Emerging evidence indicates the early growth response 1 (Egr1) plays an important role in the pathogenesis of chronic pain. However, the regulation of Egr1 expression in the DRG and spinal cord in ...neuropathic pain remains unclear. In the current study, the neuropathic pain was conducted by lumber 5 spinal nerve ligation (SNL) in rats. The role of miR‐124‐3p in Egr1 expression was examined. Our results showed that the SNL led to a significant increase in the expression of Egr1 mRNA and protein in the DRG and dorsal horn. This increased expression of Egr1 correlated with a reduction of miR‐124‐3p in the same region. Prior i.t. injection of Egr1 decoy AYX1 inhibited the expression of Egr1 and attenuated the neuropathic pain‐like hypersensitivity following SNL. The dual‐luciferase reporter assay revealed the luciferase activity of the Egr1 3′‐UTR plasmid was inhibited by the miR‐124‐3p agomir. But this inhibition was completely reversed in the mutant 3′‐UTR Egr1 group. In vivo, the SNL‐induced behavioral signs of neuropathic pain and the increases in Egr1 mRNA and protein in the DRG and dorsal horn were prevented by prior to i.t. injection of miR‐124‐3p agomir. While, i.t. injection of miR‐124‐3p antagomir in naïve rats resulted in mechanical allodynia and thermal hyperalgesia and an overexpression of Egr1 in the DRG and dorsal horn. Together, our results suggest that the miR‐124‐3p‐regulated Egr1 expression in the DRG and dorsal horn contributes to the development of neuropathic pain. Targeting miR‐124‐3p might be a promising therapeutic strategy in the treatment of chronic pain.
The Egr1 has been implicated in regulating a variety of genes’ expression. MicroRNAs (MiRNAs) have emerged as key regulators in the maladaptive plasticity of chronic pain. However, whether miRNA‐124 involves in the modulation of Egr1 expression in neuropathic pain remains elusive. In the current study, we found the spinal nerve injury‐induced down‐regulation of miR‐124‐3p via promoting Egr1 expression in the dorsal root ganglion (DRG) and the spinal dorsal horn contributed to the development of neuropathic pain. Targeting DRG and spinal miR‐124‐3p might be a promising therapeutic strategy for the prevention and treatment of chronic pain.
Engineered cartilage derived from mesenchymal stromal cells (MSCs) always fails to maintain the cartilaginous phenotype in the subcutaneous environment due to the ossification tendency. Vascular ...invasion is a prerequisite for endochondral ossification during the development of long bone. As an oral antitumor medicine, Inlyta (axitinib) possesses pronounced antiangiogenic activity, owing to the inactivation of the vascular endothelial growth factor (VEGF) signaling pathway. In this study, axitinib‐loaded poly(ε‐caprolactone) (PCL)/collagen nanofibrous membranes are fabricated by electrospinning for the first time. Rabbit‐derived MSCs‐engineered cartilage is encapsulated in the axitinib‐loaded nanofibrous membrane and subcutaneously implanted into nude mice. The sustained and localized release of axitinib successfully inhibits vascular invasion, stabilizes cartilaginous phenotype, and helps cartilage maturation. RNA sequence further reveals that axitinib creates an avascular, hypoxic, and low immune response niche. Timp1 is remarkably upregulated in this niche, which probably plays a functional role in inhibiting the activity of matrix metalloproteinases and stabilizing the engineered cartilage. This study provides a novel strategy for stable subcutaneous chondrogenesis of mesenchymal stromal cells, which is also suitable for other medical applications, such as arthritis treatment, local treatment of tumors, and regeneration of other avascular tissues (cornea and tendon).
Axitinib, an oral antiangiogenic medicine, is loaded into nanofibrous membranes by electrospinning. Engineered cartilage, derived from bone marrow mesenchymal stromal cells (BMSCs), is encapsulated in the membrane and subcutaneously implanted. The sustained and localized release of axitinib successfully inhibits the vascular invasion and creates an avascular niche. Consequently, stable chondrogenesis of BMSCs is achieved in the subcutaneous environment.
Emerging evidence has implicated an important role of synapse-associated protein-97 (SAP97)-regulated GluA1-containing AMPARs membrane trafficking in cocaine restate and in contextual episodic memory ...of schizophrenia. Herein, we investigated the role of SAP97 in neuropathic pain following lumbar 5 spinal nerve transection (SNT) in rats. Our results showed that SNT led to upregulation of SAP97, enhanced the interaction between SAP97 and GluA1, and increased GluA1-containing AMPARs membrane trafficking in the dorsal horn. Microinjection of AAV-EGFP-SAP97 shRNA in lumbar 5 spinal dorsal horn inhibited SAP97 production, decreased SAP97-GluA1 interaction, reduced the membrane trafficking of GluA1-containing AMPARs, and partially attenuated neuropathic pain following SNT. Intrathecal injections of SAP97 siRNA or NASPM, an antagonist of GluA1-containing AMPARs, also partially reversed neuropathic pain on day 7, but not on day 14, after SNT. Spinal overexpression of SAP97 by AAV-EGFP-SAP97 enhanced SAP97-GluA1 interaction, increased the membrane insertion of GluA1-containing AMPARs, and induced abnormal pain in naïve rats. In addition, treatment with SAP97 siRNA or NASPM i.t. injection alleviated SNT-induced allodynia and hyperalgesia and exhibited a longer effect in female rats. Together, our results indicate that the SNT-induced upregulation of SAP97 via promoting GluA1-containing AMPARs membrane trafficking in the dorsal horn contributes to the pathogenesis of neuropathic pain. Targeting spinal SAP97 might be a promising therapeutic strategy to treatment of chronic pain.
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•SNT induced upregulation of SAP97 and increased membrane trafficking of GluA1-containing AMPARs.•Spinal knockdown SAP97 reduced membrane insertion of GluA1-AMPARs.•Spinal knockdown SAP97 or blockade of GluA1-AMPARs alleviated neuropathic pain.
Chromosome engineering has been attempted successfully in yeast but remains challenging in higher eukaryotes, including mammals. Here, we report programmed chromosome ligation in mice that resulted ...in the creation of new karyotypes in the lab. Using haploid embryonic stem cells and gene editing, we fused the two largest mouse chromosomes, chromosomes 1 and 2, and two medium-size chromosomes, chromosomes 4 and 5. Chromatin conformation and stem cell differentiation were minimally affected. However, karyotypes carrying fused chromosomes 1 and 2 resulted in arrested mitosis, polyploidization, and embryonic lethality, whereas a smaller fused chromosome composed of chromosomes 4 and 5 was able to be passed on to homozygous offspring. Our results suggest the feasibility of chromosome-level engineering in mammals.
Designer chromosomes
One of the goals in synthetic biology is to generate complex multicellular life with designed DNA sequences. Being able to manipulate DNA at large scales, including at the chromosome level, is an important step toward this goal. So far, chromosome-level genetic engineering has been accomplished only in haploid yeast. By applying gene editing to haploid embryonic stem cells, Wang
et al
. achieved whole-chromosome ligations in mice, and successfully derived animals with 19 pairs of chromosomes, one pair fewer than is standard in this species. —DJ
The ability to perform karyotype engineering in laboratory mice has been developed using haploid stem cells and gene editing.
Glioma is the most malignant and aggressive type of brain tumour with high heterogeneity and mortality. Although some clinicopathological factors have been identified as prognostic biomarkers, the ...individual variants and risk stratification in patients with lower grade glioma (LGG) have not been fully elucidated. The primary aim of this study was to identify an efficient DNA methylation combination biomarker for risk stratification and prognosis in LGG. We conducted a retrospective cohort study by analysing whole genome DNA methylation data of 646 patients with LGG from the TCGA and GEO database. Cox proportional hazard analysis was carried out to screen and construct biomarker model that predicted overall survival (OS). The Kaplan‐Meier survival curves and time‐dependent ROC were constructed to prove the efficiency of the signature. Then, another independent cohort was used to further validate the finding. A two‐CpG site DNA methylation signature was identified by multivariate Cox proportional hazard analysis. Further analysis indicated that the signature was an independent survival predictor from other clinical factors and exhibited higher predictive accuracy compared with known biomarkers. This signature was significantly correlated with immune‐checkpoint blockade, immunotherapy‐related signatures and ferroptosis regulator genes. The expression pattern and functional analysis showed that these two genes corresponding with two methylation sites contained in the model were correlated with immune infiltration level, and involved in MAPK and Rap1 signalling pathway. The signature may contribute to improve the risk stratification of patients and provide a more accurate assessment for precision medicine in the clinic.
Lysine-specific demethylase 6B (KDM6B) serves as a key mediator of gene transcription. It regulates expression of proinflammatory cytokines and chemokines in variety of diseases. Herein, the role and ...the underlying mechanisms of KDM6B in inflammatory pain were studied.
The inflammatory pain was conducted by intraplantar injection of complete Freund's adjuvant (CFA) in rats. Immunofluorescence, Western blotting, qRT-PCR, and chromatin immunoprecipitation (ChIP)-PCR were performed to investigate the underlying mechanisms.
CFA injection led to upregulation of KDM6B and decrease in the level of H3K27me3 in the dorsal root ganglia (DRG) and spinal dorsal horn. The mechanical allodynia and thermal hyperalgesia following CFA were alleviated by the treatment of intrathecal injection of GSK-J4, and by microinjection of AAV-EGFP-KDM6B shRNA in the sciatic nerve or in lumbar 5 dorsal horn. The increased production of tumor necrosis factor-α (TNF-α) following CFA in the DRGs and dorsal horn was inhibited by these treatments. ChIP-PCR showed that CFA-induced increased binding of nuclear factor κB with TNF-α promoter was repressed by the treatment of microinjection of AAV-EGFP-KDM6B shRNA.
These results suggest that upregulated KDM6B via facilitating TNF-α expression in the DRG and spinal dorsal horn aggravates inflammatory pain.
Robust allogeneic immune reactions after transplantation impede the translational pace of human embryonic stem cells (hESCs)‐based therapies. Selective genetic editing of human leucocyte antigen ...(HLA) molecules has been proposed to generate hESCs with immunocompatibility, which, however, has not been specifically designed for the Chinese population yet. Herein, we explored the possibility of customizing immunocompatible hESCs based on Chinese HLA typing characteristics. We generated an immunocompatible hESC line by disrupting HLA‐B, HLA‐C, and CIITA genes while retaining HLA‐A*11:01 (HLA‐A*11:01‐retained, HLA‐A11R), which covers ~21% of the Chinese population. The immunocompatibility of HLA‐A11R hESCs was verified by in vitro co‐culture and confirmed in humanized mice with established human immunity. Moreover, we precisely knocked an inducible caspase‐9 suicide cassette into HLA‐A11R hESCs (iC9‐HLA‐A11R) to promote safety. Compared with wide‐type hESCs, HLA‐A11R hESC‐derived endothelial cells elicited much weaker immune responses to human HLA‐A11+ T cells, while maintaining HLA‐I molecule‐mediated inhibitory signals to natural killer (NK) cells. Additionally, iC9‐HLA‐A11R hESCs could be induced to undergo apoptosis efficiently by AP1903. Both cell lines displayed genomic integrity and low risks of off‐target effects. In conclusion, we customized a pilot immunocompatible hESC cell line based on Chinese HLA typing characteristics with safety insurance. This approach provides a basis for establishment of a universal HLA‐AR bank of hESCs covering broad populations worldwide and may speed up the clinical application of hESC‐based therapies.
Here, we first selected an human embryonic stem cell (hESC) line with homozygous human leucocyte antigen (HLA)‐A*11:01 allele, the most popular HLA‐I allele of the Chinese population, then generated a hypoimmunogenic hESC line by disrupting HLA‐B, HLA‐C, and CIITA genes while retaining HLA‐A*11:01 (named as HLA‐A11R hESCs), covering ~21% of the Chinese population. The immunocompatibility of the HLA‐A11R hESCs was verified by in vitro co‐culture assays and confirmed in humanized mice with an established human immune system. Moreover, to address safety issues, we precisely knocked a clinical trial‐grade drug‐induced caspase‐9 suicide cassette into HLA‐A11R hESCs, generating iC9‐HLA‐A11R hESCs, which were efficiently induced to apoptosis by AP1903. In summary, we customized an immunocompatible hESC cell line based on the HLA typing characteristics of the Chinese population with safety insurance, which provided the basis for the establishment of an HLA‐AR bank of hESCs covering broader populations in the world, speeding up the clinical application of a range of hESC‐based therapies.
Enterovirus C116 (EV‐C116) is a new member of the enterovirus C group which is closely associated with several infectious diseases. Although sporadic studies have detected EV‐C116 in clinical samples ...worldwide, there is currently limited information available. In this study, two EV‐C‐positive fecal specimens were detected in apparently healthy children, which harbored low abundance, through meta‐transcriptome sequencing. Based on the prototypes of several EV‐Cs, two lineages were observed. Lineage 1 included many types that could not cause EV‐like cytopathic effect in cell culture. Three genogroups of EV‐C116 were divided in the maximum likelihood tree, and the two strains in this study (XZ2 and XZ113) formed two different lineages, suggesting that EV‐C116 still diffuses worldwide. Obvious inter‐type recombination events were observed in the XZ2 strain, with CVA22 identified as a minor donor. However, another strain (XZ113) underwent different recombination situations, highlighting the importance of recombination in the formation of EV‐Cs biodiversity. The EV‐C116 strains could propagate in rhabdomyosarcoma cell cultures at low titer; however, EV‐like cytopathic effects were not observed. HEp‐2, L20B, VERO, and 293T cell lines did not provide an appropriate environment for EV‐C116 growth. These results challenge the traditional recognition of the uncultured nature of EV‐C116 strains and explain the difficulty of clinical detection.
Xpert Xpress Flu/RSV is a fast and automated real-time nucleic acid amplification tool for detecting influenza virus and respiratory syncytial virus (RSV). The aim of this study was to verify the ...accuracy of Xpert Xpress Flu/RSV for detecting influenza virus and RSV. PubMed, EMBASE, Cochrane Library, and Web of Science databases were searched up to October 2020. The quality of the original research was assessed using the Quality Assessment of Diagnostic Accuracy Studies-2 guidelines. Meta-DiSc 1.4 software was used to analyze the sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and summary receiver operating characteristic curve. Deek’s funnel plot asymmetry test was used to evaluate the publication bias using the Stata 12.0 software. Ten studies with 25 fourfold tables were included in the analysis. The sensitivity of Xpert Xpress Flu/RSV for detecting influenza A, influenza B, and RSV were 0.97, 0.98, and 0.96, respectively, and the specificities were 0.97, 1.00, and 1.00, respectively. Compared with other common clinical real-time reverse transcription-polymerase chain reaction (RT-PCR), Xpert Xpress Flu/RSV is a valuable tool for diagnosing influenza virus and RSV with high sensitivity and specificity.
Benzene exposure has been causally linked with acute myeloid leukemia (AML), but inconsistently associated with other hematopoietic, lymphoproliferative and related disorders (HLD) or solid tumors in ...humans. Many neoplasms have been described in experimental animals exposed to benzene. We used Poisson regression to estimate adjusted relative risks (RR) and the likelihood ratio statistic to derive confidence intervals for cause‐specific mortality and HLD incidence in 73,789 benzene‐exposed compared with 34,504 unexposed workers in a retrospective cohort study in 12 cities in China. Follow‐up and outcome assessment was based on factory, medical and other records. Benzene‐exposed workers experienced increased risks for all‐cause mortality (RR = 1.1, 95% CI = 1.1, 1.2) due to excesses of all neoplasms (RR = 1.3, 95% CI = 1.2, 1.4), respiratory diseases (RR = 1.7, 95% CI = 1.2, 2.3) and diseases of blood forming organs (RR = ∞, 95% CI = 3.4, ∞). Lung cancer mortality was significantly elevated (RR = 1.5, 95% CI = 1.2, 1.9) with similar RRs for males and females, based on three‐fold more cases than in our previous follow‐up. Significantly elevated incidence of all myeloid disorders reflected excesses of myelodysplastic syndrome/acute myeloid leukemia (RR = 2.7, 95% CI = 1.2, 6.6) and chronic myeloid leukemia (RR = 2.5, 95% CI = 0.8, 11), and increases of all lymphoid disorders included excesses of non‐Hodgkin lymphoma (RR = 3.9, 95%CI = 1.5, 13) and all lymphoid leukemia (RR = 5.4, 95%CI = 1.0, 99). The 28‐year follow‐up of Chinese benzene‐exposed workers demonstrated increased risks of a broad range of myeloid and lymphoid neoplasms, lung cancer, and respiratory diseases and suggested possible associations with other malignant and non‐malignant disorders.
What's new?
More than two million workers worldwide are exposed to benzene each year. In this long‐term study of Chinese workers, the authors found that chronic benzene exposure was associated with a substantial increase in the risk of myeloid and lymphoid neoplasms, lung cancer, and respiratory diseases. The results also suggest possible associations with other malignant and non‐malignant disorders.
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