Pathway Analysis Research Articles

Innovative microbial activators for enhanced bioremediation of oil-contaminated soils: mechanistic insights.

This paper developed an efficient microbial activator formula and conducted an in-depth study on its efficacy and mechanism in promoting the degradation of petroleum hydrocarbons in oil-contaminated soil. A 60-day microbial remediation experiment conducted on oily soil revealed that the microbial activators significantly boosted the activities of dehydrogenase and catalase, subsequently speeding up the degradation of petroleum hydrocarbons in the soil. The overall degradation rate reached as high as 71.23%, with the most significant degradation effect observed in asphaltenes, achieving a degradation rate of 93.98%. This was followed by aromatic hydrocarbons (90.45%), saturated hydrocarbons (84.39%), and asphaltenes (65%). Compared to traditional microbial stimulation methods, this activator demonstrated significant superiority. Microbial diversity analysis reveals that microbial activators can effectively activate microbial activity in soil targeting refractory petroleum hydrocarbon components. By comparing the changes in microbial community structure before and after the addition of microbial activators, we found that the activators promoted an increase in the abundance of microorganisms belonging to the Bacillota, Pseudomonadota, and Bacteroidetes, which have petroleum hydrocarbon degradation functions, and facilitated the evolution of microbial community structure towards a direction more conducive to petroleum hydrocarbon degradation. KEGG metabolic pathway analysis revealed that the degradation pathways for alkanes, aromatic hydrocarbons, and PAHs are primarily present in these bacterial phylum. This research not only clarifies the degradation mechanism but also supports future bioremediation efforts.

Metabolic profiling of meningioma reveals novel subgroup-specific biologic insights and outcome dependencies.

Our group and others have recently identified four molecular groups of meningioma, with unique underlying biology and outcomes. The relevance of group-specific metabolite profiles (particularly among hypermetabolic tumours), has not been explored. We performed untargeted metabolic profiling of meningiomas representing each molecular group and WHO grade. Prognostic biochemicals were identified using Cox regression and their biological importance was explored using RNA and protein-based pathway analyses. Validation was performed using targeted high performance liquid chromatography-mass spectrometry (HPLC-MS/MS). Global metabolic profiling identified 560 unique biochemicals. We identified a 21-metabolite outcome signatures which is strongly predictive of outcome after adjusting for WHO grade, extent of resection, and receipt of adjuvant radiotherapy (HR 326.49, 95%CI 16.72-6375.48, p < 0.0001). The abundance of N6-trimethyllysine was associated with earlier time to recurrence on our whole cohort (log-rank p = 0.009) and within hypermetabolic and WHO grade 2 tumours specifically; this was validated using targeted HPLC-MS/MS on two cohorts. Consensus RNA and protein expression analysis demonstrated as association between N6-trimethyllysine abundance and activation of oxidative phosphorylation pathways, which portended worse outcomes in the hypermetabolic subgroup but, interestingly, better outcomes in the proliferative subgroup. By contrast, upregulated pyruvate and lactate transporters were associated with worse outcomes in proliferative meningiomas specifically. This is the first study to demonstrate a subgroup-specific prognostic role of N6-trimethyllysine in hypermetabolic meningiomas, offering increasingly granular outcome predictions using a widely accessible technique (HPLC-MS/MS). We also suggest fundamental differences in preferred energy utilization between and a potential need for subgroup-specific therapies.

Potential prognostic and predictive biomarkers: METTL5, METTL7A, and METTL7B expression in gastrointestinal cancers.

The methyltransferase gene family is known for its diverse biological functions and critical role in tumorigenesis. This study aimed to identify these family genes in common gastrointestinal (GI) cancers using comprehensive methodologies. Gene identification involved analysis of scientific literature and insights from The Cancer Genome Atlas (TCGA) database. RNA sequencing (RNA-seq) data for colon, gastric, pancreatic, esophageal, and liver cancers were collected, processed, and normalized. Differential expression analysis was conducted using R software with the Limma package. Additionally, real-time PCR analysis was performed on 30 tumor and 30 normal tissue samples from patients with colon and gastric cancer. Pathway analysis was conducted via the EnrichR web tool, while survival analysis used Cox regression methods, and biomarker potential was assessed with the pROC package. Prognostic significance was evaluated by examining associations between gene expression, patient survival, and recurrence rates. The study also investigated diagnostic potential through receiver operating characteristic (ROC) analysis, and assessed how small molecules affect gene expression, with implications for drug resistance and sensitivity, analyzed via CCLE and GDSC datasets. Findings revealed METTL5 overexpression in colon, liver, esophagus, and pancreas cancers, while METTL7A was underexpressed in gastric, esophagus, liver, and colon cancers. METTL7B expression varied, being higher in gastric and esophagus cancers but lower in liver and colon cancers. Enrichment analysis identified pathways related to these genes, and survival analysis associated altered METTL7A and METTL5 expressions with poor prognosis and higher recurrence rates. These findings suggest that METTL genes could serve as predictive biomarkers in GI cancers, offering potential implications for patient prognosis and treatment response.

Methylaluminoxane Reactivities and Anionic Structures: From Small Oligomers to Large Sheets.

The structure and reactivity of small methylaluminoxane (MAO) species (MeAlO)n(Me3Al)m (n = 1-8) have been investigated using DFT (M06-2X), MP2, and CCSD(T) calculations. This hierarchy of methods reveals that DFT artificially stabilizes structures containing 4-coordinate oxygen atoms while higher-level calculations demonstrate a clear preference for structures with 3-coordinate oxygen and 4-coordinate aluminum centers. Analysis of ionization pathways shows these neutral MAO molecules form anions through either methide or Me2Al+ abstraction, with the latter mechanism dominant for sheet structures (n = 5-8). Smaller species (n = 1-4, m ≥ n) show minimal reactivity toward either ionization pathway. The resulting anions consistently adopt sheet structures with 3-coordinate oxygen and 4-coordinate aluminum at all levels of theory. We introduce a comprehensive stability metric incorporating both ionization energies and neutral precursor stability, which successfully explains the distribution of anions observed in ESI-MS spectra during Me3Al hydrolysis. Extension of this analysis to larger species, including the recently isolated and characterized sheet (MeAlO)26(Me3Al)9 (Luo et al., Science, 2024, 384, 1424-1428), reveals unexpected trends in reactivity.

The Cry2Aa protein is not enough to pose a threat to Pardosa astrigera.

The widespread commercialization of genetically modified (GM) crops makes it important to assess the potential impact of Bacillus thuringiensis (Bt) on non-target organisms. Pardosa astrigera is an important predator in agroforestry ecosystems, and female and male spiders may react differently to Bt toxins due to their different activity habits and nutritional requirements. In this study, we found that exposure to Cry2Aa protein did not affect the survival and body weight of P. astrigera during growth and development. However, according to 16S rRNA sequencing results of the P. astrigera adults, Cry2Aa protein not only changed the diversity of symbiont bacteria, but also changed its symbiont composition. During feeding on prey without Bt artificial feed, the dominant communities in female and male adults were Actinobacteria and Corynebacterium-1, respectively. Feeding on prey containing Cry2Aa protein, Firmicutes were the dominant phyla. At the genus level, Cry2Aa protein significantly increased the relative abundance of Enterococcus and became the dominant genus in females only. In addition, Bacillus, Weissella and other symbiotic bacteria had significant changes in females. In terms of species composition, sex differences resulted in the absence of different types of symbiotic bacteria. Functional analysis of enrichment pathways showed significant changes in various metabolic pathways such as "Carbohydrate metabolism" and "Nucleotide metabolism", and there are differences between the sexes. These findings provide new data information and support for revealing the different strategies of spiders to cope with Cry2Aa protein based on sex differences, and also provide new data information and support for environmental safety assessment of GM crops.

Lignin-coordinated niobium-based catalyst for the efficient conversion of industrial lignin in choline chloride-lactic acid integrated with ethanol deep eutectic solvent.

Catalytic depolymerization is a favorable option for the valorization of industrial lignin. In this study, a new strategy was demonstrated for the efficient reductive depolymerization of industrial lignin based on a complex solvent of choline chloride-lactic acid (ChCl-LA) DES integrated with ethanol and a C-supported N-doped niobium-based catalyst with industrial lignin as carbon source (NBC@N-LC). It was found that the introduction of ethanol significantly improved the conversion of industrial lignin in ChCl-LA. More delightfully, depolymerization of industrial lignin in the complex solvent of DES-ethanol was further promoted by using the fabricated NBC@N-LC, exhibiting the best lignin dissolution performance, the lowest undepolymerized lignin yield of 21.9 % and the highest monomers yield of 31.8 %. 4-Ethylphenol was observed to dominate the monomers with 53.6 % selectivity, revealing the directional reactivity of the catalytic depolymerization process. Reaction pathway analysis showed that the synergistic effect of NBC@N-LC and DES-ethanol led to the efficient depolymerization of lignin, in which DES acted as an excellent solvent for lignin and depolymerized intermediates, ethanol acted as an in-situ hydrogen donor, and NBC@N-LC acted as a provider of reaction sites and acid active centers. In conclusion, this work offers a good alternative compared to the available lignin depolymerization method in terms of efficiency, selectivity, and cost aspects.

Effects of rumen-degradable starch on lactation performance, gastrointestinal fermentation, and plasma metabolomic in dairy cows.

This study investigated the effects of rumen-degradable starch (RDS) on lactation performance, gastrointestinal fermentation, and plasma metabolomics in dairy cows. Six mid-lactation cows, fitted with rumen, duodenum, and ileum cannulas, were used in a duplicated 3 × 3 Latin square design with 28-day periods. The cows were fed a low RDS (LRDS; 62.18 %), medium-RDS (MRDS; 71.25 %), or high-RDS (HRDS; 80.32 %) diet. The results showed that cows fed HRDS had diet a lower milk fat content by 14.77 % (LRDS) and 11.73 % (MRDS), while increased somatic cell count compared to the LRDS and MRDS groups (34.42 and 29.38 %, respectively). Additionally, rumen fluid pH was decreased in the HRDS group than in the MRDS and LRDS groups (7.81 and 7.08 %, respectively), while microbial protein (MCP) concentration was higher in the MRDS group. The HRDS group had lower concentrations of total volatile fatty acids (TVFA) and acetate in the ileal digesta than the LRDS group. The HRDS diet decreased neutral detergent fibre (NDF) digestibility compared with LRDS and MRDS groups (7.68 and 8.50 %, respectively), and reduced plasma concentrations of superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX), while increasing plasma lipopolysaccharide (LPS) and serum amyloid-A (SAA) levels. Pathway analysis revealed that starch and sucrose metabolism, galactose metabolism, and carbohydrate digestion and absorption were upregulated in the MRDS and HRDS groups. The HRDS diet had a tendency to negatively affect linoleic acid metabolism, glycerophospholipid metabolism, and alpha-linolenic acid metabolism. These findings provide insights into optimising feed efficiency and milk quality by regulating RDS levels in dairy cow diets.

Remodeling the Proinflammatory Microenvironment in Osteoarthritis through Interleukin-1 Beta Tailored Exosome Cargo for Inflammatory Regulation and Cartilage Regeneration.

Osteoarthritis (OA) presents a significant therapeutic challenge, with few options for preserving joint cartilage and repairing associated tissue damage. Inflammation is a pivotal factor in OA-induced cartilage deterioration and synovial inflammation. Recently, exosomes derived from human umbilical cord mesenchymal stem cells (HucMSCs) have gained recognition as a promising noncellular therapeutic modality, but their use is hindered by the challenge of harvesting a sufficient number of exosomes with effective therapeutic efficacy. Given that HucMSCs are highly sensitive to microenvironmental signals, we hypothesized that priming HucMSCs within a proinflammatory environment would increase the number of exosomes secreted with enhanced anti-inflammatory properties. Subsequent miRNA profiling and pathway analysis confirmed that interleukin-1 beta (IL-1β)-induced exosomes (C-Exos) exert positive effects through miRNA regulation and signaling pathway modulation. In vitro experiments revealed that C-Exos enhance chondrocyte functionality and cartilage matrix production, as well as macrophage polarization, thereby enhancing cartilage repair. C-Exos were encapsulated in hyaluronic acid hydrogel microspheres (HMs) to ensure sustained release, leading to substantial improvements in the inflammatory microenvironment and cartilage regeneration in a rat OA model. This study outlines a strategy to tailor exosome cargo for anti-inflammatory and cartilage regenerative purposes, with the functionalized HMs demonstrating potential for OA treatment.

Mogroside V ameliorates astrocyte inflammation induced by cerebral ischemia through suppressing TLR4/TRADD pathway.

Inflammation and oxidative stress are pivotal factors in the onset and progression of secondary injury following cerebral ischemia-reperfusion (I/R). Mogroside V (MV), a primary active compound of Siraitia grosvenorii, exhibits significant anti-inflammatory and antioxidant properties. However, its specific effects in cerebral ischemia remain unclear. In this study, we evaluated the neuroprotective effects of MV in a model of focal cerebral ischemia. Male C57BL/6J mice were subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) as an in vivo model of cerebral ischemia-reperfusion injury (CIRI), while U87 cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate CIRI in vitro. MV administration was found to reduce mortality, infarct volume, cerebral edema, and alleviate neurological deficits in these I/R mice. Furthermore, MV mitigated cerebral I/R injury by decreasing oxidative stress markers, such as reactive oxygen species (ROS) and malondialdehyde (MDA), while enhancing superoxide dismutase (SOD) levels. Gene Set Enrichment Analysis (GSEA) of the KEGG pathway revealed that most differentially expressed genes (DEGs) were involved in the Toll-like receptor/NF-κB/TNF/apoptosis signaling pathway. These findings were confirmed by real-time PCR, western blotting, immunohistochemistry, and immunofluorescence co-localization which demonstrated that MV reduced astrocyte inflammatory responses by inhibiting cytokine secretion associated with the TLR4/TRADD pathway. Additionally, MV protected neurons from apoptosis, as supported by TUNEL, Nissl, and HE staining. In conclusion, MV attenuates astrocyte inflammation and exerts neuroprotective effects following cerebral I/R injury, likely through suppression of the TLR4/TRADD signaling pathway.

Growth of Hexagonal Boron Nitride from Molten Nickel Solutions: A Reactive Molecular Dynamics Study.

Metal flux methods are excellent for synthesizing high-quality hexagonal boron nitride (hBN) crystals, but the atomic mechanisms of hBN nucleation and growth in these systems are poorly understood and difficult to probe experimentally. Here, we harness classical reactive molecular dynamics (ReaxFF) to unravel the mechanisms of hBN synthesis from liquid nickel solvent over time scales up to 30 ns. These simulations mimic experimental conditions by including relatively large liquid nickel slabs containing dissolved boron and a molecular nitrogen gas phase. Overall, the reaction takes place almost exclusively on the surface of the liquid nickel, owing to the low solubility of nitrogen in bulk nickel and the intermediate species' preference for the metal-gas interface. The formation of hBN invariably begins by reaction of dinitrogen with nickel-solvated boron atoms at the surface, forming intermediate N-N-B species, which typically evolve into B-N-B units through a short-lived intermediate where a single nitrogen atom is coordinated by one nitrogen and two boron atoms. The resulting B-N-B units, in turn, coalesce with growing hBN nuclei and carry nitrogen between hBN nanocrystals in an Ostwald ripening process. The amount of hBN produced on the tens of nanosecond time scale depends critically on the boron concentration, while having a much weaker dependence on the N2 pressure for the regime considered (N2 pressures of 2.5-10 MPa, Ni-B solutions with 6-12% boron by atom fraction). The highest rate of hBN formation occurs at the lowest temperature considered (1750 K, just above the melting point of nickel), while no hBN sheets are formed at 2000 K or above. An analysis of the transition pathways for nitrogen atoms shows that the final step, incorporation of small B-N motifs into larger hBN sheets, is the rate-limiting step in the regimes considered. While raising the temperature from 1750 to 2000 K has little effect on the formation of intermediates (N-N-B, B-N-B, etc.), the lack of large hBN sheets at temperatures >1900 K is explained by decreased probability of the final step and increased probability of breakup of hBN into B-N motifs.

Identification of pain-related long non-coding RNAs for pulpitis prediction.

We investigated the recently generated RNA-sequencing dataset of pulpitis to identify the potential pain-related lncRNAs for pulpitis prediction. Differential analysis was performed on the gene expression profile between normal and pulpitis samples to obtain pulpitis-related genes. The co-expressed gene modules were identified by weighted gene coexpression network analysis (WGCNA). Then the hypergeometric test was utilized to screen pain-related core modules. The functional enrichment analysis was performed on the up- and down-regulated genes in the core module of pulpitis pain to explore the underlying mechanisms. A pain-related lncRNA-based classification model was constructed using LASSO. Consensus clustering and gene set variation analysis (GSVA) on the infiltrating immunocytes was used for pulpitis subtyping. miRanda predicts miRNA-target relationship, which was filtered by expression correlation. Hallmark pathway and enrichment analysis was performed to investigate the candidate target pathways of the lncRNAs. A total of 1830 differential RNAs were identified in pulpitis. WGCNA explored seven co-expressed modules, among which the turquoise module is pain-related with hypergeometric test. The up-regulated genes were significantly enriched in immune response related pathways. Down-regulated genes were significantly enriched in differentiation pathways. Eight lncRNAs in the pain-related module were related to inflammation. Among them, MIR181A2HG was downregulated while other seven lncRNAs were upregulated in pulpitis. The LASSO classification model revealed that MIR181A2HG and LINC00426 achieved outstanding predictive performances with perfect ROC-AUC score (AUC = 1). We differentiated the pulpitis samples into two progression subtypes and MIR181A2HG is a progressive marker for pulpitis. The miRNA-mRNA-lncRNA regulatory network of pulpitis pain was constructed, with GATA3 as a key transcription factor. NF-kappa B signaling pathway is a candidate pathway impacted by these lncRNAs. PCED1B-AS1, MIAT, MIR181A2HG, LINC00926, LINC00861, LINC00528, LINC00426 and ITGB2-AS1 may be potential markers of pulpitis pain. A two-lncRNA signature of LINC00426 and MIR181A2HG can accurately predict pulpitis, which could facilitate the molecular diagnosis of pulpitis. GATA3 might regulate these lncRNAs and downstream NF-kappa B signaling pathway. This study identified potential pain-related lncRNAs with underlying molecular mechanism analysis for the prediction of pulpitis. The classification model based on lncRNAs will facilitate the early diagnosis of pulpitis.

Inula japonica Thunb. and its active compounds ameliorate airway inflammation by suppressing JAK-STAT signaling.

Asthma, a chronic inflammatory disease, remains a global health challenge due to its complex pathophysiology and the limited treatment efficacy. This study explored the effect of Inula japonica Thunb. water extract (IJW) on asthma and its protective mechanisms. To assess the effects of IJW, we established an experimental asthma model in BALB/c mice using ovalbumin (OVA). Airway hyper-responsiveness (AHR) in response to methacholine was measured. We quantified inflammatory cell infiltration and cytokine and chemokine levels in bronchoalveolar lavage fluid (BALF), as well as total IgE levels in serum. Staining with hematoxylin and eosin and periodic acid-Schiff was used to examine the impact of IJW on lung pathology. We performed RNA sequencing to identify differentially expressed genes, which were subjected to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses. We used interleukin (IL)-4/IL-13-treated human bronchial epithelial (HBE) cells to explore the associated mechanisms. IJW showed therapeutic effects against OVA-induced asthma by alleviating AHR, peribronchial inflammation, mucus hypersecretion, and collagen fiber deposition. It reduced total IgE levels in the serum and IL-4, IL-5, IL-13, eotaxin, macrophage-derived chemokines, and periostin levels in BALF. In IL-4/IL-13-treated HBE cells, IJW and its components suppressed the Janus kinase-signal transducer and activator of the transcription (JAK-STAT) signaling. These findings support IJW's potential as a pharmacological agent for allergic airway inflammation and asthma.

Mechanisms of the Compound of Magnoliae Flos and Xanthii Fructus Essential Oils for the Treatment of Allergic Rhinitis based on the Integration of Network Pharmacology, Molecular Docking, and Animal Experiment.

Magnoliae Flos (Chinese name: Xin-Yi) and Xanthii Fructus (Chinese name: Cang-Er-Zi) are Chinese herbal medicines and have been used to treat allergic rhinitis (AR). However, the therapeutic effect, active ingredients, and probable processes of a compound of Magnoliae Flos and Xanthii Fructus in the form of essential oils (CMFXFEO) in treating AR have not been reported. This study aims to determine the efficacy of the CMFXFEO on ovalbumin (OVA)-induced AR in a rat model and to use network pharmacology and molecular docking to reveal the hub genes, biological functions, and signaling pathways of CMFXFEO against AR. Animal experiments were applied to validate the role of CMFXFEO in the treatment of AR. 20 rats were randomly divided into four groups: control group (CON, n=5), positive control group (AR, n=5), CMFXFEO-treated group (AR+CMFXFEO, n=5), and budesonide-treated group (AR+Budesonide, n=5). Rats were stimulated with OVA to induce AR. Symptom scores assessment and histo-pathomorphological evaluation was performed. The serum level of OVA-specific immunoglobulin (Ig) E was measured. Gas Chromatograph-Mass Spectrometer analysis (GC-MS) was used to identify the monomer chemical composition of CMFXFEO. The target genes of CMFXFEO were obtained by using PubChem and SwissTargetPrediction databases. The target genes of AR were screened using GeneCards, DisGeNET, and OMIM databases. The target genes were intersected using the venny2.1 website to obtain the potential therapeutic targets of CMFXFEO for treating AR and to construct the PPI network. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were used to reveal associated signaling pathways. The Sybyl tool was used to dock the CMFXFEO with key therapeutic targets molecularly. Intranasal CMFXFEO administration significantly suppressed the allergic symptoms, reduced the inflammatory cell infiltration, and the serum level of OVA-specific immunoglobulin (Ig) E. The main components of CMFXFEO obtained through the GC-MS analysis, listed as γ-terpinene (9.4908%), limonene (7.2693%), menthol (7.1821%), β-pinene (7.1190%), β-caryophyllene (7.0396%), eucalyptol (6.1367%), linalool(5.9686%), eugenol (5.0776%). A total of 398 CMFXFEO targets and 488 AR-related targets were screened, of which 42 were common targets. The GO and KEGG pathway analyses unveiled that CMFXFEO were strongly associated with several signaling pathways, including the AGE-RAGE signaling pathway, TNF signaling pathway, and Chemokine signaling pathway. PPI network construction screened six hub genes as therapeutic targets, including STAT3, IL1B, TLR4, PTGS2, ICAM1, and VCAM1. The molecular docking verification indicated that CMFXFEO have good binding activity with therapeutic targets, and β-Pinene's docking ability with TLR4 is particularly prominent. The anti-inflammatory and anti-allergic effects of CMFXFEO are to inhibit the infiltration of inflammatory cells in the OVA-induced AR rat model. The results of the network pharmacology and molecular docking deduced that the CMFXFEO may have the potential to treat AR by multiple pathways through relieving inflammatory, anti-oxidative stress response, and modulating the immune system.

Sex-specific effects of prenatal bisphenol A exposure on transcriptome-interactome profiles of autism candidate genes in neural stem cells from offspring hippocampus

Bisphenol A (BPA), an endocrine-disrupting chemical, is increasingly linked to the pathogenesis of autism spectrum disorder (ASD). This study investigates the effects of prenatal BPA exposure on neural stem cells (NSCs) from the hippocampi of rat offspring, a brain region critical for neurodevelopment and implicated in ASD. Pregnant rats were administered with BPA or vehicle control once daily via oral gavage from gestational day 1 until parturition. NSCs were isolated from the offspring’s hippocampi on postnatal day 1, and RNA sequencing was performed to examine transcriptomic alterations. Differentially expressed genes (DEGs) were identified through RNA-seq and further analyzed using Ingenuity Pathway Analysis (IPA) to explore disrupted pathways. In addition, in vitro proliferation assays were conducted, utilizing immunofluorescence staining for Sox2, a stem cell marker, and BrdU to quantify proliferating NSCs. Our results revealed that prenatal BPA exposure induced sex-specific alterations in NSC gene expression, with ASD-related genes such as Atp1a3, Nefl, and Grin1 being particularly dysregulated in male offspring. Moreover, sex-specific changes in NSC proliferation were observed. The study underscores BPA’s potential as an environmental risk factor for ASD, emphasizing the need for further research into its role in sex-specific neurodevelopmental effects.

Unveiling the signal valve specifically tuning the TGF-β1 suppression of osteogenesis: mediation through a SMAD1-SMAD2 complex

BackgroundTGF-β1 is the most abundant cytokine in bone, in which it serves as a vital factor to interdict adipogenesis and osteogenesis of bone marrow-derived mesenchymal stem cells (BM-MSCs). However, how TGF-β1 concurrently manipulates differentiation into these two distinct lineages remains elusive.MethodsTreatments with ligands or inhibitors followed by biochemical characterization, reporter assay, quantitative PCR and induced differentiation were applied to MSC line or primary BM-MSCs for signaling dissection. In vivo adipogenesis and ex vivo culture of bone explants were used to verify the functions of different SMAD complexes. Ingenuity Pathway Analysis, and analysis of transcriptomic datasets from human BM-MSCs in combination with hierarchical clustering and STRING assay were used to decipher the interplaying co-repressors. Mouse models of chronic and acute bone loss followed by biochemical assays and micro-computed tomography demonstrated the bone effects when functionally blocking the critical co-repressor HDAC1.ResultsDistinct from the TGF-β1 inhibition on adipogenesis through canonical SMAD2/3 signaling, we clarified that TGF-β1 suppresses osteogenesis by inducing the formation of previously unidentified mixed SMADs mainly composed of SMAD1 and SMAD2, in which SMAD2 recruits more TGF-β1-induced co-repressors including HDAC1, TGIF1 and ATF3, whereas SMAD1 allows directing the whole transcriptional suppression complex to the cis-elements of osteogenic genes. Depletion of the cross-activation to the mixed SMADs dismantled specifically the TGF-β1 suppression on osteogenesis without affecting its inhibition on adipogenesis. Such phenomena can be reproduced via knockdown of co-repressors such as Hdac1 or addition of HDAC1 inhibitors in TGF-β1-treated MSCs. In either the chronic or the acute bone loss model, we demonstrated that the TGF-β signaling was augmented in the bone niche during osteolysis, whereas administration of HDAC1 inhibitors significantly improved bone quality.ConclusionThis study identifies a new signal valve through which TGF-β1 can inhibit osteogenesis specifically. Functional interruption of this valve can tilt the seesaw balance of BM-MSC differentiation towards osteogenesis, highlighting the interplaying co-repressors, such as HDAC1, as promising therapeutic targets to combat diverse degenerative orthopedic diseases.

Mineral Stress Drives Loss of Heterochromatin: An Early Harbinger of Vascular Inflammaging and Calcification.

Vascular calcification is a detrimental aging pathology markedly accelerated in patients with chronic kidney disease. Prelamin A is a biomarker of vascular smooth muscle cell aging that accelerates calcification however the mechanisms remain undefined. Vascular smooth muscle cells were transduced with prelamin A using an adenoviral vector and epigenetic modifications were monitored using immunofluorescence and targeted polymerase chain reaction array. Epigenetic findings were verified in vivo using immunohistochemistry in human vessels, in a mouse model of inducible prelamin A expression, and in a rat model of chronic kidney disease-induced calcification. Transcriptomic and chromatin immunoprecipitation followed by sequencing analyses were used to identify gene targets impacted by changes in the epigenetic landscape. Molecular tools and antibody arrays were used to monitor the effects of mineral dysregulation on heterochromatin, inflammation, aging, and calcification. Here, we report that depletion of the repressive heterochromatin marks, H3K9me3 and H3K27me3, is an early hallmark of vascular aging induced by both nuclear lamina dysfunction and dysregulated mineral metabolism, which act to modulate the expression of key epigenetic writers and erasers. Global analysis of H3K9me3 and H3K27me3 marks and pathway analysis revealed deregulation of insulin signaling and autophagy pathways as well as cross-talking DNA damage and NF-κB (nuclear factor κB) inflammatory pathways consistent with early activation of the senescence-associated secretory phenotype. Expression of prelamin A in vivo induced loss of heterochromatin and promoted inflammation and osteogenic differentiation which preceded aging indices, such as DNA damage and senescence. Vessels from children on dialysis and rats with chronic kidney disease showed prelamin A accumulation and accelerated loss of heterochromatin before the onset of calcification. Dysregulated mineral metabolism drives changes in the epigenetic landscape and nuclear lamina dysfunction that together promote early induction of inflammaging pathways priming the vasculature for downstream pathological change.

Analysis and Visualization of Confounders and Treatment Pathways Leading to Amputation and Non-Amputation in Peripheral Artery Disease Patients Using Sankey Diagrams: Enhancing Explainability

Analysis and Visualization of Confounders and Treatment Pathways Leading to Amputation and Non-Amputation in Peripheral Artery Disease Patients Using Sankey Diagrams: Enhancing Explainability

Tertiary lymphoid structure signatures are associated with survival and immunotherapy response in lung adenocarcinoma

The presence of tertiary lymphoid structures (TLSs) has been correlated with improved prognosis and clinical outcomes in response to immunotherapy in certain solid tumors. However, the precise role of TLSs in lung adenocarcinoma (LUAD) remains unclear. Four datasets of LUAD were obtained from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO). The TLSs model was constructed using a multivariate Cox proportional hazards model. GO and KEGG analyses were performed to explore the biological process associated with the TLSs model. The ESTIMATE and CIBERSORT algorithms were employed to quantify immune infiltration status. TLSs signature genes (TSGs) were identified, including chemokine signature genes, TFH cell markers, TH1 cell and B cell markers, and a plasma cell marker. Diagnostic evaluations identified key genes with high diagnostic value, particularly among chemokine signature genes and TFH cells markers. Furthermore, high expression of CCL20 and IL1R2 was correlated with poorer outcomes, while other TSGs indicated more favorable prognoses. A novel TLSs score model was constructed, integrating 4 TSGs (SGPP2, MS4A1, IL1R2 and CCL20), which accurately predicted patient survival and was independently associated with prognosis. Additionally, the TLSs score served as a robust indicator for LUAD survival prediction, outperforming traditional staging systems. Comprehensive analyses of enriched pathways and immune cell infiltration patterns revealed that this score involved in metabolic processes and immune cell regulation. Furthermore, the TLSs score showed potential as an indicator of response to immunotherapy, with higher scores associated with reduced expression of immune checkpoint genes and poorer response rates. The TLSs score may serve as a predictor of prognosis and immunotherapeutic response in LUAD. These findings may offer new insights on the study of malignancy and personalized immunotherapy for LUAD patients.

Altered expression profile of plasma exosomal microRNAs in exclusive electronic cigarette adult users

Little is known about how exclusive e-cigarette use affects exosomal microRNA (miRNA) expression, which is crucial in inflammation and disease processes like cancer. We compared exosomal miRNA profiles between exclusive e-cigarette users and non-users. We used plasma samples from 15 exclusive e-cigarette users and 15 non-users from the Population Assessment of Tobacco and Health (PATH) Wave 1 study (2013–2014) and sequenced miRNAs with Illumina NextSeq 500/550. We performed differential analyses using DESeq2 in R/Bioconductor, adjusting for race, and conducted gene enrichment analyses on target genes regulated by significant miRNAs. Further, molecular-based techniques using the miRNA mimics and inhibitors were applied for the validation of the expressions of the miRNAs in vitro. We identified four miRNAs that were upregulated in exclusive e-cigarette users compared to non-users: hsa-miR-100-5p, hsa-miR-125a-5p, hsa-miR-125b-5p, and hsa-miR-99a-5p, after adjusting for the confounding effects of race. However, none of the miRNAs remained statistically significant after controlling for the false discovery rate (FDR) at 5%. Subgroup analysis of White participants only identified four miRNAs (hsa-miR-100-5p, hsa-miR-125b-5p, hsa-miR-200b-3p, and hsa-miR-99a-5p) that were also upregulated in e-cigarette users with one miRNA hsa-miR-200b-3p remaining statistical significance after controlling for the FDR at 5%. GO enrichment analysis showed that these miRNAs are involved in processes like transcription regulation and cellular protein modification. KEGG pathway analysis indicated their involvement in cancer pathways, including small cell lung cancer, renal cell carcinoma, and signaling pathways (neurotrophin, ErbB, PI3K-Akt, FoxO, Hippo, MAPK, TGF-beta). Overexpression of hsa-miR-125b-5p promoted DNA damage in bronchial epithelial cells. These findings suggest an elevation of carcinogenic cellular signaling pathways in exclusive e-cigarette users.

GRK5 is required for adipocyte differentiation through ERK activation.

Previous studies have identified G protein-coupled receptor (GPCR) kinase 5 (GRK5) as a genetic factor contributing to obesity pathogenesis, but the underlying mechanism remains unclear. We demonstrate here that Grk5 mRNA is more abundant in stromal vascular fractions of mouse white adipose tissue, the fraction that contains adipose progenitor cells, or committed preadipocytes, than in adipocyte fractions. Thus, we generated a GRK5 knockout (KO) 3T3-L1 preadipocyte to further investigate the mechanistic role of GRK5 in regulating adipocyte differentiation. During adipogenic stimulation, GRK5 KO preadipocytes had decreased lipid accumulation and delayed mature adipocyte development compared to wildtype cells coupled with suppressed adipogenic and lipogenic gene expression. Beside GPCR signaling, RNA sequencing and pathway analysis identified insulin-like growth factor 1 (IGF-1) signaling to be one of the top 5 significantly dysregulated pathways in GRK5 KO cells. GRK5 KO cells also displayed decreased insulin-stimulated ERK phosphorylation, a downstream target of insulin-stimulated IGF-1 receptor activation, suggesting that GRK5 acts through this critical pathway to impact 3T3-L1 adipocyte differentiation. To find a more translational approach, we identified a new small molecule GRK5 inhibitor that was able to reduce 3T3-L1 adipogenesis. These data suggest that GRK5 is required for adipocyte differentiation through IGF-1 receptor/ERK activation and may be a promising translational target for obesity.