Multiple Metabolic Pathways Research Articles

Extending the G1 phase improves the production of lipophilic compounds in yeast by boosting enzyme expression and increasing cell size

Cell phase engineering can significantly impact protein synthesis and cell size, potentially enhancing the production of lipophilic products. This study investigated the impact of G1 phase extension on resource allocation, metabolic functions, and the unfolded protein response (UPR) in yeast, along with the potential for enhancing the production of lipophilic compounds. In brief, the regulation of the G1 phase was achieved by deleting CLN3 (G1 cyclin) in various yeast strains. This modification resulted in a 83% increase in cell volume, a 76.9% increase in dry cell weight, a 82% increase in total protein content, a 41% increase in carotenoid production, and a 159% increase in fatty alcohol production. Transcriptomic analysis revealed significant upregulation of multiple metabolic pathways involved in acetyl-CoA (acetyl coenzyme A) synthesis, ensuring an ample supply of precursors for the synthesis of lipophilic products. Furthermore, we observed improved protein synthesis, attributed to UPR activation during the prolonged G1 phase. These findings not only enhanced our understanding and application of yeast’s capacity to synthesize lipophilic compounds in applied biotechnology but also offered unique insights into cellular behavior during the modified G1 phase, particularly regarding the UPR response, for basic research. This study demonstrates the potential of G1 phase intervention to increase the yield of hydrophobic compounds in yeast, providing a promising direction for further research.

Construction and Validation of a Novel T/NK-Cell Prognostic Signature for Pancreatic Cancer Based on Single-Cell RNA Sequencing

Background Evidence with regards to the distinction between primary and metastatic tumors in pancreatic cancer and driving factors for metastases remains limited. Methods Single-cell RNA sequencing (scRNA-seq) was conducted on metastatic pancreatic cancer. Bioinformatics analysis on relevant sequencing data was used to construct a risk model to predict patient prognosis. Furthermore, immune infiltration and metabolic differences were assessed. The biological function of key differential genes was evaluated. Results Paired primary and metastatic tumor tissues from 3 pancreatic cancer patients were collected and conducted scRNA-seq. Subsequently, the T/NK cell subgroup was the most different cell type between primary tumors and liver metastases and was selected for further analysis. Eventually, 6 specifically expressed genes of T/NK cells (B2M, ZFP36L2, ANXA1, ARL4C, TSPYL2, FYN) were used constructing the prognostic model. The stability of this model was validated by an external cohort. Meanwhile, different immune infiltration abundances occurred between high and low risk groups stratified by the model. The high-risk group had a stronger metabolic capability. Conclusions A novel prognostic T/NK-cell signature for pancreatic cancer was constructed based on scRNA-seq data and externally validated. The involved key genes may play a role in multiple metabolic pathways of metastasis and affect the tumor immune microenvironment.

Soil metagenomics reveals reduced tillage improves soil functional profiles of carbon, nitrogen, and phosphorus cycling in bulk and rhizosphere soils

Conservation tillage practices (CAT) are known to benefit soil health and soil ecosystem functions relative to conventional tillage (CVT); however, much uncertainty remains concerning microbial functional traits and their subsequent effects on soil nutrients under different tillage practices. We analyzed the functional profiles of the C, N, and P cycles in response to CAT of no-tillage (NT), reduced tillage (RT), and CVT of moldboard plowing (MP) in bulk and rhizosphere soils using shotgun sequencing. CAT induced distinct microbial functional patterns relative to CVT, and these differences were generally more evident in rhizosphere soils than in bulk soils. CAT promotes multiple metabolic pathways such as C and N decomposition, fermentation, CO oxidation, N fixation, nitrate reduction and inorganic-P and organic-P transformations in bulk and/or rhizosphere soils. Variations in these metabolic pathways were mainly driven by Bradyrhizobium, Mesorhizobium, Nitrososphaera, Phenylobacterium, Rhizobium which are affiliated with Proteobacteria, Actinobacteria, Acidobacteria, Bacteroidota and Thaumarchaeota. Furthermore, 24 high-quality metagenome-assembled genomes (MAGs) were reconstructed, of which three novel MAGs (TL100, TL46, and TL57) harbored functional genes regulating all metabolic pathways. In particular, NT-enriched MAGs (such as Sphingomonas) promote fermentation, resulting in the reduction of soil total carbon (TC) relative to MP in bulk soils. RT retained the contents of soil TC and total nitrogen (TN) well and up-regulated the phoR gene carried by Streptomyces, which promoted the regulation of P-starvation concomitantly with the increase in the contents of total phosphorous (TP) and available phosphorous (AP) in bulk soil. Additionally, assimilatory nitrate reduction coupled with organic-P mineralization was facilitated by CAT in rhizosphere soil, leading to the mitigation of N loss and the activation of soil organic-P for crop uptake. Overall, our results revealed that CAT significantly accelerated multiple metabolic potentials, and RT could sustain soil nutrient contents better than NT.

Metabolomic Analysis Reveals the Linkage between Sleep-Enhancing Effects and Metabolite Biomarkers and Pathways of Different Casein Hydrolysates in Chronic Unpredictable Mild Stressed Mice.

Casein hydrolysates have been proven to exert varying sleep-enhancing and anxiolytic effects due to their distinct release of potential peptides. However, their underlying sleep-enhancing mechanisms at the metabolic level remain unclear. This study aims to investigate the potential sleep-enhancing mechanism of casein hydrolysates through an integrated approach of untargeted and targeted metabolomics in CUMS-induced anxiety mice for the first time. The results showed seven potential biomarkers were identified and screened using orthogonal partial least-squares discriminant analysis, random forest model, and pathway analysis, including ornithine, l-proline, l-prolinamide, inhibitory neurotransmitters gamma-aminobutyric acid, 5-HIAA, fumaric acid, and 4-oxoglutaramate. Moreover, casein hydrolysates exerted sleep-enhancing effects through multiple metabolic pathways, mainly including the GABAergic system, tryptophan metabolism, and cAMP response signaling pathway, which was validated by targeted metabolomics and vital protein expressions. It was interesting that casein hydrolysates with diverse representative peptide compositions exhibited varying activity, which could be attributed to distinct alterations in metabolites via different pathways.

Integration of pharmacochemistry, pharmacodynamics and metabolomics to reveal active ingredients and mechanism of Nan Bao detox capsule alleviating methamphetamine addiction

AbstractNan Bao detox capsule (NBDC), derived from ancient Chinese opioid detox protocols, shows promising therapeutic potential in substance abuse disorders, particularly for attenuating methamphetamine (MA) addiction. This study aimed to identify active ingredients, evaluate therapeutic efficacy in an MA addiction rat model and delineate pharmacodynamic mechanisms using metabolomics. In vitro phytochemical profiling characterized 258 drug‐related compounds, with 87 prototype entities mainly identified in rat plasma. NBDC significantly attenuated METH‐induced behavioural anomalies and modulated neurotransmitter levels, notably increasing brain DA and serotonin (5‐HT) content with concomitant upregulation of D1 dopamine receptor (DRD1) and 5‐HT1A receptor (5‐HT1AR) expression, ameliorating hippocampal pathology. Metabolomic analysis identified histamine receptor as a potential target and revealed the involvement of NBDC in metabolic pathways associated with cocaine addiction, amphetamine abuse and Parkinson's disease. Conclusively, NBDC presents a promising therapeutic agent for mitigating MA addiction through a synergistic interplay of multiple constituents, pharmacological targets and metabolic pathways.

Biomaterial-Mediated Metabolic Regulation of Ferroptosis for Cancer Immunotherapy.

Ferroptosis is a lipid peroxidation-driven cell death route and has attracted enormous interest for cancer therapy. Distinct from other forms of regulated cell death, its process is involved with multiple metabolic pathways including lipids, bioenergetics, iron, and so on, which influence cancer cell ferroptosis sensitivity and communication with the immune cells in the tumor microenvironment. Development of novel technologies for harnessing the ferroptosis-associated metabolic regulatory network would profoundly improve our understanding of the immune responses and enhance the efficacy of ferroptosis-dependent immunotherapy. Interestingly, the recent advances in bio-derived material-based therapeutic platforms offer novel opportunities to therapeutically modulate tumor metabolism through the insitu delivery of molecular or material cues, which not only allows the tumor-specific elicitation of ferroptosis but also holds promise to maximize their immunostimulatory impact. In this review, we will first dissect the crosstalk between tumor metabolism and ferroptosis and its impact on the immune regulation in the tumor microenvironment, followed by the comprehensive analysis on the recent progress in biomaterial-based metabolic regulatory strategies for evoking ferroptosis-mediated antitumor immunity. A perspective section is also provided to discuss the challenges in metabolism-regulating biomaterials for ferroptosis-immunotherapy. We envision that this review may provide new insights for improving tumor immunotherapeutic efficacy in the clinic.

A Comprehensive Review on the Anti-Obesity Potential of Limonia acidissima L., and its Bioactive Compounds: Mechanisms and Applications in Weight Management

Introduction: Obesity is a global health challenge associated with numerous metabolic disorders, including diabetes and cardiovascular disease. With growing interest in natural remedies for weight management, Limonia acidissima L., has garnered attention due to its rich bioactive composition. Objectives: This systematic review evaluates the anti-obesity potential of Limonia acidissima L., and its key bioactive compounds, including fibres, phytosterols, saponins, polyphenols, flavonoids, and ascorbic acid. Methods: A comprehensive search of databases such as PubMed, Scopus, and Google Scholar was conducted, focusing on studies published between 2000 and 2023 that investigated the metabolic effects of these compounds on lipid and carbohydrate metabolism, fat absorption, and glucose regulation. Results: The results demonstrated that Limonia acidissima L., significantly reduced body weight and fat mass, improved lipid profiles, and enhanced insulin sensitivity in both animal and human studies.Phytosterols and saponins played key roles in modulating lipid metabolism, while fibres and polyphenols were found to regulate carbohydrate metabolism and improve glycemic control. Additionally, the synergistic effects of multiple bioactive compounds amplified their collective anti-obesity potential, with few reported adverse effects, suggesting a favourable safety profile. The discussion highlights the multifaceted mechanisms through which Limonia acidissima L., combats obesity, emphasizing its ability to target multiple metabolic pathways simultaneously.While the findings are promising, the need for further large-scale clinical trials to validate these effects in diverse populations is underscored. Conclusions: In conclusion, Limonia acidissima L., emerges as a potent natural remedy for obesity management, with strong potential for inclusion in weight loss supplements and functional foods.Its bioactive compounds offer a comprehensive and safe approach to addressing the global obesity epidemic, although further research is needed to establish its long-term efficacy and optimal use.

Effects of MDA-19 on Zebrafish Larval Behavior: Perspectives From Neurodevelopment, Oxidative Stress, and Metabolomics.

As global regulations on synthetic cannabinoids tighten, illicit vendors increasingly turn to new structures of synthetic cannabinoids to evade legal scrutiny. MDA-19, a novel synthetic cannabinoid, exhibited significant agonistic effects on type 2 cannabinoid receptors invivo and showed emerging trends of abuse in illicit markets. However, research on the toxicological effects of MDA-19 remains scarce. In this study, we examined the effects of MDA-19 on neurodevelopment, behavior, oxidative stress, and metabolomics by exposing zebrafish embryos to MDA-19 solutions with concentrations of 1, 10, and 20 mg/L over 5 days. Results revealed that exposure to 10 and 20 mg/L of MDA-19 accelerated hatching in zebrafish embryos but led to reduced body length without affecting mortality or malformation. Furthermore, exposure to all concentrations of MDA-19 resulted in diminished swimming ability and reduced activity time in zebrafish. Transgenic zebrafish (hb9-GFP) exposed to MDA-19 exhibited impaired development of spinal motor neurons. Notably, exposure to 20 mg/L MDA-19 increased the levels of reactive oxygen species (ROS) in zebrafish and elevated the activity of antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT), while the levels of the lipid oxidation product malondialdehyde (MDA) remained unaffected. Nontargeted metabolomics analyses showed that MDA-19 interfered with multiple metabolic pathways affecting energy metabolism, such as alanine, aspartate, and glutamate metabolism; the citric acid cycle (TCA cycle), pantothenate, and coenzyme A biosynthesis; and purine metabolism. In conclusion, the present study provided the essential evidence for the neurotoxic effects of MDA-19, which was associated with impaired neurodevelopment, dysregulation of oxidative stress homeostasis, and altered energy metabolism.

Shrinkage estimation of gene interaction networks in single-cell RNA sequencing data

BackgroundGene interaction networks are graphs in which nodes represent genes and edges represent functional interactions between them. These interactions can be at multiple levels, for instance, gene regulation, protein-protein interaction, or metabolic pathways. To analyse gene interaction networks at a large scale, gene co-expression network analysis is often applied on high-throughput gene expression data such as RNA sequencing data. With the advance in sequencing technology, expression of genes can be measured in individual cells. Single-cell RNA sequencing (scRNAseq) provides insights of cellular development, differentiation and characteristics at the transcriptomic level. High sparsity and high-dimensional data structures pose challenges in scRNAseq data analysis.ResultsIn this study, a sparse inverse covariance matrix estimation framework for scRNAseq data is developed to capture direct functional interactions between genes. Comparative analyses highlight high performance and fast computation of Stein-type shrinkage in high-dimensional data using simulated scRNAseq data. Data transformation approaches also show improvement in performance of shrinkage methods in non-Gaussian distributed data. Zero-inflated modelling of scRNAseq data based on a negative binomial distribution enhances shrinkage performance in zero-inflated data without interference on non zero-inflated count data.ConclusionThe proposed framework broadens application of graphical model in scRNAseq analysis with flexibility in sparsity of count data resulting from dropout events, high performance, and fast computational time. Implementation of the framework is in a reproducible Snakemake workflow https://github.com/calathea24/ZINBGraphicalModel and R package ZINBStein https://github.com/calathea24/ZINBStein.

Alternative sulphur metabolism in the fungal pathogen Candida parapsilosis

Candida parapsilosis is an opportunistic fungal pathogen commonly isolated from the environment and associated with nosocomial infection outbreaks worldwide. We describe here the construction of a large collection of gene disruptions, greatly increasing the molecular tools available for probing gene function in C. parapsilosis. We use these to identify transcription factors associated with multiple metabolic pathways, and in particular to dissect the network regulating the assimilation of sulphur. We find that, unlike in other yeasts and filamentous fungi, the transcription factor Met4 is not the main regulator of methionine synthesis. In C. parapsilosis, assimilation of inorganic sulphur (sulphate) and synthesis of cysteine and methionine is regulated by Met28, a paralog of Met4, whereas Met4 regulates expression of a wide array of transporters and enzymes involved in the assimilation of organosulfur compounds. Analysis of transcription factor binding sites suggests that Met4 is recruited by the DNA-binding protein Met32, and Met28 is recruited by Cbf1. Despite having different target genes, Met4 and Met28 have partial functional overlap, possibly because Met4 can contribute to assimilation of inorganic sulphur in the absence of Met28.

Microbial metabolomics in acute myeloid leukemia: From pathogenesis to treatment.

Acute myeloid leukemia (AML), the most common leukemia in adults, is a biologically heterogeneous disease arising from clonally proliferating hematopoietic stem cells. Increased appreciation of novel genetic methods has improved the understanding of AML biology. Recently, the emerging field of metabolomics has indicated qualitative and quantitative alterations in metabolic profiles in AML pathogenesis, progression and treatment. Multiple metabolic and molecular pathways regulate human metabolism and host-microbiome interactions may significantly affect this biochemical machinery. Microbiota have been found to play a significant role in hematopoietic function, metabolism and immunity, contributing to AML occurrence. A large number of studies have highlighted the importance of the composition and diversity of the gut microbiota (GM) in response to treatment and prognosis in AML. Moreover, strong evidence emphasizes the detrimental link between dysbiosis and infectious complications, a leading cause of morbidity and mortality for patients with AML. Several microbiota-related mechanisms have been linked to particular changes in host physiology so far, and microbial-derived metabolites belong to one of the most important. Circulating in the body, they modulate human conditions both locally and systemically. The extensive and diverse repertoire of bacterial metabolic functions plays a critical role in numerous processes, including leukemogenesis. Integrative analysis of microbiome and metabolome data is a promising avenue for better understanding the complex relationship between the microbiota, biochemical alterations and AML pathogenesis to effectively prevent, treat and mitigate its outcomes. This review concentrates on the pathologic roles and therapeutic implications of microbe-derived metabolites in AML settings.

Essential role of proline synthesis and the one-carbon metabolism pathways for systemic virulence of Streptococcus pneumoniae.

Virulence screens have indicated potential roles during Streptococcus pneumoniae infection for the one-carbon metabolism pathway component Fhs and proline synthesis mediated by ProABC. To define how these metabolic pathways affect S. pneumoniae virulence, we have investigated the phenotypes, transcription, and metabolic profiles of Δfhs and ΔproABC mutants. S. pneumoniae capsular serotype 6B BHN418 Δfhs and ΔproABC mutant strains had strongly reduced virulence in mouse sepsis and pneumonia models but could colonize the nasopharynx. Both mutant strains grew normally in complete media but had markedly impaired growth in chemically defined medium, human serum, and human cerebrospinal fluid. The BHN418 ΔproABC strain also had impaired growth under conditions of osmotic and oxidative stress. The virulence role of proABC was strain specific, as the D39 ΔproABC strain could still cause septicemia and grow in serum. Compared to culture in broth, in serum, the BHN418 Δfhs and ΔproABC strains showed considerable derangement in global gene transcription that affected multiple but different metabolic pathways for each mutant strain. Metabolic data suggested that Δfhs had an impaired stringent response, and when cultured in sera, BHN418 Δfhs and ΔproABC were under increased oxidative stress and had altered lipid profiles. Loss of proABC also affected carbohydrate metabolism and the accumulation of peptidoglycan synthesis precursors in the BHN418 but not the D39 background, linking this phenotype to the conditional virulence phenotype. These data identify the S. pneumoniae metabolic functions affected by S. pneumoniae one-carbon metabolism and proline biosynthesis, and the role of these genetic loci for establishing systemic infection.IMPORTANCERapid adaptation to grow within the physiological conditions found in the host environment is an essential but poorly understood virulence requirement for systemic pathogens such as Streptococcus pneumoniae. We have now demonstrated an essential role for the one-carbon metabolism pathway and a conditional role depending on strain background for proline biosynthesis for S. pneumoniae growth in serum or cerebrospinal fluid, and therefore for systemic virulence. RNAseq and metabolomic data demonstrated that the loss of one-carbon metabolism or proline biosynthesis has profound but differing effects on S. pneumoniae metabolism in human serum, identifying the metabolic processes dependent on each pathway during systemic infection. These data provide a more detailed understanding of the adaptations required by systemic bacterial pathogens in order to cause infection and demonstrate that the requirement for some of these adaptations varies between strains from the same species and could therefore underpin strain variations in virulence potential.

Integrated microbiome and metabolome analysis reveals synergistic efficacy of basil polysaccharide and gefitinib in lung cancer through modulation of gut microbiota and fecal metabolites

Emerging evidence suggests that gut microbiota and its metabolites significantly influence the effectiveness of EGFR-TKIs (e.g., gefitinib, erlotinib) in lung cancer treatment. Plant polysaccharides can interact with gut microbiota, leading to changes in the host-microbe metabolome that may affect drug metabolism and therapeutic outcomes. Our previous research demonstrated the efficacy of basil polysaccharide (BPS) in treating various cancers by regulating hypoxic microenvironment and inhibiting epithelial-mesenchymal transition process. However, the potential impact of BPS on gut microbiota has not been thoroughly explored. In this study, we employed an immunodeficient gefitinib-resistant xenograft mouse model to explore whether BPS enhances the antitumor effects of gefitinib. A multi-omics approach, including 16S rDNA amplicon sequencing and LC-MS, was used to elucidate these synergistic effects. Our findings indicate that BPS can enhance tumor responsiveness to gefitinib by modulating the gut microbiota and its metabolites through multiple metabolic pathways. These changes in gut microbiota and metabolites could potentially affect cancer related signaling pathway and lung resistance-related protein, which are pivotal in determining the efficacy of EGFR-TKIs in cancer treatment.

Integrated transcriptional and biochemical profiling suggests mechanisms associated with rapid cold hardening in adult Liriomyza trifolii (Burgess)

The leafminer Liriomyza trifolii causes severe economic damage on ornamental and horticultural crops in China. Rapid cold hardening (RCH) is a phenomenon where cold tolerance in insects can be significantly enhanced after a short-term acclimation to low temperatures. In this study, the regulation of transcription in response to cold hardening was investigated in L. trifolii adults, and fatty acids and cryoprotectant levels were measured. The composition of fatty acids changed after RCH treatment, and glucose and trehalose levels showed significant accumulation after acclimation, thus indicating that changes in fatty acids and cryoprotectants contribute to RCH in L. trifolii. RNA-seq was used to analyze transcriptional regulation after a 4 h hardening period and showed that differentially expressed genes clustered in multiple metabolic pathways, which indicates the importance of transcriptional regulation in RCH. This study expands our knowledge of biochemical and transcriptional changes in L. trifolii during cold hardening and provides a basis for further investigations aimed at understanding thermal adaptation in insects.

Targeting AMP-activated protein kinase in sepsis.

Sepsis is a global health challenge marked by limited clinical options and high mortality rates. AMP-activated protein kinase (AMPK) is a cellular energy sensor that mediates multiple crucial metabolic pathways that may be an attractive therapeutic target in sepsis. Pre-clinical experimental studies have demonstrated that pharmacological activation of AMPK can offer multiple potential benefits during sepsis, including anti-inflammatory effects, induction of autophagy, promotion of mitochondrial biogenesis, enhanced phagocytosis, antimicrobial properties, and regulation of tight junction assembly. This review aims to discuss the existing evidence supporting the therapeutic potential of AMPK activation in sepsis management.

Metabolic Pathways Associated With Obesity and Hypertension in Black Caregivers of Persons Living With Dementia.

In the U.S., Black adults have the highest prevalence of obesity and hypertension, increasing their risk of morbidity and mortality. Caregivers of persons with dementia are also at increased risk of morbidity and mortality due to the demands of providing care. Thus, Black caregivers-who are the second largest group of caregivers of persons with dementia in the U.S.-have the highest risks for poor health outcomes among all caregivers. However, the physiologic changes associated with multiple chronic conditions in Black caregivers are poorly understood. In this study, metabolomics were compared to the metabolic profiles of Black caregivers with obesity, with or without hypertension. Our goal was to identify metabolites and metabolic pathways that could be targeted to reduce obesity and hypertension rates in this group. High-resolution, untargeted metabolomic assays were performed on plasma samples from 26 self-identified Black caregivers with obesity, 18 of whom had hypertension. Logistic regression and pathway analyses were employed to identify metabolites and metabolic pathways differentiating caregivers with obesity only and caregivers with both obesity and hypertension. Key metabolic pathways discriminating caregivers with obesity only and caregivers with obesity and hypertension were butanoate and glutamate metabolism, fatty acid activation/biosynthesis, and the carnitine shuttle pathway. Metabolites related to glutamate metabolism in the butanoate metabolism pathway were more abundant in caregivers with hypertension, while metabolites identified as butyric acid/butanoate and R-(3)-hydroxybutanoate were less abundant. Caregivers with hypertension also had lower levels of several unsaturated fatty acids. In Black caregivers with obesity, multiple metabolic features and pathways differentiated among caregivers with and without hypertension. If confirmed in future studies, these findings would support ongoing clinical monitoring and culturally tailored interventions focused on nutrition (particularly polyunsaturated fats and animal protein), exercise, and stress management to reduce the risk of hypertension in Black caregivers with obesity.

Metabolomic profiling of human semen in patients with oligospermia using high performance liquid chromatography-tandem mass spectrometry

Male infertility is one of the most common reproductive dysfunctions. Despite oligospermia being a cause of infertility, few studies have been conducted on it. This study aimed to investigate differences in semen metabolic patterns in patients with oligospermia and to identify potential biomarkers associated with oligospermia. Semen samples from oligospermia patients (20 cases) and healthy controls (20 cases) were detected by high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS), and 72 and 89 metabolites were identified as potential markers in positive and negative ion modes, respectively. In addition, the results identified multiple metabolic pathways in patients with oligospermia, such as glycine serine and threonine metabolism, Synthesis and degradation of ketone bodies, Valine, leucine, and isoleucine degradation. These results described unique metabolic characteristics of semen in patients with oligospermia and provided novel insights into the mechanism of the semen disorder.

7885 Thyroid Hormone Stimulates Heaptic Lipid Metabolism in the Resolution of Liver Fibrosis

Abstract Disclosure: A. Mendoza: None. C. Wang: None. A. Lugo: None. V. Rodrigues: None. S. Houghton: None. D. Redmond: None. A. Hollenberg: None. Metabolic dysfunction-associated steatotic liver disease (MASLD) progression to steatohepatitis and fibrosis is a global health burden, leading to liver failure and death. While the pathogenesis is complex, dysregulated lipid metabolism plays a crucial role. Thyroid hormones (THs) are key metabolic regulators in the liver, promoting lipogenesis and fatty acid oxidation. Previously, we demonstrated that the carbohydrate-responsive element binding protein (ChREBP) is a critical mediator of both these processes in hepatocytes, acting downstream of the thyroid hormone receptor beta1 (TRβ1). Interestingly, while ChREBP ablation abolished TH-induced lipogenesis through downregulation of lipogenic gene expression, its role in TH-mediated fatty acid oxidation remained unclear, suggesting a multifaceted role for ChREBP in TH-regulated hepatic metabolism. Given the critical role of lipid metabolism in the progression of MASLD to metabolic dysfunction-associated steatohepatitis (MASH) and fibrosis, we aim to delineate the mechanisms by which TH promotes the regression of fibrosis. Here, we used a carbon tetrachloride (CCL4) model of liver fibrosis in TRβ1KO and ChREBPKO mice (0.4 ul/g biweekly, 9 injections). All mice in the experiments were also on a PTU-Low Iodine diet to induce hypothyroidism. In the last week of the protocol, all mice were sorted into two groups which received either water or T3 (1ug/ml). 24 hours after the last CCL4 injection all mice were sacrificed and the livers were harvested and prepared for analysis. Herein we show that T3 promotes the resolution of liver fibrosis in wild-type mice. Importantly, this resolution was abolished in both TRβ1 and ChREBP KO mice demonstrating this pathways essential role in TH-induced resolution of fibrosis. RNA-Seq analysis of TRβ1KO mice demonstrated a dual mechanism of TH action: 1. TH upregulates gene expression in multiple metabolic pathways, suggesting a rescue of hepatic metabolism towards normal liver function and 2., TH has antifibrotic action via downregulation of pathways controlling extracellular matrix synthesis and inflammation, key drivers of fibrosis. Strikingly, ChREBP was dispensable for the antifibrotic action of TH suggesting that the ChREBP-dependent regulation of hepatic metabolism is critical for the resolution of fibrosis mediated by TH. To identify novel metabolic pathways regulated by TH via ChREBP, we intersected liver mRNA-seq from TRβ1KO mice with liver-specific ChREBP Chip-Seq and identifed multiple targets regulating autophagy that require ChREBP for their activation after TH treatment. These data suggest that the ability of ChREBP to regulate TH-induced fatty-acid oxidation is mediated by the upregulation of autophagy. This supports the notion that the ability of T3 to promote the resolution of liver fibrosis is primarily dependent of it’s ability to stimulate hepatic fatty acid metabolism. Presentation: 6/1/2024

Versatile filamentous fungal host highly-producing heterologous natural products developed by genome editing-mediated engineering of multiple metabolic pathways

Natural secondary metabolites are medically, agriculturally, and industrially beneficial to humans. For mass production, a heterologous production system is required, and various metabolic engineering trials have been reported in Escherichia coli and Saccharomyces cerevisiae to increase their production levels. Recently, filamentous fungi, especially Aspergillus oryzae, have been expected to be excellent hosts for the heterologous production of natural products; however, large-scale metabolic engineering has hardly been reported. Here, we elucidated candidate metabolic pathways to be modified for increased model terpene production by RNA-seq and metabolome analyses in A. oryzae and selected pathways such as ethanol fermentation, cytosolic acetyl-CoA production from citrate, and the mevalonate pathway. We performed metabolic modifications targeting these pathways using CRISPR/Cas9 genome editing and demonstrated their effectiveness in heterologous terpene production. Finally, a strain containing 13 metabolic modifications was generated, which showed enhanced heterologous production of pleuromutilin (8.5-fold), aphidicolin (65.6-fold), and ophiobolin C (28.5-fold) compared to the unmodified A. oryzae strain. Therefore, the strain generated by engineering multiple metabolic pathways can be employed as a versatile highly-producing host for a wide variety of terpenes.

HILIC-MS and CE-MS as complementary analytical approaches to assess the impact of exposure to polychlorinated biphenyls on the polar serum metabolome of pigs

BackgroundHydrophilic interaction liquid chromatography (HILIC) and capillary electrophoresis (CE) coupled to mass spectrometry (MS) are two of the most widely used techniques to explore and characterize the polar metabolome. Both platforms have some peculiar characteristics that make them more suitable for studying different kind of metabolites and different parts of the metabolome. Consequently, their combined use is recommended to extend the coverage of the polar metabolome, particularly when multiple metabolic pathways are expected to be perturbed, as in toxicological studies related to exposure to environmental pollutants such as polychlorinated biphenyls (PCBs). It is unclear whether chronic exposure to low doses leads to effects on the most polar metabolites. ResultsIn this work, both HILIC-MS and CE-MS platforms have been used to characterize the polar metabolome of blood serum from pigs exposed to low-doses (20 ng/kg body weight (b.w.)/day) of Aroclor 1260 (a technical mixture of PCBs). The use of both platforms aims to cover large parts of the polar metabolome to better characterize the effects of PCB exposure. The results showed that the combined use of HILIC-MS and CE-MS effectively extended the coverage of the metabolome and the interpretability of the data, highlighting their complementarity in metabolic pathways analysis. The analysis of the polar metabolome evidenced a strong effect of exposure on amino acid metabolism, affecting 10 of the 20 proteic amino acids, and a consistent overflow in the urea cycle. SignificanceThis work highlights the need to implement analytical multi-platforms to better address metabolomics studies, showing the complementarity of two widely used analytical techniques (i.e. HILIC-MS and CE-MS) to study the polar metabolome in a risk assessment framework.