Conservative site-specific and single-copy transgenesis in human LINE-1 elements

Genome engineering of human cells plays an important role in biotechnology and molecular medicine. In particular, insertions of functional multi-transgene cassettes into suitable endogenous sequences will lead to novel applications. Although several tools have been exploited in this context, safety issues such as cytotoxicity, insertional mutagenesis and off-target cleavage together with limitations in cargo size/expression often compromise utility. Phage λ integrase (Int) is a transgenesis tool that mediates conservative site-specific integration of 48 kb DNA into a safe harbor site of the bacterial genome. Here, we show that an Int variant precisely recombines large episomes into a sequence, termed attH4X, found in 1000 human Long INterspersed Elements-1 (LINE-1). We demonstrate single-copy transgenesis through attH4X-targeting in various cell lines including hESCs, with the flexibility of selecting clones according to transgene performance and downstream applications. This is exemplified with pluripotency reporter cassettes and constitutively expressed payloads that remain functional in LINE1-targeted hESCs and differentiated progenies. Furthermore, LINE-1 targeting does not induce DNA damage-response or chromosomal aberrations, and neither global nor localized endogenous gene expression is substantially affected. Hence, this simple transgene addition tool should become particularly useful for applications that require engineering of the human genome with multi-transgenes.     

Studies on cytochromes of lichenized fungi under optimized culture conditions

Cytochromes are membrane-bound hemoproteins responsible for the generation of ATP via the electron transport system to fuel the metabolic processes of the organism for their growth. This study reports the properties of cytochromes present in the isolated lichenized fungi of the cultured lichen Usnea ghattensis under optimized conditions. The fungal partner of the cultured lichen Usnea ghattensis contains a, b and c types of cytochromes. The concentrations of a, b and c type cytochromes were found to be significantly high (0.0967, 0.0900, and 0.1030 mM/mg protein, respectively) in the isolated fungal symbiont of cultured lichen grown in malt-yeast extract medium supplemented with 0.01 mol/l sucrose and 0.01 mol/l polyethylglycol. The results suggest that supplementation of additional carbon sources may play a role in optimizing the growth via activating the cytochrome respiratory system in lichenized fungi.     

Antioxidant and antibacterial properties of some cultured lichens

The lichen species namely Usnea ghattensis, Heterodermia podocarpa, Arthothelium awasthii and Parmotrema tinctorum have been cultured in vitro and were screened for their antioxidant and antibacterial potential using different assay systems. The methanol extract of lichens showed antioxidant and antibacterial activities according to the order U. ghattensis>A. awasthii>H. podocarpa>P. tinctorum. The IC(50) values for the antioxidant activities of U. ghattensis and A. awasthii are less or equivalent to that of standard antioxidants. The methanolic extracts of the mycobiont and photobiont cultures of lichenU. ghattensis and A. awasthii were effective against Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis and Staphylococcus aureus. The minimum inhibitory concentration (MIC) of the extract was found between 5 and 10microg extract/ml. The results suggested that the extract of mycobiont and photobiont cultures of lichen U. ghattensis and A. awasthii could be of use as an easily accessible source of natural antioxidants and antibacterial properties for the possible food supplement or in the pharmaceutical industry.     

Antioxidant and antibacterial activities of lichen <Emphasis Type="Italic">Usnea ghattensis</Emphasis><Emphasis Type="Italic">in vitro</Emphasis>

Various solvent extracts of the lichen Usnea ghattensis showed good antioxidant activity. A methanol extract prevented lipid peroxidation by 87% followed by 65% in Trolox at 20 μg/ml. It also showed superoxide anion scavenging activity and free radical scavenging activity 56% and 73%, respectively. The known antioxidants butylated hydroxytoluene (BHT), butylated hydroxyanisol (BHA) and quercetin at similar concentrations showed superoxide anion scavenging activity of 68, 59 and 47% and free radical scavenging activity 83, 77 and 69%, respectively. In addition, these extracts were inhibitory against Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis and Staphylococcus aureus with MIC values of 5–10 μg/ml.Received after revisions 10 May 2005     

Substrate Based Ablation of Ventricular Tachycardia Through An Epicardial Approach

Ventricular tachycardia (VT) occurring late after myocardial infarction is often due to reentry circuit in the peri-infarct zone. The circuit is usually located in the sub-endocardium, though subepicardial substrates are known. Activation mapping during VT to identify target regions for ablation can be difficult if VT is non inducible or poorly tolerated. In the latter, a substrate based approach of mapping during sinus rhythm in conjunction with pace mapping helps to define the reentry circuit and select target sites for ablation in majority of patients with hemodynamically unstable VT. Percutaneous epicardial catheter ablation has been attempted as an approach where ablation by a conventional endocardial access has been unsuccessful. We report a case of post myocardial infarction scar VT which could be successfully ablated with a substrate based approach from the epicardial aspect.     

Effects of litter addition on ectomycorrhizal associates of a lodgepole pine (Pinus contorta) stand in Yellowstone National Park

Increasing soil nutrients through litter manipulation, pollution, or fertilization can adversely affect ectomycorrhizal (EM) communities by inhibiting fungal growth. In this study, we used molecular genetic methods to determine the effects of litter addition on the EM community of a Pinus contorta stand in Yellowstone National Park that regenerated after a stand-replacing fire. Two controls were used; in unmodified control plots nothing was added to the soil, and in perlite plots perlite, a chemically neutral substance, was added to maintain soil moisture and temperature at levels similar to those under litter. We found that (i) species richness did not change significantly following perlite addition (2.6 +/- 0.3 species/core in control plots, compared with 2.3 +/- 0.3 species/core in perlite plots) but decreased significantly (P < 0.05) following litter addition (1.8 +/- 0.3 species/core); (ii) EM infection was not affected by the addition of perlite but increased significantly (P < 0.001) in response to litter addition, and the increase occurred only in the upper soil layer, directly adjacent to the added litter; and (iii) Suillus granulatus, Wilcoxina mikolae, and agaricoid DD were the dominant organisms in controls, but the levels of W. mikolae and agaricoid DD decreased significantly in response to both perlite and litter addition. The relative levels of S. granulatus and a fourth fungus, Cortinariaceae species 2, increased significantly (P < 0.01 and P < 0.05, respectively) following litter addition. Thus, litter addition resulted in some negative effects that may be attributable to moisture-temperature relationships rather than to the increased nutrients associated with litter. Some species respond positively to litter addition, indicating that there are differences in their physiologies. Hence, changes in the EM community induced by litter accumulation also may affect ecosystem function.     

Experimental studies on the growth and usnic acid production in "lichen"Usnea ghattensis in vitro

The study was aimed to optimize the culture conditions for the production of usnic acid in the cultured cell aggregates composed of symbionts in lichen Usnea ghattensis in vitro. The cultured lichen tissue composed of symbionts appeared after about 2-3 weeks of inoculation in water-agar and malt-yeast extract (MYE) media and shown the production of usnic acid after 2-3 months of inoculation. However, the growth of symbionts was strongly affected by different culture conditions. The addition of excess carbon and nitrogen sources in the media has significantly enhanced the growth as well as usnic acid content. The cultured symbionts in MYE medium having 4% sucrose, 4% polyethyl glycol (PEG) gave 7.63 g dry biomass with 3.9 microg usnic acid/g dry biomass. In water-agar medium having 4% sucrose and 4% PEG gave 3.08 g dry biomass with 1.11 microg usnic acid/g dry biomass. The positive effects of medium on the growth of symbionts and the production of usnic acid are seemed to be due to nutritional factors.     

Effects of Litter Addition on Ectomycorrhizal Associates of a Lodgepole Pine (Pinus contorta) Stand in Yellowstone National Park

Increasing soil nutrients through litter manipulation, pollution, or fertilization can adversely affect ectomycorrhizal (EM) communities by inhibiting fungal growth. In this study, we used molecular genetic methods to determine the effects of litter addition on the EM community of a Pinus contorta stand in Yellowstone National Park that regenerated after a stand-replacing fire. Two controls were used; in unmodified control plots nothing was added to the soil, and in perlite plots perlite, a chemically neutral substance, was added to maintain soil moisture and temperature at levels similar to those under litter. We found that (i) species richness did not change significantly following perlite addition (2.6 ± 0.3 species/core in control plots, compared with 2.3 ± 0.3 species/core in perlite plots) but decreased significantly (P < 0.05) following litter addition (1.8 ± 0.3 species/core); (ii) EM infection was not affected by the addition of perlite but increased significantly (P < 0.001) in response to litter addition, and the increase occurred only in the upper soil layer, directly adjacent to the added litter; and (iii) Suillus granulatus, Wilcoxina mikolae, and agaricoid DD were the dominant organisms in controls, but the levels of W. mikolae and agaricoid DD decreased significantly in response to both perlite and litter addition. The relative levels of S. granulatus and a fourth fungus, Cortinariaceae species 2, increased significantly (P < 0.01 and P < 0.05, respectively) following litter addition. Thus, litter addition resulted in some negative effects that may be attributable to moisture-temperature relationships rather than to the increased nutrients associated with litter. Some species respond positively to litter addition, indicating that there are differences in their physiologies. Hence, changes in the EM community induced by litter accumulation also may affect ecosystem function.     

De novo design and synthesis of HIV-1 integrase inhibitors

Existing AIDS therapies are out of reach for most HIV-infected people in developing countries and, where available, they are limited by their toxicity and their cost. New anti-HIV agents are needed urgently to combat emerging viral resistance and reduce the side effects associated with currently available drugs. Toward this end, LeapFrog, a de novo drug design program was used to design novel, potent, and selective inhibitors of HIV-1 integrase. The designed compounds were synthesized and tested for in vitro inhibition of HIV-1 integrase. Out of the 25 compounds that were designed, and synthesized, four molecules (compounds 23, 26, 43, and 59) showed moderate to low inhibition of HIV-1 integrase for 3'-processing and 3'-strand transfer activities. Nonetheless, these compounds possess structural features not seen in known HIV-1 integrase inhibitors and thus can serve as excellent leads for further optimization of anti-HIV-1 integrase activity.