Protein sequence optimization with a pairwise decomposable penalty for buried unsatisfied hydrogen bonds

In aqueous solution, polar groups make hydrogen bonds with water, and hence burial of such groups in the interior of a protein is unfavorable unless the loss of hydrogen bonds with water is compensated by formation of new ones with other protein groups. For this reason, buried “unsatisfied” polar groups making no hydrogen bonds are very rare in proteins. Efficiently representing the energetic cost of unsatisfied hydrogen bonds with a pairwise-decomposable energy term during protein design is challenging since whether or not a group is satisfied depends on all of its neighbors. Here we describe a method for assigning a pairwise-decomposable energy to sidechain rotamers such that following combinatorial sidechain packing, buried unsaturated polar atoms are penalized. The penalty can be any quadratic function of the number of unsatisfied polar groups, and can be computed very rapidly. We show that inclusion of this term in Rosetta sidechain packing calculations substantially reduces the number of buried unsatisfied polar groups.     

Liming and deep ripping responses for a range of field crops

Grain yields were measured over 2 seasons from a range of field crops following liming and deep ripping an acid and compacted soil in north-eastern Victoria. Lime (2.5 t ha^–1) substantially reduced the level of exchangeable Al and exchangeable Mn whilst raising soil pH by about 1.0 unit. The crops grown were 7 cultivars of wheat and one cultivar each of triticale, oats, barley, rapeseed, safflower, field pea, chick pea and lupins. With the exception of lupin, liming the soil increased (p=0.05) the grain yield of all crops and cultivars. With the wheat cultivars there were 2 distinct groups with different tolerance to soil acidity. Wheat, oats, triticale and lupins had higher absolute yields than the other crops. Safflower and chick pea had very low yields without soil amendment. The magnitude of the lime response did not differ between the wheat cultivars (17%) or between any of the crop species (range 9–29%). Deep ripping the soil to break a hard compacted layer resulted in more yield for all the cereals and safflower. The results demonstrate the importance of using crops with tolerance to acid soil conditions as well as gains that can be obtained with ameliorating identifiable soil problems.     

Comparison of the effects of acylation and amidation on the antimicrobial and antiviral properties of lactoferrin

AIMS: To compare amidation and acylation of lactoferrin (LF) from bovine milk, as a means of enhancing its antimicrobial and antiviral properties. METHODS AND RESULTS: LF was chemically modified by amidation with a 1-ethyl-3-[3-(dimethylamino) propyl] carbodiimide (EDC) in the presence of ammonium ions or by acylation with either succinic or acetic anhydride. In the test systems used, amidation substantially enhanced the activity of LF against Pseudomonas fluorescens in comparison with native LF. However, increasing the net negative charge of LF by acylation had no effect on the activity of LF against P. fluorescens, and abrogated the antimicrobial activity of LF against Bacillus subtilis and Saccharomyces cerevisiae. Increasing the net negative charges of LF by acylation eliminated its antimicrobial and antiviral effects against poliovirus and feline calicivirus (nonenveloped viruses). CONCLUSIONS: The addition of positive charges to LF via amidation enhanced antimicrobial properties in contrast to increasing the negative charges by acylation, which abolished both the antimicrobial and antiviral properties of LF. SIGNIFICANCE AND IMPACT OF THE STUDY: The effects of charge alteration of LF determined in this study provides a basis for further development of LF formulations with enhanced antimicrobial effectiveness for use in food process hygiene, veterinary and health-care applications.     

Protection of HIV neutralizing aptamers against rectal and vaginal nucleases: implications for RNA-based therapeutics

RNA-based drugs are an emerging class of therapeutics. They have the potential to regulate proteins, chromatin, as well as bind to specific proteins of interest in the form of aptamers. These aptamers are protected from nuclease attack by chemical modifications that enhance their stability for in vivo usage. However, nucleases are ubiquitous, and as we have yet to characterize the entire human microbiome it is likely that many nucleases are yet to be identified. Any novel, unusual enzymes present in vivo might reduce the efficacy of RNA-based therapeutics, even when they are chemically modified. We have previously identified an RNA-based aptamer capable of neutralizing a broad spectrum of clinical HIV-1 isolates and are developing it as a vaginal and rectal microbicide candidate. As a first step we addressed aptamer stability in the milieu of proteins present in these environments. Here we uncover a number of different nucleases that are able to rapidly degrade 2'-F-modified RNA. We demonstrate that the aptamer can be protected from the nuclease(s) present in the vaginal setting, without affecting its antiviral activity, by replacement of key positions with 2'-O-Me-modified nucleotides. Finally, we show that the aptamer can be protected from all nucleases present in both vaginal and rectal compartments using Zn(2+) cations. In conclusion we have derived a stable, antiviral RNA-based aptamer that could form the basis of a pre-exposure microbicide or be a valuable addition to the current tenofovir-based microbicide candidate undergoing clinical trials.     

Additive effects of Na+ and Cl- ions on barley growth under salinity stress

Soil salinity affects large areas of the world's cultivated land, causing significant reductions in crop yield. Despite the fact that most plants accumulate both sodium (Na(+)) and chloride (Cl(-)) ions in high concentrations in their shoot tissues when grown in saline soils, most research on salt tolerance in annual plants has focused on the toxic effects of Na(+) accumulation. It has previously been suggested that Cl(-) toxicity may also be an important cause of growth reduction in barley plants. Here, the extent to which specific ion toxicities of Na(+) and Cl(-) reduce the growth of barley grown in saline soils is shown under varying salinity treatments using four barley genotypes differing in their salt tolerance in solution and soil-based systems. High Na(+), Cl(-), and NaCl separately reduced the growth of barley, however, the reductions in growth and photosynthesis were greatest under NaCl stress and were mainly additive of the effects of Na(+) and Cl(-) stress. The results demonstrated that Na(+) and Cl(-) exclusion among barley genotypes are independent mechanisms and different genotypes expressed different combinations of the two mechanisms. High concentrations of Na(+) reduced K(+) and Ca(2+) uptake and reduced photosynthesis mainly by reducing stomatal conductance. By comparison, high Cl(-) concentration reduced photosynthetic capacity due to non-stomatal effects: there was chlorophyll degradation, and a reduction in the actual quantum yield of PSII electron transport which was associated with both photochemical quenching and the efficiency of excitation energy capture. The results also showed that there are fundamental differences in salinity responses between soil and solution culture, and that the importance of the different mechanisms of salt damage varies according to the system under which the plants were grown.