Norcantharidin Enhances the Antitumor Effect of 5-Fluorouracil by Inducing Apoptosis of Cervical Cancer Cells: Network Pharmacology, Molecular Docking, and Experimental Validation.

The high recurrence rate of cervical cancer is a leading cause of cancer deaths in women. 5-Fluorouracil (5-FU) is an antitumor drug used to treat many types of cancer, but its diminishing effectiveness and side effects limit its use. Norcantharidin (NCTD), a demethylated derivative of cantharidin, exhibits various biological activities. Here, we investigated whether NCTD could potentiate 5-FU to induce cervical cancer cell death. To assess the cell viability and synergistic effects of the drugs, cell counting kit-8 and colony formation assays were performed using HR-HPV-positive cervical cancer cell lines. Annexin V-FITC/PI staining and TUNEL assays were performed to confirm the induction of apoptosis. The synergistic effect of NCTD on the antitumor activity of 5-FU was analyzed using network pharmacology, molecular docking, and molecular dynamics simulations. Apoptosis-related proteins were examined using immunoblotting. The combination of NCTD and 5-FU was synergistic in cervical cancer cell lines. Network pharmacological analysis identified 10 common targets of NCTD and 5-FU for cervical cancer treatment. Molecular docking showed the strong binding affinity of both compounds with CA12, CASP9, and PTGS1. Molecular dynamics simulations showed that the complex system of both drugs with caspase-9 could be in a stable state. NCTD enhanced 5-FU-mediated cytotoxicity by activating apoptosis-related proteins. NCTD acts synergistically with 5-FU to inhibit cervical cancer cell proliferation. NCTD enhances 5-FU-induced apoptosis in cervical cancer cell lines via the caspase-dependent pathway.

Progress on CRISPR/Cas9 system in the genetic improvement of livestock and poultry.

CRISPR/Cas9 gene editing technology, as a highly efficient genome editing method, has been extensively employed in the realm of animal husbandry for genetic improvement. With its remarkable efficiency and precision, this technology has revolutionized the field of animal husbandry. Currently, CRISPR/Cas9-based gene knockout, gene knock-in and gene modification techniques are widely employed to achieve precise enhancements in crucial production traits of livestock and poultry species. In this review, we summarize the operational principle and development history of CRISPR/Cas9 technology. Additionally, we highlight the research advancements utilizing this technology in muscle growth and development, fiber growth, milk quality composition, disease resistance breeding, and animal welfare within the livestock and poultry sectors. Our aim is to provide a more comprehensive understanding of the application of CRISPR/Cas9 technology in gene editing for livestock and poultry.

LSM14B controls oocyte mRNA storage and stability to ensure female fertility.

Controlled mRNA storage and stability is essential for oocyte meiosis and early embryonic development. However, how to regulate mRNA storage and stability in mammalian oogenesis remains elusive. Here we showed that LSM14B, a component of membraneless compartments including P-body-like granules and mitochondria-associated ribonucleoprotein domain (MARDO) in germ cell, is indispensable for female fertility. To reveal loss of LSM14B disrupted primordial follicle assembly and caused mRNA reduction in non-growing oocytes, which was concomitant with the impaired assembly of P-body-like granules. 10× Genomics single-cell RNA-sequencing and immunostaining were performed. Meanwhile, we conducted RNA-seq analysis of GV-stage oocytes and found that Lsm14b deficiency not only impaired the maternal mRNA accumulation but also disrupted the translation in fully grown oocytes, which was closely associated with dissolution of MARDO components. Moreover, Lsm14b-deficient oocytes reassembled a pronucleus containing decondensed chromatin after extrusion of the first polar body, through compromising the activation of maturation promoting factor, while the defects were restored via WEE1/2 inhibitor. Together, our findings reveal that Lsm14b plays a pivotal role in mammalian oogenesis by specifically controlling of oocyte mRNA storage and stability.

Effects of dietary oat supplementation on carcass traits, muscle metabolites, amino acid profiles, and its association with meat quality of Small-tail Han sheep.

Oat supplementation of the ruminant diet can improve growth performance and meat quality traits, but the role of muscle metabolites has not been evaluated. This study aimed to establish whether oat grass supplementation (OS) of Small-tail Han sheep improved growth performance and muscle tissue metabolites that are associated with better meat quality and flavor. After 90-day, OS fed sheep had higher live-weight and carcass-weight, and lower carcass fat. Muscle metabolomics analysis showed that OS fed sheep had higher levels of taurine, l-carnitine, inosine-5'-monophospgate, cholic acid, and taurocholic acid, which are primarily involved in taurine and hypotaurine metabolism, purine metabolism, and bile acid biosynthesis and secretion, decreased fat accumulation and they promote functional or flavor metabolites. OS also increased muscle levels of amino acids that are attributed to better quality and flavorsome mutton. These findings provided further evidence for supplementing sheep with oat grass to improve growth performance and meat quality.

Effects and mechanisms of 6-hydroxykaempferol 3,6-di-O-glucoside-7-O-glucuronide from Safflower on endothelial injury in vitro and on thrombosis in vivo.

Background: The florets of Carthamus tinctorius L. (Safflower) is an important traditional medicine for promoting blood circulation and removing blood stasis. However, its bioactive compounds and mechanism of action need further clarification. Objective: This study aims to investigate the effect and possible mechanism of 6-hydroxykaempferol 3,6-di-O-glucoside-7-O-glucuronide (HGG) from Safflower on endothelial injury in vitro, and to verify its anti-thrombotic activity in vivo. Methods: The endothelial injury on human umbilical vein endothelial cells (HUVECs) was induced by oxygen-glucose deprivation followed by reoxygenation (OGD/R). The effect of HGG on the proliferation of HUVECs under OGD/R was evaluated by MTT, LDH release, Hoechst-33342 staining, and Annexin V-FITC apoptosis assay. RNA-seq, RT-qPCR, Enzyme-linked immunosorbent assay and Western blot experiments were performed to uncover the molecular mechanism. The anti-thrombotic effect of HGG in vivo was evaluated using phenylhydrazine (PHZ)-induced zebrafish thrombosis model. Results: HGG significantly protected OGD/R induced endothelial injury, and decreased HUVECs apoptosis by regulating expressions of hypoxia inducible factor-1 alpha (HIF-1α) and nuclear factor kappa B (NF-κB) at both transcriptome and protein levels. Moreover, HGG reversed the mRNA expression of pro-inflammatory cytokines including IL-1ß, IL-6, and TNF-α, and reduced the release of IL-6 after OGD/R. In addition, HGG exhibited protective effects against PHZ-induced zebrafish thrombosis and improved blood circulation. Conclusion: HGG regulates the expression of HIF-1α and NF-κB, protects OGD/R induced endothelial dysfunction in vitro and has anti-thrombotic activity in PHZ-induced thrombosis in vivo.

Single-cell RNA sequencing of the Mongolia sheep testis reveals a conserved and divergent transcriptome landscape of mammalian spermatogenesis.

Spermatogenesis is a highly coordinated and complex process, and is pivotal for transmitting genetic information between mammalian generations. In this study, we investigated the conservation, differences, and biological functions of homologous genes during spermatogenesis in Mongolia sheep, humans, cynomolgus monkey, and mice using single-cell RNA sequencing technology. We compared X chromosome meiotic inactivation events in Mongolia sheep, humans, cynomolgus monkey, and mice to uncover the concerted activity of X chromosome genes. Subsequently, we focused on the dynamics of gene expression, key biological functions, and signaling pathways at various stages of spermatogenesis in Mongolia sheep and humans. Additionally, the ligand-receptor networks of Mongolia sheep and humans in testicular somatic and germ cells at different developmental stages were mapped to reveal conserved germ cell-soma communication using single-cell resolution. These datasets provided novel information and insights to unravel the molecular regulatory mechanisms of Mongolia sheep spermatogenesis and highlight conservation in gene expression during spermatogenesis between Mongolia sheep and humans, providing a foundation for the establishment of a large mammalian disease model of male infertility.

Three-step irradiance schedule versus two-step irradiance schedule for pain control during topical 5-aminolevulinic acid-photodynamic therapy of facial acne in Chinese patients: A prospective randomized comparative study.

BACKGROUND: A two-step irradiance schedule has been found to be useful for pain control during photodynamic therapy (PDT) on nonmelanotic skin cancer and condyloma acuminatum. OBJECTIVES: To evaluate the efficacy of a new three-step irradiance schedule derived from the psychological "peak-end rule" and two-step irradiance schedule in relieving pain during 5-aminolevulinic acid PDT (ALA-PDT) on acne. METHODS: A total of 90 moderate to severe acne patients were enrolled in our study and randomly divided into two groups with a ratio of 1:1. They were treated by a light-emitting diode light source of 633 ± 10 nm after being incubated with 5% ALA for an hour using a two-step or three-step irradiance schedule, respectively. The total irradiance intensity was 84 J/cm2 of each session and the treatment interval was 2 weeks. Pain was recorded 30 min after each PDT using a visual analog scale (VAS). Follow-up was done at baseline and 2 weeks after each treatment. The numbers of lesions were counted after the third treatment through the pictures taken before and all the side effects were recorded at each follow-up visit. RESULTS: Eighty-seven subjects completed the total three treatments (44 cases in Group A and 43 cases in Group B). The average VAS of Group B (1.61 ± 0.67) was significantly lower than that of Group A (3.14 ± 0.67), with a difference of 1.52 ± 0.08 (p < 0.0001) between them. Both groups received a similar effective rate after the total three sessions (88.64% vs. 88.37%, p > 0.05). CONCLUSIONS: The new three-step irradiance method could relieve pain during ALA-PDT more significantly than the two-step schedule with a similar effective rate.

A prospective study of the effect of fire micro-needling plus 5% topical ALA-PDT for the treatment of dissecting cellulitis of the scalp.

BACKGROUND: Different therapies have been used for dissecting cellulitis of the scalp (DCS) with poor results. Topical 5-aminolaevulinic acid photodynamic therapy (ALA-PDT) is effective for many inflammatory skin diseases. OBJECTIVES: To evaluate the efficacy and safety of fire micro-needling plus 5% ALA-PDT on DCS. METHODS: Forty-two male DCS patients were enrolled and treated by four sessions of fire micro-needling plus 5% ALA-PDT (occlusion time: 3 h, 100 mW/cm2 for 20-32 min, 633 ± 10 nm LED) with an interval of 1-2 weeks. The reduction of lesions was evaluated at each follow-up visit. The patients` symptoms were evaluated by Dermatology Life Quality Index (DLQI). All of the side effects were recorded. RESULTS: Forty-one DCS patients finished the treatments and the marked effective rate (MER) reached 65.85%. Ten patients (24.39%) were cured and 17 patients (41.46%) achieved excellent improvement. The median of lesion numbers decreased significantly from 25 to 7 (p < 0.001) with DLQI from 13 to 2 (p < 0.001). Although all the ten cured patients had a relapse during the 12-month follow-up, 5/10 patients had a remission of more than 6 months. All the patients tolerated PDT well with mild adverse effects. CONCLUSIONS: Fire micro-needling plus 5% ALA-PDT is effective for Chinese male DCS patients without the need to take systemic drugs.

[Hematoma Expansion within 24 hours of Hypertensive Intracerebral Hemorrhage and Its Association with Signs on Nonenhanced Computed Tomography].

To explore the correlation between hematoma expansion within 24 hours of hypertensive intracerebral hemorrhage and signs on nonenhanced computed tomography(NECT). Methods The clinical data and CT images of 185 patients with hypertensive intracerebral hemorrhage were retrospectively analyzed.The differences in CT parameters were compared between the expansion group and the unexpanded group.Binary logistic regression analysis was performed on the indicators with statistical significance between the two groups to identify the potential correlation between CT parameters and hematoma expansion.The roles of blend sign,lobulation sign,and black hole sign in predicting early hematoma expansion were assessed. Results The CT quantitative data including initial volume,maximum diameter,minimum diameter,maximum CT value,mean CT value,difference between maximum diameter and minimum diameter showed no significant difference between these two group(all P>0.05).CT qualitative data including blend sign,lobulation sign,and black hole sign were significantly different(P<0.05)but the differences became not significant after the hematoma broke into the ventricles.Binary logistic regression analysis showed that blend sign,lobulation sign,and black hole sign were independent risk factors for early hematoma expansion,with sensitivity,specificity,positive predictive value,and negative predictive value of the combined signs and the early hematoma expansion calculated by the four-fold table method being 78.4%,59.0%,42.1%,and 87.8%,respectively,and the Youden index was 0.374.Its Youden index was closer to 1 than the blend sign,the black hole sign,and the lobulation sign. Conclusion The blend sign,lobulation sign,and black hole sign in NECT can be used to predict hematoma expansion within 24 hours after hypertensive cerebral hemorrhage.

Effect of RIF1 on response of non-small-cell lung cancer patients to platinum-based chemotherapy by regulating MYC signaling pathway.

Platinum-based chemotherapy is used as first-line therapy for advanced non-small-cell lung cancer (NSCLC). However, there is no effective indicator to predict whether the patient would be chemo-resistant or sensitive to the therapy. In addition, it is urgent to elucidate the mechanisms of cisplatin resistance. RIF1 plays important roles in DNA replication regulation and DNA repair pathway. However, the role of RIF1 in NSCLC progression and chemotherapy response is still unknown. In this study, we found that RIF1 expression was correlated with the response of NSCLC patients to platinum-based chemotherapy (n=89, P=0.002). Among patients who have been treated with platinum chemo-therapy, those with high levels of RIF1 expression had significantly shorter survival than those with low RIF1 expression (P<0.05). RIF1 knockdown increased sensitivity to cisplatin in NSCLC patients both in vitro and in vivo. Moreover, RIF1 knockdown induced G0/G1 phase arrest and increased cisplatin-induced apoptosis and DNA damage. Further investigation showed that RIF1 regulated the expression of MYC and MYC downstream targets, including the cell cycle and double-stranded break (DSB) repair genes which might mediate the effect of RIF1 on cellular response to cisplatin. Overexpression of MYC could reverse the inhibition of MYC targets by RIF1 knockdown. Taken together, these data revealed that RIF1 played an important role in regulating MYC and MYC-activated genes, which in turn contributes to cellular response to cisplatin and NSCLC patients' response to platinum-based chemotherapy. RIF1 might serve as a novel biomarker for predicting platinum-based chemo-sensitivity and the prognosis of NSCLC patients, so as to guide the chemotherapy regimen adjustment for individual patient with NSCLC and improve their clinical outcomes.

RIF1 promotes tumor growth and cancer stem cell-like traits in NSCLC by protein phosphatase 1-mediated activation of Wnt/ß-catenin signaling.

Wnt/ß-catenin signaling is essential for proliferation and maintenance of cancer stem cell-like traits of various cancer cells. In non-small-cell lung carcinoma (NSCLC), the mechanisms underlying the hyperactivation of Wnt signaling remain unclear, as mutations in APC and ß-catenin genes are rare in NSCLC. RIF1 has been shown upregulated in breast and cervical cancer, this study intends to find out the potential effects of the expression and biological functions of RIF1 in NSCLC. Here we revealed that RIF1 was highly expressed in NCSLC at both mRNA and protein levels. RIF1 expression was significantly associated with clinical stage (P < 0.05) and prognosis (P < 0.001) of NSCLC patients. RIF1 knockdown inhibited NSCLC cell growth in vitro and in vivo, whereas overexpression of RIF1 in NSCLC cell lines promoted cell growth, cell cycle progression and cancer stem cell (CSC)-like properties via promoting PP1-AXIN interaction and thereby activating Wnt/ß-catenin signaling. Inhibition of PP1 in RIF1-overexpressed cells counteracted the effects of RIF1 on cell growth and CSC-like phenotype, as well as the Wnt/ß-catenin signaling. RIF1 expression was positively correlated with ß-catenin at the protein level in 32 NSCLC tissues. RIF1 expression closely related to MYC (r = 0.28, P < 0.001) and CCND1 (r = 0.14, P < 0.01) expression at the mRNA level in cohorts of The Cancer Genome Atlas (TCGA). These results indicated that RIF1 had an oncogenic role as a novel positive regulator of Wnt/ß-catenin signaling by directing PP1 to dephosphorylate AXIN; this novel mechanism may present a new therapeutic target for NSCLC.

RIF1 promotes human epithelial ovarian cancer growth and progression via activating human telomerase reverse transcriptase expression.

BACKGROUND: Human telomerase reverse transcriptase (hTERT) is highly expressed in over 80% of tumors, including human epithelial ovarian cancer (EOC). However, the mechanisms through which hTERT is up-regulated in EOC and promotes tumor progression remain unclear. The aim of this study is to identify RIF1 as a novel molecular target that modulate hTERT signaling and EOC growth. METHODS: RIF1 expression in ovarian cancer, benign and normal ovarian tissues was examined by immunohistochemistry. The biological role of RIF1 was revealed by MTS, colony formation and sphere formation assays. Luciferase reporter assay and chromatin immunoprecipitation (CHIP) assay were used to verify RIF1 as a novel hTERT promoter-binding protein in EOC cells. The role of RIF1 on tumorigenesis in vivo was detected by the xenograft model. RESULTS: RIF1 expression is upregulated in EOC tissues and is closely correlated with FIGO stage and prognosis of EOC patients. Functionally, RIF1 knockdown suppressed the expression and promoter activity of hTERT and consequently inhibited the growth and CSC-like traits of EOC cells. RIF1 knockdown also inhibited tumorigenesis in xenograft model. RIF1 overexpression had the opposite effect. Luciferase reporter assay and ChIP assay verified RIF1 directly bound to hTERT promoter to upregulate its expression. The rescue experiments suggested hTERT overexpression rescued the inhibition of EOC cell growth and CSC-like traits mediated by RIF1 knockdown. Consistently, hTERT knockdown abrogated the RIF1-induced promotion of EOC cell growth and CSC-like traits. CONCLUSIONS: RIF1 promotes EOC progression by activating hTERT and the RIF1/hTERT pathway may be a potential therapeutic target for EOC patients.

Downregulation of RIF1 Enhances Sensitivity to Platinum-Based Chemotherapy in Epithelial Ovarian Cancer (EOC) by Regulating Nucleotide Excision Repair (NER) Pathway.

BACKGROUND/AIMS: Rap1 interacting factor 1 (RIF1) was deemed to be involved in replication timing regulation and DNA damage response. However, little is known about the role of RIF1 in malignancies. Thus, this study aimed to investigate whether the expression of RIF1 is relevant to the response of epithelial ovarian cancer (EOC) patients to cisplatin chemotherapy and its underlying mechanism. METHODS: Immunohistochemistry was used for detecting the expression of RIF1 in 72 human ovarian cancer tissues followed by association analysis of RIF1 expression with patients' responses to platinum-based chemotherapy. The survival analysis of ovarian patients based on platinum chemotherapy was analyzed using online databases. RNA interference of RIF1 was carried out in OVCAR3 and A2780 cell lines, to determine the effect of lacking RIF1 expression on cellular responses to cisplatin by using MTS assay. The nucleotide excision repair (NER) capacity of these cells was assessed by using host-cell reactivation and UV sensitivity assay. Western Blot analysis was carried out to determine the effect of RIF1 on the proteins of NER and apoptosis signaling pathway by using RIF1 knockdown cells. BALB/c nude mice model was used for detection of response to cisplatin in vivo. RESULTS: RIF1 expression was significantly associated with the response of ovarian patients to platinum-based chemotherapy (P< 0.01). In cohorts from online databases, high expression of RIF1 was associated with higher mortality of EOC patients based on platinum chemotherapy (P < 0.01). RIF1 knockdown increased sensitivity to cisplatin in EOC in vitro and in vivo. Deletion of RIF1 impaired the NER activity by inhibiting the NER proteins in ovarian cancer cells. Besides, knockdown of RIF1 enhanced cisplatin-induced apoptosis. CONCLUSIONS: RIF1 plays an important role in regulating the expression of NER proteins, which in turn contributes to cellular response to cisplatin and EOC patients' response to platinum-based chemotherapy. RIF1 knockdown also promotes cisplatin-induced apoptosis. RIF1 may serve as a novel biomarker for predicting platinum-based chemosensitivity and the prognosis of EOC patients.

Helicase POLQ-like (HELQ) as a novel indicator of platinum-based chemoresistance for epithelial ovarian cancer.

OBJECTIVE: To investigate the role of HELQ in chemo-resistance of epithelial ovarian carcinoma (EOC), which is a critical factor of patients' prognosis. METHODS: Immunohistochemistry, survival analysis of our 87 EOC patients and bioinformatics analysis of The Cancer Genome Atlas (TCGA) datasets (Nature, 2011) disclosed the clinical importance of HELQ expression. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), and Western Blot analyses of EOC tissue were used to confirm it. Ectopic overexpression and RNA interference knockdown of HELQ were carried out in OVCAR3 and A2780 cell lines, respectively, to determine the effect of altered HELQ expression on cellular response to cisplatin by CCK8 assay. The DNA repair capacity of these cells was evaluated by using host-cell reactivation assay. Western Blot analyses were carried out to determine the effect of HLEQ on the DNA repair genes by using cells with altered HELQ expression. RESULTS: HELQ expression associates with response of EOC patients to platinum-based chemotherapy and their overall survival (OS), disease free survival (DFS). HELQ overexpression or knockdown, respectively, increased and decreased the cellular resistance to cisplatin, DNA repair activity, and expression of DNA repair proteins of Nucleotide excision repair (NER) pathway. CONCLUSIONS: HELQ plays an important role in regulating the expression of DNA repair proteins NER pathway which, in turn, contributes to cellular response to cisplatin and patients' response to platinum-based chemotherapy. Our results demonstrated that HELQ could serve as a novel indicator for chemo-resistance of EOC, which can predict the prognosis of the disease.

lncRNAs PVT1 and HAR1A are prognosis biomarkers and indicate therapy outcome for diffuse glioma patients.

Diffuse gliomas are well known malignant brain tumors. Long non-coding RNAs (lncRNAs), a type of RNA transcript with more than 200 nucleotides, involve in tumorigenesis and development of various cancers. This study focused on identifying differentially expressed lncRNAs in gliomas based on gene expression profiling, and chose certain lncRNAs PVT1, CYTOR, HAR1A and MIAT, which changed with significant differences. Further analysis of TCGA and GEO data revealed that the expressions of PVT1 and CYTOR were up-regulated, while HAR1A and MIAT expressions were down-regulated in gliomas. Their expression patterns were validated in an independent cohort containing 98 glioma specimens and 12 non-tumor tissue controls. High expression of PVT1 and CYTOR as well as low HAR1A and MIAT expression were associated with high Ki-67 level and more TP53 mutation. Kaplan-Meier survival curve and Cox regression analyses showed that glioma patients with high PVT1 expression or low HAR1A expression had poor survival outcome, aberrantly expressed PVT1 and HAR1A could be the independent prognosis biomarkers for glioma patients. Moreover, down-regulation of PVT1 and up-regulation of HAR1A contributed to improve the survival of patients who received chemotherapy and radiotherapy. These results implied that these four lncRNAs might play important role in diffuse gliomas progression, particularly, PVT1 and HAR1A could be explored as promising biomarkers for diagnosis, prognosis and target therapy of diffuse gliomas.

Silencing RIF1 decreases cell growth, migration and increases cisplatin sensitivity of human cervical cancer cells.

Replication timing regulatory factor 1 (RIF1) plays an important role in DNA replication regulation, stem cell pluripotency and DNA repair pathway. However, little is known about the molecular mechanisms and physiological significance of RIF1 in cancer and chemotherapy efficacy. In this study, we found that RIF1 is upregulated in cervical cancer tissues compared with normal tissues both at mRNA and protein levels through online databases. RIF1 knockdown reduced cervical cancer cell growth, colony formation, migration and epithelial-mesenchymal transition (EMT) markers. Flow cytometry analysis indicated that RIF1 knockdown induced apoptosis and G2 cell cycle arrest. Furthermore, RIF1 knockdown increased cisplatin sensitivity, cisplatin-induced G2/M phase arrest, apoptosis and led to defects in DNA repair in a concentration-dependent manner. In terms of mechanism research, increased CDKN1A expression and Bax/Bcl-2/caspase-3 signaling pathway might be involved in the G2/M phase arrest and increased apoptosis in RIF1-silenced cervical cancer cells. Thus, these findings indicate that RIF1 knockdown prior to chemotherapy may be a potential effective therapeutic strategy for cervical cancer.