Polymerization of Horseradish Peroxidase by a Laccase-Catalyzed Tyrosine Coupling Reaction.

The polymerization of proteins can create newly active and large bio-macromolecular assemblies that exhibit unique functionalities depending on the properties of the building block proteins and the protein units in polymers. Herein, the first enzymatic polymerization of horseradish peroxidase (HRP) is reported. Recombinant HRPs fused with a tyrosine-tag (Y-tag) through a flexible linker at the N- and/or C-termini are expressed in silkworm, Bombyx mori. Trametes sp. laccase (TL) is used to activate the tyrosine of Y-tagged HRPs with molecular O2 to form a tyrosyl-free radical, which initiates the tyrosine coupling reaction between the HRP units. A covalent dityrosine linkage is also formed through a HRP-catalyzed self-crosslinking reaction in the presence of H2 O2 . The addition of H2 O2 in the self-polymerization of Y-tagged HRPs results in lower activity of the HRP polymers, whereas TL provides site-selectivity, mild reaction conditions and maintains the activity of the polymeric products. The cocrosslinking of Y-tagged HRPs and HRP-protein G (Y-HRP-pG) units catalyzed by TL shows a higher signal in enzyme-linked immunosorbent assay (ELISA) than the genetically pG-fused HRP, Y-HRP-pG, and its polymers. This new enzymatic polymerization of HRP promises to provide highly active and functionalized polymers for biomedical applications and diagnostics probes.

Efficient and Supplementary Enzyme Cocktail from Actinobacteria and Plant Biomass Induction.

Actinobacteria plays a key role in the cycling of organic matter in soils. They secret biomass-degrading enzymes that allow it to produce the unique metabolites that originate in plant biomass. Although past studies have focused on these unique metabolites, a large-scale screening of Actinobacteria is yet to be reported to focus on their biomass-degrading ability. In the present study, a rapid and simple method is constructed for a large-scale screening, and the novel resources that form the plant biomass-degrading enzyme cocktail are identified from 850 isolates of Actinobacteria. As a result, Nonomuraea fastidiosa secretes a biomass degrading enzyme cocktail with the highest enzyme titer, although cellulase activities are lower than a commercially available enzyme. So the rich accessory enzymes are suggested to contribute to the high enzyme titer for a pretreated bagasse with a synergistic effect. Additionally, an optimized cultivation method of biomass induction caused to produce the improved enzyme cocktail indicated strong enzyme titers and a strong synergistic effect. Therefore, the novel enzyme cocktails are selected via the optimized method for large-scale screening, and then the enzyme cocktail can be improved via the optimized production with biomass-induction.

Therapeutic targeting of chemoresistant and recurrent glioblastoma stem cells with a proapoptotic variant of oncolytic herpes simplex virus.

Temozolomide (TMZ) chemotherapy, in combination with maximal safe resection and radiotherapy, is the current standard of care for patients with glioblastoma (GBM). Despite this multimodal approach, GBM inevitably relapses primarily due to resistance to chemo-radiotherapy, and effective treatment is not available for recurrent disease. In this study we identified TMZ resistant patient-derived primary and previously treated recurrent GBM stem cells (GSC), and investigated the therapeutic activity of a pro-apoptotic variant of oHSV (oHSV-TRAIL) in vitro and in vivo. We show that oHSV-TRAIL modulates cell survival and MAP Kinase proliferation signaling pathways as well as DNA damage response pathways in both primary and recurrent TMZ-resistant GSC. Utilizing real time in vivo imaging and correlative immunohistochemistry, we show that oHSV-TRAIL potently inhibits tumor growth and extends survival of mice bearing TMZ-insensitive recurrent intracerebral GSC tumors via robust and selective induction of apoptosis-mediated death in tumor cells, resulting in cures in 40% of the treated mice. In comparison, the anti-tumor effects in a primary chemoresistant GSC GBM model exhibiting a highly invasive phenotype were significant but less prominent. This work thus demonstrates the ability of oHSV-TRAIL to overcome the therapeutic resistance and recurrence of GBM, and provides a basis for its testing in a GBM clinical trial.

Tactile assessment for the reversibility of rocuronium-induced neuromuscular blockade during propofol or sevoflurane anesthesia.

We sought to determine whether tactile train-of-four (TOF) count can predict the efficacy of neostigmine administration for rocuronium-induced blockade during propofol or sevoflurane anesthesia, and to follow subsequent recovery until the TOF ratio reached 0.9. One-hundred-sixty patients, divided into eight equal groups, were randomly allocated to maintenance of anesthesia with propofol or sevoflurane. The tactile response of the adductor pollicis to TOF stimulation was evaluated on one arm, and the mechanomyographic response was recorded on the other. Neuromuscular block was induced with rocuronium 0.6 mg/kg and maintained with rocuronium to 15% of the control first twitch in TOF. Neostigmine 0.07 mg/kg was administered on reappearance of the first (Group I), second (Group II), third (Group III), or fourth (Group IV) tactile TOF response in each anesthesia. At this time, sevoflurane or the propofol dosage was reduced in each group (n = 20 in each group). The times from administration of neostigmine until the TOF ratio recovered to 0.7, 0.8, and 0.9 were recorded. The times [median (range)] to TOF ratio = 0.9 were 8.6 (4.7-18.9), 7.5 (3.4-9.8), 5.4 (1.6-8.6), and 4.7 (1.3-7.2) min in Groups I-IV during propofol anesthesia, respectively, and 28.6 (8.8-75.8), 22.6 (8.3-57.4), 15.6 (7.3-43.9), and 9.7 (5.1-26.4) min in corresponding groups during sevoflurane anesthesia, respectively (P < 0.0001). We recommend more than 2 TOF responses with propofol anesthesia and 4 TOF responses with sevoflurane anesthesia for adequate reversal within 10 and 15 min, respectively. The more tactile TOF responses present at the time of reversal achieved greater adequate recovery; however, tactile TOF responses are not a completely reliable predictor within a reasonable time period.

Virtual reality system for treatment of the fear of public speaking using image-based rendering and moving pictures.

The fear of speaking is often cited as the world's most common social phobia. The rapid growth of computer technology enabled us to use virtual reality (VR) for the treatment of the fear of public speaking. There have been two techniques used to construct a virtual environment for the treatment of the fear of public speaking: model-based and movie-based. Virtual audiences and virtual environments made by model-based technique are unrealistic and unnatural. The movie-based technique has a disadvantage in that each virtual audience cannot be controlled respectively, because all virtual audiences are included in one moving picture file. To address this disadvantage, this paper presents a virtual environment made by using image-based rendering (IBR) and chroma keying simultaneously. IBR enables us to make the virtual environment realistic because the images are stitched panoramically with the photos taken from a digital camera. And the use of chroma keying allows a virtual audience to be controlled individually. In addition, a real-time capture technique was applied in constructing the virtual environment to give the subjects more interaction, in that they can talk with a therapist or another subject.

CT and MR imaging features of adnexal torsion.

In adnexal torsion, the ovary, ipsilateral fallopian tube, or both twist with the vascular pedicle, resulting in vascular compromise. Unrelieved torsion is likely to cause hemorrhagic infarction as the degree of arterial occlusion increases. Therefore, early diagnosis is important to preserve the affected ovary. Adnexal torsion commonly accompanies an ipsilateral ovarian neoplasm or cyst but can also occur in normal ovaries, usually in children. Although ultrasonography is typically the initial emergent examination, computed tomography (CT) and magnetic resonance (MR) imaging may also be useful diagnostic tools. Common CT and MR imaging features of adnexal torsion include fallopian tube thickening, smooth wall thickening of the twisted adnexal cystic mass, ascites, and uterine deviation to the twisted side. Uncommon imaging findings in adnexal torsion that are specific to hemorrhagic infarction include hemorrhage in the thickened fallopian tube, hemorrhage within the twisted ovarian mass, and hemoperitoneum. Additional imaging findings that can suggest hemorrhagic infarction include eccentric smooth wall thickening exceeding 10 mm in a cystic ovarian mass converging on the thickened fallopian tube and lack of contrast enhancement of the internal solid component or thickened wall of the twisted ovarian mass. Early diagnosis can help prevent irreversible structural damage and may allow conservative, ovary-sparing treatment.