RNA-binding protein LIN28A upregulates transcription factor HIF1α by posttranscriptional regulation via direct binding to UGAU motifs.

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.

The tRNA pseudouridine synthase TruB1 regulates the maturation of let-7 miRNA.

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.

Mkx regulates the orthodontic tooth movement via osteoclast induction.

INTRODUCTION: The periodontal ligament (PDL) plays an important role in orthodontic tooth movement; however, the underlying molecular mechanism remains unclear. We have previously reported that the Mohawk homeobox (Mkx), a tendon-specific transcription factor, is expressed in the PDL and regulates its homeostasis. MATERIALS AND METHODS: In the present study, we examined the role of Mkx in orthodontic tooth movement via bone remodeling induced by mechanical stimulation in Mkx-deficient rats, which are widely used as experimental animals for orthodontic force application. Orthodontic tooth movement of the maxillary first molar was performed in 7-week-old male Mkx-deficient rats (n = 4) and wild-type Wistar rats (n = 4) using coil springs for 14 days. Hematoxylin and eosin (H&E) staining and tartrate-resistant acid phosphatase (TRAP) staining were performed to evaluate morphological changes and osteoclasts. Furthermore, changes in the expression of receptor activator nuclear factor-kappa B ligand (RANKL) were demonstrated using immunostaining. RESULTS: The amount of tooth movement was significantly lower in Mkx-deficient rats than in wild-type rats. The number of TRAP-positive cells was suppressed in Mkx-deficient rats on the compression side. CONCLUSION: Orthodontic tooth movement experiments in Mkx-deficient rats suggested that Mkx is involved in osteoclast induction at the alveolar bone surface on the compression side. This study reveals the possibility that Mkx plays a mechanosensory role in orthodontic tooth movement by inducing RANKL expression and osteoclastogenesis.

Safety of a short hydration method for cisplatin administration in comparison with a conventional method-a retrospective study.

OBJECTIVE: Cisplatin is administered in combination with massive hydration to avoid renal toxicity, making its administration difficult in an outpatient setting. Although a short hydration protocol for cisplatin has been recently developed, its safety is not fully understood. METHODS: Consecutive patients with lung or other cancer and an Eastern Cooperative Oncology Group performance status of 0-2 who were receiving chemotherapy containing cisplatin at a dose of ≥60 mg/m(2) in a single administration were evaluated. Seventy-four patients were treated with a short hydration protocol consisting of 1750-2250 ml of hydration with mannitol and magnesium supplementation over a period of 3.75-4.75 h on Day 1. Sixty-nine patients were treated with a conventional hydration protocol consisting of 2100-2600 ml of hydration over 6.5-7.5 h on Day 1 with pre- and post-hydration on Days 0, 2 and 3. Toxicity was then compared between the two groups. RESULTS: An elevated serum creatinine level ≥grade 1 was significantly less frequent in the group receiving the short hydration protocol than in the group receiving conventional hydration. Other toxicities were similar between the two groups. Consequently, the completion rate for the planned treatment in the short hydration group (73.0%, 54/74) was significantly higher than that in the conventional hydration group (53.6%, 37/69). CONCLUSIONS: Short hydration is safe, making cisplatin-containing chemotherapy easier to perform.

Current status of medical oncology in Japan--reality gleaned from a questionnaire sent to designated cancer care hospitals.

OBJECTIVE: Medical oncology in Japan has a relatively short history, with specialist certification starting in 2006, resulting in 867 certified medical oncologists as of 2014. Although the national government has appointed 397 Designated Cancer Care Hospitals, little is known about the actual situations of medical oncology services at these institutions. METHODS: Questionnaires regarding the presence of a medical oncology department, the number of physicians in the department, the presence of certified medical oncologists and the degree of the medical oncologists' responsibilities for drug therapies in adults with solid cancers were sent to all 397 institutions between 21 January and 1 May 2013. RESULTS: The response rate was 68.0%. Among the responses, 39.4% of the institutions had medical oncology departments with a median of three physicians. Most of the medical oncology departments were primarily responsible, as evaluated according to patient number, for the treatment of limited disease categories. The medical oncologists were significantly more responsible for molecular-targeted therapy than for chemotherapy in head and neck cancer or for cytokine therapy in renal cell carcinoma. The wide variety of adverse events associated with molecular-targeted therapy might have enhanced the roles of medical oncologists. As the proportion of hospitals with a medical oncology department increased according to the number of certified medical oncologists working at the institution, cultivating medical oncologists seems to be an urgent task for advancing medical oncology in Japan. CONCLUSIONS: The present study provides fundamental data for the future development of medical oncology in Japan. The present study is to uncover the current situation of medical oncology in Japan.

Incidence, risk factors and treatment outcomes of extravasation of cytotoxic agents in an outpatient chemotherapy clinic.

OBJECTIVE: Extravasation, the accidental leakage of an anticancer agent from a vessel into the surrounding tissues, can lead to irreversible local injuries and severe disability. Despite its considerable clinical importance, evidence-based information on extravasation in chemotherapy is lacking. This study characterized the clinical features of extravasation and identified issues to be resolved in current cancer chemotherapy performed in outpatient settings. METHODS: We retrospectively reviewed the medical charts of patients who received chemotherapy and sustained extravasation in our Outpatient Chemotherapy Clinic from April 2007 to August 2012. Chemotherapy administration and extravasation management procedures were standardized using the in-house chemotherapy guideline. RESULTS: Among 43 557 patients who received chemotherapy, 35 (0.08%) experienced extravasation. The duration between the start of infusion and extravasation was >2 h in 28 (80.0%) patients. The severity of extravasation was Grades 1, 2 and 3 in 28, 2 and 5 patients, respectively-three of whom were associated with port trouble. The contributing factor for extravasation was walking in 11 (31.4%) patients. All extravasations were cured without surgical intervention by management according to our guidelines. CONCLUSIONS: The incidence of extravasation is as low as 0.08%, using our in-house chemotherapy guideline. Extravasation from implanted ports tends to be severe.

The essential role of Mkx in periodontal ligament on the metabolism of alveolar bone and cementum.

Introduction: The periodontium is a connective tissue which consists of periodontal ligament, alveolar bone, cementum and gingiva. Periodontal ligament (PDL) is a specialized connective tissue that connects the cementum - coating the surface of the tooth - to the alveolar bone. Mohawk homeobox (Mkx) is a transcription factor that is expressed in PDL, that is known to play a vital role in the development and homeostasis of PDL. A detailed functional analysis of Mkx in the periodontal ligament for alveolar bone and cementum metabolism has not yet been conducted. Materials and methods: Alveolar bone height, bone mineral density (BMD) and bone volume fractions (Bone volume/Total volume: BV/TV) were measured and analyzed using micro-computed tomography (Micro-CT) and 3DBon on 7-week-old male wild-type (WT) (Mkx+/+) (n = 10) and Mkx-knockout (Mkx-/-) (n = 6) rats. Hematoxylin and Eosin (H&E), tartrate-resistant acid phosphatase (TRAP), alkaline phosphatase (ALP) and Masson Trichrome staining were performed on 5, 6, and 7-week-old Mkx+/+ and Mkx-/- rats. Cementum surface area and the number of TRAP-positive osteoclasts/mm were quantified, measured, and compared for 5,6 and 7-week-old Mkx+/+ and Mkx-/- rats (n = 3 each). Results: The level of alveolar bone height was significantly higher in Mkx-/- rats than in Mkx+/+ rats. On the other hand, there was significantly less BMD in Mkx-/- alveolar bone. A significant increase in cellular cementum could be observed as early as 5 weeks in Mkx-/- rats when compared with Mkx+/+ rats of the same age. More TRAP-positive osteoclasts were observed in Mkx-/- rats. Conclusion: Our findings further reveal the essential roles of Mkx in the homeostasis of the periodontal tissue. Mkx was found to contribute to bone and cementum metabolism and may be essential to the prevention of diseases such as periodontitis, and could show potential in regenerative treatments.

One-step generation of mice with gene editing by Tol2 transposon-dependent gRNA delivery.

Conditional knockout mice are valuable tools for examining the functions of targeted genes in a time- and space-specific manner. Here, we generated gene-edited mice by using the Tol2 transposon to introduce guide RNA (gRNA) into fertilized eggs obtained by crossing LSL (loxP-stop-loxP)-CRISPR-associated 9 (Cas9) mice, which express Cas9 in a Cre-dependent manner, with CAG-CreER mice. Transposase mRNA and plasmid DNA, which contained a gRNA sequence for the gene encoding tyrosinase flanked by the transposase recognition sequence, were injected together into fertilized eggs. As a result, the transcribed gRNA cleaved the target genome in a Cas9-dependent manner. Using this method, it is possible to generate conditional genome-edited mice more easily in a shorter period of time.

The mechanosensitive ion channel PIEZO1 is expressed in tendons and regulates physical performance.

How mechanical stress affects physical performance via tendons is not fully understood. Piezo1 is a mechanosensitive ion channel, and E756del PIEZO1 was recently found as a gain-of-function variant that is common in individuals of African descent. We generated tendon-specific knock-in mice using R2482H Piezo1, a mouse gain-of-function variant, and found that they had higher jumping abilities and faster running speeds than wild-type or muscle-specific knock-in mice. These phenotypes were associated with enhanced tendon anabolism via an increase in tendon-specific transcription factors, Mohawk and Scleraxis, but there was no evidence of changes in muscle. Biomechanical analysis showed that the tendons of R2482H Piezo1 mice were more compliant and stored more elastic energy, consistent with the enhancement of jumping ability. These phenotypes were replicated in mice with tendon-specific R2482H Piezo1 replacement after tendon maturation, indicating that PIEZO1 could be a target for promoting physical performance by enhancing function in mature tendon. The frequency of E756del PIEZO1 was higher in sprinters than in population-matched nonathletic controls in a small Jamaican cohort, suggesting a similar function in humans. Together, this human and mouse genetic and physiological evidence revealed a critical function of tendons in physical performance, which is tightly and robustly regulated by PIEZO1 in tenocytes.

Generation of a tendon-like tissue from human iPS cells.

Tendons and ligaments are essential connective tissues that connect the muscle and bone. Their recovery from injuries is known to be poor, highlighting the crucial need for an effective therapy. A few reports have described the development of artificial ligaments with sufficient strength from human cells. In this study, we successfully generated a tendon-like tissue (bio-tendon) using human induced pluripotent stem cells (iPSCs). We first differentiated human iPSCs into mesenchymal stem cells (iPSC-MSCs) and transfected them with Mohawk (Mkx) to obtain Mkx-iPSC-MSCs, which were applied to a newly designed chamber with a mechanical stretch incubation system. The embedded Mkx-iPSC-MSCs created bio-tendons and exhibited an aligned extracellular matrix structure. Transplantation of the bio-tendons into a mouse Achilles tendon rupture model showed host-derived cell infiltration with improved histological score and biomechanical properties. Taken together, the bio-tendon generated in this study has potential clinical applications for tendon/ligament-related injuries and diseases.

MicroRNA Expression Profiling, Target Identification, and Validation in Chondrocytes.

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.

Identification of chemical compounds regulating PD-L1 by introducing HiBiT-tagged cells.

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 1280 chemical compounds from the Library of Pharmacologically Active Compounds and identified microtubule polymerization inhibitors and thapsigargin as PD-L1 upregulators and a p97 inhibitor as a PD-L1 downregulator.

Both microRNA-455-5p and -3p repress hypoxia-inducible factor-2α expression and coordinately regulate cartilage homeostasis.

Osteoarthritis (OA), the most common aging-related joint disease, is caused by an imbalance between extracellular matrix synthesis and degradation. Here, we discover that both strands of microRNA-455 (miR-455), -5p and -3p, are up-regulated by Sox9, an essential transcription factor for cartilage differentiation and function. Both miR-455-5p and -3p are highly expressed in human chondrocytes from normal articular cartilage and in mouse primary chondrocytes. We generate miR-455 knockout mice, and find that cartilage degeneration mimicking OA and elevated expression of cartilage degeneration-related genes are observed at 6-months-old. Using a cell-based miRNA target screening system, we identify hypoxia-inducible factor-2α (HIF-2α), a catabolic factor for cartilage homeostasis, as a direct target of both miR-455-5p and -3p. In addition, overexpression of both miR-455-5p and -3p protect cartilage degeneration in a mouse OA model, demonstrating their potential therapeutic value. Furthermore, knockdown of HIF-2α in 6-month-old miR-455 knockout cartilage rescues the elevated expression of cartilage degeneration-related genes. These data demonstrate that both strands of a miRNA target the same gene to regulate articular cartilage homeostasis.

Transcriptional, epigenetic and microRNA regulation of growth plate.

Endochondral ossification is a critical event in bone formation, particularly in long shaft bones. Many cellular differentiation processes work in concert to facilitate the generation of cartilage primordium to formation of trabecular structures, all of which occur within the growth plate. Previous studies have revealed that the growth plate is tightly regulated by various transcription factors, epigenetic systems, and microRNAs. Hence, understanding these mechanisms that regulate the growth plate is crucial to furthering the current understanding on skeletal diseases, and in formulating effective treatment strategies. In this review, we focus on describing the function and mechanisms of the transcription factors, epigenetic systems, and microRNAs known to regulate the growth plate.

A case of COVID-19 with the atypical CT finding.

COVID-19 usually demonstrates the specific pattern of chest CT findings (GGO, inverted-halo sign, etc). However, some COVID-19 cases show atypical CT findings. Physicians should make comprehensive judgments.

In vitro Neo-Genesis of Tendon/Ligament-Like Tissue by Combination of Mohawk and a Three-Dimensional Cyclic Mechanical Stretch Culture System.

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.

Transcriptome analysis of sevoflurane exposure effects at the different brain regions.

BACKGROUNDS: Sevoflurane is a most frequently used volatile anesthetics, but its molecular mechanisms of action remain unclear. We hypothesized that specific genes play regulatory roles in brain exposed to sevoflurane. Thus, we aimed to evaluate the effects of sevoflurane inhalation and identify potential regulatory genes by RNA-seq analysis. METHODS: Eight-week old mice were exposed to sevoflurane. RNA from medial prefrontal cortex, striatum, hypothalamus, and hippocampus were analysed using RNA-seq. Differently expressed genes were extracted and their gene ontology terms were analysed using Metascape. These our anesthetized mouse data and the transcriptome array data of the cerebral cortex of sleeping mice were compared. Finally, the activities of transcription factors were evaluated using a weighted parametric gene set analysis (wPGSA). JASPAR was used to confirm the existence of binding motifs in the upstream sequences of the differently expressed genes. RESULTS: The gene ontology term enrichment analysis result suggests that sevoflurane inhalation upregulated angiogenesis and downregulated neural differentiation in each region of brain. The comparison with the brains of sleeping mice showed that the gene expression changes were specific to anesthetized mice. Focusing on individual genes, sevoflurane induced Klf4 upregulation in all sampled parts of brain. wPGSA supported the function of KLF4 as a transcription factor, and KLF4-binding motifs were present in many regulatory regions of the differentially expressed genes. CONCLUSIONS: Klf4 was upregulated by sevoflurane inhalation in the mouse brain. The roles of KLF4 might be key to elucidating the mechanisms of sevoflurane induced functional modification in the brain.