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Sugarcane mosaic disease poses a serious threat to the sugarcane industry. Many studies have aimed to unravel the molecular mechanism related to sugarcane and mosaic virus interaction. Quantitative reverse transcription (qRT)-PCR in combination with suitable internal reference genes has been widely used for gene expression analysis. In this study, the expression of 33 candidate reference genes and 12 candidate reference miRNAs was analyzed for first time in the leaf samples of three sugarcane genotype infected with Sorghum mosaic virus using the geNorm, NormFinder and deltaCt (deltaCq) algorithms. A comparison of the expression of eIF-4E and three virus-derived siRNA (vsiR9230S, vsiR9058A and miR16) with the normalized unstable and stable reference genes or miRNA indicated that PP2A and miR159 constituted the best single reference gene/miRNA under SrMV infection. We also suggested that both CUL + CAC and miR171+ miR1520 could serve as the most suitable reference gene/miRNA combination. The use of reliable reference genes and miRNAs should improve the accuracy of gene expression analysis in sugarcane leaves under SrMV stress.

BACKGROUND Hepatocellular carcinoma (HCC) causes a heavy disease burden worldwide. Cell division cycle 45 (Cdc45) and its encoding gene (CDC45) have been studied for a long time, but their expression patterns and roles in liver carcinogenesis and advanced HCC deterioration are still incompletely understood. This study integrated tissue microarray and bioinformatics analyses to explore the expression and clinical value of CDC45 and Cdc45 in HCC. MATERIAL AND METHODS In HCC, the expression and relationships with clinic-pathological parameters of CDC45 and Cdc45 were investigated by integrating the RNA-sequencing data, downloaded from The Cancer Genome Atlas and Oncomine databases, and tissue microarray with immunohistochemistry staining. Co-expressed genes and genetic alterations of CDC45 separately obtained from Oncomine and cBioPortal databases were identified to shed light on the potential mechanisms of CDC45 in HCC. RESULTS CDC45 and Cdc45 were both overexpressed in HCC tissues, and the CDC45 level progressively increased from stage I to III. The survival outcomes of the group with high CDC45 expression were significantly worse compared with the group with low expression. Amplification and deep deletion were 2 major significant alteration types in HCC patients, and the outcomes were worse in patients with altered versus unaltered CDC45. NUDT1, E2F1, CCNE2, MCM5, and CENPM were identified as the most significantly co-expressed genes. CONCLUSIONS CDC45 and Cdc45 were both upregulated in HCC, and increased expression levels and genetic alternations of CDC45 were correlated with worse prognosis in HCC patients. CDC45 may promote HCC by co-expressing with NUDT1, E2F1, CCNE2, MCM5, and CENPM.

Background Ten-eleven translocation (Tet) methyl-cytosine dioxygenases (including Tet1/2/3)-mediated 5mC oxidation and DNA demethylation play important roles in embryonic development and adult tissue homeostasis. The expression of Tet2 and Tet3 genes are relatively abundant in the adult murine kidneys while Tet1 gene is expressed at a low level. Although Tet3 has been shown to suppress kidney fibrosis, the role of Tet2 in kidney physiology as well as renal ischemia-reperfusion (IR) injury is still largely unknown. Results Tet2−/− mice displayed normal kidney morphology and renal function as WT mice while the expression of genes associated with tight junction and adherens junction was impaired. At 24 h post-renal IR, Tet2−/− mice showed higher SCr and BUN levels, more severe tubular damage, and elevated expression of Kim1 and Ngal genes in the kidney in comparison with WT mice. Moreover, the transcriptomic analysis revealed augmented inflammatory response in the kidneys of Tet2−/− mice. Conclusions Tet2 is dispensable for kidney development and function at baseline condition while protects against renal IR injury possibly through repressing inflammatory response. Our findings suggest that Tet2 may be a potential target for the intervention of IR-induced acute kidney injury (AKI).

Ten-Eleven Translocation (TET) proteins oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytonsie (5hmC). Our recent work found a decline in global 5hmC level in mouse kidney insulted by ischemia reperfusion (IR). However, the genomic distribution of 5hmC in mouse kidney and its relationship with gene expression remain elusive. Here, we profiled the DNA hydroxymethylome of mouse kidney by hMeDIP-seq and revealed that 5hmC is enriched in genic regions but depleted from intergenic regions. Correlation analyses showed that 5hmC enrichment in gene body is positively associated with gene expression level in mouse kidney. Moreover, IR injury-associated genes (both up- and down-regulated genes during renal IR injury) in mouse kidney exhibit significantly higher 5hmC enrichment in their gene body regions when compared to those un-changed genes. Collectively, our study not only provides the first DNA hydroxymethylome of kidney tissues but also suggests that DNA hyper-hydroxymethylation in gene body may be a novel epigenetic marker of IR injury-associated genes.

Thyroid carcinoma (THCA) is one of the most frequent endocrine cancers and has increasing morbidity. Annexin A2 (ANXA2) has been found to be highly expressed in various cancers; however, its expression level and potential mechanism in THCA remain unknown. This study investigated the clinicopathological value and primary molecular machinery of ANXA2 in THCA.Public RNA-sequencing and microarray data were obtained and analyzed with ANXA2 expression in THCA and corresponding non-cancerous thyroid tissue. A Pearson correlation coefficient calculation was used for the acquisition of ANXA2 coexpressed genes, while edgR, limma, and Robust Rank Aggregation were employed for differentially expressed gene (DEG) in THCA. The probable mechanism of ANXA2 in THCA was predicted by gene ontology and pathway enrichment. A dual-luciferase reporter assay was employed to confirm the targeting relationships between ANXA2 and its predicted microRNA (miRNA).Expression of ANXA2 was significantly upregulated in THCA tissues with a summarized standardized mean difference of 1.09 (P0.0001) based on 992 THCA cases and 589 cases of normal thyroid tissue. Expression of ANXA2 was related to pathologic stage. Subsequently, 1442 genes were obtained when overlapping 4542 ANXA2 coexpressed genes with 2248 DEGs in THCA; these genes were mostly enriched in pathways of extracellular matrix-receptor interaction, cell adhesion molecules, and complement and coagulation cascades. MiR-23b-3p was confirmed to target ANXA2 by dual-luciferase reporter assay.Upregulated expression of ANXA2 may promote the malignant biological behavior of THCA by affecting the involving pathways or being targeted by miR-23b-3p.

Background Sugarcane (Saccharum L. plant) is an important crop for sugar and bio-energy production around the world. Among sugarcane diseases, smut caused by Sporisorium scitamineum is one of the major fungal diseases causing severe losses to the sugarcane industry. The use of PCR reference genes is essential to the normalization of data on gene expression involving the sugarcane-S. scitamineum interaction system; however, no report that addresses criteria in selecting these reference genes has been published to date. Results In this study, 10 sugarcane genes and eight S. scitamineum genes were selected as candidate PCR reference genes in the sugarcane-S. scitamineum interaction system. The stability and reliability of these 18 candidate genes were analyzed in smut-resistant (NCo376) and -susceptible (YC71–374) genotypes using the statistical algorithms geNorm, NormFinder, BestKeeper, and deltaCt method. Subsequently, the relative expression levels of the sugarcane chitinase I-3 gene and S. scitamineum chorismate mutase gene were determined to validate the applicability of these sugarcane and S. scitamineum PCR reference genes, respectively. We finally found that the acyl-CoA dehydrogenase gene (ACAD), serine/arginine repetitive matrix protein 1 gene (SARMp1), or their combination (ACAD + SARMp1) could be utilized as the most suitable reference genes for normalization of sugarcane gene expression in sugarcane bud tissues after S. scitamineum infection. Similarly, the inosine 5′-monophosphate dehydrogenase gene (S10), the SEC65-signal recognition particle subunit gene (S11), or their combination (S10 + S11) were suitable for normalization of S. scitamineum gene expression in sugarcane bud tissues. Conclusions The PCR reference genes ACAD, SARMp1, S10, and S11 may be employed in gene transcriptional studies involving the sugarcane-S. scitamineum interaction system. Electronic supplementary material The online version of this article (10.1186/s12864-018-4854-z) contains supplementary material, which is available to authorized users.

Growth arrest-specific gene 7 (Gas7) is preferentially expressed in terminally differentiated brain cells and plays a crucial role during neuronal development and neurite outgrowth. Apart from that, Gas7 was found to be abundantly expressed in immune cells like murine macrophage without knowing the actual roles in immune reaction. By using the Illumina microarray analysis, we observed a clear induction of Gas7 but no other Gas family members in murine M1-polarized alveolar macrophage, which was further confirmed by RT-qPCR, Western blotting, and immunostaining analysis, suggesting a likelihood that Gas7 may participate in murine alveolar macrophage polarization. Moreover, we found that the upregulation of Gas7 in M1-polarized alveolar macrophage was almost fully blocked by IKK selective inhibitor BMS, which links Gas7 induction to nuclear factor kappa beta (NF-κB) signaling activation. Interestingly, we found that Gas7 knockdown by small interfering RNA transfection did not affect the pro-inflammatory cytokine gene Tnf and Ilb expression, whereas the expressions of canonic M1 marker gene Nos2 and other M1-dependent genes Il12b, Il6, Cxcl1, Cxcl2, and Cxcl9 were found to be reduced. Furthermore, Gas7-related M1 gene expression in alveolar macrophage was not dependent on NF-κB and STAT1 pathway. Our results demonstrate that Gas7 is potentially involved in regulation of murine M1 alveolar macrophage polarization.Gas7 was induced in LPS/IFNγ mediated M1 polarization. Gas7 are induced during time course of M1 polarization. Gas7 upregulation was dependent on NF-κB pathway in M1 polarized AMs. Gas7 knockdown reduced the M1 markers gene expression in M1 polarized AMs.

Sugarcane smut, caused by the fungus Sporisorium scitamineum, is one of the main diseases that affect sugarcane worldwide. In the present study, the cDNA-SRAP technique was used to identify genes that are likely to be involved in the response of sugarcane to S. scitamineum infection. In total, 21 bands with significant differential expression during cDNA-SRAP analysis were cloned and sequenced. Real-time qPCR confirmation demonstrated that expression of 19 of these 21 differential bands was consistent with the expression observed during cDNA-SRAP analysis, with a deduced false positive rate of 9.5%. Sequence alignment indicated that 18 of 19 differentially expressed genes showed homologies from 19% to 100% to certain genes in GenBank, including the following genes: topoisomerase (EU048780), ethylene insensitive (EU048778), and tetraspanin (EU048770). A real-time qPCR assay showed that during 0-72 h after pathogen infection, expression of the topoisomerase and the ethylene insensitive genes was upregulated, whereas expression of the tetraspanin gene was downregulated, identical to the expression patterns observed under salicylic acid treatment. Therefore, all three genes are thought to play a role during S. scitamineum challenge, but with different functions. To our knowledge, this is the first report on the application of cDNA-SRAP in differential gene expression analysis of sugarcane during a sugarcane-S. scitamineum interaction. The results obtained also contribute to a better understanding of the molecular mechanisms associated with sugarcane-S. scitamineum interactions.

Background Influenza virus infection is a major threat to human health. Small interfering RNA (siRNA) is a promising approach for the prevention and treatment of viral infections. In this study, we constructed a series of DNA vector-based short hairpin RNAs (shRNAs) that target various genes of the influenza A virus using the polymerase III U6-RNA promoter to prevent influenza virus infection in vitro and in a mouse model. Results Three sets of DNA vector-based shRNA, two targeting genes encoding the polymerase acidic protein (PA) and one targeting polymerase basic protein 2 (PB2), efficiently inhibited the replication of influenza virus A/WSN/33(H1N1) in vitro. We also successfully prevented influenza virus A/WSN/33(H1N1) infection in a C57BL/6 mouse model by intratracheal delivery of anti-PB2 shRNA. Conclusions Our findings suggest that the PB2-targeting shRNA plasmid showed potential for use as an RNAi-based therapeutic for influenza virus infection.

Background Sugarcane smut caused by Sporisorium scitamineum leads to a significant reduction in cane yield and sucrose content. MicroRNAs (miRNAs) play an important role in regulating plant responses to biotic stress. The present study was the first to use two sugarcane genotypes, YA05-179 (smut-resistant) and ROC22 (smut-susceptible), to identify differentially expressed miRNAs in sugarcane challenged with S. scitamineum by using high-throughput sequencing. Results The predicted target gene number corresponding to known differentially expressed miRNAs in YA05-179 was less than that in ROC22, however most of them were in common. Expression of differential miRNAs under S. scitamineum challenge was mostly downregulated, with similar trends in the two varieties. Gene ontology (GO) analysis showed that the target gene classification of known miRNAs was similar to that of the newly identified miRNAs. These were mainly associated with cellular processes and metabolic processes in the biological process category, as well as combination and catalytic activity in the molecular function category. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed that these predicted target genes involved in a series of physiological and biochemical pathways or disease resistance-related physiological metabolism and signal transduction pathways, suggesting that the molecular interaction mechanism between sugarcane and S. scitamineum was a complex network system. These findings also showed certain predicted target genes of miR5671, miR5054, miR5783, miR5221, and miR6478 play roles in the mitogen-activated protein kinase (MAPK) signaling pathway, plant hormone signal transduction, and plant-pathogen interaction. Quantitative real-time PCR (qRT-PCR) analysis showed that majority of the known miRNAs and its predicted target genes followed a negatively regulated mode. Seven out of eight predicted target genes showed identical expression after 12 h treatment and reached the highest degree of matching at 48 h, indicating that the regulatory role of miRNAs on the target genes in sugarcane was maximized at 48 h after S. scitamineum challenge. Conclusions Taken together, our findings serve as evidence for the association of miRNA expression with the molecular mechanism underlying the pathogenesis of sugarcane smut, particularly on the significance of miRNA levels in relation to the cultivation of smut-resistant sugarcane varieties. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-3716-4) contains supplementary material, which is available to authorized users.

Ischemia reperfusion (IR) is a frequent pathological injury to the perioperative patients. The molecular mechanism underlying IR injury is still not well characterized. In this study, we investigated the effect of IR injury on DNA hydroxymethylation in mouse kidney. Dot blot and immunochemistry analysis showed that the global level of 5-hydroxymethylcytosine (5hmC) was reduced in mouse kidney insulted by IR; however, the 5-methylcytosine (5mC) level had no significant change. hMeDIP-qPCR validated that IR injury also decreased the 5hmC enrichment at promoter regions of Cxcl10 and Ifngr2 genes. RT-qPCR analysis revealed that the mRNA levels of Cxcl10 and Ifngr2 increased in IR-injured kidney. In addition, mRNA expression of both Tet1 and Tet2 but not Tet3 was dramatically downregulated in IR-injured kidney. Taken together, our data provided the first evidence that IR injury influences DNA hydroxymethylation and Tet gene expression in mouse kidney, which may contribute to the regulation of gene transcription during renal IR injury.

Since bacterial invasion into host cells is a critical step in the infection process and the predominance of multiple-antibiotic-resistant Klebsiella (K.) pneumoniae strains, using molecular agents to interfere with K. pneumoniae invasion is an attractive approach for the prevention of infection and suppress the immune inflammatory response. In previous studies by our group, high-mobility group nucleosome-binding domain 2 (HMGN2) protein was shown to exhibit anti-bacterial activity in vitro. The objective of the present study was to investigate the effects of HMGN2 protein on the invasion of K. pneumoniae 03183 in vivo. The results showed that pre-treatment with 128 µg/ml HMGN2 significantly reduced K. pneumoniae 03183 invasion into mouse lungs and increased the mRNA expression of CXCL1 and LCN2 within 2 h. Immunohistochemical staining showed that F-actin expression was significantly decreased, and fluorescence microscopy and western blot analysis further demonstrated that HMGN2 significantly blocked K. pneumoniae 03183-induced actin polymerization. These changes implied that HMGN2 may provide protection against K. pneumoniae 03183 infection in vivo.

Heavy metal contamination has been a significant problem limiting agricultural development, but sugarcane has recently emerged as a valuable phytoremediator. To better understand the molecular mechanism behind sugarcane's metal tolerance, it is necessary to analyze the expression of a novel gene(s) by qRT-PCR. Importantly, introducing internal reference gene(s) should be selected based upon gene stable expression, the inclusion of which could enhance both the accuracy and reliability of this method. In this study, 13 candidate genes were selected and evaluated stability of each genes. The results derived by statistical algorithms were then validated by normalizing the expression of metal related gene ScMTP (GenBank Accession No. KP864146), ScMT2-1-3 (GenBank Accession No. JQ627644), ScMPP (GenBank Accession No. CA267392.1) and ScHMA1 (GenBank Accession No. CA156665.1). Collectively, our qRT-PCR results indicated that in heavy metal-exposed sugarcane, APRT was the better single internal control in expression quantification. Moreover, the combination of CAC + CUL provide for a more accurate normalization for gene transcript profiles under these same conditions. The gene expression quantification that included APRT and CAC + CUL suggested that ScMTP had a differential expression pattern, ScMT2-1-3 and ScMPP were slightly inhibited, and ScHMA1 had minimal induction of expression in response to Cd2+ and Cu2+ stresses in sugarcane. Taken together, the suitable reference genes identified in this study will benefit future work aimed at the sugarcane gene functional characterization.

Objectives This study was carried out to reveal functions and mechanisms of MEK/ERK and p38 pathways in chondrogenesis of rat bone marrow mesenchymal stem cells (BMSCs), and to investigate further any interactions between the mitogen-activated protein kinase (MAPK) and transforming growth factor-beta1 (TGF-beta1)/Smads pathway in the process. Materials and methods Chondrogenic differentiation of rat BMSCs was initiated in micromass culture, in the presence of TGF-beta1, for 2 weeks. ERK1/2 and p38 kinase activities were investigated by Western Blot analysis. Specific MAPK inhibitors PD98059 and SB20350 were employed to investigate regulatory effects of MEK/ERK and p38 signals on gene expression of chondrocyte-specific markers, and TGF-beta1 downstream pathways of Smad2/3. Results ERK1/2 was phosphorylated in a rapid but transient manner, whereas p38 was activated in a slow and sustained way. The two MAPK subtypes played opposing roles in mediating transcription of cartilage-specific genes for Col2alpha and aggrecan. TGF-beta1-stimulated gene expression of chondrogenic regulators, Sox9, Runx2 and Ihh, was also affected by activity of PD98059 and SB203580, to different degrees. However, influences of MAPK inhibitors on gene expression were relatively minor when not treated with TGF-beta1. In addition, gene transcription of Smad2/3 was significantly upregulated by TGF-beta1, but was regulated more subtly by treatment with MAPK inhibitors. Conclusions MAPK subtypes seemed to regulate chondrogenesis with a delicate balance, interacting with the TGF-beta1/Smads signalling pathway.

Reduced ileal Paneth cell alpha-defensin expression has been reported to be associated with Crohn's disease, especially in patients carrying NOD2 mutations. The aim of this study was to independently assess whether NOD2, alpha-defensins and Crohn's disease are linked.Using quantitative real-time polymerase chain reaction (RT-PCR), we measured the mRNA expression levels of key Paneth cell antimicrobial peptides (DEFA5, DEFA6, LYZ, PLA2G2A), inflammatory cytokines [interkelukin 6 (IL6) and IL8], and a marker of epithelial cell content, villin (VIL1) in 106 samples from both affected ileum (inflamed Crohn's disease cases, n = 44) and unaffected ileum (non-inflamed; Crohn's disease cases, n = 51 and controls, n = 11). Anti-human defensin 5 (HD-5) and haematoxylin/eosin immunohistochemical staining was performed on parallel sections from NOD2 wild-type and NOD2 mutant ileal Crohn's disease tissue.In Crohn's disease patients, DEFA5 and DEFA6 mRNA expression levels were 1.9- and 2.2-fold lower, respectively, in histologically confirmed inflamed ileal mucosa after adjustment for confounders (DEFA5, p0.001; DEFA6, p = 0.001). In contrast to previous studies, we found no significant association between alpha-defensin expression and NOD2 genotype. HD-5 protein data supports these RNA findings. The reduction in HD-5 protein expression appears due to surface epithelial cell loss and reduced Paneth cell numbers as a consequence of tissue damage.Reduction in alpha-defensin expression is independent of NOD2 status and is due to loss of surface epithelium as a consequence of inflammatory changes rather than being the inciting event prior to inflammation in ileal Crohn's disease.

Breast-fed infants are healthier than formula-fed infants, which may in part be due to the lack of components like human lactoferrin (HLF) in infant formula. Thus, fortification of infant formula with bioactive proteins such as recombinant HLF (rHLF) may provide health benefits. A synthetic HLF gene linked to a rice glutelin 1 promoter and signal sequence was transformed to rice cells to produce rHLF. The expression level was up to 0.5% of dehusked rice grain weight. The rHLF was specifically expressed in the rice grain but not in other tissues. Rice grains expressing rHLF were advanced for four generations and expression levels were stable. Biochemical and physical characterization showed that rHLF is identical to HLF in N-terminal sequence, pI, iron-binding capacity, antimicrobial activity against a human pathogen and resistance to protease digestion. Receptor binding activity assessed by Caco-2 cells, a human small intestinal cell line, was also retained for rHLF. Since rice may be used in infant formula, it is feasible that rice grains expressing rHLF can be directly incorporated into infant formula without purification of the rHLF, providing a convenient advantage over other expression systems.

A total of 66 transgenic rice cell lines were produced by simultaneously transforming rice callus with nine different plasmids/genes. PCR analysis indicated that the co-transformation frequency of each gene was about 70%. All the cell lines carried at least three genes and 11 cell lines carried all nine genes. Thirty-two fertile transgenic plants (R0) were generated from the transgenic cell lines and seeds of 32 transgenic R1 lines and 5 R2 lines were harvested and analyzed for gene inheritance and protein expression. Progeny segregation analysis indicated that the multiple transgenes were integrated into the same locus of the rice genome, resulting in a 3:1 segregation ratio of the transgenes. Expression analysis of all nine transgenes revealed that the transgenes were expressed in all generations (R0, R1, and R2) and about half of the transgenes from each line were expressed. The expression of one transgene appears to have no effect on the expression of another transgene. Among the 66 cell lines, six lines (9.1%) expressed seven or eight transgenes out of the nine transformed genes. All together, our results showed that multiple genes could be delivered into rice cells simultaneously and cell lines expressing multiple genes could be generated. The results and procedures reported here should be useful in designing multi-plasmid transformation experiments such as those required for plant metabolic engineering.

Stem cell-based therapy has the potential to treat an array of human diseases. However, to study the therapeutic potential and safety of these cells, a scalable cell culture medium is needed that is free of human or bovine-derived serum proteins. Thus, cost-effective recombinant serum proteins and cytokines are needed to produce such mediums. One such cytokine, leukemia inhibitory factor (LIF), has been shown to be a critical paracrine factor that maintains stem cell pluripotency in murine embryonic stem cells and human naïve stem cells while simultaneously inhibiting differentiation. We recently produced recombinant human LIF (rhLIF) in a rice-based protein expression system known as ExpressTec. (12) We described expression of rice-derived rhLIF and demonstrated its biological equivalency to E. coli-derived rhLIF in traditional and embryonic mouse stem cell systems. Here we describe the expression yield of rice-derived rhLIF and the scale up production capacity. We provide further evidence of the efficacy of rice-derived rhLIF in additional stem cell systems including human neural stem cells and mouse induced pluripotent stem (iPS) cells. The expression level, biological activity, and potential for production at commercial scale of rice-derived rhLIF provides a proof-of-principal for ExpressTec-derived proteins to produce regulatory-friendly, high performance, and dependable stem cell media.

Background.Overexpression of decoy receptor 3 (DcR3) have been reported in various classes of malignancies. However, its expression and clinicopathological contribution in gliomas has not been fully elucidated.Objective.To explore the expression and clinical significance of DcR3 protein in relation to tumor cell differentiation and proliferation in glioma cell lines and tissues.Methods.One hundred and twenty-five samples of glioma patients and 18 cases of normal brain tissues were recruited. The expression of DcR3 protein was detected using immunohistochemistry. Tumor differentiation was assessed by histologic characters and the status of glial fibrillary acidic protein (GFAP). Tumor cell labeling indexes (LIs) of Ki-67 and PCNA were also obtained. The relationship between the DcR3 level and clinicopathological features was investigated, including tumor differentiation, LIs, and survival. Meanwhile, the expression of DcR3 protein was also measured in the supernatants of 8 glioma cell lines and glioma cells freshly prepared from 8 human glioblastoma specimens by using western blot.Results.The level of DcR3 protein in gliomas was significantly higher than that in normal brain tissues (P<0.01). DcR3 expression showed positive correlations with tumor pathological grade(r=0.621,P<0.01)and negative with GFAP expression (r=-0.489,P<0.01). Furthermore, there were positive correlations between DcR3 expression and Ki-67, PCNA LIs (r=0.529,P<0.01;r=0.556,P<0.01). The survival in the DcR3 negative group was 50 ± 1.79 months, longer than that of the DcR3 positive group (48.36 ± 2.90), however, without significance(P=0.149). Different levels of DcR3 could also be detected in the culturing supernatants of all the 8 glioma cell lines and glioma cells freshly obtained from 8 human glioblastoma specimens.Conclusions.The overexpression of DcR3 might play a crucial role in the tumorigenesis, differentiation, and proliferation of glioma.

In order to evaluate rapidly the potential of various expression cassettes for transgene expression in rice grain, a transient expression assay was developed using rice immature endosperms. The promoters of several seed storage protein genes including the rice glutelins ( Gt3 , GluB-1 , GluB-2 ), rice prolamins ( PG5a , RP6 ), rice α-globulin ( Glb ) and wheat glutenin ( Bx7 ), were examined for their expression levels in rice immature endosperms isolated from the caryopses at 7–9 days after pollination. In this promoter comparison, the wheat Bx7 promoter showed the highest transcription activity, followed by the promoters of the Glb , GluB-2 , and Gt3 genes. Based on this result, further studies were conducted using the Glb and Bx7 promoters. We examined the effects of the addition of a second promoter fragment, the alfalfa mosaic virus (AMV) 5′untranslated leader sequence (UTL), and various introns and terminators from different genes. Although the addition of a second promoter fragment and the AMV 5′UTL had no effect on GUS expression levels, the insertion of the rice waxy first intron increased GUS expression of the Glb and Bx7 constructs 3 and 6-fold, respectively. The maize adh1 first intron also showed a 2–3-fold increase in the GUS expression levels of both the Glb and Bx7 reporter gene constructs. A comparison of terminator sequences revealed the nopaline synthase gene terminator from Agrobacterium and the rice waxy gene terminator were the most effective in contributing to elevated expression levels.

Transgenic seeds of rice (Oryza sativa L.) were used to investigate temporal, spatial, and hormonal regulation of a rice [alpha]-amylase gene, RAmy1A. Two overlapping segments of the RAmy1A promoter were fused to the coding region of the bacterial reporter gene, gusA. The resulting promoter-gusA fusions, pE4/GUS (-232 to +31) and pH4/GUS (-748 to +31), were used separately to transform rice protoplasts. [beta]-Glucuronidase (GUS) activity was detected in germinated transgenic seeds, although the two constructs showed no significant difference in timing or location of GUS expression. Both constructs first expressed GUS in the scutellar epithelium and then in the aleurone layer. Aleurone expression of GUS activity was strongly induced when embryoless half-seeds were treated with gibberellic acid. GUS expression in the aleurone layer was also suppressed by abscisic acid. These results indicate that the 5[prime] regulatory region from -232 to +31 is sufficient for temporal, spatial, and hormonal regulation of RAmy1A gene expression.

To investigate whether ultrasound-targeted microbubble destruction (UTMD) could transfer gene into central nervous system (CNS) following blood-brain barrier disruption (BBBD), DNA-loaded microbubbles were infused into the mice intravenously following ultrasonic exposure. Opening of the BBB, changes of mRNA and expression of enhanced green fluorescent protein (EGFP), and safety evaluation were measured. By UTMD, EGFP were substantially expressed in the cytoplasm of the neurons at the sonicated area with minor erythrocytes extravasation and the mRNA and expression of EGFP were markedly enhanced by about 15-fold and 10-fold, respectively, than that with US alone (p < 0.01). No EGFP was detected in the mice treated with DNA-loaded microbubbles or plasmid alone. The gene expression reached a climax at 48 h, gradually reduced to a much lower level thereafter. These results demonstrated UTMD could effectively enhance exogenous gene delivery and expression in CNS following BBBD, and this technique may provide a new method for CNS gene therapy.

Human β-defensin-2 (HBD-2) is an antimicrobial peptide produced by the epithelial cells that plays an important role in innate and adaptive immunity. Here we report that high mobility group protein N2 (HMGN2), a member of the high mobility group superfamily that affects chromatin function, modulates the expression of HBD-2 in A549 cells treated by lipopolysaccharide. Mechanistically, HMGN2 prolongs the retention time and enhances the accumulation of nuclear factor κB p65 in the nucleus, and promotes the acetylation of p65 through increasing histone acetyltransferase activity and enhancing p65-Ser536 phosphorylation. Additionally, chromatin immunoprecipitation reveals that HMGN2 and p65 synergistically promote their specific binding to HBD-2 promoter, thereby affecting the downstream transcription. Taken together, these results suggest that HMGN2 acts as a positive modulator of nuclear factor κB signalling to promote lipopolysaccharide-induced β-defensin expression.

Complementary DNAs encoding homologs of the tumor suppressor gene, p53, were characterized from two lepidopteran insects, Bombyx mori (Bm) and Spodoptera frugiperda (Sf). They encoded predicted proteins of 368 (41.2 kDa) (Bm) and 374 (42.5 kDa) (Sf) amino acids. The sequences shared 44% amino acid and 60% nucleotide sequence identity with each other, but exhibited less than 20% amino acid and 46% nucleotide sequence identity to Drosophila melanogaster p53. Despite the sequence diversity, conserved amino acids involved in DNA and zinc binding were present in the lepidopteran sequences. Expression of Sfp53-induced apoptosis in S. frugiperda cells, and antiserum made against recombinant Sfp53 recognized a protein whose abundance increased after treatment with DNA damaging agents.

Transferrin is an essential ingredient used in cell culture media due to its crucial role in regulating cellular iron uptake, transport, and utilization. It is also a promising drug carrier used to increase a drug’s therapeutic index via the unique transferrin receptor-mediated endocytosis pathway. Due to the high risk of contamination with blood-borne pathogens from the use of human- or animal plasma-derived transferrin, recombinant transferrin is preferred for use as a replacement for native transferrin. We expressed recombinant human transferrin in rice (Oryza sativa L.) at a high level of 1% seed dry weight (10 g/kg). The recombinant human transferrin was able to be extracted with saline buffers and then purified by a one step anion exchange chromatographic process to greater than 95% purity. The rice-derived recombinant human transferrin was shown to be not only structurally similar to the native human transferrin, but also functionally the same as native transferrin in terms of reversible iron binding and promoting cell growth and productivity. These results indicate that rice-derived recombinant human transferrin should be a safe and low cost alternative to human or animal plasma-derived transferrin for use in cell culture-based biopharmaceutical production of protein therapeutics and vaccines.

Rice seed callus expressed and secreted alpha-amylase at high levels. Twenty percent of the protein secreted by the callus was alphaamylase. The callus secreted about 840 mug alpha-amylase with 10.9 x 10(3) units of activity per gram dry weight callus per day. The alpha-amylase from callus exhibited a more complex isoform pattern than the germinating seed alpha-amylase. In addition, the level of mRNA expression by the five alpha-amylase gene groups was markedly different between callus and the germinating seed. The rice callus culture has features which it attractive as a potential system for expression proteins in plant cell fermentation systems.

Proteomic and transcriptomic platforms both play important roles in cancer research, with differing strengths and limitations. Here, we describe a proteo-transcriptomic integrative strategy for discovering novel cancer biomarkers, combining the direct visualization of differentially expressed proteins with the high-throughput scale of gene expression profiling. Using breast cancer as a case example, we generated comprehensive two-dimensional electrophoresis (2DE)/mass spectrometry (MS) proteomic maps of cancer (MCF-7 and HCC-38) and control (CCD-1059Sk) cell lines, identifying 1724 expressed protein spots representing 484 different protein species. The differentially expressed cell-line proteins were then mapped to mRNA transcript databases of cancer cell lines and primary breast tumors to identify candidate biomarkers that were concordantly expressed at the gene expression level. Of the top nine selected biomarker candidates, we reidentified ANX1, a protein previously reported to be differentially expressed in breast cancers and normal tissues, and validated three other novel candidates, CRAB, 6PGL, and CAZ2, as differentially expressed proteins by immunohistochemistry on breast tissue microarrays. In total, close to half (4/9) of our protein biomarker candidates were successfully validated. Our study thus illustrates how the systematic integration of proteomic and transcriptomic data from both cell line and primary tissue samples can prove advantageous for accelerating cancer biomarker discovery. Refereed/Peer-reviewed

Two cDNA clones, pOS103 and pOS137, were isolated which code for distinct alpha-amylase isozymes in germinating rice seeds. Sequence analysis indicated that the clones encode polypeptides of approximately 48 kDa, both of which possess a signal peptide involved in directing secretion of the protein. Comparison of the two rice alpha-amylase amino acid sequence showed that they are 76% similar to each other, while showing 85% to 90% similarity with other cereal alpha-amylases. A comparison of eleven cereal alpha-amylases also revealed three new conserved regions (I', II', and IV') not previously identified in the animal, bacterial, and fungal alpha-amylases. Regions I' and IV' are sites for intron splicing while region II' is probably involved in calcium binding. One of the rice alpha-amylase cDNAs, pOS103, encodes a protein that has two potential N-glycosylation sites, one in the signal peptide and the other in the mature portion of the protein. The cDNA clone, pOS137, encodes an alpha-amylase with a single glycosylation site in the signal peptide, suggesting that the mature OS137 isozyme is not glycosylated. Analysis of the expression of these genes in germinating rice seeds indicated that mRNA corresponding to pOS103 and pOS137 could be detected throughout a 48 h period of seed imbibition. RNA levels, however, were dramatically stimulated by treatment of embryoless half-seeds with exogenous GA3. Our results demonstrate that at least two forms of alpha-amylase are expressed in germinating rice seeds and that the expression of these genes is regulated by the phytohormone GA3.

Rice alpha-amylases are encoded by a multigene family that has previously been classified into 5 hybridization groups. DNA sequence and Southern blot analysis identified three genes (RAmy1A, RAmy1B and RAmy1C) in Group 1 with DNA sequence identity of at least 90%. Hybridization Group 2 is represented by only one gene, RAmy3D, which is identical to a previously characterized cDNA, pOS137. RAmy3D is physically linked to the sole representative of Group 5, RAmy3E. The identity between these two genes is 81.4% in the coding region but less than 50% in the 5' and 3' flanking regions. Northern blot analysis and RNA-PCR were used to detect the expression of alpha-amylase genes in various tissues. Alpha-amylase mRNA was abundant in germinating seeds and callus. Some genes were also expressed at much lower levels in roots, young leaves and immature seeds. RAmy1A and RAmy3E were expressed in all tissues while RAmy3D was expressed in all tissues except the immature seeds. RAmy1B was weakly expressed only in callus. RAmy1A transcript was most abundant in the germinating seeds, while RAmy3D and RAmy3E transcripts were most abundant in callus and immature seeds, respectively.

This study was conducted to determine the feasibility and relevant technique itinerary for the production of hBD-2 with Baculovirus Expression Vector System (BEVS).The full hBD-2/His cDNA was amplified from rpcDNA3.1/hBD-2/myc-His by using PCR with a pair of primers (hBD2p10 and hBD2p11) and was inserted into the MCS of transfer vector: pAcGHLT-A. AcNPV DNA and rpAcGHLT-A/hBD-2/His were co-transfected into Sf21 cells. Recombination would take place within the Sf21 cells between the homologous regions in the transfer vector and AcNPV DNA. After 5 days of co-transfer, both supernatant of the experimental cells and positive control cells were collected. Sf21 cells were infected with virus rAcNPVhBD-2/His and then determined by end-point dilution assay. The expression of hBD-2/His in both cell lysate and supernatant was analyzed by western blot with specific 6 poly-histamines antibody.Both enzyme cutting result and sequence analysis showed that recombinant hBD-2 with C terminal of bi-tags of myc and 6xHis had been inserted into the transfer vector of BEVS system correctly, and recombinant transfer vector rpAcGHLT-A/hBD-2/His had been constructed successfully. End-point dilution assay proved that recombinant virus rAcNPVhBD-2/His had been acquired. Western blot revealed that lysate of Sf21 cells transfected by rAcNPVhBD-2/His showed a band of relative moleculal mass about 47.5 x 10(3) which implied that a fuse peptide of hBD-2/His with up-stream of GST tag, 6xHistag, protein kinase A site and thrombin cleavage had expressed. The culture supernatant showed two bands of relative moleculal mass about 40 x 10(3) and 30 x 10(3), which were inferred to be the proceeded products of the fuse peptide during secretion process from cell into culture supernatant.These results suggested that it may be feasible to use BEVS system as a high efficient biologic reactor for producing recombinant hBD-2.

Heterologous protein expression levels in transgenic plants are of critical importance in the production of plant-made pharmaceuticals (PMPs). We studied a puroindoline b promoter and signal peptide (Tapur) driving human lysozyme expression in rice endosperm. The results demonstrated that human lysozyme expressed under the control of the Tapur cassette is seed-specific, readily extractable, active, and properly processed. Immuno-electron microscopy indicated that lysozyme expressed from this cassette is localized in protein bodies I and II in rice endosperm cells, demonstrating that this non-storage promoter and signal peptide can be used for targeting human lysozyme to rice protein bodies. We successfully employed a strategy to improve the expression of human lysozyme in transgenic rice grain by combining the Tapur cassette with our well established Gt1 expression system. The results demonstrated that when the two expression cassettes were combined, the expression level of human lysozyme increased from 5.24 +/- 0.34 mg(-1) g flour for the best single cassette line to 9.24 +/- 0.06 mg(-1) g flour in the best double cassette line, indicating an additive effect on expression of human lysozyme in rice grain.

The aim of this study is to explore the possibility and technical itinerary of establishing an mammal engineering cell line in which hBD-2 can be effectively expressed, secreted, detected, separated and purified. The full hBD2 cDNA was inserted into the multi clone site of a eukaryotic expressive plasmid pcDNA3.1/Myc-His(+) and located closely at the upstream of two tag gene (myc and 6 Poly-histidines) so as to construct another recombinant eukaryotic expressive vector of hBD-2 gene: rpcDNA3.1/Myc-His/hBD-2. By the use of RT-PCR with special primers, a band of 240 bp was amplified from COS-7 cells transfected by this recombinant plasmid, which matched full length of cDNA coding hBD2 plus myc epitope and 6 poly-histidines tags. Western blot analysis with specific anti-histidines antibody revealed that the lysate of COS-7 cells transfected by rpcDNA3.1/Myc-His/hBD-2 had a strong band with molecular weight of about 10 Kd that was approximate to the size of chiasmic peptide. Antibacterial activity assay showed that obvious bacterial inhibition occurred in both lysate and supernatant of COS-7 cells transfected by rpcDNA3.1/Myc-His/hBD-2.

A total of 66 transgenic rice cell lines were produced by simultaneously transforming rice callus with nine different plasmids/genes. PCR analysis indicated that the co-transformation frequency of each gene was about 70%. All the cell lines carried at least three genes and 11 cell lines carried all nine genes. Thirty-two fertile transgenic plants (R0) were generated from the transgenic cell lines and seeds of 32 transgenic R1 lines and 5 R2 lines were harvested and analyzed for gene inheritance and protein expression. Progeny segregation analysis indicated that the multiple transgenes were integrated into the same locus of the rice genome, resulting in a 3:1 segregation ratio of the transgenes. Expression analysis of all nine transgenes revealed that the transgenes were expressed in all generations (R0, R1, and R2) and about half of the transgenes from each line were expressed. The expression of one transgene appears to have no effect on the expression of another transgene. Among the 66 cell lines, six lines (9.1%) expressed seven or eight transgenes out of the nine transformed genes. All together, our results showed that multiple genes could be delivered into rice cells simultaneously and cell lines expressing multiple genes could be generated. The results and procedures reported here should be useful in designing multi-plasmid transformation experiments such as those required for plant metabolic engineering.

In order to understand the characteristics of recombinant protein expression and sublocalization in rice ( Oryza sativa L.) endosperm, we examined the expression level of human lysozyme protein and its subcellular location in transgenic rice seeds driven by rice glutelin and globulin promoters and signal peptides. A time course of human lysozyme expression during endosperm development was analyzed. The results showed that the expression profile of recombinant protein accumulation in endosperm paralleled that of the two storage proteins. Immunofluorescence microscopy revealed that human lysozyme and storage proteins co-localized to type-II protein bodies. Both promoter-signal peptide parings targeted recombinant protein to the protein bodies. In addition, a transgenic line with a higher lysozyme expression level exhibited morphologically different protein bodies with an unbalanced composition of lysozyme and native storage proteins. The high-level expression of recombinant protein distorted the trafficking and sorting of native storage proteins in rice endosperm and affected the expression of native storage protein.

Human alpha-1-antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon-optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice-derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant-derived AAT for treatments of various AAT-deficient diseases are discussed.

The genome-wide distribution patterns of the '6th base' 5-hydroxymethylcytosine (5hmC) in many tissues and cells have recently been revealed by hydroxymethylated DNA immunoprecipitation (hMeDIP) followed by high throughput sequencing or tiling arrays. However, it has been challenging to directly compare different data sets and samples using data generated by this method. Here, we report a new comparative hMeDIP-seq method, which involves barcoding different input DNA samples at the start and then performing hMeDIP-seq for multiple samples in one hMeDIP reaction. This approach extends the barcode technology from simply multiplexing the DNA deep sequencing outcome and provides significant advantages for quantitative control of all experimental steps, from unbiased hMeDIP to deep sequencing data analysis. Using this improved method, we profiled and compared the DNA hydroxymethylomes of mouse ES cells (ESCs) and mouse ESC-derived neural progenitor cells (NPCs). We identified differentially hydroxymethylated regions (DHMRs) between ESCs and NPCs and uncovered an intricate relationship between the alteration of DNA hydroxymethylation and changes in gene expression during neural lineage commitment of ESCs. Presumably, the DHMRs between ESCs and NPCs uncovered by this approach may provide new insight into the function of 5hmC in gene regulation and neural differentiation. Thus, this newly developed comparative hMeDIP-seq method provides a cost-effective and user-friendly strategy for direct genome-wide comparison of DNA hydroxymethylation across multiple samples, lending significant biological, physiological and clinical implications.

Sex-linked dwarfism in chickens is a form of GH resistance that resembles the Laron syndrome in humans. The dwarfism found in chickens is due to a mutant gene (dw) carried on the sex chromosome. The homozygous dwarf (dwdw) chicken is characterized by reductions in stature and plasma insulin-like growth factor-I (IGF-I) levels. Despite the absence of hepatic GH-binding activity, Southern blot analysis shows that there is no gross structural change in the gene for the GH receptor (GHR) in this strain of dwdw chicken. GH-dependent IGF-I production can be restored in cultured dwdw hepatocytes after transfection and transient expression of a chicken GHR (cGHR) cDNA, indicating that other factors that participate in GH-mediated IGF-I synthesis are intact. Northern blot analysis of liver, muscle, fat, and pituitary RNA from normal (DwDw) chickens shows a major transcript of 4.3 kilobases (kb) and three minor transcripts (0.8, 1.7, and 3.2 kb), which correspond to the cGHR. In contrast, the 0.8-kb transcript is the major cGHR transcript expressed in these tissues from dwdw chickens. Northern blot analysis with domain-specific probes shows that the 0.8-kb transcript in DwDw and dwdw liver contains only a small portion of the extracellular domain of the cGHR. A cDNA clone encoding this transcript has been isolated from a liver library prepared from a normal chicken.(ABSTRACT TRUNCATED AT 250 WORDS)

The structure and expression of the alpha-amylase-encoding gene, RAmy2A, are described. This only representative of the Amy2 subfamily in rice differs from other cereal alpha-amylase-encoding genes in several respects. It contains the largest introns of all the cereal alpha-amylase-encoding genes examined to date. Moreover, the second of three introns in this gene contains a long inverted repeat sequence that can potentially form a large and stable stem-loop structure in the unspliced RNA transcript. Finally, RAmy2A is constitutively expressed at very low levels in germinated seeds, root, etiolated leaves, immature seeds and callus. This is in marked contrast to the Amy2 genes of wheat and barley which are highly expressed in the aleurone layer of the germinated seeds.

A rice beta-glucanase gene was sequenced and its expression analyzed at the level of mRNA accumulation. This gene (Gns1) is expressed at relatively low levels in germinating seeds, shoots, leaves, panicles and callus, but it is expressed at higher levels in roots. Expression in the roots appears to be constitutive. Shoots express Gns1 at much higher levels when treated with ethylene, cytokinin, salicylic acid, and fungal elicitors derived from the pathogen Sclerotium oryzae or from the non-pathogen Saccharomyces cereviseae. Shoots also express Gns1 at higher levels in response to wounding. Expression in the shoots is not significantly affected by auxin, gibberellic acid or abscisic acid. The beta-glucanase shows 82% amino acid similarity to the barley 1,3;1,4-beta-D-glucanases, and from hybridization studies it is the beta-glucanase gene in the rice genome closest to the barley 1,3;1,4-beta-glucanase EI gene. The mature peptide has a calculated molecular mass of 32 kDa. The gene has a large 3145 bp intron in the codon for the 25th amino acid of the signal peptide. The gene exhibits a very strong codon bias of 99% G + C in the third position of the codon in the mature peptide coding region, but only 61% G + C in the signal peptide region.