STAR : Predicting recombination sites from amino acid sequence

Background Designing novel proteins with site-directed recombination has enormous prospects. By locating effective recombination sites for swapping sequence parts, the probability that hybrid sequences have the desired properties is increased dramatically. The prohibitive requirements for applying current tools led us to investigate machine learning to assist in finding useful recombination sites from amino acid sequence alone. Results We present STAR, Site Targeted Amino acid Recombination predictor, which produces a score indicating the structural disruption caused by recombination, for each position in an amino acid sequence. Example predictions contrasted with those of alternative tools, illustrate STAR'S utility to assist in determining useful recombination sites. Overall, the correlation coefficient between the output of the experimentally validated protein design algorithm SCHEMA and the prediction of STAR is very high (0.89). Conclusion STAR allows the user to explore useful recombination sites in amino acid sequences with unknown structure and unknown evolutionary origin. The predictor service is available from http://pprowler.itee.uq.edu.au/star.

Available evidence does not support serosorting as an HIV risk reduction strategy

Recent data highlighted the association between penetration of antiretrovirals in the central nervous system (CNS) and neurocognitive impairment in HIVpositive patients. Existing antiretrovirals have been ranked according to a score of neuropenetration, which was shown to be a predictor of anti-HIVactivity in the CNS and improvement of neurocognitive disorders [1]. Main factors affecting drug penetration are known to be protein binding, lipophilicity and molecular weight [2]. Moreover, active translation by membrane transporters (such as p-glycoprotein) could be a key mechanism of passage [3]. The use of raltegravir (RGV), a novel antiretroviral drug targeted to inhibit the HIV preintegrase complex, is increasing worldwide due to its efficacy and tolerability. However, penetration of RGV in the CNS has not been yet elucidated. In fact, prediction of RGV neuropenetration according to molecular characteristics is controversial. Intermediate protein binding (83%) and large volume of distribution (273 l) could suggest a high distribution beyond extracellular spaces [4]. On the contrary, low lipophilicity (oil/water partition coefficient at pH 7.4 of 2.80) and intermediate molecular weight (482.51 Da) suggest a limited diffusion. Furthermore, in-vitro studies suggest that RGV is substrate of p-glycoprotein, although this efflux pump has not been identified to significantly affect plasma pharmacokinetics [5]. In any case, no data concerning RGV passage into cerebrospinal fluid of animals or humans have yet been published.

The repertoire of nitrogen assimilation in Rhodococcus: Catalysis, pathways and relevance in biotechnology and bioremediation

The Rhodococcus genus exhibits diverse enzymatic activity that can be exploited in the conversion of natural and anthropogenic nitrogenous compounds. This catalytic response provides a selective advantage in terms of available nutrients while also serving to remove otherwise harmful xenobiotics. This review provides a critical assessment of the literature on bioconversion of organo-nitrogen compounds with a consideration of applications in bioremediation and commercial biotechnology. By examining the major nitro-organic compounds (amino acids, amines, nitriles, amides and nitroaromatics) in turn, the considerable repertoire of Rhodococcus spp. is established. The available published enzyme reaction data is coupled with genomic characterisation to provide a molecular basis for Rhodococcus enzyme activity with an assessment of the cellular properties that aid substrate accessibility and ensure stability. The metabolic gene clusters associated with the observed reaction pathways are identified and future directions in enzyme optimisation and metabolic engineering are assessed. © 2014 Society of Chemical Industry.

Crystal structures and hydrogen bonding in the anhydrous tryptaminium salts of the isomeric (2,4-dichlorophenoxy)acetic and (3,5-dichlorophenoxy)acetic acids

The anhydrous salts of 1H-indole-3-ethanamine (tryptamine) with isomeric (2,4-dichlorophenoxy)acetic acid (2,4-D) and (3,5-dichlorophenoxy)acetic (3,5-D), C10H13N2+ (C8H5Cl2O3)-, [(I) and (II), respectively] have been determined and their one-dimensional hydrogen-bonded polymeric structures are described. In the crystal of (I),the aminium H-atoms are involved in three separate inter-species N-H...O hydrogen-bonding interactions, two with carboxyl O-atom acceptors and the third in an asymmetric three-centre bidentate carboxyl O,O' chelate [graph set R2/1(4)]. The indole H-atom forms an N-H...O~carboxyl~ hydrogen bond, extending the chain structure along the b axial direction. In (II), two of the three aminium H-atoms are also involved in N-H...O(carboxyl) hydrogen bonds similar to (I) but with the third, a three-centre asymmetric interaction with carboxyl and phenoxy O-atoms is found [graph set R2/1(5)]. The chain polymeric extension is also along b. There are no pi--pi ring interactions in either of the structures. The aminium side chain conformations differ significantly between the two structures, reflecting the conformational ambivalence of the tryptaminium cation, as found also in the benzoate salts.

Exercise-induced DNA damage: Is there a relationship with inflammatory responses?

Both a systemic inflammatory response as well as DNA damage has been observed following exhaustive endurance exercise. Hypothetically, exercise-induced DNA damage might either be a consequence of inflammatory processes or causally involved in inflammation and immunological alterations after strenuous prolonged exercise (e.g. by inducing lymphocyte apoptosis and lymphocytopenia). Nevertheless, up to now only few studies have addressed this issue and there is hardly any evidence regarding a direct relationship between DNA or chromosomal damage and inflammatory responses in the context of exercise. The most conclusive picture that emerges from available data is that reactive oxygen and nitrogen species (RONS) appear to be the key effectors which link inflammation with DNA damage. Considering the time-courses of inflammatory and oxidative stress responses on the one hand and DNA effects on the other the lack of correlations between these responses might also be explained by too short observation periods. This review summarizes and discusses the recent findings on this topic. Furthermore, data from our own study are presented that aimed to verify potential associations between several endpoints of genome stability and inflammatory, immune-endocrine and muscle damage parameters in competitors of an Ironman triathlon until 19 days into recovery. The current results indicate that DNA effects in lymphocytes are not responsible for exercise-induced inflammatory responses. Furthermore, this investigation shows that inflammatory processes, vice versa, do not promote DNA damage, neither directly nor via an increased formation of RONS derived from inflammatory cells. Oxidative DNA damage might have been counteracted by training- and exercise-induced antioxidant responses. However, further studies are needed that combine advanced -omics based techniques (transcriptomics, proteomics) with state-of-the-art biochemical biomarkers to gain more insights into the underlying mechanisms.

What is the available evidence concerning relative performance of different designs of mixed-species plantings for smallholder and community forestry in the tropics? A systematic map protocol

Background There has been growing interest in mixed species plantation systems because of their potential to provide a range of socio-economic and bio-physical benefits which can be matched to the diverse needs of smallholders and communities. Potential benefits include the production of a range of forest products for home and commercial use; improved soil fertility especially when nitrogen fixing species are included; improved survival rates and greater productivity of species; a reduction in the amount of damage from pests or disease; and improved biodiversity and wildlife habitats. Despite these documented services and growing interest in mixed species plantation systems, the actual planting areas in the tropics are low, and monocultures are still preferred for industrial plantings and many reforestation programs because of perceived higher economic returns and readily available information about the species and their silviculture. In contrast, there are few guidelines for the design and management of mixed-species systems, including the social and ecological factors of successful mixed species plantings. Methods This protocol explains the methodology used to investigate the following question: What is the available evidence for the relative performance of different designs of mixed-species plantings for smallholder and community forestry in the tropics? This study will systematically search, identify and describe studies related to mixed species plantings across tropical and temperate zones to identify the social and ecological factors that affect polyculture systems. The objectives of this study are first to identify the evidence of biophysical or socio-economic factors that have been considered when designing mixed species systems for community and smallholder forestry in the tropics; and second, to identify gaps in research of mixed species plantations. Results of the study will help create guidelines that can assist practitioners, scientists and farmers to better design mixed species plantation systems for smallholders in the tropics.

Comparative analysis and distribution of Omega-3 lcPUFA Biosynthesis genes in marine molluscs

Recent research has identified marine molluscs as an excellent source of omega-3 long-chain polyunsaturated fatty acids (lcPUFAs), based on their potential for endogenous synthesis of lcPUFAs. In this study we generated a representative list of fatty acyl desaturase (Fad) and elongation of very long-chain fatty acid (Elovl) genes from major orders of Phylum Mollusca, through the interrogation of transcriptome and genome sequences, and various publicly available databases. We have identified novel and uncharacterised Fad and Elovl sequences in the following species: Anadara trapezia, Nerita albicilla, Nerita melanotragus, Crassostrea gigas, Lottia gigantea, Aplysia californica, Loligo pealeii and Chlamys farreri. Based on alignments of translated protein sequences of Fad and Elovl genes, the haeme binding motif and histidine boxes of Fad proteins, and the histidine box and seventeen important amino acids in Elovl proteins, were highly conserved. Phylogenetic analysis of aligned reference sequences was used to reconstruct the evolutionary relationships for Fad and Elovl genes separately. Multiple, well resolved clades for both the Fad and Elovl sequences were observed, suggesting that repeated rounds of gene duplication best explain the distribution of Fad and Elovl proteins across the major orders of molluscs. For Elovl sequences, one clade contained the functionally characterised Elovl5 proteins, while another clade contained proteins hypothesised to have Elovl4 function. Additional well resolved clades consisted only of uncharacterised Elovl sequences. One clade from the Fad phylogeny contained only uncharacterised proteins, while the other clade contained functionally characterised delta-5 desaturase proteins. The discovery of an uncharacterised Fad clade is particularly interesting as these divergent proteins may have novel functions. Overall, this paper presents a number of novel Fad and Elovl genes suggesting that many mollusc groups possess most of the required enzymes for the synthesis of lcPUFAs.

Super DNAging - New insights into DNA integrity, genome stability and telomeres in the oldest old

Reductions in DNA integrity, genome stability, and telomere length are strongly associated with the aging process, age-related diseases as well as the age-related loss of muscle mass. However, in people reaching an age far beyond their statistical life expectancy the prevalence of diseases, such as cancer, cardiovascular disease, diabetes or dementia, is much lower compared to “averagely” aged humans. These inverse observations in nonagenarians (90–99 years), centenarians (100–109 years) and super-centenarians (110 years and older) require a closer look into dynamics underlying DNA damage within the oldest old of our society. Available data indicate improved DNA repair and antioxidant defense mechanisms in “super old” humans, which are comparable with much younger cohorts. Partly as a result of these enhanced endogenous repair and protective mechanisms, the oldest old humans appear to cope better with risk factors for DNA damage over their lifetime compared to subjects whose lifespan coincides with the statistical life expectancy. This model is supported by study results demonstrating superior chromosomal stability, telomere dynamics and DNA integrity in “successful agers”. There is also compelling evidence suggesting that life-style related factors including regular physical activity, a well-balanced diet and minimized psycho-social stress can reduce DNA damage and improve chromosomal stability. The most conclusive picture that emerges from reviewing the literature is that reaching “super old” age appears to be primarily determined by hereditary/genetic factors, while a healthy lifestyle additionally contributes to achieving the individual maximum lifespan in humans. More research is required in this rapidly growing population of super old people. In particular, there is need for more comprehensive investigations including short- and long-term lifestyle interventions as well as investigations focusing on the mechanisms causing DNA damage, mutations, and telomere shortening.

An improved protocol for the isolation of total genomic DNA from Labyrinthulomycetes

Many protocols have been used for extraction of DNA from Thraustochytrids. These generally involve the use of CTAB, phenol/chloroform and ethanol. They also feature mechanical grinding, sonication, N2 freezing or bead beating. However, the resulting chemical and physical damage to extracted DNA reduces its quality. The methods are also unsuitable for large numbers of samples. Commercially-available DNA extraction kits give better quality and yields but are expensive. Therefore, an optimized DNA extraction protocol was developed which is suitable for Thraustochytrids to both minimise expensive and time-consuming steps prior to DNA extraction and also to improve the yield. The most effective method is a combination of single bead in TissueLyser (Qiagen) and Proteinase K. Results were conclusive: both the quality and the yield of extracted DNA were higher than with any other method giving an average yield of 8.5 µg/100 mg biomass.

Proteogenomics of selective susceptibility to endotoxin using circulating acute phase biomarkers and bioassay development in sheep: a review

Scientists have injected endotoxin into animals to investigate and understand various pathologies and novel therapies for several decades. Recent observations have shown that there is selective susceptibility to Escherichia coli lipopolysaccharide (LPS) endotoxin in sheep, despite having similar breed characteristics. The reason behind this difference is unknown, and has prompted studies aiming to explain the variation by proteogenomic characterisation of circulating acute phase biomarkers. It is hypothesised that genetic trait, biochemical, immunological and inflammation marker patterns contribute in defining and predicting mammalian response to LPS. This review discusses the effects of endotoxin and host responses, genetic basis of innate defences, activation of the acute phase response (APR) following experimental LPS challenge, and the current approaches employed in detecting novel biomarkers including acute phase proteins (APP) and micro-ribonucleic acids (miRNAs) in serum or plasma. miRNAs are novel targets for elucidating molecular mechanisms of disease because of their differential expression during pathological, and in healthy states. Changes in miRNA profiles during a disease challenge may be reflected in plasma. Studies show that gel-based two-dimensional electrophoresis (2-DE) coupled with either matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) or liquid chromatography-mass spectrometry (LC-MS/MS) are currently the most used methods for proteome characterisation. Further evidence suggests that proteomic investigations are preferentially shifting from 2-DE to non-gel based LC-MS/MS coupled with data extraction by sequential window acquisition of all theoretical fragment-ion spectra (SWATH) approaches that are able to identify a wider range of proteins. Enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qRT-PCR), and most recently proteomic methods have been used to quantify low abundance proteins such as cytokines. qRT-PCR and next generation sequencing (NGS) are used for the characterisation of miRNA. Proteogenomic approaches for detecting APP and novel miRNA profiling are essential in understanding the selective resistance to endotoxin in sheep. The results of these methods could help in understanding similar pathology in humans. It might also be helpful in the development of physiological and diagnostic screening assays for determining experimental inclusion and endpoints, and in clinical trials in future

TMIO-pyrimid hybrids are profluorescent, site-directed spin labels for nucleic acids

We report the synthesis of a new class of molecules which are hybrids of long-lived tetramethylisoindolinoxyl (TMIO) radicals and the pyrido[1,2-a]benzimidazole (PyrImid) scaffold. These compounds represent a new lead for noncovalently binding nucleic acid probes, as they interact with nucleic acids with previously unreported C (DNA) and C/U (RNA) complementarity, which can be detected by electron paramagnetic resonance (EPR) techniques. They also have promising properties for fluorimetric analysis, as their fluorescent spin-quenched derivatives exhibit a significant Stokes shift

Two-dimensional coordination polymeric structures in caesium complexes with ring-substituted phenoxyacetic acids

The two-dimensional polymeric structures of the caesium complexes with the phenoxyacetic acid analogues (4-fluorophenoxy)acetic acid, (3-chloro-2-methylphenoxy)acetic acid and the herbicidally active (2,4-dichlorophen­oxy)acetic acid (2,4-D), namely poly[[5-(4-fluorophenoxy)acetato][4-(4-fluorophenoxy)acetato]dicaesium], [Cs2(C8H6FO3)2]n, (I), poly[aqua[5-(3-chloro-2-methylphenoxy)acetato]caesium], [Cs(C9H8ClO3)(H2O)]n, (II), and poly[[7-(2,4-di­chlorophenoxy)acetato][(2,4-dichlorphenoxy)acetic acid]caesium], [Cs(C8H5Cl2O3)(C8H6Cl2O3)]n, (III), are described. In (I), the Cs+ cations of the two individual irregular coordination polyhedra in the asymmetric unit (one CsO7 and the other CsO8) are linked by bridging carboxylate O-atom donors from the two ligand molecules, both of which are involved in bidentate chelate Ocarboxy,Ophenoxy interactions, while only one has a bidentate carboxylate O,O'-chelate inter­action. Polymeric extension is achieved through a number of carboxylate O-atom bridges, with a minimum CsCs separation of 4.3231 (9) Å, giving layers which lie parallel to (001). In hydrated complex (II), the irregular nine-coordination about the Cs+ cation comprises a single monodentate water molecule, a bidentate Ocarboxy,Ophenoxy chelate interaction and six bridging carboxylate O-atom bonding interactions, giving a CsCs separation of 4.2473 (3) Å. The water mol­ecule forms intralayer hydrogen bonds within the two-dimensional layers, which lie parallel to (100). In complex (III), the irregular centrosymmetric CsO6Cl2 coordination environment comprises two O-atom donors and two ring-substituted Cl-atom donors from two hydrogen bis[(2,4-dichlorophenoxy)acetate] ligand species in a bidentate chelate mode, and four O-atom donors from bridging carboxyl groups. The duplex ligand species lie across crystallographic inversion centres, linked through a short O-HO hydrogen bond involving the single acid H atom. Structure extension gives layers which lie parallel to (001). The present set of structures of Cs salts of phenoxyacetic acids show previously demonstrated trends among the alkali metal salts of simple benzoic acids with no stereochemically favourable interactive substituent groups for formation of two-dimensional coordination polymers.

Osmolality electrolyte and carbohydrate type and oral rehydration solutions: A controlled study to compare the efficacy of two commercially available solutions (osmolalities 240 mmol/L and 340 mmol/L)

An open-label, inpatient study was undertaken to compare the efficacy of two oral rehydration solutions (ORS) given randomly to children aged 1-10 years who had acute gastroenteritis with mild or moderate dehydration (n = 45). One solution contained 60 mmol/L sodium and 1.8% glucose, total osmolality 240 mosm/l (gastrolyte, Rhone-poulenc, Rorer) and the other contained 26 mmol/l sodium, 2.7% glucose and 3.6% sucrose, total osmolality 340 mOsm/l (Glucolyte, Gilseal). Analysis of data indicated that Gastrolyte therapy resulted in significantly fewer episodes and volume of vomiting over all time periods in comparison to Glucolyte and significantly less stool volume during the first 8 h and in the 0-24 h period. The differences between treatments in degree of dehydration at each follow-up period, duration of diarrhea, and duration of hospital stay were not significant. No adverse drug reactions occurred. Six patients received intravenous rehydration treatment and were considered treatment failures. We conclude that oral rehydration therapy is safe and efficacious in the management of dehydration in acute diarrhoea and that the lower osmolar rehydration solution has clinically marginal advantages.