Rapid identification of precursor cDNAs encoding five structural classes of antimicrobial peptides from pickerel frog (Rana palustris) skin secretion by single step “shotgun” cloning.

The skin secretion of the North American pickerel frog (Rana palustris) has long been known to have pronounced noxious/toxic properties and to be highly effective in defence against predators and against other sympatric amphibians. As it consists largely of a complex mixture of peptides, it has been subjected to systematic peptidomic study but there has been little focus on molecular cloning of peptide-encoding cDNAs and by deduction, the biosynthetic precursors that they encode. Here, we demonstrate that the cDNAs encoding the five major structural families of antimicrobial peptides can be elucidated by a single step “shotgun” cloning approach using a cDNA library constructed from the source material of the peptidomic studies—the defensive skin secretion itself. Using a degenerate primer pool designed to a highly conserved nucleic acid sequence 5' to the initiation codon of known antimicrobial peptide precursor transcripts, we amplified cDNA sequences representing five major classes of antimicrobial peptides, such as esculentins, brevinins, ranatuerins, palustrins and temporins. Bioinformatic comparisons of precursor open-reading frames and nucleic acid sequences revealed high degrees of structural similarities between analogous peptides of R. palustris and the Chinese bamboo odorous frog, Rana versabilis. This approach thus constitutes a robust technique that can be used either alone or ideally, in parallel with peptidomic analysis of skin secretion, to rapidly extract primary structural information on amphibian skin secretion peptides and their biosynthetic precursors.

A Population-Based Study of IGF Axis Polymorphisms and the Esophageal Inflammation, Metaplasia, Adenocarcinoma Sequence

BACKGROUND & AIMS: Insulin-like growth factor (IGF) axis plays a key role in cell development, proliferation, and survival and is implicated in the etiology of several cancers. Few studies have examined the relationship between genetic variation of this axis and esophageal adenocarcinoma (EAC) or its precursors. METHODS: In a population-based case-control study, we investigated the association of common polymorphisms of IGF-1, IGF-2, IGF-1 receptor, IGF binding protein -3, growth hormones (GH) 1 and GH2, and GH receptor with reflux esophagitis (RE), Barrett esophagus (BE), and EAC. Two hundred and thirty RE, 224 BE, 227 EAC cases, and 260 controls were studied. Gene polymorphisms were identified using publicly available online resources; 102 IGF axis tag and putatively functional single-nucleotide polymorphisms (SNPs) were analyzed using MassARRAY iPLEX and Taqman assays. Results were analyzed using Haploview.
RESULTS: Three polymorphisms were disease-associated. IGF1 SNP rs6214 was associated with BE (adjusted P = .039). Using GG genotype as reference, odds ratio for BE in AA (wild-type) was 0.43 (95% confidence interval [CI], 0.24-0.75). GH receptor SNP rs6898743 was associated with EAC (adjusted P = .0112). With GG as reference, odds ratio for EAC in CC (wildtype) genotype was 0.42 (95% CI, 0.23-0.76). IGF1 (CA)(17) 185-bp allele was associated with RE (adjusted P = .0116). Using IGF1(non17) as reference, odds ratio for RE in IGF1(17) carriers was 7.29 (95% CI, 1.57-46.7).
CONCLUSIONS: In this study, 3 polymorphisms of IGF genes were associated with EAC or its precursors. These polymorphisms may be markers of disease risk; independent validation of our findings is required. These results suggest the IGF pathway is involved in EAC development.

The DUB/USP17 deubiquitinating enzymes: A gene family within a tandemly repeated sequence, is also embedded within the copy number variable Beta-defensin cluste

Background: The DUB/USP17 subfamily of deubiquitinating enzymes were originally identified as immediate early genes induced in response to cytokine stimulation in mice (DUB-1, DUB-1A, DUB-2, DUB-2A). Subsequently we have identified a number of human family members and shown that one of these (DUB-3) is also cytokine inducible. We originally showed that constitutive expression of DUB-3 can block cell proliferation and more recently we have demonstrated that this is due to its regulation of the ubiquitination and activity of the 'CAAX' box protease RCE1.

Results: Here we demonstrate that the human DUB/USP17 family members are found on both chromosome 4p16.1, within a block of tandem repeats, and on chromosome 8p23.1, embedded within the copy number variable betadefensin cluster. In addition, we show that the multiple genes observed in humans and other distantly related mammals have arisen due to the independent expansion of an ancestral sequence within each species. However, it is also apparent when sequences from humans and the more closely related chimpanzee are compared, that duplication events have taken place prior to these species separating.

Conclusions: The observation that the DUB/USP17 genes, which can influence cell growth and survival, have evolved from an unstable ancestral sequence which has undergone multiple and varied duplications in the species examined marks this as a unique family. In addition, their presence within the beta-defensin repeat raises the question whether they may contribute to the influence of this repeat on immune related conditions.

Genomes of "phiKMV-like viruses" of Pseudomonas aeruginosa contain localized single-strand interruptions

The "phiKMV-like viruses" comprise an important genus of T7 related phages infecting Pseudomonas aeruginosa. The genomes of these bacteriophages have localized single-strand interruptions (nicks), a distinguishing genomic trait previously thought to be unique for T5 related coliphages. Analysis of this feature in the newly sequenced phage fkF77 shows all four nicks to be localized on the non-coding DNA strand. They are present with high frequencies within the phage population and are introduced into the phage DNA at late stages of the lytic cycle. The general consensus sequence in the nicks (5'-CGACxxxxxCCTAoh pCTCCGG-3') was shown to be common among all phiKMV-related phages.

MML 53: a new low-mass, pre-main sequence eclipsing binary in the Upper Centaurus-Lupus region discovered by SuperWASP

We announce the discovery of a new low-mass, pre-main sequence eclipsing binary, MML 53. Previous observations of MML 53 found it to be a pre-main sequence spectroscopic multiple associated with the 15-22 Myr Upper Centaurus-Lupus cluster. We identify the object as an eclipsing binary for the first time through the analysis of multiple seasons of time series photometry from the SuperWASP transiting planet survey. Re-analysis of a single archive spectrum shows MML 53 to be a spatially unresolved triple system of young stars which all exhibit significant lithium absorption. Two of the components comprise an eclipsing binary with period, P = 2.097891(6) ± 0.000005 and mass ratio, q ~ 0.8. Here, we present the analysis of the discovery data.

A Novel Multilocus Sequence Typing Scheme for the Opportunistic Pathogen Propionibacterium acnes and Characterisation of Type I Cell Surface-Associated Antigens.

We have developed a novel Multilocus Sequence Typing Scheme (MLST) and database (http://pubmlst.org/pacnes/) for Propionibacterium acnes based on the analysis of seven core housekeeping genes. The scheme, which was validated against previously described antibody, single locus and Random Amplification of Polymorphic DNA (RAPD) typing methods, displayed excellent resolution and differentiated 123 isolates into 37 sequence types (ST). An overall clonal population structure was detected with six eBURST groups representing the major clades I, II and III, along with two singletons. Two highly successful and global clonal lineages, ST6 (type IA) and ST10 (type IB1), representing 65% of this current MLST isolate collection were identified. The ST6 clone and closely related single locus variants (SLV), which comprise a large clonal complex CC6, dominated isolates from patients with acne, and were also significantly associated with ophthalmic infections. Our data therefore supports an association between acne and P. acnes strains from the type IA cluster and highlights the role of a widely disseminated clonal genotype in this condition. Characterisation of type I cell surface-associated antigens that are not detected in ST10 or strains of type II and III identified two dermatan-sulphate-binding proteins with putative phase/antigenic variation signatures. We propose that the expression of these proteins by type IA organisms contributes to their role in the pathophysiology of acne and helps explain the recurrent nature of the disease. The MLST scheme and database described in this study should provide a valuable platform for future epidemiological and evolutionary studies of P. acnes.

Molecular dynamics and principal components analysis of human telomeric quadruplex multimers.

Guanine-rich DNA repeat sequences located at the terminal ends of chromosomal DNA can fold in a sequence-dependent manner into G-quadruplex structures, notably the terminal 150–200 nucleotides at the 3' end, which occur as a single-stranded DNA overhang. The crystal structures of quadruplexes with two and four human telomeric repeats show an all-parallel-stranded topology that is readily capable of forming extended stacks of such quadruplex structures, with external TTA loops positioned to potentially interact with other macromolecules. This study reports on possible arrangements for these quadruplex dimers and tetramers, which can be formed from 8 or 16 telomeric DNA repeats, and on a methodology for modeling their interactions with small molecules. A series of computational methods including molecular dynamics, free energy calculations, and principal components analysis have been used to characterize the properties of these higher-order G-quadruplex dimers and tetramers with parallel-stranded topology. The results confirm the stability of the central G-tetrads, the individual quadruplexes, and the resulting multimers. Principal components analysis has been carried out to highlight the dominant motions in these G-quadruplex dimer and multimer structures. The TTA loop is the most flexible part of the model and the overall multimer quadruplex becoming more stable with the addition of further G-tetrads. The addition of a ligand to the model confirms the hypothesis that flat planar chromophores stabilize G-quadruplex structures by making them less flexible.

The natriuretic peptide/helokinestatin precursor from Mexican beaded lizard (Heloderma horridum) venom: Amino acid sequence deduced from cloned cDNA and identification of two novel encoded helokinestatins

Natriuretic peptides are common components of reptile venoms and molecular cloning of their biosynthetic precursors has revealed that in snakes, they co-encode bradykinin-potentiating peptides and in venomous lizards, some co-encode bradykinin inhibitory peptides such as the helokinestatins. The common natriuretic peptide/helokinestatin precursor of the Gila Monster, Heloderma suspectum, encodes five helokinestatins of differing primary structures. Here we report the molecular cloning of a natriuretic peptide/helokinestatin precursor cDNA from a venom-derived cDNA library of the Mexican beaded lizard (Heloderma horridum). Deduction of the primary structure of the encoded precursor protein from this cloned cDNA template revealed that it consisted of 196 amino acid residues encoding a single natriuretic peptide and five helokinestatins. While the natriuretic peptide was of identical primary structure to its Gila Monster (H. suspectum) homolog, the encoded helokinestatins were not, with this region of the common precursor displaying some significant differences to its H. suspectum homolog. The helokinestatin-encoding region contained a single copy of helokinestatin-1, 2 copies of helokinestatin-3 and single copies of 2 novel peptides, (Phe)(5)-helokinestatin-2 (VPPAFVPLVPR) and helokinestatin-6 (GPPFNPPPFVDYEPR). All predicted peptides were found in reverse phase HPLC fractions of the same venom. Synthetic replicates of both novel helokinestatins were found to antagonize the relaxing effect of bradykinin on rat tail artery smooth muscle. Thus lizard venom continues to provide a source of novel biologically active peptides. (C) 2011 Published by Elsevier Inc.

Characterization of IS2112, a new insertion sequence from Rhodococcus, and its relationship with mobile elements belonging to the IS110 family

A new insertion sequence (IS2112) was identified in the genome of the 1-haloalkane-utilizing bacterium Rhodococcus rhodochrous NCIMB 13064. The insertion element is 1415 bp long, does not contain terminal inverted repeats, and is not flanked by directly repeated sequences. IS2112 belongs to the IS110 family of transposable elements, and forms a separate subfamily, along with IS116, Two copies of IS2112 were found in R, rhodochrous NCIMB 13064 and one, two or three copies of a similar sequence were detected in five other 1-haloalkane-degrading Rhodococcus strains. There were no sequences homologous to IS2112 found in the l-haloalkane-degrading 'Pseudomonas pavonaceae' 170 and Rhodococcus sp, HA1 or in several Rhodococcus strains which do not utilize haloalkanes, IS2112 was originally found in plasmid pRTL1 of R. rhodochrous NCIMB 13064 which harbours genes encoding utilization of l-haloalkanes, and was located 5 kbp upstream of the haloalkane dehalogenase gene (dhaA), Although the second copy of IS2112 in strain NCIMB 13064 was also present on the pRTL1 plasmid, these sequences do not apparently comprise a single composite transposon encoding haloalkane utilization. An analysis of derivatives of NCIMB 13064 revealed that IS2112 was involved in genome rearrangements. IS2112 appeared to change its location as a result of transposition and as a result of other rearrangements of the NCIMB 13064 genome.

Accelerating Multiple Sequence Alignment with the Cell BE Processor

The Cell Broadband Engine (BE) Architecture is a new heterogeneous multi-core architecture targeted at compute-intensive workloads. The architecture of the Cell BE has several features that are unique in high-performance general-purpose processors, most notably the extensive support for vectorization, scratch pad memories and explicit programming of direct memory accesses (DMAs) and mailbox communication. While these features strongly increase programming complexity, it is generally claimed that significant speedups can be obtained by using Cell BE processors. This paper presents our experiences with using the Cell BE architecture to accelerate Clustal W, a bio-informatics program for multiple sequence alignment. We report on how we apply the unique features of the Cell BE to Clustal W and how important each is in obtaining high performance. By making extensive use of vectorization and by parallelizing the application across all cores, we demonstrate a speedup of 24.4 times when using 16 synergistic processor units on a QS21 Cell Blade compared to single-thread execution on the power processing unit. As the Cell BE exploits a large number of slim cores, our highly optimized implementation is just 3.8 times faster than a 3-thread version running on an Intel Core2 Duo, as the latter processor exploits a small number of fat cores.

The Supremacy of the Sequence: Key Elements and Dimensions in the Process of Change

How single organizations manage the process of change and why only some of them are able to actually reach radical change are central questions in today’s theoretical debate. The role played by the process of change and its dimensions (namely, pace, sequence and linearity), however, has been poorly investigated. Drawing on archetype theory, this paper explores: (i) whether a specific pace of radical change exists; (ii) whether different outcomes of change are characterized by different sequences of change in key-structures and systems (iii) how the three dimensions of the process of change possibly interact. As an example of organizational change the study takes into consideration processes of accounting change in three departments of two Canadian and two Italian municipalities. The results suggest the supremacy of the sequence of implemented changes over the other two dimensions of the process in order to achieve a radical outcome of change.

Promoter region of the Escherichia coli O7-specific lipopolysaccharide gene cluster:structural and functional characterization of an upstream untranslated mRNA sequence

We report the identification of the promoter region of the Escherichia coli O7-specific lipopolysaccharide (LPS) gene cluster (wbEcO7). Typical -10 and -35 sequences were found to be located in the intervening region between galF and rlmB, the first gene of the wbEcO7 cluster. Data from RNase protection experiments revealed the existence of an untranslated leader mRNA segment of 173 bp, including the JUMPStart and two ops sequences. We characterized the structure of this leader mRNA by using the program Mfold and a combination of nested and internal deletions transcriptionally fused to a promoterless lac operon. Our results indicated that the leader mRNA may fold into a series of complex stem-loop structures, one of which includes the JUMPStart element. We have also found that one of the ops sequences resides on the predicted stem and the other resides on the loop region, and we confirmed that these sequences are essential for the RfaH-mediated regulation of the O polysaccharide cluster. A very similar stem-loop structure could be predicted in the promoter region of the LPS core operon encoding the waaQGPSBIJYZK genes. We observed another predicted stem-loop, located immediately downstream from the wbEcO7 transcription initiation site, which appeared to be involved in premature termination of transcription. This putative stem-loop is common to many other O polysaccharide gene clusters but is not present in core oligosaccharide genes. wbEcO7-lac transcriptional fusions in single copy numbers were also used to determine the effects of various environmental cues in the transcriptional regulation of O polysaccharide synthesis. No effects were detected with temperature, osmolarity, Mg2+ concentration, and drugs inducing changes in DNA supercoiling. We therefore conclude that the wbEcO7 promoter activity may be constitutive and that regulation takes place at the level of elongation of the mRNA in a RfaH-mediated manner.

Investigating Hydraulically Active Fractures in A Shallow Meta-Sedimentary Bedrock Aquifer Using Single Well Tracer Testing, Mount Stewart, Northern Ireland

Knowledge of groundwater flow/mass transport, in poorly productive aquifers which underlie over 65% of the island of Ireland, is necessary for effective management of catchment water quality and aquatic ecology. This research focuses on a fractured low-grade Ordovician/Silurian greywacke sequence which underlies approximately 25% of the northern half of Ireland. Knowledge of the unit’s hydrogeological properties remain largely restricted to localised single well open hole “transmissivity” values. Current hydrogeological conceptual models of the Greywacke view the bulk of groundwater flowing through fractures in an otherwise impermeable bedrock mass.
Core analysis permits fracture characterisation, although not all identified fractures may be involved in groundwater flow. Traditional in-situ hydraulic characterisation relies on cumbersome techniques such as packer testing or geophysical borehole logging (e.g. flowmeters). Queen’s University Belfast is currently carrying out hydraulic characterization of 16 boreholes at its Greywacke Hydrogeological Research Site at Mount Stewart, Northern Ireland.
Development of dye dilution methods, using a recently-developed downhole fluorometer, provided a portable, user-friendly, and inexpensive means of detecting hydraulically active intervals in open boreholes. Measurements in a 55m deep hole, three days following fluorescent dye injection, demonstrated the ability of the technique to detect two discrete hydraulically active intervals corresponding to zones identified by caliper and heat-pulse flowmeter logs. High resolution acoustic televiewer logs revealed the zones to correspond to two steeply dipping fractured intervals. Results suggest the rock can have effective porosities of the order of 0.1%.
Study findings demonstrate dye dilution’s utility in characterizing groundwater flow in fractured aquifers. Tests on remaining holes will be completed at different times following injection to identify less permeable fractures and develop an improved understanding of the structural controls on groundwater flow in the uppermost metres of competent bedrock.

Genomic Analysis of Pseudomonas putida Phage tf with Localized Single-Strand DNA Interruptions

The complete sequence of the 46,267 bp genome of the lytic bacteriophage tf specific to Pseudomonas putida PpG1 has been determined. The phage genome has two sets of convergently transcribed genes and 186 bp long direct terminal repeats. The overall genomic architecture of the tf phage is similar to that of the previously described Pseudomonas aeruginosa phages PaP3, LUZ24 and phiMR299-2, and 39 out of the 72 products of predicted tf open reading frames have orthologs in these phages. Accordingly, tf was classified as belonging to the LUZ24-like bacteriophage group. However, taking into account very low homology levels between tf DNA and that of the other phages, tf should be considered as an evolutionary divergent member of the group. Two distinguishing features not reported for other members of the group were found in the tf genome. Firstly, a unique end structure - a blunt right end and a 4-nucleotide 3'-protruding left end - was observed. Secondly, 14 single-chain interruptions (nicks) were found in the top strand of the tf DNA. All nicks were mapped within a consensus sequence 5'-TACT/RTGMC-3'. Two nicks were analyzed in detail and were shown to be present in more than 90% of the phage population. Although localized nicks were previously found only in the DNA of T5-like and phiKMV-like phages, it seems increasingly likely that this enigmatic structural feature is common to various other bacteriophages.

Room-temperature single phase multiferroic magnetoelectrics: Pb(Fe,M)x(Zr,Ti)(1−x)O3 [M=Ta, Nb]

We describe extensive studies on a family of perovskite oxides that are ferroelectric and ferromagnetic at ambient temperatures. The data include x-ray diffraction, Raman spectroscopy, measurements of ferroelectric and magnetic hysteresis, dielectric constants, Curie temperatures, electron microscopy
(both scanning electron microscope and transmission electron microscopy (TEM)) studies, and both longitudinal and transverse magnetoelectric constants a33 and a31. The study extends earlier work to lower Fe, Ta, and Nb concentrations at the B-site (from 15%–20% down to 5%). The magnetoelectric
constants increase supralinearly with Fe concentrations, supporting the earlier conclusions of a key role for Fe spin clustering. The room-temperature orthorhombic C2v point group symmetry inferred from earlier x-ray diffraction studies is confirmed via TEM, and the primitive unit cell size is found to be the basic perovskite Z¼1 structure of BaTiO3, also the sequence of phase transitions with increasing temperature from rhombohedral to orthorhombic to tetragonal to cubic mimics barium titanate.