A disulfidptosis-related lncRNAs signature in hepatocellular carcinoma: prognostic prediction, tumor immune microenvironment and drug susceptibility.

Disulfidptosis, a novel type of programmed cell death, has attracted researchers' attention worldwide. However, the role of disulfidptosis-related lncRNAs (DRLs) in liver hepatocellular carcinoma (LIHC) not yet been studied. We aimed to establish and validate a prognostic signature of DRLs and analyze tumor microenvironment (TME) and drug susceptibility in LIHC patients. RNA sequencing data, mutation data, and clinical data were obtained from the Cancer Genome Atlas Database (TCGA). Lasso algorithm and cox regression analysis were performed to identify a prognostic DRLs signature. Kaplan-Meier curves, principal component analysis (PCA), nomogram and calibration curve, function enrichment, TME, immune dysfunction and exclusion (TIDE), tumor mutation burden (TMB), and drug sensitivity analyses were analyzed. External datasets were used to validate the predictive value of DRLs. qRT-PCR was also used to validate the differential expression of the target lncRNAs in tissue samples and cell lines. We established a prognostic signature for the DRLs (MKLN1-AS and TMCC1-AS1) in LIHC. The signature could divide the LIHC patients into low- and high-risk groups, with the high-risk subgroup associated with a worse prognosis. We observed discrepancies in tumor-infiltrating immune cells, immune function, function enrichment, and TIDE between two risk groups. LIHC patients in the high-risk group were more sensitive to several chemotherapeutic drugs. External datasets, clinical tissue, and cell lines confirmed the expression of MKLN1-AS and TMCC1-AS1 were upregulated in LIHC and associated with a worse prognosis. The novel signature based on the two DRLs provide new insight into LIHC prognostic prediction, TME, and potential therapeutic strategies.

High expression of ENPP2 is an independent predictor of poor prognosis in liver cancer.

Ectonucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) has been identified as a potential biomarker in lung and prostate cancers; however, its expression and clinical relevance in hepatocellular carcinoma (HCC) remain incompletely understood. This study comprehensively assessed ENPP2 expression in pan-cancer using bioinformatics. We analyzed the expression of ENPP2 mRNA in primary liver cancer and adjacent tissues of patients with HCC using data from the TCGA database. Cox regression and Kaplan-Meier methods were used to identify clinicopathological factors associated with survival, and the diagnostic value of ENPP2 expression was evaluated using receiver operating characteristic curve analysis. We also validated our findings by performing real-time PCR on clinical liver cancer samples. Furthermore, we conducted gene set enrichment analysis using the Cancer Genome Atlas dataset to gain additional insights into the biological significance of ENPP2 in HCC. High ENPP2 expression in HCC patients is associated with gender and clinical stage, and is a significant prognostic factor for worse outcomes. ENPP2 expression is an independent risk factor for progression-free and disease-specific survival in both cohorts, suggesting its potential as an HCC biomarker. ENPP2's diagnostic value in HCC patients was confirmed by the area under the receiver operating characteristic curve, which was 0.806. real-time PCR analysis validated the higher expression of ENPP2 in clinical liver cancer tissues. Gene set enrichment analysis identified pathways enriched in HCC patients with high ENPP2 expression, including those related to the cell cycle, MTOR and T cell receptor signaling, and phosphatidylinositol signaling systems. We have demonstrated that ENPP2 is highly expressed in HCC and is a potential independent molecular marker for the diagnosis and prognosis of HCC.

Nucleic Acid Detection with Ion Concentration Polarization Microfluidic Chip for Reduced Cycle Numbers of Polymerase Chain Reaction.

Nucleic acid detection is widely used in disease diagnosis, food safety, environmental monitoring and many other research fields. The continuous development of rapid and sensitive new methods to detective nucleic acid is very important for practical application. In this study, we developed a rapid nucleic-acid detection method using polymerase chain reaction (PCR) combined with electrokinetic preconcentration based on ion concentration polarization (ICP). Using a Nafion film, the proposed ICP microfluidic chip is utilized to enrich the nucleic acid molecules amplified by PCR thermal cycles. To demonstrate the capability of the microfluidic device and the hybrid nucleic-acid detection method, we present an animal-derived component detection experiment for meat product identification applications. With the reduced cycle numbers of 24 cycles, the detection can be completed in about 35 min. The experimental results show that this work can provide a microfluidic device and straightforward method for rapid detection of nucleic acids with reduced cycle numbers.

Microfluidics Temperature Compensating and Monitoring Based on Liquid Metal Heat Transfer.

Microfluidic devices offer excellent heat transfer, enabling the biochemical reactions to be more efficient. However, the precision of temperature sensing and control of microfluids is limited by the size effect. Here in this work, the relationship between the microfluids and the glass substrate of a typical microfluidic device is investigated. With an intelligent structure design and liquid metal, we demonstrated that a millimeter-scale industrial temperature sensor could be utilized for temperature sensing of micro-scale fluids. We proposed a heat transfer model based on this design, where the local correlations between the macro-scale temperature sensor and the micro-scale fluids were investigated. As a demonstration, a set of temperature-sensitive nucleic acid amplification tests were taken to show the precision of temperature control for micro-scale reagents. Comparations of theoretical and experimental data further verify the effectiveness of our heat transfer model. With the presented compensation approach, the slight fluorescent intensity changes caused by isothermal amplification polymerase chain reaction (PCR) temperature could be distinguished. For instance, the probability distribution plots of fluorescent intensity are significant from each other, even if the amplification temperature has a difference of 1 °C. Thus, this method may serve as a universal approach for micro-macro interface sensing and is helpful beyond microfluidic applications.

Easy-to-Operate Co-Flow Step Emulsification Device for High-Throughput Three-Dimensional Cell Culture.

Cell culture plays an essential role in tissue engineering and high-throughput drug screening. Compared with two-dimensional (2D) in vitro culture, three-dimensional (3D) in vitro culture can mimic cells in vivo more accurately, including complex cellular organizations, heterogeneity, and cell-extracellular matrix (ECM) interactions. This article presents a droplet-based microfluidic chip that integrates cell distribution, 3D in vitro cell culture, and in situ cell monitoring in a single device. Using the microfluidic "co-flow step emulsification" approach, we have successfully prepared close-packed droplet arrays with an ultra-high-volume fraction (72%) which can prevent cells from adhering to the chip surface so as to achieve a 3D cell culture and make scalable and high-throughput cell culture possible. The proposed device could produce droplets from 55.29 ± 1.52 to 95.64 ± 3.35 µm, enabling the diverse encapsulation of cells of different sizes and quantities. Furthermore, the cost for each microfluidic CFSE chip is approximately USD 3, making it a low-cost approach for 3D cell culture. The proposed device is successfully applied in the 3D culture of saccharomyces cerevisiae cells with an occurrence rate for proliferation of 80.34 ± 3.77%. With low-cost, easy-to-operate, high-throughput, and miniaturization characteristics, the proposed device meets the requirements for 3D in vitro cell culture and is expected to be applied in biological fields such as drug toxicology and pharmacokinetics.

Easy-to-Operate Co-flow Step Emulsification Device for Droplet Digital Polymerase Chain Reaction.

Digital polymerase chain reaction (PCR) plays important roles in the detection and quantification of nucleic acid targets, while there still remain challenges including high cost, complex operation, and low integration of the instrumental system. Here, in this work, a novel microfluidic chip based on co-flow step emulsification is proposed for droplet digital PCR (ddPCR), which can achieve droplet generation, droplet array self-assembly, PCR amplification, and fluorescence detection on a single device. With the combination of single-layer lithography and punching operation, a step microstructure was constructed and it served as the key element to develop a Laplace pressure gradient at the Rayleigh-Plateau instability interface so as to achieve droplet generation. It is demonstrated that the fabrication of step microstructure is low cost, easy-to-operate, and reliable. In addition, the single droplet volume can be adjusted flexibly due to the co-flow design; thus, the ddPCR chip can get an ultrahigh upper limit of quantification to deal with DNA templates with high concentrations. Furthermore, the volume fraction of the resulting droplets in this ddPCR chip can be up to 72% and it results in closely spaced droplet arrays, makes the best of CCD camera for fluorescence detections, and is beneficial for the minimization of a ddPCR system. The quantitative capability of the ddPCR chip was evaluated by measuring template DNA at concentrations from 20 to 50 000 copies/µL. Owing to the characteristics of low cost, easy operation, excellent quantitative capability, and minimization, the proposed ddPCR chip meets the requirements of DNA molecule quantification and is expected to be applied in the point-of-care testing field.

A Microfluidic Concentration Gradient Maker with Tunable Concentration Profiles by Changing Feed Flow Rate Ratios.

Microfluidic chips-in which chemical or biological fluid samples are mixed into linear or nonlinear concentration distribution profiles-have generated enormous enthusiasm of their ability to develop patterns for drug release and their potential toxicology applications. These microfluidic devices have untapped potential for varying concentration patterns by the use of one single device or by easy-to-operate procedures. To address this challenge, we developed a soft-lithography-fabricated microfluidic platform that enabled one single device to be used as a concentration maker, which could generate linear, bell-type, or even S-type concentration profiles by tuning the feed flow rate ratios of each independent inlet. Here, we present an FFRR (feed flow rate ratio) adjustment approach to generate tens of types of concentration gradient profiles with one single device. To demonstrate the advantages of this approach, we used a Christmas-tree-like microfluidic chip as the demo. Its performance was analyzed using numerical simulation models and experimental investigations, and it showed an excellent time response (~10 s). With on-demand flow rate ratios, the FFRR microfluidic device could be used for many lab-on-a-chip applications where flexible concentration profiles are required for analysis.