Intracellular Metabolic Pathways Research Articles

A comprehensive analysis of the prognostic characteristics of microRNAs in breast cancer.

Both overall survival (OS) and disease-specific survival (DSS) are significant when determining a patient's prognosis for breast cancer (BC). The effect of DSS-related microRNAs on BC susrvival, however, is not well understood. Here, we spotted differentially expressed miRNAs (DEMs) in the TCGA database of BC DSS, identified eight DSS-related miRNAs, and constructed a risk model. AUC values at 1, 3, and 5years were 0.852, 0.861, and 0.868, respectively, indicating a risk model's excellent prognostic prediction ability. Then, we validated miRNA roles in BC OS and finally defined miR-551b as an independently prognostic miRNA in BC. According to function analysis, miR-551b is strongly linked with the emergence and spread of cancer, including protein ubiquitination, intracellular protein transport, metabolic pathways, and cancer pathways. Moreover, we confirmed the low expression of miR-551b in BC tissue and cells. After miR-551b inhibition or overexpression, cell function was either dramatically increased or diminished, respectively, indicating that miR-551b could regulate BC proliferation, invasion, and migration. In conclusion, we thoroughly clarified BC-related miRNAs on DSS and OS and verified miR-551b as a crucial regulator in the development and prognosis of cancer. These results can offer fresh ideas for BC therapy.

The Noncanonical Functions of Metabolites in Tumor Progression.

Metabolic reprogramming has emerged as a prominent hallmark of cancer, characterized by substantial alterations in nutrient uptake and intracellular metabolic pathways. Consequently, intracellular metabolite concentrations undergo significant changes which can contribute to tumorigenesis through diverse mechanisms. Beyond their classical roles in regulating metabolic pathway flux, metabolites exhibit noncanonical functions that play a crucial role in tumor progression. In this review, we delve into the nonclassical functions of metabolites in the context of tumor progression, with a particular focus on their capacity to modulate gene expression and cell signaling. Furthermore, we discuss the potential exploitation of these nonclassical functions in the enhancement of cancer therapy.

Oxidized-LDL Induces Metabolic Dysfunction in Retinal Pigment Epithelial Cells.

Recently, mitochondrial dysfunction has gained attention as a causative factor in the pathogenesis and progression of age-related macular degeneration (AMD). Mitochondrial damage plays a key role in metabolism and disrupts the balance of intracellular metabolic pathways, such as oxidative phosphorylation (OXPHOS) and glycolysis. In this study, we focused on oxidized low-density lipoprotein (ox-LDL), a major constituent of drusen that accumulates in the retina of patients with AMD, and investigated whether it could be a causative factor for metabolic alterations in retinal pigment epithelial (RPE) cells. We found that prolonged exposure to ox-LDL induced changes in fatty acid β-oxidation (FAO), OXPHOS, and glycolytic activity and increased the mitochondrial reactive oxygen species production in RPE cells. Notably, the effects on metabolic alterations varied with the concentration and duration of ox-LDL treatment. In addition, we addressed the limitations of using ARPE-19 cells for retinal disease research by highlighting their lower barrier function and FAO activity compared to those of induced pluripotent stem cell-derived RPE cells. Our findings can aid in the elucidation of mechanisms underlying the metabolic alterations in AMD.

Estrogen promotes fetal skeletal muscle mitochondrial distribution and ATP synthase activity important for insulin sensitivity in offspring.

We previously showed that offspring delivered to baboons in which levels of estradiol (E2) were suppressed during the second half of gestation exhibit insulin resistance. Mitochondria are essential for the production of ATP as the main source of energy for intracellular metabolic pathways, and skeletal muscle of type 2 diabetics exhibit mitochondrial abnormalities. Mitochondria express estrogen receptor β and E2 enhances mitochondrial function in adults. Therefore, the current study ascertained whether exposure of the fetus to E2 is essential for mitochondrial development. Levels of ATP synthase and citrate synthase and the morphology of mitochondria were determined in fetal skeletal muscle obtained near term from baboons untreated or treated daily with the aromatase inhibitor letrozole or letrozole plus E2. Specific activity and amount of ATP synthase were 2-fold lower (P < 0.05) in mitochondria from skeletal muscle of E2 suppressed letrozole-treated fetuses and restored to normal by treatment with letrozole plus E2. Immunocytochemistry showed that in contrast to the punctate formation of mitochondria in myocytes of untreated and letrozole plus E2 treated animals, mitochondria appeared to be diffuse in myocytes of estrogen-suppressed fetuses. However, citrate synthase activity and levels of proteins that control mitochondrial fission/fusion were similar in estrogen replete and suppressed animals. We suggest that estrogen is essential for fetal skeletal muscle mitochondrial development and thus glucose homeostasis in adulthood.

Detoxification of Hyperglycemia-induced Glucose Toxicity by the Hexosamine Biosynthetic Pathway.

The abnormal intermediate glucose metabolic pathways induced by elevated intracellular glucose levels during hyperglycemia often establish the metabolic abnormality that leads to cellular and structural changes in development and to progression of diabetic pathologies. Glucose toxicity generally refers to the hyperglycemia-induced irreversible cellular dysfunctions over time. These irreversible cellular dysfunctions in diabetic nephropathy include: (1) inflammatory responses, (2) mesangial expansion, and (3) podocyte dysfunction. Using these three cellular events in diabetic nephropathy as examples of glucose toxicity in the diabetic complications, this review focuses on: (1) the molecular and cellular mechanisms associated with the hexosamine biosynthetic pathway that underly glucose toxicity; and (2) the potential therapeutic tools to inhibit hyperglycemia induced pathologies. We propose novel therapeutic strategies that directly shunts intracellular glucose buildup under hyperglycemia by taking advantage of intracellular glucose metabolic pathways to dampen it by normal synthesis and secretion of hyaluronan, and/or by intracellular chondroitin sulfate synthesis and secretion. This could be a useful way to detoxify the glucose toxicity in hyperglycemic dividing cells, which could mitigate the hyperglycemia induced pathologies in diabetes.

Optimization of the quantitative protocol for organic acid in fecal samples using gas chromatography-mass spectrometry

Organic acids (OAs) play important roles in a variety of intracellular metabolic pathways, such as the tricarboxylic acid cycle, fatty acid oxidation, glycolysis. The accurate detection of OAs in fecal samples was crucial for comprehending the metabolic changes associated with various metabolic disease. However, the analytical protocol detecting OAs profiling in feces have received scant attention. In this work, an optimized protocol based on chromatography-mass spectrometry for simultaneous quantification of 23 OAs in rat feces was developed. The optimal conditions involved using a 40-mg fecal sample mixed with isopropyl alcohol, acetonitrile, and deionized water (3:2:2 vol ratio) with a total volume of 1500 μL, followed by ultrasonic extraction and a derivatization reaction with an 80 μL derivative agent. The protocol showed an acceptable linearity (R2 ≥ 0.9906), the satisfactory precision (RSD% ≤ 14.87%), the low limits of detection (0.001 to 1 μg/mL) and the limit of quantification (0.005 to 1.5 μg/mL). Moreover, the dried residues of the extracted solution showed the better stability of OAs at −20 °C, which was more suitable for a large-scale sample analysis. Finally, the developed protocol was successfully applied to compare the difference of OAs profiling in fecal samples harvested from normal and nonalcoholic fatty liver disease rats, which was beneficial to find out the metabolic change of OAs profiling and explain the related mechanism of the disease.

Genomics and metabolic characteristics of simultaneous heterotrophic nitrification aerobic denitrification and aerobic phosphorus removal by Acinetobacter indicus CZH-5

This study provides for the first time a systematic understanding of Acinetobacter indicus CZH-5 performance, metabolic pathway and genomic characteristics for aerobic nitrogen (N) and phosphorus (P) removal. Acinetobacter indicus CZH-5 showed promising performance in heterotrophic nitrification aerobic denitrification and aerobic phosphorus removal. Under optimal conditions, the maximum ammonia-N, total nitrogen and orthophosphate-P removal efficiencies were 90.17%, 86.33%, and 99.89%, respectively. The wide tolerance range suggests the strong environmental adaptability of the bacteria. The complete genome of this strain was reconstructed. Whole genome annotation was used to re-construct the N and P metabolic pathways, and related intracellular substance metabolic pathways were proposed. The transcription levels of related functional genes and enzyme activities further confirmed these metabolic mechanisms. N removal was achieved via the nitrification–denitrification pathway. Furthermore, CZH-5 exhibited significant aerobic P uptake, with phosphate diesters as the main species of intracellular P.

Oleander attenuates hepatic inflammation in a TLR4-independent manner and by favorable modulation of hepatocellular global metabolome that supports cytoprotection

Ethnopharmacological relevanceNerium oleander is used to treat liver-associated chronic metabolic diseases in traditional medicinal systems across the globe. The hepatoprotective effects of oleander are mentioned in Indian and Chinese traditional medicinal literature. Aim of the studyThe present study aimed to investigate the cellular mechanisms behind the hepatoprotective effects of a non-toxic dose of oleander (NO). Materials and methodsThe hepatoprotective effects of NO were tested against lipopolysaccharide (LPS)-treated HepG2 cells. Oxidative stress response was studied using cellular enzymatic assays, and gene expression was analyzed using qRT-PCR. HepG2 cells were pretreated with TAK-242 (pharmacological inhibitor of TLR4) to decipher the anti-inflammatory mechanisms of NO. Cell-free metabolites were analyzed using GCMS and were subjected to pathway enrichment analysis. ResultsNO reduced systemic inflammation, serum lipid peroxidation byproducts, and glucose without affecting serum transaminase levels and hepatic histopathological features. NO attenuated the inflammation-induced loss of antioxidant enzyme activities and mRNA expressions of toll-like receptor-4 (TLR4)/nuclear factor κβ (NFκβ)-dependent inflammatory genes. In TAK-242 pretreated cells, LPS was unable to induce inflammatory and oxidative responses. However, NO treatment in TAK-242 pretreated cells with LPS stimulation further reduced the signs of inflammation and improved hepatoprotective activities. A comparative analysis of the intracellular global metabolome from HepG2 cells with and without NO treatment indicated NO-mediated favorable modulation of intracellular metabolic pathways that support cytoprotective activities. ConclusionNO protects HepG2 cells from LPS-induced oxidative and inflammatory injury. The hepatoprotective effects of NO are mediated by a TLR4-independent process and through a favorable modulation of the intracellular global metabolome that supports cytoprotection.

Acetylation of xenogeneic silencer H-NS regulates biofilm development through the nitrogen homeostasis regulator in Shewanella.

Adjusting intracellular metabolic pathways and adopting suitable live state such as biofilms, are crucial for bacteria to survive environmental changes. Although substantial progress has been made in understanding how the histone-like nucleoid-structuring (H-NS) protein modulates the expression of the genes involved in biofilm formation, the precise modification that the H-NS protein undergoes to alter its DNA binding activity is still largely uncharacterized. This study revealed that acetylation of H-NS at Lys19 inhibits biofilm development in Shewanella oneidensis MR-1 by downregulating the expression of glutamine synthetase, a critical enzyme in glutamine synthesis. We further found that nitrogen starvation, a likely condition in biofilm development, induces deacetylation of H-NS and the trimerization of nitrogen assimilation regulator GlnB. The acetylated H-NS strain exhibits significantly lower cellular glutamine concentration, emphasizing the requirement of H-NS deacetylation in Shewanella biofilm development. Moreover, we discovered in vivo that the activation of glutamine biosynthesis pathway and the concurrent suppression of the arginine synthesis pathway during both pellicle and attached biofilms development, further suggesting the importance of fine tune nitrogen assimilation by H-NS acetylation in Shewanella. In summary, posttranslational modification of H-NS endows Shewanella with the ability to respond to environmental needs by adjusting the intracellular metabolism pathways.

Identification of key microbial communities and intracellular metabolic pathways to response micro-aerobic environment for enhancing degradation of N, N-dimethylformamide in refractory membrane-making wastewater

Enhanced sludge hydrolysis in micro-aerobic environment with dissolved oxygen (DO) regulation could degrade stubborn refractory N, N-dimethylformamide (DMF) in membrane-making wastewater. However, investigations on how micro-aerobic condition affects DMF conversion and microbial metabolism in biological system are still inadequate. Herein, the responses of microbial community to micro-aerobic environment, and the consequent variations of metabolic pathways in DMF degradation were unraveled. Results indicated that low concentration of DO (0.2–0.4 mg/L) could significantly enhance the ammonification rate (from 21.8 % to 52.8 %) and COD removal efficiency (from 65.2 % to 82 %) by breaking C–N and CO bonds of amide group in macromolecular organics, which help it degraded into micromolecular organics. 16S rRNA and metagenomic analysis further revealed that low DO contents prompted the changes of microbial community structure. Specifically, toxicity-tolerant fermentation bacteria (e.g. Desulfomicrobium and unclassified_c_ Deltaproteobacteria) gradually became the dominant species in micro-aerobic environment, showing positive connections with COD removal and ammonification functions. Additionally, functional genes analysis further revealed the intracellular metabolic pathways of DMF, where the upregulation of tricarboxylic acid (TCA) cycle and oxidative phosphorylation pathway was observed in micro-aerobic environment. Such regulation led to the massive synthesis of adenosine triphosphate (ATP) and NADH, which are needful in oxidative demethylation process, and thus promoting the conversion from DMF to formamide, CH4 and NH3 in Methanolobus step-by-step. Therefore, this study provided vital insights into the responses of microbial community structure and DMF intracellular metabolic pathways to micro-aerobic environment, and profoundly discussed their contributions to the efficient treatment effect on membrane-making wastewater.

Dual role of birnessite on the modulation of acid production and reinforcement of interspecific electron transfer in anaerobic digestion

Achieving efficient anaerobic digestion of highly loaded substrates is one of the most challenging problems in the field of waste resourcing. Here, the addition of birnessite (2.0 g/L) to kitchen wastewater increased the acetate and final methane yields by 40.53 and 99.18 %, respectively, while reducing the yields of propionate and butyrate by 38.17 and 48.86 %, respectively. There were two main pathways for birnessite to enhance anaerobic digestion, one of which is to act as an electron acceptor, by inducing an alteration in the ratio of reduced-state coenzyme I in the microorganism, allowing the acid production process to proceed towards deeper oxidation. Another pathway enhances the interspecific electron transfer between bacteria and archaea and improves methane yield by optimizing the metabolic relationship. The Kyoto Encyclopedia of Genes and Genomes (KEGG) functional predictions suggest that the extracellular electron transport pathway of the microorganism is enhanced with the addition of birnessite and that its intracellular metabolic pathway is biased towards the nicotinamide adenine dinucleotide (NADH) generation pathway. This work demonstrated that anaerobic digestion facilitation by metallic minerals was not monolithic; that is, different properties of the minerals were employed to intensify the different stages of anaerobic digestion and obtain an amplification cascade.

Gcn4 impacts metabolic fluxes to promote yeast chronological lifespan.

Aging is characterized by a gradual decline in physiological integrity, which impairs functionality and increases susceptibility to mortality. Dietary restriction, mimicking nutrient scarcity without causing malnutrition, is an intervention known to decelerate the aging process. While various hypotheses have been proposed to elucidate how dietary restriction influences aging, the underlying mechanisms remain incompletely understood. This project aimed to investigate the role of the primary regulator of the general amino acid control (GAAC) pathway, the transcription factor Gcn4, in the aging process of S. cerevisiae cells. Under conditions of amino acid deprivation, which activate Gcn4, the deletion of GCN4 led to a diverse array of physiological changes in the cells. Notably, the absence of Gcn4 resulted in heightened mitochondrial activity, likely contributing to the observed increase in reactive oxygen species (ROS) accumulation. Furthermore, these mutant gcn4Δ cells exhibited reduced ethanol production despite maintaining similar glucose consumption rates, suggesting a pivotal role for Gcn4 in regulating the Crabtree effect. Additionally, there was a marked reduction in trehalose, the storage carbohydrate, within the mutant cells compared to the wild-type strain. The intracellular content of free amino acids also exhibited disparities between the wild-type and GCN4-deficient strains. Taken together, our findings indicate that the absence of GCN4 disrupts cellular homeostasis, triggering significant alterations in interconnected intracellular metabolic pathways. These disruptions have far-reaching metabolic consequences that ultimately culminate in a shortened lifespan.

Statin-Induced Myopathy

Scientific relevance. Being the main class of medicinal products for dyslipidaemia treatment, statins are widely used in clinical practice in various patient populations. However, statins can cause statin-associated muscle symptoms (SAMS), which are the most frequent and, in some cases, even life-threatening adverse reactions associated with these medicinal products.Aim. The study aimed to perform a systematic review of the epidemiology, classification, and physiological pathogenesis of SAMS, risk factors for this complication, and clinical guidelines for primary care physicians regarding the identification and treatment of patients with SAMS.Discussion. SAMS is an umbrella term that covers various forms of myopathies associated with satin therapy. According to the published literature, the prevalence of SAMS varies considerably and may depend on the study design, inclusion criteria, and the medicinal product used. SAMS has multiple putative pathogenic pathways that include genetically determined processes, abnormalities in mitochondrial function, defects in intracellular signalling and metabolic pathways, and immune-mediated reactions. The main known risk factors for developing SAMS include high-dose statins, drug–drug interactions, genetic polymorphisms, female sex, older age, Asian race, history of kidney, liver, and muscle disease, and strenuous physical activity. Given the lack of universally recognised algorithms for diagnosing SAMS, clinicians should consider the clinical presentation and the temporal relationship between statin therapy and symptoms. Other factors to consider include changes in muscle-specific enzyme levels and, in some cases, the results of blood tests for antibodies to 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase.Conclusions. To ensure the safety of statin therapy, it is essential to raise clinicians’ awareness of the risk factors for SAMS, indicative clinical and laboratory findings, and the need for dynamic patient monitoring, including the involvement of clinical pharmacologists.

Engineered Bacteria: General Overview as Therapeutic Agent and a Novel Drug Delivery System.

Bacterial engineering modifies bacteria's genomic sequence using genetic engineering tools. These engineered bacteria can produce modified proteins, peptides, nucleic acids, and other biomolecules that can be used to treat various medical conditions. Engineered bacteria can target diseased tissues or organs, detect specific biomarkers in the diseased environment, and even induce specific conditions. Furthermore, a meticulously designed intracellular metabolic pathway can activate or inhibit the expression of related genes, synthesise biologically active therapeutic molecules, and precisely deliver drug payloads to diseased tissues or organs. Lactococcus (L. lactis), Salmonella (S. typhi)</i>, and E. coli (E. coli Nissle)</i> are the most studied engineered microorganisms used as drug carriers. These have been used in vaccines to treat multifactorial diseases such as cancer, autoimmune diseases, metabolic diseases, and inflammatory conditions. Other promising strains include Bifidobacterium animalis</i>, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus lugdunensis</i>, and Clostridium sporogenes</i>. Despite the low reported risk, toxic effects associated with bacterial cells, limiting their efficacy and rapid clearance due to immune responses stimulated by high bacterial concentrations, remain major drawbacks. As a result, a better and more effective method of drug delivery must be developed by combining bacterial-based therapies with other available treatments, and more research in this area is also needed.

Mitochondrial Control for Healthy and Autoimmune T Cells.

T cells are critical players in adaptive immunity, driving the tissue injury and organ damage of patients with autoimmune diseases. Consequently, investigations on T cell activation, differentiation, and function are valuable in uncovering the disease pathogenesis, thus exploring promising therapeutics for autoimmune diseases. In recent decades, accumulating studies have pinpointed immunometabolism as the fundamental determinant in controlling T cell fate. Specifically, mitochondria, as a hub of intracellular metabolism, connect glucose, lipid, and amino acid metabolic pathways. Herein, we summarize metabolic adaptations of mitochondrial oxidative phosphorylation and the relevant glucose, lipid, and amino acid metabolism during T cell activation, differentiation, and function. Further, we focused on current updates of the molecular bases for metabolic reprogramming in autoimmune T cells and advances in exploring metabolic-targeted therapeutics against autoimmune diseases. This might facilitate the in-depth understanding of autoimmune pathogeneses and the clinical management of autoimmune diseases.

Marine phytoplankton downregulate core photosynthesis and carbon storage genes upon rapid mixed layer shallowing

Marine phytoplankton are a diverse group of photoautotrophic organisms and key mediators in the global carbon cycle. Phytoplankton physiology and biomass accumulation are closely tied to mixed layer depth, but the intracellular metabolic pathways activated in response to changes in mixed layer depth remain less explored. Here, metatranscriptomics was used to characterize the phytoplankton community response to a mixed layer shallowing (from 233 to 5 m) over the course of two days during the late spring in the Northwest Atlantic. Most phytoplankton genera downregulated core photosynthesis, carbon storage, and carbon fixation genes as the system transitioned from a deep to a shallow mixed layer and shifted towards catabolism of stored carbon supportive of rapid cell growth. In contrast, phytoplankton genera exhibited divergent transcriptional patterns for photosystem light harvesting complex genes during this transition. Active virus infection, taken as the ratio of virus to host transcripts, increased in the Bacillariophyta (diatom) phylum and decreased in the Chlorophyta (green algae) phylum upon mixed layer shallowing. A conceptual model is proposed to provide ecophysiological context for our findings, in which integrated light limitation and lower division rates during transient deep mixing are hypothesized to disrupt resource-driven, oscillating transcript levels related to photosynthesis, carbon fixation, and carbon storage. Our findings highlight shared and unique transcriptional response strategies within phytoplankton communities acclimating to the dynamic light environment associated with transient deep mixing and shallowing events during the annual North Atlantic bloom.

Understanding the effect of temperature downshift on CHO cell growth, antibody titer and product quality by intracellular metabolite profiling and in vivo monitoring of redox state.

The strategy of temperature downshift has been widely used in the biopharmaceutical industry to improve antibody production and cell-specific production rate (qp ) with Chinese hamster ovary cells (CHO). However, the mechanism of temperature-induced metabolic rearrangement, especially important intracellular metabolic events, remains poorly understood. In this work, in order to explore the mechanisms of temperature-induced cell metabolism, we systematically assessed the differences in cell growth, antibody expression, and antibody quality between high-producing (HP) and low-producing (LP) CHO cell lines under both constant temperature (37°C) and temperature downshift (37°C→33°C) settings during fed-batch culture. Although the results showed that low-temperature culture during the late phase of exponential cell growth significantly reduced the maximum viable cell density (p < 0.05) and induced cell cycle arrest in the G0/G1 phase, this temperature downshift led to a higher cellular viability and increased antibody titer by 48% and 28% in HP and LP CHO cell cultures, respectively (p < 0.001), and favored antibody quality reflected in reduced charge heterogeneity and molecular size heterogeneity. Combined extra- and intra-cellular metabolomics analyses revealed that temperature downshift significantly downregulated intracellular glycolytic and lipid metabolic pathways while upregulated tricarboxylic acid (TCA)cycle, and particularly featured upregulated glutathione metabolic pathways. Interestingly, all these metabolic pathways were closely associated with the maintenance of intracellular redox state and oxidative stress-alleviating strategies. To experimentally address this, we developed two high-performance fluorescent biosensors, denoted SoNar and iNap1, for real-time monitoring of intracellular nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide + hydrogen (NAD+ /NADH) ratio and nicotinamide adenine dinucleotide phosphate (NADPH) amount, respectively. Consistent with such metabolic rearrangements, the results showed that temperature downshift decreased the intracellular NAD+ /NADH ratio, which might be ascribed to the re-consumption of lactate, and increased the intracellular NADPH amount (p < 0.01) to scavenge intracellular reactive oxygen species (ROS) induced by the increased metabolic requirements for high-level expression of antibody. Collectively, this study provides a metabolic map of cellular metabolic rearrangement induced by temperature downshift and demonstrates the feasibility of real-time fluorescent biosensors for biological processes, thus potentially providing a new strategy for dynamic optimization of antibody production processes.

3D-Printed Bioactive Scaffold Loaded with GW9508 Promotes Critical-Size Bone Defect Repair by Regulating Intracellular Metabolism.

The process of bone regeneration is complicated, and it is still a major clinical challenge to regenerate critical-size bone defects caused by severe trauma, infection, and tumor resection. Intracellular metabolism has been found to play an important role in the cell fate decision of skeletal progenitor cells. GW9508, a potent agonist of the free fatty acid receptors GPR40 and GPR120, appears to have a dual effect of inhibiting osteoclastogenesis and promoting osteogenesis by regulating intracellular metabolism. Hence, in this study, GW9508 was loaded on a scaffold based on biomimetic construction principles to facilitate the bone regeneration process. Through 3D printing and ion crosslinking, hybrid inorganic-organic implantation scaffolds were obtained after integrating 3D-printed β-TCP/CaSiO3 scaffolds with a Col/Alg/HA hydrogel. The 3D-printed β-TCP/CaSiO3 scaffolds had an interconnected porous structure that simulated the porous structure and mineral microenvironment of bone, and the hydrogel network shared similar physicochemical properties with the extracellular matrix. The final osteogenic complex was obtained after GW9508 was loaded into the hybrid inorganic-organic scaffold. To investigate the biological effects of the obtained osteogenic complex, in vitro studies and a rat cranial critical-size bone defect model were utilized. Metabolomics analysis was conducted to explore the preliminary mechanism. The results showed that 50 μM GW9508 facilitated osteogenic differentiation by upregulating osteogenic genes, including Alp, Runx2, Osterix, and Spp1 in vitro. The GW9508-loaded osteogenic complex enhanced osteogenic protein secretion and facilitated new bone formation in vivo. Finally, the results from metabolomics analysis suggested that GW9508 promoted stem cell differentiation and bone formation through multiple intracellular metabolism pathways, including purine and pyrimidine metabolism, amino acid metabolism, glutathione metabolism, and taurine and hypotaurine metabolism. This study provides a new approach to address the challenge of critical-size bone defects.

Network analysis of toxin production in Clostridioides difficile identifies key metabolic dependencies.

Clostridioides difficile pathogenesis is mediated through its two toxin proteins, TcdA and TcdB, which induce intestinal epithelial cell death and inflammation. It is possible to alter C. difficile toxin production by changing various metabolite concentrations within the extracellular environment. However, it is unknown which intracellular metabolic pathways are involved and how they regulate toxin production. To investigate the response of intracellular metabolic pathways to diverse nutritional environments and toxin production states, we use previously published genome-scale metabolic models of C. difficile strains CD630 and CDR20291 (iCdG709 and iCdR703). We integrated publicly available transcriptomic data with the models using the RIPTiDe algorithm to create 16 unique contextualized C. difficile models representing a range of nutritional environments and toxin states. We used Random Forest with flux sampling and shadow pricing analyses to identify metabolic patterns correlated with toxin states and environment. Specifically, we found that arginine and ornithine uptake is particularly active in low toxin states. Additionally, uptake of arginine and ornithine is highly dependent on intracellular fatty acid and large polymer metabolite pools. We also applied the metabolic transformation algorithm (MTA) to identify model perturbations that shift metabolism from a high toxin state to a low toxin state. This analysis expands our understanding of toxin production in C. difficile and identifies metabolic dependencies that could be leveraged to mitigate disease severity.

Adipose tissue macrophages as potential targets for obesity and metabolic diseases.

Macrophage infiltration into adipose tissue is a key pathological factor inducing adipose tissue dysfunction and contributing to obesity-induced inflammation and metabolic disorders. In this review, we aim to present the most recent research on macrophage heterogeneity in adipose tissue, with a focus on the molecular targets applied to macrophages as potential therapeutics for metabolic diseases. We begin by discussing the recruitment of macrophages and their roles in adipose tissue. While resident adipose tissue macrophages display an anti-inflammatory phenotype and promote the development of metabolically favorable beige adipose tissue, an increase in pro-inflammatory macrophages in adipose tissue has negative effects on adipose tissue function, including inhibition of adipogenesis, promotion of inflammation, insulin resistance, and fibrosis. Then, we presented the identities of the newly discovered adipose tissue macrophage subtypes (e.g. metabolically activated macrophages, CD9+ macrophages, lipid-associated macrophages, DARC+ macrophages, and MFehi macrophages), the majority of which are located in crown-like structures within adipose tissue during obesity. Finally, we discussed macrophage-targeting strategies to ameliorate obesity-related inflammation and metabolic abnormalities, with a focus on transcriptional factors such as PPARγ, KLF4, NFATc3, and HoxA5, which promote macrophage anti-inflammatory M2 polarization, as well as TLR4/NF-κB-mediated inflammatory pathways that activate pro-inflammatory M1 macrophages. In addition, a number of intracellular metabolic pathways closely associated with glucose metabolism, oxidative stress, nutrient sensing, and circadian clock regulation were examined. Understanding the complexities of macrophage plasticity and functionality may open up new avenues for the development of macrophage-based treatments for obesity and other metabolic diseases.