Binding Protein Alpha Research Articles

Forming cytoplasmic stress granules PURα suppresses mRNA translation initiation of IGFBP3 to promote esophageal squamous cell carcinoma progression

Esophageal squamous cell carcinoma (ESCC) is one of the most fatal malignancies worldwide. Recently, our group identified purine-rich element binding protein alpha (PURα), a single-stranded DNA/RNA-binding protein, to be significantly associated with the progression of ESCC. Additional immunofluorescence staining demonstrated that PURα forms cytoplasmic stress granules to suppress mRNA translation initiation. The expression level of cytoplasmic PURα in ESCC tumor tissues was significantly higher than that in adjacent epithelia and correlated with a worse patient survival rate by immunohistochemistry. Functionally, PURα strongly preferred to bind to UG-/U-rich motifs and mRNA 3´UTR by CLIP-seq analysis. Moreover, PURα knockout significantly increased the protein level of insulin-like growth factor binding protein 3 (IGFBP3). In addition, it was further demonstrated that PURα-interacting proteins are remarkably associated with translation initiation factors and ribosome-related proteins and that PURα regulates protein expression by interacting with translation initiation factors, such as PABPC1, eIF3B and eIF3F, in an RNA-independent manner, while the interaction with ribosome-related proteins is significantly dependent on RNA. Specifically, PURα was shown to interact with the mRNA 3´UTR of IGFBP3 and inhibit its expression by suppressing mRNA translation initiation. Together, this study identifies cytoplasmic PURα as a modulator of IGFBP3, which could be a promising therapeutic target for ESCC treatment.

꾸지뽕 가지 추출물의 지방세포 분화 및 지방축적 억제 효과

This study was conducted to evaluate the anti-adipogenic effects of Maclura tricuspidate twig extract (MTE) in 3T3-L1 pre-adipocytes. 3T3-L1 pre-adipocytes were differentiated with adipogenic regents (dexamethsone, 3-isobutyl-methylxanthine, insulin) by incubation for 8 days in the absence or presence of MTE. The accumulation of lipid droplets and triglycerides in cells was dose-dependently suppressed by MTE. MTE caused an increase in the release of adiponectin and inhibited the release of leptin. Consistent with these results, MTE attenuated the protein expression of peroxisome proliferator-activated receptor gamma (PPARγ), CCATT/enhancer binding protein alpha (C/EBPα), fatty acid synthase (FAS) and adipocyte Protein 2 (aP2), all of which are associated with adipogenesis in cells. Treatment with MTE also dose-dependently increased the release of free glycerol. Additionally, MTE increased the protein expression of hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL), which are associated with lipolysis. These results showed that MTE inhibits adipogenesis by regulating the expression of proteins involved in adipogenesis. Therefore, the results suggest that MTE has the potential to be used as a functional ingredient in the food industry to prevent obesity.

Development and validation of a prognostic scoring model to risk stratify childhood acute myeloid leukaemia.

SummaryTo create a personal prognostic model and modify the risk stratification of paediatric acute myeloid leukaemia, we downloaded the clinical data of 597 patients from the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database as a training set and included 189 patients from our centre as a validation set. In the training set, age at diagnosis, ‐7/del(7q) or ‐5/del(5q), core binding factor fusion genes, FMS‐like tyrosine kinase 3‐internal tandem duplication (FLT3‐ITD)/nucleophosmin 1 (NPM1) status, Wilms tumour 1 (WT1) mutation, biallelic CCAAT enhancer binding protein alpha (CEBPA) mutation were strongly correlated with overall survival and included to construct the model. The prognostic model demonstrated excellent discriminative ability with the Harrell's concordance index of 0.68, 3‐ and 5‐year area under the receiver operating characteristic curve of 0.71 and 0.72 respectively. The model was validated in the validation set and outperformed existing prognostic systems. Additionally, patients were stratified into three risk groups (low, intermediate and high risk) with significantly distinct prognosis, and the model successfully identified candidates for haematopoietic stem cell transplantation. The newly developed prognostic model showed robust ability and utility in survival prediction and risk stratification, which could be helpful in modifying treatment selection in clinical routine.

Mutational Landscape of CEBPA in Mexican Pediatric Acute Myeloid Leukemia Patients: Prognostic Implications.

BackgroundIn Mexico, the incidence of acute myeloid leukemia (AML) has increased in the last few years. Mortality is higher than in developed countries, even though the same chemotherapy protocols are used. CCAAT Enhancer Binding Protein Alpha (CEBPA) mutations are recurrent in AML, influence prognosis, and help to define treatment strategies. CEBPA mutational profiles and their clinical implications have not been evaluated in Mexican pediatric AML patients.Aim of the StudyTo identify the mutational landscape of the CEBPA gene in pediatric patients with de novo AML and assess its influence on clinical features and overall survival (OS).Materials and MethodsDNA was extracted from bone marrow aspirates at diagnosis. Targeted massive parallel sequencing of CEBPA was performed in 80 patients.ResultsCEBPA was mutated in 12.5% (10/80) of patients. Frameshifts at the N-terminal region were the most common mutations 57.14% (8/14). CEBPA biallelic (CEBPABI) mutations were identified in five patients. M2 subtype was the most common in CEBPA positive patients (CEBPAPOS) (p = 0.009); 50% of the CEBPAPOS patients had a WBC count > 100,000 at diagnosis (p = 0.004). OS > 1 year was significantly better in CEBPA negative (CEBPANEG) patients (p = 0.0001). CEBPAPOS patients (either bi- or monoallelic) had a significantly lower OS (p = 0.002). Concurrent mutations in FLT3, CSF3R, and WT1 genes were found in CEBPAPOS individuals. Their contribution to poor OS cannot be ruled out.ConclusionCEBPA mutational profiles in Mexican pediatric AML patients and their clinical implications were evaluated for the first time. The frequency of CEBPAPOS was in the range reported for pediatric AML (4.5–15%). CEBPA mutations showed a negative impact on OS as opposed to the results of other studies.

Nidus vespae Built by an Invasive Alien Hornet, Vespa velutina nigrithorax, Inhibits Adipose Tissue Expansion in High-Fat Diet-Induced Obese Mice

Simple SummaryNidus vespae (NV) has been used as an ingredient in crude drugs in Korea and China. However, the effects of NV on obesity and its mechanism have not been completely elucidated. Herein, we demonstrated the novel anti-obesity effects of NV in in vivo and in vitro systems. The administration of NV ameliorated adipose expansion and improved adipose browning in white adipose tissue from high-fat diet (HFD)-fed mice. Moreover, treatment with NV suppressed adipocyte differentiation, possibly through inactivation of the insulin signaling pathway in adipocytes.Nidus vespae, commonly known as the wasp nest, has antioxidative, anti-inflammatory, antimicrobial, and antitumor properties. However, the anti-obesity effects of Nidus vespae extract (NV) have not yet been reported. This study aimed to elucidate the potential anti-obesity effects of NV in vivo and in vitro, using a high-fat diet (HFD)-induced obese mouse model and 3T3-L1 adipocytes, respectively. NV administration to HFD-induced obese mice significantly decreased the mass and plasma lipid content of adipose tissues. Uncoupling protein-1 expression was significantly higher in the inguinal white adipose tissues of NV-treated mice than in those of HFD-fed mice. Furthermore, we found that NV inhibited the differentiation and intracellular lipid accumulation of 3T3-L1 adipocytes by regulating the insulin signaling cascade, including protein kinase B, peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein alpha, and adiponectin. These findings suggest that NV may exhibit therapeutic effects against obesity by suppressing adipose tissue expansion and preadipocyte differentiation, thereby providing critical information for the development of new drugs for disease prevention and treatment. To our knowledge, this study provides the first evidence of the anti-obesity effects of NV.

Expressional Evaluation of C/EBP Family, SREBP1, and Steroid Hormone Receptors in the Epididiymal Fat of Postnatally Developing Mouse.

The differentiation and development of preadipocyte into mature adipocyte are regulated by transcription factors, such as CCAAT enhancer binding protein (Cebp) gene family and sterol regulatory element binding transcription factor 1 (Srebp1). Steroid hormones give influences on the development and function of adipocyte. The present research examined expression patterns of CCAAT enhancer binding protein alpha (Cebpa), CCAAT enhancer binding protein beta (Cebpb), CCAAT enhancer binding protein gamma (Cebpg), sterol regulatory element binding transcription factor 1 (Srebp1), androgen receptor (Ar), and estrogen receptors (Esr) among different epididymal fat parts during postnatal period by quantitative real-time polymerase chain reaction. In the distal epididymal fat, expression of Cebpa, Cebpb, Cebpg, Srebp1, Ar, and Esr2 was increased until 12 months of age, while expression of Esr1 was decreased at 5 months of age and was not detectable after 8 months of age. In the proximal epididymal fat, transcript levels of Cebps and Srebp1 were increased at 8 months of age, followed by decreases of Cebpb and Cebpg transcript levels at 12 months of age. An additional increase of Srebp1 expression was observed at 12 months of age. Expression of Ar and Esr2 were increased until 8 months of age, followed by a drop of Ar expression level at 12 months of age. Expression pattern of Esr1 was similar to that in the distal epididymal fat. In the tail epididymal fat, expression of Cebpa, Cebpg, Srebp1, Ar, and Esr2 was increased with age. Esr1 was not detectable at all. The highest level of Cebpb was observed at 8 months of age. These data suggest the possibility of developmental and functional differentiation among the epididymal fat parts.

Effects of magnolol and honokiol blend on performance, egg quality, hepatic lipid metabolism, and intestinal morphology of hens at late laying cycle

Magnolol and its isomer honokiol are polyphenols with anti-oxidative and anti-inflammatory activities. We evaluated the effects of magnolol and honokiol supplementation alone or in combination with hen diets during the late laying cycle. A total of 540 Jingfen pink-shell laying hens (50 weeks old) were randomly assigned to six treatments: a control diet and diets supplemented with 300 mg/kg magnolol (M300), honokiol (H300), or 300 mg/kg total phenols with a magnolol/honokiol ratio of 2:1 (M200H100), 1:2 (M100H200), and 1:1 (M150H150). Compared with that of the control, all supplementation groups had higher laying rates and the M300, M100H200, and M150H150 groups showed comparatively lower feed conversion ratios. Magnolol and honokiol supplementation increased the Haugh units of fresh eggs at week 62 and alleviated the decline of the Haugh units of eggs stored for 14 days. Compared with that of the control group, the serum total antioxidant capacity of the M100H200 and M150H150 groups significantly increased, and all supplementation groups had higher total antioxidant capacity and lower malondialdehyde content in the liver. With respect to lipid metabolism, the M200H100 and M150H150 groups had lower total and relative liver weights compared with those of the control and H300 groups. The mRNA expression levels of CCAAT enhancer binding protein alpha, sterol regulatory element binding protein-1, fatty acid synthase and stearyl coenzyme A desaturase 1 involved in lipogenesis; microsomal triglyceride transfer protein and apolipoprotein B involved in fatty acid transport; and the proinflammatory cytokine interleukin-1 beta were lower in all supplementation groups compared with those in the control. With respect to gut health, the heights of the jejunum and ileum villi significantly increased in all supplementation groups compared with those of the control, and the jejunum villus heights of the M300 and M150H150 groups were higher than those of the H300 and M100H200 groups. The H300 and M150H150 groups had higher mRNA expression levels of zonula occludens-1 in the ileum compared with those in the control and M300 groups, whereas all supplementation groups had higher mRNA levels of claudin-1 than that of the control group. In conclusion, magnolol and honokiol improved hen performance and the albumen quality of fresh and stored eggs by improving the antioxidant capacity, liver lipid metabolism, and intestinal health of laying hens. The combination of magnolol and honokiol at a 1:1 ratio may be an optimal choice for hen diet supplementation.

LncRNA MIR99AHG enhances adipocyte differentiation by targeting miR-29b-3p to upregulate PPARγ

AimThe aim is to identify new long noncoding RNAs (lncRNAs) involved in adipocyte differentiation. MethodsHigh-throughput RNA sequencing of 3T3-L1 preadipocytes was carried out before and after differentiation to identify the target lncRNAs and miRNAs. The effects of lncRNA, miRNA and the network mechanism on adipocyte differentiation were evaluated in vitro and in vivo. Visceral adipose tissue (VAT) was collected from Chinese subjects with obesity or a normal body mass index (BMI), and the levels of lncRNAs, adipogenic genes and miRNAs were measured. ResultsMIR99AHG, miR-29b-3p were selected as the target lncRNA and miRNA. Short hairpin RNA against MIR99AHG inhibited the differentiation of 3T3-L1 preadipocytes, reduced the expression of the peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT enhancer-binding protein alpha (C/EBPα) and fatty acid binding protein 4 (FABP4) genes, upregulated the expression of miR-29b-3p. Overexpression of MIR99AHG showed the opposite effects. Overexpression of miR-29b-3p inhibited the differentiation of 3T3-L1 preadipocytes and decreased the PPARγ level, while inhibition of miR-29b-3p showed the opposite effects. MIR99AHG and PPARγ competed for binding to miR-29b-3p. In mice with high-fat diet-induced obesity, MIR99AHG and miR-29b-3p mRNA level were increased and decreased, respectively. Tail vein injection of adeno-associated virus 9-MIR99AHG-RNA interference (AAV9-MIR99AHG-RNAi) reduced the body weight, epididymal fat mass, MIR99AHG level and increased the expression of miR-29b-3p. The expression levels of MIR99AHG, PPARγ, C/EBPα and FABP4 in human visceral adipose tissue were higher in the obese group than in the normal weight group. ConclusionsMIR99AHG enhances adipogenesis by regulating miR-29b-3p and PPARγ, providing a new target for therapeutic intervention in obesity.

The prognostic factors in acute myeloid leukaemia with double-mutated CCAAT/enhancer-binding protein alpha (CEBPAdm).

The prognostic factors to stratify acute myeloid leukaemia (AML) with double-mutated CCAAT/enhancer-binding protein alpha (CEBPAdm) into different risk groups remains to be determined. In this retrospective study, we evaluated 171 consecutive patients with newly diagnosed AML with CEBPAdm by a Cox proportional hazards regression model. In univariate analyses, colony stimulating factor 3 receptor (CSF3R) and Wilms tumour 1 (WT1) mutations were associated with poor relapse-free survival (RFS). The induction regimens including homoharringtonine (omacetaxine mepesuccinate) or intermediate-dose cytarabine was associated with favourable RFS and overall survival (OS). The induction regimen including both homoharringtonine and intermediate-dose cytarabine was associated with the most favourable RFS (3-year RFS 84.7%) and OS (3-year OS 92.8%) compared to the conventional cytarabine and daunorubicin regimen (3-year RFS 27.7%, hazard ratio [HR] 0.126, 95% confidence interval [CI] 0.051-0.313, Wald p<0.001; and 3-year OS 56.4%, HR 0.179, 95% CI 0.055-0.586, Wald p=0.005). In multivariate analyses, the induction regimen including intermediate-dose cytarabine (HR 0.364, 95% CI 0.205-0.646, Wald p<0.001) and CSF3R mutations (HR 2.667, 95% CI 1.276-5.572, Wald p=0.009) were independently associated with RFS. Taken together, we found that induction regimen and CSF3R mutations were independent prognostic factors for AML with CEBPAdm.

Detection of TET2 Mutation in Patients with De Novo Acute Myeloid Leukemia: A Mutation Analysis of 51 Iranian Patients.

Acute myeloid leukemia (AML) is a heterogeneous clonal disease that is considered to originate from hematopoietic stem cells, which are characterized by impaired myelopoiesis and blast proliferation. TET oncogene family member 2 (TET2) mutations are frequent in myeloid malignancies and several studies have assessed the clinical importance of TET2 mutations. However, its frequency ratio has not yet been fully clarified. Method:Hence, our study was aimed to analyze TET2mut in patients with de-novo AML and their association with clinical, molecular characteristics and Nucleophosmin 1 (NPM1), Fms-like tyrosine kinase 3 (FLT3), CCAAT Enhancer Binding Protein Alpha (CEBPA) and Wilms’ tumor protein (WT1) gene expression. Fifty-one Iranian patients were screened by polymerase chain reaction (PCR) and direct sequencing to evaluate TET2 mutations frequency. Results:Out of all patients, 10 mutations in 8 patients (15.6%) were detected and closely associated with higher age and higher hemoglobin levels (p-value <0.05). Although FLT3, NPM1 and CEBPA gene expression did not show any significant correlation with TET2mut, cytogenetically normal acute myeloid leukemia (CN-AML) patients appear to bear TET2mut more frequently with lower platelet counts. Monocyte-lineages leukemia has seemed to be more linked with TET2mut in these patients. Conclusion:Our study suggests the frequency of TET2mut in our study (15.6%) is in line with previous studies and reveals the critical role of TET2 in myeloid transformation, especially in leukemia with monocytic subtypes.

C4BPA: A Novel Co-Regulator of Immunity and Fat Metabolism in the Bovine Mammary Epithelial Cells.

The C4b binding protein alpha (C4BPA) chain primarily engages in critical inflammatory and coagulation processes. The previous transcriptomic analysis showed that C4BPA is a differentially expressed gene in lower and higher fat content mammary gland cell lines from Chinese Holstein. This study aimed to investigate the effects of C4BPA on the inflammation and milk fat synthesis in bMECs by C4BPA knockdown and overexpression. The results highlighted that knockdown of C4BPA in bMECs could suppress the mRNA and protein expression of IL-6, IL-8, IL-12, and the TLR-4/NF-κB pathway-related genes and promote the expression of complement and coagulation cascade pathways related genes as well as TNF-α. Moreover, knockdown of C4BPA expression in bMECs reduced the content of triglyceride (TG) and cholesterol (CHOL) in bMECs, increased NEFA content, reduced mRNA and protein expression of ACSL1 and PPARA, and increased the mRNA and protein expression of ELOVL6, FADS1, and LPL. The bMECs, with the overexpression of C4BPA, showed the enhanced expression of TLR-4/NF-κB linked genes, IL-6, IL-8, IL-12, and mRNA and protein level while reduced mRNA expression of TNF-α, compliment, and coagulation cascade related genes was observed. In bMECs, overexpression of C4BPA enhanced the content of TG and CHOL while reducing NEFA and stimulated the mRNA and protein expression of ACSL1, PPARA, and PPARG genes while inhibiting the mRNA and protein expression of FADS1 and LPL genes. Our results show that C4BPA not only regulates the lipid metabolism through the PPAR signaling pathway in bMECs but also contributes to the inflammatory response through TLR-4/NF-κB and the complement and coagulation cascade pathways. This study, for the first time, provides the primary basis for understanding the role of C4BPA in immunity and fat metabolism, which enables the researchers for innovative direction to investigate genes associated with fat metabolism and immunity. This study also advocates that the breeders must pay attention to such type of genes with multiple functions during animal breeding.

LncRNA CEBPA-AS1 knockdown prevents neuronal apoptosis against oxygen glucose deprivation/reoxygenation by regulating the miR-455/GPER1 axis.

Ischemic stroke (IS) is a common nervous system disease, which is a major cause of disability and death in the world. In present study, we demonstrated a regulatory mechanism of CCAAT/enhancer binding protein-alpha antisense 1 (CEBPA-AS1) in oxygen glucose deprivation/reoxygenation (OGD/R)-induced SH-SY5Y cells, with a focus on neuronal apoptosis. CEBPA-AS1, miR-455, and GPER1 expressions were evaluated by using qRT-PCR and Western blotting. The binding relationship among CEBPA-AS1, miR-455, and GPER1 was determined by a dual luciferase reporter assay. Neuronal viability and apoptosis were examined using MTT and flow cytometry assays, followed by determination of apoptosis-related factors (caspase 3, caspase 8, caspase 9, Bax, and Bcl-2). CEBPA-AS1 and GPER1 levels were upregulated, and miR-455 level was downregulated in the cell model of OGD/R induced. CEBPA-AS1 knockdown increased SH-SY5Y viability and reduced OGD/R-induced apoptosis. CEBPA-AS1 could act as a sponge of miR-455, and CEBPA-AS1 knockdown was found to elevate miR-455 expression. miR-455 overexpression also promoted SH-SY5Y cell viability and rescued them from OGD/R-induced apoptosis by binding to GPER1. GPER1 overexpression or miR-455 inhibition reversed the anti-apoptotic effect of CEBPA-AS1 knockdown. These findings suggest a regulatory network of CEBPA-AS1/miR-455/GPER1 that mediates neuronal cell apoptosis in the OGD model, providing a better understanding of pathogenic mechanisms after IS.

Molecular mechanism of benzo [a] pyrene regulating lipid metabolism via aryl hydrocarbon receptor

BackgroundBenzo [a] pyrene (BaP), a potent carcinogen, has been proved that it has toxicological effects via activation the aryl hydrocarbon receptor (AhR) pathway. AhR can participate in regulating lipogenesis and lipolysis. This topic will verify whether BaP regulates lipid metabolism via AhR.Methods(1) C57BL/6 mice were gavaged with BaP for 12 weeks to detect serum lipids, glucose tolerance, and insulin resistance. Morphological changes in white adipose tissue (WAT) were detected by Hematoxylin and Eosin staining. The mRNA expression levels of adipogenesis-related factors included recombinant human CCAAT/enhancer binding protein alpha (C/EBPα), peroxisome proliferator-activated receptor gamma (PPARγ), and fatty acid binding protein 4 (FABP4) and inflammatory factors included nuclear factor kappa-B (NF-κB), monocyte chemotactic protein-1 (MCP-1), and tumor necrosis factor alpha (TNF-α) were detected using PCR. (2) Neutral lipid content changes in differentiated 3 T3-L1 adipocytes treated with BaP with and w/o AhR inhibitor were detected by Oil red staining. The protein expression levels of adipogenesis- and decomposition-related factors included PPARγ coactivator-1 alpha (PGC-1α), and peroxisome proliferation-activated receptor alpha (PPARα) were detected using western blotting. The mRNA expression levels of inflammatory factors were detected using PCR.Results(1) BaP inhibited body weight gain, decreased lipid content, increased lipid levels, and decreased glucose tolerance and insulin tolerance in mice; (2) BaP reduced the expressions of C/EBPα, PPARγ, FABP4, PGC-1α, and PPARα and increased the expressions of NF-κB, MCP-1, and TNF-α by activating AhR.ConclusionBaP inhibit fat synthesis and oxidation while inducing inflammation by activating AhR, leading to WAT dysfunction and causing metabolic complications.

Identification of a seven-gene prognostic signature using the gene expression profile of osteosarcoma.

BackgroundOsteosarcoma is a malignant bone tumor that typically occurs in adolescents or children under 20 years of age. Developing efficient clinical prognostic markers is crucial for improving the treatment of osteosarcoma patients.MethodsThree datasets related to osteosarcoma were acquired from the Gene Expression Omnibus (GEO) database. A gene signature model was established using the Limma package in the R software, univariate and multivariate survival analyses, and least absolute shrinkage and selection operator (LASSO) algorithms. The gene signature was then verified using external datasets.ResultsFrom the GEO database, 242 differentially expressed genes were identified. A total of 590 unique genes, including 380 genes from the human protein interaction network, were found to be related to biological processes such as bone development and bone cell development. Univariate Cox survival analyses revealed 43 genes that were associated with the prognosis of osteosarcoma patients. A seven-gene signature [retinitis pigmentosa 2 (RP2), polyhydroxybutyrate (PHB), myosin VI (MYO6), mutL homolog 1 (MLH1), Casein kinase 2 beta (CSNK2B), ribosomal protein L37A (RPL37A), and CCAAT/enhancer binding protein alpha (CEBPA)] was developed using LASSO regression analysis and multivariate regression analysis. This gene signature could stratify the prognostic risk of sample cases in the training set, the test set, and the external verification set (P<0.01). The area under the receiver operating characteristic curve for the 5-year survival was higher than 0.72 in both the training and verification groups.ConclusionsIn this study, a seven-gene signature was developed that is highly efficient at predicting the prognosis of patients with osteosarcoma, and therefore, this signature may be a crucial guide in the treatment of these patients.

Mechanism of CCL21/CCL19-CCR7 as a Key Regulatory Signaling Cassette for Non-Small Cell Lung Cancer Patients with Brain Metastasis

To elucidate the molecular mechanism underlying the high metastasis rate of brain for non-small cell lung cancer patients and to establish a comprehensive differential gene profile for non-small cell lung cancer brain metastasis. The differentially expressed genes for non-small cell lung cancer brain metastasis patients were systematically investigated. The enriched biological processes and signaling pathways were explored. The protein-protein interaction network was constructed. Compared with primary non-small cell lung cancer, the brain metastasis patients demonstrated 352 differentially expressed genes with the majority as down-regulated ones (342) and 10 up-regulated ones. Meanwhile, C-C motif chemokine ligand 21, C-C motif chemokine ligand 19, deleted in malignant brain tumors 1 gene, tryptase alpha/beta 1, lactotransferrin, complement component 4 binding protein alpha, C-C motif chemokine ligand 14, C-X-C motif chemokine ligand 9, cluster of differentiation 79A as well as membrane spanning 4-domains A1 were significant and differentially expressed (all down-regulated). The genes of cluster including C-C motif chemokine ligand 21, C-C motif chemokine ligand 19 played a pivotal role in non-small cell lung cancer brain metastasis. The cytokines C-C motif chemokine ligand 19 and C-C motif chemokine ligand 21 as well as their closely associated receptors, C-C chemokine receptor 7 could be detected as high expression levels in non-small cell lung cancer cell line (A549). Inhibition of C-C motif chemokine ligand 19 and C-C motif chemokine ligand 21 as well as C-C chemokine receptor 7 using small interfering ribonucleic acid obviously repressed the cell invasion and metastasis but not for cell proliferation and apoptosis. Meanwhile, the procedure was independent of micro ribonucleic acid lethal-7a regulation. This study hypothesized an innovative signaling axis (C-C motif chemokine ligand 21/C-C motif chemokine ligand 19-C-C chemokine receptor 7) for non-small cell lung cancer brain metastasis and provided a beneficial strategy for better understanding the lung metastases in clinic.

Reconstituting and Purifying Assembly Intermediates of Clathrin Adaptors AP1 and AP2.

Clathrin-coated vesicles mediate membrane cargo transportation from the plasma membrane, the trans-Golgi network, the endosome, and the lysosome. Heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) are bridges that link cargo-loaded membranes to clathrin coats. Assembly of AP2 was previously considered to be spontaneous; however, a recent study found AP2 assembly is a highly orchestrated process controlled by alpha and gamma adaptin binding protein (AAGAB). Evidence shows that AAGAB controls AP1 assembly in a similar way. Insights into the orchestrated assembly process and three-dimensional structures of assembly intermediates are only emerging. Here, we describe a protocol for reconstitution and purification of the complexes containing AAGAB and AP1 or AP2 subunits, known as AP1 and AP2 hemicomplexes. Our purification routinely yields milligrams of pure complexes suitable for structural analysis by X-ray crystallography and electron microscopy.

Oleic acid, independent of insulin, promotes differentiation of goat primary preadipocytes

Context Oleic acid together with insulin is widely used to induce preadipocyte differentiation in humans and mammals, and is also used alone in chicken preadipocytes from abdominal adipose tissue. However, it is not clear whether oleic acid alone promotes goat primary intramuscular preadipocyte differentiation. Aims The aim of the present study was to identify the role of oleic acid in regulating primary preadipocyte differentiation in goats. Methods Three healthy, 7-day-old Jianzhou goats were randomly selected. After slaughter, the longissimus dorsi tissues were collected from each goat under sterile procedures and mixed equally. The primary preadipocytes were then prepared using collagenase type I digestion, and treated with 5 mg/L insulin or different concentrations of oleic acid, including 0 μM, 50 μM, 100 μM, 150 μM and 300 μM. The results were determined using microscopy and Oil Red O staining. The expression of genes related to preadipocyte differentiation were determined by real-time fluorescence quantitative polymerase chain reaction. Results Lower concentrations of oleic acid (50 μM, 100 μM and 150 μM) did not affect the cell morphology and cell growth, whereas 300 μM oleic acid led to severe cytotoxicity compared with the control (0 μM). The treatment of oleic acid (100 μM) enhanced cellular accumulation and lipid droplets deposition significantly, which was not affected by supplementary insulin. In addition, insulin alone treatment did not alter cellular adipogenesis in goat intramuscular preadipocytes. Treatment with oleic acid significantly increased the expression of peroxisome proliferator-activated receptor gamma, CCAAT enhancer-binding protein alpha and fatty acid binding protein 4, and decreased the expression of lipoprotein esterase on Day 2 after cell differentiation, all of which decreased continually on Day 4 and Day 6. Expression of all genes increased significantly on Day 8 after oleic acid treatment in goat intramuscular preadipocytes. Conclusion The results underscore the role of oleic acid independent of insulin in promoting intramuscular preadipocytes in goats, and probably via the control of peroxisome proliferator-activated receptor gamma and CCAAT enhancer-binding protein alpha. Implications These data provide insight into the mechanism underlying preadipocyte differentiation.

Antioxidant and lipid-reducing effects of Rosa rugosa root extract in 3T3-L1 cell.

Rosa rugosa root is traditionally known to be effective in the treatment of diabetes in Korea. R. rugosa root-specific compounds also show antioxidant effects, and could reduce lipid and fat accumulation, however, the underlying mechanism has not been clarified. In present study, the antioxidant and lipid-reducing effects of a 50% ethanol extract of R. rugosa (REE) were investigated differentiated mouse preadipocytes (3T3-L1 cells). REE showed strong radical scavenging activities and inhibitory effect of total lipid accumulation and triglyceride levels in differentiated 3T3-L1 cells. In addition, REE treatment reduced the mRNA and protein levels of adipogenesis and lipogenesis markers. This REE-promoted lipid reduction was caused by downregulation of peroxisome proliferator activated receptor gamma (PPARγ), CCAAT/enhancer binding protein alpha (C/EBPα), and sterol regulatory element binding protein1 (SREBP1c) and down regulation of ERK expression. Overall, these results demonstrate the potential of REE for development of a drug in the medical treatment of lipid-associated disorders.

Genetic Alteration Profiling of Chinese Lung Adenocarcinoma and Its Effect on Targeted Therapy Efficacy.

BackgroundNon-small cell lung cancer (NSCLC) is the most common type of lung cancer and a highly heterogeneous disease with a diversity of phenotypes and genotypes in different populations. The purpose of this study is to investigate oncogenic alterations of lung adenocarcinoma (LUAD) in eastern China and their significance in targeted therapies.MethodsThis study enrolled 101 LUAD patients and used a customized DNA panel to detect molecular alterations. Comprehensive analysis of mutations and clinical application of genomic profiling was carried out.ResultsThe most commonly mutated genes were epidermal growth factor receptor (EGFR) (53%) and tumor protein p53 (TP53) (32%). The less frequently mutated genes were erb-b2 receptor tyrosine kinase 2 (ERBB2) (25%), ATR serine/threonine kinase (ATR) (20%), CCAAT enhancer binding protein alpha (CEBPA) (16%), RB transcriptional corepressor 1 (RB1) (16%), transcription factor 7 like 2 (TCF7L2) (14%), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) (12%) and spectrin alpha, erythrocytic 1 (SPTA1) (12%). Among them, the frequency of ERBB2, ATR, CEBPA, RB1 and TCF7L2 mutations was much higher than that in the databases. Seventy percent of the patients harbored at least one actionable alteration according to the OncoKB evidence. CEBPA mutations affected the efficacy of EGFR-tyrosine kinase inhibitors. ERBB2, CEBPA and TCF7L2 mutated tumors tend to have higher tumor mutation burden (TMB).ConclusionsLUAD patients from eastern China have a unique profile of mutations. The targeted DNA panel is helpful for personalized treatment decision of LUAD patients, and specific mutations may affect the efficacy of targeted therapies.

Genetic Landscape and Clonal Evolution Patterns of CEBPA-Mutated Acute Myeloid Leukaemia Based on Next-Generation Sequencing: A Retrospective Analysis

IntroductionAlthough CCAAT/enhancer binding protein alpha double mutated (CEBPADM) acute myeloid leukemia (AML) is considered a low-risk form of AML according to 2017 ELN recommendations, relapse remains a major cause of death. To assess the broader prognostic impact of other cancer-associated genes, we sequenced a panel of 40 myeloid disorders-related genes in a 25 patient cohort.Methods16 CEBPADM AML diagnosis samples along with 9 CEBPAsingle mutated (SM) were analyzed by targeted next-generation sequencing (Ion Torrent) using Oncomine Myeloid Research Assay. 4 CEBPADM and 2 CEBPASM AML relapse samples were analyzed as well. All patients received intensive chemotherapy according to 2017 ELN recommendations.ResultsWith a median follow-up of 3.2 years (range 0.4-12) 5y OS was 61% and 14% for CEBPADM and CEBPASM patients respectively. Overall, a median of 3 concurrent mutations were present at diagnosis, slightly more in CEBPA SM patients (4 vs 3 in CEBPA DMpatients). The number of somatic mutations influenced both PFS and OS (p = 0.04 and p < 0.01 respectively) independently of CEBPA mutational status. Each single unitary increase in the number of mutations increased the hazard for death of 27.7% (95% CI: -1.4-+65; p = 0.064) while passing from 4 to 5 mutations increased the odds of death by 367%. 5y OS in patients with 5 or more concurrent mutation was 14.3% vs 61.8% in patients with less than 5 co-mutated genes; 5y PFS was 0% vs 38.6%.Mutational landscape of CEBPADMand CEBPASMAML differed significantly, with GATA2, FLT3, DNMT3A and TET2 being the most frequently mutated genes in CEBPADM vs NPM1, FLT3, DNMT3A and WT1 in CEBPASM patients. NPM1(77.8% vs 6.7%; p < 0.01) and ASXL1 mutations (44.4% vs 0%; p = 0.02) were more frequent in CEBPASM patients, confirming they are mutually exclusive with CEBPA biallelic lesions. DNA methylation was the most frequently mutated pathway in biallelic patients (87%) while chromatin/cohesin complex (88%) was the most frequently mutated one in CEBPA monoallelic patients. Mutations of CEBPA, NPM1, DNMT3A, WT1, STAG2, TET2, ASXL1, IDH2, SRSF2, CALR, PRPF8, NF1, TP53, RUNX1 had the highest median variant allele frequency (VAF), more often representing founding mutations. GATA2, IDH1, KRAS, BCOR, MPL, IKZ2F1 and PTPN11 had a more borderline median VAF, variably being clonal or subclonal. Mutations in the 3 tyrosine kinases genes FLT3-ITD , CSF3R, NRAS were only subclonal.Mutations in WT1 and FLT3 were associated with increased relapse rate (p = 0.02 and p = 0.01 respectively), while patients with GATA2 mutations had a strong trend towards better 5y OS (83% vs 32%, p = 0.053).We also identified a not previously described allelic variant in the SH2B3 gene (ATGGGG/A INDEL) with an overall prevalence in our population of 58.3% (46.7% of CEBPA DM and 77.8% of CEBPA SMpatients). Patients with the SH2B3allelic variant had a significantly lower bone marrow blast percentage at diagnosis (p = 0.014) and a strong trend towards a higher number of concurrent mutations (p = 0.056). Moreover, when present at diagnosis, SH2B3 variant persisted at relapse with the same VAF. By real time PCR we demonstrated that this SH2B3 allelic variant leads to a dramatic reduction of the corresponding transcript. This gene encodes for a negative regulator of many crucial signaling pathways (SCF/c-KIT, erythropoietin/JAK2, thrombopoietin/MPL WT/ W515L, JAK2 WT/ V617F, GM-CSFR and FLT3-WT/ITD) of the hematopoietic stem cell.Matched diagnosis and relapse samples analysis suggested different features of clonal evolution: while mutations of SH2B3, WT1, DNMT3A, NPM1, and IDH1 consistently persisted at relapse, CEBPA and GATA2 mutations were unstable during disease course. ZRSR2 and PRPF8 mutations were found in relapse samples only.SummaryOur study offers insights into the genetic landscape of CEBPADM mutated AML as compared to CEBPASM AML, highlighting the contribution of NGS to risk stratification. In fact, our data show that the number and the type of concurrent mutation has a prognostic impact, possibly identifying patients eligible to first line allogeneic stem cell transplantation. We identified an allelic variant of SH2B3 that had never been functionally characterized nor associated with AML and that could represent a marker for genetic instability and a potential new target in AML treatment strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.