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Hypoxia-inducible factor 1α (HIF1α) is a transcription factor that regulates angiogenesis under hypoxic conditions. To investigate the posttranscriptional regulatory mechanism of HIF1α, we performed a cell-based screening to reveal potential cis-elements and the regulatory RNA-binding proteins that act as trans-factors. We found that LIN28A promoted HIF1α protein expression independently of the downregulation of microRNA let-7, which is also directly mediated by LIN28A. Transcriptome analysis and evaluation of RNA stability using RNA-seq and SLAM-seq analyses, respectively, revealed that LIN28A upregulates HIF1A expression via mRNA stabilization. To investigate the physical association of LIN28A with HIF1A mRNA, we performed enhanced crosslinking immunoprecipitation in 293FT cells and integrally analyzed the transcriptome. We observed that LIN28A associates with HIF1A mRNA via its cis-element motif "UGAU". The "UGAU" motifs are recognized by the cold shock domain of LIN28A, and the introduction of a loss-of-function mutation to the cold shock domain diminished the upregulatory activities performed by LIN28A. Finally, the microvessel density assay showed that the expression of LIN28A promoted angiogenesis in vivo. In conclusion, our study elucidated the role of LIN28A in enhancing the HIF1α axis at the posttranscription layer.

It is known that autophagy controls energy and nutrient balance, contributing to regulating body temperature, circadian rhythm, and food intake (Kaur and Debnath, 2015). ATG7 is an essential protein that acts upstream of the autophagy cascade. It has been reported that neuron type-specific Atg7 cKO mice exhibit distinct homeostatic phenotypes. For instance, a loss of Atg7 gene in AgRP neurons in the hypothalamus causes abnormal energy balance and food intake (Kaushik et al., 2011). In addition, POMC neuron-specific Atg7 cKO impairs neural projections to other nuclei, such as the paraventricular nucleus of the hypothalamus (PVN), the dorsomedial nucleus of the hypothalamus (DMH), and the lateral hypothalamic area (LHA) (Coupe et al., 2012), and showed a decrease in body temperature of approximately 0.5 ºC in the presence or absence of cold exposure (Martinez-Lopez et al., 2016). Notably, these hypothalamic neuron-specific Atg7 cKO mice can survive in contrast to Nestin-Cre; Atg7f/f cKO mice, usually die around one month of age (Komatsu et al., 2006). Although the biological relevance between autophagy deficiency and brain homeostasis has been investigated, the mechanisms that regulate body temperature in the postnatal stages have not been clarified. To examine this, we confirmed whether neural autophagy influences body temperature. To do this, we measured the rectal temperature in Nestin-Cre; Atg7+/+ and Nestin-Cre; Atg7f/f mice [postnatal day (P) 21] in the morning. Consistent with the previous studies (Martinez-Lopez et al., 2016), the rectal temperature of Nestin-Cre; Atg7f/f was lower at approximately 1.0 ºC than that of Nestin-Cre; Atg7+/+ mice, suggesting that autophagy is crucial for maintaining body temperature (Fig.1A). Next, to identify the specific target regulated by neural autophagy, we conducted the microarray analysis using Nestin-Cre; Atg7+/+ and Nestin-Cre; Atg7f/f brains. The global gene expression pattern significantly changed in Nestin-Cre; Atg7f/f mice compared to Nestin-Cre; Atg7+/+ (Fig.1B and Exon array dataset Table 1). We found that p62/SQSTM1 is a favorable target induced by autophagy deficiency. It is known that p62/SQSTM1 is upregulated in the Nestin-Cre; Atg7f/f brain (Komatsu et al., 2007). In addition, the transferrin receptor is an intriguing target physiologically. It has been reported that loss of autophagy causes iron deficiency due to an inhibition of ferritin degradation (Hou et al., 2016). Because ferritin is an intracellular iron storage protein, abnormal autophagy causes the accumulation of ferritin-containing iron. Furthermore, we also identified that RBM3 is significantly upregulated in Nestin-Cre; Atg7f/f brain in our transcriptome analysis (Fig.1B). It has been reported that RBM3 is one of the cold-inducible protein and regulates neurogenesis and local translation in neurons (Sertel et al., 2021; Xia et al., 2018; Zhu et al., 2019). Importantly, RBM3 is induced by body temperature drop, such as hibernation (Williams et al., 2005), and stabilizes the mRNA of circadian genes (Liu et al., 2013). Because the circadian rhythm is associated with the daily body temperature variation, these observations demonstrate that RBM3 is a potential candidate that links a defect in autophagy to lower body temperature. Then, we confirmed RBM3 gene expression in Nestin-Cre; Atg7f/f brain. The mRNA levels in Nestin-Cre; Atg7f/f brain were significantly increased compared to that in Nestin-Cre; Atg7+/+ brain (Fig.1C), indicating that loss of Atg7 gene enhances RBM3 expression in the brain. Previous studies have reported that the preoptic area (POA) is a core region that governs body temperature (Kuraoka and Nakamura, 2011). It is known that the POA neurons project to the DMH, followed by regulating a body temperature via influencing the brown adipose tissue, skeletal muscle, and blood vessels. It is also known that the POA neurons project to the arcuate nucleus (ARC) and regulates the neural circuit in the hypothalamus implicated in the body temperature and circadian rhythm (Fig. 1D). Thus, we investigated which area RBM3 was expressed in the hypothalamus. RBM3 expression was ubiquitous; however, RBM3 expression in the hypothalamus was high in Nestin-Cre; Atg7f/f brain. Especially, this phenomenon tended to be observed in the arcuate nucleus (ARC) in the hypothalamus (Fig. 1E). Interestingly, the subcellular localization of RBM3 was mainly localized in the nucleus (Fig. 1F), implying the possibility that RBM3 modulates the characteristics of target mRNA, such as splicing, stability, and export from the nucleus. In addition, the RBM3 level in the hypothalamic area of Nestin-Cre; Atg7f/f mice was significantly increased compared to that in the same area of Nestin-Cre; Atg7+/+ mice (Fig. 1G). These results suggest that RBM3 regulates neuronal activity or regional homeostasis in the hypothalamic area, leading to the control of body temperature. Taken together, our data suggest that RBM3 is a potential mediator that underlies the regulation of body temperature associated with autophagy deficiency.

In the modern world, several areas of our lives can be improved, in the form of diverse additional dimensions, in terms of quality, by machine learning. When building machine learning models, open data are often used. Although this trend is on the rise, the monetary losses since the attacks on machine learning models are also rising. Preparation is, thus, believed to be indispensable in terms of embarking upon machine learning. In this field of endeavor, machine learning models may be compromised in various ways, including poisoning attacks. Assaults of this nature involve the incorporation of injurious data into the training data rendering the models to be substantively less accurate. The circumstances of every individual case will determine the degree to which the impairment due to such intrusions can lead to extensive disruption. A modus operandi is proffered in this research as a safeguard for machine learning models in the face of the poisoning menace, envisaging a milieu in which machine learning models make use of data that emanate from numerous sources. The information in question will be presented as training data, and the diversity of sources will constitute a barrier to poisoning attacks in such circumstances. Every source is evaluated separately, with the weight of each data component assessed in terms of its ability to affect the precision of the machine learning model. An appraisal is also conducted on the basis of the theoretical effect of the use of corrupt data as from each source. The extent to which the subgroup of data in question can undermine overall accuracy depends on the estimated data removal rate associated with each of the sources described above. The exclusion of such isolated data based on this figure ensures that the standard data will not be tainted. To evaluate the efficacy of our suggested preventive measure, we evaluated it in comparison with the well-known standard techniques to assess the degree to which the model was providing accurate conclusions in the wake of the change. It was demonstrated during this test that when the innovative mode of appraisal was applied, in circumstances in which 17% of the training data are corrupt, the degree of precision offered by the model is 89%, in contrast to the figure of 83% acquired through the traditional technique. The corrective technique suggested by us thus boosted the resilience of the model against harmful intrusion.

Healthy limb joints are important for maintaining health and attaining longevity. Endochondral ossification (the replacement of cartilage with bone, occurring during skeletal development) is essential for bone formation, especially in long-axis bones. In contrast to endochondral ossification, chondrocyte populations in articular cartilage persist and maintain joint tissue into adulthood. Articular cartilage, a connective tissue consisting of chondrocytes and their surrounding extracellular matrices, plays an essential role in the mechanical cushioning of joints in postnatal locomotion. Osteoarthritis (OA) pathology relates to disruptions in the balance between anabolic and catabolic signals, that is, the loss of chondrocyte homeostasis due to aging or overuse of cartilages. The onset of OA increases with age, shortening a person’s healthy life expectancy. Although many people with OA experience pain, the mainstay of treatment is symptomatic therapy, and no fundamental treatment has yet been established. To establish regenerative or preventative therapies for cartilage diseases, further understanding of the mechanisms of cartilage development, morphosis, and homeostasis is required. In this review, we describe the general development of cartilage and OA pathology, followed by a discussion on anabolic and catabolic signals in cartilage homeostasis, mainly microRNAs.

Neat1 is a long noncoding RNA (lncRNA) that serves as an architectural component of the nuclear bodies known as paraspeckles. Two isoforms of Neat1, the short isoform Neat1_1 and the long isoform Neat1_2, are generated from the same gene locus by alternative 3’ processing. Neat1_1 is the most abundant and the best conserved isoform expressed in various cell types, whereas Neat1_2 is expressed in a small population of particular cell types, including the tip cells of the intestinal epithelium. To investigate the physiological significance of isoform switching, we created mutant mice that solely expressed Neat1_2 by deleting the upstream polyadenylation (poly-A) signal (PAS) required for the production of Neat1_1. We observed the loss of Neat1_1 and strong upregulation of Neat1_2 in various tissues and cells and the subsequent hyperformation of paraspeckles, especially in cells that normally express Neat1_2. However, the mutant mice were born at the expected Mendelian ratios and did not exhibit obvious external and histological abnormalities. These observations suggested that the hyperformation of paraspeckles does not interfere with the development and growth of these animals under normal laboratory conditions.

The periodontal ligament (PDL) comprises a fibrous tissue that connects teeth to alveolar bone and is essential for periodontal function. The transcription factor mohawk homeobox (

Logic gates using magnetic tunnel junction (MTJ)-based nonvolatile logic-in-memory (NV-LIM) architecture are designed for quantized neural networks (QNNs) for Internet-of-Things applications. The NV-LIM-based implementation reduces data transfer costs between storage and logic gate components, thereby greatly enhancing the energy efficiency of inference operations in QNNs. The impact of the proposed nonvolatile logic gates for binary and ternary neural networks on energy consumption, delay, and area overhead reduction is demonstrated through circuit evaluations based on the parameters of the measured MTJ devices.

The micronucleus is known to be a biomarker for genomic instability, which is a hallmark of tumors and aging. Normally, micronuclei are produced by segregation errors and mechanical stresses arising from dividing or migrating cells, leading to activation of the innate immune response pathway. Although micronuclei often emerge in damaged tissues, the quantitative procedure for analyzing micronuclei accurately has been problematic. Here, we introduce a novel MATLAB-based program for quantifying micronuclei (CAMDi: calculating automatic micronuclei distinction) in vitro and in vivo. CAMDi is adaptable to various experimental imaging techniques and is useful for obtaining reproducible data. CAMDi enables us to measure the accurate size of micronuclei from the three-dimensional images. Using CAMDi, we revealed a novel link between the emergence of micronuclei and neuroinflammation. We found that inflammatory stimulation does not increase the number of micronuclei in primary neurons. On the other hand, the administration of lipopolysaccharide into mice slightly increases micronuclei formation in neurons of the hippocampus region. These findings demonstrate that neuronal micronuclei formations are induced by an inflammatory response in a non-cell-autonomous manner. We provide a novel tool, CAMDi, to quantify micronuclei and demonstrate that neuronal micronuclei are produced not only by the cell-autonomous process but also by the intercellular communication associated with neuroinflammation in vivo.

MicroRNAs (miRNAs) are a class of noncoding small RNAs, which play a critical role in various biological processes including musculoskeletal formation and arthritis pathogenesis via regulating target gene expressions, raising the potentially substantial effects on gene expression networks. Over 2000 miRNAs are encoded in the human genome and a single miRNA potentially targets hundreds of genes. To examine the expression and function of miRNAs in chondrocytes and arthritis pathogenesis, we describe the protocols for the current miRNA related experiments including miRNA expression profiling by (1) Next Generation Sequencing and by TaqMan Array system, (2) miRNA target prediction by TargetScan, (3) miRNA target screening by cell-based reporter library assay, and (4) miRNA and its target interaction by HITS-CLIP (high- throughput sequencing of RNAs isolated by cross-linking immunoprecipitation) in cartilage and chondrocyte research.

Programmed death-ligand 1 (PD-L1) is a co-inhibitory molecule expressed on tumor cells. Immune checkpoint inhibitors focusing on the PD-L1 mechanism are now being studied for the treatment of various cancer types. However, the regulatory mechanism of PD-L1 is yet to be fully clarified, and a high-throughput system for comparing the abilities of small compounds in regulating PD-L1 has not yet been established. Therefore, we created a HiBiT-tagged lung adenocarcinoma cell line, PC9-KI, for easier and faster detection of changes in PD-L1 protein expression. Using PC9-KI cells, we screened 1,280 chemical compounds from the Library of Pharmacologically Active Compounds (LOPAC) and identified microtubule polymerization inhibitors and thapsigargin as PD-L1 upregulators and a p97 inhibitor as a PD-L1 downregulator.

Let‐7 is an evolutionary conserved microRNA that mediates post‐transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let‐7 biogenesis is tightly regulated by several RNA‐binding proteins, including Lin28A/B, which represses let‐7 maturation. To identify new regulators of let‐7, we devised a cell‐based functional screen of RNA‐binding proteins using a let‐7 sensor luciferase reporter and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let‐7 family members. Rather than inducing pseudouridylation of the miRNAs, high‐throughput sequencing crosslinking immunoprecipitation (HITS‐CLIP) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem‐loop structure of pri‐let‐7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri‐let‐7 and the microprocessor DGCR8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let‐7. Altogether, we reveal an unexpected function for TruB1 in promoting let‐7 maturation.

The epigenetic inheritance relies on stability of histone marks, but various diseases, including aging-related disorders, are usually associated with alterations of histone marks. Whether and how the proteasome is responsible for maintaining the histone marks during transcription and aging remain unclear. The core histones can be degraded by the atypical proteasome, which contains the proteasome activator PA200, in an acetylation-dependent manner during somatic DNA damage response and spermiogenesis. Methods: By utilizing a substitute of methionine to label proteins metabolically, we analyzed histone degradation genome-wide by sequencing the DNA fragments following pulse-chase assays. The genome-wide RNA-sequencing analysis was performed to analyze transcription and chromatin-immunoprecipitation (ChIP)-sequencing was used for analyses of histone marks. The experimental models included gene-manipulated cells (including both mouse and yeast), mouse liver, and mice. Results: Degradation of H4 or the transcription-coupled histone variant H3.3 could be suppressed by deletion of PA200 or its yeast ortholog Blm10. The histone deacetylase inhibitor accelerated the degradation rates of H3, while the mutations of the putative acetyl-lysine-binding region of PA200 abolished histone degradation in the G1-arrested cells. Deletion of PA200 dramatically altered deposition of the active transcriptional hallmarks (H3K4me3 and H3K56ac) and transcription, especially during cellular aging. Furthermore, deletion of PA200 or Blm10 accelerated cellular aging. Notably, the PA200-deficient mice displayed a range of aging-related deteriorations, including immune malfunction, anxiety-like behavior and shorter lifespan. Conclusion: PA200 promotes the transcription-coupled degradation of the core histones, and plays an important role in maintaining the stability of histone marks during transcription and aging.

The epigenetic inheritance relies on stability of histone marks, but various diseases, including aging-related diseases, are usually associated with alterations of histone marks. How the stability of histone marks is maintained still remains unclear. The core histones can be degraded by the atypical proteasome, which contains the proteasome activator PA200, in an acetylation-dependent manner during somatic DNA damage response and spermiogenesis. Here we show that PA200 promotes the transcription-coupled degradation of the core histones, and plays an important role in maintaining the stability of histone marks. Degradation of the histone variant H3.3, which is incorporated into chromatin during transcription, was much faster than that of its canonical form H3.1, which is incorporated during DNA replication. This degradation of the core histones could be suppressed by the transcription inhibitor, the proteasome inhibitor or deletion of PA200. The histone deacetylase inhibitor accelerated the degradation rates of H3 in general, especially its variant H3.3, while the mutations of the putative acetyl-lysine-binding region of PA200 abolished histone degradation in the G1-arrested cells, supporting that acetylation is involved in the degradation of the core histones. Deletion of PA200 dramatically altered deposition of the active transcriptional hallmarks (H3K4me3 and H3K56ac) and transcription, especially during cellular aging. Furthermore, deletion of PA200 or its yeast ortholog Blm10 accelerated cellular aging. Notably, the PA200-deficient mice displayed a range of aging-related deteriorations, including immune malfunction, anxiety-like behaviors and shorter lifespan. Thus, the proteasome activator PA200 is critical to the maintenance of the stability of histone marks during transcription and aging.

Let-7 is an evolutionary conserved microRNA that mediates post-transcriptional gene silencing to regulate a wide range of biological processes, including development, differentiation, and tumor suppression. Let-7 biogenesis is tightly regulated by several RNA-binding proteins, including Lin28A/B, which represses let-7 maturation. To identify new regulators of let-7, we devised a cell-based functional screen of RNA-binding proteins using a let-7 sensor luciferase reporter, and identified the tRNA pseudouridine synthase, TruB1. TruB1 enhanced maturation specifically of let-7 family members. Rather than inducing pseudouridylation of the miRNAs, HITS-CLIP (High throughput sequencing crosslinking immunoprecipitation) and biochemical analyses revealed direct binding between endogenous TruB1 and the stem-loop structure of pri-let-7, which also binds Lin28A/B. TruB1 selectively enhanced the interaction between pri-let-7 and the microprocessor DGCR-8, which mediates miRNA maturation. Finally, TruB1 suppressed cell proliferation, which was mediated in part by let-7. Altogether, we reveal an unexpected function for TruB1 in promoting let-7 maturation and function.

Chemical screening is an essential procedure for finding novel drugs of cancer treatments. Especially, cell-based screenings are mostly used for discovery of novel compounds or drug repositioning of already-used drugs. Cell viability or cell proliferation are usually used for evaluation of effects of candidate novel compounds for cancer treatments. However, high throughput system for rapid evaluation of endogenous target protein expression changes has not been established so far. Therefore, an easy and quick method for detection of endogenous target protein expression changes has been strongly needed for drug discovery. Herein, we established a novel chemical compound screening system using the HiBiT-tagging system for this aim using PD-L1 as an example. HiBiT tag is a small peptide tag composed of only 11 amino acids forming a dimer with LgBiT, and functions as a Nanoluciferase. The shortness of the peptide tag is useful for insertion of tag using genome editing technology. Thereby, we established a rapid chemical screening method using HiBiT-tagged cell lines created by CRISPR-Cas9 system. With this method, we made it possible to detect endogenous target protein expression changes within 10 minutes by just adding lytic buffer and LgBiT. For validation of this method, we performed chemical screening of PD-L1 using a HiBiT-tagged lung adenocarcinoma cell line with 1,280 compounds. As a result, we identified microtubule inhibitors as compounds upregulating PD-L1, and validated its upregulation of PD-L1 with qPCR and western blotting. In conclusion, we succeeded in establishing the method for chemical compounds detecting endogenous target protein expression changes with HiBiT-tagging system. Furthermore, we validated this method by performing chemical screening with PD-L1, and succeeded in identifying microtubules inhibitors as up-regulators of PD-L1. Citation Format: Yutaro Uchida, Takahide Matsushima, Ryota Kurimoto, Tomoki Chiba, Yuki Inutani, Hiroshi Asahara. HiBiT tagging system for high throughput chemical screening for chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 20.

In human genome, there are approximately 1,500 RNA-binding proteins (RBPs). They can regulate mRNA stability or translational efficiency via ribosomes and these processes are known as ‘post-transcriptional regulation’. Accumulating evidences indicate that post-transcriptional regulation is the determinant of the accurate levels of cytokines mRNAs. While transcriptional regulation of cytokines mRNAs has been well studied and found to be important for the rapid induction of mRNA and regulation of the acute phase of inflammation, post-transcriptional regulation by RBPs is essential for resolving inflammation in the later phase, and their dysfunction may lead to severe autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus. For post-transcriptional regulation, RBPs recognize and directly bind to cis-regulatory elements in 3′ untranslated region of mRNAs such as AU-rich or constitutive decay elements and play various roles. In this review, we summarize the recent findings regarding the role of RBPs in the regulation of inflammation.

NEAT1 is one of the most studied lncRNAs, in part because its silencing in mice causes defects in mammary gland development and corpus luteum formation and protects them from skin cancer development. Moreover, depleting NEAT1 in established cancer cell lines reduces growth and sensitizes cells to DNA damaging agents. However, NEAT1 produces two isoforms and because the short isoform, NEAT1_1, completely overlaps the 5′ part of the long NEAT1_2 isoform; the respective contributions of each of the isoforms to these phenotypes has remained unclear. Whereas NEAT1_1 is highly expressed in most tissues, NEAT1_2 is the central architectural component of paraspeckles, which are nuclear bodies that assemble in specific tissues and cells exposed to various forms of stress. Using dual RNA-FISH to detect both NEAT1_1 outside of the paraspeckles and NEAT1_2/NEAT1 inside this nuclear body, we report herein that NEAT1_1 levels are dynamically regulated during the cell cycle and targeted for degradation by the nuclear RNA exosome. Unexpectedly, however, cancer cells engineered to lack NEAT1_1, but not NEAT1_2, do not exhibit cell cycle defects. Moreover, Neat1_1-specific knockout mice do not exhibit the phenotypes observed in Neat1-deficient mice. We propose that NEAT1 functions are mainly, if not exclusively, attributable to NEAT1_2 and, by extension, to paraspeckles.

NEAT1 is one of the most studied lncRNAs, in part because its silencing in mice causes defects in mammary gland development and corpus luteum formation, and protects them from skin cancer development. Moreover, depleting NEAT1 in established cancer cell lines reduces growth and sensitizes cells to DNA damaging agents. However, NEAT1 produces two isoforms and because the short isoform, NEAT1_1, completely overlaps the 5’ part of the long NEAT1_2 isoform, the respective contributions of each of the isoforms to these phenotypes has remained unclear. Whereas NEAT1_1 is highly expressed in most tissues, NEAT1_2 is the central architectural component of paraspeckles, which are nuclear bodies that assemble in specific tissues and cells exposed to various forms of stress. Using dual RNA-FISH to detect both NEAT1_1 outside of paraspeckles and NEAT1_2/NEAT1 inside this nuclear body, we report herein that NEAT1_1 levels are dynamically regulated during the cell cycle and targeted for degradation by the nuclear RNA exosome. Unexpectedly, however, cancer cells engineered to lack NEAT1_1, but not NEAT1_2, do not exhibit cell cycle defects. Moreover, Neat1_1-specific knockout mice do not exhibit the phenotypes observed in Neat1-deficient mice. We propose that NEAT1 functions are mainly, if not exclusively, attributable to NEAT1_2 and, by extension, to paraspeckles. Graphical abstract

Excessive and constitutive activation of nuclear factor-κB (NF-κB) leads to abnormal cell proliferation and differentiation, leading to the development of malignant tumors, including lymphoma. MicroRNA 146a (miR-146a) and miR-146b, both of which carry an identical seed sequence, have been shown to contribute to inflammatory diseases and tumors by suppressing the expression of key molecules required for NF-κB activation. However, the functional and physiological differences between miR-146a and miR-146b in disease onset have not been fully elucidated. In this study, we generated miR-146b–knockout (KO) and miR-146a–KO mice by genome editing and found that both strains developed hematopoietic malignancies such as B-cell lymphoma and acute myeloid leukemia during aging. However, the B-cell lymphomas observed in miR-146a– and miR-146b–KO mice were histologically different in their morphology, and the malignancy rate is lower in miR-146b mice than miR-146a mice. Upon mitogenic stimulation, the expression of miR-146a and miR-146b was increased, but miR-146b expression was lower than that of miR-146a. Using a previously developed screening system for microRNA targets, we observed that miR-146a and miR-146b could target the same mRNAs, including TRAF6, and inhibit subsequent NF-κB activity. Consistent with these findings, both miR-146a– and miR-146b–KO B cells showed a high proliferative capacity. Taken together, sustained NF-κB activation in miR-146b KO mice could lead to the development of hematopoietic malignancy with aging.

The cyclin-dependent kinase (CDK) inhibitor p21 is an unstructured protein regulated by multiple turnover pathways. p21 abundance is tightly regulated, and its defect causes tumor development. However, the mechanisms that underlie the control of p21 level are not fully understood. Here, we report a novel mechanism by which a component of the SCF ubiquitin ligase, Fbl12, augments p21 via the formation of atypical ubiquitin chains. We found that Fbl12 binds and ubiquitinates p21. Unexpectedly, Fbl12 increases the expression level of p21 by enhancing the mixed-type ubiquitination, including not only K48- but also K63-linked ubiquitin chains, followed by promotion of binding between p21 and CDK2. We also found that proteasome activator PA28γ attenuates p21 ubiquitination by interacting with Fbl12. In addition, UV irradiation induces a dissociation of p21 from Fbl12 and decreases K63-linked ubiquitination, leading to p21 degradation. These data suggest that Fbl12 is a key factor that maintains adequate intracellular concentration of p21 under normal conditions. Our finding may provide a novel possibility that p21's fate is governed by diverse ubiquitin chains.

Microglia are resident macrophages that are critical for brain development and homeostasis. Microglial morphology is dynamically changed during postnatal stages, leading to regulating synaptogenesis and synapse pruning. Moreover, it has been well known that the shape of microglia is also altered in response to the detritus of the apoptotic cells and pathogens such as bacteria and viruses. Although the morphologic changes are crucial for acquiring microglial functions, the exact mechanism which controls their morphology is not fully understood. Here, we report that the FAT atypical cadherin family protein, FAT3, regulates the morphology of microglial cell line, BV2. We found that the shape of BV2 becomes elongated in a high-nutrient medium. Using microarray analysis, we identified that FAT3 expression is induced by culturing with a high-nutrient medium. In addition, we found that purinergic analog, hypoxanthine, promotes FAT3 expression in BV2 and mouse primary microglia. FAT3 expression induced by hypoxanthine extends the time of sustaining the elongated forms in BV2. These data suggest that the hypoxanthine-FAT3 axis is a novel pathway associated with microglial morphology. Our data provide a possibility that FAT3 may control microglial transitions involved in their morphologic changes during the postnatal stagesin vivo.

Tendons and ligaments are pivotal connective tissues that tightly connect muscle and bone. In this study, we developed a novel approach to generate tendon/ligament-like tissues with a hierarchical structure, by introducing the tendon/ligament-specific transcription factor Mohawk (MKX) into the mesenchymal stem cell (MSC) line C3H10T1/2 cells, and by applying an improved three-dimensional (3D) cyclic mechanical stretch culture system. In our developed protocol, a combination of stable Mkx expression and cyclic mechanical stretch synergistically affects the structural tendon/ligament-like tissue generation and tendon related gene expression. In a histological analysis of these tendon/ligament-like tissues, an organized extracellular matrix (ECM), containing collagen type III and elastin, was observed. Moreover, we confirmed that Mkx expression and cyclic mechanical stretch, induced the alignment of structural collagen fibril bundles that were deposited in a fibripositor-like manner during the generation of our tendon/ligament-like tissues. Our findings provide new insights for the tendon/ligament biomaterial fields.

The ubiquitin ligases, SCF complexes, consist of Cul1, Skp1, Rbx1 and the substrate recognition components F-box proteins. Previous studies have reported that one of these F-box proteins, Fbl12, which is produced by Fbxl12 gene, regulates both cell cycle and differentiation. In this paper, we show that the intronic region of Fbxl12 gene acts as an alternative promoter and induces expression of a short form of Fbl12 that lacks F-box domain (Fbl12ΔF). We also found that UV irradiation increases Fbl12ΔF mRNA in cells. Finally, Fbl12ΔF may promote the subcellular localization of Fbl12 from nucleus to cytoplasm through their binding. Our data provide the possibility that Fbl12ΔF induced by alternative promoter controls the SCFFbl12 activity in response to UV stimulation.

The proteasome is a protease complex composed of a core particle (CP) and regulatory particles (RPs) that bind to both ends of the CP. ECM29 is a protein that associates with the proteasome and is involved in the maintenance and regulation of the proteasome assembly. However, ECM29 deficient mice can form functional proteasome. In this paper we sought to identify the mechanisms and/or proteins that help and allow the maintenance of the proteasome activity in the absence of ECM29. We analyzed the proteasome components of ECM29-deficient mice and identified Hsp90 as a protein associated with the proteasome. Furthermore, the inhibition of Hsp90 led to a partial disassembly of the proteasome only in ECM29-deficient cells. Those findings attest to the importance of Hsp90 for the maintenance of the proteasome integrity in the absence of ECM29.

SRY-box 9 (SOX9) is a master transcription factor that regulates cartilage development. SOX9 haploinsufficiency resulting from breakpoints in a ∼1-Mb region upstream of SOX9 was reported in acampomelic campomelic dysplasia (ACD) patients, suggesting that essential enhancer regions of SOX9 for cartilage development are located in this long non-coding sequence. However, the cis-acting enhancer region regulating cartilage-specific SOX9 expression remains to be identified. To identify distant cartilage Sox9 enhancers, we utilized the combination of multiple CRISPR/Cas9 technologies including enrichment of the promoter-enhancer complex followed by next-generation sequencing and mass spectrometry (MS), SIN3A-dCas9-mediated epigenetic silencing, and generation of enhancer deletion mice. As a result, we could identify a critical far-upstream cis-element and Stat3 as a trans-acting factor, regulating cartilage-specific Sox9 expression and subsequent skeletal development. Our strategy could facilitate definitive ACD diagnosis and should be useful to reveal the detailed chromatin conformation and regulation.

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

To investigate the immunological changes caused by severe sepsis in elderly patients.One-year, prospective observational study.Emergency department and intensive care unit of a single university hospital.Seventy-three patients with severe sepsis and 72 healthy donors.In elderly septic patients (aged 65 yr and over), 3-month survival was significantly reduced compared with that for adult patients (18-64 yr) (60% vs. 89%, p0.01). We found that lymphopenia was prolonged for at least 21 days in elderly nonsurvivors of sepsis, while the number of lymphocytes recovered in both adult and elderly survivors of sepsis. In order to examine the immunological status of septic patients, blood samples were collected within 48 hrs of diagnosis of severe sepsis, and peripheral blood mononuclear cells were purified for flow cytometric analysis. T cell levels were significantly reduced in both adult and elderly septic patients, compared with those in healthy donors (56% and 57% reduction, respectively). Interestingly, the immunocompetent CD28+ subset of CD4+ T cells decreased, whereas the immunosuppressive PD-1+ T cells and the percentage of regulatory T cells (CD4+ T cells that are both Foxp3+ and CD25+) increased in elderly patients, especially nonsurvivors, presumably reflecting the initial signs of immunosuppression.Reduction of immunocompetent T cells followed by prolonged lymphopenia may be associated with poor prognosis in elderly septic patients.

After receiving a TCR-mediated differentiation signal, CD4 and CD8 double-positive thymocytes diverge into CD4 or CD8 single-positive T cells, for which Th-POK and Runx3 have been identified as pivotal transcription factors, respectively. The cross-antagonistic regulation of Th-POK and Runx3 seems to be essential for CD4/8 thymocyte lineage commitment. However, the process for determining which pivotal factor acts dominantly has not been established. To explore the determining process, we used an in vitro culture system in which CD4 or CD8 single-positive cells are selectively induced from CD4/8 double-positive cells. Surprisingly, we found that control of G1 cell cycle phase progression is critical for the determination. In the CD4 pathway, sustained TCR signal, as well as Th-POK, induces G1-phase extension and represses CD8 expression in a G1 extension-dependent manner. In the CD8 pathway, after receiving a transient TCR signal, the IL-7R signal, as well as Runx3, antagonizes TCR signal-mediated G1 extension and CD8 repression. Importantly, forced G1 extension cancels the functions of Runx3 to repress Th-POK and CD4 and to reactivate CD8. In contrast, it is suggested that forced G1 progression inhibits Th-POK function to repress CD8. Collectively, Th-POK and Runx3 are reciprocally involved in the control of G1-phase progression, on which they exert their functions dependently. These findings may provide novel insight into how CD4/CD8 cell lineages are determined by Th-POK and Runx3.

We explored the prey-size preference and drillhole-site selectivity in Euspira fortunei preying on Ruditapes philippinarum by means of laboratory experiments. Euspira fortunei was a size-selective predator and there was a positive correlation between predator and prey sizes. Our experiments and observations showed that prey-size limits in E. fortunei were determined by the size of its foot, which is the organ used for capturing and handling prey, as in other naticid species. Within the manipulation limits, attacks on relatively small or large prey were detected after prey of the preferred size classes became scarce. Our experiments also suggested that there was no size refuge for R. philippinarum (shell length range 0– 35 mm) from attack by E. fortunei and that R. philippinarum of shell length range 10 – 25 mm were especially vulnerable to predation. These results imply that introduction of E. fortunei probably causes significant loss in R. philippinarum stocks. The sizes of E. fortunei individuals were significantly correlated with the diameters of the drillholes left on the shells of prey. Drillhole location was generally stereotypical in size-matched prey but anomalous drillhole placements commonly occurred in size-mismatched prey. Because E. fortunei captured prey using its foot and manipulated prey in a stereotypical manner, handling of size-mismatched prey was difficult and thus attacks on such prey tended to result in anomalous drillhole placements. Euspira fortunei did not make drillholes on the edges of bivalve prey when it competed for prey.

We have developed a method to measure the amounts of cell surface-expressed membrane proteins with bioluminescence. Dinoflagellate luciferase was expressed on the surface of a mammalian cell as a chimeric fusion protein with a membrane protein of interest. Using a membrane-impermeable substrate to quantify the membrane-displayed luciferase, the expression of the membrane protein on the cell surface was determined. By inclusion of a quenching step for the luminescent activity of luciferase on the cell surface, we were able to monitor the membrane protein expression kinetics by measuring the luminescence recovery from the cell surface after quenching. The reported methods provide a convenient way to monitor the kinetics of expression and transport of membrane proteins to the cell surface. It is applicable to the high-throughput analysis of drugs or drug candidates concerning their effects on membrane protein expression.