The Antioxidant, Anti-Inflammatory and Moisturizing Effects of Camellia oleifera Oil and Its Potential Applications.

Camellia oleifera oil (CO oil) extracted from C. oleifera seeds has a 2300-year consumption history in China. However, there is relatively little research regarding its non-edible uses. This study determined the physicochemical properties of CO oil extracted via direct pressing, identified its main components using GC-MS, and evaluated its antioxidant, moisturizing, and anti-inflammatory activities. The results revealed that CO oil's acid, peroxide, iodine, and saponification values were 1.06 ± 0.031 mg/g, 0.24 ± 0.01 g/100 g, 65.14 ± 8.22 g/100 g, and 180.41 ± 5.60 mg/g, respectively. CO oil's tocopherol, polyphenol, and squalene contents were 82.21 ± 9.07 mg/kg, 181.37 ± 3.76 mg/kg, and 53.39 ± 6.58 mg/kg, respectively; its unsaturated fatty acid (UFA) content was 87.44%, and its saturated fatty acid (SFA) content was 12.56%. CO oil also demonstrated excellent moisture retention properties, anti-inflammatory effects, and certain free radical scavenging. A highly stable CO oil emulsion with competent microbiological detection was developed using formulation optimization. Using CO oil in the emulsion significantly improved the formulation's antioxidant and moisturizing properties compared with those of the emulsion formulation that did not include CO oil. The prepared emulsion was not cytotoxic to cells and could reduce cells' NO content; therefore, it may have potential nutritional value in medicine and cosmetics.

Integrated analysis of the potential roles of microRNA-messenger RNA networks in prostate adenocarcinoma.

Background: Prostate adenocarcinoma is a frequent cancer among men with high incidence and mortality rates. Biomarkers are useful for the treatment of cancers, so we need to explore the regulatory network of prostate adenocarcinoma. Method: The database from University of California Santa Cruz was used to determine expression of messenger RNAs and microRNAs. Weighted correlation network analysis was used for classifying genes. Search Tool for Recurring Instances of Neighboring Genes and Cytoscape were used for the construction of PPI network and selection of hub genes. The microRNAs were predicted in miRactDB. The relations between microRNAs and messenger RNAs were assessed by Statistical Product and Service Solutions. The prognostic value was evaluated through Kaplan-Meier method. Kyoto Encyclopedia of Genes and Genomes, Gene Ontology and Gene Set Enrichment Analysis were used for predicting potential function. Results: 10 hub genes were all overexpressed in tumor tissues compared to normal tissues, but only aurora kinase B and nucleolar and spindle associated protein 1 were both significantly related to disease-free interval and progression-free interval time.Aurora kinase B and nucleolar and spindle associated protein 1 were negatively related to hsa-miR-1-3p, hsa-miR-133a-3p, hsa-miR-133b and hsa-miR-221-3p but positively related to hsa-miR-15b-5p, hsa-miR-21-5p, hsa-miR-106b-5p, hsa-miR-183-5p, hsa-miR-191-5p, hsa-miR-210-3p, hsa-miR-425-5p and hsa-miR-653-5p. All microRNAs except has-miR-653-5p significantly were related to the disease-free interval and progression-free interval time. The functions of microRNAs were enriched in cell cycle. Conclusion: We identified hub messenger RNAs and core microRNAs and established a novel messenger RNA-microRNA network associated with the prognosis of prostate adenocarcinoma.

Proximity binding induced nucleic acid cascade amplification strategy for ultrasensitive homogeneous detection of PSA.

In this work, based on the powerful cycle amplification cascades of proximity hybridization-induced hybridization chain reaction and catalyzed hairpin assembly, we engineered a nonenzymatic and ultrasensitive method which combined the Mg2+-DNAzyme recycling signal amplification for the analysis of the human prostate specific antigen. Herein, we adopted PSA-conjugates as triggers in the self-assembly process of two hairpin DNAs (H1, H2) into the products of the CHA which could activate the HCR to induce repeated hybridization. And both ends of each adjacent sequence of the HCR products could form a unit of Mg2+-DNAzyme which in presence of cofactor Mg2+ could recognize and cyclically cleave the hairpin probes in the solution and thus generate observably enhanced fluorescent signal. Benefit from the nucleic acid circuit amplification strategy, PSA of concentration low to 0.73 pg mL-1 was detected in this system. This homogeneous sensing method in solution avoid the use of the sophisticated equipment and complex operation, as well as addition of artificial enzyme, thus greatly reducing the constraints and complexity of experimental conditions. Moreover, considering most protein biomarkers in serum don't have their corresponding aptamers, this sensing method provide a general sensing approach for homogeneous sensitive detection of these important protein biomarkers which transfer rough antigen-antibody interactivity to smart signal amplification sensing strategies, thus exhibiting a remarkable prospect in clinical application.

An enhanced plasma injection method for triggering a 10 cm-magnitude high-pressure SF6 gas gap for a megavolt ultrafast bypass switch.

Aiming at developing a megavolt ultrafast bypass switch (UFBPS) that operates at a very low working coefficient, an enhanced plasma injection (EPI) method was proposed, which ejected high-density plasmas up to several centimeters in height in a high-pressure SF6 and thus has an extremely strong triggering ability. The EPI method employed a thin polytetrafluoroethylene microcavity embedded inside the ground electrode and a metal wire electrically exploded inside the microcavity, generating a high-density metal vapor plasma that was rapidly ejected outward from the nozzle and inducing a breakdown of the residual gas gap. In this study, the ejected plasma properties by EPI and main influencing factors were examined and the EPI triggering ability was experimentally verified. The results showed that EPI evolution presented three stages: ellipsoid-shaped, mushroom-shaped, and dissipation stages. In 0.5-MPa SF6, when the trigger energy was 960 J and the exploded aluminum wire was 300 µm in radius, the EPI maximum height reached over 9 cm within 0.7 ms, with an initial evolution velocity >800 m/s. Then, an EPI cavity array was set to repetitively trigger a 10 cm-magnitude SF6 gas gap at 0.5 MPa, with a theoretical breakdown voltage >2 MV. The gas gap was successfully triggered with an average trigger delay of 538 µs when a DC 100 kV voltage (undervoltage ratio <5%) was applied. These results indicated that EPI was an effective method for triggering megavolt-magnitude high-pressure SF6 gas gaps at a low undervoltage ratio and meeting the submillisecond trigger requirement of the UFBPS.

Radiation characteristics of natural gamma-ray from coal and gangue for recognition in top coal caving.

Recognition of coal and gangue (roof rock) is a key technology to realize fully mechanized top coal caving automated mining. This paper proposes to detect the instantaneous refuse content of drawn coal and gangue mixture during top coal caving by using natural gamma-ray technology. The generating environment of coal and rock seams, the distribution characteristics of natural gamma ray from coal and roof-rock and the principle of coal-gangue recognition using natural gamma-ray method were analyzed. The natural gamma ray radiation characteristics of coal and roof-rock seams from seven different typical coal mine areas who has thick coal seams in China have been researched, and a connection between radiation intensity and refuse content was set up. The experiments on the mixed condition of roof-rock drawn from caving opening in the caving process of fully-mechanized top coal caving working face was taken and the radiative signals was real-time detected by using the self-developed coal-gangue recognition experimental system. The experiments results demonstrate the feasibility of using natural gamma-ray technology to perform real-time detection of refuse content of drawn coal and gangue mixture and the availability of self-developed coal-gangue recognition detector.

3D multi-view convolutional neural networks for lung nodule classification.

The 3D convolutional neural network (CNN) is able to make full use of the spatial 3D context information of lung nodules, and the multi-view strategy has been shown to be useful for improving the performance of 2D CNN in classifying lung nodules. In this paper, we explore the classification of lung nodules using the 3D multi-view convolutional neural networks (MV-CNN) with both chain architecture and directed acyclic graph architecture, including 3D Inception and 3D Inception-ResNet. All networks employ the multi-view-one-network strategy. We conduct a binary classification (benign and malignant) and a ternary classification (benign, primary malignant and metastatic malignant) on Computed Tomography (CT) images from Lung Image Database Consortium and Image Database Resource Initiative database (LIDC-IDRI). All results are obtained via 10-fold cross validation. As regards the MV-CNN with chain architecture, results show that the performance of 3D MV-CNN surpasses that of 2D MV-CNN by a significant margin. Finally, a 3D Inception network achieved an error rate of 4.59% for the binary classification and 7.70% for the ternary classification, both of which represent superior results for the corresponding task. We compare the multi-view-one-network strategy with the one-view-one-network strategy. The results reveal that the multi-view-one-network strategy can achieve a lower error rate than the one-view-one-network strategy.

[Effect of pH Value on Autotrophic Denitrification Process of Zero Valent Iron Substrate].

Using a substrate with zero valent iron and nitrate, the research determined the change of pH value in the iron autotrophic denitrification process and the effect of pH on zero valent iron autotrophic denitrification activated sludge using batch experiments and a continuous flow fermenter denitrification rate. Batch experiments were carried out with four reactor bottles with sludge addition. The initial pH values were 6.2, 6.7, 7.5, and 8.8, respectively, and there was an initial pH of 6.7 in a bottle without sludge addition. The results showed that the initial pH value of 6.7 obtained the highest nitrogen removal rate. For the bottle without sludge addition, the pH rose to about 10. The pH value of the four batch experiments was concentrated between 7.5-7.8 in the follow-up process, so there was no significant difference in the effects of pH on denitrifying bacteria. However, the pH value in the fermenter can be controlled stably by an acid-base pump. With five pH gradients of 6, 6.5, 7, 7.5, and 8, the adaptability and activity of microorganisms under a constant pH value were observed separately. The sludge activity was the highest at pH 6.5, and the highest nitrogen removal rate was 1.35 mg·(L·h)-1.

[Activated Sludge Mineralization and Solutions in the Process of Zero-Valent Iron Autotrophic Denitrification].

We studied the inoculation of activated sludge from domestic sewage treatment plants. The reaction of reducing nitrate by zero-valent iron was started in the up-flow anaerobic reactor. After 52 d of operation, a nitrogen removal rate of 29.3 g·(m3·d)-1 was achieved. Ferric iron and iron oxide coated on the sludge formed in the operation process caused the mineralization and slowly decreased the sludge activity. The methods of the "supplement and replacement of the sludge in the reactor" and "changing the reflux mode of the reactor" were applied. Using the method of the supplement and replacement of the sludge in the reactor, by discharging a part of the sludge in the reactor and adding the extra dosage from the anaerobic denitrifying sludge for the treatment of wastewater, after 22 d of operation, the conversion rate of nitrate-nitrogen was 33.0 g·(m3·d)-1 and the concentration of nitrite-nitrogen was 16.50 mg·L-1. The effluent average concentration of ammonia-nitrogen decreased from 12.38 mg·L-1 to 3.58 mg·L-1 and the nitrogen removal rate was recovered from 9.9 g·(m3·d)-1to 15.0 g·(m3·d)-1. The biological reaction weakened the chemical reduction of nitrate by zero-valent iron. Using the method of changing the reflux mode of the reactor, the reflux tank was arranged outside the reaction column using hydraulic circulation. The increase in the erosion of the upper layer of the sedimentation tank would bring out ferric iron and iron oxide with the outflow water and they would be deposited in the reflux tank. The corresponding ferric iron of transformation of nitrate settled in the external reflux tank was 58% at the ascending velocity of 3.49 m·h-1. The nitrate-nitrogen conversion rate was 34.3 g·(m3·d)-1, the effluent concentration of nitrite-nitrogen was 0.22 mg·L-1, and ammonia-nitrogen was 0.75 mg·L-1. Ammonia and nitrite did not extensively accumulate. The nitrogen removal rate was 33.4 g·(m3·d)-1, which solved the problem of the mineralized sludge in the long-term reactor operation. In summary, the method of reforming the reflux mode of the reactor performed better than the method of the supplement and replacement of sludge in the reactor.

Mass spectrometry based strategy for studies of binding sites and structural changes of cisplatin binding to myoglobin.

RATIONALE: It is of great significance to investigate the interaction of the metallodrug cisplatin (cis-[PtCl2 (NH3 )2 ]) with myoglobin for understanding of the mechanism of action of cisplatin and the overexpression of myoglobin in tumor cells. METHODS: The reactions of cisplatin and myoglobin were incubated under different conditions. A mass spectrometry (MS)-based strategy combining full proteolysis and limited proteolysis was developed for comprehensive studies of cisplatin-myoglobin interaction. RESULTS: The binding sites of cisplatin on myoglobin were identified as Trp14, His64, His81, His113 and His116 using electrospray ionization multiple-stage tandem mass spectrometry (ESI-MSn ) without liquid chromatography (LC) separation. The relative abundances of digested peptides from platinated myoglobin were obviously higher than those from native samples by limited proteolysis. CONCLUSIONS: An alternative and simple approach was developed to successfully monitor conformational changes of myoglobin induced by cisplatin binding using an ESI-MS-based quantification method combined with limited proteolysis. Meanwhile, His64 was firstly found to coordinate to platinum, which was likely to affect hydrogen bonds with the oxygen in the heme group. Copyright © 2016 John Wiley & Sons, Ltd.

Self-assembly of nucleic acid molecular aggregates catalyzed by a triple-helix probe for miRNA detection and single cell imaging.

We herein report a novel finding that nucleic acid molecular aggregates (NAMAs) self-assembled on graphene oxide nanoplates (GONPs) as a result of DNA rolling circle amplification (RCA) and a functionalized triple-helix probe (THP) in single cells. The functionalized THP containing the aptamer region for target recognition and the trigger DNA region for RCA was firstly used to activate RCA for miRNA imaging in single cells. Interestingly, NAMAs with the fluorescent labels were hybridized by both the RCA products and FAM-DNA, and could partly self-assemble on GONPs; meanwhile, NAMAs could extend from the GONPs, which led to the quenched fluorescence being renewed. Significantly, the NAMAs were successfully applied for low-abundance miRNA detection and imaging in single cells. The self-assembled NAMAs could generate prominent and agminated fluorescence-bright spots in single cancer cells, which will effectively drive cell imaging into a new era.

Direct determination of the binding sites of cisplatin on insulin-like growth factor-1 by top-down mass spectrometry.

Cisplatin has been widely used in the chemotherapy of a variety of tumors, and the interactions of cisplatin with proteins play very important roles in its side effects and drug resistance, as well as its pharmacokinetics and the biodistribution. Insulin-like growth factor-1 (IGF-1) was found to be associated with the drug resistance of cisplatin. Here, the interaction between cisplatin and IGF-1 was investigated using electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry. IGF-1-Pt(NH3)Cl was the main mono-adduct and the trans labilization was important to the reaction between IGF-1 and cisplatin, while another special mono-adduct IGF-1-Pt(NH3)Cl2 was observed. The rapid and sensitive top-down mass spectrometry-based approach in linear ion trap mass spectrometer has been developed to identify the binding sites of cisplatin in IGF-1 directly without tedious enzyme digestion. Three binding sites (Met59, Arg56 and Cys6) of cisplatin in IGF-1 were determined. The results not only provide a rapid and efficient way to identify the platinum binding sites in proteins, but also indicate that the binding of cisplatin could promote the fragmentation of IGF-1 and the rupture of disulfide bond.

Mass spectrometry-based analysis of glycoproteins and its clinical applications in cancer biomarker discovery.

Glycosylation is one of the most important posttranslational modifications of proteins and plays essential roles in various biological processes. Aberration in the glycan moieties of glycoproteins is associated with many diseases. It is especially critical to develop the rapid and sensitive methods for analysis of aberrant glycoproteins associated with diseases. Mass spectrometry (MS) has become a powerful tool for glycoprotein analysis. Especially, tandem mass spectrometry can provide highly informative fragments for structural identification of glycoproteins. This review provides an overview of the development of MS technologies and their applications in identification of abnormal glycoproteins and glycans in human serum to screen cancer biomarkers in recent years.

Comprehensive identification of the binding sites of cisplatin in hen egg white lysozyme.

Platinum drugs have become one of the most important kinds of chemotherapy agents, and the interactions of these drugs with proteins play very important roles in their side effects and drug resistance. However, it is still a challenge to determine the binding sites of platinum drugs in proteins with multiple disulfide bonds and stable three-dimensional structures using mass spectrometry. Here, the interaction between cisplatin and hen egg white lysozyme (HEWL), a multi-disulfide-bond-containing protein with a stable three-dimensional structure, was investigated using Fourier transform ion cyclotron resonance mass spectrometry. Typical disulfide bond reduction with dithiothreitol/tris(2-carboxyethyl)phosphine before trypsin digestion destroyed the binding of cisplatin to HEWL, and no platination sites were found. Efficient trypsin digestion methods for HEWL-cisplatin adducts were developed to avoid the loss of platinum binding to protein. At 55 °C, platinated HEWL was digested directly by trypsin in 6 h, and multiple platinated peptides were observed. In 60% acetonitrile, the digestion time of platinated HEWL was shortened to 2 h, and most of the platinated peptides were observed. In addition, the reduction of the disulfide bonds of HEWL greatly accelerated the reaction between HEWL and cisplatin, and the potential binding sites of cisplatin in reduced HEWL could be easily recognized. On the basis of the above-mentioned methods, multiple binding sites of cisplatin in HEWL were first identified by mass spectrometry.

Target-induced self-assembly of DNA nanomachine on magnetic particle for multi-amplified biosensing of nucleic acid, protein, and cancer cell.

A biosensing system is established for the multi-amplified detection of DNA or specific substrates of aptamers under isothermal conditions, which combines nicked rolling circle amplification (N-RCA) and beacon assisted amplification (BAA) with sensitive colorimetric technique by using DNAzymes as reporter units. According to the configuration, the analysis of DNA is accomplished by recognizing the target to capture nucleic acid-functionalized magnetic particles, followed by the self-assembly of the other two nucleic acids into multicomponent DNA supramolecular structure on magnetic particles. After magnetic separation, the circularization with ligase and the fragmentation with polymerase activate N-RCA and BAA in the presence of polymerase, dNTPs, and the nicking endonuclease, successively producing horseradish peroxidase (HRP)-mimicking DNAzymes that act as colorimetric reporter to catalyze the oxidation of ABTS(2-) by H2O2 in the presence of hemin. Under the optimized conditions, we obtain a wide dynamic range for DNA analysis over 6 orders of magnitude from 1.0 × 10(-14) to 1.0 × 10(-9)M with a low limit of detection of 6.8 × 10(-15)M. In the absence of a target, neither self-assembly of nucleic acids nor amplification process can be initiated, indicating an excellent selectivity of the proposed strategy. Similarly, an analogous system is activated by cancer cells or lysozyme through cooperative self-assembly of nucleic acids on magnetic particles in the presence of respective substrates of aptamers to synthesize HRP-mimicking DNAzymes that give the readout signal for the recognition events, achieving LODs of 81 Ramos cells and 7.2 × 10(-15)M lysozyme, respectively.

Probing the interaction of cisplatin with cytochrome C by electrospray ionization Fourier transform ion cyclotron resonance mass spectrometry.

As one of the most important platinum drugs, the interactions of cisplatin with proteins play very important roles in its anticancer action and side effects. Here, the interaction of cisplatin with cytochrome c was investigated using Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS). On the basis of the high-resolution data, cytochrome c-Pt(NH(3))Cl was found to be the primary monoadduct. The platinated monoadducts were related to molar ratios of cytochrome c and cisplatin, and corresponding reaction pathways were proposed. Multiple binding sites of cisplatin on cytochrome c were directly determined by FTICR MS combined with trypsin digestion without liquid chromatography (LC) separation. Four binding sites (Met65, Met80, His18, and His33) for cisplatin on cytochrome c were identified. Moreover, hydrogen/deuterium exchange (H/DX) combined with FTICR MS provides the sensitive method to insight the small conformation change of cytochrome c induced by cisplatin. This strategy can be applied to comprehensive investigation of metallodrug/protein complexes.

Studies of interaction between insulin and glutathione using electrospray ionization mass spectrometry.

RATIONALE: The interaction of glutathione (GSH) with insulin plays an important role in the degradation or regulation of insulin. The characterization of the reaction products of GSH and insulin is very important for a proper understanding of the mechanism of insulin regulation of GSH. METHODS: Solutions of insulin and glutathione were incubated under different experimental conditions in vitro. The reaction products were determined by electrospray ionization (ESI) ion trap mass spectrometry combined with Fourier transform ion cyclotron resonance (FTICR) mass spectrometry. RESULTS: The multi-reaction products were identified, including insulin A chain with two intrachain disulfides, insulin B chain with one intrachain disulfides, GSH-modified insulin, GSH-modified A chain, GSH-modified B chain, aggregates of A chain and B chain, and reduced A chain and B chain. The binding site of the B chain with insulin was determined directly by tandem mass spectrometry (MS/MS) without enzyme digestion. It was found that the reaction between GSH and insulin was pH-, O(2)- and temperature-dependent. CONCLUSIONS: The results provide insight into the interaction between GSH and insulin. It has also been demonstrated that ESI-MS combined with high-resolution FTICRMS and MS/MS provides a powerful tool for screening the reactions of proteins and small molecules.

Network target for screening synergistic drug combinations with application to traditional Chinese medicine.

BACKGROUND: Multicomponent therapeutics offer bright prospects for the control of complex diseases in a synergistic manner. However, finding ways to screen the synergistic combinations from numerous pharmacological agents is still an ongoing challenge. RESULTS: In this work, we proposed for the first time a "network target"-based paradigm instead of the traditional "single target"-based paradigm for virtual screening and established an algorithm termed NIMS (Network target-based Identification of Multicomponent Synergy) to prioritize synergistic agent combinations in a high throughput way. NIMS treats a disease-specific biological network as a therapeutic target and assumes that the relationship among agents can be transferred to network interactions among the molecular level entities (targets or responsive gene products) of agents. Then, two parameters in NIMS, Topology Score and Agent Score, are created to evaluate the synergistic relationship between each given agent combinations. Taking the empirical multicomponent system traditional Chinese medicine (TCM) as an illustrative case, we applied NIMS to prioritize synergistic agent pairs from 63 agents on a pathological process instanced by angiogenesis. The NIMS outputs can not only recover five known synergistic agent pairs, but also obtain experimental verification for synergistic candidates combined with, for example, a herbal ingredient Sinomenine, which outperforms the meet/min method. The robustness of NIMS was also showed regarding the background networks, agent genes and topological parameters, respectively. Finally, we characterized the potential mechanisms of multicomponent synergy from a network target perspective. CONCLUSIONS: NIMS is a first-step computational approach towards identification of synergistic drug combinations at the molecular level. The network target-based approaches may adjust current virtual screen mode and provide a systematic paradigm for facilitating the development of multicomponent therapeutics as well as the modernization of TCM.

Effect of additional boron on tibias of African ostrich chicks.

The aim of the present study was to find out the effects of boron on ostrich chicks fed with 0 mg/l, 100 mg/l, 200 mg/l, and 400 mg/l of additional boron in water. We measured bone mineral density (BMD), perimeter, length, weight, ash content of ostrich tibias, thickness of cortical bone, and diameter of the marrow cavity. We also analyzed the apoptosis status of paraffin sections using a TUNEL kit and examined serum levels of leptin and estradiol (E(2)). The results were dramatic. Compared with the control group, group C had a very high BMD. The serum levels of leptin in groups C and D were significantly higher than control values, and the levels of E(2) fluctuated. The perimeter, length, weight, and ash content of ostrich tibias all increased significantly with increasing dosage of boron. The cross-section analysis revealed that the bone marrow cavity shifted closer to one side in group D, which was observed on a macro-scale. This shift may be related to the toxicity of excessive boron, as indicated by the apoptosis status. According to the present data, additional boron was helpful for ostrich chick bone development, and 200 mg/l supplement boron in the drinking water appeared to be the most beneficial.

Herb network construction and co-module analysis for uncovering the combination rule of traditional Chinese herbal formulae.

BACKGROUND: Traditional Chinese Medicine (TCM) is characterized by the wide use of herbal formulae, which are capable of systematically treating diseases determined by interactions among various herbs. However, the combination rule of TCM herbal formulae remains a mystery due to the lack of appropriate methods. METHODS: From a network perspective, we established a method called Distance-based Mutual Information Model (DMIM) to identify useful relationships among herbs in numerous herbal formulae. DMIM combines mutual information entropy and "between-herb-distance" to score herb interactions and construct herb network. To evaluate the efficacy of the DMIM-extracted herb network, we conducted in vitro assays to measure the activities of strongly connected herbs and herb pairs. Moreover, using the networked Liu-wei-di-huang (LWDH) formula as an example, we proposed a novel concept of "co-module" across herb-biomolecule-disease multilayer networks to explore the potential combination mechanism of herbal formulae. RESULTS: DMIM, when used for retrieving herb pairs, achieves a good balance among the herb's frequency, independence, and distance in herbal formulae. A herb network constructed by DMIM from 3865 Collaterals-related herbal formulae can not only nicely recover traditionally-defined herb pairs and formulae, but also generate novel anti-angiogenic herb ingredients (e.g. Vitexicarpin with IC50=3.2 µM, and Timosaponin A-III with IC50=3.4 µM) as well as herb pairs with synergistic or antagonistic effects. Based on gene and phenotype information associated with both LWDH herbs and LWDH-treated diseases, we found that LWDH-treated diseases show high phenotype similarity and identified certain "co-modules" enriched in cancer pathways and neuro-endocrine-immune pathways, which may be responsible for the action of treating different diseases by the same LWDH formula. CONCLUSIONS: DMIM is a powerful method to identify the combination rule of herbal formulae and lead to new discoveries. We also provide the first evidence that the co-module across multilayer networks may underlie the combination mechanism of herbal formulae and demonstrate the potential of network biology approaches in the studies of TCM.