HGF secreted by hUC-MSCs mitigates neuronal apoptosis to repair the injured spinal cord via phosphorylation of Akt/FoxO3a pathway.

Spinal cord injury (SCI) can cause severe and permanent neurological damage, and neuronal apoptosis could inhibit functional recovery of damaged spinal cord greatly. Human umbilical cord mesenchymal stem cells (hUC-MSCs) have great potential to repair SCI because of a series of advantages, including inhibition of neuronal apoptosis and multiple differentiation. The former may play an important role. However, the detailed regulatory mechanism associated with the inhibition of neuronal apoptosis after hUC-MSCs administration has not been elucidated. In this study, proteomics analysis of precious human cerebrospinal fluid (CSF) samples collected from SCI subjects receiving hUC-MSCs delivery indicated that hepatocyte growth factor (HGF) is largely involved in SCI repair. Furthermore, overexpression of HGF derived from hUC-MSCs could decrease reactive oxygen species to prevent neuron apoptosis to the maximum, and thus lead to significant recovery of spinal cord dysfunction. Moreover, HGF could promote phosphorylation of Akt/FoxO3a pathway to decrease reactive oxygen species to reduce neuron apoptosis. For the first time, our research revealed that HGF secreted by hUC-MSCs inhibits neuron apoptosis by phosphorylation of Akt/FoxO3a to repair SCI. This study provides important clues associated with drug selection for the effective treatment of SCI in humans.

Irisin Ameliorates Intervertebral Disc Degeneration by Activating LATS/YAP/CTGF Signaling.

Unbalanced metabolism of an extracellular matrix (ECM) in nucleus pulposus cells (NPCs) is widely acknowledged as the primary cause of intervertebral disc degeneration (IDD). Irisin, a novel myokine, is cleaved from fibronectin type III domain-containing 5 (FNDC5) and has recently been proven to regulate the metabolism of ECM. However, little is known about its potential on NPCs and the development of IDD. Therefore, this study sought to examine the protective effects and molecular mechanism of irisin on IDD in vivo and in vitro. Decreased expression levels of FNDC5 and anabolism markers (COL2A1 and ACAN) but increased levels of catabolism markers (ADAMTS4) were found in degenerative nucleus pulposus (NP) tissues. In a punctured-induced rat IDD model, irisin treatment was found to significantly slow the development of IDD, and in TNF-α-stimulated NPCs, irisin treatment partly reversed the disorder of ECM metabolism. In mechanism, RNA-seq results suggested that irisin treatment affected the Hippo signaling pathway. Further studies revealed that with irisin treatment, the phosphorylation levels of key factors (LATS and YAP) were downregulated, while the expression level of CTGF was upregulated. Moreover, CTGF knockdown partially eliminated the protective effects of irisin on the metabolism of ECM in NPCs, including inhibiting the anabolism and promoting the catabolism. Taken together, this study demonstrated that the expression levels of FNDC5 were decreased in degenerative NP tissues, while irisin treatment promoted the anabolism, inhibited the catabolism of the ECM in NPCs, and delayed the progression of IDD via LATS/YAP/CTGF signaling. These results shed light on the protective actions of irisin on NPCs, leading to the development of a novel therapeutic target for treating IDD.

On the quest for novel bio-degradable plastics for agricultural field mulching.

Plasticulture, the practice of using plastic materials in agricultural applications, consumes about 6.7 million tons of plastics every year, which is about 2% of the overall global annual plastics production. For different reasons, plastic material used for agriculture is difficult to recycle. Therefore, most of it is either buried in fertile soils, thereby significantly causing deterioration of their properties, or, at best case, end in landfills where its half-life is measured in decades and even centuries. Hence, developing biodegradable plastic materials that are suitable for agricultural applications is a vital and inevitable need for the global human society. In our labs, two types of potentially biodegradable plastic polymer films were prepared and characterized imidazolium in terms of their bio-degradability. In the first approach, polymers made of ionic liquid monomers were prepared using photo radical induced polymerization. The second approach relies on formation of polyethylene-like n-alkane disulfide polymers from 1,ω-di-thiols through thermally activated air oxidation. These two families of materials were tested for their biodegradability in soils by using a simulation system that combines a controlled environment chamber equipped with a respirometer and a proton-transfer-reaction time of flight mass spectrometer (PTR-TOF-MS) system. This system provides a time-dependent and comprehensive fingerprint of volatiles emitted in the degradation process. The results obtained thus far indicate that whereas the ionic-liquid based polymer does not show significant bio-degradability under the test conditions, the building block monomer, 1,10-n-decane dithiol, as well as its disulfide-based polymer, are bio-degradable. The latter reaching, under basic soil conditions and in room temperature, ∼20% degradation within three months. These results suggest that by introduction of disulfide groups into the polyethylene backbone one may be able to render it biodegradable, thus considerably shortening its half-life in soils. Principal component analysis, PCA, of the data about the total volatiles produced during the degradation in soil indicates a distinctive volatile "fingerprint" of the disulfide-based bio-degradable products which comes from the volatile organic compounds portfolio as recorded by the PTR-TOF-MS. The biodegradation volatile fingerprint of this kind of film was different from the "fingerprint" of the soil background which served as a control. These results can help us to better understand and design biodegradable films for agricultural mulching practices.

Advanced genome-editing technologies enable rapid and large-scale generation of genetic variants for strain engineering and synthetic biology.

Targeted genome editing not only improves our understanding of fundamental rules in life sciences but also affords us versatile toolkits to improve industrially relevant phenotypes in various host cells. In this review, we summarize the recent endeavor to develop efficient genome-editing tools, and emphasize the utility of these tools to generate massive scale of genetic variants. We categorize these tools into traditional recombination-based tools, and more advanced CRISPR as well as RNA-based genome-editing tools. This diverse panel of sophisticated tools has been applied to accelerate strain engineering, upgrade biomanufacturing, and customize biosensing. In parallel with high-throughput phenotyping and AI-based optimization algorithms, we envision that genome-editing technologies will become a driving force to automate and streamline biological engineering, and empower us to address critical challenges in health, environment, energy, and sustainability.

Melatonin reverses tumor necrosis factor-alpha-induced metabolic disturbance of human nucleus pulposus cells via MTNR1B/Gαi2/YAP signaling.

Background: Intervertebral disc degeneration (IDD), the main cause of low back pain, is closely related to the inflammatory microenvironment in the nucleus pulposus (NP). Tumor necrosis factor-α (TNF-α) plays an important role in inflammation-related metabolic disturbance of NP cells. Melatonin has been proven to regulate the metabolism of NP cells, but whether it can protect NP cells from TNF-α-induced damage is still unclear. Therefore, this study aims to investigate the role and specific mechanism of melatonin on regulating the metabolism of NP cells in the inflammatory microenvironment. Methods: Western blotting, RT-qPCR and immunohistochemistry were used to detect the expression of melatonin membrane receptors (MTNR1A/B) and TNF-α in human NP tissues. In vitro, human primary NP cells were treated with or without vehicle, TNF-α and melatonin. And the metabolic markers were also detected by western blotting and RT-qPCR. The activity of NF-κB signaling and Hippo/YAP signaling were assessed by western blotting and immunofluorescence. Membrane receptors inhibitors, pathway inhibitors, lentiviral infection, plasmids transfection and immunoprecipitation were used to explore the specific mechanism of melatonin. In vivo, the rat IDD model was constructed and melatonin was injected intraperitoneally to evaluate its therapeutical effect on IDD. Results: The upregulation of TNF-α and downregulation of melatonin membrane receptors (MTNR1A/B) were observed in degenerative NP tissues. Then we demonstrated that melatonin could alleviate the development of IDD in a rat model and reverse TNF-α-impaired metabolism of NP cells in vitro. Further investigation revealed that the protective effects of melatonin on NP cells mainly rely on MTNR1B, which subsequently activates Gαi2 protein. The activation of Gαi2 could upregulate the yes-associated protein (YAP) level, resulting in anabolic enhancement of NP cells. In addition, melatonin-mediated YAP upregulation increased the expression of IκBα and suppressed the TNF-α-induced activation of the NF-κB pathway, thereby inhibiting the catabolism of NP cells. Conclusions: Our results revealed that melatonin can reverse TNF-α-impaired metabolism of NP cells via the MTNR1B/Gαi2/YAP axis and suggested that melatonin can be used as a potential therapeutic drug in the treatment of IDD.

Melatonin promotes bone marrow mesenchymal stem cell osteogenic differentiation and prevents osteoporosis development through modulating circ_0003865 that sponges miR-3653-3p.

BACKGROUND: Little is known about the implications of circRNAs in the effects of melatonin (MEL) on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteoporosis (OP) progression. The aim of our study was to investigate circRNAs in MEL-regulated BMSC differentiation and OP progression. METHODS: BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red, and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade was validated for the osteogenic differentiation of BMSCs by CCK-8, qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on OP development were tested in murine OP model. RESULTS: MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 had no effect on proliferation while promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSC osteogenic differentiation induced by MEL, but proliferation of BMSCs induced by MEL had no change whether circ_0003865 was overexpression or not. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression while BMSC proliferation was not affected. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing, but had no effect on proliferation. Finally, circ_0003865 silencing repressed OP development in mouse model. CONCLUSION: MEL promotes BMSC osteogenic differentiation and inhibits OP pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

Exosomes derived from miRNA-210 overexpressing bone marrow mesenchymal stem cells protect lipopolysaccharide induced chondrocytes injury via the NF-κB pathway.

Bone marrow mesenchymal stem cells (BMSCs)-derived exosomes (Exos) have anti-inflammatory and anti-apoptotic functions. miRNA-210 has also been confirmed to play a role in inhibiting proinflammatory cytokines. Herein, we aimed to explore the effects of Exos derived from miRNA-210-overexpressing BMSCs (BMSCs-210-Exos) and the mechanisms by which they provide protection to chondrocytes from lipopolysaccharide (LPS)-induced injury. BMSCs were transfected with or without miRNA-210. Exos substantially improved the proliferation of chondrocytes and inhibited LPS-induced cell apoptosis. Furthermore, BMSCs-210-Exos promoted the proliferation of chondrocytes and prevented LPS-induced cell apoptosis better than BMSCs-Exos not overexpressing miRNA-210. In addition, tumor necrosis factor receptor superfamily member 21 (Tnfrsf21) expression was inhibited and the NF-κB pathway was attenuated by both BMSCs-Exos and BMSCs-210-Exos during LPS-induced chondrocyte injury. Collectively, these results suggest that BMSCs-210-Exos enhance the protection of chondrocytes from LPS-induced injury via the NF-κB pathway.

Melatonin Rescued Reactive Oxygen Species-Impaired Osteogenesis of Human Bone Marrow Mesenchymal Stem Cells in the Presence of Tumor Necrosis Factor-Alpha.

Accumulation of reactive oxygen species (ROS), which can be induced by inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-α), can significantly inhibit the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs). This process can contribute to the imbalance of bone remodeling, which ultimately leads to osteoporosis. Therefore, reducing the ROS generation during osteogenesis of BMSCs may be an effective way to reverse the impairment of osteogenesis. Melatonin (MLT) has been reported to act as an antioxidant during cell proliferation and differentiation, but its antioxidant effect and mechanism of action during osteogenesis of MSCs in the inflammatory microenvironment, especially in the presence of TNF-α, remain unknown and need further study. In our study, we demonstrate that melatonin can counteract the generation of ROS and the inhibitory osteogenesis of BMSCs induced by TNF-α, by upregulating the expression of antioxidases and downregulating the expression of oxidases. Meanwhile, MLT can inhibit the phosphorylation of p65 protein and block the degradation of IκBα protein, thus decreasing the activity of the NF-κB pathway. This study confirmed that melatonin can inhibit the generation of ROS during osteogenic differentiation of BMSCs and reverse the inhibition of osteogenic differentiation of BMSCs in vitro, suggesting that melatonin can antagonize TNF-α-induced ROS generation and promote the great effect of osteogenic differentiation of BMSCs. Accordingly, these findings provide more evidence that melatonin can be used as a candidate drug for the treatment of osteoporosis.

Long noncoding RNA UCA1 promotes chondrogenic differentiation of human bone marrow mesenchymal stem cells via miRNA-145-5p/SMAD5 and miRNA-124-3p/SMAD4 axis.

Long noncoding RNA (lncRNAs) UCA1 has been known to be critical for the chondrogenic differentiation of marrow mesenchymal stem cells (MSCs). In this study, we explore the effects and mechanisms of UCA1 on the promotion of chondrogenesis of MSCs. During the processes of chondrogenic differentiation of MSCs, UCA1, miRNA-145-5p or miRNA-124-3p was overexpressed into MSCs. UCA1 substantially improved chondrogenesis of MSCs. Furthermore, UCA1 obviously down-regulated the expression of miRNA-145-5p and miRNA-124-3p, which attenuated the chondrogenic differentiation of MSCs. In addition, UCA1 significantly stimulated TGF-ß pathway member SMAD5 and SMAD4, which is targeted by miRNA-145-5p and miRNA-124-3p. Collectively, these outcomes suggest that UCA1 enhances chondrogenic differentiation of MSCs via the miRNA-145-5p/SMAD5 and miRNA-124-3p/SMAD4 axis.

Collagen type II suppresses articular chondrocyte hypertrophy and osteoarthritis progression by promoting integrin ß1-SMAD1 interaction.

Hypertrophic differentiation is not only the terminal process of endochondral ossification in the growth plate but is also an important pathological change in osteoarthritic cartilage. Collagen type II (COL2A1) was previously considered to be only a structural component of the cartilage matrix, but recently, it has been revealed to be an extracellular signaling molecule that can significantly suppress chondrocyte hypertrophy. However, the mechanisms by which COL2A1 regulates hypertrophic differentiation remain unclear. In our study, a Col2a1 p.Gly1170Ser mutant mouse model was constructed, and Col2a1 loss was demonstrated in homozygotes. Loss of Col2a1 was found to accelerate chondrocyte hypertrophy through the bone morphogenetic protein (BMP)-SMAD1 pathway. Upon interacting with COL2A1, integrin ß1 (ITGB1), the major receptor for COL2A1, competed with BMP receptors for binding to SMAD1 and then inhibited SMAD1 activation and nuclear import. COL2A1 could also activate ITGB1-induced ERK1/2 phosphorylation and, through ERK1/2-SMAD1 interaction, it further repressed SMAD1 activation, thus inhibiting BMP-SMAD1-mediated chondrocyte hypertrophy. Moreover, COL2A1 expression was downregulated, while chondrocyte hypertrophic markers and BMP-SMAD1 signaling activity were upregulated in degenerative human articular cartilage. Our study reveals novel mechanisms for the inhibition of chondrocyte hypertrophy by COL2A1 and suggests that the degradation and decrease in COL2A1 might initiate and promote osteoarthritis progression.

Active 3-D Shape Cosegmentation With Graph Convolutional Networks.

We present a novel active learning approach for shape cosegmentation based on graph convolutional networks (GCNs). The premise of our approach is to represent the collections of three-dimensional shapes as graph-structured data, where each node in the graph corresponds to a primitive patch of an oversegmented shape, and is associated with a representation initialized by extracting features. Then, the GCN operates directly on the graph to update the representation of each node based on a layer-wise propagation rule, which aggregates information from its neighbors, and predicts the labels for unlabeled nodes. Additionally, we further suggest an active learning strategy that queries the most informative samples to extend the initial training samples of GCN to generate more accurate predictions of our method. Our experimental results on the Shape COSEG dataset demonstrate the effectiveness of our approach.

Estrogen promotes the onset and development of idiopathic scoliosis via disproportionate endochondral ossification of the anterior and posterior column in a bipedal rat model.

This study aimed to verify the effects of estrogen on the onset and development of adolescent idiopathic scoliosis and the mechanisms associated with these effects by constructing a pubescent bipedal rat model. Experiments were conducted to investigate whether scoliosis progression was prevented by a Triptorelin treatment. One hundred twenty bipedal rats were divided into female, OVX (ovariectomy), OVX + E2, Triptorelin, sham, and male groups. According to a spinal radiographic analysis, the scoliosis rates and curve severity of the female and OVX + E2 groups were higher than those in the OVX, Triptorelin, and male groups. The measurements obtained from the sagittal plane of thoracic vertebrae CT confirmed a relatively slower growth of the anterior elements and a faster growth of the posterior elements between T11 and T13 in the female and OVX + E2 groups than in the OVX and Triptorelin groups. Histomorphometry and immunohistochemistry revealed a significantly longer hypertrophic zone of the vertebral cartilage growth plates that expressed more type X collagen and less type II collagen in the OVX and Triptorelin groups than in the female and OVX + E2 groups. Ki67 immunostaining confirmed an increase in the proliferation of vertebral growth plate chondrocytes in the OVX group compared with the female and OVX + E2 groups. In conclusion, estrogen obviously increased the incidence of scoliosis and curve severity in pubescent bipedal rats. The underlying mechanism may be a loss of coupling of the endochondral ossification between the anterior and posterior columns. Triptorelin decreased the incidence of scoliosis and curve magnitudes in bipedal female rats.

Fine-grained leukocyte classification with deep residual learning for microscopic images.

BACKGROUND AND OBJECTIVE: Leukocyte classification and cytometry have wide applications in medical domain, previous researches usually exploit machine learning techniques to classify leukocytes automatically. However, constrained by the past development of machine learning techniques, for example, extracting distinctive features from raw microscopic images are difficult, the widely used SVM classifier only has relative few parameters to tune, these methods cannot efficiently handle fine-grained classification cases when the white blood cells have up to 40 categories. METHODS: Based on deep learning theory, a systematic study is conducted on finer leukocyte classification in this paper. A deep residual neural network based leukocyte classifier is constructed at first, which can imitate the domain expert's cell recognition process, and extract salient features robustly and automatically. Then the deep neural network classifier's topology is adjusted according to the prior knowledge of white blood cell test. After that the microscopic image dataset with almost one hundred thousand labeled leukocytes belonging to 40 categories is built, and combined training strategies are adopted to make the designed classifier has good generalization ability. RESULTS: The proposed deep residual neural network based classifier was tested on microscopic image dataset with 40 leukocyte categories. It achieves top-1 accuracy of 77.80%, top-5 accuracy of 98.75% during the training procedure. The average accuracy on the test set is nearly 76.84%. CONCLUSIONS: This paper presents a fine-grained leukocyte classification method for microscopic images, based on deep residual learning theory and medical domain knowledge. Experimental results validate the feasibility and effectiveness of our approach. Extended experiments support that the fine-grained leukocyte classifier could be used in real medical applications, assist doctors in diagnosing diseases, reduce human power significantly.

Melatonin rescued interleukin 1ß-impaired chondrogenesis of human mesenchymal stem cells.

BACKGROUND: Osteoarthritis (OA) is a widespread arthritic disease and a primary cause of disability. Increasing evidence suggests that inflammation has a pivotal part in its pathogenesis. Interleukin-1ß (IL-1ß) is a primary mediator of local inflammatory processes in OA. Current therapies for OA mainly focus on the symptoms of the advanced stage of the disease. The possible utilization of bone marrow mesenchymal stem cells (BMSCs) to regenerate cartilage is an appealing method, but in the case of OA requires chondrogenesis to take place within an inflamed environment. Our previous study showed that melatonin (MLT) can promote chondrogenic differentiation of MSCs, but whether MLT can rescue IL-1ß-impaired chondrogenesis in human BMSCs has not yet been established. MLT, which can have anti-inflammatory and prochondrogenic effects, has demonstrated potential in defeating IL-1ß-induced inhibition of chondrogenesis and further study should be conducted. METHODS: Human bone marrow-derived MSCs were separated and cultured based on our system that was already documented. A high-density micromass culture system was used for the chondrogenic differentiation of human BMSCs, which was also described previously. Human BMSCs were induced for chondrogenesis for 7, 14, and 21 days with the treatment of IL-1ß and MLT. The cultured cartilage pellets were then evaluated by morphology, extracellular matrix accumulation, and chondrogenic, metabolic, and apoptotic marker expression. Furthermore, cell apoptosis was assessed by TUNEL assay. The phosphorylation level P65 and IκBα of the NF-κB pathway activity was explored on day 21 of chondrogenic differentiation of BMSCs. RESULTS: The current evaluation showed that MLT can save IL-1ß-impaired chondrogenesis of human BMSCs in different aspects. Firstly, MLT can restore the chondrogenic pellet size, and rescue matrix synthesis and accumulation. Secondly, MLT can upregulate chondrogenic marker COL2A1 expression at both mRNA and protein levels, and also regulate the expression levels of other chondrogenic markers like ACAN, SOX9, and COL10A1 in the presence of IL-1ß. Thirdly, MLT can maintain the metabolic balance of the chondrogenic process by suppressing expression of catabolic genes, such as MMP, MMP13, and ADAMTS4. Furthermore, MLT can subdue IL-1ß-induced cell apoptosis of BMSCs throughout chondrogenesis. Meanwhile, MLT suppressed the phosphorylation level of P65 and IκBα, which were elevated by IL-1ß treatment, indicating that MLT can attenuate the IL-1ß-induced activation of NF-κB signaling. CONCLUSION: The current evaluation showed that MLT can save IL-1ß-impaired chondrogenesis of human BMSCs by restoring the pellet size and matrix accumulation, and maintaining the metabolic balance, reducing cell apoptosis. Our study also showed that MLT can attenuate the IL-1ß-induced activation of the NF-κB signaling pathway, which is the most important pathway downstream of IL-1ß, and plays a crucial role in inflammation, apoptosis, and metabolism. Thus, MLT has prospects for treating OA due to its multifaceted functions, such as mitigating inflammation, maintaining metabolic balance, and mitigating apoptosis.

Melatonin-mediated miR-526b-3p and miR-590-5p upregulation promotes chondrogenic differentiation of human mesenchymal stem cells.

Bone marrow-derived mesenchymal stem cells (BMSCs), with inherent chondrogenic differentiation potential appear to be ideally suited for therapeutic use in cartilage regeneration. Accumulating evidence has demonstrated that melatonin can promote chondrogenic differentiation in human BMSCs. However, little is known about the mechanism. MicroRNAs (miRNAs) have been shown to regulate the differentiation of BMSCs, but their roles in melatonin-promoted chondrogenic differentiation have not been characterized. Here, we demonstrate that melatonin promoted chondrogenic differentiation of human BMSCs via upregulation of miR-526b-3p and miR-590-5p. Mechanistically, the elevated miR-526b-3p and miR-590-5p enhanced SMAD1 phosphorylation by targeting SMAD7. Additionally, administration of miR-526b-3p mimics or miR-590-5p mimics successfully promoted the chondrogenic differentiation of human BMSCs. Collectively, our study suggests that modification of BMSCs using melatonin or miRNA transduction could be an effective therapy for cartilage damage and degeneration.

Collagen type II is downregulated in the degenerative nucleus pulposus and contributes to the degeneration and apoptosis of human nucleus pulposus cells.

Degenerative disc disease (DDD) is a common degenerative condition initiated mainly within the nucleus pulposus (NP). To date, the etiopathogenesis of DDD remains unclear, and because no effective therapeutic strategies are available to target its pathological processes, DDD is still treated with symptomatic interventions that are far from adequate. Collagen type II is one of the major matrix components of the NP, and is considered to be essential to NP homeostasis. However, the specific mechanisms by which collagen type II influences NP cells remain unknown. In the present study, collagen type II expression was detected using immunohistochemistry analysis and quantitative polymerase chain reaction, and it was demonstrated to be significantly downregulated in NP tissues from patients with DDD compared with nondegenerative controls. To further explore the mechanism in vitro, interleukin (IL)­1ß stimulation was used to induce degeneration of a human NP cell line. IL­1ß stimulation upregulated both the mRNA and protein levels of the catabolic markers matrix metalloproteinase 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), while it downregulated the anabolic makers aggrecan and collagen type II. However, addition of purified collagen type II prevented this IL­1ß­induced metabolic disturbance of the NP cells. Furthermore, IL­1ß stimulation significantly promoted apoptosis in NP cells, while collagen type II treatment decreased the apoptotic rate and the protein levels of cleaved caspase­3. In conclusion, collagen type II exhibited protective effects in suppressing NP cell degeneration through its anticatabolic, proanabolic and antiapoptotic effects, suggesting that it may be a promising therapeutic agent for the prevention and treatment of DDD.

Rare coding variants in MAPK7 predispose to adolescent idiopathic scoliosis.

Adolescent idiopathic scoliosis (AIS) is a complex genetic disorder characterized by three-dimensional spinal curvatures, affecting 2%-3% of school age children, yet the causes underlying AIS are not well understood. Here, we first conducted a whole-exome sequencing and linkage analysis on a three-generation Chinese family with autosomal-dominant (AD) AIS, and then performed targeted sequencing in a discovery cohort comprising 20 AD AIS families and 86 simplex patients, and finally identified three disease-associated missense variants (c.886G> A, c.1943C> T, and c.1760C> T) in the MAPK7 gene (encoding mitogen-activated protein kinase 7). Genotyping of the three rare variants in a Chinese replication cohort comprising 1,038 simplex patients and 1,841 controls showed that their combined allele frequency was significantly over-represented in patients as compared with controls (2.0% [41/2,076] vs. 0.7% [27/3,682]; odds ratio = 2.7; P = 2.8 × 10-5 ). In vitro, we demonstrated that the three MAPK7 mutants disrupted nuclear translocation in cellular models, which is necessary for the normal function of MAPK7. In vivo, we also conducted CRISPR/Cas9-mediated deletion of mapk7 in zebrafish recapitulating the characteristic phenotype of idiopathic scoliosis. Taken together, our findings suggest that rare coding variants in MAPK7 predispose to AIS, providing clues to understanding the mechanisms of AIS.

Epstein-Barr Virus-Induced VEGF and GM-CSF Drive Nasopharyngeal Carcinoma Metastasis via Recruitment and Activation of Macrophages.

Chronic inflammation induced by persistent microbial infection plays an essential role in tumor progression. Although it is well documented that Epstein-Barr virus (EBV) infection is closely associated with nasopharyngeal carcinoma (NPC), how EBV-induced inflammation promotes NPC progression remains largely unknown. Here, we report that tumor infiltration of tumor-associated macrophages (TAM) and expression of CCL18, the cytokine preferentially secreted by TAM, closely correlate with serum EBV infection titers and tumor progression in two cohorts of NPC patients. In vitro, compared with EBV- NPC cell lines, EBV+ NPC cell lines exhibited superior capacity to attract monocytes and skew them to differentiate to a TAM-like phenotype. Cytokine profiling analysis revealed that NPC cells with active EBV replications recruited monocytes by VEGF and induced TAM by GM-CSF in an NF-κB-dependent manner. Reciprocally, TAM induced epithelial-mesenchymal transition and furthered NF-κB activation of tumor cells by CCL18. In humanized mice, NPC cells with active EBV replications exhibited increased metastasis, and neutralization of CCL18, GM-CSF, and VEGF significantly reduced metastasis. Collectively, our work defines a feed-forward loop between tumor cells and macrophages in NPC, which shows how metastatic potential can evolve concurrently with virus-induced chronic inflammation. Cancer Res; 77(13); 3591-604. ©2017 AACR.

Melatonin reversed tumor necrosis factor-alpha-inhibited osteogenesis of human mesenchymal stem cells by stabilizing SMAD1 protein.

Tumor necrosis factor-alpha (TNFα) plays a pivotal role in inflammation-related osteoporosis through the promotion of bone resorption and suppression of bone formation. Numerous drugs have been produced to treat osteoporosis by inhibiting bone resorption, but they offer few benefits to bone formation, which is what is needed by patients with severe bone loss. Melatonin, which can exert both anti-inflammatory and pro-osteogenic effects, shows promise in overcoming TNFα-inhibited osteogenesis and deserves further research. This study demonstrated that melatonin rescued TNFα-inhibited osteogenesis of human mesenchymal stem cells and that the interactions between SMURF1 and SMAD1 mediated the crosstalk between melatonin signaling and TNFα signaling. Additionally, melatonin treatment was found to downregulate TNFα-induced SMURF1 expression and then decrease SMURF1-mediated ubiquitination and degradation of SMAD1 protein, leading to steady bone morphogenetic protein-SMAD1 signaling activity and restoration of TNFα-impaired osteogenesis. Thus, melatonin has prospects for treating osteoporosis caused by inflammatory factors due to its multifaceted functions on regulation of bone formation, bone resorption, and inflammation. Further studies will focus on unveiling the specific mechanisms by which melatonin downregulates SMURF1 expression and confirming the clinical therapeutic value of melatonin in the prevention and therapy of bone loss associated with inflammation.

Exogenous Heparan Sulfate Enhances the TGF-ß3-Induced Chondrogenesis in Human Mesenchymal Stem Cells by Activating TGF-ß/Smad Signaling.

Heparan sulfate (HS) interacts with growth factors and has been implicated in regulating chondrogenesis. However, the effect of HS on TGF-ß-mediated mesenchymal stem cell (MSC) chondrogenesis and molecular mechanisms remains unknown. In this study, we explored the effects of exogenous HS alone and in combination with TGF-ß3 on chondrogenic differentiation of human MSCs and possible signal mechanisms. The results indicated that HS alone had no obvious effects on chondrogenic differentiation of human MSCs and TGF-ß/Smad2/3 signal pathways. However, the combined TGF-ß3/HS treatment resulted in a significant increase in GAG synthesis, cartilage matrix protein secretion, and cartilage-specific gene expression compared to cells treated with TGF-ß3 alone. Furthermore, HS inhibited type III TGF-ß receptors (TßRIII) expression and increased TGF-ß3-mediated ratio of the type II (TßRII) to the type I (TßRI) TGF-ß receptors and phosphorylation levels of Smad2/3. The inhibitor of the TGF-ß/Smad signal, SB431542, not only completely inhibited HS-stimulated TGF-ß3-mediated Smad2/3 phosphorylation but also completely inhibited the effects of HS on TGF-ß3-induced chondrogenic differentiation. These results demonstrate exogenous HS enhances TGF-ß3-induced chondrogenic differentiation of human MSCs by activating TGF-ß/Smad2/3 signaling.