Role of Lipocalin-2 in N1/N2 Neutrophil Polarization After Stroke.

BACKGROUND: Neutrophils and Lipocalin-2 (LCN2) play pivotal roles in cerebral ischemiareperfusion (I/R) injury. However, their contribution is not fully clarified. OBJECTIVE: This study aimed to explore the role of LCN2 and its association with neutrophil polarization in I/R injury. METHODS: A mouse model of middle cerebral artery occlusion (MCAO) was used to induce cerebral ischemia. LCN2mAb was administered 1 h and Anti-Ly6G was administered for 3d before MCAO. The role of LCN2 in the polarity transition of neutrophils was explored using an in vitro HL-60 cell model. RESULTS: LCN2mAb pretreatment had neuroprotective effects in mice. The expression of Ly6G was not significantly different, but the expression of N2 neutrophils was increased. In the in vitro study, LCN2mAb-treated N1-HL-60 cells induced N2-HL-60 polarization. CONCLUSION: LCN2 may affect the prognosis of ischemic stroke by mediating neutrophil polarization.

Glial Fibrillary Acidic Protein Levels in Post-Stroke Depression: A Prospective Ischemic Stroke Cohort.

Background and Purpose: Increased glial fibrillary acidic protein (GFAP) levels were found in cerebrovascular disease patients. The pathogenesis of depression after ischemic stroke remains largely unknown. Here, we aim to determine whether GFAP concentrations were associated with post-stroke depression (PSD) at 3 months. Methods: From March 2022 to September 2022, patients with first-ever ischemic stroke were prospectively recruited. GFAP concentrations were detected within 24 h using an enzyme-linked immunosorbent assay. The PSD was defined as a Hamilton Depression Rating Scale 24-Item score ≥ 8. Results: A total of 206 subjects with ischemic stroke (mean age: 63.6 years; 49.0% female) were enrolled. During the 90-day follow-up, 57 participants (27.7%) were observed in PSD. The median serum GFAP concentrations were 0.67 ng/mL. After adjustment for the covariates, higher increased GFAP levels were associated with increased risk of PSD (odds ratio [OR], 7.12; 95% confidence interval [CI], 3.29-15.44; P < 0.001). Also, the multivariate-adjusted OR of PSD associated with the fourth quartile of GFAP was 10.89 (95% CI, 3.53-33.60; P < 0.001) compared with the first quartile. Furthermore, the restricted cubic spline confirmed a linear association between GFAP and the risk of PSD (P for linearity < 0.001). Conclusion: Our results indicated that increased circulating GFAP concentrations were significantly correlated with the risk of PSD at 3 months. Measuring the GFAP levels after ischemic stroke may add some values for the risk stratifying of PSD.

Magnetic Microcarriers with Accurate Localization and Proliferation of Mesenchymal Stem Cell for Cartilage Defects Repairing.

Magnetic biomaterials are widely used in the field of tissue engineering because of their functions such as drug delivery and targeted therapy. In this study, a magnetically responsive composite microcarrier was prepared through in situ polymerization of dopamine with Fe3O4 (MS) to form a complex. The magnetic composite microcarriers are paramagnetic and have certain magnetic responsiveness, suitable pore size porosity for cell growth, and good blood compatibility and biocompatibility. The bone marrow mesenchyml stem cells (BMSCs) were cultured on magnetic composite microcarriers, and a static magnetic field (SMF) was applied. The results showed that BMSCs adhered to the microcarriers proliferated under the action of horizontal and vertical forces. Magnetic composite microcarriers loaded with BMSCs were implanted into the SD rat model of cartilage defect, and a magnet was added to the operative side. After 12 weeks, cartilage regeneration was observed. The results of gross observation and histological immunostaining 1 month, 2 months, and 3 mounths after operation showed that the magnetic composite microcarriers of loaded cells promoted the early maturation of cartilage and collagen secretion, and the effect of cartilage repair was significantly better than that of the control group. Gait analysis showed that implanting magnetic composite microcarriers loaded with stem cells can reduce postoperative pain and promote limb recovery in SD rats. In conclusion, this study suggests that magnetic composite microcarriers are promising tissue-engineered scaffolds for cartilage regeneration and repair.

CERS6-AS1 promotes cell proliferation and represses cell apoptosis in pancreatic cancer via miR-195-5p/WIPI2 axis.

Pancreatic cancer (PC) is a lethal malignancy that threatens human health. Long noncoding RNAs (lncRNAs) act as important mediators in PC development. Our study aimed to investigate the function and mechanism of lncRNA ceramide synthase 6 antisense RNA 1 (CERS6-AS1) in PC. As shown by RT-qPCR, CERS6-AS1 was significantly upregulated in PC cells and tissues. Silencing CERS6-AS1 suppressed PC cell viability and proliferation while enhancing cell apoptosis according to colony formation assays, EdU assays, and flow cytometry analyses. Mechanistically, CERS6-AS1 interacted with miR-195-5p to elevate the expression level of the WD repeat domain phosphoinositide interacting 2 (WIPI2), which is a downstream target gene of miR-195-5p in PC. Moreover, miR-195-5p expression was negatively associated with CERS6-AS1 expression (or WIPI2 expression) in PC tissues. Rescue assays revealed that WIPI2 overexpression rescued the effects of CERS6-AS1 deficiency on cell viability, proliferation, and apoptosis. In summary, CERS6-AS1 facilitates PC cell proliferation while inhibiting PC cell apoptosis by upregulating WIPI2 via miR-195-5p. This study might provide promising insight into the role of CERS6-AS1 in PC development.

Free thyroxine, brain frailty and clock drawing test performance in patients with acute minor stroke or transient ischaemic attack.

OBJECTIVE: Thyroid dysfunction is associated with an elevated risk of cognitive decline, but the mechanism underlying this relationship is elusive. In this study, we investigate the relationships between free thyroxine (FT4), brain frailty and clock drawing test (CDT) performance in patients with acute minor stroke or transient ischaemic attack (TIA). DESIGN, PATIENTS AND MEASUREMENTS: A total of 204 consecutive patients admitted to our hospital within 72 h after the onset of acute minor stroke or TIA were prospectively enroled and categorized in terms of quartiles of FT4 between March 2018 and August 2019. Brain frailty on magnetic resonance imaging was rated according to previously published criteria. Cognitive performance was assessed with the CDT. RESULTS: Generalized linear analysis revealed that FT4 was independently associated with higher brain frailty score after adjusting potential confounders (ß, 0.03; 95% confidence interval [CI], 0.00-0.06; p = 0.0205), which is consistent with the result of FT4 (quartile) as a categorical variable (ß, 0.34; 95% CI, 0.01-0.68; p = 0.0059; ptrend = 0.0807). A nonlinear relationship was detected between FT4 and brain frailty score, which had an inflection point of 1.19. FT4 was also associated with poor CDT performance (odds ratio, 1.15; 95% CI, 1.04-1.26; p = 0.0051). And mediation analysis found that brain frailty partially mediated the positive relationship between FT4 and poor CDT performance (indirect effect = 0.0024; 95% CI, 0.0003-0.01, p = 0.04). CONCLUSIONS: Our findings suggested that a higher FT4 level was associated with a higher brain frailty score and poorer CDT performance, and brain frailty might play an important effect on the association between FT4 and cognitive decline.

Upregulated NOX1 expression in gallbladder cancer­associated fibroblasts predicts a poor prognosis.

Gallbladder cancer (GBC) is a lethal aggressive malignant neoplasm of the biliary tract. Potential prognostic markers and therapeutic targets for this disease are urgently required. Cancer­associated fibroblasts (CAFs) play a key role in tumorigenesis and the development of cancer. Nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) expression has been reported to be involved in tumorigenesis and useful for tumor prognosis. However, NOX1 expression in the stroma of GBCs, particularly gallbladder cancer­associated fibroblasts (GCAFs), and its prognostic significance in GBC patients remains unclear. In the present study, NOX1 expression in the stroma of human gallbladder lesions in vivo was investigated, as well as in GCAFs and co­cultures of GBC­SD+GCAFs in vitro, and their correlation with clinicopathological parameters and the prognosis of GBC patients were evaluated. The results revealed that NOX1 expression was significantly upregulated in the stroma of GBCs compared with precancerous and benign lesions of the gallbladder; NOX1 expression was localized to gallbladder stromal fibroblasts expressing α­smooth muscle actin and fibroblast secreted protein­1. Furthermore, these observations were confirmed by the fact that NOX1 expression was upregulated in GCAFs as determined by Affymetrix gene profile chip analysis and reverse transcription­quantitative PCR. In addition, overexpression was observed in formed spheroids of GBC­SD+GCAF co­cultures by immunohistochemistry and western blotting in vitro. Thus, it was verified that NOX1 expression was upregulated in GCAFs. Furthermore, upregulated stromal NOX1 expression was correlated with aggressive characteristics such as differentiation degree (P=0.042), venous invasion (P=0.041), resection methods (P=0.002), and a lower survival rate (P=0.025, log­rank test) of patients with GBC. Stromal NOX1 expression (P=0.047) was an independent prognostic factor for the overall survival rate of patients with GBC. GBC patients with upregulated NOX1 expression in GCAFs had a poorer prognosis. These results revealed that stromal NOX1 may be a novel biomarker and/or target, and may contribute to the discovery of new tumor markers and potential targeted therapeutics for human GBCs.

Colorimetric immunoassay for human chorionic gonadotropin by using peroxidase-mimicking MnO2 nanorods immobilized in microplate wells.

A colorimetric immunoassay is described for the pregnancy marker human chorionic gonadotropin (HCG). The assay is based on the use of MnO2 nanorods acting as a peroxidase mimic. The nanorods were prepared by a hydrothermal method using EDTA as a template. They exhibit excellent peroxidase-like activity and stability. The nanorods were immobilized in a chitosan matrix in the wells of a BSA-modified microplate which then were further modified with streptavidin and biotinylated capture antibodies. The specific recognition between HCG and the antibodies in wells inhibit the peroxidase-like activity of the nanorods. Hence, the substrate 3,3',5,5'-tetramethylbenzidine is less efficiently catalytically oxidized by H2O2 to form a blue colored product. This results in a decrease in the absorbance at 652 nm. Response is linear in the 0.5 to 400 mIU·mL-1 HCG concentration range, and the detection limit is 0.36 mIU·mL-1 under optimized conditions. The method is highly specific, acceptably reproducible and stable. It was applied to the determination of HCG concentration in serum samples, and results were in good agreement with data obtained by the reference method." Graphical abstract Schematic presentation of colorimetric immunoassay for human chorionic gonadotropin (HCG) by using MnO2 nanorods with peroxidase-like activity immobilized in microplate wells. The specific recognition between HCG and the antibodies in wells inhibits the peroxidase-like activity of the nanorods. TMB: 3,3',5,5'-tetramethylbenzidine.

Green emitting carbon dots for sensitive fluorometric determination of cartap based on its aggregation effect on gold nanoparticles.

A fluorometric method was developed for the determination of the insecticide cartap. It is based on the use of green emitting carbon dots (CDs) and gold nanoparticles (Au NPs). The CDs were prepared from phenol and ethylene diamine by a hydrothermal route. They have excitation/emission maxima at 410/513 nm) and a fluorescence quantum yield of 29%. They were characterized by TEM, Raman, XRD, XPS, FT-IR, UV and fluorescence spectroscopies. The green fluorescence of the CDs is strongly reduced by the red-colored Au NPs because of an inner filter effect. Upon addition of cartap, it will cause the aggregation of the Au NPs owing to Au-N interaction between Au NPs and cartap to form purple colored aggregates with spectra that do not overlap the green emission of the CDs. Hence, their fluorescence is restored. Under optimum conditions, the method allows for the quantitation of cartap in the 5-300 nM concentration range, and the detection limit is 3.8 nM. The method was successfully applied to the determination of cartap in spiked real samples and gave satisfactory results. Graphical abstract Schematic presentation of green emitting carbon dots for sensitive fluorometric determination of cartap based on its aggregation effect on gold nanoparticles.

A regenerative and selective electrochemical aptasensor based on copper oxide nanoflowers-single walled carbon nanotubes nanocomposite for chlorpyrifos detection.

Chlorpyrifos is a commonly used organophosphorus pesticide in agriculture. However, its neurotoxicity poses a huge threat to human health. To detect trace amounts of chlorpyrifos, we herein developed a regenerative electrochemical aptasensor for the sensitive detection of chlorpyrifos. The nanocomposite consisting of copper oxide nanoflowers (CuO NFs) and carboxyl-functionalized single walled carbon nanotubes (c-SWCNTs) was prepared to improve the sensing performance for chlorpyrifos detection. Various characterization methods such as scanning electron microscopy (SEM), fourier transform infrared spectroscopy (FT-IR) and cyclic voltammetry (CV) were used to demonstrate the successful fabrication of biosensor. Differential pulse voltammetry (DPV) was utilized to optimize test conditions and quantify chlorpyrifos. Under optimal conditions, the biosensor obtained a good linearity for chlorpyrifos ranging from 0.1 to 150ng/mL, with a lower detection limit of 70pg/mL. This aptasensor also exhibited high selectivity and outstanding repeatability, and was successfully applied to the determination of chlorpyrifos in spiked apple and celery cabbage with satisfactory recoveries. Furthermore, the sensor can be easily regenerated by urea for continuous application. With all the features, the proposed strategy provides an excellent platform for regenerative and selective detection of chlorpyrifos.

Ring finger protein 125, as a potential highly aggressive and unfavorable prognostic biomarker, promotes the invasion and metastasis of human gallbladder cancers via activating the TGF- ß1-SMAD3-ID1 signaling pathway.

Human gallbladder cancer (GBC) is a lethal aggressive malignant neoplasm. Identification of potential molecular biomarkers and development of targeted therapeutics for GBC patients is very necessary. In this study, we firstly investigated the correlation between ring finger protein 125 (RNF125) expression and the metastasis and prognosis of GBC, and the underlying molecular mechanism. RNF125 expression in a cohort of GBC tissues was examined; its correlation with clinicopathological and prognostic factors of GBC patients was analyzed. Moreover, the metastasis-related difference expressed genes in highly and lowly aggressive GBC cell lines were identified; and the influence of RNF125 knockdown on the metastatic phenotypes and characteristic EMT markers in highly aggressive GBC NOZ cells was detected. Furthermore, the underlying molecular mechanism of RNF125 effect was explored. The results showed that RNF125 was highly expressed in GBC tissues and related with aggressive characteristics such as Nevin stage (P = 0.041) etc. and unfavorable prognosis of GBC patients (P = 0.023, log-rank test). And, RNF125 was proved to a positive metastasis-related gene in vitro. RNF125 knockdown inhibited the invasion and migration, enhanced the adhesion, upregulated E-cadherin and ß-catenin expression, and downregulated vimentin and N-cadherin expression (all P < 0.001) of NOZ cells in vitro. RNF125 promoting effect on GBC tumor progression was identified to relate with the activation of TGF-ß1-SMAD3-ID1 signaling pathway. These findings firstly confirm that high RNF125 expression is related with aggressive characteristics and unfavorable prognosis of GBC patients; RNF125 promotes the invasion and metastasis of human GBCs via activating the TGF-ß1-SMAD3-ID1 signaling pathway.

Establishment and characterization of a novel highly aggressive gallbladder cancer cell line, TJ-GBC2.

BACKGROUND: Human gallbladder cancer (GBC) is an aggressive malignant neoplasm with a poor prognosis. The development of ideal tools for example tumor cell lines for investigating biological behavior, metastatic mechanism and potential treatment in GBCs is essential. In present study, we established and characterized a GBC cell line derived from primary tumor. METHODS: Primary culture method was used to establish this cell line from a primary GBC. Light and electron microscopes, flow cytometry, chromosome analysis, heterotransplantation and immunohistochemistry were used to characterize the epidemic tumor characteristics and phenotypes of this cell line. RESULTS: A novel GBC cell line, named TJ-GBC2, was successfully established from primary GBC. This cell line had characteristic epithelial tumor morphology and phenotypes in consistent with primary GBC, such as polygon and irregular cell shape, increased CA19-9 and AFP levels, and positive expression of CK7, CK8, CK19 and E-cadherin with negative vimentin. Moreover, about 25% of the cells were in the S-G2/M phase; abnormity in structure and number of chromosome with a peak number of 90-105 and 80% hypertetraploid were observed. Furthermore, this cell line had higher invasion and highest migration abilities compared to other GBC cell lines; and metastatic-related marker MMP9 and nm23 were positively expressed. CONCLUSIONS: A novel highly aggressive GBC cell line TJ-GBC2 was successfully established from primary GBC. TJ-GBC2 cell line may be efficient tool for further investigating the biological behaviors, metastatic mechanism and potential targeted therapy of human GBC.

Effects of chronic fluoride exposure on object recognition memory and mRNA expression of SNARE complex in hippocampus of male mice.

This study aimed to investigate the effects of long-term fluoride exposure on object recognition memory and mRNA expression of soluble N-ethylmaleimidesensitive fusion protein attachment protein receptors (SNARE) complex (synaptosome-associated protein of 25 kDa (SNAP-25), vesicle-associated membrane protein 2 (VAMP-2), and syntaxin 1A) in the hippocampus of male mice. Sixty sexually matured male Kunming mice were randomly divided into four groups: control group (given distilled water), low F group (25 mg/L NaF, corresponding to 11 mg/L F(-)), medium F group (50 mg/L NaF, corresponding to 22 mg/L F(-)), and high F group (100 mg/L NaF, corresponding to 45 mg/L F(-)). After 180 days, the spontaneous locomotor behavior and object recognition memory were detected by open field test and novel object recognition (NOR) test. Results showed that compared with the control group, frequency in each zone, total distance, and line crosses were significantly increased in low F and medium F groups, suggesting fluoride enhanced excitement of mice, while there were no marked changes in high F group. Twenty-four hours after training, a deficit of long-term memory (LTM) occurred only in high F group (P < 0.05), but there was no significant change of short-term memory (STM) 1.5 h later. The mRNA expression levels of SNAP-25, VAMP-2, and syntaxin 1A were detected by real-time quantitative RT-PCR, which revealed that the mRNA expression of VAMP-2 was significantly increased in medium F and high F groups (P < 0.01). Taken together, these results indicated that long-term fluoride administration can enhance the excitement of male mice, impair recognition memory, and upregulate VAMP-2 mRNA expression, which are involved in the adverse effects of fluoride on the object recognition memory of nervous system.