The role of semaphorin 3A on chondrogenic differentiation.

Osteoblast-derived semaphorin3A (Sema3A) has been reported to be involved in bone protection, and Sema3A knockout mice have been reported to exhibit chondrodysplasia. From these reports, Sema3A is considered to be involved in chondrogenic differentiation and skeletal formation, but there are many unclear points about its function and mechanism in chondrogenic differentiation. This study investigated the pharmacological effects of Sema3A in chondrogenic differentiation. The amount of Sema3A secreted into the culture supernatant was measured using an enzyme-linked immunosorbent assay. The expression of chondrogenic differentiation-related factors, such as Type II collagen (COL2A1), Aggrecan (ACAN), hyaluronan synthase 2 (HAS2), SRY-box transcription factor 9 (Sox9), Runt-related transcription factor 2 (Runx2), and Type X collagen (COL10A1) in ATDC5 cells treated with Sema3A (1,10 and 100 ng/mL) was examined using real-time reverse transcription polymerase chain reaction. Further, to assess the deposition of total glycosaminoglycans during chondrogenic differentiation, ATDC5 cells were stained with Alcian Blue. Moreover, the amount of hyaluronan in the culture supernatant was measured by enzyme-linked immunosorbent assay. The addition of Sema3A to cultured ATDC5 cells increased the expression of Sox9, Runx2, COL2A1, ACAN, HAS2, and COL10A1 during chondrogenic differentiation. Moreover, it enhanced total proteoglycan and hyaluronan synthesis. Further, Sema3A was upregulated in the early stages of chondrogenic differentiation, and its secretion decreased later. Sema3A increases extracellular matrix production and promotes chondrogenic differentiation. To the best of our knowledge, this is the first study to demonstrate the role of Sema3A on chondrogenic differentiation.

Combined and differential roles of ADD domains of DNMT3A and DNMT3L on DNA methylation landscapes in mouse germ cells.

DNA methyltransferase 3A (DNMT3A) and its catalytically inactive cofactor DNA methyltransferase 3-Like (DNMT3L) proteins form functional heterotetramers to deposit DNA methylation in mammalian germ cells. While both proteins have an ATRX-DNMT3-DNMT3L (ADD) domain that recognizes histone H3 tail unmethylated at lysine-4 (H3K4me0), the combined and differential roles of the domains in the two proteins have not been fully defined in vivo. Here we investigate DNA methylation landscapes in female and male germ cells derived from mice with loss-of-function amino acid substitutions in the ADD domains of DNMT3A and/or DNMT3L. Mutations in either the DNMT3A-ADD or the DNMT3L-ADD domain moderately decrease global CG methylation levels, but to different degrees, in both germ cells. Furthermore, when the ADD domains of both DNMT3A and DNMT3L lose their functions, the CG methylation levels are much more reduced, especially in oocytes, comparable to the impact of the Dnmt3a/3L knockout. In contrast, aberrant accumulation of non-CG methylation occurs at thousands of genomic regions in the double mutant oocytes and spermatozoa. These results highlight the critical role of the ADD-H3K4me0 binding in proper CG and non-CG methylation in germ cells and the various impacts of the ADD domains of the two proteins.

H3K4me1 facilitates promoter-enhancer interactions and gene activation during embryonic stem cell differentiation.

Histone H3 lysine 4 mono-methylation (H3K4me1) marks poised or active enhancers. KMT2C (MLL3) and KMT2D (MLL4) catalyze H3K4me1, but their histone methyltransferase activities are largely dispensable for transcription during early embryogenesis in mammals. To better understand the role of H3K4me1 in enhancer function, we analyze dynamic enhancer-promoter (E-P) interactions and gene expression during neural differentiation of the mouse embryonic stem cells. We found that KMT2C/D catalytic activities were only required for H3K4me1 and E-P contacts at a subset of candidate enhancers, induced upon neural differentiation. By contrast, a majority of enhancers retained H3K4me1 in KMT2C/D catalytic mutant cells. Surprisingly, H3K4me1 signals at these KMT2C/D-independent sites were reduced after acute depletion of KMT2B, resulting in aggravated transcriptional defects. Our observations therefore implicate KMT2B in the catalysis of H3K4me1 at enhancers and provide additional support for an active role of H3K4me1 in enhancer-promoter interactions and transcription in mammalian cells.

One-Dimensionally Arranged Quantum-Dot Superstructures Guided by a Supramolecular Polymer Template.

Colloidal quantum dots (QDs) exhibit important photophysical properties, such as long-range energy diffusion, miniband formation, and collective photoluminescence, when aggregated into well-defined superstructures, such as three-dimensional (3D) and two-dimensional (2D) superlattices. However, the construction of one-dimensional (1D) QD superstructures, which have a simpler arrangement, is challenging; therefore, the photophysical properties of 1D-arranged QDs have not been studied previously. Herein, we report a versatile strategy to obtain 1D-arranged QDs using a supramolecular polymer (SP) template. The SP is composed of self-assembling cholesterol derivatives containing two amide groups for hydrogen bonding and a carboxyl group as an adhesion moiety on the QDs. Upon mixing the SP and dispersed QDs in low-polarity solvents, the QDs self-adhered to the SP and self-arranged into 1D superstructures through van der Waals interactions between the surface organic ligands of the QDs, as confirmed by transmission electron microscopy. Furthermore, we revealed efficient photoinduced fluorescence resonance energy transfer between the 1D-arranged QDs by an in-depth analysis of the emission spectra and decay curves.

Phylogenetic, population structure, and population demographic analyses reveal that Vicia sepium in Japan is native and not introduced.

Vicia sepium (bush vetch) is a perennial legume widely distributed throughout the Eurasian continent. However, its distribution in Japan is limited to Mt. Ibuki and small parts of central and southern Hokkaido. Therefore, each Japanese V. sepium lineage has been considered to have been introduced separately from Europe. Here, we examined whether the species was introduced or not on the basis of cpDNA sequences and genome-wide SNPs from Japanese and overseas samples. Both the cpDNA haplotype network and the nuclear DNA phylogenetic tree showed that Japanese V. sepium is monophyletic. Furthermore, although the nuclear DNA phylogenetic tree also showed that each lineage is clearly monophyletic, genetic admixture of the genetic cluster dominated in the Hokkaido lineage was also detected in the Mt. Ibuki lineage. Population divergence analysis showed that the two lineages diverged during the last glacial period. The Mt. Ibuki lineage showed a sudden population decline 300-400 years ago, indicating that some anthropogenic activity might be involved, while the Hokkaido lineage showed a gradual population decline from 5000 years ago. Consequently, these two lineages show low current genetic diversity compared with overseas lineages. These results show that the Japanese V. sepium is not introduced but is native.

The DNMT3A ADD domain is required for efficient de novo DNA methylation and maternal imprinting in mouse oocytes.

Establishment of a proper DNA methylation landscape in mammalian oocytes is important for maternal imprinting and embryonic development. De novo DNA methylation in oocytes is mediated by the DNA methyltransferase DNMT3A, which has an ATRX-DNMT3-DNMT3L (ADD) domain that interacts with histone H3 tail unmethylated at lysine-4 (H3K4me0). The domain normally blocks the methyltransferase domain via intramolecular interaction and binding to histone H3K4me0 releases the autoinhibition. However, H3K4me0 is widespread in chromatin and the role of the ADD-histone interaction has not been studied in vivo. We herein show that amino-acid substitutions in the ADD domain of mouse DNMT3A cause dwarfism. Oocytes derived from homozygous females show mosaic loss of CG methylation and almost complete loss of non-CG methylation. Embryos derived from such oocytes die in mid-to-late gestation, with stochastic and often all-or-none-type CG-methylation loss at imprinting control regions and misexpression of the linked genes. The stochastic loss is a two-step process, with loss occurring in cleavage-stage embryos and regaining occurring after implantation. These results highlight an important role for the ADD domain in efficient, and likely processive, de novo CG methylation and pose a model for stochastic inheritance of epigenetic perturbations in germ cells to the next generation.

Cleaner fish recognize self in a mirror via self-face recognition like humans.

Some animals have the remarkable capacity for mirror self-recognition (MSR), yet any implications for self-awareness remain uncertain and controversial. This is largely because explicit tests of the two potential mechanisms underlying MSR are still lacking: mental image of the self and kinesthetic visual matching. Here, we test the hypothesis that MSR ability in cleaner fish, Labroides dimidiatus, is associated with a mental image of the self, in particular the self-face, like in humans. Mirror-naive fish initially attacked photograph models of both themselves and unfamiliar strangers. In contrast, after all fish had passed the mirror mark test, fish did not attack their own (motionless) images, but still frequently attacked those of unfamiliar individuals. When fish were exposed to composite photographs, the self-face/unfamiliar body were not attacked, but photographs of unfamiliar face/self-body were attacked, demonstrating that cleaner fish with MSR capacity recognize their own facial characteristics in photographs. Additionally, when presented with self-photographs with a mark placed on the throat, unmarked mirror-experienced cleaner fish demonstrated throat-scraping behaviors. When combined, our results provide clear evidence that cleaner fish recognize themselves in photographs and that the likely mechanism for MSR is associated with a mental image of the self-face, not a kinesthetic visual-matching model. Humans are also capable of having a mental image of the self-face, which is considered an example of private self-awareness. We demonstrate that combining mirror test experiments with photographs has enormous potential to further our understanding of the evolution of cognitive processes and private self-awareness across nonhuman animals.

Systematic discovery and functional dissection of enhancers needed for cancer cell fitness and proliferation.

A scarcity of functionally validated enhancers in the human genome presents a significant hurdle to understanding how these cis-regulatory elements contribute to human diseases. We carry out highly multiplexed CRISPR-based perturbation and sequencing to identify enhancers required for cell proliferation and fitness in 10 human cancer cell lines. Our results suggest that the cell fitness enhancers, unlike their target genes, display high cell-type specificity of chromatin features. They typically adopt a modular structure, comprised of activating elements enriched for motifs of oncogenic transcription factors, surrounded by repressive elements enriched for motifs recognized by transcription factors with tumor suppressor functions. We further identify cell fitness enhancers that are selectively accessible in clinical tumor samples, and the levels of chromatin accessibility are associated with patient survival. These results reveal functional enhancers across multiple cancer cell lines, characterize their context-dependent chromatin organization, and yield insights into altered transcription programs in cancer cells.

Orthodontic Treatment for a Patient with Root Resorption of All Four Maxillary Incisors due to Bilaterally Impacted Canines.

Maxillary canines require the longest period from the generation of the tooth germ to the completion of eruption, and they have more difficulties in eruption than other teeth. The incisor roots are often resorbed due to malpositioned canines. An 11-year-old girl presented with an extremely rare case of root resorption of four maxillary incisors due to bilaterally impacted maxillary canines, which were located excessively mesially. The case was managed through oral surgery and orthodontic treatment over five years. After extracting the maxillary deciduous canines, the maxillary bilateral canines were surgically exposed. The bimaxillary lateral incisors were extracted, and the canines were orthodontically tracted over 8 months. All teeth were then aligned using edgewise brackets. No further root resorption of the central incisors was observed for 5 years after canine traction. This case demonstrates the importance of early detection of abnormally positioned canines.

Longitudinal effects of estrogen on mandibular growth and changes in cartilage during the growth period in rats.

Estrogen is a steroid hormone that induces skeletal growth and affects endochondral ossification of the long tubular bone growth plate during the growth period. However, the effects of estrogen on endochondral ossification of the mandibular condylar cartilage are unclear. In this study, ovariectomized Wistar/ST rats were used to investigate the longitudinal effects of estrogen on mandibular growth. The rats were administered different doses of estrogen. Longitudinal micro-computed tomographic scanning, histological staining and ELISA on plasma growth hormone were performed to examine the effects of estrogen on mandibular growth. The results showed that mandibular growth was suppressed throughout the growth period by estrogen in a dose-dependent manner. In addition, long-term administration of a high dose of estrogen to the rats resulted in significant increase in growth hormone throughout the growth period, significant circularization of cell nuclei in the proliferative layer, intensely staining cartilage matrix in the subchondral bone, and significant suppression of estrogen receptor (ER) alpha and beta expression in the mandibular cartilage. However, regardless of estrogen concentration, in the posterior part of the mandibular cartilage, ER expression extended to both the hypertrophic and proliferative layers. These results indicate that estrogen suppresses mandibular growth throughout the growth period. Additionally, it influences endochondral ossification via its effect on ERs.

Clinical study of a new skin antiseptic olanexidine gluconate in gastrointestinal cancer surgery.

BACKGROUND: Surgical site infection (SSI) is a common complication of gastrointestinal surgery. Olanexidine gluconate (OLG) is a novel skin antiseptic that is effective against a wide range of bacteria. The purpose of this study was to evaluate the bactericidal efficacy of OLG in gastrointestinal cancer surgery. METHODS: This retrospective study included a total of 281 patients who underwent gastrointestinal cancer surgery (stomach or colon). The patients were divided into two groups: 223 patients were treated with OLG (OLG group), and 58 patients were treated with povidone-iodine (PVP-I) (control group). The efficacy and safety outcomes were measured as the rate of SSI within 30 days after surgery. In addition, we conducted subgroup analyses according to the surgical approach (open or laparoscopic) or primary lesion (stomach or colon). RESULTS: There was a significant difference in the rate of SSI between the control group and OLG group (10.3% vs. 2.7%; p = 0.02). There was a significant difference in the SSI rate in terms of superficial infection (8.6% vs. 2.2%; p = 0.0345) but not in deep infection (1.7% vs. 0.5%; p = 0.371). There was no significant difference between the control group and OLG group in the overall rate of adverse skin reactions (5.2% vs. 1.8%; p = 0.157). CONCLUSION: This retrospective study demonstrates that OLG is more effective than PVP-I in preventing SSI during gastrointestinal cancer surgery.

Characteristics of the Maxillofacial Morphology in Patients with Idiopathic Mandibular Condylar Resorption.

Idiopathic mandibular condylar resorption (ICR) is a pathological condition characterized by idiopathic resorption of the mandibular condyle, resulting in a decrease in the size and height of the mandibular condyle. The purpose of this study was to characterize the maxillofacial morphology of ICR patients. Subjects were selected from patients that attended our orthodontic clinic between 1991 and 2019. Twenty-five patients were diagnosed with ICR by magnetic resonance imaging; however, growing patients were excluded. In total, 18 patients were finally selected. The control group comprised 18 healthy volunteers. Lateral and frontal cephalograms were also used. The ICR group had significantly more severe skeletal class II malocclusions than the control group, mainly due to retrusion of the mandible. In the ICR group, there was a tendency for a skeletal open bite due to a significantly larger clockwise rotation of the mandible than in the control group. There was no significant difference between the two groups in the inclination of the upper and lower central incisors or protrusion of the upper and lower central incisors and first molars. ICR patients have been suggested to exhibit skeletal open bite and maxillary protrusion with changes in maxillofacial morphology due to abnormal resorption of the mandibular condyle.

Further evidence for the capacity of mirror self-recognition in cleaner fish and the significance of ecologically relevant marks.

An animal that tries to remove a mark from its body that is only visible when looking into a mirror displays the capacity for mirror self-recognition (MSR), which has been interpreted as evidence for self-awareness. Conservative interpretations of existing data conclude that convincing evidence for MSR is currently restricted to great apes. Here, we address proposed shortcomings of a previous study on MSR in the cleaner wrasse Labroides dimidiatus, by varying preexposure to mirrors and by marking individuals with different colors. We found that (1) 14/14 new individuals scraped their throat when a brown mark had been provisioned, but only in the presence of a mirror; (2) blue and green color marks did not elicit scraping; (3) intentionally injecting the mark deeper beneath the skin reliably elicited spontaneous scraping in the absence of a mirror; (4) mirror-naive individuals injected with a brown mark scraped their throat with lower probability and/or lower frequency compared to mirror-experienced individuals; (5) in contrast to the mirror images, seeing another fish with the same marking did not induce throat scraping; and (6) moving the mirror to another location did not elicit renewed aggression in mirror-experienced individuals. Taken together, these results increase our confidence that cleaner fish indeed pass the mark test, although only if it is presented in ecologically relevant contexts. Therefore, we reiterate the conclusion of the previous study that either self-awareness in animals or the validity of the mirror test needs to be revised.

An atlas of gene regulatory elements in adult mouse cerebrum.

The mammalian cerebrum performs high-level sensory perception, motor control and cognitive functions through highly specialized cortical and subcortical structures1. Recent surveys of mouse and human brains with single-cell transcriptomics2-6 and high-throughput imaging technologies7,8 have uncovered hundreds of neural cell types distributed in different brain regions, but the transcriptional regulatory programs that are responsible for the unique identity and function of each cell type remain unknown. Here we probe the accessible chromatin in more than 800,000 individual nuclei from 45 regions that span the adult mouse isocortex, olfactory bulb, hippocampus and cerebral nuclei, and use the resulting data to map the state of 491,818 candidate cis-regulatory DNA elements in 160 distinct cell types. We find high specificity of spatial distribution for not only excitatory neurons, but also most classes of inhibitory neurons and a subset of glial cell types. We characterize the gene regulatory sequences associated with the regional specificity within these cell types. We further link a considerable fraction of the cis-regulatory elements to putative target genes expressed in diverse cerebral cell types and predict transcriptional regulators that are involved in a broad spectrum of molecular and cellular pathways in different neuronal and glial cell populations. Our results provide a foundation for comprehensive analysis of gene regulatory programs of the mammalian brain and assist in the interpretation of noncoding risk variants associated with various neurological diseases and traits in humans.

MUNIn: A statistical framework for identifying long-range chromatin interactions from multiple samples.

Chromatin spatial organization (interactome) plays a critical role in genome function. Deep understanding of chromatin interactome can shed insights into transcriptional regulation mechanisms and human disease pathology. One essential task in the analysis of chromatin interactomic data is to identify long-range chromatin interactions. Existing approaches, such as HiCCUPS, FitHiC/FitHiC2, and FastHiC, are all designed for analyzing individual cell types or samples. None of them accounts for unbalanced sequencing depths and heterogeneity among multiple cell types or samples in a unified statistical framework. To fill in the gap, we have developed a novel statistical framework MUNIn (multiple-sample unifying long-range chromatin-interaction detector) for identifying long-range chromatin interactions from multiple samples. MUNIn adopts a hierarchical hidden Markov random field (H-HMRF) model, in which the status (peak or background) of each interacting chromatin loci pair depends not only on the status of loci pairs in its neighborhood region but also on the status of the same loci pair in other samples. To benchmark the performance of MUNIn, we performed comprehensive simulation studies and real data analysis and showed that MUNIn can achieve much lower false-positive rates for detecting sample-specific interactions (33.1%-36.2%), and much enhanced statistical power for detecting shared peaks (up to 74.3%), compared to uni-sample analysis. Our data demonstrated that MUNIn is a useful tool for the integrative analysis of interactomic data from multiple samples.

ANGPTL2 Induces Synovial Inflammation via LILRB2.

Angiopoietin-like proteins (ANGPTLs) are circulating proteins that are expressed in various cells and tissues and are thought to be involved in the repair and remodeling of damaged tissues; however, ANGPTL2 hyperfunction has been shown to cause chronic inflammation, leading to the progression of various diseases. ANGPTL2 is known to exert cellular effects via receptors such as integrin α5ß1 and leukocyte immunoglobulin-like receptor subfamily B member 2 (LILRB2); however, their roles in ANGPTL2-induced inflammation remain unclear. In this study, we investigated the mechanisms underlying ANGPTL2-induced inflammation involving LILRB2 and various signaling pathways in human fibroblast-like synoviocytes (HFLS). The effects of ANGPTL2 and an anti-LILRB2 antibody on the gene expression of various inflammation-related factors were examined using real-time RT-PCR, while their effects on MAPK, NF-κB, and Akt phosphorylation were analyzed by western blotting. We found that the addition of ANGPTL2 enhanced the gene expression of inflammatory factors, whereas pretreatment with the anti-LILRB2 antibody for 12 h decreased the expression of these factors. Similarly, ANGPTL2 addition activated the phosphorylation of ERK, p38, JNK, NF-κB, and Akt in HFLS; however, this effect was significantly inhibited by pretreatment with the anti-LILRB2 antibody. Together, the findings of this study demonstrate that ANGPTL2 induces the expression of inflammatory factors via LILRB2 in synovial cells. Therefore, LILRB2 could be a potential therapeutic agent for treating matrix degradation in osteoarthritis.

Promoter-proximal CTCF binding promotes distal enhancer-dependent gene activation.

The CCCTC-binding factor (CTCF) works together with the cohesin complex to drive the formation of chromatin loops and topologically associating domains, but its role in gene regulation has not been fully defined. Here, we investigated the effects of acute CTCF loss on chromatin architecture and transcriptional programs in mouse embryonic stem cells undergoing differentiation to neural precursor cells. We identified CTCF-dependent enhancer-promoter contacts genome-wide and found that they disproportionately affect genes that are bound by CTCF at the promoter and are dependent on long-distance enhancers. Disruption of promoter-proximal CTCF binding reduced both long-range enhancer-promoter contacts and transcription, which were restored by artificial tethering of CTCF to the promoter. Promoter-proximal CTCF binding is correlated with the transcription of over 2,000 genes across a diverse set of adult tissues. Taken together, the results of our study show that CTCF binding to promoters may promote long-distance enhancer-dependent transcription at specific genes in diverse cell types.

Paired genetic analysis by next-generation sequencing of lung cancer and associated idiopathic pulmonary fibrosis.

The pathogenesis of lung cancer associated with idiopathic pulmonary fibrosis (IPF) has remained largely uncharacterized. To provide insight into this condition, we undertook genomic profiling of IPF-associated lung cancer as well as of adjacent fibrosing lung tissue in surgical specimens. Isolated DNA and RNA from 17 IPF-associated non-small cell lung cancer and 15 paired fibrosing lung tissue specimens were analyzed by next-generation sequencing with a panel that targets 161 cancer-related genes. Somatic genetic alterations were frequently identified in TP53 (n = 6, 35.3%) and PIK3CA (n = 5, 29.4%) genes in tumor samples as well as in EGFR (n = 7, 46.7%), PIK3CA (n = 5, 33.3%), ERBB3 (n = 4, 26.7%), and KDR (n = 4, 26.7%) in IPF samples. Genes related to the RAS-RAF signaling pathway were also frequently altered in tumor (n = 7, 41.2%) and IPF (n = 3, 20.0%) samples. The number of somatic alterations identified in IPF samples was almost as large as that detected in paired tumor samples (81 vs 90, respectively). However, only 6 of the 81 somatic alterations detected in IPF samples overlapped with those in paired tumor samples. The accumulation of somatic mutations was thus apparent in IPF tissue of patients with IPF-associated lung cancer, and the RAS-RAF pathway was implicated in lung tumorigenesis. The finding that somatic alterations were not frequently shared between tumor and corresponding IPF tissue indicates that IPF-associated lung cancer does not develop through the stepwise accumulation of somatic alterations in IPF.

CTCF mediates chromatin looping via N-terminal domain-dependent cohesin retention.

The DNA-binding protein CCCTC-binding factor (CTCF) and the cohesin complex function together to shape chromatin architecture in mammalian cells, but the molecular details of this process remain unclear. Here, we demonstrate that a 79-aa region within the CTCF N terminus is essential for cohesin positioning at CTCF binding sites and chromatin loop formation. However, the N terminus of CTCF fused to artificial zinc fingers was not sufficient to redirect cohesin to non-CTCF binding sites, indicating a lack of an autonomously functioning domain in CTCF responsible for cohesin positioning. BORIS (CTCFL), a germline-specific paralog of CTCF, was unable to anchor cohesin to CTCF DNA binding sites. Furthermore, CTCF-BORIS chimeric constructs provided evidence that, besides the N terminus of CTCF, the first two CTCF zinc fingers, and likely the 3D geometry of CTCF-DNA complexes, are also involved in cohesin retention. Based on this knowledge, we were able to convert BORIS into CTCF with respect to cohesin positioning, thus providing additional molecular details of the ability of CTCF to retain cohesin. Taken together, our data provide insight into the process by which DNA-bound CTCF constrains cohesin movement to shape spatiotemporal genome organization.

Joint profiling of DNA methylation and chromatin architecture in single cells.

We report a molecular assay, Methyl-HiC, that can simultaneously capture the chromosome conformation and DNA methylome in a cell. Methyl-HiC reveals coordinated DNA methylation status between distal genomic segments that are in spatial proximity in the nucleus, and delineates heterogeneity of both the chromatin architecture and DNA methylome in a mixed population. It enables simultaneous characterization of cell-type-specific chromatin organization and epigenome in complex tissues.