Genome-wide Identification of Structure-Forming Repeats as Principal Sites of Fork Collapse upon ATR Inhibition.

DNA polymerase stalling activates the ATR checkpoint kinase, which in turn suppresses fork collapse and breakage. Herein, we describe use of ATR inhibition (ATRi) as a means to identify genomic sites of problematic DNA replication in murine and human cells. Over 500 high-resolution ATR-dependent sites were ascertained using two distinct methods: replication protein A (RPA)-chromatin immunoprecipitation (ChIP) and breaks identified by TdT labeling (BrITL). The genomic feature most strongly associated with ATR dependence was repetitive DNA that exhibited high structure-forming potential. Repeats most reliant on ATR for stability included structure-forming microsatellites, inverted retroelement repeats, and quasi-palindromic AT-rich repeats. Notably, these distinct categories of repeats differed in the structures they formed and their ability to stimulate RPA accumulation and breakage, implying that the causes and character of replication fork collapse under ATR inhibition can vary in a DNA-structure-specific manner. Collectively, these studies identify key sources of endogenous replication stress that rely on ATR for stability.

Acetyl-CoA promotes glioblastoma cell adhesion and migration through Ca2+-NFAT signaling.

The metabolite acetyl-coenzyme A (acetyl-CoA) is the required acetyl donor for lysine acetylation and thereby links metabolism, signaling, and epigenetics. Nutrient availability alters acetyl-CoA levels in cancer cells, correlating with changes in global histone acetylation and gene expression. However, the specific molecular mechanisms through which acetyl-CoA production impacts gene expression and its functional roles in promoting malignant phenotypes are poorly understood. Here, using histone H3 Lys27 acetylation (H3K27ac) ChIP-seq (chromatin immunoprecipitation [ChIP] coupled with next-generation sequencing) with normalization to an exogenous reference genome (ChIP-Rx), we found that changes in acetyl-CoA abundance trigger site-specific regulation of H3K27ac, correlating with gene expression as opposed to uniformly modulating this mark at all genes. Genes involved in integrin signaling and cell adhesion were identified as acetyl-CoA-responsive in glioblastoma cells, and we demonstrate that ATP citrate lyase (ACLY)-dependent acetyl-CoA production promotes cell migration and adhesion to the extracellular matrix. Mechanistically, the transcription factor NFAT1 (nuclear factor of activated T cells 1) was found to mediate acetyl-CoA-dependent gene regulation and cell adhesion. This occurs through modulation of Ca2+ signals, triggering NFAT1 nuclear translocation when acetyl-CoA is abundant. The findings of this study thus establish that acetyl-CoA impacts H3K27ac at specific loci, correlating with gene expression, and that expression of cell adhesion genes are driven by acetyl-CoA in part through activation of Ca2+-NFAT signaling.

Genome-Wide Prediction of Metabolic Enzymes, Pathways, and Gene Clusters in Plants.

Plant metabolism underpins many traits of ecological and agronomic importance. Plants produce numerous compounds to cope with their environments but the biosynthetic pathways for most of these compounds have not yet been elucidated. To engineer and improve metabolic traits, we need comprehensive and accurate knowledge of the organization and regulation of plant metabolism at the genome scale. Here, we present a computational pipeline to identify metabolic enzymes, pathways, and gene clusters from a sequenced genome. Using this pipeline, we generated metabolic pathway databases for 22 species and identified metabolic gene clusters from 18 species. This unified resource can be used to conduct a wide array of comparative studies of plant metabolism. Using the resource, we discovered a widespread occurrence of metabolic gene clusters in plants: 11,969 clusters from 18 species. The prevalence of metabolic gene clusters offers an intriguing possibility of an untapped source for uncovering new metabolite biosynthesis pathways. For example, more than 1,700 clusters contain enzymes that could generate a specialized metabolite scaffold (signature enzymes) and enzymes that modify the scaffold (tailoring enzymes). In four species with sufficient gene expression data, we identified 43 highly coexpressed clusters that contain signature and tailoring enzymes, of which eight were characterized previously to be functional pathways. Finally, we identified patterns of genome organization that implicate local gene duplication and, to a lesser extent, single gene transposition as having played roles in the evolution of plant metabolic gene clusters.

Patterns of metabolite changes identified from large-scale gene perturbations in Arabidopsis using a genome-scale metabolic network.

Metabolomics enables quantitative evaluation of metabolic changes caused by genetic or environmental perturbations. However, little is known about how perturbing a single gene changes the metabolic system as a whole and which network and functional properties are involved in this response. To answer this question, we investigated the metabolite profiles from 136 mutants with single gene perturbations of functionally diverse Arabidopsis (Arabidopsis thaliana) genes. Fewer than 10 metabolites were changed significantly relative to the wild type in most of the mutants, indicating that the metabolic network was robust to perturbations of single metabolic genes. These changed metabolites were closer to each other in a genome-scale metabolic network than expected by chance, supporting the notion that the genetic perturbations changed the network more locally than globally. Surprisingly, the changed metabolites were close to the perturbed reactions in only 30% of the mutants of the well-characterized genes. To determine the factors that contributed to the distance between the observed metabolic changes and the perturbation site in the network, we examined nine network and functional properties of the perturbed genes. Only the isozyme number affected the distance between the perturbed reactions and changed metabolites. This study revealed patterns of metabolic changes from large-scale gene perturbations and relationships between characteristics of the perturbed genes and metabolic changes.

Measuring semantic similarities by combining gene ontology annotations and gene co-function networks.

BACKGROUND: Gene Ontology (GO) has been used widely to study functional relationships between genes. The current semantic similarity measures rely only on GO annotations and GO structure. This limits the power of GO-based similarity because of the limited proportion of genes that are annotated to GO in most organisms. RESULTS: We introduce a novel approach called NETSIM (network-based similarity measure) that incorporates information from gene co-function networks in addition to using the GO structure and annotations. Using metabolic reaction maps of yeast, Arabidopsis, and human, we demonstrate that NETSIM can improve the accuracy of GO term similarities. We also demonstrate that NETSIM works well even for genomes with sparser gene annotation data. We applied NETSIM on large Arabidopsis gene families such as cytochrome P450 monooxygenases to group the members functionally and show that this grouping could facilitate functional characterization of genes in these families. CONCLUSIONS: Using NETSIM as an example, we demonstrated that the performance of a semantic similarity measure could be significantly improved after incorporating genome-specific information. NETSIM incorporates both GO annotations and gene co-function network data as a priori knowledge in the model. Therefore, functional similarities of GO terms that are not explicitly encoded in GO but are relevant in a taxon-specific manner become measurable when GO annotations are limited. Supplementary information and software are available at http://www.msu.edu/~jinchen/NETSIM .

Multi-agent modeling of the South Korean avian influenza epidemic.

BACKGROUND: Several highly pathogenic avian influenza (AI) outbreaks have been reported over the past decade. South Korea recently faced AI outbreaks whose economic impact was estimated to be 6.3 billion dollars, equivalent to nearly 50% of the profit generated by the poultry-related industries in 2008. In addition, AI is threatening to cause a human pandemic of potentially devastating proportions. Several studies show that a stochastic simulation model can be used to plan an efficient containment strategy on an emerging influenza. Efficient control of AI outbreaks based on such simulation studies could be an important strategy in minimizing its adverse economic and public health impacts. METHODS: We constructed a spatio-temporal multi-agent model of chickens and ducks in poultry farms in South Korea. The spatial domain, comprised of 76 (37.5 km x 37.5 km) unit squares, approximated the size and scale of South Korea. In this spatial domain, we introduced 3,039 poultry flocks (corresponding to 2,231 flocks of chickens and 808 flocks of ducks) whose spatial distribution was proportional to the number of birds in each province. The model parameterizes the properties and dynamic behaviors of birds in poultry farms and quarantine plans and included infection probability, incubation period, interactions among birds, and quarantine region. RESULTS: We conducted sensitivity analysis for the different parameters in the model. Our study shows that the quarantine plan with well-chosen values of parameters is critical for minimize loss of poultry flocks in an AI outbreak. Specifically, the aggressive culling plan of infected poultry farms over 18.75 km radius range is unlikely to be effective, resulting in higher fractions of unnecessarily culled poultry flocks and the weak culling plan is also unlikely to be effective, resulting in higher fractions of infected poultry flocks. CONCLUSIONS: Our results show that a prepared response with targeted quarantine protocols would have a high probability of containing the disease. The containment plan with an aggressive culling plan is not necessarily efficient, causing a higher fraction of unnecessarily culled poultry farms. Instead, it is necessary to balance culling with other important factors involved in AI spreading. Better estimations for the containment of AI spreading with this model offer the potential to reduce the loss of poultry and minimize economic impact on the poultry industry.

Effect of staining solutions on discoloration of resin nanocomposites.

PURPOSE: To examine the effect of staining solutions on the discoloration of resin nanocomposites. METHODS: Three resin nanocomposites (Ceram X, Grandio, and Filtek Z350) were light cured for 40 seconds at a light intensity of 1000 mW/cm2. The color of the specimens was measured in %R (reflectance) mode before and after immersing the specimens in four different test solutions [distilled water (DW), coffee (CF), 50% ethanol (50ET) and brewed green tea (GT)] for 7 hours/day over a 3-week period. The color difference (deltaE*) was obtained based on the CIEL*a*b* color coordinate values. RESULTS: The specimens immersed in DW, 50ET and GT showed a slight increase in L* value. However, the samples immersed in CF showed a decrease in the L* value and an increase in the b* value. CF induced a significant color change (deltaE*: 3.1-5.6) in most specimens but the other solutions induced only a slight color change. Overall, coffee caused unacceptable color changes to the resin nanocomposites.

Autophagy Inhibition to Augment mTOR Inhibition: a Phase I/II Trial of Everolimus and Hydroxychloroquine in Patients with Previously Treated Renal Cell Carcinoma.

PURPOSE: Everolimus inhibits the mTOR, activating cytoprotective autophagy. Hydroxychloroquine inhibits autophagy. On the basis of preclinical data demonstrating synergistic cytotoxicity when mTOR inhibitors are combined with an autophagy inhibitor, we launched a clinical trial of combined everolimus and hydroxychloroquine, to determine its safety and activity in patients with clear-cell renal cell carcinoma (ccRCC). PATIENTS AND METHODS: Three centers conducted a phase I/II trial of everolimus 10 mg daily and hydroxychloroquine in patients with advanced ccRCC. The objectives were to determine the MTD of hydroxychloroquine with daily everolimus, and to estimate the rate of 6-month progression-free survival (PFS) in patients with ccRCC receiving everolimus/hydroxychloroquine after 1-3 prior treatment regimens. Correlative studies to identify patient subpopulations that achieved the most benefit included population pharmacokinetics, measurement of autophagosomes by electron microscopy, and next-generation tumor sequencing. RESULTS: No dose-limiting toxicity was observed in the phase I trial. The recommended phase II dose of hydroxychloroquine 600 mg twice daily with everolimus was identified. Disease control [stable disease + partial response (PR)] occurred in 22 of 33 (67%) evaluable patients. PR was observed in 2 of 33 patients (6%). PFS ≥ 6 months was achieved in 15 of 33 (45%) of patients who achieved disease control. CONCLUSIONS: Combined hydroxychloroquine 600 mg twice daily with 10 mg daily everolimus was tolerable. The primary endpoint of >40% 6-month PFS rate was met. Hydroxychloroquine is a tolerable autophagy inhibitor in future RCC or other trials.

Effect of fluoride released from fluoride-containing dental restoratives on NiTi orthodontic wires.

The present study investigated the effect of fluoride released from dental restoratives on orthodontic NiTi wires. Five different restoratives (four fluoride-containing and one non-fluoride-containing) and four different NiTi wires were examined in this study. The pH of artificial saliva (AS) was adjusted to 2.5 and 6. Content of released fluoride was determined daily for 10 days. The morphology of wires was observed using a scanning electron microscope. After immersion for 10 days, the initial microhardness of the wires decreased by 0.3-5.6% depending on the test solution. Dyract AP (DA) and F2000 (F2) (compomers) released significantly more fluoride than the other resin products (composite resins) regardless of the test solution. In pH 2.5 solution, both DA and F2 released 40-45 ppm/day fluoride for five to six days. As for the wires in contact, they did not show any visible modification in surface morphology. Therefore, despite the released fluoride, wires in contact with the fluoride-containing restoratives were not damaged regardless of the pH value of test solution.

Clinical features and treatment outcomes of septic arthritis due to Mycobacterium massiliense associated with intra-articular injection: a case report.

BACKGROUND: There are increasing reports on nosocomial Mycobacterium massiliense infection, but septic arthritis and osteomyelitis because of that microorganism is rare. This report focuses on the clinical aspects of M. massiliense arthritis outbreak concurrent with soft tissue infection. CASE PRESENTATION: An outbreak of septic arthritis among patients who had been injected at a single clinic occurred in South Korea between April and September 2012. This may be associated with repeated injection of triamcinolone contaminated with M. massiliense. Nine of the Korean patients visited our hospital complaining of painful swelling of the knees. During treatment course, patients are suffered from soft tissue abscess around the injection site. Acid-fast bacilli culture for infected tissue was positive in five patients, and polymerase chain reaction for non-tuberculous mycobacteria was positive in four patients. They were treated with antibiotics, repeated arthroscopic surgeries, incision and drainage for a long time. All patients were eventually cured but three patients have suffered from a decreased range of motion. CONCLUSION: Early clinical suspicion and microbiological diagnosis are key factors in reducing morbidity since septic arthritis with M. massiliense manifests late after the injection and treatment of it is a laborious process.

Genomic signatures of specialized metabolism in plants.

All plants synthesize basic metabolites needed for survival (primary metabolism), but different taxa produce distinct metabolites that are specialized for specific environmental interactions (specialized metabolism). Because evolutionary pressures on primary and specialized metabolism differ, we investigated differences in the emergence and maintenance of these processes across 16 species encompassing major plant lineages from algae to angiosperms. We found that, relative to their primary metabolic counterparts, genes coding for specialized metabolic functions have proliferated to a much greater degree and by different mechanisms and display lineage-specific patterns of physical clustering within the genome and coexpression. These properties illustrate the differential evolution of specialized metabolism in plants, and collectively they provide unique signatures for the potential discovery of novel specialized metabolic processes.

BoneNET: a network model of bone microstructure and dynamics.

We develop a network model of bone microstructure and dynamics, BoneNET, which is capable of quantitative assessment of Bone Mineral Density (BMD) and bone remodelling dynamics. First, we introduce a network model of bone microstructure by describing structural properties and process of bone network modelling. Secondly, we explain a mathematical model of bone microstructure by analysing the density for mineralised fibres of bone microstructure. Finally, we provide a bone remodelling dynamics among osteoblast and osteoclast and study bone networks by proposing several measurements to calculate bone strength and identify critical elements in bone microstructure.

Automatic segmentation of the spinal cord and the dural sac in lumbar MR images using gradient vector flow field.

A Computer-aided diagnosis (CAD) system aims to facilitate characterization and quantification of abnormalities as well as minimize interpretation errors caused by tedious tasks of image screening and radiologic diagnosis. The system usually consists of segmentation, feature extraction and diagnosis, and segmentation significantly affects the diagnostic performance. In this paper, we propose an automatic segmentation method that extracts the spinal cord and the dural sac from T2-weighted sagittal magnetic resonance (MR) images of lumbar spine without the need of any human intervention. Our method utilizes a gradient vector flow (GVF) field to find the candidate blobs and performs a connected component analysis for the final segmentation. MR Images from fifty two subjects were employed for our experiments and the segmentation results were quantitatively compared against reference segmentation by two medical specialists in terms of a mutual overlap metric. The experimental results showed that, on average, our method achieved a similarity index of 0.7 with a standard deviation of 0.0571 that indicated a substantial agreement. We plan to apply this segmentation method to computer-aided diagnosis of many lumbar-related pathologies.

Experience of meningovascular syphilis in human immunodeficiency virus infected patient.

Since the start of the antibiotic era, syphilis has become rare. However, in recent times, it has tended to be prevalent concomitantly with human immunodeficiency virus (HIV) infection and coinfection in North America and Europe. Now, such cases are expected to increase in elsewhere including Korea. A 40-year-old male patient visited hospital complaining of a headache for about one month. Brain computed tomography and magnetic resonance imaging, showed leptomeninged enhancing mass with edema an right porisylvian region, which was suspected to be glioma. Patient underwent a blood test and was diagnosed with syphilis and acquired immune deficiency syndrome. Partial cortical and subcortical resection were performed after small craniotomy. The dura was thick, adhered to the brain cortex, and was accompanied by hyperemic change of the cortex. The pathologic diagnosis was meningovascular syphilis (MS) in HIV infection. After the operation, the patient was treated with aqueous penicillin G. Thereafter, he had no neurological deficit except intermittent headache. At first, this case was suspected to be glioma, but it was eventually diagnosed as MS in HIV coinfection. At this point the case was judged to be worth reporting.