Protein Interaction Analysis Research Articles

Investigation of LncRNA PVT1 and MiR-21-5p Expression as Promising Novel Biomarkers for Autism Spectrum Disorder.

The characteristics of ncRNA in children with autism spectrum disorder (ASD) were observed to disclose a theoretical basis for further research on molecular markers for early warning of ASD. Children with ASD and normal control children were recruited to collect peripheral blood RNA samples. The concentration of PVT1 and miR-21-5p was quantitatively analyzed by qRT-PCR. Pearson correlation coefficient method was used to evaluate the link between PVT1 level and miR-21-5p level of the children. Receiver operating characteristic (ROC) curves were applied to reckon the predictive value of PVT1, miR-21-5p, and their combination in ASD. The interconnection of PVT1 with miR-21-5p was represented by luciferase reporter assay. The targeted genes of miR-21-5p were predicted. The enrichment and protein interaction analysis of these genes was carried out to find the important core genes and analyze their value in ASD. In the disease group, the level of PVT1 was downregulated, while the content of miR-21-5p was upregulated. The expression level of serum miR-21-5p was negatively correlated with the level of PVT1. Luciferase reporter gene assay documented that PVT1 directly targeted miR-21-5p. ROC curve showed that PVT1, miR-21-5p, and their combination showed clinical value for disease diagnosis. The functional enrichment analysis showed that the targets of miR-21-5p participated in ASD by regulating related functions and pathways. Reduced expression of PVT1 and raised miR-21-5p were good diagnostic markers for ASD, which would provide a basis for effective prevention, early diagnosis, and early intervention of ASD.

Differentially Expressed mRNA in Streptozotocin-Induced Diabetic Bladder Using RNA Sequencing Analysis.

To detect elements governing the pathogenesis of diabetic cystopathy (DC), mRNA sequencing was carried out for bladder tissues from normal rats and those with induced diabetes mellitus (DM). This research therefore offers possible underlying molecular pathways for the advancement of DC in relation to differential mRNA expression, together with visceral functional and architectural alterations noted in individuals with this condition. An intraperitoneal injection of streptozotocin (STZ) was utilized to provoke DM in male Sprague-Dawley rats. Dysregulation and significant variations between normal rats and those with induced DM were then identified by a fold change of ≥ 1.5 with a false discovery rate P < 0.05. Hierarchical clustering/heat map and Gene Ontology/DAVID reference sources were generated. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and protein-protein interaction analysis were then performed. The diabetic rodent group exhibited a greater residual urine volume (4.0 ± 0.4 mL) than their control counterparts (0.7 ± 0.2 mL, P < 0.01) at 12 weeks after diagnosis of diabetes. Expression analysis revealed 16 upregulated and 4 downregulated genes in STZDM bladder samples. A notable increase in expression was seen in PTHLH, TNFAIP6, PRC1, MAPK10, LOC686120, CASQ2, ACTG2, PDLIM3, FCHSD1, DBN1, NKD2, PDLIM7, ATF4, RBPMS2, ITGB1 and HSPB8. A notable decrease in expression was seen in SREBLF1, PBGFR1, PBLD1 and CELF1. Major genetic themes associated with mRNA upregulation and downregulation ware identified via Gene Ontology analysis and KEGG pathways. Protein to protein interaction analysis detected PDLIM3, PDLIM7, ITGB1, ACTG2 as core high frequency nodes within the network. Changes in mRNA expression together with biological process and pathways that contribute to the etiologies underlying visceral impairment of the bladder in DM are evident. Our strategy is promising for recognizing mRNAs exclusive to the bladder in DM that might offer useful targets for diagnosis and treatment.

ERH gene knockdown inhibits the proliferation and migration of ARPE-19 cells through MCM complex and EMT process

PurposeTo explore the role of the Enhancer of rudimentary homolog (ERH) gene on the proliferation and migration of ARPE-19 cells, and its mechanism. MethodsARPE-19 cells were divided into ERH gene knockdown (ERH KD) and normal ERH gene (ERH NC) groups and infected with respected virus. Cell counting kit-8 assay, wound-healing assay, and flow cytometry were performed to evaluate the effects of the ERH gene on cell proliferation, migration, and cell cycle. A 4D label-free quantitative proteomic analysis was conducted to obtain the ERH gene knockdown-related differential proteins list (DPL). Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein domain analysis, subcellular localization analysis, and protein–protein interaction (PPI) analysis were performed to explore the main downstream functions of the ERH gene. Proteins related to DNA replication, cell cycle, and epithelial-mesenchymal transition (EMT) were identified by Western blot test. ResultsThe ERH gene was successfully knocked down in ARPE-19 cells of the ERH KD group. The proliferation and migration of cells were reduced and the cell cycle was arrested at the S phase in the ERH KD group. A DPL of 47 upregulated and 108 downregulated proteins was obtained, and their functions were explored and found to be associated with the MCM complex, DNA replication, and cell cycle. Protein domain analysis, protein subcellular localization analysis, and PPI analysis showed that the MCM complex may play a key role in the proliferation of ARPE-19 cells affected by the ERH gene. DNA replication, cell cycle, and EMT-related proteins were affected when the ERH gene was knocked down. ConclusionKnockdown of ERH gene inhibits the proliferation and migration of ARPE-19 cells through the MCM complex and EMT process.

Network pharmacology -based study on the mechanism of traditional Chinese medicine in the treatment of glioblastoma multiforme

BackgroundGlioblastoma multiforme (GBM) is one of the most common primary malignant brain tumors. Yi Qi Qu Yu Jie Du Fang (YYQQJDF) is a traditional Chinese medicine (TCM) prescription for GBM. The present study aimed to use a network pharmacology method to analyze the underlying mechanism of YQQYJDF in treating GBM.MethodsGBM sample data, active ingredients and potential targets of YQQYJDF were obtained from databases. R language was used to screen differentially expressed genes (DEGs) between GBM tissues and normal tissues, and to perform enrichment analysis and weighted gene coexpression network analysis (WGCNA). The Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) database was used to perform a protein‒protein interaction (PPI) analysis. A Venn diagram was used to obtain the core target genes of YQQYJDF for GBM treatment. Molecular docking was used to verify the binding between the active ingredient molecules and the proteins corresponding to the core target genes. Cell proliferation assays and invasion assays were used to verify the effect of active ingredients on the proliferation and invasion of glioma cells.ResultsA total of 73 potential targets of YQQYJDF in the treatment of GBM were obtained. Enrichment analyses showed that the biological processes and molecular functions involved in these target genes were related to the activation of the G protein-coupled receptor (GPCR) signaling pathway and the regulation of hypoxia. The neuroactive ligand‒receptor pathway, the cellular senescence pathway, the calcium signaling pathway, the cell cycle pathway and the p53 signaling pathway might play important roles. Combining the results of WGCNA and PPI analysis, five core target genes and their corresponding four core active ingredients were screened. Molecular docking indicated that the core active ingredient molecules and the proteins corresponding to the core target genes had strong binding affinities. Cell proliferation and invasion assays showed that the core active ingredients of YQQYJDF significantly inhibited the proliferation and invasion of glioma cells (P < 0.01).ConclusionsThe present study predicted the possible active ingredients and targets of YQQYJDF in treating GBM, and analyzed its possible mechanism. These results may provide a basis and ideas for further research.

Transcriptome profiling reveals transcriptional regulation of Protegrin-1 on immune defense and development in porcine granulosa cells

Protegrin-1 (PG1) is an antimicrobial peptide (AMP) that has garnered increasing attention due to its potent immune defense activity. Our previous studies demonstrated the ability of PG1 to enhance proliferation and inhibit apoptosis of porcine granulosa cells (GCs) under oxidative stress. GCs play a crucial role in ovary follicular development. However, the specific function and underlying mechanisms of AMP in follicular development still need further elucidation. The present study aimed to comprehensively explore the biological effects of PG1 on porcine GCs using transcriptome profiling by RNA sequencing technology. Isolated GCs were incubated with or without PG1 for 24 h and transcriptome-wide analysis was exerted to identify differentially expressed genes (DEGs). The results of expression analysis revealed 1,235 DEGs, including 242 up-regulated genes and 993 down-regulated genes (|log2 (FoldChange)| > 1; adjusted P-value < 0.05). The expression levels of 7 selected DEGs were validated by quantitative reverse transcription-polymerase chain reaction (RT-qPCR) analysis, which was consistent with the RNA-sequencing data. Among the significant DEGs, several genes associated with GC function and ovarian follicle development were identified, such as estrogen receptor 2 (ESR2), growth and differentiation factor 6 (GDF6), cell division cycle 20 homolog (CDC20), Notch3, ephrin and Eph receptor system, Egl nine homolog 3 (EGLN3), and BCL2 like 14 (BCL2L14). Gene Ontology (GO) analysis revealed that the top three significant GO terms were inflammatory response, defense response, and granulocyte migration. Additionally, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis presented that DEGs were mainly enriched in the immune system, infectious disease, signaling molecules and interaction, and immune disease. Furthermore, Ingenuity Pathway Analysis (IPA) predicted that the top activated pathway was Liver X Receptor (LXR)/ Retinoid X Receptor (RXR) Activation which is known to be associated with female reproduction. Predicted protein–protein interactions (PPIs) analysis identified complement C3 (C3) as the top node with the highest degree of network connection and revealed that DEGs in the sub-networks were involved in cytokine-cytokine receptor interaction, neuroactive ligand-receptor interaction, chemokine signaling pathway, and metabolic process. In conclusion, this study expanded the understanding of the effects of PG1 on porcine GCs at the transcriptomic level and provided a theoretical basis for further investigation into the role of PG1 in immune defense and mammalian ovarian follicular development.

ABCE1 selectively promotes HIF-1α transactivation of angiogenic gene expression

BackgroundCopper (Cu), by inhibiting the factor inhibiting HIF-1 (FIH-1), promotes the transcriptional activity of hypoxia-inducible factor-1 (HIF-1). ObjectiveThe present study was undertaken to understand the molecular mechanism by which Cu inhibits FIH-1. MethodsHuman umbilical vein endothelial cells (HUVECs) were treated with dimethyloxalylglycine (DMOG) resulting in HIF-1α accumulation and the FIH-1 protein complexes were pulled down for candidate protein analysis. The metal binding sites were predicted by both MetalDetector V2.0 and Metal Ion-Binding Site Prediction Server, and then the actual ability to bind to Cu in vitro was tested by both Copper-Immobilized metal affinity chromatography (Cu-IMAC) and Isothermal Titration Calorimetry (ITC). Subsequently, subcellular localization was monitored by immunocytochemistry, GFP-fusion protein expression plasmid and Western blotting in the nuclear extract. The interaction of candidate protein with HIF-1α and FIH-1 was validated by Co-Immunoprecipitation (Co-IP). Finally, the effect of candidate protein on the FIH-1 structure and HIF-1α transcriptional activity was analyzed by the InterEvDock3 web server and real-time quantitative RT-PCR. ResultsATP-binding cassette E1 (ABCE1) was present in the FIH-1 complexes and identified as a leading Cu-binding protein as indicated by a number of possible Cu binding sites. The ability of ABCE1 to bind Cu was demonstrated in vitro. ABCE1 entered the nucleus along with FIH-1 under hypoxic conditions. Protein interaction analysis revealed that ABCE1 prevented FIH-1 to bind iron ions, inhibiting FIH-1 enzymatic activity. ABCE1 silencing suppressed the expression of Cu-dependent HIF-1 target gene BNIP3, not that of Cu-independent IGF-2. ConclusionThe results demonstrate that ABCE1, as a Cu-binding protein, enters the nucleus under hypoxic conditions and inhibits FIH-1degradation of HIF-1α, thus promoting HIF-1 transactivation of angiogenic gene expression.

Construction of a novel circRNA-miRNA-ferroptosis related mRNA network in ischemic stroke

Molecule alterations are important to explore the pathological mechanism of ischemic stroke (IS). Ferroptosis, a newly recognized type of regulated cell death, is related to IS. Identification of the interactions between circular RNA (circRNA), microRNA (miRNA) and ferroptosis related mRNA may be useful to understand the molecular mechanism of IS. The circRNA, miRNA and mRNA transcriptome data in IS, downloaded from the Gene Expression Omnibus (GEO) database, was used for differential expression analysis. Ferroptosis related mRNAs were identified from the FerrDb database, followed by construction of circRNA-miRNA-ferroptosis related mRNA network. Enrichment and protein–protein interaction analysis of mRNAs in circRNA-miRNA-mRNA network was performed, followed by expression validation by reverse transcriptase polymerase chain reaction and online dataset. A total of 694, 41 and 104 differentially expressed circRNAs, miRNAs and mRNAs were respectively identified in IS. Among which, dual specificity phosphatase 1 (DUSP1), nuclear receptor coactivator 4 (NCOA4) and solute carrier family 2 member 3 (SLC2A3) were the only three up-regulated ferroptosis related mRNAs. Moreover, DUSP1, NCOA4 and SLC2A3 were significantly up-regulated in IS after 3, 5 and 24 h of the attack. Based on these three ferroptosis related mRNAs, 4 circRNA-miRNA-ferroptosis related mRNA regulatory relationship pairs were identified in IS, including hsa_circ_0071036/hsa_circ_0039365/hsa_circ_0079347/hsa_circ_0008857-hsa-miR-122-5p-DUSP1, hsa_circ_0067717/hsa_circ_0003956/hsa_circ_0013729-hsa-miR-4446-3p-SLC2A3, hsa_circ_0059347/hsa_circ_0001414/hsa_circ_0049637-hsa-miR-885-3p-SLC2A3, and hsa_circ_0005633/hsa_circ_0004479-hsa-miR-4435-NCOA4. In addition, DUSP1 is involved in the signaling pathway of fluid shear stress and atherosclerosis. Relationship of regulatory action between circRNAs, miRNAs and ferroptosis related mRNAs may be associated with the development of IS.

CircEIF3I facilitates the recruitment of SMAD3 to early endosomes to promote TGF-β signalling pathway-mediated activation of MMPs in pancreatic cancer

BackgroundAmong digestive tract tumours, pancreatic ductal adenocarcinoma (PDAC) shows the highest mortality trend. Moreover, although PDAC metastasis remains a leading cause of cancer-related deaths, the biological mechanism is poorly understood. Recent evidence demonstrates that circular RNAs (circRNAs) play important roles in PDAC progression.MethodsDifferentially expressed circRNAs in normal and PDAC tissues were screened via bioinformatics analysis. Sanger sequencing, RNase R and actinomycin D assays were performed to confirm the loop structure of circEIF3I. In vitro and in vivo functional experiments were conducted to assess the role of circEIF3I in PDAC. MS2-tagged RNA affinity purification, mass spectrometry, RNA immunoprecipitation, RNA pull-down assay, fluorescence in situ hybridization, immunofluorescence and RNA–protein interaction simulation and analysis were performed to identify circEIF3I-interacting proteins. The effects of circEIF3I on the interactions of SMAD3 with TGFβRI or AP2A1 were measured through co-immunoprecipitation and western blotting.ResultsA microarray data analysis showed that circEIF3I was highly expressed in PDAC cells and correlated with TNM stage and poor prognosis. Functional experiments in vitro and in vivo revealed that circEIF3I accelerated PDAC cells migration, invasion and metastasis by increasing MMPs expression and activity. Mechanistic research indicated that circEIF3I binds to the MH2 domain of SMAD3 and increases SMAD3 phosphorylation by strengthening the interactions between SMAD3 and TGFβRI on early endosomes. Moreover, AP2A1 binds with circEIF3I directly and promotes circEIF3I-bound SMAD3 recruitment to TGFβRI on early endosomes. Finally, we found that circEif3i exerts biological functions in mice similar to those of circEIF3I in humans PDAC.ConclusionsOur study reveals that circEIF3I promotes pancreatic cancer progression. circEIF3I is a molecular scaffold that interacts with SMAD3 and AP2A1 to form a ternary complex, that facilitates the recruitment of SMAD3 to early endosomes and then activates the TGF-β signalling pathway. Hence, circEIF3I is a potential prognostic biomarker and therapeutic target in PDAC.

Novel CSF biomarkers for diagnosis and integrated analysis of neuropsychiatric systemic lupus erythematosus: based on antibody profiling

BackgroundNeuropsychiatric systemic lupus erythematosus (NPSLE), with various morbidities and multiple manifestations in the central nervous system, remains a limited standard for diagnosis. Our study was to discover novel biomarkers for improving the diagnostic efficiency for NPSLE.MethodsWe performed a quantitative planar protein antibody microarray to screen 1000 proteins in cerebrospinal fluid from controls, systemic lupus erythematosus (SLE, non-NPSLE) patients, and NPSLE patients. Differentially expressed proteins (DEPs) as candidate biomarkers were developed into a custom multiplexed protein antibody array for further validation in an independent larger cohort. Subsequently, we used least absolute shrinkage and selection operator regression (LASSO) analysis and multivariable logistic regression analysis for optimizing feature selection and constructing a diagnostic model. A receiver operating characteristic curve (ROC) was generated to assess the effectiveness of the models.ResultsThe expression of 29 proteins in CSF was significantly altered in the comparison of the three groups. We selected 17 proteins as candidate biomarkers in accordance with protein interaction analysis. In the larger cohort, we identified 5 DEPs as biomarkers for NPSLE, including TCN2, CST6, KLK5, L-selectin, and Trappin-2. The diagnostic model included 3 hub proteins (CST6, TCN2, KLK5) and was best at discriminating NPSLE from SLE patients. These CSF biomarkers were also highly associated with disease activity. In addition, there were 6 molecules with remarkable changes in NPSLE CSF and hippocampus, which indicated the consistency of the environment in the brain and the promising molecular targets in the pathogenesis of NPSLE.ConclusionsThe dual-chips screening strategy demonstrated KLK5, L-selectin, Trappin-2, TCN2, and CST6 as CSF biomarkers for diagnosing NPSLE.

Analysis of the Interaction of UBE2Q1 with B4GALT1 and P53: Experimental and Molecular Modeling Study.

UBE2Q1-dependent ubiquitination of key proteins including β 1,4- galactosyltransferase (GalT1), and P53 might play a pivotal role in cancer development. The present study aimed to evaluate the molecular analysis of possible interactions between UBE2Q1 with B4GALT1 and P53 proteins. We established SW1116 colorectal cancer cell line stably transfected with UBE2Q1. To verify the overexpression of UBE2Q1, we performed western blot and fluorescent microscopy analysis. Using the immunoprecipitation (IP) product of the over-expressed protein on the silver staining gel, we observed the potential interacting partners of UBE2Q1. The Molecular Operating Environment (MOE) software was also used to perform the molecular docking of the UBC domain of UBE2Q1 (2QGX) with B4GALT1 (2AGD), and P53 (tetramerization (1AIE) and DNA binding domains (1GZH)) proteins. Western blot and IP analysis detected a UBE2Q1-GFP band in transfected cells, while no band was detected for mock-transfected cells. Moreover, the overexpression of UBE2Q1 tagged with GFP was observed under fluorescent microscopy as well with about 60-70% shining. Silver staining of IP gel revealed several bands in colorectal cancer (CRC) with UBE2Q1 overexpression. Protein- Protein interaction (PPI) analysis also depicted a high affinity of the UBC domain of UBE2Q1 to the B4GALT1 and P53 (tetramerization and DNA binding domains). Molecular docking also revealed hot-spot regions for all poses. Our data suggest that UBE2Q1 as an E2 enzyme of ubiquitination system can interact with B4GALT1 and P53, and may contribute to the accumulation of misfolded important proteins and colorectal tumor development.

Pattern discovery of long non‐coding RNAs associated with the herbal treatments in breast and prostate cancers

Functionally characterized lncRNAs play critical roles in cancer progression but the potential relationship between lncRNAs and herbal medicine is yet to be known. To identify this association by RNA‐seq data for breast and prostate cancer, a co‐expression network in response to herbal medicines was performed. GO terms and pathway analyses on differential co‐expressed mRNAs revealed that lncRNAs were widely co‐expressed with metabolic process genes. On the other hand, various machine learning‐based prediction systems on the differential co‐expressed lncRNAs were implemented. Results show that the Deep Learning model could accurately forecast cancer‐related lncRNAs.BackgroundAccumulating evidence shows that long non‐coding RNAs (lncRNAs) play critical roles in cancer progression. The possible association between lncRNAs and herbal medicine is yet to be known. This study aims to identify medicinal herbs associated with lncRNAs by RNA‐seq data for breast and prostate cancer.MethodsTo develop the optimal approach for identifying cancer‐related lncRNAs, we implemented two steps: (1) applying protein–protein interaction (PPI), Gene Ontology (GO), and pathway analyses, and (2) applying attribute weighting and finding the efficient classification model of the machine learning approach.ResultsIn the first step, GO terms and pathway analyses on differential co‐expressed mRNAs revealed that lncRNAs were widely co‐expressed with metabolic process genes. We identified two hub lncRNA‐mRNA networks that implicate lncRNAs associated with breast and prostate cancer. In the second step, we implemented various machine learning‐based prediction systems (Decision Tree, Random Forest, Deep Learning, and Gradient‐Boosted Tree) on the non‐transformed and Z‐standardized differential co‐expressed lncRNAs. Based on five‐fold cross‐validation, we obtained high accuracy (91.11%), high sensitivity (88.33%), and high specificity (93.33%) in Deep Learning which reinforces the biomarker power of identified lncRNAs in this study. As data originally came from different cell lines at different durations of herbal treatment intervention, we applied seven attribute weighting algorithms to check the effects of variables on identifying lncRNAs. Attribute weighting results showed that the cell line and time had little or no effect on the selected lncRNAs list. Besides, we identified one known lncRNAs, downregulated RNA in cancer (DRAIC), as an essential feature.ConclusionsThis study will provide further insights to investigate the potential therapeutic and prognostic targets for prostate cancer (PC) and breast cancer (BC) in common.

P25-17: Modeling cellular toxicity and carcinoma reversion: Insights from In Silico protein interaction analysis

P25-17: Modeling cellular toxicity and carcinoma reversion: Insights from In Silico protein interaction analysis

A Network Landscape of HPVOPC Reveals Methylation Alterations as Significant Drivers of Gene Expression via an Immune-Mediated GPCR Signal.

HPV-associated oropharynx carcinoma (HPVOPC) tumors have a relatively low mutational burden. Elucidating the relative contributions of other tumor alterations, such as DNA methylation alterations, alternative splicing events (ASE), and copy number variation (CNV), could provide a deeper understanding of carcinogenesis drivers in this disease. We applied network propagation analysis to multiple classes of tumor alterations in a discovery cohort of 46 primary HPVOPC tumors and 25 cancer-unaffected controls and validated our findings with TCGA data. We identified significant overlap between differential gene expression networks and all alteration classes, and this association was highest for methylation and lowest for CNV. Significant overlap was seen for gene clusters of G protein-coupled receptor (GPCR) pathways. HPV16-human protein interaction analysis identified an enriched cluster defined by an immune-mediated GPCR signal, including CXCR3 cytokines CXCL9, CXCL10, and CXCL11. CXCR3 was found to be expressed in primary HPVOPC, and scRNA-seq analysis demonstrated CXCR3 ligands to be highly expressed in M2 macrophages. In vivo models demonstrated decreased tumor growth with antagonism of the CXCR3 receptor in immunodeficient but not immunocompetent mice, suggesting that the CXCR3 axis can drive tumor proliferation in an autocrine fashion, but the effect is tempered by an intact immune system. In conclusion, methylation, ASE, and SNV alterations are highly associated with network gene expression changes in HPVOPC, suggesting that ASE and methylation alterations have an important role in driving the oncogenic phenotype. Network analysis identifies GPCR networks, specifically the CXCR3 chemokine axis, as modulators of tumor-immune interactions that may have proliferative effects on primary tumors as well as a role for immunosurveillance; however, CXCR3 inhibition should be used with caution, as these agents may both inhibit and stimulate tumor growth considering the competing effects of this cytokine axis. Further investigation is needed to explore opportunities for targeted therapy in this setting.

Uncovering the interactions between PME and PMEI at the gene and protein levels: Implications for the design of specific PMEI.

Pectin methylesterase inhibitor (PMEI) can specifically bind and inhibit the activity of pectin methylesterase (PME), which has been widely used in fruit and vegetable juice processing. However, the limited three-dimensional structure, unclear action mechanism, low thermal stability and biological activity of PMEI severely limited its application. In this work, molecular recognition and conformational changes of PME and PMEI were analyzed by various molecular simulation methods. Then suggestions were proposed for improving thermal stability and affinity maturation of PMEI through semi-rational design. Phylogenetic trees of PME and PMEI were established using the Maximum likelihood (ML) method. The results show that PME and PMEI have good sequence and structure conservation in various plants, and the simulated data can be widely adopted. In this work, MD simulations were performed using AMBER20 package and ff14SB force field. Protein interaction analysis indicates that H-bonds, van der Waals forces, and the salt bridge formed of K224 with ID116 are the main driving forces for mutual molecular recognition of PME and PMEI. According to the analyses of free energy landscape (FEL), conformational cluster, and motion, the association with PMEI greatly disrupts PME's dispersed functional motion mode and biological function. By monitoring the changes of residue contact number and binding free energy, IG35M/ IG35R: IT93F and IT113W/ IT113W: ID116W mutations contribute to thermal stability and affinity maturation of the PME-PMEI complex system, respectively. This work reveals the interaction between PME and PMEI at the gene and protein levels and provides options for modifying specific PMEI.

Identification of key pathways and genes involved in psoriasis and vitiligo using bioinformatics analysis

Objective: Psoriasis and vitiligo are common immune-related skin disorders. Patients are often clinically diagnosed with both diseases. However, whether psoriasis and vitiligo share common genetic factors and aberrant signal pathways that predispose patients to both diseases remains unclear. This study was performed to clarify these factors using bioinformatics analysis. Methods: Publicly available gene expression profiles for GSE109248 and GSE53552 in psoriasis and GSE75819 and GSE65127 in vitiligo from lesional and non-lesional skin tissues were downloaded from the Gene Expression Omnibus database and analyzed to identify differentially expressed genes (DEGs). Gene Ontology terms analysis, Kyoto Encyclopedia of Genes and Genomes enrichment analysis, and protein–protein interaction analysis was also performed to elucidate the molecular mechanisms. Results: Nine overlapping DEGs were found between psoriasis and vitiligo. They are AKR1B10, CRABP1, FOXC1, GPM6B, KIT, MLPH, SOX10, TAGLN, and TUBB2B, respectively. Except for the upregulation of AKR1B10, others are downregulated. Pathway enrichment analyses revealed that these overlapping DEGs were mainly enriched in melanocyte differentiation; exocrine system development; mesenchymal cell development; stem cell differentiation and development; and neural crest cell differentiation, development, and migration. RT-qPCR was used to verify the expression of the DEGs in lesions compared to adjacent non-lesional tissues from three patients with psoriasis combined with vitiligo. Research confirmed that there were statistically significant differences among AKR1B10, FOXC1, KIT, MLPH and SOX10 in lesions compared to adjacent non-lesional tissues (P&lt;0.05), which was consistent with the bioinformatical results. Conclusion: In this study, we detected potential genes and their associated enriched pathways to help understand the molecular mechanisms underlying the simultaneous occurrence of psoriasis and vitiligo.

Establishment of protoplasts transient expression system in Pinellia ternata (Thunb.) Breit.

In this study, we established an efficient and rapid transient expression system in the protoplasts of Pinellia ternata (Thunb.) Breit. (P. ternata). The protoplasts of P. ternata were prepared from plant leaves as the source material by digesting them with the combination of 20g·l-1 cellulase and 15g·l-1 macerozyme for 6h. Based on the screening of PEG concentration, the conditions for PEG-mediated protoplast transformation were improved, and the highest transformation efficiency was found for 40% PEG 4000. Furthermore, we used the subcellular protein localization technique in P. ternata protoplasts to allow further validation of transient expression system. We present the method that can be applicable for studying both gene verification and expression in P. ternata protoplasts, thus allowing for engineering the improved varieties of P. ternata through molecular plant breeding techniques. This method can also be widely applicable for analyzing protein interactions, detecting promoter activity, for somatic cell fusion in plant breeding, as well as for other related studies.

Transcriptome profiling analysis of uterus during chicken laying periods

The avian eggshell is formed in the uterus. Changes in uterine function may have a significant effect on eggshell quality. To identify the vital genes impacting uterine functional maintenance in the chicken, uteri in three different periods (22W, 31W, 51W) were selected for RNA sequencing and bioinformatics analysis. In our study, 520, 706 and 736 differentially expressed genes (DEGs) were respectively detected in the W31 vs W22 group, W51 vs W31 group and W51 vs W22 group. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis indicated DEGs were enriched in the extracellular matrix, extracellular region part, extracellular region, extracellular matrix structural constituent, ECM receptor interaction, collagen-containing extracellular matrix and collagen trimer in the uterus (P < 0.05). Protein–protein interaction analysis revealed that FN1, LOX, THBS2, COL1A1, COL1A2, COL5A1, COL5A2, POSTN, MMP13, VANGL2, RAD54B, SPP1, SDC1, BTC, ANGPTL3 might be key candidate genes for uterine functional maintenance in chicken. This study discovered dominant genes and pathways which enhanced our knowledge of chicken uterine functional maintenance.

Identification of RNF150 as the hub gene associated with microsatellite instability in gastric cancer

Gastric cancer (GC) is a common digestive tract malignancy with the sixth global incidence and third cancer-related deaths, respectively. Microsatellite instability (MSI), accounting for one of the molecular subtypes of GC, plays an important role in GC and is affected by a sophisticated network of gene interactions. In this study, we aimed to explore the expression pattern and clinical performance of MSI related gene in GC patients. Weighted gene co-expression network analysis (WGCNA) was exploited to single out the vital module and core genes in TCGA database. We applied the protein–protein interaction (PPI) and survival analysis to propose and confirm RNF150 as the hub gene in GC. Finally, we utilized immunohistochemistry (IHC) and reverse transcription-polymerase chain reaction (RT-PCR) to explore the expression pattern of RNF150 in GC patients. With the highest weight correlation and standard correlation, RNF150 was selected as the hub gene for following validation. In validation, data obtained from the test sets showed a lower expression of RNF150 in MSI GC compared to microsatellite stability (MSS) GC. Moreover, survival analysis shows that MSI GC patients with a lower RNF150 expression level displayed the longer OS time. Compared to the expression in normal gastric tissues, the protein level of RNF150 was virtually up-regulated in ten cases of GC tissues. Furthermore, RNF150 protein level was decreased in MSI GC samples compared to MSS GC samples. When validated the mRNA expression with RT-PCR in fresh GC tissues, we also found the similar trend. RNF150 was identified as a novel MSI-related gene in GC. It is expected to be an auspicious prognostic biomarker for GC patients.

The lysosome-phagosome pathway mediates immune regulatory mechanisms in Mesocentrotus nudus against Vibrio coralliilyticus infection.

Sea urchins are a popular model species for studying invertebrate diseases. The immune regulatory mechanisms of the sea urchin Mesocentrotus nudus during pathogenic infection are currently unknown. This study aimed to reveal the potential molecular mechanisms of M. nudus during resistance to Vibrio coralliilyticus infection by integrative transcriptomic and proteomic analyses. Here, we identified a total of 135,868 unigenes and 4,351 proteins in the four infection periods of 0h, 20h, 60h and 100h in M. nudus. In the I20, I60 and I100 infection comparison groups, 10,861, 15,201 and 8,809 differentially expressed genes (DEGs) and 2,188, 2,386 and 2,516 differentially expressed proteins (DEPs) were identified, respectively. We performed an integrated comparative analysis of the transcriptome and proteome throughout the infection phase and found very a low correlation between transcriptome and proteome changes. KEGG pathway analysis revealed that most upregulated DEGs and DEPs were involved in immune strategies. Notably, "lysosome" and "phagosome" activated throughout the infection process, could be considered the two most important enrichment pathways at the mRNA and protein levels. The significant increase in phagocytosis of infected M. nudus coelomocytes further demonstrated that the lysosome-phagosome pathway played an important immunological role in M. nudus resistance to pathogenic infection. Key gene expression profiles and protein‒protein interaction analysis revealed that cathepsin family and V-ATPase family genes might be key bridges in the lysosome-phagosome pathway. In addition, the expression patterns of key immune genes were verified using qRT‒PCR, and the different expression trends of candidate genes reflected, to some extent, the regulatory mechanism of immune homeostasis mediated by the lysosome-phagosome pathway in M. nudus against pathogenic infection. This work will provide new insights into the immune regulatory mechanisms of sea urchins under pathogenic stress and help identify key potential genes/proteins for sea urchin immune responses.

Investigation of the mechanism of action of simiao pill against gout using network pharmacology and experimental validation

BackgroundGout is a chronic disease of deposition of monosodium urate crystals, characterised by hyperuricaemia and gouty arthritis, plaguing the whole world. Simiao Pill (SMP), a classic herbal formula, has been extensively used to treat arthritic symptoms in clinical practice in China for centuries. The action mode of multi-component and multi-target for traditional Chinese medicine (TCM) could be a promising treatment of gout. However, the pharmacological mechanisms of SMP still remain vague to date. Hypothesis/PurposeIn this study, network pharmacology was employed to elucidate the potential bioactive compounds and hypouricemic mechanisms of SMP. Study designAt first, we collected putative targets of SMP based on Traditional Chinese Medicine Systems Pharmacology Database databases, and relevant targets of gout were summarised by four searching database, including Disgenet, therapeutic target database, Online Mendelian Inheritance in Man, and Gene Cards. The putative targets of SMP and known therapeutic targets of gout built an interaction network, so the networks of compound targets and disease-targets were obtained soon. Then, the protein–protein interactions analysis and molecular docking verification were established. Finally, the key genes were used to find the biological pathway and explain the therapeutic mechanism by genome ontology and kyoto encyclopaedia of genes and genomes analysis. ResultsIn our study, 30 hypouricemic components and 600 hypouricemic targets of SMP were filtrated based on an online network database. Then the component-target interaction network was constructed, 10 key components and 15 key targets were further obtained. Docking studies indicated that the bio-active compounds could stably bind the pockets of target proteins. Go enrichment and and kyoto encyclopaedia of Genes and genomes pathway analysis indicated that SMP had a strong integration with gout and hyperuricemia, which might significantly contribute to multiple therapeutic effect. ConclusionsIn summary, this study used systems pharmacology to demonstrate the main components and mechanism of SMP in treating gout. Moreover, the work provided a scientific basis for the further effect research of SMP and its monomer components, but also provided a new therapeutic strategy in gout.