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As the acute lymphoblastic leukaemia (ALL) develops, expression of L-asparaginase (ASNase) protein is known to decrease. Therefore, deficiency of the ASNase protein would be regarded as one of the significant indications of the ALL. For the treatment of ALL, recombinant ASNase protein derived from bacterial origin is used which causes cytotoxicity by deprivation of Asn. However, short half-life of the protein is an obstacle for medical use. In order to overcome this limit, recombinant ASNase was fused to 30Kc19 with protein-stabilizing and cell-penetrating properties. As the 30Kc19 protein may induce steric hindrance, we further added a PLGLAG linker sequence (LK) between the ASNase and 30Kc19. The treatment of ASNase-LK-30Kc19 fusion protein demonstrated enhanced stability, cell-penetrating property, and anti-cancer activity. Intracellular delivery of both the non-cleaved and cleaved forms of the protein were observed, suggesting that ASNase acted both internally and externally, performing high anti-cancer activity by effective depletion of intracellular Asn. Additionally, ASNase-LK-30Kc19 showed high selectivity towards cancer cells. In terms of the dosage, releasable ASNase from ASNase-LK-30Kc19 reached the same half-maximal inhibitory concentration at a concentration five times lower than non-releasable ASNase-30Kc19. Altogether, the findings suggest that this fusion approach has potential applications in the treatment of ALL.

Aim. To evaluate the effectiveness of subconjunctivally injected viscoelastic material (VEM) for the self-sealing of leaking sclerotomy in transconjunctival sutureless vitrectomy (TSV). Methods. This was a prospective interventional series. Subconjunctival injection of VEM was performed in eyes showing leaking sclerotomy at the end of TSV in selected cases. This procedure was performed in 24 consecutive eyes from 24 patients scheduled for 23- or 25-gauge TSV with phacoemulsification for various vitreoretinal diseases combined with cataracts. Results. Among the 24 eyes, 13 cases were scheduled for 23-gauge TSV, while 11 cases were scheduled for 25-gauge TSV. The average number of injection sites per eye was 1.7±0.9 in the 23-gauge cases and 1.5±0.7 in the 25-gauge cases. Leakage was most commonly observed at the vitrector site of the sclerotomy, while little leakage was observed at the illuminator site. There were no cases of postoperative hypotony. Conclusion. Subconjunctival injection of VEM was simple and effective for the self-sealing of leaking sclerotomy after TSV in selected cases.

Aim. To analyze the subfoveal choroid thickness and choroidal volume in unilateral ocular ischemic syndrome (OIS). Methods. A retrospective review was conducted for all patients with unilateral OIS from October 2010 through June 2014. The subfoveal choroidal thickness (SFChT) and choroidal volume of both eyes were compared. Results. 19 unilateral OIS patients were included in this study. The mean SFChT of OIS eyes was significantly lower than that of fellow eyes (OIS eyes: 208.89 ± 82.62 μm and fellow eyes: 265.31 ± 82.77 μm, P

The physiology of a very closely related strain, Anaerocellum thermophilum DSM 6725, is described and its ability to grow on different types of defined and complex substrates is examined by focusing on switchgrass and poplar. The studies have shown that Anaerocellum thermophilum DSM 6725 has grown efficiently on both types of substrate without a chemical pretreatment step.

The effects of the mutagenesis of the substrate binding sites on the various catalytic properties of Pyrococcus furiosus thermostable amylase (TA), a cyclodextrin-degrading (CD) enzyme, are analyzed.

A study done on the gene corresponding to putative amylolytic enzyme of Pyrococcus furiosus is cloned and shown in Escherichia coli and the recombinant enzyme is purified with its enzymatic characteristics examined. Studies demonstrate that the putative amylolytic enzyme of Pyrococcus furiosus is a novel amylase, possessing characteristics of both cyclodextrin-hydrolyzing enzyme and alpha-amylase.

Purpose: To determine whether intravitreally-injected baicalin inhibits the growth of choroidal neovascularization (CNV) experimentally induced via laser photocoagulation through analysis of angiogenic factors. Materials and methods: Six CNVs were induced in the left eyes of 8-week-old male Brown Norway rats. Immediately after the induction of CNV, 4 μl of baicalin solution (0.1, 1 or 5 nmol) and 4 μl of a solution containing 100 μg of bevacizumab were slowly injected into the vitreous cavity under direct observation with an operating microscope. At 14 days after CNV induction, fluorescein angiography (FA) was performed, and choroidal flat mounts were produced for quantitative assessment of CNV. The levels of the anti-angiogenic proteins vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and matrix metalloproteinase-2 (MMP-2) were determined via Western blot analysis. Results: FA of bevacizumab- and baicalin-treated rats showed significantly reduced CNV and leakage from the CNV lesions compared to control rats at day 14. Choroidal flat mounts revealed that baicalin inhibited the growth of CNV lesions in a dose-dependent manner. Western blot analysis demonstrated that baicalin significantly attenuated the up-regulation of VEGF, PDGF and MMP-2. Conclusion: Baicalin suppressed laser-induced CNV formation in rats. These results suggest that baicalin should be considered as a candidate drug for treating exudative age-related macular degeneration. [ABSTRACT FROM AUTHOR]

To investigate laser photocoagulation and intravitreal bevacizumab efficacy and safety in patients with moderate-to-severe stage 3 retinopathy of prematurity (ROP), and to evaluate the effects of treatment on the development of peripheral retinal vessels. A retrospective chart review of 15 premature babies, all of whom were diagnosed with stage 3 ROP, was conducted. Patients with moderate-to-severe stage 3 ROP, thus with a vascular-active ROP, received intravitreal injections of bevacizumab (0.5 mg/0.02 ml) and laser photocoagulation, whereas those with relatively inactive ROP received laser photocoagulation only. Patients were examined 1, 2, 4, and 8 weeks after treatment, or until peripheral retinal vessel growth over the laser scar was noted. Both eyes of 15 patients diagnosed with moderate-to-severe stage 3 ROP were evaluated. Eight patients ( n = 16 eyes) received intravitreal bevacizumab injection and laser photocoagulation, and seven patients ( n = 14 eyes) received laser photocoagulation only. During the follow-up period, regression of plus disease and peripheral retinal vessel development appeared significantly more rapidly in patients who received both intravitreal bevacizumab injection and laser photocoagulation. Peripheral retinal vessel development over the laser scar was identified 1–2 weeks after treatment. No systemic or significant ocular complications, such as vitreous hemorrhage, retinal detachment, or endophthalmitis, were noted during follow-up after treatment. A combination of laser photocoagulation and intravitreal bevacizumab injection seems to be a safe and effective therapy in patients with moderate-to-severe stage 3 ROP, promulgating rapid development of peripheral retinal vessels. [ABSTRACT FROM AUTHOR]

We present an efficient pipeline enabling high-throughput analysis of protein structure in solution with small angle X-ray scattering (SAXS). Our SAXS pipeline combines automated sample handling of microliter volumes, temperature and anaerobic control, rapid data collection and data analysis, and couples structural analysis with automated archiving. We subjected 50 representative proteins, mostly from Pyrococcus furiosus, to this pipeline and found that 30 were multimeric structures in solution. SAXS analysis allowed us to distinguish aggregated and unfolded proteins, define global structural parameters and oligomeric states for most samples, identify shapes and similar structures for 25 unknown structures, and determine envelopes for 41 proteins. We believe that high-throughput SAXS is an enabling technology that may change the way that structural genomics research is done. [ABSTRACT FROM AUTHOR]

A gene that encodes the enzyme Pyrococcus furiosus cyclodextrin glucanotransferase (PFCGT) was cloned in Escherichia coli. PFCGT was highly expressed in recombinant E. coli after compensation for codon usage bias using the pRARE plasmid. Purified PFCGT was extremely thermostable with an optimal temperature and pH of 95°C and 5.0, respectively, retaining 97% of its activity at 100°C. Incubation at 60°C for 20 min during the purification process led to a 1.5-fold increase in enzymatic activity. A time course assay of the PFCGT reaction with starch indicated that cyclic α-1,4-glucans with DPs greater than 20 were produced at the beginning of the incubation followed by an increase in β-CD. The major final product of PFCGT cyclization was β-CD, and thus the enzyme is a β-CGTase. [ABSTRACT FROM AUTHOR]

DNA shuffling was used to improve the thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2. Two highly thermostable mutants, III-1 and III-2, were generated after three rounds of shuffling and recombination of mutations. Their optimal reaction temperatures were all 80 °C, which was 10 °C higher than that of the wild-type. The mutant enzyme III-1 carried seven mutations: N147D, F195L, N263S, D311G, A344V, F397S, and N508D. The half-life of III-1 was about 20 times greater than that of the wild-type at 78 °C. The mutant enzyme III-2 carried M375T in addition to the mutations in III-1, which was responsible for the decrease in specific activity. The half-life of III-2 was 568 min while that of the wild-type was <1 min at 80 °C. The melting temperatures of III-1 and III-2, as determined by differential scanning calorimetry, increased by 6.1 °C and 11.4 °C, respectively. Hydrogen bonding, hydrophobic interaction, electrostatic interaction, proper packing, and deamidation were predicted as the mechanisms for the enhancement of thermostability in the enzymes with the mutations. [ABSTRACT FROM AUTHOR]

Genomic analysis of the hyperthermophilic archaeon Pyrococcus furiosus revealed the presence of an open reading frame (ORF PF1939) similar to the enzymes in glycoside hydrolase family 13. This amylolytic enzyme, designated PFTA (Pyrococcusfuriosus thermostable amylase), was cloned and expressed in Escherichia coli. The recombinant PFTA was extremely thermostable, with an optimum temperature of 90°C. The substrate specificity of PFTA suggests that it possesses characteristics of both α-amylase and cyclodextrin-hydrolyzing enzyme. Like typical α-amylases, PFTA hydrolyzed maltooligosaccharides and starch to produce mainly maltotriose and maltotetraose. However, it could also attack and degrade pullulan and β-cyclodextrin, which are resistant to α-amylase, to primarily produce panose and maltoheptaose, respectively. Furthermore, acarbose, a potent α-amylase inhibitor, was drastically degraded by PFTA, as is typical of cyclodextrin-hydrolyzing enzymes. These results confirm that PFTA possesses novel catalytic properties characteristic of both α-amylase and cyclodextrin-hydrolyzing enzyme. [ABSTRACT FROM AUTHOR]

The article describes a new orthodontic appliance for orthopedic correction of Class III malocclusion. It introduces and evaluates the Tandem Traction Bow Appliance (TTBA) for treatment of growing Class III patients. It discusses the construction of the appliance. It also presents a case study to determine the efficacy of TTBA.

A letter to the editor is presented in response to the article on the therapeutic use of intravitreal bevacizumab and photodynamic therapy in treating chronic central serous chorioretinopathy.

"Anaerocellum thermophilum" DSM 6725 is a strictly anaerobic bacterium that grows optimally at 75°C. It uses a variety of polysaccharides, including crystalline cellulose and untreated plant biomass, and has potential utility in biomass conversion. Here we report its complete genome sequence of 2.97 Mb, which is contained within one chromosome and two plasmids (of 8.3 and 3.6 kb). The genome encodes a broad set of cellulolytic enzymes, transporters, and pathways for sugar utilization and compared to those of other saccharolytic, anaerobic thermophiles is most similar to that of Caldicellulosiruptor saccharolyticus DSM 8903. [ABSTRACT FROM AUTHOR]

Most fungi and bacteria degrade plant cell walls by secreting free, complementary enzymes that hydrolyze cellulose; however, some bacteria use large enzymatic assemblies called cellulosomes, which recruit complementary enzymes to protein scaffolds. The thermophilic bacterium Caldicellulosiruptor bescii uses an intermediate strategy, secreting many free cellulases that contain multiple catalytic domains. One of these, CelA, comprises a glycoside hydrolase family 9 and a family 48 catalytic domain, as well as three type III cellulose-binding modules. In the saccharification of a common cellulose standard, Avicel, CelA outperforms mixtures of commercially relevant exo- and endoglucanases. From transmission electron microscopy studies of cellulose after incubation with CelA, we report morphological features that suggest that CelA not only exploits the common surface ablation mechanism driven by general cellulase processivity, but also excavates extensive cavities into the surface of the substrate. These results suggest that nature's repertoire of cellulose digestion paradigms remain only partially discovered and understood. [ABSTRACT FROM AUTHOR]