Implementation of Organ Culture storage of donor corneas: a 3 year study of its impact on the corneal transplant wait list at the Lions New South Wales Eye Bank

Organ Culture corneal storage offers an extended storage time and increased donor pool and tissue assessment opportunities. In September 2011, the Lions New South Wales Eye Bank (LNSWEB) moved from hypothermic storage to Organ Culture corneal storage. This study evaluates the impact of implementation of Organ Culture on donor eye retrieval and the corneal transplant waiting list over a 3 year period in NSW, Australia. Retrospective review of the LNSWEB data from September 2011 to August 2014. Tissue collection, waiting list and tissue utilization data were recorded. The data from September 2008 to August 2011 for Optisol-GS storage was used for comparison. The annual donor and cornea collection rate increased 35 % and 44 % respectively with Organ Culture compared to Optisol-GS storage. The utilization rate of corneal tissue increased from 73.4 % with hypothermic storage to 77.2 % with Organ Culture storage. The transplant wait list decreased by 77.3 % from September 2011 to August 2014 and correlated with the increased rate of corneal transplantation (r = ?0.9381, p < 0.0001). No other factors impacting the wait list changed over this period. Corneas not used from either storage method were due to unacceptable endothelial cell density/viability. The contamination rate of corneas stored in Organ Culture medium was low at 1.74 %. The Organ Culture storage method increases the corneal donor pool available to Eye banks. The practical benefits of the extended storage time and increased donor assessment opportunities have directly led to an increase in corneal utilization rate and a significant decrease in recipient wait list time.     

Polarization of human donor corneas

To investigate the de-orientation effect of DSAEK grafts by observing the cross patterns and polarization power of human donor corneas using a polarizing device (Lumaxis^®). Forty human donor corneas were placed in small petri-plates with epithelial side facing up. Polarizing power (arbitrary unit) and crosses were monitored and recorded by the software. The tissue was marked at ‘Superior’ position to ensure that the base and the polarizer are in alignment with each other after the cut. The anterior lamellar cut was performed using microkeratome. The lenticule was placed back in the same position as marked to mimic the alignment. The tissue was further rotated by 45° ensuring that the base of the cornea and the polarizer were in alignment. The polarization power and ‘crosses’ were identified at each step. The average of forty corneas from pre-cut to post-45° angular change showed statistically significant difference (p < 0.05) in terms of polarizing power. The cross-shaped pattern deformed and lost the sharpness towards 45° angle. However, multiple variances in terms of ‘cross-patterns’ were observed throughout the study. Lumaxis^® was able to determine the worst quality tissue in terms of polarization (no black zone and crosses). Despite the quality of cross pattern which can be used as an additional objective parameter to evaluate the optical properties of the corneal tissue, this preliminary study needs to be further justified in terms of clinical relevance whether polarization changes with oriented or de-oriented grafts have any effects and consequences on the visual acuity.     

Non-invasive measurement of transparency,arcus senilis,and scleral rim diameter of corneas during eye banking

We developed a non-invasive device to quantify transparency (T), clear corneal diameter (CCD) excluding arcus senilis, and scleral rim diameter (SRD) of stored corneas. The T value (expressed in % on a relative scale), based on the modulation transfer function principle, referred to the ratio of local contrasts of a special LED backlit chart measured with and without cornea. CCD and SRD (in mm) were automatically calculated by morphologic operations. Firstly, we assessed measurement reproducibility. We then determined the agreement of T and CCD values with 3-level scores given independently by three experts on 179 scientific corneas. Thirdly, an eye bank was equipped with the device, and 358 consecutive organ-cultured (OC) corneas were tested for donor- and storage- related factors possibly influencing T and CCD. Reproducibility of T, CCD and SRD measurements was high, with intraclass correlation coefficients of 0.982, 0.886, and 0.999 respectively. Capacity to discriminate the three levels of transparency and arcus senilis was good, with T of 20.0 (10.0–33.6), 38.3 (24.3–75.4) and 57.9 (33.9–90.0) % respectively for T deemed poor, average, and good (P < 0.001), and CCD of 9.8 (7.3–10.6), 10.5 (8.2–11.5), and 11.1 (9.9–12.0) mm respectively for arcus senilis deemed prominent, moderate or absent (P < 0.001). T was correlated with neither donor age nor endothelial cell density nor storage time, but slightly worsened during OC for corneas assessed twice. In conclusion, the device, which can be easily integrated in the facilities of an eye bank, provides reliable objective measurement of T, CCD, and SRD. This could be a useful tool for standardizing quality assessment of stored corneas and consequently optimizing their selection for penetrating, endothelial or anterior lamellar keratoplasty.     

Changeability of sperm chromatin structure during liquid storage of ovine semen in milk-egg yolk- and soybean lecithin-based extenders and their relationships to field-fertility

The aim of this experiment was to study the effect of semen extender on sperm chromatin structure and to correlate chromatin integrity with field-fertility of preserved ram semen. Ejaculates of at least 2 × 10⁹ sperm/ml and 70 % progressive motility were collected using an artificial vagina from Chios rams (n = 11, 4–6 years old), split-diluted to 1 × 10⁹ sperm/ml with milk-egg yolk- and soybean lecithin (Ovixcell^®)-based extenders, packaged in 0.5-ml straws and examined after 6, 24 and 48 h of storage at 5 ± 1 °C. Evaluation endpoints were computer-assisted sperm motion analysis, fluorescence-based analysis of chromatin structure by chromomycin A₃ and acridine orange assays, and 65-day pregnancy rate (PR) of 34- to 36-h preserved semen after intra-cervical insemination of ewes (n = 154) in progestagen-synchronized estrus. Neither extender nor storage time had any influence on incidence of decondensed chromatin. Unlike Ovixcell^® extender, deterioration of sperm motility (P < 0.01) and chromatin stability (P < 0.005) was detected after 48 h of storage in milk-egg yolk extender. Sperm motility accounted for 14.4–18.5 % of variations in chromatin integrity (P < 0.001). No significant difference was found in PR of Ovixcell^®- and milk-egg yolk-stored semen. Nevertheless, PR differed between rams (14.3–71.4 %; P < 0.025). Chromatin integrity explained 10.2–56.3 % of variations in PR (P < 0.05–0.01). A pronounced decline in PR (19.1 %) was observed when percentages of decondensed and destabilized chromatin have reached thresholds of 10.5–30 % and 4–9 %, respectively. In conclusion, Ovixcell^® is superior to milk-egg yolk extender in preserving chromatin stability and motility. Chromatin defects are negatively associated with sperm fertility.     

Effects of adjuvant and conidial concentration on the efficacy of <Emphasis Type="Italic">Beauveria bassiana</Emphasis> for the control of the two spotted spider mite, <Emphasis Type="Italic">Tetranychus urticae</Emphasis>

Greenhouse experiments were conducted on various crops (cucumber, tomato, eggplant, green bean) to ascertain the effects of Break-thru^® (polyether-polymethylsiloxane-copolymer, a silicone surfactant) and an oil emulsion, on Beauveria bassiana (Balsamo) Vuillemin (Bb) applications for the control of the two spotted spider mite, Tetranychus urticae Koch. The objectives were to compare a) the efficacy of Bb control when applied in aqueous Break-thru^® or an oil emulsion; b) the effects of various concentrations of Bb conidia, as affected by each surfactant; and c) the effects of Break-thru^® on the activity of the fungus. Conidia were suspended either in an aqueous Break-thru^® or an emulsifiable formulation at different conidial concentrations (1.05 × 10⁶, 2.1 × 10⁶ and 4.2 × 10⁶ conidia ml^?1) and sprayed onto leaves 2 weeks after artificial pest inoculation. Two sprays were performed, with an interval of one week from one spray to another, and T. urticae population counts (both motile and egg stages) were made on plant leaves 7 days after each spray. Bb conidia in Break-thru^® were more efficacious than conidia in emulsifiable formulation. With the highest rate of conidia (4.2 × 10⁶ conidia ml^?1), mortality of adult mites ranged from 60 ± 4.2 (mean ± SE) to 85.7 ± 4.3% in the Break-thru^® suspension and 39.4 ± 7 to 61.3 ± 6% in the oil emulsion. Leaf damage index was also substantially reduced from 70% in the unsprayed control to 40% by the application of Bb conidia at the highest rate with Break-thru^®. Break-thru^® can be combined with Bb in the integrated management of T. urticae and Isolate R444 is a promising candidate for the control of the pest.     

Abscisic acid,gibberellins and brassinosteroids in Kelpak®, a commercial seaweed extract made from Ecklonia maxima

The seaweed extract Kelpak^® made from the kelp Ecklonia maxima is registered as a biostimulant for use in agriculture. It elicits many beneficial responses including improved root and shoot growth, higher yields and greater resistance to abiotic and biotic stresses. Previously, cytokinins, auxins and polyamines were identified in Kelpak^®. The aim of the present study was to quantify other groups of plant growth regulators (PGRs)—abscisic acid (ABA), gibberellins (GAs) and brassinosteroids—that may be present in E. maxima and Kelpak^®. Kelpak^® samples harvested between 2008 and 2010 and stored for up to 26 months were analysed using ultra performance liquid chromatography tandem mass spectrometry. ABA levels were below the limits of detection in E. maxima but were detected in low concentrations in Kelpak^®, ranging from 0.31 to 20.70 pg mL^?1 Kelpak^®. Eighteen GAs were found in E. maxima and Kelpak^® with concentrations from 187.54 to 565.96 pg mL^?1 Kelpak^®. The biologically active GAs (GA₁, GA₃, GA₄, GA₅, GA₆ and GA₇) comprised less than 3 % in Kelpak^®. Although GA₁₃ (a final product in the metabolic pathway) was present in low concentrations in E. maxima, very high concentrations were present in Kelpak^®. The brassinosteroids brassinolide (BL) and castasterone (CS) were identified in E. maxima and Kelpak^®. Concentrations varied with harvest and storage time, ranging from 384.72 to 793.23 pg BL mL^?1 Kelpak^® and 62.84 to 567.51 pg CS mL^?1 Kelpak^®. It is likely that this cocktail of natural PGRs present in Kelpak^® may act individually or in concert and thus contribute to the numerous favourable physiological responses elicited by Kelpak^® application to plants.     

Measurements of donor endothelial keratoplasty lenticules prepared from fresh donated whole eyes by using ultrasound and optical coherence tomography

This study was conducted to analyze the profile and thickness of endothelial keratoplasty lenticules prepared from fresh donated whole eyes with Visante optical coherence tomography (V-OCT) compared to measurements obtained from ultrasound pachymetry (USP) at the Central Eye Bank of Iran. Microkeratome-assisted precut corneas were prepared for Descemet stripping automated endothelial keratoplasty by using standard eye bank protocol. Central posterior lenticule thickness (CPLT) on fresh whole eye, before excising corneoscleral disc and transferring to Optisol-GS, was measured by USP. V-OCT was used to measure central, paracentral, and midperipheral thicknesses of lenticules after transferring the tissue to Optisol-GS. Chi Square and Bonferroni tests were respectively used to uncover the differences between the USP and V-OCT measurements and also the thickness profile of lenticules. Postoperative reports for the entire transplanted lenticules were recorded. Accordingly, on evaluation of 312 enrolled precut corneas, CPLT measurements by V-OCT versus USP were statistically different (mean: 136 µm vs 165 µm, respectively; P = 0.008). Thickness profile of the posterior lenticules revealed increased thickness from the central to the peripheral parts of the cornea (mean increase of 16 µm at the pericentral and 64.2 µm at the peripheral locations, respectively); however, the increase in the thickness was relatively symmetrical. Postoperative reports of transplanted lenticules were unremarkable, since there were no posterior flap detachments. In essence, V-OCT measurements of microkeratome-assisted precut lenticules prepared from fresh donated whole eyes averaged 29 μm thinner than USP measurements and revealed a significant but symmetric increase of thickness towards the peripheral parts of the corneas. However, the variation in the thickness profile did not affect the attachment or the clarity of transplanted precut lenticlues.     

Physicochemical and Preclinical Evaluation of a Novel Buccal Measles Vaccine

The aim of this study is to develop an orally disintegrating film (ODF) containing a microparticulate measles vaccine formulation for buccal delivery. The measles vaccine microparticles were made with biocompatible and biodegradable bovine serum albumin (BSA) and processed by spray drying. These vaccine microparticles were incorporated in the ODF, consisting of Lycoat RS720®, Neosorb P60W® and Tween 80. The yield of the microparticles was approximately 85–95%, w/w. The mean size of the vaccine microparticles was 3.65?±?1.89 μm and had a slightly negative surface charge of 32.65?±?2.4 mV. The vaccine particles were nontoxic to normal cells at high concentrations (500 μg/2.5?×?10⁵ cells) of vaccine particles. There was a significant induction of innate immune response by vaccine microparticles which was observed in vitro when compared to blank microparticles (P?<?0.05). The vaccine microparticles also significantly increased the antigen presentation and co-stimulatory molecules expression on antigen presenting cells, which is a prerequisite for Th1 and Th2 immune responses. When the ODF vaccine formulation was dosed in juvenile pigs, significantly higher antibody titers were observed after week 2, with a significant increase at week 4 and plateauing through week 6 comparative to naïve predose titers. The results suggest that the ODF measles vaccine formulation is a viable dosage form alternative to noninvasive immunization that may increase patient compliance and commercial distribution.     

Seaweed-Derived Biostimulant (Kelpak<Superscript>®</Superscript>) Influences Endogenous Cytokinins and Bioactive Compounds in Hydroponically Grown <Emphasis Type="Italic">Eucomis autumnalis</Emphasis>

The effect of a seaweed-derived biostimulant (Kelpak^® at 1, 2.5 and 5 % dilution; v/v) on the growth, endogenous cytokinin (CK) and phytochemical content in Eucomis autumnalis (Mill.) Chitt. under hydroponic conditions was evaluated. After 4 months, the stimulatory effect of Kelpak^® treatments was more noticeable in the underground organs than in the aerial organs. Total endogenous CK was also higher in plants treated with Kelpak^® (c.a. 1000–1200 pmol g^?1 DW) compared to control plants (860 pmol g^?1 DW). Isoprenoid CKs (which mainly accumulated in the aerial organs) were more dominant than aromatic-type CKs across all the treatments. A total of 11 bioactive chemicals (8 phenolic acids and 3 flavonoids) and eucomic acid known for their diverse biological activities were quantified in the samples. The most abundant compound was p-coumaric acid (6.5 µg g^?1 DW) and it was approximately sevenfold higher in 2.5 % Kelpak^®-treated plants than in the control. It was also noteworthy that syringic acid only occurred in the underground organs of 5 % Kelpak^®-treated plants. Eucomic acid which is a major bioactive compound in E. autumnalis was significantly enhanced in Kelpak^® treatments, and the leaves accounted for more than 70 % of the overall content. Thus, Kelpak^® elicited a significant influence on the growth, endogenous CK and phytochemical content in E. autumnalis. These findings provide additional evidence of the enormous potential of Kelpak^® as a useful biostimulant with practical applications in various agricultural endeavours.     

Influence of the application methods and doses on the susceptibility of black vine weevil larvae Otiorhynchus sulcatus to Metarhizium anisopliae in field-grown strawberries

Entomopathogenic fungi are commercially available for the control of insect pests, including the black vine weevil (BVW) Otiorhynchus sulcatus Fabricius (Coleoptera:Curculionidae). However, Metarhizium anisopliae (Metsch.) Sorokin (Clavicipitaceae) has not been used to control BVW in field-grown strawberries. Field trials were conducted in different locations in the UK during 2009–2010 to evaluate the different formulations (granular vs. drench) and application methods (premixed, drench, bare root treatment) of commercial strain of M. anisopliae Met52^® (=F52), the entomopathogenic nematodes and the organophosphate insecticide Cyren^® against BVW. The highest dose (10¹⁴ cfu ha^?1) tested provided significantly better control than the intermediate (10¹³ cfu ha^?1) or low (10¹² cfu ha^?1) doses. BVW larval control at the high, intermediate and low doses was 71–96, 40–75 and 6–11 %, respectively. Premixing, drench or bare root treatment with Met52^® gave similar levels of BVW control. Irrespective of the application methods or soil types, the high dose rate of Met52^® provided the best control. Significantly high larval control was achieved (78–97 %) when chlorpyrifos was applied at planting than eight weeks post planting (53 %). There were significant differences in BVW control between Met52^® (88 %) and reduced doses of Heterorhabditis bacteriophora Poinar (20–29 %) or Steinernema kraussei Steiner (39–75 %) when applied alone. However, when used together, low dose of S. kraussei and Met52^® provided 100 % control of BVW larvae. This study shows that Met52^® has considerable potential for the control of BVW larvae in commercial field-grown strawberry, thereby offering an environmentally benign alternative to chemical insecticides.     

The effect of phosphorus binding clay (Phoslock®) in mitigating cyanobacterial nuisance: a laboratory study on the effects on water quality variables and plankton

This laboratory study examined the lanthanum modified clay Phoslock^® for its effectiveness to bind soluble reactive phosphorus (SRP), release of nutrients from this modified clay, its influence on water quality variables (pH, oxygen saturation %, conductivity and turbidity), effects on phytoplankton growth (green alga Scenedesmus obliquus, cyanobacteria Microcystis aeruginosa and Anabaena sp.), and, lastly, its effect on the population growth of the rotifer Brachionus calyciflorus. A clear dose–response for SRP binding by the modified clay was observed. A small amount of ammonium is released from Phoslock^®. We found no effect of Phoslock^® on pH or oxygen saturation. Conductivity increased with the increasing concentration of Phoslock^®. An application of Phoslock^® caused a transient increase of turbidity up to 211 NTU. However, due to rapid settlement, turbidity fell below 13 NTU (~1 m Secchi depth), after 6 h. Phoslock^® addition caused a reduction in growth of all phytoplankton species tested that we attribute to the combined effects of light limitation, flocculation with the bentonite and binding of SRP to Phoslock^®. We estimated the EC₅₀ of Phoslock^® on the population growth of rotifer B. calyciflorus to be 0.15 g Phoslock^® l^?1. Overall, the results of our study indicate Phoslock^® seems to be suitable for field applications.     

Comparison of organ cultured precut corneas versus surgeon-cut corneas for Descemet’s stripping automated endothelial keratoplasty

To compare precut and surgeon-cut organ cultured donor corneas for DSAEK. A total of 119 consecutive eyes treated with DSAEK were retrospectically identified. 65 grafts were cut by the surgeon (Moria, ALTK System) prior to DSAEK and 54 grafts were precut by laboratory technicians from the Danish Eye Bank (Horizon single-use system). 1 year after surgery, tomographic images were obtained with the Pentacam HR. Endothelial cell density (ECD) and best-corrected visual acuity (BCVA) was determined. Graft thickness and graft asymmetry was evaluated in the centre and 1 mm from the edge of the graft in 6 semi-meridians. 1 year after surgery, the ECD loss was similar in the two groups, averaging 25.9 ± 14 % in surgeon-cut, and 22.9 ± 17 % in precut group (p = 0.33). Mean central graft thickness was 172 ± 6 μm in surgeon-cut grafts and 182 ± 6 μm in precut grafts (p = 0.30). BCVA was similar in surgeon-cut and precut corneas; being 0.25 ± 0.02 logMAR and 0.24 ± 0.02 logMAR, respectively (p = 0.59). The graft asymmetry index was 1.48 ± 0.02 for surgeon-cut and 1.44 ± 0.02 for precut grafts. There were no significant differences in complications rate in both groups. No correlations between BCVA and central graft thickness or graft asymmetry index in both groups were observed. Organ cultured precut donor corneas are comparable with surgeon-cut grafts with respect to ECD, graft thickness and asymmetry, and postoperative complication rate.     

Toxicity of non-ionic surfactants and interactions with fungal entomopathogens toward Bemisia tabaci biotype B

Several non-ionic surfactants can be used to enhance insecticidal performance of various bio-based products and agrochemicals. In this study, we describe the toxicity of four commercial non-ionic surfactants against immature Bemisia tabaci (Gennadius) biotype B whiteflies along with their spreading ability. Results revealed that trisiloxane-based surfactants (Break-thru^® and Silwet^® L-77) exhibited the highest toxicity to 1st–2nd (early) and 3rd–4th (late) instar nymphs as well as the greatest wetting performance. We also investigated whether sub-lethal concentrations of Break-thru^® and Silwet^® L-77 improved the insecticidal efficacy of the fungal entomopathogens Beauveria bassiana (Balsamo) Vuillemin (strain CG1229) and Isaria fumosorosea Wise (strain CG1228) conidial suspensions against whiteflies. Germination of hydrophobic conidia of both isolates were unaffected by these surfactants over the range 100–1000 ppm. The combinations of both fungi with trisiloxane carriers significantly increased nymphal mortality with mostly additive and synergistic effects on early and late nymphs, respectively. In screenhouse trials, both fungi (10⁷ conidia ml^?1) mixed with 200 ppm of Silwet^® L-77 significantly improved effectiveness against early nymphs (72–74 % mortality) compared with controls treated with water and Silwet^® L-77 alone. In addition, reduced volume rates of I. fumosorosea in Silwet^® L-77 (equivalent to 100 l ha^?1) were at least as effective against late nymphs as higher volume rates (200 l ha^?1) at equivalent conidial doses. Our findings underline the compatibility and enhanced activity of silicon-based surfactants with B. bassiana and I. fumosorosea for use in integrated whitefly management programs.     

A portable device for measuring donor corneal transparency in eye banks

To develop a portable device for measuring the donor corneal transparency and validate its efficacy for corneal evaluation in the eye-banks and for research. The transparency device (TD) has a light source, a detachable system for corneal insertion and a base for light transmission. The probe detects the transmitted light which is measured by a lux-meter. A contact lens was set as ‘control’ to reduce the light scattering concern, an empty petri-plate as ‘blank’ and the cornea as ‘sample’. Two experts and non-experts (masked) observed the corneas for subjective analysis which was then compared using the TD. The parameters observed were scars, foreign-body, stromal-deformities, folds, thickness and opacity which were then converted to a relative overall percentage by the observer. Twenty corneas were evaluated for correlation, five tissues to obtain standard-deviation and twenty-four pairs for a comparative study. Experts mimicked the eye-banks with long-term experience while non-experts mimicked the emerging eye-banks. Subjective values by the experts closely resembled the measurements by TD. The average correlation between the experts and the non-experts to TD was 0.985 and 0.960 respectively. TD showed higher reproducibility than experts followed by the non-experts. The comparative study showed that increase in thickness reduces the transparency. TD is portable, easy, efficient, maintains sterility and less expensive hence the emerging eye-banks and researchers can use to raise their standards and evaluate the transparency for in vitro tests and comparative studies. The suitable transparency for the cornea deemed for clinical applications was found to be >75 %.     

Comparative toxicity of an acetogenin-based extract and commercial pesticides against citrus red mite

Acetogenins, a class of natural compounds produced by some Annonaceae species, are potent inhibitors of mitochondrial electron transport systems. Although the cellular respiration processes are an important biochemical site for the acaricidal action of compounds, few studies have been performed to assess the bioactivity of acetogenin-based biopesticides on spider mites, mainly against species that occur in orchards. Using residual contact bioassays, this study aimed to evaluate the bioactivity of an ethanolic extract from Annona mucosa seeds (ESAM) (Annonaceae) against the citrus red mite Panonychus citri (McGregor) (Acari: Tetranychidae), an important pest of the Brazilian citriculture. ESAM is a homemade biopesticide which was previously characterized by its high concentration of acetogenins. It caused both high mortality of P. citri females (LC₅₀ = 7,295, 4,662, 3,463, and 2,608 mg l^?1, after 48, 72, 96, and 120 h of exposure, respectively) and significant oviposition deterrence (EC₅₀ = 3.194,80 mg l^?1). However, there was no effect on P. citri female fertility (hatching rate). In addition, the ESAM efficacy (in terms of its LC₉₀) was compared with commercial acaricides/insecticides (at its recommended rate) of both natural [Anosom^® 1 EC (annonin), Derisom^® 2 EC (karanjin), and Azamax^® 1.2 EC (azadirachtin + 3-tigloylazadirachtol)] and synthetic origin [Envidor^® 24 SC (spirodiclofen)]. Based on all of the analyzed variables, the ESAM exhibited levels of activity superior to other botanical commercial acaricides and similar to spirodiclofen. Thus, our results indicate that ESAM may constitute a biorational acaricide for citrus red mite integrated pest management in Brazilian citrus orchards, particularly for local use.     

Micropropagation of the pistachio and its rootstocks by temporary immersion system

Although several studies have been reported on the micropropagation of the pistachio and its rootstocks, to date none of them had been efficient on the mass production of these plants in bioreactor systems. Thus, the micropropagation of juvenile pistachio shoot tips and nodal buds was investigated in a temporary immersion bioreactor system (RITA^®) and on a conventional semi-solid medium. Among the tested immersion conditions, immersion for 24 min every 16 h reduced vitrification and improved proliferation in the pistachio. Interactions were evident in immersion time and frequency in nodal segments. Nodal buds were better than shoot tips as the highest multiple shoot formation was recorded in MS medium containing 4 mg L^?1 BA and 0.1 mg L^?1 GA₃ in RITA^®. Although shoot tip necrosis (STN) was observed in shoots proliferated on semi-solid MS medium, such a symptom did not occur in shoots sprouted in the RITA^®. Additionally, these optimized conditions were applied to nodal buds of mature male pistachio ‘Atl?’ and Pistacia rootstocks (P. khinjuk Stocks and P. atlantica Desf.), and the micropropagation in the bioreactor system, in comparison to the semi-solid medium, was also improved. Furthermore, in vitro rooting of pistachio plantlets, despite the lower range (27.5 %), was also achieved in RITA^®. However, rooting was better on semi-solid medium for all tested species (ranged between 50 and 70 %). The results of this study showed that RITA^® could be used for the mass propagation of pistachio and its rootstocks, as well as for other woody plant species.     

Cellulases from Penicillium funiculosum: production, properties and application to cellulose hydrolysis

The objective of this work is to investigate the utilization of two abundant agricultural residues in Brazil for the production and application of cellulolytic enzymes. Different materials obtained after pretreatment of sugarcane bagasse, as well as pure synthetic substrates, were considered for cellulase production by Penicillium funiculosum. The best results for FPase (354 U L^?1) and β-glucosidase (1,835 U L^?1) production were observed when sugarcane bagasse partially delignified cellulignin (PDC) was used. The crude extract obtained from PDC fermentation was then partially characterized. Optimal temperatures for cellulase action ranged from 52 to 58°C and pH values of around 4.9 contributed to maximum enzyme activity. At 37°C, the cellulases were highly stable, losing less than 15% of their initial activity after 23 h of incubation. There was no detection of proteases in the P. funiculosum extract, but other hydrolases, such as endoxylanases, were identified (147 U L^?1). Finally, when compared to commercial preparations, the cellulolytic complex from P. funiculosum showed more well-balanced amounts of β-glucosidase, endo- and exoglucanase, resulting in the desired performance in the presence of a lignocellulosic material. Cellulases from this filamentous fungus had a higher glucose production rate (470 mg L^?1 h^?1) when incubated with corn cob than with Celluclast^®, GC 220^® and Spezyme^® (312, 454 and 400 mg L^?1 h^?1, respectively).     

Self-Nanoemulsifying Drug Delivery System of Nifedipine: Impact of Hydrophilic–Lipophilic Balance and Molecular Structure of Mixed Surfactants

A simple but novel mixed surfactant system was designed to fabricate a self-nanoemulsifying drug delivery system (SNEDDS) based on hydrophilic–lipophilic balance (HLB) value. The impacts of HLB and molecular structure of surfactants on the formation of SNEDDS were investigated. After screening various oils and surfactants, nifedipine (NDP)-loaded liquid SNEDDS was formulated with Imwitor^® 742 as oil and Tween^®/Span^® or Cremophor^®/Span^® as mixed surfactant. Droplet size of the emulsions obtained after dispersing SNEDDS containing Tween^®/Span^® in aqueous medium was independent of the HLB of a mixed surfactant. The use of the Cremophor^®/Span^® blend gave nanosized emulsion at higher HLB. The structure of the surfactant was found to influence the emulsion droplet size. Solid SNEDDS was then prepared by adsorbing NDP-loaded liquid SNEDDS comprising Cremophor^® RH40/Span^® 80 onto Aerosil^® 200 or Aerosil^® R972 as inert solid carrier. Solid SNEDDS formulations using higher amounts (30–50% w/w) of Aerosil^® 200 exhibited good flow properties with smooth surface and preserved the self-emulsifying properties of liquid SNEDDS. Differential scanning calorimetry and X-ray diffraction studies of solid SNEDDS revealed the transformation of the crystalline structure of NDP due to its molecular dispersion state. In vitro dissolution study demonstrated higher dissolution of NDP from solid SNEDDS compared with NDP powder.     

Interclass small leucine-rich repeat proteoglycan interactions regulate collagen fibrillogenesis and corneal stromal assembly

The corneal stroma is enriched in small leucine-rich proteoglycans (SLRPs), including both class I (decorin and biglycan) and class II (lumican, keratocan and fibromodulin). Transparency is dependent on the assembly and maintenance of a hierarchical stromal organization and SLRPs are critical regulatory molecules. We hypothesize that cooperative interclass SLRP interactions are involved in the regulation of stromal matrix assembly. We test this hypothesis using a compound Bgn^−/0/Lum^−/− mouse model and single Lum^−/− or Bgn^−/0 mouse models and wild type controls. SLRP expression was investigated using immuno-localization and immuno-blots. Structural relationships were defined using ultrastructural and morphometric approaches while transparency was analyzed using in vivo confocal microscopy. The compound Bgn^−/0/Lum^−/− corneas demonstrated gross opacity that was not seen in the Bgn^−/0 or wild type corneas and greater than that in the Lum^−/− mice. The Bgn^−/0/Lum^−/− corneas exhibited significantly increased opacity throughout the stroma compared to posterior opacity in the Lum^−/− and no opacity in Bgn^−/0 or wild type corneas. In the Bgn^−/0/Lum^−/− corneas there were abnormal lamellar and fibril structures consistent with the functional deficit in transparency. Lamellar structure was disrupted across the stroma with disorganized fibrils, and altered fibril packing. In addition, fibrils had larger and more heterogeneous diameters with an abnormal structure consistent with abnormal fibril growth. This was not observed in the Bgn^−/0 or wild type corneas and was restricted to the posterior stroma in Lum^−/− mice. The data demonstrate synergistic interclass regulatory interactions between lumican and biglycan. These interactions are involved in regulating both lamellar structure as well as collagen fibrillogenesis and therefore, corneal transparency.     

Biodegradation of epoxyconazole and piraclostrobin fungicides by Klebsiella sp. from soil

Three bacterial strains have been isolated from soil in which soybean had been continuously cropped and treated with Opera^®, a fungicide containing epoxyconazole and pyraclostrobin. The three strains (1,805, 2,801 and 3,803), obtained from soil at 80–100 cm depth, were selected on medium containing 0.03% Opera^®. Morphological examination revealed that the strains were Gram-negative, and two of them (1,805 and 2,801) exhibited polymorphism. The growth profiles demonstrated that 1,805 and 3,803 were more efficient growing in the presence of Opera^® than 2,801. Maximum growth was reached between 24 and 48 h, however, 2,801 was not able to survive after this period. The total protein content produced by 1,805, 2,801 and 3,803 in liquid selective medium containing Opera^® were 111.0 ± 0.02, 80.0 ± 0.05 and 130.5 ± 0.07 μg/ml, respectively. According to its biochemical and molecular features, strain 1,805 was identified as Klebsiella sp. On the basis of the characteristics presented (facultative anaerobic nature, polymorphic character and capacity of growing in the presence of Opera^®) strain 1,805 seems to be able to degrade the epoxyconazole and pyraclostrobin.