Differentiation of Rice Tungro Bacilliform Virus Strains by Restriction Analysis and DNA Hybridization

Rice tungro bacilliform virus (RTBV) is one of the two viruses that cause tungro disease. Four RTBV strains maintained in the greenhouse for 4 years, G1, G2, Ic, and L, were differentiated by restriction fragment length polymorphism (RFLP) analysis of the native viral DNA. Although strains G1 and Ic had identical restriction patterns when cleaved with Pst1, BamHI, EcoRI, and EcoRV, they can be differentiated from strains G2 and L by EcoRI and EcoRV digestion. These same endonucleases also differentiate strain G2 from strain L. When total DNA extracts from infected plants were used instead of viral DNA, and digested with EcoRV, identical restriction patterns for each strain (G2 and L) were obtained from roots, leaves, and leaf sheaths of infected plants. The restriction patterns were consistent from plant to plant, in different varieties, and at different times after inoculation. This technique can be used to differentiate RTBV strains and determine the variability of a large number of field samples.

Assessment of commercial network RTK user positioning performance over long inter-station distances

The paper provides an assessment of the performance of commercial Real Time Kinematic (RTK) systems over longer than recommended inter-station distances. The experiments were set up to test and analyse solutions from the i-MAX, MAX and VRS systems being operated with three triangle shaped network cells, each having an average inter-station distance of 69km, 118km and 166km. The performance characteristics appraised included initialization success rate, initialization time, RTK position accuracy and availability, ambiguity resolution risk and RTK integrity risk in order to provide a wider perspective of the performance of the testing systems. ----- ----- The results showed that the performances of all network RTK solutions assessed were affected by the increase in the inter-station distances to similar degrees. The MAX solution achieved the highest initialization success rate of 96.6% on average, albeit with a longer initialisation time. Two VRS approaches achieved lower initialization success rate of 80% over the large triangle. In terms of RTK positioning accuracy after successful initialisation, the results indicated a good agreement between the actual error growth in both horizontal and vertical components and the accuracy specified in the RMS and part per million (ppm) values by the manufacturers. ----- ----- Additionally, the VRS approaches performed better than the MAX and i-MAX when being tested under the standard triangle network with a mean inter-station distance of 69km. However as the inter-station distance increases, the network RTK software may fail to generate VRS correction and then may turn to operate in the nearest single-base RTK (or RAW) mode. The position uncertainty reached beyond 2 meters occasionally, showing that the RTK rover software was using an incorrect ambiguity fixed solution to estimate the rover position rather than automatically dropping back to using an ambiguity float solution. Results identified that the risk of incorrectly resolving ambiguities reached 18%, 20%, 13% and 25% for i-MAX, MAX, Leica VRS and Trimble VRS respectively when operating over the large triangle network. Additionally, the Coordinate Quality indicator values given by the Leica GX1230 GG rover receiver tended to be over-optimistic and not functioning well with the identification of incorrectly fixed integer ambiguity solutions. In summary, this independent assessment has identified some problems and failures that can occur in all of the systems tested, especially when being pushed beyond the recommended limits. While such failures are expected, they can offer useful insights into where users should be wary and how manufacturers might improve their products. The results also demonstrate that integrity monitoring of RTK solutions is indeed necessary for precision applications, thus deserving serious attention from researchers and system providers.

Recent advances in cancer therapy targeting proteins involved in DNA double-strand break repair

Damage to genetic material represents a persistent and ubiquitous threat to genomic stability. Once DNA damage is detected, a multifaceted signaling network is activated that halts the cell cycle, initiates repair, and in some instances induces apoptotic cell death. In this article, we will review DNA damage surveillance networks, which maintain the stability of our genome, and discuss the efforts underway to identify chemotherapeutic compounds targeting the core components of DNA double-strand breaks (DSB) response pathway. The majority of tumor cells have defects in maintaining genomic stability owing to the loss of an appropriate response to DNA damage. New anticancer agents are exploiting this vulnerability of cancer cells to enhance therapeutic indexes, with limited normal tissue toxicity. Recently inhibitors of the checkpoint kinases Chk1 and Chk2 have been shown to sensitize tumor cells to DNA damaging agents. In addition, the treatment of BRCA1- or BRCA2-deficient tumor cells with poly(ADP-ribose) polymerase (PARP) inhibitors also leads to specific tumor killing. Due to the numerous roles of p53 in genomic stability and its defects in many human cancers, therapeutic agents that restore p53 activity in tumors are the subject of multiple clinical trials. In this article we highlight the proteins mentioned above and catalog several additional players in the DNA damage response pathway, including ATM, DNA-PK, and the MRN complex, which might be amenable to pharmacological interventions and lead to new approaches to sensitize cancer cells to radio- and chemotherapy. The challenge is how to identify those patients most receptive to these treatments.

Spatiotemporal investigations of DNA damage repair using microbeams

Cellular response to radiation damage is made by a complex network of pathways and feedback loops whose spatiotemporal organisation is still unclear despite its decisive role in determining the fate of the damaged cell. Revealing the dynamic sequence of the repair proteins is therefore critical in understanding how the DNA repair mechanisms work. There are also still open questions regarding the possible movement of damaged chromatin domains and its role as trigger for lesion recognition and signalling in the DNA repair context. The single-cell approach and the high spatial resolution offered by microbeams provide the perfect tool to study and quantify the dynamic processes associated with the induction and repair of DNA damage. We have followed the development of radiation-induced foci for three DNA damage markers (i.e. γ-H2AX, 53BP1 and hSSB1) using normal fibroblasts (AG01522), human breast adenocarcinoma cells (MCF7) and human fibrosarcoma cells (HT1080) stably transfected with yellow fluorescent protein fusion proteins following irradiation with the QUB X-ray microbeam (carbon X-rays <2 µm spot). The size and intensity of the foci has been analysed as a function of dose and time post-irradiation to investigate the dynamics of the above-mentioned DNA repair processes and monitor the remodelling of chromatin structure that the cell undergoes to deal with DNA damage.

Multiple human single-stranded DNA binding proteins function in genome maintenance : structural, biochemical and functional analysis

DNA exists predominantly in a duplex form that is preserved via specific base pairing. This base pairing affords a considerable degree of protection against chemical or physical damage and preserves coding potential. However, there are many situations, e.g. during DNA damage and programmed cellular processes such as DNA replication and transcription, in which the DNA duplex is separated into two singlestranded DNA (ssDNA) strands. This ssDNA is vulnerable to attack by nucleases, binding by inappropriate proteins and chemical attack. It is very important to control the generation of ssDNA and protect it when it forms, and for this reason all cellular organisms and many viruses encode a ssDNA binding protein (SSB). All known SSBs use an oligosaccharide/oligonucleotide binding (OB)-fold domain for DNA binding. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating strand-exchange proteins and helicases, and mediation of protein–protein interactions. Recently two additional human SSBs have been identified that are more closely related to bacterial and archaeal SSBs. Prior to this it was believed that replication protein A, RPA, was the only human equivalent of bacterial SSB. RPA is thought to be required for most aspects of DNA metabolism including DNA replication, recombination and repair. This review will discuss in further detail the biological pathways in which human SSBs function.

Physical and functional interaction of the archaeal single-stranded DNA-binding protein SSB with RNA polymerase

Archaeal transcription utilizes a complex multisubunit RNA polymerase and the basal transcription factors TBP and TF(II)B, closely resembling its eukaryal counterpart. We have uncovered a tight physical and functional interaction between RNA polymerase and the single-stranded DNA-binding protein SSB in Sulfolobus solfataricus. SSB stimulates transcription from promoters in vitro under TBP-limiting conditions and supports transcription in the absence of TBP. SSB also rescues transcription from repression by reconstituted chromatin. We demonstrate the potential for promoter melting by SSB, suggesting a plausible basis for the stimulation of transcription. This stimulation requires both the single-stranded DNA-binding domain and the acidic C-terminal tail of the SSB. The tail forms a stable interaction with RNA polymerase. These data reveal an unexpected role for single-stranded DNA-binding proteins in transcription in archaea.

hSSB1 interacts directly with the MRN complex stimulating its recruitment to DNA double-strand breaks and its endo-nuclease activity

hSSB1 is a recently discovered single-stranded DNA binding protein that is essential for efficient repair of DNA double-strand breaks (DSBs) by the homologous recombination pathway. hSSB1 is required for the efficient recruitment of the MRN complex to sites of DSBs and for the efficient initiation of ATM dependent signalling. Here we explore the interplay between hSSB1 and MRN. We demonstrate that hSSB1 binds directly to NBS1, a component of the MRN complex, in a DNA damage independent manner. Consistent with the direct interaction, we observe that hSSB1 greatly stimulates the endo-nuclease activity of the MRN complex, a process that requires the C-terminal tail of hSSB1. Interestingly, analysis of two point mutations in NBS1, associated with Nijmegen breakage syndrome, revealed weaker binding to hSSB1, suggesting a possible disease mechanism.

hSSB1 rapidly binds at the sites of DNA double-strand breaks and is required for the efficient recruitment of the MRN complex

hSSB1 is a newly discovered single-stranded DNA (ssDNA)-binding protein that is essential for efficient DNA double-strand break signalling through ATM. However, the mechanism by which hSSB1 functions to allow efficient signalling is unknown. Here, we show that hSSB1 is recruited rapidly to sites of double-strand DNA breaks (DSBs) in all interphase cells (G1, S and G2) independently of, CtIP, MDC1 and the MRN complex (Rad50, Mre11, NBS1). However expansion of hSSB1 from the DSB site requires the function of MRN. Strikingly, silencing of hSSB1 prevents foci formation as well as recruitment of MRN to sites of DSBs and leads to a subsequent defect in resection of DSBs as evident by defective RPA and ssDNA generation. Our data suggests that hSSB1 functions upstream of MRN to promote its recruitment at DSBs and is required for efficient resection of DSBs. These findings, together with previous work establish essential roles of hSSB1 in controlling ATM activation and activity, and subsequent DSB resection and homologous recombination (HR).

INTS3 controls the hSSB1-mediated DNA damage response

Human SSB1 (single-stranded binding protein 1 [hSSB1]) was recently identified as a part of the ataxia telangiectasia mutated (ATM) signaling pathway. To investigate hSSB1 function, we performed tandem affinity purifications of hSSB1 mutants mimicking the unphosphorylated and ATM-phosphorylated states. Both hSSB1 mutants copurified a subset of Integrator complex subunits and the uncharacterized protein LOC58493/c9orf80 (henceforth minute INTS3/hSSB-associated element [MISE]). The INTS3–MISE–hSSB1 complex plays a key role in ATM activation and RAD51 recruitment to DNA damage foci during the response to genotoxic stresses. These effects on the DNA damage response are caused by the control of hSSB1 transcription via INTS3, demonstrating a new network controlling hSSB1 function.

Involvement of Exo1b in DNA damage-induced apoptosis

Apoptosis is essential for the maintenance of inherited genomic integrity. During DNA damage-induced apoptosis, mechanisms of cell survival, such as DNA repair are inactivated to allow cell death to proceed. Here, we describe a role for the mammalian DNA repair enzyme Exonuclease 1 (Exo1) in DNA damage-induced apoptosis. Depletion of Exo1 in human fibroblasts, or mouse embryonic fibroblasts led to a delay in DNA damage-induced apoptosis. Furthermore, we show that Exo1 acts upstream of caspase-3, DNA fragmentation and cytochrome c release. In addition, induction of apoptosis with DNA-damaging agents led to cleavage of both isoforms of Exo1. The cleavage of Exo1 was mapped to Asp514, and shown to be mediated by caspase-3. Expression of a caspase-3 cleavage site mutant form of Exo1, Asp514Ala, prevented formation of the previously observed fragment without any affect on the onset of apoptosis. We conclude that Exo1 has a role in the timely induction of apoptosis and that it is subsequently cleaved and degraded during apoptosis, potentially inhibiting DNA damage repair.

Single-stranded DNA-binding protein hSSB1 is critical for genomic stability

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair1. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein–protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer1. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response.

Phosphorylation of Exo1 modulates homologous recombination repair of DNA double-strand breaks

DNA double-strand break (DSB) repair via the homologous recombination pathway is a multi-stage process, which results in repair of the DSB without loss of genetic information or fidelity. One essential step in this process is the generation of extended single-stranded DNA (ssDNA) regions at the break site. This ssDNA serves to induce cell cycle checkpoints and is required for Rad51 mediated strand invasion of the sister chromatid. Here, we show that human Exonuclease 1 (Exo1) is required for the normal repair of DSBs by HR. Cells depleted of Exo1 show chromosomal instability and hypersensitivity to ionising radiation (IR) exposure. We find that Exo1 accumulates rapidly at DSBs and is required for the recruitment of RPA and Rad51 to sites of DSBs, suggesting a role for Exo1 in ssDNA generation. Interestingly, the phosphorylation of Exo1 by ATM appears to regulate the activity of Exo1 following resection, allowing optimal Rad51 loading and the completion of HR repair. These data establish a role for Exo1 in resection of DSBs in human cells, highlighting the critical requirement of Exo1 for DSB repair via HR and thus the maintenance of genomic stability.

Queer/error : gay media systems and processes of abjection

Gay community media functions as a system with three nodes, in which the flows of information and capital theoretically benefit all parties: the gay community gains a sense of cohesion and citizenship through media; the gay media outlets profit from advertisers’ capital; and advertisers recoup their investments in lucrative ‘pink dollar’ revenue. But if a necessary corollary of all communication systems is error or noise, where—and what—are the errors in this system? In this paper we argue that the ‘error’ in the gay media system is Queerness, and that the gay media system ejects (in a process of Kristevan abjection) these Queer identities in order to function successfully. We examine the ways in which Queer identities are excluded from representation in such media through a discourse and content analysis of The Sydney Star Observer (Australia’s largest gay and lesbian paper). First, we analyse the way Queer bodies are excluded from the discourses that construct and reinforce both the ideal gay male body and the notions of homosexual essence required for that body to be meaningful. We then argue that abject Queerness returns in the SSO’s discourses of public health through the conspicuous absence of the AIDS-inflicted body (which we read as the epitome of the abject Queer), since this absence paradoxically conjures up a trace of that which the system tries to expel. We conclude by arguing that because the ‘Queer error’ is integral to the SSO, gay community media should practise a politics of Queer inclusion rather than exclusion.

Wavefront aberrations and the depth of focus of the human eye

The depth of focus (DOF) can be defined as the variation in image distance of a lens or an optical system which can be tolerated without incurring an objectionable lack of sharpness of focus. The DOF of the human eye serves a mechanism of blur tolerance. As long as the target image remains within the depth of focus in the image space, the eye will still perceive the image as being clear. A large DOF is especially important for presbyopic patients with partial or complete loss of accommodation (presbyopia), since this helps them to obtain an acceptable retinal image when viewing a target moving through a range of near to intermediate distances. The aim of this research was to investigate the DOF of the human eye and its association with the natural wavefront aberrations, and how higher order aberrations (HOAs) can be used to expand the DOF, in particular by inducing spherical aberrations ( 0 4 Z and 0 6 Z ). The depth of focus of the human eye can be measured using a variety of subjective and objective methods. Subjective measurements based on a Badal optical system have been widely adopted, through which the retinal image size can be kept constant. In such measurements, the subject.s tested eye is normally cyclopleged. Objective methods without the need of cycloplegia are also used, where the eye.s accommodative response is continuously monitored. Generally, the DOF measured by subjective methods are slightly larger than those measured objectively. In recent years, methods have also been developed to estimate DOF from retinal image quality metrics (IQMs) derived from the ocular wavefront aberrations. In such methods, the DOF is defined as the range of defocus error that degrades the retinal image quality calculated from the IQMs to a certain level of the possible maximum value. In this study, the effect of different amounts of HOAs on the DOF was theoretically evaluated by modelling and comparing the DOF of subjects from four different clinical groups, including young emmetropes (20 subjects), young myopes (19 subjects), presbyopes (32 subjects) and keratoconics (35 subjects). A novel IQM-based through-focus algorithm was developed to theoretically predict the DOF of subjects with their natural HOAs. Additional primary spherical aberration ( 0 4 Z ) was also induced in the wavefronts of myopes and presbyopes to simulate the effect of myopic refractive correction (e.g. LASIK) and presbyopic correction (e.g. progressive power IOL) on the subject.s DOF. Larger amounts of HOAs were found to lead to greater values of predicted DOF. The introduction of primary spherical aberration was found to provide moderate increase of DOF while slightly deteriorating the image quality at the same time. The predicted DOF was also affected by the IQMs and the threshold level adopted. We then investigated the influence of the chosen threshold level of the IQMs on the predicted DOF, and how it relates to the subjectively measured DOF. The subjective DOF was measured in a group of 17 normal subjects, and we used through-focus visual Strehl ratio based on optical transfer function (VSOTF) derived from their wavefront aberrations as the IQM to estimate the DOF. The results allowed comparison of the subjective DOF with the estimated DOF and determination of a threshold level for DOF estimation. Significant correlation was found between the subject.s estimated threshold level for the estimated DOF and HOA RMS (Pearson.s r=0.88, p<0.001). The linear correlation can be used to estimate the threshold level for each individual subject, subsequently leading to a method for estimating individual.s DOF from a single measurement of their wavefront aberrations. A subsequent study was conducted to investigate the DOF of keratoconic subjects. Significant increases of the level of HOAs, including spherical aberration, coma and trefoil, can be observed in keratoconic eyes. This population of subjects provides an opportunity to study the influence of these HOAs on DOF. It was also expected that the asymmetric aberrations (coma and trefoil) in the keratoconic eye could interact with defocus to cause regional blur of the target. A dual-Badal-channel optical system with a star-pattern target was used to measure the subjective DOF in 10 keratoconic eyes and compared to those from a group of 10 normal subjects. The DOF measured in keratoconic eyes was significantly larger than that in normal eyes. However there was not a strong correlation between the large amount of HOA RMS and DOF in keratoconic eyes. Among all HOA terms, spherical aberration was found to be the only HOA that helped to significantly increase the DOF in the studied keratoconic subjects. Through the first three studies, a comprehensive understanding of DOF and its association to the HOAs in the human eye had been achieved. An adaptive optics system was then designed and constructed. The system was capable of measuring and altering the wavefront aberrations in the subject.s eye and measuring the resulting DOF under the influence of different combination of HOAs. Using the AO system, we investigated the concept of extending the DOF through optimized combinations of 0 4 Z and 0 6 Z . Systematic introduction of a targeted amount of both 0 4 Z and 0 6 Z was found to significantly improve the DOF of healthy subjects. The use of wavefront combinations of 0 4 Z and 0 6 Z with opposite signs can further expand the DOF, rather than using 0 4 Z or 0 6 Z alone. The optimal wavefront combinations to expand the DOF were estimated using the ratio of increase in DOF and loss of retinal image quality defined by VSOTF. In the experiment, the optimal combinations of 0 4 Z and 0 6 Z were found to provide a better balance of DOF expansion and relatively smaller decreases in VA. Therefore, the optimal combinations of 0 4 Z and 0 6 Z provides a more efficient method to expand the DOF rather than 0 4 Z or 0 6 Z alone. This PhD research has shown that there is a positive correlation between the DOF and the eye.s wavefront aberrations. More aberrated eyes generally have a larger DOF. The association of DOF and the natural HOAs in normal subjects can be quantified, which allows the estimation of DOF directly from the ocular wavefront aberration. Among the Zernike HOA terms, spherical aberrations ( 0 4 Z and 0 6 Z ) were found to improve the DOF. Certain combinations of 0 4 Z and 0 6 Z provide a more effective method to expand DOF than using 0 4 Z or 0 6 Z alone, and this could be useful in the optimal design of presbyopic optical corrections such as multifocal contact lenses, intraocular lenses and laser corneal surgeries.

Note on similarity solutions for viscous flow over an impermeable and non-linearly (quadratic) stretching sheet

Similarity solutions for flow over an impermeable, non-linearly (quadratic) stretching sheet were studied recently by Raptis and Perdikis (Int. J. Non-linear Mech. 41 (2006) 527–529) using a stream function of the form ψ=αxf(η)+βx2g(η). A fundamental error in their problem formulation is pointed out. On correction, it is shown that similarity solutions do not exist for this choice of ψ