MiR-18 inhibitor promotes the differentiation of bovine skeletal muscle-derived satellite cells by increasing MEF2D expression.

Skeletal muscle is composed of muscle fibers formed from myoblast differentiation. Recently, numerous researchers have demonstrated that microRNAs (miRNAs) play an essential role in modulating the proliferation and differentiation of myoblasts. Our previous study has shown that among the miR-17-92 cluster members, miR-17 and miR-20a together with miR-19b can efficiently promote the differentiation of murine C2C12 and bovine primary myoblasts. However, the role of miR-18 in this process remains elusive. In this study, we revealed that miR-18 inhibited the differentiation of bovine skeletal muscle-derived satellite cells (bMDSCs), whereas an miR-18 inhibitor significantly promoted cell differentiation (p < 0.001). Then, a target gene of miR-18 was found to be myocyte enhancer factor 2D (MEF2D), which is critical for myoblast differentiation. Furthermore, we found that the combination of the miR-18 inhibitor and miR-19 significantly improved the formation of bMDSCs-derived muscle fibers (p < 0.001). This study revealed the role of miR-18 in bovine skeletal muscle differentiation and contributed to the understanding of the regulatory mechanism of mammalian myogenic differentiation.

Beef is a beneficial food source, and improving muscle yield and quality has become a hot topic in the beef industry. Therefore, our study aimed to explore effective methods to improve bovine muscle cell differentiation to increase beef production. The study revealed that microRNA-18 (miR-18) inhibitor could promote the differentiation of bovine skeletal muscle-derived satellite cells (bMDSCs) by increasing the expression of myocyte enhancer factor 2D (MEF2D), a critical gene for myoblast differentiation. Furthermore, we found that combined inhibitors of miR-18 and miR-19 could significantly improve bMDSCs differentiation. Our study demonstrated the role of a new regulatory factor that may enhance beef production level and contributed to elucidating the mechanism of muscle differentiation.

Establishment of a specific in vivo Cu(â ) reporting system based on metallothionein screening.

Copper is one of the indispensable trace metal elements in organisms, but excess copper means cytotoxicity. Cells protect themselves by storing excess copper in copper-binding proteins. Metallothioneins (MTs) are a group of low-molecular-weight, cysteine-rich proteins, which are well known for sensing and binding the overcharged Zn(Ⅱ), Cd(Ⅱ), and Cu(Ⅰ) in cells. However, there are only few reports on MTs that can specifically respond to intracellular copper ions in mammals in real-time. Here, we screened copper-response MTs in pancreatic cancer cells through data-mining, RNA-seq, and qPCR analysis. We found that MT1E, MT1F, and MT1X mRNA were significantly upregulated after exogenous copper ion induction. By constructing the stable cell lines with MT1E, MT1F, or MT1X promoter-driven EGFP as reporters, we found that only PMT1F-EGFP could specifically and stably report the intracellular Cu(Ⅰ) changes in multiple cell lines including Panc-1, 8988T, 293T, HepG2, and normal hepatic cells, indicating that PMT1F-EGFP is an ideal in vivo Cu(Ⅰ) reporter. Using the PMT1F-EGFP reporter, we found that MEK inhibitors (U0126) and Astragaloside IV could significantly increase intracellular copper ions. According to these results, PMT1F-EGFP reporter can sense intracellular copper change and can be used to screen copper-target drugs and study copper-related cellular physiology and pathology.

MiR-7 mediates mitochondrial impairment to trigger apoptosis and necroptosis in Rhabdomyosarcoma.

BACKGROUND: Rhabdomyosarcoma (RMS) is a pediatric cancer with rhabdomyoblastic phenotype and mitochondria act as pivotal regulators of its growth and progression. While miR-7-5p (miR-7) is reported to have a tumor-suppressive role, little is yet known about its antitumor activity in RMS. METHODS: The effects of miR-7 on RMS were analyzed both in vitro and in vivo. Cell death modalities induced by miR-7 were identified. Influence on mitochondria was evaluated through RNA sequencing data, morphological observation and mitochondrial functional assays, including outer membrane permeability, bioenergetics and redox balance. Dual-luciferase assay and phenotype validation after transient gene silencing were performed to identify miR-7 targets in RMS. RESULTS: MiR-7 executed anti-tumor effect in RMS beyond proliferation inhibition. Morphologic features and molecular characteristics with apoptosis and necroptosis were found in miR-7-transfected RMS cells. Chemical inhibitors of apoptosis and necroptosis were able to prevent miR-7-induced cell death. Further, we identified that mitochondrial impairment mainly contributed to these phenomena and mitochondrial proteins SLC25A37 and TIMM50 were crucial targets for miR-7 to induce cell death in RMS. CONCLUSION: Our results extended the mechanism of miR-7 antitumor role in rhabdomyosarcoma cancer, and provided potential implications for its therapy.

Copper promotes sheep pancreatic duct organoid growth by activation of an antioxidant protein 1-dependent MEK-ERK pathway.

Proper amounts of copper supplemented in livestock feed improve the physical growth and traits of farm animals. The pancreas is an important organ with both exocrine and endocrine portions. To investigate the role and mechanism of copper in the sheep pancreas, we first established sheep pancreatic duct organoids (sPDOs). We found that an appropriate amount of copper benefited the formation and growth of sPDOs, whereas excess or deficient copper damaged sPDOs. We found that the proliferation-stimulating effect of copper was related to the copper chaperone antioxidant protein 1 (ATOX1)-dependent activation of MEK-ERK1/2 signaling. Atox1 knockdown suppressed the cell proliferation of sPDOs, even in the presence of the MEK activator. These results indicate that moderate concentrations of copper promote sPDO growth through ATOX1-regulated cell proliferation by activation of MEK-ERK. Moreover, our study indicates that organoids may be a useful model to study organ growth mechanisms in livestock.

Molecular chaperone HspB2 inhibited pancreatic cancer cell proliferation via activating p53 downstream gene RPRM, BAI1, and TSAP6.

Heat shock proteins (HSPs) were known as the molecular chaperones, which play a pivotal role in the protein quality control system, ensuring correct folding of proteins, and facilitating the correct refolding of damaged proteins via the transient interaction with their substrate proteins. They also practice in the regulation of cell cycles and are involved in apoptosis. We found that HspB2 was almost completely silent in pancreatic cancer and few studies investigated the role of HspB2 in cancer cells, particularly in pancreatic cancer. Here, we reported that HspB2 effectively inhibited cell proliferation in Panc-1 cells. Specifically, we demonstrated that HspB2 could combine mut-p53 and change the DNA binding site of mutant p53, subsequently upregulated the expression of RPRM, BAI-1, and TSAP6 which were the downstream genes of wt-p53, participate in mediating downstream responses to p53, including inhibiting cell proliferation and angiogenesis. The main aim of this study is to investigate the relationship between HspB2 and p53, and provide a novel treatment strategy for pancreatic cancer.

EGF suppresses the expression of miR-124a in pancreatic ß cell lines via ETS2 activation through the MEK and PI3K signaling pathways.

Diabetes mellitus is characterized by pancreatic ß cell dysfunction. Previous studies have indicated that epidermal growth factor (EGF) and microRNA-124a (miR-124a) play opposite roles in insulin biosynthesis and secretion by beta cells. However, the underlying mechanisms remain poorly understood. In the present study, we demonstrated that EGF could inhibit miR-124a expression in beta cell lines through downstream signaling pathways, including mitogen-activated protein kinase kinase (MEK) and phosphatidylinositol 3-kinase (PI3K) cascades. Further, the transcription factor ETS2, a member of the ETS (E26 transformation-specific) family, was identified to be responsible for the EGF-mediated suppression of miR-124a expression, which was dependent on ETS2 phosphorylation at threonine 72. Activation of ETS2 decreased miR-124a promoter transcriptional activity through the putative conserved binding sites AGGAANA/TN in three miR-124a promoters located in different chromosomes. Of note, ETS2 played a positive role in regulating beta cell function-related genes, including miR-124a targets, Forkhead box a2 (FOXA2) and Neurogenic differentiation 1 (NEUROD1), which may have partly been through the inhibition of miR-124 expression. Knockdown and overexpression of ETS2 led to the prevention and promotion of insulin biosynthesis respectively, while barely affecting the secretion ability. These results suggest that EGF may induce the activation of ETS2 to inhibit miR-124a expression to maintain proper beta cell functions and that ETS2, as a novel regulator of insulin production, is a potential therapeutic target for diabetes mellitus treatment.

Zinc chelator TPEN induces pancreatic cancer cell death through causing oxidative stress and inhibiting cell autophagy.

The essential trace element zinc (Zn) is widely required in cellular functions, and abnormal Zn homeostasis causes a variety of health problems including immunodeficiency and sensory dysfunctions. Previous studies had shown that Zn availability was also important for tumor growth and progression. The aim of the present study was to investigate the potential mechanisms of N,N,N,N-Tetrakis(2-pyridylmethyl)-ethylenediamine (TPEN) (a membrane permeable zinc chelator) induced pancreatic cancer cell death. The text of inductively coupled plasma-mass spectrometry (ICP-MS) showed in human pancreatic cancer samples that the zinc content in cancer was higher than that in adjacent tissues. The pancreatic cancer cell lines Panc-1, 8988T, BxPc-3, and L3.6 were used in this study. Our results indicated that TPEN markedly induced cell death, via increasing reactive oxygen species (ROS) and restraining autophagy. Our data also indicated that TPEN-stimulated mitochondrial metabolism produced much ROS. Meanwhile, TPEN reduced the levels of glutathione (GSH) and triggered ROS outbreak, which were the main causes of cell death. In addition, cell autophagy was significantly depressed in Panc-1 cells treated by TPEN, which was due to the ability of disrupting lysosomal by TPEN. Thus, we thought zinc depletion by TPEN was a potential therapeutic strategy for pancreatic cancer.

Guanine-rich RNA binding protein GRSF1 inhibits myoblast differentiation through repressing mitochondrial ROS production.

Guanine-rich RNA sequence binding factor 1 (GRSF1) is a member of the RNA-binding protein (RBP) family. GRSF1 regulates RNA metabolism through RNA processing, transport and translation in the cytoplasm and mitochondria. However, its role in myogenesis has not been investigated. Here, we demonstrated that the expression of mitochondrial GRSF1 was negatively related to the differentiation of mouse skeletal myoblasts. Interference with GRSF1 promotes myogenesis both in vitro and in vivo without affecting MyoD expression or cell proliferation. Further studies illustrated that GRSF1 regulated myogenic differentiation through direct targeting of mitochondrial GPX4, a key regulator of the cellular redox status, leading to the modulation of ROS levels, which is important for myogenesis. Our findings underscore a critical function for GRSF1 during skeletal myogenesis linked to its regulation of muscle redox homeostasis.

Expression characterisation of cyclophilin BrROC1 during light treatment and abiotic stresses response in Brassica rapa subsp. rapa 'Tsuda'.

ROC1 is a prototypic peptidyl prolyl cis/trans isomerase (PPIase) of the plant cytosol belonging to the large subfamily of cyclophilins that are associated with diverse functions through foldase, scaffolding, chaperoning or other unknown activities. Although many functions of plant cyclophilins have been reported, the molecular basis of stress-responsive expression of plant cyclophilins is still largely unknown. To characterise the roles of BrROC1 during light treatment and their responses in various abiotic stresses, we identified BrROC1 genes and characterised their expression patterns in Brassica rapa subsp. rapa 'Tsuda'. Our results showed that BrROC1 genes are multi-family genes. Transcript level analysis showed BrROC1-2 expressed higher than BrROC1-1 in 0 to 6-day-old seedlings under natural light. Moreover, BrROC1-2 genes were also induced to highly express in the cotyledon, upper hypocotyls and lower hypocotyls of seedlings under UV-A and blue-light treatment. In addition, the transcript level of BrROC1-1 was higher in pigment tissues than that in unpigment tissues (cotyledon and lower hypocotyl) under UV-A and blue-light treatment. Furthermore, when the unpigment epidermis (shaded light) of 2-month-old 'Tsuda' turnip roots was exposed to UV-A light, transcript levels of the BrROC1-1 and BrROC1-2 were significantly increased with time prolongation. These two BrROC1 genes might be involved in UV-A-induced anthocyanin synthesis in the root epidermis of 'Tsuda' turnip, which accumulates high levels of anthocyanin. These two BrROC1 genes were also induced to be regulated by abiotic stresses such as high or low temperature, dehydration, osmotic and salt stresses. Then, the results indicate that BrROC1 genes are involved in light induction response and may play important roles in adaptation of plants to various environmental stresses.

Increased activity of ß-glucuronidase variants produced by site-directed mutagenesis.

ß-glucuronidase (BGus) is an essential glycosyl hydrolase which has been widely used in biological and biomedical applications. In this paper, we report the construction and screening of nineteen Escherichia coli BGus (EBGus) mutants using site-directed mutagenesis. The mutants G559N, G559S and G559T showed a 3-5 fold increase in enzyme activity in comparison to wild type EBGus. In particular, G559S, with the highest activity, showed 2-6 fold enhanced activity compared to abalone and snail BGus extracts. Moreover, the glycine to serine mutagenesis for the same site in Staphylococcus sp. RLH1 BGus (StBGus) exhibited significantly enhanced activity, which indicated the importance of the G559→S mutation on BGus function. Based on this structural analysis, we postulate that the mutation at G559 plays an important role in the stabilization of the enzyme conformation, and thereby facilitates the effective binding of substrate.

Baicalein inhibits pancreatic cancer cell proliferation and invasion via suppression of NEDD9 expression and its downstream Akt and ERK signaling pathways.

Baicalein, a flavone ingredient of Scutellaria baicalensis Georgi, is a promising anti-cancer agent. However, its potential anti-pancreatic cancer effects and the underlying mechanisms are still unclear. In this study, we showed that Baicalein not only induced apoptosis, but also suppressed proliferation, migration and invasion of two pancreatic cancer cell lines BxPC-3 and PANC-1 in a dose- and time-dependent manner. Notably, Baicalein exhibited low toxicity to normal human liver or kidney cells. We further discovered that Baicalein suppressed BxPC-3 and PANC-1 cell proliferation and invasion through targeting the expression of NEDD9, a Cas scaffolding protein, to decrease Akt and ERK activities. Especially, Baicalein decreased Akt phosphorylation at T-308 via lowering NEDD9-dependent PDK1 expression. Overexpression of NEDD9 effectively rescued proliferation and invasion of BxPC-3 and PANC-1 cells dampened by Baicalein. Taken together, our findings suggest that Baicalein is a potent remedy applied to pancreatic cancer treatment in the future.

Polymer-Protein Core-Shell Nanoparticles for Enhanced Antigen Immunogenicity.

Nanoengineered vaccine platforms can be modeled after viruses and other pathogens with highly organized and repetitive structures that trigger the host immune system. Here we demonstrated a pyridine-grafted poly(ε-caprolactone)-based polymer-protein core-shell nanoparticles (PPCS-NPs) platform can effectively trigger the host immune system and lead to significantly higher antibody titers.

Proteome analysis of dormancy-released seeds of Fraxinus mandshurica Rupr. in response to re-dehydration under different conditions.

Desiccation tolerance is the ability of orthodox seeds to achieve equilibrium with atmospheric relative humidity and to survive in this state. Understanding how orthodox seeds respond to dehydration is important for improving quality and long-term storage of seeds under low temperature and drought stress conditions. Long-term storage of seeds is an artificial situation, because in most natural situations a seed that has been shed may not remain in a desiccated state for very long, and if dormant it may undergo repeated cycles of hydration. Different types of seeds are differentially sensitive to desiccation and this directly affects long-term storage. For these reasons, many researchers are investigating loss of desiccation tolerance during orthodox seed development to understand how it is acquired. In this study, the orthodox seed proteome response of Fraxinus mandshurica Rupr. to dehydration (to a relative water content of 10%, which mimics seed dehydration) was investigated under four different conditions viz. 20 °C; 20 °C with silica gel; 1 °C; and 1 °C after pretreatment with Ca2+. Proteins from seeds dehydrated under different conditions were extracted and separated by two-dimensional difference gel electrophoresis (2D-DIGE). A total of 2919 protein spots were detected, and high-resolution 2D-DIGE indicated there were 27 differentially expressed. Seven of these were identified using MALDI TOF/TOF mass spectrometry. Inferences from bioinformatics annotations of these proteins established the possible involvement of detoxifying enzymes, transport proteins, and nucleotide metabolism enzymes in response to dehydration. Of the seven differentially abundant proteins, the amounts of six were down-regulated and one was up-regulated. Also, a putative acyl-coenzyme A oxidase of the glyoxylate cycle increased in abundance. In particular, the presence of kinesin-1, a protein important for regulation and cargo interaction, was up-regulated in seeds exposed to low temperature dehydration. Kinesin-1 is present in all major lineages, but it is rarely detected in seed desiccation tolerance of woody species. These observations provide new insight into the proteome of seeds in deep dormancy under different desiccation conditions.

[Preparation of anti-HBsAg epitope monoclonal antibodies and establishment of a sandwich ELISA for HBV serotype detection].

OBJECTIVE: To prepare the monoclonal antibodies against HBsAg epitopes and establish a sandwich ELISA system for HBV serotype detection. METHODS: The BALB/c mice were immunized with HBsAg subtype proteins. The hybridoma cells were cultured in serum-free medium after cell fusion and high throughput screening ELISA (HTS-ELISA). Monoclonal antibodies were purified with protein A. The specificity, affinity, isotype, and epitope of the mAbs were characterized respectively and the sandwich ELISA system was established. RESULTS: The titers of mouse anti-sera reached 1:10(5);. After the cell fusion and HTS-ELISA, the four hybridoma cell lines were obtained. The mAbs were purified and named #2-4, #18-23, #7-7, #56-71, respectively. The mAbs had a high affinity (over 10(9); L/mol). Indirect ELISA showed that #2-4, #18-23, #7-7 and #56-71 could recognize HBsAg "d, y, r, w" epitopes, respectively. The sandwich ELISA was established through using #3-11 (Anti-HBsAg "a" epitope) as the coating antibody while the HRP labeled mAb as the secondary antibody. The optimized sandwich ELISA was proved to have a good specificity by testing different antibody-antigen reactions. CONCLUSION: The mAbs against HBsAg epitopes we prepared had a good affinity and specificity. The sandwich ELISA for HBV serotype detection we established successfully provided a basis for HBV serotype detection and disease diagnosis.

[Preparation of monoclonal antibodies against human Apo B100 and establishment of a sandwich ELISA system].

AIM: To prepare the monoclonal antibodies (mAbs) against human Apo B100 and establish a double-antibody sandwich ELISA system for human Apo B100 detection. METHODS: The BALB/c mice were immunized with human Apo B100 antigen. After cell fusion and screening, the hybridoma cells were cultured in serum-free medium and the supernatant was purified to obtain mAbs using protein A. The affinity, subtype, specificity and epitope of the mAbs were characterized and the sandwich ELISA system was established. RESULTS: Four hybridoma cell lines 4-1-2, 4-2-2, 4-3-2 and 4-6-3 were obtained. The affinity of the anti-Apo B100 mAbs was up to 1×109 L/mol and nearly had no cross reaction with other relevant proteins. Linear detection of the sandwich ELISA system using 4-3-2 and 4-6-3 covered a range from (1.3-80) ng/mL, and its sensitivity was 1.24 ng/mL. The intra-assay coefficient of variation (CV) and inter-assay CV were less than 10% and 15%, respectively, and the recovery rate was above 90%. CONCLUSION: The mAbs against human Apo B100 have been prepared and a sandwich ELISA system for human Apo B100 detection has been established successfully, which provide a basis for human Apo B100 detection and disease diagnosis.

[The application of two-dimensional liquid phase chromatography to separation of total protein of Leymus chinensis shoot].

The total proteins of Leymus chinensis were extracted from eight-week-old seedlings grown in the greenhouse by TCA-acetone precipitation method. After the lysis buffer was replaced by the start buffer, proteins were fractionated along the first dimension using chromatofocusing (CF). Subsequently the fractions with pI values between 8.5 and 4.0 collected after the first dimension separation were further fractionated by nonporous silica reverse-phase high-performance liquid chromatography (NPS-RP-HPLC, HPRP). With ProteoVue software the pI/UV map was generated to show the protein expression profile of Leymus chinensis. Some experiments were tested to optimize the fraction procedure. Three different elution gradients were employed to get the optimal chromatogram. Comparison of the protein expression pattern between 2D-PAGE and 2D-LC indicated that 2D-LC was a powerful tool in protein fraction. Reproducibility and veracity of the protein patterns were confirmed in different injections of the same sample. A method to fractionate the total protein of Leymus chinensis shoot with two-dimensional liquid chromatography (2D-LC) was founded.