Human single-stranded DNA binding proteins are essential for maintaining genomic stability

The double-stranded conformation of cellular DNA is a central aspect of DNA stabilisation and protection. The helix preserves the genetic code against chemical and enzymatic degradation, metabolic activation, and formation of secondary structures. However, there are various instances where single-stranded DNA is exposed, such as during replication or transcription, in the synthesis of chromosome ends, and following DNA damage. In these instances, single-stranded DNA binding proteins are essential for the sequestration and processing of single-stranded DNA. In order to bind single-stranded DNA, these proteins utilise a characteristic and evolutionary conserved single-stranded DNA-binding domain, the oligonucleotide/oligosaccharide-binding (OB)-fold. In the current review we discuss a subset of these proteins involved in the direct maintenance of genomic stability, an important cellular process in the conservation of cellular viability and prevention of malignant transformation. We discuss the central roles of single-stranded DNA binding proteins from the OB-fold domain family in DNA replication, the restart of stalled replication forks, DNA damage repair, cell cycle-checkpoint activation, and telomere maintenance.

‘All in a day’s work’ : the lifeworld of older people in New Zealand rest homes

This doctoral thesis contributes to critical gerontology research by investigating the lived experiences of residents in the everyday world of New Zealand rest homes. There is a need to understand how frail rest home residents experience "age". This study focuses on describing and understanding residents lived experiences. As the New Zealand population is ageing, this phenomenological focus adds clarity to the poorly understood lived experiences about being aged in rest homes. Policy initiatives such as the Positive Ageing Strategy with its emphasis on keeping older people living in the community largely ignore the life practices of the increasing proportions of frail older people who require long-term residential care. My mixed-methods modified framework approach draws on the lifeworld as understood by Max van Manen (1990) and Alfred Schütz (1972). The lifeworld is made up of thematic strands of lived experience: these being lived space, lived time, lived body and lived relations with others, which are both the source and object of phenomenological research (van Manen, 1990). These strands are temporarily unravelled and considered in-depth for 27 residents who took part in audio-recorded interviews, before being interwoven through a multiple-helix model, into an integrated interpretation of the residents‟ lifeworld. Supplementing and backgrounding the interviews with these residents, are descriptive data including written interview summaries and survey findings about the relationships and pastimes of 352 residents living in 21 rest homes, which are counted and described. The residents day-to-day use of rest home space, mediated temporal order, self-managed bodies and minds, and negotiated relationships are interpreted. The mythology of the misery of rest home life is challenged, and a more constructive critical gerontology approach is offered. Findings of this research reveal how meanings around daily work practices are constructed by the residents. These elders participate in daily rest home life, from the sidelines or not at all, as they choose or are able, and this always involves work for the residents. They continue to actively manage satisfactory and fulfilling pastimes and relationships, because in their ordinary, everyday lifeworld it is “all in a day‟s work”.

Sterilizing tissue-materials using pulsed power plasma

This paper investigates the potential of pulsed power to sterilize hard and soft tissues and its impact on their physico-mechanical properties. It hypothesizes that pulsed plasma can sterilize both vascular and avascular tissues and the transitive layers in between without deleterious effects on their functional characteristics. Cartilage/bone laminate was chosen as a model to demonstrate the concept, treated at low temperature, at atmospheric pressure, in short durations and in buffered environment using a purposed-built pulsed power unit. Input voltage and time of exposure were assigned as controlling parameters in a full factorial design of experiment to determine physical and mechanical alteration pre- and post-treatment. The results demonstrated that, discharges of 11 kV sterilized samples in 45 s, reducing intrinsic elastic modules from 1.4 ± 0.9 to 0.9 ± 0.6 MPa. There was a decrease of 14.1 % in stiffness and 27.8 % in elastic-strain energy for the top quartile. Mechanical impairment was directly proportional to input voltage (P value < 0.05). Bacterial inactivation was proportional to treatment time for input voltages above 32 V (P < 0.001; R Sq = 0.98). Thermal analysis revealed that helix-coil transition decelerated with exposure time and collagen fibrils were destabilized as denaturation enthalpy reduced by 200 μV. We concluded by presenting a safe operating threshold for pulsed power plasma as a feasible protocol for effective sterilization of connective tissues with varying level of loss in mechanical robustness which we argue to be acceptable in certain medical and tissue engineering application.

Knowledge unlatched : encouraging innovation in academic publishing through collective action and Open Access licensing

This case study examines the way in which Knowledge Unlatched is combining collective action and open access licenses to encourage innovation in markets for specialist academic books. Knowledge Unlatched is a not for profit organisation that has been established to help a global community of libraries coordinate their book purchasing activities more effectively and, in so doing, to ensure that books librarians select for their own collections become available for free for anyone in the world to read. The Knowledge Unlatched model is an attempt to re-coordinate a market in order to facilitate a transition to digitally appropriate publishing models that include open access. It offers librarians an opportunity to facilitate the open access publication of books that their own readers would value access to. It provides publishers with a stable income stream on titles selected by libraries, as well as an ability to continue selling books to a wider market on their own terms. Knowledge Unlatched provides a rich case study for researchers and practitioners interested in understanding how innovations in procurement practices can be used to stimulate more effective, equitable markets for socially valuable products.

Higher Education and Sustainable Development : A Model for Curriculum Renewal

Responding to the global and unprecedented challenge of capacity building for twenty-first century life, this book is a practical guide for tertiary education institutions to quickly and effectively renew the curriculum towards education for sustainable development. The book begins by exploring why curriculum change has been so slow. It then describes a model for rapid curriculum renewal, highlighting the important roles of setting timeframes, formal and informal leadership, and key components and action strategies. The second part of the book provides detailed coverage of six core elements that have been trialled and peer reviewed by institutions around the world: - raising awareness among staff and students - mapping graduate attributes - auditing the curriculum - developing niche degrees, flagship courses and fully integrated programs - engaging and catalysing community and student markets - integrating curriculum with green campus operations. With input from more than seventy academics and grounded in engineering education experiences, this book will provide academic staff with tools and insights to rapidly align program offerings with the needs of present and future generations of students.

Expanding horizons : scientific frontiers, legal regulation, and globalization

In the six decades since the discovery of the double helix structure of DNA by Watson and Crick in 1953, developments in genetic science have transformed our understanding of human health and disease. These developments, along with those in other areas such as computer science, biotechnology, and nanotechnology, have opened exciting new possibilities for the future. In addition, the increasing trend for technologies to converge and build upon each other potentially increases the pace of change, constantly expanding the boundaries of the scientific frontier. At the same time, however, scientific advances are often accompanied by public unease over the potential for unforeseen, negative outcomes. For governments, these issues present significant challenges for effective regulation. This Article analyzes the challenges associated with crafting laws for rapidly changing science and technology. It considers whether we need to regulate, how best to regulate for converging technologies, and how best to ensure the continued relevance of laws in the face of change.

Mechanical model and superelastic properties of carbon microcoils with circular cross-section

Here we report on an unconventional Ni-P alloy-catalyzed, high-throughput, highly reproducible chemical vapor deposition of ultralong carbon microcoils using acetylene precursor in the temperature range 700-750 °C. Scanning electron microscopy analysis reveals that the carbon microcoils have a unique double-helix structure and a uniform circular cross-section. It is shown that double-helix carbon microcoils have outstanding superelastic properties. The microcoils can be extended up to 10-20 times of their original coil length, and quickly recover the original state after releasing the force. A mechanical model of the carbon coils with a large spring index is developed to describe their extension and contraction. Given the initial coil parameters, this mechanical model can successfully account for the geometric nonlinearity of the spring constants for carbon micro- and nanocoils, and is found in a good agreement with the experimental data in the whole stretching process.

UpaH Is a newly identified autotransporter protein That contributes to biofilm formation and bladder colonization by Uropathogenic Escherichia coli CFT073

Escherichia coli is the primary cause of urinary tract infection (UTI) in the developed world. The major factors associated with virulence of uropathogenic E. coli (UPEC) are fimbrial adhesins, which mediate specific attachment to host receptors and trigger innate host responses. Another group of adhesins is represented by the autotransporter (AT) subgroup of proteins. In this study, we identified a new AT-encoding gene, termed upaH, present in a 6.5-kb unannotated intergenic region in the genome of the prototypic UPEC strain CFT073. Cloning and sequencing of the upaH gene from CFT073 revealed an intact 8.535-kb coding region, contrary to the published genome sequence. The upaH gene was widely distributed among a large collection of UPEC isolates as well as the E. coli Reference (ECOR) strain collection. Bioinformatic analyses suggest β-helix as the predominant structure in the large N-terminal passenger (α) domain and a 12-strand β-barrel for the C-terminal β-domain of UpaH. We demonstrated that UpaH is expressed at the cell surface of CFT073 and promotes biofilm formation. In the mouse UTI model, deletion of the upaH gene in CFT073 and in two other UPEC strains did not significantly affect colonization of the bladder in single-challenge experiments. However, in competitive colonization experiments, CFT073 significantly outcompeted its upaH isogenic mutant strain in urine and the bladder.

Detection of DNA mutations using novel SERS (Surface-Enhanced Raman Spectroscopy) diagnostic platform

This article describes the detection of DNA mutations using novel Au-Ag coated GaN substrate as SERS (surface-enhanced Raman spectroscopy) diagnostic platform. Oligonucleotide sequences corresponding to the BCR-ABL (breakpoint cluster region-Abelson) gene responsible for development of chronic myelogenous leukemia were used as a model system to demonstrate the discrimination between the wild type and Met244Val mutations. The thiolated ssDNA (single-strand DNA) was immobilized on the SERS-active surface and then hybridized to a labeled target sequence from solution. An intense SERS signal of the reporter molecule MGITC was detected from the complementary target due to formation of double helix. The SERS signal was either not observed, or decreased dramatically for a negative control sample consisting of labeled DNA that was not complementary to the DNA probe. The results indicate that our SERS substrate offers an opportunity for the development of novel diagnostic assays.

Making the profitable transition towards sustainable business practice

The last three decades have been difficult for companies and industry. In an increasingly competitive international business climate with shifting national environmental regulations, higher standards are being demanded by the consumer and community groups, not-to-mention the escalating cost of primary resources such as water, steel and minerals. The cause of these pressures is the traditional notion held by business executives and engineers that there is an inherent trade off between eco-efficiency and improving the economic bottom line. However there is significant evidence and examples of best practice to show that there is in fact no trade-off between the environment and the economy if sustainable development through continual improvement is adopted. It is highly possible therefore for companies to make a profitable transition towards sustainable business practice, where along the transition significant business opportunities can be taken advantage of. Companies are by their very nature dynamic, influential and highly capable of adapting to change. Making an organisational transformation to a sustainable business is not outside the capacity of the typical company, who know much of what is needed already to change their activities to satisfy current market demands while achieving competitiveness. However in order to make the transition towards sustainable business practice companies require some key mechanisms such as accurate information on methodologies and opportunities, understanding of the financial and non-financial incentives, permission from stakeholders and shareholders, understanding of the emerging market opportunities, a critical mass of leaders in their sector and demonstrated case studies, and awarding appropriate risk-taking activities undertaken by engineers and CEOs. Satisfying these requirements will adopt an innovative culture within the company that strives for continual improvement and successfully transforms itself to achieve competitiveness in the 21st Century. This paper will summarise the experiences of The Natural Edge Project (TNEP) and its partners in assisting organisations to make a profitable transition towards sustainable business practice through several initiatives. The Natural Advantage of Nations publication provides the critical information required by business leaders and engineers to set the context of sustainable business practice. The Profiting in a Carbon Constrained World report, developed with Natural Capitalism Inc led by Hunter Lovins, summarises the opportunities available to companies to take advantage of the carbon trading market mechanisms such as the Chicago Climate Exchange and European Climate Exchange. The Sustainability Helix then guides the company through the transition by identifying the key tools and methodologies required by companies to reduce environmental loading while dramatically improving resource productivity and achieving competitiveness. Finally, the Engineering Sustainable Solutions Program delivers the key engineering information required by companies and university departments to deliver sustainable engineering solutions. The initiatives are of varying complexity and level of application, however all are designed to provide key staff the critical information required to make a profitable transition towards sustainable business practice. It is then their responsibility to apply and teach their knowledge to the rest of the organisation.

Two monoclonal antibodies to precisely the same epitope of type ii collagen select non-crossreactive phage clones by phage display: Implications for autoimmunity and molecular mimicry

Two monoclonal antibodies (mAb) CB268 and CII-C1 to type II collagen (CII) react with precisely the same conformational epitope constituted by the residues ARGLT on the three chains of the CII triple helix. The antibodies share structural similarity, with most differences in the complementarity determining region 3 of the heavy chain (HCDR3). The fine reactivity of these mAbs was investigated by screening two nonameric phage-displayed random peptide libraries. For each mAb, there were phage clones (phagotopes) that reacted strongly by ELISA only with the selecting mAb, and inhibited binding to CII only for that mAb, not the alternate mAb. Nonetheless, a synthetic peptide RRLPFGSQM corresponding to an insert from a highly reactive CII-C1-selected phagotope, which was unreactive (and non-inhibitory) with CB268, inhibited the reactivity of CB268 with CII. Most phage-displayed peptides contained a motif in the first part of the molecule that consisted of two basic residues adjacent to at least one hydrophobic residue (e.g. RRL or LRR), but the second portion of the peptides differed for the two mAbs. We predict that conserved CDR sequences interact with the basic-basic-hydrophobic motif, whereas non-conserved amino acids in the binding sites (especially HCDR3) interact with unique peptide sequences and limit cross-reactivity. The observation that two mAbs can react identically with a single epitope on one antigen (CII), but show no cross-reactivity when tested against a second (phagotope) indicates that microorganisms could exhibit mimics capable of initiating autoimmunity without this being evident from conventional assays.

Mimotopes identified by phage display for the monoclonal antibody CII- C1 to type II collagen

The characterization of B cell epitopes has been advanced by the use of random peptide libraries displayed within the coat protein of bacteriophage. This technique was applied to the monoclonal antibody (mAb) C1 to type II collagen (CII-C1). CII-C1 is known to react with a conformational epitope on type II collagen that includes residues 359-363. Three rounds of selection were used to screen two random nonameric phage libraries and 18 phagotopes were isolated. CII-C1 reacted by ELISA with 17 of the 18 phagotopes: one phagotope contained a stop codon. Of the eight most reactive phage, seven inhibited the reactivity by ELISA of CII-C1 with type II collagen. Of the 18 phage isolated, 11 encoded the motif F-G-x-Q with the sequence F-G-S-Q in 6, 2 encoded F-G-Q, and one the reverse motif Q-x-y-F. Most phagotopes that inhibited the reactivity of CII-C1 encoded two particular motifs consisting of two basic amino acid residues and a hydrophobic residue in the first part of the insert and the F-G-x-Q or F-G-Q motif ill the second part; phagotopes which contained only one basic residue in the first part of the sequence were less reactive. These motifs are not represented in the linear sequence of type II collagen and thus represent mimotopes of the epitope for CII-C1 on type II collagen. There were five phagotopes with peptide inserts containing the sequence RLPFG occurring in the Epstein-Barr virus nuclear antigen, EBNA- 1. This is of interest because EBV has been implicated in the initiation of rheumatoid arthritis (RA) by reason of increased reactivity to EBNA-1 in RA sera. In conclusion, the phage display technique disclosed mimotopes for a conformational epitope of type II collagen, and revealed an interesting homology with a sequence of the EBNA-1 antigen from Epstein Barr virus.

In vivo evaluation of folate decorated cross-linked micelles for the delivery of platinum anticancer drugs

The biodistribution of micelles with and without folic acid targeting ligands were studied using a block copolymer consisting of acrylic acid (AA) and polyethylene glycol methyl ether acrylate (PEGMEA) blocks. The polymers were prepared using RAFT polymerization in the presence of a folic acid functionalized RAFT agent. Oxoplatin was conjugated onto the acrylic acid block to form amphiphilic polymers which, when diluted in water, formed stable micelles. In order to probe the in vivo stability, a selection of micelles were cross-linked using 1,8-diamino octane. The sizes of the micelles used in this study range between 75 and 200 nm, with both spherical and worm-like conformation. The effects of cross-linking, folate conjugation and different conformation on the biodistribution were studied in female nude mice (BALB/c) following intravenous injection into the tail vein. Using optical imaging to monitor the fluorophore-labeled polymer, the in vivo biodistribution of the micelles was monitored over a 48 h time-course after which the organs were removed and evaluated ex vivo. These experiments showed that both cross-linking and conjugation with folic acid led to increased fluorescence intensities in the organs, especially in the liver and kidneys, while micelles that are not conjugated with folate and not cross-linked are cleared rapidly from the body. Higher accumulation in the spleen, liver, and kidneys was also observed for micelles with worm-like shapes compared to the spherical micelles. While the various factors of cross-linking, micelle shape, and conjugation with folic acid all contribute separately to prolong the circulation time of the micelle, optimization of these parameters for drug delivery devices could potentially overcome adverse effects such as liver and kidney toxicity.

Molecular dynamics simulations of CXCL-8 and its interactions with a receptor peptide, heparin fragments, and sulfated linked cyclitols

CXCL-8 (Interleukin 8) is a CXC chemokine with a central role in the human immune response. We have undertaken extensive in silico analyses to elucidate the interactions of CXCL-8 with its various binding partners, which are crucial for its biological function. Sequence and structure analyses showed that residues in the thirdq β-sheet and basic residues in the heparin binding site are highly variable, while residues in the second β-sheet are highly conserved. Molecular dynamics simulations in aqueous solution of dimeric CXCL-8 have been performed with starting geometries from both X-ray and NMR structures showed shearing movements between the two antiparallel C-terminal helices. Dynamic conservation analyses of these simulations agreed with experimental data indicating that structural differences between the two structures at quaternary level arise from changes in the secondary structure of the N-terminal loop, the 310-helix, the 30s, 40s, and 50s loops and the third β-sheet, resulting in a different interhelical separation. Nevertheless, the observation of these different states indicates that CXCL-8 has the potential to undergo conformational changes, and it seems likely that this feature is relevant to the mode of binding of glycosaminoglycan (GAG) mimetics such as cyclitols. Simulations of the receptor peptide fragment−CXCL-8 complex identified several specific interactions of the receptor peptide with CXCL-8 that could be exploited in the structure-based design of competitive peptides and nonpeptidic molecules targeting CXCL-8 for combating inflammatory diseases. Simulations of the CXCL-8 dimer complexed with a 24-mer heparin fragment and of the CXCL-8−receptor peptide complex revealed that Arg60, Lys64, and Arg68 in the dimer bind to cyclitols in a horseshoe pattern, defining a region which is spatially distinct from the receptor binding site. There appears to be an optimum number of sulfates and an optimum length of alkyl spacers required for the interaction of cyclitol inhibitors with the dimeric form of CXCL-8. Calculation of the binding affinities of cyclitol inhibitors reflected satisfactorily the ranking of experimentally determined inhibitory potencies. The findings of these molecular modeling studies will help in the search for inhibitors which can modulate various CXCL-8 biological activities and serve as an excellent model system to study CXC-inhibitor interactions.

Molecular dynamics simulation of the phosphorylation-induced conformational changes of a tau peptide fragment

Aggregation of the microtubule associated protein tau (MAPT) within neurons of the brain is the leading cause of tauopathies such as Alzheimer's disease. MAPT is a phospho-protein that is selectively phosphorylated by a number of kinases in vivo to perform its biological function. However, it may become pathogenically hyperphosphorylated, causing aggregation into paired helical filaments and neurofibrillary tangles. The phosphorylation induced conformational change on a peptide of MAPT (htau225−250) was investigated by performing molecular dynamics simulations with different phosphorylation patterns of the peptide (pThr231 and/or pSer235) in different simulation conditions to determine the effect of ionic strength and phosphate charge. All phosphorylation patterns were found to disrupt a nascent terminal β-sheet pattern (226VAVVR230 and 244QTAPVP249), replacing it with a range of structures. The double pThr231/pSer235 phosphorylation pattern at experimental ionic strength resulted in the best agreement with NMR structural characterization, with the observation of a transient α-helix (239AKSRLQT245). PPII helical conformations were only found sporadically throughout the simulations. Proteins 2014; 82:1907–1923. © 2014 Wiley Periodicals, Inc.