Host modulation therapy for improving the osseointegration of dental implants under bone healing-suppressed conditions: a preclinical rodent-model experiment.

PURPOSE: Placing dental implants in areas with low bone density or in conditions where bone healing is suppressed is challenging for clinicians. An experiment using a rodent model was performed with the aim of determining the efficacy of host modulation by increasing the systemic level of cholesterol sulfate (CS) using Irosustat in the context of the bone healing process around dental implants. METHODS: In 16 ovariectomised female Sprague-Dawley rats, 2 implant fixtures were placed in the tibial bones (1 fixture on each side). At 1 week after surgery, the high-CS group (n=8) received Irosustat-mixed feed, while the control group (n=8) was fed conventionally. Block specimens were obtained at 5 weeks post-surgery for histologic analysis and the data were evaluated statistically (P<0.05). RESULTS: Unlike the high-CS group, half of the specimens in the control group demonstrated severe bone resorption along with a periosteal reaction in the cortex. The mean percentages of bone-to-implant contact (21.5%) and bone density (28.1%) near the implant surface were significantly higher in the high-CS group than in the control group (P<0.05), as was the number of Haversian canals (by 5.3). CONCLUSIONS: Host modulation by increasing the CS level may enhance the osseointegration of dental implants placed under conditions of impaired bone healing.

Epithelial plasticity enhances regeneration of committed taste receptor cells following nerve injury.

Taste receptor cells are taste bud epithelial cells that are dependent upon the innervating nerve for continuous renewal and are maintained by resident tissue stem/progenitor cells. Transection of the innervating nerve causes degeneration of taste buds and taste receptor cells. However, a subset of the taste receptor cells is maintained without nerve contact after glossopharyngeal nerve transection in the circumvallate papilla in adult mice. Here, we revealed that injury caused by glossopharyngeal nerve transection triggers the remaining differentiated K8-positive taste receptor cells to dedifferentiate and acquire transient progenitor cell-like states during regeneration. Dedifferentiated taste receptor cells proliferate, express progenitor cell markers (K14, Sox2, PCNA) and form organoids in vitro. These data indicate that differentiated taste receptor cells can enter the cell cycle, acquire stemness, and participate in taste bud regeneration. We propose that dedifferentiated taste receptor cells in combination with stem/progenitor cells enhance the regeneration of taste buds following nerve injury.

Microtubule-associated protein 1 A and tubby act independently in regulating the localization of stereocilin to the tips of inner ear hair cell stereocilia.

Tubby mice exhibit hearing impairment due to the loss of stereocilin from the tip regions that connect the tallest stereocilia of the outer hair cells (OHCs) to the tectorial membrane. Stereocilin is an essential stereociliary protein in the OHCs, the mutation of which in humans causes autosomal recessive non-syndromic deafness. Map1a is a modifier of tubby hearing (moth1), and its wild-type allele, rather than the moth1 allele from the C57BL/6 J strain, restores stereocilin localization to the stereocilia and rescues the hearing impairment of tubby mice. The mechanism by which MAP1A accomplishes this is unclear, partly due to ambiguity regarding whether the tubby mutation is a true null. We therefore generated Tub-null (Tub-/-) mice by deleting exon 3 and found that they exhibit hearing impairment like that of tubby mice, suggesting the tubby mutation is a loss-of-function mutation with regard to hearing. When we crossed Tub-/- mice with AKR mice that have wild-type Map1a alleles, we found that wild-type MAP1A restores stereocilin localization to the tips of stereocilia and rescues hearing impairment. These data suggest MAP1A does not require interaction with tubby protein in maintaining stereocilin at the tips of stereocilia and that OHCs use two independent molecules-MAP1A and tubby-to doubly ensure proper stereocilin localization.

Fine-tuning of epithelial taste bud organoid to promote functional recapitulation of taste reactivity.

Taste stem/progenitor cells from posterior mouse tongues have been used to generate taste bud organoids. However, the inaccessible location of taste receptor cells is observed in conventional organoids. In this study, we established a suspension-culture method to fine-tune taste bud organoids by apicobasal polarity alteration to form the accessible localization of taste receptor cells. Compared to conventional Matrigel-embedded organoids, suspension-cultured organoids showed comparable differentiation and renewal rates to those of taste buds in vivo and exhibited functional taste receptor cells and cycling progenitor cells. Accessible taste receptor cells enabled the direct application of calcium imaging to evaluate the taste response. Moreover, suspension-cultured organoids can be genetically altered. Suspension-cultured taste bud organoids harmoniously integrated with the recipient lingual epithelium, maintaining the taste receptor cells and gustatory innervation capacity. We propose that suspension-cultured organoids may provide an efficient model for taste research, including taste bud development, regeneration, and transplantation.

Single-Cell RNA Sequencing Analysis of Human Dental Pulp Stem Cell and Human Periodontal Ligament Stem Cell.

INTRODUCTION: Many studies have investigated the transcriptome profiles of dental stem cells for regenerative medicine. However, such studies use bulk RNA and do not consider cell-level heterogeneity. Here, we investigated the characteristics and heterogeneity of human dental pulp stem cells (hDPSCs) and human periodontal ligament stem cells (hPDLSCs) at the single-cell level and examined the differences between them. METHODS: hDPSCs and hPDLSCs were obtained from caries-free premolars (n = 2). Single-cell RNA sequencing of hDPSCs and hPDLSCs was performed using a Single Emulsion Systems (Dolomite Microfluidics, Royston, UK) droplet microfluidic device and the Illumina NextSeq550 system (Illumina, San Diego, CA). Data alignment was performed using STAR v2.4.0, and further analysis was performed using the Seurat, singleR, and clusterProfiler packages in R (R Foundation for Statistical Computing, Vienna, Austria). RESULTS: hDPSCs and hPDLSCs were separated into 3 clusters. hDPSCs mainly exhibited osteogenic and neurogenic cell populations. The main populations of hPDLSCs comprised osteogenic and myofibroblastic populations. hPDLSCs showed high "scores" for osteogenic gene expression, whereas hDPSCs had high neurogenic and endogenic scores. CONCLUSIONS: Single-cell RNA sequencing of hDPSCs and hPDLSCs revealed that the genes were expressed in specific clusters. The results of these analyses can be used as reference databases and valuable resources for further research in dental therapeutics.

Whole-Brain Mapping of the Expression Pattern of T1R2, a Subunit Specific to the Sweet Taste Receptor.

Chemosensory receptors are expressed primarily in sensory organs, but their expression elsewhere can permit ligand detection in other contexts that contribute to survival. The ability of sweet taste receptors to detect natural sugars, sugar alcohols, and artificial sweeteners suggests sweet taste receptors are involved in metabolic regulation in both peripheral organs and in the central nervous system. Our limited knowledge of sweet taste receptor expression in the brain, however, has made it difficult to assess their contribution to metabolic regulation. We, therefore, decided to profile the expression pattern of T1R2, a subunit specific to the sweet taste receptor complex, at the whole-brain level. Using T1r2-Cre knock-in mice, we visualized the overall distribution of Cre-labeled cells in the brain. T1r2-Cre is expressed not only in various populations of neurons, but also in glial populations in the circumventricular organs and in vascular structures in the cortex, thalamus, and striatum. Using immunohistochemistry, we found that T1r2 is expressed in hypothalamic neurons expressing neuropeptide Y and proopiomelanocortin in arcuate nucleus. It is also co-expressed with a canonical taste signaling molecule in perivascular cells of the median eminence. Our findings indicate that sweet taste receptors have unidentified functions in the brain and suggest that they may be a novel therapeutic target in the central nervous system.

Differential Roles of Tubby Family Proteins in Ciliary Formation and Trafficking.

Cilia are highly specialized organelles that extend from the cell membrane and function as cellular signaling hubs. Thus, cilia formation and the trafficking of signaling molecules into cilia are essential cellular processes. TULP3 and Tubby (TUB) are members of the tubby-like protein (TULP) family that regulate the ciliary trafficking of G-protein coupled receptors, but the functions of the remaining TULPs (i.e., TULP1 and TULP2) remain unclear. Herein, we explore whether these four structurally similar TULPs share a molecular function in ciliary protein trafficking. We found that TULP3 and TUB, but not TULP1 or TULP2, can rescue the defective cilia formation observed in TULP3-knockout (KO) hTERT RPE-1 cells. TULP3 and TUB also fully rescue the defective ciliary localization of ARL13B, INPP5E, and GPR161 in TULP3 KO RPE-1 cells, while TULP1 and TULP2 only mediate partial rescues. Furthermore, loss of TULP3 results in abnormal IFT140 localization, which can be fully rescued by TUB and partially rescued by TULP1 and TULP2. TUB's capacity for binding IFT-A is essential for its role in cilia formation and ciliary protein trafficking in RPE-1 cells, whereas its capacity for PIP2 binding is required for proper cilia length and IFT140 localization. Finally, chimeric TULP1 containing the IFT-A binding domain of TULP3 fully rescues ciliary protein trafficking, but not cilia formation. Together, these two TULP domains play distinct roles in ciliary protein trafficking but are insufficient for cilia formation in RPE-1 cells. In addition, TULP1 and TULP2 play other unknown molecular roles that should be addressed in the future.

Recent Advances in Understanding Peripheral Taste Decoding I: 2010 to 2020.

Taste sensation is the gatekeeper for direct decisions on feeding behavior and evaluating the quality of food. Nutritious and beneficial substances such as sugars and amino acids are represented by sweet and umami tastes, respectively, whereas noxious substances and toxins by bitter or sour tastes. Essential electrolytes including Na+ and other ions are recognized by the salty taste. Gustatory information is initially generated by taste buds in the oral cavity, projected into the central nervous system, and finally processed to provide input signals for food recognition, regulation of metabolism and physiology, and higher-order brain functions such as learning and memory, emotion, and reward. Therefore, understanding the peripheral taste system is fundamental for the development of technologies to regulate the endocrine system and improve whole-body metabolism. In this review article, we introduce previous widely-accepted views on the physiology and genetics of peripheral taste cells and primary gustatory neurons, and discuss key findings from the past decade that have raised novel questions or solved previously raised questions.

Identification of a novel NPR1 homolog gene, OsNH5N16, which contributes to broad-spectrum resistance in rice.

Over half of the earth's population consumes rice as the primary food crop for dietary calories. However, severe loss of rice yield occurs due to bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) and bakanae disease caused by Fusarium fujikuroi (F. fujikuroi). Therefore, broad-spectrum resistance (BSR) to these pathogens is essential for rice cultivation. The Nonexpressor of Pathogenesis-Related Genes1 (NPR1), which is related to the signal molecule salicylic acid (SA) and the expression of pathogenesis-related (PR) genes, is a key regulator of systemic acquired resistance (SAR). Although five NPR1 homologs (NHs) have been identified in rice thus far, their cellular and biological functions remain largely unexplored. In this study, we identified a novel rice NH gene from Oryza sativa L. cv. Dongjin. The genetic variation of single nucleotide polymorphisms in OsNH5 caused a single amino acid substitution of asparagine for serine at residue 16. OsNH5N16 was mainly located in the nucleus, and its transcription was induced by Xoo. We generated transgenic rice lines constitutively expressing OsNH5N16 to investigate its function. Plants that overexpressed OsNH5N16 displayed enhanced BSR to Xoo and F. fujikuroi compared with wild varieties, and the transcription of PR genes such as OsPR1, GLUC, and CHIT2 was considerably upregulated. Moreover, we revealed that SA increases the transcription of OsNH5N16 and the promoter activity of OsPR1 regulated by OsNH5N16. These results showed that OsNH5N16 enhances BSR by regulating the expression of PR genes related to SAR and it is controlled by SA at the transcriptional and post-translational levels. This is the first report on the innate immune response conferring BSR associated with NH5.

Multisensory interactions regulate feeding behavior in Drosophila.

The integration of two or more distinct sensory cues can help animals make more informed decisions about potential food sources, but little is known about how feeding-related multimodal sensory integration happens at the cellular and molecular levels. Here, we show that multimodal sensory integration contributes to a stereotyped feeding behavior in the model organism Drosophila melanogaster Simultaneous olfactory and mechanosensory inputs significantly influence a taste-evoked feeding behavior called the proboscis extension reflex (PER). Olfactory and mechanical information are mediated by antennal Or35a neurons and leg hair plate mechanosensory neurons, respectively. We show that the controlled delivery of three different sensory cues can produce a supra-additive PER via the concurrent stimulation of olfactory, taste, and mechanosensory inputs. We suggest that the fruit fly is a versatile model system to study multisensory integration related to feeding, which also likely exists in vertebrates.

Analysis of personal and national factors that influence depression in individuals during the COVID-19 pandemic: a web-based cross-sectional survey.

BACKGROUND: The World Health Organization (WHO) declared coronavirus disease (COVID-19) a pandemic on March 11, 2020. Previous studies of infectious diseases showed that infectious diseases not only cause physical damage to infected individuals but also damage to the mental health of the public. Therefore this study aims to analyze the factors that affected depression in the public during the COVID-19 pandemic to provide evidence for COVID-19-related mental health policies and to emphasize the need to prepare for mental health issues related to potential infectious disease outbreaks in the future. RESULTS: This study performed the following statistical analyses to analyze the factors that influence depression in the public during the COVID-19 pandemic. First, to confirm the level of depression in the public in each country, the participants' depression was plotted on a Boxplot graph for analysis. Second, to confirm personal and national factors that influence depression in individuals, a multi-level analysis was conducted. As a result, the median Patient Health Questionnaire-9 (PHQ-9) score for all participants was 6. The median was higher than the overall median for the Philippines, Indonesia, and Paraguay, suggesting a higher level of depression. In personal variables, depression was higher in females than in males, and higher in participants who had experienced discrimination due to COVID-19 than those who had not. In contrast, depression was lower in older participants, those with good subjective health, and those who practiced personal hygiene for prevention. In national variables, depression was higher when the Government Response Stringency Index score was higher, when life expectancy was higher, and when social capital was higher. In contrast, depression was lower when literacy rates were higher. CONCLUSIONS: Our study reveals that depression was higher in participants living in countries with higher stringency index scores than in participants living in other countries. Maintaining a high level of vigilance for safety cannot be criticized. However, in the current situation, where coexisting with COVID-19 has become inevitable, inflexible and stringent policies not only increase depression in the public, but may also decrease resilience to COVID-19 and compromise preparations for coexistence with COVID-19. Accordingly, when establishing policies such as social distancing and quarantine, each country should consider the context of their own country.

COVID-19 screening center models in South Korea.

In a recent report, the British Broadcasting Company (BBC) introduced South Korea's measures to manage COVID-19 as role model for the world. Screening centers serve as frontiers for preventing community transmission of infectious diseases. COVID-19 screening centers in Korea operate 24 h a day, always open for individuals with suspected COVID-19 symptoms. South Korea concentrated COVID-19 screening centers around cities with high population density. Advanced screening centers (models C, D, and E) proved more effective and efficient in the prevention of COVID-19 than the traditional screening centers (models A and B). Particularly, screening centers at Incheon Airport in South Korea prevent transmission through imported cases effectively. It will be important elsewhere, as in South Korea, to establish an infectious disease delivery system that can lead to 'Test-Treat-Track' using an adequate model of screening centers.

Ventromedial hypothalamic primary cilia control energy and skeletal homeostasis.

Dysfunction of primary cilia is related to dyshomeostasis, leading to a wide range of disorders. The ventromedial hypothalamus (VMH) is known to regulate several homeostatic processes, but those modulated specifically by VMH primary cilia are not yet known. In this study, we identify VMH primary cilia as an important organelle that maintains energy and skeletal homeostasis by modulating the autonomic nervous system. We established loss-of-function models of primary cilia in the VMH by either targeting IFT88 (IFT88-KOSF-1) using steroidogenic factor 1-Cre (SF-1-Cre) or injecting an adeno-associated virus Cre (AAV-Cre) directly into the VMH. Functional impairments of VMH primary cilia were linked to decreased sympathetic activation and central leptin resistance, which led to marked obesity and bone-density accrual. Obesity was caused by hyperphagia, decreased energy expenditure, and blunted brown fat function and was also associated with insulin and leptin resistance. The effect of bone-density accrual was independent of obesity, as it was caused by decreased sympathetic tone resulting in increased osteoblastic and decreased osteoclastic activities in the IFT88-KOSF-1 and VMH primary cilia knockdown mice. Overall, our current study identifies VMH primary cilia as a critical hypothalamic organelle that maintains energy and skeletal homeostasis.

Single-cell transcriptome maps of myeloid blood cell lineages in Drosophila.

The Drosophila lymph gland, the larval hematopoietic organ comprised of prohemocytes and mature hemocytes, has been a valuable model for understanding mechanisms underlying hematopoiesis and immunity. Three types of mature hemocytes have been characterized in the lymph gland: plasmatocytes, lamellocytes, and crystal cells, which are analogous to vertebrate myeloid cells, yet molecular underpinnings of the lymph gland hemocytes have been less investigated. Here, we use single-cell RNA sequencing to comprehensively analyze heterogeneity of developing hemocytes in the lymph gland, and discover previously undescribed hemocyte types including adipohemocytes, stem-like prohemocytes, and intermediate prohemocytes. Additionally, we identify the developmental trajectory of hemocytes during normal development as well as the emergence of the lamellocyte lineage following active cellular immunity caused by wasp infestation. Finally, we establish similarities and differences between embryonically derived- and larval lymph gland hemocytes. Altogether, our study provides detailed insights into the hemocyte development and cellular immune responses at single-cell resolution.

Chemical Controllable Gene Drive in Drosophila.

Gene drive systems that propagate transgenes via super-Mendelian inheritance can potentially control insect-borne diseases and agricultural pests. However, concerns have been raised regarding unforeseen ecological consequences, and methods that prevent undesirable gene drive effects have been proposed. Here, we report a chemical-induced control of gene drive. We prepared a CRISPR-based gene drive system that can be removed by a site-specific recombinase, Rippase, the expression of which is induced by the chemical RU486 in fruit flies. Exposure of fruit flies to RU486 resulted in 7-12% removal of gene drive elements at each generation, leading to a significant reduction in gene drive-fly propagation. Mathematical modeling and simulation suggest that our system offers several advantages over a previously reported gene drive control system. Our chemical control system can provide a proof-of-principle for the reversible control of gene drive effects depending on ecological status and human needs.

Loss of Sirtuin 6 in osteoblast lineage cells activates osteoclasts, resulting in osteopenia.

Adult bone homeostasis requires a fine-tuned balance between the activity of osteoblasts and osteoclasts. This osteoblast-osteoclast coupling is therapeutically important because it limits the efficacy of most anabolic or anti-resorptive treatments for osteoporosis. Sirtuin6 (SIRT6), a histone deacetylase, was implicated recently as an important regulator in bone homeostasis, but its in vivo function in osteoblast lineage cells remains unclear, mainly due to a lack of in vivo experiments with osteoblast lineage-specific Sirt6 knockout mice. Here, we show that Sirt6 in mature osteoblasts and/or osteocytes inhibits osteoclastogenesis via a paracrine mechanism. We found that osteoblast/osteocyte-specific Sirt6 knockout mice show reduced bone mass due to increased osteoclast formation. Mechanistically, we attribute this increased osteoclastogenesis to decreased osteoprotegerin expression in Sirt6-null osteoblasts and osteocytes. This loss of Sirt6 in osteoblasts and osteocytes does not, however, alter bone formation parameters in vivo. It does accelerate osteogenic differentiation in ex vivo culture, indicating that the osteoblast/osteocyte-autonomous functions of SIRT6 have minor effects on the osteopenic phenotype. These results establish a critical role for SIRT6 in mature osteoblasts and osteocytes in adult bone homeostasis as a negative paracrine regulator of osteoclastogenesis.

Distinct roles of stereociliary links in the nonlinear sound processing and noise resistance of cochlear outer hair cells.

Outer hair cells (OHCs) play an essential role in hearing by acting as a nonlinear amplifier which helps the cochlea detect sounds with high sensitivity and accuracy. This nonlinear sound processing generates distortion products, which can be measured as distortion-product otoacoustic emissions (DPOAEs). The OHC stereocilia that respond to sound vibrations are connected by three kinds of extracellular links: tip links that connect the taller stereocilia to shorter ones and convey force to the mechanoelectrical transduction channels, tectorial membrane-attachment crowns (TM-ACs) that connect the tallest stereocilia to one another and to the overlying TM, and horizontal top connectors (HTCs) that link adjacent stereocilia. While the tip links have been extensively studied, the roles that the other two types of links play in hearing are much less clear, largely because of a lack of suitable animal models. Here, while analyzing genetic combinations of tubby mice, we encountered models missing both HTCs and TM-ACs or HTCs alone. We found that the tubby mutation causes loss of both HTCs and TM-ACs due to a mislocalization of stereocilin, which results in OHC dysfunction leading to severe hearing loss. Intriguingly, the addition of the modifier allele modifier of tubby hearing 1 in tubby mice selectively rescues the TM-ACs but not the HTCs. Hearing is significantly rescued in these mice with robust DPOAE production, indicating an essential role of the TM-ACs but not the HTCs in normal OHC function. In contrast, the HTCs are required for the resistance of hearing to damage caused by noise stress.

Neural regulation of energy and bone homeostasis by the synaptic adhesion molecule Calsyntenin-3.

Neuronal regulation of energy and bone metabolism is important for body homeostasis. Many studies have emphasized the importance of synaptic adhesion molecules in the formation of synapses, but their roles in physiology still await further characterization. Here, we found that the synaptic adhesion molecule Calsyntenin-3 (CLSTN3) regulates energy and bone homeostasis. Clstn3 global knockout mice show reduced body mass with improved leptin sensitivity and increased energy expenditure compared to their wild-type littermates. In addition, Clstn3 knockout mice show reduced marrow volume and cortical bone mass without alteration of trabecular bone microarchitecture. This reduced bone mass is not bone cell-autonomous because neither osteoblast- nor osteoclast-specific Clstn3 knockout mice show bone defects; similarly, in vitro cultures of both Clstn3 knockout osteoblasts and osteoclasts do not show any defects. These reduced body and bone mass phenotypes can be attributed instead to neuronal CLSTN3 because they are recapitulated by pan-neuronal but not sympathetic neuron-specific deletion of Clstn3. This study reveals novel physiological functions of neuronal Clstn3 as a key regulator of energy and bone homeostasis.

Vibration Control of Scanning Electron Microscopes with Experimental Approaches for Performance Enhancement.

A vibration isolator embedded in precision equipment, such as a scanning electron microscope (SEM), wafer inspection equipment, and nanoimprint lithography equipment, play a critical role in achieving the maximum performance of the equipment during the fabrication of nano/micro-electro-mechanical systems. In this study, the factors that degrade the performance of SEM equipment with isolation devices are classified and discussed, and improvement measures are proposed from the viewpoints of the measured image patterns and vibrations in comparison with the relevant vibration criteria. In particular, this study quantifies the image patterns measured using SEMs, and the results are discussed along with the measured vibration. A guide for the selection of mounting equipment is presented by performing vibration analysis on the lower mount of the dual elastic mount configuration applied to the SEM, as well as the image patterns analyzed with that configuration. In addition, design modifications for the mount and its arrangement are suggested based on impact tests and numerical simulations.