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Objective To assess the efficacy and safety of ultrasonic bone curette‐assisted dome‐like laminoplasty in the treatment of ossification of longitudinal ligament (OPLL) involving C2. Methods A total of 64 patients with OPLL involving C2 level were enrolled. Thirty‐eight patients who underwent ultrasonic bone curette‐assisted dome‐like laminoplasty were defined as ultrasonic bone curette group (UBC), and 28 patients who underwent traditional high‐speed drill‐assisted dome‐like laminoplasty were defined as high‐speed drill group (HSD). Patient characteristics such as age, sex, body mass index (BMI), symptomatic duration, and other information like the type of OPLL, the time of surgery, blood loss, C2–C7 Cobb angle change and complications were all recorded and compared. The Japanese Orthopaedic Association (JOA) score, the nerve root functional improvement rate (IR), and the visual analogue scale (VAS) were used to assess neurological recovery and pain relief. The change of the distance between the apex of ossification and a continuous line connecting the anterior edges of the lamina was measured to assess the spinal expansion extent. The measured data were statistically processed and analyzed using SPSS 21.0 software, and the measurement data were expressed as mean ± SD. Results In ultrasonic bone curette (UBC) group and high‐speed drill group (HSD) group, the average time for laminoplasty was 52.3 ± 18.2 min and 76.0 ± 21.8 min and the mean bleeding loss volume was 155.5 ± 41.3 mL and 177.4 ± 54.7 mL, respectively, with a statistically significant difference between the groups. Both groups demonstrated a significant improvement in neurological function. However, the VAS score in UBC group was lower than in HSD group at the 6‐month follow‐up (P 0.05). Only one patient in the UBC group and five patients in the HSD group displayed cerebrospinal fluid (CSF) leakag. Conclusions With the use of ultrasonic bone curette in OPLL dome‐like decompression, the decompression surgery could be completed relatively safely and quickly. It effectively reduced the amount of intraoperative blood loss and complications, and had better initial recovery of neck pain., We assessed the efficacy and safety of ultrasonic bone curette‐assisted dome‐like laminoplasty in the treatment of C2 involved OPLL. We found that the decompression surgery could be completed relatively safely and with less intraoperative blood loss and fewer postoperative complications by using the ultrasonic bone curette.

Background context Laminoplasty is a common surgical method used to treat patients with cervical ossification of the posterior longitudinal ligament (OPLL). Although laminoplasty is preferred over traditional laminectomy, the factors affecting the complications and outcomes are unclear. Recently, sagittal balance indexes have been revealed to be predictors of clinical outcomes in patients with cervical degenerative diseases, but their relationships with laminoplasty-treated OPLL outcomes remains unknown. Purpose The purpose of this study is to evaluate the relationship of preoperative cervical sagittal balance indexes and clinical outcome in laminoplasty treated OPLL patients. Study design This is a retrospective case study. Patient population Between January 2015 and January 2017, 181 consecutively included patients who underwent cervical laminoplasty for OPLL were enrolled (male:female ratio=126:75; mean age=60.2 years). Cervical spine lateral radiographs in neutral, flexion, and extension positions were taken before and 2 years after the surgery. Outcome measures The C2–C7 Cobb angle, T1 slope, C1–C7 sagittal vertical axis (SVA), C2–C7 SVA, CGH (center of gravity of the head)-C7 SVA, cervical JOA (Japanese Orthopedic Association) score, and neck VAS (visual analogue scale) score were measured preoperatively and postoperatively at the 2-year follow-up. Methods The patients were divided into two groups according to changes in the lordotic angle or the recovery rate of the JOA score. The relationships between the postoperative lordosis loss or the clinical outcome and the preoperative variables, including the patient's age, JOA score, C2-C7 Cobb angle, T1 slope, C1-C7 SVA, C2-C7 SVA, and CGH-C7 SVA, were investigated. Results The patients were divided into two groups according to the postoperative change in the C2-C7 Cobb angle. There were no differences in the age, preoperative C2-C7 Cobb angle, C1-C7 SVA, or C2-C7 SVA; there was only a difference in the preoperative CGH-C7 SVA and T1 slope level (p=.038, p=.042). The postoperative JOA and JOA recovery rate were related to the postoperative lordosis loss in cervical alignment (p=.048, p=.031). We again divided the patients into two groups according to the JOA recovery rate and found that only the preoperative CGH-C7 SVA and C1–C7 SVA were related to the neurological outcome (p=.011, p=.047). According to the multivariate logistic regression analysis, higher preoperative CGH-C7 SVA levels were significantly associated with decreases in the lordosis angle postoperatively and the clinical outcome (p=.018, OR=1.225; p=.034, OR=1.654). The ROC (receiver operating characteristic) analysis revealed that the proper cutoff value of preoperative CGH-C7 SVA for predicting the postoperative loss of lordosis and clinical outcomes is 3.8 cm. Conclusion Preoperative cervical sagittal balance indexes are related to the outcomes of OPLL patients after laminoplasty. Patients with high preoperative CGH-C7 SVA levels have a high probability of developing sagittal imbalances and neurological symptoms of the cervical spine, and this measurement can be used as a predictor of outcomes in laminoplasty-treated cervical OPLL patients.

Purpose To investigate transitions in resistance mechanisms, virulence characteristics and molecular epidemiology of carbapenem-resistant Klebsiella pneumoniae (CRKP) during 2003-2016 in a major Eastern Chinese medical center. Patients and methods From a total of 2299 K. pneumoniae clinical strains collected from 2003 to 2016, 214 were found to be CRKP isolates and were selected for further study. Characterization of these was conducted by molecular detection of antibiotic resistance markers and virulence determinants, modified carbapenem inactivation method and multilocus sequence typing (MLST). Results In this study, the prevalence of CRKP was increasing over the 14-year period, mirroring a national trend. These CRKP strains were resistant to most of the tested, clinically relevant drugs. The majority of these CRKP strains were positive for carbapenemases, with the Klebsiella pneumoniae carbapenemase (KPC) found to be the dominant type (207/210, 98.6%). The carrier rates of virulence genes uge, entB, fimH, mrkD and ureA increased in 2016, while the ybtA, iucA and irp2 showed a relatively constant trend. From MLST data, ST11 (88.8%, 190/214) was the preponderant sequence type (ST), followed by ST15 (1.9%, 4/214) and ST656 (1.4%, 3/214). Several strains with less common STs (ST690, ST895, ST1823 and ST1384) were also detected, and these too showed high levels of antimicrobial resistance. Conclusion The average national rise in CRKP across China is mirrored in this in-depth analysis of a single hospital, while the prevalence of hypervirulent CRKP (such as ST15) was relatively low as of 2016. Continuous monitoring is necessary to keep track of CRKP and should include the prospect of newly emerging strains with less common STs and the prospect of detecting carbapenem-resistant, carbapenemase-negative Klebsiella pneumoniae.

Objectives The aim of this study was to investigate the mechanisms of fosfomycin resistance and epidemiological characteristics in fosfomycin-resistant enterococci in China. Methods A collection of 761 enterococcal clinical isolates from a teaching hospital in Wenzhou, China were studied. The fosfomycin susceptibility of the isolates was investigated by the agar dilution method. The isolates were also analysed for mechanisms of re fosfomycin resistance by PCR and quantitative real-time PCR. Furthermore, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed to analyse the molecular epidemiological characteristics of the fosfomycin-resistant isolates. Results In this study, 0.3% (1/372) of Enterococcus faecalis and 4.9% (19/389) of Enterococcus faecium clinical isolates were found to be resistant to fosfomycin. Among the 20 fosfomycin-resistant isolates, 5 harboured the fosB gene, 10 carried multiple amino acid substitutions in MurA, and 6 showed high-level expression of the fosX gene; of note, 1 isolate simultaneously carried fosB and amino acid mutation in MurA. Furthermore, a high degree of homology in the fosfomycin-resistant enterococci was confirmed using MLST and PFGE. Conclusion These finding demonstrate that the fosB gene, mutations in the fosfomycin target enzyme MurA, and a high expression level of fosX were the resistance mechanisms in these fosfomycin-resistant enterococci.

Objectives: Ossification of the posterior longitudinal ligament (OPLL) presents as the development of heterotopic ossification in the posterior longitudinal ligament of the spine. The etiology of OPLL is genetically linked, as shown by its high prevalence in Asian populations. However, the molecular mechanism of the disease remains obscure. In this study, we explored the function and mechanism of OPLL-specific microRNAs. Methods: The expression levels of the ossification-related OPLL-specific miR-181 family were measured in normal or OPLL ligament tissues. The effect of miR-181a on the ossification of normal or pathogenic ligament cells was tested using real-time polymerase chain reaction (PCR), Western blot, alizarin red staining and alkaline phosphatase (ALP) staining. The candidate targets of miR-181 were screened using a dual luciferase reporter assay and functional analysis. The link between miR-181a and its target PBX1 was investigated using chromatin immunoprecipitation, followed by real-time PCR detection. Histological and immunohistochemical analysis as well as micro-CT scanning were used to evaluate the effects of miR-181 and its antagonist using both tip-toe-walking OPLL mice and in vivo bone formation assays. Results: Using bioinformatic analysis, we found that miR-181a-5p is predicted to play important roles in the development of OPLL. Overexpression of miR-181a-5p significantly increased the expression of ossification-related genes, staining level of alizarin red and ALP activity, while the inhibition of miR-181a-5p by treatment with an antagomir had the opposite effects. Functional analysis identified PBX1 as a direct target of miR-181a-5p, and we determined that PBX1 was responsible for miR-181a-5p's osteogenic phenotype. By chromatin immunoprecipitation assay, we found that miR-181a-5p controls ligament cell ossification by regulating PBX1-mediated modulation of histone methylation and acetylation levels in the promoter region of osteogenesis-related genes. Additionally, using an in vivo model, we confirmed that miR-181a-5p can substantially increase the bone formation ability of posterior ligament cells and cause increased osteophyte formation in the cervical spine of tip-toe-walking mice. Conclusions: Our data unveiled the mechanism by which the miR-181a-5p/PBX1 axis functions in the development of OPLL, and it revealed the therapeutic effects of the miR-181a-5p antagomir in preventing OPLL development both in vivo and in vitro. Our work is the first to demonstrate that microRNA perturbation could modulate the development of OPLL through epigenetic regulation.

Aims: To explore the role of the Sphingosine 1-Phosphate (S1P)/Receptor2 (S1PR2) pathway in thrombin-induced hyperpermeability (TIP) and to test whether bivalirudin can reverse TIP via the S1P-S1PRs pathway.Methods and Results: Using western blot, we demonstrated that Human umbilical vein endothelial cells (HUVECs) that were cultured with 2 U/ml thrombin showed significantly increased S1PR2 expression while S1PR1and three kept unchanged. Such increment was attenuated by JTE-013 pretreatment and by presence of bivalirudin. Exposure of 2 U/ml of thrombin brought a higher level of S1P both intracellularly and extracellularly within the HUVECs by using ELISA detecting. Thrombin induced S1P and S1PR2 increment was restored by usage of PF543 and bivalirudin. Bivalirudin alone did not influenced the level of S1P and S1PR1,2, and S1PR3 compare to control group. As a surrogate of cytoskeleton morphology, phalloidin staining and immunofluorescence imaging were used. Blurry cell edges and intercellular vacuoles or spaces were observed along thrombin-exposed HUVECs. Presence of JTE-013 and bivalirudin attenuated such thrombin-induced permeability morphological change and presence of heparin failed to show the protective effect. Transwell chamber assay and probe assay were used to measure and compare endothelial permeability in vitro. An increased TIP was observed in HUVECs cultured with thrombin, and coculture with bivalirudin, but not heparin, alleviated this increase. JTE-013 treatment yielded to similar TIP alleviating effect. In vivo, an Evans blue assay was used to test subcutaneous and organ microvascular permeability after the treatment of saline only, thrombin + saline, thrombin + bivalirudin, thrombin + heparin or thrombin + JTE-013. Increased subcutaneous and organ tissue permeability after thrombin treatment was observed in thrombin + saline and thrombin + heparin groups while treatment of bivalirudin and JTE-013 absent this effect.Conclusion: S1P/S1PR2 mediates TIP by impairing vascular endothelial barrier function. Unlike heparin, bivalirudin effectively blocked TIP by inhibiting the thrombin-induced S1P increment and S1PR2 expression, suggesting the novel endothelial protective effect of bivalirudin under pathological procoagulant circumstance.

Although several lines of evidence existed suggesting that Mortalin was linked with survival in malignant tumors; it has been barely described regarding the prognostic involvement of its expression in hepatocellular carcinoma (HCC). Herein, to understand the prognostic meaning of Mortalin expression, Immunohistochemistry was undertaken to observe the immunohistochemical characteristics of Mortalin on HCC tissue microarray consisting of 90 cases of HCC and its paired normal control dots, followed by detailed statistical analysis with the accompanying clinicopathological variables available, including gender, age, tumor size, differentiation, cirrhosis, vascular invasion, clinical stage, T classification and intrahepatic metastases. Meanwhile, Kaplan-Meier survival curve was plotted to analyze the prognostic difference for patients with high and low expression of Mortalin. It was exhibited that Mortalin was over-expressed in HCC tissues relative to paired normal control and elevated Mortalin significantly correlated with vascular invasion, clinical stage and intrahepatic metastasis. Kaplan-Meier survival analysis revealed that Mortalin was remarkably associated with overall survival and disease-free survival. Multivariate Cox regression analysis showed that expression of Mortalin was an independent prognostic factor in HCC. Collectively, the data we provided here support the prognostic prediction value of Mortalin in HCC.

Highlights • Dissecting basilar artery aneurysm (DBAA) is relatively rare. • We report the first case of a DBAA manifesting as sudden sensorineural hearing loss. • This case report adds to the symptom spectrum of DBAA., Dissecting aneurysms of the basilar artery are rare and severe entities with high hemorrhage and mortality rates. Common clinical symptoms of this condition include cerebral ischemia, subarachnoid hemorrhage, and neurological deficits induced by space-occupying effects. Vertigo, headache, and cranial nerve paralysis are also frequent symptoms. We report a case of dissecting basilar artery aneurysm manifesting as sudden sensorineural hearing loss. A 53-year-old man presented with vertigo, left-sided tinnitus, and sudden sensorineural hearing loss for 1 hour. A physical examination showed left-sided hearing loss and bilateral horizontal nystagmus, and a radiological examination showed a dissecting aneurysm originating from the basilar artery. A diagnosis of a dissecting aneurysm of the basilar artery with sudden sensorineural hearing loss was made. The aneurysm was treated using stent-assisted coil embolization. Postoperatively, the vertigo and tinnitus, as well as the left hearing loss, greatly improved. Follow-up digital subtraction angiography 10 months after the operation showed complete disappearance of the dissecting aneurysm of the basilar artery. In patients with sudden sensorineural hearing loss, clinicians should be aware of the potential of a dissecting basilar artery aneurysm. An interdisciplinary approach is important in diagnosis and treatment of this rare condition.

Parkinson's disease (PD), one of the motor system disorders, is characterized by the loss of dopamine-producing brain cells. Accumulating evidence has highlighted the involvement of microRNAs (miRs) in the development and progression of PD. Hence, we aimed at exploring possible effects of miR-326 on the progression of PD in mice in an attempt to elucidate the underlying mechanism associated with the kallikrein-related peptidase 7 (KLK7)-mediated mitogen-activated protein kinase (MAPK) signaling pathway. In order to identify the regulatory relationship between miR-326 and KLK7 and its biological significance in PD, PD mouse models were established and subsequently treated with mimics or inhibitors of miR-326 or siRNA-KLK7. The content of striatal dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 3-methoxytyrosine (3-MT), superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and malondialdehyde (MDA); positive expression of tyrosine hydroxylase (TH) and inducible nitric oxide synthase (iNOS); and the levels of IL-1, IL-6, TNF-α, INF-γ, and MAPK signaling pathway-related genes were determined accordingly. The results obtained indicated that KLK7 was negatively targeted by miR-326, with lower miR-326 and higher KLK7 detected among PD mice. The overexpression of miR-326 or silencing of KLK7 was demonstrated to increase the content of DA, DOPAC, HVA, 3-MT, SOD, GSH-Px, and TH positive expression, while reducing iNOS positive expression, MDA content and cell apoptosis, as well as inhibited levels of IL-1, IL-6, TNF-α, INF-γ, and mRNA and protein levels of p38, ERK, JNK, and caspase-3. Taken together, these results provided evidence suggesting that miR-326 could inhibit iNOS activation and apoptosis of dopaminergic neurons through inhibiting the MAPK signaling pathway and negatively regulating KLK7 in mice with PD. These findings highlight the potential of miR-326 as a novel target for future PD treatment.

Chlorhexidine is widely used as a disinfectant in hospitals, which may impose a selective pressure on bacteria. This study aimed to determine whether continuous exposure to chlorhexidine could lead to adaptive resistance and cross-resistance as well as investigating potential resistance mechanisms. Three clinical Klebsiella pneumoniae strains susceptible to conventional antimicrobials were selected and were continuously cultured in broth with gradually increasing concentrations of chlorhexidine. Antimicrobial susceptibility was determined. Mechanisms of acquired resistance to chlorhexidine and colistin were analysed by PCR and reverse transcription quantitative PCR (RT-qPCR). Furthermore, fitness was assessed through growth curve assays. Increased resistance to chlorhexidine and colistin was observed in all strains. Expression of the cepA gene was upregulated in the adapted strains, suggesting that hyperexpression of CepA was probably the main mechanism of adaptive resistance to chlorhexidine. The amino acid substitutions Leu82Arg and Arg256Gly in PmrB were detected in all of the adapted strains, whilst Leu344Pro was only identified in one adapted strain, indicating that the PmrB substitution was responsible for the cross-resistant phenotype. Moreover, chlorhexidine adaptation might have an effect on bacterial growth.

Although liver regeneration has been intensively studied in various ways, the mechanisms underlying liver regeneration remain elusive. Apoptosis-stimulating protein two of p53 (ASPP2) was discovered as a binding partner of p53 and plays an important role in regulating cell apoptosis and growth. However, the role of ASPP2 in hepatocyte proliferation and liver regeneration has not been reported. The expression profile of ASPP2 was measured in a mouse model with 70% partial hepatectomy (PHX). Liver regeneration and hepatocyte proliferation were detected in wild-type (ASPP2+/+) and ASPP2 haploinsufficient (ASPP2+/−) mice with PHX. The mammalian target of rapamycin (mTOR) and autophagy pathways were analyzed in the ASPP2+/+ and ASPP2+/− mice with PHX. After rapamycin or 3-methyladenine (3-MA) treatment, hepatocyte proliferation and liver regeneration were analyzed in the ASPP2+/+ and ASPP2+/− mice with PHX. ASPP2 expression was shown to be upregulated at the early stage and downregulated at the late stage. Compared to the ASPP2+/+ mice, liver regeneration was enhanced in ASPP2+/− mice with 70% PHX. In addition, compared to the ASPP2+/+ mice, the mTORC1 pathway was significantly upregulated and the autophagic pathway was downregulated in ASPP2+/−mice with 70% PHX. Inhibition of the mTORC1 pathway significantly suppressed liver regeneration in ASPP2+/− mice with 70% PHX. In contrast, disruption of the autophagic pathway further enhanced liver regeneration in ASPP2+/− mice with 70% PHX. ASPP2 deficiency can promote liver regeneration through activating the mTORC1 pathway, which further regulates downstream molecules, such as those related to autophagy and p70S6K expression in mouse model post-PHX.

Shanji Nan, Peng Wang, Yizhi Zhang, Jia Fan Department of Neurology, The Second Hospital of Jilin University, Changchun, 130041, People’s Republic of ChinaCorrespondence: Jia FanDepartment of Neurology, The Second Hospital of Jilin University, No. 218, Ziqiang Street, Changchun, 130041, Jilin Province, People’s Republic of ChinaTel +86-13843028656Email fanjia@jlu.edu.cnPurpose: Recent evidence has highlighted the anti-inflammatory properties of the constituent of Green Tea Polyphenols (GTP), epigallocatechin-3-gallate (EGCG) which has been suggested to exert a neuroprotective effect on Alzheimer’s disease (AD). The current study aimed to elucidate the effect of EGCG on memory function in rats with AD.Methods: AD rat models were initially established through an injection with Aβ 25– 35 solution, followed by gavage with EGCG at varying doses to determine the effect of EGCG on learning and cognitive deficits in AD. Morris water maze test was conducted to evaluate the spatial memory function of the rats. Immunohistochemistry and Western blot analysis were performed to identify Tau phosphorylation. The expression of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) mRNA and protein in rat hippocampus was measured by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis. Acetylcholinesterase (AchE) activity, Aβ 1-42 expression and Ach content were all detected using enzyme-linked immunosorbent assay (ELISA).Results: EGCG intervention brought about a decrease in the escape latency period while increasing the time at the target quadrant among the AD rats. EGCG decreased the hyperphosphorylation of Tau in hippocampus. BACE1 expression and activity as well as the expression of Aβ 1-42 were suppressed by EGCG. Moreover, EGCG promoted Ach content by diminishing the activity of AchE.Conclusion: The current study demonstrates that EGCG may diminish the hyperphosphorylation of the Tau protein, downregulate BACE1 and Aβ 1-42 expression to improve the antioxidant system and learning and memory function of rats with AD.Keywords: Aβ 1-42, ACh, AChE, BACE1, epigallocatechin-3-gallate, learning and memory function, tau hyperphosphorylation

Background Acute coronary syndrome (ACS) is a cardiac emergency with high mortality. Exposure to high copper (Cu) concentration has been linked to ACS. However, whether DNA methylation contributes to the association between Cu and ACS is unclear. Methods We measured methylation level at > 485,000 cytosine-phosphoguanine sites (CpGs) of blood leukocytes using Human Methylation 450 Bead Chip and conducted a genome-wide meta-analysis of plasma Cu in a total of 1243 Chinese individuals. For plasma Cu-related CpGs, we evaluated their associations with the expression of nearby genes as well as major cardiovascular risk factors. Furthermore, we examined their longitudinal associations with incident ACS in the nested case-control study. Results We identified four novel Cu-associated CpGs (cg20995564, cg18608055, cg26470501 and cg05825244) within a 5% false discovery rate (FDR). DNA methylation level of cg18608055, cg26470501, and cg05825244 also showed significant correlations with expressions of SBNO2, BCL3, and EBF4 gene, respectively. Higher DNA methylation level at cg05825244 locus was associated with lower high-density lipoprotein cholesterol level and higher C-reactive protein level. Furthermore, we demonstrated that higher cg05825244 methylation level was associated with increased risk of ACS (odds ratio [OR], 1.23; 95% CI 1.02–1.48; P = 0.03). Conclusions We identified novel DNA methylation alterations associated with plasma Cu in Chinese populations and linked these loci to risk of ACS, providing new insights into the regulation of gene expression by Cu-related DNA methylation and suggesting a role for DNA methylation in the association between copper and ACS.

Neurodegeneration in Alzheimer's disease (AD) results in microglial activation, which may participate in the inflammatory cascade accelerating tissue damage. In this study, we sought to characterize the alleviatory role of microRNA-711 (miR-711) encapsulated in microglia-derived extracellular vesicles (EVs) in a model of AD. Ultracentrifugation was employed to extract EVs from microglia (BV2 cells), which were identified using Western blot analysis of the EVs marker proteins Alix and CD63. A repetitive mild traumatic brain injury (rmTBI) mouse model was induced by controlled cortical impact. After overexpressing miR-711 or 1,4,5-trisphosphate 3-kinase B (Itpkb) in BV2 cells, we evaluated the inflammation in BV2 cells and the ratio of microglia M2/M1. Further, we injected BV2 cell-secreted EVs with overexpressed miR-711 or Itpkb into rmTBI mice through a tail vein to investigate the inflammation markers in mouse serum and, the M2/M1 phenotype ratio of microglia in brain tissues, and to evaluate neurological deficit and cognitive function. The EVs obtained by ultracentrifugation were verified by the presence of Alix and CD63 expression. Mechanistic studies suggested that miR-711 targeted and inhibited Itpkb, thereby repressing Tau phosphorylation and increasing the ratio of M2/M1. Furthermore, miR-711-containing EVs reduced the score of neurological deficits and improved cognitive function in rmTBI mice. The administration of microglia-derived EVs loaded with miR-711, which mediated the hyperphosphorylation of Tau protein in the Itpkb pathway, effectively alleviated neurodegenerative changes and cognitive dysfunction in AD.

Background Bipolar disorder (BPD) is a common mood disorder that is often goes misdiagnosed or undiagnosed. Recently, machine learning techniques have been combined with neuroimaging methods to aid in the diagnosis of BPD. However, most studies have focused on the construction of classifiers based on single-modality MRI. Hence, in this study, we aimed to construct a support vector machine (SVM) model using a combination of structural and functional MRI, which could be used to accurately identify patients with BPD. Methods In total, 44 patients with BPD and 36 healthy controls were enrolled in the study. Clinical evaluation and MRI scans were performed for each subject. Next, image pre-processing, VBM and ReHo analyses were performed. The ReHo values of each subject in the clusters showing significant differences were extracted. Further, LASSO approach was recruited to screen features. Based on selected features, the SVM model was established, and discriminant analysis was performed. Results After using the two-sample t-test with multiple comparisons, a total of 8 clusters were extracted from the data (VBM = 6; ReHo = 2). Next, we used both VBM and ReHo data to construct the new SVM classifier, which could effectively identify patients with BPD at an accuracy of 87.5% (95%CI: 72.5–95.3%), sensitivity of 86.4% (95%CI: 64.0–96.4%), and specificity of 88.9% (95%CI: 63.9–98.0%) in the test data (p = 0.0022). Conclusions A combination of structural and functional MRI can be of added value in the construction of SVM classifiers to aid in the accurate identification of BPD in the clinic.

Background: Coronavirus disease (COVID-19) is a current global public health emergency. However, current research on the blood test results of pregnant women with COVID-19 is insufficient. Methods: A case-control study was carried out based on clinical blood test results. Pregnant COVID-19 patients, pregnant COVID-19 patients with diabetes, and pregnant COVID-19 patients with hypertension, were assessed in this study. Also, 120 controls were matched by age, parity, fetus number, and presence of chronic disease. T-tests, Chi-square tests, Wilcoxon signed-rank tests, and Kruskal-Wallis tests were used to compare data from the blood tests and liver function indices among the selected groups. Results: Between January 24 and March 14, 2020, 60 pregnant COVID-19 patients delivered at the Maternal and Child Health Hospital of Hubei Province. The average maternal age of pregnant COVID-19 patients was 30.97 years and the mean gestational period was 37.87 weeks. 71.67% (43/60) of pregnant COVID-19 patients gave birth by cesarean delivery. In total, 21.67% (13/60) were diagnosed with diabetes and 18.33% (11/60) were diagnosed with hypertension during pregnancy. Compared to controls, pregnant COVID-19 patients showed significantly lower numbers of blood lymphocytes and higher numbers of neutrophils, as well as higher levels of C-reactive protein and total bilirubin. Among the three groups, pregnant COVID-19 patients with diabetes had significantly higher levels of neutrophils and lower levels of total protein. Aspartate transaminase levels were higher in pregnant COVID-19 patients with hypertension than in pregnant COVID-19 patients with no comorbidities and controls with hypertension. Interpretations: Blood and liver function indices indicate that chronic complications, including hypertension and diabetes, could increase the risk of inflammation and liver injury in pregnant COVID-19 patients.

Identifying cognitive dysfunction in the early stages of Bipolar Disorder (BD) can allow for early intervention. Previous studies have shown a strong correlation between cognitive dysfunction and number of manic episodes. The objective of this study was to apply machine learning (ML) techniques on a battery of cognitive tests to identify first-episode BD patients (FE-BD). Two cohorts of participants were used for this study. Cohort #1 included 74 chronic BD patients (CHR-BD) and 53 healthy controls (HC), while the Cohort #2 included 37 FE-BD and 18 age- and sex-matched HC. Cognitive functioning was assessed using the Cambridge Neuropsychological Test Automated Battery (CANTAB). The tests examined domains of visual processing, spatial memory, attention and executive function. We trained an ML model to distinguish between chronic BD patients (CHR-BD) and HC at the individual level. We used linear Support Vector Machines (SVM) and were able to identify individual CHR-BD patients at 77% accuracy. We then applied the model to Cohort #2 (FE-BD patients) and achieved an accuracy of 76% (AUC = 0.77). These results reveal that cognitive impairments may appear in early stages of BD and persist into later stages. This suggests that the same deficits may exist for both CHR-BD and FE-BD. These cognitive deficits may serve as markers for early BD. Our study provides a tool that these early markers can be used for detection of BD.

Aims Nonalcoholic fatty liver disease (NAFLD) is a lipid metabolism disorder. Naringin (a main active ingredient in Ganshuang granules) is a flavanone that has been demonstrated to exert hepatoprotective and antifibrotic effects. The present study aimed to use a novel tissue-engineered fatty liver model to assess the effects and mechanisms of naringin on NAFLD. Main methods Intracellular triglyceride (TG) was examined by oil red O staining and commercial kits. The proteins associated with lipid metabolism were measured by western blotting and/or qPCR. Very low-density lipoprotein (VLDL) was measured by ELISA. A CCK8 assay was used to assess the cytotoxicity of naringin. Molecular docking was used to predict the interactions and binding patterns between naringin and target proteins. Key findings Naringin significantly reduced intracellular TG accumulation by 52.7% in tissue-engineered fatty (TEF) livers, and also the level of pyruvate dehydrogenase kinase 4. Naringin downregulated CD36 and proliferator activated-receptor γ expression, reducing the uptake of FFAs; naringin also downregulated de novo liposynthetases by reducing acetyl CoA carboxylase, fatty acid synthetase etc. in TEF livers. Moreover, naringin increased the expression of proliferator activated-receptor α (PPAR-α) and carnitine palmitoyltransferase 1 to improve the oxidation of fatty acids. The levels of VLDL secreted from TEF livers were reduced by 24.7% after naringin treatment. Molecular docking analyses determined the bioactivity of naringin through its specific binding to CD36 and PPAR-α. Significance Naringin improved lipid metabolism disorders in TEF livers by reducing fatty acid uptake and de novo lipogenesis and increasing fatty acid oxidation. CD36 and PPAR-α might be specific targets of naringin.

Background Accumulating evidence has reported the role of microRNA (miR) on ischemic brain injury. We aim to investigate the mechanism of miR-376b-5p/Sex-determining region Y-box 7 (SOX7)/Wnt/β-catenin axis in mice with ischemic brain injury. Methods Transient middle cerebral artery occlusion (tMCAO) model was established by suture method. Expression levels of miR-376b-5p, SOX7, and Wnt/β-catenin pathway-related proteins (Wnt3a and β-catenin) in brain tissues of tMCAO mice were determined by RT-qPCR and western blot analysis. The target relationship between miR-376b-5p and SOX7 was tested by bioinformatics analysis and luciferase activity assay. The neurological scores of mice were recorded and their behaviors were observed. Moreover, the brain damage, oxidative stress indices, hemoglobin (Hb) content, content of brain water, infarct area, TUNEL positive cells, blood-brain barrier permeability and the number of intact neurons in the ischemic-side brain tissues of tMCAO mice were detected via upregulated miR-376b-5p or downregulated SOX7. Results In mice with ischemic brain injury, miR-376b-5p targeted and downregulated SOX7 in brain tissues, thus activating the Wnt/β-catenin pathway. The tMCAO mice showed higher neurological scores, severe brain damage, decreased number of neurons, as well as elevated blood-brain barrier permeability, Hb content, cerebral edema and infarct area. These symptoms could be alleviated by miR-376b-5p elevation or SOX7 inhibition. SOX7 overexpression reversed the effects of miR-376b-5p elevation on tMCAO mice. Conclusion Our study suggests that miR-376b-5p could improve the blood-brain barrier permeability, relieve brain edema and decrease infarct area, thus improve ischemic brain injury via the inhibition of SOX7 and activation of Wnt/β-catenin pathway.

Testing for effects of pesticides on nontarget organisms is an integral part of ecological risk assessment. In the present study, the acute toxicity of sulfoxaflor to earthworms was evaluated using an artificial soil toxicity test, and sublethal effects were assessed through oxidative stress and metabolomics. Sulfoxaflor is a supertoxic pollutant to earthworms that easily bioaccumulates in earthworms, and contains LC2, LC10, and LC50 values of 0.08 (0.04–0.13), 0.19 (0.11–0.25), and 0.54 (0.45–0.65) mg/kg, respectively. Sublethal doses of sulfoxaflor resulted in oxidative damage to earthworms in which antioxidant enzymatic activities including SOD, CAT, and GST were significantly inhibited and MDA content accumulated. Metabolomics analysis suggested that the energy metabolism and the urea cycle in earthworms were significantly activated, while nucleotide metabolism was depressed, which could cause DNA damage. The results suggest earthworms have the potential to be a new entry point for sulfoxaflor into th...

Most of our knowledge on the mechanisms underlying diatom-bacterial interactions has been acquired through studies involving isolation of culturable partners. Here, we established a laboratory model of intermediate complexity between complex natural communities and laboratory pure culture models. We investigated the whole community formed by the freshwater diatom Asterionella formosa and its associated bacteria in a laboratory context, including both culturable and unculturable bacteria. Combining cellular and molecular approaches, we showed that in laboratory cultures, A. formosa microbiome was dynamic and comprised of numerous bacterial species (mainly Proteobacteria and Bacteroidetes). Using metagenomics, we explored several metabolic potentials present within the bacterial community. Our analyses suggested that bacteria were heterotrophic although a third of them (Alpha- and Beta-proteobacteria) could also be phototrophic. About 60% of the bacteria, phylogenetically diverse, could metabolize glycolate. The capacity to synthesize molecules such as B vitamins appeared unevenly distributed among bacteria. Altogether, our results brought insights into the bacterial diversity found in diatom-bacterial communities and hinted at metabolic interdependencies within the community that could result in diatom-bacterial and bacterial-bacterial interactions. The present work allowed us to explore the functional architecture of the bacterial community associated with A. formosa in culture and is complementary to field studies. This article is protected by copyright. All rights reserved.

The intestine is the main reservoir of bacterial pathogens in most organisms. Klebsiella pneumoniae is an important opportunistic pathogen associated with nosocomial bacterial infections. Intestinal colonization with K. pneumoniae has been shown to be associated with an increased risk of subsequent infections. However, not all K. pneumoniae strains in the intestine cause further infection, and the distinction of the difference among strains that cause infection after colonization and the ones causing only asymptomatic colonization is unclear. In this study, we report a case of a hospitalized patient from the ICU. We screened out two intestine colonization strains (FK4111, FK4758) to analyze the subsequent infection conditions. We set up infection models of zebrafish and Galleria mellonella to establish the differences in the potential for causing subsequent infection and the immunological specificities after K. pneumoniae intestine colonization. Sudan Black B and neutral red staining results indicated that FK4758 was more responsive to neutrophil recruitment and phagocytosis of macrophages than FK4111. The results of the assessment of the organ bacterial load revealed that FK4111 and FK4758 both had the highest bacterial loads in the zebrafish intestine compared to those in other organs. However, in the zebrafish spleen, liver, and heart, the FK4758 load was significantly higher than that of FK4111. The ST37 strain FK4111, which does not produce carbapenemase, did not cause infection after colonization, whereas the ST11 strain FK4758, which produces carbapenemase, caused infection after intestinal colonization. Our finding demonstrated that not all intestinal colonization of K. pneumoniae subsequently caused infections, and the infections of K. pneumoniae after colonization are different. Therefore, the infection models we established provided possibility for the estimation of host-microbial interactions.

The aim of this study was to investigate mechanisms of nitrofurantoin resistance and epidemiological characteristics in Escherichia coli clinical isolates. From a total of 1444 E. coli clinical isolates collected from our hospital in 2015, 18 (1.2%) nitrofurantoin-resistant E. coli isolates were identified with nitrofurantoin minimum inhibitory concentrations (MICs) ranging from 128 µg/mL to ≥512 µg/mL. The prevalence of the nfsA gene in nitrofurantoin-resistant, -intermediate and -susceptible isolates was 88.9%, 88.9% and 100%, respectively, and the prevalence of the nfsB gene was 66.7%, 61.1% and 100%, respectively. Eight nitrofurantoin-resistant isolates and two nitrofurantoin-intermediate isolates possessed oqxAB genes. In nitrofurantoin-resistant isolates, mutations in NfsA (the majority of mutated sites were I117T and G187D, accounting for 38.9%) and/or NfsB were detected, whereas only NfsA mutations were found in intermediate isolates and no sequence changes were detected in susceptible isolates. A ≥4-fold decrease in MIC was observed in eight nitrofurantoin-resistant isolates following addition of the efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone (CCCP). The mean expression level of oqxB in nitrofurantoin-resistant isolates increased ca. 7-fold compared with intermediate isolates. Multilocus sequence typing (MLST) categorised the 18 nitrofurantoin-resistant isolates into 11 different sequence types. Pulsed-field gel electrophoresis (PFGE) analysis revealed that homology among the nitrofurantoin-resistant isolates was low and sporadic. In conclusion, mutations in nfsA and nfsB were the main mechanisms leading to nitrofurantoin resistance, and overexpression of the oqxAB gene might help to further increase the MIC of nitrofurantoin.

P-glycoprotein (P-gp) is an important multidrug resistance (MDR) regulator for leukemia to mediate its development and thus can be considered as a powerful reference for the diagnosis of MDR. The detection of P-gp is of vital significance and has attracted considerable concerns. In this study, we proposed a surface-enhanced Raman scattering (SERS) method for the evaluation of P-gp expression levels in leukemia cell lines. Basically, we utilized an aqueous phase sandwich-type immunoassay to analyze the expression of P-gp. First, anti-CD45-decorated magnetic beads (MBs) and P-gp antibody-decorated SERS probes were fabricated. CD45 is a common protein expressed in all leukemia cells. As a result, a sandwich immunocomplex can be formed by the MBs, P-gp-overexpressed leukemia cells, and SERS probes. The expression level of P-gp determines the amount of SERS probes that can be captured. Consequently, the SERS intensity of the immunocomplex can be used to evaluate the expression level of P-gp. In a typical procedure, we measured the P-gp expression of an MDR leukemia cell line (K562/ADM) as well as unprocessed whole-blood samples. The SERS intensity of K562/ADM cells was highly correlated with the extent of MDR or the incubation time of adriamycin (which is an MDR inducing drug). In addition, the SERS intensity of the refractory/relapsing group was about sixfolds of that of the control group ( P < 0.01). These results demonstrated that the proposed method holds excellent sensitivity, specificity, reliability, and application potential in assessing both cultured cells and clinical samples. With these outstanding features, we anticipated that such a SERS-based method could be very helpful for the clinical diagnosis of early-stage MDR in leukemia.

Linezolid is considered as a last-resort antimicrobial agent, the resistance of which is of great concern. The aim of this study was to investigate the mechanisms and transferability of linezolid resistance and molecular epidemiology of linezolid-resistant enterococcal isolates in Wenzhou, China. A collection of 1623 enterococcal strains, including 789 Enterococcus faecalis and 834 Enterococcus faecium, were isolated from our hospital during 2011-2016. Antimicrobial susceptibility testing and clinical data analysis were performed. Molecular mechanisms of linezolid resistance, including the existence of resistance genes cfr and optrA, as well as the mutations in 23S rRNA and ribosomal proteins L3, L4, and L22, were investigated by PCR and sequencing. Conjugation experiments were conducted, and epidemiological characteristics were analyzed by PFGE and MLST. In our study, 31 (3.93%) E. faecalis and 2 (0.24%) E. faecium exhibited resistance to linezolid. Risk factors correlated with linezolid-resistant enterococcal infections included gastrointestinal surgery hospitalization, urogenital disorders, tumor, diabetes, and polymicrobial infections. Among these isolates, 6 (18.18%) harbored cfr, 9 (27.27%) harbored optrA, and 18 (54.55%) co-harbored cfr and optrA. However, mutational mechanisms were not found in this study. Conjugation experiments demonstrated the transferability of cfr and optrA between Gram-positive and Gram-negative bacteria. The clone of these isolates was diverse and scattered. It is noteworthy that cfr and optrA were the main mechanisms of linezolid resistance in this study, posing a potential risk of spread of linezolid resistance. Strikingly, it reported firstly that the two transferable resistance genes cfr and optrA coexisted in the same E. faecalis isolates.

The blood brain barrier (BBB) maintains the basic stability of the brain tissue under physiological conditions, while destroys and exaggerates brain edema and inflammatory response after ischemic stroke. In this study, we researched S-oxiracetam (S-ORC), a nootropic drug, alleviates BBB dysfunction and protects against ischemic stroke in rats. Middle cerebral artery occlusion(MCAO)/reperfusion in rats is applied to mimic ischemic stroke. One hour after reperfusion, rats are administered intravenously with different dose (0.12, 0.24, or 0.48 g/kg) of S-ORC for continuative three days. Seventy-two hours after MCAO, TTC staining, hematoxylin and eosin (H&E) staining, brain water content, immunohistochemical staining, EB extravasation, western blot are provided to evaluate the protective effect and possible mechanism of S-ORC on BBB dysfunction. Furthermore, brain concentration of verapamil (P-glycoprotein substrate) and atenolol (paracellular transport marker) were assayed by UPLC-MS/MS co administration with or without S-ORC. The results show that post-treatment of S-ORC decreases cerebral infarct size, lessens brain edema, inhibits neutrophil infiltration and cytokines releasing. Furthermore, S-ORC treatment decreases EB leakage, downregulates MMP-9, upregulates occludin and claudin-5, and decreases brain concentration of verapamil and atenolol after MCAO surgery. In conclusion, the present study demonstrates that post-treatment of S-ORC alleviates BBB dysfunction by regulating tight junction proteins (TJPs), upregulating P-glycoprotein function, and protects against ischemic stroke as result.

Tumor antigens is at the core of cancer immunotherapy, however, the ideal antigen selection is difficult especially in poorly immunogenic tumors. In this study, we designed a strategy to modify hepatocellular carcinoma (HCC) cells by surface expressing anti-CD3scfv within the tumor site strictly, which depended on the E1A-engineered human umbilical cord mesenchymal stem cells (HUMSC.E1A) delivery system. Subsequently, membrane-bound anti-CD3scfv actived the lymphocytes which lysed HCC cells bypassing the expression of antigens or MHC restriction. First, we constructed the anti-CD3scfv gene driven by human α-fetoprotein (AFP) promoter into an adenoviral vector and the E1A gene into the lentiviral vector. Our results showed that anti-CD3scfv could specifically express on the surface of HCC cells and activate the lymphocytes to kill target cells effectively in vitro. HUMSC infected by AdCD3scfv followed by LentiR.E1A could support the adenoviral replication and packaging in vitro 36 h after LentiR.E1A infection. Using a subcutaneous HepG2 xenograft model, we confirmed that AdCD3scfv and LentiR.E1A co-transfected HUMSC could migrate selectively to the tumor site and produce considerable adenoviruses. The new generated AdCD3scfv infected and modified tumor cells successfully. Mice injected with the MSC.E1A.AdCD3scfv and lymphocytes significantly inhibited the tumor growth compared with control groups. Furthermore, 5-fluorouracil (5-FU) could sensitize adenovirus infection at low MOI resulting in improved lymphocytes cytotoxicity in vitro and in vivo. In summary, this study provides a promising strategy for solid tumor immunotherapy.

When performing immunofluorescent labeling of microtubules, Triton X-100 (TX100) is commonly used as the cell membrane permeabilization agent to improve the accessibility of antigens. Usually, before immunofluorescent labeling, cells are fixed first by aldehydes, followed by permeabilization with TX100. Here, we report an innovative immunofluorescent labeling strategy for microtubules with a meaningful alteration, that is, to treat cells with TX100 first and fix with aldehydes later. We proved that this subtle change can greatly improve the specificity of microtubular immunolabeling. However, treating cells first with TX100 can also severely disrupt the integrity of microtubules if an excessive amount of TX100 is used. Hence, TX100 is a “double-edged sword” in immunofluorescent labeling of microtubules and elaborative control of its dosage is required. In the experiment, we compared different immunofluorescent labeling protocols using various cell lines and found that treating cells with 0.02% TX100 before fixation is an optimal solution. Confocal laser scanning microscopy (CLSM), atomic force microscopy (AFM) and single molecule localization microscopy (SMLM) are utilized to verify the immunofluorescent labeling results performed via the presented unusual protocol. It is possible that such a modified immunofluorescent labeling protocol of microtubules can be generalized as a universal strategy.

Background Klebsiella pneumoniae-induced pyogenic liver abscess (KP-PLA) has emerged as a life-threatening disease worldwide. However, to date, a limited number of scholars have attempted to systematically elucidate the characteristics of KP-PLA. The aim of the present study was to analyze clinical, microbiological, and molecular epidemiological characteristics of KP-PLA patients in Southeastern China. Methods The KP-PLA cases from a tertiary teaching hospital in China from January 2016 to December 2017 were systemically studied and elucidated comprehensively. The virulence factors, resistant spectrum, and clones of K. pneumoniae isolates were identified with string test, polymerase chain reaction (PCR), antimicrobial susceptibility test, and multilocus sequence typing. Moreover, the characteristics in KP-PLA patients with and without other hepatobiliary diseases (OHD) were also been compared. Results A total of 163 KP-PLA cases were enrolled, in which the majority of those cases were senior males, and often associated with multiple underlying diseases, including diabetes (49.7%). The remaining cases belonged to healthy individuals (50.3%). The clinical symptoms were common but nonspecific, characterized by increased inflammatory parameters and abnormal liver function parameters. The abscess was often right-sided solitary presentation (58.3%). Cephalosporin or carbapenem plus metronidazole combined with percutaneous puncture or catheter drainage were favorable therapeutics. Although low resistance rates of commonly used antimicrobial drugs (iroN (24.5%) and magA (45.4%), the high prevalence of virulence genes (e.g. aerobactin, rmpA, mrkD, fimH, uge, ureA, entB, ybtA, kfuBC, and wcaG) was identified (68.7–100.0%). Additionally, ST23 was found as a predominant sequence type (ST; 38.7%), and three novel STs (ST3507, ST3508 and ST3509) were noted as well. Conclusions The present study reported the abundant hvKp strains in KP-PLA, as well as convergence of hypervirulent and MDR K. pneumoniae isolates from the KP-PLA patients, particularly those cases with OHD. Given the various clinical manifestations and destructive pathogenicity, determination of the comprehensive characteristics of such isolates is highly essential to effectively carry out for optimal management and treatment of KP-PLA.

Carbapenem-resistant, hypervirulent Klebsiella pneumoniae (CR-hvKP) has recently emerged as a significant threat to public health. In this study, 29 Klebsiella pneumoniae isolates were isolated from eight patients admitted to the intensive care unit (ICU) of a comprehensive teaching hospital located in China from March 2017 to January 2018. Clinical information of patients was the basis for the further analyses of the isolates including antimicrobial susceptibility tests, identification of antibiotic resistance and virulence gene determinants, multilocus sequence typing (MLST), XbaI-macrorestriction by pulsed-field gel electrophoresis (PFGE). Selected isolates representing distinct resistance profiles and virulence phenotypes were screened for hypervirulence in a Galleria mellonella larvae infection model. In the course of the outbreak, the overall mortality rate of patients was 100% (n=8) attributed to complications arising from CR-hvKP infections. All isolates except one (28/29, 96.6%) were resistant to multiple antimicrobial agents, and harbored diverse resistance determinants that included the globally prevalent carbapenemase blaKPC-2. Most isolates had hypervirulent genotypes being positive for nineteen virulence-associated genes, including iutA (25/29, 86.2%), rmpA (27/29, 93.1%), ybtA (27/29, 93.1%), entB (29/29, 100%), fimH (29/29, 100%) and mrkD (29/29, 100%). MLST revealed ST11 for the majority of isolates (26/29, 89,7%). Infection assays demonstrated high mortality in the Galleria mellonella model with the highest LD50 values for three isolates (less than 105 CFU/mL) demonstrating the degree of hypervirulence of these CR-hvKP isolates, and is discussed relative to previous outbreaks of CR-hvKP.

Intervertebral disc degeneration (IDD) is the main cause of low back pain and the mechanism of which is far from fully revealed. Although inflammation directed nucleus pulposus (NP) extracellular matrix metabolism dysregulation is known to be the main cause of the degeneration process, few is known about the protective factors. Using high-throughput label-free proteomics, we found that inflammation-related autocrine factor Chitinase-3-like protein 1 (CHI3L1, or YKL-40) is highly expressed in the NP cells during degeneration. Immunohistochemical analysis show that the expression of CHI3L1 is NP tissue specific, and increase significantly during degeneration. Overexpression of CHI3L1 significantly decrease the catabolism, and increase the anabolism of extracellular matrix. Knockdown of CHI3L1 using siRNAs show the opposite results, which imply that the protective role of CHI3L1 in IDD. Using high-throughput RNA sequencing and functional analyses, we find that AKT3 expression and its phosphorylation is mainly regulated by CHI3L1. And lastly, the mechanism of which is also validated using human and mouse degenerated NP tissues. In summary, our findings show that the inflammation-related autocrine factor CHI3L1 is NP specific, and it protects IDD by promoting the AKT3 signaling, which may serve as a potential therapeutic target in intervertebral disc degeneration.

Inonotus obliquus polysaccharide (IOPS) was initially separated and purified via precipitation from an aqueous extract with 80% alcohol, a DEAE-52 cellulose anion exchange column, and a Sephadex G-100 gel permeation chromatography system. IOPS was found to have a molecular weight of 111.9 kDa. In L-glutamic acid (L-Glu)-damaged HT22 cells, a 3-h pre-incubation with IOPS enhanced cell viability, inhibited apoptosis and caspase-3 activity, reduced the release of lactate dehydrogenase, restored the dissipated mitochondrial membrane potential, and suppressed the excess accumulation of intracellular reactive oxygen species. Compared with L-Glu-exposed cells, IOPS pre-treated cells exhibited reduced levels of Bcl-2 associated X protein (Bax) and Kelch-like ECH-associated protein 1 (Keap1) and enhanced levels of B-cell lymphoma-2 (Bcl-2), NF-E2p45-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), superoxide dismutase-1 (SOD-1), and cysteine ligase catalytic subunit. In amyloid precursor protein/presenilin 1 (APP/PS1) transgenic mice, an 8-week course of IOPS improved the pathological behaviors related to memory and cognition, reduced the deposition of β-amyloid peptides and neuronal fiber tangles induced by enhanced phosphor-Tau in the brain, and modulated the levels of anti- and pro-oxidative stress enzymes. Additionally, IOPS enhanced the expression levels of Nrf2 and its downstream proteins, including HO-1 and SOD-1, in the brains of APP/PS1 mice. The present study successfully demonstrated the protective effect of IOPS against AD and revealed the possible mechanism underlying the ability of IOPS to modulate oxidative stress, especially Nrf2 signaling, and mediate mitochondrial apoptosis.

Discovering novel drugs for cancer immunotherapy requires a robust in vitro drug screening platform that allows for straightforward probing of cell–cell communications. Here, we combined surface-enhanced Raman scattering (SERS) nanoprobes with microfluidic networks to monitor in situ the cancer–immune system intercellular communications. The microfluidic platform links up immune cells with cancer cells, where the cancer-cell secretions act as signaling mediators. First, gold@silver core–shell nanorods were employed to fabricate SERS immunoprobes for analysis of the signaling molecules. Multiple cancer secretions in a tumor microenvironment were quantitatively analyzed by a SERS-assisted three-dimensional (3D) barcode immunoassay with high sensitivity (1 ng/mL). Second, in an on-chip cell proliferation assay, multiple immunosuppressive proteins secreted by cancer cells were found to inhibit activation of immune cells, indicating that the platform simulates the physiological process of cancer–immune system communications. Furthermore, potential drug candidates were tested on this platform. A quantitative SERS immunoassay was performed to evaluate drug efficacy at regulating the secretion behavior of cancer cells and the activity of immune cells. This assay showed the suitability of this platform for in vitro drug screening. It is expected that the fully integrated and highly automated SERS-microfluidic platform will become a powerful analytical tool for probing intercellular communications and should accelerate the discovery and clinical validation of novel drugs.

Currently, it is a key challenge to remove the postsurgical residuals and metastasis of hepatocellular carcinoma (HCC). Oncolytic adenoviral virotherapy is an attractive treatment modality for cancer; however, the difficulty remains regarding its intravenous administration. The aim of this study was to develop a targeted therapeutic system which has great potential to overcome the postsurgical residuals and metastasis of HCC. In this system, we developed a conditionally replicative adenovirus (CRAd) loaded on human umbilical cord-derived mesenchymal stem cells (HUMSCs), in which the CRAd contained an adenovirus E1A gene dual regulated by α-fetoprotein promoter and microRNA-122 target sequence. When HUMSCs homed to the tumor sites and differentiated into hepatocyte-like cells within tumor microenvironment, the CRAds were packaged and released strictly to the local tumor. Subsequently, the CRAd lysed tumor cells selectively with the post-infection regulation. The study showed the specific oncolytic effect of the CRAd to HCC cells and the production of the CRAd by differentiated HUMSCs in vitro. Furthermore, we proved the hepatocyte-like transformation of HUMSC in the microenvironment of orthotopic or heterotopic hepatoma. Finally, this therapeutic system exhibited dramatic tumor inhibition on both orthotopic and subcutaneous hepatic xenograft tumor model mice with less toxicity on normal organs. The study results have demonstrated that this targeted therapeutic strategy is a promising method to resolve the problem of postsurgical residuals and metastasis of HCC.

Exosomes are small membrane vesicles secreted by cells and enriched with plenty of proteins. Considering their significant roles in different physical activities and potential value for diagnostic drug delivery, researchers have put great efforts in in vitro tracking and content analysis of exosomes. Recently, the emergence of different kinds of super-resolution microscopy provides powerful tools for exosome study. Here, we demonstrate the application of single-molecule localization based super-resolution imaging technique in the imaging and tracking of cancer-derived exosomes. In the experiment, first, cancer-derived exosomes are extracted from the culture media of tumor cells. Then the exosome membrane receptors are labeled with photoswitchable probes, which allow super-resolution imaging of these membrane receptors via photoactivated localization microscopy (PALM) or stochastic optical reconstruction microscopy (STORM). By using human breast cancer cell-derived exosomes, we demonstrated simultaneous dual-color PALM/STORM imaging of two kinds of membrane receptors on the exosome membrane. Moreover, the successful labeling and imaging of exosomes make it possible to observe the interaction between cancer-derived exosomes and normal cells. Meanwhile, we realized the colocalization of cancer-derived exosomes and lysosomes in recipient cells with PALM/STORM imaging. Since exosomes play a vital role in intercellular communications, we anticipate that the presented PALM/STORM-based imaging and tracking of exosomes holds a great potential in the investigation of the mechanism of exosome-mediated cancer metastasis.

Albiflorin (AF), one of important bioactive constituents of Paeonia lactiflora Radix, possesses neuro-protective effect. The present study aims to investigate the antinociceptive activities of AF and possible mechanisms. AF suppressed acetic acid-caused writhing, lengthened the latency period of mouse in hot plate test, and reduced the licking and biting response time of the injected mouse paw during phase I and phase II, and it suggested that AF exerted the antinociceptive activity mainly through central nervous system. Nimodipine, a commonly used calcium channels blocker, strongly lengthened AF-enhanced latency period of mouse in hot plate test. Compared with control group, AF reduced the levels of euronal nitric oxide synthase (nNOS), and enhanced the levels of serotonin (5-HT) in serum and/or hypothalamus before and after 30-s thermal stimuli. The reduced activation of calmodulin-dependent protein kinase II and c-Jun N-terminal kinase in hypothalamus was observed in AF-treated mice. Collectively, AF-mediated antinociceptive activities were, at least partially, related to calcium channels.

Background: Smoking is a risk factor for many human diseases. DNA methylation has been related to smoking, but genome-wide methylation data for smoking in Chinese populations is limited. Objectives: We aimed to investigate epigenome-wide methylation in relation to smoking in a Chinese population. Methods: We measured the methylation levels at > 485,000 CpG sites (CpGs) in DNA from leukocytes using a methylation array and conducted a genome-wide meta-analysis of DNA methylation and smoking in a total of 596 Chinese participants. We further evaluated the associations of smoking-related CpGs with internal polycyclic aromatic hydrocarbon (PAH) biomarkers and their correlations with the expression of corresponding genes. Results: We identified 318 CpGs whose methylation levels were associated with smoking at a genome-wide significance level (false discovery rate < 0.05), among which 161 CpGs annotated to 123 genes were not associated with smoking in recent studies of Europeans and African Americans. Of these smoking-related CpGs, methylation levels at 80 CpGs showed significant correlations with the expression of corresponding genes (including RUNX3, IL6R, PTAFR, ANKRD11, CEP135 and CDH23), and methylation at 15 CpGs was significantly associated with urinary 2-hydroxynaphthalene, the most representative internal monohydroxy-PAH biomarker for smoking. Conclusion: We identified DNA methylation markers associated with smoking in a Chinese population, including some markers that were also correlated with gene expression. Exposure to naphthalene, a byproduct of tobacco smoke, may contribute to smoking-related methylation. Citation: Zhu X, Li J, Deng S, Yu K, Liu X, Deng Q, Sun H, Zhang X, He M, Guo H, Chen W, Yuan J, Zhang B, Kuang D, He X, Bai Y, Han X, Liu B, Li X, Yang L, Jiang H, Zhang Y, Hu J, Cheng L, Luo X, Mei W, Zhou Z, Sun S, Zhang L, Liu C, Guo Y, Zhang Z, Hu FB, Liang L, Wu T. 2016. Genome-wide analysis of DNA methylation and cigarette smoking in Chinese. Environ Health Perspect 124:966–973; http://dx.doi.org/10.1289/ehp.1509834

Foxp3-expressing Treg cells have been well documented to provide immune regulation by promoting immune tolerance and suppressing immune over-reaction. Cimetidine (CIM), used to inhibit stomach acid secretion, has been reported to promote immune responses and suppress Treg cell function in several studies. However, the underlying mechanism is unknown. To investigate CIM effects on the suppressive function of Treg and Foxp3, here we used CIM to stimulate human CD4+CD25+ Treg cells and Jurkat T cells and evaluated changes of Foxp3 expression and stability. Our data showed that CIM leads to a reduction of Foxp3 via E3 ligase Stub1-mediated proteosomal degradation, which is dependent on an activated PI3K-AKT-mTOR pathway. Thus, CIM affects the suppressive function of Treg cells by destabilizing their Foxp3 expression.

The critical and ubiquitous enzyme adenylate kinase (ADK) catalyzes the nucleotide phosphoryl exchange reaction: 2ADP ↔ ATP + AMP. The ADK3 in the chloroplasts of the green alga Chlamydomonas reinhardtii, bears an unusual C-terminal extension that is similar to the C-terminal end of the intrinsically disordered protein CP12. In this study, we report that this enzyme, when oxidized but not when reduced, is able to interact with the chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forming a stable complex as shown by native electrophoresis and mass spectrometry. In this bienzyme complex, the activity of ADK3 is unchanged while the NADPH-dependent activity of GAPDH is significantly inhibited. Moreover ADK3, like CP12, can protect GAPDH against thermal inactivation and aggregation. The ADK3-GAPDH bienzyme complex is unable to recruit phosphoribulokinase (PRK), in contrast with the ternary complex formed between GAPDH-CP12 and PRK. The interaction between ADK3 and GAPDH might be a mechanism to regulate the crucial ATP: NADPH ratio within chloroplasts to optimize the Calvin-Benson cycle during rapid fluctuation in environmental resources. ENZYMES Adenylate kinase (EC 2.7.4.3), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.13), phosphoribulokinase (PRK, EC 2.7.1.19).

The ability to arrange thoughts and actions in an appropriate serial order (the problem of serial order) is essential to complex behaviors such as language, reasoning and cognitive planning. Patients with Parkinson's disease (PD) perform poorly in tasks that rely on the successful rearrangement of working memory representations. We hypothesized that serial ordering is impaired in nondemented patients with mild PD. We recruited 49 patients with mild idiopathic PD (Hoehn and Yahr Scale 1-2.5) and 51 matched healthy adults. Nineteen patients had normal global cognition (PD-NC, Montreal Cognitive Assessment, MoCA≥26/30) and thirty patients had mild cognitive impairment (PD-MCI, 21≤MoCA≤25). All participants underwent three working memory assessments: two experimental tests that require reordering random digits following a particular rule (adaptive digit ordering test and digit span backward test) and a control test that requires maintaining but no reordering (digit span forward test). PD-NC and PD-MCI patients performed significantly worse (with lower test scores and larger ordering costs) than healthy controls in both digit ordering and backward tests, although they performed normally in the forward test. The ordering cost increased as a function of age across groups, indicating an aging-related decline in the ability of serial ordering. However, individual patients' task performances were not correlated with their severity or duration of motor symptoms, or daily exposure to dopaminergic drugs. These results suggested that serial ordering deficits exist in early stages of PD, prior to subtle changes in global cognition and in parallel with motor symptoms.

Exosomes are small membrane vesicles secreted by a wide variety of cells. Studies have demonstrated that exosomal miRNAs can influence the biological processes of recipient cells. Therefore, direct imaging and tracking of exosomal miRNAs in living recipient cells are essential for exosome functional analysis. However, the moderate spatial resolution of conventional fluorescence microscopy limits the precise imaging and tracking of exosomes considering their relatively small size (

Grifola frondosa is an edible fungus with a variety of potential pharmacological activities. This study investigates the hypoglycemic, anti-diabetic nephritic, and antioxidant properties of G. frondosa polysaccharides in diet-streptozotocin-induced diabetic rats. After a 4-week treatment with 100 mg/kg of metformin and 200 mg/kg of one of four different G. frondosa polysaccharide mixtures (especially GFPS3 and GFPS4), diabetic rats had enhanced body weight and suppressed plasma glucose, indicating the hypoglycemic activities of the G. frondosa polysaccharides. G. frondosa polysaccharides regulated the level of serum creatinine, blood urea nitrogen, N-acetyl-β-d-glucosaminidase, and albuminuria; inhibited the serum levels of interleukin (IL)-2, IL-6, and TNF-α; and enhanced the serum levels of matrix metalloproteinase 9 and interferon-α, confirming their anti-diabetic nephritic activities. G. frondosa polysaccharides ameliorated the pathological alterations in the kidneys of diabetic rats. Moreover, G. frondosa polysaccharides modulated the serum levels of oxidant factors such as superoxide dismutase, glutathione peroxidase, catalase, malondialdehyde, and reactive oxygen species, revealing their antioxidant properties. Furthermore, the administration of G. frondosa polysaccharides inhibited nuclear factor kappa B activities in the serum and kidneys. All of the data revealed that the activation of nuclear factor kappa B plays a central role in G. frondosa polysaccharide-mediated anti-diabetic and anti-nephritic activities.

International audience; Among intrinsically disordered proteins, conditionally disordered proteins undergo dramatic structural disorder rearrangements upon environmental changes and/or post-translational modifications that directly modulate their function. Quantifying the dynamics of these fluctuating proteins is extremely challenging but paramount to understanding the regulation of their function. The chloroplast protein CP12 is a model of such proteins, and acts as a redox switch by formation/disruption of its two disulfide bridges. It regulates the Calvin cycle by forming, in oxidized conditions, a supramolecular complex with glyceraldehyde-3phosphate dehydrogenase (GAPDH) and then phosphoribulokinase. In this complex both enzymes are inactive. The highly dynamic nature of CP12 has so far hindered structural characterization explaining its mode of action. Thanks to a synergistic combination of smallangle X-ray scattering, nuclear magnetic resonance and circular dichroism that drove the molecular modeling of structural ensembles, we deciphered the structural behavior of Chlamydomonas reinhardtii oxidized CP12 (CP12ox) alone and in the presence of GAPDH. Contrary to sequence-based structural predictions, the N-terminal region is unstable, oscillates at the ms timescale between helical and random conformations, and is connected through a disordered linker to its C-terminus, which forms a stable helical turn. Upon binding to GAPDH, CP12ox undergoes an induced unfolding of its N-terminus. This phenomenon called cryptic disorder contributes to decrease the entropy cost and explains CP12 unusual high affinity for its partners.

Background: Aging is related to an increased risk of morbidity and mortality and is affected by environmental factors. Exposure to polycyclic aromatic hydrocarbons (PAHs) is associated with adverse health outcomes; but the association of such exposure with DNA methylation aging, a novel aging marker, is unclear. Objectives: Our aim was to investigate the association of PAH exposure with methylation aging. Methods: We trained and validated a methylation age predictor suitable for Chinese populations using whole blood methylation data in 989 Chinese and 160 Caucasians. We defined two aging indicators: Δage, as methylation age minus chronological age; and aging rate, the ratio of methylation to chronological age. The association of PAH exposure with aging indicators was evaluated using linear regressions in three panels of healthy Chinese participants (N=539, among the aforementioned 989 Chinese participants) whose exposure levels were assessed by 10 urinary monohydroxy-PAH metabolites. Results: We developed a methylation age predictor providing accurate predictions in both Chinese individuals and Caucasian persons (R=0.94–0.96, RMSE=3.8–4.3). Among the 10 urinary metabolites that we measured, 1-hydroxypyrene and 9-hydroxyphenanthrene were associated with methylation aging independently of other OH-PAHs and risk factors; 1-unit increase in 1-hydroxypyrene was associated with a 0.53-y increase in Δage [95% confidence interval (CI): 0.18, 0.88; false discovery rate (FDR) FDR=0.004] and 1.17% increase in aging rate (95% CI: 0.36, 1.98; FDR=0.02), whereas for 9-hydroxyphenanthrene, the increase was 0.54-y for Δage (95% CI: 0.17, 0.91; FDR=0.004), and 1.15% for aging rate (95% CI: 0.31, 1.99; FDR=0.02). The association direction was consistent across the three Chinese panels with the association magnitude correlating with the panels’ exposure levels; the association was validated by methylation data of purified leukocytes. Several cytosine-phosphoguanines, including those located on FHL2 and ELOVL2, were found associated with both aging indicators and monohydroxy-PAH levels. Conclusions: We developed a methylation age predictor specific for Chinese populations but also accurate for Caucasian populations. Our findings suggest that exposure to PAHs may be associated with an adverse impact on human aging and epigenetic alterations in Chinese populations. https://doi.org/10.1289/EHP2773

Background Epidemiologic studies have found that moderate alcohol consumption was associated with a decreased risk of coronary heart disease (CHD) incidence. Nevertheless, whether the drinking pattern is associated with CHD incidence still remains inconclusive. Methods We included 8,469 Chinese men aged 45–81 years, who were free of CHD, stroke, or cancer at baseline from Dongfeng-Tongji cohort. A semi-structured questionnaire was used to collect information on alcohol consumption and other covariates. Cox proportional hazard regression model was applied to estimate the multivariable-adjusted hazard rations (HRs) and 95% confidence intervals (95% CIs). Results During an average of 4.36 years of follow-up, we identified 959 incident CHD events. Compared with non-drinkers, the multivariable-adjusted HR (95% CI) of CHD incidence was 0.84 (0.71–0.98) in current drinkers. With respect to drinking pattern, men who consumed 20.01–40 grams ethanol once a time had a 24% lower risk of incident CHD (HR = 0.76, 95% CI = 0.62, 0.94) compared with non-drinkers. The adjusted HRs (95% CI) of CHD incidence were 0.80 (0.65, 0.99), 1.02 (0.84, 1.22), and 0.75 (0.59–0.96) in subjects who consumed 0.01–10, 10.01–30, and > 30 grams ethanol per day, respectively. Participants who consumed 20.01–40 grams ethanol per time with less than 5 times per week had the lowest risk of CHD incidence (HR = 0.73, 95% CI = 0.52, 0.96). No significant associations were observed between type or frequency of alcohol consumption and CHD incidence. Conclusions Drinking was associated with a lower risk of CHD incidence in middle-aged and older Chinese men and moderate quantity of ethanol amounts once a time with lower frequency could been considered as a healthy drinking pattern, which might modify the relationship between alcohol consumption and incident CHD.

Background Although blinatumomab, a bispecific T cell engaging antibody, exhibits high clinical response rates in patients with relapsed or refractory B-precursor acute lymphoblastic leukemia (B-ALL) and B cell non-Hodgkin’s lymphoma (B-NHL), it still has some limitations because of its short half-life. Mesenchymal stromal cells (MSCs) represent an attractive approach for delivery of therapeutic agents to cancer sites owing to their tropism towards tumors, but their immunosuppression capabilities, especially induced by indoleamine 2,3-dioxygenase (IDO), should also be taken into consideration. Methods Human umbilical cord-derived MSCs (UC-MSCs) were genetically modified to secrete Tandab (CD3/CD19), a tetravalent bispecific tandem diabody with two binding sites for CD3 and two for CD19. The tropism of MSCs towards Raji cells in vitro was determined by migration assays, and the homing property of MSCs in vivo was analyzed with firefly luciferase-labeled MSCs (MSC-Luc) by bioluminescent imaging (BLI). The cytotoxicity of T cells induced by MSC-secreting Tandab (CD3/CD19) was detected in vitro and in vivo in combination with d-1-methyl-tryptophan (D-1MT), an IDO pathway inhibitor. Results The purified Tandab (CD3/CD19) was functional with high-binding capability both for CD3-positive cells and CD19-positive cells and was able to induce specific lysis of CD19-positive cell lines (Raji, Daudi, and BJAB) in the presence of T cells. Additionally, results from co-culture killing experiments demonstrated that Tandab (CD3/CD19) secreted from MSCs was also effective. Then, we confirmed that D-1MT could enhance the cytotoxicity of T cells triggered by MSC-Tandab through reversing T cell anergy with down-regulation of CD98 and Jumonji and restoring the proliferation capacity of T cells. Furthermore, MSC-Luc could selectively migrate to tumor site in a BALB/c nude mouse model with Raji cells. And mice injected with MSC-Tandab in combination with D-1MT significantly inhibited the tumor growth. Conclusions These results suggest that UC-MSCs releasing Tandab (CD3/CD19) is an efficient therapeutic tool for the treatment of B cell lymphoma when combined with D-1MT. Electronic supplementary material The online version of this article (doi:10.1186/s13045-017-0397-z) contains supplementary material, which is available to authorized users.

RhoB, a member of the Ras homolog gene family and GTPase, regulates intracellular signaling pathways by interfacing with epidermal growth factor receptor (EGFR), Ras, and phosphatidylinositol 3-kinase (PI3K)/Akt to modulate responses in cellular structure and function. Notably, the EGFR, Ras, and PI3K/Akt pathways can lead to downregulation of RhoB, while simultaneously being associated with an increased propensity for tumorigenesis. Functionally, RhoB, part of the Rho GTPase family, regulates intracellular signaling pathways by interfacing with EGFR, RAS, and PI3K/Akt/mammalian target of rapamycin (mTOR), and MYC pathways to modulate responses in cellular structure and function. Notably, the EGFR, Ras, and PI3K/Akt pathways can lead to downregulation of RhoB, while simultaneously being associated with an increased propensity for tumorigenesis. RHOB expression has a complex regulatory backdrop consisting of multiple histone deacetyltransferase (HDACs 1 and 6) and microRNA (miR-19a, -21, and -223)-mediated mechanisms of modifying expression. The interwoven nature of RhoB’s regulatory impact and cellular roles in regulating intracellular vesicle trafficking, cell motion, and the cell cycle lays the foundation for analyzing the link between loss of RhoB and tumorigenesis within the context of age-related decline in RhoB. RhoB appears to play a tissue-specific role in tumorigenesis, as such, uncovering and appreciating the potential for restoration of RHOB expression as a mechanism for cancer prevention or therapeutics serves as a practical application. An in-depth assessment of RhoB will serve as a springboard for investigating and characterizing this key component of numerous intracellular messaging and regulatory pathways that may hold the connection between aging and tumorigenesis.

Rationale: Acute coronary syndrome (ACS) is a leading cause of death worldwide. Immune functions play a vital role in ACS development; however, whether epigenetic modulation contributes to the regulation of blood immune cells in this disease has not been investigated. Objective: We conducted an epigenome-wide analysis with circulating immune cells to identify differentially methylated genes in ACS. Methods and Results: We examined genome-wide methylation of whole blood in 102 ACS patients and 101 controls using HumanMethylation450 array, and externally replicated significant discoveries in 100 patients and 102 controls. For the replicated loci, we further analyzed their association with ACS in 6 purified leukocyte subsets, their correlation with the expressions of annotated genes, and their association with cardiovascular traits/risk factors. We found novel and reproducible association of ACS with blood methylation at 47 cytosine-phosphoguanine sites (discovery: false discovery rate P + T cells, CD4 + T cells, and B cells. Blood methylation of 26 replicated cytosine-phosphoguanine sites showed significant correlation with expressions of annotated genes (including IL6R , FASLG , and CCL18 ; P −4 ), and differential gene expression in case versus controls corroborated the observed differential methylation. The replicated loci suggested a role in ACS-relevant functions including chemotaxis, coronary thrombosis, and T-cell–mediated cytotoxicity. Functional analysis using the top ACS-associated methylation loci in purified T and B cells revealed vital pathways related to atherogenic signaling and adaptive immune response. Furthermore, we observed a significant enrichment of the replicated cytosine-phosphoguanine sites associated with smoking and low-density lipoprotein cholesterol ( P enrichment ≤1×10 −5 ). Conclusions: Our study identified novel blood methylation alterations associated with ACS and provided potential clinical biomarkers and therapeutic targets. Our results may suggest that immune signaling and cellular functions might be regulated at an epigenetic level in ACS.

Grifola frondosa, a type of food and medical fungus, has been shown to exhibit various pharmacological activities, including anticancer effects. As the most typical cancer diagnosed among female patients, breast cancer remains a huge concern threatening human health globally. In the present study, the anti-breast cancer effects of Grifola frondosa polysaccharides (GFPs) and the underlying mechanisms were investigated in MCF-7 and MDA-MB-231 cells, as well as in nude mice bearing MCF-7 tumor xenografts. GFPs exerted cytotoxic effects on the cells, as indicated by a decrease in cell viability, and an increase in the apoptototic rate, lactate dehydrogenase release and reactive oxygen species accumulation, inducing mitochondrial dysfunction. The increased expression of Bax, cleaved caspase-3 and caspase-8, and the reduced levels of B-cell lymphoma 2 (Bcl-2) and Bcl-extra large (Bcl-xL) were observed in the cells incubated with GFPs and in the tumor tissues of the mice treated with GFPs. Moreover, the GFPs significantly suppressed the phosphorylation of AKT/glycogen synthase kinase-3β and extracellular signal-regulated kinases in a time-dependent manner. Finally, the inhibition of MCF-7 tumor xenograft growth further confirmed the anti-breast cancer effects of GFPs. All these findings revealed that GFPs induced human breast cancer cell apoptosis via the mitochondrial-dependent apoptotic pathway, and provide experimental evidence to support the use of Grifola frondosa as a potential treatment for breast cancer.