Molecular mechanism of high glucose-induced mitochondrial DNA damage in retinal ganglion cells.

Mitochondrial DNA damage in retinal ganglion cells (RGCs) may be closely related to lesions of glaucoma. RGCs were cultured with different concentrations of glucose and grouped into 3 groups, namely normal control (NC) group, Low-Glu group, and High-Glu group. Cell viability was measured with cell counting kit-8, and cell apoptosis was measured using flow cytometry. The DNA damage was measured with comet assay, and the morphological changes of damaged mitochondria in RGCs were observed using TEM. Western blot analyzed the expression of MRE11, RAD50, and NBS1 protein. Cell viability of RGCs in Low-Glu and High-Glu groups were lower than that of NC group in 48 and 96 h. The cell apoptosis in NC group was 4.9%, the Low-Glu group was 12.2% and High-Glu group was 24.4%. The comet imaging showed that NC cells did not have tailings, but the low-Glu and high-Glu group cells had tailings, indicating that the DNA of RGCs had been damaged. TEM, mitochondrial membrane potential, ROS, mitochondrial oxygen consumption, and ATP content detection results showed that RGCs cultured with high glucose occurred mitochondrial morphology changes and dysfunction. MRE11, RAD50, and NBS1 protein expression associated with DNA damage repair pathway in High-Glu group declined compared with Low-Glu group. Mitochondrial DNA damage caused by high glucose will result in apoptosis of retinal ganglion cells in glaucoma.

Identification of a novel ferroptosis-related gene signature associated with retinal degeneration induced by light damage in mice.

Background: Neurodegenerative retinal diseases such as retinitis pigmentosa are serious disorders that may cause irreversible visual impairment. Ferroptosis is a novel type of programmed cell death, and the involvement of ferroptosis in retinal degeneration is still unclear. This study aimed to investigate the related ferroptosis genes in a mice model of retinal degeneration induced by light damage. Methods: A public dataset of GSE10528 deriving from the Gene Expression Omnibus database was analyzed to identify the differentially expressed genes (DEGs). Gene set enrichment analysis between light damage and control group was conducted. The differentially expressed ferroptosis-related genes (DE-FRGs) were subsequently identified by intersecting the DEGs with a ferroptosis genes dataset retrieved from the FerrDb database. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were further performed using the DE-FRGs. A protein-protein interaction (PPI) network was constructed to identify hub ferroptosis-related genes (HFRGs). The microRNAs (miRNAs)-HFRGs, transcription factors (TFs)-HFRGs networks as well as target drugs potentially interacting with HFRGs were analyzed utilizing bioinformatics algorithms. Results: A total of 932 DEGs were identified between the light damage and control group. Among these, 25 genes were associated with ferroptosis. GO and KEGG analyses revealed that these DE-FRGs were mainly enriched in apoptotic signaling pathway, response to oxidative stress and autophagy, ferroptosis, necroptosis and cytosolic DNA-sensing pathway. Through PPI network analysis, six hub ferroptosis-related genes (Jun, Stat3, Hmox1, Atf3, Hspa5 and Ripk1) were ultimately identified. All of them were upregulated in light damage retinas, as verified by the GSE146176 dataset. Bioinformatics analyses predicated that 116 miRNAs, 23 TFs and several potential therapeutic compounds might interact with the identified HFRGs. Conclusion: Our study may provide novel potential biomarkers, therapeutic targets and new insights into the ferroptosis landscape in retinal neurodegenerative diseases.

IS-Seq: a bioinformatics pipeline for integration sites analysis with comprehensive abundance quantification methods.

BACKGROUND: Integration site (IS) analysis is a fundamental analytical platform for evaluating the safety and efficacy of viral vector based preclinical and clinical Gene Therapy (GT). A handful of groups have developed standardized bioinformatics pipelines to process IS sequencing data, to generate reports, and/or to perform comparative studies across different GT trials. Keeping up with the technological advances in the field of IS analysis, different computational pipelines have been published over the past decade. These pipelines focus on identifying IS from single-read sequencing or paired-end sequencing data either using read-based or using sonication fragment-based methods, but there is a lack of a bioinformatics tool that automatically includes unique molecular identifiers (UMI) for IS abundance estimations and allows comparing multiple quantification methods in one integrated pipeline. RESULTS: Here we present IS-Seq a bioinformatics pipeline that can process data from paired-end sequencing of both old restriction sites-based IS collection methods and new sonication-based IS retrieval systems while allowing the selection of different abundance estimation methods, including read-based, Fragment-based and UMI-based systems. CONCLUSIONS: We validated the performance of IS-Seq by testing it against the most popular  analytical workflow available in the literature (INSPIIRED) and using different scenarios. Lastly, by performing extensive simulation studies and a comprehensive wet-lab assessment of our IS-Seq pipeline we could show that in clinically relevant scenarios, UMI quantification provides better accuracy than the currently most widely used sonication fragment counts as a method for IS abundance estimation.

Clinical Significance of Visual Training in Improving Visual Function After Retinal Detachment in Adolescents.

Context: Epidemiological data has shown that retinal detachment (RD) can occur at any age but has a poor prognosis in the adolescent population, which can cause huge obstacles to their life and learning. Although medications can achieve some curative effect, they have potential side effects and differences in individual efficacy. Objective: • The study intended to explore the clinical significance of visual training in improving recovery of postoperative visual function after external reduction of retinal detachment in adolescent patients. Design: The research team designed a prospective randomized controlled study. Setting: The study took place at Guiyang First People's Hospital in Guiyang, Guizhou, China. Participants: Participants were 110 adolescents with retinal detachments who underwent external reduction surgery, each on one eye for 110 eyes in total, and who were patients at the hospital between June 2015 and June 2019. Intervention: The research team assigned 52 participants to the visual-function training group, the intervention group, and 58 participants to the control group, according to the random-number-table method. Each group participated in training method for six months. Outcome Measures: To compare the groups, the research team measured visual function using the Visual Function scale (VF), binocular fusion function using the Worth Four Light Test (W4LT), and stereoscopic vision using the Titmus Stereo Test. The team obtained preoperative and postoperative data-at baseline, one day before surgery and postoperatively at one month and 3 months, and postintervention at 6 months. Results: Both groups had visual impairment after surgery. For visual function, the intervention group's scores after surgery increased gradually and were significantly higher than those of the control group at each follow-up time (P < .05). No significant difference in binocular fusion function existed between the groups at baseline or at one month after surgery (P > .05). At 3 months after surgery and postintervention, the proportions of participants in the intervention group with normal binocular fusion function were 86.54% and 88.46%, respectively, compared to that of the control group, at 68.97% and 70.69%, respectively. The intervention group's recovery was significantly better than that of the control group at 3 months after surgery and postintervention, at P < .028 and P < .022, respectively. No significant difference existed between the groups in stereoscopic vision at baseline, at 9.62% and 12.07%, respectively (P > .05). The proportion of participants in the intervention group with normal, binocular, stereoscopic vision increased gradually, and at one and 3 months after surgery and postintervention was 67.31%, 82.69%, and 88.46%, respectively. The intervention group's recovery was significantly better than that of the control group at one and 3 months after surgery and postintervention, at P < .028, P < .010, and P < .013, respectively. Conclusions: Visual training can effectively promote the recovery of visual function after external reduction of RD in adolescents and improve patients' prognoses. Moreover, long-term persistence can achieve significant effects, making the training worthy of clinical promotion.

Explore the molecular mechanism of angle-closure glaucoma in elderly patients induced telomere shortening of retinal ganglion cells through oxidative stress.

Senile glaucoma is a common ophthalmological disease in the elderly. It is a disease of visual papillary perfusion caused by elevated intraocular pressure, complicated by visual dysfunction. Glaucoma can cause serious damage to the normal vision of the elderly. Therefore, exploring the related molecular mechanisms of glaucoma is of great significance to the diagnosis and treatment of glaucoma. This is an exploratory study. Establish a mouse model and conduct experimental groupings. After one week of adaptive feeding, the mice were intraperitoneally injected with an anesthetic mixture: ketamine + xylazine. Then the mice were sacrificed by neck dissection, and the eyeball tissues were immediately dissected. HE staining was used to analyze the histopathological characteristics of the retina of each group of mice. MitoSOX fluorescent probe was used to analyze the content of ROS in retinal tissue. The ELISA analysis was used to detect the activation of ß-galactosidase for the aging characteristics of retinal ganglion cells in retinal tissues. Immunohistochemistry experiments were used to analyze the expression of telomerase TERT in retinal tissues. Western blot analysis was used to determine the expression of proteins POT1, TERF1, TERF2, and TINF2 in retinal tissues. The HE staining experiment showed that the damage of retinal tissue decreased from group Glaucoma to group Old, group Old to group Young. The experimental results of MitoSOX fluorescent probe show that ROS content is positively correlated with the degree of tissue damage. ELISA analysis results showed that the expression trend of ß-galactosidase was the same as the ROS content. The protein expression levels related to telomere protection (TRET, POT1, TREF1, TREF2 and TINF2) all increased from group Glaucoma to group Old, group Old to group Young. The increase in ROS content, the decrease in telomere protection-related protein expression (telomere shortening) induced by ROS, and the increase of the expression of ß-galactosidase, are all potential molecular mechanisms for the occurrence of angle-closure glaucoma in elderly patients.

Hyperglycemia induces retinal ganglion cell endoplasmic reticulum stress to the involvement of glaucoma in diabetic mice.

Glaucoma is a neurodegenerative disease leading to visual loss. Since glaucoma is associated with chronic renal diseases (RDs) their rate is higher in patients with RDs, and end-stage RDs (ESRDs) than in the general population and kidney transplant recipients. OBJECTIVE: To explore the molecular mechanism of diabetic internal environment in regulating the endoplasmic reticulum stress of the retinal ganglion cells (RGCs). METHODS: Thirty-six SPF grade type 2 diabetes models were divided into 3 groups: Diabetes mellitus (DM), DM + glaucoma and 4-phenylbutyric acid-DM (4-PBA-DM) + glaucoma group. C57BL6 mice of the same week age were taken as the negative control (NC) group. The morphology of RGCs and their axon in the 4 groups were labeled by fluorescent reactive dye Dil. The apoptosis situation of RGCs was determined by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay. The protein expression values of RTN4IP1, Protein kinase R-like endoplasmic reticulum kinase (PERK), eukaryotic initiation factor 2A (eIF2a) and X-box-binding Protein 1 (XBP1) were determined by western blot. The relative mRNA levels of cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP), Caspase12 and Bax were determined by quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Glaucoma promotes the apoptosis of RGCs. The protein expression values of RTN4IP1, PERK and XBP1 in DM mouse models with glaucoma were much higher compared to only DM mouse models. Further injection of endoplasmic reticulum stress inhibitor 4-PBA decreased the expression values. The relative mRNA levels of CHOP, Cysteine aspartic acid specific protease12 (Caspase12) and BCL2-associated X protein (Bax) in DM + glaucoma were significantly higher compared to those in DM group. Further injection of endoplasmic reticulum stress inhibitor 4-PBA decreased the mRNA levels. CONCLUSION: Endoplasmic reticulum stress (ERS) is the underlying cause of glaucoma, which could promote the apoptosis of RGCs in diabetic mice.

Asymmetric cryptosystem based on optical scanning cryptography and elliptic curve algorithm.

We propose an asymmetric cryptosystem based on optical scanning cryptography (OSC) and elliptic curve cryptography (ECC) algorithm. In the encryption stage of OSC, an object is encrypted to cosine and sine holograms by two pupil functions calculated via ECC algorithm from sender's biometric image, which is sender's private key. With the ECC algorithm, these holograms are encrypted to ciphertext, which is sent to the receiver. In the stage of decryption, the encrypted holograms can be decrypted by receiver's biometric private key which is different from the sender's private key. The approach is an asymmetric cryptosystem which solves the problem of the management and dispatch of keys in OSC and has more security strength than the conventional OSC. The feasibility of the proposed method has been convincingly verified by numerical and experiment results.

High-throughput analysis of hematopoietic stem cell engraftment after intravenous and intracerebroventricular dosing.

Hematopoietic stem/progenitor cell gene therapy (HSPC-GT) has shown clear neurological benefit in rare diseases, which is achieved through the engraftment of genetically modified microglia-like cells (MLCs) in the brain. Still, the engraftment dynamics and the nature of engineered MLCs, as well as their potential use in common neurogenerative diseases, have remained largely unexplored. Here, we comprehensively characterized how different routes of administration affect the biodistribution of genetically engineered MLCs and other HSPC derivatives in mice. We generated a high-resolution single-cell transcriptional map of MLCs and discovered that they could clearly be distinguished from macrophages as well as from resident microglia by the expression of a specific gene signature that is reflective of their HSPC ontogeny and irrespective of their long-term engraftment history. Lastly, using murine models of Parkinson's disease and frontotemporal dementia, we demonstrated that MLCs can deliver therapeutically relevant levels of transgenic protein to the brain, thereby opening avenues for the clinical translation of HSPC-GT to the treatment of major neurological diseases.

Dynamic transcriptional activity and chromatin remodeling of regulatory T cells after varied duration of interleukin-2 receptor signaling.

Regulatory T (Treg) cells require (interleukin-2) IL-2 for their homeostasis by affecting their proliferation, survival and activation. Here we investigated transcriptional and epigenetic changes after acute, periodic and persistent IL-2 receptor (IL-2R) signaling in mouse peripheral Treg cells in vivo using IL-2 or the long-acting IL-2-based biologic mouse IL-2-CD25. We show that initially IL-2R-dependent STAT5 transcription factor-dependent pathways enhanced gene activation, chromatin accessibility and metabolic reprogramming to support Treg cell proliferation. Unexpectedly, at peak proliferation, less accessible chromatin prevailed and was associated with Treg cell contraction. Restimulation of IL-2R signaling after contraction activated signature IL-2-dependent genes and others associated with effector Treg cells, whereas genes associated with signal transduction were downregulated to somewhat temper expansion. Thus, IL-2R-dependent Treg cell homeostasis depends in part on a shift from more accessible chromatin and expansion to less accessible chromatin and contraction. Mouse IL-2-CD25 supported greater expansion and a more extensive transcriptional state than IL-2 in Treg cells, consistent with greater efficacy to control autoimmunity.

A phase sensitive x-ray breast tomosynthesis system: Preliminary patient images with cancer lesions.

Phase-sensitive x-ray imaging continues to attract research for its ability to visualize weakly absorbing details like those often encountered in biology and medicine. We have developed and assembled the first inline-based high-energy phase sensitive breast tomosynthesis (PBT) system, which is currently undergoing patient imaging testing at a clinical site. The PBT system consists of a microfocus polychromatic x-ray source and a direct conversion-based flat panel detector coated with a 1 mm thick amorphous selenium layer allowing a high detective quantum efficiency at high energies. The PBT system scans a compressed breast over 15° with 9 angular projection views. The high-energy scan parameters are carefully selected to ensure similar or lower mean glandular dose levels to the clinical standard of care systems. Phase retrieval and data binning are applied to the phase contrast angular projection views and a filtered back-projection algorithm is used to reconstruct the final images. This article reports the distributions of radiation dose versus thickness of the compressed breasts at 59 and 89 kV and sample PBT images acquired from 3 patients. Preliminary PBT images demonstrate the feasibility of this new imaging modality to acquire breast images at lower radiation dose as compared to the clinical digital breast tomosynthesis system with enhanced lesion characteristics (i.e. lesion spiculation and margins).

Dual-energy phase retrieval algorithm for inline phase sensitive x-ray imaging system.

Phase retrieval is vital for quantitative x-ray phase contrast imaging. This work presents an iterative method to simultaneously retrieve the x-ray absorption and phase images from a single x-ray exposure. The proposed approach uses the photon-counting detectors' energy-resolving capability in providing multiple spectrally resolved phase contrast images from a single x-ray exposure. The retrieval method is derived, presented, and experimentally tested with a multi-material phantom in an inline phase contrast imaging setup. By separating the contributions of photoelectric absorption and Compton scattering to the attenuation, the authors divide the phase contrast image into two portions, the attenuation map arises from photoelectric absorption and a pseudo phase contrast image generated by electron density. This way one can apply the Phase Attenuation Dualiby (PAD) algorithm and Fresnel propagation for the iteration. The retrieval results from the experimental images show that this iterative method is fast, accurate, robust against noise, and thus yields noticeable enhancement in contrast to noise ratios.

Overexpression of HTRA1 increases the proliferation and migration of retinal pigment epithelium cells.

BACKGROUND: Age-related macular degeneration (AMD) mainly affects the central region of retina and has many late-stage manifestations. OBJECTIVES: Age-related macular degeneration is a leading cause of irreversible blindness in older people. The main feature of AMD is retinal pigment epithelium (RPE) degeneration. In this study, we aimed to explore the influence of HTRA1 expression on the proliferation and migration of RPE cells. MATERIAL AND METHODS: Human ARPE-19 cells were transfected with an HTRA1 overexpression lentivirus or HTRA1 siRNA to silence HtrA1 expression. Quantitave reverse-transcription polymerase chain reaction (qRT-PCR) and western blotting were used to verify the relative level of HTRA1 mRNA and expression of HTRA1 protein of transfected human ARPE-19 cells. The MTT clone formation and transwell assays were used to confirm the effect of HTRA1 expression on the proliferation, colony forming ability and migration of ARPE-19 cells. RESULTS: The proliferation capacity (shown as optical density value) of ARPE-19 cells in the HTRA1-overexpressing group at culture times of 24 h and 48 h were 0.595 ±0.032 and 0.867 ±0.037 respectively, which were much higher than in the mock group. However, the proliferative capacity of cells in the HTRA1-silenced group decreased with increasing time of culture, compared with the mock group. The number of cloned and migrating cells in the HTRA1-overexpressing group were much higher than in the mock group, whereas the numbers in the HTRA1-silenced group were significantly lower. CONCLUSIONS: Overexpression of HTRA1 promotes proliferation and migration of RPE cells, which can help maintain the function of sensory neurons in the retina. Therefore, HTRA1 may be a suitable target for AMD treatments.

Development and preclinical evaluation of a patient-specific high energy x-ray phase sensitive breast tomosynthesis system.

BACKGROUND: This article reports the first x-ray phase sensitive breast tomosynthesis (PBT) system that is aimed for direct translation to clinical practice for the diagnosis of breast cancer. PURPOSE: To report the preclinical evaluation and comparison of the newly built PBT system with a conventional digital breast tomosynthesis (DBT) system. METHODS AND MATERIALS: The PBT system is developed based on a comprehensive inline phase contrast theoretical model. The system consists of a polyenergetic microfocus x-ray source and a flat panel detector mounted on an arm that is attached to a rotating gantry. It acquires nine projections over a 15° angular span in a stop-and-shoot manner. A dedicated phase retrieval algorithm is integrated with a filtered back-projection method that reconstructs tomographic slices. The American College of Radiology (ACR) accreditation phantom, a contrast detail (CD) phantom and mastectomy tissue samples were imaged at the same glandular dose levels by both the PBT and a standard of care DBT system for image quality characterizations and comparisons. RESULTS: The PBT imaging scores with the ACR phantom are in good to excellent range and meet the quality assurance criteria set by the Mammography Quality Standard Act. The CD phantom image comparison and associated statistical analyses from two-alternative forced-choice reader studies confirm the improvement offered by the PBT system in terms of contrast resolution, spatial resolution, and conspicuity. The artifact spread function (ASF) analyses revealed a sizable lateral spread of metal artifacts in PBT slices as compared to DBT slices. Signal-to-noise ratio values for various inserts of the ACR and CD phantoms further validated the superiority of the PBT system. Mastectomy sample images acquired by the PBT system showed a superior depiction of microcalcifications vs the DBT system. CONCLUSION: The PBT imaging technology can be clinically employed for improving the accuracy of breast cancer screening and diagnosis.

Role of mammalian target of rapamycin in regulating HIF-1α and vascular endothelial growth factor signals in glaucoma.

Hypoxia inducible factor subtype 1α (HIF-1α) in retinal tissues is involved in the development of glaucoma. This study examined the role played by mammalian target of rapamycin (mTOR) in regulating expression of HIF-1α and its downstream pathway, vascular endothelial growth factor (VEGF). Glaucoma was induced by chronic elevation of intraocular pressure using laser burns in rats. ELISA and western blot analysis were employed to determine the levels of HIF-1α, VEGF and mTOR in retinal tissues of eyes with high intraocular pressure. In results, HIF-1α, VEGF and VEGF receptor subtype 2 were increased in laser eyes. The p-mTOR, mTOR-mediated phosphorylation of 4E-binding protein 4, p70 ribosomal S6 protein kinase 1 were also amplified in retina of laser eyes. Blocking mTOR using rapamycin attenuated HIF-1α-VEGF pathways, accompanied with downregulation of apoptotic Caspase-3. Our data revealed potential signalling pathways engaged in the development of glaucoma, including the activation of mTOR and HIF-1α-VEGF mechanism.

Madecassoside protects retinal pigment epithelial cells against hydrogen peroxide-induced oxidative stress and apoptosis through the activation of Nrf2/HO-1 pathway.

Age-related macular degeneration (AMD) is a progressive and degenerative ocular disease associated with oxidative stress. Madecassoside (MADE) is a major bioactive triterpenoid saponin that possesses antioxidative activity. However, the role of MADE in AMD has never been investigated. In the current study, we aimed to evaluate the protective effect of MADE on retinal pigment epithelium (RPE) cells under oxidative stress condition. We used hydrogen peroxide (H2O2) to induce oxidative damage in human RPE cells (ARPE-19 cells). Our results showed that H2O2-caused significant decrease in cell viability and increase in lactate dehydrogenase (LDH) release were dose-dependently attenuated by MADE. MADE treatment also attenuated H2O2-induced reactive oxygen species (ROS) and malondialdehyde (MDA) production in RPE cells. The reduced glutathione (GSH) level and superoxide dismutase (SOD) activity in H2O2-induced ARPE-19 cells were elevated after MADE treatment. MADE also suppressed caspase-3 activity and bax expression, as well as increased bcl-2 expression. Furthermore, H2O2-induced increase in expression levels of HO-1 and nuclear Nrf2 were enhanced by MADE treatment. Finally, knockdown of Nrf2 reversed the protective effects of MADE on H2O2-induced ARPE-19 cells. In conclusion, these findings demonstrated that MADE protected ARPE-19 cells from H2O2-induced oxidative stress and apoptosis by inducing the activation of Nrf2/HO-1 signaling pathway.

Predicting fringe visibility in dual-phase grating interferometry with polychromatic X-ray sources.

Dual phase grating X-ray interferometry is radiation dose-efficient as compared to common Talbot-Lau grating interferometry. The authors developed a general quantitative theory to predict the fringe visibility in dual-phase grating X-ray interferometry with polychromatic X-ray sources. The derived formulas are applicable to setups with phase gratings of any phase modulation and with either monochromatic or polychromatic X-rays. Numerical simulations are presented to validate the derived formulas. The theory provides useful tools for design optimization of dual-phase grating X-ray interferometers.

Human Neutrophil Elastase Activated Fluorescent Probe for Pulmonary Diseases Based on Fluorescence Resonance Energy Transfer Using CdSe/ZnS Quantum Dots.

There is an increasing demand for effective noninvasive diagnosis against common pulmonary diseases, which are rising sharply due to the serious air pollution. Human neutrophil elastase (HNE), a typical protease highly involved in pulmonary inflammatory diseases and lung cancer, is a potential predictor for disease progression. Currently, few of the HNE-targeting probes are applicable in vivo due to the limitation in sensitivity and biocompatibility. Herein, we reported the achievement of in vitro detection and in vivo imaging of HNE by incorporating the HNE-specific peptide substrate, quantum dots (QDs), and organic dyes into the fluorescence resonance energy transfer (FRET) system. The refined nanoprobe, termed QDP, could specifically measure the HNE with excellent sensitivity of 7.15 pM in aqueous solution and successfully image the endogenous and exogenous HNE in living cells. In addition, this nanoprobe enabled HNE imaging in mouse models of lung cancer and acute lung injury, and the HNE activity at high temporal and spatial resolution was continuously monitored. Most importantly, QDP successfully discriminated the serums of patients with lung diseases from those of the healthy controls based on the HNE activity determination. Overall, this study demonstrates the advantages of a FRET-system-based nanoprobe in imaging performance and provides an applicable tool for in vivo HNE detection and pulmonary disease diagnosis.

Distinct features of nucleolus-associated domains in mouse embryonic stem cells.

Heterochromatin in eukaryotic interphase cells frequently localizes to the nucleolar periphery (nucleolus-associated domains (NADs)) and the nuclear lamina (lamina-associated domains (LADs)). Gene expression in somatic cell NADs is generally low, but NADs have not been characterized in mammalian stem cells. Here, we generated the first genome-wide map of NADs in mouse embryonic stem cells (mESCs) via deep sequencing of chromatin associated with biochemically purified nucleoli. As we had observed in mouse embryonic fibroblasts (MEFs), the large type I subset of NADs overlaps with constitutive LADs and is enriched for features of constitutive heterochromatin, including late replication timing and low gene density and expression levels. Conversely, the type II NAD subset overlaps with loci that are not lamina-associated, but in mESCs, type II NADs are much less abundant than in MEFs. mESC NADs are also much less enriched in H3K27me3 modified regions than are NADs in MEFs. Additionally, comparision of MEF and mESC NADs revealed enrichment of developmentally regulated genes in cell-type-specific NADs. Together, these data indicate that NADs are a developmentally dynamic component of heterochromatin. These studies implicate association with the nucleolar periphery as a mechanism for developmentally regulated gene expression and will facilitate future studies of NADs during mESC differentiation.

Genomic Characterization of Endothelial Enhancers Reveals a Multifunctional Role for NR2F2 in Regulation of Arteriovenous Gene Expression.

RATIONALE: Significant progress has revealed transcriptional inputs that underlie regulation of artery and vein endothelial cell fates. However, little is known concerning genome-wide regulation of this process. Therefore, such studies are warranted to address this gap. OBJECTIVE: To identify and characterize artery- and vein-specific endothelial enhancers in the human genome, thereby gaining insights into mechanisms by which blood vessel identity is regulated. METHODS AND RESULTS: Using chromatin immunoprecipitation and deep sequencing for markers of active chromatin in human arterial and venous endothelial cells, we identified several thousand artery- and vein-specific regulatory elements. Computational analysis revealed that NR2F2 (nuclear receptor subfamily 2, group F, member 2) sites were overrepresented in vein-specific enhancers, suggesting a direct role in promoting vein identity. Subsequent integration of chromatin immunoprecipitation and deep sequencing data sets with RNA sequencing revealed that NR2F2 regulated 3 distinct aspects related to arteriovenous identity. First, consistent with previous genetic observations, NR2F2 directly activated enhancer elements flanking cell cycle genes to drive their expression. Second, NR2F2 was essential to directly activate vein-specific enhancers and their associated genes. Our genomic approach further revealed that NR2F2 acts with ERG (ETS-related gene) at many of these sites to drive vein-specific gene expression. Finally, NR2F2 directly repressed only a small number of artery enhancers in venous cells to prevent their activation, including a distal element upstream of the artery-specific transcription factor, HEY2 (hes related family bHLH transcription factor with YRPW motif 2). In arterial endothelial cells, this enhancer was normally bound by ERG, which was also required for arterial HEY2 expression. By contrast, in venous endothelial cells, NR2F2 was bound to this site, together with ERG, and prevented its activation. CONCLUSIONS: By leveraging a genome-wide approach, we revealed mechanistic insights into how NR2F2 functions in multiple roles to maintain venous identity. Importantly, characterization of its role at a crucial artery enhancer upstream of HEY2 established a novel mechanism by which artery-specific expression can be achieved.

Sample phase gradient and fringe phase shift in triple phase grating X-ray interferometry.

Triple phase grating X-ray interferometry is a promising new technique of grating based X-ray differential phase contrast imaging. Accurate retrieval of sample phase gradients from measured interference fringe shifts is a key task in X-ray interferometry. To fulfill this task in triple phase grating X-ray interferometry with monochromatic X-ray sources, the authors derived exact formulas relating sample phase gradient to fringe phase shift. These formulas not only provide a design optimization tool for triple phase grating interferometry, but also lay a foundation for quantitative phase contrast imaging.