Kassorins: Novel innate immune system peptides from skin secretions of the African hyperoliid frogs, Kassina maculata and Kassina senegalensis

From defensive skin secretions acquired from two species of African hyperoliid frogs, Kassina maculata and Kassina senegalensis, we have isolated two structurally related, C-terminally amidated tridecapeptides of novel primary structure that exhibit a broad spectrum of biological activity. In reflection of their structural novelty and species of origin, we named the peptides kassorin M (FLEGLLNTVTGLLamide; 1387.8 Da) and kassorin S (FLGGILNTITGLLamide; 1329.8 Da), respectively. The primary structure and organisation of the biosynthetic precursors of kassorins M and S were deduced from cloned skin secretion-derived cDNA. Both open-reading frames encoded a single copy of kassorin M and S, respectively, located at the C-terminus. Kassorins display limited structural similarities to vespid chemotactic peptides (7/13 residues), temporin A (5/13 residues), the N-terminus of Lv-ranaspumin, a foam nest surfactant protein of the frog, Leptodactylus vastus, and an N-terminal domain of the equine sweat surfactant protein, latherin. Both peptides elicit histamine release from rat peritoneal mast cells. However, while kassorin S was found to possess antibacterial activity against Staphylococcus aureus, kassorin M was devoid of such activity. In contrast, kassorin M was found to contract the smooth muscle of guinea pig urinary bladder (EC50 = 4.66 nM) and kassorin S was devoid of this activity. Kassorins thus represent the prototypes of a novel family of peptides from the amphibian innate immune system as occurring in defensive skin secretions.

Targets of genome copy number reduction in primary breast cancers identified by integrative genomics

The identification of specific oncogenes and tumor suppressor genes in regions of recurrent aneuploidy is a major challenge of molecular cancer research. Using both oligonucleotide single-nucleotide polymorphism and mRNA expression arrays, we integrated genomic and transcriptional information to identify and prioritize candidate cancer genes in regions of increased and decreased chromosomal copy number in a cohort of primary breast cancers. Confirming the validity of this approach, several regions of previously-known copy number (CN) alterations in breast cancer could be successfully reidentified. Focusing on regions of decreased CN, we defined a prioritized list of eighteen candidate genes, which included ARPIN, FBNI, and LZTSI, previously shown to be associated with cancers in breast or other tissue types, and novel genes such as P29, MORF4LI, and TBCID5. One such gene, the RUNX3 transcription factor, was selected for further study. We show that RUNX3 is present at reduced CNs in proportion to the rest of the tumor genome and that RUNX3 CN reductions can also be observed in a breast cancer series from a different center. Using tissue microarrays, we demonstrate in an independent cohort of over 120 breast tissues that RUNX3 protein is expressed in normal breast epithelium but not fat and stromal tissue, and widely down-regulated in the majority of breast cancers (> 85%). In vitro, RUNX3 overexpression suppressed the invasive potential of MDA-MB-231 breast cancer cells in a matrigel assay. Our results demonstrate the utility of integrative genomic approaches to identify novel potential cancer-related genes in primary tumors. This article contains Supplementary Material available at http:// www.interscience.wiley.com/jpages/1045-2257/suppmat. (c) 2006 Wiley-Liss, Inc.

Methods to increase response to postal and electronic questionnaires

Background: Postal and electronic questionnaires are widely used for data collection in epidemiological studies but non-response reduces the effective sample size and can introduce bias. Finding ways to increase response to postal and electronic questionnaires would improve the quality of health research. Objectives: To identify effective strategies to increase response to postal and electronic questionnaires. Search strategy: We searched 14 electronic databases to February 2008 and manually searched the reference lists of relevant trials and reviews, and all issues of two journals. We contacted the authors of all trials or reviews to ask about unpublished trials. Where necessary, we also contacted authors to confirm methods of allocation used and to clarify results presented. We assessed the eligibility of each trial using pre-defined criteria. Selection criteria: Randomised controlled trials of methods to increase response to postal or electronic questionnaires. Data collection and analysis: We extracted data on the trial participants, the intervention, the number randomised to intervention and comparison groups and allocation concealment. For each strategy, we estimated pooled odds ratios (OR) and 95% confidence intervals (CI) in a random-effects model. We assessed evidence for selection bias using Egger's weighted regression method and Begg's rank correlation test and funnel plot. We assessed heterogeneity among trial odds ratios using a Chi 2 test and the degree of inconsistency between trial results was quantified using the I 2 statistic. Main results: Postal We found 481 eligible trials.The trials evaluated 110 different ways of increasing response to postal questionnaires.We found substantial heterogeneity among trial results in half of the strategies. The odds of response were at least doubled using monetary incentives (odds ratio 1.87; 95% CI 1.73 to 2.04; heterogeneity P < 0.00001, I 2 = 84%), recorded delivery (1.76; 95% CI 1.43 to 2.18; P = 0.0001, I 2 = 71%), a teaser on the envelope - e.g. a comment suggesting to participants that they may benefit if they open it (3.08; 95% CI 1.27 to 7.44) and a more interesting questionnaire topic (2.00; 95% CI 1.32 to 3.04; P = 0.06, I 2 = 80%). The odds of response were substantially higher with pre-notification (1.45; 95% CI 1.29 to 1.63; P < 0.00001, I 2 = 89%), follow-up contact (1.35; 95% CI 1.18 to 1.55; P < 0.00001, I 2 = 76%), unconditional incentives (1.61; 1.36 to 1.89; P < 0.00001, I 2 = 88%), shorter questionnaires (1.64; 95%CI 1.43 to 1.87; P < 0.00001, I 2 = 91%), providing a second copy of the questionnaire at follow up (1.46; 95% CI 1.13 to 1.90; P < 0.00001, I 2 = 82%), mentioning an obligation to respond (1.61; 95% CI 1.16 to 2.22; P = 0.98, I 2 = 0%) and university sponsorship (1.32; 95% CI 1.13 to 1.54; P < 0.00001, I 2 = 83%). The odds of response were also increased with non-monetary incentives (1.15; 95% CI 1.08 to 1.22; P < 0.00001, I 2 = 79%), personalised questionnaires (1.14; 95% CI 1.07 to 1.22; P < 0.00001, I 2 = 63%), use of hand-written addresses (1.25; 95% CI 1.08 to 1.45; P = 0.32, I 2 = 14%), use of stamped return envelopes as opposed to franked return envelopes (1.24; 95% CI 1.14 to 1.35; P < 0.00001, I 2 = 69%), an assurance of confidentiality (1.33; 95% CI 1.24 to 1.42) and first class outward mailing (1.11; 95% CI 1.02 to 1.21; P = 0.78, I 2 = 0%). The odds of response were reduced when the questionnaire included questions of a sensitive nature (0.94; 95% CI 0.88 to 1.00; P = 0.51, I 2 = 0%). Electronic: We found 32 eligible trials. The trials evaluated 27 different ways of increasing response to electronic questionnaires. We found substantial heterogeneity among trial results in half of the strategies. The odds of response were increased by more than a half using non-monetary incentives (1.72; 95% CI 1.09 to 2.72; heterogeneity P < 0.00001, I 2 = 95%), shorter e-questionnaires (1.73; 1.40 to 2.13; P = 0.08, I 2 = 68%), including a statement that others had responded (1.52; 95% CI 1.36 to 1.70), and a more interesting topic (1.85; 95% CI 1.52 to 2.26). The odds of response increased by a third using a lottery with immediate notification of results (1.37; 95% CI 1.13 to 1.65), an offer of survey results (1.36; 95% CI 1.15 to 1.61), and using a white background (1.31; 95% CI 1.10 to 1.56). The odds of response were also increased with personalised e-questionnaires (1.24; 95% CI 1.17 to 1.32; P = 0.07, I 2 = 41%), using a simple header (1.23; 95% CI 1.03 to 1.48), using textual representation of response categories (1.19; 95% CI 1.05 to 1.36), and giving a deadline (1.18; 95% CI 1.03 to 1.34). The odds of response tripled when a picture was included in an e-mail (3.05; 95% CI 1.84 to 5.06; P = 0.27, I 2 = 19%). The odds of response were reduced when "Survey" was mentioned in the e-mail subject line (0.81; 95% CI 0.67 to 0.97; P = 0.33, I 2 = 0%), and when the e-mail included a male signature (0.55; 95% CI 0.38 to 0.80; P = 0.96, I 2 = 0%). Authors' conclusions: Health researchers using postal and electronic questionnaires can increase response using the strategies shown to be effective in this systematic review. Copyright © 2009 The Cochrane Collaboration. Published by John Wiley & Sons, Ltd.


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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.

Cache-Integrated Network Interfaces: Flexible On-Chip Communication and Synchronization for Large-Scale CMPs

Per-core scratchpad memories (or local stores) allow direct inter-core communication, with latency and energy advantages over coherent cache-based communication, especially as CMP architectures become more distributed. We have designed cache-integrated network interfaces, appropriate for scalable multicores, that combine the best of two worlds – the flexibility of caches and the efficiency of scratchpad memories: on-chip SRAM is configurably shared among caching, scratchpad, and virtualized network interface (NI) functions. This paper presents our architecture, which provides local and remote scratchpad access, to either individual words or multiword blocks through RDMA copy. Furthermore, we introduce event responses, as a technique that enables software configurable communication and synchronization primitives. We present three event response mechanisms that expose NI functionality to software, for multiword transfer initiation, completion notifications for software selected sets of arbitrary size transfers, and multi-party synchronization queues. We implemented these mechanisms in a four-core FPGA prototype, and measure the logic overhead over a cache-only design for basic NI functionality to be less than 20%. We also evaluate the on-chip communication performance on the prototype, as well as the performance of synchronization functions with simulation of CMPs with up to 128 cores. We demonstrate efficient synchronization, low-overhead communication, and amortized-overhead bulk transfers, which allow parallelization gains for fine-grain tasks, and efficient exploitation of the hardware bandwidth.

FMRFamide-like peptides in root knot nematodes and their potential role in nematode physiology

FMRFamide-like peptides (FLPs) are a diverse group of neuropeptides that are expressed abundantly in nematodes. They exert potent physiological effects on locomotory, feeding and reproductive musculature and also act as neuromodulators. However, little is known about the specific expression patterns and functions of individual peptides. The current study employed rapid amplification of cDNA ends-polymerase chain reaction (RACE-PCR) to characterize flp genes from infective juveniles of the root knot nematodes, Meloidogyne incognita and Meloidogyne minor. The peptides identified from these transcripts are sequelogs of FLPs from the free-living nematode, Caenorhabditis elegans; the genes have therefore been designated as Mi-flp-1, Mi-flp-7, Mi-flp-12, Mm-flp-12 and Mi-flp-14. Mi-flp-1 encodes five FLPs with the common C-terminal moiety, NFLRFamide. Mi-flp-7 encodes two copies of APLDRSALVRFamide and APLDRAAMVRFamide and one copy of APFDRSSMVRFamide. Mi-flp-12 and Mm-flp-12 encode the novel peptide KNNKFEFIRFamide (a longer version of RNKFEFIRFamide found in C. elegans). Mi-flp-14 encodes a single copy of KHEYLRFamide (commonly known as AF2 and regarded as the most abundant nematode FLP), and a single copy of the novel peptide KHEFVRFamide. These FLPs share a high degree of conservation between Meloidogyne species and nematodes from other clades, including those of humans and animals, perhaps suggesting a common neurophysiological role which may be exploited by novel drugs. FLP immunoreactivity was observed for the first time in Meloidogyne, in the circumpharyngeal nerve ring, pharyngeal nerves and ventral nerve cord. Additionally, in situ hybridization revealed Mi-flp-12 expression in an RIR-like neuron and Mi-flp-14 expression in SMB-like neurons, respectively. These localizations imply physiological roles for FLP-12 and FLP-14 peptides, including locomotion and sensory perception.

Photochemical reduction of oxygen adsorbed to nanocrystalline TiO2 films: A transient absorption and oxygen scavenging study of different TiO2 preparations

Transient absorption spectroscopy (TAS) has been used to study the interfacial electron-transfer reaction between photogenerated electrons in nanocrystalline titanium dioxide (TiO2) films and molecular oxygen. TiO2 films from three different starting materials (TiO2 anatase colloidal paste and commercial anatase/rutile powders Degussa TiO2 P25 and VP TiO2 P90) have been investigated in the presence of ethanol as a hole scavenger. Separate investigations on the photocatalytic oxygen consumption by the films have also been performed with an oxygen membrane polarographic detector. Results show that a correlation exists between the electron dynamics of oxygen consumption observed by TAS and the rate of oxygen consumption through the photocatalytic process. The highest activity and the fastest oxygen reduction dynamics were observed with films fabricated from anatase TiO2 colloidal paste. The use of TAS as a tool for the prediction of the photocatalytic activities of the materials is discussed. TAS studies indicate that the rate of reduction of molecular oxygen is limited by interfacial electron-transfer kinetics rather than by the electron trapping/detrapping dynamics within the TiO2 particles.

Preparation and characterisation of novel thick sol-gel titania film photocatalysts

The preparation and characterization of thick (9 mum), clear, mechanically robust and photocatalytically active films of nanocrystalline anatase titania are described. XRD and SEM analysis show the films comprise 13 nm particles of anatase TiO2. Thin (54 nm) films of the 'paste' TiO2, along with sol-gel titania films made by a more traditional route are also prepared and characterised. All titania films mediate the photocatalytic destruction of stearic acid with a quantum yield of 0.0016 +/- 0.0003. using either 365 nm (i.e. BLB) or 254 nm (germicidal) light. P25 TiO2 films also appear to mediate the same process with a similar formal quantum efficiency. Of all the films tested, the thick paste TiO2 films are the most ideally suited for use with near UV light, for reasons which are discussed. All the titania films tested exhibit photoinduced superhydrophilicity.

Development and clinical validation of a loop-mediated isothermal amplification method for the rapid detection of Neisseria meningitidis

Loop-mediated isothermal amplification (LAMP) is an innovative technique that allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, optimization, and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detection of capsular Neisseria meningitidis were described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross-reactivity with other Neisseria spp. or with a comprehensive panel of other common human pathogens. The lower limit of detection was 6 ctrA gene copies detectable in 48 min, with positive reactions readily identifiable visually via a simple color change. Higher copy numbers could be detected in as little as 16 min. When applied to a total of 394 clinical specimens, the LAMP assay in comparison to a conventional TaqMan® based real-time polymerase chain reaction system demonstrated a sensitivity of 100% and a specificity of 98.9% with a ? coefficient of 0.942. The LAMP method represents a rapid, sensitive, and highly specific technique for the detection of N. meningitidis and has the potential to be used as a point-of-care molecular test and in resource-poor settings.

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.

Hopanoid Production Is Required for Low-pH Tolerance, Antimicrobial Resistance, and Motility in Burkholderia cenocepacia

Hopanoids are pentacyclic triterpenoids that are thought to be bacterial surrogates for eukaryotic sterols, such as cholesterol, acting to stabilize membranes and to regulate their fluidity and permeability. To date, very few studies have evaluated the role of hopanoids in bacterial physiology. The synthesis of hopanoids depends on the enzyme squalene-hopene cyclase (Shc), which converts the linear squalene into the basic hopene structure. Deletion of the 2 genes encoding Shc enzymes in Burkholderia cenocepacia K56-2, BCAM2831 and BCAS0167, resulted in a strain that was unable to produce hopanoids, as demonstrated by gas chromatography and mass spectrometry. Complementation of the Delta shc mutant with only BCAM2831 was sufficient to restore hopanoid production to wild-type levels, while introducing a copy of BCAS0167 alone into the Delta shc mutant produced only very small amounts of the hopanoid peak. The Delta shc mutant grew as well as the wild type in medium buffered to pH 7 and demonstrated no defect in its ability to survive and replicate within macrophages, despite transmission electron microscopy (TEM) revealing defects in the organization of the cell envelope. The Delta shc mutant displayed increased sensitivity to low pH, detergent, and various antibiotics, including polymyxin B and erythromycin. Loss of hopanoid production also resulted in severe defects in both swimming and swarming motility. This suggests that hopanoid production plays an important role in the physiology of B. cenocepacia.

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.

Influence of Different European Cements (CEM) on the Hydration of Cover-Zone Concrete during the Curing and Post Curing Periods

The durability of reinforced concrete structures depends, in the main, on the performance of the cover-zone concrete as it is this which protects the steel from the external environment. This paper focusses on the use of discretised electrical property measurements to study depth-related features during both the curing and post-curing period thereby allowing an integrated assessment of the protective properties of the cover region. In the current work, use is made of a small, multi-electrode array embedded within the surface 75mm of concrete specimens. Concretes were manufactured with different European cements (CEM) and water/binder ratios representing mixes which satisfied the minimum requirements for a range of environmental exposure classes including exposure to chlorides. Electrical resistance measurements were taken over a period in excess of 300 days which showed on-going hydration, pozzolanic reaction and pore-structure refinement; in addition, in the post-curing period, when exposed to a cyclic chloride ponding regime, measurements could be used to study the convective zone and ionic enrichment of the surface layer.

Multi-Modal CTL:Completeness, Complexity, and an Application

We define a multi-modal version of Computation Tree Logic (ctl) by extending the language with path quantifiers E and A where d denotes one of finitely many dimensions, interpreted over Kripke structures with one total relation for each dimension. As expected, the logic is axiomatised by taking a copy of a ctl axiomatisation for each dimension. Completeness is proved by employing the completeness result for ctl to obtain a model along each dimension in turn. We also show that the logic is decidable and that its satisfiability problem is no harder than the corresponding problem for ctl. We then demonstrate how Normative Systems can be conceived as a natural interpretation of such a multi-dimensional ctl logic. © 2009 Springer Science+Business Media B.V.

Characterization of physio-chemical processes and hydration kinetics in concretes containing supplementary cementitious materials using electrical property measurements

The current study monitors both the short- and long-term hydration characteristics of concrete using discretized conductivity measurements from initial gauging, through setting and hardening, the latter comprising both the curing and post-curing periods. In particular, attention is directed to the near-surface concrete as it is this zone which protects the steel from the external environment and has a major influence on durability, performance and service-life. A wide range of concrete mixes is studied comprising both plain Portland cement concretes and concretes containing fly-ash and ground granulated blast furnace slag. The parameter normalised conductivity was used to identify four distinct stages in the hydration process and highlight the influence of supplementary cementitious materials (SCM) on hydration and hydration kinetics. A relationship has been presented to account for the temporal decrease in conductivity, post 10-days hydration. The testing procedure and methodology presented lend itself to in-situ monitoring of reinforced concrete structures. (c) 2013 Elsevier Ltd. All rights reserved.