A mouse model to distinguish NLRP6-mediated inflammasome-dependent and -independent functions.

The NOD-like receptor (NLR) family pyrin domain containing 6 (NLRP6) serves as a sensor for microbial dsRNA or lipoteichoic acid (LTA) in intestinal epithelial cells (IECs), and initiating multiple pathways including inflammasome pathway and type I interferon (IFN) pathway, or regulating nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways. NLRP6 can exert its function in both inflammasome-dependent and inflammasome-independent manners. However, there is no tool to distinguish the contribution of individual NLRP6-mediated pathway to the physiology and pathology in vivo. Here, we validated that Arg39 and Trp50 residues in the pyrin domain (PYD) of murine NLRP6 are required for ASC recruitment and inflammasome activation, but are not important for the RNA binding and PYD-independent NLRP6 oligomerization. We further generated the Nlrp6R39E&W50E mutant mice, which showed reduced inflammasome activation in either steady state intestine or during viral infection. However, the type I IFN production in cells or intestine tissue from Nlrp6R39E&W50E mutant mice remain normal. Interestingly, NLRP6-mediated inflammasome activation or the IFN-I production seems to play distinct roles in the defense responses against different types of RNA viruses. Our work generated a useful tool to study the inflammasome-dependent role of NLRP6 in vivo, which might help to understand the complexity of multiple pathways mediated by NLRP6 in response to the complicated and dynamic environmental cues in the intestine.

Single-cell RNA sequencing reveals the evolution of the immune landscape during perihematomal edema progression after intracerebral hemorrhage.

BACKGROUND: Perihematomal edema (PHE) after post-intracerebral hemorrhage (ICH) has complex pathophysiological mechanisms that are poorly understood. The complicated immune response in the post-ICH brain constitutes a crucial component of PHE pathophysiology. In this study, we aimed to characterize the transcriptional profiles of immune cell populations in human PHE tissue and explore the microscopic differences between different types of immune cells. METHODS: 9 patients with basal ganglia intracerebral hemorrhage (hematoma volume 50-100 ml) were enrolled in this study. A multi-stage profile was developed, comprising Group1 (n = 3, 0-6 h post-ICH, G1), Group2 (n = 3, 6-24 h post-ICH, G2), and Group3 (n = 3, 24-48 h post-ICH, G3). A minimal quantity of edematous tissue surrounding the hematoma was preserved during hematoma evacuation. Single cell RNA sequencing (scRNA-seq) was used to map immune cell populations within comprehensively resected PHE samples collected from patients at different stages after ICH. RESULTS: We established, for the first time, a comprehensive landscape of diverse immune cell populations in human PHE tissue at a single-cell level. Our study identified 12 microglia subsets and 5 neutrophil subsets in human PHE tissue. What's more, we discovered that the secreted phosphoprotein-1 (SPP1) pathway served as the basis for self-communication between microglia subclusters during the progression of PHE. Additionally, we traced the trajectory branches of different neutrophil subtypes. Finally, we also demonstrated that microglia-produced osteopontin (OPN) could regulate the immune environment in PHE tissue by interacting with CD44-positive cells. CONCLUSIONS: As a result of our research, we have gained valuable insight into the immune-microenvironment within PHE tissue, which could potentially be used to develop novel treatment modalities for ICH.

Characterization of Quantitative Trait Loci for Leaf Rust Resistance from CI 13227 in Three Winter Wheat Populations.

Leaf rust is a widespread foliar wheat disease causing substantial yield losses worldwide. Slow-rusting is "adult plant" resistance that significantly slows epidemic development and thereby reduces yield loss. Wheat accession CI 13227 was previously characterized as having slow-rusting resistance. To validate the quantitative trait loci (QTL) and develop diagnostic markers for slow rusting resistance in CI 13227, a new population of recombinant inbred lines (RILs) of CI 13227 × Everest was evaluated for latent period (LP), final severity (FS), area under disease progress curve (AUDPC), and infection type (IT) in greenhouses and genotyped using genotyping-by-sequencing (GBS). Four QTL were identified on chromosome arms 2BL, 2DS, 3BS, and 7BL, explaining 6.82 to 28.45% of the phenotypic variance for these traits. Seven kompetitive allele specific polymorphism (KASP) markers previously reported to be linked to the QTL in two other CI 13227 populations were validated. In addition, the previously reported QLr.hwwg-7AL was remapped to 2BL (renamed QLr.hwwg-2BL) after adding new markers in this study. Phenotypic data showed that the RILs harboring two or three of the QTL had a significantly longer LP. QLr.hwwg-2DS on 2DS showed a major effect on all rust resistance traits and was finely mapped to a 2.7 Mb interval by two newly developed flanking markers from exome capture. Three disease-resistance genes and two transporter genes were identified as the putative candidates for QLr.hwwg-2DS. The validated QTL can be used as slow rusting resistance resources and the markers developed in this study will be useful for marker-assisted selection.

Emission Wavelength-Tunable Bicyclic Dioxetane Chemiluminescent Probes for Precise In Vitro and In Vivo Imaging.

Chemiluminescent probes have become increasingly popular in various research areas including precise tumor imaging and immunofluorescence analysis. Nevertheless, previously developed chemiluminescence probes are mainly limited to studying oxidation reaction-associated biological events. This study presents the first example of bioimaging applicable bicyclic dioxetane chemiluminescent probes with tunable emission wavelengths that range from 525 to 800 nm. These newly developed probes were able to detect the analytes of ß-Gal, H2O2, and superoxide with high specificity and a limit of detection of 77 mU L-1, 96, and 28 nM, respectively. The bioimaging application of the probes was verified in ovarian and liver cancer cells and macrophage cells, allowing the detection of the content of ß-Gal, H2O2, and superoxide inside the cells. The high specificity allowed us to image the xenografted tumor in mice. We expect that our probes will receive extensive applications in recording complex biomolecular events using noninvasive imaging techniques.

Quantitative trait loci for rolled leaf in a wheat EMS mutant from Jagger.

KEY MESSAGE: Two QTLs with major effects on rolled leaf trait were consistently detected on chromosomes 1A (QRl.hwwg-1AS) and 5A (QRl.hwwg-5AL) in the field experiments. Rolled leaf (RL) is a morphological strategy to protect plants from dehydration under stressed field conditions. Identification of quantitative trait loci (QTLs) underlining RL is essential to breed drought-tolerant wheat cultivars. A mapping population of 154 recombinant inbred lines was developed from the cross between JagMut1095, a mutant of Jagger, and Jagger to identify quantitative trait loci (QTLs) for the RL trait. A linkage map of 3106 cM was constructed with 1003 unique SNPs from 21 wheat chromosomes. Two consistent QTLs were identified for RL on chromosomes 1A (QRl.hwwg-1AS) and 5A (QRl.hwwg-5AL) in all field experiments. QRl.hwwg-1AS explained 24-56% of the phenotypic variation and QRl.hwwg-5AL explained up to 20% of the phenotypic variation. The combined percent phenotypic variation associated with the two QTLs was up to 61%. Analyses of phenotypic and genotypic data of recombinants generated from heterogeneous inbred families of JagMut1095 × Jagger delimited QRl.hwwg-1AS to a 6.04 Mb physical interval. This work lays solid foundation for further fine mapping and map-based cloning of QRl.hwwg-1AS.

An injectable and thermosensitive hydrogel with nano-aided NIR-II phototherapeutic and chemical effects for periodontal antibacteria and bone regeneration.

Periodontitis is a common public health problem worldwide and an inflammatory disease with irregular defect of alveolar bone caused by periodontal pathogens. Both antibacterial therapy and bone regeneration are of great importance in the treatment of periodontitis. In this study, injectable and thermosensitive hydrogels with 3D networks were used as carriers for controlled release of osteo-inductive agent (BMP-2) and Near Infrared Region-II (NIR-II) phototherapy agents (T8IC nano-particles). T8IC nano-particles were prepared by reprecipitation and acted as photosensitizer under 808 nm laser irradiation. Besides, we promoted photodynamic therapy (PDT) through adding H2O2 to facilitate the antibacterial effect instead of increasing the temperature of photothermal therapy (PTT). Hydrogel + T8IC + Laser + BMP-2 + H2O2 incorporated with mild PTT (45 °C), enhanced PDT and sustained release of BMP-2. It was present with excellent bactericidal effect, osteogenic induction and biosafety both in vitro and in vivo. Besides, immunohistochemistry staining and micro-CT analyses had confirmed that PTT and PDT could promote bone regeneration through alleviating inflammation state. Altogether, this novel approach with synergistic antibacterial effect, anti-inflammation and bone regeneration has a great potential for the treatment of periodontitis in the future.

Sirt6 Alleviated Liver Fibrosis by Deacetylating Conserved Lysine 54 on Smad2 in Hepatic Stellate Cells.

BACKGROUNDS AND AIMS: Activation of hepatic stellate cells (HSCs) is a central driver of fibrosis. This study aimed to elucidate the role of the deacetylase sirtuin 6 (Sirt6) in HSC activation and liver fibrosis. APPROACH AND RESULTS: Gain-of-function and loss-of-function models were used to study the function of Sirt6 in HSC activation. Mass spectrometry was used to determine the specific acetylation site. The lecithin retinol acyltransferase-driven cyclization recombination recombinase construct (CreERT2) mouse line was created to generate HSC-specific conditional Sirt6-knockout mice (Sirt6△HSC ). We found that Sirt6 is most abundantly expressed in HSCs as compared with other liver cell types. The expression of Sirt6 was decreased in activated HSCs and fibrotic livers of mice and humans. Sirt6 knockdown and Sirt6 overexpression increased and decreased fibrogenic gene expression, respectively, in HSCs. Mechanistically, Sirt6 inhibited the phosphorylation and nuclear localization of mothers against decapentaplegic homolog (Smad) 2. Further study demonstrated that Sirt6 could directly interact with Smad2, deacetylate Smad2, and decrease the transcription of transforming growth factor ß/Smad2 signaling. Mass spectrometry revealed that Sirt6 deacetylated conserved lysine 54 on Smad2. Mutation of lysine 54 to Arginine in Smad2 abolished the regulatory effect of Sirt6. In vivo, specific ablation of Sirt6 in HSCs exacerbated hepatocyte injury and cholestasis-induced liver fibrosis in mice. With targeted delivery of the Sirt6 agonist MDL-800, its concentration was 9.28-fold higher in HSCs as compared with other liver cells and alleviated hepatic fibrosis. CONCLUSIONS: Sirt6 plays a key role in HSC activation and liver fibrosis by deacetylating the profibrogenic transcription factor Smad2. Sirt6 may be a potential therapeutic target for liver fibrosis.

Sirtuin 6 supra-physiological overexpression in hypothalamic pro-opiomelanocortin neurons promotes obesity via the hypothalamus-adipose axis.

Sirtuin 6 (Sirt6), a member of the Sirtuin family, has important roles in maintaining glucose and lipid metabolism. Our previous studies demonstrated that the deletion of Sirt6 in pro-opiomelanocortin (POMC)-expressing cells by the loxP-Cre system resulted in severe obesity and hepatic steatosis. However, whether overexpression of Sirt6 in hypothalamic POMC neurons could ameliorate diet-induced obesity is still unknown. Thus, we generated mice specifically overexpressing Sirt6 in hypothalamic POMC neurons (PSOE) by stereotaxic injection of Cre-dependent adeno-associated viruses into the arcuate nucleus of Pomc-Cre mice. PSOE mice showed increased adiposity and decreased energy expenditure. Furthermore, thermogenesis of BAT and lipolysis of WAT were both impaired, caused by reduced sympathetic nerve innervation and activity in adipose tissues. Mechanistically, Sirt6 overexpression decreasing STAT3 acetylation, thus lowering POMC expression in the hypothalamus underlined the observed phenotypes in PSOE mice. These results demonstrate that Sirt6 overexpression specifically in the hypothalamic POMC neurons exacerbates diet-induced obesity and metabolic disorders via the hypothalamus-adipose axis.

Multifunctional photodynamic/photothermal nano-agents for the treatment of oral leukoplakia.

Oral leukoplakia (OLK) has gained extensive attention because of the potential risk for malignant transformation. Photosensitizers (PSs) played an indispensable role in the photodynamic therapy (PDT) of OLK, but the poor light sensitivity greatly hampered its clinical application. Herein, a novel organic photosensitive ITIC-Th nanoparticles (ITIC-Th NPs) were developed for OLK photodynamic/photothermal therapy (PTT). ITIC-Th NPs present both high photothermal conversion efficiency (~ 38%) and suitable reactive oxygen species (ROS) generation ability under 660 nm laser irradiation, making them possess excellent PDT and PTT capability. In 4-nitroquinoline 1-oxide (4NQO)-induced oral precancerous animal models, ITIC-Th NPs effectively suppress the OLK's cancerization without apparent topical or systemic toxicity in vivo. This study offers a promising therapeutic strategy for PDT and PTT in OLK treatment, and this study is the first interdisciplinary research in the field of multimodal therapy for OLK.

Quantitative assessment of wheat quality using near-infrared spectroscopy: A comprehensive review.

Wheat is one of the most widely cultivated crops throughout the world. A great need exists for wheat quality assessment for breeding, processing, and products production purposes. Near-infrared spectroscopy (NIRS) is a rapid, low-cost, simple, and nondestructive assessment method. Many advanced studies associated with NIRS for wheat quality assessment have been published recently, either introducing new chemometrics or attempting new assessment parameters to improve model robustness and accuracy. This review provides a comprehensive overview of NIRS methodology including its principle, spectra pretreatments, spectral wavelength selection, outlier disposal, dataset division, regression methods, and model evaluation. More importantly, the applications of NIRS in the determination of analytical parameters, rheological parameters, and end product quality of wheat are summarized. Although NIRS showed great potential in the quantitative determination of analytical parameters, there are still challenges in model robustness and accuracy in determining rheological parameters and end product quality for wheat products. Future model development needs to incorporate larger databases, integrate different spectroscopic techniques, and introduce cutting-edge chemometrics methods. In addition, calibration based on external factors should be considered to improve the predicted results of the model. The NIRS application in micronutrients needs to be extended. Last, the idea of combining standard product sensory attributes and spectra for model development deserves further study.

A comprehensive review of wheat phytochemicals: From farm to fork and beyond.

The health benefits of whole wheat consumption can be partially attributed to wheat's phytochemicals, including phenolic acids, flavonoids, alkylresorcinols, carotenoids, phytosterols, tocopherols, and tocotrienols. It is of increasing interest to produce whole wheat products that are rich in bioactive phytochemicals. This review provides the fundamentals of the chemistry, extraction, and occurrence of wheat phytochemicals and includes critical discussion of several long-lasting issues: (1) the commonly used nomenclature on distribution of wheat phenolic acids, namely, soluble-free, soluble-conjugated, and insoluble-bound phenolic acids; (2) different extraction protocols for wheat phytochemicals; and (3) the chemistry and application of in vitro antioxidant assays. This review further discusses recent advances on the effects of genotypes, environments, field management, and processing techniques including ultrafine grinding, germination, fermentation, enzymatic treatments, thermal treatments, and food processing. These results need to be interpreted with care due to varied sample preparation protocols and limitations of in vitro assays. The bioaccessibility, bioavailability, metabolism, and potential health benefits of wheat phytochemicals are also reviewed. This comprehensive and critical review will benefit scientific researchers in the field of bioactive compounds of cereal grains and also those in the cereal food industry to produce high-quality functional foods.

Mesencephalic astrocyte-derived neurotrophic factor alleviates alcohol induced hepatic steatosis via activating Stat3-mediated autophagy.

Alcoholic fatty liver disease (AFLD) is induced by alcohol consumption and may progress to more severe liver diseases such as alcoholic steatohepatitis, fibrosis and cirrhosis, and even hepatocellular carcinoma. Mesencephalic astrocyte-derived neurotrophic factor (MANF) participates in maintaining lipid homeostasis. However, the role of MANF in the pathogenesis of AFLD remains unclear. We established an AFLD mouse model following the US National Institute on Alcohol Abuse and Alcoholism procedure. Both mRNA and protein levels of MANF were significantly increased in the chronic binge alcohol feeding model. Liver-specific knockout of MANF aggravated hepatic lipid accumulation. Similarly, liver-specific overexpression of MANF alleviated AFLD in mouse livers. MANF affected hepatic lipid metabolism by modulating autophagy. The levels of LC3-II and Atg5-Atg12 were decreased in mouse livers with MANF liver-specific knockout and increased with MANF liver-specific overexpression. Furthermore, MANF changed the phosphorylation of Stat3 and its nuclear localization. MANF may have a protective role in the development of AFLD.

Abnormal expression of TGFBR2, EGF, LRP10, and IQGAP1 is involved in the pathogenesis of coronary artery disease.

Coronary artery disease (CAD) is the most common cardiovascular disease worldwide. In this study, we investigated the pathogenesis of CAD. We downloaded the GSE98583 dataset, including 12 CAD samples and 6 normal samples, from the Gene Expression Omnibus (GEO) database and screened differentially expressed genes (DEGs) in CAD versus normal samples. Next, we performed functional enrichment analysis, protein-protein interaction (PPI) network, and functional module analyses to explore potential functions and regulatory functions of identified DEGs. Next, transcription factors (TFs) and microRNAs (miRNAs) targeting DEGs were predicted. In total, 456 DEGs were identified in CAD and normal samples, including 175 upregulated and 281 downregulated genes. These genes were enriched in the intestinal immune network for immunoglobulin A production and the mitogen-activated protein kinase signaling pathway (e.g., TGFBR2 and EGF). The PPI network contained 212 genes, and HIST1H2BJ, HIST1H2AC, EGF, and EP300 were hub genes with degrees higher than 10. Four significant modules were identified from the PPI network, with genes in the modules mainly enriched in the inflammatory response, protein ubiquitination involved in ubiquitin-dependent protein catabolic processes, protein transport, and mitochondrial translational elongation, respectively. Two TFs (E2F1 and FOXK1) and five miRNAs (miR-122A, miR-516-5P, miR-507, miR-342, and miR-520F) were predicted to target 112 DEGs. miR-122A reportedly targets both LRP10 and IQGAP1 in the TF-miRNA target regulatory network. The abnormal expression of TGFBR2, EGF, LRP10, and IQGAP1 may be implicated in CAD pathogenesis. Our study provides targets and potential regulators for investigating CAD pathogenesis.

Targeting Interstitial Myofibroblast-Expressed Integrin αvß3 Alleviates Renal Fibrosis.

Renal fibrosis is the final manifestation of various chronic kidney diseases. Interstitial myofibroblasts, which are reported to highly express integrin αvß3, are the effector cells in renal fibrogenesis. Since current therapies do not efficiently target these cells, there is no effective therapeutic method for preventing or mitigating the disease. Here, we modified sterically stable PEGylated liposomes with the pentapeptide cRGDfC (RGD-Lip), which has a high affinity for αvß3, to specifically deliver drug to renal interstitial myofibroblasts. Our results showed that attaching cRGDfC to liposomes significantly increased their uptake by activated renal fibroblasts NRK-49F cells, and this effect was greatly abolished by adding excess-free cRGDfC or a knockdown of αvß3. Systemic administration of RGD-Lip gave rise to significant accumulation in a fibrotic kidney, which is ascribed to the specific recognition with integrin αvß3 on interstitial myofibroblasts. When loaded with celastrol, RGD-guided liposomes dramatically depressed the proliferation and activation of NRK-49F cells in vitro. Additionally, celastrol-loaded RGD-Lip markedly attenuated renal fibrosis, injury, and inflammation induced by unilateral ureteral obstruction (UUO) in mice, without inducing significant systemic toxicity. Thus, this liposomal system shows great promise for delivering therapeutic agents to interstitial myofibroblasts for renal fibrosis treatment with minimal side effects.

High-Affinity Anti-VISTA Antibody Protects against Sepsis by Inhibition of T Lymphocyte Apoptosis and Suppression of the Inflammatory Response.

BACKGROUND: B7 family members and ligands have been identified as critical checkpoints in orchestrating the immune response during sepsis. V-domain Ig suppressor of T cell activation (VISTA) is a new inhibitory immune checkpoint involved in restraining T cell response. Previous studies demonstrated that VISTA engagement on T cells and myeloid cells could transmit inhibitory signals, resulting in reduced activation and function. The current study was designed to determine the potential therapeutic effects of a high-affinity anti-VISTA antibody (clone MH5A) in a murine model of sepsis. METHODS: Polymicrobial sepsis was induced in male C57BL/6 mice via cecal ligation and puncture. Expression profiles of VISTA on T lymphocytes and macrophage were examined at 24 and 72 h postsurgery. The effects of anti-VISTA mAb on the 7-day survival, lymphocyte apoptosis, cytokine expression, bacterial burden, and vital organ damage were determined. Furthermore, the effects of anti-VISTA mAb on CD3+ T cell apoptosis and macrophage activation were determined in vitro. RESULTS: VISTA was substantially expressed on T cells and macrophages in sham-operated mice; septic peritonitis did not induce significant changes in the expression profiles. Treatment with MH5A improved the survival of septic mice, accompanied by reduced lymphocyte apoptosis, decreased cytokine expression, and enhanced bacterial clearance. Engagement of VISTA receptor with MH5A mitigated CD3+ T cell apoptosis cultured from CLP mice and suppressed LPS-induced cytokine production by macrophage in vitro. CONCLUSION: The present study identified VISTA as a novel immune checkpoint in the regulation of T cell and macrophage response during sepsis. Modulation of the VISTA pathway might offer a promising opportunity in the immunotherapy for sepsis.

Telmisartan attenuates obesity-induced insulin resistance via suppression of AMPK mediated ER stress.

Telmisartan is a known angiotensin II (Ang II) AT1 receptor blocker (ARB). While the beneficial effect of Telmisartan on glucose and lipid metabolism has been reported, the underlying molecular mechanism remained unclear. The endoplasmic reticulum (ER) stress is considered as one of important factors contributing to insulin resistance. In this study, we found that Telmisartan alleviated diet-induced obesity and insulin resistance, suppressed inflammation in adipose tissue, and alleviated hepatic steatosis. Furthermore, we showed that Telmisartan suppressed ER stress by activating AMP-activated protein kinase (AMPK) signaling pathway in vivo. In differentiated 3T3-L1 adipocytes, Telmisartan also improved palmitate acid (PA) induced ER stress. Compound C, an AMPK inhibitor, could abolish beneficial effect of Telmisartan on ER stress. Our data indicated Telmisartan improved obesity-induced insulin resistance through suppression of ER stress by activation of AMPK. These results provided the evidence that Telmisartan may have therapeutic potential for the treatment of obesity and type II diabetes.

SB431542-Loaded Liposomes Alleviate Liver Fibrosis by Suppressing TGF-ß Signaling.

Liver fibrosis is a common outcome of most chronic liver diseases, but there is no clinically approved drug for its treatment. Previous studies have reported the potential of SB431542 as an inhibitor of TGF-ß signaling in the treatment of liver fibrosis, but it shows poor water solubility and low bioavailability. Here, we improve these characteristics of SB431542 by loading it into liposomes (SB-Lips) with two FDA-approved excipients: soya phosphatidyl S100 and Solutol HS15. In vitro, SB-Lips had stronger inhibitory effects on the proliferation and activation of hepatic stellate cells LX-2 than free SB. After an intravenous injection in a CCl4-induced liver fibrosis mouse model, SB-Lips accumulated preferentially in the liver, its area under the concentration-time curve was significantly higher than that of free SB431542, and it alleviated hepatic fibrosis significantly more than free drug, which was associated with greater inhibition of TGF-ß signaling. Furthermore, SB-Lips did not cause significant injury to other organs. These results suggest that our liposomal system is safe and effective for delivering SB431542 to fibrotic liver.

An identified ensemble within a neocortical circuit encodes essential information for genetically-enhanced visual shape learning.

Advanced cognitive tasks are encoded in distributed neocortical circuits that span multiple forebrain areas. Nonetheless, synaptic plasticity and neural network theories hypothesize that essential information for performing these tasks is encoded in specific ensembles within these circuits. Relatively simpler subcortical areas contain specific ensembles that encode learning, suggesting that neocortical circuits contain such ensembles. Previously, using localized gene transfer of a constitutively active protein kinase C (PKC), we established that a genetically-modified circuit in rat postrhinal cortex, part of the hippocampal formation, can encode some essential information for performing specific visual shape discriminations. However, these studies did not identify any specific neurons that encode learning; the entire circuit might be required. Here, we show that both learning and recall require fast neurotransmitter release from an identified ensemble within this circuit, the transduced neurons; we blocked fast release from these neurons by coexpressing a Synaptotagmin I siRNA with the constitutively active PKC. During learning or recall, specific signaling pathways required for learning are activated in this ensemble; during learning, calcium/calmodulin-dependent protein kinase II, MAP kinase, and CREB are activated; and, during recall, dendritic protein synthesis and CREB are activated. Using activity-dependent gene imaging, we showed that during learning, activity in this ensemble is required to recruit and activate the circuit. Further, after learning, during image presentation, blocking activity in this ensemble reduces accuracy, even though most of the rest of the circuit is activated. Thus, an identified ensemble within a neocortical circuit encodes essential information for performing an advanced cognitive task.

miRNA-125b Regulates Osteogenic Differentiation of Periodontal Ligament Cells Through NKIRAS2/NF-κB Pathway.

BACKGROUND/AIMS: Osteogenesis of periodontal ligament cells (PDLCS) is essential for alveolar bone repair. Varieties of factors have been found involved in the regulation of PDLCs osteoblast differentiation. Aim of this study was to identify microRNA as a regulator of the os-teogenic differentiation of PDLCs. METHODS: The CD markers were analyzed by flow cytometry analysis. Osteoblast differentiation of PDLCs was induced by treatment with dexamethasone, ß-glycerol phosphate and α-ascorbic acid. The expression of osteoblastic phenotype was evaluated after the induction by simultaneous monitoring of alkaline phosphatase activity, the expression of genes involved in osteoblastic differentiation by RT-qPCR and Western Blot, and mineralization at the same time. MicroRNA and NKIRAS2 expression was determined by RT-qPCR. Luciferase reporter assays were performed to test whether miR-125b is capable of interacting with the 3'UTR sequence of NKIRAS2. The possible signaling pathway was determined by Western Blot. RESULTS: In this study, we found that the expression of miR-125b was down regulated during the process of ostoblast differentiation of PDLCs. When the expression of miR-125b was up regulated, the osteogenic differentiation of PDLCs was inhibited. During this process, the over-expressed miR-125b led to the activation of NF-κB. NF-κB inhibitor interacting RAS-like 2 (NKIRAS2) is one of target gene of miR-125b, and it is a regulator of NF-κB signaling that plays various roles in osteoblastic differentiation. We demonstrate thatmiR-125b is involved in osteogenic differentiation of PDLCs. CONCLUSION: Our data support the hypothesis that that miR-125b attenuates PDLCs osteoblastic differentiation by targeting NKIRAS2 and enhancing NF-κB signaling.