although the incidence and prevalence of CD have increased over time, the diagnostic methods and treatment options are still limited. Therefore, it is urgent to investigate the potential biomarkers and targeted drugs for CD. Methods Gene expression data was downloaded from GEO datasets. Differential gene expression analysis was performed to identify the dysregulated immune-related genes. Multiple machine algorithms, including randomForest, SVM-RFE, and LASSO, were used to select the hub immune-related genes (HIGs). The immune-related genes score (IG score) and artificial neural network (ANN) were constructed based on HIGs. Potential drugs targeting HIGs were identified by using the Enrichr platform and molecular docking method. Results We identified the dysregulated immune-related genes at a genome-wide level and demonstrated their roles in CD-related immune pathways. The hub genes (MR1, CCL25, and TNFSF13B) were further screened by integrating several machine algorithms. Meanwhile, the CD patients were divided into distinct subtypes with either high- or low-immunoactivity using single-sample gene set enrichment analysis (ssGSEA) and consensus clustering. By constructing IG score based on HIGs, we found that patients with high IG score were mainly attributed to high-immunoactivity subgroups, which suggested a strong link between HIGs and immunoactivity of CD patients. In addition, the novel constructed ANN model showed the sound diagnostic ability of HIGs. Mechanistically, we validated that the HIGs play pivotal roles in regulating CD's immune and inflammatory state. Through targeting the HIGs, we also found potential drugs for anti-CD treatment by using the Enrichr platform and molecular docking method. Conclusions This study unveils the HIGs and elucidates the networks regulated by these genes in the context of CD. It underscores the pivotal significance of HIGs in accurately predicting the presence or absence of CD in patients. Consequently, this research offers promising prospects for the development of diagnostic biomarkers and therapeutic targets for CD

Additional file 2 of Developing neural network diagnostic models and potential drugs based on novel identified immune-related biomarkers for celiac disease

Additional file 2: Fig. S1. GO and KEGG analysis of 58 differentially expressed immune-related genes. A GO enrichment results in differentially expressed immune-related genes. B KEGG enrichment results in differentially expressed immune-related genes. Fig. S2. Heatmap shows the overall landscape of CD patients' ssGSEA score of 28 immune gene sets. Fig. S3. Consensus matrix heatmap when K = 3–9. It is related to Fig. 3D. Fig. S4. The box plot shows the ssGSEA score of immune cells of the C1 and C2 groups. (ns, no significance, *P < 0.05, **P < 0.01, ***P < 0.001). Fig. S5. Validation of the IG score in the GSE164883 set. A The violin plot shows the IG score between the control and CD groups. B The ROC curve of the IG score in the GSE164883 validation set. Fig. S6. ROC analysis validated the diagnostic performance of HIGs. ROC curves of the indicated HIGs in the GSE11501 training set (A) and GSE164883 validation set (B). Fig. S7. Construction of artificial neural network (ANN) based on HIGs. A The construction of an artificial neural network (ANN) based on MR1, TNFSF13B, and CCL25. B The AUC of the training cohort with a value of 0.824. C The AUC of the test cohort with a value of 0.733. Fig. S8. 3D (left) and 2D (right) structure of complexes of HIGs and drugs. It is related to Fig. 7

Acyl Chain Specificity of Marine Streptomyces klenkii PhosPholipase D and Its Application in Enzymatic Preparation of Phosphatidylserine

Mining of phospholipase D (PLD) with altered acyl group recognition except its head group specificity is also useful in terms of specific acyl size phospholipid production and as diagnostic reagents for quantifying specific phospholipid species. Microbial PLDs from Actinomycetes, especially Streptomyces, best fit this process requirements. In the present studies, a new PLD from marine Streptomyces klenkii (SkPLD) was purified and biochemically characterized. The optimal reaction temperature and pH of SkPLD were determined to be 60 °C and 8.0, respectively. Kinetic analysis showed that SkPLD had the relatively high catalytic efficiency toward phosphatidylcholines (PCs) with medium acyl chain length, especially 12:0/12:0-PC (67.13 S−1 mM−1), but lower catalytic efficiency toward PCs with long acyl chain (&gt;16 fatty acids). Molecular docking results indicated that the different catalytic efficiency was related to the increased steric hindrance of long acyl-chains in the substrate-binding pockets and differences in hydrogen-bond interactions between the acyl chains and substrate-binding pockets. The enzyme displayed suitable transphosphatidylation activity and the reaction process showed 26.18% yield with L-serine and soybean PC as substrates. Present study not only enriched the PLD enzyme library but also provide guidance for the further mining of PLDs with special phospholipids recognition properties

Additional file 1 of Developing neural network diagnostic models and potential drugs based on novel identified immune-related biomarkers for celiac disease

Additional file 1: Table S1. 896 differentially expressed genes. Table S2. Results of three machine algorithms. Table S3. ssGSEA score of 28 immune gene sets in celiac disease patients. Table S4. IG score for GSE11501 training set based on HIGs. Table S5. IG score for GSE164883 validation set based on HIGs. Table S6. ANN diagnosis effect for the grouping of immune characteristics of celiac disease subtypes. Table S7. 2483 immune genes from the ImmPort database. Table S8. 28 immune gene sets from the TISIDB database

Easily-injectable shear-thinning hydrogel provides long-lasting submucosal barrier for gastrointestinal endoscopic surgery

Submucosal injection material has shown protective effect against gastrointestinal injury during endoscopic surgery in clinic. However, the protective ability of existing submucosal injection material is strictly limited by their difficult injectability and short barrier time. Herein, we report a shear-thinning gellan gum hydrogel that simultaneously has easy injectability and long-lasting barrier function, together with good hemostatic property and biocompatibility. Shear-thinning property endows our gellan gum hydrogel with excellent endoscopic injection performance, and the injection pressure of our gellan gum hydrogel is much lower than that of the small molecule solution (50 wt% dextrose) when injected through the endoscopic needle. More importantly, our gellan gum hydrogel shows much stronger barrier retention ability than normal saline and sodium hyaluronate solution in the ex vivo and in vivo models. Furthermore, our epinephrine-containing gellan gum hydrogel has a satisfactory hemostatic effect in the mucosal lesion resection model of pig. These results indicate an appealing application prospect for gellan gum hydrogel utilizing as a submucosal injection material in endoscopic surgery

Developmental and Activity-Dependent Expression of LanCL1 Confers Antioxidant Activity Required for Neuronal Survival

SummaryProduction of reactive oxygen species (ROS) increases with neuronal activity that accompanies synaptic development and function. Transcription-related factors and metabolic enzymes that are expressed in all tissues have been described to counteract neuronal ROS to prevent oxidative damage. Here, we describe the antioxidant gene LanCL1 that is prominently enriched in brain neurons. Its expression is developmentally regulated and induced by neuronal activity, neurotrophic factors implicated in neuronal plasticity and survival, and oxidative stress. Genetic deletion of LanCL1 causes enhanced accumulation of ROS in brain, as well as development-related lipid, protein, and DNA damage; mitochondrial dysfunction; and apoptotic neurodegeneration. LanCL1 transgene protects neurons from ROS. LanCL1 protein purified from eukaryotic cells catalyzes the formation of thioether products similar to glutathione S-transferase. These studies reveal a neuron-specific glutathione defense mechanism that is essential for neuronal function and survival