Dual-ligand modification of PEGylated liposomes shows better cell selectivity and efficient gene delivery

The objective of this study was to develop an efficient dual-ligand based PEGylated liposomal delivery system that had target specificity as well as properties that would enhance cellular uptake. PEGylated liposomes (PEG-LP) were prepared by the lipid film hydration method by adding distearoyl phosphoethanolamine-polyethylene-glycol-2000 conjugate (DSPE-PEG2000) to a lipid mixture. The cyclic RGD (Arg-Gly-Asp) peptide, a specific ligand with affinity for Integrin α_vβ₃ was coupled to the distal end of the PEG on the PEG-LP (RGD-PEG-LP). Stearylated octaarginine (STR-R8) was incorporated on the surface of the RGD-PEG-LP as dual-ligand (R8/RGD-PEG-LP) that functions as a cell penetrating peptide (CPP). RGD-PEG-LP and R8/RGD-PEG-LP were preferentially taken up by caveolae-mediated and clathrin-mediated endocytosis pathways, respectively. Compared to PEG-LP, R8/RGD-PEG-LP showed an enhanced cellular uptake as well as a higher transfection efficiency in Integrin α_vβ₃ expressing cells. However, the amount of cellular uptake or gene expression by the single ligand versions was negligible, even in Integrin α_vβ₃ expressing cells. No remarkable difference in cellular uptake or gene expression was observed for cells in which the expression of targeted receptors was absent. It can be concluded that dual-ligand modified PEG-LP possesses a strong capability for the efficient internalization of PEG-LP and consequently would be an effective tool for the targeted delivery of macromolecules or chemotherapeutics through accelerated cellular uptake.     

Critical role for Gab2 in transformation by BCR/ABL

The BCR/ABL oncogene causes chronic myelogenous leukemia (CML) in humans and a CML-like disease, as well as lymphoid leukemia, in mice. p210 BCR/ABL is an activated tyrosine kinase that phosphorylates itself and several cellular signaling proteins. The autophosphorylation site tyrosine 177 binds the adaptor Grb2 and helps determine the lineage and severity of BCR/ABL disease: Tyr177 mutation (BCR/ABL-Y177F) dramatically impairs myeloid leukemogenesis, while diminishing lymphoid leukemogenesis. The critical signal(s) from Tyr177 has remained unclear. We report that Tyr177 recruits the scaffolding adaptor Gab2 via a Grb2/Gab2 complex. Compared to BCR/ABL-expressing Ba/F3 cells, BCR/ABL-Y177F cells exhibit markedly reduced Gab2 tyrosine phosphorylation and association of phosphatidylinositol-3 kinase (PI3K) and Shp2 with Gab2 and BCR/ABL, and decreased PI3K/Akt and Ras/Erk activation, cell proliferation, and spontaneous migration. Remarkably, bone marrow myeloid progenitors from Gab2 (-/-) mice are resistant to transformation by BCR/ABL, whereas lymphoid transformation is diminished as a consequence of markedly increased apoptosis. BCR/ABL-evoked PI3K/Akt and Ras/Erk activation also are impaired in Gab2 (-/-) primary myeloid and lymphoid cells. Our results identify Gab2 and its associated proteins as key determinants of the lineage and severity of BCR/ABL transformation.     

Association of CYP3A4, CYP3A5 polymorphisms with lung cancer risk in Bangladeshi population

The rate of direct smoking, second hand smoking, and smokeless tobacco users as well as the amount of environmental pollutant like polycyclic aromatic hydrocarons is increasing in Bangladesh. Therefore, the prevalence of lung cancer is increasing day by day. To the best of our knowledge, no pharmacogentic study of CYP3A4, CYP3A5 genes has been reported on Bangladeshi population relating those with lung cancer. The present study was conducted to determine the association of CYP3A4, CYP3A5 gene polymorphisms and tobacco smoking in the development of lung cancer in Bangladeshi population. A case–control study was carried out on 106 lung cancer patients and 116 controls to investigate three allelic variants—CYP3A4*1B, CYP3A5*3, and CYP3A5*6 using Polymerase Chain Reaction Restriction Fragment Length Polymorphism. Risk of lung cancer was estimated as odds ratio (OR) and 95 % confidence interval (CI) using unconditional logistic regression models. The variant allele frequencies for CYP3A4*1B (*1A/*1B?+?*1B/*1B) were 2.83 % and 0.86 % and that of CYP3A5*3 (*1A/*3?+?*3/*3) were 88.68 % and 85.34 % in cases and controls, respectively. Individual carrying at least one variant allele of CYP3A4*1B (CYP3A4*1A/1B?+?*1B/1B) has a 3.35 times more risk (OR?=?3.35, 95 % Cl?=?0.34-32.71, p?=?0.271) for developing lung cancer whereas individual carrying at least one variant allele of CYP3A5 (CYP3A5*1A/3?+?*3/3) has a 1.26 times more risk (OR?=?1.35, 95 % Cl?=?0.61–2.97) and both are statistically non-significant (p?>?0.05). CYP3A5*6 was absent in the study population. No association of lung cancer with the mentioned polymorphisms was found both in heavy and light smokers. In the cases of all three major types of lung cancer—squamous cell carcinoma, adenocarcinoma, and small cell carcinoma—significantly strong relationships (p???0.05) have been found. To confirm the association of lung cancer with the mentioned polymorphisms, large number volunteers (patients and controls) will be required.     

Frequency of epidermal growth factor receptor mutations in Bangladeshi patients with adenocarcinoma of the lung

Background

Worldwide studies on lung adenocarcinoma have demonstrated a genetic divergence of the epidermal growth factor receptor (EGFR) pathway according to ethnicity, such as higher frequency of activated EGFR mutations among East Asian patients. However, such information is still lacking in some developing countries.

Methods

We investigated the frequency of EGFR mutations among Bangladeshi patients with adenocarcinoma of the lung. Fine-needle aspiration tissue samples were collected from 61 Bangladeshi patients. Polymerase chain reaction–single-strand conformation polymorphism was performed on extracted DNA for mutational analysis of EGFR exons 19 and 21.

Results

EGFR mutations were found in 14 of 61 (23.0 %) Bangladeshi patients. There was no significant difference in EGFR mutation rate with regard to patient’s age, sex, smoking history, clinical stage of lung cancer, subtypes of adenocarcinoma, and tumor differentiation.

Conclusion

The present study revealed that the EGFR mutation rate in Bangladeshi patients with adenocarcinoma of the lung was higher than in African–American, Arabian, and white Caucasian patients, and was lower than in East Asia.     

Cytotoxicity of the rhizome of medicinal plants

Objective

To investigate the cytotoxicity of the crude ethanol extract of the rhizome of Zingiber zerumbet (Z. zerumbet) (L) Smith. and Curcuma zedoaria (C. zedoaria) Rosc. against Artemia salina Leach.

Methods

Fresh rhizomes of Z. zerumbet (L) Smith. and C. zedoaria Rosc. were extracted separately in cold with ethanol (2.5 L) and after concentration a brownish syrupy suspension of ethanol extracts of Z. zerumbet (L) Smith. and C. zedoaria Rosc. was obtained. The cytotoxic effect of the crude ethanol extracts of both plants was determined by brine shrimp lethality bioassay.

Results

Crude ethanol extracts of the rhizome of Z. zerumbet (L) Smith. showed the highest cytotoxicity (LC₅₀ was 1.24 µg/mL) against brine shrimp nauplii as compared with C. zedoaria Rosc. (LC₅₀ was 33.593 µg/mL) after 24 h of exposure.

Conclusions

It can be concluded that the rhizome of Z. zerumbet (L) Smith. and C. zedoaria Rosc. can be used as a source of cytotoxic agent.     

Determination of abdominal fat thickness using dual electrode separation in the focused impedance method (FIM)

Subcutaneous fat layer thickness in the abdomen is a risk indicator of several diseases and disorders like diabetes and heart problems and could be used as a measure of fitness. Skinfold measurement using mechanical calipers is simple but prone to error. Ultrasound scanning techniques are yet to be established as accurate methods for this purpose. magnetic resonance imaging (MRI) and computed tomography (CT) scans can provide the answer but are expensive and not available widely. Some initiatives were made earlier to use electrical impedance to this end, but had inadequacies. In the first part of this paper, a 4-electrode focused impedance method (FIM) with different electrode separations has been studied for its possible use in the determination of abdominal fat thickness in a localized region. For this, a saline phantom was designed to provide different electrode separations and different layers of resistive materials adjacent to the electrodes. The background saline simulated the internal organs having low impedance while the resistive layers simulated the subcutaneous fat. The plot of the measured impedance with electrode separation had different 'slopes' for different thicknesses of resistive layers, which offered a method to obtain an unknown thickness of subcutaneous fat layer. In the second part, measurements were performed on seven human subjects using two electrode separations. Fat layer thickness was measured using mechanical calipers. A plot of the above 'slope' against fat thickness could be fitted using a straight line with an R(2) of 0.93. Then this could be used as a calibration curve for the determination of unknown fat thickness. Further work using more accurate CT and MRI measurements would give a better calibration curve for practical use of this non-invasive and low-cost technique in abdominal fat thickness measurement.