The leaves of Diospyros kaki exert beneficial effects on a benzalkonium chloride-induced murine dry eye model.

PURPOSE: In this study, the beneficial effects of the oral administration of ethanol extract of Diospyros kaki (EEDK) were tested on a mouse dry eye model induced by benzalkonium chloride (BAC). METHODS: A solution of 0.2% BAC was administered topically to mouse eyes for 14 days, twice daily, to induce dry eye. Various concentrations of EEDK were administrated daily by oral gavage for 14 days after BAC treatment. Preservative-free eye drops were instilled in the positive-control group. The tear secretion volume (Schirmer's test), tear break-up time (BUT), and fluorescein score were measured on the ocular surface. BAC-induced corneal damage was tested with hematoxylin-eosin staining. Moreover, apoptotic cell death in the corneal epithelial layer was investigated with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. The protein expression level of interleukin-1alpha (IL-1α), IL-1ß, IL-6, tumor necrosis factor- alpha (TNF-α), and monocyte chemotactic protein-1 (MCP-1) was determined with western blot analysis. Furthermore, squamous metaplasia in the corneal epithelial layer was detected with immunofluorescent staining for cytokeratine-10. The cellular proliferation in the cornea was examined with immunohistochemical staining for Ki-67. RESULTS: EEDK treatment resulted in prolonged BUT, decreased fluorescein score, increased tear volume, and smoother epithelial cells compared with BAC treatment alone in the cornea. Moreover, EEDK treatment inhibited the inflammatory response and corneal epithelial cell death in a BAC-induced murine dry eye model, and changes in squamous cells were inhibited. Proliferative activity in the corneal epithelium cells was improved with EEDK. CONCLUSIONS: EEDK could be a potential therapeutic agent in the clinical treatment of dry eye.

Effect of combined cataract surgery and ranibizumab injection in postoperative macular edema in nonproliferative diabetic retinopathy.

PURPOSE: To evaluate whether intravitreal ranibizumab injection at cataract surgery prevents postoperative diabetic macular edema (PME) in patients with stable diabetic retinopathy without significant macular edema. METHODS: Eighty patients with cataract, stable diabetic retinopathy, and no significant macular edema were randomized to a sham group (cataract surgery only) or a group undergoing cataract surgery plus intraoperative ranibizumab injection. Best-corrected visual acuities, central subfield thickness, and total macular volume were assessed at baseline and 1 week, 1, 3, and 6 months postoperatively by spectral domain optical coherence tomography. Clinically meaningful PME (central subfield thickness increase >60 µm relative to baseline) was computed. RESULTS: The groups did not differ in baseline best-corrected visual acuity, central subfield thickness, and total macular volume. Compared with the ranibizumab injection group, the sham group had significantly larger central subfield thickness increases relative to baseline at 1 week and 1 month; larger total macular volume increases at all time points (P = 0.012, P = 0.005, P < 0.001, P < 0.001, P = 0.005, P = 0.017, respectively); higher PME frequency at 1 month (P = 0.019); and poorer best-corrected visual acuity improvement from baseline to 6 months after surgery (P = 0.046). CONCLUSION: In patients with stable diabetic retinopathy without significant macular edema, intravitreal ranibizumab injection at cataract surgery may prevent the postoperative worsening of macular edema and may improve the final visual outcome without affecting safety.

Effects of ultraviolet rays and particulate matter on hair porosity in damaged hair.

BACKGROUND: With growing interest in hair health, researchers are exploring aspects beyond the surface qualities of hair, such as its porous inner structure. While previous studies have focused on the effects of treatments such as perming and hair dying on hair porosity, less emphasis has been paid to the effects of harmful environmental factors such as ultraviolet (UV) rays and particulate matter on the porous nature of hair. AIMS: The aim of this study was to bridge this gap by investigating how UV rays and particulate matter affect hair porosity in different ways. Our study could help elucidate how these external factors influence hair health and shed light on previously unknown aspects of hair porosity. METHODS: Hair tresses were bleached, cut into 1 cm-long sections, and stained with methylene blue. The sections were then irradiated with UV light or exposed to particulate matter. RESULTS: Bleached hair absorbed more methylene blue than normal hair. UV radiation-induced hair porosity occurred at 3 h after irradiation and increased with time. Particulate matter alone did not affect the porosity of the damaged hair; however, in combination with UV irradiation, it substantially increased hair porosity. CONCLUSION: Environmental challenges such as a depleted ozone layer and increasing pollution may increase hair porosity, which can be prevented by maintaining healthy hair.

Insights into plant biomass conversion from the genome of the anaerobic thermophilic bacterium Caldicellulosiruptor bescii DSM 6725.

Caldicellulosiruptor bescii DSM 6725 utilizes various polysaccharides and grows efficiently on untreated high-lignin grasses and hardwood at an optimum temperature of ∼ 80 °C. It is a promising anaerobic bacterium for studying high-temperature biomass conversion. Its genome contains 2666 protein-coding sequences organized into 1209 operons. Expression of 2196 genes (83%) was confirmed experimentally. At least 322 genes appear to have been obtained by lateral gene transfer (LGT). Putative functions were assigned to 364 conserved/hypothetical protein (C/HP) genes. The genome contains 171 and 88 genes related to carbohydrate transport and utilization, respectively. Growth on cellulose led to the up-regulation of 32 carbohydrate-active (CAZy), 61 sugar transport, 25 transcription factor and 234 C/HP genes. Some C/HPs were overproduced on cellulose or xylan, suggesting their involvement in polysaccharide conversion. A unique feature of the genome is enrichment with genes encoding multi-modular, multi-functional CAZy proteins organized into one large cluster, the products of which are proposed to act synergistically on different components of plant cell walls and to aid the ability of C. bescii to convert plant biomass. The high duplication of CAZy domains coupled with the ability to acquire foreign genes by LGT may have allowed the bacterium to rapidly adapt to changing plant biomass-rich environments.

Robust, high-throughput solution structural analyses by small angle X-ray scattering (SAXS).

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.

Recurrent submacular hemorrhage in patients with neovascular age-related macular degeneration.

PURPOSE: To describe the incidence, risk factors for, and long-term visual outcomes of recurrent submacular hemorrhage in the context of age-related macular degeneration. METHODS: Medical records of patients with neovascular age-related macular degeneration with or without polypoidal choroidal vasculopathy showing submacular hemorrhage at their first visit to our institution were reviewed. The required minimum follow-up period was 24 months, and any newly developed submacular hemorrhage larger than 1 disk area after near-complete resolution of initial hemorrhage was defined as recurrence. RESULTS: A total of 47 eyes of 47 patients were eligible for inclusion. Twenty-four patients showed recurrent submacular hemorrhage during the follow-up period (Group I). Patients without recurrent submacular hemorrhage were included in Group II. The time to recurrent submacular hemorrhage in Group I patients was 21.4 ± 9.2 months. Polypoidal choroidal vasculopathy was present in 50% of Group I patients (n = 12) and 13% of Group II patients (n = 3) (P = 0.025). Intravitreal anti-vascular endothelial growth factor injection was performed during the follow-up period in 70.8% of Group I patients (n = 17) and 95.7% of Group II patients (n = 22) (P = 0.048). Visual acuity change during the follow-up period did not significantly differ between the two groups. CONCLUSION: In patients with neovascular age-related macular degeneration presenting with submacular hemorrhage at their first visit, the incidence of recurrent submacular hemorrhage was 51.1% in our retrospective long-term follow-up study. The presence of polypoidal choroidal vasculopathy was associated with an increased risk of recurrent submacular hemorrhage. Use of anti-vascular endothelial growth factor agents was correlated with a reduced risk of such hemorrhage. Visual acuity was stably maintained over 2 years regardless of hemorrhage recurrence.

Enhanced anti-cancer effect using MMP-responsive L-asparaginase fused with cell-penetrating 30Kc19 protein.

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.

Effects of intravitreal bevacizumab and laser in retinopathy of prematurity therapy on the development of peripheral retinal vessels.

OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Genome sequence of the anaerobic, thermophilic, and cellulolytic bacterium "Anaerocellum thermophilum" DSM 6725.

"Anaerocellum thermophilum" DSM 6725 is a strictly anaerobic bacterium that grows optimally at 75 degrees 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.

Efficient degradation of lignocellulosic plant biomass, without pretreatment, by the thermophilic anaerobe "Anaerocellum thermophilum" DSM 6725.

Very few cultivated microorganisms can degrade lignocellulosic biomass without chemical pretreatment. We show here that "Anaerocellum thermophilum" DSM 6725, an anaerobic bacterium that grows optimally at 75 degrees C, efficiently utilizes various types of untreated plant biomass, as well as crystalline cellulose and xylan. These include hardwoods such as poplar, low-lignin grasses such as napier and Bermuda grasses, and high-lignin grasses such as switchgrass. The organism did not utilize only the soluble fraction of the untreated biomass, since insoluble plant biomass (as well as cellulose and xylan) obtained after washing at 75 degrees C for 18 h also served as a growth substrate. The predominant end products from all growth substrates were hydrogen, acetate, and lactate. Glucose and cellobiose (on crystalline cellulose) and xylose and xylobiose (on xylan) also accumulated in the growth media during growth on the defined substrates but not during growth on the plant biomass. A. thermophilum DSM 6725 grew well on first- and second-spent biomass derived from poplar and switchgrass, where spent biomass is defined as the insoluble growth substrate recovered after the organism has reached late stationary phase. No evidence was found for the direct attachment of A. thermophilum DSM 6725 to the plant biomass. This organism differs from the closely related strain A. thermophilum Z-1320 in its ability to grow on xylose and pectin. Caldicellulosiruptor saccharolyticus DSM 8903 (optimum growth temperature, 70 degrees C), a close relative of A. thermophilum DSM 6725, grew well on switchgrass but not on poplar, indicating a significant difference in the biomass-degrading abilities of these two otherwise very similar organisms.

Transconjunctival sutureless vitrectomy for the treatment of vitreoretinal complications in patients with diabetes mellitus.

BACKGROUND AND OBJECTIVE: To compare the safety and effectiveness of 25-gauge transconjunctival sutureless vitrectomy (TSV) with conventional 20-gauge pars plana vitrectomy (PPV) for the treatment of vitreoretinal complications in patients with diabetic retinopathy. PATIENTS AND METHODS: Forty-five eyes of 44 consecutive patients with diabetes mellitus who had undergone 25-gauge TSV for the treatment of vitreous hemorrhage, traction retinal detachment, or diabetic macular edema and 45 control patients with diabetes mellitus who had undergone conventional 20-gauge PPV were studied. RESULTS: The mean duration of surgery in the 25-gauge TSV group was statistically significantly shorter than that in the 20-gauge PPV group. The mean visual acuity at both 1 week and 1 month after surgery was statistically significantly better in the 25-gauge TSV group than in the 20-gauge PPV group. Complications in the 25-gauge TSV group were similar to those in the 20-gauge PPV group. CONCLUSION: A shorter operation time and faster recovery of postoperative visual acuity were the main advantages of using 25-gauge TSV. Despite its limitations, 25-gauge TSV can be safely used for the treatment of vitreoretinal complications in patients with diabetes mellitus.

Molecular cloning and biochemical characterization of the first archaeal maltogenic amylase from the hyperthermophilic archaeon Thermoplasma volcanium GSS1.

Maltogenic amylases (MAases), a subclass of cyclodextrin (CD)-hydrolyzing enzymes belonging to glycoside hydrolase family 13, have been studied extensively, but their physiological roles in microbes and evolutionary relationships with other amylolytic enzymes remain unclear. Here, we report the biochemical properties of a thermostable archaeal MAase from Thermoplasma volcanium GSS1 (TpMA) for the first time. The primary structure and catalytic properties of TpMA were similar to those of MAases, such as possession of an extra domain at its N-terminal and preference for CD over starch. TpMA showed high thermostability and optimal activity at 75 degrees C and 80 degrees C for beta-CD and soluble starch, respectively. The recombinant TpMA exists as a high oligomer in a solution and the oligomeric TpMA was dissociated into dimer and monomer mixture by a high concentration of NaCl. The substrate preference and thermostability of TpMA were significantly dependent on the oligomeric state of the enzyme. However, TpMA exhibited distinguishable characteristics from those of bacterial MAases. The transglycosylation pattern of TpMA was opposite to that of bacterial MAases. TpMA formed more alpha-1,4-glycosidic linked transfer product than alpha-1,6-linked products. Like as alpha-amylases, notably, TpMA has a longer subsite structure than those of other CD-degrading enzymes. Our findings in this study suggest that TpMA, the archaeal MAase, shares characteristics of both bacterial MAases and alpha-amylases, and locates in the middle of the evolutionary process between alpha-amylases and bacterial MAases.

A standardized extract of Rhynchosia volubilis Lour. exerts a protective effect on benzalkonium chloride-induced mouse dry eye model.

ETHNOPHARMACOLOGICAL RELEVANCE: In contrast to other leguminous plants generally used as food, Rhynchosia volubilis Loureiro, a small soybean with a black seed coat, has been used as a traditional oriental remedy for various human diseases in Eastern Asia. In this study, we demonstrated the protective effect of R. volubilis against dry eye disease. AIM OF THE STUDY: We aimed to investigate whether a standardized ethanol extract of R. volubilis (EERV) can protect the cornea in a benzalkonium chloride (BAC)-induced mouse dry eye model. MATERIALS AND METHODS: Experimental dry eye was induced by the instillation of 0.2% BAC on mouse cornea. A standardized ethanol extract of R. volubilis (EERV) was orally administered following BAC treatment. The positive control group was treated with commercial eye drops. Fluorescein staining, tear break-up time (BUT), and hematoxylin and eosin staining were evaluated on the ocular surface. Squamous metaplasia and apoptosis in the corneal epithelial layer were detected by immunostaining. Furthermore, the protein expression of cytochrome c, Bcl-2, and Bax was determined. RESULTS: EERV treatment significantly improved fluorescein scoring, BUT, and smoothness in the cornea compared to the vehicle group. In addition, EERV inhibited squamous metaplasia and apoptosis in the cornea. The expression of cytochrome c and Bax was upregulated, while that of Bcl-2 was downregulated in the vehicle group compared with that in the control group. However, EERV treatment inhibited the expression of cytochrome c and Bax, while that of Bcl-2 was improved. CONCLUSION: Standardized EERV could be a beneficial candidate for the treatment of dry eye disease.

Contribution of W229 to the transglycosylation activity of 4-alpha-glucanotransferase from Pyrococcus furiosus.

A W229H mutant of 4-alpha-glucanotransferase (4-alpha-GTase) from Pyrococcus furiosus was constructed and its catalytic properties were studied to investigate the role of W229 in the catalytic specificities of the enzyme. Various activities and kinetic parameters were determined for the wild-type and W229H mutant enzymes. The transglycosylation factor and transglycosylation activity of the mutant enzyme markedly decreased, but its hydrolysis activity was scarcely affected. It was discovered that the k(cat)/K(m) value of transglycosylation activity significantly decreased to about 15% of that of the wild type, while k(cat)/K(m) value of hydrolysis activity changed little for the mutant enzyme. The hydrophobicity of W229 was thought to be critical to the transglycosylation activity of the enzyme based on the enzyme's modeled tertiary structures.

Enhancing thermostability of maltogenic amylase from Bacillus thermoalkalophilus ET2 by DNA shuffling.

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 degrees C, which was 10 degrees 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 degrees 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 degrees C. The melting temperatures of III-1 and III-2, as determined by differential scanning calorimetry, increased by 6.1 degrees C and 11.4 degrees 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.

Enzymatic preparation of maltohexaose, maltoheptaose, and maltooctaose by the preferential cyclomaltooligosaccharide (cyclodextrin) ring-opening reaction of Pyrococcus furiosus thermostable amylase.

Specific-length maltooligosaccharides, particularly maltohexaose, maltoheptaose, and maltooctaose, were prepared from cyclomaltooligosaccharides (cyclodextrins, CDs) by the preferential cyclodextrin ring-opening reaction of an amylolytic enzyme from Pyrococcus furiosus. The enzyme primarily produces maltohexaose, maltoheptaose, and maltooctaose by hydrolyzing alpha-, beta-, and gamma-CD, respectively. This study aims to develop a high-efficiency synthesis of specific maltooligosaccharides at high-purity. [formula: see text]

Subconjunctival Injection of Viscoelastic Material for Leaking Sclerotomy in Transconjunctival Sutureless Vitrectomy.

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.

Effect of Posterior Subtenon Triamcinolone Acetonide Injection on Diabetic Macular Edema Refractory to Intravitreal Bevacizumab Injection.

PURPOSE: To evaluate the effects of posterior subtenon triamcinolone acetonide injection on refractory diabetic macular edema (DME) after intravitreal bevacizumab (IVB) injection failure. METHODS: Patients with DME and central subfield thickness (CST) >300 µm who did not respond to IVB injections were retrospectively included. Specifically, we enrolled patients who were diagnosed with refractory DME and who experienced an increase in CST after 1 to 2 IVB injections or no decrease after ≥3 consecutive IVB injections. One clinician injected 20 mg of triamcinolone acetonide into the posterior subtenon space. All patients received ophthalmic examinations at baseline and at 2, 4, and 6 months post-baseline. Examinations included Snellen visual acuity, intraocular pressure, and spectral-domain optical coherence tomography. RESULTS: Forty eyes of 34 patients were included. The average baseline CST was 476 µm. The average CST decreased to 368 µm at 2 months, 374 µm at 4 months, and 427 µm at 6 months (p < 0.001 for all results, Wilcoxon signed-rank test). The average intraocular pressure increased from 15.50 to 16.92 mmHg at 2 months but decreased to 16.30 mmHg at 4 months and 15.65 mmHg at 6 months. Logarithm of the minimum angle of resolution visual acuity improved from 0.56 to 0.50 at 2 months (p = 0.023), 0.50 at 4 months (p = 0.083), and 0.48 at 6 months (p = 0.133, Wilcoxon signed-rank test). No complications were detected. CONCLUSIONS: Posterior subtenon triamcinolone acetonide is an effective and safe treatment for reducing CST in DME refractory to IVB.

Diospyros kaki Extract Inhibits Alkali Burn-Induced Corneal Neovascularization.

The purpose of this study was to evaluate the effect of ethanol extract of Diospyros kaki (EEDK) leaves on corneal neovascularization (CoNV) in rats. One week after the alkali burns in the corneas, the CoNV area coverage in the CoNV-positive control group, 100 mg/kg EEDK group, and 200 mg/kg EEDK group was 43.3% ± 5.5%, 337.7% ± 2.5%, and 27.2% ± 4.3%, respectively. The areas of CoNV in the EEDK-treated groups were significantly different from those of the CoNV group. EEDK significantly attenuated the upregulation of vascular endothelial growth factor, fibroblast growth factor, interleukin-6, and matrix metalloproteinase-2 (MMP-2) protein levels. Orally administrated D. kaki inhibited CoNV development in rats.