Versatile Mitogenic and Differentiation-Inducible Layer Formation by Underwater Adhesive Polypeptides.

Artificial materials have no biological functions, but they are important for medical devices such as artificial organs and matrices for regenerative medicine. In this study, mitogenic and differentiation-inducible materials are devised via the simple coating of polypeptides, which contain the sequence of epidermal growth factor or insulin-like growth factor with a key amino acid (3,4-dihydroxyphenylalanine) of underwater adhesive proteins. The adhesive polypeptides prepared via solid-phase synthesis form layers on various substrates involving organic and inorganic materials to provide biological surfaces. Through the direct activation of cognate receptors on interactive surfaces, the materials enable increased cell growth and differentiation compared to that achieved by soluble growth factors. This superior growth and differentiation are attributed to the long-lasting signal transduction (triggered by the bound growth factors), which do not cause receptor internalization and subsequent downregulation.

Strategy to Immobilize Peptide Probe Selected through In Vitro Ribosome Display for Electrochemical Aptasensor Application.

In this study, we demonstrated an electrochemical aptasensor for calmodulin (CaM) detection and the peptide sequence (YWDKIKDFIGG) is obtained from in vitro ribosome display selection. To immobilize this peptide probe on the electrode surface, cystine was incorporated at the end of this peptide sequence. After a maleimide-functionalized poly(3,4-ethylenedioxythiophene), poly(EODT-MI), film was electropolymerized on the electrode, the peptide probe was immobilized through thiol-ene conjugation with the cystine end. Four peptides with different linkers were used for the binding test of bovine serum albumin and CaM using a quartz crystal microbalance. The zwitterionic linker EKEKEKEKEKEK provided good antifouling properties and the highest CaM binding. Furthermore, the immobilization of the peptide with this zwitterionic linker resulted in a minimal increase in the electrochemical impedance. By immobilizing the peptide with the selected zwitterionic linker, we successfully demonstrated an electrochemical aptasensor with a linear detection range for CaM from 0.01 to 10 mg/L and a detection limit of 0.001 mg/L.

Phosphorogenic and spontaneous formation of tris(bipyridine)ruthenium in peptide scaffolds.

Redox-active ruthenium complexes have been widely used in various fields; however, the harsh conditions required for their synthesis are not always conducive to their subsequent use in biological applications. In this study, we demonstrate the spontaneous formation of a derivative of tris(bipyridine)ruthenium at 37°C through the coordination of three bipyridyl ligands incorporated into a peptide to a ruthenium ion. Specifically, we synthesized six bipyridyl-functionalized peptides with randomly chosen sequences. The six peptides bound to ruthenium ions and exhibited similar spectroscopic and electrochemical features to tris(bipyridine)ruthenium, indicating the formation of ruthenium complexes as we anticipated. The photo-excited triplet state of the ruthenium complex formed in the peptides exhibited an approximately 1.6-fold longer lifetime than that of tris(bipyridine)ruthenium. We also found that the photo-excited state of the ruthenium complexes was able to transfer an electron to methyl viologen, indicating that the ruthenium complexes formed in the peptides had the same ability to transfer charge as tris(bipyridine)ruthenium. We believe that this strategy of producing ruthenium complexes in peptides under mild conditions will pave the way for developing new metallopeptides and metalloproteins containing functional metal-complexes.

Controlled quercetin release from high-capacity-loading hyperbranched polyglycerol-functionalized graphene oxide.

PURPOSE: An efficient drug-delivery system was prepared based on graphene oxide using a facile and one-step strategy for controlling the release of anticancer drugs. METHODS: Fabrication of single-layer graphene oxide (GO) sheets was carried out by both modified and improved Hummers method. Biocompatible hyperbranched polyglycerol (HPG) was grafted on the surface of GO through the ring-opening hyperbranched polymerization of glycidol. Various ratios of GO and glycidol were used for polymer grafting. An anticancer drug, quercetin (Qu), was loaded into modified GO via noncovalent interactions. RESULTS: Polymer grafting on the surface of GO sheets was confirmed by results obtained from Fourier-transform infrared and Raman spectroscopy, thermogravimetric analysis, energy-dispersive X-ray and X-ray spectroscopy, scanning electron microscopy, and atomic force microscopy. It was revealed that polymerization increased d-spacing between the basal planes. In addition, as a hydrophilic polymer, HPG improved the stability and dispersion of GO sheets in biological solutions and endowed extra drug-loading capacity for the sheets. The effect of hyperbranched structure on drug loading and release was investigated by comparing drug loading and release for HPG-modified GO and linear PPO-modified GO. Our experiments indicated high drug-loading capacity (up to 185%), and excellent encapsulation efficiency (up to 93%) for HPG-GO compared to linear PO-grafted GO. The release profile of Qu under various pH levels exhibited controlled and sustained drug release without an initial burst effect for HPG-GO, suggesting that an acidic solution could facilitate drug release. HPG-GO did not show any cytotoxicity on the MCF7 cell line in different concentrations during 72 hours' incubation. Uptake and entrance of HPG-GO into the cells were verified by determining the intracellular amount of Qu by high-performance liquid chromatography. CONCLUSION: A combination of the unique properties of GO and the biodegradable polymer polyglycerol revealed high drug-loading capacity, pH-dependent drug release, and cytocompatibility with HPG-GO, thus introducing it as a promising nanocarrier for anticancer drug delivery.

In vitro selection of electrochemical peptide probes using bioorthogonal tRNA for influenza virus detection.

An electrosensitive peptide probe has been developed from an in vitro selection technique using biorthogonal tRNA prepared with an electroreactive non-natural amino acid, 3,4-ethylenedioxythiophene-conjugated aminophenylalanine. The selected probe quantitatively detected the influenza virus based on a signal "turn-on" mechanism. The developed strategy could be used to develop electrochemical biosensors toward a variety of targets.

Fluorogenic Enhancement of an in Vitro-Selected Peptide Ligand by Replacement of a Fluorescent Group.

To prepare a fluorogenic peptide ligand which binds to an arbitrary target, we previously succeeded in seeking a fluorogenic ligand to calmodulin using in vitro selection. In this study the environment-sensitive fluorescent group in the selected peptide ligand was replaced with other fluorescent groups to find the possibility to increase the fluorogenic activity. Surface plasmon resonance measurement exhibited that the binding affinity was held even after the replacement. However, the replacement significantly affected the fluorogenic activity. It depended on the kind of incorporated fluorophors and linker length. As a result, the incorporation of 4-N,N-dimethylamino-1,8-naphthalimide enhanced the fluorescence intensity over 100-fold in the presence of target calcium-bound calmodulin. This study demonstrated that the functionality of in vitro selected peptide can be tuned with keeping the binding affinity.

Two site genetic incorporation of varying length polyethylene glycol into the backbone of one peptide.

Polyethylene glycol (PEG) of different lengths was genetically incorporated into the backbone of a polypeptide using stop-anticodon and frameshift anticodon-containing tRNAs, which were acylated with PEG-containing amino acids.

In vitro selection of a photoresponsive peptide aptamer to glutathione-immobilized microbeads.

Photoresponsive peptide aptamer to glutathione-immobilized microbeads was in vitro selected using ribosome display incorporated with tRNA carrying an amino acid coupled with an azobenzene.

Direct in vitro selection of titanium-binding epidermal growth factor.

Epidermal growth factor (EGF) with affinity to TiO2 surfaces was obtained by direct in vitro selection. A random peptide library was generated for fusion to the N-terminal of EGF, and polypeptides exhibiting affinity were selected in vitro by ribosome display. The best-performing polypeptide sequence was selected for synthesis using a solid-phase method and showed high affinity to TiO2 after refolding. Molecular dynamic simulations indicated that the interaction of the selected peptide segment with the TiO2 surface was comparable to that of a previously reported titanium-binding peptide, TBP-1. The hydroxyl groups in the selected peptide segment were found to be critical for the binding interaction. NIH3T3 cell culture for two days in the presence of the TiO2-binding EGF showed that it was able to enhance cell proliferation as much as unmodified EGF in solution. As a result, the selected EGF construct was able to induce cell proliferation on titanium surfaces. This direct in vitro selection technique should extend the possibilities for the design of other surface-binding growth factors.

A fluorogenic peptide probe developed by in vitro selection using tRNA carrying a fluorogenic amino acid.

A peptide that binds and emits fluorescence in response to conformational change in a target protein was developed by in vitro selection using tRNA carrying a fluorogenic amino acid. This technology could prove to be useful for the development of separation-free immunoassays and bio-imaging analyses.

Bio-orthogonal and combinatorial approaches for the design of binding growth factors.

Merrifield chemistry enables the convenient synthesis of oligonucleotides and peptides, while recombinant DNA technology has facilitated protein engineering. Recently, protein engineering has been extended into bio-orthogonal protein engineering by the development of specific chemical or enzymatic modification technologies. The combinatorial approach of molecular evolutionary engineering (or in vitro selection) has also provided a new design tool for functional peptides. These methodologies have enabled the development of various new proteinaceous materials for biological and medical applications. Here, we will discuss recent progress in the molecular design of proteins with respect to the preparation of binding growth factors, which are of increasing importance in the biomaterials field.

Genetic PEGylation.

Polyethylene glycol (PEG) was genetically incorporated into a polypeptide. Stop-anticodon-containing tRNAs were acylated with PEG-containing amino acids and were then translated into polypeptides corresponding to DNA sequences containing the stop codons. The molecular weights of the PEG used were 170, 500, 700, 1000, and 2000 Da, and the translation was confirmed by mass spectrometry. The PEG incorporation ratio decreased as the molecular weight of PEG increased, and PEG with a molecular weight of 1000 Da was only slightly incorporated. Although improvement is required to increase the efficiency of the process, this study demonstrates the possibility of genetic PEGylation.

Design and synthesis of binding growth factors.

Growth factors play important roles in tissue regeneration. However, because of their instability and diffusible nature, improvements in their performance would be desirable for therapeutic applications. Conferring binding affinities would be one way to improve their applicability. Here we review techniques for conjugating growth factors to polypeptides with particular affinities. Conjugation has been designed at the level of gene fusion and of polypeptide ligation. We summarize and discuss the designs and applications of binding growth factors prepared by such conjugation approaches.

Carbonate apatite-facilitated intracellularly delivered siRNA for efficient knockdown of functional genes.

Gene therapy through intracellular delivery of a functional gene or a gene-silencing element is a promising approach to treat critical diseases. Elucidation of the genetic basis of human diseases with complete sequencing of human genome revealed many vital genes as possible targets in gene therapy programs. RNA interference (RNAi), a powerful tool in functional genomics to selectively silence messenger RNA (mRNA) expression, can be harnessed to rapidly develop novel drugs against any disease target. The ability of synthetic small interfering RNA (siRNA) to effectively silence genes in vitro and in vivo, has made them particularly well suited as a drug therapeutic. However, since naked siRNA is unable to passively diffuse through cellular membranes, delivery of siRNA remains the major hurdle to fully exploit the potential of siRNA technology. Here pH-sensitive carbonate apatite has been developed to efficiently deliver siRNA into the mammalian cells by virtue of its high affinity interactions with the siRNA and the desirable size of the resulting siRNA/apatite complex for effective cellular endocytosis. Moreover, following internalization by cells, siRNA was found to be escaped from the endosomes in a time-dependent manner and finally, more efficiently silenced reporter genes at a low dose than commercially available lipofectamine. Knockdown of cyclin B1 gene with only 10nM of siRNA delivered by carbonate apatite resulted in the significant death of cancer cells, suggesting that the new method of siRNA delivery is highly promising for pre-clinical and clinical cancer therapy.

Successful surgical repair for Emery-Dreifuss muscular dystrophy valvular disease with long-term follow-up.

Emery-Dreifuss muscular dystrophy (EDMD) is an X-linked muscular dystrophy in which cardiac involvement can be serious. The disease progresses rapidly and the prognosis is strongly associated with cardiac involvement. We present the case of a 49-year-old man who was admitted with shortness of breath. Echocardiography revealed a huge right atrium and severe tricuspid regurgitation caused by annular dilatation and destruction of leaflets and chordae by pacemaker leads. Tricuspid valve replacement, right atrial plication and implantation of epicardial pacemaker leads were performed. The patient is in a good condition seven years after surgery. Cardiac transplantation is the desired therapy for valvular disease in EDMD cardiomyopathy, but this case indicates that valve replacement is also acceptable if performed with the appropriate timing.

The efficacy of human atrial natriuretic peptide in patients with renal dysfunction undergoing cardiac surgery.

OBJECTIVES: The purpose of this study was to investigate the efficacy of carperitide (human atrial natriuretic peptide [h-ANP]) in perioperative management in patients with renal dysfunction, especially its kidney-protecting effects. PATIENTS AND METHODS: The subjects were 18 patients who underwent elective cardiac surgery using cardiopulmonary bypass (CPB) with a preoperative serum creatinine (Cr) level of 1.2 mg/dl or more. The subjects were prospectively assigned to 2 groups: an h-ANP-treated group (Group H, n = 10) and a non-h-ANP-treated group (Group N, n = 8). At the beginning of surgery, h-ANP administration was initiated and continued for 5 days or more. The central dose was 0.02 microg/kg/min. The primary end point included the serum Cr level and creatinine clearance (Ccr). RESULTS: In Group H, Cr level significantly decreased after surgery compared to the preoperative level. The Ccr values were significantly higher 2 and 3 days after surgery than the preoperative values. And the intraoperative urine volume significantly increased. In Group H, an increase in urinary N-acetyl-beta-D-glucosaminidase (NAG) level the day after surgery was significantly inhibited in comparison with Group N. CONCLUSION: The results of this study suggest that in patients with renal dysfunction before cardiac surgery, continuous low-dose h-ANP therapy maintains renal function, preventing its deterioration.

Isolated undersized mitral annuloplasty for functional mitral regurgitation in non-ischemic dilated cardiomyopathy: reconsideration of the relationship between preoperative coaptation depth and persistent mitral regurgitation.

BACKGROUND: A preoperative coaptation depth (CD) >or=11 mm is apparently a predictive factor for persistent mitral regurgitation (MR) after undersized mitral annuloplasty for functional MR. The results of studies of isolated undersized mitral annuloplasty in non-ischemic dilated cardiomyopathy (DCM) are reported, including the relationship between the preoperative CD and recurrent MR. METHODS AND RESULTS: Six patients (mean age, 61 years) with severe functional MR in non-ischemic DCM underwent isolated undersized mitral annuloplasty. There were no hospital deaths. At intermediate follow-up of 2.2+/-1.9 years, New York Heart Association functional class improved significantly from 3.3+/-0.5 before surgery to 2.2+/-0.4 after surgery (p=0.0016). At a mean echocardiographic follow-up of 1.9+/-1.7 years, MR grade improved significantly from 4.0+/-0.0 before surgery to 1.0+/-0.6 after surgery (p<0.001). In 4 of 5 patients with a preoperative CD >or=11 mm, functional MR improved to mild or less than mild after surgery. CONCLUSIONS: Isolated undersized mitral annuloplasty improved clinical symptoms and functional MR in non-ischemic DCM. These results suggest that preoperative CD >or=11 mm does not always predict recurrent MR after isolated undersized mitral annuloplasty for functional MR in cases of non-ischemic DCM.

A case of Takotsubo cardiomyopathy complicated by ventricular septal perforation.

The patient was a 73-year-old female who developed chest pain and dyspnea 16 days after her husband passed away. ST segment elevation was detected on V2-5 by electrocardiography and emergency coronary arteriography was done for suspected acute myocardial infarction. No coronary arterial stenosis was present and ventricular septal perforation and takotsubo cardiomyopathy were diagnosed by left ventriculography. The perforation was closed and the patient was discharged 23 days after surgery. This patient had a very rare case of takotsubo cardiomyopathy, which was complicated by ventricular septal perforation and was saved by surgical treatment.

Comparison of the effects of aortic valve replacement using 19-mm Carpentier-Edwards Perimount bioprosthesis and 19-mm Medtronic Mosaic bioprosthesis.

OBJECTIVE: Short (< or =3 months)- and middle (> or =4 months)-term results of aortic valve replacement (AVR) using 19-mm Carpentier-Edwards Perimount (CEP) bioprosthetic valves and 19-mm Medtronic Mosaic (MM) bioprosthetic valves in patients with small aortic annulus were compared. PATIENTS AND METHODS: At our facility, AVR was performed using bioprostheses in 110 patients from April 1999 to March 2006. Of these patients, 40 were treated using 19-mm CEP (Group C), and 9 using 19-mm MM (Group M). Evaluation by inquiry, physical examination, and echocardiography was performed before, a short term after, and a middle term after surgery, and the effects of AVR were compared. RESULTS: The New York Heart Association (NYHA) functional class grade showed improvements in both groups. The aortic valve peak pressure gradient was 29.8 +/- 10.1 mmHg in Group C and 53.8 +/- 17.3 mmHg in Group M, being higher in Group M, a middle term after surgery. However, the left ventricular mass index (LVMI) showed improvements in both groups compared with the values before surgery, and the left ventricular ejection fraction (LVEF) was maintained. During the middle term after surgery, the frequency of cardiac events showed no significant difference between the two groups. CONCLUSIONS: In the patients treated with 19-mm MM, the aortic valve peak pressure gradient was higher than in those treated with 19-mm CEP, but acceptable improvements in the LVMI, maintenance of the LVEF, and avoidance of cardiac events were observed in both groups.

Biocompatibility of poly2methoxyethylacrylate coating for cardiopulmonary bypass.

The systemic inflammatory response to cardiopulmonary bypass (CPB) may contribute to the development of postoperative complications. Heparin-coated circuits and poly2methoxyethylacrylate (PMEA)-coated circuits have been developed to reduce the risk of such complications. We compared the biocompatibility of these circuits. Twelve patients scheduled to undergo elective coronary artery bypass grafting (CABG) with CPB were assigned to CPB with a PMEA-coated circuit (PMEA-coated group, n=6) or a heparin-coated circuit (heparin-coated group, n=6). The plasma concentrations of the following inflammatory markers were measured before CPB and just after, 4 hours after, and 24 hours after the termination of CPB: cytokines (interleukin [IL]-6, IL-8, IL-10), complement factor (C3a), polymorphonuclear elastase (PMNE), and coagulofibrinolytic factors (thrombin-antithrombin III complex [TAT], D-dimer). Postoperative clinical response was evaluated on the basis of respiratory index, blood loss, and the postoperative and preoperative body-weight percent ratio. There were no significant differences between the groups in the plasma concentrations of IL-6, IL-10, C3a, PMNE, TAT, or D-dimer. Plasma IL-8 concentrations were below the assay detection limits at all time points in both groups. Clinical variables did not differ significantly between the groups. In conclusion, PMEA-coated CPB circuits are as biocompatible as heparin-coated CPB circuits and prevent postoperative organ dysfunction in patients undergoing elective CABG with CPB.