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Generation of the stable cell lines is a highly efficient tool in functional studies of certain genes or proteins, where the particular genes or proteins are inducibly expressed. The KRAB-associated protein-1 (KAP1) is an important transcription regulatory protein, which is investigated in several molecular biological studies. In this study, we have aimed to generate a stable cell line for inducing KAP1 expression. The recombinant plasmid pcDNA5/FRT/TO-KAP1 was constructed at first, which was then transfected into Flp-In™T-REx™-HEK293 cells to establish an inducible pcDNA5/FRT/TO-KAP1-HEK293 cell line. The Western blot analysis showed that the protein level of KAP1 is over-expressed in the established stable cell line by doxycycline induction, both dose and time dependently. Thus we have successfully established stable pcDNA5/FRT/TO-KAP1-HEK293 cell line, which can express KAP1 inducibly. This inducible cell line might be very useful for KAP1 functional studies.

The effect of methionine deprivation (methionine stress) on the proliferation, survival, resistance to chemotherapy, and regulation of gene and protein expression in pancreatic tumor lines is examined. Methionine stress prevents successful mitosis and promotes cell cycle arrest and accumulation of cells with multiple micronuclei with decondensed chromatin. Inhibition of mitosis correlates with CDK1 down-regulation and/or inhibition of its function by Tyr(15) phosphorylation or Thr(161) dephosphorylation. Inhibition of cell cycle progression correlates with loss of hyperphosphorylated Rb and up-regulation of p21 via p53 and/or transforming growth factor-beta (TGF-beta) activation depending on p53 status. Although methionine stress-induced toxicity is not solely dependent on p53, the gain in p21 and loss in CDK1 transcription are more enhanced in wild-type p53 tumors. Up-regulation of SMAD7, a TGF-beta signaling inhibitor, suggests that SMAD7 does not restrict the TGF-beta-mediated induction of p21, although it may prevent up-regulation of p27. cDNA oligoarray analysis indicated a pleiotropic response to methionine stress. Cell cycle and mitotic arrest is in agreement with up-regulation of NF2, ETS2, CLU, GADD45alpha, GADD45beta, and GADD45gamma and down-regulation of AURKB, TOP2A, CCNA, CCNB, PRC1, BUB1, NuSAP, IFI16, and BRCA1. Down-regulation of AREG, AGTR1, M-CSF, and EGF, IGF, and VEGF receptors and up-regulation of GNA11 and IGFBP4 signify loss of growth factor support. PIN1, FEN1, and cABL up-regulation and LMNB1, AREG, RhoB, CCNG, TYMS, F3, and MGMT down-regulation suggest that methionine stress sensitizes the tumor cells to DNA-alkylating drugs, 5-fluorouracil, and radiation. Increased sensitivity of pancreatic tumor cell lines to temozolomide is shown under methionine stress conditions and is attributed in part to diminished O(6)-methylguanine-DNA methyltransferase and possibly to inhibition of the cell cycle progression.

Fiber length (FL) and strength (FS) are the core indicators for evaluating cotton fiber quality. The corresponding stages of fiber elongation and secondary wall thickening are of great significance in determining FL and FS formation, respectively. QTL mapping and high-throughput sequencing technology have been applied to dissect the molecular mechanism of fiber development. In this study, 15 cotton chromosome segment substitution lines (CSSLs) with significant differences in FL and FS, together with their recurrent parental Gossypium hirsutum line CCRI45 and donor parent G. barbadense line Hai1, were chosen to conduct RNA-seq on developing fiber samples at 10 days post anthesis (DPA) and 20 DPA. Differentially expressed genes (DEGs) were obtained via pairwise comparisons among all 24 samples (each one with three biological repeats). A total of 969 DEGs related to FL-high, 1285 DEGs to FS-high, and 997 DEGs to FQ-high were identified. The functional enrichment analyses of them indicated that the GO terms of cell wall structure and ROS, carbohydrate, and phenylpropanoid metabolism were significantly enriched, while the GO terms of glucose and polysaccharide biosynthesis, and brassinosteroid and glycosylphosphatidylinositol metabolism could make great contributions to FL and FS formation, respectively. Weighted gene co-expressed network analyses (WGCNA) were separately conducted for analyzing FL and FS traits, and their corresponding hub DEGs were screened in significantly correlated expression modules, such as EXPA8, XTH, and HMA in the fiber elongation and WRKY, TDT, and RAC-like 2 during secondary wall thickening. An integrated analysis of these hub DEGs with previous QTL identification results successfully identified a total of 33 candidate introgressive DEGs with non-synonymous mutations between the Gh and Gb species. A common DEG encoding receptor-like protein kinase 1 was reported to likely participate in fiber secondary cell thickening regulation by brassionsteroid signaling. Such valuable information was conducive to enlightening the developing mechanism of cotton fiber and also provided an abundant gene pool for further molecular breeding. [ABSTRACT FROM AUTHOR]

Objective: To identify differentially expressed long noncoding RNAs (lncRNAs) in condyloma acuminatum (CA) and to explore their probable regulatory mechanisms by establishing coexpression networks. Methods: High-throughput RNA sequencing was performed to assess genome-wide lncRNA expression in CA and paired adjacent mucosal tissue. The expression of candidate lncRNAs and their target genes in larger CA specimens was validated using real-time quantitative reverse transcriptase polymerase chain reaction (RT‒qPCR). Furthermore, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were used for the functional enrichment analysis of these candidate lncRNAs and differential mRNAs. The coexpressed mRNAs of the candidate lncRNAs, calculated by Pearson's correlation coefficient, were also analysed using GO and KEGG analysis. In addition, the interactions among differentially expressed lncRNAs (DElncRNAs)-cis-regulatory transcription factors (cisTFs)-differentially expressed genes (DEGs) were analysed and their network was constructed. Results: A total of 546 lncRNAs and 2553 mRNAs were found to be differentially expressed in CA compared to the paired control. Functional enrichment analysis revealed that the DEGs coexpressed with DElncRNAs were enriched in the terms of cell adhesion and keratinocyte differentiation, and the pathways of ECM-receptor interaction, local adhesion, PI3K/AKT and TGF-ß signaling. We further constructed the network among DElncRNAs-cisTFs-DEGs and found that these 95 DEGs were mainly enriched in GO terms of epithelial development, regulation of transcription or gene expression. Furthermore, the expression of 3 pairs of DElncRNAs and cisTFs, EVX1-AS and HOXA13, HOXA11-AS and EVX1, and DLX6-AS and DLX5, was validated with a larger number of specimens using RT‒qPCR. Conclusion: CA has a specific lncRNA profile, and the differentially expressed lncRNAs play regulatory roles in mRNA expression through cis-acting TFs, which provides insight into their regulatory networks. It will be useful to understand the pathogenesis of CA to provide new directions for the prevention, clinical treatment and efficacy evaluation of CA. [ABSTRACT FROM AUTHOR]

Objective: We aimed to screen novel gene signatures for ovarian cancer (OC) and explore the role of biomarkers in OC via regulating pyroptosis using bioinformatics analysis. Methods: Differentially expressed genes (DEGs) of OC were screened from GSE12470 and GSE16709 datasets. Hub genes were determined from protein–protein interaction networks after bioinformatics analysis. The role of Centromeric protein M (CENPM) in OC was assessed by subcutaneous tumor experiment using hematoxylin–eosin and immunohistochemical staining. Tumor metastasis was evaluated by detecting epithelial-mesenchymal transition-related proteins. The proliferation, migration, and invasion were determined using cell counting kit and transwell assay. Enzyme-linked immunosorbent assay was applied to measure inflammatory factors. The mRNA and protein expression were detected using real-time quantitative PCR and western blot. Results: We determined 9 hub genes (KIFC1, PCLAF, CDCA5, KNTC1, MCM3, OIP5, CENPM, KIF15, and ASF1B) with high prediction value for OC. In SKOV3 and A2780 cells, the expression levels of hub genes were significantly up-regulated, compared with normal ovarian cells. CENPM was selected as a key gene. Knockdown of CENPM suppressed proliferation, migration, and invasion of OC cells. Subcutaneous tumor experiment revealed that CENPM knockdown significantly suppressed tumor growth and metastasis. Additionally, pyroptosis was promoted in OC cells and xenograft tumors after CENPM knockdown. Furthermore, CENPM knockdown activated cGAS-STING pathway and the pathway inhibitor reversed the inhibitory effect of CENPM knockdown on viability, migration, and invasion of OC cells. Conclusion: CENPM was a novel biomarker of OC, and knockdown of CENPM inhibited OC progression by promoting pyroptosis and activating cGAS-STING pathway. [ABSTRACT FROM AUTHOR]

MYB98 is master regulator of the molecular network involved in pollen tube attraction. Until recently, it was unclear how this gene exhibits exclusively synergid cell-specific expression in ovule. Our recent study has established that a 16-bp-long SaeM element is crucial for its synergid cell-specific expression in ovule, and an 84-bp-long fragment harboring SaeM is sufficient to drive the process. In this study, we have developed a workflow to predict functional roles of potential transcription factors (TFs) putatively binding to the promoter region, taking MYB98 promoter as a test subject. After sequential assessment of co-expression pattern, network analysis, and potential master regulator identification, we have proposed a multi-TF model for MYB98 regulation. Our study suggests that ANL2, GT-1, and their respective homologs could be direct regulators of MYB98 and indicates that TCP15, TCP16, FRS9, and HB34 are likely master regulators of the majority of the TFs involved in its regulation. Comprehensive studies in the future are expected to offer more insights into such propositions. Developed workflow can be used while designing similar regulome-related studies for any other species and genes. [ABSTRACT FROM AUTHOR]

Background: Cuproptosis is a novel mode of cell death, which is strongly related to energy metabolism in mitochondria and regulated by protein lipoylation. Currently, the molecular mechanisms of cuproptosis-related genes (CRGs) involved in systemic lupus erythematosus (SLE) largely remained unclear, our study is aimed to explore the mechanisms of cuproptosis and CRGs involved in SLE. Methods: Bulk RNA-seq datasets were collected to display the expressions of CRGs in peripheral blood mononuclear cells (PBMCs) of SLE and healthy individuals, and then ROC analysis was used to establish the diagnostic models of CRGs. Next, the immune infiltration analyses were applied to reveal the difference of immune cells infiltration in LIAS-low and LIAS-high group. Additionally, WGCNA analysis was performed to find the gene modules significantly correlated with the LIAS expression level. We also performed the functional enrichment analyses for LIAS-related gene modules to determine the potential pathways involved in the development of SLE. Finally, scRNA-seq dataset was used to cluster immune cell subsets, reveal the activated pathways, and study cell-cell interactions in LIAS-low and LIAS-high cells. Result: We found CDKN2A was significantly increased and LIAS was significantly decreased in SLE patients compared with healthy individuals. The AUC score showed that LIAS had a great diagnostic value than other CRGs. Additionally, the results of immune infiltration analyses showed that immune cells proportion were diverse in LIAS-low and LIAS-high samples. The gene sets related to LIAS expression level were involved in dephosphorylation of JAK1 by SHP1, phosphorylation of STAT2, cytokine signaling in immune system, expression of interferon-alpha and beta, inhibition of JAK kinase activity by SOCS1/3, and so on. Finally, the results of cell-cell communication showed that CCL- (CCL5 + CCR1) and ANNEXIN- (ANXA1 + FPR1) might play an essential role in the communication network between LIAS-low and LIAS-high cells. Conclusion: Above findings inferred that LIAS-mediated cuproptosis might involve in a comprehensive cellular and molecular mechanism to cause the occurrence and development of SLE. [ABSTRACT FROM AUTHOR]

Background: Atherosclerosis (AS) is a chronic inflammatory disease, as a main cause leading to vascular diseases worldwide. Although increasing studies have focused on macrophages in AS, the exact relating mechanism is still largely unclear. Our study aimed to explore the pathogenic role and diagnostic role of macrophage autophagy related genes (MARGs) in AS. Methods: All datasets were downloaded from Gene Expression Omnibus database and Human Autophagy Database. The differential expression analysis and cross analysis were performed to identify candidate MARGs. GO and KEGG enrichment analyses were conducted to obtain the functional information. Moreover, we analyzed the correlation between target gene and macrophage polarization in AS. The correlation between target gene and plaque instability, different stages of AS were also analyzed. Results: Compared with normal samples, a total of 575 differentially expressed genes (DEGs) were identified in AS samples. A total of 12 overlapped genes were obtained after cross-analysis of the above 575 DEGs and autophagy related genes (ARGs). Then, 10 MARGs were identified in AS samples, which were significantly enriched in 22 KEGG pathways and 61 GO terms. The expression of HSPB8 was significantly down-regulated in atherosclerotic samples compared with normal samples (with largest fold change). Meanwhile, the proportion of M-CSF in low HSPB8 expression AS group was higher than high expression AS group. Furthermore, the expression of HSPB8 was negatively correlated with most inflammatory factors. Conclusion: The downregulation of MARG HSPB8 probably involves in the M2 macrophage polarization in AS samples. HSPB8 is a promising diagnostic marker for AS patients. [ABSTRACT FROM AUTHOR]

Glutathione peroxidase 4 (GPX4) plays an important effect on ferroptosis. Down-regulating the expression of GPX4 mRNA can decrease the content of GPX4. In this work, a gold nanoflare (AuNF) probe loaded with anti-sense sequences targeting for GPX4 mRNA was designed to monitor and down-regulate intracellular GPX4 mRNA using fluorescence imaging in situ and using anti-sense technology. The results revealed that there was a marked difference for the expression of GPX4 mRNA in different cell lines, and the survival rate of cancer cells was not significantly effected when the relative mRNA and protein expression levels of GPX4 was down-regulated by AuNF probes. However, when co-treated with AuNF probes, the low expression of GPX4 strengthened erastin-induced ferroptosis, and this synergy showed a better effect on inhibiting the proliferation of cancer cells. [ABSTRACT FROM AUTHOR]

The late embryonic development abundant protein (LEA) is a family of proteins widely present in the body and related to osmoregulation. Saussurea involucrata is an extremely cold-tolerant plant. In our previous studies, we found that the LEAs gene in Saussurea involucrata has up-regulated expression under low temperature. To evaluate the biological function of SiLEA5 protein under low-temperature stress and its potential in agricultural breeding, we isolated the SiLEA5 gene from Saussurea involucrata, constructed a plant overexpression vector, and transformed tomato. We found that SiLEA5 protein significantly increased the yield of transgenic tomatoes by increasing their photosynthetic capacity, including net photosynthetic rate, stomatal conductance, and intercellular CO2 concentration. Under low-temperature stress, the SiLEA5 protein can regulate proline metabolism and oxidative stress, which confers transgenic tomatos with cold resistance. Thus, our work provided evidence for the role of SiLEA5 protein in low-temperature stress resistance in plants, as well as potential applications in crop breeding and cold stress resistance research. [ABSTRACT FROM AUTHOR]

As a cellular protease, transmembrane serine protease 2 (TMPRSS2) plays roles in various physiological and pathological processes, including cancer and viral entry, such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Herein, we conducted expression, mutation, and prognostic analyses for the TMPRSS2 gene in pan-cancers as well as in COVID-19-infected lung tissues. The results indicate that TMPRSS2 expression was highest in prostate cancer. A high expression of TMPRSS2 was significantly associated with a short overall survival in breast invasive carcinoma (BRCA), sarcoma (SARC), and uveal melanoma (UVM), while a low expression of TMPRSS2 was significantly associated with a short overall survival in lung adenocarcinoma (LUAD), demonstrating TMPRSS2 roles in cancer patient susceptibility and severity. Additionally, TMPRSS2 expression in COVID-19-infected lung tissues was significantly reduced compared to healthy lung tissues, indicating that a low TMPRSS2 expression may result in COVID-19 severity and death. Importantly, TMPRSS2 mutation frequency was significantly higher in prostate adenocarcinoma (PRAD), and the mutant TMPRSS2 pan-cancer group was significantly associated with long overall, progression-free, disease-specific, and disease-free survival rates compared to the wild-type (WT) TMPRSS2 pan-cancer group, demonstrating loss of functional roles due to mutation. Cancer cell lines were treated with small molecules, including cordycepin (CD), adenosine (AD), thymoquinone (TQ), and TQFL12, to mediate TMPRSS2 expression. Notably, CD, AD, TQ, and TQFL12 inhibited TMPRSS2 expression in cancer cell lines, including the PC3 prostate cancer cell line, implying a therapeutic role for preventing COVID-19 in cancer patients. Together, these findings are the first to demonstrate that small molecules, such as CD, AD, TQ, and TQFL12, inhibit TMPRSS2 expression, providing novel therapeutic strategies for preventing COVID-19 and cancers. [ABSTRACT FROM AUTHOR]

Motivation: Studies have shown that classifying cancer subtypes can provide valuable information for a range of cancer research, from aetiology and tumour biology to prognosis and personalized treatment. Current methods usually adopt gene expression data to perform cancer subtype classification. However, cancer samples are scarce, and the high-dimensional features of their gene expression data are too sparse to allow most methods to achieve desirable classification results. Results: In this paper, we propose a deep learning approach by combining a convolutional neural network (CNN) and bidirectional gated recurrent unit (BiGRU): our approach, DCGN, aims to achieve nonlinear dimensionality reduction and learn features to eliminate irrelevant factors in gene expression data. Specifically, DCGN first uses the synthetic minority oversampling technique algorithm to equalize data. The CNN can handle high-dimensional data without stress and extract important local features, and the BiGRU can analyse deep features and retain their important information; the DCGN captures key features by combining both neural networks to overcome the challenges of small sample sizes and sparse, high-dimensional features. In the experiments, we compared the DCGN to seven other cancer subtype classification methods using breast and bladder cancer gene expression datasets. The experimental results show that the DCGN performs better than the other seven methods and can provide more satisfactory classification results. [ABSTRACT FROM AUTHOR]

Epigenetic modifications viz. DNA methylation, histone modifications, and RNA-based alterations play a crucial role in the development of cardiovascular diseases. In this study, we investigated DNA methylation with an aim to reveal the epigenetic etiology of heart failure. Sprague–Dawley rats surviving myocardial infarction developed acute heart failure in 1 week. Genomic DNA methylation changes were profiled by bisulfite sequencing, and gene expression levels were analyzed by RNA-seq in failing and sham-operation hearts. A total of 3480 differentially methylated genes in the promoter regions including transcriptional start site and 1934 transcriptome-altered genes were identified in the defected hearts. Common differential genes were enriched by the gene ontology, Kyoto Encyclopedia of Genes and Genomes pathway, and protein–protein interaction for HF phenotypes. Among these, Mettl11b, HDAC3, HDAC11, ubiquitination-related genes, and snoRNAs are new epigenetic classifiers that had not been reported yet, which may be important regulators in HF. [ABSTRACT FROM AUTHOR]

Aim. Hypertension is a complicated disorder with multifactorial etiology and high heritability. Our previous work has identified L3MBTL4 as a novel susceptibility gene for the development of essential hypertension, accompanied with activation of p38/JNK. Yet, little evidence has been reported whether p38/JNK contributed directly to L3MBTL4-induced vascular remodeling and exploring the potential mechanism of L3MBTL4 in vascular smooth muscle cells (VSMCs). Methods. We evaluated the contribution of L3MBTL4 on proliferation, migration, and phenotype changes of VSMCs and further explored the critical role of p38 and JNK signaling pathway underlying. Results. In L3MBTL4 transgenic rats, we found that the elevated blood pressure, increased left ventricular hypertrophy, and thickened vascular media layer were significantly relieved by both p38 and JNK inhibitors. Meanwhile, increased cell proliferation, advanced cell cycle progression, greater migratory capability, and synthetic phenotype were observed in L3MBTL4 overexpressed VSMCs, which could be blocked by either p38 or JNK inhibitor. Conclusions. Our findings pinpointed that p38 and JNK were required for the proliferation and phenotype changes of VSMCs induced by L3MBTL4 in hypertension. These novel findings yield new insights into the genetic and biological basis of hypertension and are fundamental for further studies to explore the intervention strategies targeting L3MBTL4 and p38/JNK to counteract the progression of hypertension. [ABSTRACT FROM AUTHOR]

BosR, a Fur family member, is essential for the pathogenesis of the Lyme disease pathogen, Borrelia burgdorferi. Unlike typical Fur proteins in which DNA binding represses gene expression, binding of BosR to the rpoS promoter directly activates rpoS transcription in B. burgdorferi. However, virtually nothing is known concerning potential structural features and amino acid residues of BosR that are important for protein function and virulence regulation in B. burgdorferi. Particularly, it remains unknown what structural motifs or residues of BosR coordinate Zn, although previous analyses have indicated that the function of BosR may depend on Zn. To address these information gaps, we herein introduced mutations into four conserved cysteine residues in two putative CXXC motifs of BosR. Our data showed that the ability of BosR to bind Zn was dramatically reduced when the CXXC motifs were mutated. Moreover, we found that the two CXXC motifs contributed to the ability of BosR to form dimers. By using a trans -complementation genetic approach, we additionally demonstrated that both CXXC motifs of BosR were essential for in vivo gene expression regulation. Mutation of any of the four cysteines abolished the transcriptional activation of rpoS. In contrast to wild type BosR, each mutant protein was incapable of binding the rpoS promoter in electrophoretic mobility shift assays. The combined data strongly support that the two CXXC motifs and four cysteines constitute the structural site essential for Zn-coordination, protein dimerization, and the unique regulatory activity of BosR. [ABSTRACT FROM AUTHOR]

Myocardial ischemia/reperfusion (I/R) injury is a key factor in deterioration of myocardial function. The c-Jun NH2-terminal kinase (JNK) activation and the transcription factor nuclear factor-kappaB (NF-κB) nuclear translocation have been found in I/R injury. 6-Gingerol, an important bioactive ingredient of ginger, has been reported to have cardiovascular pharmacological effects. However, the molecular mechanism through which it is beneficial is unclear. In this work, I/R induced the increase in the apoptosis and reactive oxygen species level in AC16 cardiomyocytes. 6-Gingerol administration decreased cardiomyocyte apoptosis and improved oxidative stress indexes. 6-Gingerol administration also inhibited I/R-induced HMGB2 expression upregulation and JNK activation and reduced Cleaved Poly(ADP-ribose) polymerases (PARP) and Caspase-3 expression. HMGB2 treatment mimicked the effect of I/R-induced cell damage, which was reversed by 6-gingerol administration. On the other hand, transcriptional activity of NF-κB was reduced in 6-gingerol treated cells. Thus, overall results indicated that 6-gingerol administration protected I/R-induced cardiomyocytes apoptosis via JNK/NF-κB pathway in the regulation of HMGB2. This work supported the efficacy of 6-gingerol on cardiovascular disease and partially revealed its mechanism, which was helpful for understanding the therapeutic effects of this natural drug. [ABSTRACT FROM AUTHOR]

Reducing sympathetic neurohormone expression is a key therapeutic option in attenuating cardiac remodelling. Present study tested the feasibility of attenuating cardiac remodelling through reducing sympathetic neurohormone level by partial cardiac sympathetic denervation in a rat model of chronic volume overload. Male Sprague‐Dawley rats were randomized into sham group (S, n = 7), aortocaval fistula group (AV, n = 7), and aortocaval fistula with bilateral sympathetic stellate ganglionectomy group (AD, n = 8). After 12 weeks, myocardial protein expression of sympathetic neurohormones, including tyrosine hydroxylase, neuropeptide Y, growth associated protein 43, and protein gene product 9.5, were significantly up‐regulated in AV group compared to S group, and down‐regulated in AD group. Cardiac remodelling was aggravated in AV group compared to S group and attenuated in AD group. The myocardial deposition of extracellular matrix, including collagen I and III, was enhanced in AV group, which was reduced in AD group. Myocardial angiotensin II and aldosterone expressions were significantly up‐regulated in AV group and down‐regulated in AD group. Our results show that bilateral sympathetic stellate ganglionectomy could attenuate cardiac remodelling and fibrosis by down‐regulating sympathetic neurohormones expression in this rat model of chronic volume overload. [ABSTRACT FROM AUTHOR]

Circular RNAs (circRNAs) are molecular sponges that are involved in regulation of multiple types of cancer. The present study aimed to screen and explore the key circRNA/microRNA (miRNA or miR)/mRNA interactions in head and neck squamous cell carcinoma (HNSCC) using bioinformatics. A total of six pairs of cancerous and adjacent healthy tissue were obtained from patients with HNSCC and genome-wide transcriptional sequencing was performed. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway enrichment analyses were performed on differentially expressed genes (DEGs). Moreover, expression levels of DEGs were verified in HNSCC cells and tissues using reverse transcription-quantitative (RT-q)PCR. A molecular regulatory network consisting of three circRNAs, seven miRNAs and seven mRNAs was constructed, resulting in identification of two signaling axes, hsa_circ_0035431/hsa-miR-940/fucosyltransferase 6 (FUT6) and hsa_circ_0035431/hsa-miR-940/cingulin-like 1 (CGNL1). FUT6 and CGNL1 were downregulated in HNSCC compared with adjacent healthy tissue and the expression levels of these genes were associated with tumor stage. Low FUT6 and CGNL1 expression levels were associated with lower overall survival rate and progression-free intervals in HNSCC. RT-qPCR demonstrated that hsa_circ_0035431, FUT6 and CGNL1 were downregulated in HNSCC cells and tissue and hsa-miR-940 was upregulated. Notably, these results were consistent with those obtained using high-throughput sequencing. In conclusion, hsa_circ_0035431 may participate in regulation of FUT6 and CGNL1 expression by sponging hsa-miR-940, thus, impacting the occurrence, development and prognosis of HNSCC. [ABSTRACT FROM AUTHOR]

Several microRNAs (miRNAs) have been suggested as novel biomarkers for diagnosing gastric cancer (GC) at an early stage, but the single-marker strategy may ignore the co-regulatory relationships and lead to low diagnostic specificity. Thus, multi-target modular diagnostic biomarkers are urgently needed. In this study, a Zsummary and NetSVM-based method was used to identify GC-related hub miRNAs and activated modules from clinical miRNA co-expression networks. The NetSVM-based sub-network consisting of the top 20 hub miRNAs reached a high sensitivity and specificity of 0.94 and 0.82. The Zsummary algorithm identified an activated module (miR-486, miR-451, miR-185, and miR-600) which might serve as diagnostic biomarker of GC. Three members of this module were previously suggested as biomarkers of GC and its 24 target genes were significantly enriched in pathways directly related to cancer. The weighted diagnostic ROC AUC of this module was 0.838, and an optimized module unit (miR-451 and miR-185) obtained a higher value of 0.904, both of which were higher than that of individual miRNAs. These hub miRNAs and module have the potential to become robust biomarkers for early diagnosis of GC with further validations. Moreover, such modular analysis may offer valuable insights into multi-target approaches to cancer diagnosis and treatment. [ABSTRACT FROM AUTHOR]

Xiao-Er-An-Shen Decoction (XEASD), a Chinese herbal formula, has been used in clinic for treating insomnia and mental excitement in children and adolescents. However, less of scientific data supports its effectiveness in clinic. Here, we aim to study the role of XEASD in regulating neuron differentiation and antioxidant activity. An HPLC-MS was used to chemically standardize herbal extract of XEASD. The standardized herbal extracts of XEASD (0.3–3.0 mg/mL) were applied onto cultured PC12 cells for 48 hours. The treatment with XEASD extract induced neurite outgrowth of PC12 cells in a dose-dependent manner, having the highest response by ~50% of differentiated cells. Application of XEASD extract dose dependently stimulated expressions of NF68, NF160, and NF200 in cultured PC12 cells. Furthermore, XEASD activated the phosphorylation of cAMP responsive element binding protein on PC12 cells, the effect of which was blocked by H89, a protein kinase A inhibitor. Moreover, XEASD showed free radical scavenging activity and stimulated the transcriptional activity of ARE. These results supported the neurobeneficial effects of XEASD in the induction of neurite outgrowth and protection against oxidative stress and could be useful for neurological diseases, in which neurotrophin deficiency and oxidation insult are involved. [ABSTRACT FROM AUTHOR]

Abstract: Background: Radiofrequency catheter ablation (RFCA) of atrial fibrillation (AF) is known to induce left atrial remodeling and prothrombotic response. Aims: This study aimed to evaluate the effect of remote ischemic preconditioning (RIPC) on left atrial remodeling and prothrombotic response induced by RFCA of AF. Methods: Forty‐four patients with drug‐refractory paroxysmal AF undergoing RFCA were randomized into RIPC (four short episodes of forearm ischemia) and control groups before the procedure. Blood samples were collected before RIPC/sham RIPC, and 24 and 72 hours later after the procedure. The atrial remodeling marker matrix metalloproteinase‐9 (MMP‐9) and endothelial damage marker von Willebrand factor (vWF) were measured using enzyme‐linked immunosorbent assay. Platelet activation was evaluated by flow cytometric measurements of the expression of platelet P‐selectin (CD62P) and active glycoprotein IIb/IIIa receptor (PAC‐1). The early recurrence of atrial fibrillation (ERAF) in the two groups was observed over the subsequent 3 months. Results: RFCA resulted in a significant increase in MMP‐9 and vWF in both the groups, which persisted for 72 hours. However, the expression of CD62P and PAC‐1 showed less increase during RFCA in either group. The RIPC group showed a lower increase in MMP‐9 and vWF compared with the control group. In contrast, no significant differences were found in the trend of expression of CD62P and PAC‐1 during RFCA between the two groups. The AF recurrence in the 3 months after the ablation was significantly lower in the RIPC group than in the control group. Conclusions: RIPC before RFCA for paroxysmal AF significantly reduces the increase in markers of left atrial remodeling and endothelial damage associated with the procedure, and results in a lower ERAF. [ABSTRACT FROM AUTHOR]

To investigate the impact of cytochrome P450 (CYP) genetic polymorphisms CYP2B6, CYP2C19, and CYP3A5 on mRNA expression, cyclophosphamide/4-hydroxycyclophosphamide pharmacokinetics, and treatment outcomes of the R-CHOP regimen (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone) in Chinese patients with non-Hodgkin's lymphoma, 567 cases were investigated. Plasma concentrations of cyclophosphamide/4-hydroxycyclophosphamide were determined using liquid chromatography-tandem mass spectrometry and pharmacokinetic parameters calculated. The frequencies of CYP2B6*1/*29 and CYP2B6*1/*30 were 18 (3.2%) and 30 (5.3%), respectively. Liver samples with CYP2B6*29/*30, CYP2C19*2, CYP2B6*6, or CYP3A5*3 had significantly lower target mRNA expression. Samples with CYP2B6*6 and/or CYP2C19*2 had lower exposure to 4-hydroxycyclophosphamide, whereas those with CYP2B6*1G/*1H had higher exposure. Multivariate model showed that samples with CYP2C19*2 or CYP2B6 785A>raG had worse treatment response than samples with CYP3A5*3. CYP2C19*2, CYP2B6*6, CYP2B6*29, and CYP2B6*30 were protective factors for toxicity in the multivariate model. The best model for gene-gene interactions for treatment response contained CYP2C19*2, CYP2B6 785A>G, and CYP3A5*3 (cross-validation consistency [CVC], 8/10; P = .0035). The best prediction model for grade 2-4 toxicities had CYP2C19*2, CYP2B6 785A>G, and 516 G>T (CVC, 10/10; P < .0001). In previous reports, we were the first to report that the frequency of copy-number variations in CYP2B6 is higher among Chinese than among other ethnic populations. Genetic variations in CYP2B6, CYP2C19, and CYP3A5 dramatically affected the mRNA expression of proteins, the pharmacokinetics of 4-hydroxycyclophosphamide, and the R-CHOP treatment response in Chinese subjects. Patients carrying CYP3A5*3 were more likely to have a better treatment response, whereas patients with CYP2C19*2 or CYP2B6 516G>T, or CYP2B6 785A>G had higher tolerance to treatment. [ABSTRACT FROM AUTHOR]

GADD45A (growth arrest and DNA damage 45 A) is the first stress-inducible gene identified to be a target of p53. However, no studies to date have assessed variants of the GADD45 gene and their potential relationship to tumor susceptibility. We investigated the association of the GADD45A (1506T>C) polymorphism with ovarian cancer development in 258 ovarian cancer patients and 332 age-matched healthy women as controls using sequence analysis. We found a statistically significant difference in the GADD45A (1506T>C) genotype distributions between the case and control groups (TT vs. TC vs. CC, P = 0.0021) and found that variant 1506T>C was significantly associated with an increased risk of ovarian cancer (P<0.001, OR = 1.71, 95% CI [1.28–2.29]). We observed a statistically significant effect between tumor histology (P = 0.032) and CA125 status (P = 0.021). Carrying the C allele (TC+CC) was associated with an increased risk of positive CA125 (OR = 3.20, 95% CI [1.15–8.71). Carrying the T allele (TT+TC) showed a significant correlation with both higher GADD45A mRNA expression and longer ovarian cancer RFS (relapse-free survival) and OS (overall survival). We are the first group to demonstrate that the GADD45A (1506T>C) polymorphism is associated with ovarian cancer susceptibility and prognosis. These data suggest that GADD45A (1506T>C) is a new tumor susceptibility gene and could be a useful molecular marker for assessing ovarian cancer risk and for predicting ovarian cancer patient prognosis. [ABSTRACT FROM AUTHOR]

This study was designed to investigate the effect of melatonin on the expression of aortic inflammatory cytokines and its underlying mechanisms in rats. Melatonin deficiency rats (Px, N = 16) were created by pinealectomy and were fed with normal diet for 16 weeks after the surgery, and compared with sham-operated rats (Con, N = 14). Serum lipid profile, glucose metabolism parameters, serum oxidative stress and inflammatory biomarkers were evaluated. The expression of inflammatory cytokines in the aorta endothelia was analyzed. To evaluate the signal transduction pathways of melatonin on the expression of cytokines, rat aortic endothelial cell lines (RAECs) were treated with melatonin, and their protein expressions of inflammatory cytokines and phosphorylation levels of relevant signal pathways were detected. At the 16th week after surgery in Px rats, their serum triglyceride, very low density lipoprotein cholesterol, free fatty acid and glucose levels were prominently elevated (all P < 0.05); serum oxidative stress biomarker malondialdehyde, serum inflammatory biomarkers oxidized low-density lipoprotein, tumor necrosis factor-α, interleukin-6 and C reactive protein were also significantly increased. Meanwhile, the expression of inflammatory cytokines: monocyte chemotactic protein-1 (MCP-1), vascular adhesion molecule 1 (VCAM-1) and matrix metalloproteinase-9 (MMP-9) of the aorta endothelia in Px rats were significantly up-regulated (all P < 0.05). In vitro, melatonin significantly decreased the expression of MCP-1, VCAM-1 and MMP-9 proteins, along with the suppression of phosphorylation levels of nuclear factor κB (NF-κB)/P65 and p38 mitogen-activated protein kinase (P38-MAPK) in RAECs. Melatonin deficiency elevates the serum inflammatory biomarkers and increases aortic inflammatory responses. Melatonin regulates these inflammatory responses by NF-κB and P38-MAPK involved pathways. [ABSTRACT FROM AUTHOR]

Objective: This study was to explore the role of EFEMP1 in ovarian tumor progression and its relationship with prognosis of ovarian carcinoma. Methods: EFEMP1 mRNA and protein expressions in normal ovarian tissue, ovarian tumor, high invasive subclones and low invasive subclones were evaluated by immunohistochemistry and real time RT-PCR. Serum EFEMP1 levels in patients with ovarian tumor were measured by ELISA assay. To assess the angiogenic properties of EFEMP1, VEGF and tumor microvessel density were analyzed in ovarian carcinoma by immunohistochemistry. Results: EFEMP1 expression was up-regulated in ovarian carcinoma, positively correlated with MVD and VEGF, and its overexpression and high serum levels were significantly associated with high stage, low differentiation, lymph node metastasis and poor prognosis of ovarian cancer. EFEMP1 expression was also found to be over-expressed in the highly invasive subclones compared with the low invasive subclones. Conclusion: EFEMP1 is a newly identified gene over-expressed in ovarian cancer, associated with poor clinicopathologic features and promotes angiogenesis. This study shows that EFEMP1 may serve as a new prognostic factor and a therapeutic target for patients with ovarian cancer in the future. [ABSTRACT FROM AUTHOR]

Chelator-assisted phytoremediation is an efficacious method for promoting the removal efficiency of heavy metals (HMs). The effects of N, N-bis(carboxymethyl)-L-glutamic acid (GLDA) and polyaspartic acid (PASP) on Cd uptake and pyrene removal by Solanum nigrum L. (S. nigrum) were compared in this study. Using GLDA or PASP, the removal efficiency of pyrene was over 98%. And PASP observably raised the accumulation and transport of Cd by S. nigrum compared with GLDA. Meanwhile, both GLDA and PASP markedly increased soil dehydrogenase activities (DHA) and microbial activities. DHA and microbial activities in the PASP treatment group were 1.05 and 1.06 folds of those in the GLDA treatment group, respectively. Transcriptome analysis revealed that 1206 and 1684 differentially expressed genes (DEGs) were recognized in the GLDA treatment group and PASP treatment group, respectively. Most of the DEGs found in the PASP treatment group were involved in the metabolism of carbohydrates, the biosynthesis of brassinosteroid and flavonoid, and they were up-regulated. The DEGs related to Cd transport were screened, and ABCG3 , ABCC4 , ABCG9 and Nramp5 were found to be relevant with the reduction of Cd stress in S. nigrum by PASP. Furthermore, with PASP treated, transcription factors (TFs) related to HMs such as WRKY , bHLH , AP2/ERF , MYB were down-regulated, while more MYB and bZIP TFs were up-regulated. These TFs associated with plant stress resistance would work together to induce oxidative stress. The above results indicated that PASP was more conducive for phytoremediation of Cd-pyrene co-contaminated soil than GLDA. [Display omitted] • Polyaspartic acid was more beneficial to improve phytoextraction of Cd. • Enhanced Cd transport by chelators was closely related to transcriptional regulation. • Polyaspartic acid regulated brassinosteroid, flavone and flavonol biosynthesis. • Chelators regulated transporter proteins and transcription factors gene expressions. [ABSTRACT FROM AUTHOR]

Abstract: Myostatin (MSTN), belonging to transforming growth factor (TGF) β superfamily, is a negative regulator of muscular development in mammals and fish. The expression of MSTN can be regulated by hormone and environmental factors. Sex steroids, androgen and estrogen can affect sex differentiation and also can affect muscle development. However, few documents are concerned about the effects of sex steroids on MSTN expression in fish. Here, we report the cloning and expressional analyses of MSTN in rare minnow (Gobiocypris rarus), an emerging model fish in China. The MSTN cDNA of rare minnow that has an open reading frame of 1128 base pairs (bp) encodes a protein of 375 amino acids belonging to fish MSTN1 with typical structures like a prodomain, a mature MSTN domain, a conservative hydrolytic site (RIRR) and 9 conservative cysteine residues in mature MSTN. MSTN was detectable in all the examined tissues by RT-PCR that indicated the ubiquitous expression of MSTN in adult tissues. MSTN was detectable from zygote till hatching showed the maternal deposition of MSTN mRNA from oocytes and continuous expression during embryogenesis. Treatment of rare minnow with 1μg/L 17α-methyl testosterone (MT) or 17α-ethynyl estradiol (EE2) significantly impaired the body growth and increased the expression of MSTN mRNA detected by quantitative real time PCR. However, low dosage of MT (10ng/L) had no significant effects on body growth and MSTN expression. The results suggest that the effects of exogenous sex steroids on growth can be mediated through MSTN. It is also suggested that low concentration of sex steroid (less than10ng/L) may have little effect on MSTN expression. [Copyright &y& Elsevier]

Objective Epilepsy-aphasia spectrum (EAS) are a group of epilepsy syndromes denoting an association between epilepsy, speech disorders and the EEG signature of centrotemporal spikes. Mutations in the GRIN2A gene, encoding the NMDA glutamate receptor α2 subunit were reported in focal epilepsy with speech disorder. We aimed to explore the role of GRIN2A mutations in patients with centrotemporal spikes related epileptic syndromes in a Chinese cohort. Methods Patients with Landau-Kleffner syndrome (LKS), epileptic encephalopathy with continuous spike-and-wave during sleep (ECSWS), atypical benign partial epilepsy (ABPE), and benign epilepsy with centrotemporal spikes (BECTS) were recruited. GRIN2A mutation screening was performed using PCR and Sanger sequencing. Results 122 patients, including 9 LKS, 26 ECSWS, 42 ABPE and 45 BECTS were enrolled. The mean age of seizure or aphasia onset was 5 years, ranging from 10 months to 11 years. Heterozygous GRIN2A mutations were detected in four patients (G760S, D1385Y, C455Y and C231R) GRIN2A mutation was found in 11.1% (1 out of 9 cases) of LKS, and in 7.1% (3 out of 42 cases) of ABPE, but in none with ECSWS and BECTS. No GRIN2A mutation was found in patients with a family history of febrile seizures or epilepsy. Conclusion GRIN2A mutation is a genetic cause in less than 11% patients with LKS or ABPE. GRIN2A gene is a rare causative gene in Chinese patients with EAS, suggesting the possibility of other gene involved in the pathogenesis. [ABSTRACT FROM AUTHOR]

The corticotropin releasing factor (CRF) plays a central role in regulating the activities of hypothalamic-pituitary-adrenal (HPA) axis in the presence of a variety of stressful stimuli via binding to its type 1 receptors (CRFR1). Despite that many peptidic or non-peptidic antagonists of CRFR1 have been developed to serve as therapeutic tools to CRF-related pathologies, none of them have been utilized clinically. Targeting the extracellular domain 1 (EC1) of CRFR1, the CRF-binding site, represents a new strategy to inhibit the function of the receptor. However, no such agents have been identified up to now. Herein, by using an 87-amino acid fragment corresponding to the EC1 region as the bait, we screened the binding polypeptides from a phage display (Ph.D.-12) peptide library. After 3-round biopanning, positive clones were selected and the polypeptides carried by them were identified. 5 polypeptides were found to bind with the target specifically. Among them, the P7 exhibited the highest affinity. By evaluating the cAMP accumulation in the CRFR1 or CRFR2-expressing HEK293 cells, we demonstrated that P7 blocking the function of CRFR1, but not CRFR2. In addition, we also found that P7 and CRF act on CRFR1 competitively. Taken together, we reveal that P7, a novel polypeptide identified from phage display library, inhibits the function of CRFR1 effectively and specifically by binding at its EC1 domain. The new polypeptide might provide a promising agent for diagnostic or therapeutic utilities in CRF-related disorders. [ABSTRACT FROM AUTHOR]

Abstract: C-reactive protein (CRP) was reported to be a predictor for left ventricular (LV) remodeling. Matrix metalloproteinase (MMP)-10 participates in the LV remodeling process. However, the intrinsic relationship between CRP and MMP-10 in cardiomyocytes remains unclear. The purpose of this study is to observe whether CRP may promote MMP-10 expression, and if so, to clarify signaling pathways to be involved in CRP-induced MMP-10 expression in cardiomyocytes. We observed in cultured cardiomyocytes that CRP at a dose of 5μg/ml increased MMP-10 expression and activity in a time-dependent manner, as measured by real-time polymerase chain reaction (PCR), western blots, and casein zymography analysis. We hypothesized that signal pathways of mitogen-activated protein kinases (MAPKs) and Janus kinases (JAKs)/signal transducers and activators of transcription (STATs) might be involved in CRP-induced MMP-10 expression. Our results showed that CRP markedly activated c-Raf/MEK/ERK and JAK1/ERK signaling pathways but not JAK1/STAT3 signaling pathway by using the phosphor-specific antibodies against these pathways, and blockages of c-Raf/MEK/ERK and JAK1/ERK signaling pathways by the specific ERK1/2 inhibitor U0126 and JAK1 inhibitor piceatannol could significantly decrease CRP-induced MMP-10 expression. In addition, we demonstrated that the DNA binding sites of AP-1 and STAT3 in the nucleus of cardiomyocytes mediated CRP-induced MMP-10 expression. In conclusion, we demonstrated that CRP promoted MMP-10 expression and activity in cardiomyocytes, and clarified that c-Raf/MEK/ERK and JAK1/ERK signaling pathways were involved in MMP-10 expression regulation via activation of DNA binding sites for AP-1 and STAT3 in cardiomyocytes. Our findings suggest that CRP acts as a predictor for LV remodeling might be associated with its promotion effect on MMP-10 expression and activity. [Copyright &y& Elsevier]

Abstract: Gobiocypris rarus is an emerging fish model for aquatic toxicology in China as it is sensitive to environmental hormone disruptors. Exogenous sex steroids can affect sex differentiation and the expression of sex-related genes. Foxl2, a member of forkhead-box transcription factor family, is the key gene for ovary development and its mutation causes the blepharophimosis ptosis epicanthus inversus syndrome in human. We find that two foxl2 genes exist in fish genome, one is foxl2, and the other is foxl2b. Here, we reported the isolation and expression of foxl2 in G. rarus. G. rarus foxl2 cDNA is 1700bp in length with a 921bp of open reading frame encoding 306 amino acids containing the typical FH-domain. Semi-quantitative RT-PCR revealed its predominant expression in the eye, brain, gill and gonads. Moreover, the expression level in the ovary was significantly higher than that in the testis. Quantitative RT-PCR showed that foxl2 was up regulated after treatment with estradiol and was down regulated with 2-methyl-testosterone. These results suggested that Foxl2 plays an important role in female development of G. rarus, foxl2 mRNA expression is regulated by downstream sex hormones, and foxl2 can be used as a molecular indicator monitoring the environmental endocrine disruptors. [Copyright &y& Elsevier]