SDF-1 (stromal cell derived factor-1) has been found to be widely expressed during dental pulp inflammation, while hDPSCs (human dental pulp stem cells) contribute to the repair of dental pulp. We showed that the migration of hDPSCs was induced by SDF-1 in a concentration-dependent manner and could be inhibited with siCXCR4 (C-X-C chemokine receptor type 4) and siCDC42 (cell division control protein 42), as well as drug inhibitors such as AMD3100 (antagonist of CXCR4), LY294002 (inhibitor of PI3K) and PF573228 (inhibitor of FAK). It was also confirmed that SDF-1 regulated the phosphorylation of FAK (focal adhesion kinases) on cell membranes and the translocation of β-catenin into the cell nucleus. Subsequent experiments confirmed that the expression of CXCR4 and β-catenin and the phosphorylation of FAK, PI3K (phosphoinositide 3-kinase), Akt and GSK3β (glycogen synthase kinase-3β) were altered significantly with SDF-1 stimulation. FAK and PI3K worked in coordination during this process. Our findings provide direct evidence that SDF-1/CXCR4 axis induces hDPSCs migration through FAK/PI3K/Akt and GSK3β/β-catenin pathways, implicating a novel mechanism of dental pulp repair and a possible application of SDF-1 for the treatment of pulpitis.

Leucine-rich-repeat-containing G protein-coupled receptors (LGRs) have been widely found to be implicated with development and progression in multiple cancer types. However, the clinical significance and biological functions of LGR6 in ovarian cancer remains unclear. In this study, LGR6 expression was mainly examined by immunohistochemistry. Functional assays in vitro and animal experiments in vivo were carried out to explore the effect of LGR6 on cancer stem cell (CSC) characteristics and chemotherapeutic responses in ovarian cancer cells. Luciferase assays and GSEA were used to discern the underlying mechanisms contributing to the roles of LGR6 in ovarian cancer. Here, we reported that LGR6 was upregulated in ovarian cancer, which positively correlated with poor chemotherapeutic response and progression survival in ovarian cancer patients. Loss-of-function assays showed that downregulating LGR6 abrogated the CSC-like phenotype and chemoresistance in vitro. More importantly, silencing LGR6 improved the chemoresistance of ovarian cancer cells to cisplatin in vivo. Mechanistic investigation further revealed that silencing LGR6 inhibited stemness and chemoresistance by repressing Wnt/β-catenin signaling. Collectively, our results uncover a novel mechanism contributing to LGR6-induced chemotherapeutic resistance in ovarian cancer, providing the evidence for LGR6 as a potential therapeutic target in ovarian cancer.

Infection induces the production of proinflammatory cytokines and chemokines such as interleukin-8 (IL-8) and interleukin-6 (IL-6). Although they facilitate local antiviral immunity, their excessive release leads to life-threatening cytokine release syndrome, exemplified by the severe cases of coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In the present study, we found that interleukin-8 (IL-8) was upregulated by PDCoV infection. We then demonstrated that PDCoV E protein induced IL-8 production and that the TM domain and the C-terminal domain of the E protein were important for IL-8 production. Subsequently, we showed here that deleting the AP-1 and NF-κB binding motif in porcine IL-8 promoter abrogated its activation, suggesting that IL-8 expression was dependent on AP-1 and NF-κB. Furthermore, PDCoV E induced IL-8 production, which was also dependent on the NF-κB pathway through activating nuclear factor p65 phosphorylation and NF-κB inhibitor alpha (IκBα) protein phosphorylation, as well as inducing the nuclear translocation of p65, eventually resulting in the promotion of IL-8 production. PDCoV E also activated c-fos and c-jun, both of which are members of the AP-1 family. These findings provide new insights into the molecular mechanisms of PDCoV-induced IL-8 production and help us further understand the pathogenesis of PDCoV infection.

Tremendous efforts have been made to explore biomarkers for classifying and grading glioma. However, the majority of the current understanding is based on public databases that might not accurately reflect the Asian population. Here, we investigated the genetic landscape of Chinese glioma patients using a validated multigene next-generation sequencing (NGS) panel to provide a strong rationale for the future classification and prognosis of glioma in this population.

Epithelial-mesenchymal transition (EMT) plays an important role in the invasion and metastasis of colorectal cancer, which is mediated by FAK and EGF. However, whether FAK participates in EMT in colorectal cancer cells through the EGF/EGFR signaling pathway remains unknown. The aim of this study was to investigate the effector mechanisms of FAK in the process of EGF-induced EMT in colorectal cancer cells and to determine whether miR-217 is involved in this process. Caco-2 cancer cells were routinely cultured with and without treatment with 100 ng/mL EGF, and changes in cell morphology were observed using an inverted microscope. In addition, a transwell assay was used to detect cell migration under the condition of EGF treatment. The expression of FAK, pFAK, E-cadherin, vimentin, and β actin was assessed by western blotting, and the expression of miR-217 was assessed using real-time PCR. We found that EGF induced EMT in colorectal cancer cells and enhanced cell migration and invasion ability. Moreover, FAK was involved in the EGF-induced EMT of colorectal cancer cells. EGF upregulated the expression of E-cadherin in colorectal cancer cells by activating FAK, and miR-217 was found to participate in EGF-induced EMT in colorectal cancer cells. Our findings indicate that EGF induces EMT in colorectal cancer cells by activating FAK, and miR-217 is involved in the EGF/FAK/E-cadherin signaling pathway.

As the ozone hole in the North and South poles continues to increase, the entire ecosystem will face an environmental crisis caused by enhanced UV-B radiation. Considering the function of TiO2 and the application of nanomaterials in agriculture, the effect of TiO2-NPs on UV-B stress tolerance in Arabidopsis was investigated. The phenotype of plants was determined, and the expression patterns of antioxidant systems and related genes were analyzed. Modification of the antioxidant system and changes in the flavonoid content of plants were observed by histochemical staining. The effects of TiO2-NPs and UV-B on mitosis were observed at the cellular level, and the degree of DNA damage was analyzed by the detection of CPDs content. The effects of TiO2-NPs and UV-B on SOD isozymes were detected by SOD isozyme Native-PAGE electrophoresis. A laser confocal microscope was used to explore the protective mechanism of TiO2-NPs against UV-B radiation. Results showed that pretreatment of TiO2-NPs significantly alleviated the stress of UV-B radiation on plants. TiO2-NPs activated the antioxidant system of plants, improved the activity of antioxidant enzymes, and promoted the synthesis of flavonoids. Moreover, TiO2-NPs could effectively shield UV-B radiation to prevent the depolymerization of microtubules in plant cells. 10 mg/L of TiO2-NPs is a safe and effective application dose, which has no biological toxicity to plants. Our research results reported for the first time that pretreatment of TiO2-NPs could effectively alleviate UV-B stress to plants, providing new ideas for the application of nanomaterials in agriculture.

Methylprednisolone (MP) is a synthetic glucocorticoid used for the treatment of spinal cord injury (SCI). Soluble Nogo-66 receptor (NgR) ectodomain is a novel experimental therapy for SCI that promotes axonal regeneration by blocking the growth inhibitory effects of myelin constituents in the adult central nervous system. To evaluate the potential complementarity of these mechanistically distinct pharmacological reagents we compared their effects alone and in combination after thoracic (T7) dorsal hemisection in the rat. Treatment with an ecto-domain of the rat NgR (27-310) fused to a rat IgG [NgR(310)ecto-Fc] (50 microm intrathecal, 0.25 microL/h for 28 days) or MP alone (30 mg/kg i.v., 0, 4 and 8 h postinjury) improved the rate and extent of functional recovery measured using Basso, Beattie, Bresnahan (BBB) scoring and footprint analysis. The effect of MP treatment on BBB score was apparent the day after SCI whereas the effect of NgR(310)ecto-Fc was not apparent until 2 weeks after SCI. NgR(310)ecto-Fc or MP treatment resulted in increased axonal sprouting and/or regeneration, quantified by counting biotin dextran amine-labeled corticospinal tract axons, and increased the number of axons contacting motor neurons in the ventral horn gray matter caudal to the lesion. Combined treatment with NgR(310)ecto-Fc and MP had a more pronounced effect on recovery of function and axonal growth compared with either treatment alone. The data demonstrate that NgR(310)ecto-Fc and MP act in a temporally and mechanistically distinct manner and suggest that they may have complementary effects.

Upon migrating to the injured sites, bone marrow mesenchymal stem cells (BMSCs) play critical roles in the repair of bone lesion caused by chronic apical periodontitis. Emerging evidences have shown that Enterococcus faecalis is always associated with apical periodontitis, especially refractory apical periodontitis. But the mechanism underlying how Enterococcus faecalis affects the migration of BMSCs remains unclear.

The incidence of early stage multiple primary lung cancer (MPLC) has been increasing in recent years, while the ideal strategy for its diagnosis and treatment remains controversial. The present study conducted genomic analysis to identify a new molecular classification method for accurately predicting the diagnosis and therapy for patients with early stage MPLC.

The endoplasmic reticulum (ER) plays a pivotal role in protein synthesis, folding, and modification. Under stress conditions such as oxidative stress and inflammation, the ER can become overwhelmed, leading to an accumulation of misfolded proteins and ensuing ER stress. This triggers the unfolded protein response (UPR) designed to restore ER homeostasis. Alcoholic liver disease (ALD), a spectrum disorder resulting from chronic alcohol consumption, encompasses conditions from fatty liver and alcoholic hepatitis to cirrhosis. Metabolites of alcohol can incite oxidative stress and inflammation in hepatic cells, instigating ER stress. Prolonged alcohol exposure further disrupts protein homeostasis, exacerbating ER stress which can lead to irreversible hepatocellular damage and ALD progression. Elucidating the contribution of ER stress to ALD pathogenesis may pave the way for innovative therapeutic interventions. This review delves into ER stress, its basic signaling pathways, and its role in the alcoholic liver injury.

Postoperative monitoring for patients with colorectal cancer (CRC) requires sensitive biomarkers that are associated with medical response and adjuvant therapy following surgery. Conventional tumor biomarkers [including carcinoembryonic antigen (CEA), CA19-9 and CA125] are widely used, but none of the markers provide high sensitivity or specificity. Previous studies indicated that circulating tumor DNA (ctDNA) is useful for postoperative monitoring of patients with cancer. However, the majority of previous studies involved patients with lung cancer, and therefore further studies are required which investigate patients with CRC. The present study enrolled 11 patients with CRC. All patients underwent surgery, and a number of patients were treated with postoperative chemotherapy. Tumor tissues and serial blood samples were collected from each patient, and somatic mutations of each sample were obtained using next-generation sequencing. The mutation landscape and dynamic changes in mutations for each patient were analyzed, and these results were compared with the changes of CEA levels. A number of driver genes were selected, including tumor protein P53 (TP53), APC and KRAS, to monitor the postoperative outcome of the 11 patients with CRC. Driver mutations were detected in preoperative plasma in 7 patients, with markedly decreased mutation rates detected in postoperative plasma compared with preoperative plasma. Driver mutations were not detected in 4 patients in the preoperative or postoperative plasma. In 1 patient with metastatic rectal cancer, the rate of TP53 mutation increased from 8.95 (preoperative) to 71.4% (postoperative), and a new phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α mutation emerged. This patient succumbed to mortality six months following surgery, however there were no marked changes in CEA levels during periodic detection of CEA levels. In summary, ctDNA has a high sensitivity and specificity in prediction of the prognosis of patients with CRC.

In Arabidopsis, HESO1 and URT1 act cooperatively on unmethylated miRNA and mRNA uridylation to induce their degradation. Their collaboration significantly impacts RNA metabolism in plants. However, the molecular mechanism determining the functional difference and complementarity of these two enzymes remains unclear. We previously solved the three-dimensional structure of URT1 in the absence and presence of UTP. In this study, we further determined the structure of URT1 in complex with a 5'-AAAU-3' RNA stretch that mimics the post-catalytic state of the mRNA poly(A) tail after the addition of the first uridine. Structural analysis and enzymatic assays revealed that L527 and Y592 endow URT1 with a preference to interact with purine over pyrimidine at the -1 RNA binding position, thus controlling the optimal number of uridine added to the 3' extremity of poly(A) as two. In addition, we observed that a large-scale conformational rearrangement in URT1 occurs upon binding with RNA from an 'open' to a 'closed' state. Molecular dynamic simulation supports an open-closed conformational selection mechanism employed by URT1 to interact with RNA substrates and maintain distributive enzymatic activity. Based on the above results, a model regarding the catalytic cycle of URT1 is proposed to explain its di-uridylation activity.

Here, we present a protocol for the detection of the two STING isoforms (erSTING and pmSTING) in human peripheral blood mononuclear cells or mouse splenocytes using Western blot and PCR. We detail steps to construct plasmids encoding each isoform and transfer them into mouse and human cell lines. Finally, we describe how to detect cell membrane localization of pmSTING using flow cytometry, immunoprecipitation, and immunofluorescence. This protocol is applicable for proteins with well-predicted topological structures. For complete details on the use and execution of this protocol, please refer to Li et al.1.

Coronaviruses (CoVs) are a family of the largest RNA viruses that typically cause respiratory, enteric, and hepatic diseases in animals and humans, imposing great threats to the public safety and animal health. Porcine deltacoronavirus (PDCoV), a newly emerging enteropathogenic coronavirus, causes severe diarrhea in suckling piglets all over the world and poses potential risks of cross-species transmission. Here, we use PDCoV as a model of CoVs to illustrate the reciprocal regulation between CoVs infection and host antiviral responses. In this study, downregulation of DNA polymerase delta interacting protein 3 (POLDIP3) was confirmed in PDCoV infected IPEC-J2 cells by isobaric tags for relative and absolute quantification (iTRAQ) and Western blotting analysis. Overexpression of POLDIP3 inhibits PDCoV infection, whereas POLDIP3 knockout (POLDIP3-/-) by CRISPR-Cas9 editing significantly promotes PDCoV infection, indicating POLDIP3 as a novel antiviral regulator against PDCoV infection. Surprisingly, an antagonistic strategy was revealed that PDCoV encoded nonstructural protein 5 (nsp5) was responsible for POLDIP3 reduction via its 3C-like protease cleavage of POLDIP3 at the glutamine acid 176 (Q176), facilitating PDCoV infection due to the loss of antiviral effects of the cleaved fragments. Consistent with the obtained data in IPEC-J2 cell model in vitro, POLDIP3 reduction by cleavage was also corroborated in PDCoV infected-SPF piglets in vivo. Collectively, we unveiled a new antagonistic strategy evolved by PDCoV to counteract antiviral innate immunity by nsp5-mediated POLDIP3 cleavage, eventually ensuring productive virus replication. Importantly, we further demonstrated that nsp5s from PEDV and TGEV harbor the conserved function to cleave porcine POLDIP3 at the Q176 to despair POLDIP3-mediated antiviral effects. In addition, nsp5 from SARS-CoV-2 also cleaves human POLDIP3. Therefore, we speculate that coronaviruses employ similar POLDIP3 cleavage mechanisms mediated by nsp5 to antagonize the host antiviral responses to sustain efficient virus infection.

Social distancing measures (SDMs) are community-level interventions that aim to reduce person-to-person contacts in the community. SDMs were a major part of the responses first to contain, then to mitigate, the spread of SARS-CoV-2 in the community. Common SDMs included limiting the size of gatherings, closing schools and/or workplaces, implementing work-from-home arrangements, or more stringent restrictions such as lockdowns. This systematic review summarized the evidence for the effectiveness of nine SDMs. Almost all of the studies included were observational in nature, which meant that there were intrinsic risks of bias that could have been avoided were conditions randomly assigned to study participants. There were no instances where only one form of SDM had been in place in a particular setting during the study period, making it challenging to estimate the separate effect of each intervention. The more stringent SDMs such as stay-at-home orders, restrictions on mass gatherings and closures were estimated to be most effective at reducing SARS-CoV-2 transmission. Most studies included in this review suggested that combinations of SDMs successfully slowed or even stopped SARS-CoV-2 transmission in the community. However, individual effects and optimal combinations of interventions, as well as the optimal timing for particular measures, require further investigation. This article is part of the theme issue 'The effectiveness of non-pharmaceutical interventions on the COVID-19 pandemic: the evidence'.

It is well established that stimulating delta-opioid receptor (DOR) with its specific agonists elicits neuroprotection against hypoxia/ischemia. Mitochondrial dysfunction plays a key role in hypoxic neuronal injury, but the effects of DOR activation on mitochondrial dysfunction in neurons are poorly elucidated. In this investigation, we studied the effects of [D-Ala2, D-Leu5] enkephalin (DADLE), a potent DOR agonist, on acute mitochondrial dysfunction and ensuing cell damage induced by sodium azide in primary rat cortical neuronal cultures, and explored possible mechanisms underlying. Here, we show that DADLE reverses NaN(3)-induced acute mitochondrial dysfunction by selectively activating DOR, mainly including mitochondrial membrane depolarization, mitochondrial Ca(2+) overload and reactive oxygen species generation. DOR stimulation also inhibits cytochrome c release and caspase-3 activation, and attenuates neuronal death caused by acute NaN(3) insults. Furthermore, DOR activation with DADLE protects neurons from acute NaN(3) insults mainly through PKC-ERK pathway, and mitochondrial ERK activation is especially required for DOR neuroprotection against acute mitochondrial dysfunction.

The growth of injured axons in the adult mammalian CNS is limited after injury. Three myelin proteins, Nogo, MAG (myelin-associated glycoprotein), and OMgp (oligodendrocyte myelin glycoprotein), bind to the Nogo-66 receptor (NgR) and inhibit axonal growth in vitro. Transgenic or viral blockade of NgR function allows axonal sprouting in vivo. Here, we administered the soluble function-blocking NgR ectodomain [aa 27-310; NgR(310)ecto] to spinal-injured rats. Purified NgR(310)ecto-Fc protein was delivered intrathecally after midthoracic dorsal over-hemisection. Axonal sprouting of corticospinal and raphespinal fibers in NgR(310)ecto-Fc-treated animals correlates with improved spinal cord electrical conduction and improved locomotion. The ability of soluble NgR(310)ecto to promote axon growth and locomotor recovery demonstrates a therapeutic potential for NgR antagonism in traumatic spinal cord injury.

In recent years, a novel, highly virulent variant of porcine epidemic diarrhea virus (PEDV) has emerged, causing substantial economic losses to the pork industry worldwide. In this study, a PEDV strain named LNsy was successfully isolated in China. Phylogenetic analysis based on the whole genome revealed that PEDV LNsy belonged to the G2 subtype. For the first time, a unique four amino acids (4-aa) insertion was identified in the COE region of the spike (S) protein (residues 499-640), resulting in an extra alpha helix in the spatial structure of the COE region. To determine changes in virus-neutralization (VN) antibody reactivity of the virus, polyclonal antibodies (PAbs) against the S protein of different subtypes were used in a VN test. Both PAbs against the S protein of the G1 and G2 subtype showed reduced VN reactivity to PEDV LNsy. Further, recombination analyses revealed that PEDV LNsy was the result of recombination between PEDV GDS13 and GDS46 strains at the genomic breakpoints (nt 17,959-20,594 in the alignment) in the ORF1b gene of the genomes. Pathological examination showed gross morphological pathological changes in the gut, including significant villus atrophy and shedding of the infected piglets. These results indicated that a 4-aa insertion in the COE region of the S protein may have partly altered the profiles of VN antibodies and thus it will be important to develop vaccine candidates to resist wild virus infection and to monitor the genetic diversity of PEDV.

Porcine deltacoronavirus (PDCoV) is a recently discovered enteropathogenic coronavirus and has caused significant economic impacts on the pork industry. Although studies have partly uncovered the molecular mechanism of PDCoV-host interaction, it requires further research. In this study, we explored the roles of Stromal Antigen 2 (STAG2) in PDCoV infection. We found that STAG2-deficient cells inhibited infection with vesicular stomatitis virus (VSV) and PDCoV, whereas restoration of STAG2 expression in STAG2-depleted (STAG2-/-) IPEC-J2 cells line restored PDCoV infection, suggesting that STAG2 is involved in the PDCoV replication. Furthermore, we found that STAG2 deficiency results in robust interferon (IFN) expression. Subsequently, we found that STAG2 deficiency results in the activation of JAK-STAT signaling and the expression of IFN stimulated gene (ISG), which establish an antiviral state. Taken together, the depletion of STAG2 activates the JAK-STAT signaling and induces the expression of ISG, thereby inhibiting PDCoV replication. Our study provides new insights and potential therapeutic targets for unraveling the mechanism of PDCoV replication.

Periodontitis is the most prevalent oral infection disease, which causes the destruction of periodontal supporting tissues and eventual tooth loss. This study aimed to investigate the molecular mechanism of miRNA-23b (miR-23b) in regulating the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in an inflammatory environment. Results revealed that tumor necrosis factor-α (TNF-α), a notoriously inflammatory cytokine, remarkably attenuated the osteogenic differentiation of hPDLSCs, which were partially rescued by SKL2001 (Wnt/β-catenin agonist). We further explored the underlying roles of miRNAs involved in TNF-α-inhibited osteogenesis of hPDLSCs. The miR-23b significantly increased with TNF-α stimulation, which was abolished by SKL2001. Similar to the effect of TNF-α, miR-23b agonist (agomir-23b) dramatically reduced the expression of runt-related transcription factor 2 (Runx2) and suppressed the osteogenic differentiation of hPDLSCs. The inhibition of miR-23b significantly increased Runx2, which is the major transcription factor during osteogenesis, thereby indicating that miR-23b was an endogenous regulator of Runx2 in hPDLSCs. Bioinformatic analysis and dual luciferase reporter assays confirmed that Runx2 was a target gene of miR-23b. Furthermore, the gain function assay of Runx2 revealed that the Runx2 overexpression efficiently reversed the suppression of the osteogenic differentiation of hPDLSCs with miR-23b agonist, suggesting that the suppressing effect of miR-23b on osteogenesis was mediated by Runx2 inhibition. Our study clarified that miR-23b mediated the TNF-α-inhibited osteogenic differentiation of hPDLSCs by targeting Runx2. Therefore, the expanded function of miR-23b in the osteogenesis of hPDLSCs under inflammatory conditions. This study might provide new insights and a novel therapeutic target for periodontitis.