Enhancing single-cell encapsulation in droplet microfluidics with fine-tunable on-chip sample enrichment.

Single-cell encapsulation in droplet microfluidics is commonly hindered by the tradeoff between cell suspension density and on-chip focusing performance. In this study, we introduce a novel droplet microfluidic chip to overcome this challenge. The chip comprises a double spiral focusing unit, a flow resistance-based sample enrichment module with fine-tunable outlets, and a crossflow droplet generation unit. Utilizing a low-density cell/bead suspension (2 × 106 objects/mL), cells/beads are focused into a near-equidistant linear arrangement within the double spiral microchannel. The excess water phase is diverted while cells/beads remain focused and sequentially encapsulated in individual droplets. Focusing performance was assessed through numerical simulations and experiments at three flow rates (40, 60, 80 µL/min), demonstrating successful focusing at 40 and 80 µL/min for beads and cells, respectively. In addition, both simulation and experimental results revealed that the flow resistance at the sample enrichment module is adjustable by punching different outlets, allowing over 50% of the aqueous phase to be removed. YOLOv8n-based droplet detection algorithms realized the counting of cells/beads in droplets, statistically demonstrating single-cell and bead encapsulation rates of 72.2% and 79.2%, respectively. All the results indicate that this on-chip sample enrichment approach can be further developed and employed as a critical component in single-cell encapsulation in water-in-oil droplets.

Oxidase-like Fe-N/C single atom nanozyme enables sensitive detection of ascorbic acid and acid phosphatase.

The development of cost-effective and easy-to-use strategies for the detection of ascorbic acid (AA) and acid phosphatase (ACP) is in high demand but challenging. Thus, we report a novel colorimetric platform based on Fe-N/C single atom nanozyme with efficient oxidase mimicking activity for their highly sensitive detection. The designed Fe-N/C single atom nanozyme can directly oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to produce a blue oxidation product (oxTMB) in the absence of H2O2. In addition, L-ascorbic acid 2-phosphate can be hydrolyzed to ascorbic acid in the presence of ACP, which inhibits the oxidation reaction and results in a significant bleaching of the blue color. Based on these phenomena, a novel colorimetric assay with high catalytic activity was developed for the determination of ascorbic acid and acid phosphatase with detection limits of 0.092 µM and 0.048 U/L, respectively. Notably, this strategy was successfully applied to the determination of ACP in human serum samples and evaluate ACP inhibitors, indicating its potential as a valuable tool for clinical diagnosis and research.

IGF2BP3 Regulates TMA7-mediated Autophagy and Cisplatin Resistance in Laryngeal Cancer via m6A RNA Methylation.

Translation machinery associated 7 homolog (TMA7) is closely related to proliferation-related diseases. However, the function and regulatory mechanism of TMA7 in laryngeal squamous cell carcinoma (LSCC) remain unclear. The present study aimed to investigate the effect of TMA7 on the occurrence and development of LSCC and to study the mechanism of TMA7. TMA7 is upregulated in LSCC tissues and associated with poor prognosis. After TMA7 downregulation, the autophagy level was increased, and the proliferation, migration, and invasion of LSCC cells were inhibited. The m6A methylated reader IGF2BP3 enhanced the stability of TMA7 and reduced the level of autophagy. TMA7 interacted directly with UBA2. Furthermore, the activation of the IGF2BP3-regulated TMA7-UBA2-PI3K pathway is the primary mechanism by which TMA7 inhibits autophagy and promotes the progression of LSCC. The current study revealed that IGF2BP3-mediated TMA7 m6A modification promotes LSCC progression and cisplatin-resistance through UBA2-PI3K pathway, providing new insights into the autophagy-related mechanism, potential biomarkers, and therapeutic targets for LSCC.

A novel probe for tetracyclines detection and its applications in cell imaging based on fluorescent WS2 quantum dots.

In this study, a novel fluorescence sensor for tetracyclines (TCs) detection was designed using WS2 quantum dots (WS2 QDs). WS2 QDs could be quenched by TCs through the inner filter effect (IFE). The limit of detection of this proprosed method is 39 nM, 52 nM, and 28 nM for tetracycline (TC), doxycycline (DC), and oxytetracycline (OTC), respectively. The as-proposed strategy was successfully applied to detect TC in milk samples and human serum samples. The WS2 QDs were highly biocompatible and showed lower toxicity. Moreover, the WS2 QDs was successfully applied to imaging TC in HeLa cells owing to its excellent optical performance and great biocompatibility.

A colorimetric smartphone-based platform for pesticides detection using Fe-N/C single-atom nanozyme as oxidase mimetics.

In this study, a novel highly sensitive colorimetric platform has been designed for malathion assay based on Fe-N/C SAzyme. The as-synthesized SAzyme can directly oxidize 3,3´,5,5´-tetramethylbenzidine (TMB) to generate blue colored oxidized TMB. L-ascorbic acid-2-phosphate (AA2P), a substrate of acid phosphatase (ACP), could be hydrolyzed to AA, thereafter inhibit the oxidization reaction of TMB, leading to a conspicuous blue color fading. With the addition of malathion hindered the ACP activity and limited the AA production, resulting in the recovery of the catalytic activity of single-atom nanozyme. Under optimized operational conditions, a novel colorimetric assay has been designed for malathion detection with LOD of 0.42 nM. Besides, quantification of malathion in environmental and food samples was achieved based on the proposed strategy. In addition, the successfully integrated paper/smartphone sensor provided sensitive, and rapid, reliable detection of malathion with a LOD of 1 nM.

MoS2 quantum dots as fluorescent probe for methotrexate detection.

Herein, we developed a unique fluorescence biosensor for methotrexate assay based on MoS2 quantum dots, which were synthesized in one step using sodium molybdate and cysteine as raw materials. The fluorescence of MoS2 QDs could be quenched when encountered with methotrexate, which was attributed to the inner filter effect (IFE). Furthermore, this present IFE-based method showed the linearity between the MoS2 QDs fluorescence intensity and the methotrexate concentration in the range of 0.05-1 µM with the LOD of 42 nM. The practical applicability of this strategy was successfully demonstrated by detecting methotrexate in real samples. Results indicated that the proposed method could be a promising sensing platform for methotrexate analysis.

LncRNA TP73-AS1 promotes nasopharyngeal carcinoma progression through targeting miR-342-3p and M2 polarization via exosomes.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a deadly cancer, mainly presenting in southeast and east Asia. Long noncoding RNAs (lncRNAs) play essential roles in cancer progression. Exosomes are critical for intercellular communication. Thus, the aim of this study was to identify the functional lncRNAs in NPC and its relevant mechanisms. METHODS: Data from public databases were utilized to screen for functional lncRNAs in NPC. Functional and mechanical experiments were performed to determine the role of lncRNAs in NPC and its relative molecular mechanisms. Exosomes derived from NPC cells were isolated to determine their function in tumor-associated macrophages. RESULTS: LncRNA TP73-AS1 was increased in NPC cells and tissues and was associated with a poor prognosis. TP73-AS1 overexpression promoted proliferation, colony formation, and DNA synthesis of NPC cells while TP73-AS1 knockdown showed opposite roles. TP73-AS1 could directly bind with miR-342-3p. MiR-342-3p overexpression attenuated the effect of TP73-AS1 in NPC cells. Furthermore, TP73-AS1 was transferred by exosomes to promote M2 polarization of macrophages. Lastly, exosomal TP73-AS1 enhanced the motility and tube formation of macrophages. CONCLUSIONS: Together, this study suggests that TP73-AS1 promotes NPC progression through targeting miR-342-3p and exosome-based communication with macrophages and that TP73-AS1 might be an emerging biomarker for NPC.

RBM15 facilitates laryngeal squamous cell carcinoma progression by regulating TMBIM6 stability through IGF2BP3 dependent.

BACKGROUND: Laryngeal cancer has the highest mortality rate among head and neck tumours. RNA N6-methyladenosine (m6A) is the most plentiful and variable in mammalian mRNA. Yet, the m6A regulatory mechanism underlying the carcinogenesis or progression of LSCC remains poorly understood. METHODS: The m6A RNA methylation quantification kit was used to detect tissue methylation levels. m6A microarray analysis, mRNA transcriptomic sequencing (mRNA-seq), and proteomics were used to determine RBM15, TMBIM6, and IGF2BP3. Immunohistochemical (IHC), quantitative real-time PCR (qRT-PCR) and Western blot were used to investigate RBM15, TMBIM6, and IGF2BP3 expression in tissue samples and cell lines. The biological effects of RBM15 were detected both in vitro and in vivo. The combination relationship between RBM15/IGF2BP3 and TMBIM6 was verified by RNA immunoprecipitation (RIP) assay, Methylated RNA immunoprecipitation sequencing (MeRIP-seq), RNase Mazf, and luciferase report assay. RNase Mazf was used to determine the methylation site on TMBIM6 mRNA. Hoechst staining assay was used to confirm the apoptotic changes. The actinomycin D verified TMBIM6 stability. RESULTS: The global mRNA m6A methylation level significantly increased in LSCC patients. RBM15, as a "writer" of methyltransferase, was significantly increased in LSCC and was associated with unfavorable prognosis. The knockdown of RBM15 reduced the proliferation, invasion, migration, and apoptosis of LSCC both in vitro and in vivo. The results were reversed after overexpressing RBM15. Mechanically, TMBIM6 acted as a downstream target of RBM15-mediated m6A modification. Furthermore, RBM15-mediated m6A modification of TMBIM6 mRNA enhanced TMBIM6 stability through IGF2BP3-dependent. CONCLUSION: Our results revealed the essential roles of RBM15 and IGF2BP3 in m6A methylation modification in LSCC, thus identifying a novel RNA regulatory mechanism.

Facile synthesis of biomass waste-derived fluorescent N, S, P co-doped carbon dots for detection of Fe3+ ions in solutions and living cells.

Fluorescent carbon dots derived from natural biomass have received widespread attention in recent years due to their superior optical and chemical properties. In this work, we proposed a method to synthesize fluorescent nitrogen, sulfur, and phosphorus co-doped carbon dots (NSP-CDs) using biomass waste as a precursor. The blue emitting carbon dots were prepared from the seeds of green pepper, and Fe3+ ions could quench the fluorescence of NSP-CDs. Therefore, a fluorescent "turn-off" sensor based on NSP-CDs was constructed for the detection of Fe3+ ions. Further, NSP-CDs were evaluated as a fluorescent biosensor for the detection of Fe3+ in tap water and lake water samples, showing their potential value in practical applications. The cytotoxicity test further confirmed that NSP-CDs have good biocompatibility and can be extended to cell imaging and intracellular Fe3+ detection. The proposed method is simple, economical and green, which can meet the requirements of environmental monitoring and biological imaging.

Circular RNA 103862 Promotes Proliferation and Invasion of Laryngeal Squamous Cell Carcinoma Cells Through the miR-493-5p/GOLM1 Axis.

Accumulating evidence suggests that circular RNAs (circRNAs) may be a key contributor to oncogenesis. Yet, the function of circRNAs in laryngeal squamous cell carcinoma (LSCC) is still not clear. In this study, we examined the function of circRNA_103862 in LSCC progression by analyzing the tissue specimens collected from a patient with LSCC by using different LSCC cell models in vitro and an LSCC xenograft model in nude mice. We found that circRNA_103862 was frequently upregulated in the tissues of LSCC and was correlated with metastasis and prognosis of LSCC patients. Furthermore, circRNA_103862 downregulation could reduce proliferation, migration, and invasion ability of LSCC cells. In terms of mechanism exploration, miR-493-5p was sponged by circRNA_103862. Rescue experiments also showed that circRNA_103862 could achieve a carcinogenic effect by regulating miR-493-5p. Moreover, a luciferase reporter analysis showed that Golgi membrane protein 1 (GOLM1) is a downstream effector of miR-493-5p. In conclusion, our data suggested that circRNA_103862 promotes the proliferation of LSCC through targeting the miR-493-5p/GOLM1 axis, and it might serve as a potential prognosis marker and therapy target for LSCC.

LncRNA MSC-AS1 aggravates nasopharyngeal carcinoma progression by targeting miR-524-5p/nuclear receptor subfamily 4 group A member 2 (NR4A2).

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a subtype of head and neck cancer with dismal prognosis and high relapse rate. The role of long non-coding RNAs (lncRNAs) in NPC has become a research hotspot in recent years. This study aimed to interrogate the function and mechanism of lncRNA MSC antisense RNA 1 (MSC-AS1) in NPC. METHODS: MSC-AS1 level in NPC tissues and cells were detected by RT-qPCR. Function of MSC-AS1 in NPC cells was assessed by CCK-8, EdU, TUNEL, caspase-3 activity, and transwell invasion assay. Interaction of microRNA-524-5p (miR-524-5p) with MSC-AS1 and nuclear receptor subfamily 4 group A member 2 (NR4A2) was determined by RIP and luciferase reporter assays. RESULTS: MSC-AS1 was upregulated in NPC tissues and cells. Functional assays indicated that MSC-AS1 exacerbated cell proliferation, hindered apoptosis, and facilitated invasion and epithelial-to-mesenchymal transition (EMT) in NPC. Mechanistically, MSC-AS1 sequestered miR-524-5p to upregulate NR4A2 expression in NPC cells. Finally, NR4A2 was conformed as an oncogene in NPC, and overexpressed NR4A2 could restore MSC-AS1 knockdown-mediated inhibition on NPC progression. CONCLUSIONS: Our study firstly showed that lncRNA MSC-AS1 aggravated NPC progression by sponging miR-524-5p to increase NR4A2 expression, indicating MSC-AS1 as a novel target for the lncRNA-targeted therapy in NPC.

Regulation of laryngeal squamous cell cancer progression by the lncRNA RP11-159K7.2/miR-206/DNMT3A axis.

Long non-coding RNAs (lncRNAs), which are longer than 200 nt, have been proved to play a role in promoting or inhibiting cancer progression. The following study investigated the role and underlying mechanisms of lncRNA RP11-159K7.2 in laryngeal squamous cell carcinoma (LSCC) progression. Briefly, in situ hybridization (ISH) and real-time quantitative PCR (RT-qPCR) showed higher expression of RP11-159K7.2 in LSCC tissues and cell lines. Patients with low expression level of RP11-159K7.2 lived longer compared to those with high expression of RP11-159K7.2 (χ2  = 39.111, ***P < 0.001). Multivariate Cox regression analysis suggested that lncRNA RP11-159K7.2 was an independent prognostic factor for LSCC patients (HR = 2.961, ***P < 0.001). Furthermore, to investigate the potential involvement of RP11-159K7.2 in the development of LSCC, we knocked out the expression of endogenous RP11-159K7.2 in TU-212 cells and AMC-HN-8 cells via CRISPR/Cas9 double vector lentiviral system. RP11-159K7.2 knockout decreased LSCC cell growth and invasion both in vitro and in vivo. Mechanically, we found that RP11-159K7.2 could positively regulate the expression of DNMT3A by sponging miR-206. In addition, a feedback loop was also discovered between DNMT3A and miR-206. To sum up, these findings suggest that lncRNA RP11-159K7.2 could be used as a potential biomarker for prognosis and treatment of LSCC.

Alkaline-promoted regulation of the peroxidase-like activity of Ni/Co LDHs and development bioassays.

Nanozymes' activities could be regulated by a simple and effective pH change in an in situ manner. In this work, for the first time, the peroxidase-like activity of Ni/Co layered double hydroxides (LDHs) was regulated via the alkaline-promoted reaction of fluorogenic substrate homovanillic acid and H2O2, and a promising tool for pH sensing was developed over the pH range of 8.3-9.6. As peroxidase nanozyme model, Ni/Co LDHs showed ease of preparation, low-cost, and water-solubility, which played an important role in this luminescence system. Based on the pH-dependent regulation of the Ni/Co LDHs activity, we constructed the bioassay platform for the determination urea, urease, penicillin G, and penicillinase with a wide linear range of 17-1000 µM, 3.3-270 mU mL-1, 3.3-1300 µM and 3.3-100 mU mL-1, respectively. This study not only demonstrated the alkaline-promoted modulation the nanozymes' activities, but also established a facile approach to develop novel bioassays.

A Novel LncRNA, AC091729.7 Promotes Sinonasal Squamous Cell Carcinomas Proliferation and Invasion Through Binding SRSF2.

Long non-coding RNAs (lncRNAs) play important roles in various biological progresses of carcinogenesis. However, the function of lncRNAs in human sinonasal squamous cell carcinoma (SNSCC) remains greatly unclear. In the current study, lncRNA AC091729.7 expression was examined in SNSCC samples by using microarray, RNA in situ hybridization (ISH) and real-time fluorescence quantitative PCR (qRT-PCR). Cell viability, colony-formation, wound-healing, and transwell assays were applied to SNSCC cells. Xenograft mouse models were employed to evaluate the role of AC091729.7 in growth of SNSCC in vivo. Human protein microarray (HuprotTM Protoarray) and RNA immunoprecipitation (RIP) were used for identifying AC091729.7 binding proteins in SNSCC. Results showed AC091729.7 was upregulated and closely connected with the survival of the SNSCC patients. Knockdown of AC091729.7 suppressed SNSCC cell migration, proliferation, invasion in vitro. Furthermore, downregulation of AC091729.7 could inhibit the growth of SNSCC in vivo. Moreover, Human protein microarray and RIP suggested that AC091729.7 directly combine with the serine/arginine rich splicing factor 2 (SRSF2). Our results suggest that in the cell progression of SNSCC, lncRNA AC091729.7 plays a carcinogenic role and serves as a novel biomarker and latent curative target in SNSCC patients.

A three-lncRNA expression signature predicts survival in head and neck squamous cell carcinoma (HNSCC).

Increasing evidence has shown that long non-coding RNAs (lncRNAs) have important biological functions and can be used as a prognostic biomarker in human cancers. However, investigation of the prognostic value of lncRNAs in head and neck squamous cell carcinoma (HNSCC) is in infancy. In the present study, we analyzed the lncRNA expression data in a large number of HNSCC patients (n=425) derived from The Cancer Genome Atlas (TCGA) to identify an lncRNA expression signature for improving the prognosis of HNSCC. Three lncRNAs are identified to be significantly associated with survival in the training dataset using Cox regression analysis. Three lncRNAs were integrated to construct an lncRNA expression signature that could stratify patients of training dataset into the high-risk group and low-risk group with significantly different survival time (median survival 1.85 years vs. 5.48 years; P=0.0018, log-rank test). The prognostic value of this three-lncRNA signature was confirmed in the testing and entire datasets, respectively. Further analysis revealed that the prognostic power of three-lncRNA signature was independent of clinical features by multivariate Cox regression and stratified analysis. These three lncRNAs were significantly associated with known genetic and epigenetic events by means of functional enrichment analysis. Therefore, our results indicated that the three-lncRNA expression signature can predict HNSCC patients' survival.

Expression of long non-coding RNA HOXA11-AS is correlated with progression of laryngeal squamous cell carcinoma.

Long noncoding RNA HOXA11 antisense RNA (HOXA11-AS) is involved in tumorigenesis and development of some human cancers. However, the role of HOXA11-AS in human laryngeal squamous cell cancer (LSCC) is yet unclear. In this study, we firstly investigated the expression of HOXA11-AS in LSCC. Microarray and qRT-PCR showed that the level of HOXA11-AS was significantly higher in LSCC than that in the corresponding adjacent non-neoplastic tissues. ISH revealed that HOXA11-AS was strongly expressed in the nucleus and closely related to the T grade, neck nodal metastasis, and clinical stage. Patients with T3-4 grade, neck nodal metastasis, or advanced clinical stage presented a high HOXA11-AS expression. Kaplan-Meier analysis showed that high HOXA11-AS expression could predict a poor prognosis in LSCC patients. Furthermore, HOXA11-AS knockdown significantly inhibited the growth, migration, and invasion of LSCC cells. Taken together, the current data indicated that HOXA11-AS plays an oncogenic role in the cellular processes of LSCC and serve as a novel marker and a potential therapeutic target in LSCC patients.