System analysis based on the T cell exhaustion­related genes identifies CD38 as a novel therapy target for ovarian cancer.

Ovarian cancer (OV) is highly heterogeneous tumor with a very poor prognosis. Studies increasingly show that T cell exhaustion is prognostically relevant in OV. The aim of this study was to dissect the heterogeneity of T cell subclusters in OV through single cell transcriptomic analysis. The single RNA-sequencing (scRNA-seq) data of five OV patients were analyzed, and six major cell clusters were identified after threshold screening. Further clustering of T cell-associated clusters revealed four subtypes. Pathways related to oxidative phosphorylation, G2M checkpoint, JAK-STAT and MAPK signaling were significantly activated, while the p53 pathway was inhibited in the CD8+ exhausted T cells. The standard marker genes of CD8+ T cell exhaustion were screened to develop a T-cell related gene score (TRS) based on random forest plots in TCGA cohort. The patients with low TRS have better prognosis compared to the patients with high TRS in both TCGA and GEO. In addition, most genes included in the TRS showed significant differences in expression levels between the high- and low-risk groups. Immune cell infiltration was analyzed using the MCPcounter and xCell algorithms, which revealed significant differences between the two risk groups, indicating that the different prognoses may stem from the respective immune landscapes. In addition, CD38 knockdown in OV cell lines increased apoptosis and inhibited invasion in vitro. Finally, we performed a drug sensitivity analysis and identified six potential drug candidates for OV. To summarize, we identified the heterogeneity and clinical significance of T cell exhaustion in OV and built a superior prognostic model based on T cell exhaustion genes, which can contribute to the development of more precise and effective therapies.

Tumor cuproptosis and immune infiltration improve survival of patients with hepatocellular carcinoma with a high expression of ferredoxin 1.

Background: Cuproptosis is a novel cell death pathway dependent on cellular copper ions and ferredoxin 1 (FDX1). Hepatocellular carcinoma (HCC) is derived from healthy liver as a central organ for copper metabolism. It remains no conclusive evidence whether cuproptosis is involved in survival improvement of patients with HCC. Method: A 365-liver hepatocellular carcinoma (LIHC) cohort with RNA sequencing data and paired clinical and survival information was obtained from the The Cancer Genome Atlas (TCGA) dataset. A retrospective cohort of 57 patients with HCC with stages I/II/III was collected by Zhuhai People's Hospital from August 2016 to January 2022. Low- or high-FDX1 groups were divided according to the median value of FDX1 expression. Cibersort, single-sample gene set enrichment analysis, and multiplex immunohistochemistry analyzed immune infiltration in LIHC and HCC cohorts. Cell proliferation and migration of HCC tissues and hepatic cancer cell lines were evaluated using the Cell Counting Kit-8. Quantitative real-time PCR and RNA interference measured and downregulated FDX1 expression. Statistical analysis was conducted by R and GraphPad Prism software. Results: High FDX1 expression significantly enhanced survival of patients with LIHC from the TCGA dataset, which was also demonstrated through a retrospective cohort with 57 HCC cases. Immune infiltration was different between the low- and high-FDX1 expression groups. Natural killer cells, macrophages, and B cells were significantly enhanced, and PD-1 expression was low in the high-FDX1 tumor tissues. Meanwhile, we found that a high expression of FDX1 decreased cell viability in HCC samples. HepG2 cells with FDX1 expression are sensitive to Cu2+, and interference of FDX1 promoted proliferation and migration of tumor cells. The consistent results were also demonstrated in Hep3B cells. Conclusion: This study reveals that cuproptosis and tumor immune microenvironment were together involved in improvement of survival in patients with HCC with a high expression of FDX1.

Case report: Transvaginal single-port extraperitoneal laparoscopic sacrospinous ligament fixation for apical prolapse: A single-center case series.

Background: Sacrospinous ligament fixation (SSLF) is a minimally invasive and effective procedure for the treatment of apical prolapse. Because intraoperative exposure of the sacrospinous ligament is difficult, SSLF is difficult. The aim of our article is to determine the safety and feasibility of single-port extraperitoneal laparoscopic SSLF for apical prolapse. Methods: This single-center, single-surgeon case series study included 9 patients with pelvic organ prolapse quantification (POP-Q) III or IV apical prolapse who underwent single-port laparoscopic SSLF. Additionally, transobturator tension-free vaginal tap (TVT-O) was performed in 2 patients, and anterior pelvic mesh reconstruction was performed in 1 patient. Results: The operative time ranged from 75 to 105 (mean, 88.9 ± 10.2) min, and blood loss ranged from 25 to 100 (mean, 43.3 ± 22.6) ml. No serious operative complications, blood transfusions, visceral injuries, or postoperative gluteal pain were reported for these patients. After 2-4 months of follow-up, no recurrence of POP, gluteal pain, urinary retention/incontinence, or other complications was observed. Conclusion: Transvaginal single-port SSLF is a safe, effective, and easy-to-master operation for apical prolapse.

Transcriptome- and metabolome-based candidate mechanism of BCR-ABL-independent resistance to olverembatinib in Philadelphia chromosome-positive acute lymphoblastic leukemia.

Olverembatinib represents the third-generation breakpoint cluster region protein-Abelson-murine leukemia 1 (BCR-ABL1) tyrosine kinase inhibitor with oral bioavailability, which can be used to overcome the T315I mutation in Philadelphia chromosome-positive (Ph +) leukemia. BCR-ABL-independent resistance to olverembatinib has been reported among patients in various clinical cases. However, the mechanism of olverembatinib resistance has rarely been reported. This study has illustrated bone marrow cell transcriptome and metabolome profiles among Ph + acute lymphoblastic leukemias (ALL) cases pre- and post-olverembatinib resistance. The transcriptome studies demonstrated that PI3K/AKT, purine metabolism, and other signaling pathways could play a vital role in olverembatinib resistance. As suggested by metabolomics, olverembatinib resistance in Ph + ALL was associated with purine metabolism alterations. Subsequently, high-performance liquid chromatography along with real-time quantitative PCR was utilized to measure purine metabolism-related mRNA levels and metabolism expression levels between olverembatinib resistance and sensitive cell lines. Our results elucidate the mechanism of olverembatinib resistance in Ph + ALL at transcriptome and metabolome levels, which facilitate a better understanding of olverembatinib resistance and hence may prove crucial in identifying novel drugs to tackle this conundrum.

A keratin/chitosan sponge with excellent hemostatic performance for uncontrolled bleeding.

Uncontrolled bleeding leads to a higher fatality rate in the situation of surgery, traffic accidents and warfare. Traditional hemostatic materials such as bandages are not ideal for uncontrolled or incompressible bleeding. Therefore, it is of great significance to develop a new medical biomaterial with excellent rapid hemostatic effect. Keratin is a natural, biocompatible and biodegradable protein which contains amino acid sequences that induce cell adhesion. As a potential biomedical material, keratin has been developed and paid attention in tissue engineering fields such as promoting wound healing and nerve repair. Herein, a keratin/chitosan (K/C) sponge was prepared to achieve rapid hemostasis. The characterizations of K/C sponge were investigated, including SEM, TGA, liquid absorption and porosity, showing that the high porosity up to 90.12 ± 2.17 % resulted in an excellent blood absorption. The cytotoxicity test and implantation experiment proved that the K/C sponge was biocompatible and biodegradable. Moreover, the prepared K/C sponge showed better hemostatic performance than chitosan sponge (CS) and the commercially available gelatin sponge in both rat tail amputation and liver trauma bleeding models. Further experiments showed that K/C sponge plays a hemostatic role through the endogenous coagulation pathway, thus shortening the activated partial thromboplastin time (APTT) effectively. Therefore, this study provided a K/C sponge which can be served as a promising biomedical hemostatic material.

Dexmedetomidine inhibits cell malignancy in osteosarcoma cells via miR-520a-3p-YOD1 interactome.

BACKGROUND: Osteosarcoma is a common malignant tumor in adolescents with a low 5-year survival rate. Dexmedetomidine (DEX) has been widely used for surgery of osteosarcoma patients. MiR-520a-3p and YOD1 expression was abnormal in osteosarcoma cells. However, whether DEX affects osteosarcoma progression via miR-520a-3p-YOD1 interactome needs to be explored. METHODS: We detected osteosarcoma cells biological behavior by CCK-8 assay, BrdU assay, cell adhesion assay, and apoptosis assay, respectively. The miR-520a-3p and YOD1 levels was explored in osteosarcoma cell lines by RT-qPCR or western blotting assay. RESULTS: In this study, we found that DEX treating osteosarcoma cells inhibited cell viability, proliferation and adhesion, while it promoted cell apoptosis. Moreover, miR-520a-3p targeting to YOD1 also functionally repressed cell malignancy in osteosarcoma cells. Notably, DEX treatment could inhibit YOD1 expression via upregulating miR-520a-3p, thereby suppressing cell malignancy in osteosarcoma. CONCLUSIONS: Our study first revealed that DEX inhibited malignancy of osteosarcoma cells via miR-520a-3p/YOD1 axis.

Increased Serum Levels of Trypsin Inhibitor Kazal1 in Patients with HBV-Related Hepatocellular Carcinoma Predict a Poor Prognosis.

BACKGROUND: Trypsin Inhibitor Kazal1 (SPINK1) is overexpressed in various tumors, but its role in hepatitis B virus (HBV) related hepatocellular carcinoma (HCC) is unclear. The aim of this study was to investigate SPINK1 levels during the chronic progression of HBV infection and their association with the prognosis of HBV-related HCC. METHODS: This study enrolled 102 patients with chronic hepatitis B (CHB), 95 patients with HBV-related liver cirrhosis (LC), 104 patients with HBV-related HCC, 25 patients with intrahepatic cholangiocarcinoma (ICC), and 98 healthy controls (HCs). The serum expression of SPINK1 in each group was compared. SPINK1 levels in the supernatant of HepG2.2.15, HepG2, Huh7, and LO2 cells were determined by ELISA. The diagnostic efficacy of SPINK1 for HBV-related HCC was evaluated. Hazard ratios (HRs) for the short-term prognosis of HBV-related HCC were assessed. RESULTS: SPINK1 levels were the highest in the HBV-related HCC group compared with the HC, CHB, HBV-related LC, and ICC groups (3.19 ± 1.11 versus 1.09 ± 0.38, 1.75 ± 0.55, 2.09 ± 0.62, and 2.40 ± 0.85 ng/mL, p < 0.01). SPINK1 levels in the supernatant of HepG2.2.15 cells were higher than those in HepG2, Huh7, and LO2 cells (2.85 ± 0.03 versus 1.54 ± 0.04, 1.50 ± 0.04, 0.9 ± 0.04 ng/mL, p < 0.001). The best cutoff point for the SPINK1 level was 2.48 ng/mL. The high SPINK1 expression group (≥ 2.48 ng/mL) had a larger tumor size, poorer Child-Pugh classification and more HBV DNA than the low expression group (< 2.48 ng/mL) (all p < 0.05). In the HBV-related HCC group, a SPINK1 level ≥ 2.48 ng/mL along with a high alpha-fetoprotein (AFP) level, large tumor size and poor Child-Pugh grade predicted poorer overall survival (HR 4.65, 95% confidence interval (CI): 2.07 - 10.43, p < 0.001). CONCLUSIONS: Serum SPINK1 had a high diagnostic efficacy for predicting HBV-related HCC. The presence of HBV-related HCC with a high serum SPINK1 level (≥ 2.48 ng/mL) may be associated with a poor short-term prognosis.

MicroRNA-204-5p mediates sevoflurane-induced cytotoxicity in HT22 cells by targeting brain-derived neurotrophic factor.

BACKGROUND: Sevoflurane is widely used as an inhalational anesthetic in clinical practice. However, sevoflurane can cause cytotoxicity and induce learning capacity decline in patients. A previous publication indicated that miR-204-5p might have a close relationship with sevoflurane-induced neurotoxicity. When exposed to sevoflurane, the expression of miR-204-5p in neonatal hippocampus of rats was significantly increased. Hence, we aimed to investigate the role of miR-204-5p in sevoflurane-induced neurotoxicity using a mouse hippocampal neuronal cell line (HT22). METHODS: The levels of miR-204-5p in HT22 cells were detected by RT-qPCR. In addition, the effects of miR-204-5p on cell viability, apoptosis and proliferation were evaluated by CCK-8, flow cytometric, and immunofluorescence assay, respectively. Western blotting was used to detect expressions of Bax, Bcl-2, active caspase 3, BDNF, TrkB, p-TrkB, Akt and p-Akt in HT22 cells. ELISA assay was used to examine the levels of total superoxide dismutase (SOD), reduced glutathione (GSH), malondialdehyde (MDA) and reactive oxygen species (ROS) in cells. Meanwhile, the dual luciferase reporter system assay was employed to explore the interaction of miR-204-5p and BDNF in cells. RESULTS: The level of miR-204-5p was increased in sevoflurane-treated HT22 cells. Moreover, downregulation of miR-204-5p inhibited sevoflurane-induced apoptosis and promoted cell proliferation by upregulating the proteins of Bcl-2 and downregulating the expressions of Bax and active caspase-3 in HT22 cells. In addition, inhibition of miR-204-5p alleviated sevoflurane-induced oxidative injuries in HT22 cells via decline of ROS and MDA and upregulation of SOD and GSH. Furthermore, bioinformatics and dual luciferase assay demonstrated that miR-204-5p can inhibit the TrkB/Akt pathway by targeting BDNF. CONCLUSION: Our findings indicated that downregulation of miR-204-5p can decrease oxidative status in HT22 cells and alleviate sevoflurane-induced cytotoxicity through stimulating the BDNF/TrkB/Akt pathway. Therefore, miR-204-5p might be a potential biomarker and therapeutic target for the treatment of sevoflurane-induced neurotoxicity.

ROS Is a Factor Regulating the Increased Polysaccharide Production by Light Quality in the Edible Cyanobacterium Nostoc flagelliforme.

To explore the regulatory factor of light quality affecting exopolysaccharide (EPS) production, transcriptome analysis of Nostoc flagelliforme cells exposed to red light (R), blue light (B), and mixed light (B/R = 15:7) (BR) with white fluorescent light as control was performed. The differentially expressed genes mainly enriched in carbohydrate metabolism and energy metabolism. Significant enrichment in the oxidation-reduction process and energy metabolism indicated that intracellular redox homeostasis was disrupted. An assay of reactive oxygen species (ROS) and malondialdehyde contents demonstrated light quality induced oxidative stress. To illustrate the relationship between ROS level and EPS accumulation, the effects of the exogenous addition of ROS scavenger N-acetyl cysteine and inducer H2O2 on the oxidation-reduction level and EPS production were compared. The results revealed that light quality regulated EPS biosynthesis via the intracellular ROS level directly other than oxidative stress. Understanding such relationships might provide guidance for efficient EPS production to regulate the intracellular redox level.

Comparative proteomic analysis of Nostoc flagelliforme reveals the difference in adaptive mechanism in response to different ultraviolet-B radiation treatments.

Nostoc flagelliforme is a pioneer organism in the desert and highly resistant to ultraviolet B (UV-B) radiation, while the involved adaptive mechanism has not been fully explored yet. To elucidate the responsive mechanism, two doses of UV-B radiation (low: 1 W/m2 and high: 5 W/m2) were irradiated for 6 h and 48 h, respectively, and their effects on global metabolism in N. flagelliforme were comprehensively investigated. In this study, we used iTRAQ-based proteomic approach to explore the proteomes of N. flagelliforme, and 151, 172, 124 and 148 differentially expressed proteins were identified under low and high UV-B doses for 6 h and 48 h, respectively. Functional classification analysis showed these proteins were mainly involved in photosynthesis, amino acid metabolism, antioxidant activity and carbohydrate metabolism. Further analysis revealed that UV-B imposed restrictions on primary metabolism including photosynthesis, Calvin cycle, and amino acid metabolism, and cells started defense mechanism through repair of DNA and protein damage, increasing antioxidant activity, and accumulating extracellular polysaccharides to minimize the damage. Moreover, high UV-B dose imposed more severe restrictions and activated stronger defense mechanism compared with low dose. The results would improve the understanding of molecular mechanisms of UV-B-stress adaption in N. flagelliforme.

The physiological responses of terrestrial cyanobacterium Nostoc flagelliforme to different intensities of ultraviolet-B radiation.

Nostoc flagelliforme is a pioneer organism in the desert and exerts important ecological functions. The habitats of N. flagelliforme are characterized by intense solar radiation, while the ultraviolet B (UV-B) tolerance has not been fully explored yet. To evaluate the physiological responses of N. flagelliforme to UV-B radiation, three intensities (1 W m-2, 3 W m-2 and 5 W m-2) were used, and the changes in photosynthetic pigments, cell morphology, mycosporine-like amino acids (MAAs) synthesis and cell metabolism were comparatively investigated. Under high UV-B intensity or long term radiation, chlorophyll a, allophycocyanin and phycocyanin were greatly decreased; scanning electron microscope observations showed that cell morphology significantly changed. To reduce the damage, cells synthesized a large amount of carotenoid. Moreover, three kinds of MAAs were identified, and their concentrations varied with the changes of UV-B intensity. Under 1 W m-2 radiation, cells synthesized shinorine and porphyra-334 against UV-B, while with the increase of intensity, more shinorine turned into asterine-330. Metabolite profiling revealed the contents of some cytoprotective metabolites were greatly increased under 5 W m-2 radiation. The principal component analysis showed cells exposed to UV-B were metabolically distinct from the control sample, and the influence on metabolism was particularly dependent on intensity. The results would improve the understanding of physiological responses of N. flagelliforme to UV-B radiation and provide an important theoretical basis for applying this organism to control desertification.