Search

Histone H3K4 methylation is catalysed by the multi-protein complex known as the Set1/COMPASS or MLL/COMPASS-like complex, an element that is highly evolutionarily conserved from yeast to humans. However, the components and mechanisms by which the COMPASS-like complex targets the H3K4 methylation of plant pathogenic genes in fungi remain elusive. Here we present a comprehensive analysis combining biochemical, molecular, and genome-wide approaches to characterize the roles of the COMPASS-like family in Magnaporthe oryzae, a model plant fungal pathogen. We purified and identified six conserved subunits of COMPASS from the rice blast fungus M. oryzae, i.e., MoBre2 (Cps60/ASH2L), MoSpp1 (Cps40/Cfp1), MoSwd2 (Cps35), MoSdc1 (Cps25/DPY30), MoSet1 (MLL/ALL) and MoRbBP5 (Cps50), using an affinity tag on MoBre2. We determined the SPRY domain of MoBre2 can recognize directly with DPY30 domain of MoSdc1 in vitro. Furthermore, we found that deletion of the genes encoding COMPASS subunits of MoBre2, MoSpp1 and MoSwd2 caused similar defects regarding invasive hyphal development and pathogenicity. Genome-wide profiling of H3K4me3 revealed that the it has remarkable co-occupancy at the TSS regions of target genes. Significantly, these target genes are often involved in spore germination and pathogenesis. Decreased gene expression caused by the deletion of MoBre2, MoSwd2 or MoSpp1 gene was highly correlated with decrease in H3K4me3. Taken together, these results suggest that MoBre2, MoSpp1, and MoSwd2 function as a whole COMPASS complex, contributing to fungal development and pathogenesis by regulating H3K4me3-targeted genes in M. oryzae.

Class 2 CRISPR–Cas proteins have been widely developed as genome editing and transcriptional regulating tools. Class 1 type I CRISPR–Cas constitutes ~60% of all the CRISPR–Cas systems. However, only type I–B and I–E systems have been used to control mammalian gene expression and for genome editing. Here we demonstrate the feasibility of using type I–F system to regulate human gene expression. By fusing transcription activation domain to Pseudomonas aeruginosa type I–F Cas proteins, we activate gene transcription in human cells. In most cases, type I–F system is more efficient than other CRISPR-based systems. Transcription activation is enhanced by elongating the crRNA. In addition, we achieve multiplexed gene activation with a crRNA array. Furthermore, type I–F system activates target genes specifically without off-target transcription activation. These data demonstrate the robustness and programmability of type I–F CRISPR–Cas in human cells. Class 1 type I CRISPR–Cas systems have not been as extensively developed for genome engineering as Class 2 systems. Here the authors modify the Type I–F CRISPR–Cas system for transcriptional activation of gene expression.

Background The skin harbors complex communities of resident microorganisms, yet little is known of their physiological roles and the molecular mechanisms that mediate cutaneous host-microbe interactions. Here, we profiled skin transcriptomes of mice reared in the presence and absence of microbiota to elucidate the range of pathways and functions modulated in the skin by the microbiota. Results A total of 2820 genes were differentially regulated in response to microbial colonization and were enriched in gene ontology (GO) terms related to the host-immune response and epidermal differentiation. Innate immune response genes and genes involved in cytokine activity were generally upregulated in response to microbiota and included genes encoding toll-like receptors, antimicrobial peptides, the complement cascade, and genes involved in IL-1 family cytokine signaling and homing of T cells. Our results also reveal a role for the microbiota in modulating epidermal differentiation and development, with differential expression of genes in the epidermal differentiation complex (EDC). Genes with correlated co-expression patterns were enriched in binding sites for the transcription factors Klf4, AP-1, and SP-1, all implicated as regulators of epidermal differentiation. Finally, we identified transcriptional signatures of microbial regulation common to both the skin and the gastrointestinal tract. Conclusions With this foundational approach, we establish a critical resource for understanding the genome-wide implications of microbially mediated gene expression in the skin and emphasize prospective ways in which the microbiome contributes to skin health and disease. Electronic supplementary material The online version of this article (10.1186/s40168-018-0404-9) contains supplementary material, which is available to authorized users.

Colorectal cancer (CRC) cells undergo apoptosis in the presence of the small-molecule inhibitor ABT-263 by up-regulating antiapoptotic Bcl-2 family members. However, the resistance to ABT-263 gradually developed in most solid tumors due to its low affinity to Mcl-1. Here, we found the BET-Bromodomain inhibitor JQ1, when combined with ABT-263, synergistically reduced Mcl-1 protein level, induced apoptosis, and decreased cell viability in the CRC HCT-15, HT-29 and SW620 cells. The subsequent mechanism study revealed that a pathway of c-Myc/miR-1271-5p/Noxa/Mcl-1 underlies the synergistic effect of such combination treatment. We discovered that miR-1271-5p, the key mediator for the synergistic effect, is transcriptionally activated by c-Myc, and binds to the 3'-UTR of noxa to inhibit its protein production. The combination treatment of JQ1 and ABT-263 inhibited c-Myc protein level and also c-Myc-driven expression of miR-1271-5p, subsequently increased the protein level of Noxa, and finally promotes the degradation of Mcl-1. Our findings provide an alternative strategy to resolve the resistance during treatment of CRC by JQ1, and also discovered a novel miR-1271-5p-dependent regulatory mechanism for gene expression of noxa.

Mammalian cell expression systems are the most commonly used platforms for producing biotherapeutic proteins. However, development of recombinant mammalian cell lines is often hindered by the unstable and variable transgene expression associated with random integration. We have developed an efficient strategy for site-specific integration of genes of interest (GOIs). This method enables rapid and precise insertion of a gene expression cassette at defined loci in mammalian cells, resulting in homogeneous transgene expression. We identified the Hipp11 (H11) gene as a "safe harbor" locus for gene knock-in in CHO-S cells. Using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 mediated homologous recombination, we knocked in a DNA cassette (the landing pad) that includes a pair of PhiC31 bacteriophage attP sites and genes facilitating integrase-based GOI integration. A master cell line, with the landing pad inserted correctly in the H11 locus, was established. This master cell line was used for site-specific, irreversible recombination, catalyzed by PhiC31 integrase. Using this system, an integration efficiency of 97.7% was achieved with green fluorescent protein (GFP) after selection. The system was then further validated in HEK293T cells, using an analogous protocol to insert the GFP gene at the ROSA26 locus, resulting in 90.7% GFP-positive cells after selection. In comparison, random insertion yielded 0.68% and 1.32% GFP-positive cells in the CHO-S and HEK293T cells, respectively. Taken together, these findings demonstrated an accurate and effective protocol for generating recombinant cell lines to provide consistent protein production. Its likely broad applicability was illustrated here in two cell lines, CHO-S and HEK293T, using two different genomic loci as integration sites. Thus, the system is potentially valuable for biomanufacturing therapeutic proteins.

Halogenated dipeptides, 3, 5-di-I-tyrosylalanine (DIYA), have been identified as novel disinfection byproducts (DBPs), following chloramination of authentic water. However, little is known about their toxicity. Zebrafish embryos were used to assess the toxicity of novel iodinated DBPs (I-DBPs). Although DIYA did not exhibit high acute toxicity to embryonic zebrafish (LC50 > 2 mM), it significantly inhibited pigmentation of melanophores and xanthophores on head, trunk and tail at 500 μM as determined by photographic analysis. Whereas N-phenylthiourea (PTU) as a pigment inhibitor did not inhibit development of yellow pigments. Colorimetric detection of melanin further confirmed these results. Quantitative real time polymerase chain reaction (qRT-PCR) measurements indicated that genes (dct, slc24a5, tyr, tyrp1a, tyrp1b, silva) associated with the melanogenesis pathway were dramatically down-regulated following exposure to 500 μM DIYA. In addition, enzymatic activity of tyrosinase (TYR) decreased, also demonstrating that the underlying mechanism of hypopigmentation was attributed to the disruption of melanogenesis pathway. Transcription levels of xanthophore genes (gch2, bnc2, csf1a, csf1b, pax7a and pax7b) were also monitored by qRT-PCR assay. DIYA exposure up-regulated expression of gch2 and bnc2, but not csf1 and pax7. Tested DIYA analogues, brominated tyrosine was unlikely to inhibit pigmentation, indicating that the iodine substitution and dipeptides structure are of important structural feature for the inhibition of pigmentation. In this study, we observed that DIYA inhibited melanogenesis related genes, which might contribute to pigmentation defects. Moreover, as an emerging I-DBPs, the developmental toxicity of aromatic dipeptides should be further studied.

Hybrid triploid loaches (Misgurnus anguillicaudatus) were generated from natural tetraploid and diploid loaches. We studied the gonads of parents and offspring from direct and reciprocal crosses through histological and transcriptome analyses. The triploid offspring had inferior ability to form sperm and egg cells compared with diploid forms. After sequencing the transcriptomes, 655,109,158 clean reads were obtained, and 62,821 unigenes and 178,962 transcripts were assembled. Of these unigenes, 23,189 were annotated in the GO database, 18,525 in the KEGG database and 24,661 in the KOG database. 36 fertility-related genes were found to be differentially expressed between the direct cross (2n × 4n) progenies and their parents, while 53 fertility-related genes between the reciprocal cross (4n × 2n) progenies and their parents. Following protein-protein interaction network analyses, 54 differentially expressed genes, including PLCB4, cyp17a1 and Pla2g4d, were mined, yielding candidate genes involved in the poor fertility of hybrid triploid loaches. This is the first report of transcriptomes of gonads of hybrid triploid loaches and their parents, offering a substantial contribution to sequence resources for this species and providing a deep insight into the molecular mechanism controlling the fertility of hybrid triploid fish.

Pb can damage the physiological function of human organs by entering the human body via food-chain enrichment. Revealing the mechanisms of maize tolerance to Pb is critical for preventing this. In this study, a Pb-tolerant maize inbred line, 178, was used to analyse transcription factors (TFs) expressed under Pb stress based on RNA sequencing data. A total of 464 genes expressed in control check (CK) or Pb treatment samples were annotated as TFs. Among them, 262 differentially expressed transcription factors (DETs) were identified that responded to Pb treatment. Furthermore, the DETs were classified into 4 classes according to their expression patterns, and 17, 12 and 2 DETs were significantly annotated to plant hormone signal transduction, basal transcription factors and base excision repair, respectively. Seventeen DETs were found to participate in the plant hormone signal transduction pathway, where basic leucine zippers (bZIPs) were the most significantly enriched TFs, with 12 members involved. We further obtained 5 Arabidopsis transfer DNA (T-DNA) mutants for 6 of the maize bZIPs, among which the mutants atbzip20 and atbzip47, representing ZmbZIP54 and ZmbZIP107, showed obviously inhibited growth of roots and above-ground parts, compared with wild type. Five highly Pb-tolerant and 5 highly Pb-sensitive in maize lines were subjected to DNA polymorphism and expression level analysis of ZmbZIP54 and ZmbZIP107. The results suggested that differences in bZIPs expression partially accounted for the differences in Pb-tolerance among the maize lines. Our results contribute to the understanding of the molecular regulation mechanisms of TFs in maize under Pb stress.

Tetrabromobisphenol A (TBBPA), hexabromocyclododecane (HBCD) and decabromodiphenyl ether (BDE 209), suspected ubiquitous contaminants, account for the largest volume of brominated flame retardants (BFRs) since penta-BDE and octa-BDE have been phased out globally. In this paper, the growth inhibition and gene transcript levels of antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT)) and the stress-response gene involved in the prevention of oxidative stress (Hsp70) of earthworms (Eisenia fetida) exposed to TBBPA, HBCD and BDE 209 were measured to identify the toxicity effects of selected BFRs on earthworms. The growth of earthworms treated by TBBPA at 200 and 400 mg/kg dw were inhibited at rate of 13.7% and 22.0% respectively, while there was no significant growth inhibition by HBCD and BDE 209. A significant (P

Circular RNAs (circRNAs) represent a class of endogenous noncoding RNAs that have recently been recognized as important regulators of gene expression and pathological networks. However, their transcriptional activities and functional mechanisms in cancer remain largely unknown. Here, we present results from a global circRNA expression and functional analysis of patients with hepatocellular carcinoma (HCC). Using a circRNA microarray, we identified 226 differentially expressed circRNAs, of which 189 were significantly upregulated and 37 were downregulated. High expression of circRNA_100338, one of the upregulated circRNAs in HCC, is closely correlated with a low cumulative survival rate and metastatic progression in HCC patients with hepatitis B. Furthermore, our in silico and experimental analyses identified miR-141-3p as a direct target of circRNA_100338. Thus, circRNA_100338 functions as an endogenous sponge for miR-141-3p in HCC. In addition, we identified the crucial antagonistic roles of circRNA_100338 and miR-141-3p in the regulation of invasive potential in liver cancer cells. Overall, the differential expression of multiple circRNAs in HCC tissues and their clinical significance in hepatitis B-related HCC patients as revealed by our study suggests that circRNA_100338 is a potentially valuable biomarker for HCC diagnosis and target for HCC therapeutics.

Long non-coding RNAs (lncRNAs) play important roles in almost all biological processes. However, there is little information on the effects of lncRNAs on ovulation and lambing rates. In the present study, we used high-throughput RNA sequencing to identify differentially expressed lncRNAs between the ovaries of multiparous (Mul) and uniparous (Uni) Anhui White goats. Among the 107,255,422 clean reads, 183,754 lncRNAs were significantly differentially expressed between the Uni and Mul. Among them, 455 lncRNAs were co-expressed between the two samples, whereas, 157,523 lncRNAs were uniquely expressed in the Uni, and 25,776 uniquely lncRNAs were expressed in the Mul. Through Cis role analysis, 24 lncRNAs were predicted to overlap with cis-regulatory elements, which involved in Progesterone-mediated oocyte maturation, Steroid biosynthesis, Oocyte meiosis, and gonadotropin-releasing hormone (GnRH) signaling pathway. These 4 pathways were related to ovulation, and the KEGG pathway analysis on target genes of the differentially expressed lncRNAs confirmed this results. In addition, 10 lncRNAs harbored precursors of 40 miRNAs, such as TCONS_00320849 related to a mature miRNA sequence, miR-365a, which was reported to be related to proliferation, were annotated in the precursor analysis of miRNAs. The present expand the understanding of lncRNA biology and contribute to the annotation of the goat genome. The study will provide a resource for lncRNA studies of ovulation and lambing.

The mycobacterial peptidoglycan has structure and biosynthetic pathways to similar those of other bacteria. UDP-N-acetylglucosamine enolpyruvyle transferase (MurA) catalyzes the first reaction in the biosynthesis of peptidoglycan. The MurA enzyme has been identified from various bacterial species, but the in-depth biochemical properties of mycobacterial MurA have not been characterized. In this study, both Mycobacterium tuberculosis MurA protein and Mycobacterium smegmatis MurA protein were overexpressed in Escherichia coli and purified by affinity chromatography. MurA activity was detected by HPLC. A colorimetric assay of MurA activity was also developed and the kinetic properties of Mtb MurA and Msm MurA were determined using this colorimetric assay. A conditional murA gene knockout strain was constructed by DNA homologous recombination. The disruption of murA in the genome of M. smegmatis led to loss of viability at a non-permissive temperature. Drastic morphological and structural alterations in the M. smegmatis murA knockout strain were observed by scanning electron microscopy and transmission electron microscopy. These results demonstrated that murA was an essential gene for growth of M. smegmatis. Therefore, MurA is a potential target for developing new anti-tuberculosis drugs.

Enhancer elements are essential for tissue-specific gene regulation during mammalian development. Although these regulatory elements are often distant from their target genes, they affect gene expression by recruiting transcription factors to specific promoter regions. Because of this long-range action, the annotation of enhancer element-target promoter pairs remains elusive. Here, we developed a novel analysis methodology that takes advantage of Hi-C data to comprehensively identify these interactions throughout the human genome. To do this, we used a geometric distribution-based model to identify DNA-DNA interaction hotspots that contact gene promoters with high confidence. We observed that these promoter-interacting hotspots significantly overlap with known enhancer-associated histone modifications and DNase I hypersensitive sites. Thus, we defined thousands of candidate enhancer elements by incorporating these features, and found that they have a significant propensity to be bound by p300, an enhancer binding transcription factor. Furthermore, we revealed that their target genes are significantly bound by RNA Polymerase II and demonstrate tissue-specific expression. Finally, we uncovered that these elements are generally found within 1 Mb of their targets, and often regulate multiple genes. In total, our study presents a novel high-throughput workflow for confident, genome-wide discovery of enhancer-target promoter pairs, which will significantly improve our understanding of these regulatory interactions.

In order to investigate how cells recognize biomaterials, mRNA that was expressed in attached Intestinal epithelial cells (IEC-6) on various suture substrates was evaluated. The expressed cell cycle regulators (cyclin D1, CDK4 and p21) mRNA were then isolated and detected using the real time- polymerase chain reaction (PCR) method. As a result, cyclin D1 gene expression was affected by cell-polymer adhesion and was associated with cell proliferation. In addition, CDK4 gene expression was affected by cell proliferation rather than by cell-biomaterial interaction. The p21 mRNA gene expression was higher in cells on more hydrophilic surfaces than on hydrophobic surfaces. Further, the cyclin D1, CDK4 and p21 gene expression were also influenced by the surface chemistry of suture materials. We concluded that the expression of cyclin D1, CDK4 and p21 mRNA was a powerful method for studying cell-biomaterial interactions or the evaluation of the carcinogenic activity of biomaterials.

Background Numerous studies have demonstrated that the NDV-mediated gene therapy is a promising new approach for treatment of cancers. P53 plays a vital role in tumor suppression and surveillance. Therefore, we hypothesize that a recombinant NDV expressing P53 would be an ideal agent for the hepatoma therapy. Results In the essay, the human P53 gene was incorporated into the genome of a lentogenic strain (named rNDV-P53), which did not affect viral replication kinetics and magnitude in HepG2 cells. Compared to the vehicle virus, rNDV-P53 increased cell growth suppressor ratio and early apoptosis by 2 folds, and decreased the mitochondrial membrane potential in HepG2 cells. In vivo studies, treatment with rNDV-P53 reduced tumor volume of tumor-bearing mice by more than 4 folds, tumor weight by more than 5 folds comparing with rNDV. The 120-day survival rate of rNDV-P53-treated mice was 75 %, survival rate of rNDV-treated mice was 12.5 %. TUNEL analysis showed a significant increase in the apoptosis rate in the tumor tissues of rNDV-P53-treated mice than that of rNDV-treated mice. Moreover, serum chemistries revealed an insignificant change of blood urea nitrogen (BUN), creatinine levels, alanine aminotransferase (ALT) and aspartate transaminase (AST) in rNDV-P53-treated group compared to normal mice, suggesting treatment with the recombinant virus was not toxic. Conclusion rNDV-P53 is a potent candidate for carcinoma therapy especially for hepatocarcinoma.

Accurate mapping of next-generation sequencing (NGS) reads to reference genomes is crucial for almost all NGS applications and downstream analyses. Various repetitive elements in human and other higher eukaryotic genomes contribute in large part to ambiguously (non-uniquely) mapped reads. Most available NGS aligners attempt to address this by either removing all non-uniquely mapping reads, or reporting one random or "best" hit based on simple heuristics. Accurate estimation of the mapping quality of NGS reads is therefore critical albeit completely lacking at present. Here we developed a generalized software toolkit "AlignerBoost", which utilizes a Bayesian-based framework to accurately estimate mapping quality of ambiguously mapped NGS reads. We tested AlignerBoost with both simulated and real DNA-seq and RNA-seq datasets at various thresholds. In most cases, but especially for reads falling within repetitive regions, AlignerBoost dramatically increases the mapping precision of modern NGS aligners without significantly compromising the sensitivity even without mapping quality filters. When using higher mapping quality cutoffs, AlignerBoost achieves a much lower false mapping rate while exhibiting comparable or higher sensitivity compared to the aligner default modes, therefore significantly boosting the detection power of NGS aligners even using extreme thresholds. AlignerBoost is also SNP-aware, and higher quality alignments can be achieved if provided with known SNPs. AlignerBoost's algorithm is computationally efficient, and can process one million alignments within 30 seconds on a typical desktop computer. AlignerBoost is implemented as a uniform Java application and is freely available at https://github.com/Grice-Lab/AlignerBoost.

Germ cells implement elaborate mechanisms to protect their genetic material and to regulate gene expression during differentiation. Piwi proteins bind Piwi-interacting RNAs (piRNAs), small germline RNAs whose biogenesis and functions are still largely elusive. We used high-throughput sequencing after cross-linking and immunoprecipitation (HITS-CLIP) coupled with RNA-sequencing (RNA-seq) to characterize the genome-wide target RNA repertoire of Mili (Piwil2) and Miwi (Piwil1), two Piwi proteins expressed in mouse postnatal testis. We report the in vivo pathway of primary piRNA biogenesis and implicate distinct nucleolytic activities that process Piwi-bound precursor transcripts. Our studies indicate that pachytene piRNAs are the end products of RNA processing. HITS-CLIP demonstrated that Miwi binds spermiogenic mRNAs directly, without using piRNAs as guides, and independent biochemical analyses of testis mRNA ribonucleoproteins (mRNPs) established that Miwi functions in the formation of mRNP complexes that stabilize mRNAs essential for spermiogenesis.

In this study, intestinal epithelial cells (IEC)-6 were cultured in different concentration extracts of Mg–6Zn alloys for different time periods. We studied the indirect effects of Mg–6Zn alloys on cell cycle of IEC-6 cells. The cell cycle of IEC-6 cells was measured using flow cytometry. And, the cell cycle of IEC-6 cells was evaluated by investigating the expression of cyclin D1, CDK4, and P21 using real-time polymerase chain reaction (PCR) and Western blotting tests. It was found that the IEC-6 cells displayed better cell functions in 20% extract of the Mg–6Zn alloy extracts, compared to the 100% or 60% extract. The in vitro results indicated that the conspicuous alkaline environment that is a result of rapid corrosion of Mg–6Zn alloys is disadvantageous to cell cycle of IEC-6 cells.

To explore the role of Med19, a component of the Mediator complex that coactivates DNA-binding transcription factors, in the proliferation and tumorigenesis of human hepatocellular carcinoma cells.The human hepatocellular carcinoma cell lines HepG2 and Hep3B were infected with lentiviral vectors encoding interfering RNA (RNAi) targeting the Med19 gene. To further confirm the inhibitory effects of RNAi vectors on Med19 gene expression, quantitative real-time RT-PCR and Western blotting assays were used. The proliferation of HepG2 and Hep3B cells after transduction with the Med19-RNAi-Lentivirus vector was evaluated by MTT conversion, BrdU incorporation, colony formation, and cell-cycle assays in vitro. In addition, the ability of the Med19-RNAi-Lentivirus vector-infected Hep3B cells to form tumors after inoculation into nude mice was determined.Recombinant lentiviral vectors expressing small interfering RNA (siRNA) against Med19 were constructed and were found to efficiently downregulate Med19 mRNA and protein levels in HepG2 and Hep3B cells. Furthermore, the inhibition of Med19 by RNAi dramatically reduced hepatocellular carcinoma cell proliferation, induced cell-cycle arrest in the G(0)/G(1) phase, and suppressed tumor formation.These results provide new evidence of an important role for Med19 in the development of hepatocellular carcinomas, suggesting that lentivirus-mediated RNAi to target Med19 is a potential tool for inhibiting cancer cell proliferation and tumorigenesis.

MicroRNAs (miRNAs or miRs) are endogenous non-coding RNAs that negatively regulate gene expression by binding to the 3' non-coding regions of target mRNAs, resulting in their cleavage or blocking their translation. miRNAs may have an impact on cell differentiation, proliferation, and survival, and their deregulation can be inclined to diseases and cancers, including thyroid tumors. The purpose of this review is to summarize the existing findings of deregulated miRNAs in different types of thyroid tumors and to exhibit their potential target genes, especially to demonstrate those involved in tumor invasion and metastasis. In addition, new findings of circulating miRNA expression profiles, single nucleotide polymorphism (SNP) in thyroid tumors, and the correlation of somatic mutations with deregulated miRNA expression in thyroid tumors were all included in this review.

To investigate the influence of hepatitis B virus (HBV)-encoded small surface protein (SHBs) on hepatic cell expression of host genes related to lipid metabolism.The full-length SHBs gene was amplified from HBV genotype C by polymerase chain reaction (PCR) and cloned into the pcDNA3.1(+) expression vector for stable transfection into HepG2 cells (selected by G418 screening); cells transfected with empty vector served as control. The SHBs mRNA and protein levels were detected by reverse transcription-PCR and enzyme-linked immunosorbent assay. SHBs effects on expression of genes and proteins related to lipid metabolism were detected by real-time quantitative (q)PCR and western blotting, respectively.The stably transfected cell line HepG2-pn3.1-SHBs was established successfully. qPCR showed that the HepG2-pn3.1-SHBs cells had significantly down-regulated transcription of the ECHS1, APOA1 and LPL genes (0.161+/-0.043 vs. control cells: 0.210+/-0.022, t = 2.479; 0.031+/-0.007 vs. 0.094+/-0.055, t = 2.752; 0.770+/-0.036 vs. 0.982+/-0.031, t = 10.914), but significantly up-regulated ACC and SREBP-1c genes (0.113+/-0.027 vs. 0.059+/-0.022, t = -3.757; 0.019+/-0.002 vs. 0.015+/-0.001, t = -4.330). The CPT1a and PPARa genes' expression was slightly, but not significantly, down-regulated in the HepG2-pn3.1-SHBs cells (0.028+/-0.005 vs. 0.030+/-0.004, t = 1.022; 0.014+/-0.004 vs. 0.015+/-0.002, t = 0.758). Western blotting showed similar expression trends for the corresponding proteins.SHBs alters the expression of some host genes with known functions in fatty acid synthesis and decomposition; however, it remains unclear whether the hepatitis B surface antigen can directly contribute to development of hepatic steatosis.

microRNAs (miRNAs) are involved in the pathogenesis of diverse human cancers through its target genes, including papillary thyroid cancer (PTC). However, there are few studies regarding associations between clinicopathological features of PTC with the expression of specific miRNAs and its potential target genes. In the present study, analysis of miRNA was integrated with mRNA expression profiles in aggressive PTC. miRNA and gene expression arrays were used to identify a subset of differentially expressed miRNAs and mRNAs between aggressive and non-aggressive PTCs. These miRNAs and mRNAs were further validated by qPCR in a cohort of 20 PTCs with extrathyroidal invasion and/or distant metastases, and 20 PTCs with no extrathyroidal invasion. The target of these miRNAs was determined by luciferase reporter and bioinformatic analysis. The miRNA arrays identified 14 upregulated miRNAs and 10 downregulated miRNAs in aggressive compared with non-aggressive PTCs. Significant miRNA deregulation was confirmed in the validation cohort, with upregulation of miR-146b-5p and miR-221/222 and downregulation of miR-16 and miR-613 in aggressive PTCs. The gene arrays identified 2000 differentially expressed genes, in which TIMP3, ZNFR3, FN1 and ITGA2 were observed to be target genes inversely correlated with miR-221/222, miR-146b-5p, miR-613 and miR-16, respectively. The results of the present study indicated the potential importance of miR-221/222, miR-146b-5p, miR-16 and miR-613 in determining the aggressive properties of PTC by targeting TIMP3, ZNFR3, FN1 and ITGA2, respectively. Additional studies should be conducted to confirm the results.

Papillary thyroid cancer (PTC) is the most common form of well-differentiated thyroid carcinoma. Although the incidence of PTC has been on the increase in China, the precise causes of the disease remain unknown and a prognostic index has yet to be identified. In the present study, quantitative real‑time PCR and western blot analysis were used to detect the mRNA and protein expression levels, respectively, of forkhead box E1 (FOXE1) in 30 PTC tissue specimens matched with adjacent non-tumor thyroid tissues. FOXE1 expression in 81 PTC paraffin-embedded blocks was also evaluated retrospectively by immunohistochemistry (IHC) and its correlation with the clinicopathological parameters of PTC patients was analyzed. Compared with adjacent non-tumor tissues, the mRNA expression levels of the FOXE1 gene in tumor tissues were significantly higher in 65% of PTC tissue specimens. The results from western blotting and IHC analysis were consistent with FOXE1 mRNA expression. Furthermore, the expression of FOXE1 was significantly correlated with the extra-capsular invasion of tumor cells, lymph node metastasis and tumor stage (P=0.048, P=0.036 and P=0.009, respectively). Thus, our results demonstrated that the expression of FOXE1 was significantly increased in PTC tumor tissues and correlated with clinical prognosis. Therefore, FOXE1 is a potential biomarker for PTC prognosis as well as a new therapeutic target.

Transcriptional gene silencing controls transposons and other repetitive elements through RNA-directed DNA methylation (RdDM) and heterochromatin formation. A key component of the Arabidopsis RdDM pathway is ARGONAUTE4 (AGO4), which associates with siRNAs to mediate DNA methylation. Here, we show that AGO4 preferentially targets transposable elements embedded within promoters of protein-coding genes. This pattern of AGO4 binding cannot be simply explained by the sequences of AGO4-bound siRNAs; instead, AGO4 binding to specific gene promoters is also mediated by long non-coding RNAs (lncRNAs) produced by RNA polymerase V. lncRNA-mediated AGO4 binding to gene promoters directs asymmetric DNA methylation to these genomic regions and is involved in regulating the expression of targeted genes. Finally, AGO4 binding overlaps sites of DNA methylation affected by the biotic stress response. Based on these findings, we propose that the targets of AGO4-directed RdDM are regulatory units responsible for controlling gene expression under specific environmental conditions.

MicroRNAs (miRNAs) are short non-coding RNAs that negatively modulate gene expression at the post-transcriptional level and are known to be involved in the cross-talk between the host and virus. Using a standard plaque assay and real-time PCR method, we found that ectopic expression of miR-101 could significantly suppress herpes simplex virus-1 (HSV-1) replication, and that blocking endogenous miR-101 could increase viral progeny without affecting cell viability. Bioinformatics analysis indicates the 3' untranslated region (UTR) of mitochondrial ATP synthase subunit beta (ATP5B) has a putative binding site for miR-101. MiR-101 can directly bind to ATP5B 3'UTR and negatively regulate ATP5B expression. Using a RNA interference technique, knockdown of ATP5B significantly inhibited HSV-1 replication, indicating that ATP5B functions as a pro-viral factor. The ectopic expression of ATP5B lacking the 3'UTR could override the suppressive effect of miR-101 on HSV-1 replication. A concordant inverse correlation between miR-101 and ATP5B was observed in HSV-1-infected HeLa cells. Up-regulation of miR-101 expression may play a role in repressing productive HSV-1 replication by targeting ATP5B. Exploring the role of host-encoded miRNA in the regulation of viral infection would enable us to better understand the intricate networks of host-pathogen interactions.

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) large subunit (LS) Nepsilon-methyltransferase (Rubisco LSMT, EC 2.1.1.127) catalyzes methylation of the LS of Rubisco. A pea (Pisum sativum L. cv Laxton's Progress No. 9) Rubisco LSMT cDNA was expressed in Escherichia coli, but most of the expressed protein was found in the insoluble fraction as an inclusion body. Expression at lower temperatures increased the level of soluble Rubisco LSMT and the associated enzymatic activity. However, the soluble form of Rubisco LSMT occurred as two molecular mass forms with the lower molecular mass suggestive of N-terminal processing at Ser-37. Deletion of 108 nucleotides from the 5' end encoding the N-terminal 36 amino acids of Rubisco LSMT resulted in a 10-fold increase in solubility and activity. Further addition of a 3' nucleotide sequence coding for a hexahistidyl carboxy-terminal peptide enabled purification of the N-terminally truncated Rubisco LSMT to homogeneity. Five milligrams of pure recombinant Rubisco LSMT was obtained from a 1-liter E. coli cell culture. The apparent kinetic constants for recombinant Rubisco LSMT for spinach Rubisco and AdoMet were only slightly different from the constants determined using affinity-purified native Rubisco LSMT from pea chloroplasts. However, there was a 6- to 7-fold reduction in the kcat for Rubisco LSMT, which was apparently a consequence of catalytic inactivation due to exposure to NiSO4 during purification. The availability of larger quantities of purified Rubisco LSMT should enable studies of the structure-function relationships in Rubisco LSMT and moreover its interaction with Rubisco.

Background The genetic closeness and divergent muscle growth rates of broilers and layers make them great models for myogenesis study. In order to discover the molecular mechanisms determining the divergent muscle growth rates and muscle mass control in different chicken lines, we systematically identified differentially expressed genes between broiler and layer skeletal muscle cells during different developmental stages by microarray hybridization experiment. Results Taken together, 543 differentially expressed genes were identified between broilers and layers across different developmental stages. We found that differential regulation of slow-type muscle gene expression, satellite cell proliferation and differentiation, protein degradation rate and genes in some metabolic pathways could give great contributions to the divergent muscle growth rates of the two chicken lines. Interestingly, the expression profiles of a few differentially expressed genes were positively or negatively correlated with the growth rates of broilers and layers, indicating that those genes may function in regulating muscle growth during development. Conclusion The multiple muscle cell growth regulatory processes identified by our study implied that complicated molecular networks involved in the regulation of chicken muscle growth. These findings will not only offer genetic information for identifying candidate genes for chicken breeding, but also provide new clues for deciphering mechanisms underlining muscle development in vertebrates.