Common co-lipids, in synergy, impart high gene transfer properties to transfection-incompetent cationic lipids

Efficacious cationic transfection lipids usually need either DOPE or cholesterol as co-lipid to deliver DNA inside the cell cytoplasm in non-viral gene delivery. If both of these co-lipids fail in imparting gene transfer properties, the cationic lipids are usually considered to be transfection inefficient. Herein, using both the reporter gene assay in CHO, COS-1 and HepG2 cells and the whole cell histochemical X-gal staining assay in representative CHO cells, we demonstrate that common co-lipids DOPE, Cholesterol and DOPC, when act in synergy, are capable of imparting improved gene transfer properties to a novel series of cationic lipids (1-5). Contrastingly, lipids 1-5 became essentially transfection-incompetent when used in combination with each of the pure co-lipid components alone.     

RNAi-mediated silencing of the Bmi-1 gene causes growth inhibition and enhances doxorubicin-induced apoptosis in MCF-7 cells

The oncogene Bmi-1 is a member of the Polycomb group gene family. Its expression is found to be greatly increased in a number of malignant tumors including breast cancer. This could suggest Bmi-1 as a potent therapeutic target. In this study, RNAi was introduced to down-regulate the expression of Bmi-1 in a highly malignant breast adenocarcinoma cell line, MCF-7. A thorough study of the biological behavior and chemosensitivity changes of the MCF-7 cells was carried out in context to the therapeutic potential of Bmi-1. The results obtained indicated that siRNA targeting of Bmi-1 could lead to an efficient and specific inhibition of endogenous Bmi-1 activity. The mRNA and protein expression of Bmi-1 were determined by RT-PCR and Western blot, respectively. Furthermore, silencing of Bmi-1 resulted in a drastic inhibition of the growth of MCF-7 cells as well as G(1) /S phase transition. The number of target cells was found to increase in phase G (0) /G (1) and decrease in the S phase, but no increase in the basal level of apoptosis was noticed. On the other hand, a reduction in the expression of cyclin D1 and an increase in the expression of p21 were also noticed. Silencing of Bmi-1 made the MCF-7 cells more sensitive to the chemotherapeutic agent doxorubicin and induced a significantly higher percentage of apoptotic cells. Here, we report on a study regarding the RNAi-mediated silencing of the Bmi-1 gene in breast cancer.     

Activation of Alpha Chymotrypsin by Three Phase Partitioning Is Accompanied by Aggregation

Precipitation of alpha chymotrypsin in the simultaneous presence of ammonium sulphate and t-butanol (three phase partitioning) resulted in preparations which showed self aggregation of the enzyme molecules. Precipitation with increasing amounts of ammonium sulphate led to increasing size of aggregates. While light scattering estimated the hydrodynamic diameter of these aggregates in the range of 242–1124 nm; Nanoparticle tracking analysis (NTA) gave the value as 130–462 nm. Scanning electron microscopy and gel filtration on Sephadex G-200 showed extensive aggregation in these preparations. Transmission electron microscopy showed that the aggregates had irregular shapes. All the aggregates had about 3× higher catalytic activity than the native enzyme. These aggregates did not differ in λ_max of fluorescence emission which was around 340 nm. However, all the aggregates showed higher fluorescence emission intensity. Far-UV and near-UV circular dichroism also showed no significant structural changes as compared to the native molecule. Interestingly, HPLC gel filtration (on a hydroxylated silica column) gave 14 nm as the diameter for all preparations. Light scattering of preparations in the presence of 10% ethylene glycol also dissociated the aggregates to monomers of 14 nm. Both these results indicated that hydrophobic interactions were the driving force behind this aggregation. These results indicate: (1) Even without any major structural change, three phase partitioning led to protein molecules becoming highly prone to aggregation. (2) Different methods gave widely different estimates of sizes of aggregates. It was however possible to reconcile the data obtained with various approaches. (3) The nature of the gel filtration column is crucial and use of this technique for refolding and studying aggregation needs a rethink.     

Anatomical Variations in the Sinoatrial Nodal Artery: A Meta-Analysis and Clinical Considerations

ResultsSixty-six studies (n = 21455 hearts) were included in the meta-analysis. The SANa usually arose as a single vessel with a pooled prevalence of 95.5% (95%CI:93.6–96.9). Duplication and triplication of the artery were also observed with pooled prevalence of 4.3% (95%CI:2.8–6.0) and 0.3% (95%CI:0–0.7), respectively. The most common origin of the SANa was from the right coronary artery (RCA), found in 68.0% (95%CI:55.6–68.9) of cases, followed by origin from the left circumflex artery, and origin from the left coronary artery with pooled prevalence of 22.1% (95%CI:15.0–26.2) and 2.7 (95%CI:0.7–5.2), respectively. A retrocaval course of the SANa was the most common course of the artery with a pooled prevalence of 47.1% (95%CI:36.0–55.5). The pooled prevalence of an S-shaped SANa was 7.6% (95%CI:2.9–14.1).ConclusionsThe SANa is most commonly reported as a single vessel, originating from the RCA, and taking a retrocaval course to reach the SAN. Knowledge of high risk anatomical variants of the SANa, such as an S-shaped artery, must be taken into account by surgeons to prevent iatrogenic injuries. Specifically, interventional or cardiosurgical procedures, such as the Cox maze procedure for atrial fibrillation, open heart surgeries through the right atrium or intraoperative cross-clamping or dissection procedures during mitral valve surgery using the septal approach can all potentiate the risk for injury in the setting of high-risk morphological variants of the SANa.     

Silencing of Bmi-1 gene by RNA interference enhances sensitivity to doxorubicin in breast cancer cells

The oncogene Bmi-1 is highly up-regulated in breast carcinoma and is found to be efficient in preventing apoptosis of the cancer cells. Doxorubicin is an important chemotherapeutic agent against breast carcinoma. However, the effective therapeutic response to doxorubicin is often associated with severe toxicity. The present study is targetted at developing a strategy to increase doxorubicin sensitivity to lower doses without compromising its efficacy. A stable cell line with a persistent silencing of Bmi-1 was established. MTT assay was performed to evaluate 50% inhibitory concentration (IC50) values of doxorubicin. Apoptosis was detected by FCM and the expression of related genes [phosphor-Akt (pAkt), totle-Akt (tAkt), Bcl-2 and Bax] was studied by Western blot. In vivo, the sensitivity of the tumor tissues against doxorubicin was evaluated by transplanted MCF-7 nude mice model and the apoptosis of tissue cells was detected by TUNEL assay. The expression of pAkt and Bcl-2 was down-regulated, whereas Bax was up-regulated in Bmi-1 silencing cells. The results obtained indicated that silencing of Bmi-1 can render MCF-7 cells more sensitive to doxorubicin which induced a significantly higher percentage of apoptosis cells in vitro and in vivo. All together these results clearly demonstrate that Bmi-1 siliencing combined treatment of doxorubicin might be a new strategy for biological treatment on breast cancer.     

The Prevalence of Anatomical Variations of the Median Nerve in the Carpal Tunnel: A Systematic Review and Meta-Analysis

ResultsThirty-one studies (n = 3918 hands) were included in the meta-analysis. The pooled prevalence rates of the extraligamentous, subligamentous, and transligamentous courses were 75.2% (95%CI:55.4%-84.7%), 13.5% (95%CI:3.6%-25.7%), and 11.3% (95%CI:2.4%-23.0%), respectively. The prevalence of Lanz group 2, 3, and 4 were 4.6% (95%CI:1.6%-9.1%), 2.6% (95%CI:0.1%-2.8%), and 2.3% (95%CI:0.3%-5.6%), respectively. Ulnar side of branching of the TMB was found in 2.1% (95%CI:0.9%-3.6%) of hands. The prevalence of hypertrophic thenar muscles over the transverse carpal ligament was 18.2% (95%CI:6.8%-33.0%). A transligamentous course of the TMB was more commonly found in hands with hypertrophic thenar muscles (23.4%, 95%CI:5.0%-43.4%) compared to those without hypertrophic musculature (1.7%, 95%CI:0%-100%). In four studies (n = 423 hands), identical bilateral course of the TMB was found in 72.3% (95%CI:58.4%-84.4%) of patients.ConclusionsAnatomical variations in the course of the TMB and the MN in the carpal tunnel are common in the population. Thus, we recommend an ulnar side approach to carpal tunnel release, with a careful layer by layer dissection, to avoid iatrogenic damage to the TMB.     

Neuroprotective Role of Quercetin on Rotenone-Induced Toxicity in SH-SY5Y Cell Line Through Modulation of Apoptotic and Autophagic Pathways

The detrimental impact on the food chain due to the overuse of rotenone is partly responsible for alpha-synuclein (α-syn) mediated neurotoxicity. It is hypothesized that rotenone overdose leads to cytosolic proteopathy resulting in modulation of apoptosis and autophagic pathways. The aim of our study is to explore the neuroprotective role of quercetin, a beneficial polyphenol against rotenone-induced neurotoxicity in dopaminergic human SH-SY5Y cell lines. In our study we demonstrated the correlation of rotenone-induced neurotoxicity through elevation of intracellular reactive oxygen species (ROS) and imbalance in the mitochondrial membrane potential (MMP). Moreover, the morphological distortion of cell, condensation of nuclei, externalization of the inner phosphatidylserine, cleavage of caspase 3, and Poly ADP Ribose Polymerase (PARP) confirmed apoptosis. However, all these lethal effects were ameliorated by treatment of quercetin to the cells. On the other hand rotenone has a strong effect on autophagy which is a regulated degrading and recycling cellular process to remove dysfunctional proteins. Indeed, rotenone-mediated autophagy resulted in the enhancement of autophagosome-bound microtubule-associated protein light chain-3 (LC3-II) expression. Furthermore, excess accumulation of acidic vesicles was detected in presence of rotenone. Lysosome associated membrane protein (LAMP-2A) is yet another crucial protein that recruits overexpressed or misfolded proteins into the lumen of lysosome to trigger autophagy. In all cases the impact of rotenone on the cells acquired significant protection through quercetin treatment. In the present work we therefore opine the prospects of quercetin as a therapeutic candidate against neurotoxicity.

     

Gene transfer efficacies of novel cationic amphiphiles with alanine, beta-alanine, and serine headgroups: a structure-activity investigation

Herein, we report on the relative in vitro efficacies of nine novel non-glycerol based cationic amphiphiles with increasing hydrophobic tails and the amino acids serine, alanine and beta-alanine as the headgroup functionalities (lipids 1-9, Scheme 1) in transfecting multiple cultured cells including CHO, COS-1, MCF-7, and HepG2. The gene transfer efficiencies of lipids 1-9 were evaluated using the reporter gene assays in all the four cell lines and the whole cell histochemical X-gal staining assays in representative CHO cells. In CHO, HepG2, and MCF-7 cells, cationic lipids with alanine (4-6) and beta-alanine (7-9) headgroups were found to be remarkably more transfection efficient than their serine headgroup counterparts (1-3). Most notably, in CHO, HepG2, and MCF-7 cells, in combination with cholesterol as auxiliary lipid, the transfection efficiencies of the cationic lipids with alanine and beta-alanine headgroups and myristyl and palmityl tails (lipids 4, 5, 7 and 8) were significantly higher (2-3-fold) than that of LipofectAmine-2000, a widely used commercially available liposomal tranfection vectors. Surprisingly, in COS-1 cells, although cationic lipids with beta-alanine headgroups (7-9) were strikingly transfection efficient (3-4-fold more efficacious than LipofectAmine-2000), the gene transfer properties of both their structural isomers (4-6) and their serine headgroup counterparts (1-3) were adversely affected. In summary, the present structure-activity investigation demonstrate that high gene delivery efficacies of cationic amphiphiles containing alanine or beta-alanine headgroups can get seriously compromised by substituting the alanine or beta-alanine with serine presumably due to the enhanced sensitivity of DNA associated with such serine-head-containing cationic lipids.     

Comparative genomics analysis of statistically significant genomic islands of Helicobacter pylori strains for better understanding the disease prognosis

Bacterial virulence factors are often located in their genomic islands (GIs). Helicobacter pylori, a highly diverse organism is reported to be associated with several gastrointestinal diseases like, gastritis, gastric cancer (GC), peptic ulcer, duodenal ulcer (DU) etc. A novel similarity score (Sm)-based comparative analysis with GIs of 50 H. pylori strains revealed clear idea of the various factors which promote disease progression. Two putative pathogenic GIs in some of the H. pylori strains were identified. One GI, having a putative labile enterotoxin and other dynamin-like proteins (DLPs), is predicted to increase the release of toxin by membrane vesicular formation. Another island contains a virulence-associated protein D (vapD) which is a component of a type-II toxin–antitoxin system (TAs), leads to enhance the severity of the H. pylori infection. Besides the well-known virulence factors like Cytotoxin-associated gene A (CagA) and vacA, several GIs have been identified which showed to have direct or indirect impact on H. pylori clinical outcomes. One such GI, containing lipopolysaccharide (LPS) biosynthesis genes was revealed to be directly connected with disease development by inhibiting the immune response. Another collagenase-containing GI worsens ulcers by slowing down the healing process. GI consisted of fliD operon was found to be connected to flagellar assembly and biofilm production. By residing in biofilms, bacteria can avoid antibiotic therapy, resulting in chronic infection. Along with well-studied CagA and vacuolating toxin A (vacA) virulent genes, it is equally important to study these identified virulence factors for better understanding H. pylori-induced disease prognosis.