Efficient deformation algorithm for plasmid DNA simulations

Background

Plasmid DNA molecules are closed circular molecules that are widely used in life sciences, particularly in gene therapy research. Monte Carlo methods have been used for several years to simulate the conformational behavior of DNA molecules. In each iteration these simulation methods randomly generate a new trial conformation, which is either accepted or rejected according to a criterion based on energy calculations and stochastic rules. These simulation trials are generated using a method based on crankshaft motion that, apart from some slight improvements, has remained the same for many years.

Results

In this paper, we present a new algorithm for the deformation of plasmid DNA molecules for Monte Carlo simulations. The move underlying our algorithm preserves the size and connectivity of straight-line segments of the plasmid DNA skeleton. We also present the results of three experiments comparing our deformation move with the standard and biased crankshaft moves in terms of acceptance ratio of the trials, energy and temperature evolution, and average displacement of the molecule. Our algorithm can also be used as a generic geometric algorithm for the deformation of regular polygons or polylines that preserves the connections and lengths of their segments.

Conclusion

Compared with both crankshaft moves, our move generates simulation trials with higher acceptance ratios and smoother deformations, making it suitable for real-time visualization of plasmid DNA coiling. For that purpose, we have adopted a DNA assembly algorithm that uses nucleotides as building blocks.     

Pharmacological inhibition of GSK-3 in a guinea pig model of LPS-induced pulmonary inflammation: II. Effects on skeletal muscle atrophy

Background

Chronic obstructive pulmonary disease (COPD) is accompanied by pulmonary inflammation and associated with extra-pulmonary manifestations, including skeletal muscle atrophy. Glycogen synthase kinase-3 (GSK-3) has been implicated in the regulation of muscle protein- and myonuclear turnover; two crucial processes that determine muscle mass. In the present study we investigated the effect of the selective GSK-3 inhibitor SB216763 on muscle mass in a guinea pig model of lipopolysaccharide (LPS)-induced pulmonary inflammation-associated muscle atrophy.

Methods

Guinea pigs were pretreated with either intranasally instilled SB216763 or corresponding vehicle prior to each LPS/saline challenge twice weekly. Pulmonary inflammation was confirmed and indices of muscle mass were determined after 12 weeks. Additionally, cultured skeletal muscle cells were incubated with tumor necrosis factor α (TNF-α) or glucocorticoids (GCs) to model the systemic effects of pulmonary inflammation on myogenesis, in the presence or absence of GSK-3 inhibitors.

Results

Repeated LPS instillation induced muscle atrophy based on muscle weight and muscle fiber cross sectional area. Intriguingly, GSK-3 inhibition using SB216763 prevented the LPS-induced muscle mass decreases and myofiber atrophy. Indices of protein turnover signaling were unaltered in guinea pig muscle. Interestingly, inhibition of myogenesis of cultured muscle cells by TNF-α or synthetic GCs was prevented by GSK-3 inhibitors.

Conclusions

In a guinea pig model of LPS-induced pulmonary inflammation, GSK-3 inhibition prevents skeletal muscle atrophy without affecting pulmonary inflammation. Resistance to inflammation- or GC-induced impairment of myogenic differentiation, imposed by GSK-3 inhibition, suggests that sustained myogenesis may contribute to muscle mass maintenance despite persistent pulmonary inflammation. Collectively, these results warrant further exploration of GSK-3 as a potential novel drug target to prevent or reverse muscle wasting in COPD.     

Phase II protocol for the evaluation of new treatments in patients with advanced gastric carcinoma: results of ECOG 5282

The study was a Phase II randomized study to evaluate the efficacy of new agents for the treatment of advanced gastric carcinoma. Patients were randomized to receive single agent chemotherapy with mitoxantrone, etoposide, aclacinomycin-A or spirogermanium. The patients were stratified by prior use of chemotherapy, prior doxorubicin use and ECOG performance status. Patients with a history of cardiac disease or prior doxorubicin exceeding a dose of 400 mg/m² were restrictively randomized to sopirogermanium or etoposide only. One hundred and fourteen patients were registered for the study. Among 98 evaluable patients there were only two partial responses (both in the etoposide arm), and one complete response in the mitoxantrone arm. The median survival on the study was 3.3 months. One hundred and six patients were analyzable for toxicity. There were four treatment-related deaths and four life-threatening toxicities. Because of low response rates and relatively high toxicities the studied compounds were not deemed worth further investigation for advanced gastric cancer.     

Electrotransformation studies in Clostridium cellulolyticum

Electropermeabilization of Clostridium cellulolyticum was optimized using ATP leakage assays. Electrotransformation was then performed under optimized conditions (6 to 7.5 kV cm⁻¹ field strength applied during 5 ms to a mixture containing methylated plasmids and late exponential phase cell suspensions (10 molecules:1 cell) in a sucrose-containing buffer). Transformants were only obtained when 7 or 7.5 kV cm⁻¹ pulses were applied. Transformation efficiencies evaluated from the growth curves of transformed cells were between 10⁵ and 10⁷ transformants per microgram of plasmid DNA for five different replicon-based plasmids. Restriction nuclease digestion patterns of pJIR418 purified from transformed Clostridia and Escherichia coli were indistinguishable, indicating that heterologous DNA was structurally stable in the Clostridium strain. Copy numbers of 130, 70 and 10 were estimated from purification yield for pCTC1, pKNT19 and pJIR418, respectively. Journal of Industrial Microbiology & Biotechnology (2001) 27, 271–274. Received 12 September 2000/ Accepted in revised form 25 November 2000     

A comparison of the illness beliefs of people with angina and their peers: a questionnaire study

Background  

What people believe about their illness may affect how they cope with it. It has been suggested that such beliefs stem from those commonly held within society . This study compared the beliefs held by people with angina, regarding causation and coping in angina, with the beliefs of their friends who do not suffer from angina.     

Water transport by aquaporins in the extant plant Physcomitrella patens

Although aquaporins (AQPs) have been shown to increase membrane water permeability in many cell types, the physiological role of this increase was not always obvious. In this report, we provide evidence that in the leafy stage of development (gametophore) of the moss Physcomitrella patens, AQPs help to replenish more rapidly the cell water that is lost by transpiration, at least if some water is in the direct vicinity of the moss plant. Three AQP genes were cloned in P. patens: PIP2;1, PIP2;2, and PIP2;3. The water permeability of the membrane was measured in protoplasts from leaves and protonema. A significant decrease was measured in protoplasts from leaves and protonema of PIP2;1 or PIP2;2 knockouts but not the PIP2;3 knockout. No phenotype was observed when knockout plants were grown in closed petri dishes with ample water supply. Gametophores isolated from the wild type and the pip2;3 mutant were not sensitive to moderate water stress, but pip2;1 or pip2;2 gametophores expressed a water stress phenotype. The knockout mutant leaves were more bent and twisted, apparently suffering from an important loss of cellular water. We propose a model to explain how the AQPs PIP2;1 and PIP2;2 delay leaf dessication in a drying atmosphere. We suggest that in ancestral land plants, some 400 million years ago, APQs were already used to facilitate the absorption of water.     

Evolutionary transfer of ORF-containing group I introns between different subcellular compartments (chloroplast and mitochondrion)

We describe here a case of homologous introns containing homologous open reading frames (ORFs) that are inserted at the same site in the large subunit (LSU) rRNA gene of different organelles in distantly related organisms. We show that the chloroplast LSU rRNA gene of the green alga Chlamydomonas pallidostigmatica contains a group I intron (CpLSU.2) encoding a site-specific endonuclease (I-CpaI). This intron is inserted at the identical site (corresponding to position 1931-1932 of the Escherichia coli 23S rRNA sequence) as a group I intron (AcLSU.m1) in the mitochondrial LSU rRNA gene of the amoeboid protozoon Acanthamoeba castellanii. The CpLSU.2 intron displays a remarkable degree of nucleotide similarity in both primary sequence and secondary structure to the AcLSU.m1 intron; moreover, the Acanthamoeba intron contains an ORF in the same location within its secondary structure as the CpLSU.2 ORF and shares with it a strikingly high level of amino acid similarity (65%; 42% identity). A comprehensive survey of intron distribution at site 1931 of the chloroplast LSU rRNA gene reveals a rather restricted occurrence within the polyphyletic genus Chlamydomonas, with no evidence of this intron among a number of non- Chlamydomonad green algae surveyed, nor in land plants. A parallel survey of homologues of a previously described and similar intron/ORF pair (C. reinhardtii chloroplast CrLSU/A. castellanii mitochondrial AcLSU.m3) also shows a restricted occurrence of this intron (site 2593) among chloroplasts, although the intron distribution is somewhat broader than that observed at site 1931, with site-2593 introns appearing in several green algal branches outside of the Chlamydomonas lineage. The available data, while not definitive, are most consistent with a relatively recent horizontal transfer of both site-1931 and site- 2593 introns (and their contained ORFs) between the chloroplast of a Chlamydomonas-type organism and the mitochondrion of an Acanthamoeba- like organism, probably in the direction chloroplast to mitochondrion. The data also suggest that both introns could have been acquired in a single event.