Additional file 2: Figure S1. of Identification and characterization of conserved lncRNAs in human and rat brain

The expression of conserved lncRNAs compared with the expression of non-conserved lncRNAs and protein-coding genes in human brain. (PDF 53 kb

Additional file 1: of Identification and characterization of conserved lncRNAs in human and rat brain

List of conserved lncRNAs and Table S1. (DOCX 33 kb

Additional file 3: Figure S2. of Identification and characterization of conserved lncRNAs in human and rat brain

Multiple rat lncRNAs locate in the orthologous region of a human lncRNA RP11-472I20.3–001. (PDF 120 kb

Expression and purification of L-asparaginase from <i>Escherichia coli</i> and the inhibitory effects of cyclic dipeptides

<p>L-asparaginase, a key enzyme involved in nitrogen metabolism, is an effective anti-tumour agent. Cyclic dipeptides, a group of compounds, contain several important biological functions. In this paper, we proposed a novel method for L-asparaginase expression and purification from <i>Echerichia coli</i> and determined the effect of cyclic dipeptides on the enzymatic activity of recombinant L-asparaginase. The gene <i>ansB</i> encoding L-asparaginase was amplified from the genome of <i>E. coli</i> BL21 (DE3) by polymerase chain reaction and sub-cloned into pET-15b vector to construct expressing plasmid pET-15b-ansB. The expression of recombinant protein was purified by affinity chromatography using a nickel resin followed by anion exchange chromatography. The purity and quality of the recombinant L-asparaginase were optimised. The results indicated that km for the recombinant L-asparaginase was 3.02 × 10⁻⁴ mol/L. Both cyclo-(Pro-Tyr) and cyclo-(Pro-Phe) could inhibit the activity of recombinant L-asparaginase at the level of 10⁻⁵ mol/L.</p

Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells

<div><p>Mechanisms of the progression from Barrett’s esophagus (BE) to esophageal adenocarcinoma (EA) are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK) inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H₂O₂ production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H₂O₂ production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H₂O₂ production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.</p></div

Isolation and transcription start site mapping of the <i>Xenopus</i> ALF promoter.

<p>(A) PCR reactions were performed with <i>X. laevis</i> genomic DNA that had been digested with EcoRV, HincII, PvuII, and StuI. The gene specific primer (GSP) was located 70 base pairs downstream of the 5′ end of ALF mRNA. AP is the adaptor primer. (B) The 1.7 kb HincII ALF promoter fragment contains a DNA transposon and two other repeats, examples of which are aligned in the figure. (C) To map the start site, RT-PCR reactions were performed with oocyte RNA using primers located at various locations throughout the ALF promoter region (S0-S6). The results show strong bands with primers S5 and S6 (lanes 6, 7), weaker bands with primers S3 and S4 (lanes 4, 5), and no bands with primers S1 and S2 (lanes 2, 3). (D) Sequence analysis of nearly 40 RACE clones shows the distribution of start sites throughout the promoter region. The number of hits observed at each position is indicated. Locations of the ATG and primers S3-S6 are indicated. (E) Sodium bisulfite methylation analysis of the ALF promoter shows a high degree of methylation (filled circles) in liver tissue where the gene is normally off, and little to no methylation (open circles) in oocytes where the ALF gene is normally on. Filled cirlces represent methylation while open circles represent no methylation.</p

Data_Sheet_1_A novel Toxoplasma gondii TGGT1_316290 mRNA-LNP vaccine elicits protective immune response against toxoplasmosis in mice.ZIP

Toxoplasma gondii (T. gondii) can infect almost all warm-blooded animals and is a major threat to global public health. Currently, there is no effective drug or vaccine for T. gondii. In this study, bioinformatics analysis on B and T cell epitopes revealed that TGGT1_316290 (TG290) had superior effects compared with the surface antigen 1 (SAG1). TG290 mRNA-LNP was constructed through the Lipid Nanoparticle (LNP) technology and intramuscularly injected into the BALB/c mice, and its immunogenicity and efficacy were explored. Analysis of antibodies, cytokines (IFN-γ, IL-12, IL-4, and IL-10), lymphocytes proliferation, cytotoxic T lymphocyte activity, dendritic cell (DC) maturation, as well as CD4+ and CD8+ T lymphocytes revealed that TG290 mRNA-LNP induced humoral and cellular immune responses in vaccinated mice. Furthermore, T-Box 21 (T-bet), nuclear factor kappa B (NF-kB) p65, and interferon regulatory factor 8 (IRF8) subunit were over-expressed in the TG290 mRNA-LNP-immunized group. The survival time of mice injected with TG290 mRNA-LNP was significantly longer (18.7 ± 3 days) compared with the survival of mice of the control groups (p 7) of mice immunized with TG290 mRNA-LNP significantly prolonged the survival time of these mice. This study demonstrates that TG290 mRNA-LNP induces specific immune response against T. gondii and may be a potential toxoplasmosis vaccine candidate for this infection.</p

Effects of activating and repressing regulatory factors on endogenous and introduced promoter DNA in somatic cells and germ cells.

<p>Silencing factors are red/black while activating factors are green. Solid lines show paths of function while dashed lines indicate either absence or failure to function. (A) Silencing factors do not repress an introduced germ cell gene promoter in somatic cells, allowing activating factors to (inappropriately) drive transcription. (B) Silencing factors are absent or do not function on endogenous and introduced promoters in germ cells, while activating factors result in transcription from both types of DNA.</p

Identification of oocyte proteins that interact with the A and B elements.

<p>(A) A 96 bp fragment from the ALF85+ promoter was labeled and used as the probe in EMSA assays with oocyte extracts. A summary of the position of the A and B elements and the factor binding sites are shown in the two top lines. Beneath this is shown the relative locations of a series of overlapping oligonocleotide competitors. (B) An additional set of oligonucleotide competitors that contained specific mutations were also used as competitors in the binding assays. (C) Bandshift analysis shows the ALF promoter forms several protein-DNA complexes using oocyte-derived cell-free extracts. The main complexes are indicated by the labels 1, 2, and 3. The P2-05 competitor selectively abolishes complex 1 (lane 2), while the P2-89 competitor selectively abolishes complex 3 and to a lesser extent complex 2 (lane 6). (D) Additional competition assays show that P2-05-M1 but not P2-05-M2 is able to compete for complex 3 (compare lanes 3 and 4). Similarly, the P2-89-M2 competitor but not P2-89-M1 is able to compete for binding of complexes 2 and 3 (compare lanes 7 and 8). (E) Competition with mutant oligos P2-05-M3, P2-89-M3, and P2-89-M4 further refines the binding site to the positions noted in the ‘Complex Formation’ line in (A).</p

NOX5-S expression and H₂O₂ production in FLO-1 EA cells after ROCK2 overexpression.

<p>(A) A typical example of three Western blot analysis showed that ROCK2 protein was successfully overexpressed in FLO-1 cells after transfection with ROCK2 expression plasmid. (B) A typical example of three Western blot analysis and (C) summarized data showed that overexpression of constitutively active ROCK2 significantly increased NOX5-S protein expression. (D) Overexpression of constitutively active ROCK2 significantly increased H₂O₂ production in FLO-1 EA cells. The data suggest that ROCK2 may contribute to acid-induced increase in NOX5-S expression and H₂O₂ production. N = 3, ANOVA,* P<0.03, ** P<0.001, compared with control plasmid group.</p