Enhanced Glucose Uptake in Phenylbutyric Acid-Treated 3T3-L1 Adipocytes

Diabetes Mellitus is a chronic metabolic disease marked by altered glucose homeostasis and insulin resistance. The phosphatase PTEN antagonizes the insulin-induced-PI3K-driven cascade that normally leads to GLUT4 membrane translocation. This study investigates the effect of Phenylbutyric Acid (PBA), a chemical chaperone and a potential mediator of PTEN activity, on glucose uptake in differentiated 3T3-L1 adipocytes. Adipocyte differentiation status was quantified by Oil Red O staining and the expression of AP2. Baseline and insulin-induced adipocyte glucose uptake were assayed with and without PBA treatment. Expression of GLUT1, GLUT4, PIP3, pAkt, pPTEN, and PARK-7 was examined by western blot. Plasma membrane expression of GLUT4 was determined using immunofluorescence. Leptin and adiponectin secretion was measure by enzyme-linked immunosorbent assay. PBA treatment, alone or with insulin induction, significantly increased glucose uptake in 3T3-L1 adipocytes. PBA significantly increased GLUT1 but not GLUT4 total protein expression. However, a significant increase in membrane GLUT4 protein translocation was observed. The expression of PIP3 and pAkt increased indicating enhanced PI3k pathway activity. There was a significant decrease in PTEN activity as evident by a rise in the phosphorylated form of this protein. PARK7 protein expression increased with PBA. Treating differentiated adipocytes with PBA did not alter their differentiation status, but decreased the leptin to adiponectin ratio. Conclusion: this study showed that PBA enhances adipocyte glucose uptake potentially through its effect on glucose transporter expression and/or trafficking via the PI3K signaling pathway; suggesting PBA as a possible candidate for the ancillary management of diabetes.     

High doses of sodium tungstate can promote mitochondrial dysfunction and oxidative stress in isolated mitochondria

Tungstate (W) is recognized as an agent of environmental pollution and a substitute to depleted uranium. According to some preliminary studies, tungstate toxicity is related to the formation of reactive oxygen species (ROS) under abnormal pathological conditions. The kidneys and liver are the main tungstate accumulation sites and important targets of tungstate toxicity. Since the mitochondrion is the main ROS production site, we evaluated the mechanistic toxicity of tungstate in isolated mitochondria for the first time, following a two‐step ultracentrifugation method. Our findings demonstrated that tungstate‐induced mitochondrial dysfunction is related to the increased formation of ROS, lipid peroxidation, and potential membrane collapse, correlated with the amelioration of adenosine triphosphate and glutathione contents. The present study indicated that mitochondrial dysfunction was associated with disruptive effects on the mitochondrial respiratory chain and opening of mitochondrial permeability transition (MPT) pores, which is correlated with cytochrome c release. Our findings suggest that high concentrations of tungstate (2 mM)‐favored MPT pore opening in the inner membranes of liver and kidney mitochondria of rats. Besides, the results indicated higher tungstate susceptibility in the kidneys, compared with the liver.     

Spectroscopic,biological, and molecular modeling studies on the interactions of [Fe(III)-meloxicam] with G-quadruplex DNA and investigation of its release from bovine serum albumin (BSA) nanoparticles

The guanine-rich sequence, specifically in DNA, telomeric DNA, is a potential target of anticancer drugs. In this work, a mononuclear Fe(III) complex containing two meloxicam ligands was synthesized as a G-quadruplex stabilizer. The interaction between the Fe(III) complex and G-quadruplex with sequence of 5′-G₃(T₂AG₃)₃-3′ (HTG21) was investigated using spectroscopic methods, molecular modeling, and polymerase chain reaction (PCR) assays. The spectroscopic methods of UV–vis, fluorescence, and circular dichroism showed that the metal complex can effectively induce and stabilize G-quadruplex structure in the G-rich 21-mer sequence. Also, the binding constant between the Fe(III) complex and G-quadruplex was measured by these methods and it was found to be 4.53(±0.30)?×?10⁵ M^?1). The PCR stop assay indicated that the Fe(III) complex inhibits DNA amplification. The cell viability assay showed that the complex has significant antitumor activities against Hela cells. According to the UV–vis results, the interaction of the Fe(III) complex with duplex DNA is an order of magnitude lower than G-quadruplex. Furthermore, the release of the complex incorporated in bovine serum albumin nanoparticles was also investigated in physiological conditions. The release of the complex followed a bi-phasic release pattern with high and low releasing rates at the first and second phases, respectively. Also, in order to obtain the binding mode of the Fe(III) complex with G-quadruplex, molecular modeling was performed. The molecular docking results showed that the Fe(III) complex was docked to the end-stacked of the G-quadruplex with a π–π interaction, created between the meloxicam ligand and the guanine bases of the G-quadruplex.     

Dosimetric characteristics of the INTRABEAM ® system with spherical applicators in the presence of air gaps and tissue heterogeneities

The main aim of this study was to investigate the dosimetric characteristics of the INTRABEAM ® system in the presence of air gaps between the surface of applicators (APs) and tumor bed. Additionally, the effect of tissue heterogeneities was another focus. Investigating the dosimetric characteristics of the INTRABEAM® system is essential to deliver the required dose to the tumor bed correctly and reduce the delivered dose to the ribs and lung. Choosing the correct AP size and fitting it to the lumpectomy cavity is essential to remove the effect of air gaps and avoid inaccurate dose delivery. Consequently, the Geant4 toolkit was used to simulate the INTRABEAM ® system with spherical APs of various sizes. The wall effect of the ion chamber (IC) PTW 34013 used in the present study was checked. The simulations were validated in comparison with measurements, and then used to calculate any inaccuracies in dose delivery in the presence of 4- and 10-mm air gaps between the surface of the APs and the tumor bed. Also, the doses received due to tissue heterogeneities were characterized. It turned out that measurements and simulations were approximately in agreement (± 2%) for all sizes of APs. The perturbation factor introduced by the IC due to differences in graphite-coated polyethylene and air as compared to the phantom material was approximately equal to one for all AP. The greatest relative dose delivery difference was observed for an AP with a diameter of 1.5 cm, i.e., 44% and 70% in the presence of 4- and 10-mm air gaps, respectively. In contrast, the lowest relative dose delivery difference was observed for an AP with a diameter of 5 cm, i.e., 24% and 42% in the presence of 4- and 10-mm air gaps, respectively. Increasing APs size showed a decrease in relative dose delivery difference due to the presence of air gaps. In addition, the undesired dose received by the ribs turned out to be higher when a treatment site closer to the ribs was assumed. The undesired dose received by the ribs increased as the AP size increased. The lung dose turned out to be decreased due to the shielding effect of the ribs, small lung density, and long separation distance from the AP surface.     

Comparison of dosimetric characteristics of physical wedge and enhanced dynamic wedge in inhomogeneous medium using Monte Carlo simulations

BackgroundWidely used physical wedges in clinical radiotherapy lead to beam intensity attenuation as well as the beam hardening effect, which must be considered. Dynamic wedges devised to overcome the physical wedges (PWs) problems result in dosimetry complications due to jaw movement while the beam is on. This study was aimed to investigate the usability of physical wedge data instead of enhanced dynamic wedge due to the enhanced dynamic wedge (EDW) dosimetry measurement hardships of Varian 2100CD in inhomogeneous phantom by Monte Carlo code as a reliable method in radiation dosimetry.Materials and methodsA PW and EDW-equipped-linac head was simulated using BEAMnrc code. DOSXYZnrc was used for three-dimensional dosimetry calculation in the CIRS phantom.ResultsBased on the isodose curves, EDW generated a less scattered as well as lower penumbra width compared to the PW. The depth dose variations of PWs and EDWs were more in soft tissue than the lung tissue. Beam profiles of PW and EDW indicated good coincidence in all points, except for the heel area.ConclusionResults demonstrated that it is possible to apply PW data instead of EDW due to the dosimetry and commissioning hardships caused by EDW in inhomogeneous media.     

Recombinant Production and Intein-Mediated Purification of an Antimicrobial Peptide,BR2

Nowadays, targeted therapy of cancer is under intensive focus of many investigations due to severe side effects imposed by various cancer chemotherapeutics. BR2 is a modified antimicrobial cell penetrating peptide with confirmed capability of delivering various cargos specifically to cancerous cells. However, because of its small size, its recombinant production by conventional methods is difficult, and its chemical synthesis imposes high cost. Hence, the aim of the present study was to evaluate if recombinant production and intein-mediated purification of this peptide is possible and finally evaluate its safety as a suitable targeted drug delivery vector on cancer and normal cell lines. In this regard, the coding sequence of BR2 was cloned in pTBX1 to be expressed in-frame with GyrA intein and then subjected to inducible protein expression using IPTG. Afterwards, the expressed protein was transferred to chitin-loaded columns and the peptide was purified according to manufacrure’s instruction. SDS–PAGE and Western blot analysis confirmed the expression of BR2-GyrA fusion protein by showing a band of approximately 31 kDa. Moreover, SDS–PAGE of the purified peptide showed a band of approximately 3 kDa, confirming the successful purification of BR2. Finally, in order to evaluate the safety of the produced peptide, its effects was evaluated on MCF-7 and HEK-293 cell lines by MTT assay, and compared to the effects of chemically synthesized BR2. Statistical analysis of the MTT assay results showed that the recombinantly produced peptide had no significant toxic effects on MCF-7 and HEK 293 cells, comparing to negative control, and this was similar to the effects of the synthetic BR2. Hence, the recombinant BR2 can be used for production of novel vehicles for targeted delivery of cytotoxic cargos in the future.     

Excessive R-loops trigger an inflammatory cascade leading to increased HSPC production

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