Silencing of transgene expression in mammalian cells by DNA methylation and histone modifications in gene therapy perspective

DNA methylation and histone modifications are vital in maintaining genomic stability and modulating cellular functions in mammalian cells. These two epigenetic modifications are the most common gene regulatory systems known to spatially control gene expression. Transgene silencing by these two mechanisms is a major challenge to achieving effective gene therapy for many genetic conditions. The implications of transgene silencing caused by epigenetic modifications have been extensively studied and reported in numerous gene delivery studies. This review highlights instances of transgene silencing by DNA methylation and histone modification with specific focus on the role of these two epigenetic effects on the repression of transgene expression in mammalian cells from integrative and non-integrative based gene delivery systems in the context of gene therapy. It also discusses the prospects of achieving an effective and sustained transgene expression for future gene therapy applications.     

Acute Toxicity and Gastroprotection Studies of a New Schiff Base Derived Copper (II) Complex against Ethanol-Induced Acute Gastric Lesions in Rats

Background

Copper is an essential element in various metabolisms. The investigation was carried out to evaluate acute gastroprotective effects of the Copper (II) complex against ethanol-induced superficial hemorrhagic mucosal lesions in rats.

Methodology/Principal Findings

Rats were divided into 7 groups. Groups 1 and 2 were orally administered with Tween 20 (10% v/v). Group 3 was orally administered with 20 mg/kg omeprazole (10% Tween 20). Groups 4–7 received 10, 20, 40, and 80 mg/kg of the complex (10% Tween 20), respectively. Tween 20 (10% v/v) was given orally to group 1 and absolute ethanol was given orally to groups 2–7, respectively. Rats were sacrificed after 1 h. Group 2 exhibited severe superficial hemorrhagic mucosal lesions. Gastric wall mucus was significantly preserved by the pre-treatment complex. The results showed a significant increase in glutathione (GSH), superoxide dismutase (SOD), nitric oxide (NO), and Prostaglandin E2 (PGE₂) activities and a decrease in malondialdehyde (MDA) level. Histology showed marked reduction of hemorrhagic mucosal lesions in groups 4–7. Immunohistochemical staining showed up-regulation of Hsp70 and down-regulation of Bax proteins. PAS staining of groups 4–7 showed intense stain uptake of gastric mucosa. The acute toxicity revealed the non-toxic nature of the compound.

Conclusions/Significance

The gastroprotective effect of the Copper (II) complex may possibly be due to preservation of gastric wall mucus; increase in PGE₂ synthesis; GSH, SOD, and NO up-regulation of Hsp70 protein; decrease in MDA level; and down-regulation of Bax protein.     

FRET-based detection of M1 muscarinic acetylcholine receptor activation by orthosteric and allosteric agonists

Background and Objective

Muscarinic acetylcholine receptors (mAChRs) are 7-transmembrane, G protein-coupled receptors that regulate a variety of physiological processes and represent potentially important targets for therapeutic intervention. mAChRs can be stimulated by full and partial orthosteric and allosteric agonists, however the relative abilities of such ligands to induce conformational changes in the receptor remain unclear. To gain further insight into the actions of mAChR agonists, we have developed a fluorescently tagged M₁ mAChR that reports ligand-induced conformational changes in real-time by changes in Förster resonance energy transfer (FRET).

Methods

Variants of CFP and YFP were inserted into the third intracellular loop and at the end of the C-terminus of the mouse M₁ mAChR, respectively. The optimized FRET receptor construct (M₁-cam5) was expressed stably in HEK293 cells.

Results

The variant CFP/YFP-receptor chimera expressed predominantly at the plasma membrane of HEK293 cells and displayed ligand-binding affinities comparable with those of the wild-type receptor. It also retained an ability to interact with Gα_q/11 proteins and to stimulate phosphoinositide turnover, ERK1/2 phosphorylation and undergo agonist-dependent internalization. Addition of the full agonist methacholine caused a reversible decrease in M₁ FRET (F_EYFP/F_ECFP) that was prevented by atropine pre-addition and showed concentration-dependent amplitude and kinetics. Partial orthosteric agonists, arecoline and pilocarpine, as well as allosteric agonists, AC-42 and 77-LH-28-1, also caused atropine-sensitive decreases in the FRET signal, which were smaller in amplitude and significantly slower in onset compared to those evoked by methacholine.

Conclusion

The M₁ FRET-based receptor chimera reports that allosteric and orthosteric agonists induce similar conformational changes in the third intracellular loop and/or C-terminus, and should prove to be a valuable molecular reagent for pharmacological and structural investigations of M₁ mAChR activation.     

Molecular characterisation of Vibrio cholerae O1 strains carrying an SXT/R391-like element from cholera outbreaks in Kenya: 1994-2007

Background  

Over the last decade, cholera outbreaks in parts of Kenya have become common. Although a number of recent studies describe the epidemiology of cholera in Kenya, there is pauCity of information concerning the diversity and occurrence of mobile genetic elements in Vibrio cholerae strains implicated in these outbreaks. A total of 65 Vibrio cholerae O1 El Tor serotype Inaba isolated between 1994 and 2007 from various outbreaks in Kenya were investigated for mobile genetic elements including integrons, transposons, the integrating conjugative elements (ICEs), conjugative plasmids and for their genotypic relatedness.     

Grey water characteristics and treatment options for rural areas in Jordan

Low water consumption in rural areas in Jordan had resulted in the production of concentrated grey water. Average COD, BOD and TSS values were 2568mg/l, 1056mg/l and 845mg/l, respectively. The average grey water generation was measured to be 14L/c.d. Three different treatment options were selected based on certain criterions, and discussed in this article. The examined treatment systems are septic tank followed by intermittent sand filter; septic tank followed by wetlands; and UASB-hybrid reactor. Advantages and disadvantages of each system are presented. It was concluded that UASB-hybrid reactor would be the most suitable treatment option in terms of compactness and simplicity in operation. The volume of UASB-hybrid reactor was calculated to be 0.268m(3) with a surface area of 0.138m(2) for each house having 10 inhabitants on average. Produced effluent is expected to meet Jordanian standards set for reclaimed water reuse in irrigating fruit trees.     

More topics from the tropics: additional thoughts to Mammides et al.

Most studies on tropical conservation questions are conducted by researchers of developed countries from the north. This geographic disconnection was recently criticised by Mammides et al. Here, we reflect on their findings and add further views from scientist’s and journal editor’s perspectives. We argue that journals are, a priori, most strongly interested in research questions and approaches that will likely increase their scientific impact and prestige. This is rarely compatible with publishing articles on questions with restricted global impact or based on single taxa. We question whether small changes in the editorial policy of international conservation journals will considerably improve the geographic diversity in key conservation publications. Rather, thematic scopes of the leading conservation journals should be modified, preferably in close collaboration with leading conservationists from the south. We are convinced that long-term investments in the tropics will create a stronger local scientific community, thus bolstering academic morale, and finally may lead to an increase in the submission and acceptance rate of articles written from scientists from these regions.     

Contrasting resistance and resilience to light variation of the coupled oxic and anoxic components of an experimental microbial ecosystem

Understanding how microbial communities of aquatic ecosystems respond to environmental change remains a critical challenge in microbial ecology. In this study, we used light‐dependent oxic–anoxic micro‐ecosystems to understand how the functioning and diversity of aerobic and anaerobic lake analog communities are affected by a pulse light deprivation. Continuous measurements of oxygen concentration were made and a time series of full‐length 16S rRNA sequencing was used to quantify changes in alpha‐ and beta diversity. In the upper oxic layer, oxygen concentration decreased significantly under light reduction, but showed resilience in daily mean, minimum, and maximum after light conditions were restored to control level. Only the amplitude of diurnal fluctuations in oxygen concentrations did not recover fully, and instead tended to remain lower in treated ecosystems. Alpha diversity of the upper oxic layer communities showed a delayed increase after light conditions were restored, and was not resilient in the longer term. In contrast, alpha diversity of the anoxic lower layer communities increased during the light reduction, but was resilient in the longer term. Community composition changed significantly during light reduction, and showed resilience in the oxic layer and lack of resilience in the anoxic layer. Alpha diversity and the amplitude of daily oxygen fluctuations within and among treatments were strongly correlated, suggesting that higher diversity could lead to less variable oxygen concentrations, or vice versa. Our experiment showed that light deprivation induces multifaceted responses of community function (oxygen respiration) and structure, hence focusing on a single stability component could potentially be misleading.     

Influence of Abscisic acid on K+ absorption by leaf discs ofSolanum tuberosum

It is shown that, contrary to what is generally found, treatment with abscisic acid (ABA) of potato leaf tissues resulted in an increase of K⁺ uptake. Comparison with other hormones was made: BAP induced an inhibition and GA₃ a stimulation of K⁺ uptake. The uptake was sensitive to several metabolic inhibitors, external pH and ATPase inhibitors while p-chloromercuribenzenesulfonic acid (PCMBS) had no effect. Uptake kinetics revealed the presence of both saturable and linear components which were both stimulated by ABA treatment. Our data are consistent with an effect of ABA on the active and passive components of K⁺ uptake. These results are discussed in relation to the action of ABA on foliar senescence and the action on ion partitioning in the whole plant.     

Partial purification and characterization of a folatebinding protein from human choroid plexus

A folate-binding protein (binder) from human choroid plexus was solubilized with Triton X-100 and partially purified in three steps: (1) affinity chromatography, (2) Sephadex G-200 column chromatography, and (3) polyacrylamide gel electrophoresis. When the partially purified binder was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the binding activity was located in the region of the gel with a molecular weight between 45,000 and 60,000. The specific activity of the binder after the three purification steps was 1.2 g folic acid/mg protein, a 316-fold purification. Binding activity of the partially purified binder decreased below pH 6.0 and above pH 8.0 was unaffected by treatment with ribonuclease or deoxyribonuclease, but was abolished with trypsin, chymotrypsin, or protease (Streptomyces griesus). The binding of folic acid to the human binder was inhibited by folate > H₄-folate > methyl-H₄-folate dihydrofolate pteroic acid methotrexate aminopterin.     

Colorimetric determination of free and total cholesterol by flow injection analysis with a fiber optic detector.

A flow injection method for the determination of total and free cholesterol is presented. Cholesterol esterase and cholesterol oxidase are immobilized on aminoalkyl glass beads. The beads are packed into a tubular glass reactor. The cholesterol esters traversing through the esterase reactor are cleaved to cholesterol and fatty acids. The oxidase reactor converts cholesterol to cholest-4-en-3-one and hydrogen peroxide is generated. The sample stream is merged with reagent streams consisting of a peroxidase solution and a solution of 2,2'-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) diammonium salt, and a hydrogen peroxide-dependent color reaction takes place in a short coiled reactor. The signal is monitored by means of fiber optic instrumentation. Cholesterol concentration can be related to the absorption of the oxidized dye form at a wavelength of 425 nm. The working range is 0.5-0.8 mmol l-1, and the sample throughputs are 60 and 30 h-1 for free and total cholesterol, respectively.