Metformin Promotes Antitumor Immunity via Endoplasmic-Reticulum-Associated Degradation of PD-L1.

Metformin has been reported to possess antitumor activity and maintain high cytotoxic T lymphocyte (CTL) immune surveillance. However, the functions and detailed mechanisms of metformin's role in cancer immunity are not fully understood. Here, we show that metformin increases CTL activity by reducing the stability and membrane localization of programmed death ligand-1 (PD-L1). Furthermore, we discover that AMP-activated protein kinase (AMPK) activated by metformin directly phosphorylates S195 of PD-L1. S195 phosphorylation induces abnormal PD-L1 glycosylation, resulting in its ER accumulation and ER-associated protein degradation (ERAD). Consistently, tumor tissues from metformin-treated breast cancer patients exhibit reduced PD-L1 levels with AMPK activation. Blocking the inhibitory signal of PD-L1 by metformin enhances CTL activity against cancer cells. Our findings identify a new regulatory mechanism of PD-L1 expression through the ERAD pathway and suggest that the metformin-CTLA4 blockade combination has the potential to increase the efficacy of immunotherapy.

Portable particle mass spectrometer.

Particle pollutants in air have been confirmed to damage human health. The PM10 concentration is an important parameter for air quality determination. In this study, a portable quadrupole ion trap mass spectrometer (QIT-MS) was developed and used to quantitate microparticles and particulate standards. The instrument can be used to perform online analysis of various microsized particles. The instrument can be used to analyze various sizes of disperse particles with accurate mass by a histogram profile. The overall detection efficiencies of particles in the sample for polystyrene were obtained. PM10-like reference materials were used for calibration to analyze the size and mass distribution of an environmental sample. The instrument shows the potential for quantitation of different particles of an unknown sample.

Comprehensive Workflow for Mapping Disulfide Linkages Including Free Thiols and Error Checking by On-Line UV-Induced Precolumn Reduction and Spiked Control.

Mapping highly complicated disulfide linkages and free thiols via liquid chromatography-tandem mass spectrometry (LC-MS2) is challenging because of the difficulties in optimizing sample preparation to acquire critical MS data and detecting mispairings. Herein, we report a highly efficient and comprehensive workflow using an on-line UV-induced precolumn reduction tandem mass spectrometry (UV-LC-MS2) coupled with two-stage data analysis and spiked control. UV-LC-MS2 features a gradient run of acetonitrile containing a tunable percentage of photoinitiators (acetone/alcohol) that drives the sample to the MS through a UV-flow cell and reverse phase column to separate UV-induced products for subsequent fragmentation via low energy collision-induced dissociation. This allowed the alkylated thiol-containing and UV-reduced cysteine-containing peptides to be identified by a nontargeted database search. Expected or unexpected disulfide/thiol mapping was then carried out based on the search results, and data were derived from partially reduced species by photochemical reaction. Complete assignments of native and scrambled disulfide linkages of insulin, α-lactalbumin, and bovine serum albumin (BSA) as well as the free C34-BSA were demonstrated using none or single enzyme digestion. This workflow was applied to characterize unknown disulfide/thiol patterns of the recombinant cyclophilin 1 monomer (rTvCyP1 mono) from the human pathogen Trichomonas vaginalis. α-Lactalbumin was judiciously chosen as a spiked control to minimize mispairings due to sample preparation. rTvCyP1 was determined to contain a high percentage of thiol (>80%). The rest of rTvCyP1 mono were identified to contain two disulfide/thiol patterns, of which C41-C169 linkage was confirmed to exist as C53-C181 in rTvCyP2, a homologue of rTvCyP1. This platform identifies heterogeneous protein disulfide/thiol patterns in a de-novo fashion with artifact control, opening up an opportunity to characterize crude proteins for many applications.

Development of a focused high-energy macromolecular ion beam.

In this work, we report the development of a focused macromolecular ion beam with kinetic energy of up to 110 keV. The system consists of a quadrupole ion trap (QIT), einzel lens and linear accelerator (LINAC). Based on the combination of matrix-assisted laser desorption ionization (MALDI) and quadrupole ion trapping (QIT), ions were desorbed from the surface and trapped with an ion trap to form biomolecular ion packets. Positive- and negative-pulsed voltages were applied on each end-cap electrode of the QIT to extract the ion packets and form an ion beam that was subsequently focused via an einzel lens and accelerated by stepwise pulsed voltages. The tabletop instrument was designed and successfully demonstrated via measurements of molecular ions of insulin, cytochrome c and bovine serum albumin (BSA) with mass-to-charge ratios (m/z) ranging from ∼5.8 to 66.5 k. This is the first report of both a focused and high-kinetic-energy protein ion beam. In addition, both secondary ions and electrons were observed from the surface by hypervelocity ion beam bombardment. This focused macromolecular ion beam has demonstrated its potential in the study of interactions between large molecular ions with other molecules either in the gas phase or upon a surface.

Ash2l interacts with Oct4-stemness circuitry to promote super-enhancer-driven pluripotency network.

Pluripotency and cell fates can be modulated through the regulation of super-enhancers; however, the underlying mechanisms are unclear. Here, we showed a novel mechanism in which Ash2l directly binds to super-enhancers of several stemness genes to regulate pluripotency and self-renewal in pluripotent stem cells. Ash2l recruits Oct4/Sox2/Nanog (OSN) to form Ash2l/OSN complex at the super-enhancers of Jarid2, Nanog, Sox2 and Oct4, and further drives enhancer activation, upregulation of stemness genes, and maintains the pluripotent circuitry. Ash2l knockdown abrogates the OSN recruitment to all super-enhancers and further hinders the enhancer activation. In addition, CRISPRi/dCas9-mediated blocking of Ash2l-binding motifs at these super-enhancers also prevents OSN recruitment and enhancer activation, validating that Ash2l directly binds to super-enhancers and initiates the pluripotency network. Transfection of Ash2l with W118A mutation to disrupt Ash2l-Oct4 interaction fails to rescue Ash2l-driven enhancer activation and pluripotent gene upregulation in Ash2l-depleted pluripotent stem cells. Together, our data demonstrated Ash2l formed an enhancer-bound Ash2l/OSN complex that can drive enhancer activation, govern pluripotency network and stemness circuitry.

A portable multiple ionization source biological mass spectrometer.

In the past, matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI), used for large biomolecule detection, were usually installed in two separate mass spectrometers. In this study, they were equipped in the same mass spectrometer. This portable biological mass spectrometer has multiple ionization capabilities in the same mass spectrometer and shares the same mass analyzer and detector. This mass spectrometer can be operated under low vacuum (∼10-3 Torr) and can use air as the buffer gas. Therefore, the demand for pumping is reduced and rare gas feeding is no longer essential. A small scroll pump, employed to assist a miniature turbo pump, is sufficient to maintain the operational pressure. The mass spectra of biomolecules were obtained using frequency scanning instead of voltage ramping. Therefore, a wider mass-to-charge ratio (m/z) range was achieved. Furthermore, the design also couples a conversion dynode with a channeltron to enhance the mass detection range. This homemade mass spectrometer has the capability to measure charged particles with very large m/z values (m/z > 100 000). The concentrations of the studied compounds (angiotensin, insulin, cytochrome C, bovine serum albumin (BSA), immunoglobulin G, and immunoglobulin A) are from 5 femtomole to 100 picomole, and the mass resolutions are from 30 to 260. The mass range of this portable mass spectrometer was comparable with a commercial linear time-of-flight mass spectrometer owing to the use of the frequency scan.

IKKα activation of NOTCH links tumorigenesis via FOXA2 suppression.

Proinflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report a TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.

High accuracy differentiating autoimmune pancreatitis from pancreatic ductal adenocarcinoma by immunoglobulin G glycosylation.

BACKGROUND: Misdiagnosis of autoimmune pancreatitis (AIP) as pancreatic cancer (PDAC) or vice versa can cause dismal patents' outcomes. Changes in IgG glycosylation are associated with cancers and autoimmune diseases. This study investigated the IgG glycosylation profiles as diagnostic and prognostic biomarkers in PDAC and AIP. METHODS: Serum IgG-glycosylation profiles from 86 AIP patients, 115 PDAC patients, and 57 controls were analyzed using liquid chromatography-electrospray ionization mass spectrometry. Classification and regression tree (CART) analysis was applied to build a decision tree for discriminating PDAC from AIP. The result was validated in an independent cohort. RESULTS: Compared with AIP patients and controls, PDAC patients had significantly higher agalactosylation, lower fucosylation, and sialylation of IgG1, a higher agalactosylation ratio of IgG1 and a higher agalactosylation ratio of IgG2. AIP patients had significantly higher fucosylation of IgG1 and a higher sialylation ratio of IgG subclasses 1, 2 and 4. Using the CART analysis of agalactosylation and sialylation ratios in the IgG to discriminate AIP from PDAC, the diagnostic accuracy of the glycan markers was 93.8% with 94.6% sensitivity and 92.9% specificity. There were no statistically significant difference of IgG-glycosylation profiles between diffuse type and focal type AIP. CONCLUSIONS: AIP and PDAC patients have distinct IgG-glycosylation profilings. IgG-glycosylation could different PDAC from AIP with high accuracy.

A quadrupole ion trap mass spectrometer for dry microparticle analysis.

In this work, we report a new design of a charge detection quadrupole ion trap mass spectrometer (QIT-MS) for the analysis of micro-sized dry inorganic and bioparticles including red blood cells (RBCs) and different sizes of MCF-7 breast cancer cells. The developed method is one of the fastest methods to measure the mass of micro-sized particles. This system allows the online analysis of various micro-sized particles up to 1 × 1017 Da. The calibration of the mass spectrometer has been done by using different sizes of polystyrene (PS) particles (2-15 µm). The measured masses of RBCs were around 1.8 × 1013 Da and MCF-7 cancer cells were between 1 × 1014 and 4 × 1014 Da. The calculated mass distribution profiles of the particles and cells were given as histogram profiles. The statistical data were summarized after Gaussian type fitting to the experimental histogram profiles. The new method gives very promising results for the analysis of particles and has very broad application.

EGFR modulates microRNA maturation in response to hypoxia through phosphorylation of AGO2.

MicroRNAs (miRNAs) are generated by two-step processing to yield small RNAs that negatively regulate target gene expression at the post-transcriptional level. Deregulation of miRNAs has been linked to diverse pathological processes, including cancer. Recent studies have also implicated miRNAs in the regulation of cellular response to a spectrum of stresses, such as hypoxia, which is frequently encountered in the poorly angiogenic core of a solid tumour. However, the upstream regulators of miRNA biogenesis machineries remain obscure, raising the question of how tumour cells efficiently coordinate and impose specificity on miRNA expression and function in response to stresses. Here we show that epidermal growth factor receptor (EGFR), which is the product of a well-characterized oncogene in human cancers, suppresses the maturation of specific tumour-suppressor-like miRNAs in response to hypoxic stress through phosphorylation of argonaute 2 (AGO2) at Tyr 393. The association between EGFR and AGO2 is enhanced by hypoxia, leading to elevated AGO2-Y393 phosphorylation, which in turn reduces the binding of Dicer to AGO2 and inhibits miRNA processing from precursor miRNAs to mature miRNAs. We also identify a long-loop structure in precursor miRNAs as a critical regulatory element in phospho-Y393-AGO2-mediated miRNA maturation. Furthermore, AGO2-Y393 phosphorylation mediates EGFR-enhanced cell survival and invasiveness under hypoxia, and correlates with poorer overall survival in breast cancer patients. Our study reveals a previously unrecognized function of EGFR in miRNA maturation and demonstrates how EGFR is likely to function as a regulator of AGO2 through novel post-translational modification. These findings suggest that modulation of miRNA biogenesis is important for stress response in tumour cells and has potential clinical implications.

Whole-genome methylation profiling of peripheral blood mononuclear cell for acute exacerbations of chronic obstructive pulmonary disease treated with corticosteroid.

OBJECTIVE: Although association studies in the general population may be relevant for determining susceptibility to chronic obstructive pulmonary disease (COPD), they may be less applicable for pharmacogenetics research in participants who have already acquired the disease. PATIENTS AND METHODS: A genome-wide methylation profiling (generated by HumanMethylation450 BeadChips study was performed on peripheral blood mononuclear cells of 24 patients with AECOPD (acute exacerbation COPD), with good and poor responsiveness to standard corticosteroid treatment. Pyrosequencing was used to replicate the selected CpG sites in 50 patients with AECOPD with standard corticosteroid treatment. RESULTS: The results showed the patients with AECOPD with good and poor response to standard corticosteroid treatment have a distinct DNA methylation pattern. A total of 23 CpG loci located in 19 known gene regions, including gene-body and promoter, appeared to be significantly differentially methylated. Replication by pyrosequencing revealed that one CpG site in PSMD8 showed the same trend of differential methylation and reached to statistical significance as the microarray result. CONCLUSION: Our preliminary findings provide evidence for molecular heterogeneity in patients with AECOPD, which may contribute to significant differences in their response to COPD treatment.

Biomolecular Clusters Distribution up to Mega Dalton Region Using MALDI-Quadrupole Ion Trap Mass Spectrometer.

We present the first report on complete cluster distributions of cytochrome c (molecular weight of 12.4 kDa) and bovine serum albumin ((BSA), molecular weight of 66.4 kDa) with mass-to-charge ratio (m/z) reaching 350,000 and 1,400,000, respectively, by matrix-assisted laser desorption/ionization (MALDI). Large cluster distributions of the analytes were measured by our homemade frequency-scanned quadrupole ion trap (QIT) mass spectrometer with a charge detector. To our knowledge, we report the highest m/z clusters of these two biomolecules. The quantitative results indicate that large clusters ions of cytochrome c and BSA follow the power law (r² > 0.99) with cluster size distribution, which provides experimental evidence for the laser ablation studies of MALDI.

ESI MS for Microsized Bioparticles.

An ESI ion trap mass spectrometer was designed for high-throughput and rapid mass analysis of large bioparticles. Mass calibration of the instrument was performed using commercially available polystyrene (PS) microparticles with a size comparable to cancer cells. Different sizes of MCF-7 breast cancer cells (8 to 15 µm) were used in this study. The masses of different cancer cells were measured. This system allows for the analysis of all types of particles.

Human CLEC18 Gene Cluster Contains C-type Lectins with Differential Glycan-binding Specificity.

The human C-type lectin 18 (clec18) gene cluster, which contains three clec18a, clec18b, and clec18c loci, is located in human chromosome 16q22. Although the amino acid sequences of CLEC18A, CLEC18B, and CLEC18C are almost identical, several amino acid residues located in the C-type lectin-like domain (CTLD) and the sperm-coating protein/Tpx-1/Ag5/PR-1/Sc7 (SCP/TAPS) domain, also known as the cysteine-rich secretory proteins/antigen 5/pathogenesis-related 1 proteins (CAP) domain, are distinct from each other. Genotyping by real-time PCR and sequencing further shows the presence of multiple alleles in clec18a/b/c loci. Flow cytometry analysis demonstrates that CLEC18 (CLEC18A, -B, and -C) are expressed abundantly in human peripheral blood cells. Moreover, CLEC18 expression is further up-regulated when monocytes differentiate into macrophages and dendritic cells. Immunofluorescence staining reveals that CLEC18 are localized in the endoplasmic reticulum, Golgi apparatus, and endosome. Interestingly, CLEC18 are also detectable in human sera and culture supernatants from primary cells and 293T cells overexpressing CLEC18. Moreover, CLEC18 bind polysaccharide in Ca(2+)-independent manner, and amino acid residues Ser/Arg(339) and Asp/Asn(421) in CTLD domain contribute to their differential binding abilities to polysaccharides isolated from Ganoderma lucidum (GLPS-F3). The Ser(339) (CLEC18A) → Arg(339) (CLEC18A-1) mutation completely abolishes CLEC18A-1 binding to GLPS-F3, and a sugar competition assay shows that CLEC18 preferentially binds to fucoidan, ß-glucans, and galactans. Because proteins with the SCP/TAPS/CAP domain are able to bind sterol and acidic glycolipid, and are involved in sterol transport and ß-amyloid aggregation, it would be interesting to investigate whether CLEC18 modulates host immunity via binding to glycolipids, and are also involved in glycolipid transportation and protein aggregation in the future.

Inactivation of the particulate methane monooxygenase (pMMO) in Methylococcus capsulatus (Bath) by acetylene.

Acetylene (HCCH) has a long history as a mechanism-based enzyme inhibitor and is considered an active-site probe of the particulate methane monooxygenase (pMMO). Here, we report how HCCH inactivates pMMO in Methylococcus capsulatus (Bath) by using high-resolution mass spectrometry and computational simulation. High-resolution MALDI-TOF MS of intact pMMO complexes has allowed us to confirm that the enzyme oxidizes HCCH to the ketene (C2H2O) intermediate, which then forms an acetylation adduct with the transmembrane PmoC subunit. LC-MS/MS analysis of the peptides derived from in-gel proteolytic digestion of the protein subunit identifies K196 of PmoC as the site of acetylation. No evidence is obtained for chemical modification of the PmoA or PmoB subunit. The inactivation of pMMO by a single adduct in the transmembrane PmoC domain is intriguing given the complexity of the structural fold of this large membrane-protein complex as well as the complicated roles played by the various metal cofactors in the enzyme catalysis. Computational studies suggest that the entry of hydrophobic substrates to, and migration of products from, the catalytic site of pMMO are controlled tightly within the transmembrane domain. Support of these conclusions is provided by parallel experiments with two related alkynes: propyne (CH3CCH) and trifluoropropyne (CF3CCH). Finally, we discuss the implication of these findings to the location of the catalytic site in pMMO.

An aptamer targeting shared tumor-specific peptide antigen of MAGE-A3 in multiple cancers.

A DNA aptamer was identified against the shared tumor-specific MAGE-A3111-125 peptide antigen. The dissociation constant between the aptamer and the peptide was measured at 57 nM. Binding of the aptamer to seven types of cancer cells, melanoma, breast, colorectal, liver, lung, pancreas and oral cancer, was confirmed with flow cytometry and fluorescence imaging. Cy3-conjugated aptamers signals were specifically localized to the surface of those cancer cells. The results indicate that the DNA aptamer against the shared tumor-specific MAGE-A3 peptide can be used in cancer cell targeting and has the potential for developing into new modalities for the diagnosis of multiple cancers.

Investigation of non-covalent complexations of Ca(II) and Mg(II) ions with insulin by using electrospray ionization mass spectrometry.

RATIONALE: Insulin is a peptide hormone secreted by pancreatic ß-cells. Ca(II) and Mg(II) ions play an important role in the secretion of insulin. There is no study about a direct complexation of Ca(II) or Mg(II) with insulin and their equilibrium constants. Electrospray ionization mass spectrometry (ESI-MS) is a practical method for the monitoring of non-covalent complexes such as Ca(II)-insulin and Mg(II)-insulin. Here, the equilibrium constants of Ca(II)-insulin and Mg(II)-insulin non-covalent complexes have been calculated after ESI-MS measurements in aqueous solutions. METHODS: The effects of pH, competitive binding, ion exchange, and Na(I) and K(I) ions on Ca(II)-insulin and Mg(II)-insulin complexation have been examined by measuring by ESI-MS. The dissociation equilibrium constants (K1 and K2 ) of Ca(II)-insulin and Mg(II)-insulin complexes were calculated from the binomial graph derived from the ESI-MS normalized peak intensities. The MS/MS spectra of the complexes have been examined. RESULTS: The dissociation equilibrium constants were found to K1 : 1.29 × 10(-4)  M and K2 : 9.69 × 10(-4)  M for the Ca(II)-insulin complexes, and K1 : 1.37 × 10(-4)  M and K2 : 9.12 × 10(-4)  M for Mg(II)-insulin complexes. Ca(II) ions have higher complexation capability with insulin than Mg(II) ions. CONCLUSIONS: The binding equilibrium constants of Ca(II)- and Mg(II)-insulin non-covalent complexes have been determined successfully by ESI-MS. Ca(II) and Mg(II) ions are involved in the insulin secretion by forming non-covalent complexes. Copyright © 2016 John Wiley & Sons, Ltd.

Near-Infrared Light-Mediated Photodynamic Therapy Nanoplatform by the Electrostatic Assembly of Upconversion Nanoparticles with Graphitic Carbon Nitride Quantum Dots.

Photodynamic therapy (PDT) is a promising antitumor treatment that is based on photosensitizers. This therapy kills cancer cells by generating reactive oxygen species (ROS) after irradiation with specific laser wavelengths. Being a potential photosensitizer, graphitic carbon nitride (g-C3N4) quantum dots (QDs) are noncytotoxic. Although the use of g-C3N4 QDs is challenged by the limited tissue penetration of UV light, g-C3N4 QDs display excellent ultraviolet (UV) light-triggered cytotoxicity. The g-C3N4 QDs were synthesized using a solid-phase hydrothermal method. The well-distributed hydrophilic g-C3N4 can be combined with NaYF4:Yb3+/Tm3+ upconversion nanoparticles via the positive ligand poly(l-lysine) to produce the final nanocomposite, NaYF4:Yb/Tm-PLL@g-C3N4. Upconversion nanoparticles can transfer IR light into UV light and promote g-C3N4 to release blue-to-green visible light to generate different images. Moreover, g-C3N4 is a promising photosensitizer in PDT because g-C3N4 can transfer oxygen into toxic ROS. The singlet oxygen formed by g-C3N4 displays great potential for use in the treatment of cancer.

Selective killing of cancer cells by nanoparticle-assisted ultrasound.

BACKGROUND: Intense ultrasound, such as that used for tumor ablation, does not differentiate between cancerous and normal cells. A method combining ultrasound and biocompatible gold or magnetic nanoparticles (NPs) was developed under in vitro conditions using human breast and lung epithelial cells, which causes ultrasound to preferentially destroy cancerous cells. RESULTS: Co-cultures of BEAS-2B normal lung cells and A549 cancerous lung cells labeled with green and red fluorescent proteins, respectively, were treated with focused ultrasound beams with the addition of gold and magnetic nanoparticles. There were significantly more necrotic A549 cells than BEAS-2 cells when gold nanoparticles were added to the culture medium [(50.6 ± 15.1) vs. (7.4 ± 2.9) %, respectively, P < 0.01]. This selective damage to cancer cells was also observed for MDA-MB231 breast cancer cells relative to MCF-10A normal breast cells after treatment with magnetic nanoparticles. CONCLUSIONS: The data obtained for different cell lines indicate that nanoparticle-assisted ultrasound therapy (NAUT) could be an effective new tool for cancer-specific treatment and could potentially be combined with conventional methods of cancer diagnosis and therapy to further increase the overall cancer cure rate.

Proteomics-Based Analysis of Protein Complexes in Pluripotent Stem Cells and Cancer Biology.

A protein complex consists of two or more proteins that are linked together through protein-protein interactions. The proteins show stable/transient and direct/indirect interactions within the protein complex or between the protein complexes. Protein complexes are involved in regulation of most of the cellular processes and molecular functions. The delineation of protein complexes is important to expand our knowledge on proteins functional roles in physiological and pathological conditions. The genetic yeast-2-hybrid method has been extensively used to characterize protein-protein interactions. Alternatively, a biochemical-based affinity purification coupled with mass spectrometry (AP-MS) approach has been widely used to characterize the protein complexes. In the AP-MS method, a protein complex of a target protein of interest is purified using a specific antibody or an affinity tag (e.g., DYKDDDDK peptide (FLAG) and polyhistidine (His)) and is subsequently analyzed by means of MS. Tandem affinity purification, a two-step purification system, coupled with MS has been widely used mainly to reduce the contaminants. We review here a general principle for AP-MS-based characterization of protein complexes and we explore several protein complexes identified in pluripotent stem cell biology and cancer biology as examples.