Large DNA Methylation Nadirs Anchor Chromatin Loops Maintaining Hematopoietic Stem Cell Identity.

Higher-order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we find that large (>7.3 kb) DNA methylation nadirs (termed "grand canyons") can form long loops connecting anchor loci that may be dozens of megabases (Mb) apart, as well as inter-chromosomal links. The interacting loci cover a total of ∼3.5 Mb of the human genome. The strongest interactions are associated with repressive marks made by the Polycomb complex and are diminished upon EZH2 inhibitor treatment. The data are suggestive of the formation of these loops by interactions between repressive elements in the loci, forming a genomic subcompartment, rather than by cohesion/CTCF-mediated extrusion. Interestingly, unlike previously characterized subcompartments, these interactions are present only in particular cell types, such as stem and progenitor cells. Our work reveals that H3K27me3-marked large DNA methylation grand canyons represent a set of very-long-range loops associated with cellular identity.

Dnmt3a loss and Idh2 neomorphic mutations mutually potentiate malignant hematopoiesis.

Mutations in the epigenetic regulators DNMT3A and IDH1/2 co-occur in patients with acute myeloid leukemia and lymphoma. In this study, these 2 epigenetic mutations cooperated to induce leukemia. Leukemia-initiating cells from Dnmt3a-/- mice that express an IDH2 neomorphic mutant have a megakaryocyte-erythroid progenitor-like immunophenotype, activate a stem-cell-like gene signature, and repress differentiated progenitor genes. We observed an epigenomic dysregulation with the gain of repressive H3K9 trimethylation and loss of H3K9 acetylation in diseased mouse bone marrow hematopoietic stem and progenitor cells (HSPCs). HDAC inhibitors rapidly reversed the H3K9 methylation/acetylation imbalance in diseased mouse HSPCs while reducing the leukemia burden. In addition, using targeted metabolomic profiling for the first time in mouse leukemia models, we also showed that prostaglandin E2 is overproduced in double-mutant HSPCs, rendering them sensitive to prostaglandin synthesis inhibition. These data revealed that Dnmt3a and Idh2 mutations are synergistic events in leukemogenesis and that HSPCs carrying both mutations are sensitive to induced differentiation by the inhibition of both prostaglandin synthesis and HDAC, which may reveal new therapeutic opportunities for patients carrying IDH1/2 mutations.

Haploinsufficiency of cohesin protease, Separase, promotes regeneration of hematopoietic stem cells in mice.

Cohesin recently emerged as a new regulator of hematopoiesis and leukemia. In addition to cohesin, whether proteins that regulate cohesin's function have any direct role in hematopoiesis and hematologic diseases have not been fully examined. Separase, encoded by the ESPL1 gene, is an important regulator of cohesin's function. Canonically, protease activity of Separase resolves sister chromatid cohesion by cleaving cohesin subunit-Rad21 at the onset of anaphase. Using a Separase haploinsufficient mouse model, we have uncovered a novel role of Separase in hematopoiesis. We report that partial disruption of Separase distinctly alters the functional characteristics of hematopoietic stem/progenitor cells (HSPCs). Although analyses of peripheral blood and bone marrow of Espl1+/Hyp mice broadly displayed unperturbed hematopoietic parameters during normal hematopoiesis, further probing of the composition of early hematopoietic cells in Espl1+/Hyp bone marrow revealed a mild reduction in the frequencies of the Lin- Sca1+ Kit- (LSK) or LSK CD48+ CD150- multipotent hematopoietic progenitors population without a significant change in either long-term or short-term hematopoietic stem cells (HSCs) subsets at steady state. Surprisingly, however, we found that Separase haploinsufficiency promotes regeneration activity of HSCs in serial in vivo repopulation assays. In vitro colony formation assays also revealed an enhanced serial replating capacity of hematopoietic progenitors isolated from Espl1+/Hyp mice. Microarray analysis of differentially expressed genes showed that Separase haploinsufficiency in HSCs (SP-KSL) leads to enrichment of gene signatures that are upregulated in HSCs compared to committed progenitors and mature cells. Taken together, our findings demonstrate a key role of Separase in promoting hematopoietic regeneration of HSCs.

Energy Efficient Pupil Tracking Based on Rule Distillation of Cascade Regression Forest.

As the demand for human-friendly computing increases, research on pupil tracking to facilitate human-machine interactions (HCIs) is being actively conducted. Several successful pupil tracking approaches have been developed using images and a deep neural network (DNN). However, common DNN-based methods not only require tremendous computing power and energy consumption for learning and prediction; they also have a demerit in that an interpretation is impossible because a black-box model with an unknown prediction process is applied. In this study, we propose a lightweight pupil tracking algorithm for on-device machine learning (ML) using a fast and accurate cascade deep regression forest (RF) instead of a DNN. Pupil estimation is applied in a coarse-to-fine manner in a layer-by-layer RF structure, and each RF is simplified using the proposed rule distillation algorithm for removing unimportant rules constituting the RF. The goal of the proposed algorithm is to produce a more transparent and adoptable model for application to on-device ML systems, while maintaining a precise pupil tracking performance. Our proposed method experimentally achieves an outstanding speed, a reduction in the number of parameters, and a better pupil tracking performance compared to several other state-of-the-art methods using only a CPU.

Light-Weight Student LSTM for Real-Time Wildfire Smoke Detection.

As the need for wildfire detection increases, research on wildfire smoke detection combining low-cost cameras and deep learning technology is increasing. Camera-based wildfire smoke detection is inexpensive, allowing for a quick detection, and allows a smoke to be checked by the naked eye. However, because a surveillance system must rely only on visual characteristics, it often erroneously detects fog and clouds as smoke. In this study, a combination of a You-Only-Look-Once detector and a long short-term memory (LSTM) classifier is applied to improve the performance of wildfire smoke detection by reflecting on the spatial and temporal characteristics of wildfire smoke. However, because it is necessary to lighten the heavy LSTM model for real-time smoke detection, in this paper, we propose a new method for applying the teacher-student framework to deep LSTM. Through this method, a shallow student LSTM is designed to reduce the number of layers and cells constituting the LSTM model while maintaining the original deep LSTM performance. As the experimental results indicate, our proposed method achieves up to an 8.4-fold decrease in the number of parameters and a faster processing time than the teacher LSTM while maintaining a similar detection performance as deep LSTM using several state-of-the-art methods on a wildfire benchmark dataset.

DOT1L as a therapeutic target for the treatment of DNMT3A-mutant acute myeloid leukemia.

Mutations in DNA methyltransferase 3A (DNMT3A) are common in acute myeloid leukemia and portend a poor prognosis; thus, new therapeutic strategies are needed. The likely mechanism by which DNMT3A loss contributes to leukemogenesis is altered DNA methylation and the attendant gene expression changes; however, our current understanding is incomplete. We observed that murine hematopoietic stem cells (HSCs) in which Dnmt3a had been conditionally deleted markedly overexpress the histone 3 lysine 79 (H3K79) methyltransferase, Dot1l. We demonstrate that Dnmt3a(-/-) HSCs have increased H3K79 methylation relative to wild-type (WT) HSCs, with the greatest increases noted at DNA methylation canyons, which are regions highly enriched for genes dysregulated in leukemia and prone to DNA methylation loss with Dnmt3a deletion. These findings led us to explore DOT1L as a therapeutic target for the treatment of DNMT3A-mutant AML. We show that pharmacologic inhibition of DOT1L resulted in decreased expression of oncogenic canyon-associated genes and led to dose- and time-dependent inhibition of proliferation, induction of apoptosis, cell-cycle arrest, and terminal differentiation in DNMT3A-mutant cell lines in vitro. We show in vivo efficacy of the DOT1L inhibitor EPZ5676 in a nude rat xenograft model of DNMT3A-mutant AML. DOT1L inhibition was also effective against primary patient DNMT3A-mutant AML samples, reducing colony-forming capacity (CFC) and inducing terminal differentiation in vitro. These studies suggest that DOT1L may play a critical role in DNMT3A-mutant leukemia. With pharmacologic inhibitors of DOT1L already in clinical trials, DOT1L could be an immediately actionable therapeutic target for the treatment of this poor prognosis disease.

Driver's Facial Expression Recognition in Real-Time for Safe Driving.

In recent years, researchers of deep neural networks (DNNs)-based facial expression recognition (FER) have reported results showing that these approaches overcome the limitations of conventional machine learning-based FER approaches. However, as DNN-based FER approaches require an excessive amount of memory and incur high processing costs, their application in various fields is very limited and depends on the hardware specifications. In this paper, we propose a fast FER algorithm for monitoring a driver's emotions that is capable of operating in low specification devices installed in vehicles. For this purpose, a hierarchical weighted random forest (WRF) classifier that is trained based on the similarity of sample data, in order to improve its accuracy, is employed. In the first step, facial landmarks are detected from input images and geometric features are extracted, considering the spatial position between landmarks. These feature vectors are then implemented in the proposed hierarchical WRF classifier to classify facial expressions. Our method was evaluated experimentally using three databases, extended Cohn-Kanade database (CK+), MMI and the Keimyung University Facial Expression of Drivers (KMU-FED) database, and its performance was compared with that of state-of-the-art methods. The results show that our proposed method yields a performance similar to that of deep learning FER methods as 92.6% for CK+ and 76.7% for MMI, with a significantly reduced processing cost approximately 3731 times less than that of the DNN method. These results confirm that the proposed method is optimized for real-time embedded applications having limited computing resources.

Genomic hypomethylation in the human germline associates with selective structural mutability in the human genome.

The hotspots of structural polymorphisms and structural mutability in the human genome remain to be explained mechanistically. We examine associations of structural mutability with germline DNA methylation and with non-allelic homologous recombination (NAHR) mediated by low-copy repeats (LCRs). Combined evidence from four human sperm methylome maps, human genome evolution, structural polymorphisms in the human population, and previous genomic and disease studies consistently points to a strong association of germline hypomethylation and genomic instability. Specifically, methylation deserts, the ~1% fraction of the human genome with the lowest methylation in the germline, show a tenfold enrichment for structural rearrangements that occurred in the human genome since the branching of chimpanzee and are highly enriched for fast-evolving loci that regulate tissue-specific gene expression. Analysis of copy number variants (CNVs) from 400 human samples identified using a custom-designed array comparative genomic hybridization (aCGH) chip, combined with publicly available structural variation data, indicates that association of structural mutability with germline hypomethylation is comparable in magnitude to the association of structural mutability with LCR-mediated NAHR. Moreover, rare CNVs occurring in the genomes of individuals diagnosed with schizophrenia, bipolar disorder, and developmental delay and de novo CNVs occurring in those diagnosed with autism are significantly more concentrated within hypomethylated regions. These findings suggest a new connection between the epigenome, selective mutability, evolution, and human disease.

Fast human detection for intelligent monitoring using surveillance visible sensors.

Human detection using visible surveillance sensors is an important and challenging work for intruder detection and safety management. The biggest barrier of real-time human detection is the computational time required for dense image scaling and scanning windows extracted from an entire image. This paper proposes fast human detection by selecting optimal levels of image scale using each level's adaptive region-of-interest (ROI). To estimate the image-scaling level, we generate a Hough windows map (HWM) and select a few optimal image scales based on the strength of the HWM and the divide-and-conquer algorithm. Furthermore, adaptive ROIs are arranged per image scale to provide a different search area. We employ a cascade random forests classifier to separate candidate windows into human and nonhuman classes. The proposed algorithm has been successfully applied to real-world surveillance video sequences, and its detection accuracy and computational speed show a better performance than those of other related methods.

Flow cytometry analysis of murine hematopoietic stem cells.

Hematopoietic stem cells (HSCs) remain the most well-characterized adult stem cell population both in terms of markers for purification and assays to assess functional potential. However, despite over 40 years of research, working with HSCs in the mouse remains challenging because of the relative abundance (or lack thereof) of these cells in the bone marrow. The frequency of HSCs in murine bone marrow is about 0.01% of total nucleated cells and ∼5,000 can be isolated from an individual mouse depending on the age, sex, and strain of mice as well as purification scheme utilized. Adding to the challenge is the continual reporting of new markers for HSC purification, which makes it difficult for the uninitiated in the field to know which purification strategies yield the highest proportion of long-term, multilineage HSCs. In this updated version of our review from 2009, we review different strategies for hematopoietic stem and progenitor cell identification and purification. We will also discuss methods for rapid flow cytometric analysis of peripheral blood cell types, and novel strategies for working with rare cell populations such as HSCs in the analysis of cell cycle status by BrdU, Ki-67, and Pyronin Y staining. The purpose of updating this review is to provide insight into some of the recent experimental and technical advances in mouse hematopoietic stem cell biology.

Improvement of the efficiency of Si solar cells using nano-microscale combination patterns.

Solar cells have a high reported energy conversion efficiency level according to various techniques. The energy conversion efficiency, however, is limited by the reflection of the light at the packaging glass surface. For this reason, some research groups are focused on nano/micro scale patterns. Nano/micro scale patterns can increase the energy conversion efficiency by means of light absorption. In this paper, we used three types of master stamps 300 nm pitch of a moth-eye pattern, a micro lens, and a hole pattern with a 200 nm diameter. These patterns were fabricated using UV-nanoimprint lithography on a cover glass which served as a protective layer of a Si solar cell. In additional, the transmittance and conversion efficiency of single-crystal Si solar cell were measured. Consequently, the conversion efficiency of a Si solar cell was found to increase from 16.69% to 18.16%.

A new mold structure to replicate patterns over 1 microm in depth for substrate conformal imprint lithography.

This paper shows an improved mold replication process that uses polyurethane acrylate (PUA) and polyethylene terephthalate (PET) for the fabrication of an ultraviolet (UV) imprinting mold used in substrate conformal imprint lithography (SCIL). With the conventional replication process, which uses hard polydimethylsiloxane (h-PDMS) as a pattern layer, it is difficult to detach the mold from a silicon master for metal oxide semiconductor field effect transistor (MOSFET) that has patterns with over 1-micron depth. However, the method proposed in this paper allows us to easily replicate patterns that have more than 1-micron depth. The key idea of this method is to use PET film as a bonding layer to attach the PUA layer to the polydimethylsiloxane (PDMS) cushion layer to overcome the weak the adhesion force between the PUA and PDMS layer. We demonstrate how to make the modified replica mold and present imprinting results obtained using this replica mold in the SCIL process.

Exercise reprograms the inflammatory landscape of multiple stem cell compartments during mammalian aging.

Exercise has the ability to rejuvenate stem cells and improve tissue regeneration in aging animals. However, the cellular and molecular changes elicited by exercise have not been systematically studied across a broad range of cell types in stem cell compartments. We subjected young and old mice to aerobic exercise and generated a single-cell transcriptomic atlas of muscle, neural, and hematopoietic stem cells with their niche cells and progeny, complemented by whole transcriptome analysis of single myofibers. We found that exercise ameliorated the upregulation of a number of inflammatory pathways associated with old age and restored aspects of intercellular communication mediated by immune cells within these stem cell compartments. Exercise has a profound impact on the composition and transcriptomic landscape of circulating and tissue-resident immune cells. Our study provides a comprehensive view of the coordinated responses of multiple aged stem cells and niche cells to exercise at the transcriptomic level.

VDUP1 exacerbates bacteremic shock in mice infected with Pseudomonas aeruginosa.

Vitamin-D3 upregulated protein-1 (VDUP1) is a stress response protein. Pseudomonas aeruginosa (P. aeruginosa) infection is a leading cause of death. Mice infected with live P. aeruginosa exhibit significantly decreased VDUP1 expression. However, the function of VDUP1 during P. aeruginosa-induced mouse bacteremic shock is unknown. To address the function of VDUP1 in P. aeruginosa-infected mice, we constructed a bacteremic shock model wherein both wild-type and VDUP1-deficient mice were infected intra-peritoneally with live P. aeruginosa. We found that VDUP1-deficient mice were more resistant to P. aeruginosa-induced bacteremic shock than wild-type mice, as shown by the increased survival, accelerated bacterial clearance and suppression of cytokine overproduction of the VDUP1-deficient mice. VDUP1 promoted the recruitment of neutrophils into the peritoneal cavities of infected mice. VDUP1 impeded the phagocytosis of non-opsonized P. aeruginosa via phosphatidylinositide 3-kinase (PI3K) pathway in macrophages. P. aeruginosa infection induced the generation of reactive oxygen species (ROS), and the increased production of ROS by the peritoneal cells of VDUP1-deficient mice was advantageous in clearing the bacteria. Overall, VDUP1 aggravates bacteremic shock; thus, VDUP1 can be considered a target molecule for the inhibition of P. aeruginosa-induced bacteremic shock.

Suppressor of cytokine signaling 2 regulates IL-15-primed human NK cell function via control of phosphorylated Pyk2.

NK cells are capable of killing virus-infected or tumor cells and producing IFN-gamma. Resting NK cells, however, have only minimal cytolytic activity and secrete a low level of IFN-gamma. The cytokine IL-15 can promote the expression of effector functions by resting NK cells. In this study, we demonstrate that suppressor of cytokine signaling 2 (SOCS2) has a novel role in IL-15-primed human NK cell function. SOCS2 expression was upregulated in NK cells following stimulation with IL-15. During IL-15-mediated NK cell priming, SOCS2 interacted with phosphorylated proline-rich tyrosine kinase 2 (Pyk2) at tyrosine 402 (p-Pyk2(Tyr402)) and induced the proteasome-mediated degradation of p-Pyk2(Tyr402) via ubiquitination. Knockdown of SOCS2 resulted in the accumulation of p-Pyk2(Tyr402) and blocked NK cell effector functions. In addition, NK cell cytolytic activity and IFN-gamma production were inhibited by overexpression of the wild-type of Pyk2 but not by the overexpression of tyrosine 402 mutant of Pyk2. These results suggest that SOCS2 regulates human NK cell effector functions via control of phosphorylated Pyk2 depending on IL-15 existence.

Pattern uniformity in large-area ultraviolet nano-imprinting by a cylindrically inflated flexible mold under low pressure.

This paper shows a novel nano-imprint method with a polydimethylsiloxane (PDMS) replica mold that was bonded on a cylindrically inflated polycarbonate (PC) film via a low air pressure. The PDMS mold, which was deformed in terms of its cylindrical shape, made a line contact with a substrate from the center region and the contact region, then expanded gradually to the outside of the substrate when the contact force increased. This contact procedure squeezed the resin that was dropped on the substrate from the center to the outside, which prevented the trapping of air bubbles while the cavities were filled with the patterns on the PDMS mold. The main characteristic of the proposed process was that the nano-imprint can be realized under a low pressure, compared to conventional processes. We will show the system that was implemented under the proposed process concept and the patterns that were transferred in an ultraviolet curable resin under pressure conditions of less than 5 kPa.

The disordered N-terminal domain of DNMT3A recognizes H2AK119ub and is required for postnatal development.

DNA methyltransferase 3a (DNMT3A) plays a crucial role during mammalian development. Two isoforms of DNMT3A are differentially expressed from stem cells to somatic tissues, but their individual functions remain largely uncharacterized. Here we report that the long isoform DNMT3A1, but not the short DNMT3A2, is essential for mouse postnatal development. DNMT3A1 binds to and regulates bivalent neurodevelopmental genes in the brain. Strikingly, Dnmt3a1 knockout perinatal lethality could be partially rescued by DNMT3A1 restoration in the nervous system. We further show that the intrinsically disordered N terminus of DNMT3A1 is required for normal development and DNA methylation at DNMT3A1-enriched regions. Mechanistically, a ubiquitin-interacting motif embedded in a putative α-helix within the N terminus binds to mono-ubiquitinated histone H2AK119, probably mediating recruitment of DNMT3A1 to Polycomb-regulated regions. These data demonstrate an isoform-specific role for DNMT3A1 in mouse postnatal development and reveal the N terminus as a necessary regulatory domain for DNMT3A1 chromatin occupancy and functions in the nervous system.

Pseudomonas aeruginosa eliminates natural killer cells via phagocytosis-induced apoptosis.

Pseudomonas aeruginosa (PA) is an opportunistic pathogen that causes the relapse of illness in immunocompromised patients, leading to prolonged hospitalization, increased medical expense, and death. In this report, we show that PA invades natural killer (NK) cells and induces phagocytosis-induced cell death (PICD) of lymphocytes. In vivo tumor metastasis was augmented by PA infection, with a significant reduction in NK cell number. Adoptive transfer of NK cells mitigated PA-induced metastasis. Internalization of PA into NK cells was observed by transmission electron microscopy. In addition, PA invaded NK cells via phosphoinositide 3-kinase (PI3K) activation, and the phagocytic event led to caspase 9-dependent apoptosis of NK cells. PA-mediated NK cell apoptosis was dependent on activation of mitogen-activated protein (MAP) kinase and the generation of reactive oxygen species (ROS). These data suggest that the phagocytosis of PA by NK cells is a critical event that affects the relapse of diseases in immunocompromised patients, such as those with cancer, and provides important insights into the interactions between PA and NK cells.

RasGRP1 is required for human NK cell function.

Cross-linking of NK activating receptors activates phospholipase-gamma and subsequently induces diacylglycerol and Ca(2+) as second messengers of signal transduction. Previous studies reported that Ras guanyl nucleotide-releasing protein (RasGRP) 1, which is activated by diacylglycerol and Ca(2+), is crucial for TCR-mediated Ras-ERK activation. We now report that RasGRP1, which can also be detected in human NK cells, plays an essential role in NK cell effector functions. To examine the role of RasGRP1 in NK cell functions, the expression of RasGRP1 was suppressed using RNA interference. Knockdown of RasGRP1 significantly blocked ITAM-dependent cytokine production as well as NK cytotoxicity. Biochemically, RasGRP1-knockdown NK cells showed markedly decreased ability to activate Ras, ERK, and JNK. Activation of the Ras-MAPK pathway was independently shown to be indispensable for NK cell effector functions via the use of specific pharmacological inhibitors. Our results reveal that RasGRP1 is required for the activation of the Ras-MAPK pathway leading to NK cell effector functions. Moreover, our data suggest that RasGRP1 might act as an important bridge between phospholipase-gamma activation and NK cell effector functions via the Ras-MAPK pathway.

Thioredoxin-interacting protein regulates hematopoietic stem cell quiescence and mobilization under stress conditions.

Hematopoietic stem cells (HSCs) are maintained in a quiescent state in bone marrow (BM) niches by intrinsic and extrinsic signals. The mechanisms regulating the quiescence and mobilization of HSCs, however, remain unclear. In this study, we report that the expression of thioredoxin-interacting protein (TXNIP) is decreased during HSC activation. In Txnip(-/-) mice, the long-term reconstituting HSC population is decreased and exhausted, and its capacity to repopulate is rapidly lost. These effects are associated with hyperactive Wnt signaling, an active cell cycle, and reduced p21 expression under conditions of stress. TXNIP deficiency reduced the CXCL12- and osteopontin-mediated interaction between HSCs and the bone marrow, and impaired homing and retention in the osteoblastic niche, resulting in mobilized HSCs. Therefore, we propose that TXNIP is essential for maintaining HSC quiescence and the interaction between HSCs and the BM niche.