Global metabolomics revealed deviations from the metabolic aging clock in colorectal cancer patients.

Background: Markers of aging hold promise in the context of colorectal cancer (CRC) care. Utilizing high-resolution metabolomic profiling, we can unveil distinctive age-related patterns that have the potential to predict early CRC development. Our study aims to unearth a panel of aging markers and delve into the metabolomic alterations associated with aging and CRC. Methods: We assembled a serum cohort comprising 5,649 individuals, consisting of 3,002 healthy volunteers, 715 patients diagnosed with colorectal advanced precancerous lesions (APL), and 1,932 CRC patients, to perform a comprehensive metabolomic analysis. Results: We successfully identified unique age-associated patterns across 42 metabolic pathways. Moreover, we established a metabolic aging clock, comprising 9 key metabolites, using an elastic net regularized regression model that accurately estimates chronological age. Notably, we observed significant chronological disparities among the healthy population, APL patients, and CRC patients. By combining the analysis of circulative carcinoembryonic antigen levels with the categorization of individuals into the "hypo" metabolic aging subgroup, our blood test demonstrates the ability to detect APL and CRC with positive predictive values of 68.4% (64.3%, 72.2%) and 21.4% (17.8%, 25.9%), respectively. Conclusions: This innovative approach utilizing our metabolic aging clock holds significant promise for accurately assessing biological age and enhancing our capacity to detect APL and CRC.

Targeted Therapy of Lung Adenocarcinoma by the Nanoplatform Based on Milk Exosomes Loaded with Paclitaxel.

Lung cancer is the most common cancer throughout the world. Currently, most lung cancer therapies are still limited by serious side effects caused. This paper reports a biocompatible drug delivery system that utilizes milk-derived exosomes to deliver paclitaxel to treat lung adenocarcinoma. First, milk-derived exosomes were modified with integrin αVß3, αVß5-binding peptide iRGD so that they could successfully target lung adenocarcinoma cells. Then, iRGD modified exosomes were loaded with paclitaxel (PAC) via electroporation and used for tumor therapy. These modified exosomes proved effective in killing lung adenocarcinoma cells, and the exosome-based nanoplatform showed no obvious toxicity to normal cells. Further more, the exosome-based nanoplatform could effectively penetrate the interior of the 3D tumor sphere, reaching more tumor cells and demonstrating that it is a promising tool for lung adenocarcinoma therapy.

Multi-Omics Signatures Link to Ticagrelor Effects on Vascular Function in Patients With Acute Coronary Syndrome.

BACKGROUND: Long-term antiplatelet agents including the potent P2Y12 antagonist ticagrelor are indicated in patients with a previous history of acute coronary syndrome. We sought to compare the effect of ticagrelor with that of aspirin monotherapy on vascular endothelial function in patients with prior acute coronary syndrome. METHODS: This was a prospective, single center, parallel group, investigator-blinded randomized controlled trial. We randomized 200 patients on long-term aspirin monotherapy with prior acute coronary syndrome in a 1:1 fashion to receive ticagrelor 60 mg BD (n=100) or aspirin 100 mg OD (n=100). The primary end point was change from baseline in brachial artery flow-mediated dilation at 12 weeks. Secondary end points were changes to platelet activation marker (CD41_62p) and endothelial progenitor cell (CD34/133) count measured by flow cytometry, plasma level of adenosine, IL-6 (interleukin-6) and EGF (epidermal growth factor), and multi-omics profiling at 12 weeks. RESULTS: After 12 weeks, brachial flow-mediated dilation was significantly increased in the ticagrelor group compared with the aspirin group (ticagrelor: 3.48±3.48% versus aspirin: -1.26±2.85%, treatment effect 4.73 [95% CI, 3.85-5.62], P<0.001). Nevertheless ticagrelor treatment for 12 weeks had no significant effect on platelet activation markers, circulating endothelial progenitor cell count or plasma level of adenosine, IL-6, and EGF (all P>0.05). Multi-omics pathway assessment revealed that changes in the metabolism and biosynthesis of amino acids (cysteine and methionine metabolism; phenylalanine, tyrosine, and tryptophan biosynthesis) and phospholipids (glycerophosphoethanolamines and glycerophosphoserines) were associated with improved brachial artery flow-mediated dilation in the ticagrelor group. CONCLUSIONS: In patients with prior acute coronary syndrome, ticagrelor 60 mg BD monotherapy significantly improved brachial flow-mediated dilation compared with aspirin monotherapy and was associated with significant changes in metabolomic and lipidomic signatures. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT03881943.

A prospective study of serum metabolomic and lipidomic changes in myopic children and adolescents.

BACKGROUND: Myopia is a prevalent eye disorder, especially among children and adolescents in eastern Asian countries. Multiple measures have already been taken to prevent and treat myopia, including atropine and dopamine. However, the serum metabolic picture of myopia has not yet been studied as a whole and remains largely unclear. In this paper, a prospective and panoramic study was carried out to find out the whole serum metabolomic and lipidomic picture of myopia. METHODS: With untargeted mass spectrometry (MS), myopia among 211 children and adolescents was studied. The MS features were first grouped across the samples. Then, compound annotation was carried out based on these features. Finally, the metabolite features were mapped to pathways, whose biological functions in myopia were studied and discussed. RESULTS: A total of 275 metabolite features were derived from 92 aligned MS peak groups with significant fold changes, and then mapped to 33 pathways. By a comprehensive consideration of significance, fold change, importance score and appearance in different omics, 9 pathways were selected, and their biological functions were further analyzed. Among these selected pathways, 5 pathways were related with oxidative stress, a validated phenomenon during myopia development, while 5 pathways were related with dopamine receptor D2, whose molecular function in myopia treatment is not fully understood. A total of 177 metabolite features from 45 peak groups were related with the studied pathways. CONCLUSION: This prospective study shed light on the whole picture of metabolomic mechanism underlying myopia and provided guidance to further elucidation of compounds and pathways in this whole picture.

A "quasi" confocal droplet reader based on laser-induced fluorescence (LIF) cytometry for highly-sensitive and contamination-free detection.

Highly-sensitive and contamination-free droplet digital PCR (ddPCR) is an enabling technology and widely needed for accurate quantification of nucleic acid in clinical applications. In this paper, a novel droplet reader was developed by combining a "quasi" confocal laser-induced fluorescence (LIF) cytometry with a delicate microfluidic chip design. The droplets with a size of 90 µm was illuminated at an out-of-focus position by two aligned laser beams to generate maximum fluorescent signal. Additionally, the lateral offset position of the microfluidic chip should be precisely tuned so that the bandwidth of the FAM and VIC channels were configured at the matching sizes. Then, PMT gain voltages and pneumatic pressures were optimized for better droplet detection efficiencies. An aerosol adsorption experiment was performed to demonstrate that there was no aerosol contamination, and detected copy numbers of both mutants and wild types scaled linearly with the expected input copy numbers (r2>0.998) with a LoB of 0.0 copies and LoD of 3.0 copies. The results demonstrated that this droplet reader with the delicate chip is a convenient, highly-sensitive and contamination-free to detect fluorescence signals inside droplets after ddPCR, which is highly promising for broad applications of ddPCR in clinical diagnosis.

A density-watershed algorithm (DWA) method for robust, accurate and automatic classification of dual-fluorescence and four-cluster droplet digital PCR data.

Droplet digital PCR (ddPCR) is a single-molecule amplification technology with broad applications in precision medicine and clinical diagnosis. Dual-fluorescence and four-cluster ddPCR (two/four-ddPCR) assay is an effective way to quantify copy numbers. Currently, two/four-ddPCR data are usually classified with manual thresholds. For clinical applications, automatic and accurate methods are required to avoid subjectivity in diagnosis. Although there are some automatic classification algorithms, their accuracy and robustness still need to be improved to meet the needs of clinical diagnosis. Therefore, a new method is in high demand to automatically classify two/four-ddPCR data in an accurate and robust way. Here, a novel density-watershed algorithm (DWA) method was developed for the accurate, automatic and unsupervised classification of two/four-ddPCR data. First, data gridding was applied to a scatter plot of the fluorescence signal intensity to calculate data densities. Based on the data densities, the watershed algorithm was used to divide the gridded scatter plot into isolated regions automatically. Next, an optimal cluster pattern was determined based on these isolated regions, and excess regions were merged. Finally, the two/four-ddPCR data were classified based on the merged regions, and DNA template copy numbers were calculated accordingly. Using the DWA method for the quantification of both wild types and mutants of epidermal growth factor receptor (EGFR) L858R and T790M, the classification results were highly consistent with expectations, and significantly better than commonly-used automatic algorithms for now. The computed template copy numbers scaled proportionally to the relative concentration of input templates (r2 > 0.998) in four orders of magnitude with a good reproducibility, and achieved a limit of detection over 40 times lower than the commonly-used automatic algorithms. Furthermore, the DWA method was validated on 254 clinical DNA samples derived from frozen tissues, formalin-fixed paraffin-embedded tissues and peripheral blood. In most cases, the DWA method derived accurate and valid classification results. This highly effective DWA method may be widely used for automatically classifying two/four-ddPCR data, and it will greatly promote the application of ddPCR in clinical diagnosis.

A multiplex droplet digital PCR assay for non-invasive prenatal testing of fetal aneuploidies.

Higher multiplexing in droplet digital PCR (ddPCR) can simplify the detection process of ddPCR-based non-invasive prenatal testing (NIPT) and improve its reliability, making it a practical approach in clinical practice. However, a high level of multiplex ddPCR-based NIPT has rarely been reported. In this study, we developed a multiplex ddPCR assay using universal locked nucleic acid (LNA) probes to reliably identify fetal aneuploidies. We first performed statistical analysis based on the Poisson distribution to evaluate the required number of target DNA molecules and the total number of droplets for a ddPCR assay. Next, we designed two sets of primers and probes to quantify cfDNA from chromosomes 21 and 18 and then determined the disease status of a sample. Finally, we evaluated our multiplex ddPCR assay with 60 clinical plasma samples. All of the 60 clinical samples were correctly identified. The accessibility and cost-effectiveness of our multiplex ddPCR-based NIPT make it a competitive prenatal testing method in clinical use.

Rapid and efficient isolation and detection of extracellular vesicles from plasma for lung cancer diagnosis.

Extracellular vesicles (EVs) are cell-derived nanoscale vesicles that provide promising biomarkers for the non-invasive diagnosis of cancer because they carry important cancer-related DNA, RNA and protein biomarkers. However, the clinical application of EVs is limited by tedious and non-standardized isolation methods that require bulky instrumentation. Here, we propose an easy-to-operate, simple dielectrophoretic (DEP) method for EV isolation with higher recovery efficiency (>83%) and higher purity than ultracentrifugation (UC). The DEP chip reduces the isolation procedure from 8 h to 30 min. To facilitate subsequent analysis, our DEP chip achieved integration of EV isolation and in situ lysis of EVs for the first time. Our chip also achieved on-chip siRNA delivery to EVs isolated by DEP. We found that EVs isolated from the plasma of lung cancer patients contained higher levels of miR-21, miR-191 and miR-192 compared to those from healthy people. With on-chip detection, EGFR in EVs could distinguish lung cancer patients from healthy people. Overall, this study provides an efficient and practical approach to the isolation and detection of EVs, which could be used for the early diagnosis of lung cancer.

A Cosine Similarity Algorithm Method for Fast and Accurate Monitoring of Dynamic Droplet Generation Processes.

Droplet microfluidics has attracted significant interests in functional microcapsule synthesis, pharmaceuticals, fine chemicals, cosmetics and biomedical research. The low variability of performing chemical reactions inside droplets could benefit from improved homogeneity and reproducibility. Therefore, accurate and convenient methods are needed to monitor dynamic droplet generation processes. Here, a novel Cosine Similarity Algorithm (CSA) method was developed to monitor the droplet generation frequency accurately and rapidly. With a microscopic droplet generation video clip captured with a high-speed camera, droplet generation frequency can be computed accurately by calculating the cosine similarities between the frames in the video clip. Four kinds of dynamic droplet generation processes were investigated including (1) a stable condition in a single microfluidic channel, (2) a stable condition in multiple microfluidic channels, (3) a single microfluidic channel with artificial disturbances, and (4) microgel fabrication with or without artificial disturbances. For a video clip with 5,000 frames and a spatial resolution of 512 × 62 pixels, droplet generation frequency up to 4,707.9 Hz can be calculated in less than 1.70 s with an absolute relative calculation error less than 0.08%. Artificial disturbances in droplet generation processes can be precisely determined using the CSA method. This highly effective CSA method could be a powerful tool for further promoting the research of droplet microfluidics.

Noninvasive and Accurate Detection of Hereditary Hearing Loss Mutations with Buccal Swab Based on Droplet Digital PCR.

Hereditary hearing loss is a common clinical neurosensory disorder in humans and has a high demand for genetic screening. Current screening techniques using peripheral blood or dried blood spots (DBSs) are invasive. Therefore, this study aims to develop a noninvasive and accurate detection method for eight hotspot deafness-associated mutations based on buccal swab and droplet digital PCR (ddPCR). First, this method was evaluated for analytic performance including specificity, detection limit, dynamic range using plasmid DNA. The specificity was 100% and the detection limit was 5 copies. The dynamic range of this ddPCR-based method was from 10 to 105 copies/µL. Next, the method was found to accurately quantify mitochondrial gene heteroplasmy rate as low as 1% for both m.1494C > T and m.1555A > G sites. Then, we demonstrated that buccal swab was a reliable sample. DNA can be extracted and accurately quantified after a buccal swab had been stored for 90 days at either room temperature or -20 °C. Finally, clinical samples (23 DBSs and 42 buccal swabs) were tested to further evaluate the accuracy and clinical applicability of this method. All clinical samples were accurately quantified and genotyped. This noninvasive and accurate method is highly promising as a genetic screening method for deafness-associated mutations due to its high sensitivity and accuracy.

Functional Variants Identified Efficiently through an Integrated Transcriptome and Epigenome Analysis.

Although genome-wide association studies (GWAS) have identified numerous genetic loci associated with complex diseases, the underlying molecular mechanisms of how these loci contribute to disease pathogenesis remain largely unknown, due to the lack of an efficient strategy to identify these risk variants. Here, we proposed a new strategy termed integrated transcriptome and epigenome analysis (iTEA) to identify functional genetic variants in non-coding elements. We considered type 2 diabetes mellitus as a model and identified a well-known diabetic risk variant rs35767 using iTEA. Furthermore, we discovered a new functional SNP, rs815815, involved in glucose metabolism. Our study provides an approach to directly and quickly identify functional genetic variants in type 2 diabetes mellitus, and this approach can be extended to study other complex diseases.