Thermally Reversible Gel-Sol Transition of Hydrogels via Dissociation and Association of an Artificial Protein Nanocage.

Oligomeric protein nanocages often disassemble into their subunits and reassemble by external stimuli. Thus, using these nanocages as cross-linkers for hydrogel network structures is a promising approach to allow hydrogels to undergo stimuli-responsive gel-sol transitions or self-healing. Here, we report hydrogels that show a reversible gel-sol transition resulting from the heat-induced dissociation and reassociation of protein nanocages. The hydrogel contained the 60-mer artificial protein nanocage, TIP60, as a supramolecular cross-linker for polyethylene glycol network structures. The hydrogel showed a gel-to-sol transition upon heating at a temperature above the melting point of TIP60 and immediately returned to a gel state upon cooling to room temperature. During the heating and cooling treatment of the hydrogel, small-angle X-ray scattering analysis suggested the dissociation and reassociation of TIP60. Furthermore, we demonstrated redox-responsive cargo release from TIP60 in the hydrogel. These results showed the potential of TIP60 as a component of multi-stimuli-responsive hydrogels.

Reversible Assembly of an Artificial Protein Nanocage Using Alkaline Earth Metal Ions.

Protein nanocages are of increasing interest for use as drug capsules, but the encapsulation and release of drug molecules at appropriate times require the reversible association and dissociation of the nanocages. One promising approach to addressing this challenge is the design of metal-dependent associating proteins. Such designed proteins typically have Cys or His residues at the protein surface for connecting the associating proteins through metal-ion coordination. However, Cys and His residues favor interactions with soft and borderline metal ions, such as Au+ and Zn2+, classified by the hard and soft acids and bases concept, restricting the types of metal ions available to drive association. Here, we show the alkaline earth (AE) metal-dependent association of the recently designed artificial protein nanocage TIP60, which is composed of 60-mer fusion proteins. The introduction of a Glu (hard base) mutation to the fusion protein (K67E mutant) prevented the formation of the 60-mer but formed the expected cage structure in the presence of Ca, Sr, or Ba ions (hard acids). Cryogenic electron microscopy (cryo-EM) analysis indicated a Ba ion at the interface of the subunits. Furthermore, we demonstrated the encapsulation and release of single-stranded DNA molecules using this system. Our results provide insights into the design of AE metal-dependent association and dissociation mechanisms for proteins.

Alcanivorax bacteria as important polypropylene degraders in mesopelagic environments.

IMPORTANCE: PP biodegradation has not been clearly shown (it has been uncertain whether the PP structure is actually biodegraded or not). This is the first report on the obvious biodegradation of PP. At the same time, this study shows that Alcanivorax bacteria could be major degraders of PP in mesopelagic environments. Moreover, PP biodegradation has been investigated by using solid PP as the sole carbon source. However, this study shows that PP would not be used as a sole carbon and energy source. Our data thus provide very important and key knowledge for PP bioremediation.

Column-free purification of an artificial protein nanocage, TIP60.

Protein nanocages, which have inner cavities and surface pores, are attractive materials for various applications, such as in catalysts and medicine. Recently, we produced an artificial protein nanocage, TIP60, and demonstrated its potential as a stimuli-responsive nanocarrier. In the present study, we report a simple purification method for TIP60 that can replace time-consuming and costly affinity chromatography purification. TIP60, which has an anionic surface charge, aggregated at mildly acidic pH and redissolved at neutral pH, maintaining its cage structure. This pH-responsive reversible precipitation allowed us to purify TIP60 from soluble fractions of the E. coli cell lysate by controlling the pH. Compared with conventional Ni-NTA column purification, the pH-responsive precipitation method provided purified TIP60 with similar purity (∼80%) and higher yield. This precipitation purification method should facilitate the large-scale investigation and practical use of TIP60 nanocages.

Natriuretic peptide receptor-C releases and activates guanine nucleotide-exchange factor H1 in a ligand-dependent manner.

Although natriuretic peptide receptor-C (NPR-C) is involved in the clearance of natriuretic peptides from plasma, it also possesses other physiological functions, such as inhibition of adenylyl cyclase activity through Gαi. However, the physiological roles and intracellular signaling pathways of NPR-C have yet been not fully elucidated. In this study, we identified a RhoA-specific guanine nucleotide-exchange factor, GEF-H1, as a novel binding protein of NPR-C. We demonstrated that endogenous NPR-C interacted with GEF-H1 in HeLa cells, and that the interaction between NPR-C and GEF-H1 was dependent on a 37-amino acid cytoplasmic region of NPR-C. In contrast, another natriuretic peptide receptor, NPR-A, which includes the kinase homology and guanylyl cyclase domains in the intracellular region, did not interact with GEF-H1. We also revealed that the ligands of NPR-C (i.e., ANP, CNP, and osteocrin) caused dissociation of GEF-H1 from NPR-C. Furthermore, osteocrin treatment induced phosphorylation of GEF-H1 at Ser-886, enhanced the interaction of GEF-H1 with 14-3-3, and increased the amount of activated GEF-H1. These findings strongly supported that NPR-C may be involved in diverse physiological roles by regulating GEF-H1 signaling.

Outcomes of idiopathic pulmonary arterial hypertension in Japanese children: a retrospective cohort study.

Recently, targeted therapy has been developed for idiopathic pulmonary arterial hypertension (IPAH). Studies evaluating the prognosis of IPAH have been conducted in adults. However, there is no nationwide survey of pediatric patients with IPAH regarding the long-term prognosis in Japan. Therefore, we investigated the clinical outcomes of Japanese pediatric patients with IPAH and risk factors for a poor prognosis. This multi-center, retrospective cohort study included pediatric patients with IPAH under the age of 15 years, who were gleaned from the nationwide network of Japanese Society of Pediatric Cardiology and Cardiac Surgery (JSPCCS). The questionnaire was sent to members of JSPCCS in 2015. Patients who were diagnosed with IPAH from 1994 to 2014 were included. The primary endpoint was death or lung transplantation. Ninety-five patients were finally enrolled. Both the mean age at diagnosis and the mean follow-up duration were 7 years. Ninety-five percent of patients had received targeted therapy for IPAH during follow-up. The overall 1, 3, 5, and 10-year event free rate, estimated using Kaplan-Meier survival estimate, was 96, 91, 83, and 74%, respectively. The prognosis was significantly poorer in patients with increased right ventricular systolic pressure (RVp), mean pulmonary artery pressure (mPAP) (≥ 52 mmHg), cardiothoracic ratio (≥ 55%), and levels of B-type natriuretic peptide (BNP) during follow-up (≥ 300 pg/mL) than in those without these parameters. In conclusion, in Japanese children with IPAH, the event-free rate for death or lung transplantation was found to be good. Greater RVp, mPAP, BNP levels during follow-up, and cardiothoracic ratio may be predictive indicators for a poor prognosis.

Postinfectious Bronchiolitis Obliterans Misdiagnosed as Bronchial Asthma in a Pediatric Patient.

Bronchiolitis obliterans is a chronic obstructive respiratory disease involving stenosis or occlusion of the bronchioles and smaller airways. The prognosis of bronchiolitis obliterans is poor, and the patient might require home oxygen therapy and/or lung transplantation. Bronchiolitis obliterans has various etiologies; in children, the most common causes are infections by respiratory pathogens like adenoviruses. In such cases, the condition is termed as postinfectious bronchiolitis obliterans. A 7-year-old girl was diagnosed with bronchial asthma at the age of 1 year and was on a regimen of a leukotriene receptor antagonist and an inhaled corticosteroid. At 1 year of age, she was admitted to our hospital with a respiratory syncytial virus infection, and despite continued treatment with the above drugs, she required frequent readmissions. At the age of 7 years, she was diagnosed with postinfectious bronchiolitis obliterans based on the following findings: mosaic perfusion on high-resolution chest computed tomography and ventilation-perfusion mismatch on ventilation-perfusion scintigraphy. A lung biopsy was not performed due to its invasiveness. It has been suggested that appropriate treatment during the early stage improves the prognosis of bronchiolitis obliterans. This disease might be misdiagnosed as bronchial asthma because of the clinical similarities. In patients who do not respond to the treatment for bronchial asthma, pediatricians should consider other diseases with similar signs and symptoms, such as bronchiolitis obliterans, in the differential diagnosis.

Chemoenzymatic Cascade Synthesis of Optically Pure Alkanoic Acids by Using Engineered Arylmalonate Decarboxylase Variants.

Arylmalonate decarboxylase (AMDase) catalyzes the cofactor-free asymmetric decarboxylation of prochiral arylmalonic acids and produces the corresponding monoacids with rigorous R selectivity. Alteration of catalytic cysteine residues and of the hydrophobic environment in the active site by protein engineering has previously resulted in the generation of variants with opposite enantioselectivity and improved catalytic performance. The substrate spectrum of AMDase allows it to catalyze the asymmetric decarboxylation of small methylvinylmalonic acid derivatives, implying the possibility to produce short-chain 2-methylalkanoic acids with high optical purity after reduction of the nonactivated C=C double bond. Use of diimide as the reductant proved to be a simple strategy to avoid racemization of the stereocenter during reduction. The developed chemoenzymatic sequential cascade with use of R- and S-selective AMDase variants produced optically pure short-chain 2-methylalkanoic acids in moderate to full conversion and gave both enantiomers in excellent enantiopurity (up to 83 % isolated yield and 98 % ee).

PFG acted as an inducer of premature senescence in TIG-1 normal diploid fibroblast and an inhibitor of mitosis in the HeLa cells.

Our previous work has reported an anti-proliferative compound from moutan cortex, paeoniflorigenone which can induce cancer-selective apoptosis. However, its anti-proliferative mechanism is still unknown. According to morphology changes (hypertrophy and flattening), we hypothesized that PFG can induce senescence or inhibit cell mitosis. Here we show that PFG can induce cellular senescence, evidenced by the expression of senescence-associated ß-galactosidase, G0/G1 cell cycle arrest and permanent loss of proliferative ability, in normal TIG-1 diploid fibroblast but not cancerous HeLa cells. In cancerous HeLa cells, PFG inhibited proliferation by inducing S and G2/M cell cycle arrest and mitosis inhibition. DNA damage response was activated by PFG, interestingly the reactive oxygen species level was suppressed instead of escalated. To sum up, we report 3 new roles of PFG as, 1. inducer of premature senescence in normal TIG-1 cells, 2. inhibitor of mitosis in cancerous HeLa cells, 3. ROS scavenger. Abbreviations: PFG: Paeoniflorigenone; ROS: reactive oxygen species; ATM: ataxia telangiectasia mutated; t-BHP: tert-butyl hydroperoxide; SA-ß-gal: senescence-associatedß-galactosidase; DNA-PKcs: DNA-dependent protein kinase; γ-H2AX: H2AX phosphoryla-tion at Ser-139.

Pharmacological effects of N-[2-[[2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetyl]oxy]ethyl]hyaluronamide (diclofenac Etalhyaluronate, SI-613), a novel sodium hyaluronate derivative chemically linked with diclofenac.

BACKGROUND: Osteoarthritis (OA) is the most common joint disorder worldwide and one of the leading causes of disability in the elderly. We have investigated the novel sodium hyaluronate derivative chemically linked with diclofenac (DF), diclofenac etalhyaluronate (SI-613), which is a potentially safer and more effective treatment for OA knee pain. In this study, we evaluated the pharmacological effects of SI-613 in experimental arthritis models. METHODS: We compared the analgesic and anti-inflammatory effects of intra-articularly administered SI-613, hyaluronic acid (HA), and of orally administered diclofenac sodium (DF-Na) in rat silver nitrate-induced arthritis model and rabbit antigen-induced arthritis model. RESULTS: A single intra-articular (IA) administration of SI-613 significantly suppressed pain responses in rats in a dose-dependent manner. The analgesic effects were greater than those of HA, a mixture of DF-Na and HA, or an oral once-daily administration of DF-Na. In the rabbit arthritis model, SI-613 significantly reduced knee joint swelling compared with that in the control group on day 1 after a single IA injection. This significant anti-inflammatory effect was observed until day 28. In the pharmacokinetic study, the DF concentration in the synovium after SI-613 administration reached its maximum concentration of 311.6 ng/g on day 1, and gradually declined to 10 ng/g by day 28. It fell below the lower limit of quantification on day 35. Thus, a clear correlation was found between pharmacokinetics and pharmacodynamics. These results demonstrate that SI-613 exerts its long-lasting and potent anti-inflammatory effect by sustainable release of DF in the knee joint tissues. CONCLUSION: A single IA injection of SI-613 was shown to exert analgesic and anti-inflammatory effects for 28 days in non-clinical pharmacological studies, suggesting that SI-613 will be a promising candidate in the treatment of osteoarthritis pain.

Design of Hollow Protein Nanoparticles with Modifiable Interior and Exterior Surfaces.

Protein-based nanoparticles hold promise for a broad range of applications. Here, we report the production of a uniform anionic hollow protein nanoparticle, designated TIP60, which spontaneously assembles from a designed fusion protein subunit based on the geometric features of polyhedra. We show that TIP60 tolerates mutation and both its interior and exterior surfaces can be chemically modified. Moreover, TIP60 forms larger structures upon the addition of a cationic protein. Therefore, TIP60 can be used as a modifiable nano-building block for further molecular assembly.

Type II cGMP-dependent protein kinase negatively regulates fibroblast growth factor signaling by phosphorylating Raf-1 at serine 43 in rat chondrosarcoma cells.

Although type II cGMP-dependent protein kinase (PKGII) is a major downstream effector of cGMP in chondrocytes and attenuates the FGF receptor 3/ERK signaling pathway, its direct target proteins have not been fully explored. In the present study, we attempted to identify PKGII-targeted proteins, which are associated with the inhibition of FGF-induced MAPK activation. Although FGF2 stimulation induced the phosphorylation of ERK1/2, MEK1/2, and Raf-1 at Ser-338 in rat chondrosarcoma cells, pretreatment with a cell-permeable cGMP analog strongly inhibited their phosphorylation. On the other hand, Ser-43 of Raf-1 was phosphorylated by cGMP in a dose-dependent manner. Therefore, we examined the direct phosphorylation of Raf-1 by PKGII. Wild-type PKGII phosphorylated Raf-1 at Ser-43 in a cGMP-dependent manner, but a PKGII D412A/R415A mutant, which has a low affinity for cGMP, did not. Finally, we found that a phospho-mimic mutant, Raf-1 S43D, suppressed FGF2-induced MAPK pathway. These results suggest that PKGII counters FGF-induced MEK/ERK activation through the phosphorylation of Raf-1 at Ser-43 in chondrocytes.

Protein Nanoparticle Formation Using a Circularly Permuted α-Helix-Rich Trimeric Protein.

We here report the production of highly spherical protein nanoparticles based on the domain-swapping oligomerization of a circularly permuted trimeric protein, major histocompatibility complex (MHC) class II associated chaperonin. The size distribution of the nanoparticles can be adjusted to between 40 and 100 nm in diameter, and thus, these particles are suitable as drug carriers following purification under basic conditions. Our approach involves no harsh treatments and could provide an alternative approach for protein nanoparticle formation.

Apoptosis-inducing activity and antiproliferative effect of Paeoniflorigenone from moutan cortex.

Ninety samples from the extracts of plants from traditional Chinese medicines were screened for antitumor activity. Paeoniflorigenone (PFG) was isolated as an active ingredient from the root of moutan cortex, which showed the strongest activity. In addition, our data indicated that PFG was cytotoxic and induced apoptosis selectively in the cancer cell lines. These effects were cancelled by the addition of caspase inhibitor Z-VAD-FMK, suggesting that it was mediated by caspase-3 activation.

Low temperature plasma equipment applied on surgical hemostasis and wound healings.

Low temperature plasma (LTP) coagulation equipment, which avoids causing burn injuries to patients, has been introducing into minimally invasive surgery. The mechanism by which this equipment stops bleeding is to directly occupy the injury with the formed blood clots, and different from the mechanism of the common electrical hemostatic devices that cauterize the tissues around the bleeding to stem the blood flow. A noteworthy point is that LTP treatment with our equipment is not confined only to the blood coagulation system, but it has significant effects on the other blood components to form clots with or without hemolysis, and that there is a plasma current threshold that determines whether the treatment makes stable clots. In this review, we introduce the clinical benefits of LTP current and describe the clot formation it facilitates.

Identification and characterization of two new 5-keto-4-deoxy-D-Glucarate Dehydratases/Decarboxylases.

BACKGROUND: Hexuronic acids such as D-galacturonic acid and D-glucuronic acid can be utilized via different pathways within the metabolism of microorganisms. One representative, the oxidative pathway, generates α-keto-glutarate as the direct link entering towards the citric acid cycle. The penultimate enzyme, keto-deoxy glucarate dehydratase/decarboxylase, catalyses the dehydration and decarboxylation of keto-deoxy glucarate to α-keto-glutarate semialdehyde. This enzymatic reaction can be tracked continuously by applying a pH-shift assay. RESULTS: Two new keto-deoxy glucarate dehydratases/decarboxylases (EC 4.2.1.41) from Comamonas testosteroni KF-1 and Polaromonas naphthalenivorans CJ2 were identified and expressed in an active form using Escherichia coli ArcticExpress(DE3). Subsequent characterization concerning K m, k cat and thermal stability was conducted in comparison with the known keto-deoxy glucarate dehydratase/decarboxylase from Acinetobacter baylyi ADP1. The kinetic constants determined for A. baylyi were K m 1.0 mM, k cat 4.5 s-1, for C. testosteroni K m 1.1 mM, k cat 3.1 s-1, and for P. naphthalenivorans K m 1.1 mM, k cat 1.7 s-1. The two new enzymes had a slightly lower catalytic activity (increased K m and a decreased k cat) but showed a higher thermal stability than that of A. baylyi. The developed pH-shift assay, using potassium phosphate and bromothymol blue as the pH indicator, enables a direct measurement. The use of crude extracts did not interfere with the assay and was tested for wild-type landscapes for all three enzymes. CONCLUSIONS: By establishing a pH-shift assay, an easy measurement method for keto-deoxy glucarate dehydratase/decarboxylase could be developed. It can be used for measurements of the purified enzymes or using crude extracts. Therefore, it is especially suitable as the method of choice within an engineering approach for further optimization of these enzymes.

Analysis of mobile fronthaul bandwidth and wireless transmission performance in split-PHY processing architecture.

We analyze the mobile fronthaul (MFH) bandwidth and the wireless transmission performance in the split-PHY processing (SPP) architecture, which redefines the functional split of centralized/cloud RAN (C-RAN) while preserving high wireless coordinated multi-point (CoMP) transmission/reception performance. The SPP architecture splits the base stations (BS) functions between wireless channel coding/decoding and wireless modulation/demodulation, and employs its own CoMP joint transmission and reception schemes. Simulation results show that the SPP architecture reduces the MFH bandwidth by up to 97% from conventional C-RAN while matching the wireless bit error rate (BER) performance of conventional C-RAN in uplink joint reception with only 2-dB signal to noise ratio (SNR) penalty.

Red blood cell coagulation induced by low-temperature plasma treatment.

Low-temperature plasma (LTP) treatment promotes blood clot formation by stimulation of the both platelet aggregation and coagulation factors. However, the appearance of a membrane-like structure in clots after the treatment is controversial. Based on our previous report that demonstrated characteristics of the form of coagulation of serum proteins induced by LTP treatment, we sought to determine whether treatment with two plasma instruments, namely BPC-HP1 and PN-110/120TPG, formed clots only from red blood cells (RBCs). LTP treatment with each device formed clots from whole blood, whereas LTP treatment with BPC-HP1 formed clots in phosphate-buffered saline (PBS) containing 2 × 10(9)/mL RBCs. Light microscopic analysis results showed that hemolysis formed clots consisting of materials with membrane-like structures from both whole blood and PBS-suspended RBCs. Moreover, electron microscopic analysis results showed a monotonous material with high electron density in the formed clots, presenting a membrane-like structure. Hemolysis disappeared with the decrease in the current through the targets contacting with the plasma flare and clot formation ceased. Taken together, our results and those of earlier studies present two types of blood clot formation, namely presence or absence of hemolysis capability depending on the current through the targets.

Arylmalonate decarboxylase-a highly selective bacterial biocatalyst with unknown function.

Bacterial arylmalonate decarboxylase (AMDase) shows high enantioselectivity and a broad substrate spectrum in the asymmetric synthesis of optically pure arylaliphatic carboxylic acids. The determination of the structure of AMDase has greatly extended the understanding of the catalytic mechanism of this unique cofactor-free decarboxylase and allowed the generation of tailor-made enzyme variants with improved catalytic properties. Despite this increase in knowledge and applicability, the natural role of the enzyme remains unknown. This mini-review summarizes the recent findings on the molecular mechanism and the synthetic application of the enzyme.

Prognostic value of multiple biomarkers for cardiovascular mortality in adult congenital heart disease: comparisons of single-/two-ventricle physiology, and systemic morphologically right/left ventricles.

Although there are many biomarkers for heart failure, limited data are available regarding their prognostic value in adult congenital heart disease (ACHD). We investigated the potential of various biomarkers to predict ACHD mortality in a single-center, retrospective cohort study. Blood levels of neurohormones [angiotensin II, endothelin-1 (ET-1), norepinephrine (NE), aldosterone, and plasma renin activity]; inflammatory biomarkers [high-sensitivity C-reactive protein (hs-CRP), high-sensitivity tumor necrosis factor, soluble TNF receptor type I and II (sTNF-RI and sTNF-RII), and interleukin-6 (IL-6)]; and brain natriuretic peptide (BNP) were measured in 103 ACHD patients (median age 28 years). Subjects were divided into patients with single-ventricle physiology (SV group, n = 61) and those with two-ventricle physiology (TV group, n = 42); and into patients with a systemic right ventricle (SRV group, n = 25) and those with a systemic left ventricle (SLV group, n = 78). During a median follow-up period of 6.5 years, 12 patients (11 %) died of acute decompensated heart failure (ADHF). Predictive biomarkers, which are related to the New York Heart Association class and cardiothoracic ratio, were as follows: elevated levels of BNP, ET-1, sTNF-RI, NE, and IL-6 in the overall patient group; IL-6, NE, hs-CRP, BNP, and ET-1 in the TV group; BNP and ET-1 in the SV group; BNP, NE, hs-CRP, sTNF-RI, IL-6, and ET-1 in the SLV group. Elevated levels of ET-1 in SRV groups were slightly although not significantly associated with these. Various clinical biomarkers are associated with ADHF mortality in ACHD patients. The most prominent mortality predictors in biomarker profiles may vary according to differences in ventricular physiology and systemic ventricle morphology.