Molecular Characterization of Cuproptosis-related lncRNAs: Defining Molecular Subtypes and a Prognostic Signature of Ovarian Cancer.

Cuproptosis, a newly discovered form of programmed cell death, relies on mitochondrial respiration, the chain of which has been found to be altered in ovarian cancer (OC). The current work probed into the effects of Cuproptosis on the prognosis, immune microenvironment and therapeutic response of OC based on Cuproptosis-related lncRNAs. Data on OC gene expression and clinical characteristics were collected from TCGA, ICGC and GEO databases, and mRNA and lncRNA were distinguished. Cuproptosis-related lncRNAs were screened for consensus clustering analysis. Differentially expressed lncRNAs (DElncRNAs) were identified between clusters, and least absolute shrinkage and selection operator (LASSO) and Cox regression analysis were performed to establish a prognostic signature. Its potential value in OC was evaluated by Gene Set Enrichment Analysis (GSEA), tumor cell mutation and immune microenvironment analysis, and response to immunotherapy and antineoplastic drugs. According to the classification scheme of Cuproptosis-related lncRNAs, OC was divided into four molecular subtypes, which were different in survival time, immune characteristics and somatic mutation. The prognostic signature between subtypes included 10 lncRNAs, which were significantly correlated with the prognosis, immune microenvironment related indexes, the expression of immune checkpoint molecules and the sensitivity of antineoplastic drug Paclitaxel and Gefitinib of OC. We examined the expression of ten LncRNAs in OC cell lines and found that LINC00189, ZFHX4-AS1, RPS6KA2-IT1 and C9orf106 were expressed elevated in OC cell lines, and LINC00861, LINC00582, DEPDC1-AS1, LINC01556, LEMD1-AS1, TYMSOS expression was decreased in OC cell lines. The results of CCK8 showed that the cell viability of OC cells decreased after inhibition of C9orf106, whereas the cell viability of OC cells increased after inhibition of LEMD1-AS1. This work revealed new Cuproptosis-related lncRNA molecular subtypes exhibiting tumor microenvironment (TME) heterogeneity for OC and proposed a prognostic signature that may have benefits in understanding the prognosis, pathological features and immune microenvironment of OC patients.

Identifying a Novel Eight-NK Cell-related Gene Signature for Ovarian Cancer Prognosis Prediction.

BACKGROUND: Ovarian cancer (OVC) is the most common and costly tumor in the world with unfavorable overall survival and prognosis. This study is aimed to explore the prognostic value of natural killer cells related genes for OVC treatment. METHODS: RNA-seq and clinical information were acquired from the TCGA-OVC dataset (training dataset) and the GSE51800 dataset (validation dataset). Genes linked to NK cells were obtained from the immPort dataset. Moreover, ConsensusClusterPlus facilitated the screening of molecular subtypes. Following this, the risk model was established by LASSO analysis, and immune infiltration and immunotherapy were then detected by CIBERSORT, ssGSEA, ESTIMATE, and TIDE algorithms. RESULTS: Based on 23 NK cell-related genes with prognosis, TCGA-OVC samples were classified into two clusters, namely C1 and C2. Of these, C1 had better survival outcomes as well as enhanced immune infiltration and tumor stem cells. Additionally, it was more suitable for immunotherapy and was also sensitive to traditional chemotherapy drugs. The eight-gene prognosis model was constructed and verified via the GSE51800 dataset. Additionally, a high infiltration level of immune cells was observed in low-risk patients. Low-risk samples also benefited from immunotherapy and chemotherapy drugs. Finally, a nomogram and ROC curves were applied to validate model accuracy. CONCLUSION: The present study identified a RiskScore signature, which could stratify patients with different infiltration levels, immunotherapy, and chemotherapy drugs. Our study provided a basis for precisely evaluating OVC therapy and prognosis.

Tanshinone IIA protects mouse testes from heat stress injury by inhibiting apoptosis and TGFß1/Smad2/Smad3 signaling pathway.

Heat stress can cause testicular damage and affect male fertility. Tanshinone IIA (TSA) is a monomer substance derived from plants, with antioxidant and anti-apoptotic effects. Whether it can repair testicular damage caused by heat stress is unclear. This study aims to construct a mouse testicular heat stress injury model and intervene with TSA. Various methods such as histopathology, high-throughput sequencing, bioinformatics analysis, and molecular biology were used to investigate whether TSA can alleviate heat stress-induced testicular injury and its mechanism. Results showed that heat stress significantly reduced the diameter of the mouse seminiferous tubules, increased cell apoptosis in the testicular tissue, and significantly decreased testosterone levels. After TSA intervention, testicular morphology and cell apoptosis improved significantly, and testosterone secretion function was restored. High-throughput transcriptome sequencing found that key differentially expressed genes between the HS group and the control and TSA groups clustered in the apoptosis and TGFß signaling pathways. Using western blot technology, we found that the HS group upregulated TGFß1/Smad2/Smad3 pathway protein expression, causing cell apoptosis, testicular tissue organic lesions, and affecting testicular secretion function. Through TSA intervention, we found that it can inhibit TGFß1/Smad2/Smad3 pathway protein expression, thereby restoring testicular damage caused by heat stress. This study confirms that TSA can effectively restore testicular damage caused by heat stress in mice, possibly by inhibiting the TGFß1/Smad2/Smad3 pathway to suppress apoptosis.

Therapeutic Effect of Seawater Pearl Powder on UV-Induced Photoaging in Mouse Skin.

The objective of this study was to investigate the therapeutic effect of seawater pearl powder (SPP) on ultraviolet (UV) irradiation-induced photoaging in mouse skin. The protein and trace elements in SPP were detected by liquid chromatography-mass spectrometry, atomic fluorescence spectrometry, and inductively coupled plasma-atomic emission spectrometry. The effect of SPP on treating skin damage resulting from UV-induced photoaging was observed by gross physical appearance and histopathological analysis. Oxidative stress and melanin synthesis were analyzed using biochemical method. Western blotting was applied to analyze the phosphorylation and expression levels of matrix metalloproteinase-1 (MMP-1), collagen I, and proteins involved in the mitogen-activated protein kinase (MAPK) signaling pathways (p38, ERK, and JNK). The results show that SPP has a significant therapeutic effect on UV-induced photoaging of skin and improves and restores appearance and tissue structure of mouse skin. The major mechanism may be related to reduction of expression level of MMP-1 and enhancement of collagen I production via inhibition of MAPK signaling pathway after scavenging of excess reactive oxygen species (ROS) in the UV-induced photoaged skin of mice. Meanwhile, it may also be involved in reducing melanin content by inhibiting tyrosinase activity after scavenging excess ROS in the UV-induced photoaged skin of mice. Therefore, SPP could be a good substance to treat photoaging skin. Taking cost-effectiveness and efficacy into consideration, the optimal concentration of SPP for treating photoaging skin could be 100 mg/g.

Desensitization of TRPV1 Involved in the Antipruritic Effect of Osthole on Histamine-Induced Scratching Behavior in Mice.

Osthole has been isolated from the fruits of Cnidium monnieri (L.) Cusson, which has been used in Chinese traditional medicine to treat pruritic disorders for a long time. However, the antipruritic mechanism of osthole is not fully understood. In the present study, using calcium imaging, molecular docking, and animal scratching behavior, we analyzed the pharmacological effects of osthole on transient receptor potential vanilloid 1 (TRPV1). The results showed that osthole significantly induced calcium influx in a dose-dependent manner in dorsal root ganglion (DRG) neurons. Osthole-induced calcium influx was inhibited by AMG9810, an antagonist of TRPV1. Osthole and the TRPV1 agonist capsaicin-induced calcium influx were desensitized by pretreatment with osthole. Furthermore, molecular docking results showed that osthole could bind to TRPV1 with a hydrogen bond by anchoring to the amino acid residue ARG557 in the binding pocket of TRPV1. In addition, TRPV1 is a downstream ion channel for the histamine H1 and H4 receptors to transmit itch signals. Osthole attenuated scratching behavior induced by histamine, HTMT (histamine H1 receptor agonist), and VUF8430 (histamine H4 receptor agonist) in mice. These results suggest that osthole inhibition of histamine-dependent itch may be due to the activation and subsequent desensitization of TRPV1 in DRG neurons.

Computational and In Vitro Analysis of Plumbagin's Molecular Mechanism for the Treatment of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor and the second leading cause of cancer-related death in the world. Plumbagin (PL) is a small molecule naphthoquinone compound isolated from Plumbago zeylanica L. that has important anticancer properties, but its mechanism requires further investigation. In this study, we used a comprehensive network pharmacology approach to study the mechanism of action of PL for the treatment of HCC. The method includes the construction of multiple networks; moreover, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to identify biological processes and signaling pathways. Subsequently, in vitro experiments were performed to verify the predicted molecular mechanisms obtained from the network pharmacology-based analysis. Network pharmacological analysis showed that PL may exert anti-HCC effects by enhancing reactive oxygen species (ROS) production to generate oxidative stress and by regulating the PI3K/Akt and MAPK signaling pathways. In vitro experiments confirmed that PL mainly mediates the production of ROS, regulates the PI3K/Akt and MAPK signaling pathways to promote apoptosis and autophagy, and shows significant therapeutic effects on HCC. In conclusion, our work proposes a comprehensive systems pharmacology approach to explore the potential mechanism of PL for the treatment of HCC.

Ubiquitome analysis reveals the involvement of lysine ubiquitination in the spermatogenesis process of adult buffalo (Bubalus bubalis) testis.

Protein ubiquitination, a major and conserved post-translational modification, is known to play a critical regulatory role in many biological processes in eukaryotes. Although several ubiquitinated proteins have been found in buffalo (Bubalus bubalis) testis in our previous studies, large-scale profiling of buffalo testis ubiquitome has not been reported to date. In the present study, we first identified a global profiling of lysine ubiquitination of adult buffalo testis using a highly sensitive LC-MS/MS coupled with immune-affinity enrichment of ubiquitinated peptides. In total, 422 lysine ubiquitination sites were identified in 262 proteins in adult buffalo testis tissue. Bioinformatics analysis showed that the ubiquitinated proteins are involved in a variety of biological processes and diverse subcellular localizations. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein interaction network analysis indicated that proteasome, glycolysis/gluconeogenesis and gap junction pathways are modulated by protein ubiquitination in testis. Besides, 44 ubiquitinated proteins may involve in spermatogenesis according to the SpermatogenesisOnline database, of which, the ubiquitination of HSPA2 and UCHL1 were confirmed by Immunoprecipitation (IP)/Western blot analysis. Taken together, these data provide a global view of ubiquitome in buffalo testis for the first time, and serve as an important resource for exploring the physiological role especially spermatogenesis of lysine ubiquitination in testis in mammals.

Pearl powder reduces sleep disturbance stress response through regulating proteomics in a rat model of sleep deprivation.

AIMS: This study aimed to explore whether pearl could help prevent cognitional morbidity and improve the metabolic processes of hippocampus. METHODS: Rats were divided into group of control (CTL), sleep deprivation (SD) and pearl powder (PP). The sleeplessness was introduced to all rats except control. Before and after administration with vehicle or pearl powder, cognition was evaluated by Morris water maze (MWM). The protein expression in hippocampus among all groups was examined using iTRAQ-based global proteomic analysis. RESULTS: Morris water maze tests revealed improvements of insomnia-induced cognitive deficit in both PP- and ES-treated rats, as compared to SD rats. However, proteomic analysis indicates that the pharmacological impact on gene expression of these two medicines is quite different: pearl is more capable of correcting aberrant gene expression caused by SD than estazolam. Therefore, pearl is more suitable for treatment of insomnia. These data, together with protein-protein interaction analysis, indicate that several pathways, affected by sleep deprivation, may be rescued by pearl powder: retrograde endocannabinoid signalling pathway, and the protein interaction or network enrich in oxidative phosphorylation Parkinson's disease and Huntington disease, etc CONCLUSIONS: Sleep deprivation can mimic cognition decline caused by insomnia with altered protein expression in the hippocampus; such behavioural and pathological changes can be significantly ameliorated by pearl powder.

Plumbagin Restrains Hepatocellular Carcinoma Angiogenesis by Stromal Cell-Derived Factor (SDF-1)/CXCR4-CXCR7 Axis.

BACKGROUND Anti-angiogenic therapy has recently emerged as a highly promising therapeutic strategy for treating hepatocellular carcinoma (HCC). MATERIAL AND METHODS We assessed cellular proliferation, invasion, and activation of growth factors (VEGF and IL-8) with SDF-1 induced in the hepatocellular carcinoma cell line SMMC-7721, and this progression was limited by plumbagin (PL). The human umbilical vein endothelial cell line HUVEC was co-cultured with SDF-1-induced SMMC-7721, and the expressions of CXCR7, CXCR4, and PI3K/Akt pathways after PL treatment were detected by RT-PCR and Western blot analysis. RESULTS The treatment of the hepatoma cell line SMMC-7721 with SDF-1 resulted in enhanced secretion of the angiogenic factors, IL-8 and VEGF, and shows that these stimulatory effects are abolished by PL. The study further demonstrated that PL not only abolishes SDF-1-induced formation of endothelial tubes, but also inhibits expression of CXCR4 and CXCR7, and partially prevents activation of angiogenic signaling pathways. CONCLUSIONS The effect of PL on the SDF-1-CXCR4/CXCR7 axis has become an attractive target for inhibiting angiogenesis in hepatoma cells. Our results provide more evidence for the clinical application of PL as part of traditional Chinese medicine in modern cancer treatment.

Proteomic profiles of buffalo spermatozoa and seminal plasma.

A comprehensive identification of the proteins of spermatozoa and seminal plasma enables the full characterization of sperm biology and is essential to understand how seminal plasma influences sperm fertility. In this study, proteomics of buffalo spermatozoa and seminal plasma were analyzed using a bottom-up approach. A total of 2147 and 864 proteins were identified from mature spermatozoa and seminal plasma, respectively. Of these, 371 proteins-42.9% in the seminal plasma set-were common to both spermatozoa and seminal plasma. ODF2, AKAP4, and TUBB were the most highly abundant proteins of spermatozoa whereas the three most highly abundant proteins in seminal plasma were ALB, CLU, and AZGP1. Gene Ontology (GO) analysis indicated that most sperm proteins were involved in transport, phosphorylation and macromolecule localization. More seminal plasma proteins were related to proteolysis, response to stimulus and homeostatic process. Pathways associated with energy metabolism, protein processing and RNA transport were significantly enriched in spermatozoa. In buffalo seminal plasma, three distinguishing GO pathways-proteasome activity, lysosomal activity, complement and coagulation cascades-were found to play important roles in sperm protection. Protein-protein interaction (PPI) network analysis indicated that 531 and 620 proteins participated in buffalo sperm and seminal plasma PPI networks, respectively. Sub-network mining elaborated detailed biological events. This study is the first to provide an in-depth proteomic analysis of buffalo spermatozoa and seminal plasma. The results extend our understanding of male buffalo reproduction and provide clues for determining the relationships between sperm biology and seminal plasma.

Analyzing bovine OCT4 and NANOG enhancer activity in pluripotent stem cells using fluorescent protein reporters.

Green fluorescent protein (GFP) reporters controlled by the regulatory region of OCT4 and NANOG-two master regulators for pluripotency are widely used in studies of pluripotent stem cell establishment and embryo development. Alongside the challenge in establishing bovine pluripotent stem cells, the application of bovine-specific gene reporters has rarely been explored. Using lentivirus-based GFP reporter, we investigated the upstream regulatory regions of bovine OCT4 and NANOG. These reporters show activity in both naïve- and primed-state pluripotency when infected into mouse and human embryonic stem cells (ESCs), respectively. Consistent with what is found in humans and mice, the bovine OCT4-distal enhancer (bOCT4-DE) but not the proximal enhancer (bOCT4-PE) region is preferentially activated in naïve-state pluripotency. Furthermore, the bOCT4-DE region is silenced upon conversion of naive-state ESCs into primed-state epiblast stem cells (EpiSCs). Co-infection of mouse fibroblasts with the reprograming factors for induced pluripotent stem cell (iPSC) induction leads to the generation of GFP positive colonies, demonstrating that these GFP reporters can serve as live indicators for induced pluripotent cell establishment. We further proved that the bovine OCT4 distal enhancer is active in bovine blastocysts. We established the lentiviral-based fluorescent reporters controlled by bovine OCT4 and NANOG enhancer sequences. These reporter constructs show activity in naïve- and primed-pluripotent states. These reporters may serve as versatile tools for bovine ESC/iPSC generation and identification, as well as for developmental studies of bovine embryos.

JAK/STAT3 regulated global gene expression dynamics during late-stage reprogramming process.

BACKGROUND: The generation of induced pluripotent stem cells (iPSCs) has underdefined mechanisms. In addition, leukemia inhibitory factor (LIF) activated Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway is the master regulator for naïve-state pluripotency achievement and maintenance. However, the regulatory process to attain naïve pluripotent iPSCs is not well understood. RESULTS: We performed transcriptome analysis to dissect the genomic expression during mouse iPSC induction, with or without blocking the JAK/STAT3 activity. We describe JAK/STAT3 signaling-specific biological events such as gametogenesis, meiotic/mitotic cell cycle, and DNA repair, and JAK/STAT3-dependent expression of key transcription factors such as the naïve pluripotency-specific genes, developmental pluripotency associated (Dppa) family, along with histone modifiers and non-coding RNAs in reprogramming. We discover that JAK/STAT3 activity does not affect early phase mesenchymal to epithelial transition (MET) but is necessary for proper imprinting of the Dlk1-Dio3 region, an essential event for pluripotency achievement at late-reprogramming stage. This correlates with the JAK/STAT3-dependent stimulation of Dppa3 and Polycomb repressive complex 2 (PRC2) genes. We further demonstrate that JAK/STAT3 activity is essential for DNA demethylation of pluripotent loci including Oct4, Nanog, and the Dlk1-Dio3 regions. These findings correlate well with the previously identified STAT3 direct targets. We further propose a model of pluripotency achievement regulated by JAK/STAT3 signaling during the reprogramming process. CONCLUSIONS: Our study illustrates novel insights for JAK/STAT3 promoted pluripotency establishment, which are valuable for further improving the naïve-pluripotent iPSC generation across different species including humans.

LIF-activated Jak signaling determines Esrrb expression during late-stage reprogramming.

The regulatory process of naïve-state induced pluripotent stem cell (iPSC) generation is not well understood. Leukemia inhibitory factor (LIF)-activated Janus kinase/signal transducer and activator of transcription 3 (Jak/Stat3) is the master regulator for naïve-state pluripotency achievement and maintenance. The estrogen-related receptor beta (Esrrb) serves as a naïve-state marker gene regulating self-renewal of embryonic stem cells (ESCs). However, the interconnection between Esrrb and LIF signaling for pluripotency establishment in reprogramming is unclear. We screened the marker genes critical for complete reprogramming during mouse iPSC generation, and identified genes including Esrrb that are responsive to LIF/Jak pathway signaling. Overexpression of Esrrb resumes the reprogramming halted by inhibition of Jak activity in partially reprogrammed cells (pre-iPSCs), and leads to the generation of pluripotent iPSCs. We further show that neither overexpression of Nanog nor stimulation of Wnt signaling, two upstream regulators of Esrrb in ESCs, stimulates the expression of Esrrb in reprogramming when LIF or Jak activity is blocked. Our study demonstrates that Esrrb is a specific reprogramming factor regulated downstream of the LIF/Jak signaling pathway. These results shed new light on the regulatory role of LIF pathway on complete pluripotency establishment during iPSC generation.

Enhanced human somatic cell reprogramming efficiency by fusion of the MYC transactivation domain and OCT4.

The development of human induced pluripotent stem cells (iPSCs) holds great promise for regenerative medicine. However the iPSC induction efficiency is still very low and with lengthy reprogramming process. We utilized the highly potent transactivation domain (TAD) of MYC protein to engineer the human OCT4 fusion proteins. Applying the MYC-TAD-OCT4 fusion proteins in mouse iPSC generation leads to shorter reprogramming dynamics, with earlier activation of pluripotent markers in reprogrammed cells than wild type OCT4 (wt-OCT4). Dramatic enhancement of iPSC colony induction efficiency and shortened reprogramming dynamics were observed when these MYC-TAD-OCT4 fusion proteins were used to reprogram primary human cells. The OCT4 fusion proteins induced human iPSCs are pluripotent. We further show that the MYC Box I (MBI) is dispensable while both MBII and the linking region between MBI/II are essential for the enhanced reprogramming activity of MYC-TAD-OCT4 fusion protein. Consistent with an enhanced transcription activity, the engineered OCT4 significantly stimulated the expression of genes specifically targeted by OCT4-alone, OCT4/SOX2, and OCT4/SOX2/KLF4 during human iPSC induction, compared with the wt-OCT4. The MYC-TAD-OCT4 fusion proteins we generated will be valuable tools for studying the reprogramming mechanisms and for efficient iPSC generation for humans as well as for other species.

Akt3 is responsible for the survival and proliferation of embryonic stem cells.

The phosphatidylinositol 3-kinase (PI3K)/protein kinase B (PKB/Akt) pathway plays an important role in regulating cell proliferation, metabolism, and survival. However, the distinct roles of Akt isoforms (Akt1, Akt2, and Akt3) in pluripotent stem cell maintenance are not fully defined. Using mouse embryonic stem cells (ESCs), we show that direct inhibition of Akt activity leads to ESC apoptosis. The Akt3, but not Akt1 or Akt2, activity specifically regulates this effect. Inhibiting Akt3 also leads to a cell cycle arrest at G1 phase. These regulatory roles of Akt3 are dependent on its kinase activity. Blocking the expression of Akt1 plus Akt2 in ESCs does not affect cell survival or proliferation, although blocking Akt1 aggravates the apoptotic effect induced by depletion of Akt3. We further show that blocking Akt3 in ESCs results in significant nuclear accumulation of p53, as well as the activation of its downstream targets, such as Mdm2, p21, and Fas. Inhibiting p53 and its downstream targets partially rescued the effects caused by Akt3-depletion. Our results revealed an Akt3 isoform-specific mechanism for ESC survival and proliferation involving the control of p53 activity.

Proteome Profile and Quantitative Proteomic Analysis of Buffalo (Bubalusbubalis) Follicular Fluid during Follicle Development.

Follicular fluid (FF) accumulates in the antrum of the ovarian follicle and provides the microenvironment for oocyte development. FF plays an important role in follicle growth and oocyte maturation. The FF provides a unique window to investigate the processes occurring during buffalo follicular development. The observed low quality of buffalo oocytes may arise from the poor follicular microenvironment. Investigating proteins found in buffalo FF (BFF) should provide insight into follicular development processes and provide further understanding of intra-follicular maturation and oocytes quality. Here, a proteomic-based approach was used to analyze the proteome of BFF. SDS-PAGE separation combined with mass spectrometry was used to generate the proteomic dataset. In total, 363 proteins were identified and classified by Gene Ontology terms. The proteins were assigned to 153 pathways, including signaling pathways. To evaluate difference in proteins expressed between BFF with different follicle size (small, <4 mm; and large, >8 mm), a quantitative proteomic analysis based on multi-dimensional liquid chromatography pre-fractionation tandem Orbitrap mass spectrometry identification was performed. Eleven differentially expressed proteins (six downregulated and five upregulated in large BFF) were identified and assigned to a variety of functional processes, including serine protease inhibition, oxidation protection and the complement cascade system. Three differentially expressed proteins, Vimentin, Peroxiredoxin-1 and SERPIND1, were verified by Western blotting, consistent with the quantitative proteomics results. Our datasets offers new information about proteins present in BFF and should facilitate the development of new biomarkers. These differentially expressed proteins illuminate the size-dependent protein changes in follicle microenvironment.