Hierarchical superstructure aerogels for in situ biofluid metabolomics.

High-throughput biofluid metabolomics analysis for screening life-threatening diseases is urgently needed. However, the high salt content of biofluid samples, which introduces severe interference, can greatly limit the analysis throughput. Here, a new 3-D interconnected hierarchical superstructure, namely a "plasmonic gold-on-silica (Au/SiO2) double-layered aerogel", integrating distinctive features of an upper plasmonic gold aerogel with a lower inert silica aerogel was successfully developed to achieve in situ separation and storage of inorganic salts in the silica aerogel, parallel enrichment of metabolites on the surface of the functionalized gold aerogel, and direct desorption/ionization of enriched metabolites by the photo-excited gold aerogel for rapid, sensitive, and comprehensive metabolomics analysis of human serum/urine samples. By integrating all these unique advantages into the hierarchical aerogel, multifunctional properties were introduced in the SALDI substrate to enable its effective utilization in clinical metabolomics for the discovery of reliable metabolic biomarkers to achieve unambiguous differentiation of early and advanced-stage lung cancer patients from healthy individuals. This study provides insight into the design and application of superstructured nanomaterials for in situ separation, storage, and photoexcitation of multi-components in complex biofluid samples for sensitive analysis.

Serum Lipidomic Fingerprints Encode Early Diagnosis and Staging of Lung Cancer on a Novel PbS/Au-Layered Substrate.

Lung cancer (LC) is a major cause of mortality among malignant tumors. Early diagnosis through lipidomic profiling can improve prognostic outcomes. In this study, a uniform PbS/Au-layered substrate that enhances the laser desorption/ionization process, an interfacial process triggered on the substrate surface upon laser excitation, was designed to efficiently characterize the lipidomic profiles of LC patient serum. By controlling the stacking arrangement and particle sizes of PbS QDs and AuNPs, the optimized substrate promotes the generation of excited electrons and creates an enhanced electric field that polarizes analyte molecules, facilitating ion adduction formation ([M + Na]+ and [M + K]+) and enhancing detection sensitivity down to the femtomole level. Combining multivariate statistics and machine learning, a distinct lipidomic biomarker panel is successfully identified for the early diagnosis and staging of LC, with an accurate prediction validated by an area under the curve of 0.9479 and 0.9034, respectively. We also found that 18 biomarkers were significantly correlated with six metabolic pathways associated with LC. These results demonstrate the potential of this innovative PbS/Au-layered substrate as a sensitive platform for accurate diagnosis of LC and facilitate the development of lipidomic-based diagnostic tools for other cancers.

Covalent Bonding and Coulomb Repulsion-Guided AuNP Array: A Tunable and Reusable Substrate for Metabolomic Characterization of Lung Cancer Patient Sera.

Surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) has gained increased attention in the metabolic characterization of human biofluids. However, the stability and reproducibility of nanoparticle-based substrates remain two of the biggest challenges in high-salt environments. Here, by controlling the extent of Coulomb repulsion of 26 nm positively charged AuNPs, a homogeneous layer of covalently bonded AuNPs on a coverslip with tunable interparticle distances down to 16 nm has been successfully fabricated to analyze small biomolecules in human serum. Compared with the self-assembled AuNP array, the covalently bonded AuNP array showed superior performances on stability, reproducibility, and sensitivity in high-salt environments. The stable attachment of AuNPs maintained a detection reproducibility with a RSD less than 12% and enabled the reusability of the array for 10 experiments without significant signal deterioration (<15%) and carryover effects. Moreover, the closely positioned AuNPs allowed the coupling of photoinduced plasmons to generate an enhanced electric field, which promotes the generation of excited electrons to facilitate the desorption/ionization processes instead of the heat dissipation, thus enhancing the detection sensitivity with detection limits down to the femtomole level. Combined with machine learning methods, the AuNP array has been successfully applied to discover seven biomarkers for differentiating early-stage lung cancer patients from healthy controls. It is anticipated that this simple approach of developing robust AuNP arrays can also be extended to other types of NP arrays for wider applications of SALDI-MS technology.

Combining MALDI-MS with machine learning for metabolomic characterization of lung cancer patient sera.

An increasing amount of evidence has proven that serum metabolites can instantly reflect disease states. Therefore, sensitive and reproducible detection of serum metabolites in a high-throughput manner is urgently needed for clinical diagnosis. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a high-throughput platform for metabolite detection, but it is hindered by significant signal fluctuations because of the "sweet spot" effect of organic matrices. Here, by screening two transformation methods and four normalization techniques to reduce the significant signal fluctuations of the DHB matrix, an integrated MALDI-MS data processing approach combined with machine learning methods was established to reveal metabolic biomarkers of lung cancer. In our study, 13 distinctive features with statistically significant differences (p < 0.001) between 34 lung cancer patients and 26 healthy controls were selected as significant potential biomarkers of lung cancer. 6 out of the 13 distinctive features were identified as intact metabolites. Our results demonstrate the potential for clinical application of MALDI-MS in serum metabolomics for biomarker screening in lung cancer.

Expression and significance of Cystatin-C in clear cell renal cell carcinoma.

PURPOSE: Cystatin-C (Cys-C) has been studied as a valuable prognostic indicator in several malignancies. The goal of this study is to explore the expression and prognostic significance of Cys-C in clear cell renal cell carcinoma (CCRCC). METHODS: Immunohistochemistry and western blot assays were performed to evaluate the level of Cys-C expression in CCRCC tissue. Expression levels of Cys-C in CCRCC tissue samples in relation to clinicopathological characteristics of the tumors were assessed. Their prognostic significance was analyzed by univariate and multivariate analyses. In addition, the expression of Cys-C in 786-O cell lines was inhibited by using CRISPR/Case9 and the effects of Cys-C knockout on 786-O cells in vitro were evaluated using MTT method, colony formation assay, cell cycle assay, and cell migration and invasive assay. RESULTS: The expression level of Cys-C was lower in CCRCC tissues (n = 253) than in paired adjacent non-cancerous tissues (n = 164) by immunohistochemistry (P < 0.001). Among the 253 patients, the results showed that patients with low Cys-C expression level in cancer tissue has longer overall survival (OS) than that with high Cys-C level. Furthermore, knockout of Cys-C in 786-O cell line has ability to suppress cell proliferation, induce G0/G1 phase arrest, inhibited cell invasion, decreased phosphorylation of ERK1/2, STAT-3 and enhanced phosphorylated JNK expression. CONCLUSIONS: A decrease in serum Cys-C is a favorable prognostic indicator for CCRCC patients. Inhibition of Cys-C suppressed RCC 786-O cell proliferation and invasion. These results indicated that Cys-C could serve as an ideal prognostic biomarker in patients with CCRCC.

Hepatocellular toxicity of oxalicumone A via oxidative stress injury and mitochondrial dysfunction in healthy human liver cells.

The marine­derived oxalicumone A (POA) has been demonstrated as a potent anti­tumor bioactive agent for a variety of human carcinoma, but to the best of our knowledge, remains to be evaluated in healthy liver cells. As many drugs distribute preferentially in the liver, the present study aimed to investigate the effects of POA on apoptosis, oxidative stress and mitochondrial function in L­02 healthy liver cells. A Cell­Counting kit­8 assay demonstrated that POA inhibits the proliferation of L­02 cells in a dose­ and time­dependent manner. Furthermore, POA induced apoptosis by increasing the percentage of cells in early apoptosis and the sub­G1 cell cycle, along with causing S­phase arrest in L­02 cells. Additionally, POA activated caspase 3, increased the protein expression levels of Fas ligand and B­cell lymphoma X­associated protein, and decreased the expression of the anti­apoptotic protein B­cell lymphoma 2. POA additionally reduced the content of GSH and the activity of superoxide dismutase, elevated malondialdehyde and nitric oxide levels, increased reactive oxygen species production and the levels of alanine aminotransferase and aspartate aminotransferase, which suggested that POA induced lipid peroxidation injury in L­02 cells and that oxidative stress serves an important role. Furthermore, POA caused alternations of mitochondrial function, including an abrupt depletion of adenosine triphosphate synthesis, mitochondrial permeability transition pore opening and depletion of mitochondrial membrane potential in L­02 cells. These data suggested that POA exerts cytotoxicity, at least in part, by inducing oxidative stress, mitochondrial dysfunction, and eventually apoptosis. Changes in mitochondrial function and oxidative stress by POA may therefore be critical in POA­induced toxicity in L­02 cells.

Preoperative serum cystatin-C as a potential biomarker for prognosis of renal cell carcinoma.

PURPOSE: The prognostic value of serum cystatin-C (Cys-C) in renal cell carcinoma (RCC) remains unknown. The purpose of this study is to explore the prognostic value of Cys-C for RCC patients. PATIENTS AND METHODS: The levels of preoperative Cys-C, creatinine (CRE) and estimated glomerular filtration rate (e-GFR) were retrospectively collected in 325 RCC patients undergoing surgery. The cutoff values of Cys-C, CRE and e-GFR were determined by the standardized Cutoff Finder algorithm. The receiver operating characteristic (ROC) curve and pairwise comparison were performed to compare the three variables. Univariate and multivariate Cox regression analyses were performed to investigate the prognostic value of serum Cys-C in RCC. RESULTS: Based on the analysis of Cutoff Finder algorithm, ROC curve and pairwise comparison, the preoperative Cys-C was superior to CRE and e-GFR as a predictive factor in RCC. Multivariate Cox regression analyses showed that high preoperative Cys-C (>1.09 mg/L) was significantly associated with shorter overall survival (OS) in all RCC patients (hazard ratio [HR], 1.59; P = 0.012), patients at pT1-2 (P<0.001), pN0 (P<0.001) and pM0 stages (P<0.001). Moreover, Multivariate Cox regression analyses also showed that in the 306 patients without metastasis, high preoperative Cys-C was also associated with shorter disease-free survival (DFS) (HR, 3.50; P = 0.013). CONCLUSIONS: An elevated preoperative Cys-C level was demonstrated to be related with worse survival in patients with RCC. Measuring preoperative serum Cys-C might be a simple way for finding poor prognostic patients and patients with elevated preoperative Cys-C level should be more closely followed up.

Modified lingguizhugan decoction incorporated with dietary restriction and exercise ameliorates hyperglycemia, hyperlipidemia and hypertension in a rat model of the metabolic syndrome.

BACKGROUND: Modified Lingguizhugan Decoction (MLD) came from famous Chinese medicine Linggui Zhugan Decoction. The MLD is used for the treatment of metabolic syndrome in the clinical setting. Our study focuses on the comprehensive treatment of MLD incorporated with dietary restriction and exercise in a rat model of the metabolic syndrome (MS). METHODS: Rats were divided into five groups: control group (Cont), high-fat diet group (HFD), high-fat diet incorporated with dietary restriction group (HFD-DR), exercise incorporated with dietary restriction group (HFD-DR-Ex) and MLD incorporated with dietary restriction and exercise group (HFD-DR-Ex-MLD). Treatments were conducted for 1 week after feeding high-fat diet for 12 weeks. The effects of treatments on high fat diet-induced obesity, hyperglycemia, hyperlipidemia, hypertension, hepatic injury and insulin resistance in rats of MS were examined. In addition, the tumor necrosis factor-α (TNF-α), leptin and protein kinase B (PKB) in rats serum and liver were also examined by enzyme-linked immunosorbent assay (ELISA). RESULTS: After a week's intervention by dietary restriction, dietary restriction incorporated with exercise or MLD, compared with HFD rats, the relative weight of liver and fat, levels of triglyceride, total cholesterol, low-density lipoprotein, free fatty acid, aspartate aminotransferase, glutamic-pyruvic transaminase and alkaline phosphatase, insulin, were significantly decreased (p < 0.05 or 0.01). This treatment also inhibited abnormal increases of TNF-α, leptin and PKB in serum and liver. CONCLUSION: MLD incorporated with dietary restriction and exercise treatment exhibit effects in alleviating high-fat diet-induced obesity, hyperglycemia, hyperlipidemia, hypertension, hepatic injury and insulin resistance, which are possibly due to the down-regulation of TNF-α, leptin and PKB.

Toxicity study of oxalicumone A, derived from a marine-derived fungus Penicillium oxalicum, in cultured renal epithelial cells.

Oxalicumone A (POA), a novel dihydrothiophene-condensed chromone, was isolated from the marine­derived fungus Penicillium oxalicum. Previous reports demonstrated that POA exhibits strong activity against human carcinoma cells, thus it has been suggested as a bioactive anticancer agent. To research the toxic effect of POA on cultured normal epithelial human kidney­2 (HK­2) cells and evaluate its clinical safety, cell survival was evaluated by the Cell Counting Kit-8 assay and apoptosis was evaluated by Hoechst 33258 staining, flow cytometry, caspase­3 activity assay and western blotting. 2',7'-Dichlorofluorescin diacetate and JC­1 dye staining was used to evaluate reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP), respectively. The results indicated that POA inhibited HK-2 cell growth and promoted apoptosis, by increasing levels of Fas cell surface cell receptor and the B­cell lymphoma 2 associated protein X apoptosis regulator (Bax)/B­cell lymphoma 2 apoptosis regulator (Bcl-2) ratio. POA treatment also induced release of ROS and loss of MMP in HK­2 cells. Compared with untreated control, a significant decrease was also demonstrated in superoxide dismutase activity and glutathione content with POA treatment, accompanied by enhanced release of N­acetyl­ß­D­glucosaminidase, increased leakage of lactate dehydrogenase, increased malondialdehyde formation and increased release of nitric oxide. In conclusion, the present in vitro study revealed that POA exhibits antiproliferation activity on HK­2 cells, through stimulation of apoptosis and oxidative stress injury, which may be relevant to its clinical application. The present study may, therefore, offer valuable new information regarding the use of POA as a candidate novel antitumor drug for clinical use.