High-throughput Analysis Research Articles

PD-1 interactome in osteosarcoma: identification of a novel PD-1/AXL interaction conserved between humans and dogs

The PD-1/PDL-1 immune checkpoint inhibitors revolutionized cancer treatment, yet osteosarcoma remains a therapeutic challenge. In some types of cancer, PD-1 receptor is not solely expressed by immune cells but also by cancer cells, acting either as a tumor suppressor or promoter. While well-characterized in immune cells, little is known about the role and interactome of the PD-1 pathway in cancer. We investigated PD-1 expression in human osteosarcoma cells and studied PD-1 protein–protein interactions in cancer. Using U2OS cells as a model, we confirmed PD-1 expression by western blotting and characterized its intracellular as well as surface localization through flow cytometry and immunofluorescence. High-throughput analysis of PD-1 interacting proteins was performed using a pull-down assay and quantitative mass spectrometry proteomic analysis. For validation and molecular modeling, we selected tyrosine kinase receptor AXL—a recently reported cancer therapeutic target. We confirmed the PD-1/AXL interaction by immunoblotting and proximity ligation assay (PLA). Molecular dynamics (MD) simulations uncovered binding affinities and domain-specific interactions between extracellular (ECD) and intracellular (ICD) domains of PD-1 and AXL. ECD complexes exhibited strong binding affinity, further increasing for the ICD complexes, emphasizing the role of ICDs in the interaction. PD-1 phosphorylation mutant variants (Y223F and Y248F) did not disrupt the interaction but displayed varying strengths and binding affinities. Using bemcentinib, a selective AXL inhibitor, we observed reduced binding affinity in the PD-1/AXL interaction, although it was not abrogated. To facilitate the future translation of this finding into clinical application, we sought to validate the interaction in canine osteosarcoma. Osteosarcoma spontaneously occurs at significantly higher frequency in dogs and shares close genetic and pathological similarities with humans. We confirmed endogenous expression of PD-1 and AXL in canine osteosarcoma cells, with PD-1/AXL interaction preserved in the dog cells. Also, the interacting residues remain conserved in both species, indicating an important biological function of the interaction. Our study shed light on the molecular basis of the PD-1/AXL interaction with the implication for its conservation across species, providing a foundation for future research aimed at improving immunotherapy strategies and developing novel therapeutic approaches.

IFlpMosaics enable the multispectral barcoding and high-throughput comparative analysis of mutant and wild-type cells.

To understand gene function, it is necessary to compare cells carrying the mutated target gene with normal cells. In most biomedical studies, the cells being compared are in different mutant and control animals and, therefore, do not experience the same epigenetic changes and tissue microenvironment. The experimental induction of genetic mosaics is essential to determine a gene cell-autonomous function and to model the etiology of diseases caused by somatic mutations. Current technologies used to induce genetic mosaics in mice lack either accuracy, throughput or barcoding diversity. Here we present the iFlpMosaics toolkit comprising a large set of new genetic tools and mouse lines that enable recombinase-dependent ratiometric induction and single-cell clonal tracking of multiple fluorescently labeled wild-type and Cre-mutant cells within the same time window and tissue microenvironment. The labeled cells can be profiled by multispectral imaging or by fluorescence-activated flow cytometry and single-cell RNA sequencing. iFlpMosaics facilitate the induction and analysis of genetic mosaics in any quiescent or progenitor cell, and for any given single or combination of floxed genes, thus enabling a more accurate understanding of how induced genetic mutations affect the biology of single cells during tissue development, homeostasis and disease.

Design and implementation of a scalable and high-throughput EEG acquisition and analysis system

Recent advances in neuroscience, neuromorphic intelligence, and brain–computer interface (BCI) technologies have created a need for fast, efficient, and convenient electroencephalogram (EEG) data acquisition systems. However, the existing equipment was limited in its flexibility, restricting non-invasive studies to research or medical settings. To address this issue, low-cost, compact EEG acquisition devices have been developed, allowing for frequent and flexible brain data acquisition in various scenarios. This paper introduces a scalable and high-throughput EEG signal acquisition and analysis system based on field-programmable gate array (FPGA) technology. The proposed system offers electrode scalability, on-chip computing, and optional wireless functionality extension. These features are achieved through the design of a highly scalable underlying EEG acquisition module and an FPGA central module that enables software-defined high-throughput expansion and high-speed data exchange between software and hardware. The paper presents two implementation cases that demonstrate the potential of the proposed system. The first case introduces a wearable wireless EEG system, enabling the deployment of effective and user-friendly steady-state visual evoked potential (SSVEP)-BCI applications in consumer-grade scenarios. The second case integrates an FPGA central module with multiple basic EEG acquisition modules to construct a high-throughput BCI system for cost-effective and real-time EEG data acquisition and processing. This configuration allows for flexible deployment in research and clinical applications, including attention index, SSVEP, motor imagery (MI), and emotion recognition. This combination further demonstrates the potential of scalable EEG systems and emphasizes the need for further integration or chipization. These implementations validate the feasibility of compact and efficient EEG devices and highlight the promising applications of scalable BCI system in various fields.

Development and application of a LC-HRMS method for simultaneous determination of ten illicit antifungal drugs in herbal bacteriostatic products.

The widespread use of sub-therapeutic antibiotics may lead to treatment failures, increased resistance to antibiotics, and even the encouragement of the formation of superbugs. Fraudulent herbal medicines that actually contain unlabeled active ingredients are a global problem. Therefore, streamlined and accurate analytical techniques that can identify and quantify a variety of antimicrobials in suspected illegal products are required. In the present work, we developed a sensitive, robust, and accurate method that involves multi-step extraction, EMR-lipid-based pass-through cleanup, and UHPLC-Q/HRMS with an orbital ion trap instrument analysis for the simultaneous detection of ten illicit antifungal drugs (flucytosine, fluconazole, ketoconazole, naftifine hydrochloride, griseofulvin, bifonazole, econazole nitrate, elubiol, clotrimazole, and miconazole nitrate) in herbal disinfection products. This validated method was then applied to 210 authentic samples, resulting in 25% of the samples being positive for the drugs. The results suggested the need to strengthen controls in this field to detect illicit antifungal drugs in herbal bacteriostatic products, which represents a potential health risk for the consumer. On a global scale, more and more frequent routine monitoring is being carried out in this area. This study provides valuable insights into large-scale complex sample chemical profiling, and the method developed appears to be promising and applicable for high-throughput routine multiple antibiotic analysis in a complex sample matrix.

High-Throughput Single-Cell Analysis of Local Nascent Protein Deposition in 3D Microenvironments via Extracellular Protein Identification Cytometry (EPIC).

Extracellular matrix (ECM) guides cell behavior and tissue fate. Cell populations are notoriously heterogeneous leading to large variations in cell behavior at the single-cell level. Although insights into population heterogeneity are valuable for fundamental biology, regenerative medicine, and drug testing, current ECM analysis techniques only provide either averaged population-level data or single-cell data from a limited number of cells. Here, extracellular protein identification cytometry (EPIC) is presented as a novel platform technology that enables high-throughput measurements of local nascent protein deposition at single-cell level. Specifically, human primary chondrocytes are microfluidically encapsulated in enzymatically crosslinked microgels of 16 picoliter at kHz rates, forming large libraries of discrete 3D single-cell microniches in which ECM can be deposited. ECM proteins are labeled using fluorescence immunostaining to allow for nondestructive analysis via flow cytometry. This approach reveals population heterogeneity in matrix deposition at unprecedented throughput, allowing for the identification and fluorescent activated cell sorting-mediated isolation of cellular subpopulations. Additionally, it is demonstrated that inclusion of a second cell into microgels allows for studying the effect of cell-cell contact on matrix deposition. In summary, EPIC enables high-throughput single-cell analysis of nascent proteins in 3D microenvironments, which is anticipated to advance fundamental knowledge and tissue engineering applications.

Reprogramming of iPSCs to NPCEC-like cells by biomimetic scaffolds for zonular fiber reconstruction

Ectopia lentis (EL), characterised by impaired zonular fibers originating from non-pigmented ciliary epithelial cells (NPCEC), presents formidable surgical complexities and potential risks of visual impairment. Cataract surgery is the only treatment method for EL, but it leads to the loss of accommodative power of the lens post-operatively. Furthermore, the challenge of repairing zonular ligaments in situ remains a significant global issue. Ocular tissue and aqueous humour samples from patients with EL were subjected to RNA sequencing and Olink high-throughput proteomic analysis, revealing the downregulation of pathogenic genes (FBN1, MFAP2) and upregulation of secretory proteins (IL-12, MMP-1). The high expression of FBN1 and MFAP2 in NPCECs suggests their potential as candidates for zonular fiber construction; however, the limited availability of donor sources restricts the feasibility of NPCEC transplantation therapy. The reprogramming and directional differentiation of induced pluripotent stem cells (iPSC) to NPCEC was successfully achieved using the developed biomimetic scaffolds that mimic the microstructures of natural radial zonular fibers. Excitingly, the single injection of induced NPCEC-like cells significantly contributed to restoring and enhancing mechanical properties in zonular fiber structures in a rabbit model with EL. This proposed in situ iPSC-based regeneration technique might serve as an innovative therapeutic strategy for clinical EL patients, reduce the cataract surgery rate, and retain the adjustment capacity of inherent lentis.

NMR metabolomics as a complementary tool to brix-acid tests for navel orange quality control of long-term cold storage.

Quality control plays a crucial role in maintaining the reputation of agricultural organizations by ensuring that their products meet the expected standards and preventing any loss during the packaging process. A significant responsibility of quality control is conducting periodic product assessments. However, subjective interpretation during physical inspections of fruits can lead to variability in reporting. To counter this, assessing total soluble solids (Brix) and percent acidity (Acid) can provide a more objective approach. Nevertheless, it is essential to note that many fruit metabolites can impact these parameters. Nuclear magnetic resonance (NMR) spectroscopy, particularly 1H-NMR, has become a popular tool for quality control in recent years due to its precision, sample preservation, and high throughput analysis. This manuscript investigates if the standard Brix/Acid tests are directly related to the levels of metabolites during cold storage. Using citrus as the model system, a metabolomics analysis was conducted to identify patterns in the cold storage metabolite profiles of the juice, albedo, and flavedo tissues. The results show that Brix (or total dissolved solids) correlates well with sucrose, glucose, and fructose levels and moderately with choline levels. Acid(percent acidity) levels displayed a negative correlation with both fructose and choline levels. Interestingly, the formate levels were susceptible to storage time and directly related to Acid measurements. This study suggests metabolomics could be a complementary technique to quality control of fruits in cold storage, especially with cost-effective desktop NMR spectrometers.

Proteomics analysis in rats reveals convergent mechanisms between major depressive disorder and dietary zinc deficiency

Abstract Background Preclinical and clinical studies have shown that dietary zinc deficiency can lead to symptoms similar to those observed in major depressive disorder (MDD). However, the underlying molecular mechanisms remain unclear. To investigate these mechanisms, we examined proteomic changes in the prefrontal cortex (PFC) and hippocampus (HP) of rats, two critical brain regions implicated in the pathophysiology of depression. Methods Rats were fed diets either adequate in zinc (ZnA, 50 mg Zn/kg) or deficient in zinc (ZnD, <3 mg/kg) for four weeks. High-throughput proteomic analysis was used to detect changes in protein expression, supplemented by enzyme activity assay for mitochondrial complexes I and IV, examining their functional impacts. Results ZnD led to significant alterations in protein expression related to zinc transport and mitochondrial function. Proteomic analysis revealed changes in zinc transporter family members such as Slc30a1 (6.64 log2FC), Slc30a3 (-2.32 log2FC), Slc30a4 (2.87 log2FC), Slc30a5 (5.90 log2FC), Slc30a6 (1.50 log2FC), and Slc30a7 (2.17 log2FC) in the PFC, and Slc30a3 (-1.02 log2FC), Slc30a5 (-1.04 log2FC), and Slc30a7 (1.08 log2FC) in the HP of rats subjected to ZnD. Furthermore, ZnD significantly affected essential mitochondrial activity proteins, including Atp5pb (3.25 log2FC), Cox2 (2.28 log2FC), Atp5me (2.04 log2FC), Cyc1 (2.30 log2FC), Cox4i1 (1.23 log2FC), Cox7c (1.63 log2FC), and Cisd1 (1.55 log2FC), with a pronounced decrease in complex I activity in the PFC. Conclusions Our study demonstrates that ZnD leads to significant proteomic changes in the PFC and HP of rats. Specifically, ZnD alters the expression of zinc transporter proteins and proteins critical for mitochondrial function. The significant decrease in complex I activity in the PFC further underscores the impact of ZnD on mitochondrial function. These results highlight the molecular mechanisms by which ZnD can influence brain function and contribute to symptoms similar to those observed in depression.

Alteration of Immune Function and Gut Microbiome Composition in Carassius auratus Challenged With Rahnella aquatilis.

Rahnella aquatilis as an emerging pathogen can cause bacterial enteritis in cyprinid fish such as crucian carp Carassius auratus. Currently, the characterisation of immune function and gut microbiome composition in C. auratus orally challenged by R. aquatilis were yet unknown. In this study, we therefore investigated the changes of histopathology, white blood cells (i.e., LEU, NEU and LYM), serum biochemical indicators (e.g., CRE, CK and CHO) and digestive enzyme activity (e.g., LYS, AST, ALT, GSH-Px and AKP), as well as complements and immune-related genes (e.g., C3, F2, LysC, TLR3, MyD88, TGF-β, TNF-α and IL-15) that were significantly altered after the oral administration of R. aquatilis KCL-5. Moreover, the gut microbiome composition and diversity were analysed by using 16S rRNA gene high-throughput sequencing analysis. The correlation analysis showed that the high abundance of phylum Proteobacteria, Actinobacteria, Firmicutes and Fusobacteria was related to the pathogenesis of enteritis caused by oral infection. KEGG enrichment analysis indicated that fatty acid, carbon and pyruvate metabolism were significantly increased pathways (p < 0.05). To our best knowledge, this is a rare report of physicochemical properties and gut microbiome in C. auratus by R. aquatilis infection, which will provide a scientific reference for the clinical diagnosis and prevention of bacterial enteritis in cyprinid fish.

Identification and validation of lung adenocarcinoma (LUAD)-associated targets for monascin from the extracts of Monascus purpureus-fermented rice: Compound preparation, high-throughput genome sequencing and bioinformatics analysis

Despite the remarkable bioactivities exhibited by monascin against several tumors, its therapeutic targets remain unexplored. In this study, our objective is to identify therapeutic targets based on the antitumor activity of monascin against lung adenocarcinoma (LUAD) A549 cells. The compound monascin was derived from extracts of Monascus purpureus-fermented rice. Its chemical structure was determined using spectroscopic methods, while the physicochemical properties of monascin were investigated through thermal behavior analysis. Herein, antitumor studies indicated that monascin significantly inhibited the viability of A549 cells, with an IC50 value of 2.05 μM; RNA-sequencing revealed 12 up-regulated and 29 down-regulated genes as differentially expressed genes (DEGs) for A549 cells in response to monascin; RT-qPCR validation supported the plausibility of the RNA-sequencing results. Moreover, utilizing data from the Cancer Genome Atlas (TCGA) database, univariate and multivariate Cox regression analyses provided evidence supporting GPR37 and FAM83A as potential candidate genes for predicting disease progression in LUAD patients. Additionally, the LASSO Cox regression and nomogram analyses confirmed that FAM83A and GPR37 genes had the potential to predict overall survival for LUAD patients. Finally, molecular docking studies suggested that monascin could interact with both GPR37 and FAM83A proteins through the hydrogen bonding and hydrophobic interactions; Western blotting assays indicated that monascin inhibited the expression levels of both GPR37 and FAM83A proteins. Overall, the clinical prognosis of GPR37 and FAM83A highlights the rationale for targeting these specific genes with monascin. Additionally, there findings provide compelling evidence for the ploypharmacological properties of monascin against LUAD.

Colonization of phthalate-degrading endophytic bacterial consortium altered bacterial community and enzyme activity in plants

Phthalates (PAEs) are widely distributed hazardous organic compounds that pose threats to ecosystems and human health. Endophytic bacteria can effectively eliminate PAEs contamination risk. However, limited information is available regarding the impact of endophytic bacterial colonization on bacterial communities within plants. In this study, the endophytic bacterial consortium EN was colonized in lettuce by seed soaking, root irrigation, leaf spraying, and combined spraying-irrigation, resulting in a marked improvement in plant growth. The findings revealed that consortium EN colonization through combined spraying-irrigation exhibited superior degradation capability with 40.54% PAEs removal from soil. Meanwhile, the residual PAEs in lettuce decreased by 94.05% compared with the uninoculated treatment. High-throughput sequencing analysis indicated that colonization of consortium EN altered the bacterial community in lettuce. Specifically, the relative abundance of the dominant genus Pseudomonas was significantly higher than that in the uninoculated control (P < 0.01). Additionally, colonization enhanced the activities of peroxidase and catalase in lettuce, thereby improving plant resistance. This work offers a theoretical foundation for comprehending the mechanism underlying the bioremediation of PAEs contamination by endophytic bacteria in soil-plant system.

Comprehensive lipid profile analysis of three fish roe by untargeted lipidomics using ultraperformance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry

Fish roe is a significant nutrition source from marine byproducts, containing rich contents of long-chain omega-3 fatty acids (n 3-LC PUFAs). However, investigations focused on the nutritional values and lipid composition of various fish roe species are limited. This research aims to perform a high-throughput lipidomics analysis to comprehensively evaluate and compare the lipid profiles of three consumed marine fish roe using ultraperformance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF-MS/MS). Results showed notable differences in three fish roe species in terms of sphingolipids (SLs), phospholipids (PLs), and glycerolipids (GLs) compositions. Furthermore, the high percentages of n-3 PUFA-PLs in yellow croaker and mackerel roe indicate their significant nutritional value. In total, 449 lipid molecular species of 13 lipid subclasses were identified; of them, 31 lipid molecular species could serve as potential biomarkers for the differentiation of three fish roe. This study provides comprehensive lipid profiles of three fish roe, demonstrating their potential values in functional food industries.

Study on the relationship between microbial community succession and physicochemical factors in the fermentation of rice-flavor Baijiu based on high-throughput and redundancy analysis techniques

Fermented grains are the main carrier of microbial fermentation and the direct source of Baijiu flavor substances, but so far, there are few studies on the changes in the fermented grains of rice-flavor Baijiu. This experiment systematically analyzed the correlation between microbial community succession and physicochemical factors in the fermentation process of two different types of rice (japonica: M and indica: H) fermented grains. The results showed that during the fermentation process, the changes in total acid, pH, starch, and total phenols of H and M were consistent. However, M and H showed different trends in terms of reducing sugar content and alcohol content. At the phylum level, both types of fermented grains had Firmicutes and Proteobacteria as the absolute dominant phyla. At the genus level, there was a slight difference in the dominant genera of M and H in the early stages, but the main genera in the later stages of fermentation were different. RDA analyses showed different correlations between dominant genera and physicochemical factors with some interesting results. The results of this study will provide a theoretical basis for the regulation of rice-flavor Baijiu fermentation.

Distinct microbial communities associated with health-relevant wild berries.

Lingonberries (Vaccinium vitis-idaea L.), rowanberries (Sorbus aucuparia L.) and rosehips (Rosa canina L.) positively affect human health due to their healing properties, determined by a high content of bioactive compounds. The consumption of unprocessed wild berries is relevant and encouraged, making their in-depth microbiological characterization essential for food safety. This study presents the first high-throughput sequencing analysis of bacterial and fungal communities distributed on the surface of lingonberries, rowanberries and rosehips. Significant plant-defined differences in the taxonomic composition of prokaryotic and eukaryotic microbiota were observed. The bacterial community on rosehips was shown to be prevalent by Enterobacteriaceae, lingonberries by Methylobacteriaceae and rowanberries by Sphingomonadaceae representatives. Among the fungal microbiota, Dothioraceae dominated on rosehips and Exobasidiaceae on lingonberries; meanwhile, rowanberries were inhabited by a similar level of a broad spectrum of fungal families. Cultivable yeast profiling revealed that lingonberries were distinguished by the lowest amount and most distinct yeast populations. Potentially pathogenic to humans or plants, as well as beneficial and relevant biocontrol microorganisms, were identified on tested berries. The combination of metagenomics and a cultivation-based approach highlighted the wild berries-associated microbial communities and contributed to uncovering their potential in plant health, food and human safety.

RXRα/MR signaling promotes diabetic kidney disease by facilitating renal tubular epithelial cells senescence and metabolic reprogramming

Cell senescence and metabolic reprogramming are significant features of diabetic kidney disease (DKD). However, the underlying mechanisms between cell senescence and metabolic reprogramming are poorly defined. Here, we report that retinoid X receptor α (RXRα), a key nuclear receptor transcription factor, regulates cell senescence and metabolic reprogramming in DKD. Through high-throughput sequencing, bioinformatic analysis and experimental validation, we confirmed the critical role of RXRα in promoting cell senescence and metabolic dysregulation in renal tubular epithelial cells (RTECs) induced by lipid overload. In vivo, in situ injection of AAV9-shRxra into the kidney reduced proteinuria, RTECs senescence and insulin resistance in DKD mice. In vitro, knockdown of RXRα markedly improved G2/M phase arrest and suppressed the expression of senescence-associated secretory phenotypes (SASPs). Protein-protein interaction (PPI) analysis and unbiased bioinformatics were employed to identify the direct interactions between RXRα and the mineralocorticoid receptor (MR), which were subsequently validated through coimmunoprecipitation. Gene network analysis revealed the collaborative regulatory role of RXRα and MR in RTECs senescence. In an accelerated aging mouse model, treatment with a MR antagonist has been shown to inhibite the RXRα/MR signaling, improve RTECs senescence, and reduce interstitial fibrosis and lipid deposition in the kidneys. These findings indicate that inhibition of RXRα/MR signaling could alleviate cell senescence during metabolic disorders. Thus, our study revealed that RXRα/MR signaling serves as a critical regulatory factor mediating the crosstalk between cell senescence and metabolic reprogramming, shedding light on a novel mechanism for targeting cell senescence and metabolic dysregulation in DKD.

A Diverse Vibrio Community in Ría de Vigo (Northwestern Spain)

The genus Vibrio is genetically and ecologically diverse, resulting in severe economic losses in aquacultural macroalgae and animals. Studies on vibrios will contribute to the conservation of aquatic aquaculture in Ría de Vigo (Spain), which is famous for its shellfish farming activity. However, limited research focused on the diversity and distribution of Vibrio spp. in Ría de Vigo has been reported. Here, a slight increase in the abundance of Vibrio spp. was recorded from inshore to the open sea by quantitative PCR (qPCR). Vibrios were more abundant in the free-living (FL) than in the particle-associated (PA) fraction, showing that FL might be their preferential lifestyle. Photobacterium piscicola, Vibrio japonicus and Vibrio harveyi, which are serious pathogens for fish and invertebrates in mariculture, were found to be the dominant species across all samples by high-throughput sequencing and analyses and were mainly affected by stochastic processes. More colony-forming units and species of vibrios were recorded at 16 °C (18 species) than at 28 °C (6 species), and five isolates (5.05% of the total isolates) showed low 16S rRNA gene similarity (&lt;98.65%) to the known strains. The results indicate that culture conditions closer to the in situ temperature may help to isolate more Vibrio species. Our findings may contribute to local aquatic aquaculture research, providing background information and novel insights for management and pathogen prevention in aquaculture, and more studies should focus on Ría de Vigo to acquire novel or pathogenic microbes in the future.

Chromosome-level genome assembly of the Suminoe oyster Crassostrea ariakensis in south China

The Suminoe oyster Crassostrea ariakensis (Fujita, 1913) is one of the most important ecological and fishery bivalve mollusks with a worldwide distribution. Here, we reported an improved high-quality chromosomal-level genome assembly of C. ariakensis inhabiting the South China Sea, using Nanopore technology, Illumina sequencing, and high-throughput chromosomal conformation capture analysis. The assembled genome size is 631.73 Mb, with contig N50 length of 5.36 Mb and scaffold N50 length of 61.15 Mb, and is assigned to 10 chromosomes. A total of 29,357 protein-coding genes are predicted, 96.68% of which are functionally annotated. The genome contains 347.11 Mb (54.94%) of repetitive elements and 1130 noncoding RNAs. This improved genome assembly of south C. ariakensis is an important resource for understanding oyster diversity and evolution, and provides insights into genetic improvement, protection and management of oyster resource.

Urinary metabolomics provide insights into coronary artery disease in individuals with type 1 diabetes.

Type 1 diabetes increases the risk of coronary artery disease (CAD). High-throughput metabolomics may be utilized to identify metabolites associated with disease, thus, providing insight into disease pathophysiology, and serving as predictive markers in clinical practice. Urine is less tightly regulated than blood, and therefore, may enable earlier discovery of disease-associated markers. We studied urine metabolomics in relation to incident CAD in individuals with type 1 diabetes. We prospectively studied CAD in 2501 adults with type 1 diabetes from the Finnish Diabetic Nephropathy Study. 209 participants experienced incident CAD within the 10-year follow-up. We analyzed the baseline urine samples with a high-throughput targeted urine metabolomics platform, which yielded 54 metabolites. With the data, we performed metabolome-wide survival analyses, correlation network analyses, and metabolomic state profiling for prediction of incident CAD. Urinary 3-hydroxyisobutyrate was associated with decreased 10-year incident CAD, which according to the network analysis, likely reflects younger age and improved kidney function. Urinary xanthosine was associated with 10-year incident CAD. In the network analysis, xanthosine correlated with baseline urinary allantoin, which is a marker of oxidative stress. In addition, urinary trans-aconitate and 4-deoxythreonate were associated with decreased 5-year incident CAD. Metabolomic state profiling supported the usage of CAD-associated urinary metabolites to improve prediction accuracy, especially during shorter follow-up. Furthermore, urinary trans-aconitate and 4-deoxythreonate were associated with decreased 5-year incident CAD. The network analysis further suggested glomerular filtration rate to influence the urinary metabolome differently between individuals with and without future CAD. We have performed the first high-throughput urinary metabolomics analysis on CAD in individuals with type 1 diabetes and found xanthosine, 3-hydroxyisobutyrate, trans-aconitate, and 4-deoxythreonate to be associated with incident CAD. In addition, metabolomic state profiling improved prediction of incident CAD.

High-throughput metagenomics analysis exposes the presence of gladiolus mosaic virus and a novel Potyvirus from Iris versicolor showing mosaic symptoms

High-throughput metagenomics analysis exposes the presence of gladiolus mosaic virus and a novel Potyvirus from Iris versicolor showing mosaic symptoms

Large-Volume Injection and Assessment of Reference Standards for n-Alkane δD and δ13C Analysis via Gas Chromatography Isotope Ratio Mass Spectrometry.

Compound-specific stable isotope analysis of hydrogen (δD) and carbon (δ13C) in organic compounds is a valuable tool in biogeochemical research. A key limitation of this method is the relatively large amount of sample required to achieve desirable precision. We developed a large-volume (20 μL) injection method that allows for high throughput analysis of less concentrated samples and tested it for δ13C and δD measurements of n-alkanes. We also conducted a comparison of reference standards and assessed several methods to normalize and correct n-alkane δD and δ13C measurements. The mean precision of the δD method based on 233 environmental n-alkane samples (two to three replications per sample) is 4.0‰ (1σ, estimated from the weighted mean of the pooled unbiased standard deviations) and 0.46‰ (1σ) for δ13C from 37 environmental samples (two to three replications per sample). The evaluation of reference standards shows that the use of n-alkane standards with large offsets in δD values in adjacent n-alkane chains can lead to biases in measurement correction. The large-volume injection method shows good reproducibility of δ13C and δD measurements of n-alkanes and reduces the required sample concentration by about 80%. We propose that for δD measurements, a reference standard set should be used in which each reference standard has a limited range of δD values and no adjacent n-alkane chains, to minimize memory effects.