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BackgroundHepatocellular carcinoma (HCC) is one of the most prevalent causes of cancer-related morbidity and mortality worldwide. Late-stage detection and the complex molecular mechanisms driving tumor progression contribute significantly to its poor prognosis. Dysregulated R-loops, three-stranded nucleic acid structures associated with genome instability, play a key role in the malignant characteristics of various tumors. However, the detailed role and mechanism of R-loops in HCC progression remain elusive and require further exploration. This study aimed to construct an R-loop scoring signature centered on prognosis and lipid metabolism, thereby enhancing our understanding of HCC progression and identifying potential therapeutic targets.MethodsIn this study, we utilized the single-cell RNA-sequencing (scRNA-seq) data from HCC patients (GSE149614 and CRA002308) to construct an R-loop scoring model based on the identified R-loop regulator genes (RLRGs) related to HBV infection through WGCNA analysis. We also explored the tumor microenvironment and intercellular communication related to R-loop score. Additionally, a prognostic risk model based on the fatty acid metabolism-associated RLRGs was constructed using data from the TCGA database, and its association with immune infiltration, mutations, and drug sensitivity was analyzed. In vitro and in vivo experiments were performed to investigate the role of RLRG CLTC in lipid metabolism and HCC progression.ResultsUsing scRNA-seq data from HCC, we established an R-loop scoring model based on identified RLRGs related to HBV infection. Moreover, the more suppressive tumor immune microenvironment and stronger intercellular communication were displayed in malignant cells with high R-loop scores. The cell trajectory and cellular metabolism analysis exhibited a significant association between lipid metabolism and RLRGs. Additionally, we constructed a prognostic risk model consisting of 8 RLRGs related to fatty acid metabolism, which effectively evaluated the prognostic value, status of tumor immune microenvironment, gene mutations, and chemotherapeutic drug sensitivity for HCC patients. Notably, validation experiments suggested that CLTC could regulate lipid metabolism through R-loop formation and facilitate tumor progression in HCC.ConclusionCollectively, our study proposes an R-loop scoring model associated with tumor immune microenvironment, lipid metabolism and prognostic value. CLTC, an R-loop regulator, emerges as a promising prognostic biomarker and therapeutic target, offering new insights into potential treatment strategies for HCC patients.

Abstract Background Vitamin B12 (VB12) has a wide range of applications and high economic value. In this study, a new strain with high VB12 production potential, Ensifer adhaerens S305, was identified in sewage. Because E. adhaerens strains have become the main strains for VB12 production via fermentation in recent years, the directional modification of the S305 strain to obtain a strain suitable for the industrial production of VB12 has great potential and commercial value. Results 16S rRNA and genome-wide phylogenetic tree analysis combined with average nucleotide identity (ANI) analysis showed that the high-yielding VB12 strain was a E. adhaerens strain and that its VB12 synthesis pathway genes were highly similar to related genes of strains of this and other species, including E. adhaerens Casida A, Pseudomonas denitrificans SC 510, and E. adhaerens Corn53. High-pressure liquid chromatography (HPLC) results indicated that the VB12 yields of the S305 strain were more than double those of the Casida A strain under different medium components. Multiple genes with significantly upregulated and downregulated transcription were identified by comparing the transcription intensity of different genes through transcriptome sequencing. KEGG enrichment analysis of the porphyrin metabolism pathway identified 9 significantly upregulated and downregulated differentially expressed genes (DEGs) in the VB12 synthesis pathway, including 7 transcriptionally upregulated genes (cobA, cobT, hemA, cobJ, cobN, cobR, and cobP) that were episomally overexpressed in the Casida A strain. The results showed that the VB12 yield of the overexpressed strain was higher than that of the wild-type strain. Notably, the strains overexpressing the cobA and cobT genes exhibited the most significant increases in VB12 yield, i.e., 31.4% and 24.8%, respectively. The VB12 yield of the S305 strain in shake-flask culture was improved from 176.6 ± 8.21 mg/L to 245.6 ± 4.36 mg/L by integrating the cobA and cobT genes into the strain. Conclusion Phylogenetic tree and ANI analysis showed that the Ensifer and Sinorhizobium strains were quite different at the genome level; the overexpression and integrated expression of significantly upregulated genes in the VB12 synthesis pathway could increase the yield of VB12, further improving the VB12 yield of the E. adhaerens S305 strain.

Hybrid two-phase closed thermosyphon (TPCT) artificial ground freezing technique is an effective cooling strategy adaptable to different geological and meteorological conditions in permafrost regions. Previous studies have focused on the feasibility of hybrid TPCT with little attention given to the cooling characteristics and long-term effects of this technique under actual environmental conditions. In this study, a novel structured semiconductor refrigeration device, with simple structure, small size, and wide operating temperature range, was designed and manufactured to act as the active condenser of a hybrid TPCT, and the cooling effect of this device was investigated during a two-year field test under stable power supply. This study showed that: 1) The minimum (maximum) average temperature of the semiconductor refrigeration two-phase closed thermosyphon (SRTPCT) evaporator was 0.5 (2.1) °C lower than the TPCT evaporator. The average ground temperature decreased by 0.3 °C, and the permafrost table rose to 0.2 m at 1 m from the evaporators around SRTPCT. The semiconductor refrigeration device added more than 9.75 MJ of cold storage to the soil around the TPCT evaporator during an operating cycle. 2) The optimal working period for the semiconductor refrigeration device was from June to October, and the cooling effect was better when the ambient temperature was low in the warm season. 3) As active condenser of hybrid TPCT in permafrost regions, semiconductor refrigeration device has a larger heat transfer power enhancement for TPCTs than vapor compression refrigeration device in the warm season, and vice versa in the cold season, as well as a higher heat transfer power enhancement for TPCTs than adsorption refrigeration device. The results of this study provide relevant parameters and references for the design and engineering application of future SRTPCT.

Stevioside is a secondary metabolite of diterpenoid glycoside production in plants. It has been used as a natural sweetener in various foods because of its high sweetness and low-calorie content. In this study, we constructed a Saccharomyces cerevisiae strain for the complete synthesis of stevioside using a metabolic engineering strategy. Firstly, the synthesis pathway of steviol was modularly constructed in S. cerevisiae BY4742, and the precursor pathway was strengthened. The yield of steviol was used as an indicator to investigate the expression effect of different sources of diterpene synthases under different combinations, and the strains with further improved steviol yield were screened. Secondly, glycosyltransferases were heterologously expressed in this strain to produce stevioside, the sequence of glycosyltransferase expression was optimized, and the uridine diphosphate-glucose (UDP-Glc) supply was enhanced. Finally, the results showed that the strain SST-302III-ST2 produced 164.89 mg/L of stevioside in a shake flask experiment, and the yield of stevioside reached 1104.49 mg/L in an experiment employing a 10 L bioreactor with batch feeding, which was the highest yield reported. We constructed strains with a high production of stevioside, thus laying the foundation for the production of other classes of steviol glycosides and holding good prospects for application and promotion.

Abstract Purpose Nectin-4 is specifically up-regulated in various tumors, exert crucial effects on tumor occurrence and development. Nevertheless, the role and molecular mechanism of Nectin-4 in osteosarcoma (OS) are rarely studied. Methods The expression of Nectin-4 and its relationship with clinical characteristics of OS were investigated using OS clinical tissues, tissue microarrays, TCGA, and GEO databases. Moreover, the effect of Nectin-4 on cell growth and mobility was detected by CCK-8, colony formation, transwell, and wound-healing assays. The RT-qPCR, Western blotting, and luciferase reporter assays were performed to explore molecular mechanisms through which Nectin-4 mediates the expression of miR-520c-3p, thus modulating PI3K/AKT/NF-κB signaling. In vivo mice models constructed by subcutaneous transplantation and tail vein injection were used to validate the functional roles of Nectin-4 and miR-520c-3p. Results Nectin-4 displayed a higher expression in OS tumor tissues compared with normal tissues, and its overexpression was positively associated with tumor stage and metastasis in OS patients. Functionally, Nectin-4 enhanced OS cells growth and mobility in vitro. Mechanistically, Nectin-4 down-regulated the levels of miR-520c-3p that directly targeted AKT-1 and P65, thus leading to the stimulation of PI3K/AKT/NF-κB signaling. In addition, the expression of miR-520c-3p was apparently lower in OS tissues than in normal tissues, and its low expression was significantly related to tumor metastasis. Furthermore, ectopic expression of miR-520c-3p markedly blocked the effect of Nectin-4 on OS cell growth and mobility. Knockdown of Nectin-4 could suppress the tumorigenesis and metastasis in vivo, which could be remarkably reversed by miR-520c-3p silencing. Conclusions Nectin-4 as an oncogene can promote OS progression and metastasis by activating PI3K/AKT/NF-κB signaling via down-regulation of miR-520c-3p, which could represent a novel avenue for identifying a potential therapeutic target for improving patient outcomes.

Two-phase closed thermosyphons (TPCTs) have been widely used in permafrost regions because of their good cooling effect. The variation in the inclination angle of TPCTs significantly affects the heat transfer process and temperature distribution inside TPCTs, which is still not fully understood. This study experimentally investigated the heat transfer performance of TPCTs with inclination angles varied from 0° to 90° with 10° intervals. In addition to the outer wall temperature and heat flux, the thermal regime of the working medium inside TPCT was simultaneously monitored based on the core-tube. Experimental results show that the temperature distributions inside TPCT along the axial direction vary regularly with inclination angle. The heat transfer rates of the evaporator and condenser sections change nonmonotonically and nonlinearly with the inclination angle. The influence of the inclination angle on the thermal resistance is greater in the evaporator and the adiabatic sections than in the condenser section. Consequently, a TPCT at 10° produces the best heat transfer performance, corresponding to the best isothermal characteristics, the maximum heat transfer rates of the evaporator and condenser sections, and the minimum total thermal resistance. This study provides a reference of monitoring method for further research on TPCT.

ObjectivesTo explore the direct and indirect heat damage zone of radiofrequency ablation (RFA) in porcine vertebrae and to verify the safety of RFA in a vascularized vertebral tumor model.MethodsRFA was performed in the porcine lumbar vertebrae. Magnetic resonance (MR) imaging, hematoxylin and eosin (HE), and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) were used to assess the extent of direct and indirect injuries after RFA. The cavity of lumbar vertebrae was made, and the adjacent muscle flap was used to fill the cavity to make a vertebrae tumor model. RFA was performed in the vascularized vertebral tumor model. ResultsT1-weighted images showed a hypointensive region in the center surrounded by a more hypointensive rim on day 0 and 14. T2-weighted images showed that RFA zone was hypointensive on day 0. On day 7, hypointensity was detected in the center surrounded by a hyperintensive rim. HE showed that the RFA zone could be clearly observed on day 14. Thin bone marrow loss areas were seen around the RFA zone, which was consistent with the hyperintensive rim on the T2-weighted images. TUNEL showed a large number of apoptotic cells in the RFA zone. During RFA in the vertebral tumor model, the temperature of all monitoring positions was less than 45 °C.ConclusionUsing in vivo experiments, the effective zone of RFA was evaluated by MR imaging and pathology, and the direct and indirect damage range were obtained. The safety of RFA was verified by RFA in a vascularized vertebral tumor model.

For a long time, it takes a lot of time and energy for psychological workers to classify the psychological problems of college students. In order to quickly and efficiently understand the common psychological problems of college students in the region for real-time analysis in the post-epidemic era, 2,000 college students’ psychological problems were selected as research data in the community question section of the “Su Xin” application, a psychological self-help and mutual aid platform for college students in Jiangsu Province. First, word segmentation, removal of stop words, establishment of word vectors, etc. were used for the preprocessing of research data. Secondly, it was divided into 9 common psychological problems by LDA clustering analysis, which also combined with previous researches. Thirdly, the text information was processed into word vectors and transferred to the Attention-Based Bidirectional Long Short-Term Memory Networks (AB-LSTM). The experimental results showed that the proposed model has a higher test accuracy of 78% compared with other models.

Hepatocellular carcinoma (HCC) with high heterogeneity is a common malignancy worldwide, but effective treatments are limited. Ferroptosis plays a critical role in tumors as a novel iron-dependent and reactive oxygen species-reliant type of cell death. Several studies have shown that long non-coding RNAs (lncRNAs) can drive HCC initiation and progression. However, the prognostic value of ferroptosis-related lncRNAs in patients with HCC has not been explored comprehensively. Gene set variation analysis (GSVA) based on gene set and RNA-seq profiles obtained from public databases indicated that ferroptosis is suppressed in HCC patients. Ferroptosis-related differentially expressed lncRNAs were screened by Pearson’s test. Univariate Cox regression, least absolute shrinkage and selection operator (LASSO) regression, and multivariate Cox regression were performed to establish a novel five ferroptosis-related lncRNA signature in the training cohort with 60% patients, which was further verified in the testing cohort with 40% patients. Dimensionality reduction analysis, Kaplan–Meier curve, receiver operating characteristic (ROC) curve, independent prognostic analysis, and stratification analysis confirmed that our signature had a high clinical application value in predicting the overall survival of HCC patients. Compared to the clinicopathological factors and the other four published HCC prognostic signatures, the current risk model had a better predictive value. The comparison results of functional enrichment, tumor immune microenvironment, genomic heterogeneity, and drug sensitivity between the high- and low-risk groups showed that the risk score is associated with extensive genomic alterations, immunosuppressive tumor microenvironment, and clinical treatment response. Finally, cell experiments showed that silencing LNCSRLR expression inhibited the growth, proliferation, migration, and invasion of the HCC cell line. Thus, the model can function as an efficient indicator for predicting clinical prognosis and treatment of anticancer drugs in HCC patients.