Legitimate Target Research Articles

Anti-Obese Hormone Adiponectin Regulates Emergency Hematopoiesis and Antibacterial Response Through Downregulation Of Socs3 In Hematopoietic Progenitor Cells

Anti-obesity countermeasures attract rising attention because obesity induces multiple health complications such as metabolic syndrome, predisposition to cancer, heart diseases, stroke, infections, etc. One of the key features of obesity is decreased adiponectin, a critical anti-diabetic hormone secreted by adipocytes to modulate a number of metabolic processes including glucose regulation and fatty acid oxidation. Hematopoietic stem and progenitor cells (HSPCs) expand in size against infections, especially bacterial infections through immune response. In spite of the emerging clinical and basic reports of adiponectin, precise mechanisms of adiponectin for infectious control, especially for hematopoietic response, are largely unknown.As adipocytes are one of the major cellular components of bone marrow microenvironment which is critical for the proliferation and maintenance of hematopoietic cells, we hypothesized that obesity-related dysfunction of adipocytes in bone marrow might cause compromised hematopoietic response leading to increased susceptibility to infection. With the use of high-fat-fed mice, a well-known obesity model, we revealed that obesity had no apparent impact on steady-state hematopoiesis except for a slightly hypercellular bone marrow. Upon G-CSF administration to mimic harmful infection in vivo, high-fat-fed mice showed impaired expansion of HSPCs and myeloid cells in the bone marrow, along with the reduction of myeloid cells in peripheral circulation, i.e. diminished “emergency hematopoiesis”. In a Listeria monocytogenes peritonitis model, high-fat-fed mice also confirmed reduced expansion of HSPCs and myeloid cells in the bone marrow, attenuated leukocytosis and infiltration of leucocytes into the peritoneal cavity, resulting in a delayed bacterial clearance in vivo. Interestingly, the concentration of adiponectin was significantly reduced in the bone marrow of high-fat-fed mice compared to control mice, possibly reflecting a high relevance of adiponectin to hematopoietic response. To assess the exact role of adiponectin in emergency hematopoiesis, we utilized adiponectin-deficient (adipo-/-) mice. Genetic ablation of adiponectin caused no change in steady-state hematopoiesis, whereas G-CSF administration in adipo-/- mice showed attenuated HSPC expansion in the bone marrow, consistent with a series of high-fat-fed mice. Reciprocal transplants with adipo+/+ and adipo -/- mice demonstrated that adiponectin from bone marrow environment of the recipient, not from the donor hematopoietic cells, is essential for the efficient expansion of HSPCs in response to G-CSF. Furthermore, adipo-/- mice showed reduced hematopoietic response against Listeria monocytogenes infection, similar to the phenotype of high-fat-fed mice. Importantly, we confirmed that intravenous administration of recombinant adiponectin restored the responsiveness to G-CSF and the bacterial clearance on Listeria infection, both in adipo-/- and high-fat-fed mice, which affirm the importance of adiponectin treatment against infection. In HSPCs and myeloid cells from adipo-/- mice or high-fat-fed mice, phosphorylation of Stat3, a key component for emergency hematopoiesis, was suppressed, underscoring a role of adiponectin in emergency hematopoiesis at the molecular level. We revealed that those cells aberrantly expressed Socs3, a major inhibitory factor of Stat3, and that was reverted by intravenous administration of adiponectin, indicating that adiponectin potentiated G-CSF-dependent Stat3 phosphorylation in vitro and in vivo. These data revealed that adiponectin regulates hematopoietic response against infections, highlighting adiponectin as a legitimate target against infectious diseases in obese patients. From our findings breakthrough drugs to increase adiponectin are highly warranted. Disclosures:No relevant conflicts of interest to declare.

BRD4 Sustains Melanoma Proliferation and Represents a New Target for Epigenetic Therapy

Metastatic melanoma remains a mostly incurable disease. Although newly approved targeted therapies are efficacious in a subset of patients, resistance and relapse rapidly ensue. Alternative therapeutic strategies to manipulate epigenetic regulators and disrupt the transcriptional program that maintains tumor cell identity are emerging. Bromodomain and extraterminal domain (BET) proteins are epigenome readers known to exert key roles at the interface between chromatin remodeling and transcriptional regulation. Here, we report that BRD4, a BET family member, is significantly upregulated in primary and metastatic melanoma tissues compared with melanocytes and nevi. Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. RNA sequencing of BET inhibitor-treated cells followed by Gene Ontology analysis showed a striking impact on transcriptional programs controlling cell growth, proliferation, cell-cycle regulation, and differentiation. In particular, we found that, rapidly after BET displacement, key cell-cycle genes (SKP2, ERK1, and c-MYC) were downregulated concomitantly with the accumulation of cyclin-dependent kinase (CDK) inhibitors (p21 and p27), followed by cell-cycle arrest. Importantly, BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status, opening the possibility of using these small-molecule compounds to treat patients for whom no effective targeted therapy exists. Collectively, our study reveals a critical role for BRD4 in melanoma tumor maintenance and renders it a legitimate and novel target for epigenetic therapy directed against the core transcriptional program of melanoma.

Joint reactive jammer detection and localization in an enterprise WiFi network

The deployment of wireless networks for Internet connectivity has been rapid during the last decade. Following this trend, enterprises utilize multiple access points (APs) in order to provide wireless connectivity to authorized users within its premises. However, wireless networks are extremely vulnerable to PHY/MAC layer attacks such as jamming. A jammer transmits electromagnetic energy on the medium in order to either block the access to any legitimate transmitter or cause collisions at the receiver (or both). One of the most advanced jamming models is that of reactive jamming. A reactive jammer does not constantly transmit energy on the air, but only jams when a legitimate (target) packet is on the medium, aiming at its collision at the receiver. Previous studies have shown that reactive jamming is one of the most difficult attack models to detect. In this work, we propose a scheme that performs both detection and localization of a reactive jammer in an enterprise WiFi network. In brief, a reactive jammer can virtually increase the interference range of the target AP and thus increase the busy times of nearby APs that use the same frequency. By quantifying this effect we are able to accurately detect the presence of a reactive jammer and perform a coarse grain localization of the jammer. Our simulation results show that our scheme can achieve high true positives and low false positives simultaneously. In addition, our coarse grain localization scheme exhibits error on the order of the AP coverage area.

Distribution of National Institutes of Health Stroke Scale in the Cincinnati/Northern Kentucky Stroke Study

Little is known about the distribution of National Institutes of Health Stroke Scale (NIHSS) scores from patients with ischemic stroke sampled from population-based studies. We describe the distribution of NIHSS in ischemic stroke cases from the Cincinnati/Northern Kentucky Stroke Study. Within a biracial population of 1.3 million, all strokes among area residents in 2005 were ascertained by screening discharge records at local hospitals and outpatient clinics. A sampling scheme was developed to ascertain additional cases presenting to physician offices and nursing homes, not identified through the other sources. All confirmed ischemic stroke cases underwent chart abstraction, and a retrospective NIHSS (rNIHSS) score (range, 0-42) was generated on the basis of initial physician examination findings. There were 2233 ischemic stroke cases identified during the 12-month study. The overall median rNIHSS score was 3 (interquartile range, 1-7). Median rNIHSS score was 3, 7, and 1, respectively, for stroke cases ascertained through the admitted, in-hospital, and out-of-hospital sources. Median rNIHSS was significantly higher in subjects ≥80 years compared with younger cases (4 versus 3). More than half of all ischemic stroke cases have mild symptom severity on initial presentation (ie, rNIHSS≤3). Monitoring trends in NIHSS represents a legitimate target for population-based surveillance efforts.

Restricting Justice: Political Interventions in the Home and in the Market

Restricting Justice: Political Interventions in the Home and in the Market

High-density lipoproteins: A consensus statement from the National Lipid Association

For >4 decades it has been recognized that elevated serum levels of high-density lipoprotein cholesterol (HDL-C) are associated with reduced risk of cardiovascular disease (CVD) and its sequelae. Many prospective observational studies performed around the world have confirmed an inverse relationship between HDL-C and cardiovascular risk in people irrespective of sex, race, or ethnicity. Consequently, it was assumed that, by extension, raising HDL-C through lifestyle modification and pharmacologic intervention would reduce risk of CVD. Animal studies are consistent with this assumption. Lipid treatment guidelines around the world promoted the recognition of HDL-C as a therapeutic target, especially in high-risk patients. Some post hoc analyses from randomized controlled trials also suggest that raising HDL-C beneficially affects the risk of CVD. However, a number of recent randomized studies putatively designed to test the "HDL hypothesis" have failed to show benefit. The results of these trials have caused many clinicians to question whether HDL-C is a legitimate therapeutic target. In response to the many questions and uncertainties raised by the results of these trials, the National Lipid Association convened an expert panel to evaluate the current status of HDL-C as a therapeutic target; to review the current state of knowledge of HDL particle structure, composition, and function; and to identify the salient questions yet to be answered about the role of HDL in either preventing or contributing to atherosclerotic disease. The expert panel's conclusions and clinical recommendations are summarized herein. The panel concludes that, although low HDL-C identifies patients at elevated risk, and much investigation suggests that HDL may play a variety of antiatherogenic roles, HDL-C is not a therapeutic target at the present time. Risk stratified atherogenic lipoprotein burden (low-density lipoprotein cholesterol and non-HDL-C) should remain the primary and secondary targets of therapy in patients at risk, as described by established guidelines. The National Lipid Association emphasizes that rigorous research into the biology and clinical significance of low HDL-C should continue. The development of novel drugs designed to modulate the serum levels and functionality of HDL particles should also continue. On the basis of an enormous amount of basic scientific and clinical investigation, a considerable number of reasons support the need to continue to investigate the therapeutic effect of modulating HDL structure and function.

Clean fake: Authenticating documents and persons in migrant Moscow

ABSTRACTWhat does it mean for Kyrgyzstani migrant workers in contemporary Russia to be legally legible to the state when informal agencies market fictive residency documents and “clean fake” work permits? Examining the uncertainty around being authentically documented provides insight into a mode of governance in urban Russia that thrives less on rendering subjects legible than on working the space of ambiguity between life and law. This dynamic has significant social consequences for the way certain bodies come to be scrutinized as particularly untrustworthy, particularly liable to fakery, and, thus, particularly legitimate targets for document checks, fines, and threats of deportation. The ambiguity with which migrants are forced to live highlights the need to explore how documentary regimes, structures of feeling, and racializing practices coincide.

“Law, Liberty and Morality”: Fifty Years On

In 1962 H.L.A. Hart delivered the prestigious Harry Camp Lectures at Stanford University. The three lectures were published the following year with the title of Law, Liberty and Morality and were soon recognized as one of Hart’s most important contributions to legal and political philosophy. The context in which the lectures were written is well known. In the autumn of 1957 the Report of the Committee on Homosexual Offences and Prostitution, known as the ‘Wolfenden Report’, was published in London. The report stated that homosexual behaviour between consenting adults in private should not be a criminal offence, and that there ‘must remain a realm of private morality and immorality which is, in brief and crude terms, not the law’s business’. Lord Devlin, a prominent conservative judge, criticized the Report by arguing that since a society’s existence depends on the maintenance of a core set of shared political and moral values, any conduct that threatens these values thereby threatens the very existence of the society built around them. Conduct of this sort, Devlin argued, cannot be considered purely private and therefore is the legitimate target of criminal law sanctions. Law, Liberty and Morality was written as a response to Devlin’s arguments. Hart criticized the connection instituted by Devlin between immorality and ‘social disintegration’ and defended the view that the only purposes for which criminal law sanctions can be legitimately inflicted are preventing harm or offense to others and (in some cases) preventing harm to self. Hart’s view is widely recognized, both by its defenders and by its critics, as the most powerful formulation that the liberal position had received since J.S. Mill’s On Liberty, and its influence on the philosophical debate of the last 50 years can hardly be overestimated. The contributions to this symposium take a fresh look at this debate with the aim of critically engaging with the position originally defended by Hart as well as exploring new solutions to some of the issues that were raised in his exchange with Devlin. The first paper, by Jeffrie Murphy, closely focuses on Law, Liberty and Morality, raising some

Abstract A10: BRD4 is a new therapeutic target in melanoma

Abstract Metastatic melanoma remains a mostly incurable disease. Although newly approved targeted therapies are efficacious in a subset of patients, resistance and relapse rapidly ensue. Alternative therapeutic strategies to manipulate epigenetic regulators and disrupt the transcriptional program that maintains tumor cell identity are emerging. Bromodomain and extraterminal domain (BET) family of proteins consists of BRD2, BRD3, BRD4, and testis- specific BRDT, and are epigenome readers known to exert key roles at the interface between chromatin remodeling and transcriptional regulation. We investigated the role of BET proteins in melanoma tumor maintenance and assessed their value as therapeutic targets. Data mining of our previously published gene expression profile of melanoma cell lines and immunostaining of melanoma tissue microarray revealed that BRD4 is significantly upregulated in primary and metastatic melanoma tissues compared to melanocytes and nevi, thus suggesting a potential role for BET family proteins in promoting melanoma tumorigenesis. Treatment with BET inhibitors impaired melanoma cell proliferation and colony formation in vitro. Moreover, tumor growth and metastatic behavior assessed by a xenograft model also revealed impairment of melanoma proliferation in vivo. These effects were mostly recapitulated by individual silencing of BRD4, and not of other BET family members. RNA sequencing of BET inhibitor-treated cells followed by gene ontology analysis showed a striking impact on transcriptional programs controlling cell growth, proliferation, cell-cycle regulation and differentiation. In particular, we found that, rapidly after BET displacement, key cell cycle genes (SKP2, ERK1 and c-MYC) were downregulated concomitantly with the accumulation of CDK inhibitors (p21, p27), followed by melanoma cell cycle arrest. However, single genetic manipulation of these cell cycle genes did not rescue the cytostatic effect of BET inhibition, suggesting that BET inactivation leads to a non-redundant, simultaneous regulation of multiple cell cycle effectors. Interestingly, SKP2 and ERK1 mRNA levels directly correlated with those of BRD4 in a panel of melanoma tissues, suggesting that these two factors may be direct BRD4 targets. Importantly, the effects of the BET inhibitor were not influenced by BRAF or NRAS mutational status, opening the possibility of using these small molecule compounds to treat patients for whom no effective targeted therapy currently exists. Collectively, our results strongly support a critical role for BRD4 in melanoma tumor maintenance, and render it a legitimate and novel target for epigenetic therapy directed against the core transcriptional program of melanoma. Citation Format: Barbara Fontanals-Cirera, Miguel F. Segura, Avital Gaziel-Sovran, Maria V. Guijarro, Doug Hanniford, Pilar Gonzalez-Gomez, Weijia Zhang, Guantao Zhang, Farbod Darvishian, Michael Ohlmeyer, Iman Osman, Ming-Ming Zhou, Eva Hernando. BRD4 is a new therapeutic target in melanoma. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A10.

Editorial (Hot Topic: MiRNAs as Legitimate Targets for Cancer Therapy)

Editorial (Hot Topic: MiRNAs as Legitimate Targets for Cancer Therapy)

Molecular changes in the phosphatidylinositide 3‐kinase (PI3K) pathway are common in gastric cancer

The phosphatidylinositide 3-kinase (PI3K) pathway is an important signalling pathway that is frequently activated in cancer cells. This has led to the emergence of PI3K inhibitors as potential new treatment modalities for many cancers. We have investigated the frequency of molecular changes in the PI3K pathway in gastric cancer. A series of sixty one human gastric cancer specimens and nine human gastric cancer cell lines were screened for PIK3CA mutations and copy number gain by direct sequencing and multiplex ligation-dependent probe amplification (MLPA), respectively. PTEN protein levels were assessed by immunohistochemistry. Alterations in the PI3K pathway were found in 33 of 61 (54%) gastric tumours. PIK3CA mutation and copy number gain were detected in 3 (4.9%) and 8 (13.1%), respectively, of 61 gastric cancer samples while PTEN loss was detected in 24 (39%) of the tumours. Two tumours had both PTEN loss and PIK3CA copy number gain. There were no significant associations between these PI3K pathway changes and the clinical features of the tumours. Alterations in the PI3K pathway are frequent in gastric tumours implicating this pathway as a legitimate therapeutic target in gastric cancer.

Lipid profiles of non-diabetic healthy and ischaemic heart disease patients.

To find any difference in the fasting lipid profile in patients with history of ischaemic heart disease (IHD) and established atherosclerotic plaques on angiography and in subjects with no known history of IHD. Observational, cross-sectional study. Immunology Department of University of Health Sciences, Lahore, from January 2007 to January 2009. In this study, 200 male subjects, between 40 and 60 years of age were recruited. Serum cholesterol and triglycerides were determined by enzymatic CHOD-PAP and GPO-PAP calorimetric method, HDL-C by accelerator selective detergent method and LDL-C by direct homogeneous enzymatic method. Serum ox-LDL contents were determined by using quantitative sandwich enzyme immuno-assay kits. The total serum cholesterol, triglyceride and LDL-C levels were within the normal range in control and patient groups whereas HDL-C levels were significantly higher in the control group compared to the patient group (p = 0.001). A significant difference (p = 0.001) for HDL-C levels was observed between smokers and non-smokers. Serum ox-LDL levels were higher in patient group as compared to the control group but the difference was not statistically significant. The significantly lower HDL levels in patient group with normal cholesterol, LDL and triglyceride levels may suggest low HDL-C has a greater role in IHD. Raising plasma HDL-cholesterol through weight loss, healthy diet, increased physical activity and by proper pharmacotherapy is, therefore, a legitimate therapeutic target for the optimal prevention of CHD in native population.

Turning Points of World War I: An Interview with Ian F.W. Beckett

Turning Points of World War I: An Interview with Ian F.W. Beckett Donald A. Yerxa AS WE APPROACH THE CENTENARY OF THE OUTBREAK OF World War I, there will be no dearth of books dealing with the conflict. And in these pages we will be highlighting some of the best of these, especially those that look at the war in a new light. In The Making of the First World War (Yale University Press, 2013) Ian F.W. Beckett discusses twelve pivotal moments that not only changed the outcome of the war, but also the global history of the 20th century. Senior editor Donald A. Yerxa interviewed Beckett in January 2013. Donald A. Yerxa: Would you provide our readers with a summary of what you are doing inThe Making of the First World War? Ian F.W. Beckett: I was asked to comment on a manuscript suggesting various turning points in the Second World War. While they were clearly significant, the chosen events were all either political or military, and of immediate impact. I suggested that one should look beyond purely political and military episodes. I was then invited, or rather challenged, to show what I meant in terms of the Great War. Accordingly, I am trying to illustrate a broad range of events between 1914 and 1919 that might be considered important turning points or pivotal moments, not just at the precise moment they occurred but also for the future unfolding of historical events. I also wanted to illustrate social and cultural themes, and to be consciously global rather than purely Eurocentric in my choices. Yerxa: How did you go about selecting the episodes you discuss in the book? Beckett: I began with a fairly long list and whittled it down in the end to twelve illustrative episodes. Some events of lasting global significance had to be included, such as the fall of the Romanovs, the division of the Ottoman Empire in the Middle East—I deal with Turkish entry to the war in 1914 as well as the Balfour Declaration in 1917—and Woodrow Wilson’s impact on peacemaking. I felt that what might be termed “decisive battles”—and even more so the opening days of prolonged campaigns— were a cul-de-sac to avoid. Consider the continuing popular British obsession with July 1, 1916, the opening day of the battle of the Somme. This has had a cultural impact in Britain, but only since the 1960s; the Anzac landing on Gallipoli in April 1915 had a cultural impact at the time, and it continues to this day in terms of Australian perceptions. I also wanted to illustrate aspects of the “totality” of the war such as economic mobilization and the ways in which civilians became legitimate targets for attack. I wanted to choose some less obvious events, such as the Belgians opening the floodgates to close off the last open sector of what was to become the Western Front in 1914. The symbolism of the death of Emperor Franz Joseph also seemed an interesting case to examine. Above all, I wanted to provoke a debate on what constitutes historical turning points that might be useful for students as well as interesting to general readers. Click for larger view View full resolution A scene from the Somme in The Devil is Coming, episode 13 of the BBC's Great War series (1964). Yerxa: What prompted you to write this particular book on World War I? Beckett: I had more or less decided that I had written enough on the First World War after completing the second edition of my general text, The Great War, 1914–18, in 2007. But it is such a fascinating and complex conflict that I though it worth revisiting one more time, and it seemed a way perhaps of reaching an audience that would not have been necessarily exposed to some of the academic revisionism that has revolutionized historians’ views of the war in the last decade or so. In Britain in particular there is still this popular image of futility that needs to be challenged. Yerxa: What was the impact of the first true war documentary,The Battle of the Somme...

Decrease of α-Chains in β-Thalassemia

In the pathophysiology of β-thalassemia, globin chain imbalance plays a central role in predicting red blood cell (RBC) life span and disease severity. Strategies to improve globin chain imbalance are therefore a legitimate target in the management of this incurable genetic disorder. Classical gene addition with the available retroviral vectors can alter one of the two variables while combined reduction of achains could provide a more potent therapeutic effect. We developed foamy virus (FV) vectors for the production of β-globin and vectors targeting the a-globin transcript using the shRNA technology. Using FVderived vectors, we expressed human anti-a-globin short hairpin RNAs, off a potent PolIII promoter (H1); of the 4 different shRNAs tested, α-globin mRNA reduction varied from 6.3 to 54% of the control CD34+ cells. Similarly, vectors developed for the mouse α-globin, resulted in a significant reduction (range 15-28% of the control) of aglobin in erythroid colonies of Lin- cells. To assay vector performance in vivo, we investigated the hematological parameters in thal3+/- mice transpalnted with FV-transduced Lin- cells, transduced with anti-alpha-globin shRNA. Despite low chimerism and low vector copy numbers (<0.5 per cell), we observed a 10% reduction in red cell distribution width, a marker for distorted erythropoiesis. We finally developed a combination FV vector expressing β-globin off a HS40 enhancer and anti-α-globin shRNA and tested its performance in human CD34+ cells from a thalassemic patient. Globin chain imbalance was ameliorated from a β/α ratio of 0.12 to the level of 0.5, clearly indicating a therapeutic benefit. Overall, shRNA control of α-globin excess is a feasible target but requires improvements since the RNAi effect is tough to predict and should ideally be combined with controllable elements.

“Don’t Know” Responses to Risk Perception Measures

Risk perceptions are legitimate targets for behavioral interventions because they can motivate medical decisions and health behaviors. However, some survey respondents may not know (or may not indicate) their risk perceptions. The scope of "don't know" (DK) responding is unknown. Examine the prevalence and correlates of responding DK to items assessing perceived risk of colorectal cancer. Two nationally representative, population-based, cross-sectional surveys (2005 National Health Interview Survey [NHIS]; 2005 Health Information National Trends Survey [HINTS]), and one primary care clinic-based survey comprised of individuals from low-income communities. Analyses included 31,202 (NHIS), 1,937 (HINTS), and 769 (clinic) individuals. Five items assessed perceived risk of colorectal cancer. Four of the items differed in format and/or response scale: comparative risk (NHIS, HINTS); absolute risk (HINTS, clinic), and "likelihood" and "chance" response scales (clinic). Only the clinic-based survey included an explicit DK response option. "Don't know" responding was 6.9% (NHIS), 7.5% (HINTS-comparative), and 8.7% (HINTS-absolute). "Don't know" responding was 49.1% and 69.3% for the "chance" and "likely" response options (clinic). Correlates of DK responding were characteristics generally associated with disparities (e.g., low education), but the pattern of results varied among samples, question formats, and response scales. The surveys were developed independently and employed different methodologies and items. Consequently, the results were not directly comparable. There may be multiple explanations for differences in the magnitude and characteristics of DK responding. "Don't know" responding is more prevalent in populations affected by health disparities. Either not assessing or not analyzing DK responses could further disenfranchise these populations and negatively affect the validity of research and the efficacy of interventions seeking to eliminate health disparities.

Surgeon Vice Admiral Sir Godfrey Milton-Thompson 1930 – 2012

Drones enhance military capability and form a potent element of force protection, allowing humans to be removed from hazardous environments and tedious jobs. However, there are moral, legal, and political dangers associated with their use. Although a time may come when it is possible to develop a drone that is able to autonomously and ethically engage a legitimate target with greater reliability than a human, until then military drones demand a crawl-walk-run development methodology, consent by military personnel for weapon use, and continued debate about the complex issues surrounding their deployment.

CLIP (Cross-Linking and Immunoprecipitation) Identification of RNAs Bound by a Specific Protein

Guilt by association remains a powerful argument for assigning function in biological processes: Proteins that bind a specific RNA are strongly implicated in modulating the fate and action of that RNA. Detecting such associations is a major goal in understanding RNA function. The cross-linking and immunoprecipitation (CLIP) method described here allows assignments of RNA-binding proteins to their RNA targets by using ultraviolet (UV) cross-linking to covalently capture close (near-covalent bond distances) association of RNA with protein, followed by immunopurification of the protein partner with extraction and subsequent characterization of the RNA partner sequence(s). Pools of target sequences can be captured and their genomic origins are studied so that regulatory and other functional relationships between the protein and its RNA targets can be inferred. The advantage of this protocol is that it captures only intimately associated RNAs and proteins, and so is expected to be highly specific. Some investigators report capturing cross-links between rRNA and nonribosomal proteins in cases where ribosomes are not efficiently cleared from the lysate before immunopurification of the target protein, so the limits of specificity for the method are yet to be fully explored. Its main disadvantages are its many steps and that it may fail to work for all proteins or capture all legitimate targets of any given protein if the necessary chemical groups are not optimally arranged for photocross-linking in the complex. However, properly performed, this method should provide many useful insights into the nature of the RNA ligands for an RNA-binding protein of interest.

Cyber Warfare: Applying the Principle of Distinction in an Interconnected Space

While the rules of the jus in bello are generally operative in cyberspace, it appears to be problematic to apply the fundamental principle of distinction because of the systemic interconnection of military and civilian infrastructure in the cyber realm. In this regard, the application of the accepted legal definition of military objectives will make various components of the civilian cyber infrastructure a legitimate military objective. In order to avoid serious repercussions for the civilian population that might follow from this inherent interconnectedness, different concepts are analysed that could provide potential solutions for a clearer separation of legitimate military targets and protected civilian installations and networks. The approaches discussed range from the exemption of central cyber infrastructure components that serve important civilian functions, to the creation of ‘digital safe havens’ and possible precautionary obligations regarding the segregation of military and civilian networks. As a solution, the authors propose a dynamic interpretation of the wording ‘damage to civilian objects’ within the principle of proportionality of Article 51(5)(b) of Additional Protocol I, an interpretation that would comprise the degradation of the functionality of systems that serve important civilian functions.

The Image before the Weapon

For centuries, philosophers and publicists have sought to formalize the distinction between combatants and civilians under what is known as the principle of distinction. Although this principle has long been viewed as stable and relatively straightforward, Helen M. Kinsella demonstrates in The Image before Weapon that it is anything but. From ancient philosophers to modern scholars, Kinsella illustrates how the discourses of gender, civilization, and innocence have essentialized what is often an arbitrary line between legitimate and illegitimate targets of violence during war. Moreover, these lines have been drawn in such a way that they serve the interests of the dominant military power at any given time, while at the same time reproducing and naturalizing political systems of dominance and control. Kinsella begins by interrogating the notions of immunity and just conduct of war that evolved in early Western thought. She argues that the notion of who may be legitimately targeted versus who must be spared did not, contrary to common belief, form the basis for universal rights for the innocent (generally understood to be women, children, and old men), but to serve the interests of the dominant powers. For Thomas Acquinas, for example, the concern was more “with the innocence of the soldier than with the innocence of the one who may be killed” (35). The distinction played an important function in the noble knights' chivalric code, which gave the Crusades their legitimacy and allowed them to “reconcile the warring way of life…with spiritual renewal and grace—as well as to gain substantial economic and social rewards.” At the same time, it allowed the Catholic Church to “assert its sacred authority and to reclaim the Holy Land” (42). To show how feeble these protections were, Kinsella notes that the protection of civilians did not extend to the uncivilized or “heathen” peoples. Instead, it established a bright line between the civilized and the uncivilized, reifying the right of Christian armies to conquer non-Western populations.

Alcohol-Impaired Driving in the United States: Contributors to the Problem and Effective Countermeasures

Objective: To review the effectiveness of current countermeasures in alcohol-impaired driving. Method: This article provides an overview of the contributors to the alcohol-impaired driving problem in the United States and reviews the effectiveness of alcohol-impaired driving countermeasures. Results: Many effective countermeasures have been used during the past few decades both to deter drivers from driving when they are over the legal limit for alcohol and to discourage driving while intoxicated (DWI) offenders from reoffending once they have been caught and convicted. In recent years, greater attention has been given to the problem of “hardcore” drinking drivers, a term coined to refer to those who repeatedly drive with high blood alcohol concentrations and are resistant to changing their behavior. Although such individuals are a legitimate target for attention, focusing predominantly on this group will result in missed opportunities to address a large portion of alcohol-impaired driving crashes. This article provides a review of the primary countermeasures that have been used to reduce alcohol-impaired driving and summarizes evidence for their effectiveness. It asks the question of where, in an environment of limited resources, attention should be focused. Conclusions: General deterrent approaches, such as frequent and highly publicized sobriety checkpoints, have the greatest potential to save lives and should be the mainstay of state and local efforts. Specific deterrent approaches, aimed at deterring DWI offenders from reoffending, such as alcohol ignition interlocks, should be applied to all apprehended drivers, whatever their drinking history. Evidence suggests that they could benefit from them. In the future, advanced in-vehicle technologies that would prevent vehicles from being driven when their drivers are over the legal limit may hold the key to drastically reducing the alcohol-impaired driving problem.