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Purpose: Problems in management of oral cancers or precancers include identification of patients at risk for metastasis, tumor recurrence, and second primary tumors or risk for progression of precancers (dysplasia) to cancer. Thus, the objective of this study was to clarify the role of genomic aberrations in oral cancer progression and metastasis.Experimental Design: The spectrum of copy number alterations in oral dysplasia and squamous cell carcinomas (SCC) was determined by array comparative genomic hybridization. Associations with clinical characteristics were studied and results confirmed in an independent cohort.Results: The presence of one or more of the chromosomal aberrations +3q24-qter, -8pter-p23.1, +8q12-q24.2, and +20 distinguishes a major subgroup (70%–80% of lesions, termed 3q8pq20 subtype) from the remainder (20%–30% of lesions, non-3q8pq20). The 3q8pq20 subtype is associated with chromosomal instability and differential methylation in the most chromosomally unstable tumors. The two subtypes differ significantly in clinical outcome with risk for cervical (neck) lymph node metastasis almost exclusively associated with the 3q8pq20 subtype in two independent oral SCC cohorts.Conclusions: Two subtypes of oral lesions indicative of at least two pathways for oral cancer development were distinguished that differ in chromosomal instability and risk for metastasis, suggesting that +3q,–8p, +8q, and +20 constitute a biomarker with clinical utility for identifying patients at risk for metastasis. Moreover, although increased numbers of genomic alterations can be harbingers of progression to cancer, dysplastic lesions lacking copy number changes cannot be considered benign as they are potential precursors to non-3q8pq20 locally invasive, yet not metastatic oral SCC. Clin Cancer Res; 17(22); 7024–34. ©2011 AACR.

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Receptor-interacting protein kinase 1 (RIPK1), a key component of the cellular necroptosis pathway, has gained recognition as an important therapeutic target. Pharmacologic inhibition or genetic inactivation of RIPK1 has shown promise in animal models of disease ranging from acute ischemic conditions, chronic inflammation, and neurodegeneration. We present here a class of RIPK1 inhibitors that is distinguished by a lack of a lipophilic aromatic group present in most literature inhibitors that typically occupies a hydrophobic back pocket of the protein active site. Despite not having this ubiquitous feature of many known RIPK1 inhibitors, we were able to obtain compounds with good potency, kinase selectivity, and pharmacokinetic properties in rats. The use of the lipophilic yet metabolically stable pentafluoroethyl group was critical to balancing the potency and properties of optimized analogs.

The kinase RIP1 acts in multiple signaling pathways to regulate inflammatory responses and it can trigger both apoptosis and necroptosis. Its kinase activity has been implicated in a range of inflammatory, neurodegenerative, and oncogenic diseases. Here, we explore the effect of inhibiting RIP1 genetically, using knock-in mice that express catalytically inactive RIP1 D138N, or pharmacologically, using the murine-potent inhibitor GNE684. Inhibition of RIP1 reduced collagen antibody-induced arthritis, and prevented skin inflammation caused by mutation of Sharpin, or colitis caused by deletion of Nemo from intestinal epithelial cells. Conversely, inhibition of RIP1 had no effect on tumor growth or survival in pancreatic tumor models driven by mutant Kras, nor did it reduce lung metastases in a B16 melanoma model. Collectively, our data emphasize a role for the kinase activity of RIP1 in certain inflammatory disease models, but question its relevance to tumor progression and metastases.

Nucleophilic addition of organometallic reagents to ketimines and hydrazones can be a challenging transformation. Here we report the use of fluorenone-derived mixed azines which promote facile addition of Grignard reagents. The fluorenylidene activating group is easily installed and removed, thereby offering practical access to highly substituted amines and hydrazines.

Using adenine base editors, we aim to treat sickle cell disease by generating single nucleotide polymorphisms in human CD34+ hematopoietic stem and progenitor cells (HSPCs) at specific target sites by mediating A-T to G-C base conversions. While ex vivo gene editing approaches show great therapeutic promise, access is limited due to the requirement of an autologous hematopoietic stem cell (HSC) transplant to deliver the ex vivo edited cells. To further increase the number of patients eligible for base editing therapy, we are developing an alternative approach to directly deliver base editors to HSCs in vivo through non-viral delivery methods. Lipid Nanoparticles (LNPs) are a clinically validated, non-viral approach that enables the delivery of nucleic acid payloads, which may avoid the challenges associated with ex vivo approaches including the transplantation of edited CD34+ HSPCs. Here we describe the development and characterization of LNPs for the delivery of messenger RNA (mRNA) to HSPCs in vivo in both mice and cynomolgus macaques. By screening >1,000 chemically distinct LNPs in vivo utilizing a DNA barcoding technology, we identified several hit LNPs capable of biodistribution to HSPCs. Upon individual validation of these hit LNPs by delivery of Cre recombinase mRNA in a Cre-reporter mouse model (Ai14), which expresses the fluorescent protein tdTomato under a constitutive CAG promoter following Cre-meditate gene editing, we confirmed that several LNPs efficiently delivered Cre recombinase mRNA to mouse Lin-Sca-1+c-Kit+ (LSK) HSPCs. We next confirmed the most potent hit LNP (LNP-HSC1) identified from the in vivo screen to transfect LSK HSPCs in a dose-dependent manner between 0.1 and 1.0 mg/kg Cre recombinase mRNA, transfecting over 40% of LSK HSPCs in Ai14 mice at 1.0mg/kg. In a transfection durability study using Ai14 mice, we observed maintenance of tdTomato+ LSK HSPCs levels in the bone marrow at 10 weeks post-LNP delivery. As LNP-HSC1 had been identified and validated in mice of a C57BL6/j background, we next confirmed its ability to transfect a reporter mRNA into HSPCs in Balb/c mice and in 5 cynomolgus macaques. LNP-HSC1 efficiently transfected LSK HSPCs in Balb/c mice at doses ranging from 0.3 to 1.0 mg/kg. In 5 cynomolgus macaques (n=5 across two experiments), we observed a dose-dependent increase in reporter mRNA delivery with an average of 19% of bone marrow-derived CD34+ HSPCs (n=3) expressing the reporter protein at the highest dose tested. Taken together, these data demonstrate the value of our in vivo high-throughput LNP screening approach to identify novel LNPs capable of delivering to HSPCs, providing a promising delivery platform for an in vivo HSC gene editing approach for the treatment of hemoglobinopathies. Disclosures Sago: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Campbell: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lutz: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Patwardhan: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hamilton: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Wong: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Keating: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Murray: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Singh: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company, Current holder of individual stocks in a privately-held company, Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees.

Mutationally activated Ras is one of the most common oncogenic drivers found across all malignancies, and its selective inhibition has long been a goal in both pharma and academia. One of the oldest and most validated methods to inhibit overactive Ras signaling is by interfering with its post-translational processing and subsequent cellular localization. Previous attempts to target Ras processing led to the development of farnesyltransferase inhibitors, which can inhibit H-Ras localization but not K-Ras due to its ability to bypass farnesyltransterase inhibition through alternative prenylation by geranylgeranyltransferase. Here, we present the creation of a neo-substrate for farnesyltransferase that prevents the alternative prenlation by geranylgeranyltransferase and mislocalizes oncogenic K-Ras in cells.

Community Child Health (community paediatrics) is a challenging and fulfilling career, working with vulnerable children with developmental disorders, disabilities, those who are at risk of abuse, children looked after by the Local Authority or who are being adopted. Community paediatricians work in a variety of settings, provide personal continuity of care and excel in multi-agency working with statutory and other services. Alongside their clinical skills, they also play a vital role in planning and implementing local health improvements for all children in their area. This review summarizes how community paediatrics developed in the UK, the current training programme for consultant community paediatricians and the kind of posts available. Community paediatrics is a shortage speciality and there will be plenty of posts in the UK for the foreseeable future. We hope that this article will help medical students and paediatricians in training to make better informed career choices and offer encouragement to readers who are considering subspecialising in this dynamic and fulfilling area of paediatrics.

In an examination of Wajdi Mouawad's play, Incendies (2003) and the 2010 film version of the play directed and scripted by Denis Villeneuve, this article explores the quest narrative as it underpins these texts, with particular reference to exile, trauma and reparation. The suggestive trope of “home” as this intersects with identity, and the threat to identity posed by war and suffering, as well as the palimpsestic construction and reconstruction of memory, and its ability (and inability) to palliate the traumatised “housing” of the body (in particular the maternal body of Nawal Marwan, the character who originates the theatrical and filmic chronotopes) is also investigated in relation to the reworking of the Oedipus myth that informs both play and film. Through the theoretical lens of Derridean and Lacanian poststructuralism and the imbrication of gender and trauma studies, the article interrogates the complex staging of traumatic re- enactment as manifested in the interstitial spaces between the...

Despite the tremendous advances of the past four decades, chemists are far from being able to use chiral catalysts to control the stereoselectivity of any desired reaction. New concepts for the construction and mode of operation of chiral catalysts have the potential to open up previously inaccessible reaction space. The recognition and categorization of distinct approaches seems to play a role in triggering rapid exploration of new territory. This Review both reflects on the origins as well as details a selection of the latest examples of an area that has advanced considerably within the past five years or so: the use of chiral anions in asymmetric catalysis. Defining reactions as involving chiral anions is a difficult task owing to uncertainties over the exact catalytic mechanisms. Nevertheless, we attempt to provide an overview of the breadth of reactions that could reasonably fall under this umbrella.

Purpose This three-year group randomized controlled trial assessed whether a multi-age, multi-level bullying prevention and intervention with staff capacity building, can reduce bullying among primary school children. Methods This study comprised two intervention and one comparison conditions. Student self-report data were collected from 2552 Grades 4 and 6 students at baseline and three posttests. Results The high intervention was more effective than the moderate and low interventions, with significant positive effects for ‘being bullied’ in the Grades 4 and 6 cohorts, for ‘bullying others’ in the Grade 4 cohort and ‘telling if bullied’ in the Grade 6 cohort. Conclusions Comprehensive whole-school programmes that include capacity building and parental involvement appear to reduce bullying behaviour more than programmes without these components.

The discovery of distinct modes of asymmetric catalysis has the potential to rapidly advance chemists' ability to build enantioenriched molecules. As an example, the use of chiral cation salts as phase-transfer catalysts for anionic reagents has enabled a vast set of enantioselective transformations. A largely overlooked analogous mechanism wherein a chiral anionic catalyst brings a cationic species into solution is itself a powerful method. The concept is broadly applicable to a number of different reaction pathways, including to the enantioselective fluorocyclization of olefins, and dearomatization of aromatic systems with a cationic electrophile-transferring (e.g., fluorinating) agent and a chiral phosphate catalyst. The reactions proceed in high yield and stereoselectivity. The compounds and methods of the invention are of particular value, especially considering the scarcity of alternative approaches.

Chiral Brønsted acids have become an invaluable tool for achieving a variety of asymmetric chemical transformations under catalytic conditions while avoiding the use of toxic and expensive metals1–8. While the catalysts developed so far are remarkably effective at activating polarized functional groups, chemists have not yet been able to use organic Brønsted acids to catalyze highly enantioselective transformations of unactivated carbon-carbon multiple bonds. This deficiency persists despite the fact that racemic acid-catalyzed “Markovnikov” additions to olefins are a well-established part of the chemist’s toolbox. Here we show that chiral dithiophosphoric acids catalyze the intramolecular hydroamination and hydroarylation of dienes and allenes to generate heterocyclic products in exceptional yield and enantiomeric excess. To help rationalize the unique success of this catalytic system, we present a mechanistic hypothesis that involves the addition of the acid catalyst to the diene followed by SN2′ displacement of the resulting dithiophosphate intermediate. Mass spectrometry and deuterium labelling studies are presented in support of the proposed mechanism. The catalysts and concepts revealed in this study should prove applicable to other asymmetric functionalizations of unsaturated systems.

A group randomized controlled trial tested the efficacy of the Friendly Schools program to reduce student bullying behaviour. This socio‐ecological intervention targeted the whole school, classroom, family, and individual students to reduce bullying behaviour. Self‐report data were collected in 29 schools over three years from a cohort of 1968 eight to nine‐year‐olds. Surveys measured frequency of being bullied, bullying others, telling if bullied and observing bullying. Results indicate that intervention students were significantly less likely to observe bullying at 12, 24 and 36 months and be bullied after 12 and 36 months, and significantly more likely to tell if bullied after 12 months than comparison students. No differences were found for self‐reported perpetration of bullying. The findings suggest whole‐of‐school programs that engage students in their different social contexts appear to reduce their experiences of being bullied and increase their likelihood of telling someone if they are bullied.

Traditionally, transition metal–catalyzed enantioselective transformations rely on chiral ligands tightly bound to the metal to induce asymmetric product distributions. Here we report high enantioselectivities conferred by a chiral counterion in a metal-catalyzed reaction. Two different transformations catalyzed by cationic gold(I) complexes generated products in 90 to 99% enantiomeric excess with the use of chiral binaphthol–derived phosphate anions. Furthermore, we show that the chiral counterion can be combined additively with chiral ligands to enable an asymmetric transformation that cannot be achieved by either method alone. This concept of relaying chiral information via an ion pair should be applicable to a vast number of metal-mediated processes.

Substitution of the C-11 aniline of mifepristone can provide compounds with altered pharmacokinetic and pharmacodynamic (PK/PD) profiles that may find use for new indications. The development of new steroid intermediates and specialized library synthesis methods were required to enable the efficient preparation of structurally complex C-11 modified mifepristone analogs.

The continued growth of unmanned aircraft systems (UAS) is driving innovation from delivery techniques to military surveillance. Cost and knowledge barriers are major limiting factors to general UAS adoption. A UAS controlled by an onboard smartphone is proposed to leverage consumer understanding and transform the emergent industry. Based upon user-supplied GPS flight path coordinates, the UAS can subsequently take flight, collect relevant data, and safely land without further instruction. This project consists of a 1.5-meter wingspan airplane carrying an Android smartphone connected via IOIO interface to servos that actuate the control surfaces and motor. With the exception of the initial GPS coordinates, no live direction is necessary for operation. Primarily, this level of autonomy was achieved by aggregating a diverse set of known algorithms to collectively validate the feasibility of smartphone-controlled autonomous systems.

Aims To determine the impact of a school-based harm minimization smoking intervention compared to traditional abstinence-based approaches. Design, setting and participants A school-based cluster randomized trial was conducted in Perth, Western Australia in 30 government high schools from 1999 to 2000. Over 4000 students were recruited to participate and schools were assigned randomly to either the harm minimization intervention or a standard abstinence-based programme. Intervention The harm minimization intervention comprised eight 1-hour lessons over 2 years, quitting support from school nurses and enactment of policies to support programme components. Comparison schools implemented standard abstinence-based programmes and policies. Measures Cigarette smoking was categorized at two levels: regular smoking, defined as smoking on 4 or more days in the previous week; and 30-day smoking as any smoking within the previous month. Findings At immediate post-test (20 months post-baseline), after accounting for baseline differences, school-level clustering effects, socio-economic status, gender and family smoking, intervention students were less likely to smoke regularly [OR = 0.51, 95% confidence interval (CI) = 0.36, 0.71] or to have smoked within the previous 30 days (OR = 0.69, 95% CI = 0.53, 0.91). Conclusion The school-based adolescent harm minimization intervention appears to have been more effective than the abstinence-based social influences programme at reducing regular smoking.

There exists limited empirical evidence of the effectiveness of universal school-based interventions to reduce or prevent children's bullying. The Friendly Schools project was a 3-year longitudinal randomised control trial designed to determine the efficacy of a universal holistic intervention to prevent or reduce bullying among primary school children. The trial involved 1968 Western Australian children, their parents and their teachers. This paper discusses the components of the Friendly Schools program, and how empirical, theoretical and “promising” mechanisms of change (mediators) were operationalised to develop a whole-of-school approach to reduce bullying. This paper concludes that further research is needed to determine empirically: (a) which mechanisms of change mediate improvements in children's bullying behaviour and mental health status and (b) the relative contribution of the various components of a whole-of-school approach to bullying prevention and reduction in children.

Reactions proceeding through cationic intermediates that lack a Lewis or Brønsted basic site present a challenge for traditional asymmetric catalysis based on chiral metals or organocatalysts. We present an enantioselective ring opening of tetrasubstituted meso-aziridinium ions with alcohol nucleophiles proceeding through a chiral ion pair with a binaphthol-phosphate anion. The reaction is initiated by silver-induced ring closure of beta-chloroamines using the Ag salt of the chiral anion as in situ generated catalyst. Use of insoluble Ag2CO3 as silver source is essential to obtain high enantioselectivity; we believe the chiral phosphate acts as a "chiral anion phase transfer catalyst" to bring silver ion into the organic phase. The chiral anion concept can also be extended to the related asymmetric opening of meso-episulfonium ions generated by protonation of trichloroacetimidates vicinal to sulfides.

The selective dealkylative arylation of trialkyl amines, N-alkyl pyrrolidines, N-alkyl piperidines and dialkyl anilines with electron deficient heteroaryl chlorides was investigated. Efficient and practical reaction conditions were determined for a range of substrates.

Novel concepts in asymmetric catalysis have the potential to open up previously inaccessible reaction space. This Review reflects on the origins of an area that has undergone dramatic recent advancement: the use of chiral anions in asymmetric catalysis. Details of a selection of the latest examples are also given.

Purpose: Problems in management of oral cancers or precancers include identification of patients at risk for metastasis, tumor recurrence, and second primary tumors or risk for progression of precancers (dysplasia) to cancer. Thus, the objective of this study was to clarify the role of genomic aberrations in oral cancer progression and metastasis. Experimental Design: The spectrum of copy number alterations in oral dysplasia and squamous cell carcinomas (SCC) was determined by array comparative genomic hybridization. Associations with clinical characteristics were studied and results confirmed in an independent cohort. Results: The presence of one or more of the chromosomal aberrations +3q24-qter, -8pter-p23.1, +8q12-q24.2, and +20 distinguishes a major subgroup (70%–80% of lesions, termed 3q8pq20 subtype) from the remainder (20%–30% of lesions, non-3q8pq20). The 3q8pq20 subtype is associated with chromosomal instability and differential methylation in the most chromosomally unstable tumors. The two subtypes differ significantly in clinical outcome with risk for cervical (neck) lymph node metastasis almost exclusively associated with the 3q8pq20 subtype in two independent oral SCC cohorts. Conclusions: Two subtypes of oral lesions indicative of at least two pathways for oral cancer development were distinguished that differ in chromosomal instability and risk for metastasis, suggesting that +3q,–8p, +8q, and +20 constitute a biomarker with clinical utility for identifying patients at risk for metastasis. Moreover, although increased numbers of genomic alterations can be harbingers of progression to cancer, dysplastic lesions lacking copy number changes cannot be considered benign as they are potential precursors to non-3q8pq20 locally invasive, yet not metastatic oral SCC. Clin Cancer Res; 17(22); 7024–34. ©2011 AACR.

The author argues that director Gary Marshall's film, Pretty Woman, like Shakespeare's The Winter's Tale, although drawing upon the Greek myth of Pygmalion and Galatea, actually subverts it. In the original myth, Galatea is created through the metamorphosis of the statue of feminine ideality which Pygmalion moulds. This myth embodies the stereotype of man as active creator and woman as passive adjunct to male fantasy. In Pretty Woman and Shakespeare's play, however, it is the woman who is the creator, re-moulding and re-educating her husband/lover towards a new, less truncated selfhood. Both Pretty Woman and The Winter's Tale also establish a dialectical relationship between art and life, between fantasy and reality, myth and anti-myth. These are shown, through a manipulation of the boundaries between them, to be, not mutually exclusive categories, but mutually defining and mutually enriching.

Although Australia's restrictive tobacco control policies have made it an international leader in reducing smoking prevalence, and only 7% of teens smoke weekly, cessation efforts are still needed among adolescents. Of the campaigns and programs implemented in Australia to reduce this problem among adolescents, most have provided only abstinence messages and few have been effectively evaluated and translated into policy and practice. This article describes the translation of a harm minimization cessation program for teens, the Smoking Cessation for Youth Project (SCYP), derived from an approach developed with adults. In addition, the article describes the unique sociopolitical context of Australia in which the SCYP program was developed and the significant conceptual, contextual, and methodological factors that enabled and limited this program's effective implementation and translation.

Declines in adolescent smoking prevalence have slowed recently, resulting in increased interest and literature in tobacco harm minimisation. To date, harm reduction strategies have focused largely on modifying the product and alternative (safer) mechanisms of nicotine delivery. There has been little exploration of primary harm minimisation to prevent the onset of regular smoking among young people. A major concern expressed about harm reduction interventions and young people is that they may increase experimentation among non-users.The Smoking Cessation for Youth Project was a 2-year school-based cluster randomised controlled trial conducted in 30 Western Australian schools. Results on the primary outcome showed a significant reduction in regular smoking among 4636 13-15-year-olds receiving a harm minimisaton versus standard intervention. This paper addresses the intervention effects on 2078 students who had not smoked at baseline.At 20-month follow-up, smoking initiation was slightly lower among intervention students than comparison students (who received a largely abstinence-based intervention), although this difference did not attain statistical significance (OR=0.86; 95% confidence interval: 0.68, 1.09).This study provided limited evidence to suggest that harm minimisation is a superior approach to abstinence-based interventions for non-smokers. However, this intervention did not contribute to increased experimentation among non-smokers. Although more trials are required, these results indicate that fears of potential negative iatrogenic effects from school-based harm minimisation interventions may be unwarranted.

A novel series of pyrrolidine-constrained phenethylamines were developed as dipeptidyl peptidase IV (DPP4) inhibitors for the treatment of type 2 diabetes. The cyclohexene ring of lead-like screening hit 5 was replaced with a pyrrolidine to enable parallel chemistry, and protein co-crystal structural data guided the optimization of N-substituents. Employing this strategy, a >400x improvement in potency over the initial hit was realized in rapid fashion. Optimized compounds are potent and selective inhibitors with excellent pharmacokinetic profiles. Compound 30 was efficacious in vivo, lowering blood glucose in ZDF rats that were allowed to feed freely on a mixed meal.

Libraries of mifepristone analogs, MP-Acids, were designed and synthesized to increase the chances of identifying GR antagonists that possess liver-selective pharmacological profiles. MP-Acids were uniformly potent GR antagonists in binding and in cell-based functional assays. A high throughput pharmacokinetic selection strategy that employs the cassette dosing of MP-Acids was developed to identify liver-targeting compounds. Thus, resource-intensive in vivo assays to measure liver-selective pharmacology were enriched with GR antagonists that achieve high concentrations in the liver.

Legalization of assisted suicide in Oregon ushered in a new approach to evaluating suicidal patients with serious medical illnesses. Two competing paradigms—the traditional model (1–4) and the assisted suicide competency model (5)—now exist. This report compares the traditional approach to evaluating and treating suicidal symptoms with the assisted suicide competency model delineated in an assisted suicide guidebook used in Oregon (5), the only state where such a practice is legal.

This study examined the degree to which different policy-based whole-school smoking actions were associated with cigarette smoking among 4,697 ninth-grade students in 31 schools in Perth, Australia. Smoking is banned in all schools in Western Australia. Cross-sectional self-report surveys measured student smoking prevalence and intensity and its correlates. Simultaneously, school policies and practices related to cigarette smoking were assessed by interviews with principals from each of the 31 schools. The relationship between school policies and practices and cigarette smoking were evaluated using logistic regression. After controlling for socioeconomic status, family smoking, and gender, no differences in "ever smoking" and "regular smoking" rates were seen among schools that reported having a school health policy, a school drug policy, or school health committee. Both ever smoking (p

Microarray measurements offer the potential to compare the abundances of numerous nucleic acid sequences in parallel. Using linker-adapter PCR products from mapped BAC clones we have made arrays that permit scanning the human genome for single copy gains and losses of DNA sequence, which requires reliable detection of 50 percent changes. The DNA is printed at high concentration on amino-silane or chromium coated surface using a custom-built capillary pin printing system. Spots are printed on 130 micrometers centers or closer to minimize the size of the arrays. Hybridization occurs in a dextran sulfate/formamide buffer at 37 degrees C, using slow rocking to mix the reaction. The entire array is imaged in a single CCD frame using a custom built system that employs mercury arc illumination. Up to four fluorochromes can be imaged from a single array with adequate spectral separation. Typically we use DAPI to stain the DNA in the array spots to facilitate automatic image segmentation during analysis, and fluorescein, Cy3, and Cy5 or their spectral equivalents, for labeling specimen nucleic acids. Array spots are segmented and quantitative fluorescence intensities and intensity ratios are automatically calculated in < 1 minute per approximately 8000 element array using the custom software UCSF SPOT.© (2002) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.