搜索结果：Metabolic engineering

Characterizing the dynamics of systemic metabolic pathways during cancer progression could enable the development of prognostic tools and therapeutic strategies. However, achieving this goal requires analytical platforms optimized for liquid biospecimens together with interpretive frameworks capable of identifying robust serum-based biomarkers. Here, we establish a network-based metabolic profiling framework using 1 9F NMR-based serum amino acid analysis to characterize systemic metabolic remodeling during colorectal cancer (CRC) progression. Using an analytical protocol optimized for clinical serum samples, we quantified circulating amino acids from 152 CRC patients and implemented a ratio-based normalization strategy to mitigate cohort variability in concentration-based approaches. We systematically evaluated both individual amino acid changes and correlation structures across tumor stages. Advanced-stage CRC exhibited a distinct metabolic shift characterized by decreased valine and increased glycine levels. Correlation network analysis further revealed stage-dependent remodeling of circulating amino acid interactions, leading to the emergence of a glycine-centered metabolic architecture. Importantly, machine-learning models integrating individual amino acid levels with network-derived features significantly improved the prediction of recurrence or metastasis compared with models using either feature type alone and outperformed conventional biomarker carcinoembryonic antigen (AUROC = 0.806). These findings highlight the remodeling of circulating amino acid network as a promising strategy for prognostic stratification and postoperative monitoring in CRC.

Inflammatory bowel disease (IBD) is a chronic relapsing inflammatory condition that has a rapidly changing global epidemiology. IBD has been traditionally viewed as a primary immune system dysfunction, but emerging evidence more accurately describes IBD as a perturbance of the intricate balance between host immunity, the intestinal microbiome, and intestinal metabolism. Although genetic and environmental components have long been recognized as contributors, accumulating evidence increasingly highlights the pivotal role of microbial dysbiosis in the pathogenesis of IBD. In patients with IBD, intestinal dysbiosis, which is often characterized by reduced Firmicutes and increased pro-inflammatory bacteria, triggers a cascade of pathogenic events. These pathogenic events include impaired epithelial barrier function, dysregulated immune activation against luminal antigens, and immune reprogramming. Central to these processes are functional changes in microbial metabolism, particularly in pathways involving short-chain fatty acids (SCFAs), bile acids, and redox homeostasis, which critically contribute to the development of chronic mucosal inflammation. The current therapeutic backbone of IBD-including aminosalicylates, biologics, and immunomodulators-largely targets the inflammatory response. However, the challenges such as primary non-response, secondary loss of response, and systemic side effects are often problematic. Consequently, there is an urgent need to develop novel therapeutic and preventive strategies that target the underlying microbial and metabolic causes of the disease rather than modulating immune responses. This review integrates the pathomechanistic implications of the microbiome-metabolic axis in the maintenance of gut homeostasis and its disruption in IBD, with particular emphasis on the global epidemiology of the disease. We further evaluate emerging therapeutic and preventive strategies aimed at restoring the microbiome-metabolic axis, including fecal microbiota transplantation (FMT), probiotic therapy, bacteriophage therapy, and helminth-based therapies. In addition, we explore the potential of advanced approaches such as microbiome engineering and precision genome editing to enable highly personalized therapeutic paradigms. By bridging microbial ecology with clinical pathology, this review highlights the transformative potential of targeting the host-microbiota interface to achieve improved long-term outcomes in IBD.

Biodegradable plastics are often promoted as an eco-sustainable alternative to conventional polymers. However, their potential to degrade into microplastics still poses significant health risks. Commonly used materials such as polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) have been widely adopted across various industries. While the toxicity of PLA microplastics has been studied extensively, the biological effects of PLGA microplastics remain largely unknown. Through metagenomic sequencing and untargeted metabolomic profiling, we evaluated the impacts of both PLA and PLGA microplastics on gut bacteria, fungi, virulence factors, microbial metabolic pathways, and metabolites in feces, serum, and liver tissue in this study. Our results demonstrate that both types of biodegradable microplastics disrupt gut microbiota and host metabolic homeostasis. PLA exposure provoked more pronounced changes in gut bacteria, fungi, virulence factors, and fecal and hepatic metabolites. In contrast, microbial metabolic pathways and serum metabolites were more strongly affected by PLGA. Several altered features were common to both microplastics, including enrichment of hepatic metabolic pathways related to valine, leucine, and isoleucine biosynthesis; one-carbon pool by folate; glycine, serine, and threonine metabolism; pantothenate and CoA biosynthesis; taurine and hypotaurine metabolism; and cysteine and methionine metabolism. Other disturbances were material-specific, such as UMP biosynthesis pathways, which were altered exclusively by PLA, while palmitate biosynthesis and unsaturated fatty acid biosynthesis were affected only by PLGA. These findings advance our understanding of the distinct and shared health risks posed by different biodegradable microplastics, providing a clearer basis for assessing their long-term safety.

With the rapid development of synthetic biology and metabolic engineering, compartmentalized biosynthesis of natural products has attracted extensive attention, in which peroxisomes present unique advantages over other subcellular organelles. Herein, the peroxisomes cofactor balance was systematically engineered in Saccharomyces cerevisiae BY4741 to achieve efficient biosynthesis of α‑humulene. Firstly, the cytoplasmic mevalonate (MVA) pathway and AcHS2 were targeted to peroxisomes using the ScPEX5*-oPTS1* orthogonal transport system, resulting in strain LM07 with an α-humulene titer of 978.92 mg/L. Subsequently, co-expression of POX1 and FOX2 enhanced peroxisomal acetyl-CoA supply, knockout of MDH3 increased NADH availability, and overexpression of PCD1, Cv1693, PXN, and ANT1 established a peroxisomal CoA metabolic cycle, which increased the titer by 47.97% to 1448.46 mg/L. Furthermore, we conducted a screening of α-humulene synthases from different sources and enhanced the α-humulene titer in yeast by 110.71%. Finally, a cytoplasm-peroxisome dual-compartment engineering strategy was implemented to optimize the cytoplasmic α-humulene synthetic pathway. The engineered strain LM27 produced 2502.69 mg/L α-humulene in shake-flask fermentation. This regulatory strategy provides a valuable technical reference for the efficient biosynthesis of other terpenoids in S. cerevisiae.

Rheumatoid arthritis (RA) is a chronic autoimmune disorder characterized by dysregulated T cell responses and metabolic disturbances. Mesenchymal stromal cells (MSCs) have shown therapeutic promise, but their mechanisms, particularly concerning T cell metabolism, remain incompletely defined. This study investigated whether human umbilical cord-derived MSCs (hUC-MSCs) ameliorate collagen-induced arthritis (CIA) by modulating T cell metabolism and differentiation. CIA was induced in DBA/1 mice. Animals received PBS or hUC-MSCs on day 28. Arthritis index (AI), joint histology, serum cytokines (TNF-α, IL-6, IL-17, and TGF-β), and metabolites (lactate and pyruvate) were assessed. Splenic T cell transcription factors (FOXP3, RORγt, and PU.1) and glycolytic genes (GLUT1, G6PD, and PFKFB3) were analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) and western blot. In vitro, human CD4+ T cells were cocultured with hUC-MSCs under T-helper 17 (Th17)-polarizing conditions. T cell subsets, glycolytic metabolites, and gene/protein expression were evaluated by flow cytometry, colorimetric assays, RT-qPCR, and western blot. MSC treatment significantly attenuated arthritis severity, joint destruction, and splenomegaly in CIA mice. It reduced serum pro-inflammatory cytokines and normalized elevated lactate and pyruvate levels. In the spleen, MSCs suppressed RORγt and PU.1 while enhancing FOXP3 expression, and downregulated GLUT1 and G6PD mRNA. Positive correlations were found between glycolytic markers (GLUT1 and G6PD) and pro-inflammatory transcription factors (RORγt and PU.1), and between serum lactate and inflammatory cytokines. In vitro, hUC-MSCs directly inhibited Th17 differentiation and promoted Treg generation in human CD4+ T cells. This metabolic reprogramming was functionally coupled to a shift in T cell differentiation: a suppression of pro-inflammatory Th17 cells and a promotion of regulatory T (Treg) generation in human CD4+ T cells. This was accompanied by reduced lactate production and significant downregulation of GLUT1, G6PD, and PFKFB3 at both mRNA and protein levels. hUC-MSCs ameliorate CIA by restoring the Th17/Treg balance through metabolic reprogramming of T cells, specifically by suppressing glycolysis. This immunometabolic mechanism highlights the therapeutic potential of MSCs in RA.

Plastic pollution remains a major global environmental challenge due to the persistence and recalcitrance of synthetic polymers, particularly polyolefins such as polyethylene and polypropylene. Conventional management strategies, including landfilling, incineration, and chemical recycling, are energy-intensive and often fail to achieve complete degradation. Biological approaches offer a promising alternative, yet microbial systems alone are frequently limited by low degradation efficiency, incomplete mineralization, and challenges in scalability. In this context, insect-microbiome systems have emerged as integrated biological platforms in which host physiology and gut microbial communities interact to facilitate polymer transformation. This review synthesizes current knowledge on plastic degradation in insect larvae, with a focus on mechanistic insights into host-microbe interactions. Evidence suggests a multi-step process involving mechanical fragmentation, oxidative modification of polymer chains, microbial depolymerization, and metabolic processing of intermediates. However, the relative contributions of host-derived enzymes and gut microbiota, as well as the extent of true biodegradation versus partial transformation, remain incompletely resolved. Advances in multi-omics approaches have provided important insights into these systems by linking physicochemical changes in polymers to underlying molecular and metabolic processes. At the same time, these data are largely correlative, and direct experimental validation of specific enzymatic roles remains limited. Building on these insights, emerging enzyme engineering and synthetic biology strategies aim to replicate the coordinated, multi-step processes observed in insect systems for improved degradation efficiency. This review highlights insect-microbiome systems as valuable models for understanding complex biodegradation mechanisms and guiding the development of integrated, enzyme-based strategies for sustainable plastic waste management.

Rheumatoid arthritis is a chronic autoimmune disorder characterized by persistent synovial inflammation, oxidative stress, and metabolic reprogramming. Emerging evidence highlights miRNAs as key post-transcriptional regulators linking these pathogenic processes. Among them, miR-200a, miR-1, and miR-548-3p have been implicated in oxidative stress responses and glucose metabolism; however, their roles in RA remain largely unexplored. This study integrates bioinformatic prediction and experimental validation to examine the expression profiles of these miRNAs, identify their target genes related to oxidative stress and metabolism, and elucidate their contribution to RA pathogenesis. RT-PCR analysis revealed significant upregulation of miR-200a and downregulation of miR-1 and miR-548-3p in RA patients. RA-associated DEGs were identified from publicly available GEO microarray datasets. Further, genes linked to oxidative stress and glucose metabolism were retrieved from GeneCards and MSigDB. Overlapping genes across datasets were subjected to GO and KEGG enrichment analyses, followed by the construction of a PPI network using STRING. Five hub genes-MAPK1, FOXP1, CDC42, RUNX1, and ETS1-were identified as central nodes within the PPI network. Functional enrichment indicated their involvement in myeloid cell differentiation, regulation of apoptosis, and peptidyl-tyrosine phosphorylation, while KEGG mapping associated them with cellular senescence, circadian rhythm pathways, viral carcinogenesis, and neurotrophins signaling. Collectively, these findings suggest that miR-200a, miR-1, and miR-548-3p potentially involved in oxidative and metabolic pathways in RA, offering candidate biomarkers and therapeutic targets for disease management. Further, RT-PCR for hub genes showed elevated expression of RUNX1, MAPK1, ETS1, FOXP1, and CDC42 in RA patients. Overall, our findings reveal a potential miRNA-mediated regulatory axis underlying oxidative and metabolic disturbances in RA, offering new insights into disease mechanisms and laying the groundwork for the development of novel biomarkers and therapeutic targets.

In this study, the interaction models between an extract of red grape pomace obtained by extraction with eutectic solvents and three enzymes, responsible for pro-inflammatory (lipoxygenase) and metabolic syndrome responses (α-amylase and α-glucosidase), were systematically investigated through experiments and computational simulations. The chromatographic analysis allowed the identification of thirty-two polyphenolic compounds in the extract, predominantly phenolic acids (73%), with a total polyphenol content of 575.58 mg/kg. The main identified compounds were vanillic acid, azelaic acid, chlorogenic acid, suberic acid, abietic acid, phlorizin, polydatin, and t-resveratrol. SwissADME and ProTox-3.0 bioinformatic tools were employed to assess the absorption, distribution, metabolism, and excretion, highlighting that 23.23% of the identified compounds may exert good oral bioavailability in humans and neuroprotective or cognitive-enhancing effects within the central nervous system. A high inhibition rate of 99% for amylase and glucosidase and 82% for lipoxygenase was suggested. The thermodynamic analysis suggested that hydrogen bonding, van der Waals, and hydrophobic forces primarily drove the interactions. The molecular docking results revealed details on the potential mechanisms involved in the inhibition of the metabolic syndrome-associated enzymes exerted by the phenolic compounds from red grape extract. Different phenolic compounds were identified to attach to the active site of the enzymes, interfering with their activity. The anti-diabetic activity might be assigned to vanillic acid and suberic acid, which bind to α-amylase and α-glucosidase respectively, whereas suberic acid and resveratrol appeared to be responsible for the direct inhibitory mechanism of lipoxygenase.

Maternal diet high in saturated fatty acids (SFA) promote infant gut dysbiosis and impairs metabolic and neurocognitive outcomes; however, the protective potential of maternal polyunsaturated fatty acids (PUFA), particularly omega-3 (n3), remains unclear. This study examined how maternal diets enriched in SFA (20% milk fat), omega-6 (n6; 20% corn oil), or omega-3 (n3; 19% olive oil + 1% fish oil) influence neonatal metabolism, neurodevelopment, the gut microbiome, the gut-blood-brain metabolomes, and the brain lipidome in C57BL/6 mice. The offspring were exposed to these diets only during gestation and lactation and then maintained on a Western-style diet for 10 weeks. Compared to SFA, maternal PUFA-rich diets induced distinct and persistent microbiome signatures and reshaped the gut and systemic metabolomic profiles into adulthood. The offspring of n3-fed dams displayed higher lean-to-fat mass ratios, improved ileal morphology, and enhanced gut epithelial integrity. Chronic low-grade inflammation (MCP-1) along the gut-blood-brain axis was markedly reduced in n3 offspring. Moreover, maternal n3 intake enhanced synaptic plasticity, suppressed neuroinflammation, and enriched brain lipids and metabolites associated with membrane integrity, neuronal signaling, and anti-inflammatory pathways. Overall, maternal omega-3 intake confers long-term neuroprotective effects by modulating brain lipid remodeling and the gut-brain-immune axis.

Members of the genus Pestalotiopsis have been reported from symptomatic, asymptomatic, and dead plant tissues, and are therefore frequently described as phytopathogens, endophytes, or saprobes. This study focused on two closely related species, Pestalotiopsis formosana and P. neolitseae, which have been isolated from saprobic, endophytic, and pathogenic contexts in recent studies, raising questions regarding the basis for these diverse observed lifestyles. To clarify this ambiguity, we sequenced six new genomes representing strains of these species spanning these observed lifestyles and compared them with four publicly available, curated plant-associated genomes. Despite contrasting field observations, strains of P. formosana and P. neolitseae showed nearly identical genome features, sharing a core genome of 12,021 orthologous proteins with almost identical secretome content, effectors, CAZyme repertoires, and secondary metabolite gene clusters. Carbon-use assays (Biolog FF and minimal media) further showed broadly overlapping metabolic capabilities, although some strain-level differences were observed. CAZyme-based trophic prediction (CATAStrophy) also placed all analysed strains within the same broad trophic prediction space. Taken together, these results do not support clear genome-scale differentiation corresponding to the assigned lifestyle categories within the present sampling framework. Instead, the data are consistent with the interpretation that these fungi share a broadly conserved genomic toolkit, while ecological expression may depend on regulatory, physiological, host-related, and environmental factors. These findings provide a comparative framework for future studies integrating transcriptomics, metabolomics, and experimental infection assays to clarify how ecological behaviour is expressed in Pestalotiopsis.

Several studies have developed predictive models for venous thromboembolism (VTE) after metabolic and bariatric surgery (MBS), but none have incorporated complications that increase VTE risk. This study aimed to evaluate the risk factors for VTE following MBS using machine learning (ML) algorithms, using models that either excluded or included time-aware postoperative complication variables-these were engineered to include only complications that occurred before the VTE event. We used the MBSAQIP database from 2020 to 2023. XGBoost, random forest classifiers, support vector machines (SVMs), artificial neural networks (ANNs), and logistic regression models were used to predict VTE after MBS in models with and without considering other postoperative complications that occurred before VTE occurrence. The study included 2198 and 698,284 VTE and non-VTE cases, respectively. For models excluding complications, the area under the curve (AUC) values were as follows: XGBoost (0.668), RandomForestClassifier (0.660), SVM (0.649), ANN (0.657), and logistic regression (0.669). Including time-adjusted complications improved all model performances, with AUCs of XGBoost (0.680), RandomForestClassifier (0.664), SVM (0.666), ANN (0.697), and logistic regression (0.690). The strongest postoperative predictor of VTE was the need for reoperation. Other postoperative predictors included prolonged length of stay, ICU admission, reintervention, organ-space surgical site infection, sepsis, and anastomotic leak. Postoperative complications and prolonged length of stay were strongly associated with VTE after MBS, suggesting that patients with these risk factors may benefit from enhanced prophylactic strategies.

Immunogenic cell death (ICD) represents a promising approach to convert "cold tumor" into "hot tumor". However, its induction is often hampered by the immunosuppressive tumor microenvironment fueled by aberrant glucose metabolism. Here, we report a novel "metabolic dual-clamp" strategy inspired by the cooperative pincer movement of crabs to achieve glucose metabolism blockade. Glucose oxidase (GOx) serves as the "external clamp" to deplete extracellular glucose, while 2-deoxy-D-glucose (2-DG) acts as the "internal clamp" to inhibit intracellular glucose metabolism. This synergistic system is co-delivered by a self-assembled DNA nanocage, AS1411-Mn/2-DG-GOx (AMDG). The AMDG not only inhibits glucose metabolism to reverse immunosuppression but also initiates a cascade reaction: Mn2+ catalyzes H2O2 to O2, promoting GOx activity and generating more H2O2 to enhance Mn2+-mediated CDT, and ultimately leading to cascaded amplification of ICD. Furthermore, Mn2+ acts as a STING agonist to activate the cGAS-STING pathway, promoting dendritic cell maturation and cytotoxic T lymphocyte infiltration. Both in vitro and in vivo studies demonstrate that this strategy effectively inhibits tumor glucose metabolism, thereby enhancing ICD-induced antitumor immunity. This study presents the first exploration of the GOx and 2-DG combination, establishing a powerful synergistic treatment paradigm based on glucose metabolism reprogramming.

Prediabetes represents an intermediate metabolic condition between normal blood glucose and diabetes, characterized by insulin resistance, metabolic dysfunction, and low-grade inflammation, collectively contributing to early renal injury. However, manifestations such as glomerular hyperfiltration, increased urinary protein excretion, and subclinical tubular injury are often overlooked during this stage. Therefore, elucidating the underlying molecular mechanisms and implementing early interventions may significantly attenuate subsequent renal function decline. In this context, although Modified Huanglian Wendan Decoction (MHWD) has demonstrated efficacy in regulating glucose and lipid metabolism, its effects on renal injury during prediabetes have not been fully elucidated. UPLC-MS analysis was conducted to characterize the main active compounds of MHWD. Subsequently, a prediabetic rat model was established using a high-fat diet in combination with streptozotocin to explore the effects of MHWD on insulin sensitivity, body weight, blood glucose, and lipid profiles. Transcriptomic and proteomic analyses of the kidneys were subsequently performed to explore MHWD's molecular mechanisms, which were further validated in vivo. In vitro studies in HK-2 cells exposed to high glucose were conducted to explore MHWD mechanisms, with autophagy's role in anti-inflammatory effects assessed using chloroquine and the levels of autophagy-related proteins and inflammatory cytokines determined. Metformin, an AMPK activator, was used as a positive control to assess MHWD's effects on the AMPK/ULK1 pathway, autophagy, and inflammatory responses. The AMPK inhibitor Compound C was subsequently applied to assess whether these protective effects were dependent on the AMPK/ULK1 pathway. UPLC-MS analysis identified 15 principal chemical constituents of MHWD. In prediabetic rats, MHWD treatment alleviated metabolic abnormalities, including hyperglycemia, insulin resistance, dyslipidemia, and weight gain, while attenuating renal injury and systemic as well as renal inflammation. Transcriptomic and proteomic analyses indicated that MHWD exerted its core effects through metabolic regulation, restoration of autophagy, and anti-inflammatory actions. Transmission electron microscopy, immunofluorescence, and Western blot analyses confirmed that MHWD restores renal autophagy and suppresses inflammation via the AMPK/ULK1 pathway. These effects were recapitulated in high-glucose-exposed HK-2 cells and were comparable to those of metformin. Inhibition by chloroquine or Compound C suppressed the protective effects of MHWD, indicating that its renal protective and anti-inflammatory benefits were mediated via AMPK/ULK1-dependent autophagy, thereby mitigating metabolic dysregulation and renal injury in prediabetic states. MHWD ameliorates metabolic disorders and preserves renal function in prediabetes by restoring autophagic homeostasis, enhancing energy metabolism, and suppressing inflammation via the AMPK/ULK1 pathway, offering a mechanistic basis for early kidney protection and potential clinical application.

Cardiovascular-kidney-metabolic syndrome (CKM) contributes substantially to death, but the extent of malnutrition documentation among CKM-attributed deaths is unclear. We quantified temporal trends and disparities in US CKM-related deaths involving malnutrition from 1999 to 2023. Using Centers for Disease Control and Prevention Wide-Ranging Online Data for Epidemiologic Research Multiple Cause of Death data, we identified deaths with CKM as the underlying cause and malnutrition as a contributing cause. We calculated age-adjusted mortality rates (AAMRs) per 100 000 using the 2000 US standard population, assessed trends with joinpoint regression, quantified malnutrition involvement using an AAMR-based share, and modeled the death-count-based share with weighted binomial regression. National AAMRs followed a U-shaped pattern, declining from 2.31 in 1999 to about 0.9 in the early 2010s, then increasing to 3.78 (95% CI, 3.71-3.85) in 2023 (average annual percentage change, 2.1%). Despite declining overall CKM death, the AAMR-based share increased from 0.67% to 1.40%. Adults aged ≥65 years accounted for most deaths and had higher AAMRs than younger adults, although younger decedents showed faster adjusted annual increases in malnutrition documentation. Black adults had the highest AAMRs throughout, whereas relative increases in malnutrition involvement were steeper among White adults. The West had higher baseline odds and the most rapid acceleration. Malnutrition involvement in CKM-attributed deaths increased over time despite declining overall CKM deaths, highlighting malnutrition as an underrecognized component of end-of-life vulnerability in CKM and supporting integration of nutritional assessment and supportive care in high-burden populations and regions.

Etoposide, a semisynthetic derivative of podophyllotoxin originally isolated from the rhizomes of mayapple (Podophyllum peltatum) and indian Podophyllum (P. hexandrum) plants, is one of the most powerful chemotherapeutic agents used to treat various types of solid tumors and blood malignancies. Despite its clinical importance, its supply is recurrently constrained due to a heavy reliance on plant extraction, where low natural precursor abundance and increasing climate-related pressures limit production scalability. Developing alternative manufacturing routes has therefore become a major objective, though reconstruction of this complex biosynthetic pathway has long posed significant challenges, even with recent advances in synthetic biology and metabolic engineering. Yeast has emerged as a robust cellular chassis for reconstituting, either partially or entirely, plant secondary metabolite pathways, and enabling cost-effective bioproduction. Here, we established an integrated biotechnological strategy for the sustainable production of advanced etoposide intermediates using engineered yeast cell factories. By combining pathway refactoring, gene copy number optimization, and tailored co-enzyme compatibility, we established an efficient heterologous pathway converting yatein into (-)-4'-desmethyl-epipodophyllotoxin (4'dEPT) in yeast. Iterative strain engineering improved metabolic flux distribution, leading to enhanced titers and accelerated production kinetics, while process engineering proved essential to maximizing overall system performance. Finally, we also demonstrated the viability of coupling bioproduction in cell factories with downstream, semisynthetic conversion by successfully isolating bioreactor-derived 4'dEPT and converting it into etoposide. In parallel, identifying resilient plant resources that can accumulate high levels of YAT provides a complementary strategy for securing the precursor supply at scale. Overall, this report validates the concept of a hybrid etoposide production platform integrating controlled plant biomass sourcing, engineered yeast cell factories, and chemical transformation steps.

Cytotoxicity testing is a critical step in the preclinical evaluation of biomaterials and medical devices, with extracellular matrix (ECM)-based biomaterials constituting a significant category. The current ISO 10993-5:2009 standard, Biological Evaluation of Medical Devices-Part 5: Tests for In Vitro Cytotoxicity, delineates four metabolic assays that primarily rely on colorimetric methods and colony formation. However, relying solely on colorimetric assays or colony formation fails to provide precise insights into cell function/activity and may yield false-positive results, contributing to interlaboratory discrepancies. This study systematically evaluated ISO 10993-5-recommended assays for ECM-based commercial products, specifically assessing the impact of key assay variables including cell types, contact mode (test extracts versus test material itself), and media components (with or without serum) on biological outcomes. These evaluations support the development of more accurate and robust test methods. While all four assays indicated the noncytotoxic nature of the test samples, metabolic activity readings varied substantially depending on the serum presence, cell types, and assay method employed. To address these limitations and achieve more precise insights into cellular activity, cell membrane integrity (live/dead staining), cell-ECM attachment (actin cytoskeleton), proliferation (Ki67), and apoptosis (annexin V) were analyzed. Notably, despite observing increased metabolic activity (100%-150%) under serum-free conditions measured using MTT and XTT assays, live/dead and actin staining showed no corresponding changes in cell viability or attachment, and Ki67 indicated only ∼15% proliferation. Annexin V staining was detected only in human primary dermal fibroblasts, highlighting their greater reliability over L929 cells for detecting apoptosis. These findings provide a valuable reference for researchers, regulatory bodies, and industry stakeholders in refining cytotoxicity testing protocols and guiding future ISO 10993-5 revisions for more reliable assessment of biomaterials and medical devices.

Diabetic cardiomyopathy (DbCM) impairs cardiac performance through complex mechanisms, which limits effective therapies. We investigated the protein networks of calmodulin (CaM), a striatin (STRN)-binding protein implicated in DbCM, and compared them with the STRN network from the same tissues. Using CaM as bait, we precipitated protein clusters from left ventricles (LVs) of diabetic rat hearts at 8 and 24 weeks post-streptozotocin (post-STZ). Diabetic rats exhibited pathological remodeling, evidenced by increased heart-to-body weight ratio, β-MHC protein, and ANF mRNA expression. Western blotting showed elevated STRN, but not PP2A-A or -C subunits, in chronic stages. Proteomic analysis at 24 weeks revealed 49 differentially interacting proteins (DIPs) with CaM: 18 enhanced and 31 diminished in diabetic LVs. Functional annotation highlighted the recruitment of proteins in metabolic pathways (fatty acid elongation and lipid metabolism) and PPAR signaling, alongside reduced interactions in lipid response, glucocorticoid response, calcium signaling, and amino acid biosynthesis. Comparative analysis of CaM and STRN interactomes converged to 231 proteins implicated mainly in metabolic processes. Out of these, three proteins (Hnrnpm, Acot7, and Gsta3) surfaced as regulators of metabolic, oxidative, and post-transcriptional remodeling. These findings reveal novel functional roles for the CaM/STRN complex in DbCM and identify a shared signaling hub that may guide therapeutic strategies for diabetes-associated cardiac deterioration.

The anaerobic/aerobic/anoxic (AOA) process involves the inevitable consumption of intracellular carbon sources during the aerobic phase, which limits the nitrogen removal efficiency of subsequent endogenous denitrification. Therefore, elucidating the effects of different aeration patterns on endogenous metabolic mechanisms and microbial interactions is crucial. In this study, the characteristics of intracellular carbon source utilization under continuous and intermittent aeration patterns were comparatively analyzed in continuous-flow suspended sludge reactors treating low C/N municipal wastewater. During 210 days of operation, both the continuous aeration system (AOAC) and intermittent aeration system (AOAI) achieved a total inorganic nitrogen (TIN) removal efficiency of approximately 90%. The results demonstrated that the AOAI system effectively reduced the oxidation of intracellular carbon sources within the aerobic zone. However, intermittent aeration was prone to introducing inter-zonal dissolved oxygen (DO) interference, which caused a metabolic lag in the anoxic phase and consequently constrained the rapid utilization of electron donors. Conversely, the prolonged continuous anoxic conditions in the AOAC system enriched the hydrolytic-fermentative bacterium Caldithrix. This enrichment of Caldithrix enhanced the utilization of extracellular polymers and cellular decay products, providing additional electron donors for endogenous denitrification. Furthermore, glycogen was identified as the core carbon source driving endogenous denitrification in both systems, with the dominant genus Rubrivivax playing a pivotal role. In conclusion, this study elucidated the impact of aeration patterns on the endogenous metabolic networks of the AOA process from the perspective of carbon utilization mechanisms, providing valuable insights for advancing low-energy, high-efficiency nitrogen removal.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is highly prevalent in people with type 2 diabetes and represents a major contributor to liver-related and extrahepatic morbidity and mortality. Despite this strong epidemiological overlap, the clinical course of MASLD in diabetes is highly heterogeneous and not fully explained by conventional metabolic risk factors. Human genetic studies have provided important insights into this variability by revealing that hepatic steatosis and its downstream consequences can arise through biologically distinct pathways. Genome-wide association studies have identified multiple genetic loci influencing liver fat accumulation, disease severity and progression. These loci implicate diverse mechanisms, including liver-intrinsic defects in lipid handling, enhanced hepatic lipogenesis, and systemic metabolic dysfunction related to insulin resistance and adipose tissue biology. Genetic evidence indicates that similar degrees of hepatic steatosis may therefore reflect different underlying biological processes, with differing implications for glycaemic management, cardiovascular risk and liver disease progression. In this review, we examine the genetic architecture of hepatic steatosis and MASLD in the context of diabetes. We discuss evidence from family studies, genome-wide association analyses, imaging genetics and Mendelian randomisation, with an emphasis on cautious causal interpretation. We also explore gene-diabetes and gene-environment interactions that modify disease expression and may contribute to variability in clinical outcomes and treatment response. Finally, we consider the translational implications of MASLD genetics, including risk stratification, therapeutic target discovery and emerging genotype-informed approaches to clinical management. We highlight key research gaps, particularly the need for ancestry-diverse studies, improved phenotyping in diabetes populations, and integration of genetic analyses into prospective clinical trials. Together, current genetic evidence supports a mechanism-based framework for understanding MASLD heterogeneity in diabetes and provides a foundation for more precise approaches to risk assessment and management.