Development of an enhanced immunoassay based on protein nanoparticles displaying an IgG-binding domain and luciferase.

Detection of small amounts of target molecules with high sensitivity is important for the diagnosis of many diseases, including cancers, and is particularly important to detect early stages of disease. Here, we report the development of a temperature-responsive fusion protein (ELP-DCN) comprised of an elastin-like polypeptide (ELP), poly-aspartic acid (D), antibody-binding domain C (C), and NanoLuc luciferase (N). ELP-DCN proteins form nanoparticles above a certain threshold temperature that display an antibody-binding domain and NanoLuc luciferase on their surface. ELP-DCN nanoparticles can be applied for enhancement of immunoassay systems because they provide more antibody-binding sites and an increased number of luciferase molecules, resulting in an increase in assay signal. Here, we report the detection of human serum albumin (HSA) as a model protein using anti-HSA and ELP-DCN proteins. Upon formation of ELP-DCN nanoparticles, the detection limit improved tenfold compared to the monomeric form of ELP-DCN.

Temperature-Responsive Multifunctional Protein Hydrogels with Elastin-like Polypeptides for 3-D Angiogenesis.

Supramolecular protein hydrogels with tunable properties represent promising candidates for advanced designer extracellular matrices (ECMs). To control cellular functions, ECMs should be able to spatiotemporally regulate synergistic signaling between transmembrane receptors and growth factor (GF) receptors. In this study, we developed genetically engineered temperature-responsive multifunctional protein hydrogels. The designed hydrogel was fabricated by combining the following four peptide blocks: thermosensitive elastin-like polypeptides (ELPs), a polyaspartic acid (polyD) chain to control aggregation and delivery of GFs, a de novo-designed helix peptide that forms antiparallel homotetrameric coiled-coils, and a biofunctional peptide. The resultant coiled-coil unit bound ELPs (CUBEs) exhibit a controllable sol-gel transition with tunable mechanical properties. CUBEs were functionalized with bone sialoprotein-derived RGD (bRGD), and human umbilical vein endothelial cells (HUVECs) were three-dimensionally cultured in bRGD-modified CUBE (bRGD-CUBE) hydrogels. Proangiogenic activity of HUVECs was promoted by bRGD. Moreover, heparin-binding angiogenic GFs were immobilized to bRGD-CUBEs via electrostatic interactions. HUVECs cultured in GF-tethered bRGD-CUBE hydrogels formed three-dimensional (3-D) tubulelike structures. The designed CUBE hydrogels may demonstrate utility as advanced smart biomaterials for biomedical applications. Further, the protein hydrogel design strategy may provide a novel platform for constructing designer 3-D microenvironments for specific cell types.

Construction of DNA-displaying nanoparticles by enzymatic conjugation of DNA and elastin-like polypeptides using a replication initiation protein.

DNA-displaying nanoparticles comprised of conjugates of single-stranded DNA (ssDNA) and elastin-like polypeptide (ELP) were developed. ssDNA was enzymatically conjugated to ELPs via a catalytic domain of Porcine Circovirus type 2 replication initiation protein (pRep) fused to ELPs. Nanoparticles were formed upon heating to temperatures above the phase transition temperature due to the hydrophobicity of ELPs and the hydrophilicity of conjugated ssDNA. We demonstrated the applicability of the resultant nanoparticles as drug carriers with tumor-targeting properties by conjugating a DNA aptamer, which is known to bind to Mucin 1 (MUC1), to ELPs. DNA aptamer-displaying nanoparticles encapsulating the anti-cancer drug paclitaxel were able to bind to cells overexpressing MUC1 and induce cell death.

Development of drug-loaded protein nanoparticles displaying enzymatically-conjugated DNA aptamers for cancer cell targeting.

Modification of protein-based drug carriers with tumor-targeting properties is an important area of research in the field of anticancer drug delivery. To this end, we developed nanoparticles comprised of elastin-like polypeptides (ELPs) with fused poly-aspartic acid chains (ELP-D) displaying DNA aptamers. DNA aptamers were enzymatically conjugated to the surface of the nanoparticles via genetic incorporation of Gene A* protein into the sequence of the ELP-D fusion protein. Gene A* protein, derived from bacteriophage ϕX174, can form covalent complexes with single-stranded DNA via the latter's recognition sequence. Gene A* protein-displaying nanoparticles exhibited the ability to deliver the anticancer drug paclitaxel (PTX), whilst retaining activity of the conjugated Gene A* protein. PTX-loaded protein nanoparticles displaying DNA aptamers known to bind to the MUC1 tumor marker resulted in increased cytotoxicity with MCF-7 breast cancer cells compared to PTX-loaded protein nanoparticles without the DNA aptamer modification.

Construction of DNA-NanoLuc luciferase conjugates for DNA aptamer-based sandwich assay using Rep protein.

OBJECTIVE: We developed a DNA-NanoLuc luciferase (NnaoLuc) conjugates for DNA aptamer-based sandwich assay using the catalytic domain of the replication initiator protein derived from porcine circovirus type 2 (pRep). RESULTS: For construction of DNA aptamer and NanoLuc conjugate using the catalytic domain of Rep from PCV2. pRep fused to NanoLuc was genetically constructed and expressed in E. coli. After purification, the activities of fused pRep and NanoLuc were evaluated, and DNA-NanoLuc conjugates were constructed via the fused pRep. Finally, constructed DNA-NanoLuc conjugates were applied for use in a DNA aptamer-based sandwich assay. Here, pRep was used not only for conjugation of the NanoLuc to the detection aptamer, but also for immobilization of the capture aptamer on the plate surface. CONCLUSION: We have demonstrated that DNA-NanoLuc conjugates via the catalytic domain of PCV2 Rep could be applied for DNA aptamer-based sandwich assay system.

A DNA-scaffold platform enhances a multi-enzymatic cycling reaction.

OBJECTIVE: We explored the co-localization of multiple enzymes on a DNA backbone via a DNA-binding protein, Gene-A* (A*-tag) to increase the efficiency of cascade enzymatic reactions. RESULTS: Firefly luciferase (FLuc) and pyruvate orthophosphate dikinase (PPDK) were genetically fused with A*-tag and modified with single-stranded (ss) DNA via A*-tag. The components were assembled on ssDNA by hybridization, thereby enhancing the efficiency of the cascading bioluminescent reaction producing light emission from pyrophosphate. The activity of A*-tag in each enzyme was investigated with dye-labeled DNA. Co-localization of the enzymes via hybridization was examined using a gel shift assay. The multi-enzyme complex showed significant improvement in the overall efficiency of the cascading reaction in comparison to a mixture of free enzymes. CONCLUSION: A*-tag is highly convenient for ssDNA modification of versatile enzymes, and it can be used for construction of functional DNA-enzyme complexes.

Microfluidic-Nanofiber Hybrid Array for Screening of Cellular Microenvironments.

Cellular microenvironments are generally sophisticated, but crucial for regulating the functions of human pluripotent stem cells (hPSCs). Despite tremendous effort in this field, the correlation between the environmental factors-especially the extracellular matrix and soluble cell factors-and the desired cellular functions remains largely unknown because of the lack of appropriate tools to recapitulate in vivo conditions and/or simultaneously evaluate the interplay of different environment factors. Here, a combinatorial platform is developed with integrated microfluidic channels and nanofibers, associated with a method of high-content single-cell analysis, to study the effects of environmental factors on stem cell phenotype. Particular attention is paid to the dependence of hPSC short-term self-renewal on the density and composition of extracellular matrices and initial cell seeding densities. Thus, this combinatorial approach provides insights into the underlying chemical and physical mechanisms that govern stem cell fate decisions.

Construction of a Defined Biomimetic Matrix for Long-Term Maintenance of Mouse Induced Pluripotent Stem Cells.

The existing in vitro culture systems often use undefined and animal-derived components for the culture of pluripotent stem cells. Artificial bioengineered peptides have the potential to become alternatives to these components of extracellular matrix (ECM). Integrins and cadherins are two cell adhesion proteins important for stem cell self-renewal, differentiation, and phenotype stability. In the present study, we sought to mimic the physico-biochemical properties of natural ECMs that allow self-renewal of mouse induced pluripotent stem cells (iPSCs). We develop a genetically engineered ECM protein (ERE-CBP) that contains (i) an integrin binding peptide sequence (RGD/R), (ii) an E-/N-cadherin binding peptide sequence (SWELYYPLRANL/CBP), and (iii) 12 repeats of APGVGV elastin-like polypeptides (ELPs/E).While ELPs allow efficient coating by binding to nontreated hydrophobic tissue culture plates, RGD/R and CBP support integrin- and cadherin-dependent cell attachment, respectively. Mouse iPSCs on this composite matrix exhibit a more compact phenotype compared to cells on control gelatin substrate. We also demonstrated that the ERE-CBP supports proliferation and long-term self-renewal of mouse iPSCs for up to 17 passages without GSK3ß (CHIR99021) and Erk (PD0325901) inhibitors. Overall, our engineered ECM protein, which is cost-effective to produce in prokaryotic origin and flexible to modify with other cell adhesion peptides or growth factors, provides a novel approach for expansion of mouse iPSCs in vitro.

Design of luciferase-displaying protein nanoparticles for use as highly sensitive immunoassay detection probes.

In this study, we developed a protein nanoparticle-based immunoassay to detect cancer biomarkers using a bioluminescent fusion protein. This method relies on the use of protein nanoparticles comprised of genetically-engineered elastin-like polypeptides (ELPs) fused with poly-aspartic acid tails (ELP-D), previously developed in our lab. The sizes of the self-assembled ELP-D nanoparticles can be regulated at the nanoscale by charged repulsion of the poly-aspartic acid chains. To improve the sensitivity of enzyme-linked immunosorbent assays (ELISAs), we herein demonstrate the multivalent display of NanoLuc® (Nluc) luciferase and a biotin acceptor peptide (BAP) on the surfaces of ELP-D nanoparticles, and demonstrate the sensitivity of these multivalent nanoparticles as detection probes. The fusion protein comprised of ELP-D and Nluc-BAP (ELP-D-Nluc-BAP) was found to form nanoparticles with Nluc and BAP displayed multivalently on their surfaces. Moreover, the use of the nanoparticles in ELISA resulted in a detection sensitivity for α-fetoprotein (AFP) about 10 times higher than that of an assay relying on the use of the monomeric version of the fusion protein. Taken together, ELP-D-based nanoparticles displaying multivalent luciferases on their surfaces enable the construction of an ELISA with enhanced sensitivity.

3D printing of soft lithography mold for rapid production of polydimethylsiloxane-based microfluidic devices for cell stimulation with concentration gradients.

Three-dimensional (3D) printing is advantageous over conventional technologies for the fabrication of sophisticated structures such as 3D micro-channels for future applications in tissue engineering and drug screening. We aimed to apply this technology to cell-based assays using polydimethylsiloxane (PDMS), the most commonly used material for fabrication of micro-channels used for cell culture experiments. Useful properties of PDMS include biocompatibility, gas permeability and transparency. We developed a simple and robust protocol to generate PDMS-based devices using a soft lithography mold produced by 3D printing. 3D chemical gradients were then generated to stimulate cells confined to a micro-channel. We demonstrate that concentration gradients of growth factors, important regulators of cell/tissue functions in vivo, influence the survival and growth of human embryonic stem cells. Thus, this approach for generation of 3D concentration gradients could have strong implications for tissue engineering and drug screening.

Assessment of Stress and Well-Being of Japanese Employees Using Wearable Devices for Sleep Monitoring Combined With Ecological Momentary Assessment: Pilot Observational Study.

BACKGROUND: Poor sleep quality can elevate stress levels and diminish overall well-being. Japanese individuals often experience sleep deprivation, and workers have high levels of stress. Nevertheless, research examining the connection between objective sleep assessments and stress levels, as well as overall well-being, among Japanese workers is lacking. OBJECTIVE: This study aims to investigate the correlation between physiological data, including sleep duration and heart rate variability (HRV), objectively measured through wearable devices, and 3 states (sleepiness, mood, and energy) assessed through ecological momentary assessment (EMA) and use of rating scales for stress and well-being. METHODS: A total of 40 office workers (female, 20/40, 50%; mean age 40.4 years, SD 11.8 years) participated in the study. Participants were asked to wear a wearable wristband device for 8 consecutive weeks. EMA regarding sleepiness, mood, and energy levels was conducted via email messages sent by participants 4 times daily, with each session spaced 3 hours apart. This assessment occurred on 8 designated days within the 8-week timeframe. Participants' stress levels and perception of well-being were assessed using respective self-rating questionnaires. Subsequently, participants were categorized into quartiles based on their stress and well-being scores, and the sleep patterns and HRV indices recorded by the Fitbit Inspire 2 were compared among these groups. The Mann-Whitney U test was used to assess differences between the quartiles, with adjustments made for multiple comparisons using the Bonferroni correction. Furthermore, EMA results and the sleep and HRV indices were subjected to multilevel analysis for a comprehensive evaluation. RESULTS: The EMA achieved a total response rate of 87.3%, while the Fitbit Inspire 2 wear rate reached 88.0%. When participants were grouped based on quartiles of well-being and stress-related scores, significant differences emerged. Specifically, individuals in the lowest stress quartile or highest subjective satisfaction quartile retired to bed earlier (P<.001 and P=.01, respectively), whereas those in the highest stress quartile exhibited greater variation in the midpoint of sleep (P<.001). A multilevel analysis unveiled notable relationships: intraindividual variability analysis indicated that higher energy levels were associated with lower deviation of heart rate during sleep on the preceding day (ß=-.12, P<.001), and decreased sleepiness was observed on days following longer sleep durations (ß=-.10, P<.001). Furthermore, interindividual variability analysis revealed that individuals with earlier midpoints of sleep tended to exhibit higher energy levels (ß=-.26, P=.04). CONCLUSIONS: Increased sleep variabilities, characterized by unstable bedtime or midpoint of sleep, were correlated with elevated stress levels and diminished well-being. Conversely, improved sleep indices (eg, lower heart rate during sleep and earlier average bedtime) were associated with heightened daytime energy levels. Further research with a larger sample size using these methodologies, particularly focusing on specific phenomena such as social jet lag, has the potential to yield valuable insights. TRIAL REGISTRATION: UMIN-CTR UMIN000046858; https://center6.umin.ac.jp/cgi-open-bin/ctr/ctr_view.cgi?recptno=R000053392.

Construction of semisynthetic DNA-protein conjugates with Phi X174 Gene-A* protein.

DNA-protein conjugates have frequently been used as versatile molecular tools for a variety of applications in biotechnology to harness synergistic effects of DNA and protein functions. With applications for DNA-protein conjugates growing, easy-to-use and economical methods for the synthesis of DNA-protein conjugates are required. In this study, we developed a method for site-specific labeling of single-stranded DNA (ssDNA) to a recombinant protein of interest (POI) through the Gene-A* protein (Gene-A*) from bacteriophage phi X174, without any chemical modifications of ssDNA. Gene-A* protein is an enzyme that site-selectively cleaves an oligodeoxyribonucleotide (ODN) containing a Gene-A* recognition sequence, at which point a tyrosine residue of Gene-A* is bonded to the 5'-phosphoryl group of the cleavage site via a stable phosphotyrosine linkage. Here, we constructed three kinds of recombinant proteins fused to Gene-A*: N-terminally Gene-A*-fused enhanced green fluorescent protein (EGFP), C-terminally Gene-A*-fused EGFP, and N-terminally Gene-A*-fused firefly luciferase (FLuc). The reaction yields of DNA-protein conjugation catalyzed by the Gene-A* moiety reached 80-90% in the three proteins, and kinetic study revealed that the reaction achieved a steady state after 10 min. Moreover, dot blot analyses were performed to evaluate the hybridization and aptamer-forming ability of ssDNA conjugated to the Gene-A* moiety of a recombinant Gene-A*-FLuc protein. This study demonstrated that a strategy using recombinant proteins fused to Gene-A* could offer a versatile, rapid, easy-to-use, and economical platform for producing DNA-protein conjugates.

Detection of small RNA molecules by a combination of branched rolling circle amplification and bioluminescent pyrophosphate assay.

Aberrant expression of miRNAs often correlates with various human diseases. Therefore, miRNAs have been focused as disease biomarkers. Here, a novel application of a bioluminescence (BL) assay for small RNA quantification is described. The assay is based on detecting pyrophosphate (PPi) molecules released during branched rolling circle amplification (BRCA) with a second primer in the presence of target RNA molecules. The number of released PPi molecules is correlated with the target RNA copy number. This assay was capable of detecting at least 20 amol of target RNA molecules, and the dynamic range extended over at least three orders of magnitude. Appropriate use of a second primer allowed for sensitive detection of RNA molecules with a high S/N ratio in less time. Moreover, the assay could specifically detect as low as 0.1 fmol of a target small RNA within a total RNA extract with high reproducibility. These data suggest that our assay has the potential to become a simple, rapid, and highly sensitive method to detect miRNA. Furthermore, this method combined with a BL assay, which utilizes a widely used inexpensive luminometer, could be used for a wider, versatile range of applications.

Construction of multifunctional fusion proteins with a laminin-derived short peptide to promote neural differentiation of mouse induced pluripotent stem cells.

There is growing interest in the functional roles of the extracellular matrix (ECM) in regulating the fate of pluripotent stem cells (PSCs). An artificially bioengineered ECM provides an excellent model for studying the molecular mechanisms underlying self-renewal and differentiation of PSCs, without multiple unknown and variable factors associated with natural substrates. Here, we have engineered multifunctional fusion proteins that are based on peptides from laminin, including p20, RGD, and elastin-like polypeptide (ELP), where laminin peptides work as cell adhesion molecules (CAMs) and ELP to promote anchorage. The functionality of these chimeric proteins, referred to as ERE-p20 and E-p20, was assessed by determining their ability to immobilize cells on a hydrophobic polystyrene surface, improve mouse induced pluripotent stem cells (miPSCs) attachment, and promote miPSC differentiation to neural progenitors. ERE-p20 and E-p20 proteins showed hydrophobic binding saturation to the polystyrene plates around 500 nM (2.39 µg/cm2 ) and 750 nM (2.27 µg/cm2 ) protein concentrations, respectively. The apparent maximum cell binding to ERE-p20 and E-p20 was approximately 81% and 73%, respectively, relative to gelatin. For neural precursors, neurite outgrowth was enhanced by the presence of RGD and p20 peptides. The expression levels of neuronal marker protein MAP2 were upregulated approximately 2.5-fold and threefold by ERE-p20 and E-p20, respectively, relative to laminin. Overall, we have shown that elastin-mimetic fusion proteins consisting of p20 with and without RGD peptides are able to induce neuronal differentiation. In conclusion, our newly designed bioengineered fusion proteins allow preparation of specific bioactive matrices or coating/scaffold for miPSCs differentiation.

Fabrication of a Multiplexed Artificial Cellular MicroEnvironment Array.

Cellular microenvironments consist of a variety of cues, such as growth factors, extracellular matrices, and intercellular interactions. These cues are well orchestrated and are crucial in regulating cell functions in a living system. Although a number of researchers have attempted to investigate the correlation between environmental factors and desired cellular functions, much remains unknown. This is largely due to the lack of a proper methodology to mimic such environmental cues in vitro, and simultaneously test different environmental cues on cells. Here, we report an integrated platform of microfluidic channels and a nanofiber array, followed by high-content single-cell analysis, to examine stem cell phenotypes altered by distinct environmental factors. To demonstrate the application of this platform, this study focuses on the phenotypes of self-renewing human pluripotent stem cells (hPSCs). Here, we present the preparation procedures for a nanofiber array and the microfluidic structure in the fabrication of a Multiplexed Artificial Cellular MicroEnvironment (MACME) array. Moreover, overall steps of the single-cell profiling, cell staining with multiple fluorescent markers, multiple fluorescence imaging, and statistical analyses, are described.

Design of bFGF-tethered self-assembling extracellular matrix proteins via coiled-coil triple-helix formation.

Self-assembling peptides are attractive materials for tissue engineering applications because of their functionality including high biocompatibility and biodegradability. Modification of self-assembling peptides with functional motifs, such as the cell-adhesive tripeptide sequence RGD leads to functional artificial extracellular matrices (ECMs). In this study, we developed an artificial self-assembling ECM protein tethered with a growth factor via heterotrimer triple-helix (helix A/B/C) formation. The helix A and helix C peptides, which are capable of forming a heterodimer coiled-coil structure, were fused to both ends of a matrix protein composed of the elastin-derived structural unit (APGVGV)12 with an RGD motif. The helix B peptide, which constituents the third helix of the triple-helix structure, was fused with basic fibroblast growth factor (bFGF) for tethering to the artificial ECM proteins. Each recombinant protein exhibited cell adhesion and cell proliferation activities similar to the original, while the designed bFGF-tethered ECM protein exhibited superior cell proliferation activity. These results demonstrate that the approach of creating growth factor-tethered self-assembling proteins via triple-helix formation can be applied to develop functional ECMs for tissue engineering applications.

Characterization of Phenotypic and Transcriptional Differences in Human Pluripotent Stem Cells under 2D and 3D Culture Conditions.

Human pluripotent stem cells hold great promise for applications in drug discovery and regenerative medicine. Microfluidic technology is a promising approach for creating artificial microenvironments; however, although a proper 3D microenvironment is required to achieve robust control of cellular phenotypes, most current microfluidic devices provide only 2D cell culture and do not allow tuning of physical and chemical environmental cues simultaneously. Here, the authors report a 3D cellular microenvironment plate (3D-CEP), which consists of a microfluidic device filled with thermoresponsive poly(N-isopropylacrylamide)-ß-poly(ethylene glycol) hydrogel (HG), which enables systematic tuning of both chemical and physical environmental cues as well as in situ cell monitoring. The authors show that H9 human embryonic stem cells (hESCs) and 253G1 human induced pluripotent stem cells in the HG/3D-CEP system maintain their pluripotent marker expression under HG/3D-CEP self-renewing conditions. Additionally, global gene expression analyses are used to elucidate small variations among different test environments. Interestingly, the authors find that treatment of H9 hESCs under HG/3D-CEP self-renewing conditions results in initiation of entry into the neural differentiation process by induction of PAX3 and OTX1 expression. The authors believe that this HG/3D-CEP system will serve as a versatile platform for developing targeted functional cell lines and facilitate advances in drug screening and regenerative medicine.

Microfluidic Image Cytometry for Single-Cell Phenotyping of Human Pluripotent Stem Cells.

A microfluidic human pluripotent stem cell (hPSC) array has been developed for robust and reproducible hPSC culture methods to assess chemically defined serum- and feeder-free culture conditions. This microfluidic platform, combined with image cytometry, enables the systematic analysis of multiple simultaneously detected marker expression in individual cells, for screening of various chemically defined media across hPSC lines, and the study of phenotypic responses.

Delivery of bFGF for Tissue Engineering by Tethering to the ECM.

Delivery of growth factors to target cells is an important subject in tissue engineering. Towards that end, we have developed a growth factor-tethered extracellular matrix (ECM). Here, basic fibroblast growth factor (bFGF) was tethered to extracellular matrix noncovalently. The designed ECM was comprised of 12 repeats of the APGVGV peptide motif derived from elastin as a stable structural unit and included the well-known cell adhesive RGD peptide as an active functional unit. To bind bFGF to the ECM, an acidic amino acid-rich sequence was introduced at the C-terminus of the ECM protein. It consisted of 5 repeats of 4 aspartic acids and a serine, DDDDS. bFGF has a highly basic amino acid domain. Therefore, bFGF was tethered to the ECM protein by electrostatic interaction. Cells cultured on bFGF-tethered ECM were well attached to the ECM and induced proliferation without addition of soluble bFGF.