Lab on the Microneedles: A Wearable Metal-organic Frameworks-Based Sensor for Visual Monitoring of Stress Hormone.

Abnormal secretion and dysrhythmias of cortisol (CORT) are associated with various diseases such as sleep disorders, depression, and chronic fatigue. Wearable devices are a cutting-edge technology for point-of-care detection and dynamic monitoring of CORT with inspiring convenience. Herein, we developed a minimally invasive skin-worn device with the advanced integration of both interstitial fluid (ISF) sampling and target molecule sensing for simultaneous detection of CORT via a microneedle-based sensor with high sensitivity, excellent efficiency, and outstanding reproducibility. In the microneedle patch, swellable hydrogel was employed as the adsorption matrix for ISF extraction. Meanwhile, europium metal-organic frameworks (Eu-MOF) wrapped in the matrix played a vital role in CORT recognition and quantitative analysis. The wearable and label-free Eu-MOF-loaded microneedle patch exhibited high sensitivity in CORT detection with the detection limit reaching 10-9 M and excellent selectivity. Molecular dynamics simulation-driven mechanism exploration revealed that the strong interface interaction promoted fluorescence quenching of Eu-MOF. Moreover, in vitro and in vivo investigation confirmed the feasibility and reliability of the sensing method, and excellent biocompatibility was validated. Overall, a sensitive approach based on the wearable Eu-MOF microneedle (MN) patch was established for the simultaneous detection of CORT via visible fluorescence quenching with exciting clinical-translational ability.

A Unique Approach: Biomimetic Graphdiyne-Based Nanoplatform to Treat Prostate Cancer by Combining Cuproptosis and Enhanced Chemodynamic Therapy.

Purpose: Current treatment approaches for Prostate cancer (PCa) often come with debilitating side effects and limited therapeutic outcomes. There is urgent need for an alternative effective and safe treatment for PCa. Methods: We developed a nanoplatform to target prostate cancer cells based on graphdiyne (GDY) and a copper-based metal-organic framework (GDY-CuMOF), that carries the chemotherapy drug doxorubicin (DOX) for cancer treatment. Moreover, to provide GDY-CuMOF@DOX with homotypic targeting capability, we coated the PCa cell membrane (DU145 cell membrane, DCM) onto the surface of GDY-CuMOF@DOX, thus obtaining a biomimetic nanoplatform (DCM@GDY-CuMOF@DOX). The nanoplatform was characterized by using transmission electron microscope, atomic force microscope, X-ray diffraction, etc. Drug release behavior, antitumor effects in vivo and in vitro, and biosafety of the nanoplatform were evaluated. Results: We found that GDY-CuMOF exhibited a remarkable capability to load DOX mainly through π-conjugation and pore adsorption, and it responsively released DOX and generated Cu+ in the presence of glutathione (GSH). In vivo experiments demonstrated that this nanoplatform exhibits remarkable cell-killing efficiency by generating lethal reactive oxygen species (ROS) and mediating cuproptosis. In addition, DCM@GDY-CuMOF@DOX effectively suppresses tumor growth in vivo without causing any apparent side effects. Conclusion: The constructed DCM@GDY-CuMOF@DOX nanoplatform integrates tumor targeting, drug-responsive release and combination with cuproptosis and chemodynamic therapy, offering insights for further biomedical research on efficient PCa treatment.

ACOX2 Serves as a Favorable Indicator Related to Lipid Metabolism and Oxidative Stress for Biochemical Recurrence in Prostate Cancer.

Given the heterogeneity of tumors, there is an urgent need for accurate prognostic parameters in prostate cancer (PCa) patients. Lipid metabolism (LM) reprogramming and oxidative stress (OS) play a vital role in the progression of PCa. In this work, we identified five LM-OS-related genes (including ACOX2, PPRAGC1A, PTGS1, PTGS2, and HAO1) associated with the biochemical recurrence (BCR) of PCa. Subsequently, a prognostic signature was established based on these five genes. Kaplan-Meier survival estimates, receiver operating characteristic curves, and relationship analysis between risk score and clinical characters were applied to measure the robustness of the signature in an external cohort. A nomogram of risk score combined with clinical characteristics was constructed for clinical application. Functional enrichment analysis suggested that the underlying mechanism related to the signature included the calcium signaling, lipid transport, and cell cycle signaling pathways. Furthermore, WEE1 inhibitor was identified as a potential agent related to the cell cycle for high-risk patients. The mRNA expression and the prognostic value of the five genes were determined, and ACOX2 was identified as the key gene related to the prognostic signature. The protein expression of ACOX2 was measured in a prostate tissue microarray through an immunohistochemistry assay, confirming the bioinformatics results. By constructing the ACOX2-overexpressing PCa cell lines PC-3 and 22Rv1, the biological function of PCa cells was investigated. The cell viability, colony formation, migration, and invasion ability of PCa cell lines overexpressing ACOX2 were hindered. Decreased cellular lipid content and elevated cellular ROS content were observed in ACOX2-overexpressing PCa cell lines with reduced G2/M phases. In conclusion, this work presents the first prognostic signature specifically focused on LM-OS for PCa. ACOX2 could serve as a favorable indicator for the BCR in PCa. Further experiments are required to identify the potential underlying mechanism.

Lateral flow strip assay of a gene segment in the COVID-19 virus with combined dual readout mode and preliminary multisite hybrid chain reaction amplification.

The past and present scenario of COVID-19 has revealed the necessity of simple point-of-care tests. When combined with the great advantages of amplification, lateral flow assay nucleic acid analysis represents a more sensitive molecular diagnostic technique compared to universal protein analysis. Room temperature operation, an enzyme-free nature, and in situ elongation make hybrid chain reaction amplification (HCR) a good candidate for amplified combined lateral flow assays (LFAs). Since dual modes of detection can not only satisfy different application scenarios, but also reduce the false-negative rate, in this paper, visual and fluorescent detection based on labelling with colloidal gold nanoparticles and fluorescence labelling were incorporated into a HCR integrated with a LFA. The detection assay was finished in 30 minutes. The linear relationship between the signal and the concentration of the characteristic segment in the COVID-19 ORF gene was demonstrated. The obtained detection limits of as low as 10 fM (6.02 × 103 copies per mL) and 1 fM (6.02 × 102 copies per mL), respectively, were comparable with those in the literature. The multi-site HCR amplification integrated with LFA of a 1053 bp nucleic acid chain was also preliminarily studied, and tri-site amplification was found to exhibit higher signal intensity than single-site amplification. This study provides a promising strategy for simple, sensitive, and wide-ranging detection of pathogenic bacteria.

Triterpenoid saponins from Bupleurum marginatum and their anti-liver fibrotic activities.

A new triterpenoid saponin (1), along with five known compounds (2-6), was isolated from Bupleurum marginatum Wall. ex DC, of which compounds 2-4 were obtained for the first time from this plant. The structures were confirmed by the analysis of 1D, 2D NMR, and HR-ESIMS data, and comparison with previous spectral data. Anti-liver fibrotic activities of the isolates were determined as proliferation inhibition of LPS-induced activation of HSC-T6 in vitro.

Water and Air Stable Copper(I) Complexes of Tetracationic Catenane Ligands for Oxidative C-C Cross-Coupling.

Aqueous soluble and stable Cu(I) molecular catalysts featuring a catenane ligand composed of two dicationic, mutually repelling but mechanically interlocked macrocycles are reported. The ligand interlocking not only fine-tunes the coordination sphere and kinetically stabilizes the Cu(I) against air oxidation and disproportionation, but also buries the hydrophobic portions of the ligands and prevents their dissociation which are necessary for their good water solubility and a sustained activity. These catenane Cu(I) complexes can catalyze the oxidative C-C coupling of indoles and tetrahydroisoquinolines in water, using H2O2 as a green oxidant with a good substrate scope. The successful use of catenane ligands in exploiting aqueous Cu(I) catalysis thus highlights the many unexplored potential of mechanical bond as a design element for exploring transition metal catalysis under challenging conditions.

Machine learning-based integration develops a stress response stated T cell (Tstr)-related score for predicting outcomes in clear cell renal cell carcinoma.

BACKGROUND: Establishment of a reliable prognostic model and identification of novel biomarkers are urgently needed to develop precise therapy strategies for clear cell renal cell carcinoma (ccRCC). Stress response stated T cells (Tstr) are a new T-cell subtype, which are related to poor disease stage and immunotherapy response in various cancers. METHODS: 10 machine-learning algorithms and their combinations were applied in this work. A stable Tstr-related score (TCs) was constructed to predict the outcomes and PD-1 blockade treatment response in ccRCC patients. A nomogram based on TCs for personalized prediction of patient prognosis was constructed. Functional enrichment analysis and TimiGP algorithm were used to explore the underlying role of Tstr in ccRCC. The key TCs-related gene was identified by comprehensive analysis, and the bioinformatics results were verified by immunohistochemistry using a tissue microarray. RESULTS: A robust TCs was constructed and validated in four independent cohorts. TCs accurately predicted the prognosis and PD-1 blockade treatment response in ccRCC patients. The novel nomogram was able to precisely predict the outcomes of ccRCC patients. The underlying biological process of Tstr was related to acute inflammatory response and acute-phase response. Mast cells were identified to be involved in the role of Tstr as a protective factor in ccRCC. TNFS13B was shown to be the key TCs-related gene, which was an independent predictor of unfavorable prognosis. The protein expression analysis of TNFSF13B was consistent with the mRNA analysis results. High expression of TNFSF13B was associated with poor response to PD-1 blockade treatment. CONCLUSIONS: This study provides a Tstr cell-related score for predicting outcomes and PD-1 blockade therapy response in ccRCC. Tstr cells may exert their pro-tumoral role in ccRCC, acting against mast cells, in the acute inflammatory tumor microenvironment. TNFSF13B could serve as a key biomarker related to TCs.

Neuroinflammatory response on a newly combinatorial cell-cell interaction chip.

Neuroinflammation is a common feature in various neurological disorders. Understanding neuroinflammation and neuro-immune interactions is of significant importance. However, the intercellular interactions in the inflammatory model are intricate. Microfluidic chips, with their complex micrometer-scale structures and real-time observation capabilities, offer unique advantages in tackling these complexities compared to other techniques. In this study, microfluidic chip technology was used to construct a microarray physical barrier structure with 15 µm spacing, providing well-defined cell growth areas and clearly delineated interaction channels. Moreover, an innovative hydrophilic treatment process on the glass surface facilitated long-term co-culture of cells. The developed neuroinflammation model on the chip revealed that SH-SY5Y cytotoxicity was predominantly influenced by co-cultured THP-1 cells. The co-culture model fostered complex interactions that may exacerbate cytotoxicity, including irregular morphological changes of cells, cell viability reduction, THP-1 cell migration, and the release of inflammatory factors. The integration of the combinatorial cell-cell interaction chip not only offers a clear imaging detection platform but also provides diverse data on cell migration distance, migration direction, and migration angle. Furthermore, the designed ample space for cell culture, along with microscale channels with fluid characteristics, allow for the study of inflammatory factor distribution patterns on the chip, offering vital theoretical data on biological relevance that conventional experiments cannot achieve. The fabricated user-friendly, reusable, and durable co-culture chip serves as a valuable in vitro tool, providing an intuitive platform for gaining insights into the complex mechanisms underlying neuroinflammation and other interacting models.

Corrigendum: Altered expression of inflammation-associated molecules in striatum: an implication for sensitivity to heavy ion radiations.

[This corrects the article DOI: 10.3389/fncel.2023.1252958.].

Microbiome in a ground-based analog cabin of China Space Station during a 50-day human occupation.

Dead-corner areas in space station that untouched by the clean-up campaign often experience microorganisms outbreaks, but the microbiome of these areas has never been studied. In this study, the microbiome in a ground-based analog ``Tianhe'' core module of China Space Station was first investigated during a 50-day three-crew occupation. Dead-corner areas were receiving attention by adopting a new sampling method. Results indicate that the astronauts occupation did not affect the dominant bacteria community, but affected a small proportion. Due to the frequent activity of astronauts in the work and sleep areas, the biomarkers in these two areas are common human skin surface and gut microorganisms, respectively. For areas that astronaut rarely visits, the biomarkers in which are common environmental microbial groups. Fluorescence counting showed that 70.12-84.78% of bacteria were alive, with a quantity of 104-105 cells/100 cm2. With the occupation time extension, the number of microorganisms increased. At the same sampling time, there was no significant bioburden difference in various locations. The cultivable bioburden ranged from 101 to 104 colony forming unit (CFU)/100 cm2, which are the following eight genera Penicillium, Microsphaeropsis, Stachybotrys, Humicola, Cladosporium, Bacillus, Planomicrobium, and Acinetobacter. Chryseomicrobium genus may be a key focus for future microbial prevention and control work.

From Fluorescence-Transfer-Lightening-Printing-Assisted Conductive Adhesive Nanocomposite Hydrogels toward Wearable Interactive Optical Information-Electronic Strain Sensors.

The interactive flexible device, which monitors the human motion in optical and electrical synergistic modes, has attracted growing attention recently. The incorporation of information attribute within the optical signal is deemed advantageous for improving the interactive efficiency. Therefore, the development of wearable optical information-electronic strain sensors holds substantial promise, but integrating and synergizing various functions and realizing strain-mediated information transformation keep challenging. Herein, an amylopectin (AP) modified nanoclay/polyacrylamide-based nanocomposite (NC) hydrogel and an aggregation-induced-emission-active ink are fabricated. Through the fluorescence-transfer printing of the ink onto the hydrogel film in different strains with nested multiple symbolic information, a wearable interactive fluorescent information-electronic strain sensor is developed. In the sensor, the nanoclay plays a synergistic "one-stone-three-birds" role, contributing to "lightening" fluorescence (≈80 times emission intensity enhancement), ionic conductivity, and excellent stretchability (>1000%). The sensor has high biocompatibility, resilience (elastic recovery ratio: 97.8%), and strain sensitivity (gauge factor (GF): 10.9). Additionally, the AP endows the sensor with skin adhesiveness. The sensor can achieve electrical monitoring of human joint movements while displaying interactive fluorescent information transformation. This research poses an efficient strategy to develop multifunctional materials and provides a general platform for achieving next-generation interactive devices with prospective applications in wearable devices, human-machine interfaces, and artificial intelligence.

Chromosomal level genome assemblies of two Malus crabapple cultivars Flame and Royalty.

Malus hybrid 'Flame' and Malus hybrid 'Royalty' are representative ornamental crabapples, rich in flavonoids and serving as the preferred materials for studying the coloration mechanism. We generated two sets of high-quality chromosome-level and haplotype-resolved genome of 'Flame' with sizes of 688.2 Mb and 675.7 Mb, and those of 'Royalty' with sizes of 674.1 Mb and 663.6 Mb, all anchored to 17 chromosomes and with a high BUSCO completeness score nearly 99.0%. A total of 47,833 and 47,307 protein-coding genes were annotated in the two haplotype genomes of 'Flame', and the numbers of 'Royalty' were 46,305 and 46,920 individually. The assembled high-quality genomes offer new resources for studying the origin and adaptive evolution of crabapples and the molecular basis of the accumulation of flavonoids and anthocyanins, facilitating molecular breeding of Malus plants.

Microfluidic chip immunoassay based on rolling circle amplification and G-quadruplex/Thioflavin T for multiplex detection of CTX I.

A label-free immunoassay based on rolling circle amplification (RCA) and G-quadruplex/Thioflavin T (G4/ThT) is proposed to realize the sensitive detection of carboxy-terminal cross-linked fragment of type I collagen (CTX I) for bone loss. Under the optimal conditions, as low as 38.02 pg/mL of CTX I can be detected. To improve the detecting throughput and simplify the operation, a microfluidic chip was designed, fabricated, and used for CTX I detection based on the proposed assay. The detection can be completed with only a single on-chip magnetic separation step, which was easy to operate, less time-consuming, and has only low reagent consumption. The limit of detection was 131.83 pg/mL by observing with fluorescence microscope. With further improvement of detection equipment, the sensitivity of on-chip detection can be improved. It can be expected that the proposed RCA/G4/ThT immunoassay for sensitive and high-throughput automated detection of CTX I might be chosen as a potential analytical tool for clinical osteoporosis diagnosis and in-orbit bone loss detection.

Cancer-cell-specific Self-Reporting Photosensitizer for Precise Identification and Ablation of Cancer Cells.

Cancer-cell-specific fluorescent photosensitizers (PSs) are highly desired molecular tools for cancer ablation with minimal damage to normal cells. However, such PSs that can achieve cancer specification and ablation and a self-reporting manner concurrently are rarely reported and still an extremely challenging task. Herein, we have proposed a feasible strategy and conceived a series of fluorescent PSs based on simple chemical structures for identifying and killing cancer cells as well as monitoring the photodynamic therapy (PDT) process by visualizing the change of subcellular localization. All of the constructed cationic molecules could stain mitochondria in cancer cells, identify cancer cells specifically, and monitor cancer cell viability. Among these, IVP-Br has the strongest ability to produce ROS, which serves as a potent PS for specific recognition and killing of cancer cells. IVP-Br could translocate from mitochondria to the nucleolus during PDT, self-reporting the entire therapeutic process. Mechanism study confirms that IVP-Br with light irradiation causes cancer cell ablation via inducing cell cycle arrest, cell apoptosis, and autophagy. The efficient ablation of tumor through PDT induced by IVP-Br has been confirmed in the 3D tumor spheroid chip. Particularly, IVP-Br could discriminate cancer cells from white blood cells (WBCs), exhibiting great potential to identify circulating tumor cells (CTCs).

FKBP10 promotes clear cell renal cell carcinoma progression and regulates sensitivity to the HIF2α blockade by facilitating LDHA phosphorylation.

Renal cell carcinoma (RCC) is one of the three major malignant tumors of the urinary system and originates from proximal tubular epithelial cells. Clear cell renal cell carcinoma (ccRCC) accounts for approximately 80% of RCC cases and is recognized as a metabolic disease driven by genetic mutations and epigenetic alterations. Through bioinformatic analysis, we found that FK506 binding protein 10 (FKBP10) may play an essential role in hypoxia and glycolysis pathways in ccRCC progression. Functionally, FKBP10 promotes the proliferation and metastasis of ccRCC in vivo and in vitro depending on its peptidyl-prolyl cis-trans isomerase (PPIase) domains. Mechanistically, FKBP10 binds directly to lactate dehydrogenase A (LDHA) through its C-terminal region, the key regulator of glycolysis, and enhances the LDHA-Y10 phosphorylation, which results in a hyperactive Warburg effect and the accumulation of histone lactylation. Moreover, HIFα negatively regulates the expression of FKBP10, and inhibition of FKBP10 enhances the antitumor effect of the HIF2α inhibitor PT2385. Therefore, our study demonstrates that FKBP10 promotes clear cell renal cell carcinoma progression and regulates sensitivity to HIF2α blockade by facilitating LDHA phosphorylation, which may be exploited for anticancer therapy.

The pyroptosis mediated biomarker pattern: an emerging diagnostic approach for Parkinson's disease.

BACKGROUND: Parkinson's disease (PD) affects 1% of people over 60, and long-term levodopa treatment can cause side effects. Early diagnosis is of great significance in slowing down the pathological process of PD. Multiple pieces of evidence showed that non-coding RNAs (ncRNAs) could participate in the progression of PD pathology. Pyroptosis is known to be regulated by ncRNAs as a key pathological feature of PD. Therefore, evaluating ncRNAs and pyroptosis-related proteins in serum could be worthy biomarkers for early diagnosis of PD. METHODS: NcRNAs and pyroptosis/inflammation mRNA levels were measured with reverse transcriptase quantitative polymerase chain reaction (RT-qPCR). Luciferase assays were performed to confirm GSDME as a target of miR-675-5p and HMGB1 as a target of miR-1247-5p. In the serum of healthy controls (n = 106) and PD patients (n = 104), RT-qPCR was utilized to assess miR-675-5p, miR-1247-5p, and two related ncRNAs (circSLC8A1and lncH19) levels. The enzyme-linked immunosorbent assay measured serum levels of pyroptosis-related proteins in controls (n = 54) and PD patients (n = 70). RESULTS: Our data demonstrated that miR-675-5p and miR-1247-5p significantly changed in PD neuron and animal models. Overexpressed miR-675-5p or downregulated miR-1247-5p could regulate pyroptosis and inflammation in PD neuron models. Using the random forest algorithm, we constructed a classifier based on PD neuron-pyroptosis pathology (four ncRNAs and six proteins) having better predictive power than single biomarkers (AUC = 92%). Additionally, we verified the performance of the classifier in early-stage PD patients (AUC ≥ 88%). CONCLUSION: Serum pyroptosis-related ncRNAs and proteins could serve as reliable, inexpensive, and non-invasive diagnostic biomarkers for PD. LIMITATIONS: All participants were from the same region. Additionally, longitudinal studies in the aged population are required to explore the practical application value of the classifier.

Epidemiology of respiratory pathogens in patients with acute respiratory tract infection in Xiamen, China: A retrospective survey from 2020 to 2022.

Acute respiratory tract infections (ARTI) are caused by respiratory pathogens and range from asymptomatic infections to severe respiratory diseases. These diseases can be life threatening with high morbidity and mortality worldwide. Under the pandemic of coronavirus disease 2019 (COVID-19), little has been reported about the pathogen etiologies and epidemiology of patients suffering from ARTI of all age in Xiamen. Region-specific surveillance in individuals with ARTI of all ages was performed in Xiamen from January 2020 to October 2022. Here, we observed the epidemiological characteristics of thirteen pathogens within ARTI patients and further revealed the difference of that between upper respiratory tract infections (URTI) and lower respiratory tract infections (LRTI). In total 56.36 % (2358/4184) of the ARTI patients were positive for at least one respiratory pathogen. Rhinovirus (RVs, 29.22 %), influenza A (FluA, 19.59 %), respiratory syncytial virus (RSV, 18.36 %), metapneumovirus (MPV, 13.91 %), and adenovirus (ADV, 10.31 %) were the five leading respiratory pathogens. Respiratory pathogens displayed age- and season-specific patterns, even between URTI and LRTI. Compared with other groups, a higher proportion of FluA (52.17 % and 68.75 %, respectively) infection was found in the adult group and the elder group, while the lower proportion of RVs (14.11 % and 11.11 %) infection was also observed in them. Although ARTI cases circulated throughout the year, RVs, FluB, and BoV peaked in autumn, and FluA circulated more in summer. Besides, the co-infectious rate was 8.7 % with the most common for RVs. Logistic regression analyses revealed the correlations between respiratory pathogens and disease types. These results are essential for replenishing epidemiological characteristics of common respiratory pathogens that caused ARTI in Xiamen during the epidemic of COVID-19, and a better understanding of it might optimize the local prevention and clinical control.

Altered expression of inflammation-associated molecules in striatum: an implication for sensitivity to heavy ion radiations.

Background and objective: Heavy ion radiation is one of the major hazards astronauts face during space expeditions, adversely affecting the central nervous system. Radiation causes severe damage to sensitive brain regions, especially the striatum, resulting in cognitive impairment and other physiological issues in astronauts. However, the intensity of brain damage and associated underlying molecular pathological mechanisms mediated by heavy ion radiation are still unknown. The present study is aimed to identify the damaging effect of heavy ion radiation on the striatum and associated underlying pathological mechanisms. Materials and methods: Two parallel cohorts of rats were exposed to radiation in multiple doses and times. Cohort I was exposed to 15 Gy of 12C6+ ions radiation, whereas cohort II was exposed to 3.4 Gy and 8 Gy with 56Fe26+ ions irradiation. Physiological and behavioural tests were performed, followed by 18F-FDG-PET scans, transcriptomics analysis of the striatum, and in-vitro studies to verify the interconnection between immune cells and neurons. Results: Both cohorts revealed more persistent striatum dysfunction than other brain regions under heavy ion radiation at multiple doses and time, exposed by physiological, behavioural, and 18F-FDG-PET scans. Transcriptomic analysis revealed that striatum dysfunction is linked with an abnormal immune system. In vitro studies demonstrated that radiation mediated diversified effects on different immune cells and sustained monocyte viability but inhibited its differentiation and migration, leading to chronic neuroinflammation in the striatum and might affect other associated brain regions. Conclusion: Our findings suggest that striatum dysfunction under heavy ion radiation activates abnormal immune systems, leading to chronic neuroinflammation and neuronal injury.

Prevention of STAT3-related pathway in SK-N-SH cells by natural product astaxanthin.

PURPOSE: Neuroblastoma (NB) is the most common solid malignancy in children. Despite current intensive treatment, the long-term event-free survival rate is less than 50% in these patients. Thus, patients with NB urgently need more valid treatment strategies. Previous research has shown that STAT3 may be an effective target in high-risk NB patients. However, there are no effective inhibitors in clinical evaluation with low toxicity and few side effects. Astaxanthin is a safe and natural anticancer product. In this study, we investigated whether astaxanthin could exert antitumor effects in the SK-N-SH neuroblastoma cancer cell line. METHOD: MTT and colony formation assays were used to determine the effect of astaxanthin on the proliferation and colony formation of SK-N-SH cells. Flow cytometry assays were used to detect the apoptosis of SK-N-SH cells. The migration and invasion ability of SK-N-SH cells were detected by migration and invasion assays. Western blot and RT-PCR were used to detect the protein and mRNA levels. Animal experiments were carried out and cell apoptosis in tissues were assessed using a TUNEL assay. RESULT: We confirmed that astaxanthin repressed proliferation, clone formation ability, migration and invasion and induced apoptosis in SK-N-SH cells through the STAT3 pathway. Furthermore, the highest inhibitory effect was observed when astaxanthin was combined with si-STAT3. The reason for this may be that the combination of astaxanthin and si-STAT3 can lower STAT3 expression further than astaxanthin or si-STAT3 alone. CONCLUSION: Astaxanthin can exert anti-tumor effect on SK-N-SH cells. The inhibitory effect was the higher when astaxanthin was combined with si-STAT3.

[Characterization and identification of primarily cultured astrocytes and microglia].

The aim of this study was to investigate the growth characteristics of primarily cultured astrocytes and microglia of different generations and then optimize the method for obtaining primary astrocytes and microglia effectively. Primarily cultured microglia were isolated and purified from the cortices of neonatal mice. The proliferation curve of mixed glia cells was measured by Cell Counting Kit-8 (CCK-8) assay, the proportion of astrocytes and microglia was detected by flow cytometry, and the polarization of the two types of glia cells was identified by immunofluorescence staining. Cell growth results showed that the mixed glia cells of P0 and P1 generation had the best proliferative activity; 97.3% of the high purity microglia could be obtained by mechanical shaking at 170 r/min for 30 min, and there was no significant difference in the morphology of ionized calcium-binding adapter molecule 1 (Iba-1) positive microglia and the proportion of M1 and M2 phenotype among the P0, P1 and P2 generations of microglia isolated by the above methods. Moreover, 95.7 % of the high purity astrocytes could be obtained by astrocyte cell surface antigen-2 (ACSA-2) magnetic beads separation, and there was no significant difference in the morphology of glial fibrillary acidic protein (GFAP) positive astrocyte and the proportion of A1 and A2 phenotype among the P0, P1 and P2 generations of astrocyte isolated by the above methods. Taken together, this study observed the growth characteristics of primarily cultured microglia and astrocyte in vitro, and then proved the best generations for purifying microglia and astrocytes. Finally, we optimized the methods of obtaining microglia and astrocyte, and verified that continuous culture within 2 generations will not affect the functional phenotypes of glia cells. These results provide technical support for studying the molecular mechanism of inflammation-associated diseases in nervous system.