Global landscape of replicative DNA polymerase usage in the human genome.

The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequencing (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide.

Bio-functionalized titanium surfaces with modified silk fibroin carrying titanium binding motif to enhance the ossific differentiation of MC3T3-E1.

Silk fibroin (SF) from Bombyx mori has superior properties as both a textile and a biomaterial, and has been used to functionalize the surfaces of various medical inorganic materials including titanium (Ti). In this study, we endowed SF with reversible binding ability to Ti by embedding a titanium binding motif (minTBP-1 and RKLPDA). Artificial SF proteins were first created by conjugating gene cassettes for SF motif (AGSGAG) and minTBP-1 motif with different ratios, which have been shown to bind reversibly to Ti surfaces in quartz crystal microbalance analyses.

Pentapartite fractionation of particles in oral fluids by differential centrifugation.

Oral fluids (OFs) contain small extracellular vesicles (sEVs or exosomes) that carry disease-associated diagnostic molecules. However, cells generate extracellular vesicles (EVs) other than sEVs, so the EV population is quite heterogeneous. Furthermore, molecules not packaged in EVs can also serve as diagnostic markers.

New Role for Growth/Differentiation Factor 15 in the Survival of Transplanted Brown Adipose Tissues in Cooperation with Interleukin-6.

To identify factors involved in the earliest phase of the differentiation of human embryonic stem cells (hESCs) into brown adipocytes (BAs), we performed multi-time point microarray analyses. We found that growth/differentiation factor 15 (GDF15) expressions were specifically upregulated within three days of differentiation, when expressions of immature hESC markers were sustained. Although GDF15 expressions continued to increase in the subsequent differentiation phases, -deficient hESCs differentiated into mature BAs (Day 10) without apparent abnormalities.

Subtypes of tumour cell-derived small extracellular vesicles having differently externalized phosphatidylserine.

Phosphatidylserine (PS) has skewed distributions in the plasma membrane and is preferentially located in the inner leaflet of normal cells. Tumour cells, however, expose PS at the outer leaflet of cell surfaces, thereby potentially modulating the bio-signalling of cells. Interestingly, exosomes - or, more properly, small extracellular vesicles (sEVs) - which are secreted from tumour cells, are enriched with externalized PS, have been proposed as being involved in the progression of cancers, and could be used as a marker for tumour diagnostics.

Host Cell Prediction of Exosomes Using Morphological Features on Solid Surfaces Analyzed by Machine Learning.

Exosomes are extracellular nanovesicles released from any cells and found in any body fluid. Because exosomes exhibit information of their host cells (secreting cells), their analysis is expected to be a powerful tool for early diagnosis of cancers. To predict the host cells, we extracted multidimensional feature data about size, shape, and deformation of exosomes immobilized on solid surfaces by atomic force microscopy (AFM).

Encryption of agonistic motifs for TLR4 into artificial antigens augmented the maturation of antigen-presenting cells.

Adjuvants are indispensable for achieving a sufficient immune response from vaccinations. From a functional viewpoint, adjuvants are classified into two categories: "physical adjuvants" increase the efficacy of antigen presentation by antigen-presenting cells (APC) and "signal adjuvants" induce the maturation of APC. Our previous study has demonstrated that a physical adjuvant can be encrypted into proteinous antigens by creating artificial proteins from combinatorial assemblages of epitope peptides and those peptide sequences having propensities to form certain protein structures (motif programming).

Isolation of human salivary extracellular vesicles by iodixanol density gradient ultracentrifugation and their characterizations.

Diagnostic methods that focus on the extracellular vesicles (EVs) present in saliva have been attracting great attention because of their non-invasiveness. EVs contain biomolecules such as proteins, messenger RNA (mRNA) and microRNA (miRNA), which originate from cells that release EVs, making them an ideal source for liquid biopsy. Although there have been many reports on density-based fractionation of EVs from blood and urine, the number of reports on EVs from saliva has been limited, most probably because of the difficulties in separating EVs from viscous saliva using density gradient centrifugation.

Combinatorial contextualization of peptidic epitopes for enhanced cellular immunity.

Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our "motif-programming" approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures.

Suppression of Aggrus/podoplanin-induced platelet aggregation and pulmonary metastasis by a single-chain antibody variable region fragment.

Almost all highly metastatic tumor cells possess high platelet aggregating abilities, thereby form large tumor cell-platelet aggregates in the microvasculature. Embolization of tumor cells in the microvasculature is considered to be the first step in metastasis to distant organs. We previously identified the platelet aggregation-inducing factor expressed on the surfaces of highly metastatic tumor cells and named as Aggrus.

Autonomous silica encapsulation and sustained release of anticancer protein.

We present a novel method for preparing a silica carrier for the sustained release of a proteinaceous pharmaceutical. This method makes use of the silicification activity of the protein itself, which autonomously formed a protein-silica composite upon simple incubation with a silica precursor. The composite was dissolved, and the encapsulated protein was released into a culture medium, thereby sustaining the protein's activity for a long period of time.

Synthesis of functional signaling domains by combinatorial polymerization of phosphorylation motifs.

The adaptor protein Cas contains a core substrate domain with multiple YXXP motifs that are phosphorylated by Src and other tyrosine kinases. Here, we used a synthetic strategy to determine the importance of the arrangement, spacing, and identity of the YXXP motifs. By polymerizing short DNA sequences encoding two phosphorylation motifs, we created a panel of Cas mutants in which the entire substrate domain was replaced by synthetic domains containing random numbers and arrangements of the motifs.

Effect of motif-programmed artificial proteins on the calcium uptake in a synthetic hydrogel.

Motif-programmed artificial proteins with mineralization-related activity were covalently immobilized onto the surface of a hydrogel, poly(2-hydroxyethyl methacrylate) (PHEMA). We investigated the influence of assaying conditions upon the ability of three selected proteins (PS64, PS382 and PS458) to modulate calcification in vitro. A long-term assay measuring the real amount of calcium phosphate phase in the protein-modified PHEMA showed that all proteins enhanced the uptake of calcium by the hydrogel.

Motif-programmed artificial protein induces apoptosis in several cancer cells by disrupting mitochondria.

By combinatorially assembling two natural motifs, respectively, associated with protein transduction (PTD) and induction of apoptosis (BH3), we previously synthesized an artificial protein (#284) that is taken up into cells, where it induces apoptosis. Here we used cluster analysis of GI(50) (average concentration required for 50% growth inhibition), as well as immunohistochemical and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling analyses to further characterize the capacity of #284 to induce apoptosis in a panel of 39 cancer cell lines. Our results showed that #284 preferentially inhibited the growth of several cancer cells with a GI(50) of approximately 5 microM, which is in the range of conventional anticancer drugs such as cisplatin and etoposide.

A synthesis approach to understanding repeated peptides conserved in mineralization proteins.

We created artificial proteins that contained repeats of a short peptide motif, Asn-Gly-Asx. In nature this motif is repeated within shell proteins as an idiosyncratic domain, while in vitro it has been shown to suppress calcification. The motif was embedded within peptide sequences that did or did not have the ability to form secondary structures, which provided the motif with a variety of physicochemical properties.

Structural properties of an artificial protein that regulates the nucleation of inorganic and organic crystals.

Technological advances have facilitated the generation of artificial proteins that possess the capabilities of recognizing and binding to inorganic solids and/or controlling nucleation processes that form inorganic solids. However, very little is known regarding the structure of these interesting polypeptides and how their structure contributes to functionality. To address this deficiency, we report structural investigations of an artificial protein, p288, that self-assembles and controls the nucleation of simple salts and organic compounds into dendrite-like crystals.

Motif programming: a microgene-based method for creating synthetic proteins containing multiple functional motifs.

The presence of peptide motifs within the proteins provides the synthetic biologist with the opportunity to fabricate novel proteins through the programming of these motifs. Here we describe a method that enables one to combine multiple peptide motifs to generate a combinatorial protein library. With this method, a set of sense and antisense oligonucleotide primers were prepared.

Synthesis of functional proteins by mixing peptide motifs.

Here, we describe a synthetic approach for generating artificial proteins by the assemblage of naturally occurring peptide motifs. Two motifs respectively related to apoptosis induction and protein transduction were encrypted into different reading frames of an artificial gene (microgene), which was then polymerized; random frame shifts at the junctions between the microgene units yielded combinatorial polymers of three reading frames. Among the proteins created, #284 was found to penetrate through cell membranes and exert a strong apoptotic effect on several cancer cell lines.

Distinct macroscopic structures developed from solutions of chemical compounds and periodic proteins.

By controlling the growth of inorganic crystals, macro-biomolecules, including proteins, play pivotal roles in modulating biomineralization. Natural proteins that promote biomineralization are often composed of simple repeats of peptide sequences; however, the relationship between these repetitive structures and their functions remains largely unknown. Here we show that an artificial protein containing a repeated peptide sequence allows NaCl, KCl, CuSO(4) and sucrose to form a variety of macroscopic structures, as represented by their dendritic configurations.