Integrative taxonomy and phylogenetic relationships between representatives of genera within Merliniinae (Nematoda: Merliniidae), with new data on fourteen known and one unknown species.

The application of integrative taxonomic approaches is useful to species delineation based on a combination of distinct types of characters, here morphological features and ribosomal DNA sequences. In this study, we surveyed ectoparasitic nematodes of the subfamily Merliniinae in cultivated and natural environments in Iran. Results of morphological and morphometrical studies, light and scanning electron microscopic observations, and molecular analyses allowed us the identification of fourteen known and one unknown species including representatives of the genera Amplimerlinius (five species), Geocenamus (one species), Merlinius (three species), Nagelus (two species), Paramerlinius (one species), Scutylenchus (two species), and Telomerlinius (one species). The unknown species, Scutylenchus sp., characterized by having 35-50 incisures at mid-body; lateral field with 6 longitudinal incisures; lip region slightly offset by a constriction, flattened at front end; bearing 5-7 annuli; cephalic framework not refractive; stylet robust, 18.3-27 µm long; post anal intestinal sac absent; tail elongate conical, dorsally convex, with 24 (19-28) annuli in ventral side, ending to a smooth terminus and males common; spicules 24.5-31 µm long. The phylogenetic analyses were carried out using molecular data from nuclear ribosomal DNA (rDNA) genes viz. D2-D3 expansion segments of the large ribosomal subunit (28S rRNA), partial small ribosomal subunit (18S rRNA), and internal transcribed spacer (ITS). The molecular variability of D2-D3 expansion segments of the 28S rRNA and partial 18S rRNA was low in this family in comparison to the ITS region, which could be a more helpful molecular marker in species and genus identification.

Taxonomical considerations and molecular phylogeny of the closely related genera Bitylenchus, Sauertylenchus and Tylenchorhynchus (Nematoda: Telotylenchinae), with one new and four known species from Iran.

During several nematological surveys in cultivated and natural habitats in Khuzestan and Zanjan provinces of Iran, a new species, Bitylenchus parvulus n. sp., two new records for Iran - namely, Tylenchorhynchus agri and Tylenchorhynchus graciliformis - and a population of Bitylenchus parvus and one of Sauertylenchus maximus were recovered and characterized based upon morphological and molecular approaches. The new species is characterized by lip region with five to seven annuli, stylet 17.7 (17.0-18.5) µm long, sub-cylindrical tail narrowing abruptly near terminus giving a bluntly digitate shape to the tail tip, cuticle near anterior part of vulva wrinkled and post-rectal sac occupies whole of tail cavity. The phylogenetic analyses were carried out using molecular data from D2-D3 expansion segments of large ribosomal subunit (28S rRNA) for all studied species and the partial small ribosomal subunit (18S rRNA) for the new species. The representatives of Bitylenchus and Sauertylenchus formed distinct clades from Tylenchorhynchus members, supporting the hypothesis in which Bitylenchus and Sauertylenchus could be considered as valid genera, but rejecting the 'large-genus' concept for Tylenchorhynchus. Also, Sauertylenchus ibericus was proposed as a junior synonym of S. maximus based on the results from morphological and phylogenetic analysis. Furthermore, an identification key for all known species included in the three genera Bitylenchus, Tylenchorhynchus and Sauertylenchus is presented herein. The number of transverse annuli on the lip region and presence/absence of post-rectal sac were considered as the main diagnostic characters for classifying the species into seven groups, and other morphological and morphometric characters were subsequently used for distinguishing species in each group.

Morphological and molecular data of two species of the rare genera Thada Thorne, 1941 and Tenunemellus Siddiqi, 1986 (Nematoda: Tylenchidae) from Iran.

Thada populus n. sp. was found in the rhizosphere of Populus euphratica in the city of Dezful, south-western Iran. The new species is characterized by its 365-453 µm body length, cuticle with transverse annuli 0.9-1.2 µm wide, lateral fields with four lines, lip region low with one or two annuli, 7.0-8.5 µm wide at base, longitudinal or slightly sigmoid amphidial slit, delicate stylet 8.4-9.8 µm long with posteriorly directed knobs, dorsal pharyngeal gland opening at 1.0-1.5 µm posterior to the stylet knobs, non-muscular and valve-less median bulb, pyriform and offset basal bulb, almost oval spermatheca, posterior position of vulva (V = 75-79%) without lateral membrane, short post-vulval uterine sac and conical tail with finely to broadly rounded terminus. Morphological differences of the new species with those of the type species, Thada striata, are discussed. Molecular phylogenetic studies of the new species using partial sequences of small subunit ribosomal DNA revealed that the new species formed a clade with Malenchus spp., Filenchus facultativus, F. fungivorus and Filenchus sp. in Bayesian inference. Morphological and molecular phylogenetic studies were also performed on Tenunemellus indicus, the second recovered species, the latter analysis using partial sequences of large subunit ribosomal DNA. In the inferred Bayesian tree, T. indicus formed a clade with Labrys fuzhouensis, Lelenchus leptosoma from the Netherlands, Malenchus spp. and Filenchus discrepans.

Pain management during needle insertion with low level laser.

AIM: To date, there is no safe, effective, and rapid method to eliminate the pain associated with needle insertion. It is noticed that using a low-energy laser before intradermal and intramuscular injection would help rapid local anaesthesia with a lidocaine product. However, there is no valuable data on the effect of the concomitant use of laser and benzocaine as a pre-treatment option, compared to local application of benzocaine products alone before needle insertion. MATERIALS AND METHODS: A prospective single-blind clinical trial was designed to assess pain perception during needle insertion into mucosa in 66 healthy volunteer dental students. They received a topical anaesthetic agent on one side (control) applied for one minute and a topical anaesthetic agent plus low-power laser on the other side (experimental) prior to needle insertion. The first pre-treatment method was chosen based on block randomisation table. A dentist recorded the subjects' reaction based on the visual analogue scale (VAS) during insertion of needle in the two groups. SPSS version 15 was used for data analysis. The statistical significant level was defined at P-value< 0.01. RESULTS: The mean scores of VAS for the experimental and control groups was 21± and 19± 2.70 respectively, with a statistically significant higher VAS score in the experimental group, although it was not clinically significant. CONCLUSION: Simultaneous application of laser with local anaesthetic products containing benzocaine in the buccal mucosa before dental needle insertion does not reduce pain perception.

Application of neural network method to detect type of uranium contamination by estimation of activity ratio in environmental alpha spectra.

The discrimination of the composition of environmental and non-environmental materials by the estimation of the (234)U/(238)U activity ratio in alpha-particle spectrometry is important in many applications. If the interfering elements are not completely separated from the uranium, they can interfere with the determination of (234)U. Thickness as a result of the existence of iron in the source preparation phase and their alpha lines can broaden the alpha line of (234)U in alpha spectra. Therefore, the asymmetric broadening of the alpha line of (234)U and overlapping of peaks make the analysis of the alpha particle spectra and the interpretation of the results difficult. Applying Artificial Neural Network (ANN) to a spectrometry system is a good idea because it eliminates limitations of classical approaches by extracting the desired information from the input data. In this work, the average of a partial uranium raw spectrum, were considered. Each point that its slope was of the order of 0-1% per 10 channels, was used as input to the multi-layer feed forward error-back propagation network. The network was trained by an alpha spectrum library which has been developed in the present work. The training data in this study was actual spectral data with any reasonable thickness and interfering elements. According to the results, the method applied to estimate the activity ratio in this work, can examine the alpha spectrum for peaks which would not be expected for a source of given element and provide the clues about composition of uranium contamination in the environmental samples in a fast screening and classifying procedures.

Selective preconcentration of ultra trace copper(II) using octadecyl silica membrane disks modified by a recently synthesized glyoxime derivative.

A new simple and reliable method has been developed to selectively separate and concentrate ultra trace amounts of copper ion in aqueous samples for subsequent measurement by atomic absorption spectrometry (AAS). The Cu(2+) ions are adsorbed selectively and quantitatively during passage of aqueous solutions through octadecyl silica membrane disks modified with bis(2-hydroxyphenylamino) glyoxime. The retained copper ions then stripped from the disk with a minimal amount of 0.2M nitric acid solution as eluent, and determined by AAS. The proposed method permitted large enrichment factors of about 100 or higher. The limit of detection of the proposed method is 0.004ngml(-1). The maximum capacity of the membrane disks modified with 25mg of ligand was found to be 280+/-32mug of copper(II). The effects of various cationic interferences on the percent recovery of copper in binary mixtures were studied. The method was successfully applied to the recovery and determination of copper in several water samples.

A new method for preparing biodegradable microparticles and entrapment of hydrocortisone in DL-PLG microparticles using supercritical fluids.

An improved process for the production of polymeric microparticles, based on solution-enhanced dispersion by supercritical fluids (SEDS) using a combination of supercritical N(2) and CO(2), was evaluated. The biodegradable polymers, poly(D,L-lactide-co-glycolide): copolymer composition 50:50 (DL-PLG), poly(L-lactide) (L-PLA), poly(D,L-lactide) (DL-PLA) and polycaprolactone, were used for preparation of microparticles by a modified SEDS process. Solutions of the polymers in organic solvents were dispersed and the solvent was extracted with supercritical CO(2) and N(2). The morphology, the size distributions and degree of hydrocortisone entrapment were determined. The combination of supercritical N(2) and CO(2) led to a more efficient dispersion of the polymer solutions than CO(2) alone. This resulted in a reduction of particle size of the microparticles produced from all of the amorphous polymers. Discrete spherical microparticles with a mean volumetric diameter of less than 10 microm were produced from DL-PLG, DL-PLA and L-PLA. Hydrocortisone was successfully entrapped within the DL-PLG microparticles. The modified SEDS process improved the dispersion of amorphous polymer solutions resulting in formation of small spherical microparticles. The SEDS process can be used for incorporation of drugs into the DL-PLG microparticles.

Preparation of biodegradable microparticles using solution-enhanced dispersion by supercritical fluids (SEDS).

PURPOSE: We have evaluated a new process, involving solution-enhanced dispersion by supercritical fluids (SEDS), for the production of polymeric microparticles. METHODS: The biodegradable polymers, Poly (DL-lactide-co-glycolide): copolymer composition 50:50 (DL-PLG), Poly (L-lactide) (L-PLA), Poly (DL-lactide) (DL-PLA) and Polycaprolactone (PCL), were used for preparation of microparticles using SEDS. Solutions of the polymers in organic solvents were dispersed and sprayed with supercritical CO2. Extraction of the organic solvents resulted in the formation of solid microparticles. The amounts of highly toxic solvents such as dichloromethane (MC) were reduced in the process. RESULTS: Microparticles were obtained from all polymers. The mean particle size and shape varied with the polymer used. The morphology of the particles was strongly affected by the choice of polymer solvent. Discrete spherical microparticles of DL-PLG were produced with a mean volumetric diameter of 130 microm. The microparticles of the L-PLA were almost spherical, and their size increased from 0.5 to 5 microm as the density of supercritical CO2 decreased. PCL formed microparticles with diameters of 30-210 microm and showed a strong tendency to form films at high pressure. CONCLUSIONS: The SEDS process appears a promising method for production of microparticles from biodegradable polymers without the use of toxic solvents.

Encapsulation of rotavirus into poly(lactide-co-glycolide) microspheres.

Two small-scale double emulsion techniques for incorporation of formaldehyde-inactivated rotavirus particles (FRRV) into poly(lactide-co-glycolide) (PLG) microspheres were developed and optimised. The effects of high-speed homogenisation versus vortex mixing on the double emulsion stability, microsphere size, entrapment efficiency and in vitro release of FRRV in the second emulsification step were studied. A stable double emulsion was verified only when using vortex mixing in this step. Slow removal of the organic phase allowed measurement of the size of the emulsion droplets and subsequent prediction of the size of the resulting microspheres. Microspheres in the size range of 1-10 microm were prepared using both techniques. The homogenisation technique was sensitive to changes in the operating time, the emulsification energy and the volume of the outer aqueous phase, while the vortex technique was more robust. Rotavirus was released in vitro in a triphasic manner with both techniques. The more robust vortex technique was selected for preparation of PLG microspheres containing rotavirus for in vivo studies. After immunisation of mice with a single intramuscular injection, the PLG-FRRV microspheres elicited an IgG antibody response in serum detected by ELISA equally high as that elicited with FRRV alone. These results indicate that the antigenicity of FFRV was retained after incorporation into PLG microspheres using the vortex technique.

Biological activity of lysozyme after entrapment in poly(d,l-lactide-co-glycolide)-microspheres.

PURPOSE: The purpose of this study was to investigate the process of preparing microspheres for maximising entrapment efficiently (EE) and retained biological activity (RBA) of peptides and proteins. METHODS: A controlled-release formulation based on poly(d,l-lactide-co-glycolide) was designed and produced using a small-scale double emulsion method. These PLG microspheres contained a model peptide, lysozyme. The retained bioactivity of the incorporated lysozyme was determined by bacterial assay. The size distributions and the morphology of the microspheres were characterized. RESULTS: The RBA and EE improved when the PLG concentration in the organic phase of the emulsion was increased. A high lysozyme concentration in the inner water phase of the emulsion resulted in decreased EE and an increase in the proportion of fragmented particles. The RBA of lysozyme in the microspheres varied between 30 and 80% with changes to the process. CONCLUSIONS: The study shows that the RBA of lysozyme in PLG microspheres is strongly dependent on the experimental conditions for preparing the microspheres. Measurement of the EE alone, without the RBA is insufficient to evaluate the efficacy of the designed delivery system.