Edible oil degradation by using yeast coculture of <Emphasis Type="Italic">Rhodotorula pacifica</Emphasis> ST3411 and <Emphasis Type="Italic">Cryptococcus laurentii</Emphasis> ST3412

To develop a microbial treatment of edible oil-contaminated wastewater, microorganisms capable of rapidly degrading edible oil were screened. The screening study yielded a yeast coculture comprising Rhodotorula pacifica strain ST3411 and Cryptococcus laurentii strain ST3412. The coculture was able to degrade efficiently even at low contents of nitrogen ([NH₄–N] = 240 mg/L) and phosphorus sources ([PO₄–P] = 90 mg/L). The 24-h degradation rate of 3,000 ppm mixed oils (salad oil/lard/beef tallow, 1:1 w/w) at 20°C was 39.8% ± 9.9% (means ± standard deviations of eight replicates). The highest degradation rate was observed at 20°C and pH 8. In a scaled-up experiment, the salad oil was rapidly degraded by the coculture from 671 ± 52.0 to 143 ± 96.7 ppm in 24 h, and the degradation rate was 79.4% ± 13.8% (means ± standard deviations of three replicates). In addition, a repetitive degradation was observed with the cell growth by only pH adjustment without addition of the cells.     

Quercetin in Lyotropic Liquid Crystalline Formulations: Physical, Chemical and Functional Stability

The purpose of this study was to develop a lyotropic liquid crystalline formulation using the emulsifier vitamin E TPGS and evaluate its behavior after incorporation of a flavonoid, quercetin. The physical (macro and microscopic), chemical (determination of quercetin content by the HPLC method) and functional (determination of quercetin antioxidant activity by DPPH^• assay) stability of the lamellar liquid crystalline formulation containing flavonoid was evaluated when stored at 4 ± 2 °C; 30 ± 2 °C/70 ± 5% RH (relative humidity) and 40 ± 2 °C/70 ± 5% RH during 12 months. The lamellar liquid crystalline structure of the formulation was maintained during the experiment, however chemical and functional stability results showed a great influence of the storage period in all conditions tested. A significant decrease in quercetin content (approximately 40%) was detected during the first month of storage and a similar significant loss in antioxidant activity was detected after 6 months. The remaining flavonoid content was unchanged during the final 6 months of the experimental period. The results suggest possible interactions between quercetin and the liquid crystalline formulation, which could inhibit or reduce the quercetin activity incorporated in the system. In conclusion, the present study demonstrated that incorporation of quercetin (1%) did not affect the liquid crystalline structure composed of vitamin E TPGS/IPM/PG–H₂O (1:1) at 63.75/21.25/15 (w/w/w). Nevertheless, of the total quercetin incorporated in the system only 60% was free to act as an antioxidant.     

Development of Lipid-Based Nanoparticles for Enhancing the Oral Bioavailability of Paclitaxel

The current research work investigates the potential of solid lipid nanoparticles (SLNs) in improving the oral bioavailability of paclitaxel. Paclitaxel-loaded SLNs (PTX-SLNs) were prepared by modified solvent injection method using stearylamine as lipid, soya lecithin and poloxamer 188 as emulsifiers. SLNs were characterized in terms of surface morphology, size and size distribution, surface chemistry and encapsulation efficiency. Pharmacokinetics and bioavailability studies were conducted in male Swiss albino mice after oral administration of PTX-SLNs. SLNs exhibited spherical shape with smooth surface as analyzed by transmission electron microscopy (TEM). The mean particle size of SLNs was 96 ± 4.4 nm with a low polydispersity index of 0.162 ± 0.04 and zeta potential of 39.1 ± 0.8 mV. The drug entrapment efficiency was found to be 75.42 ± 1.5% with a loading capacity of 31.5 ± 2.1% (w/w). Paclitaxel showed a slow and sustained in vitro release profile and followed Higuchi kinetic equations. After oral administration of the PTX-SLNs, drug exposure in plasma and tissues was ten- and twofold higher, respectively, when compared with free paclitaxel solution. PTX-SLNs produced a high mean C _max (10,274 ng/ml) compared with that of free paclitaxel solution (3,087 ng/ml). The absorbed drug was found to be distributed in liver, lungs, kidneys, spleen, and brain. The results suggested that PTX-SLNs dispersed in an aqueous environment are promising novel formulations that enhanced the oral bioavailability of hydrophobic drugs, like paclitaxel and were quite safe for oral delivery of paclitaxel as observed by in vivo toxicity studies.     

Cavamax W7 composite ethosomal gel of clotrimazole for improved topical delivery: development and comparison with ethosomal gel

The present research work was aimed to formulate clotrimazole encapsulated Cavamax W7 composite ethosomes by injection method for improved delivery across epidermis. 3² factorial design was used to design nine formulations (F1-F9) and compared with ethosomal formulations (F10-F12). F9 with vesicle size of 202.8 ± 4.8 nm, highest zeta potential (−83.6 ± 0.96 mV) and %EE of 98.42 ± 0.15 was selected as optimized composite ethosome and F12 as reference ethosomal formulation. As revealed by transmission electron microscopy F9 vesicles were more condensed, uniformly spherical in shape than F12 vesicles. Vesicular stability studies indicated F9 to be more stable as compared to F12. Both F9 and F12 were incorporated in carbopol 934 gel base to get G1–G8 gel formulations and evaluated for in vitro skin permeability. Cavamax W7 composite ethosomal optimized gel (G5) showed higher in vitro percent cumulative drug permeation (88.53 ± 2.10%) in 8 h and steady state flux (J _ss) of 3.39 ± 1.45 μg/cm²/min against the J _ss of 1.57 ± 0.23 μg/cm²/min for ethosomal gel (G1) and 1.13 ± 0.06 μg/cm²/min for marketed formulation. The J _ss flux of G5 was independent of amount of drug applied/unit area of skin. In vivo confocal laser scanning microscopic study of G5 depicted uniform and deeper penetration of rhodamine B (marker) in epidermis from Cavamax W7 composite ethosomal gel in comparison to G1. Finally, G5 demonstrated better (p < 0.05) antifungal activity against Candida albicans and Aspergillus niger than G1 thus, signifying that Cavamax W7 composite ethosomes present a superior stable and efficacious vesicular system than ethosomal formulation for topical delivery of clotrimazole.     

Investigation of the Physicochemical and Physicomechanical Properties of a Novel Intravaginal Bioadhesive Polymeric Device in the Pig Model

The purpose of this study was to develop and evaluate the bioadhesivity, in vitro drug release, and permeation of an intravaginal bioadhesive polymeric device (IBPD) loaded with 3′-azido-3′-deoxythymidine (AZT) and polystyrene sulfonate (PSS). Modified polyamide 6,10, poly(lactic-coglycolic acid), polyacrylic acid, polyvinyl alcohol, and ethylcellulose were blended with model drugs AZT and PSS as well as radio-opaque barium sulfate (BaSO4) and then compressed into caplet devices on a tableting press. One set of devices was coated with 2% w/v pentaerythritol polyacrylic acid (APE-PAA) while another remained uncoated. Thermal analysis was performed on the constituent polymers as well the IBPD. The changes in micro-environmental pH within the simulated human vaginal fluid due to the presence of the IBPD were assessed over a period of 30 days. Textural profile analysis indicated that the bioadhesivity of the APE-PAA-coated devices (3.699 ± 0.464 N; 0.0098 ± 0.0004 J) was higher than that of the uncoated devices (1.198 ± 0.150 N; 0.0019 ± 0.0001 J). In addition, BaSO4-facilitated X-ray imaging revealed that the IBPD adhered to pig vaginal tissue over the experimental period of 30 days. Controlled drug release kinetics was obtained over 72 days. During a 24-h permeation study, an increase in drug flux for both AZT (0.84 mg cm⁻² h⁻¹) and PSS (0.72 mg cm⁻² h⁻¹) was realized up to 12 h and thereafter a steady-state was achieved. The diffusion and dissolution dynamics were mechanistically deduced based on a chemometric and molecular structure modeling approach. Overall, results suggested that the IBPD may be sufficiently bioadhesive with desirable physicochemical and physicomechanical stability for use as a prolonged intravaginal drug delivery device.     

Use of soybean vinasses as a germinant medium for a Geobacillus stearothermophilus ATCC 7953 sterilization biological indicator

A novel low-cost medium was developed from by-products and wastes from the ethanol agro-industry to replace commercial media in the production of a steam sterilization biological indicator (BI). Various recovery media were developed using soybean or sugarcane molasses and vinasse to prepare a self-contained BI. Media performance was evaluated by viability and heat resistance (D _121 °C value) according to regulatory standards. A medium produced with a soybean vinasse ratio of 1:70 (1.4%) (w/v) produced the results, with D _121 °C = 2.9 ± 0.5 min and Usk = 12.7 ± 2.1 min. The addition of 0.8% (w/v) yeast extract improved the germination of heat-damaged spores. The pH variation from 6.0 to 7.3 resulted in a gradual increase in the D _121 °C value. The absence of calcium chloride resulted in a decrease in germination, while no significant differences were observed with starch addition. Soybean vinasses may thus be used as the main component of a culture medium to substitute for commercial media in the production of self-contained biological indicators. The use of ethanol production waste in this biotechnological process realized a reliable performance, minimized the environmental impact, and decreased BI production costs while producing a high quality product.     

Ochratoxin A production in aniseed-based media by selected fungal strains and in anise fruits (<Emphasis Type="Italic">Pimpinella anisum</Emphasis> L.)

The growth conditions and ochratoxin A (OTA) production of Aspergillus strains were studied in aniseed (Pimpinella anisum L.)-based media. The results showed that methanol/NaHCO₃ (50:50, v/v) mixture for extraction and competitive direct ELISA analytical method are capable of detecting low OTA concentrations in this raw material, which were confirmed by HPLC with fluorescence detection (R ² = 0.994). In aniseed meal extract agar artificially contaminated with selected fungi, the higher OTA values obtained were 283.8 ± 28.1 μg L^-1 for A. carbonarius and between 1.7 ± 0.1 μg L^-1 and 16.5 ± 12.8 μg L^-1 for A. steynii strains. While the optimal conditions of growth for A. carbonarius and A. steynii are 28°C and 0.98 a _w, the optimal production of OTA was observed at 0.99 a _w for both A. carbonarius and A. steynii but at 22°C and 28°C, respectively. Except in one sample, all the aniseed samples analysed were negative for OTA natural contamination. This study demonstrates that aniseed can be a matrix capable to contamination with OTA, at least produced by A. carbonarius and A. steynii strains, regardless of the antimicrobial properties of aniseed essential oil.     

Rice germ oil as multifunctional excipient in preparation of self-microemulsifying drug delivery system (SMEDDS) of tacrolimus

Surmounting the constraints of limited solubilization efficiency and prime requisite of antioxidant for conventional lipid formulations, the research work explores an edge over formulation utilizing potential applicability of rice germ oil (RGO) as a multifunctional excipient. Self-microemulsifying drug delivery system (SMEDDS) of tacrolimus (TAC) was formulated with RGO, an indigenous source of gamma-oryzanol. Being the same biological source, RGO and rice bran oil (RBO) were compared and it was found that RGO have more solubilization potential for TAC (2.2-fold) as well as higher antioxidant activity (8.06-fold) than the RBO. TAC-SMEDDS was prepared using RGO/Capmul PG8 (2:3) as an oil phase, Cremophore EL as a surfactant, and Transcutol P as a cosurfactant. The approximate particle size of TAC-SMEDDS was found to be 38 nm by dynamic light scattering and 12 nm by small angle neutron scattering. The in vitro dissolution studies showed complete and rapid drug release in 30 min compared to a plain drug (<5%) and marketed capsule (<50%). AUC and C _max were found to be 45.05 ± 15.64 ng h/ml and 3.91 ± 1.2 ng/ml for TAC-SMEDDS, 12.59 ± 5.54 ng h/ml and 0.48 ± 0.12 ng/ml for plain TAC, and 30.23 ± 10.34 ng h/ml and 2.31 ± 0.68 ng/ml for marketed formulation, respectively. The improved pharmacokinetic profile of TAC-SMEDDS is correlating to the dissolution results. Thus, gamma-oryzanol-enriched RGO acts as a potential multifunctional excipient for lipid formulations.     

Synthesis of a citrulline-rich cyanophycin by use of <Emphasis Type="Italic">Pseudomonas putida</Emphasis> ATCC 4359

Synthesis of cyanophycin (multi-l-arginyl-poly-l-aspartic acid, CGP) in recombinant organisms is an important option to obtain sufficiently large amounts of this polymer with a designed composition for use as putative precursors for biodegradable technically interesting chemicals. Therefore, derivates of CGP, harbouring a wider range of constituents, are of particular interest. As shown previously, cyanophycin synthetases with wide substrate ranges incorporate other amino acids than arginine. Therefore, using an organism, which produces the required supplement by itself, was the next logical step. Former studies showed that Pseudomonas putida strain ATCC 4359 is able to produce large amounts of l-citrulline from l-arginine. By expressing the cyanophycin synthetase of Synechocystis sp. PCC 6308, synthesis of CGP was observed in P. putida ATCC 4359. Using an optimised medium for cultivation, the strain was able to synthesise insoluble CGP amounting up to 14.7 ± 0.7% (w/w) and soluble CGP amounting up to 28.7 ± 0.8% (w/w) of the cell dry matter, resulting in a total CGP content of the cells of 43.4% (w/w). HPLC analysis of the soluble CGP showed that it was composed of 50.4 ± 1.3 mol % aspartic acid, 32.7 ± 2.8 mol % arginine, 8.7 ± 1.6 mol % citrulline and 8.3 ± 0.4 mol % lysine, whereas the insoluble CGP contained less than 1 mol % of citrulline. Using a mineral salt medium with 1.25 or 2% (w/v) sodium succinate, respectively, plus 23.7 mM l-arginine, the cells synthesised insoluble CGP amounting up to 25% to 29% of the CDM with only a very low citrulline content.     

Home range,range overlap,and site fidelity of introduced Siberian chipmunks in a suburban French forest

Home range size, range overlap, and multiyear site fidelity were investigated for introduced Siberian chipmunks (Tamias sibiricus) in a French suburban forest from bimonthly trapping sessions for 4 years (2004–2007). Annual home range sizes (100% minimum convex polygon, ±SE) were estimated from 39 trapping histories of 28 different adult residents. Males (N = 13, 1.86 ± 0.32 ha) had a home range 2.5 times larger than females (N = 26, 0.71 ± 0.08 ha); a male home range included significantly more trapping centers (arithmetic mean of capture locations) of females (5.5 ± 0.7) than of males (2.3 ± 0.5). Chipmunks exhibited strong multiyear site fidelity: mean distance between annual trapping centers of individuals trapped over two successive years was small (N = 82, 26 ± 2 m) compared to the largest home range length (ranging from 36 to 281 m); overlap between annual home range sizes of residents was 84 ± 5% (N = 11). These results improve our understanding of the space occupation of this unknown species in a novel environment.     

A novel cold-adapted lipase from <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. XMZ-26: gene cloning and characterisation

Acinetobacter sp. XMZ-26 (ACCC 05422) was isolated from soil samples obtained from glaciers in Xinjiang Province, China. The partial nucleotide sequence of a lipase gene was obtained by touchdown PCR using degenerate primers designed based on the conserved domains of cold-adapted lipases. Subsequently, a complete gene sequence encoding a 317 amino acid polypeptide was identified. Our novel lipase gene, lipA, was overexpressed in Escherichia coli. The recombinant protein (LipA) was purified by Ni-affinity chromatography, and then deeply characterised. The LipA resulted to hydrolyse pNP esters of fatty acids with acyl chain length from C2 to C16, and the preferred substrate was pNP octanoate showing a k _cat = 560.52 ± 28.32 s⁻¹, K _m = 0.075 ± 0.008 mM, and a k _cat/K _m = 7,377.29 ± 118.88 s⁻¹ mM⁻¹. Maximal LipA activity was observed at a temperature of 15°C and pH 10.0 using pNP decanoate as substrate. That LipA peaked at such a low temperature and remained most activity between 5°C and 35°C indicated that it was a cold-adapted enzyme. Remarkably, this lipase retained much of its activity in the presence of commercial detergents and organic solvents, including Ninol, Triton X-100, methanol, PEG-600, and DMSO. This cold-adapted lipase may find applications in the detergent industry and organic synthesis.     

Extraction and characterisation of alginate from brown seaweeds (Fucales,Phaeophyceae) collected from Port Dickson,Peninsular Malaysia

Four species of brown seaweeds, namely Sargassum baccularia, Sargassum binderi, Sargassum siliquosum and Turbinaria conoides, harvested from Port Dickson, Negeri Sembilan, Malaysia were analysed for ash content, alginate yield and alginate properties. Seaweeds calcined at 450°C were found to have low amount of non-combustible residue as these were not contaminated by calcareous animals. Alginate was extracted from these seaweeds by two methods: hot and cold. In the hot method, the storing time was 3 h and the processing temperature was 50°C, whilst in the cold method, the sample was stored overnight at room temperature. Higher yield of alginate was obtained by the hot method compared to the cold method, but alginate extracted by the cold method gave higher molecular weight. In the hot method, 49.9% of alginate was extracted from S. siliquosum, followed by T. conoides (41.4%), S. binderi (38.9%) and S. baccularia (26.7%). Alginate extracted from T. conoides has an average molecular weight, M _w, of 8.06 × 10⁵ g mol⁻¹, whereas alginate from S. siliquosum was the lowest in M _w (4.81 × 10⁵ g mol⁻¹) when the extraction was done at room temperature. Alginate extracted from S. baccularia was found to be very heat-sensitive. Its M _w has dropped more than 83%, from 7.52 × 10⁵ to 1.23 × 10⁵ g mol⁻¹, when the extraction temperature was raised. The effect of heat on the extent of depolymerisation of the alginate molecule of the other three brown seaweed species was less significant, with decrease in molecular weight ranging between 13% and 16%.     

Preparation and <Emphasis Type="Italic">In Vitro</Emphasis>/<Emphasis Type="Italic">In Vivo</Emphasis> Evaluation of Microparticle Formulations Containing Meloxicam

In this study, we have formulated chitosan-coated sodium alginate microparticles containing meloxicam (MLX) and aimed to investigate the correlation between in vitro release and in vivo absorbed percentages of meloxicam. The microparticle formulations were prepared by orifice ionic gelation method with two different sodium alginate concentrations, as 1% and 2% (w/v), in order to provide different release rates. Additionally, an oral solution containing 15 mg of meloxicam was administered as the reference solution for evaluation of in vitro/in vivo correlation (ivivc). Following in vitro characterization, plasma levels of MLX and pharmacokinetic parameters [elimination half-life (t _1/2), maximum plasma concentration (C _max), time for C _max (t _max)] after oral administration to New Zealand rabbits were determined. Area under plasma concentration–time curve (AUC_0–∞) was calculated by using trapezoidal method. A linear regression was investigated between released% (in vitro) and absorbed% (in vivo) with a model-independent deconvolution approach. As a result, increase in sodium alginate content lengthened in vitro release time and in vivo t _max value. In addition, for ivivc, linear regression equations with r ² values of 0.8563 and 0.9402 were obtained for microparticles containing 1% and 2% (w/v) sodium alginate, respectively. Lower prediction error for 2% sodium alginate formulations (7.419 ± 4.068) compared to 1% sodium alginate formulations (9.458 ± 5.106) indicated a more precise ivivc for 2% sodium alginate formulation.     

Development of Spray Dried Liposomal Dry Powder Inhaler of Dapsone

This investigation was undertaken to evaluate practical feasibility of site specific pulmonary delivery of liposomal encapsulated Dapsone (DS) dry powder inhaler for prolonged drug retention in lungs as an effective alternative in prevention of Pneumocystis carinii pneumonia (PCP) associated with immunocompromised patients. DS encapsulated liposomes were prepared by thin film evaporation technique and resultant liposomal dispersion was passed through high pressure homogenizer. DS nano-liposomes (NLs) were separated by ultra centrifugation and characterized. NLs were dispersed in phosphate buffer saline (PBS) pH 7.4 containing different carriers like lactose, sucrose, and hydrolyzed gelatin, and 15% l-leucine as antiadherent. The resultant dispersion was spray dried and spray dried formulation were characterized to ascertain its performance. In vitro pulmonary deposition was assessed using Andersen Cascade Impactor as per USP. NLs were found to have average size of 137 ± 15 nm, 95.17 ± 3.43% drug entrapment, and zeta potential of 0.8314 ± 0.0827 mV. Hydrolyzed gelatin based formulation was found to have low density, good flowability, particle size of 7.9 ± 1.1 μm, maximum fine particle fraction (FPF) of 75.6 ± 1.6%, mean mass aerodynamic diameter (MMAD) 2.2 ± 0.1 μm, and geometric standard deviation (GSD) 2.3 ± 0.1. Developed formulations were found to have in vitro prolonged drug release up to 16 h, and obeys Higuchi's Controlled Release model. The investigation provides a practical approach for direct delivery of DS encapsulated in NLs for site specific controlled and prolonged release behavior at the site of action and hence, may play a promising role in prevention of PCP.     

Heterologous expression and characterization of Choline Oxidase from the soil bacterium Arthrobacter nicotianae

In the course of a microbial screening of soil samples for new oxidases, different enrichment strategies were carried out. With choline as the only carbon source, a microorganism was isolated and identified as Arthrobacter nicotianae. From this strain, a gene coding for a choline oxidase was isolated from chromosomal DNA. This gene named codA was cloned in Escherichia coli BL21-Gold and the protein (An_CodA) heterologously overexpressed as a soluble intracellular protein of 59.1 kDa. Basic biochemical characterization of purified protein revealed a pH optimum of 7.4 and activity over a broad temperature range (15–70 °C). Specific activities were determined toward choline chloride (4.70 ± 0.12 U/mg) and the synthetic analogs bis(2-hydroxyethyl)-dimethylammonium chloride (0.05 ± 0.45 × 10^–2 U/mg) and tris-(2-hydroxyethyl)-methylammonium methylsulfate (0.01 ± 0.12 × 10^–2 U/mg). With increasing number of oxidizable groups, a significant decrease in activity was noted. Determination of kinetic parameters in atmorspheric oxygen resulted in K _M = 1.51 ± 0.09 mM and V _max = 42.73 ± 0.42 mU/min for choline chloride and K _M = 4.77 ± 0.76 mM and V _max = 48.40 ± 2.88 mU/min for the reaction intermediate betaine aldehyde respectively. Nuclear magnetic resonance spectroscopic analysis of the products formed during the enzyme reaction with choline chloride showed that in vitro the intermediate betaine aldehyde exists also free in solution.     

Effect of Plasticizer on Release Kinetics of Diclofenac Sodium Pellets Coated with Eudragit RS 30 D

The present study was designed to investigate the effect of two plasticizers, i.e., triethyl citrate (TEC) and polyethylene glycol 6000 (PEG 6000) on the in vitro release kinetics of diclofenac sodium from sustained-release pellets. Ammonio methacrylate copolymer type B (Eudragit RS 30 D) is used as the release-retarding polymer. Both plasticizers were used at 10% and 15% (w/w) of Eudragit RS 30 D. Pellets were prepared by powder layering technology and coated with Eudragit RS 30 D by air suspension technique. Thermal properties of drug and drug-loaded beads were studied using differential scanning calorimeter (DSC). DSC thermogram represented the identity of raw materials and exhibited no interaction or complexation between the active and excipients used in the pelletization process. Dissolution study was performed by using USP apparatus 1. No significant difference was observed among the physical properties of the coated pellets of different batches. When dissolution was performed as pure drug, about 8.22% and 90% drug was dissolved at 2 h in 0.1 N HCl and at 30 min in buffer (pH 6.8), respectively. From all formulations, the release of drug in acid media was very negligible (maximum 1.8 ± 0.08% at 2 h) but in buffer only 12% and 30% drug was released at 10 h from coated pellets containing TEC and PEG 6000, respectively, indicating that Eudragit RS 30 D significantly retards the drug release rate and that drug release was varied according to the type and amount of plasticizers used. The amount of TEC in coating formulation significantly effected drug release (p < 0.001), but the effect of PEG 6000 was not significant. Formulations containing PEG 6000 released more drug (98.35 ± 2.35%) than TEC (68.01 ± 1.04%) after 24 h. Different kinetic models like zero order, first order, and Higuchi were used for fitting drug release pattern. Zero order model fitted best for diclofenac release in all formulations. Drug release mechanism was derived with Korsmeyer equation.     

Modified Nanoprecipitation Method for Preparation of Cytarabine-Loaded PLGA Nanoparticles

The present investigation was aimed at developing cytarabine-loaded poly(lactide-coglycolide) (PLGA)-based biodegradable nanoparticles by a modified nanoprecipitation which would have sustained release of the drug. Nine batches were prepared as per 3² factorial design to optimize volume of the co-solvent (0.22–0.37 ml) and volume of non-solvent (1.7–3.0 ml). A second 3² factorial design was used for optimization of drug: polymer ratio (1:5) and stirring time (30 min) based on the two responses, mean particle size (125 ± 2.5 nm), and percentage entrapment efficiency (21.8 ± 2.0%) of the Cyt-PLGA nanoparticles. Optimized formulation showed a zeta potential of −29.7 mV indicating good stability; 50% w/w of sucrose in Cyt-PLGA NP was added successfully as cryoprotectant during lyophilization for freeze-dried NPs and showed good dispersibility with minimum increase in their mean particle sizes. The DSC thermograms concluded that in the prepared PLGA NP, the drug was present in the amorphous phase and may have been homogeneously dispersed in the PLGA matrix. In vitro drug release from the pure drug was complete within 2 h, but was sustained up to 24 h from PLGA nanoparticles with Fickian diffusion. Stability studies showed that the developed PLGA NPs should be stored in the freeze-dried state at 2–8°C where they would remain stable in terms of both mean particle size and drug content for 2 months.     

A new manganese superoxide dismutase identified from Beauveria bassiana enhances virulence and stress tolerance when overexpressed in the fungal pathogen

A superoxide dismutase (SOD) was characterized from Beauveria bassiana, a fungal entomopathogen widely applied to insect control. This 209-aa enzyme (BbSod2) showed no more than 71% sequence identity to other fungal Mn-SODs, sharing all conserved residues with the Mn-SOD family and lacking a mitochondrial signal. The SOD activity of purified BbSod2 was significantly elevated by Mn²⁺, suppressed by Cu²⁺ and Zn²⁺ but inhibited by Fe³⁺. Overexpressing the enzyme in a BbSod2-absent B. bassiana strain enhanced its SOD activity (107.2 ± 6.1 U mg⁻¹ protein) by 4–10-fold in different transformants analyzed. The best BbSod2-transformed strain with the SOD activity of 1,157.9 ± 74.7 U mg⁻¹ was 93% and 61% more tolerant to superoxide-generating menadione in both colony growth (EC₅₀ = 2.41 ± 0.03 versus 1.25 ± 0.01 mM) and conidial germination (EC₅₀ = 0.89 ± 0.06 versus 0.55 ± 0.07 mM), and 23% more tolerant to UV-B irradiation (LD₅₀ = 0.49 ± 0.02 versus 0.39 ± 0.01 J cm⁻²). Its virulence to Spodoptera litura larvae was enhanced by 26% [LT₅₀ = 4.5 (4.2–4.8) versus 5.7 (5.2–6.4) days]. Our study highlights for the first time that the Mn²⁺-cofactored, cytosolic BbSod2 contributes significantly to the virulence and stress tolerance of B. bassiana and reveals possible means to improving field persistence and efficacy of a fungal formulation by manipulating the antioxidant enzymes of a candidate strain.     

Microemulsion-based gel of terbinafine for the treatment of onychomycosis: optimization of formulation using D-optimal design

The aim of the present investigation was to evaluate microemulsion as a vehicle for dermal drug delivery and to develop microemulsion-based gel of terbinafine for the treatment of onychomycosis. D-optimal mixture experimental design was adopted to optimize the amount of oil (X ₁), Smix (mixture of surfactant and cosurfactant; X ₂) and water (X ₃) in the microemulsion. The formulations were assessed for globule size (in nanometers; Y ₁) and solubility of drug in microemulsion (in milligrams per milliliter; Y ₂). The microemulsion containing 5.75% oil, 53.75% surfactant–cosurfactant mixture and 40.5% water was selected as the optimized batch. The globule size and solubility of the optimized batch were 18.14 nm and 43.71 mg/ml, respectively. Transmission electron microscopy showed that globules were spherical in shape. Drug containing microemulsion was converted into gel employing 0.75% w/w carbopol 934P. The optimized gel showed better penetration and retention in the human cadaver skin as compared to the commercial cream. The cumulative amount of terbinafine permeated after 12 h was 244.65 ± 18.43 μg cm⁻² which was three times more than the selected commercial cream. Terbinafine microemulsion in the gel form showed better activity against Candida albicans and Trichophyton rubrum than the commercial cream. It was concluded that drug-loaded gel could be a promising formulation for effective treatment of onychomycosis.     

Tracking the Ovarian Cycle in Black-and-Gold Howlers (<Emphasis Type="Italic">Alouatta caraya</Emphasis>) by Measuring Fecal Steroids and Observing Vaginal Bleeding

A better understanding of a species’ reproductive physiology can help conservation programs to manage primates in the wild and develop assisted reproductive technologies in captivity. We investigated whether measurements of fecal progestin and estrogen metabolites obtained by a radioimmunoassay could be used to monitor the ovarian cycle of Alouatta caraya. We also compared the occurrence of vaginal bleeding with the hormone profiles. We collected fecal samples from 3 adult and 1 subadult captive female over 5 mo and performed vaginal cytology for the adults. The interval between fecal progestin surges in the adult females was 19.11 ± 2.14 d (n = 18 cycles). Fecal progestin concentrations remained at basal values for 9.83 ± 2.21 d (n = 18) and rose to elevated values for 9.47 ± 0.72 d (n = 19). The subadult female showed basal levels of fecal estrogen and progestin concentrations throughout the study, suggesting that our hormone measurements are valid to monitor the ovarian cycle. Bleeding periods coincided with basal levels of fecal estrogens and progestin at intervals of 19.8 ± 0.9 d and lasted for 4.1 ± 1.0 d. Although we obtained these data from only 3 individuals, the results indicate that this species likely has a menstrual-type ovarian cycle. These data provide the first endocrine profile for the Alouatta caraya ovarian cycle and are similar to results obtained for other howler species. This similarity is important for comparative studies of howlers, allowing for a better understanding of their reproductive physiology and contributing to a critical information base for managing Alouatta species.