Promotion of Homologous Recombination and Genomic Stability by RAD51AP1 via RAD51 Recombinase Enhancement

Homologous recombination (HR) repairs chromosome damage and is indispensable for tumor suppression in humans. RAD51 mediates the DNA strand-pairing step in HR. RAD51 associated protein 1 (RAD51AP1) is a RAD51-interacting protein whose function has remained elusive. Knockdown of RAD51AP1 in human cells by RNA interference engenders sensitivity to different types of genotoxic stress, and RAD51AP1 is epistatic to the HR protein XRCC3. Moreover, RAD51AP1-depleted cells are impaired for the recombinational repair of a DNA double-strand break and exhibit chromatid breaks both spontaneously and upon DNA-damaging treatment. Purified RAD51AP1 binds both dsDNA and a D loop structure and, only when able to interact with RAD51, greatly stimulates the RAD51-mediated D loop reaction. Biochemical and cytological results show that RAD51AP1 functions at a step subsequent to the assembly of the RAD51-ssDNA nucleoprotein filament. Our findings provide evidence that RAD51AP1 helps maintain genomic integrity via RAD51 recombinase enhancement.

Mechanistic insights into RAD51-associated protein 1 (RAD51AP1) action in homologous DNA repair

Homologous recombination catalyzed by the RAD51 recombinase is essential for maintaining genome integrity upon the induction of DNA double strand breaks and other DNA lesions. By enhancing the recombinase activity of RAD51, RAD51AP1 (RAD51-associated protein 1) serves a key role in homologous recombination-mediated chromosome damage repair. We show here that RAD51AP1 harbors two distinct DNA binding domains that are both needed for maximal protein activity under physiological conditions. We have finely mapped the two DNA binding domains in RAD51AP1 and generated mutant variants that are impaired in either or both of the DNA binding domains. Examination of these mutants reveals that both domains are indispensable for RAD51AP1 function in cells. These and other results illuminate the mechanistic basis of RAD51AP1 action in homologous DNA repair.

Diabetic peripheral neuropathy and retinal tissue thickness

Using retinal imaging, the nature and extent of compromise of retinal structural integrity has been characterized in individuals suffering from diabetic peripheral neuropathy. These findings extend our understanding of the pathological processes involved in diabetic neuropathy and offer novel ophthalmic approaches to the diagnosis and monitoring of this debilitating condition.

Software watermarking resilient to debugging attacks

In 2006, Gaurav Gupta and Josef Pieprzyk presented an attack on the branch-based software watermarking scheme proposed by Ginger Myles and Hongxia Jin in 2005. The software watermarking model is based on replacing jump instructions or unconditional branch statements (UBS) by calls to a fingerprint branch function (FBF) that computes the correct target address of the UBS as a function of the generated fingerprint and integrity check. If the program is tampered with, the fingerprint and/or integrity checks change and the target address is not computed correctly. Gupta and Pieprzyk's attack uses debugger capabilities such as register and address lookup and breakpoints to minimize the requirement to manually inspect the software. Using these resources, the FBF and calls to the same is identified, correct displacement values are generated and calls to FBF are replaced by the original UBS transferring control of the attack to the correct target instruction. In this paper, we propose a watermarking model that provides security against such debugging attacks. Two primary measures taken are shifting the stack pointer modification operation from the FBF to the individual UBSs, and coding the stack pointer modification in the same language as that of the rest of the code rather than assembly language to avoid conspicuous contents. The manual component complexity increases from O(1) in the previous scheme to O(n) in our proposed scheme.

Senataxin plays an essential role with DNA damage response proteins in meiotic recombination and gene silencing

Senataxin, mutated in the human genetic disorder ataxia with oculomotor apraxia type 2 (AOA2), plays an important role in maintaining genome integrity by coordination of transcription, DNA replication, and the DNA damage response. We demonstrate that senataxin is essential for spermatogenesis and that it functions at two stages in meiosis during crossing-over in homologous recombination and in meiotic sex chromosome inactivation (MSCI). Disruption of the Setx gene caused persistence of DNA double-strand breaks, a defect in disassembly of Rad51 filaments, accumulation of DNA:RNA hybrids (R-loops), and ultimately a failure of crossing-over. Senataxin localised to the XY body in a Brca1-dependent manner, and in its absence there was incomplete localisation of DNA damage response proteins to the XY chromosomes and ATR was retained on the axial elements of these chromosomes, failing to diffuse out into chromatin. Furthermore persistence of RNA polymerase II activity, altered ubH2A distribution, and abnormal XY-linked gene expression in Setx⁻/⁻ revealed an essential role for senataxin in MSCI. These data support key roles for senataxin in coordinating meiotic crossing-over with transcription and in gene silencing to protect the integrity of the genome.

Diagnostics of rolling element bearings for the traction system of high speed trains : experimental evidences

Monitoring of the integrity of rolling element bearings in the traction system of high speed trains is a fundamental operation in order to avoid catastrophic failures and to implement effective condition-based maintenance strategies. Diagnostics of rolling element bearings is usually based on vibration signal analysis by means of suitable signal processing techniques. The experimental validation of such techniques has been traditionally performed by means of laboratory tests on artificially damaged bearings, while their actual effectiveness in industrial applications, particularly in the field of rail transport, remains scarcely investigated. This paper will address the diagnostics of bearings taken from the service after a long term operation on a high speed train. These worn bearings have been installed on a test-rig, consisting of a complete full-scale traction system of a high speed train, able to reproduce the effects of wheel-track interaction and bogie-wheelset dynamics. The results of the experimental campaign show that suitable signal processing techniques are able to diagnose bearing failures even in this harsh and noisy application. Moreover, the most suitable location of the sensors on the traction system is also proposed.

Experimental evidences in bearing diagnostics for traction system of high speed trains

Rolling element bearings are the most critical components in the traction system of high speed trains. Monitoring their integrity is a fundamental operation in order to avoid catastrophic failures and to implement effective condition based maintenance strategies. Generally, diagnostics of rolling element bearings is usually performed by analyzing vibration signals measured by accelerometers placed in the proximity of the bearing under investigation. Several papers have been published on this subject in the last two decades, mainly devoted to the development and assessment of signal processing techniques for diagnostics. The experimental validation of such techniques has been traditionally performed by means of laboratory tests on artificially damaged bearings, while their actual effectiveness in specific industrial applications, particularly in rail industry, remains scarcely investigated. This paper is aimed at filling this knowledge gap, by addressing the diagnostics of bearings taken from the service after a long term operation on the traction system of a high speed train. Moreover, in order to test the effectiveness of the diagnostic procedures in the environmental conditions peculiar to the rail application, a specific test-rig has been built, consisting of a complete full-scale train traction system, able to reproduce the effects of wheeltrack interaction and bogie-wheelset dynamics. The results of the experimental campaign show that suitable signal processing techniques are able to diagnose bearing failures even in this harsh and noisy application. Moreover, the most suitable location of the sensors on the traction system is proposed, in order to limit their number.

Celebrating and sustaining Indigenous knowledges through higher education

In its intervention at the 10th session of the United Nations Permanent Forum on Indigenous Issues 2010, the World Indigenous Network Higher Education Consortium (WINHEC) acknowledged that despite a history of protracted but limited attempts by Governments globally to address the low participation and graduation rates of Indigenous peoples from higher education at post graduate level, this continues to be an area of considerable concern. This paper speaks to the development of an innovative academic process that profiles the ground breaking work of WINHEC and a cohort of Indigenous academics in developing academic programs designed to address this systemic failure. The concept of these programs was endorsed in 2006 at a WINHEC conference where Indigenous representatives from across the world met to discuss in part, historical and contempory impediments to Indigenous success within higher education. The goal of WINHEC has been to develop a nested suite of inventive postgraduate awards founded within the scholarship of Indigenous Knowledge which encapsulates an epistemological approach. This has been a ground breaking process that has included collaborative and intellectual contributions of Indigenous academics from diverse cultural nations across the globe and, in particular, Australia. In 2012 the culmination of this dream and the suite of courses developed, honours and embrace the uniqueness of Indigenous Knowledge and the cultural integrity of Indigenous Leadership.

Introduction : Understanding Connections between Time, Space and the Body

The experiences and constructs of time, space and bodies saturate human discourse—naturally enough, since they are fundamental to existence—yet there has long been a tendency for the terms to be approached somewhat independently, belying the depth of their interconnections. It was a desire to address that apparent shortcoming that inspired this book, and the interdisciplinary meetings from which it was born, the 1st Global Conferences on ‘Time, Space and the Body’ and ‘Body Horror’ held in Sydney in February 2013. Following the lively, often provocative, exchange of ideas throughout those meetings, the writing here crosses conventional boundaries inhabiting everyday life and liminal experiences, across cultures, life circumstances, and bodily states. Through numerous theoretical frameworks and with reference to a variety of media, the authors problematize or deconstruct commonplace assumptions to reveal challenging new perspectives on the diverse cultures and communities which make our world. If there is an overarching theme of this collection it is diversity itself. The writers here come from numerous academic fields, but a good number of them also draw on first-hand cultural production in the arts: photography, sculpture and fine art instillation, for example. Of course, however laudable it might be, there is a potential problem in such diversity: does it produce fruitful dialogue moving toward creative, workable syntheses or simply a cacophony of competing, incomprehensible, barely comprehending voices? To a large degree this depends upon the intellectual, existential ambitions as well as the old-fashioned goodnatured tolerance of both writers and readers. But we hope three unifying characteristics are discernable in the following chapters viewed as a whole: firstly, a genuine concern for the world humans inhabit and the communities they form as bodies in space and time; secondly, an emphasis upon the experience of the human subject, exemplified perhaps by the number of chapters drawing on phenomenology; thirdly, an adventurous, explorative impulse associated with an underlying sense that being, since it is inseparable from the body’s temporality, is always becoming, and here the presence of poststructuralist influences is unmistakable, often explicit. Our challenge as editors has been to present the enormous variety of subjects and views in a way that would render the book coherent and at the same time encourage readers to make explorations themselves into realms they might usually consider beyond their field of interest. To that end we have divided the book into six sections around loosely defined themes, each offering different angles on how time and/or space unfold in and around bodies.

MAS NMR measurements of intact articular bovine cartilage

Articular cartilage (AC), an avascular connective tissue lining articulating surfaces of the long bones, comprises extracellular biopolymers. In functionally compromised states such as osteoarthritis, thinned or lost AC causes reduced mobility and increased health-care costs. Understanding of the characteristics responsible for the load bearing efficiency of AC and the factors leading to its degradation are incomplete. DTI shows the structural alignment of collagen in AC [1] and T2 relaxation measurements suggest that the average director of reorientational motion of water molecules depends on the degree of alignment of collagen in AC [2]. Information on the nature of the chemical interactions involved in functional AC is lacking. The need for AC structural integrity makes solid state NMR an ideal tool to study this tissue. We examined the contribution of water in different functional ‘compartments’ using 1H-MAS, 13C-MAS and 13C-CPMAS NMR of bovine patellar cartilage incubated in D2O. 1H-MAS spectra signal intensity was reduced due to H/D exchange without a measureable redistribution of relative signal intensity. Chemical shift anisotropy was estimated by lineshape analysis of multiple peaks in the 1H-MAS spinning sidebands. These asymmetrical sidebands suggested the presence of multiple water species in AC. Therefore, water was added in small aliquots to D2O saturated AC and the influence of H2O and D2O on organic components was studied with 13C-MAS-NMR and 13C-CPMAS-NMR. Signal intensity in 13C-MAS spectra showed no change in relative signal intensity throughout the spectrum. In 13C-CPMAS spectra, displacement of water by D2O resulted in a loss of signal in the aliphatic region due to a reduction in proton availability for cross-polarization. These results complement dehydration studies of cartilage using osmotic manipulation [3] and demonstrate components of cartilage that are in contact with mobile water.

Solid-state MAS NMR measurements of intact articular bovine cartilage

Articular cartilage (AC), an avascular connective tissue lining articulating surfaces of the long bones, comprises extracellular biopolymers. In functionally compromised states such as osteoarthritis, thinned or lost AC causes reduced mobility and increased health-care costs. Understanding of the characteristics responsible for the load bearing efficiency of AC and the factors leading to its degradation are incomplete. DTI shows the structural alignment of collagen in AC [1] and T2 relaxation measurements suggest that the average director of reorientational motion of water molecules depends on the degree of alignment of collagen in AC [2]. Information on the nature of the chemical interactions involved in functional AC is lacking. The need for AC structural integrity makes solid state NMR an ideal tool to study this tissue. We examined the contribution of water in different functional ‘compartments’ using 1H-MAS, 13C-MAS and 13C-CPMAS NMR of bovine patellar cartilage incubated in D2O. 1H-MAS spectra signal intensity was reduced due to H/D exchange without a measureable redistribution of relative signal intensity. Chemical shift anisotropy was estimated by lineshape analysis of multiple peaks in the 1H-MAS spinning sidebands. These asymmetrical sidebands suggested the presence of multiple water species in AC. Therefore, water was added in small aliquots to D2O saturated AC and the influence of H2O and D2O on organic components was studied with 13C-MAS-NMR and 13C-CPMAS-NMR. Signal intensity in 13C-MAS spectra showed no change in relative signal intensity throughout the spectrum. In 13C-CPMAS spectra, displacement of water by D2O resulted in a loss of signal in the aliphatic region due to a reduction in proton availability for cross-polarization. These results complement dehydration studies of cartilage using osmotic manipulation [3] and demonstrate components of cartilage that are in contact with mobile water.

Centrobin regulates the assembly of functional mitotic spindles

The proper function of the spindle is crucial to the high fidelity of chromosome segregation and is indispensable for tumor suppression in humans. Centrobin is a recently identified centrosomal protein that has a role in stabilizing the microtubule structure. Here we functionally characterize the defects in centrosome integrity and spindle assembly in Centrobin-depleted cells. Centrobin-depleted cells show a range of spindle abnormalities including unfocused poles that are not associated with centrosomes, S-shaped spindles and mini spindles. These cells undergo mitotic arrest and subsequently often die by apoptosis, as determined by live cell imaging. Co-depletion of Mad2 relieves the mitotic arrest, indicating that cells arrest due to a failure to silence the spindle checkpoint in metaphase. Consistent with this, Centrobin-depleted metaphase cells stained positive for BubR1 and BubR1 S676. Staining with a panel of centrosome markers showed a loss of centrosome anchoring to the mitotic spindle. Furthermore, these cells show less cold-stable microtubules and a shorter distance between kinetochore pairs. These results show a requirement of Centrobin in maintaining centrosome integrity, which in turn promotes anchoring of mitotic spindle to the centrosomes. Furthermore, this anchoring is required for the stability of microtubule–kinetochore attachments and biogenesis of tension-ridden and properly functioning mitotic spindle.

Maintaining the ethic of service [Editorial]

This issue of Precedent is concerned with professional legal ethics. ln my view, professional ethics are rules about how you do your job, based on moral principles. By virtue of the nature of the work they do, the reputation of the institution through which they are admitted to practice (the court), and the consequences that can flow if they act inappropriately or incompetently, lawyers are under constant scrutiny in all aspects of their lives. Errors, omissions or misdeeds in both their professional and their personal lives have the potential to damage them, their clients, the profession itself and the court. We ought never to take for granted the trust the public places in us to preserve the integrity of the legal system itself, especially in times when that system may be under threat, either from without or from within.

Duty to the court and the administration of justice : some examples, implications and clarifications

No liberal democracy can survive without popular trust in its judicial system. The legal profession and the judiciary enjoy a level of independence and autonomy from the executive that makes them both powerful and privileged. A UNIQUE AND ORGANIC DUTY: So long as the courts are seen to fulfil their duty to guard against encroachments by the executive on the freedoms and rights of individual citizens with integrity and credibility, they maintain enough public support to retain their normative authority. But support for those with power and privilege is easily undermined. It is contingent upon trust. Lawyers who breach that trust in ways that go to the heart of the legal system ought to expect to be made examples of and to suffer severe penalties. The good news is that the sorts of breach discussed here should be neither difficult to anticipate nor to avoid – in theory. In practice, smart and honest lawyers sometimes fall foul of these duties for all sorts of understandable (if not condonable) reasons. Law does not get practised in a social or cultural vacuum. Lawyers are people, and people have weaknesses, failings and stresses...

In pursuit of an architecture realism

The noble idea of studying seminal works to ‘see what we can learn’ has turned in the 1990s into ‘let’s see what we can take’ and in the last decade a more toxic derivative ‘what else can’t we take’. That is my observation as a student of architecture in the 1990s, and as a practitioner in the 2000s. In 2010, the sense that something is ending is clear. The next generation is rising and their gaze has shifted. The idea of classification (as a means of separation) was previously rejected by a generation of Postmodernists; the usefulness of difference declined. It’s there in the presence of plurality in the resulting architecture, a decision to mine history and seize in a willful manner. This is a process of looking back but never forward. It has been a mono-culture of absorption. The mono-culture rejected the pursuit of the realistic. It is a blanket suffocating all practice of architecture in this country from the mercantile to the intellectual. Independent reviews of Australia’s recent contributions to the Venice Architecture Biennales confirm the malaise. The next generation is beginning to reconsider classification as a means of unification. By acknowledging the characteristics of competing forces it is possible to bring them into a state of tension. Seeking a beautiful contrast is a means to a new end. In the political setting, this is described by Noel Pearson as the radical centre[1]. The concept transcends the political and in its most essential form is a cultural phenomenon. It resists the compromised position and suggests that we can look back while looking forward. The radical centre is the only demonstrated opportunity where it is possible to pursue a realistic architecture. A realistic architecture in Australia may be partially resolved by addressing our anxiety of permanence. Farrelly’s built desires[2] and Markham’s ritual demonstrations[3] are two ways into understanding the broader spectrum of permanence. But I think they are downstream of our core problem. Our problem, as architects, is that we are yet to come to terms with this place. Some call it landscape others call it country. Australian cities were laid out on what was mistaken for a blank canvas. On some occasions there was the consideration of the landscape when it presented insurmountable physical obstacles. The architecture since has continued to work on its piece of a constantly blank canvas. Even more ironic is the commercial awards programs that represent a claim within this framework but at best can only establish a dialogue within itself. This is a closed system unable to look forward. It is said that Melbourne is the most European city in the southern hemisphere but what is really being described there is the limitation of a senseless grid. After all, if Dutch landscape informs Dutch architecture why can’t the Australian landscape inform Australian architecture? To do that, we would have to acknowledge our moribund grasp of the meaning of the Australian landscape. Or more precisely what Indigenes call Country[4]. This is a complex notion and there are different ways into it. Country is experienced and understood through the senses and seared into memory. If one begins design at that starting point it is not unreasonable to think we can arrive at an end point that is a counter trajectory to where we have taken ourselves. A recent studio with Masters students confirmed this. Start by finding Country and it would be impossible to end up with a building looking like an Aboriginal man’s face. To date architecture in Australia has overwhelmingly ignored Country on the back of terra nullius. It can’t seem to get past the picturesque. Why is it so hard? The art world came to terms with this challenge, so too did the legal establishment, even the political scene headed into new waters. It would be easy to blame the budgets of commerce or the constraints of program or even the pressure of success. But that is too easy. Those factors are in fact the kind of limitations that opportunities grow out of. The past decade of economic plenty has, for the most part, smothered the idea that our capitals might enable civic settings or an architecture that is able to looks past lot line boundaries in a dignified manner. The denied opportunities of these settings to be prompted by the Country they occupy is criminal. The public realm is arrested in its development because we refuse to accept Country as a spatial condition. What we seem to be able to embrace is literal and symbolic gestures usually taking the form of a trumped up art installations. All talk – no action. To continue to leave the public realm to the stewardship of mercantile interests is like embracing derivative lending after the global financial crisis.Herein rests an argument for why we need a resourced Government Architect’s office operating not as an isolated lobbyist for business but as a steward of the public realm for both the past and the future. New South Wales is the leading model with Queensland close behind. That is not to say both do not have flaws but current calls for their cessation on the grounds of design parity poorly mask commercial self interest. In Queensland, lobbyists are heavily regulated now with an aim to ensure integrity and accountability. In essence, what I am speaking of will not be found in Reconciliation Action Plans that double as business plans, or the mining of Aboriginal culture for the next marketing gimmick, or even discussions around how to make buildings more ‘Aboriginal’. It will come from the next generation who reject the noxious mono-culture of absorption and embrace a counter trajectory to pursue an architecture of realism.