Differential Regulation of Intracisternally Injected Angiotensin II-Induced Mechanical Allodynia and Thermal Hyperalgesia in Rats.

The present study examined the underlying mechanisms of mechanical allodynia and thermal hyperalgesia induced by the intracisternal injection of angiotensin (Ang) II. Intracisternal Ang II injection decreased the air puff threshold and head withdrawal latency. To determine the operative receptors for each distinct type of pain behavior, we intracisternally injected Ang II receptor antagonists 2 h after Ang II injection. Losartan, an Ang II type 1 receptor (AT1R) antagonist, alleviated mechanical allodynia. Conversely, PD123319, an Ang II type 1 receptor (AT2R) antagonist, blocked only thermal hyperalgesia. Immunofluorescence analyses revealed the co-localization of AT1R with the astrocyte marker GFAP in the trigeminal subnucleus caudalis and co-localization of AT2R with CGRP-positive neurons in the trigeminal ganglion. Intracisternal pretreatment with minocycline, a microglial inhibitor, did not affect Ang II-induced mechanical allodynia, whereas L-α-aminoadipate, an astrocyte inhibitor, significantly inhibited Ang II-induced mechanical allodynia. Furthermore, subcutaneous pretreatment with botulinum toxin type A significantly alleviated Ang II-induced thermal hyperalgesia, but not Ang II-induced mechanical allodynia. These results indicate that central Ang II-induced nociception is differentially regulated by AT1R and AT2R. Thus, distinct therapeutic targets must be regulated to overcome pain symptoms caused by multiple underlying mechanisms.

Single-cell RNA sequencing reveals heterogeneity of adipose tissue-derived mesenchymal stem cells under chondrogenic induction.

This study investigated how adipose tissue-derived mesenchymal stem cells (AT-MSCs) respond to chondrogenic induction using droplet-based single-cell RNA sequencing (scRNA-seq). We analyzed 37,219 high-quality transcripts from control cells and cells induced for 1 week (1W) and 2 weeks (2W). Four distinct cell clusters (0-3), undetectable by bulk analysis, exhibited varying proportions. Cluster 1 dominated in control and 1W cells, whereas cluster 3, 2, and 0 exclusively dominated in control, 1W, and 2W cells, respectively. Furthermore, heterogeneous chondrogenic markers expression within clusters emerged. Gene ontology (GO) enrichment analysis of differentially expressed genes unveiled cluster-specific variations in key biological processes (BP): (1) Cluster 1 exhibited upregulation of GO-BP terms related to ribosome biogenesis and translational control, crucial for maintaining stem cell properties and homeostasis; (2) Additionally, cluster 1 showed upregulation of GO-BP terms associated with mitochondrial oxidative metabolism; (3) Cluster 3 displayed upregulation of GO-BP terms related to cell proliferation; (4) Clusters 0 and 2 demonstrated similar upregulation of GO-BP terms linked to collagen fibril organization and supramolecular fiber organization. However, only cluster 0 showed a significant decrease in GO-BP terms related to ribosome production, implying a potential correlation between ribosome regulation and the differentiation stages of AT-MSCs. Overall, our findings highlight heterogeneous cell clusters with varying balances between proliferation and differentiation before and after chondrogenic stimulation. This provides enhanced insights into the single-cell dynamics of AT-MCSs during chondrogenic differentiation.

The RNA ligation method using modified splint DNAs significantly improves the efficiency of circular RNA synthesis.

Circular RNA (circRNA) is a non-coding RNA with a covalently closed loop structure and usually more stable than messenger RNA (mRNA). However, coding sequences (CDSs) following an internal ribosome entry site (IRES) in circRNAs can be translated, and this property has been recently utilized to produce proteins as novel therapeutic tools. However, it is difficult to produce large proteins from circRNAs because of the low circularization efficiency of lengthy RNAs. In this study, we report that we successfully synthesized circRNAs with the splint DNA ligation method using RNA ligase 1 and the splint DNAs, which contain complementary sequences to both ends of precursor linear RNAs. This method results in more efficient circularization than the conventional enzymatic method that does not use the splint DNAs, easily generating circRNAs that express relatively large proteins, including IgG heavy and light chains. Longer splint DNA (42 nucleotide) is more effective in circularization. Also, the use of splint DNAs with an adenine analog, 2,6-diaminopurine (DAP), increase the circularization efficiency presumably by strengthening the interaction between the splint DNAs and the precursor RNAs. The splint DNA ligation method requires 5 times more splint DNA than the precursor RNA to efficiently produce circRNAs, but our modified splint DNA ligation method can produce circRNAs using the amount of splint DNA which is equal to that of the precursor RNA. Our modified splint DNA ligation method will help develop novel therapeutic tools using circRNAs, to treat various diseases and to develop human and veterinary vaccines.

In Vivo Stem Cell Imaging Principles and Applications.

Stem cells are the foundational cells for every organ and tissue in our body. Cell-based therapeutics using stem cells in regenerative medicine have received attracting attention as a possible treatment for various diseases caused by congenital defects. Stem cells such as induced pluripotent stem cells (iPSCs) as well as embryonic stem cells (ESCs), mesenchymal stem cells (MSCs), and neuroprogenitors stem cells (NSCs) have recently been studied in various ways as a cell-based therapeutic agent. When various stem cells are transplanted into a living body, they can differentiate and perform complex functions. For stem cell transplantation, it is essential to determine the suitability of the stem cell-based treatment by evaluating the origin of stem, the route of administration, in vivo bio-distribution, transplanted cell survival, function, and mobility. Currently, these various stem cells are being imaged in vivo through various molecular imaging methods. Various imaging modalities such as optical imaging, magnetic resonance imaging (MRI), ultrasound (US), positron emission tomography (PET), and single-photon emission computed tomography (SPECT) have been introduced for the application of various stem cell imaging. In this review, we discuss the principles and recent advances of in vivo molecular imaging for application of stem cell research.

Emerging roles of tRNA-derived small RNAs in cancer biology.

Transfer RNAs (tRNAs) play an essential role in mRNA translation by delivering amino acids to growing polypeptide chains. Recent data demonstrate that tRNAs can be cleaved by ribonucleases, and the resultant cleavage products, tRNA-derived small RNAs (tsRNAs), have crucial roles in physiological and pathological conditions. They are classified into more than six types according to their size and cleavage positions. Since the initial discovery of the physiological functions of tsRNAs more than a decade ago, accumulating data have demonstrated that tsRNAs play critical roles in gene regulation and tumorigenesis. These tRNA-derived molecules have various regulatory functions at the transcriptional, post-transcriptional, and translational levels. More than a hundred types of modifications are found on tRNAs, affecting the biogenesis, stability, function, and biochemical properties of tsRNA. Both oncogenic and tumor suppressor functions have been reported for tsRNAs, which play important roles in the development and progression of various cancers. Abnormal expression patterns and modification of tsRNAs are associated with various diseases, including cancer and neurological disorders. In this review, we will describe the biogenesis, versatile gene regulation mechanisms, and modification-mediated regulation mechanisms of tsRNA as well as the expression patterns and potential therapeutic roles of tsRNAs in various cancers.

Accelerometer-measured stepping cadence patterns in Korean adults: an analysis of data from the 2014-2015 Korea National Health and Nutrition Examination Survey.

OBJECTIVES: The aim of this study was to identify the stepping cadence patterns in Korean adults by using objectively measured accelerometer data to analyze the time spent in each cadence category by sex and age. METHODS: During the 2014-2015 Korean National Health and Nutrition Examination Survey, 1,703 males and females aged 19-64 years provided at least 1 valid day of data (wearing an accelerometer ≥ 10 hr/d). The mean cumulative time and percentage per day in 8 cadence categories (0, 1-19, 20-29, 30-59, 60-79, 80-99, 100-119, and ≥ 120 steps/min) by sex and age group were calculated. RESULTS: Cumulative time and percentage per day decreased across the incremental cadence categories. Participants spent 360.08± 2.56 min/d in the non-movement cadence category and 361.50± 2.28 min/d in the incidental movement cadence category. However, they spent only about 18.1 min/d (2.1%) at ≥ 100 steps/min. Males spent significantly more time in the cadence categories of sporadic movement, purposeful steps, slow walking, and medium walking, but the other categories, except for brisk walking, had higher values in females (p< 0.001). The older age group spent less time in non-movement cadence categories, and the youngest and oldest groups spent more time at a higher cadence (≥ 100 steps/min) than the other age groups. Similar patterns were found in a subgroup analysis by sex. CONCLUSIONS: Korean adults spent most of their time at a low cadence and only a few minutes at a high cadence (≥100 steps/min); this trend was consistent across sex and age groups.

The 3'tsRNAs are aminoacylated: Implications for their biogenesis.

Emerging evidence indicates that tRNA-derived small RNAs (tsRNAs) are involved in fine-tuning gene expression and become dysregulated in various cancers. We recently showed that the 22nt LeuCAG3´tsRNA from the 3´ end of tRNALeu is required for efficient translation of a ribosomal protein mRNA and ribosome biogenesis. Inactivation of this 3´tsRNA induced apoptosis in rapidly dividing cells and suppressed the growth of a patient-derived orthotopic hepatocellular carcinoma in mice. The mechanism involved in the generation of the 3´tsRNAs remains elusive and it is unclear if the 3´-ends of 3´tsRNAs are aminoacylated. Here we report an enzymatic method utilizing exonuclease T to determine the 3´charging status of tRNAs and tsRNAs. Our results showed that the LeuCAG3´tsRNA, and two other 3´tsRNAs are fully aminoacylated. When the leucyl-tRNA synthetase (LARS1) was inhibited, there was no change in the total tRNALeu concentration but a reduction in both the charged tRNALeu and LeuCAG3´tsRNA, suggesting the 3´tsRNAs are fully charged and originated solely from the charged mature tRNA. Altering LARS1 expression or the expression of various tRNALeu mutants were also shown to affect the generation of the LeuCAG3´tsRNA further suggesting they are created in a highly regulated process. The fact that the 3´tsRNAs are aminoacylated and their production is regulated provides additional insights into their importance in post-transcriptional gene regulation that includes coordinating the production of the protein synthetic machinery.

tRNA-Derived Small RNAs: Novel Epigenetic Regulators.

An epigenetic change is a heritable genetic alteration that does not involve any nucleotide changes. While the methylation of specific DNA regions such as CpG islands or histone modifications, including acetylation or methylation, have been investigated in detail, the role of small RNAs in epigenetic regulation is largely unknown. Among the many types of small RNAs, tRNA-derived small RNAs (tsRNAs) represent a class of noncoding small RNAs with multiple roles in diverse physiological processes, including neovascularization, sperm maturation, immune modulation, and stress response. Regarding these roles, several pioneering studies have revealed that dysregulated tsRNAs are associated with human diseases, such as systemic lupus, neurological disorder, metabolic disorder, and cancer. Moreover, recent findings suggest that tsRNAs regulate the expression of critical genes linked with these diseases by a variety of mechanisms, including epigenetic regulation. In this review, we will describe different classes of tsRNAs based on their biogenesis and will focus on their role in epigenetic regulation.

Transfer RNA-Derived Small RNAs: Another Layer of Gene Regulation and Novel Targets for Disease Therapeutics.

Decades after identification as essential for protein synthesis, transfer RNAs (tRNAs) have been implicated in various cellular processes beyond translation. tRNA-derived small RNAs (tsRNAs), referred to as tRNA-derived fragments (tRFs) or tRNA-derived, stress-induced RNAs (tiRNAs), are produced by cleavage at different sites from mature or pre-tRNAs. They are classified into six major types representing potentially thousands of unique sequences and have been implicated to play a wide variety of regulatory roles in maintaining normal homeostasis, cancer cell viability, tumorigenesis, ribosome biogenesis, chromatin remodeling, translational regulation, intergenerational inheritance, retrotransposon regulation, and viral replication. However, the detailed mechanisms governing these processes remain unknown. Aberrant expression of tsRNAs is found in various human disease conditions, suggesting that a further understanding of the regulatory role of tsRNAs will assist in identifying novel biomarkers, potential therapeutic targets, and gene-regulatory tools. Here, we highlight the classification, biogenesis, and biological role of tsRNAs in regulatory mechanisms of normal and disease states.

A tRNA-Derived Small RNA Regulates Ribosomal Protein S28 Protein Levels after Translation Initiation in Humans and Mice.

tRNA-derived small RNAs (tsRNAs) have been implicated in many cellular processes, yet the detailed mechanisms are not well defined. We previously found that the 3' end of Leu-CAG tRNA-derived small RNA (LeuCAG3'tsRNA) regulates ribosome biogenesis in humans by maintaining ribosomal protein S28 (RPS28) levels. The tsRNA binds to coding (CDS) and non-coding 3' UTR sequence in the RPS28 mRNA, altering its secondary structure and enhancing its translation. Here we report that the functional 3' UTR target site is present in primates while the CDS target site is present in many vertebrates. We establish that this tsRNA also regulates mouse Rps28 translation by interacting with the CDS target site. We further establish that the change in mRNA translation occurred at a post-initiation step in both species. Overall, our results suggest that LeuCAG3'tsRNA might maintain ribosome biogenesis through a conserved gene regulatory mechanism in vertebrates.

Transfer RNA-Derived Small Non-Coding RNA: Dual Regulator of Protein Synthesis.

Transfer RNA-derived small RNAs (tsRNAs) play a role in various cellular processes. Accumulating evidence has revealed that tsRNAs are deeply implicated in human diseases, such as various cancers and neurological disorders, suggesting that tsRNAs should be investigated to develop novel therapeutic intervention. tsRNAs provide more complexity to the physiological role of transfer RNAs by repressing or activating protein synthesis with distinct mechanisms. Here, we highlight the detailed mechanism of tsRNA-mediated dual regulation in protein synthesis and discuss the necessity of novel sequencing technology to learn more about tsRNAs.

miR-122 removal in the liver activates imprinted microRNAs and enables more effective microRNA-mediated gene repression.

miR-122 is a highly expressed liver microRNA that is activated perinatally and aids in regulating cholesterol metabolism and promoting terminal differentiation of hepatocytes. Disrupting expression of miR-122 can re-activate embryo-expressed adult-silenced genes, ultimately leading to the development of hepatocellular carcinoma (HCC). Here we interrogate the liver transcriptome at various time points after genomic excision of miR-122 to determine the cellular consequences leading to oncogenesis. Loss of miR-122 leads to specific and progressive increases in expression of imprinted clusters of microRNAs and mRNA transcripts at the Igf2 and Dlk1-Dio3 loci that could be curbed by re-introduction of exogenous miR-122. mRNA targets of other abundant hepatic microRNAs are functionally repressed leading to widespread hepatic transcriptional de-regulation. Together, this reveals a transcriptomic framework for the hepatic response to loss of miR-122 and the outcome on other microRNAs and their cognate gene targets.

Bidirectional Transcriptome Analysis of Rat Bone Marrow-Derived Mesenchymal Stem Cells and Activated Microglia in an In Vitro Coculture System.

Microglia contribute to the regulation of neuroinflammation and play an important role in the pathogenesis of brain diseases. Thus, regulation of neuroinflammation triggered by activated microglia in brain diseases has become a promising curative strategy. Bone marrow-derived mesenchymal stem cells (BM-MSCs) have been shown to have therapeutic effects, resulting from the regulation of inflammatory conditions in the brain. In this study, we investigated differential gene expression in rat BM-MSCs (rBM-MSCs) that were cocultured with lipopolysaccharide- (LPS-) stimulated primary rat microglia using microarray analysis and evaluated the functional relationships through Ingenuity Pathway Analysis (IPA). We also evaluated the effects of rBM-MSC on LPS-stimulated microglia using a reverse coculture system and the same conditions of the transcriptomic analysis. In the transcriptome of rBM-MSCs, 67 genes were differentially expressed, which were highly related with migration of cells, compared to control. The prediction of the gene network using IPA and experimental validation showed that LPS-stimulated primary rat microglia increase the migration of rBM-MSCs. Reversely, expression patterns of the transcriptome in LPS-stimulated primary rat microglia were changed when cocultured with rBM-MSCs. Our results showed that 65 genes were changed, which were highly related with inflammatory response, compared to absence of rBM-MSCs. In the same way with the aforementioned, the prediction of the gene network and experimental validation showed that rBM-MSCs decrease the inflammatory response of LPS-stimulated primary rat microglia. Our data indicate that LPS-stimulated microglia increase the migration of rBM-MSCs and that rBM-MSCs reduce the inflammatory activity in LPS-stimulated microglia. The results of this study show complex mechanisms underlying the interaction between rBM-MSCs and activated microglia and may be helpful for the development of stem cell-based strategies for brain diseases.

A transfer-RNA-derived small RNA regulates ribosome biogenesis.

Transfer-RNA-derived small RNAs (tsRNAs; also called tRNA-derived fragments) are an abundant class of small non-coding RNAs whose biological roles are not well understood. Here we show that inhibition of a specific tsRNA, LeuCAG3'tsRNA, induces apoptosis in rapidly dividing cells in vitro and in a patient-derived orthotopic hepatocellular carcinoma model in mice. This tsRNA binds at least two ribosomal protein mRNAs (RPS28 and RPS15) to enhance their translation. A decrease in translation of RPS28 mRNA blocks pre-18S ribosomal RNA processing, resulting in a reduction in the number of 40S ribosomal subunits. These data establish a post-transcriptional mechanism that can fine-tune gene expression during different physiological states and provide a potential new target for treating cancer.

Increased precursor microRNA-21 following status epilepticus can compete with mature microRNA-21 to alter translation.

MicroRNA-21 (miR-21) is consistently up-regulated in various neurological disorders, including epilepsy. Here, we show that the biogenesis of miR-21 is altered following pilocarpine-induced status epilepticus (SE) with an increase in precursor miR-21 (pre-miR-21) in rats. We demonstrate that pre-miR-21 has an energetically favorable site overlapping with the miR-21 binding site and competes with mature miR-21 for binding in the 3'UTR of TGFBR2 mRNA, but not NT-3 mRNA in vitro. This binding competition influences miR-21-mediated repression in vitro and correlates with the increase in TGFBR2 and decrease in NT-3 following SE. Polysome profiling reveals co-localization of pre-miR-21 in the ribosome fraction with translating mRNAs in U-87 cells. The current work suggests that pre-miR-21 may post-transcriptionally counteract miR-21-mediated suppression following SE and could potentially lead to prolonged TGF-ß receptor expression impacting epileptogenesis. The study further supports that the ratio of the pre to mature miRNA may be important in determining the regulatory effects of a miRNA gene.

Peptoniphilus mikwangii sp. nov., isolated from a clinical specimen of human origin.

A novel Gram-positive, anaerobic, and coccus-shaped bacterium, designated as strain ChDC B134(T), was isolated from a human postoperative infectious lesion in the right maxilla between the lateral incisor and canine and was characterized by polyphasic taxonomic analysis. 16S rRNA gene sequence analysis revealed that the strain ChDC B134(T) belonged to the genus Peptoniphilus, as it showed sequence similarities to Peptoniphilus indolicus KCTC 15023(T) (94.0 %) and Peptoniphilus asaccharolyticus KCTC 3321(T) (93.8 %). The prevalent fatty acids of of strain ChDC B134(T) were C16:0 (20.3 %), C18:1 cis 9 (34.3 %), and C18:0 (13.2 %). The DNA G+C content was 30.9 mol%. The cell wall diamino acid was D-ornithine, which is a property shared by other reference type strains of the genus Peptoniphilus. Based on the results of phenotypic, chemotaxonomic, and phylogenetic analysis, strain ChDC B134(T) (=KCOM 1628(T) = KCTC 15227(T) = JCM 30223(T)) should be classified as the type strain of a novel species of genus Peptoniphilus, for which the name Peptoniphilus mikwangii sp. nov. is proposed.

Five-year retrospective radiographic follow-up study of dental implants with sandblasting with large grit, and acid etching-treated surfaces.

OBJECTIVES: The purpose of this study is to evaluate five-year radiographic follow-up results of the Korean sandblasting with large grit, and acid etching (SLA)-treated implant system. MATERIALS AND METHODS: The subjects of the study are 54 patients who have been followed-up to date, of the patients who underwent implant surgery from May 1, 2009 to April 30, 2011. In all, 176 implant placements were performed. Radiographs were taken before the first surgery, immediately after the first and second surgeries, immediately and six months after the final prosthesis installation, and every year after that. Bone loss was evaluated by the method suggested by Romanos and Nentwig. RESULTS: A total of 176 implant placements were performed-122 in men and 54 in women. These patients have been followed-up for an average of 4.9 years. In terms of prosthetic appliances, there were 156 bridges and 20 single prostheses. Nine implants installed in the maxillary molar area, three in the mandibular molar area and two in the maxillary premolar area were included in group M, with bone loss less than 2 mm at the crestal aspect of the implant. Of these, eight implants were single prostheses. In all, six implants failed-four in the mandible and two in the maxilla. All of these failures occurred in single-implant cases. The implant survival rate was 98.1% on the maxilla and 94.3% on the mandible, with an overall survival of 96.6%. CONCLUSION: Within the limitations of this study, implants with the SLA surface have a very superior survival rate in relatively poor bone environments such as the maxilla.

Draft Genome Sequence of the Novel Peptoniphilus sp. Strain ChDC B134, Isolated from a Human Periapical Abscess Lesion.

The genus Peptoniphilus comprises butyrate-producing, nonsaccharolytic species that use peptone and amino acids as major energy sources. The novel Peptoniphilus sp. strain ChDC B134 (=KCOM 1628) was isolated from a human periapical abscess lesion. Here, we report the draft genome sequence of the strain.

Postoperative maxillary cyst after maxillary sinus augmentation.

The posterior edentulous maxilla is a critical anatomic region for dental implant therapy. Because of severe alveolar bone resorption and maxillary sinus pneumatization, low bone volume is often presented clinically. Although maxillary sinus augmentation has been developed to promote bone reconstruction and oral rehabilitation, complications have been reported. Possible complications include paranasal sinusitis, loss of the graft, and displacement of an implant into the antrum. In this study, we present an observed rare complication of maxillary sinus augmentation, a postoperative maxillary cyst that occurred 10 years after treatment.

Antibody-mediated osseous regeneration: the early events in the healing response.

Bone engineering strategies often exploit modulation of the extracellular environment, including delivery of cell and growth factors to repair and regenerate damaged tissues. During bone healing, the expression of endogenous bone morphogenetic proteins is an essential component of the healing response. However, in some situations, the inherent reparative capacity available in the local microenvironment is exceeded by the requirements of the defects. We have recently reported on a novel strategy, that exploits the specificity of antibodies to capture and make available endogenous osteogenic growth factors, referred to as "antibody-mediated osseous regeneration" (AMOR). The objective of the present study was to identify some of the cellular and molecular events involved in AMOR in an effort to begin to elucidate the mechanism of AMOR. The rat critical-sized calvarial defect model was used, where anti-bone morphogenetic protein (BMP)-2 monoclonal antibody (mAb), isotype-control mAb, or recombinant human (rh)BMP-2 were immobilized on absorbable collagen calvarial sponge (ACS) by adsorption, and then implanted into calvarial defects. The results demonstrated persistence of implanted mAbs for short term from 1 to 2 weeks after implantation. Increased cell infiltration was found in defects treated with anti-BMP-2 mAb. Examination of proteins on ACS scaffolds retrieved from defect sites demonstration increased levels of BMP-2, BMP-4, and BMP-7 proteins in sites implanted with anti-BMP-2 mAb. Moreover, BMP-2, BMP-4, and BMP-7 gene expression levels were increased in sites implanted with anti-BMP-2 mAb. Micro-computed tomography and histological analysis demonstrated that the bone within calvarial defects was fully regenerated in sites implanted with either anti-BMP-2 mAb or rhBMP-2. However, rhBMP-2-regenerated bone exhibited aberrant histomorphology with dystrophic calcification and invasion of subjacent areas. Altogether, the results revealed evidence for anti-BMP-2 mAbs to form an immune complex with BMP-2, BMP-4, and BMP-7, and bind to cells to mediate osteogenesis bone regeneration in vivo. This approach suggests a significant role for antibodies in regenerative orthopedic medicine.