Overexpression of TSPAN8 in consensus molecular subtype 3 colorectal cancer.

BACKGROUND: Recently, consensus molecular subtypes (CMSs) have been proposed as a robust transcriptome-based classification system for colorectal cancer (CRC). Tetraspanins (TSPANs) are transmembrane proteins. They have been associated with the development of numerous malignancies, including CRC, through their role as "master organizers" for multi-molecular membrane complexes. No previous study has investigated the correlation between TSPANs and CMS classification. Herein, we investigated the expression of TSPANs in patient-derived primary CRC tissues and their CMS classifications. METHODS: RNA samples were derived from primary CRC tissues (n = 100 patients diagnosed with colorectal adenocarcinoma) and subjected to RNA sequencing for transcriptome-based CMS classification and TSPAN-relevant analyses. Immunohistochemistry (IHC) and immunofluorescence (IF) stains were conducted to observe the protein expression level. To evaluate the relative biological pathways, gene-set enrichment analysis was performed. RESULTS: Of the highly expressed TSPAN genes in CRC tissues (TSPAN8, TSPAN29, and TSPAN30), TSPAN8 was notably overexpressed in CMS3-classified primary tissues. The overexpression of TSPAN8 protein in CMS3 CRC was also observed by IHC and IF staining. As a result of gene-set enrichment analysis, TSPAN8 may potentially play a role in organizing signaling complexes for kinase-based metabolic deregulation in CMS3 CRC. CONCLUSIONS: The present study reports the overexpression of TSPAN8 in CMS3 CRC. This study proposes TSPAN8 as a subtype-specific biomarker for CMS3 CRC. This finding provides a foundation for future CMS-based studies of CRC, a complex disease and the second leading cause of cancer mortality worldwide.

Volatile Organic Compound Detection by Graphene Field-Effect Transistors Functionalized with Fly Olfactory Receptor Mimetic Peptides.

An olfactory receptor mimetic peptide-modified graphene field-effect transistor (gFET) is a promising solution to overcome the principal challenge of low specificity graphene-based sensors for volatile organic compound (VOC) sensing. Herein, peptides mimicking a fruit fly olfactory receptor, OR19a, were designed by a high-throughput analysis method that combines a peptide array and gas chromatography for the sensitive and selective gFET detection of the signature citrus VOC, limonene. The peptide probe was bifunctionalized via linkage of a graphene-binding peptide to facilitate one-step self-assembly on the sensor surface. The limonene-specific peptide probe successfully achieved highly sensitive and selective detection of limonene by gFET, with a detection range of 8-1000 pM, while achieving facile sensor functionalization. Taken together, our target-specific peptide selection and functionalization strategy of a gFET sensor demonstrates advancement of a precise VOC detection system.

Adenosine A2B receptor down-regulates metabotropic glutamate receptor 5 in astrocytes during postnatal development.

Metabotropic glutamate receptor 5 (mGluR5) in astrocytes is a key molecule for controlling synapse remodeling. Although mGluR5 is abundant in neonatal astrocytes, its level is gradually down-regulated during development and is almost absent in the adult. However, in several pathological conditions, mGluR5 re-emerges in adult astrocytes and contributes to disease pathogenesis by forming uncontrolled synapses. Thus, controlling mGluR5 expression in astrocyte is critical for several diseases, but the mechanism that regulates mGluR5 expression remains unknown. Here, we show that adenosine triphosphate (ATP)/adenosine-mediated signals down-regulate mGluR5 in astrocytes. First, in situ Ca2+ imaging of astrocytes in acute cerebral slices from post-natal day (P)7-P28 mice showed that Ca2+ responses evoked by (S)-3,5-dihydroxyphenylglycine (DHPG), a mGluR5 agonist, decreased during development, whereas those evoked by ATP or its metabolite, adenosine, increased. Second, ATP and adenosine suppressed expression of the mGluR5 gene, Grm5, in cultured astrocytes. Third, the decrease in the DHPG-evoked Ca2+ responses was associated with down-regulation of Grm5. Interestingly, among several adenosine (P1) receptor and ATP (P2) receptor genes, only the adenosine A2B receptor gene, Adora2b, was up-regulated in the course of development. Indeed, we observed that down-regulation of Grm5 was suppressed in Adora2b knockout astrocytes at P14 and in situ Ca2+ imaging from Adora2b knockout mice indicated that the A2B receptor inhibits mGluR5 expression in astrocytes. Furthermore, deletion of A2B receptor increased the number of excitatory synapse in developmental stage. Taken together, the A2B receptor is critical for down-regulation of mGluR5 in astrocytes, which would contribute to terminate excess synaptogenesis during development.

Proteomic Exploration of Membrane Curvature Sensors Using a Series of Spherical Supported Lipid Bilayers.

Membrane curvature-sensing (MCS) proteins recognize and regulate the morphologies of biological membranes. As these proteins lack characteristic sequence motifs in their primary structure, they are not instantly recognizable by genomic databases. Overcoming this technological challenge toward the agile identification of new proteins can promote the elucidation of membrane morphological regulation. Here, for the selective identification of MCS proteins, comparative proteomic analysis was performed using different sizes of the spherical supported lipid bilayer (SSLB), which consists of spherical SiO2 particles covered with a lipid bilayer. Because of the presence of SiO2 core, the curvature of the surrounding membrane is well-controlled and stable even on a micron scale. To prove this concept, known membrane curvature-sensing protein domains, Bin/Amphiphysin/Rvs (BAR) and Epsin N-terminal homology (ENTH), were evaluated by performing a binding assay using SSLBs, and the preferential binding to the highly curved membrane was confirmed. Peripheral membrane proteins obtained from normal human dermal fibroblast (NHDF) and human breast cancer (MDA-MB-231) cells were used in shotgun proteomic analysis, and 786 and 949 proteins were identified from SSLBs as lipid membrane binders, respectively. Statistical quantitative analyses of proteins detected from each SSLB with a different size revealed 118 candidate proteins, including 23 proteins unique to MDA-MB-231 cells, as membrane curvature sensors, including some previously reported curvature sensors. Functional clustering analysis based on the KEGG orthology database revealed that the protein-binding property to specific high or low membrane curvature correlated with their functions. Further investigation of candidate proteins will lead to the identification of new MCS proteins as well as cancer biomarkers.

Peptide-Functionalized Quantum Dots for Rapid Label-Free Sensing of 2,4,6-Trinitrotoluene.

Explosive compounds, such as 2,4,6-trinitrotoluene (TNT), pose a great concern in terms of both global public security and environmental protection. There are estimated to be hundreds of TNT contaminated sites all over the world, which will affect the health of humans, wildlife, and the ecosystem. Clearly, the ability to detect TNT in soils, water supplies, and wastewater is important for environmental studies but also important for security, such as in ports and boarders. However, conventional spectroscopic detection is not practical for on-site sensing because it requires sophisticated equipment and trained personnel. We report a rapid and simple chemical sensor for TNT by using TNT binding peptides which are conjugated to fluorescent CdTe/CdS quantum dots (QDs). QDs were synthesized in the aqueous phase, and the peptide was attached directly to the surface of the QDs by using thiol groups. The fluorescent emission from the QDs was quenched in response to the addition of TNT. The response could even be observed by the naked eye. The limit of detection from fluorescence spectroscopic measurement was estimated to be approximately 375 nM. In addition to the rapid response (within a few seconds), selective detection was demonstrated. We believe this label-free chemical sensor contributes to progress for the on-site explosive sensing.

Systematic Screening and Deep Analysis of CoPt Binding Peptides Leads to Enhanced CoPt Nanoparticles Using Designed Peptides.

Using protein and peptide additives to direct the crystallization of inorganic materials is a very attractive and environmentally friendly strategy to access complex and sometimes inaccessible mineral phases. CoPt is a very desirable high-magnetoanisotropic material in its L10 phase, but this is acquired by annealing at high temperatures which is incompatible with delicate nanomaterial assembly. Previous studies identified one peptide with high affinity to CoPt and four peptides with high affinity to FePt L10 phase nanoparticles (NPs) through phage display biopanning selection. While synthesis mediated by these peptides offered a small degree of L10 character to the NPs, they do not have the magnetoanistropy required for applications. In this study, we improve the activity of peptide directed crystallization by designing second generation peptides. We use the five literature sequences (LS) to probe the binding affinity deeper through dissection (alanine scanning), reduction (truncations), and substitution of the LS to find key amino acids and motifs. This is performed using a SPOT peptide array, importantly probing interactions at three stages of NP formation: with precursor, during synthesis, and with NPs. We found four universal features: 1) the importance of basic residues, particularly lysine flanking both ends of the sequence; 2) the importance of methionine; 3) shorter sequences show higher affinity than longer ones; and 4) acidic residues have a negative impact on binding with aspartic acid less favorable than glutamic acid. However, an acidic amino acid benefits, presumably to balance charge. The short motif KSLS had high affinity in all assays. Three sequences were selected from the screening, and three sequences were designed from the rules above. These were used to mediate a green synthesis of CoPt nanoparticles. The screened peptides mediated the formation of NPs with improved coercivity (90-110 Oe) compared to the LS (30-80 Oe), while the designed peptides facilitated formation of CoPt NPs with the highest coercivity (109 to 132 Oe), representing a massive improvement on L10 character. This result along with deeper insight this methodology brings offers vast potential for the future.

Array-Based Screening of Silver Nanoparticle Mineralization Peptides.

The use of biomolecules in nanomaterial synthesis has received increasing attention, because they can function as a medium to produce inorganic materials in ambient conditions. Short peptides are putative ligands that interact with metallic surfaces, as they have the potential to control the synthesis of nanoscale materials. Silver nanoparticle (AgNP) mineralization using peptides has been investigated; however, further comprehensive analysis must be carried out, because the design of peptide mediated-AgNP properties is still highly challenging. Herein, we employed an array comprising 200 spot synthesis-based peptides, which were previously isolated as gold nanoparticle (AuNP)-binding and/or mineralization peptides, and the AgNP mineralization activity of each peptide was broadly evaluated. Among 10 peptides showing the highest AgNP-synthesis activity (TOP10), nine showed the presence of EE and E[X]E (E: glutamic acid, and X: any amino acid), whereas none of these motifs were found in the WORST25 (25 peptides showing the lowest AgNP synthesis activity) peptides. The size and morphology of the particles synthesized by TOP3 peptides were dependent on their sequences. These results suggested not only that array-based techniques are effective for the peptide screening of AgNP mineralization, but also that AgNP mineralization regulated by peptides has the potential for the synthesis of AgNPs, with controlled morphology in environmentally friendly conditions.

Identifying Single Viruses Using Biorecognition Solid-State Nanopores.

Immunosensing is a bioanalytical technique capable of selective detections of pathogens by utilizing highly specific and strong intermolecular interactions between recognition probes and antigens. Here, we exploited the molecular mechanism in artificial nanopores for selective single-virus identifications. We designed hemagglutinin antibody mimicking oligopeptides with a weak affinity to influenza A virus. By functionalizing the pore wall surface with the synthetic peptides, we rendered specificity to virion-nanopore interactions. The ligand binding thereof was found to perturb translocation dynamics of specific viruses in the nanochannel, which facilitated digital typing of influenza by the resistive pulse bluntness. As amino acid sequence degrees of freedom can potentially offer variety of recognition ability to the molecular probes, this peptide nanopore approach can be used as a versatile immunosensor with single-particle sensitivity that promises wide applications in bioanalysis including bacterial and viral screening to infectious disease diagnosis.

Identification of Individual Bacterial Cells through the Intermolecular Interactions with Peptide-Functionalized Solid-State Pores.

Bioinspired pore sensing for selective detection of flagellated bacteria was investigated. The Au micropore wall surface was modified with a synthetic peptide designed from toll-like receptor 5 (TLR5) to mimic the pathogen-recognition capability. We found that intermolecular interactions between the TLR5-derived recognition peptides and flagella induce ligand-specific perturbations in the translocation dynamics of Escherichia coli, which facilitated the discrimination between the wild-type and flagellin-deletion mutant (ΔfliC) by the resistive pulse patterns thereby demonstrating the sensing of bacteria at a single-cell level. These results provide a novel concept of utilizing weak intermolecular interactions as a recognition probes for single-cell microbial identification.

Oil accumulation by the oleaginous diatom Fistulifera solaris as revealed by the genome and transcriptome.

Oleaginous photosynthetic organisms such as microalgae are promising sources for biofuel production through the generation of carbon-neutral sustainable energy. However, the metabolic mechanisms driving high-rate lipid production in these oleaginous organisms remain unclear, thus impeding efforts to improve productivity through genetic modifications. We analyzed the genome and transcriptome of the oleaginous diatom Fistulifera solaris JPCC DA0580. Next-generation sequencing technology provided evidence of an allodiploid genome structure, suggesting unorthodox molecular evolutionary and genetic regulatory systems for reinforcing metabolic efficiencies. Although major metabolic pathways were shared with nonoleaginous diatoms, transcriptome analysis revealed unique expression patterns, such as concomitant upregulation of fatty acid/triacylglycerol biosynthesis and fatty acid degradation (ß-oxidation) in concert with ATP production. This peculiar pattern of gene expression may account for the simultaneous growth and oil accumulation phenotype and may inspire novel biofuel production technology based on this oleaginous microalga.

An SPR Sensor Chip Based on Peptide-Modified Single-Walled Carbon Nanotubes with Enhanced Sensitivity and Selectivity in the Detection of 2,4,6-Trinitrotoluene Explosives.

In this study, we developed a surface plasmon resonance (SPR) sensor chip based on 2,4,6-trinitrotoluene (TNT) recognition peptide-modified single-walled carbon nanotubes (SWCNTs). The carboxylic acid-functionalized SWCNTs were immobilized on a 3-aminopropyltriethoxysilane (APTES)-modified SPR Au chip surface. Through π-stacking between the aromatic amino acids and SWCNTs, the TNT recognition peptide TNTHCDR3 was immobilized onto the surface of the SWCNTs. The peptide⁻SWCNTs-modified sensor surface was confirmed and evaluated by atomic force microscope (AFM) observation. The peptide⁻SWCNTs hybrid SPR sensor chip exhibited enhanced sensitivity with a limit of detection (LOD) of 772 ppb and highly selective detection compared with commercialized carboxymethylated dextran matrix sensor chips.

Carbenoxolone inhibits TRPV4 channel-initiated oxidative urothelial injury and ameliorates cyclophosphamide-induced bladder dysfunction.

Carbenoxolone (CBX) is a clinically prescribed drug for the treatment of digestive ulcer and inflammation. It is also a widely used pharmacological inhibitor of several channels in basic research. Given that the overactivity of several channels, including those inhibitable by CBX, underlies bladder dysfunction, we tested the potential therapeutic application and mechanism of CBX in the treatment of voiding dysfunction. In a mouse model of cystitis induced by cyclophosphamide (CYP), CBX administration prevented the CYP-elicited increase in bladder weight, oedema, haemorrhage, and urothelial injury. CBX also greatly improved micturition pattern, as manifested by the apparently decreased micturition frequency and increased micturition volume. Western blot results showed that CBX suppressed CYP-induced increase in protein carbonyls, COX-2, and iNOS. Further analysis using cultured urothelial cells revealed that acrolein, the major metabolite of CYP, caused protein oxidation, p38 activation, and urothelial injury. These effects of acrolein were reproduced by TRPV4 agonists and significantly prevented by antioxidant NAC, p38 inhibitor SB203580, TRPV4 antagonist RN-1734, and CBX. Further studies showed that CBX potently suppressed TRPV4 agonist-initiated calcium influx and subsequent cell injury. CBX attenuated CYP-induced cystitis in vivo and reduced acrolein-induced cell injury in vitro, through mechanisms involving inhibition of TRPV4 channels and attenuation of the channel-mediated oxidative stress. CBX might be a promising agent for the treatment of bladder dysfunction.

Rational Design of Peptide-Functionalized Surface Plasmon Resonance Sensor for Specific Detection of TNT Explosive.

In this study, a rationally-designed 2,4,6-trinitrotoluene (TNT) binding peptide derived from an amino acid sequence of the complementarity-determining region (CDR) of an anti-TNT monoclonal antibody was used for TNT detection based on a maleimide-functionalized surface plasmon resonance (SPR) sensor. By antigen-docking simulation and screening, the TNT binding candidate peptides were obtained as TNTHCDR1 derived from the heavy chain of CDR1, TNTHCDR2 derived from CDR2, and TNTHCDR3 from CDR3 of an anti-TNT antibody. The binding events between candidate peptides and TNT were evaluated using the SPR sensor by direct determination based on the 3-aminopropyltriethoxysilane (APTES) surface. The TNT binding peptide was directly immobilized on the maleimide-functionalized sensor chip surface from N-γ-maleimidobutyryl-oxysuccinimide ester (GMBS). The results demonstrated that peptide TNTHCDR3 was identified and selected as a TNT binding peptide among the other two candidate peptides. Five kinds of TNT analogues were also investigated to testify the selectivity of TNT binding peptide TNTHCDR3. Furthermore, the results indicated that the APTES-GMBS-based SPR sensor chip procedure featured a great potential application for the direct detection of TNT.

Comparative Subcellular Localization Analysis of Magnetosome Proteins Reveals a Unique Localization Behavior of Mms6 Protein onto Magnetite Crystals.

UNLABELLED: The magnetosome is an organelle specialized for inorganic magnetite crystal synthesis in magnetotactic bacteria. The complex mechanism of magnetosome formation is regulated by magnetosome proteins in a stepwise manner. Protein localization is a key step for magnetosome development; however, a global study of magnetosome protein localization remains to be conducted. Here, we comparatively analyzed the subcellular localization of a series of green fluorescent protein (GFP)-tagged magnetosome proteins. The protein localizations were categorized into 5 groups (short-length linear, middle-length linear, long-length linear, cell membrane, and intracellular dispersing), which were related to the protein functions. Mms6, which regulates magnetite crystal growth, localized along magnetosome chain structures under magnetite-forming (microaerobic) conditions but was dispersed in the cell under nonforming (aerobic) conditions. Correlative fluorescence and electron microscopy analyses revealed that Mms6 preferentially localized to magnetosomes enclosing magnetite crystals. We suggest that a highly organized spatial regulation mechanism controls magnetosome protein localization during magnetosome formation in magnetotactic bacteria. IMPORTANCE: Magnetotactic bacteria synthesize magnetite (Fe3O4) nanocrystals in a prokaryotic organelle called the magnetosome. This organelle is formed using various magnetosome proteins in multiple steps, including vesicle formation, magnetosome alignment, and magnetite crystal formation, to provide compartmentalized nanospaces for the regulation of iron concentrations and redox conditions, enabling the synthesis of a morphologically controlled magnetite crystal. Thus, to rationalize the complex organelle development, the localization of magnetosome proteins is considered to be highly regulated; however, the mechanisms remain largely unknown. Here, we performed comparative localization analysis of magnetosome proteins that revealed the presence of a spatial regulation mechanism within the linear structure of magnetosomes. This discovery provides evidence of a highly regulated protein localization mechanism for this bacterial organelle development.

Biomagnetic Recovery and Bioaccumulation of Selenium Granules in Magnetotactic Bacteria.

UNLABELLED: Using microorganisms to remove waste and/or neutralize pollutants from contaminated water is attracting much attention due to the environmentally friendly nature of this methodology. However, cell recovery remains a bottleneck and a considerable challenge for the development of this process. Magnetotactic bacteria are a unique group of organisms that can be manipulated by an external magnetic field due to the presence of biogenic magnetite crystals formed within their cells. In this study, we demonstrated an account of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria alongside and independent of magnetite crystal biomineralization when grown in a medium containing selenium oxyanion (SeO3 (2-)). Quantitative analysis shows that magnetotactic bacteria accumulate the largest amount of target molecules (Se) per cell compared with any other previously reported nonferrous metal/metalloid. For example, 2.4 and 174 times more Se is accumulated than Te taken up into cells and Cd(2+) adsorbed onto the cell surface, respectively. Crucially, the bacteria with high levels of Se accumulation were successfully recovered with an external magnetic field. The biomagnetic recovery and the effective accumulation of target elements demonstrate the potential for application in bioremediation of polluted water. IMPORTANCE: The development of a technique for effective environmental water remediation is urgently required across the globe. A biological remediation process of waste removal and/or neutralization of pollutant from contaminated water using microorganisms has great potential, but cell recovery remains a bottleneck. Magnetotactic bacteria synthesize magnetic particles within their cells, which can be recovered by a magnetic field. Herein, we report an example of accumulation and precipitation of amorphous elemental selenium nanoparticles within magnetotactic bacteria independent of magnetic particle synthesis. The cells were able to accumulate the largest amount of Se compared to other foreign elements. More importantly, the Se-accumulating bacteria were successfully recovered with an external magnetic field. We believe magnetotactic bacteria confer unique advantages of biomagnetic cell recovery and of Se accumulation, providing a new and effective methodology for bioremediation of polluted water.

Co-ordinated functions of Mms proteins define the surface structure of cubo-octahedral magnetite crystals in magnetotactic bacteria.

Magnetotactic bacteria synthesize magnetosomes comprised of membrane-enveloped single crystalline magnetite (Fe3 O4 ). The size and morphology of the nano-sized magnetite crystals (< 100 nm) are highly regulated and bacterial species dependent. However, the control mechanisms of magnetite crystal morphology remain largely unknown. The group of proteins, called Mms (Mms5, Mms6, Mms7, and Mms13), was previously isolated from the surface of cubo-octahedral magnetite crystals in Magnetospirillum magneticum strain AMB-1. Analysis of an mms6 gene deletion mutant suggested that the Mms6 protein plays a major role in the regulation of magnetite crystal size and morphology. In this study, we constructed various mms gene deletion mutants and characterized the magnetite crystals formed by the mutant strains. Comparative analysis showed that all mms genes were involved in the promotion of crystal growth in different manners. The phenotypic characterization of magnetites also suggested that these proteins are involved in controlling the geometries of the crystal surface structures. Thus, the co-ordinated functions of Mms proteins regulate the morphology of the cubo-octahedral magnetite crystals in magnetotactic bacteria.

Peptide array screening with anti-GLP-1 monoclonal antibody: Discovery of cysteine-containing DPP-IV inhibitory peptides.

Inhibition of dipeptidyl peptidase IV (DPP-IV) is an effective pharmacotherapy for the management of type 2 diabetes. Recent findings have suggested that various dietary proteins can serve as precursors to peptides that inhibit DPP-IV. Although several DPP-IV inhibitory peptides derived from food materials have been reported, more effective inhibitory peptides remain to be discovered. This study aimed to identify potent DPP-IV inhibitory peptides that earlier approaches had overlooked by employing a screening method that combined peptide arrays and neutralizing antibodies. Octa-peptides covering the complete amino acid sequences of four casein proteins and two whey proteins were synthesized on arrays via a solid-phase method. These peptides were then reacted with a monoclonal antibody specifically engineered to recognize glucagon-like peptide 1 (GLP-1), a substrate of DPP-IV. The variable region of the anti-GLP-1 monoclonal antibody is utilized to mimic the substrate-binding region of DPP-IV, enabling the antibody to bind to peptides that interact with DPP-IV. Based on this feature, 26 peptides were selected as DPP-IV inhibitory peptide candidates, 11 of which showed strong DPP-IV inhibitory activity. Five of these peptides consistently contained cysteines positioned two to four residues from the N-terminus. Treatment with disulfide formation decreased the DPP-IV inhibitory activity of these cysteine-containing peptides, while the inhibitory activity of α-lactalbumin hydrolysates increased with reducing treatment. These results revealed that the thiol group is important for DPP-IV inhibitory activity. This study provides a useful screen for DPP-IV inhibitory peptides and indicates the importance of reductive cysteine residues within DPP-IV inhibitory peptides.

Osmotic stress induces the formation of migrasome-like vesicles.

Migrasomes are extracellular vesicles that form on the retraction fibers of migrating cells. In this study, we report the formation of migrasome-like vesicles enriched in tetraspanin 4 and containing cytoplasmic components in response to hypoosmotic stress. When migrating cells were subjected to hypoosmotic stress, vesicles with a size distribution of 0.5 to 2 µm formed on the retraction fibers, and vanished in a few minutes. The vesicles are rich in cholesterol, and their number was reduced when cells were pretreated with lipoprotein-deficient serum. The formation of migrasome-like vesicles upon hypoosmotic stress may provide biophysical cues regarding the cellular response to this external stimulus in cells and tissues.

Screening of novel DPP-IV inhibitory peptides derived from bovine milk proteins using a peptide array platform.

Dipeptidyl peptidase IV (DPP-IV) has become an important target in the prevention and treatment of diabetes. Although many DPP-IV inhibitory peptides have been identified by a general approach involving the repeated fractionation of food protein hydrolysates, the obtained results have been dependent on the content of each peptide and fractionation conditions. In the present study, a peptide array that provides comprehensive assays of peptide sequences was used to identify novel DPP-IV inhibitory peptides derived from bovine milk proteins; these peptides were then compared with those identified using the general approach. While the general approach identified only known peptides that were abundant in the hydrolysate, the peptide array-based approach identified 10 novel DPP-IV inhibitory peptides, all of which had proline at the second residue from the N-terminus. The proper or combined use of these two approaches, which have different advantages, will enable the efficient development of novel bioactive foods and drugs.

Synthesis of atom-precise supported metal clusters via solid-phase peptide synthesis.

While the utility of supported metal and alloy clusters as catalytic materials is widely recognized, their precise synthesis remains a challenge. Here, we demonstrate the precise synthesis of these clusters via metallopeptides. This technique is characterized by its ability to be automated using Merrifield's solid-phase peptide synthesis (SPPS). Metallopeptides with iron and platinum complexes in their side chains have been prepared using this SPPS. These metallopeptides were successfully transformed into the corresponding supported metal clusters by heating in a hydrogen atmosphere.