Early Breast Cancer Detection Using Untargeted and Targeted Metabolomics.

Breast cancer (BC) is a common cause of morbidity and mortality, particularly in women. Moreover, the discovery of diagnostic biomarkers for early BC remains a challenging task. Previously, we [Jasbi et al. J. Chromatogr. B. 2019, 1105, 26-37] demonstrated a targeted metabolic profiling approach capable of identifying metabolite marker candidates that could enable highly sensitive and specific detection of BC. However, the coverage of this targeted method was limited and exhibited suboptimal classification of early BC (EBC). To expand the metabolome coverage and articulate a better panel of metabolites or mass spectral features for classification of EBC, we evaluated untargeted liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QTOF-MS) data, both individually as well as in conjunction with previously published targeted LC-triple quadruple (QQQ)-MS data. Variable importance in projection scores were used to refine the biomarker panel, whereas orthogonal partial least squares-discriminant analysis was used to operationalize the enhanced biomarker panel for early diagnosis. In this approach, 33 altered metabolites/features were detected by LC-QTOF-MS from 124 BC patients and 86 healthy controls. For EBC diagnosis, significance testing and analysis of the area under receiver operating characteristic (AUROC) curve identified six metabolites/features [ethyl (R)-3-hydroxyhexanoate; caprylic acid; hypoxanthine; and m/z 358.0018, 354.0053, and 356.0037] with p < 0.05 and AUROC > 0.7. These metabolites informed the construction of EBC diagnostic models; evaluation of model performance for the prediction of EBC showed an AUROC = 0.938 (95% CI: 0.895-0.975), with sensitivity = 0.90 when specificity = 0.90. Using the combined untargeted and targeted data set, eight metabolic pathways of potential biological relevance were indicated to be significantly altered as a result of EBC. Metabolic pathway analysis showed fatty acid and aminoacyl-tRNA biosynthesis as well as inositol phosphate metabolism to be most impacted in response to the disease. The combination of untargeted and targeted metabolomics platforms has provided a highly predictive and accurate method for BC and EBC diagnosis from plasma samples. Furthermore, such a complementary approach yielded critical information regarding potential pathogenic mechanisms underlying EBC that, although critical to improved prognosis and enhanced survival, are understudied in the current literature. All mass spectrometry data and deidentified subject metadata analyzed in this study have been deposited to Mendeley Data and are publicly available (DOI: 10.17632/kcjg8ybk45.1).

Idiopathic pulmonary fibrosis and diabetes mellitus: a meta-analysis and systematic review.

BACKGROUND: Idiopathic pulmonary fibrosis (IPF) is a chronic diffuse interstitial lung disease, of which the etiology has been poorly understood. Several studies have focused on the relationship between IPF and diabetes mellitus (DM) in the past years but have failed to reach a consensus. This meta-analysis aimed to examine the association between diabetes to IPF. METHODS: We accumulated studies investigating the association between DM and IPF from databases including Medline, Cochrane Library, Embase, Web of Science, and China National Knowledge Infrastructure. RevMan 5.3 and the Newcastle-Ottawa Scale (NOS) were utilized to analyze the data and assess the quality of the included studies. The value of odds ratio (OR) with 95% confidence interval (CI) was used as the measure to estimate the risk of DM in IPF. Heterogeneity was assessed by I2 statistics. We also performed subgroup analysis, meta-regression, and Egger's test for bias analysis. RESULTS: Nine case-control studies with 5096 IPF patients and 19,095 control subjects were included in the present meta-analysis, which indicated a positive correlation between DM and IPF (OR 1.65, 95% CI 1.30-2.10; P < 0.0001). Meta-regression and subgroup analysis negated the influence of covariates like cigarette smoking, age and gender, but the heterogeneity existed and could not be fully explained. CONCLUSION: IPF and DM may be associated, but the causal relationship remains indeterminate till now. Further rigorously designed studies are required to confirm the present findings and investigate the possible mechanisms behind the effect of DM on IPF.

Comprehensive Isotopic Targeted Mass Spectrometry: Reliable Metabolic Flux Analysis with Broad Coverage.

Metabolic flux analysis (MFA) is highly relevant to understanding metabolic mechanisms of various biological processes. While the pace of methodology development in MFA has been rapid, a major challenge the field continues to witness is limited metabolite coverage, often restricted to a small to moderate number of well-known compounds. In addition, isotopic peaks from an enriched metabolite tend to have low abundances, which makes liquid chromatography tandem mass spectrometry (LC-MS/MS) highly useful in MFA due to its high sensitivity and specificity. Previously we have built large-scale LC-MS/MS approaches that can be routinely used for measurement of up to ∼1,900 metabolite/feature levels [Gu et al. Anal. Chem. 2015, 87, 12355-12362. Shi et al. Anal. Chem. 2019, 91, 13737-13745.]. In this study, we aim to expand our previous studies focused on metabolite level measurements to flux analysis and establish a novel comprehensive isotopic targeted mass spectrometry (CIT-MS) method for reliable MFA analysis with broad coverage. As a proof-of-principle, we have applied CIT-MS to compare the steady-state enrichment of metabolites between Myc(oncogene)-On and Myc-Off Tet21N human neuroblastoma cells cultured with U-13C6-glucose medium. CIT-MS is operationalized using multiple reaction monitoring (MRM) mode and is able to perform MFA of 310 identified metabolites (142 reliably detected, 46 kinetically profiled) selected from >35 metabolic pathways of strong biological significance. Further, we developed a novel concept of relative flux, which eliminates the requirement of absolute quantitation in traditional MFA and thus enables comparative MFA under the pseudosteady state. As a result, CIT-MS was shown to possess the advantages of broad coverage, easy implementation, fast throughput, and more importantly, high fidelity and accuracy in MFA. In principle, CIT-MS can be easily adapted to track the flux of other labeled tracers (such as 15N-tracers) in any metabolite detectable by LC-MS/MS and in various biological models (such as mice). Therefore, CIT-MS has great potential to bring new insights to both basic and clinical metabolism research.

2,2',4,4'-tetrabromodiphenyl ether (BDE-47) induces wide metabolic changes including attenuated mitochondrial function and enhanced glycolysis in PC12 cells.

Polybrominated diphenyl ethers (PBDEs) are extensively used as brominated flame retardants in various factory products. As environmental pollutants, the adverse effects of PBDEs on human health have been receiving considerable attention. However, the precise fundamental mechanisms of toxicity induced by PBDEs are still not fully understood. In this study, the mechanism of cytotoxicity induced by 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) was investigated by combining Seahorse XFp analysis and mass spectrometry-based metabolomics and flux approaches in PC12 cells, one of the most widely used neuron-like cell lines for investigating cytotoxic effects. The Seahorse results suggest that BDE-47 significantly attenuated mitochondrial respiration and enhanced glycolysis in PC12 cells. Additionally, metabolomics results revealed the reduction of TCA metabolites such as citrate, succinate, aconitate, malate, fumarate, and glutamate after BDE-47 exposure. Metabolic flux analysis showed that BDE-47 exposure reduced the oxidative metabolic capacity of mitochondria in PC12 cells. Furthermore, various altered metabolites were found in multiple metabolic pathways, especially in glycine-serine-threonine metabolism and glutathione metabolism. A total of 17 metabolic features were determined in order to distinguish potentially disturbed metabolite markers of BDE-47 exposure. Our findings provide possible biomarkers of cytotoxic effects induced by BDE-47 exposure, and elicit a deeper understanding of the intramolecular mechanisms that could be used in further studies to validate the potential neurotoxicity of PBDEs in vivo. Based on our results, therapeutic approaches targeting mitochondrial function and the glycolysis pathway may be a promising direction against PBDE exposure.

Database-Assisted Globally Optimized Targeted Mass Spectrometry (dGOT-MS): Broad and Reliable Metabolomics Analysis with Enhanced Identification.

Targeted mass spectrometry (MS) is an important measurement approach in metabolomics with strong analytical performance, given its specificity, sensitivity, and quantitative capacity. However, traditional targeted-MS relies heavily on chemical standards for the development of various detection panels; thus, its metabolite coverage is often limited to those well-known and commercially available compounds. To address this fundamental gap, we previously developed a novel approach [ H. Gu et al. Anal. Chem. 2015 , 87 , 12355 - 12362 ], globally optimized targeted (GOT)-MS, which enables reliable metabolic analysis with broad coverage using a single triple quadrupole instrument. In the present study, we further developed and optimized an innovative targeted MS approach, database-assisted globally optimized targeted (dGOT)-MS, which utilizes the HMDB and METLIN databases to significantly improve both identification and metabolite coverage. As it is well-known, these metabolomics databases have a comprehensive collection of metabolites and their tandem MS spectra; therefore, in this study, multiple reaction monitoring transitions (MRMs) were directly obtained from the databases and, after optimizing MS parameters for those MRMs, 927 metabolites were measured from a plasma aqueous extract sample with high reliability by dGOT-MS. Of these, 310 were confirmed using pure chemical standards while the rest were annotated by identification level using database entries. Furthermore, using breast cancer diagnosis as a proof-of-principle metabolomics application, we showed dGOT-MS to significantly outperform a traditional large-scale targeted MS assay for potential biomarker discovery. In principle, dGOT-MS is able to cover all metabolites (including lipids) that have been characterized in these comprehensive metabolomics databases from various types of biological samples. Therefore, dGOT-MS opens a novel avenue for MS measurements and may play an important role in many future metabolomics studies.

AAV8-Mediated Gene Therapy Rescues Retinal Degeneration Phenotype in a Tlcd3b Knockout Mouse Model.

Purpose: The purpose of this study was to assess the therapeutic efficacy of rAAV8-hGRK1-Tlcd3b in a Tlcd3b-/- mouse model of retinal generation and validate TLCD3B's role as a ceramide synthase in vivo. Methods: Using Tlcd3b-/- mice as an inherited retinal disease animal model, we performed subretinal injection of rAAV8-hGRK1-Tlcd3b and evaluated the efficacy of gene replacement therapy. Tlcd3b-/- mice were treated at two time points: postnatal day 21 (P21) and postnatal day 120 (P120) with various dosages. Results: Tlcd3b overexpression rescued retinal degeneration in the mutant mice, as indicated by significantly improved photoreceptor function and preservation of photoreceptor cells over the course of 1 year. Although Tlcd3b is expressed in all cell types in the retina, photoreceptor cell-specific expression of Tlcd3b is sufficient to rescue the phenotype, indicating the primary function of TLCD3B is in photoreceptors. Consistent with the idea that TLCD3B is a ceramide synthase, mass spectrometry analyses of the mutant retina indicate the reduction of C16-, C18-, and C20-ceramides in the retina, which are restored with Tlcd3b overexpression. Conclusions: Our findings demonstrated the therapeutic efficacy of gene therapy in treating Tlcd3b mutant retina, laying the foundation for developing future therapy for TLCD3B retinopathy.

Batesian mimicry in the nonrewarding saprophytic orchid Danxiaorchis yangii.

Batesian mimicry, a type of deceptive pollination, is a complicated strategy used by nonrewarding plants to attract pollinators, but some hypotheses concerning this have not been systematically verified. In order to show in detail a case of Batesian mimicry on saprophytic orchid Danxiaorchis yangii, the ecological relationship between Danxiaorchis yangii, Lysimachia alfredi and Dufourea spp. was explored. Lysimachia alfredi could provide a reward to Dufourea sp., whereas Danxiaorchis yangii not. The floral morphology and geographical distribution of these two plants were highly overlapping, and the fruit set rate of Danxiaorchis yangii was significantly positively correlated with the number of nearby L. alfredi individuals. In a glass cylinder experiment, Danxiaorchis yangii and L. alfredi attracted Dufourea spp. through visual signals, but the insect could not distinguish between flowers of the two plants before landing on flowers. The ultraviolet reflection spectra of flowers between the two plant species were highly similar. In the hexagonal color models constructed according to the visual characteristics of bees, the flower color signals of these two plant species highly overlap, indicating that the visual signals of the flowers of the two plants to the pollinator were greatly similar. All of these results provided evidence that Danxiaorchis yangii simulated the visual signals of L. alfredi through Batesian mimicry, thereby deceptively attracting Dufourea spp.

Huisheng Oral Solution Adjunct With Platinum-Based Chemotherapy for the Treatment of Advanced Non-Small Cell Lung Cancer: A Meta-Analysis and Systematic Review.

BACKGROUND: Platinum-based chemotherapy is one of the first line therapies for the advanced non-small cell lung cancer (NSCLC), even though its high toxicity and limited clinical effects cannot be neglected. Huisheng oral solution (HSOS) has been widely used as an adjuvant chemotherapy drug for NSCLC in China. To systematically analyze the therapeutic effects of the combination of HSOS and platinum-based chemotherapy, a comprehensive meta-analysis was performed. OBJECTIVES: This systematic review and meta-analysis aimed to evaluate the effectiveness and safety of HSOS for NSCLC. METHODS: The randomized controlled trials (RCTs) were selected on seven medical databases up to June 2020, including advanced NSCLC treatment using HSOS plus platinum-based chemotherapy versus chemotherapy alone. We followed the PRISMA checklist in general, applying Cochrane handbook 5.1.0, GRADE Pro GDT, RevMan 5.3, Stata12.0, and TSA 0.9.5.10 Beta to evaluate the quality of the study and analyze the data. RESULTS: Based on Cochrane handbook 5.1.0, 15 RCTs consisting 1165 patients met the criteria and were selected for further analysis. Compared to chemotherapy alone, the chemotherapy combined with HSOS significantly improved objective tumor response (ORR) [RR = 1.38, 95% CI (1.19, 1.59), P < 0.0001], disease control rate (DCR) [RR = 1.10, 95% CI (1.04, 1.16), P = 0.0006], and Karnofsky performance status (KPS) [RR = 1.79, 95% CI (1.41, 2.26), P < 0.00001]. However, there was no evidence of improvement in the 1-year survival rate [RR = 1.37, 95% CI (0.98, 1.92), P = 0.07]. In terms of the side effects, HSOS administered concurrently with chemotherapy resulted in a serial of substantial benefits: lower toxicity to white blood cell [RR = 0.30, 95% CI (0.20, 0.43), P < 0.00001], lower platelet toxicity [RR = 0.56, 95% CI (0.34, 0.92), P = 0.02], and reduced incidence of vomiting [RR = 0.52, 95% CI (0.29, 0.92), P = 0.03]. CONCLUSIONS: The meta-analysis indicated that HSOS plus platinum-based chemotherapy was more beneficial for patients, as the therapy could synergize antitumor activity and could attenuate toxicity. The finding requires confirmation by further rigorously designed RCTs.