Lipid Transport Research Articles

Mycobacterium tuberculosis Mce3R TetR-like Repressor Forms an Asymmetric Four-Helix Bundle and Binds a Nonpalindrome Sequence†.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a major global health concern. TetR family repressors (TFRs) are important for Mtb's adaptation to the human host environment. Our study focuses on one notable Mtb repressor, Mce3R, composed of an unusual double TFR motif. Mce3R-regulated genes encode enzymes implicated in cholesterol metabolism, resistance against reactive oxygen species, and lipid transport activities important for Mtb survival and persistence in the host and for the cellular activity of a 6-azasteroid derivative. Here, we present the structure of Mce3R bound to its DNA operator, unveiling a unique asymmetric assembly previously unreported. We obtained a candidate DNA-binding motif through MEME motif analysis, comparing intergenic regions of mce3R orthologues and identifying nonpalindromic regions conserved between orthologues. Using an electrophoretic mobility shift assay (EMSA), we confirmed that Mce3R binds to a 123-bp sequence that includes the predicted motif. Using scrambled DNA and DNA oligonucleotides of varying lengths with sequences from the upstream region of the yrbE3A (mce3) operon, we elucidated the operator region to be composed of two Mce3R binding sites, each a 25-bp asymmetric sequence separated by 53 bp. Mce3R binds with a higher affinity to the downstream site with a Kd of 2.4 ± 0.7 nM. The cryo-EM structure of Mce3R bound to the 123-bp sequence was refined to a resolution of 2.51 Å. Each Mce3R monomer comprises 21 α-helices (α1-α21) folded into an asymmetric TFR-like structure with a core asymmetric four-helix bundle. This complex has two nonidentical HTH motifs and a single ligand-binding domain. The two nonidentical HTHs from each TFR bind within the high-affinity, nonpalindromic operator motif, with Arg53 and Lys262 inserted into the major groove. Site-directed mutagenesis of Arg53 to alanine abrogated DNA binding, validating the Mce3R/DNA structure obtained. Among 811,645 particles, 63% were Mce3R homodimer bound to two duplex oligonucleotides. Mce3R homodimerizes primarily through α15, and each monomer binds to an identical site in the DNA duplex oligonucleotide.

Adipocyte associated glucocorticoid signaling regulates normal fibroblast function which is lost in inflammatory arthritis.

Fibroblasts play critical roles in tissue homeostasis, but in pathologic states they can drive fibrosis, inflammation, and tissue destruction. Little is known about what regulates the homeostatic functions of fibroblasts. Here, we perform RNA sequencing and identify a gene expression program in healthy synovial fibroblasts characterized by enhanced fatty acid metabolism and lipid transport. We identify cortisol as the key driver of the healthy fibroblast phenotype and that depletion of adipocytes, which express high levels of Hsd11b1, results in loss of the healthy fibroblast phenotype in mouse synovium. Additionally, fibroblast-specific glucocorticoid receptor Nr3c1 deletion in vivo leads to worsened arthritis. Cortisol signaling in fibroblasts mitigates matrix remodeling induced by TNF and TGF-β1 in vitro, while stimulation with these cytokines represses cortisol signaling and adipogenesis. Together, these findings demonstrate the importance of adipocytes and cortisol signaling in driving the healthy synovial fibroblast state that is lost in disease.

Proteomic Profiling of COVID-19 Patients Sera: Differential Expression with Varying Disease Stage and Potential Biomarkers

Background/Objectives: SARS-CoV-2 is one of the viruses that caused worldwide health issues. This effect is mainly due to the wide range of disease prognoses it can cause. The aim of this study is to determine protein profiles that can be used as potential biomarkers for patients’ stratification, as well as potential targets for drug development. Methods: Eighty peripheral blood samples were collected from heathy as well as SARS-CoV-2 patients admitted at a major tertiary care center in Riyadh, Saudi Arabia. A label-free quantitative mass spectrometry-based proteomic analysis was conducted on the extracted sera. Protein–protein interactions and functional annotations of identified proteins were performed using the STRING. Results: In total, two-hundred-eighty-eight proteins were dysregulated among all four categories. Dysregulated proteins were mainly involved in the network map of SARS-CoV-2, immune responses, complement activation, and lipid transport. Compared to healthy subjects, the most common upregulated protein in all three categories were CRP, LGALS3BP, SAA2, as well as others involved in SARS-CoV-2 pathways such as ZAP70 and IGLL1. Notably, we found fifteen proteins that significantly discriminate between healthy/recovered subjects and moderate/under medication patients, among which are the SERPINA7, HSPD1 and TTC41P proteins. These proteins were also significantly downregulated in under medication versus moderate patients. Conclusions: Our results emphasize the possible association of specific proteins with the SARS-CoV-2 pathogenesis and their potential use as disease biomarkers and drug targets. Our study also gave insights about specific proteins that are likely increased upon infection but are likely restored post recovery.

Exploring the interplay between yeast cell membrane lipid adaptation and physiological response to acetic acid stress.

In the present study, we successfully engineered a yeast strain that could grow under high acetic acid stress by regulating its diacylglycerol metabolism. We compared how the plasma membrane and total cell membranes responded to acetic acid by adjusting their lipid content. By combining physiological and lipidomics analyses in cells cultivated in the absence or presence of acetic acid, we found that the capacity of the membrane to adapt lipid composition together with sufficient energy supply influenced membrane properties in response to stress. We suggest that potentiating the intracellular energy system or enhancing lipid transport to destination membranes should be taken into account when designing membrane engineering strategies. The findings highlight new directions for future yeast cell factory engineering.

Divergent roles of RIPK3 and MLKL in high-fat diet-induced obesity and MAFLD in mice.

Cell death frequently occurs in the pathogenesis of obesity and metabolic dysfunction-associated fatty liver disease (MAFLD). However, the exact contribution of core cell death machinery to disease manifestations remains ill-defined. Here, we show via the direct comparison of mice genetically deficient in the essential necroptotic regulators, receptor-interacting protein kinase-3 (RIPK3) and mixed lineage kinase domain-like (MLKL), as well as mice lacking apoptotic caspase-8 in myeloid cells combined with RIPK3 loss, that RIPK3/caspase-8 signaling regulates macrophage inflammatory responses and drives adipose tissue inflammation and MAFLD upon high-fat diet feeding. In contrast, MLKL, divergent to RIPK3, contributes to both obesity and MAFLD in a manner largely independent of inflammation. We also uncover that MLKL regulates the expression of molecules involved in lipid uptake, transport, and metabolism, and congruent with this, we discover a shift in the hepatic lipidome upon MLKL deletion. Collectively, these findings highlight MLKL as an attractive therapeutic target to combat the growing obesity pandemic and metabolic disease.

Nonvesicular lipid transfer drives myelin growth in the central nervous system.

Oligodendrocytes extend numerous cellular processes that wrap multiple times around axons to generate lipid-rich myelin sheaths. Myelin biogenesis requires an enormously productive biosynthetic machinery for generating and delivering these large amounts of newly synthesized lipids. Yet, a complete understanding of this process remains elusive. Utilizing volume electron microscopy, we demonstrate that the oligodendroglial endoplasmic reticulum (ER) is enriched in developing myelin, extending into and making contact with the innermost myelin layer where growth occurs. We explore the possibility of transfer of lipids from the ER to myelin, and find that the glycolipid transfer protein (GLTP), implicated in nonvesicular lipid transport, is highly enriched in the growing myelin sheath. Mice with a specific knockout of Gltp in oligodendrocytes exhibit ER pathology, hypomyelination and a decrease in myelin glycolipid content. In summary, our results demonstrate a role for nonvesicular lipid transport in CNS myelin growth, revealing a cellular pathway in developmental myelination.

TMIC-52. DECODING METABOLIC INTERACTIONS IN GLIOBLASTOMA TUMOR MICROENVIRONMENT THROUGH COMPREHENSIVE SPATIAL TRANSCRIPTOMIC AND PROTEOMIC ANALYSIS

Abstract INTRODUCTION Within the GBM microenvironment, distinct micro-niches arise from independent cancer cell lineages with unique metabolic needs, characterized as Fast Cycling Cells (FCCs) and Slow Cycling Cells (SCCs). FCCs preferentially utilize aerobic glycolysis for energy, whereas treatment-resistant SCCs depend on lipid metabolism. Our study decodes the molecular and spatial heterogeneity within the GBM microenvironment, focusing on the dichotomy of immune infiltrates and the metabolic interactions between SCCs and the immune compartment in human GBM patients. METHODS We performed spatial transcriptomics and proteomics combined with geospatially resolved single-cell neighborhood analysis of IHC-stained GBM tissues using CellProfiler and the SNAQ algorithm that we developed to investigate the specific immune microenvironment of SCCs and FCCs. Holotomography and fluorescence-based time-lapse imaging coupled with flow cytometry were used to study lipid transfer between immune cells and GBM cells. Finally, the effects of pharmacological and genetic targeting of lipid transfer were investigated both in vitro and in vivo. RESULTS Our study establishes a connection between spatial metabolic heterogeneity and immune diversity, highlighting how specific tumor cell lineages correlate with characteristic immune micro-niches. In particular, SCCs exploit the metabolic properties of recruited immunosuppressive myeloid-derived cells, which metabolically support tumor cells by facilitating lipid transport and supporting SCC metabolism. Our results show that inhibiting lipid transfer affect SCCs, remodels the immune microenvironment, delays tumor growth and improves disease outcome. Our study also demonstrates a correlation between predicted sensitivity to lipid-modifying drugs, such as statins, and the SCC phenotype. This correlation could enable stratification and selection of patients most likely to benefit from such treatments. CONCLUSIONS This study establishes critical connections between the TME, cellular metabolism, anti-tumor immunity, and treatment vulnerabilities. Our research provides novel insights into how metabolic exchanges contribute to tumor progression and suggests that targeting metabolic interactions could be an effective therapeutic strategy for GBM.

Comparative analysis of gut microbiota in incident and prevalent peritoneal dialysis patients with peritoneal fibrosis, correlations with peritoneal equilibration test data in the peritoneal fibrosis cohort.

The connection between peritoneal function and gut microbiota in peritoneal fibrosis (PF) patients remains uncertain. Peritoneal equilibration test (PET) was employed to evaluate peritoneal function in patients with peritoneal fibrosis (PF). 16S rRNA sequencing was used to analyze the gut flora in incident peritoneal dialysis (PD) and PF groups, identifying differential microbial communities. Spearman's correlation analysis, conducted using SPSS 26.0, was employed to explore the microbial associations with PF. In the PF group, the PET showed a 4-h dialysate-to-plasma creatinine ratio (D/PCr) ratio of 0.62 ± 1.01 and a 4-h ultrafiltration (UF) volume of 41.73 ± 76.71 mL with a 2.5% glucose dwell. The alpha diversity between the PD and PF groups did not exhibit a significant difference. The PD and PF groups were predominantly composed of Firmicutes, Tenericutes, and Ignavibacteriae. Linear discriminant analysis effect size (LEfse) analysis indicates that the Firmicutes, Lactobacillales, and Bacilli were significantly enriched in the PD group, whereas Lachnospiraceae, Phenylobacterium, and Caulobacteraceae were enriched among the PF group. The functional prediction of gut microbiota genes revealed variations in metabolic pathways related to lipid transport, energy metabolism, and post-translational protein modifications between the two cohorts. In the PF group, Ignavibacteriae and Bdellovibrio correlated positively with the 4-h D/PCr ratio (p <0.05), while Prevotella negatively correlated with the 4-h UF from 2.5% glucose dwell (p <0.05). The intestinal microbiota of patients newly initiated on peritoneal dialysis significantly differs from that of those with long-term peritoneal dialysis complicated by peritoneal fibrosis, with the latter's peritoneal function potentially linked to Prevotella, Ignavibacteriae, and Bdellovibrio.

Adipocytes reprogram the proteome of breast cancer cells in organotypic three-dimensional cell cultures.

While epidemiological evidence has long linked obesity with an increased risk of breast cancer, the intricate interactions between adipocytes and cancer cells within the tumor microenvironment remain largely uncharted territory. The use of organotypic three-dimensional (3D) cell cultures that more accurately mimic the spatial architecture of tumors represents an innovative approach to this complex issue. In the present study, we investigated the effects of adipocytes on the proteome of Hs578t breast cancer cells cultured in a 3D microenvironment. Using different treatments, we rigorously optimized the experimental conditions to induce the optimal differentiation of 3T3-L1 fibroblasts into mature adipocytes. Then, we grow the Hs578t cells in a simulated microenvironment using an on-top model for organotypic 3D cultures. Our data showed that cancer cells formed 3D stellate-like architectures when grown over an extracellular matrix proteins-enriched scaffold for 48h. Proteomic profiling using LC-MS/MS mass spectrometry of Hs578t cells grown in 3D conditions with or without the adipocyte-enriched culture discovered 916 unique proteins. Of these, 605 showed no significant changes in abundance, whereas 87 proteins were significantly upregulated and 224 downregulated after interaction with fat cells (p < 0.05, FC > 2.0). Bioinformatic analysis of upregulated proteins indicated that the most enriched GO terms and molecular functions were related to lipids transport, cell differentiation, hypoxia response, and cell junctions. In addition, several modulated proteins have been previously associated with breast cancer progression. Interestingly, lipid transport proteins, including PITPNM2, ATP2C1, ABCA12, HDLBP, and APOB, showed perturbations in their expression, which were also associated with low overall survival in breast cancer patients. Functional studies showed that the knockdown of apolipoprotein B (APOB) expression in Hs578t cells reduced the size of 3D cellular structures. Moreover, APOB-knocked cells cocultured with adipocytes for 48h exhibited a significant decrease of intracellular lipids, whereas an increase in the adipocytes was found. Our results indicate that the 3D microenvironment and the adipocytes crosstalk reprogram the proteome of breast cancer cells. These data help us understand the environmental effects in gene expression and contribute to discovering novel tumor proteins with potential intervention in breast cancer therapy.

Genetic Polymorphisms in Obesity: How Variants in Lipid Metabolism Genes Contribute to Hyperlipidemia

Obesity is a complex metabolic disorder influenced by both environmental and genetic factors. Among the genetic contributors, polymorphisms in lipid metabolism genes play a significant role in regulating lipid profiles and promoting hyperlipidemia. This review aims to explore the impact of genetic variants on lipid metabolism in individuals with obesity, focusing on genes such as APOE, LPL, FTO, PCSK9, CETP, and FABP. These polymorphisms alter lipid processing, transportation, and storage, leading to aberrant lipid levels, which contribute to hyperlipidemia and subsequent cardiometabolic complications. We will also discuss the gene-environment interactions that modulate these genetic effects and review potential therapeutic interventions targeting lipid metabolism to manage hyperlipidemia in obese individuals. Keywords: Genetic polymorphisms, obesity, lipid metabolism, hyperlipidemia, gene-environment interactions, cardiometabolic disease

Integrative analyses of genetic mechanisms responsible for bone-fat imbalance in osteoporosis.

Osteoporosis manifests through adipocyte accrual and osteoblast diminution within bone marrow. However, the precise mechanisms driving the shift from osteogenesis to adipogenesis in bone marrow mesenchymal stem cells (BMSCs) remain largely undefined. In this study, we harnessed the power of bioinformatic tools to analyze gene expression patterns of BMSCs during adipogenic differentiation and osteoporosis using the data from Gene Expression Omnibus (GEO) repositories (GSE113253 and GSE35956), complemented by in vitro and in vivo experiments to validate the findings. Five distinct expression profiles of differentially expressed genes across the adipogenic timeline were identified. The initial phase is marked by ribosome biogenesis and rRNA processing, which is followed by the metabolism of organic acids and processing of inorganic ions. In contrast, the terminal phase is characterized by lipid transport, accumulation, and metabolism, alongside inorganic cation metabolism, thereby underscoring unique transcriptional signatures during the early and late stages of adipogenic differentiation. In BMSCs derived from osteoporotic samples, there is a notable decline in cellular proliferation and a diminished osteogenic capacity. Critically, the genes common to both adipogenesis and osteoporosis in BMSCs are predominantly involved in the negative regulation of Wnt signaling and cellular proliferation. Key genes including SOCS1, MYC, CEBPB, FYN, AXIN2, and RXRA are identified and show downregulation in BMSCs from aged mice. Subsequent in vitro experiments have validated the regulatory influence of RXRA on both adipogenic and osteogenic differentiations of BMSCs, highlighting its crucial role as a central modulator in bone formation and the pathophysiology of osteoporosis.

Serum Lipoprotein Profiling by NMR Spectroscopy Reveals Alterations in HDL-1 and HDL-2 Apo-A2 Subfractions in Alzheimer's Disease.

Identifying biomarkers for Alzheimer's disease (AD) is crucial, due to its complex pathology, which involves dysfunction in lipid transport, contributing to neuroinflammation, synaptic loss, and impaired amyloid-β clearance. Nuclear magnetic resonance (NMR) is able to quantify and stratify lipoproteins. The study investigated lipoproteins in blood from AD patients, aiming to evaluate their diagnostic potential. Serum and plasma were collected from AD patients (n = 25) and healthy individuals (n = 25). We conducted a comprehensive lipoprotein profiling on serum samples using NMR spectroscopy, analysing 112 lipoprotein subfractions. In plasma, we measured unspecific markers of neuronal damage and AD hallmark proteins using single molecule array technology. Additionally, clinical data and cerebrospinal fluid biomarker levels were also collected to enrich our data. Our findings, after adjusting for age and sex differences, highlight significant alterations in two specific lipoproteins; high-density lipoprotein (HDL)-1 Apo-A2 (H1A2) and HDL-2 Apo-A2 (H2A2), both with area under the curve (AUC) values of 0.67, 95% confidence interval (CI) = 0.52-0.82). These results indicate that these lipoprotein subfractions may have potential as indicators of AD-related metabolic changes.

ApoE: The Non-Protagonist Actor in Neurological Diseases

Background: Apolipoprotein E (APOE = gene, ApoE = protein) is a glycoprotein involved in the biological process of lipid transportation and metabolism, contributing to lipid homeostasis. APOE has been extensively studied for its correlation with neurodegenerative diseases, in particular Alzheimer’s disease (AD), where the possession of the epsilon 4 (E4) allele is established as a risk factor for developing AD in non-familiar sporadic forms. Recently, evidence suggests a broad involvement of E4 also in other neurological conditions, where it has been shown to be a predictive marker for worse clinical outcomes in Parkinson’s disease (PD), brain trauma, and disturbances of consciousness. The mechanisms underlying these associations are complex and involve amyloid-β (Aβ) peptide accumulation and neuroinflammation, although many others have yet to be identified. Objectives: The aim of this review is to overview the current knowledge on ApoE as a non-protagonist actor in processes underlying neurodegenerative diseases and its clinical significance in AD, PD, acquired brain trauma, and Disorders of Consciousness (DoC). Ethical implications of genetic testing for APOE variants and information disclosure will also be briefly discussed.

Transcriptome and targeted metabolome analysis of lipid profiles, nutrients compositions and volatile compounds in longissimus dorsi of different pig breeds.

Improving meat quality is important for commercial production and breeding. The molecular mechanism of intramuscular fat (IMF) deposition and meat characteristics remain further study. This study aimed to study the mechanism of IMF deposition and meat characteristics including redox potential, nutrients compositions and volatile compounds in longissimus dorsi (LD) by comparing with different pig breeds including Shanghai white (SW), Duroc x (Landrace Yorkshire) (DLY) and Laiwu (LW) pigs. Results showed that the contents of IMF, triglyceride (TG), total cholesterol (TC), and redox potential parameters were lower, while the content of MDA and activity of lactate dehydrogenase (LDH) were higher in LD of SW pigs compared with LW pigs (p<0.05). No differences were observed about these parameters between SW and DLY pigs. Also, the contents of medium-long chain fatty acids and γ-aminobutyric acid (GABA) were higher, while Asp was lower in LD of SW pigs compared with LW pigs (p<0.05). Volatile compounds results showed that 6 ketones, 4 alkenes, 11 alkanes, 2 aldehydes, 1 alcohol were increased and cholesterol was decreased in SW pigs compared with LW pigs. Transcriptome results showed that differential expressed genes involved in lipid synthesis, metabolism and transport in LD between SW and LW pigs, which were further verified by qPCR. Spearman correlation showed that HSL and Nedd4 were positively related to contents of TG and IMF, while negatively related to volatile compounds and fatty acids (p<0.05). Plin3 and Mgll were negatively related to contents of TG, IMF and cholesterol, while positively related to MDA, LDH, and volatile compounds (p<0.05). PPARA was negatively related to contents of TC and IMF, and activity of SOD, while positively related to volatile compounds (p<0.05). Our study provided new insights into potential mechanisms of IMF deposition, nutrients composition and volatile compounds of muscular tissues of different pig breeds.

Adipose tissue/ heart lipid transport determines cardiac diastolic function

Abstract Introduction Heart failure with preserved ejection fraction (HFpEF) is characterized by diastolic dysfunction and often occurring in obese individuals. Previous research has demonstrated that the modification of lipid metabolism can prevent the onset of diastolic dysfunction. In our project we have investigated how lipid transport influences the onset of diastolic dysfunction utilizing heart- and adipose tissue-specific knockdowns of the fatty acid (FA) transport protein 1 (FatP1) in Drosophila melanogaster. Methods We generated Drosophila with heart-specific (+/+; Hand4.2-GAL4/UAS-FatP1; tdtK/+) (cFatP1-KD) and adipose tissue-specific (+/+; ppl-GAL4/UAS-FatP1; tdtK/+) (atFat-P1-KD) FatP1 knockdowns using the UAS/GAL4 system. The flies' cardiomyocytes endogenously express tdTomato, facilitating fluorescence-based live imaging of the heart by high-resolution video fluorescence microscopy, as previously described. Cardiac morphology and cardiac lipid accumulation was monitored by confocal microscopy. Additionally, mitochondrial function was evaluated using the glycolytic Seahorse assay of whole hearts. Results Blocking FA-uptake into adipose tissue in atFatP1-KD flies resulted in a highly significant reduction in relaxation velocity (p=0.0009, WT vs. atFatP1-KD), indicative of diastolic dysfunction in flies, while contraction velocity and fractional shortening (systolic function) remained unchanged. Diastolic dysfunction was associated with a significant increase of cardiac lipid droplet (LD) quantity and size in flies lacking FatP1 in adipose tissue (p=0.0275 and p&amp;lt;0.0001, respectively, WT vs. atFatP1-KD). Cardiac mitochondrial function was significantly impaired in atFatP1-KD flies compared to WT flies. Reducing cardiac FA uptake in heart-specific FatP1 KD flies led to a decrease in end-diastolic diameter and a significant reduction in relaxation and contraction velocity (p&amp;lt;0.0001 and p=0.0001, respectively, WT vs. cFatP1-KD), suggesting impairment in both diastolic and systolic function. Interestingly, the number of cardiac LDs increased in cFATP1-KD, however their size decreased significantly resulting in an overall unchanged lipid content compared to WT flies (p=0.0475 and p=0.0367, respectively, WT vs. atFatP1-KD). Mitochondrial function showed no significant alterations. Conclusion This study demonstrates that reducing FA-uptake in adipose or cardiac tissue results in cardiac dysfunction in Drosophila melanogaster. Reduction of adipose tissue FA-uptake results exclusively in diastolic dysfunction likely mediated by cardiac lipid overload and subsequent mitochondrial dysfunction. In contrast, blockade of Fatp1-mediated cardiac FA-uptake induces diastolic/ systolic dysfunction which was unrelated to alterations in cardiac lipid content and mitochondrial dysfunction suggesting alternative mechanisms. These study identifies FatP1-mediated FA-uptake in adipose tissue as a potential therapeutic target for the treatment of diastolic dysfunction.

Intravenous administration of gold nanoparticles in rats exhibit alterations in sphingomyelins, bile acids, sphingolipids, and cholesterol esters levels

AbstractNanoparticles (NP) have gained significant attention in biomedical research due to their unique properties and potential applications in drug delivery, imaging, and diagnostics. Gold (AuNPs) and silver (AgNPs) NPs are among the important nanoplatforms that received extensive attention recently for various biomedical applications. Understanding the complex interaction of these NP in biological systems is essential to unveil their pharmacological, Pharmacokinetic and toxicological attributes. Metabolomics has proven invaluable in providing detailed insights into NP’s biodistribution, metabolic effects, and potential toxicity. This study aims to investigate the underlying metabolic pathways affected by in vivo exposure to NP using a robust metabolomics approach. In this work, spherical polyethylene glycol (PEG) modified AuNPs (13 nm, diameter) or AgNPs (20 nm, diameter) were synthesized and dosed into rats via intravenous route to study the associated metabolic changes. Rats (n = 14) were divided into three groups: control (n = 2), AuNPs (n = 6) and AgNPs (n = 6), to mimic potential biomedical exposure scenarios. Duplicate serum samples were collected 24 h post-dosing, and comprehensive metabolite profiling was performed using liquid chromatography-mass spectrometry (LC-MS/MS) and flow injection analysis-mass spectrometry (FIA-MS/MS). Metabolite extraction followed the MxP Quant 500 Kit protocol, with chromatographic separation using the Xevo TQS system. Metabolite identification and quantification were conducted with isotopically labelled internal standards and multiple reaction monitoring (MRM), utilizing optimized conditions under mass spectrometry (MS) as provided by Biocrates. Annotation of metabolites was determined by retention times and specific MRMs for each compound. Results indicate that AuNPs treatment significantly impacted several metabolic pathways. Notably, there was an increase in sphingomyelin (SM 34:2) levels (estimate 0.23, p ≤ 0.001), which are critical for cell membrane structure and signalling. Additionally, a decrease in glycochenodeoxycholic acid levels was also triggered by treatment with AuNPs, suggesting modulation of bile acid metabolism with potential effects on lipid homeostasis. Furthermore, treatment with AuNPs caused significant alterations in cholesterol ester levels, essential for lipid storage and transport, indicating disruptions in these mechanisms. These metabolic changes suggest that gold nanoparticles can disrupt fatty acid metabolism, pyrimidine/purine metabolism, and amino acid synthesis. These findings contribute to the growing understanding of nanoparticle toxicity profile and underscore the need for further research to ensure the safe application of nanoparticles in biomedical and other fields.

RNA-seq and whole-genome re-sequencing reveal Micropterus salmoides growth-linked gene and selection signatures under carbohydrate-rich diet and varying temperature

This study was performed on Micropterus salmoides to determine growth-linked gene and single nucleotide polymorphism (SNP) markers under carbohydrate diet and varying temperature through RNA-seq and whole-genome re-sequencing. The results showed that growth-related genes were primarily enriched in the fat digestion and absorption signaling pathway, playing a role in lipid transport and metabolism. Fatty acid binding protein 6, bile salt-activated lipase-like, lysophosphatidylcholine acyltransferase 2, phospholipase A2, minor isoenzyme-like, and phospholipase A2, group IB (pancreas) were identified as the crucial genes. The differentially expressed genes between high and low temperatures were enriched in the pentose phosphate pathway, with carbohydrate transport and metabolism were most affected by temperature. Major facilitator superfamily domain containing 10, phosphogluconate dehydrogenase, fructose-1,6-bisphosphatase 1a, and spinster homolog 3 transcript variant X1(spns3) were the shared genes affected by temperature. From all the common genes, 10 growth-associated SNP markers were identified. The TT genotype at rs7781 was associated with lower body weight, while AA genotype at rs31434173 and CC genotype at rs31435313 showed positive correlation with body weight. Analogously, the GG genotype at rs31436887 and AA genotype at rs31438769 also found to characterize better growth performance. At low temperature, individuals with the AA genotype at rs11506587 and rs31435313 exhibited the slowest growth. For the genotypes labeled with rs11510589, the GG individual grew faster than the AA individual, whereas the opposite phenomenon occurred in these genotypes when labeled with rs11511314. The genotypes at rs32970704 and rs32967921 showed no growth correlation. The AA genotype at rs31435313 in spns3 had the slowest growth under a carbohydrate-rich diet regardless of the temperature. Our study presents candidate genes and SNP markers associated with growth influenced by carbohydrate and temperature, providing basis for the development of M. salmoides strain that better accepts carbohydrate diets at varying temperature.

Structure and function of Mycobacterium tuberculosis EfpA as a lipid transporter and its inhibition by BRD-8000.3

EfpA, the first major facilitator superfamily (MFS) protein identified in Mycobacterium tuberculosis (Mtb), is an essential efflux pump implicated in resistance to multiple drugs. EfpA-inhibitors have been developed to kill drug-tolerant Mtb. However, the biological function of EfpA has not yet been elucidated. Here, we present the cryo-EM structures of EfpA complexed with lipids or the inhibitor BRD-8000.3 at resolutions of 2.9 Å and 3.4 Å, respectively. Unexpectedly, EfpA forms an antiparallel dimer. Functional studies reveal that EfpA is a lipid transporter and BRD-8000.3 inhibits its lipid transport activity. Intriguingly, the mutation V319F, known to confer resistance to BRD-8000.3, alters the expression level and oligomeric state of EfpA. Based on our results and the observation of other antiparallel dimers in the MFS family, we propose an antiparallel-function model of EfpA. Collectively, our work provides structural and functional insights into EfpA's role in lipid transport and drug resistance, which would accelerate the development of antibiotics against this promising drug target.

The Importance of Gut Microbiota on Choline Metabolism in Neurodegenerative Diseases

The gut microbiota is a complex ecosystem that influences digestion, immune response, metabolism, and has been linked to health and well-being. Choline is essential for neurotransmitters, lipid transport, cell-membrane signaling, methyl-group metabolism and is believed to have neuroprotective properties. It is found in two forms, water-soluble and lipid-soluble, and its metabolism is different. Long-term choline deficiency is associated with many diseases, and supplements are prescribed for improved health. Choline supplements can improve cognitive function in adults but not significantly. Choline is a precursor of phospholipids and an acetylcholine neurotransmitter precursor and can be generated de novo from phosphatidylcholine via phosphatidylethanolamine-N-methyltransferase and choline oxidase. Choline supplementation has been found to have a beneficial effect on patients with neurodegenerative diseases, such as Alzheimer’s disease (AD), by increasing amyloid-β, thioflavin S, and tau hyper-phosphorylation. Choline supplementation has been shown to reduce amyloid-plaque load and develop spatial memory in an APP/PS1 mice model of AD. Choline is necessary for normative and improved function of brain pathways and can reduce amyloid-β deposition and microgliosis. Clinical research suggests that early neurodegenerative diseases (NDs) can benefit from a combination of choline supplements and the drugs currently used to treat NDs in order to improve memory performance and synaptic functioning.

Beneficial effects of AMP Scy-hepc on the gut microbiota contribute to the potential growth of Larimichthys crocea

BackgroundIn light of the global ban on antibiotics in animal feed, antimicrobial peptides (AMPs) have emerged as a compelling substitute, garnering considerable interest for their potential as feed additives. Our previous study revealed that the extended (one-year) daily administration of the AMP Scy-hepc substantially boosted the growth of Larimichthys crocea. However, the exact influence of dietary supplementation with AMPs on the gut microbiota and the potential beneficial mechanisms remain unclear. Inspired by this, the present study endeavors to examine the alterations in gut microbiota at various gut sites in L. crocea following a 60-day Scy-hepc feeding regimen, building upon our prior research efforts.ResultsUtilizing 16S rRNA sequencing, we found that dietary supplementation with Scy-hepc significantly promoted the growth of L. crocea, which may be caused by the remarkable changes in the microbial communities within the foregut and midgut. Notable changes were observed in Tenericutes, Firmicutes, Proteobacteria, Cyanobacteria and Spirochaetes. The bacterial load trends in both the foregut and midgut demonstrated a notable increase following a 60-day Scy-hepc feeding, as determined by absolute quantitative PCR analysis. Moreover, Scy-hepc supplementation increased the abundance of potential probiotics (Rhodobiaceae and Planococcaceae) and reduced the abundance of opportunistic pathogens (Flavobacteriia and Mollicutes). This led to a more intricate microbial network with enhanced metabolism-related functions, especially in lipid transport and metabolism, signal transduction mechanisms, and coenzyme transport and metabolism. And the microbial resistance (Rs) exhibits no significant change between the Scy-hepc and control groups, indicating a minimal level of toxicity to the gut microbiota of L. crocea.ConclusionsIn summary, this study provides compelling evidences supporting the beneficial alteration of gut microbiota in the foregut and midgut as an underlying mechanism by which Scy-hepc feeding promotes host growth. These findings offer a novel perspective for investigating the advantageous effects of AMPs on fish health and the advancement of aquaculture.