N6-methyladenine DNA modification in Drosophila.

DNA N(6)-methyladenine (6mA) modification is commonly found in microbial genomes and plays important functions in regulating numerous biological processes in bacteria. However, whether 6mA occurs and what its potential roles are in higher-eukaryote cells remain unknown. Here, we show that 6mA is present in Drosophila genome and that the 6mA modification is dynamic and is regulated by the Drosophila Tet homolog, DNA 6mA demethylase (DMAD), during embryogenesis. Importantly, our biochemical assays demonstrate that DMAD directly catalyzes 6mA demethylation in vitro. Further genetic and sequencing analyses reveal that DMAD is essential for development and that DMAD removes 6mA primarily from transposon regions, which correlates with transposon suppression in Drosophila ovary. Collectively, we uncover a DNA modification in Drosophila and describe a potential role of the DMAD-6mA regulatory axis in controlling development in higher eukaryotes.

Stella safeguards the oocyte methylome by preventing de novo methylation mediated by DNMT1.

Postnatal growth of mammalian oocytes is accompanied by a progressive gain of DNA methylation, which is predominantly mediated by DNMT3A, a de novo DNA methyltransferase1,2. Unlike the genome of sperm and most somatic cells, the oocyte genome is hypomethylated in transcriptionally inert regions2-4. However, how such a unique feature of the oocyte methylome is determined and its contribution to the developmental competence of the early embryo remains largely unknown. Here we demonstrate the importance of Stella, a factor essential for female fertility5-7, in shaping the oocyte methylome in mice. Oocytes that lack Stella acquire excessive DNA methylation at the genome-wide level, including in the promoters of inactive genes. Such aberrant hypermethylation is partially inherited by two-cell-stage embryos and impairs zygotic genome activation. Mechanistically, the loss of Stella leads to ectopic nuclear accumulation of the DNA methylation regulator UHRF18,9, which results in the mislocalization of maintenance DNA methyltransferase DNMT1 in the nucleus. Genetic analysis confirmed the primary role of UHRF1 and DNMT1 in generating the aberrant DNA methylome in Stella-deficient oocytes. Stella therefore safeguards the unique oocyte epigenome by preventing aberrant de novo DNA methylation mediated by DNMT1 and UHRF1.

Comparison of Flavonoid Content, Antioxidant Potential, Acetylcholinesterase Inhibition Activity and Volatile Components Based on HS-SPME-GC-MS of Different Parts from Matteuccia struthiopteris (L.) Todaro.

Matteuccia struthiopteris is one of the most globally consumed edible ferns and widely used in folk medicine. Reports mainly focus on young fronds and the rhizome which are common edible medicinal parts. However, there are few detailed reports on other parts. Therefore, the volatile components of different parts based on HS-SPME-GC-MS were identified, and total flavonoid contents, antioxidant activities and acetylcholinesterase inhibitory activities were compared in order to reveal the difference of volatile components and potential medicinal value of different parts. The results showed that total flavonoid contents, antioxidant activities and volatile components of different parts were obviously different. The crozier exhibited the strongest antioxidant activities, but only underground parts exhibited a dose-dependent inhibition potential against AChE. Common volatile compounds were furfural and 2-furancarboxaldehyde, 5-methyl-. In addition, it was found that some volatile components from adventitious root, trophophyll, sporophyll and petiole were important ingredients in food, cosmetics, industrial manufacturing and pharmaceutical applications.

A PCB image segmentation model based on rotational X-ray computed laminography imaging.

BACKGROUND: The rapid development of industrialization in printed circuit board (PCB) warrants more complexity and integrity, which entails an essential procedure of PCB inspection. X-ray computed laminography (CL) enables inspection of arbitrary regions for large-sized flat objects with high resolution. PCB inspection based on CL imaging is worthy of exploration. OBJECTIVE: This work aims to extract PCB circuit layer information based on CL imaging through image segmentation technique. METHODS: In this work, an effective and applicable segmentation model for PCB CL images is established for the first time. The model comprises two components, with one integrating edge diffusion and l0 smoothing to filter CL images with aliasing artifacts, and the other being the fuzzy energy-based active contour model driven by local pre-fitting energy to segment the filtered images. RESULT: The proposed model is able to suppress aliasing artifacts in the PCB CL images and has good performance on images of different circuit layers. CONCLUSIONS: Results of the simulation experiment reveal that the method is capable of accurate segmentation under ideal scanning condition. Testing of different PCBs and comparison of different segmentation methods authenticate the applicability and superiority of the model.

Elucidating Flavonoid and Antioxidant Activity in Edible and Medicinal Herbs Woodwardia japonica (L.f.) Sm. Based on HPLC-ESI-TOF-MS and Artificial Neural Network Model: Response to Climatic Factors.

Woodwardia japonica is a kind of great potential edible and medicinal fern. In a previous study, it was found that flavonoid and antioxidant activity of W. japonica from different sites were different. However, the cause of the differences has still been unclear, which has restricted the utilization of W. japonica. In this paper, flavonoid and antioxidant activity of W. japonica from nine different regions were determined with the method of a colorimetric assay with UV-VIS spectrophotometry and HPLC-ESI-TOF-MS, and the effects of climate factors on flavonoids and antioxidant activities were evaluated by mathematical modeling and statistical methods. The results showed: (1) total flavonoid content (TFC) of W. japonica from Wuyi Mountain (Jiangxi) was the highest, which might be related to the low temperature; (2) the differences of antioxidant activities of W. japonica might be related to precipitation; (3) five flavonols, two flavones and one isoflavone were tentatively identified in W. japonica; (4) flavonol and isoflavone might be affected by sunshine duration, and flavones were probably related to temperature. In conclusion, the effects of climate factors on flavonoids and antioxidants are significant, which would provide an important basis for further exploring the mechanism of climate affecting secondary metabolites.

Correction of motion artifact in CL based on MAFusNet.

Computed laminography (CL) is one of the best methods for nondestructive testing of plate-like objects. If the object and the detector move continually while the scanning is being done, the data acquisition efficiency of CL will be significantly increased. However, the projection images will contain motion artifact as a result. A multi-angle fusion network (MAFusNet) is presented in order to correct the motion artifact of CL projection images considering the properties of CL projection images. The multi-angle fusion module significantly increases the ability of MAFusNet to deblur by using data from nearby projection images, and the feature fusion module lessens information loss brought on by data flow between the encoders. In contrast to conventional deblurring networks, the MAFusNet network employs synthetic datasets for training and performed well on realistic data, proving the network's outstanding generalization. The multi-angle fusion-based network has a significant improvement in the correction effect of CL motion artifact through ablation study and comparison with existing classical deblurring networks, and the synthetic training dataset can also significantly lower the training cost, which can effectively improve the quality and efficiency of CL imaging in industrial nondestructive testing.

Comparison of Constituents and Antioxidant Activity of Above-Ground and Underground Parts of Dryopteris crassirhizoma Nakai Based on HS-SPME-GC-MS and UPLC/Q-TOF-MS.

Dryopteris crassirhizoma Nakai is a Chinese traditional medicinal fern plant for heat-clearing and detoxifying, promoting blood circulation and dissipating blood stasis. Previous researches showed that many factors could influence the components of medicinal plants, and the plant part is one of the main factors. So far, only the underground part of D. crassirhizoma, called "Mianma Guanzhong", has been widely sold in the market. However, the above-ground part was usually at low utilization, resulting in a waste of medicinal resources. In order to further develop and utilize the medicinal resources of D. crassirhizoma, the constituents, total flavonoid contents and antioxidant activity of the above-ground and underground parts of D. crassirhizoma were tentatively analyzed and compared based on HS-SPME-GC-MS and UPLC/Q-TOF-MS. The results showed that (1) the volatile components were mainly focused in the above-ground part of D. crassirhizoma, including 3-carene, isoledene, ionene, 4-amino-1-naphthol and furfural. (2) Nonvolatile components of the underground part of D. crassirhizoma contained phenolic acid, flavonoids, phloroglucinol and less fatty acid. (3) The common compounds of the above-ground and underground parts of D. crassirhizoma were phenolic acid and flavaspidic acid AB. (4) Antioxidant activity of the underground part was stronger than that of the above-ground part of D. crassirhizoma. In conclusion, both the above-ground and underground parts of D. crassirhizoma are important medicinal resources worthy of further development.

Detection of N6-Methyladenine in Eukaryotes.

DNA N6-methyladenine (6mA) is a chemical modification at the N6-positon of adenine. In the last decades, 6mA had been found in genome from numerous prokaryotic species, but only existed in a few lower eukaryotes. In prokaryotes, 6mA plays an important role in restriction-modification, DNA replication, and DNA mismatch repair. Because of the too low abundance of 6mA, it was long-stalled whether 6mA existed in multicellular eukaryotes and playing any functions, particularly in mammals. In recent years, partially benefitting from the advances in analytical methods, 6mA was found in the genomes from Drosophila melanogaster, Chlamydomonas algae, Caenorhabditis elegans, zebrafish, Xenopus laevis and mouse embryonic stem cells and even in the human genome. The 6mA was dynamic changed in early embryonic development of fly and zebrafish and much more enriched in gene body of transposons in fly, repetitive regions in zebrafish, around the transcription start sites in Chlamydomonas, and widespread distribution in C. elegans, indicating 6mA probably playing different functions in different species. Meanwhile, 6mA methylases and demethylases were found in fly, worm, and Chlamydomonas. In this chapter, we will briefly review the distribution, regulation, and function of 6mA in eukaryotes and focus on the advances of 6mA analysis methods, especially LC-MS/MS, immunoprecipitation, next-generation sequencing, and single-molecule real-time sequencing technology.

Roles of the CSE1L-mediated nuclear import pathway in epigenetic silencing.

Epigenetic silencing can be mediated by various mechanisms, and many regulators remain to be identified. Here, we report a genome-wide siRNA screening to identify regulators essential for maintaining gene repression of a CMV promoter silenced by DNA methylation. We identified CSE1L (chromosome segregation 1 like) as an essential factor for the silencing of the reporter gene and many endogenous methylated genes. CSE1L depletion did not cause DNA demethylation. On the other hand, the methylated genes derepressed by CSE1L depletion largely overlapped with methylated genes that were also reactivated by treatment with histone deacetylase inhibitors (HDACi). Gene silencing defects observed upon CSE1L depletion were linked to its nuclear import function for certain protein cargos because depletion of other factors involved in the same nuclear import pathway, including KPNAs and KPNB1 proteins, displayed similar derepression profiles at the genome-wide level. Therefore, CSE1L appears to be critical for the nuclear import of certain key repressive proteins. Indeed, NOVA1, HDAC1, HDAC2, and HDAC8, genes known as silencing factors, became delocalized into cytosol upon CSE1L depletion. This study suggests that the cargo specificity of the protein nuclear import system may impact the selectivity of gene silencing.

Stella protein facilitates DNA demethylation by disrupting the chromatin association of the RING finger-type E3 ubiquitin ligase UHRF1.

Stella is a maternal gene required for oogenesis and early embryogenesis. Stella overexpression in somatic cells causes global demethylation. As we have recently shown, Stella sequesters nuclear ubiquitin-like with PHD and RING finger domains 1 (UHRF1), a RING finger-type E3 ubiquitin ligase essential for DNA methylation mediated by DNA methyltransferase 1 and triggers global demethylation. Here, we report an overexpressed mutant Stella protein without nuclear export activity surprisingly retained its ability to cause global demethylation. By combining biochemical interaction assays, isothermal titration calorimetry, immunostaining, and live-cell imaging with fluorescence recovery after photobleaching, we found that Stella disrupts UHRF1's association with chromatin by directly binding to the plant homeodomain of UHRF1 and competing for the interaction between UHRF1 and the histone H3 tail. Consistently, overexpression of Stella mutants that do not directly interact with UHRF1 fails to cause genome-wide demethylation. In the presence of nuclear Stella, UHRF1 could not bind to chromatin and exhibited increased dynamics in the nucleus. Our results indicate that Stella employs a multilayered mechanism to achieve robust UHRF1 inhibition, which involves the dissociation from chromatin and cytoplasmic sequestration of UHRF1.

The proinflammatory cytokine TNFα induces DNA demethylation-dependent and -independent activation of interleukin-32 expression.

IL-32 is a cytokine involved in proinflammatory immune responses to bacterial and viral infections. However, the role of epigenetic events in the regulation of IL-32 gene expression is understudied. Here we show that IL-32 is repressed by DNA methylation in HEK293 cells. Using ChIP sequencing, locus-specific methylation analysis, CRISPR/Cas9-mediated genome editing, and RT-qPCR (quantitative RT-PCR) and immunoblot assays, we found that short-term treatment (a few hours) with the proinflammatory cytokine tumor necrosis factor α (TNFα) activates IL-32 in a DNA demethylation-independent manner. In contrast, prolonged TNFα treatment (several days) induced DNA demethylation at the promoter and a CpG island in the IL-32 gene in a TET (ten-eleven translocation) family enzyme- and NF-κB-dependent manner. Notably, the hypomethylation status of transcriptional regulatory elements in IL-32 was maintained for a long time (several weeks), causing elevated IL-32 expression even in the absence of TNFα. Considering that IL-32 can, in turn, induce TNFα expression, we speculate that such feedforward events may contribute to the transition from an acute inflammatory response to chronic inflammation.

Review of CT image reconstruction open source toolkits.

Computed tomography (CT) has been widely applied in medical diagnosis, nondestructive evaluation, homeland security, and other science and engineering applications. Image reconstruction is one of the core CT imaging technologies. In this review paper, we systematically reviewed the currently publicly available CT image reconstruction open source toolkits in the aspects of their environments, object models, imaging geometries, and algorithms. In addition to analytic and iterative algorithms, deep learning reconstruction networks and open codes are also reviewed as the third category of reconstruction algorithms. This systematic summary of the publicly available software platforms will help facilitate CT research and development.

Maternal Sall4 Is Indispensable for Epigenetic Maturation of Mouse Oocytes.

Sall4 (Splat-like 4) plays important roles in maintaining pluripotency of embryonic stem cells and in various developmental processes. Here, we find that Sall4 is highly expressed in oocytes and early embryos. To investigate the roles of SALL4 in oogenesis, we generated Sall4 maternal specific knock-out mice by using CRISPR/Cas9 system, and we find that the maternal deletion of Sall4 causes developmental arrest of oocytes at germinal vesicle stage with non-surrounded nucleus, and the subsequent meiosis resumption is prohibited. We further discover that the loss of maternal Sall4 causes failure in establishment of DNA methylation in oocytes. Furthermore, we find that Sall4 modulates H3K4me3 and H3K27me3 modifications by regulating the expression of key histone demethylases coding genes Kdm5b, Kdm6a, and Kdm6b in oocytes. Moreover, we demonstrate that the aberrant H3K4me3 and H3K27me3 cause mis-expression of genes that are critical for oocytes maturation and meiosis resumption. Taken together, our study explores a pivotal role of Sall4 in regulating epigenetic maturation of mouse oocytes.

Metabolically Generated Stable Isotope-Labeled Deoxynucleoside Code for Tracing DNA N6-Methyladenine in Human Cells.

DNA N6-methyl-2'-deoxyadenosine (6mdA) is an epigenetic modification in both eukaryotes and bacteria. Here we exploited stable isotope-labeled deoxynucleoside [15N5]-2'-deoxyadenosine ([15N5]-dA) as an initiation tracer and for the first time developed a metabolically differential tracing code for monitoring DNA 6mdA in human cells. We demonstrate that the initiation tracer [15N5]-dA undergoes a specific and efficient adenine deamination reaction leading to the loss the exocyclic amine 15N, and further utilizes the purine salvage pathway to generate mainly both [15N4]-dA and [15N4]-2'-deoxyguanosine ([15N4]-dG) in mammalian genomes. However, [15N5]-dA is largely retained in the genomes of mycoplasmas, which are often found in cultured cells and experimental animals. Consequently, the methylation of dA generates 6mdA with a consistent coding pattern, with a predominance of [15N4]-6mdA. Therefore, mammalian DNA 6mdA can be potentially discriminated from that generated by infecting mycoplasmas. Collectively, we show a promising approach for identification of authentic DNA 6mdA in human cells and determine if the human cells are contaminated with mycoplasmas.

Interior tomography with curvelet-based regularization.

The interior problem, i.e. reconstruction from local truncated projections in computed tomography (CT), is common in practical applications. However, its solution is non-unique in a general unconstrained setting. To solve the interior problem uniquely and stably, in recent years both the prior knowledge- and compressive sensing (CS)-based methods have been developed. Those theoretically exact solutions for the interior problem are called interior tomography. Along this direction, we propose here a new CS-based method for the interior problem based on the curvelet transform. A curvelet is localized in both radial and angular directions in the frequency domain. A two-dimensional (2D) image can be represented in a curvelet frame. We employ the curvelet transform coefficients to regularize the interior problem and obtain a curvelet frame based regularization method (CFRM) for interior tomography. The curvelet coefficients of the reconstructed image are split into two sets according to their visibility from the interior data, and different regularization parameters are used for these two sets. We also presents the results of numerical experiments, which demonstrate the feasibility of the proposed approach.

Simultaneous algebraic reconstruction technique based on guided image filtering.

The challenge of computed tomography is to reconstruct high-quality images from few-view projections. Using a prior guidance image, guided image filtering smoothes images while preserving edge features. The prior guidance image can be incorporated into the image reconstruction process to improve image quality. We propose a new simultaneous algebraic reconstruction technique based on guided image filtering. Specifically, the prior guidance image is updated in the image reconstruction process, merging information iteratively. To validate the algorithm practicality and efficiency, experiments were performed with numerical phantom projection data and real projection data. The results demonstrate that the proposed method is effective and efficient for nondestructive testing and rock mechanics.

Boronic acid-mediated polymerase chain reaction for gene- and fragment-specific detection of 5-hydroxymethylcytosine.

The gene- or fragment-specific detection of newly recognized deoxyribonucleic acid (DNA) base 5-hydroxymethylcytosine (5hmC) will provide insights into its critical functions in development and diseases, and is also important for screening 5hmC-rich genes as an indicator of epigenetic states, pathogenic processes and pharmacological responses. Current analytical technologies for gene-specific detection of 5hmC are heavily dependent on glucosylated 5hmC-resistant restriction endonuclease cleavage. Here, we find that boronic acid (BA) can inhibit the amplification activity of Taq DNA polymerase for replicating glucosylated 5hmC bases in template DNA by interacting with their glucose moiety. On the basis of this finding, we propose for the first time a BA-mediated polymerase chain reaction (PCR) assay for rapid and sensitive detection of gene- or fragment-specific 5hmC without restriction-assay-like sequence limitations. To optimize the BA-mediated PCR assay, we further tested BA derivatives and show that one BA derivative, 2-(2'-chlorobenzyloxy) phenylboronic acid, displays the highest inhibitory efficiency. Using the optimized assay, we demonstrate the enrichment of 5hmC in an intron region of Pax5 gene (a member of the paired box family of transcription factors) in mouse embryonic stem cells. Our work potentially opens a new way for the screening and identification of 5hmC-rich genes and for high throughput analysis of 5hmC in mammalian cells.

Surrogate arterial phase imaging using a long duration (≈1.5 min) radial acquisition T1-weighted sequence: an alternative in patients unable to breath-hold.

BACKGROUND: Pediatric and adult patients unable to suspend respiration generally undergo magnetic resonance (MR) examinations that lack arterial phase imaging, which is a phase that provides substantial information on disease processes. An MR strategy that provides this type of information may be of considerable value. PURPOSE: To describe and assess the feasibility and enhancement quality of early-phase imaging utilizing long-duration radial 3D-GRE imaging by initiating the sequence prior to starting contrast injection. MATERIAL AND METHODS: Thirty-three consecutive patients (10 men, 23 women; 50.7 ± 25.5 years) underwent free-breathing gadolinium-enhanced radial 3D-GRE, with sequence initiation 30 s prior to contrast injection. Late hepatic arterial (LHA) phase was chosen for comparison. Images were evaluated for enhancement and overall image quality. Organ enhancement was calculated. Sub-group analysis was performed. RESULTS: Twenty-two examinations of radial 3D-GRE sequences were acquired during the LHA phase. Organ enhancement scores were of satisfactory to good quality (range, 3.32-3.82). There was a significant trend of superior overall enhancement quality scores in pediatrics and examinations performed at 3 T (P = 0.0225 and 0.0001, respectively). CONCLUSION: Arterial phase abdominal MR imaging is feasible using conventional radial 3D-GRE by adopting this simplistic proposed approach, which may allow arterial-phase imaging in patients unable to breath-hold.

Utilizing gemstone spectral CT imaging to evaluate the therapeutic efficacy of radiofrequency ablation in lung cancer.

PURPOSE: To evaluate the therapeutic efficacy of radiofrequency ablation (RFA), quantitative water-based, and iodine-based images of gemstone spectral computed tomography (CT) were analyzed. PATIENTS AND METHODS: 30 patients underwent lung RFAs from March 2012 to March 2013. Through enhanced chest scans, we obtained the tumor size values by conventional CT images, and quantitatively analyzed the densities of iodine and water in lung tumors from water-based and iodine-based material decomposition images. RESULTS: Tumors in 22 cases increased in size after RFA while there was no detectable change in the remaining 8 cases. Through water-based material decomposition images, the water content in the tumors increased from (1014.76 ± 6.83 mg/mL) to (1022.71 ± 10.16 mg/mL) after RFA, and this difference was significant (t = -2.329, p < 0.05). Through iodine-based material decomposition images, the iodine content in the tumors was 2.49 ± 0.74 mg/mL before RFA. The tumors were mostly or completely necrotized after RFA and the iodine content in the area of necrosis reduced to 0.45 ± 0.29 mg/m (t = 11.072, p = 0.000). CONCLUSION: By comparing the tumor size, water content and iodine content before and after RFA, we can visualize the morphology and metabolic states of the tumors and evaluate the therapeutic efficiency.

X-ray CT geometrical calibration via locally linear embedding.

For X-ray computed tomography (CT), geometric calibration and rigid patient motion compensation are inter-related issues for optimization of image reconstruction quality. Non-calibrated system geometry and patient movement during a CT scan will result in streak-like, blurring and other artifacts in reconstructed images. In this paper, we propose a locally linear embedding based calibration approach to address this challenge under a rigid 2D object assumption and a more general way than what has been reported before. In this method, projections are linearly represented by up-sampled neighbors via locally linear embedding, and CT system parameters are iteratively estimated from projection data themselves. Numerical and experimental studies show that images reconstructed with calibrated parameters are in excellent agreement with the counterparts reconstructed with the true parameters.