Effect of temperature and propylene glycol as a cosolvent on dissolution of clotrimazole.

Herein, the solubility study of clotrimazole was performed in a propylene glycol+water system. The solubility values were fitted to various cosolvency equations. The model accuracies were studied with the computation of the mean relative deviations. The thermodynamic behavior was investigated according to the van't Hoff and Gibbs equations for clotrimazole in the propylene glycol+water system. Furthermore, the density data for clotrimazole were determined in mixtures of propylene glycol+water and fitted to the Jouyban-Acree equation.

Phylogenetic Diversity of Dermanyssus gallinae (Dermanyssidae) based on Mitochondrial Cytochrome Oxidase-1 Gene Sequence Collected from Different Bird Species in Iran.

A wide range of hosts, especially birds, can be infested with Dermanyssus gallinae (D. gallinae), as an obligate hematophagous mite. In this study, cytochrome oxidase 1 (CO1) gene sequences were employed to perform molecular and phylogenetic analyses of D. gallinae collected from different bird species in Iran. Adult mites were collected from the body surface and cage material of ornamental and wild birds in industrial farms located in the Western and Northwestern regions of Iran. The infestation was identified in layer poultry farming by inspecting the eggs and the whole surfaces of the birds' bodies. The holding area and body surface of the ornamental and wild birds were also thoroughly examined. The D. gallinae samples were assigned to two subgroups of haplogroup A (i.e., A1 and A2). The phylogenetic tree suggested that the D. gallinae samples collected from wild birds in the A1 sub-haplogroup should be placed beside Japanese, Norwegian, Italian, and French samples isolated from wild birds in the A2 sub-haplogroup. Additionally, the highest phylogenetic similarity in the A2 sub-group was observed between mites isolated from ornamental and industrial birds in Australia. The findings of the present study suggest that crows and sparrows may play an important role in the transmission of D. gallinae infestation to other species of wild birds due to their high population, as well as their presence in most areas.

A dual-signaling electrochemical ratiometric strategy combining "signal-off" and "signal-on" approaches for detection of MicroRNAs.

A dual-signaling electrochemical ratio metric strategy was developed for detection microRNA-18a based on the duplex-specific nuclease-assisted target recycling and electrochemical atom transfer radical polymerization signal amplification. In the presence of target microRNA, RNA/DNA duplexes are formed, which become the substrate of the duplex-specific nuclease-assisted target recycling. Hence only the DNA strand is cleaved by duplex-specific nuclease enzyme, resulting in the throw away of methylene blue (MB) from the electrode (signal off) accompanied by releasing of target microRNA, which can be recycled in the next hybridization. The remaining piece of capture DNAs on the electrode surface hybridize with the Azide labeled-signal DNAs. "Click reactions" were carried out between 3-Butynyl-2-bromoisobutyrate and Azide to initiate the electrochemical atom transfer radical polymerization reaction. This process could bring a great number of ferrocenylmethyl methacrylate (FMMA) on the surface of electrode (signal on). The IFMMA/IMB value was proportionate to the microRNA-18a concentration and measured by square wave voltammetry. The promising potential of the proposed biosensor in clinical analyses was exhibited by its remarkable features such as strong performance, high specificity, agreeable storage stability, and notable selectivity in real sample evaluation with no pretreatment or amplification. Finally, our biosensing method offers such an application to be used for the early clinical diagnosis of Pancreatic Cancer.

An enzyme-free electrochemical biosensor for simultaneous detection of two hemophilia A biomarkers: Combining target recycling with quantum dots-encapsulated metal-organic frameworks for signal amplification.

A sensitive and selective electrochemical method for simultaneous detection of two hemophilia A-related microRNAs (miR-1246 and miR-4521) was developed. This detection tactic was based on gold nanoparticles (AuNPs), heavy metals quantum dots-encapsulated metal-organic frameworks (QDs@ZIF-8), and catalytic hairpin assembly (CHA) for signal application. Primarily, hairpins H1 and H2 were hybridized with targets miR-1246 (T1) and miR-4521 (T2) for forming H1-T1 and H2-T2 duplex stranded DNAs (dsDNAs) that were able to open the hairpins H3 and H4 for the formation of H1-H3 and H2-H4 dsDNAs. Meanwhile, lots of H1-H3 and H2-H4 dsDNAs were created by releasing the target to take part in the next cycle for signal amplification. And then single stranded fragments of H1-H3 and H2-H4 dsDNAs were utilized for hybridizing the PbS@ZIF-8-S1 and CdS@ZIF-8-S2 in order to amplify the electrochemical signal. The diagnosis of corresponding target miRs using differential pulse voltammetry has been possible by releasing Pb (II) and Cd (II) ions from PbS@ZIF-8 and CdS@ZIF-8 tags by HCI leaching. In this context, encapsulation of heavy metals quantum dots (QDs) was done in zeolitic imidazolate framework-8 (ZIF-8) to form QDs@ZIF-8 muti-core-shell particles by in situ growth of ZIF-8 in the presence of QDs. Since the quantity of QDs tagged to each target miRs grows massively, being resulted from a huge number of QDs that encapsulated in each QDs@ZIF-8 label, the sensitivity of the biosensor using QDs@ZIF-8 particles as signal tags is about 15 times that of a biosensor using QDs as signal tags. Several conditions of determination like incubation time for labeling and capture probe, HCl leaching time, and reaction time of CHA were optimized. Under the optimized conditions, this assay allowed the detection of target miRs in the range of 1 fM to 1  µM with detection limits of 0.19 fM and 0.28 fM for miR-1246 and miR-4521 (S/N = 3). The biosensor can discriminate complementary, 1-base mismatched and non-complementary sequences quite well, according to the catalytic hairpin assembly. Furthermore, the biosensor was utilized efficiently for quick and direct analysis of microRNAs in human serum. Thus, this tactic presents an innovative platform for microRNAs expression profiling in biomedical research and clinical diagnosis.

Modification of Source Strength in Low-Dose-Rate Lung Brachytherapy with 125I and 103Pd Seeds.

BACKGROUND: A new treatment approach for most patients who have undergone early stage non-small-cell lung carcinoma (NSCLC) is wedge resection plus permanent implant brachytherapy. However, the specification of dose to medium at low energies especially in heterogeneous lung is unclear yet. OBJECTIVE: The present study aims to modify source strength for different configurations of 125I and 103Pd seeds used in lung permanent implant brachytherapy. METHODS: Different arrays of 125I and 103Pd seeds were simulated by MCNPX code in protocol-based water vs. actual 3D lung environments. Absorbed dose was, then, scored in both mediums. Dose differences between both environments were calculated and source strength was modified for the prescription point. In addition, lung-to-water absorbed dose ratio was obtained and presented by precise equations. RESULTS: Due to significant differences in prescription dose, source strength was modified 16%-19% and 37%-43% for different configurations of 125I and 103Pd seeds, respectively. In addition, depth-dependent dose differences were observed between the actual lung and protocol-based water mediums (dose difference as a function of depth). CONCLUSION: Modification of source strength is essential for different arrangements of 125I and 103Pd seeds in lung implantation. Modified source strength and presented equations are recommended to be considered in future studies based on lung brachytherapy.

Microsatellite diversity of the Nordic type of goats in relation to breed conservation: how relevant is pure ancestry?

In the last decades, several endangered breeds of livestock species have been re-established effectively. However, the successful revival of the Dutch and Danish Landrace goats involved crossing with exotic breeds and the ancestry of the current populations is therefore not clear. We have generated genotypes for 27 FAO-recommended microsatellites of these landraces and three phenotypically similar Nordic-type landraces and compared these breeds with central European, Mediterranean and south-west Asian goats. We found decreasing levels of genetic diversity with increasing distance from the south-west Asian domestication site with a south-east-to-north-west cline that is clearly steeper than the Mediterranean east-to-west cline. In terms of genetic diversity, the Dutch Landrace comes next to the isolated Icelandic breed, which has an extremely low diversity. The Norwegian coastal goat and the Finnish and Icelandic landraces are clearly related. It appears that by a combination of mixed origin and a population bottleneck, the Dutch and Danish Land-races are separated from the other breeds. However, the current Dutch and Danish populations with the multicoloured and long-horned appearance effectively substitute for the original breed, illustrating that for conservation of cultural heritage, the phenotype of a breed is more relevant than pure ancestry and the genetic diversity of the original breed. More in general, we propose that for conservation, the retention of genetic diversity of an original breed and of the visual phenotype by which the breed is recognized and defined needs to be considered separately.

Estimation from moments measurements for amyloid depolymerisation.

Estimating reaction rates and size distributions of protein polymers is an important step for understanding the mechanisms of protein misfolding and aggregation, a key feature for amyloid diseases. This study aims at setting this framework problem when the experimental measurements consist in the time-dynamics of a moment of the population (i.e. for instance the total polymerised mass, as in Thioflavin T measurements, or the second moment measured by Static Light Scattering). We propose a general methodology, and we solve the problem theoretically and numerically in the case of a depolymerising system. We then apply our method to experimental data of depolymerising oligomers, and conclude that smaller aggregates of ovPrP protein should be more stable than larger ones. This has an important biological implication, since it is commonly admitted that small oligomers constitute the most cytotoxic species during prion misfolding process.

Role of ruminants in the epidemiology of Echinococcus granulosus in Tabriz area, Northwest of Iran.

Hydatidosis is a widespread zoonotic disease with a worldwide distribution. This disease produces significant financial losses and it remains a serious health problem in a number of countries. Hydatidosis results from ingesting the eggs of Echinococcus granulosus, and the developing larvae envelop themselves in a cyst which forms in the organs of the intermediate hosts. The aim of this study was to investigate the prevalence of cystic echinococcosis in sheep, cattle, buffalo, and goats, slaughtered in the Tabriz Abattoir, Northwest of Iran. From April 2012 to April 2013, out of 14828 slaughtered animals examined for CE (hydatidosis) in liver and lungs (5000 sheep, 6125 cattle, 1103 buffaloes and 2600 goats), 25.57% were infected. The rates of CE in lungs and liver were 15.2% and 7.18% in sheep, respectively, 15.30% and 9.73% in goats, respectively, 18.71% and 9.61% in cattle, respectively and 15.68% and 11.24% in buffaloes, respectively. The infection rate was higher in lungs and was more pronounced in cattle (P<0.05). There were significant differences found in the prevalence rates of the various ruminants, and also between the sexes (P<0.05). The findings of the present study revealed that lungs were the main infected tissue, and male animals were less likely to be infected than female animals. The infection rates of the different ruminants increased significantly with age, (P<0.05).

HLA class II susceptibility pattern for type 1 diabetes (T1D) in an Iranian population.

This study aimed to determine the HLA-DRB1/HLA-DQB1 susceptibility and protection pattern for type 1 diabetes (T1D) in a population from Hamadan, north-west of Iran. A total of 133 patients with T1D were tested for HLA-DRB1 and HLA-DQB1 alleles using PCR-SSP compared to 100 ethnic-matched healthy controls. Alleles and haplotypes frequencies were compared between both groups. The most susceptible alleles for disease were HLA-DRB1*03:01, DRB1*04:02, DQB1*02:01 and DQB1*03:02, and protective alleles were HLA-DRB1*07:01, *11:01, *13:01, *14:01 and DRB1*15 and HLA-DQB1*06:01, *06:02 and *06:03. Haplotype analysis revealed that patients with T1D had higher frequencies of DRB1*03:01-DQB1*02:01 (OR = 4.86, P < 10(-7) ) and DRB1*04:02-DQB1*03:02 (OR = 9.93, P < 10(-7) ) and lower frequencies of DRB1*07:01-DQB1*02:01 (P = 0.0005), DRB1*11:01-DQB1*03:01 (P = 0.001), DRB1*13:01-DQB1*06:03 (P = 0.002) and DRB1*15-DQB1*06:01 (P = 0.001) haplotypes compared to healthy controls. Heterozygote combination of both susceptible haplotypes (DR3/DR4) confers the highest risk for T1D (RR = 18.80, P = 4 × 10(-5) ). Additionally, patients with homozygote diplotype, DR3/DR3 and DR4/DR4, showed a similar risk with less extent to heterozygote combination (P = 0.0004 and P = 0.01, respectively). Our findings not only confirm earlier reports from Iranians but also are in line with Caucasians and partly with Asians and some African patients with T1D. Remarkable differences were the identification of DRB1*04:01-DQB1*03:02, DRB1*07:01-DQB1*03:03 and DRB1*16-DQB1*05:02 as neutral and DRB1*13:01-DQB1*06:03 as the most protective haplotypes in this study.

Computational-aided design of molecularly imprinted polymer for selective extraction of methadone from plasma and saliva and determination by gas chromatography.

The main objective of this research was computational designing of an imprinted polymer for selective solid phase extraction (SPE) of methadone from plasma and saliva samples analyzed by gas chromatography-flam ionization detector (GC-FID). The density functional theory (DFT) at B3LYP/6-31G+ (d, p) level and Gaussian 2003 package was used to calculate the interaction energy of template-monomers (ΔE). The effect of polymerization solvent was also studied using polarizable continuum model (PCM). It was shown that, methacrylic acid (MAA) gave the largest ΔE in acetonitrile as a polymerization solvent. To examine the validity of this approach, two MIP were synthesized for methadone as template molecule and methacrylic acid as functional monomer in acetonitrile (AN) and methanol (MeOH), respectively. The performance of each polymer was evaluated by using imprinting effect. As it is expected, the best results were obtained for the molecularly imprinted polymer (MIP) which was prepared in AN. For the optimized method, the linearity between responses (peak areas) and concentration of methadone in plasma and saliva samples were found over the range of 3.6-40,000 ng mL(-1) (R(2)=0.997) and 3.0-40,000 ng mL(-1) (R(2)=0.998), respectively. The limit of detection (LOD) and limit of quantification (LOQ) for methadone in plasma were calculated to be 2.45 and 3.6 ng mL(-1), respectively. The LOD and LOQ for methadone in saliva were 2.14 and 3.0 ng mL(-1), respectively. The relative standard deviation (RSD; n=4) for plasma samples containing 10, 100, 500, 1000 ng mL(-1)of methadone were 5.98, 5.78, 5.52, 4.78, 4.74, and the RSD (n=4) for saliva sample containing 5, 20, 100, 1000 ng mL(-1) of methadone were 4.74, 5.1, 5.9, 5.6, respectively.

The prevalence of Linguatula serrata nymphs in goats slaughtered in Tabriz, Iran.

During 1 year (April 2009-April 2010), the prevalence of Linguatula serrata (L. serrata) nymphs in liver and mesenteric and mediastinal lymph nodes (MLN) of 740 native goats of different sex and ages were examined in Tabriz, north west of Iran. Initially the organs were macroscopically examined for nymphal stage of L. serrata and then were digested by acid-pepsin method to further investigation. The prevalence rate was obtained 55.27%. The mesenteric lymph nodes (MLN) in 334 (45.13%), the lymph nodes (LN) in 102 (13.78%) and the livers in 8 (1.08%) goats out of 740 were infected with L. serrata nymphs. The results indicated a high rate of infection of LN in goats in this region of Iran.

Proteinase K-resistant material in ARR/VRQ sheep brain affected with classical scrapie is composed mainly of VRQ prion protein.

Classical scrapie is a prion disease in sheep and goats. In sheep, susceptibility to disease is genetically influenced by single amino acid substitutions. Genetic breeding programs aimed at enrichment of arginine-171 (171R) prion protein (PrP), the so-called ARR allele, in the sheep population have been demonstrated to be effective in reducing the occurrence of classical scrapie in the field. Understanding the molecular basis for this reduced prevalence would serve the assessment of ARR adaptation. The prion formation mechanism and conversion of PrP from the normal form (PrP(C)) to the scrapie-associated form (PrP(Sc)) could play a key role in this process. Therefore, we investigated whether the ARR allele substantially contributes to scrapie prion formation in naturally infected heterozygous 171Q/R animals. Two methods were applied to brain tissue of 171Q/R heterozygous sheep with natural scrapie to determine the relative amount of the 171R PrP fraction in PrP(res), the proteinase K-resistant PrP(Sc) core. An antibody test differentiating between 171Q and 171R PrP fragments showed that PrP(res) was mostly composed of the 171Q allelotype. Furthermore, using a novel tool for prion research, endoproteinase Lys-C-digested PrP(res) yielded substantial amounts of a nonglycosylated and a monoglycosylated PrP fragment comprising codons 114 to 188. Following two-dimensional gel electrophoresis, only marginal amounts (<9%) of 171R PrP(res) were detected. Enhanced 171R(res) proteolytic susceptibility could be excluded. Thus, these data support a nearly zero contribution of 171R PrP in PrP(res) of 171R/Q field scrapie-infected animals. This is suggestive of a poor adaptation of classical scrapie to this resistance allele under these natural conditions.

Illustration of effective parameters on growth of ZnO micro/nano rods, on the borosilicate glass via hydrothermal process.

Hexagonal micro/nanorods of ZnO were synthesized via mild hydrothermal growth method under different conditions. The growth of the rods was accomplished in two manners: firstly, on bare borosilicate glass, and secondly, on ZnO seed layer prepared by sol-gel spin coating process. All the obtained surfaces were studied by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The studies demonstrate that, although with the decrease of concentration of precursor solution on bare borosilicate glass, the diameter of the rods decreases, but the orientation will deteriorate and the density of the rods will decrease. On the other hand, hydrothermal growth on the seed layer causes the decrease in the diameter of the rods, while maintaining the orientation along the c-axis; therefore, the presence of seed layer plays an important role in decreasing the diameter of the rods; so that at a constant concentration, compared with the case without seed layer, the diameter of rods decreased 10 times from approximately 500 nm to approximately 50 nm.

Vesicle permeabilization by purified soluble oligomers of prion protein: a comparative study of the interaction of oligomers and monomers with lipid membranes.

The conversion of normal cellular prion protein (PrP) into its pathological isoform, scrapie PrP, may occur at the cell surface or, more probably, in late endosomes. The early events leading to the structural conversion of PrP appear to be related to the presence of more or less stable soluble oligomers, which might mediate neurotoxicity. In the current study, we investigate the interaction of alpha-rich PrP monomers and beta-rich size-exclusion-chromatography-purified PrP oligomers with lipid membranes. We compare their structural properties when associated with lipid bilayers and study their propensities to permeabilize the membrane at physiological pH. We also study the influence of the N-terminal flexible region (residues 24-103) by comparing full-length PrP(24-234) and N-terminally truncated PrP(104-234) oligomers. We showed that both 12-subunit oligomers cause an immediate and large increase in the permeability of the membrane, whereas equivalent amounts of monomeric forms cause no detectable leakage. Although the two monomeric PrP constructs undergo an alpha-to-beta conformational change when bound to the negatively charged membrane, only the full-length form of monomeric PrP has a weak fusogenic effect. Finally, the oligomers affect the integrity of the membrane differently from the monomers, independently of the presence of the N-terminal flexible domain. As for other forms of amyloidogenesis, a reasonable mechanism for the toxicity arising from PrP fibrillization must be associated with low-molecular-weight oligomeric intermediates, rather than with mature fibrils. Knowledge of the mechanism of action of these soluble oligomers would have a high impact on the development of novel therapeutic targets.

Prion protein oligomerization.

The PrP propensity to adopt different structures is tightly linked to transmissible spongiform encephalopathies (TSE) which include Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scjeinker (GSS) and Kuru syndrome. In most cases, TSE is associated with the accumulation in the brain of an abnormally folded protease-resistant protein, PrP Sc or PrPres, which is derived from a cellular host-encoded protease-sensitive conformer, designated PrP C. The prion propagation in the brain is postulated to occur via a conformational change of PrP C into the amyloidogenic form PrP Sc, characterized by a high beta sheet content. The characterization of PrP SC oligomers as well as their biological activity is currently an area of active research. Indeed, PrP Sc structural diversity was proposed several years ago as a hypothesis to explain the origin of "prion strain" diversity. As prion pathologies belong to protein miss-assembly diseases, investigation of PrP conformational dynamics and, more precisely, oligomerization pathways exploration will help to achieve a better understanding of the pathological events at the molecular level.

Are cattle, sheep, and goats endangered species?

For about 10 000 years, farmers have been managing cattle, sheep, and goats in a sustainable way, leading to animals that are well adapted to the local conditions. About 200 years ago, the situation started to change dramatically, with the rise of the concept of breed. All animals from the same breed began to be selected for the same phenotypic characteristics, and reproduction among breeds was seriously reduced. This corresponded to a strong fragmentation of the initial populations. A few decades ago, the selection pressures were increased again in order to further improve productivity, without enough emphasis on the preservation of the overall genetic diversity. The efficiency of modern selection methods successfully increased the production, but with a dramatic loss of genetic variability. Many industrial breeds now suffer from inbreeding, with effective population sizes falling below 50. With the development of these industrial breeds came economic pressure on farmers to abandon their traditional breeds, and many of these have recently become extinct as a result. This means that genetic resources in cattle, sheep, and goats are highly endangered, particularly in developed countries. It is therefore important to take measures that promote a sustainable management of these genetic resources; first, by in situ preservation of endangered breeds; second, by using selection programmes to restore the genetic diversity of industrial breeds; and finally, by protecting the wild relatives that might provide useful genetic resources.

Characterization of a complex formed between human plasminogen and recombinant sheep prion: pressure and thermal sensitivity of complex formation.

Scrapie is thought to be caused by one or more conformations of a proteinacious particle called a prion. The infectious form(s) is referred to as the scrapie form of the prion protein (PrPsc) whereas a benign form, the cellular conformer, is referred to as PrPC. The cellular conformation of the sheep prion protein formed a 1:1 complex with human plasminogen. The complex precipitated at 0 degrees C (Ksp = 17* 10(-12) M2). This precipitation reaction was sensitive to both temperature and pressure. When subjected to hydrostatic pressure the precipitate dissolved. At 25 degrees C the complex was soluble with a dissociation constant of about 10(-7) M as determined by isothermal titration calorimetry. Absorption, fluorescence and circular dichroism spectroscopy showed that neither protein, in the complex, underwent a detectable structural change so long as proteolytic inhibitors were present. In the absence of proteolytic inhibitors, plasminogen slowly cleaved the prion protein.

CD4+ T cell-mediated immunity against prion proteins.

The prion protein (PrP(C)) is essential for susceptibility to transmissible spongiform encephalopathies. A specific conformer of this protein (PrP(Sc)) is, according to the 'protein only' hypothesis, the principal or only component of the infectious agent, designated prion. Transmission of prions between species is often inefficient, resulting in low attack rates and/or prolonged incubation times and is ascribed to a 'species barrier' caused by differences in the amino acid sequence of PrP between recipient and donor. In this report, we demonstrate that these differences in amino acid sequence result in presentation of distinct peptides on major histocompatibility complex class II molecules. These peptides result in activation of specific CD4+ T cells which leads to the induction of an effective immune response against foreign PrP as demonstrated by antibody production. Therefore, CD4+ T cells represent a crucial component of the immune system to distinguish between foreign and self PrP.

Selective prion protein binding to synaptic components is modulated by oxidative and nitrosative changes induced by copper(II) and peroxynitrite in cholinergic synaptosomes, unveiling a role for calcineurin B and thioredoxin.

Choline acetyltransferase (ChAT) and choline transport are decreased after nitrosative stress. ChAT activity is altered in scrapie-infected neurons, where oxidative stress develops. Cellular prion protein (PrPc) may play a neuroprotective function in participating in the redox control of neuronal environment and regulation of copper metabolism, a role impaired when PrPc is transformed into PrPSc in prion pathologies. The complex cross-talk between PrPc and cholinergic neurons was analyzed in vitro using peroxynitrite and Cu2+ treatments on nerve endings isolated from Torpedo marmorata, a model of the motoneuron pre-synaptic element. Specific interactions between solubilized synaptic components and recombinant ovine prion protein (PrPrec) could be demonstrated by Biacore technology. Peroxynitrite abolished this interaction in a concentration-dependent way and induced significant alterations of neuronal targets. Interaction was restored by prior addition of peroxynitrite trapping agents. Cu2+ (in the form of CuSO4) treatment of synaptosomes triggered a milder oxidative effect leading to a bell-shaped increase of PrPrec binding to synaptosomal components, counteracted by the natural thiol agents, glutathione and thioredoxin. Copper(II)-induced modifications of thiols in several neuronal proteins. A positive correlation was observed between PrPrec binding and immunoreactive changes for calcineurin B and its partners, suggesting a synergy between calcineurin complex and PrP for copper regulation.

A new synthesis of 1-chloroalkynes.

4-Unsubstituted isoxazolinones derived from the corresponding beta-ketoesters can be chlorinated and converted into 1-chloroalkynes upon treatment with sodium nitrite and ferrous sulfate in aqueous acetic acid.