Caffeic acid phenethyl ester inhibits neuro-inflammation and oxidative stress following spinal cord injury by mitigating mitochondrial dysfunction via the SIRT1/PGC1α/DRP1 signaling pathway.

BACKGROUND: The treatment of spinal cord injury (SCI) has always been a significant research focus of clinical neuroscience, with inhibition of microglia-mediated neuro-inflammation as well as oxidative stress key to successful SCI patient treatment. Caffeic acid phenethyl ester (CAPE), a compound extracted from propolis, has both anti-inflammatory and anti-oxidative effects, but its SCI therapeutic effects have rarely been reported. METHODS: We constructed a mouse spinal cord contusion model and administered CAPE intraperitoneally for 7 consecutive days after injury, and methylprednisolone (MP) was used as a positive control. Hematoxylin-eosin, Nissl, and Luxol Fast Blue staining were used to assess the effect of CAPE on the structures of nervous tissue after SCI. Basso Mouse Scale scores and footprint analysis were used to explore the effect of CAPE on the recovery of motor function by SCI mice. Western blot analysis and immunofluorescence staining assessed levels of inflammatory mediators and oxidative stress-related proteins both in vivo and in vitro after CAPE treatment. Further, reactive oxygen species (ROS) within the cytoplasm were detected using an ROS kit. Changes in mitochondrial membrane potential after CAPE treatment were detected with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide. Mechanistically, western blot analysis and immunofluorescence staining were used to examine the effect of CAPE on the SIRT1/PGC1α/DRP1 signaling pathway. RESULTS: CAPE-treated SCI mice showed less neuronal tissue loss, more neuronal survival, and reduced demyelination. Interestingly, SCI mice treated with CAPE showed better recovery of motor function. CAPE treatment reduced the expression of inflammatory and oxidative mediators, including iNOS, COX-2, TNF-α, IL-1ß, 1L-6, NOX-2, and NOX-4, as well as the positive control MP both in vitro and in vivo. In addition, molecular docking experiments showed that CAPE had a high affinity for SIRT1, and that CAPE treatment significantly activated SIRT1 and PGC1α, with down-regulation of DRP1. Further, CAPE treatment significantly reduced the level of ROS in cellular cytoplasm and increased the mitochondrial membrane potential, which improved normal mitochondrial function. After administering the SIRT1 inhibitor nicotinamide, the effect of CAPE on neuro-inflammation and oxidative stress was reversed.On the contrary, SIRT1 agonist SRT2183 further enhanced the anti-inflammatory and antioxidant effects of CAPE, indicating that the anti-inflammatory and anti-oxidative stress effects of CAPE after SCI were dependent on SIRT1. CONCLUSION: CAPE inhibits microglia-mediated neuro-inflammation and oxidative stress and supports mitochondrial function by regulating the SIRT1/PGC1α/DRP1 signaling pathway after SCI. These effects demonstrate that CAPE reduces nerve tissue damage. Therefore, CAPE is a potential drug for the treatment of SCI through production of anti-inflammatory and anti-oxidative stress effects.

ROS: Executioner of regulating cell death in spinal cord injury.

The damage to the central nervous system and dysfunction of the body caused by spinal cord injury (SCI) are extremely severe. The pathological process of SCI is accompanied by inflammation and injury to nerve cells. Current evidence suggests that oxidative stress, resulting from an increase in the production of reactive oxygen species (ROS) and an imbalance in its clearance, plays a significant role in the secondary damage during SCI. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a crucial regulatory molecule for cellular redox. This review summarizes recent advancements in the regulation of ROS-Nrf2 signaling and focuses on the interaction between ROS and the regulation of different modes of neuronal cell death after SCI, such as apoptosis, autophagy, pyroptosis, and ferroptosis. Furthermore, we highlight the pathways through which materials science, including exosomes, hydrogels, and nanomaterials, can alleviate SCI by modulating ROS production and clearance. This review provides valuable insights and directions for reducing neuronal cell death and alleviating SCI through the regulation of ROS and oxidative stress.

Perceptual contrast reduces the judgments of learning of small-font words and increases the judgments of learning of large-font words compared with the no-contrast conditions.

The font-size effect on judgments of learning (JOLs) refers to large-font words being rated as more memorable than small ones when required to predict memory performance during the study phase. This study examines perceptual contrast as the prerequisite for this font-size effect on JOLs and explores how perceptual contrast leads to this effect. In Experiment 1, perceptual contrast was achieved by inserting words with one font (e.g., 18 pt) into a series of words with another font (e.g., 70 pt) at a particular proportion (1:4). In Experiment 2, perceptual contrast was manipulated by presenting two different font words up and down in a pair. The results of both experiments showed that: (1) participants rated higher JOLs for large than small fonts under the contrast conditions, but the JOL difference between the two fonts was not significant under the no-contrast conditions; (2) the JOLs of small-font words under the contrast conditions was reduced compared with the no-contrast conditions, but the JOLs of large-font words under the contrast conditions was increased compared with the no-contrast conditions. These results indicated that perceptual contrast was the prerequisite for the font-size effect on JOLs. The reason for this effect is that, compared to no-contrast conditions, perceptual contrast reduces the JOLs of small-font words while increasing the JOLs of large-font words. This study may deepen researchers' understanding of the mechanism of the font-size effect on JOLs and help educators effectively guide students to learn.

OCCURRENCE OF PATHOGENIC CHYTRID FUNGI BATRACHOCHYTRIUM SALAMANDRIVORANS AND BATRACHOCHYTRIUM DENDROBATIDIS IN THE HONG KONG NEWT (PARAMESOTRITON HONGKONGENSIS) AND OTHER WILD AND IMPORTED AMPHIBIANS IN A SUBTROPICAL ASIAN REGION.

One of the major threats for the massive loss in global amphibian diversity is chytridiomycosis, caused by chytrid fungi Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal). Following its discovery in 2013, Bsal has emerged as a severe threat to the global survival of urodelans. In 2018, a study reported a high prevalence of Bsal (65.6%) in the Hong Kong newts (Paramesotriton hongkongensis, Near Threatened) of a southern China population adjacent to Hong Kong (HK). Uncertainty regarding the Bsal infection status of P. hongkongensis inhabiting HK raised deep concern over the risk of introducing Bsal from that population. We screened the skin swabs from wild individuals of P. hongkongensis, 15 sympatric amphibian species, and 16 imported amphibian species in HK for chytrids. We found that both Bsal and Bd occur in low prevalences in P. hongkongensis (Bsal 1.7%, 5/293; Bd 0.34%, 1/293), Hong Kong cascade frog, Amolops hongkongensis, family Ranidae (Bsal only, 5.26%, 1/19), and Asian common toad, Duttaphrynus melanostictus, family Bufonidae (Bsal only, 5.88%, 1/17), populations of HK, with infected individuals being asymptomatic, suggesting a potential role of these species as reservoirs of Bsal. Conversely, Bd, but not Bsal, was present on 13.2% (9/68) of imported amphibians, indicating a high chytrid introduction risk posed by international amphibian trade. Long-term surveillance of the presence of Bd and Bsal in wild and captive amphibians would be advisable, and we recommend that import and export of nonnative chytrid carriers should be prevented, especially to those regions with amphibian populations naïve to Bd and Bsal.

Nucleus Pulposus Cells Induce M2 Polarization of RAW264.7 via CX3CL1/CX3CR1 Pathway and M2 Macrophages Promote Proliferation and Anabolism of Nucleus Pulposus Cells.

Background: The mechanisms underlying M2 macrophage polarization induced by nucleus pulposus (NP) cells are unclear. The effects that M2-polarized macrophages have on NP cells are also controversial. Methods: Transcriptome sequencing was performed to detect the gene change profiles between NP cells from ruptured intervertebral disc (IVD) and normal IVD. The main difference on biological activities between the two cell groups were analyzed by GO analysis and KEGG analysis. Virus transduction, flow cytometry, immunofluorescence, RT-PCR, western blot, CCK-8, TUNEL staining, and AO/EB staining were performed to explore the interactions between NP cells and RAW264.7 macrophages. Statistics were performed using SPSS26. Results: 801 upregulated and 276 downregulated genes were identified in NP cells from ruptured IVD in mouse models. According to GO and KEGG analysis, we found that the differentially expressed genes (DEG) were dominantly enriched in inflammatory response, extracellular matrix degradation, blood vessel morphogenesis, immune effector process, ossification, chemokine signaling pathway, macrophage activation, etc. CX3CL1 was one of the top 20% DEG, and we confirmed that both NP tissue and cells expressed remarkably higher level of CX3CL1 in mouse models (p < 0.001∗). Besides, we further revealed that both the recombinant CX3CL1 and NP cells remarkably induced M2 polarization of RAW264.7 (p < 0.001∗), respectively, while this effect was significantly reversed by si-CX3CL1 or JMS-17-2 (p < 0.001∗). Furthermore, we found that M2 macrophages significantly decreased the apoptosis rate (p < 0.001∗) and the catabolic gene levels (p < 0.001∗) of NP cells, while increased the viability, proliferation as well as the anabolic gene levels of NP cells (p < 0.01∗). Conclusions: Via regulating CX3CL1/CX3CR1 pathway, NP cells can induce the M2 macrophage polarization. M2 polarized macrophages can further promote NP cell viability, proliferation, and anabolism, while inhibit NP cell apoptosis and catabolism.

Role of regulatory T cells in spinal cord injury.

Spinal cord injury is an intricate process involving a series of multi-temporal and multi-component pathological events, among which inflammatory response is the core. Thus, it is crucial to find a way to prevent the damaging effects of the inflammatory response. The research has found that Treg cells can suppress the activation, proliferation, and effector functions of many parenchymal cells by multiple mechanisms. This review discusses how Treg cells regulate the inflammatory cells to promote spinal cord recovery. These parenchymal cells include macrophages/microglia, oligodendrocytes, astrocytes, and others. In addition, we discuss the adverse role of Treg cells, the status of treatment, and the prospects of cell-based therapies after spinal cord injury. In conclusion, this review provides an overview of the regulatory role of Treg cells in spinal cord injury. We hope to offer new insights into the treatment of spinal cord injury.

Role of peripheral immune cells in spinal cord injury.

Secondary spinal cord injury is caused by an inflammatory response cascade, and the process is irreversible. The immune system, as a mediator of inflammation, plays an important role in spinal cord injury. The spinal cord retains its immune privilege in a physiological state. Hence, elucidating the mechanisms by which peripheral immune cells are recruited to the lesion site and function after spinal cord injury is meaningful for the exploration of clinical therapeutic targets. In this review, we provide an overview of the multifaceted roles of peripheral immune cells in spinal cord injury.

Glioblastoma: two immune subtypes under the surface of the cold tumor.

Glioblastoma is classified as an immunocompromised tumor. The immune pattern beneath the cold tumor surface, however, has yet to be confirmed. Understanding the immune pattern of glioblastoma will aid in the development of effective treatment strategies. We performed weighted gene co-expression network analysis on all immune-related genes in TCGA-GBM transcriptional data and screened 35 prognosis-related immune genes. Unsupervised consistent clustering of these genes was used to analyze the immunological pattern of GBM. A glioblastoma immune prognostic score was developed by using 13 genes discovered by cox regression methods and verified with the GEO dataset to assess the immune profile, prognosis, and immunotherapy effects in individual patients. Glioblastoma has two immune modalities, immune tolerance and immunodeficiency, with distinct immune microenvironments, tumor-associated macrophages being one of the most promising new therapeutic targets. GIPS is a promising biomarker for assessing immune evasion mechanisms, immunotherapy responses, and prognosis in patients.

N6-Methyladenosine Regulator-Mediated Immue Patterns and Tumor Microenvironment Infiltration Characterization in Glioblastoma.

Background: Epigenetic modifications, according to emerging evidence, perform a critical role for cellular immune response and tumorigenesis. Nonetheless, the role of N6-methyladenosine modification in shaping of the glioblastoma tumor microenvironment is unknown. Methods: N6-methyladenosine(m6A) methylation patterns in GBM patients were evaluated via multiple omics analysis of 15 m6A regulators and systematically correlated with tumor immune features. For quantification of N6-methyladenosine methylation patterns of individual patients, GM-score was developed and correlated with clinical and immunological characteristics. Results: Glioblastoma has two different m6A methylation patterns that are strongly associated with TME characteristics, tumor subtype, immunotherapy response, and patient prognosis. High-GM-score is associated with an immune tolerance phenotype dominated by the IDH1 wild molecular subtype and the Mesenchymal tissue subtype, as well as a high infiltration of immune cells and stromal cells and a poor prognosis. Furthermore, despite higher immune checkpoint expression, individuals with a high-GM-score have a poorer response to anti-CTLA4 immunotherapy regimens due to T-cells dysfunctional. Low-GM-score individuals had an immunodeficient phenotype dominated by IDH mutant molecular subtypes and Proneural tissue subtypes, with less immune cell infiltration and a better prognosis. Furthermore, patients with low-GM-scores had higher microsatellite instability (MSI) and t-cell exclusion scores, as well as a better response to anti-CTLA4 immunotherapy regimens. Conclusion: This study demonstrated that m6A modification patterns play an important role in the shaping of TME complexity and diversity. The GM-score could identify m6A modification patterns in individual patients, resulting in a more personalization and efficacious anti-tumor immunotherapy strategy.

Identification and characterization of L-type lectin receptor-like kinases involved in Glycine max-Phytophthora sojae interaction.

MAIN CONCLUSION: Soybean contains a group of 64 L-type lectin receptor-like kinases. Three LecRKs were involved in the interactions with Phytophthora sojae and Bradyrhizobium diazoefficiens. L-type lectin receptor-like kinases (LecRKs) comprise an important class of membrane-localized receptor-like kinases that are involved in plant adaptation. In this study, we performed an inventory analysis of LecRKs in Glycine max (soybean). In total, 64 GmLecRKs containing the canonical LecRK feature were identified. Phylogenetic analysis revealed that 48 GmLecRKs have close orthologs in Arabidopsis or Solanum lycopersicum, while 16 are likely present only in the leguminous plant species. Transcriptome analyses revealed that expressions of multiple GmLecRK genes are either induced or suppressed during infection by the soybean root rot pathogen Phytophthora sojae. In addition, overexpression of the three LecRKs (Glyma.17G085000, Glyma.05G041300 or Glyma.17G224600) in the soybean hairy roots enhanced resistance to P. sojae. Upon inoculation with Bradyrhizobium diazoefficiens, overexpression of Glyma.17G085000 in the soybean hairy roots does not significantly influence the nodulation, while overexpression of Glyma.05G041300 or Glyma.17G224600 slightly reduced the number and dry weight of nodules. This study highlights the importance of LecRKs in regulating plant-microbe interactions and provides new knowledge on the deployment of LecRKs to increase resistance in soybean.

Inhibiting Phase Transfer of Protein Nanoparticles by Surface Camouflage-A Versatile and Efficient Protein Encapsulation Strategy.

Engineering a system with a high mass fraction of active ingredients, especially water-soluble proteins, is still an ongoing challenge. In this work, we developed a versatile surface camouflage strategy that can engineer systems with an ultrahigh mass fraction of proteins. By formulating protein molecules into nanoparticles, the demand of molecular modification was transformed into a surface camouflage of protein nanoparticles. Thanks to electrostatic attractions and van der Waals interactions, we camouflaged the surface of protein nanoparticles through the adsorption of carrier materials. The adsorption of carrier materials successfully inhibited the phase transfer of insulin, albumin, ß-lactoglobulin, and ovalbumin nanoparticles. As a result, the obtained microcomposites featured with a record of protein encapsulation efficiencies near 100% and a record of protein mass fraction of 77%. After the encapsulation in microcomposites, the insulin revealed a hypoglycemic effect for at least 14 d with one single injection, while that of insulin solution was only ∼4 h.

GIT1 protects traumatically injured spinal cord by prompting microvascular endothelial cells to clear myelin debris.

The clearance of myelin debris is a critical step in the functional recovery following spinal cord injury (SCI). As phagocytes do, microvascular endothelial cells (MECs) participate in myelin debris clearance at the injury site within one week. Our group has verified that G protein-coupled receptor kinase 2 interacting protein-1 (GIT1) is essential in autophagy and angiogenesis, both of which are tightly related to the uptake and degradation of myelin debris by MECs. Here, we analyzed the performance and mechanism of GIT1 in myelin debris clearance after SCI. The SCI contusion model was established and in vitro MECs were treated with myelin debris. Better recovery from traumatic SCI was observed in the GIT1 WT mice than in the GIT1 KO mice. More importantly, we found that GIT1 prompted MECs to clear myelin debris and further enhanced MECs angiogenesis in vivo and in vitro. Mechanistically, GIT1-mediated autophagy contributed to the clearance of myelin debris by MECs. In this study, we demonstrated that GIT1 may prompt MECs to clear myelin debris via autophagy and further stimulate MECs angiogenesis via upregulating VEGF. Our results indicate that GITI may serve as a promising target for accelerating myelin debris clearance and improving SCI recovery.

Initiation of PI3K/AKT pathway by IGF-1 decreases spinal cord injury-induced endothelial apoptosis and microvascular damage.

AIM: Apoptosis of endothelial cells (ECs) is a crucial factor in blood-spinal cord barrier (BSCB) disruption post spinal cord injury (SCI). Insulin-like growth factor-1 (IGF-1) is a protective cytokine that plays an important role in multiple diseases, whereas the distinct role in SCI-induced remains critical questions to address. Here we designed to explore the role and underlying mechanism of IGF-1 in endothelial damage after SCI. MAIN METHODS: In the current study, we established mouse microvascular endothelial cells (MVECs) injury model via LPS and cDNA of IGF-1 was transfected into MVECs. In vivo SCI mice, overexpression of IGF-1 (SCI-IGF-1) and its corresponding empty vehicle (SCI-NC) were conducted using lentivirus, then apoptosis degree, component of tight junction, and inflammatory damage were evaluated. KEY FINDINGS: IGF-1 treatment in MVECs displayed a milder apoptosis and cell damage under LPS insult. IGF-1 increased the level of PI3K/AKT pathway, which impeded the procedure of apoptosis. Blocking of PI3K/AKT pathway markedly neutralized the effect of IGF-1 treatment. Transfection of excess IGF-1 into SCI mice significantly corrected microenvironment of neural tissue repair, reduced area of injured core and improved functional recovery with greater activation of PI3K/AKT pathway. SIGNIFICANCE: The results above argue that the promising roles played by IGF-1 is potentially vital for developing effective future therapies in SCI.

Analysis of Correlation Between Age and Cervical Facet Joint Degeneration and Modic Changes in Patients with Cervical Spondylotic Myelopathy.

BACKGROUND Because facet joints move with the disc, changes in vertebral bodies occur simultaneously with progression of degeneration of cervical facet joints. This study investigated age-related differences in cervical facet joint abnormalities and multi-dimensional characteristics of MCs in patients with cervical spondylotic myelopathy. MATERIAL AND METHODS Forty-five patients underwent both magnetic resonance imaging (MRI) and computed tomography (CT) of the cervical spine. Axial and sagittal parameter changes from C3 to C7, including facet orientation (FO) and facet tropism (FT), and Modic changes (MCs), were evaluated and documented preoperatively, and we also measured the heights and diameters of MCs and performed correlation analysis and established linear regression models. RESULTS The axial facet orientation increased slightly from C3 66.5 (11.4) to C7 89.9 (19). The sagittal facet orientation and facet tropism increased between C3-C4 and C6-C7, but it decreased between C4 to C6. The MCs volume decreased from C3 to C4 and increased from C4 to C7. There was a gradual decrease of FO and FT from C3 to C5 and a gradual increase of these 2 angles from C5 to C7 in all age groups. The lowest values of FO and FT were detected at C5, while the highest values of FO and FT were detected at C7. CONCLUSIONS Age was negatively correlated with the axial, sagittal, and coronal cervical facet orientation, especially at C4/5 level. The FT with respect to the axial and sagittal plane from C5 to C6 increased with age.

Relationship between Endplate Defects, Modic Change, Disc Degeneration, and Facet Joint Degeneration in Patients with Low Back Pain.

PURPOSE: The endplate defects (EDs), Modic changes (MCs), disc degeneration (DD), facet orientation (FO), and facet tropism (FT) were demonstrated to be related to the low back pain (LBP). The aim of this study was to investigate possible correlations between them. METHODS: 75 patients were reviewed to evaluate the degenerative change in vertebral bodies (EDs and MCs), intervertebral discs (DD), and facet joint degeneration (FO and FT). All patients were categorized into four groups based on the grade of EDs. Clinical outcomes were evaluated with the visual analog scale (VAS) and Oswestry disability index (ODI) before and after surgery. RESULTS: There was no difference between the four groups in baseline characteristics except for gender and weight. FT is positively correlated with FO. The same rule exists between EDs, the size of MCs II, FO (left) and FO (right), and VAS and ODI. The grade of EDs is positively correlated with the grade of DD. L4-L5 can bear more load than other levels; thus, the grade of EDs is higher than that of other lumbar levels. The preoperative LBP was relieved in all groups in varying degrees. The change of pain and dysfunction is inversely proportional to the grade of EDs in the general trend. CONCLUSION: The relationship between weight, gender, and disc degeneration provided a mechanism by which increasing weight can predispose to DD. Different grades of EDs had different effects on patients with LBP. There was a significant correlation between EDs, MCs II, DD, FT, and FO.

C7 slope and its association with serum lipid levels and Modic changes in patients with cervical spondylotic myelopathy.

Background: Several studies have substituted the T1 slope (T1S) with the C7 slope (C7S) because the C7 endplate is clearer on radiographs. Further, abnormal serum lipid levels have been proven to be related with the development of disc degeneration. The aim of this study was to explore the relationship between C7S, serum lipid levels, cervical parameters related to cervical sagittal balance and Modic changes (MCs) in patients with multisegment cervical spondylotic myelopathy (CSM). Methods: Between January 2014 and January 2017, 75 patients with multisegment CSM were enrolled in our retrospective study. Gender, age, history of smoking status and alcohol consumption, and laboratory test data were recorded. The cervical sagittal balance parameters C7S, T1S, cervical lordosis (CL), neck tilt (NT), thoracic inlet angle (TIA), C2-C7 sagittal vertical axis (SVA), and T1S-CL were analyzed with Spearman correlation tests and multiple linear regression analysis. We diagnosed MCs through computed tomography or magnetic resonance imaging of the cervical spine. Patients were divided into four subgroups according to the presence or absence of MCs and their C7S values. Results: 75 patients were included in our study. Age, gender, C7S, and T1S were significantly different between the two groups. However, there was no statistical difference with regard to smoking status, alcohol consumption, lipoprotein(a), high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, albumin, globulin, triglycerides, total cholesterol, Ca2+, CL, T1S, TIA, NT, and T1S-CL. The correlation between HDL-C, LDL-C, ALB, GLB, Ca2+, C7S, T1S, MCs, NT, TIA, and C2-C7 SVA was statistically significant. Conclusion: Significant correlations were observed between MCs and TG (as well as other preoperative sagittal parameters), which may accelerate the development of degeneration of the cervical spine. Therefore, alcohol consumption, TG, and sagittal parameters, such as C7S, and T1S could be a promising candidate for the assessment of cervical sagittal balance and predicting neck pain.

Lack of endogenous parathyroid hormone delays fracture healing by inhibiting vascular endothelial growth factor­mediated angiogenesis.

Intermittent low­dose injections of parathyroid hormone (PTH) have been reported to exert bone anabolic effects and to promote fracture healing. As an important proangiogenic cytokine, vascular endothelial growth factor (VEGF) is secreted by bone marrow mesenchymal stem cells (BMSCs) and osteoblasts, and serves a crucial regulatory role in the process of vascular development and regeneration. To investigate whether lack of endogenous PTH causes reduced angiogenic capacity and thereby delays the process of fracture healing by downregulating the VEGF signaling pathway, a PTH knockout (PTHKO) mouse fracture model was generated. Fracture healing was observed using X­ray and micro­computerized tomography. Bone anabolic and angiogenic markers were analyzed by immunohistochemistry and western blot analysis. The expression levels of VEGF and associated signaling pathways in murine BMSC­derived osteoblasts were measured by quantitative polymerase chain reaction and western blot analysis. The expression levels of protein kinase A (PKA), phosphorylated­serine/threonine protein kinase (pAKT), hypoxia­inducible factor­1α (HIF1α) and VEGF were significantly decreased in BMSC­derived osteoblasts from PTHKO mice. In addition, positive platelet endothelial cell adhesion molecule staining was reduced in PTHKO mice, as determined by immunohistochemistry. The expression levels of HIF1α, VEGF, runt­related transcription factor 2, osteocalcin and alkaline phosphatase were also decreased in PTHKO mice, and fracture healing was delayed. In conclusion, lack of endogenous PTH may reduce VEGF expression in BMSC­derived osteoblasts by downregulating the activity of the PKA/pAKT/HIF1α/VEGF pathway, thus affecting endochondral bone formation by causing a reduction in angiogenesis and osteogenesis, ultimately leading to delayed fracture healing.

Leucine-rich repeat receptor-like gene screen reveals that Nicotiana RXEG1 regulates glycoside hydrolase 12 MAMP detection.

Activation of innate immunity by membrane-localized receptors is conserved across eukaryotes. Plant genomes contain hundreds of such receptor-like genes and those encoding proteins with an extracellular leucine-rich repeat (LRR) domain represent the largest family. Here, we develop a high-throughput approach to study LRR receptor-like genes on a genome-wide scale. In total, 257 tobacco rattle virus-based constructs are generated to target 386 of the 403 identified LRR receptor-like genes in Nicotiana benthamiana for silencing. Using this toolkit, we identify the LRR receptor-like protein Response to XEG1 (RXEG1) that specifically recognizes the glycoside hydrolase 12 protein XEG1. RXEG1 associates with XEG1 via the LRR domain in the apoplast and forms a complex with the LRR receptor-like kinases BAK1 and SOBIR1 to transduce the XEG1-induced defense signal. Thus, this genome-wide silencing assay is demonstrated to be an efficient toolkit to pinpoint new immune receptors, which will contribute to developing durable disease resistance.

ERK inhibition sensitizes CZ415-induced anti-osteosarcoma activity in vitro and in vivo.

mTOR is a valuable oncotarget for osteosarcoma. The anti-osteosarcoma activity by a novel mTOR kinase inhibitor, CZ415, was evaluated. We demonstrated that CZ415 potently inhibited survival and proliferation of known osteosarcoma cell lines (U2OS, MG-63 and SaOs2), and primary human osteosarcoma cells. Further, CZ415 provoked apoptosis and disrupted cell cycle progression in osteosarcoma cells. CZ415 treatment in osteosarcoma cells concurrently blocked mTORC1 and mTORC2 activation. Intriguingly, ERK-MAPK activation could be a major resistance factor of CZ415. ERK inhibition (by MEK162/U0126) or knockdown (by targeted ERK1/2 shRNAs) dramatically sensitized CZ415-induced osteosarcoma cell apoptosis. In vivo, CZ415 oral administration efficiently inhibited U2OS tumor growth in mice. Its activity was further potentiated with co-administration of MEK162. Collectively, we demonstrate that ERK inhibition sensitizes CZ415-induced anti-osteosarcoma activity in vitro and in vivo. CZ415 could be further tested as a promising anti-osteosarcoma agent, alone or in combination of ERK inhibition.

Endogenous Parathyroid Hormone Promotes Fracture Healing by Increasing Expression of BMPR2 through cAMP/PKA/CREB Pathway in Mice.

BACKGROUND/AIMS: Endogenous parathyroid hormone (PTH) plays an important role in fracture healing. This study investigated whether endogenous PTH regulates fracture healing by bone morphogenetic protein (BMP) and/or the transforming growth factor-ß (TGF-ß) signaling pathway. METHODS: Eight-week-old wild-type (WT) and PTH-knockout (PTH KO) male mice were selected, and models of open right-femoral fracture were constructed. Fracture healing and callus characteristics of mice in the two groups were compared by X-ray, micro-computed tomography, histological, and immunohistochemical examinations. Bone marrow mesenchymal stem cells (BMMSCs) of 8-week-old WT and PTHKO male mice were obtained and induced into osteoblasts and chondrocytes. RESULTS: We found that expression levels of Runt-related transcription factor (RUNX2), bone morphogenetic protein-receptor-type Ⅱ (BMPR2), phosphorylated Smad 1/5/8, and phosphorylated cyclic adenosine monophosphate-responsive element binding protein (CREB) in the callus of PTHKO mice were significantly decreased, whereas no significant difference in expression of SOX9, TGF-ßR2,or pSMAD2/3 was observed between PTHKO and WT mice. Additionally, the activity of osteoblast alkaline phosphatase was low at 7 days post-induction, and was upregulated by addition of PTH or dibutyryl cyclic adenosine monophosphate (dbcAMP) to the cell culture. Furthermore, H89 (protein kinase A inhibitor)eliminated the simulating effects of PTH and dbcAMP, and a low concentration of cyclic adenosine monophosphate (cAMP) was observed in PTHKO mouse BMMSCs. CONCLUSION: These results suggested that endogenous PTH enhanced BMPR2 expression by a cAMP/PKA/CREB pathway in osteoblasts, and increased RUNX2 expression through transduction of the BMP/pSMAD1/5/8 signaling pathway.