Functional Clustering Analysis Research Articles

Characterization of Changes in Ripening Process of Volatile Apple Compounds Based on HS-SPME-GC-MS Analysis

The aim of this study was to identify the aromatic compounds present in different apple varieties and to gain insights into the changes in the aromatic compounds during ripening. Three apple varieties (“Red Astrachan”, “Ning Qiu”, and “Golden Delicious”) at different stages of ripening were selected for this study; their peel and pulp were analyzed via headspace solid-phase microextraction (HS-SPME)–gas chromatography–mass spectrometry (HS-SPME-GC-MS), and 30 volatile compounds were identified. The samples’ differences were analyzed using heat map cluster analysis, principal component analysis (PCA), and an independent samples t-test. The results showed that the content of aromatic compounds in the peel was higher than that in the pulp. The relative content of esters in the aromatic compounds of the three apple varieties followed the order of pulp > peel and “Ning Qiu” > “Golden Delicious” > “Red Astrachan”. This suggests that “Ning Qiu” combines the advantages of its parents in terms of its aroma content. The highest concentrations of aroma compounds in “Red Astrachan” and “Ning Qiu” accumulate before the ripening stage, and care should be taken to choose an appropriate harvesting time according to the different needs during production. The main compounds of “Red Astrachan” are aldehydes and C8 esters, while those of “Ning Qiu” and “Golden Delicious” are alkenes and esters. After analyzing the relative odor activity values (rOAVs) of key volatile compounds and their aroma descriptors during the harvest period, acetic acid pentyl ester, butanoic acid hexyl ester, hexanoic acid hexyl ester, and 2-ethyl-1-Hexanol were found to contribute the most to the overall flavor of the peel and pulp. “Ning Qiu” combines the parental advantages of the concentrated peel of “Red Astrachan” and the astringent pulp of “Golden Delicious”, with compounds in its composition that give a pleasing aroma. Mature “Ning Qiu” fruits have a more intense aroma and fruity flavor. The development of flavor-specific varieties has provided the theoretical basis for future research in molecular hybridization, molecular-assisted breeding, and the molecular biology of apple flavor synthesis and metabolism.

Upregulation of miRNA-450b-5p targets ACTB to affect drug resistance and prognosis of ovarian cancer via the PI3K/Akt signaling pathway.

Ovarian cancer (OC) is the most malignant gynecologic cancer, and chemoresistance is a major cause of treatment failure in patients with OC. The understanding of microRNA (miRNA) in cancer is limited, and the role of miRNA (miR)-450b-5p in cancer drug resistance is unknown. In this study, we aim to evaluate the role of miR-450b-5p in drug-resistant OC and its underlying mechanisms. MiR-450b-5p expression was assessed in drug-sensitive and resistant OC cells via quantitative real-time polymerase chain reaction. Cell viability was evaluated using the Cell Counting Kit-8 assay. Progression-free survival (PFS) and overall survival (OS) curves were generated using the Kaplan-Meier method and the log-rank test. Target genes of miR-450b-5p were identified from the Cancer MIRNome database. Co-expressed genes were obtained from The Cancer Genome Atlas and Cancer Genome cBioportal for pathway enrichment and functional clustering analysis. The miRNA-450b-5p expression was significantly increased in A2780 and SKOV3 OC-resistant cells and significantly increased by 17-fold in the A2780-CBP-Lv-miR-450b-5p cells compared to A2780-CBP and A2780-CBP-Lv-NC cells. The up-regulated expression of miR-450b-5p increased the cell viability and half maximal inhibitory concentration (IC50) of A2780 platinum-resistant cells and was associated with poor OS. We obtained 33 potential target genes of miR-450b-5p and beta-actin (ACTB) might be a potential target of miR-450b-5p. Low expression of ACTB predicted poor OS and PFS. We obtained 362 common genes co-expressed with ACTB, which involved 4 critical pathways. PI3K acted as an upstream pathway of the other three pathways, which ultimately responded to drug resistance regulation in OC. The genes enriched in four pathways were cross-analyzed and 13 overlapping genes were obtained. These 13 genes were also significantly and positively co-expressed with ACTB at both protein and mRNA levels. High expression of miRNA-450b-5p might affect drug resistance and prognosis in OC by targeting 13 co-expressed genes of ACTB directly through the PI3K/Akt signaling pathway. Thus, miR-450b-5p might provide a new therapeutic target for drug resistance in OC.

Abstract P103: Assessment of Angiotensin II-induced Calcium Signals in Primary Human Adrenocortical Cells

Background: Cytosolic and mitochondrial Ca 2+ concentrations are key signals for triggering the secretion of aldosterone in primary aldosteronism (PA), the most common form of secondary hypertension. Aim: To investigate [Ca 2+ ] i transients and oscillations in human CD56 + cells isolated from aldosterone producing adenoma (APA) and normal surrounding adrenocortical (AAC) cells. Methods: We assessed [Ca 2+ ] i in CD56 + APA and APA-adjacent cells (AAC) in basal conditions and after angiotensin II (Ang II) stimulation using Fura-2 dye, live confocal microscopy, and an ad hoc developed custom-made pipeline to analyze [Ca 2+ ] i signals in terms of frequency and peak parameters as amplitude, full width at half maximum (FWHM) and area under the curve (AUC). As the two-pore domain K + TASK- 2 channels are under expressed in APA, we used quinidine (20 μM), 20’ prior to [Ca 2+ ] i imaging acquisition to examine the effect of the pharmacological inhibition of this channel. Results: Resting [Ca 2+ ] i basal levels were higher in AAC (n=11) (0.75, IQR;0.90-0.56) than in APA (n=11) (0.36 IQR;0.69-0.33), (p < 0.02). We detected spontaneous Ca 2+ activity, during resting conditions, in both cell types without significant difference in peak parameters. Upon 1 nM Ang II simulation, AAC showed elevated FWHM, amplitude, and AUC peak parameters of first Ca 2+ transients. However, APA cells exhibited elevated peak parameters of subsequent Ca 2+ oscillations. Ca 2 oscillations frequency and degree of oscillating cells were significantly higher in APA cells than in AAC. After 24 hours in culture, both AAC and APA cells developed rosette-like cell structure resembling the “glomeruli” of the intact zona glomerulosa clusters. Functional Statistics and clustering analysis showed synchronous behavior of cells belonging to the same cluster without significant differences between AAC and APA. In AAC, Task-2 Inhibition with quinidine tended to increase spontaneous activity and altered peak’s parameters but did not influence frequency of oscillation. Conclusion: Quantitative assessment of [Ca 2+ ] i signals unveiled significant differences in [Ca 2+ ] i dynamics between human AAC and APA cells. These results suggest the concurrent modulation of frequency and amplitude of [Ca 2+ ] I subsequent oscillations as a potential driver of increased rate of aldosterone production.

Phospholipid production and signaling by a plant defense inducer against Podosphaera xanthii is genotype-dependent.

Biotrophic phytopathogenic fungi such as Podosphaera xanthii have evolved sophisticated mechanisms to adapt to various environments causing powdery mildews leading to substantial yield losses. Today, due to known adverse effects of pesticides, development of alternative control means is crucial and can be achieved by combining plant protection products with resistant genotypes. Using plant defense inducers, natural molecules that stimulate plant immune system mimicking pathogen attack is sustainable, but information about their mode of action in different hosts or host genotypes is extremely limited. Reynoutria sachalinensis extract, a known plant defense inducer, especially through the Salicylic acid pathway in Cucurbitaceae crops against P. xanthii, was employed to analyze the signaling cascade of defense activation. Here, we demonstrate that R. sachalinensis extract enhances phospholipid production and signaling in a Susceptible to P. xanthii courgette genotype, while limited response is observed in an Intermediate Resistance genotype due to genetic resistance. Functional enrichment and cluster analysis of the upregulated expressed genes revealed that inducer application promoted mainly lipid- and membrane-related pathways in the Susceptible genotype. On the contrary, the Intermediate Resistance genotype exhibited elevated broad spectrum defense pathways at control conditions, while inducer application did not promote any significant changes. This outcome was obvious and at the metabolite level. Main factor distinguishing the Intermediate Resistance form the Susceptible genotype was the epigenetic regulated increased expression of a G3P acyltransferase catalyzing phospholipid production. Our study provides evidence on phospholipid-based signaling after plant defense inducer treatment, and the selective role of plant's genetic background.

Potential correlation between chronic periodontitis and Parkinson's disease.

This study aims to investigate possible hub genes, associated pathways, and transcription factors between chronic periodontitis (CP) and Parkinson's disease (PD). Gene expression profiles of CP (GSE16134, GSE23586, and GSE10334) and PD (GSE20141 and GSE49036) were downloaded from the gene expression omnibus (GEO) database for differential expression analysis and functional clustering analysis. The protein-protein interaction (PPI) network was constructed, and hub genes were screened by four topological analysis algorithms and modular segmentation. Functional clustering analysis was performed. The hub genes were validated by external datasets of CP and PD, and causal relation was further assessed by Mendelian randomization (MR). After merging the data, 1 211 differentially expressed genes (DEGs) were screened in the CP datasets; of which, 551 were upregulated and 660 were downregulated. A total of 2 407 DEGs were screened in the PD dataset, of which, 1 438 were upregulated and 969 were downregulated. The PPI network included 145 nodes and 126 edges. Four hub genes (FCGR3B, PRF1, IL18, and CD33) and three transcription factors (HSF1, HSF2, and HSF4) were finally screened. The relevant pathway was predominantly natural killer (NK) cell-mediated toxic effects. The MR results suggest a possible positive causal relationship between CP and the risk of developing PD. This study indicated the probably shared pathophysiology and possible causal relationship between CP and PD and may offer novel concepts and therapeutic targets for future mechanistic investigations.

Argonaute protein assisted drug discovery for miRNA-181c-5p and target gene ATM translation repression: a computational approach.

The miRNA binds to AGO's seed region, prompting the exploration of small molecules that can offset miRNA repression of target mRNA. This miRNA-181c-5p was found to be upregulated in the chronic traumatic encephalopathy, a prevalent neurodegenerative disease in contact sports and military personals. The research aimed to identify compounds that disrupt the AGO-assisted loop formation between miRNA-181c-5p and ATM, consequently repressing the translation of ATM. Target genes from commonly three databases (DIANA-microT-CDS, miRDB, RNA22 and TargetScan) were subjected to functional annotation and clustering analysis using DAVID bioinformatics tool. Haddock server were employed to make miRNA-181c-5p:ATM-AGO complex. A total of 2594 small molecules were screened using Glide XP based on their highest binding affinity towards the complex, through a three-phase docking approach. The top 5 compounds (DB00674-Galantamine, DB00371-Meprobamate, DB00694-Daunorubicin, DB00837-Progabide, and DB00851-Dacarbazine) were further analyzed for stability in the miRNA-181c-5p:ATM-AGO-ligand complex interaction using GROMACS (version 2023.2). Hence, these findings suggest that these molecules hold potential for facilitating AGO-assisted repression of ATM gene translation.

Mechanism of electroacupuncture for vascular dementia based on proteomics

To explore the potential mechanism of electroacupuncture (EA) for vascular dementia (VD) using tandem mass tag (TMT) quantitative proteomics technology. Among 80 male SPF SD rats, 78 rats which met the selection criteria through the Morris water maze test were selected and randomly divided into a sham surgery group (18 rats) and a surgery group (60 rats). VD model was established by four-vessel occlusion (4-VO) method in the surgery group, and 36 rats with successful modeling were randomly assigned to a model group (18 rats) and an EA group (18 rats). Each group was further divided into three subgroups based on intervention duration, with each subgroup containing 6 rats. Seven days after model establishment, the EA group received EA intervention at left and right "Sishencong" (EX-HN 1) and bilateral "Fengchi" (GB 20), with continuous wave at a frequency of 2 Hz and current intensity of 1 mA, daily for 30 min, with subgroups receiving EA for 7, 14, or 21 d respectively. Cognitive function before and after interventions was assessed using Morris water maze. Proteomic analysis was conducted on the optimal EA subgroup and corresponding sham surgery and model subgroups, identifying differentially expressed proteins and analyzing them through bioinformatics. Differentially expressed target proteins was performed using parallel reaction monitoring (PRM) and Western blot techniques. Compared to the sham surgery group, the model group exhibited prolonged escape latency and reduced number of platform crossings (P<0.01); compared with model group, the EA group showed reductions in escape latency and increased platform crossings after 7, 14, and 21 days of intervention (P<0.01, P<0.05). Compared to the 7 and 14-day intervention, the rats in the EA group of 21-day intervention showed the most significant improvements in reductions of escape latency and increased platform crossings (P<0.01, P<0.05), and was selected for further proteomic, PRM analyses, and Western blot validation. Compared to the sham surgery group, the model group displayed 71 differentially expressed proteins, with 50 up-regulated and 21 down-regulated proteins; compared to the model group, the EA group had 54 differentially expressed proteins, with 30 up-regulated and 24 down-regulated proteins. Functional enrichment and clustering analyses indicated that these proteins were primarily associated with cellular processes, metabolic processes, phagocytosis recognition, immune response, and regulation of extracellular matrix, etc. Enrichment was observed in the mammalian target of rapamycin (mTOR) signaling pathway and neurotrophic factors signaling pathways, involving glycogen synthase kinase 3β (GSK3β) and mitogen-activated protein kinase kinase 2 (Map2k2), with PRM and Western blot findings consistent with the proteomic results. Which meant that compared with the model group, the protein expression of GSK3β and Map2k2 of hippocampus was increased in the EA group (P<0.01, P<0.05). EA at "Sishencong" (EX-HN 1) and "Fengchi" (GB 20) could improve cognitive function in VD rats, with the mechanism involving multiple targets and pathways, potentially related to GSK3β, Map2k2 proteins, and the mTOR and neurotrophic factor signaling pathways.

Comparative proteomics reveals that fatty acid metabolism is involved in myocardial adaptation to chronic hypoxic injury.

Congenital heart disease (CHD) is the most serious form of heart disease, and chronic hypoxia is the basic physiological process underlying CHD. Some patients with CHD do not undergo surgery, and thus, they remain susceptible to chronic hypoxia, suggesting that some protective mechanism might exist in CHD patients. However, the mechanism underlying myocardial adaptation to chronic hypoxia remains unclear. Proteomics was used to identify the differentially expressed proteins in cardiomyocytes cultured under hypoxia for different durations. Western blotting assays were used to verify protein expression. A Real-Time Cell Analyzer (RTCA) was used to analyze cell growth. In this study, 3881 proteins were identified by proteomics. Subsequent bioinformatics analysis revealed that proteins were enriched in regulating oxidoreductase activity. Functional similarity cluster analyses showed that chronic hypoxia resulted in proteins enrichment in the mitochondrial metabolic pathway. Further KEGG analyses found that the proteins involved in fatty acid metabolism, the TCA cycle and oxidative phosphorylation were markedly upregulated. Moreover, knockdown of CPT1A or ECI1, which is critical for fatty acid degradation, suppressed the growth of cardiomyocytes under chronic hypoxia. The results of our study revealed that chronic hypoxia activates fatty acid metabolism to maintain the growth of cardiomyocytes.

Global compositional and functional states of the human gut microbiome in health and disease.

The human gut microbiota is of increasing interest, with metagenomics a key tool for analyzing bacterial diversity and functionality in health and disease. Despite increasing efforts to expand microbial gene catalogs and an increasing number of metagenome-assembled genomes, there have been few pan-metagenomic association studies and in-depth functional analyses across different geographies and diseases. Here, we explored 6014 human gut metagenome samples across 19 countries and 23 diseases by performing compositional, functional cluster, and integrative analyses. Using interpreted machine learning classification models and statistical methods, we identified Fusobacterium nucleatum and Anaerostipes hadrus with the highest frequencies, enriched and depleted, respectively, across different disease cohorts. Distinct functional distributions were observed in the gut microbiomes of both westernized and nonwesternized populations. These compositional and functional analyses are presented in the open-access Human Gut Microbiome Atlas, allowing for the exploration of the richness, disease, and regional signatures of the gut microbiota across different cohorts.

Morphological variation in Schizothorax oconnori, Schizothorax waltoni (Teleostei: Cyprinidae: Schizothoracinae), and their natural hybrids from the middle Yarlung Zangbo River, Tibet.

The morphological variation in Schizothorax oconnori, Schizothorax waltoni, and their natural hybrids was examined using conventional and image-based analysis approaches. In total, 38 specimens of S. oconnori, 35 of S. waltoni, and 37 natural hybrids were collected from the Shigatse to the Lhasa section of the Yarlung Zangbo River during June and July 2021. A total of 21 morphometric, 4 meristic, and 27 truss variables were employed for the classification of S. oconnori, S. waltoni, and natural hybrids. Principal component analysis (PCA) and factor analysis (FA), as well as discriminant function analysis (DFA) and cluster analysis (CA), were conducted to identify differences based on traditional and truss measurements. Four principal components explained 75.92% of the variation among the morphometric characters, while five principal components accounted for 79.69% of the variation among the truss distances. FA results showed that factor 1 was associated with head shape, and factor 2 was associated with fins based on morphometric characters. Among the truss characters, factor 1 was related to head shape, and factor 2 was related to chest shape. In DFA, morphometric measurements achieved higher accuracy (100%) compared to truss distances (94.55%). The head morphology of hybrids exhibited intermediate traits between S. oconnori and S. waltoni. Both morphometry-based and truss-based clustering indicated that the morphology of natural hybrids leaned toward S. oconnori. In conclusion, the combination of morphometric and truss analysis is beneficial for classifying S. oconnori, S. waltoni, and their natural hybrids. The presence of natural hybrids could be considered an evolutionary response to the differentiation of nutritional and spatial niches in the middle Yarlung Zangbo River.

The Microglial Transcriptome of Age-Associated Deep Subcortical White Matter Lesions Suggests a Neuroprotective Response to Blood-Brain Barrier Dysfunction.

Age-associated deep-subcortical white matter lesions (DSCLs) are an independent risk factor for dementia, displaying high levels of CD68+ microglia. This study aimed to characterize the transcriptomic profile of microglia in DSCLs and surrounding radiologically normal-appearing white matter (NAWM) compared to non-lesional control white matter. CD68+ microglia were isolated from white matter groups (n = 4 cases per group) from the Cognitive Function and Ageing Study neuropathology cohort using immuno-laser capture microdissection. Microarray gene expression profiling, but not RNA-sequencing, was found to be compatible with immuno-LCM-ed post-mortem material in the CFAS cohort and identified significantly differentially expressed genes (DEGs). Functional grouping and pathway analysis were assessed using the Database for Annotation Visualization and Integrated Discovery (DAVID) software, and immunohistochemistry was performed to validate gene expression changes at the protein level. Transcriptomic profiling of microglia in DSCLs compared to non-lesional control white matter identified 181 significant DEGs (93 upregulated and 88 downregulated). Functional clustering analysis in DAVID revealed dysregulation of haptoglobin-haemoglobin binding (Enrichment score 2.5, p = 0.017), confirmed using CD163 immunostaining, suggesting a neuroprotective microglial response to blood-brain barrier dysfunction in DSCLs. In NAWM versus control white matter, microglia exhibited 347 DEGs (209 upregulated, 138 downregulated), with significant dysregulation of protein de-ubiquitination (Enrichment score 5.14, p < 0.001), implying an inability to maintain protein homeostasis in NAWM that may contribute to lesion spread. These findings enhance understanding of microglial transcriptomic changes in ageing white matter pathology, highlighting a neuroprotective adaptation in DSCLs microglia and a potentially lesion-promoting phenotype in NAWM microglia.

Genetic Variability and Multivariate Analysis of Physio-Biochemical Traits in Coloured Grape Genotypes

Twenty-two coloured grape accessions were screened for various physio-biochemical traits. The genetic variability and multivariate analysis were carried out for growth and biochemical parameters of grape accessions grown under Pune conditions. High genotypic and phenotypic coefficient of variations were observed in average bunch weight, berry weight, proline and total tannin. All the biochemical parameters showed very high estimated heritability for broad sense (h2b.s.). Higher genetic gain was recorded for average bunch weight, total tannin, proline and protein while low genetic gain was observed in berry diameter and seeds/berry. The multivariate analysis revealed that biochemical parameters could be potentially used for discrimination of grape genotype. The heat map cluster analysis showed higher biochemical parameters in Kali Sahebi, Kishmish Maldovski, Red Ficosa, H-516 and Omania Black. Among the accessions studied, Kali Sahebi performed better concerning growth attributes like average bunch weight, berry diameter, berry and seed weight, days to harvest and biochemical constituents like total phenol, anthocyanin and colour intensity. A very close association was observed between phenotypic and genotypic variation, particularly for biochemical parameters. Therefore, very high heritability and genetic advance were recorded in these parameters. It can be concluded that the parameters governing the very close association between phenotypic and genotypic variation can contribute to the varietal selection and trait-specific varietal development programmes.

Bioinformatics integration reveals key genes associated with mitophagy in myocardial ischemia-reperfusion injury

BackgroundMyocardial ischemia is a prevalent cardiovascular disorder associated with significant morbidity and mortality. While prompt restoration of blood flow is essential for improving patient outcomes, the subsequent reperfusion process can result in myocardial ischemia–reperfusion injury (MIRI). Mitophagy, a specialized autophagic mechanism, has consistently been implicated in various cardiovascular disorders. However, the specific connection between ischemia–reperfusion and mitophagy remains elusive. This study aims to elucidate and validate central mitophagy-related genes associated with MIRI through comprehensive bioinformatics analysis.MethodsWe acquired the microarray expression profile dataset (GSE108940) from the Gene Expression Omnibus (GEO) and identified differentially expressed genes (DEGs) using GEO2R. Subsequently, these DEGs were cross-referenced with the mitophagy database, and differential nucleotide sequence analysis was performed through enrichment analysis. Protein–protein interaction (PPI) network analysis was employed to identify hub genes, followed by clustering of these hub genes using cytoHubba and MCODE within Cytoscape software. Gene set enrichment analysis (GSEA) was conducted on central genes. Additionally, Western blotting, immunofluorescence, and quantitative polymerase chain reaction (qPCR) analyses were conducted to validate the expression patterns of pivotal genes in MIRI rat model and H9C2 cardiomyocytes.ResultsA total of 2719 DEGs and 61 mitophagy-DEGs were identified, followed by enrichment analyses and the construction of a PPI network. HSP90AA1, RPS27A, EEF2, EIF4A1, EIF2S1, HIF-1α, and BNIP3 emerged as the seven hub genes identified by cytoHubba and MCODE of Cytoscape software. Functional clustering analysis of HIF-1α and BNIP3 yielded a score of 9.647, as determined by Cytoscape (MCODE). In our MIRI rat model, Western blot and immunofluorescence analyses confirmed a significant elevation in the expression of HIF-1α and BNIP3, accompanied by a notable increase in the ratio of LC3II to LC3I. Subsequently, qPCR confirmed a significant upregulation of HIF-1α, BNIP3, and LC3 mRNA in the MIRI group. Activation of the HIF-1α/BNIP3 pathway mediates the regulation of the degree of Mitophagy, thereby effectively reducing apoptosis in rat H9C2 cardiomyocytes.ConclusionsThis study has identified seven central genes among mitophagy-related DEGs that may play a pivotal role in MIRI, suggesting a correlation between the HIF-1α/BNIP3 pathway of mitophagy and the pathogenesis of MIRI. The findings highlight the potential importance of mitophagy in MIRI and provide valuable insights into underlying mechanisms and potential therapeutic targets for further exploration in future studies.

The characterization of protein lactylation in relation to cardiac metabolic reprogramming in neonatal mouse hearts

In mammals, the neonatal heart can regenerate upon injury within a short time after birth, while adults lose this ability. Metabolic reprogramming has been demonstrated to be critical for cardiomyocyte proliferation in the neonatal heart. Here, we reveal that cardiac metabolic reprogramming could be regulated by altering global protein lactylation. By performing 4D label-free proteomics and lysine lactylation (Kla) omics analyses in mouse hearts at postnatal days 1, 5, and 7, 2297 Kla sites from 980 proteins are identified, among which 1262 Kla sites from 409 proteins are quantified. Functional clustering analysis reveals that the proteins with altered Kla sites are mainly involved in metabolic processes. The expression and Kla levels of proteins in glycolysis show a positive correlation while a negative correlation in fatty acid oxidation. Furthermore, we verify the Kla levels of several differentially modified proteins, including ACAT1, ACADL, ACADVL, PFKM, PKM, and NPM1. Overall, our study reports a comprehensive Kla map in the neonatal mouse heart, which will help to understand the regulatory network of metabolic reprogramming and cardiac regeneration.

Machine learning and experimental validation identified autophagy signature in hepatic fibrosis.

The molecular mechanisms of hepatic fibrosis (HF), closely related to autophagy, remain unclear. This study aimed to investigate autophagy characteristics in HF. Gene expression profiles (GSE6764, GSE49541 and GSE84044) were downloaded, normalized, and merged. Autophagy-related differentially expressed genes (ARDEGs) were determined using the limma R package and the Wilcoxon rank sum test and then analyzed by GO, KEGG, GSEA and GSVA. The infiltration of immune cells, molecular subtypes and immune types of healthy control (HC) and HF were analyzed. Machine learning was carried out with two methods, by which, core genes were obtained. Models of liver fibrosis in vivo and in vitro were constructed to verify the expression of core genes and corresponding immune cells. A total of 69 ARDEGs were identified. Series functional cluster analysis showed that ARDEGs were significantly enriched in autophagy and immunity. Activated CD4 T cells, CD56bright natural killer cells, CD56dim natural killer cells, eosinophils, macrophages, mast cells, neutrophils, and type 17 T helper (Th17) cells showed significant differences in infiltration between HC and HF groups. Among ARDEGs, three core genes were identified, that were ATG5, RB1CC1, and PARK2. Considerable changes in the infiltration of immune cells were observed at different expression levels of the three core genes, among which the expression of RB1CC1 was significantly associated with the infiltration of macrophage, Th17 cell, natural killer cell and CD56dim natural killer cell. In the mouse liver fibrosis experiment, ATG5, RB1CC1, and PARK2 were at higher levels in HF group than those in HC group. Compared with HC group, HF group showed low positive area in F4/80, IL-17 and CD56, indicating decreased expression of macrophage, Th17 cell, natural killer cell and CD56dim natural killer cell. Meanwhile, knocking down RB1CC1 was found to inhibit the activation of hepatic stellate cells and alleviate liver fibrosis. ATG5, RB1CC1, and PARK2 are promising autophagy-related therapeutic biomarkers for HF. This is the first study to identify RB1CC1 in HF, which may promote the progression of liver fibrosis by regulating macrophage, Th17 cell, natural killer cell and CD56dim natural killer cell.

Genomic analysis reveals genes that encode the synthesis of volatile compounds by a Bacillus velezensis-based biofungicide used in the treatment of grapes to control Aspergillus carbonarius

Fungal control strategies based on the use of Bacillus have emerged in agriculture as eco-friendly alternatives to replace/reduce the use of synthetic pesticides. Bacillus sp. P1 was reported as a new promising strain for control of Aspergillus carbonarius, a known producer of ochratoxin A, categorized as possible human carcinogen with high nephrotoxic potential. Grape quality can be influenced by vineyard management practices, including the use of fungal control agents. The aim of this study was to evaluate, for the first time, the quality parameters of Chardonnay grapes exposed to an antifungal Bacillus-based strategy for control of A. carbonarius, supporting findings by genomic investigations. Furthermore, genomic tools were used to confirm that the strain P1 belongs to the non-pathogenic species Bacillus velezensis and also to certify its biosafety. The genome of B. velezensis P1 harbors genes that are putatively involved in the production of volatiles and hydrolytic enzymes, which are responsible for releasing the free form of aroma compounds. In addition to promote biocontrol of phytopathogenic fungi and ochratoxins, the treatment with B. velezensis P1 did not change the texture (hardness and firmness), color and pH of the grapes. Heat map and hierarchical clustering analysis (HCA) of volatiles evaluated by GC/MS revealed that Bacillus-treated grapes showed higher levels of compounds with a pleasant odor descriptions such as 3-hydroxy-2-butanone, 2,3-butanediol, 3-methyl-1-butanol, 3,4-dihydro-β-ionone, β-ionone, dihydroactinidiolide, linalool oxide, and β-terpineol. The results of this study indicate that B. velezensis P1 presents desirable properties to be used as a biocontrol agent.

Inhibition of anti-tumour reactivity of immune cells in the salivary gland cancer: A proteomic approach

BackgroundAdenoid cystic carcinoma (ACC), mucoepidermoid carcinoma (MEC), and oral squamous cell carcinoma (OSCC) respond differently to immunotherapy. Pembrolizumab, an immune checkpoint inhibitor, has been approved by the Food and Drug Administration for the treatment of squamous cell carcinomas of the head and neck region. While MEC has shown some response to pembrolizumab; however, ACC is the least responsive. At the molecular level, head and neck cancers produce immunosuppressive molecules, resulting in immune evasion. Therefore, we hypothesised that salivary gland cancer cells produce a higher number of immunosuppressive proteins that cause suppression of the immune system's anti-tumour reactivity. MethodTo determine differential protein expressions in OSCC, MEC, and ACC, we constructed cancer–immune cell co-culture models using different oral and salivary gland cancer cells. We performed SWATH, proteome profilers, gene ontology biological function, functional annotation clustering and protein interaction network analysis of all cancer samples in the co-culture models. ResultsAnalysis of the acquired data showed that the overexpressed proteins in the OSCC cells and participated more in metabolic process, while in the salivary gland cancer cells, overexpressed proteins participated more in immune processes, immune checkpoint pathway. Upon protein function analysis of salivary gland cells, the overexpressed proteins found negatively affecting immune process and checkpoint pathway proteins. ConclusionOverall, we conclude that salivary gland cancer is less responsive to immunotherapy, possibly because of the high presence of immunosuppressive proteins. However, further analysis is needed to verify the biological functions and interactive partners of each differentially expressed protein in ACC cells.

Specific regional features of modern euroscepticism: A comparative analysis

This study investigated the topical issue of regional variation of Euroscepticism in the modern EU. The growing opposition to European integration in different parts of the continent threatens the unity and legitimacy of the European project, which necessitates a thorough study of the specifics of this phenomenon in a comparative perspective. The pur- pose of this study was to carry out a comprehensive analysis of the specific regional features of modern Euroscepticism based on theoretical understanding and empirical verification of its determinants and manifestations in different con- texts. The study employed methods of comparison, structural, and functional analysis, cluster analysis, discourse analysis, and case studies to identify common patterns and differences in the nature and dynamics of Euroscepticism in different countries and regions. The findings of the study suggest that Euroscepticism is a multidimensional and context-specific phenomenon, the intensity and nature of which is determined by a complex of historical, socio-economic, political, and cultural factors. The comparative analysis has found three main regional models of Euroscepticism – Central European, Southern European, and Northwestern – each with a distinctive profile and determinants. Despite the differences, a com- mon pattern is the link between the dynamics of Euroscepticism and the asymmetric effects of integration at the local level – the growth of Euroscepticism is most pronounced in peripheral regions and communities that have been margin- alised by economic development and feel threatened by their own identity. At the same time, the penetration of moderate Euroscepticism into the political mainstream poses risks of normalising populist criticism of the EU

Umbilical Cord Blood-Derived Cells Can Reconstruct Hematopoiesis in an Aplastic Anemia Animal Model.

To explore the efficacy and the mechanism of the umbilical cord-derived cells combined with cyclosporine A (CsA) in treating aplastic anemia (AA) in mice. Immune-mediated AA model mice were treated with CsA + UC mesenchymal stem cells (UC-MSC), CsA + umbilical cord blood regulatory T cells (UCB-Treg), UC-MSC, UCB-Treg, CsA alone, or blank control, respectively (n = 9 mice/group). CsA and the cell infusion was administered on d0. Routine peripheral blood testing was performed once weekly; bone marrow colony culture, bone marrow cell flow cytometry, peripheral blood T cell subsets, and serum inflammatory cytokines tests were performed on d14. Transcriptome sequencing was performed for cells from CsA + UC-MSC, CsA + UCB-Treg, and CsA groups to detect the possible related genes. Gene function cluster and signal pathway enrichment analysis were also performed. Blank control mice died due to pancytopenia within 21 days, whereas mice in other groups survived for >28 days. On d14, the CsA + UC-MSC and CsA + UCB-Treg groups had higher white blood cell (WBC) counts than the other groups (p < 0.05), along with higher burst-forming unit (BFU) and colony-forming unit-granulocyte, macrophage (CFU-GM) counts (p < 0.01). The CsA + UC-MSC group had the highest BFU count (p < 0.01). The CsA + UC-MSC and CsA + UCB-Treg groups exhibited the highest bone marrow CD34+ cell proportion (9.68% ± 1.35% and 8.17% ± 0.53%, respectively; p < 0.01). Tumor necrosis factor (TNF)-α and interleukin (IL)-2 levels in the CsA + UC-MSC group (p < 0.05) and TNF-α, interleukin-2, and interferon (INF)-γ levels in the CsA + UC-Treg group (p < 0.01) were lower than those in the CsA group. Compared with CsA treatment, CsA + UC-MSC significantly downregulated the histone methylation pathway (p < 0.05), whereas CsA + UCB-Treg significantly upregulated energy metabolism processes (p < 0.05). Treatment with CsA + UC-MSC upregulated superoxide dismutase activity compared with CsA + UCB-Treg treatment. Adding UC-MSC or UCB-Treg to CsA markedly enhanced the reconstruction of hematopoiesis in AA mice, with UC-MSC eliciting greater efficiency than UCB-Treg. Accordingly, the addition of these cells could further improve immune abnormalities.

Quantitative proteomic analysis reveals regulatory networks of extracellular matrix receptor interaction pathways in endothelial cells after myocardial infarction

Cardiac fibrosis, a significant pathological alteration following myocardial infarction (MI), remains enigmatic with respect to the role of cardiac endothelial cells (ECs). To elucidate the proteomic shifts in cardiac ECs accompanying MI-induced cardiac fibrosis, a standard MI mice model was established through ligation of the left anterior descending branch. Following 14 days of effective modeling, we isolated primary ECs from the hearts of both sham and MI models utilizing the CD31 microbeads sorting technique. Quantitative proteomics and bioinformatics methodologies, including tandem mass spectrometry, were employed to discern proteomic alterations in the primary endothelial cells of the experimental groups. Comprehensive analyses, including Gene Ontology analysis, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, functional enrichment analysis, and functional enrichment cluster analysis, revealed an up-regulation of proteins associated with extracellular matrix-receptor interaction pathway in cardiac fibrosis post-MI. Subsequent Western blot analysis confirmed the up-regulation of specific proteins involved in this pathway, namely collagen type VI alpha 2 (Col6&amp;alpha;2), vitronectin (Vtn), and integrin beta (Itg&amp;beta;). We conclude that the expression levels of Col6&amp;alpha;2, Vtn, and Itg&amp;beta; in primary ECs during the early stage of cardiac fibrosis, 14 days post-MI, were significantly elevated compared to the sham group (P &lt; 0.05). This observation suggests that ECM-receptor interaction could potentially influence the progression of cardiac fibrosis following MI.