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2.  Tightly integrated single- and multi-crystal data collection strategy calculation and parallelized data processing in JBluIce beamline control system 
Journal of Applied Crystallography  2014;47(Pt 6):1992-1999.
Single- and multi-crystal data collection strategy and automated data processing have been tightly integrated into the JBluIce graphical user interface. Grid Engine is used to distribute these processes into multiple workstations to make efficient use of all available computing resources.
The calculation of single- and multi-crystal data collection strategies and a data processing pipeline have been tightly integrated into the macromolecular crystallographic data acquisition and beamline control software JBluIce. Both tasks employ wrapper scripts around existing crystallographic software. JBluIce executes scripts through a distributed resource management system to make efficient use of all available computing resources through parallel processing. The JBluIce single-crystal data collection strategy feature uses a choice of strategy programs to help users rank sample crystals and collect data. The strategy results can be conveniently exported to a data collection run. The JBluIce multi-crystal strategy feature calculates a collection strategy to optimize coverage of reciprocal space in cases where incomplete data are available from previous samples. The JBluIce data processing runs simultaneously with data collection using a choice of data reduction wrappers for integration and scaling of newly collected data, with an option for merging with pre-existing data. Data are processed separately if collected from multiple sites on a crystal or from multiple crystals, then scaled and merged. Results from all strategy and processing calculations are displayed in relevant tabs of JBluIce.
PMCID: PMC4248568  PMID: 25484844
automated data processing; multi-crystal data collection strategies; X-ray crystallography; Grid Engine
3.  A genetic and computational approach to structurally classify neuronal types 
Nature communications  2014;5:3512.
The importance of cell types in understanding brain function is widely appreciated but only a tiny fraction of neuronal diversity has been catalogued. Here, we exploit recent progress in genetic definition of cell types in an objective structural approach to neuronal classification. The approach is based on highly accurate quantification of dendritic arbor position relative to neurites of other cells. We test the method on a population of 363 mouse retinal ganglion cells. For each cell, we determine the spatial distribution of the dendritic arbors, or “arbor density” with reference to arbors of an abundant, well-defined interneuronal type. The arbor densities are sorted into a number of clusters that is set by comparison with several molecularly defined cell types. The algorithm reproduces the genetic classes that are pure types, and detects six newly clustered cell types that await genetic definition.
PMCID: PMC4164236  PMID: 24662602
4.  Novel mTOR inhibitory activity of ciclopirox enhances parthenolide antileukemia activity 
Experimental hematology  2013;41(9):10.1016/j.exphem.2013.04.012.
Ciclopirox, an antifungal agent commonly used for the dermatologic treatment of mycoses, has been shown recently to have antitumor properties. Although the exact mechanism of ciclopirox is unclear, its antitumor activity has been attributed to iron chelation and inhibition of the translation initiation factor eIF5A. In this study, we identify a novel function of ciclopirox in the inhibition of mTOR. As with other mTOR inhibitors, we show that ciclopirox significantly enhances the ability of the established preclinical antileukemia compound, parthenolide, to target acute myeloid leukemia. The combination of parthenolide and ciclopirox demonstrates greater toxicity against acute myeloid leukemia than treatment with either compound alone. We also demonstrate that the ability of ciclopirox to inhibit mTOR is specific to ciclopirox because neither iron chelators nor other eIF5A inhibitors affect mTOR activity, even at high doses. We have thus identified a novel function of ciclopirox that might be important for its antileukemic activity.
PMCID: PMC3809917  PMID: 23660068
5.  Vitamin C deficiency improves somatic embryo development through distinct gene regulatory networks in Arabidopsis  
Journal of Experimental Botany  2014;65(20):5903-5918.
Depletion of cellular vitamin C improves somatic embryogenesis in Arabidopsis. Improved embryo number and quality is through changes in gene regulatory network activation and cellular architecture.
Changes in the endogenous ascorbate redox status through genetic manipulation of cellular ascorbate levels were shown to accelerate cell proliferation during the induction phase and improve maturation of somatic embryos in Arabidopsis. Mutants defective in ascorbate biosynthesis such as vtc2-5 contained ~70 % less cellular ascorbate compared with their wild-type (WT; Columbia-0) counterparts. Depletion of cellular ascorbate accelerated cell division processes and cellular reorganization and improved the number and quality of mature somatic embryos grown in culture by 6-fold compared with WT tissues. To gain insight into the molecular mechanisms underlying somatic embryogenesis (SE), we profiled dynamic changes in the transcriptome and analysed dominant patterns of gene activity in the WT and vtc2-5 lines across the somatic embryo culturing process. Our results provide insight into the gene regulatory networks controlling SE in Arabidopsis based on the association of transcription factors with DNA sequence motifs enriched in biological processes of large co-expressed gene sets. These data provide the first detailed account of temporal changes in the somatic embryo transcriptome starting with the zygotic embryo, through tissue dedifferentiation, and ending with the mature somatic embryo, and impart insight into possible mechanisms for the improved culture of somatic embryos in the vtc2-5 mutant line.
PMCID: PMC4203126  PMID: 25151615
Arabidopsis thaliana; ascorbic acid; gene regulatory networks; redox; somatic embryogenesis; transcriptome
7.  Towards protein-crystal centering using second-harmonic generation (SHG) microscopy 
The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-­ray diffraction of protein crystals has been explored.
The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-­ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using β2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-­ray minibeam. SHG imaging was found to provide about 2 µm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed.
PMCID: PMC3640472  PMID: 23633594
second-harmonic generation microscopy; crystal centering; imaging
8.  Area-dependent time courses of brain activation during video-induced symptom provocation in social anxiety disorder 
Previous functional imaging studies using symptom provocation in patients with social anxiety disorder (SAD) reported inconsistent findings, which might be at least partially related to different time-dependent activation profiles in different brain areas. In the present functional magnetic resonance imaging study, we used a novel video-based symptom provocation design in order to investigate the magnitude and time course of activation in different brain areas in 20 SAD patients and 20 healthy controls.
The disorder-related videos induced increased anxiety in patients with SAD as compared to healthy controls. Analyses of brain activation to disorder-related versus neutral video clips revealed amygdala activation during the first but not during the second half of the clips in patients as compared to controls. In contrast, the activation in the insula showed a reversed pattern with increased activation during the second but not during the first half of the video clips. Furthermore, a cluster in the anterior dorsal anterior cingulate cortex showed a sustained response for the entire duration of the videos.
The present findings suggest that different regions of the fear network show differential temporal response patterns during video-induced symptom provocation in SAD. While the amygdala is involved during initial threat processing, the insula seems to be more involved during subsequent anxiety responses. In accordance with cognitive models of SAD, a medial prefrontal region engaged in emotional-cognitive interactions is generally hyperactivated.
PMCID: PMC4052290  PMID: 24921039
Social anxiety disorder; Symptom provocation; Functional magnetic resonance imaging (fMRI); Amygdala; Insula; Medial prefrontal cortex
9.  Highly Sensitive Detection of Urinary Cadmium to Assess Personal Exposure 
Analytica chimica acta  2013;773:45-51.
A series of Boron-Doped Diamond (BDD) ultramicroelectrode arrays were fabricated and investigated for their performance as electrochemical sensors to detect trace level metals such as cadmium. The steady-state diffusion behavior of these sensors was validated using cyclic voltammetry followed by electrochemical detection of cadmium in water and in human urine to demonstrate high sensitivity (>200 μA/ppb/cm2) and low background current (<4 nA). When an array of ultramicroelectrodes was positioned with optimal spacing, these BDD sensors showed a sigmoidal diffusion behavior. They also demonstrated high accuracy with linear dose dependence for quantification of cadmium in a certified reference river water sample from the National Institute of Standards and Technology (NIST) as well as in a human urine sample spiked with 0.25–1 ppb cadmium.
PMCID: PMC3622219  PMID: 23561905
Boron-Doped Diamond (BDD); ultramicroelectrode array; electrochemical sensors; urinary trace metal detection; cadmium; differential pulse stripping voltammetry (DPSV)
10.  BCL-2 inhibition targets oxidative phosphorylation and selectively eradicates quiescent human leukemia stem cells 
Cell stem cell  2013;12(3):329-341.
Most forms of chemotherapy employ mechanisms involving induction of oxidative stress, a strategy that can be effective due to the elevated oxidative state commonly observed in cancer cells. However, recent studies have shown that relative redox levels in primary tumors can be heterogeneous, suggesting that regimens dependent on differential oxidative state may not be uniformly effective. To investigate this issue in hematological malignancies, we evaluated mechanisms controlling oxidative state in primary specimens derived from acute myelogenous leukemia (AML) patients. Our studies demonstrate three striking findings. First, the majority of functionally-defined leukemia stem cells (LSCs) are characterized by relatively low levels of reactive oxygen species (termed “ROS-low”). Second, ROS-low LSCs aberrantly over-express BCL-2. Third, BCL-2 inhibition reduced oxidative phosphorylation and selectively eradicated quiescent LSCs. Based on these findings, we propose a model wherein the unique physiology of ROS-low LSCs provides an opportunity for selective targeting via disruption of BCL-2-dependent oxidative phosphorylation.
PMCID: PMC3595363  PMID: 23333149
Acute myeloid leukemia; AML; leukemia; leukemia stem cells; LSCs; oxidative phosphorylation; glycolysis; BCL-2; ABT-263; reactive oxygen species; ROS; oxidative state; energy metabolism
11.  Pegloticase immunogenicity: the relationship between efficacy and antibody development in patients treated for refractory chronic gout 
The efficacy of pegloticase, a polyethylene glycol (PEG)-conjugated mammalian recombinant uricase, approved for chronic refractory gout, can be limited by the development of antibodies (Ab). Analyses from 2 replicate, 6-month, randomized controlled trials were performed to characterize Ab responses to pegloticase.
Anti-pegloticase, anti-PEG, and anti-uricase Ab were determined by validated enzyme-linked immunosorbent assays. Ab titers were analyzed for possible relationships with serum pegloticase concentrations, serum uric acid (sUA) lowering, and risk of infusion reactions (IRs).
Sixty-nine (41%) of 169 patients receiving pegloticase developed high titer anti-pegloticase Ab (> 1:2430) and 40% (67/169) developed anti-PEG Ab; 1 patient receiving placebo developed high titer anti-pegloticase Ab. Only 14% (24/169) of patients developed anti-uricase Ab, usually at low titer. In responders, patients showing sustained UA lowering, mean anti-pegloticase titers at week 25 (1:837 ± 1687 with biweekly and 1:2025 ± 4506 with monthly dosing) were markedly lower than in nonresponders (1:34,528 ± 42,228 and 1:89,658 ± 297,797, respectively). Nonresponder status was associated with reduced serum pegloticase concentrations. Baseline anti-pegloticase Ab, evident in 15% (31/212) of patients, did not predict subsequent loss of urate-lowering response. Loss of sUA response preceded IRs in 44 of 56 (79%) pegloticase-treated patients.
Loss of responsiveness to pegloticase is associated with the development of high titer anti-pegloticase Ab that increase clearance of pegloticase and are associated with a loss of the sUA lowering effect and increased IR risk. Pre-infusion sUA can be used as a surrogate for the presence of deleterious anti-pegloticase Ab.
Trial registration
NCT00325195. Registered 10 May 2006, NCT01356498. Registered 27 October 2008.
PMCID: PMC4060440  PMID: 24588936
12.  Prostaglandin E2 increases hematopoietic stem cell survival and accelerates hematopoietic recovery after radiation injury 
Stem cells (Dayton, Ohio)  2013;31(2):372-383.
Hematopoietic stem and progenitor cells (HSPCs), which continuously maintain all mature blood cells, are regulated within the marrow microenvironment. We previously reported that pharmacologic treatment of naïve mice with prostaglandin E2 (PGE2) expands HSPCs. However, the cellular mechanisms mediating this expansion remain unknown. Here we demonstrate that PGE2 treatment in naïve mice inhibits apoptosis of HSPCs without changing their proliferation rate. In a murine model of sub-lethal total body irradiation (TBI), in which HSPCs are rapidly lost, treatment with a long-acting PGE2 analogue (dmPGE2) reversed the apoptotic program initiated by TBI. dmPGE2 treatment in vivo decreased the loss of functional HSPCs following radiation injury, as demonstrated both phenotypically and by their increased reconstitution capacity. The antiapoptotic effect of dmPGE2 on HSPCs did not impair their ability to differentiate in vivo, resulting instead in improved hematopoietic recovery after TBI. dmPGE2 also increased microenvironmental cyclooxygenase-2 expression and expanded the α-SMA+ subset of marrow macrophages, thus enhancing the bone marrow microenvironmental response to TBI. Therefore, in vivo treatment with PGE2 analogues may be particularly beneficial to HSPCs in the setting of injury by targeting them both directly and also through their niche. The current data provide rationale for in vivo manipulation of the HSPC pool as a strategy to improve recovery after myelosuppression.
PMCID: PMC3580384  PMID: 23169593
13.  Tophus measurement as an outcome measure for clinical trials of chronic gout: progress and research priorities 
The Journal of rheumatology  2011;38(7):10.3899/jrheum.110272.
Despite the recognition that tophus regression is an important outcome measure in clinical trials of chronic gout, there is no agreed method of tophus measurement. A number of methods have been used in clinical trials of chronic gout, from simple physical measurement techniques to complex advanced imaging methods. This paper summarises the methods of tophus measurement that have been used and discusses the properties of these methods. Physical measurement using Vernier calipers fulfils most aspects of the Outcomes Measures in Rheumatology (OMERACT) filter. Rigorous testing of the complex methods, particularly with respect to reliability and sensitivity to change is needed, to determine the appropriate use of these methods. Further information is also required regarding which method of physical measurement is best for use in future clinical trials. The need to develop and test a patient reported measure of tophus burden is also highlighted.
PMCID: PMC3882031  PMID: 21724716
14.  Genomic dissection of the seed 
Seeds play an integral role in the global food supply and account for more than 70% of the calories that we consume on a daily basis. To meet the demands of an increasing population, scientists are turning to seed genomics research to find new and innovative ways to increase food production. Seed genomics is evolving rapidly, and the information produced from seed genomics research has exploded over the past two decades. Advances in modern sequencing strategies that profile every molecule in every cell, tissue, and organ and the emergence of new model systems have provided the tools necessary to unravel many of the biological processes underlying seed development. Despite these advances, the analyses and mining of existing seed genomics data remain a monumental task for plant biologists. This review summarizes seed region and subregion genomic data that are currently available for existing and emerging oilseed models. We provide insight into the development of tools on how to analyze large-scale datasets.
PMCID: PMC4162360  PMID: 25309563
Arabidopsis; next generation sequencing; oilseed; RNA seq; seed; soybean; transcriptome
15.  LORD-Q: a long-run real-time PCR-based DNA-damage quantification method for nuclear and mitochondrial genome analysis 
Nucleic Acids Research  2013;42(6):e41.
DNA damage is tightly associated with various biological and pathological processes, such as aging and tumorigenesis. Although detection of DNA damage is attracting increasing attention, only a limited number of methods are available to quantify DNA lesions, and these techniques are tedious or only detect global DNA damage. In this study, we present a high-sensitivity long-run real-time PCR technique for DNA-damage quantification (LORD-Q) in both the mitochondrial and nuclear genome. While most conventional methods are of low-sensitivity or restricted to abundant mitochondrial DNA samples, we established a protocol that enables the accurate sequence-specific quantification of DNA damage in >3-kb probes for any mitochondrial or nuclear DNA sequence. In order to validate the sensitivity of this method, we compared LORD-Q with a previously published qPCR-based method and the standard single-cell gel electrophoresis assay, demonstrating a superior performance of LORD-Q. Exemplarily, we monitored induction of DNA damage and repair processes in human induced pluripotent stem cells and isogenic fibroblasts. Our results suggest that LORD-Q provides a sequence-specific and precise method to quantify DNA damage, thereby allowing the high-throughput assessment of DNA repair, genotoxicity screening and various other processes for a wide range of life science applications.
PMCID: PMC3973301  PMID: 24371283
16.  Patient-reported Outcomes in Chronic Gout: A Report from OMERACT 10 
The Journal of rheumatology  2011;38(7):10.3899/jrheum.110271.
To summarize the endorsement of measures of patient-reported outcome (PRO) domains in chronic gout at the 2010 Outcome Measures in Rheumatology Meeting (OMERACT 10).
During the OMERACT 10 gout workshop, validation data were presented for key PRO domains including pain [pain by visual analog scale (VAS)], patient global (patient global VAS), activity limitation [Health Assessment Questionnaire-Disability Index (HAQ-DI)], and a disease-specific measure, the Gout Assessment Questionnaire version 2.0 (GAQ v2.0). Data were presented on all 3 aspects of the OMERACT filters of truth, discrimination, and feasibility. One PRO, health-related quality of life measurement with the Medical Outcomes Study Short-form 36 (SF-36), was previously endorsed at OMERACT 9.
One measure for each of the 3 PRO of pain, patient global, and activity limitation was endorsed by > 70% of the OMERACT delegates to have appropriate validation data. Specifically, pain measurement by VAS was endorsed by 85%, patient global assessment by VAS by 73%, and activity limitation by HAQ-DI by 71%. GAQ v2.0 received 30% vote and was not endorsed due to several concerns including low internal consistency and lack of familiarity with the measure. More validation studies are needed for this measure.
With the endorsement of one measure each for pain, patient global, SF-36, and activity limitation, all 4 PRO for chronic gout have been endorsed. Future validation studies are needed for the disease-specific measure, GAQ v2.0. Validation for PRO for acute gout will be the focus of the next validation exercise for the OMERACT gout group.
PMCID: PMC3850171  PMID: 21724715
17.  A Revised Estimate of the Burden of Illness of Gout☆ 
Gout is a chronic, inflammatory arthritis characterized by painful and debilitating acute/episodic flares. Until recently, gout has been regarded as a minor medical problem, in part because the associated economic burden has not been appreciated. Previous literature on this subject focused on the costs associated with acute episodes of gout rather than on the long-term medical and economic implications of this chronic disorder.
Our aim was to estimate the current impact of gout in the United States with respect to disability and economic costs.
The following data sources were used: published data on the incremental economic burden of gout; statistics from the US Census Bureau and the US Bureau of Labor Statistics; and recent epidemiological and clinical literature concerning the course, treatment, and outcomes of the disease. Disability is expressed as days of lost productivity. Charges for gout-related treatments were used as direct cost inputs.
Gout affects an estimated 8 million Americans, among whom those working have an average of almost 5 more absence days annually than workers without gout. On average, the incremental annual cost of care for a gout patient is estimated at >$3000 compared with a nongouty individual. Even though comorbidities common in gout patients account for a portion of this increased economic burden, the total annual cost attributable to gout patients in the United States is likely in the tens of billions of dollars and comparable to those of other major chronic disorders, such as migraine and Parkinson’s disease.
The economic burden of gout is most readily assessable in patients whose acute arthritic flares result in emergency department visits, bedridden days, and episodic loss of productivity. Chronic progression of the disease can also result in long-term impairment of function and health-related quality of life, but the contribution of chronic gout to the economic burden is more difficult to quantitate because gout is frequently associated with serious cardiovascular, metabolic, and renal comorbidities. Recent demonstration that successful gout management can reverse functional deficits in many chronic gout patients, however, supports the views that chronic gout contributes substantially to the medical and thus economic costs of these patients and that early and aggressive efforts to improve gout outcomes are likely to reduce the associated economic burden.
PMCID: PMC3898191  PMID: 24465034
burden of illness; gout
18.  Tophus burden reduction with pegloticase: results from phase 3 randomized trials and open-label extension in patients with chronic gout refractory to conventional therapy 
Arthritis Research & Therapy  2013;15(5):R137.
Two replicate randomized, placebo-controlled six-month trials (RCTs) and an open-label treatment extension (OLE) comprised the pegloticase development program in patients with gout refractory to conventional therapy. In the RCTs, approximately 40% of patients treated with the approved dose saw complete response (CR) of at least one tophus. Here we describe the temporal course of tophus resolution, total tophus burden in patients with multiple tophi, tophus size at baseline, and the relationship between tophus response and urate-lowering efficacy.
Baseline subcutaneous tophi were analyzed quantitatively using computer-assisted digital images in patients receiving pegloticase (8 mg biweekly or monthly) or placebo in the RCTs, and pegloticase in the OLE. Tophus response, a secondary endpoint in the trials, was evaluated two ways. Overall tophus CR was the proportion of patients achieving a best response of CR (without any new/enlarging tophi) and target tophus complete response (TT-CR) was the proportion of all tophi with CR.
Among 212 patients randomized in the RCTs, 155 (73%) had ≥1 tophus and 547 visible tophi were recorded at baseline. Overall tophus CR was recorded in 45% of patients in the biweekly group (P = 0.002 versus placebo), 26% in the monthly group, and 8% in the placebo group after six months of RCT therapy. TT-CR rates at six months were 28%, 19%, and 2% of tophi, respectively. Patients meeting the primary endpoint of sustained urate-lowering response to therapy (responders) were more likely than nonresponders to have an overall tophus CR at six months (54% vs 20%, respectively and 8% with placebo).
Both overall tophus CR and TT-CRs increased with treatment duration in the OLE, reaching 70% (39/56) of patients and 55% (132/238) of target tophi after one year of treatment in patients receiving pegloticase during both the RCTs and OLE. At that time point, more tophi had resolved in responders (102/145 or 70% of tophi) than nonresponders (30/93; 32%).
Pegloticase reduced tophus burden in patients with refractory tophaceous gout, especially those achieving sustained urate-lowering. Complete resolution of tophi occurred in some patients by 13 weeks and in others with longer-term therapy.
Trial registrations
NCT00325195, NCT01356498
PMCID: PMC3979037  PMID: 24286509
19.  High-dose therapy and autologous stem cell transplant for transformed non-Hodgkin lymphoma in the rituximab era 
Leukemia & lymphoma  2011;53(5):830-835.
The impact of rituximab on outcome of high dose therapy and autologous stem cell transplantation (HD-ASCT) for transformed NHL has not been previously described. We analyzed eighteen consecutive patients with indolent NHL who transformed to diffuse large B-cell lymphoma (DLBCL), received rituximab-containing therapy either before or after transformation and underwent subsequent HD-ASCT. With a median follow-up of 40 months, the 2-year PFS was 59% and the 2-year OS was 82%. Six patients did not receive rituximab pre-transformation; this group had a significantly better PFS at 2 years post HD-ASCT compared to 12 patients who were exposed to rituximab pre-transformation (p=0.03). HD-ASCT remains an effective therapeutic option for transformed NHL in the rituximab era. However, patients exposed to rituximab pre-transformation appear to have inferior HD-ASCT outcomes, and thus may benefit from novel conditioning and maintenance regimens in the setting of HD-ASCT.
PMCID: PMC3653616  PMID: 22023518
HD-ASCT; Transformed NHL; Rituximab; Transplant
20.  Integrated nonlinear optical imaging microscope for on-axis crystal detection and centering at a synchrotron beamline 
Journal of Synchrotron Radiation  2013;20(Pt 4):531-540.
Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction for combined single-platform analysis, examining the viability of NLO microscopy as an alternative to the conventional X-ray raster scan for the purposes of sample centering. Second-harmonic generation microscopy and two-photon excited ultraviolet fluorescence microscopy were evaluated for crystal detection, and assessed by X-ray raster scanning.
Nonlinear optical (NLO) instrumentation has been integrated with synchrotron X-ray diffraction (XRD) for combined single-platform analysis, initially targeting applications for automated crystal centering. Second-harmonic-generation microscopy and two-photon-excited ultraviolet fluorescence microscopy were evaluated for crystal detection and assessed by X-ray raster scanning. Two optical designs were constructed and characterized; one positioned downstream of the sample and one integrated into the upstream optical path of the diffractometer. Both instruments enabled protein crystal identification with integration times between 80 and 150 µs per pixel, representing a ∼103–104-fold reduction in the per-pixel exposure time relative to X-ray raster scanning. Quantitative centering and analysis of phenylalanine hydroxylase from Chromobacterium violaceum cPAH, Trichinella spiralis deubiquitinating enzyme TsUCH37, human κ-opioid receptor complex kOR-T4L produced in lipidic cubic phase (LCP), intimin prepared in LCP, and α-cellulose samples were performed by collecting multiple NLO images. The crystalline samples were characterized by single-crystal diffraction patterns, while α-cellulose was characterized by fiber diffraction. Good agreement was observed between the sample positions identified by NLO and XRD raster measurements for all samples studied.
PMCID: PMC3682636  PMID: 23765294
XRD; NLO; SHG; SONICC; centering; protein; TPE-UVF; microscopy; LCP; two-photon
21.  Late Relapses Following High-Dose Autologous Stem Cell Transplantation (HD-ASCT) for Hodgkin Lymphoma (HL) in the ABVD Therapeutic era 
Salvage chemotherapy followed by high dose autologous stem cell transplantation (HD-ASCT) is the standard of care for patients who have relapsed or refractory Hodgkin Lymphoma (HL). Few trials have had long-term follow-up post HD-ASCT in the ABVD era of treatment. We reviewed 95 consecutive patients who received HD-ASCT for relapsed or refractory HL following ABVD failure between 1990 and 2006 at the University of Rochester. Median follow-up for survivors was 8.2 years. All patients received HD-ASCT following up-front ABVD (or equivalent) failure. At 5 years, overall survival (OS) and event-free survival (EFS) were 54% and 37%, respectively. In total, 54 patients have died; 37 of these patients died directly of HL. Notably, there were 19 deaths > 3 years post HD-ASCT and 13 of these late deaths are directly attributable to HL. Furthermore, there were 51 documented relapses, 9 of which occurred >3 years post HD-ASCT. In contrast to other studies, we did not observe a plateau in EFS following transplantation. Patients appear to be at continuous risk of recurrence beyond 3 years after HD-ASCT. Our results emphasize the importance of long-term follow-up for both toxicity and recurrence, and have important implications in defining success of post-transplant maintenance strategies.
PMCID: PMC3269502  PMID: 21871246
Hodgkin lymphoma; hematopoietic stem cells; late effects of therapy; relapsed and refractory disease; HD-ASCT
22.  AMASS: Algorithm for MSI Analysis by Semi-supervised Segmentation 
Journal of proteome research  2011;10(10):4734-4743.
Mass Spectrometric Imaging (MSI) is a molecular imaging technique that allows the generation of 2D ion density maps for a large complement of the active molecules present in cells and sectioned tissues. Automatic segmentation of such maps according to patterns of co-expression of individual molecules can be used for discovery of novel molecular signatures (molecules that are specifically expressed in particular spatial regions). However, current segmentation techniques are biased towards the discovery of higher abundance molecules and large segments; they allow limited opportunity for user interaction and validation is usually performed by similarity to known anatomical features.
We describe here a novel method, AMASS (Algorithm for MSI Analysis by Semi-supervised Segmentation). AMASS relies on the discriminating power of a molecular signal instead of its intensity as a key feature, uses an internal consistency measure for validation, and allows significant user interaction and supervision as options. An automated segmentation of entire leech embryo data images resulted in segmentation domains congruent with many known organs, including heart, CNS ganglia, nephridia, nephridiopores, and lateral and ventral regions, each with a distinct molecular signature. Likewise, segmentation of a rat brain MSI slice data set yielded known brain features, and provided interesting examples of co-expression between distinct brain regions. AMASS represents a new approach for the discovery of peptide masses with distinct spatial features of expression.
PMCID: PMC3190602  PMID: 21800894
Mass Spectrometry Imaging; MALDI Imaging; Segmentation
23.  Chromosomal translocations are guided by the spatial organization of the genome 
Cell  2012;148(5):908-921.
The extent to which the three dimensional organization of the genome contributes to chromosomal translocations is an important question in cancer genomics. We now have generated a high resolution Hi-C spatial organization map of the G1-arrested mouse pro-B cell genome and mapped translocations from target DNA double strand breaks (DSBs) within it via high throughput genome-wide translocation sequencing. RAG endonuclease-cleaved antigen-receptor loci are dominant translocation partners for target DSBs regardless of genomic position, reflecting high frequency DSBs at these loci and their co-localization in a fraction of cells. To directly assess spatial proximity contributions, we normalized genomic DSBs via ionizing-radiation. Under these conditions, translocations were highly enriched in cis along single chromosomes containing target DSBs and within other chromosomes and sub-chromosomal domains in a manner directly related to pre-existing spatial proximity. Our studies reveal the power of combining two high-throughput genomic methods to address long-standing questions in cancer biology.
PMCID: PMC3320767  PMID: 22341456
Translocations; 3D nuclear organization; DNA double-strand breaks; genome stability
24.  Automated sample-scanning methods for radiation damage mitigation and diffraction-based centering of macromolecular crystals 
Journal of Synchrotron Radiation  2011;18(Pt 5):717-722.
Two sample-scanning features have been implemented for the macromolecular crystallography beamlines at APS sector 23: automated diffraction-based rastering employing multiple polygon-shaped two-dimensional grids overlaid on a sample to locate and center small and invisible crystals or to find the best-diffracting regions in a larger crystal, and automated data collection along a three-dimensional vector to mitigate the effects of radiation damage.
Automated scanning capabilities have been added to the data acquisition software, JBluIce-EPICS, at the National Institute of General Medical Sciences and the National Cancer Institute Collaborative Access Team (GM/CA CAT) at the Advanced Photon Source. A ‘raster’ feature enables sample centering via diffraction scanning over two-dimensional grids of simple rectangular or complex polygonal shape. The feature is used to locate crystals that are optically invisible owing to their small size or are visually obfuscated owing to properties of the sample mount. The raster feature is also used to identify the best-diffracting regions of large inhomogeneous crystals. Low-dose diffraction images taken at grid positions are automatically processed in real time to provide a quick quality ranking of potential data-collection sites. A ‘vector collect’ feature mitigates the effects of radiation damage by scanning the sample along a user-defined three-dimensional vector during data collection to maximize the use of the crystal volume and the quality of the collected data. These features are integrated into the JBluIce-EPICS data acquisition software developed at GM/CA CAT where they are used in combination with a robust mini-beam of rapidly changeable diameter from 5 µm to 20 µm. The powerful software–hardware combination is being applied to challenging problems in structural biology.
PMCID: PMC3161817  PMID: 21862850
macromolecular crystallography; beamline automation; data acquisition; high-throughput crystallography; crystal centering; radiation damage; rastering
25.  Fast fluorescence techniques for crystallography beamlines 
Journal of Applied Crystallography  2011;44(Pt 4):772-778.
On-the-fly adaptive edge scanning and shuttle on-the-fly rastering fluorescence techniques have been developed to improve the efficiency of macromolecular crystallography beamlines.
This paper reports on several developments of X-ray fluorescence techniques for macromolecular crystallography recently implemented at the National Institute of General Medical Sciences and National Cancer Institute beamlines at the Advanced Photon Source. These include (i) three-band on-the-fly energy scanning around absorption edges with adaptive positioning of the fine-step band calculated from a coarse pass; (ii) on-the-fly X-ray fluorescence rastering over rectangular domains for locating small and invisible crystals with a shuttle-scanning option for increased speed; (iii) fluorescence rastering over user-specified multi-segmented polygons; and (iv) automatic signal optimization for reduced radiation damage of samples.
PMCID: PMC3247932  PMID: 21808424
macromolecular crystallography; beamline automation; data acquisition; high-throughput crystallography; X-ray fluorescence; multi-wavelength anomalous diffraction

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