An improved PCR-based amplification of unknown homologous DNA sequences

The PCR primers used for cloning of evolutionary conserved genes or homologous DNA sequences are usually guessmer oligonucleotides. We introduce a simple way using Pfu polymerase to overcome possible PCR amplification failure because of 3'-end mismatches of guessed primers with the target DNA.     

Genotyping of Acanthamoeba isolates from clinical and environmental specimens in Iran

In this study, 15 Acanthamoeba isolates from AK patients and 10 environmental samples (water, soil and animal-origin samples) were classified at the genotype level based on the sequence analysis of the Diagnostic Fragment 3 (DF3) of Acanthamoeba small subunit rRNA gene. The obtained results revealed that most of these Acanthamoeba strains belonged to genotype T4 both in clinical and environmental samples. The presence T11 genotype in clinical samples was also revealed after the genotyping analysis and to our knowledge this is the first report of T11 genotype in Iran. Moreover, the isolation of T4 genotype from cow faeces in this study highlights a possible transmission of Acanthamoeba through animal faeces in Iran.Overall, the widespread distribution of pathogenic Acanthamoeba T4 across the environmental sources and the increasing number of contact lens wearers in Iran, demands more awareness within the public and health professionals as this pathogen is emerging as a risk for human health in Iran and worldwide.     

TSG-6 regulates bone remodeling through inhibition of osteoblastogenesis and osteoclast activation

TSG-6 is an inflammation-induced protein that is produced at pathological sites, including arthritic joints. In animal models of arthritis, TSG-6 protects against joint damage; this has been attributed to its inhibitory effects on neutrophil migration and plasmin activity. Here we investigated whether TSG-6 can directly influence bone erosion. Our data reveal that TSG-6 inhibits RANKL-induced osteoclast differentiation/activation from human and murine precursor cells, where elevated dentine erosion by osteoclasts derived from TSG-6(-/-) mice is consistent with the very severe arthritis seen in these animals. However, the long bones from unchallenged TSG-6(-/-) mice were found to have higher trabecular mass than controls, suggesting that in the absence of inflammation TSG-6 has a role in bone homeostasis; we have detected expression of the TSG-6 protein in the bone marrow of unchallenged wild type mice. Furthermore, we have observed that TSG-6 can inhibit bone morphogenetic protein-2 (BMP-2)-mediated osteoblast differentiation. Interaction analysis revealed that TSG-6 binds directly to RANKL and to BMP-2 (as well as other osteogenic BMPs but not BMP-3) via composite surfaces involving its Link and CUB modules. Consistent with this, the full-length protein is required for maximal inhibition of osteoblast differentiation and osteoclast activation, although the isolated Link module retains significant activity in the latter case. We hypothesize that TSG-6 has dual roles in bone remodeling; one protective, where it inhibits RANKL-induced bone erosion in inflammatory diseases such as arthritis, and the other homeostatic, where its interactions with BMP-2 and RANKL help to balance mineralization by osteoblasts and bone resorption by osteoclasts.     

Simultaneous Desorption and Desorption Kinetics of Phenanthrene,Anthracene, and Heavy Metals from Kaolinite with Different Organic Matter Content

Soils contaminated simultaneously with polycyclic aromatic hydrocarbons (PAHs) and heavy metals pose major threat to human health and environment by getting released from soil into water environment. The purpose of this study was to evaluate simultaneous desorption and desorption kinetics of PAHs (phenanthrene and anthracene) and heavy metals (lead, nickel, and zinc) from artificially contaminated kaolinite soils with different organic matter content. Batch desorption tests were conducted using single and combined enhancing agents containing Triton X-100 and Tween 80 as non-ionic surfactants, Ethylenediaminetetraacetic acid (EDTA) as a chelating agent, and citric acid as an organic acid. The solution with the highest removal efficiency was the combined solution of Triton X-100 (10% w/w) + EDTA (0.01 M). Removal levels around 92, 46, 92, 95, and 96% were obtained for phenanthrene, anthracene, lead, nickel, and zinc, respectively, by using this combination. Batch desorption kinetics experiments were performed using the mentioned combination. During the first 24 h, desorption kinetics were rapid, followed by a plateau until the end. The data obtained from desorption kinetics experiments were fitted with four kinetics models: pseudo-second-order equation, empirical power function, elovich, and parabolic diffusion. The correlation coefficient of the pseudo-second-order equation was higher than that of other functions. Moreover, batch experiments have showed inverse correlations between removal efficiency and organic matter content of soil.     

Mechanisms of oncostatin M-induced pulmonary inflammation and fibrosis

Oncostatin M (OSM), an IL-6 family cytokine, has been implicated in a number of biological processes including the induction of inflammation and the modulation of extracellular matrix. In this study, we demonstrate that OSM is up-regulated in the bronchoalveolar lavage fluid of patients with idiopathic pulmonary fibrosis and scleroderma, and investigate the pathological consequences of excess OSM in the lungs. Delivery of OSM to the lungs of mice results in a significant recruitment of inflammatory cells, as well as a dose-dependent increase in collagen deposition in the lungs, with pathological correlates to characteristic human interstitial lung disease. To better understand the relationship between OSM-induced inflammation and OSM-induced fibrosis, we used genetically modified mice and show that the fibrotic response is largely independent of B and T lymphocytes, eosinophils, and mast cells. We further explored the mechanisms of OSM-induced inflammation and fibrosis using both protein and genomic array approaches, generating a "fibrotic footprint" for OSM that shows modulation of various matrix metalloproteinases, extracellular matrix components, and cytokines previously implicated in fibrosis. In particular, although the IL-4/IL-13 and TGF-beta pathways have been shown to be important and intertwined of fibrosis, we show that OSM is capable of inducing lung fibrosis independently of these pathways. The demonstration that OSM is a potent mediator of lung inflammation and extracellular matrix accumulation, combined with the up-regulation observed in patients with pulmonary fibrosis, may provide a rationale for therapeutically targeting OSM in human disease.     

Hydrogen peroxide biosensor based on a myoglobin/hydrophilic room temperature ionic liquid film

The composite film based on Nafion and hydrophilic room temperature ionic liquid (RTIL) 1-butyl-3-methyl-imidazolium chloride ([bmim]Cl) was used as an immobilization matrix to entrap myoglobin (Mb). The study of ionic liquid (IL)-Mb interaction by ultraviolet-visible (UV-vis) spectroscopy showed that Mb retains its native conformation in the presence of IL. The immobilized Mb displayed a pair of well-defined cyclic voltammetric peaks with a formal potential (E_o^’) of −0.35 V in a 0.1 M phosphate buffer solution (PBS) of pH 7.0. The immobilized Mb exhibited excellent electrocatalytic response to the reduction of hydrogen peroxide, based on which a mediator-free amperometric biosensor for hydrogen peroxide was designed. The linear range for the determination of hydrogen peroxide was from 1.0 to 180 μM with a detection limit of 0.14 μM at a signal/noise ratio of 3. The apparent Michaelis constant () for the electrocatalytic reaction was 22.6 μM. The stability, repeatability, and selectivity of the sensor were evaluated. The proposed biosensor has a lower detection limit than many other IL-heme protein-based biosensors and is free from common interference in hydrogen peroxide biosensors.     

Synthesis of biologically stable gold nanoparticles using imidazolium-based amino acid ionic liquids

A novel double-step reduction procedure for the synthesis of gold nanoparticles (AuNPs) using amino acid ionic liquids has been employed. 1-Dodecyl-3-methyl imidazolium tryptophan ([C(12)mim]Trp) and 1-ethyl-3-methyl imidazolium tryptophan ([C(2)mim]Trp) were used for this synthesis. The synthesized AuNPs were characterized by UV-vis spectroscopy, transmission electron microscopy and dynamic light scattering. The behavior of these AuNPs were also probed in a biological media. It was proven that AuNPs synthesized at [C(12)mim]Trp have more stability than AuNPs synthesized at [C(2)mim]Trp due to the longer alkyl chain of the imidazolium moiety. The solubility test shows that the resultant AuNPs have a hydrophilic nature. Finally, it was seen that due to the presence of a biomolecule, namely Trp, in the structure of AuNPs protecting shell, higher stability and biocompatibility was achieved in the biological media.     

Enhanced Optical Transmission Through Metallic Holes Array: Role of TE Polarization in SPP Excitation

Optical transmission through double-layer metallic subwavelength holes array is studied under oblique incidence by split-field finite-difference time-domain method. Both TM and TE polarizations are investigated. It is proved that the transmission peaks can also be observed for TE polarization due to the excitation of surface plasmon polaritons (SPP) through diffraction orders. By changing the incident angle, these transmission peaks follow the SPP wavelength shift. The field profiles, even for the field components not present in the incident field, clearly show the SPP excitation. The mechanism of enhanced transmission will be fully discussed.     

Evidence for a KATP Channel in Rough Endoplasmic Reticulum (rerKATP Channel) of Rat Hepatocytes

We report in a previous study the presence of a large conductance K⁺ channel in the membrane of rough endoplasmic reticulum (RER) from rat hepatocytes incorporated into lipid bilayers. Channel activity in this case was found to decrease in presence of ATP 100 µM on the cytoplasmic side and was totally inhibited at ATP concentrations greater than 0.25 mM. Although such features would be compatible with the presence of a K_ATP channel in the RER, recent data obtained from a brain mitochondrial inner membrane preparation have provided evidence for a Maxi-K channel which could also be blocked by ATP within the mM concentration range. A series of channel incorporation experiments was thus undertaken to determine if the ATP-sensitive channel originally observed in the RER corresponds to K_ATP channel. Our results indicate that the gating and permeation properties of this channel are unaffected by the addition of 800 nM charybdotoxin and 1 µM iberiotoxin, but appeared sensitive to 10 mM TEA and 2.5 mM ATP. Furthermore, adding 100 µM glibenclamide at positive potentials and 400 µM tolbutamide at negative or positive voltages caused a strong inhibition of channel activity. Finally Western blot analyses provided evidence for Kir6.2, SUR1 and/or SUR2B, and SUR2A expression in our RER fractions. It was concluded on the basis of these observations that the channel previously characterized in RER membranes corresponds to K_ATP, suggesting that opening of this channel may enhance Ca²⁺ releases, alter the dynamics of the Ca²⁺ transient and prevent accumulation of Ca²⁺ in the ER during Ca²⁺ overload.     

Determination of the axial and circumferential mechanical properties of the skin tissue using experimental testing and constitutive modeling

The skin, being a multi-layered material, is responsible for protecting the human body from the mechanical, bacterial, and viral insults. The skin tissue may display different mechanical properties according to the anatomical locations of a body. However, these mechanical properties in different anatomical regions and at different loading directions (axial and circumferential) of the mice body to date have not been determined. In this study, the axial and circumferential loads were imposed on the mice skin samples. The elastic modulus and maximum stress of the skin tissues were measured before the failure occurred. The nonlinear mechanical behavior of the skin tissues was also computationally investigated through a suitable constitutive equation. Hyperelastic material model was calibrated using the experimental data. Regardless of the anatomic locations of the mice body, the results revealed significantly different mechanical properties in the axial and circumferential directions and, consequently, the mice skin tissue behaves like a pure anisotropic material. The highest elastic modulus was observed in the back skin under the circumferential direction (6.67 MPa), while the lowest one was seen in the abdomen skin under circumferential loading (0.80 MPa). The Ogden material model was narrowly captured the nonlinear mechanical response of the skin at different loading directions. The results help to understand the isotropic/anisotropic mechanical behavior of the skin tissue at different anatomical locations. They also have implications for a diversity of disciplines, i.e., dermatology, cosmetics industry, clinical decision making, and clinical intervention.