Identification of miR-30e* Regulation of Bmi1 Expression Mediated by Tumor-Associated Macrophages in Gastrointestinal Cancer

Bmi1 is overexpressed in a variety of human cancers including gastrointestinal cancer. The high expression level of Bmi1 protein is associated with poor prognosis of gastrointestinal cancer patients. On the other hand, tumor-associated macrophages (TAMs) contribute to tumor growth, invasion, and metastasis by producing various mediators in the tumor microenvironment. The aim of this study was to investigate TAM-mediated regulation of Bmi1 expression in gastrointestinal cancer. The relationship between TAMs and Bmi1 expression was analyzed by immunohistochemistry and quantitative real-time PCR (qRT-PCR), and results showed a positive correlation with tumor-infiltrating macrophages (CD68 and CD163) and Bmi1 expression in cancer cells. Co-culture with TAMs triggered Bmi1 expression in cancer cell lines and enhanced sphere formation ability. miRNA microarray analysis of a gastric cancer cell line co-cultured with macrophages was conducted, and using in silico methods to analyze the results, we identified miR-30e* as a potential regulator of Bmi1 expression. Luciferase assays using miR-30e* mimic revealed that Bmi1 was a direct target for miR-30e* by interactions with the putative miR-30e* binding sites in the Bmi1 3′ untranslated region. qRT-PCR analysis of resected cancer specimens showed that miR-30e* expression was downregulated in tumor regions compared with non-tumor regions, and Bmi1 expression was inversely correlated with miR-30e* expression in gastric cancer tissues, but not in colon cancer tissues. Our findings suggest that TAMs may cause increased Bmi1 expression through miR-30e* suppression, leading to tumor progression. The suppression of Bmi1 expression mediated by TAMs may thus represent a possible strategy as the treatment of gastrointestinal cancer.     

Direct and Indirect Control of the Initiation of Meiotic Recombination by DNA Damage Checkpoint Mechanisms in Budding Yeast

Meiotic recombination plays an essential role in the proper segregation of chromosomes at meiosis I in many sexually reproducing organisms. Meiotic recombination is initiated by the scheduled formation of genome-wide DNA double-strand breaks (DSBs). The timing of DSB formation is strictly controlled because unscheduled DSB formation is detrimental to genome integrity. Here, we investigated the role of DNA damage checkpoint mechanisms in the control of meiotic DSB formation using budding yeast. By using recombination defective mutants in which meiotic DSBs are not repaired, the effect of DNA damage checkpoint mutations on DSB formation was evaluated. The Tel1 (ATM) pathway mainly responds to unresected DSB ends, thus the sae2 mutant background in which DSB ends remain intact was employed. On the other hand, the Mec1 (ATR) pathway is primarily used when DSB ends are resected, thus the rad51 dmc1 double mutant background was employed in which highly resected DSBs accumulate. In order to separate the effect caused by unscheduled cell cycle progression, which is often associated with DNA damage checkpoint defects, we also employed the ndt80 mutation which permanently arrests the meiotic cell cycle at prophase I. In the absence of Tel1, DSB formation was reduced in larger chromosomes (IV, VII, II and XI) whereas no significant reduction was found in smaller chromosomes (III and VI). On the other hand, the absence of Rad17 (a critical component of the ATR pathway) lead to an increase in DSB formation (chromosomes VII and II were tested). We propose that, within prophase I, the Tel1 pathway facilitates DSB formation, especially in bigger chromosomes, while the Mec1 pathway negatively regulates DSB formation. We also identified prophase I exit, which is under the control of the DNA damage checkpoint machinery, to be a critical event associated with down-regulating meiotic DSB formation.     

Rates of Serious Intracellular Infections in Autoimmune Disease Patients Receiving Initial Glucocorticoid Therapy

Background/Aims

The Japanese National Hospital Organization evidence-based medicine (EBM) Study group for Adverse effects of Corticosteroid therapy (J-NHOSAC) is a Japanese hospital-based cohort study investigating the safety of the initial use of glucocorticoids (GCs) in patients with newly diagnosed autoimmune diseases. Using the J-NHOSAC registry, the purpose of this observational study is to analyse the rates, characteristics and associated risk factors of intracellular infections in patients with newly diagnosed autoimmune diseases who were initially treated with GCs.

Methodology/Principal Findings

A total 604 patients with newly diagnosed autoimmune diseases treated with GCs were enrolled in this registry between April 2007 and March 2009. Cox proportional-hazards regression was used to determine independent risk factors for serious intracellular infections with covariates including sex, age, co-morbidity, laboratory data, use of immunosuppressants and dose of GCs. Survival was analysed according to the Kaplan-Meier method and was assessed by the log-rank test. There were 127 serious infections, including 43 intracellular infections, during 1105.8 patient-years of follow-up. The 43 serious intracellular infections resulted in 8 deaths. After adjustment for covariates, diabetes (Odds ratio [OR]: 2.5, 95% confidence interval [95% CI] 1.1–5.9), lymphocytopenia (≦1000/μl, OR: 2.5, 95% CI 1.2–5.2) and use of high-dose (≧30 mg/day) GCs (OR: 2.4, 95% CI 1.1–5.3) increased the risk of intracellular infections. Survival curves showed lower intracellular infection-free survival rate in patients with diabetes, lymphocytopaenia and high-dose GCs treatments.

Conclusions/Significance

Patients with newly diagnosed autoimmune diseases were at high risk of developing intracellular infection during initial treatment with GCs. Our findings provide background data on the risk of intracellular infections of patients with autoimmune diseases. Clinicians showed remain vigilant for intracellular infections in patients with autoimmune diseases who are treated with GCs.     

Increasing cell-free gene expression yields from linear templates in Escherichia coli and Vibrio natriegens extracts by using DNA-binding proteins

In crude extract-based cell-free protein synthesis (CFPS), DNA templates are transcribed and translated into functional proteins. Although linear expression templates (LETs) are less laborious and expensive to generate, plasmid templates are often desired over polymerase chain reaction-generated LETs due to increased stability and protection against exonucleases present in the extract of the reaction. Here we demonstrate that addition of a double stranded DNA-binding protein to the CFPS reaction, termed single-chain Cro protein (scCro), achieves terminal protection of LETs. This CroP-LET (scCro-based protection of LET) method effectively increases superfolder green fluorescent protein (sfGFP) expression levels from LETs in Escherichia coli CFPS reactions by sixfold. Our yields are comparable to other strategies that provide chemical and enzymatic DNA stabilization in E. coli CFPS. Notably, we also report that the CroP-LET method successfully enhanced yields in CFPS platforms derived from nonmodel organisms. Our results show that CroP-LET increased sfGFP yields by 18-fold in the Vibrio natriegens CFPS platform. With the fast-expanding applications of CFPS platforms, this method provides a practical and generalizable solution to protect linear expression DNA templates.     

Gibberellin mediates the development of gelatinous fibres in the tension wood of inclined Acacia mangium seedlings

Background and Aims

Gibberellin stimulates negative gravitropism and the formation of tension wood in tilted Acacia mangium seedlings, while inhibitors of gibberellin synthesis strongly inhibit the return to vertical growth and suppress the formation of tension wood. To characterize the role of gibberellin in tension wood formation and gravitropism, this study investigated the role of gibberellin in the development of gelatinous fibres and in the changes in anatomical characteristics of woody elements in Acacia mangium seedlings exposed to a gravitational stimulus.

Methods

Gibberellin, paclobutrazol and uniconazole-P were applied to the soil in which seedlings were growing, using distilled water as the control. Three days after the start of treatment, seedlings were inclined at 45 ° to the vertical and samples were harvested 2 months later. The effects of the treatments on wood fibres, vessel elements and ray parenchyma cells were analysed in tension wood in the upper part of inclined stems and in the opposite wood on the lower side of inclined stems.

Key Results

Application of paclobutrazol or uniconazole-P inhibited the increase in the thickness of gelatinous layers and prevented the elongation of gelatinous fibres in the tension wood of inclined stems. By contrast, gibberellin stimulated the elongation of these fibres. Application of gibberellin and inhibitors of gibberellin biosynthesis had only minor effects on the anatomical characteristics of vessel and ray parenchyma cells.

Conclusions

The results suggest that gibberellin is important for the development of gelatinous fibres in the tension wood of A. mangium seedlings and therefore in gravitropism.     

Bacillus brevis,a Host Bacterium for Efficient Extracellular Production of Useful Proteins

Abstract

Since its discovery in 1998 RNA interference (RNAi), a potent and highly selective gene silencing mechanism, has revolutionized the field of biological science. The ability of RNAi to specifically down-regulate the expression of any cellular protein has had a profound impact on the study of gene function in vitro. This property of RNAi also holds great promise for in vivo functional genomics and interventions against a wide spectrum of diseases, especially those with “undruggable” therapeutic targets. Despite the enormous potential of RNAi for medicine, development of in vivo applications has met with significant problems, particularly in terms of delivery. For effective gene silencing to occur, silencing RNA must reach the cytoplasm of the target cell. Consequently, various strategies using chemically modified siRNA, liposomes, nanoparticles and viral vectors are being developed to deliver silencing RNA. These approaches, however, can be expensive and in many cases they lack target cell specificity or clinical compatibility. Recently, we have shown that RNAi can be activated in vitro and in vivo by non-pathogenic bacteria engineered to manufacture     

Single Quantum Dot Tracking Reveals that an Individual Multivalent HIV-1 Tat Protein Transduction Domain Can Activate Machinery for Lateral Transport and Endocytosis

The mechanisms underlying the cellular entry of the HIV-1 Tat protein transduction domain (TatP) and the molecular information necessary to improve the transduction efficiency of TatP remain unclear due to the technical limitations for direct visualization of TatP''s behavior in cells. Using confocal microscopy, total internal reflection fluorescence microscopy, and four-dimensional microscopy, we developed a single-molecule tracking assay for TatP labeled with quantum dots (QDs) to examine the kinetics of TatP initially and immediately before, at the beginning of, and immediately after entry into living cells. We report that even when the number of multivalent TatP (mTatP)-QDs bound to a cell was low, each single mTatP-QD first locally induced the cell''s lateral transport machinery to move the mTatP-QD toward the center of the cell body upon cross-linking of heparan sulfate proteoglycans. The centripetal and lateral movements were linked to the integrity and flow of actomyosin and microtubules. Individual mTatP underwent lipid raft-mediated temporal confinement, followed by complete immobilization, which ultimately led to endocytotic internalization. However, bivalent TatP did not sufficiently promote either cell surface movement or internalization. Together, these findings provide clues regarding the mechanisms of TatP cell entry and indicate that increasing the valence of TatP on nanoparticles allows them to behave as cargo delivery nanomachines.     

Effects of combined centrifugation and ultraviolet irradiation of eggs on pattern formation in Chironomus embryos

Summary

Combined mild centrifugation and uv irradiation of Chironomus embryos modified the developmental types expected from centrifugation alone, somewhat differently from the combined strong centrifugation and uv irradiation of Smittia embryos. The modifications changed with the stages irradiated. The change caused by anterior irradiation may depend on whether or not a part of the cytoplasmic zone is irradiated simultaneously with the anterior yolky end; because most of the cytoplasm lies in the posterior half of egg at early irradiation, while the tip of the cytoplasm redistributes near the anterior end by the late irradiation. Early uv irradiation of the anterior end of centrifuged eggs, causing the formation of a double abdomen (DA) or an inverted embryo, is not photoreversible, while the uv damage to the anterior end of uncentrifuged eggs, inducing DA, is. These facts suggest that there is another photoirreversible uv target in addition to the photoreversible target for DA induction or the anterior determinant shown in Smittia. Other changes, such as the induction of a double cephalon by late irradiation of the centrifuged egg, are photoreversible, but in an unusual way in that the level of photorecovery is similar to the result of incubation in the dark after early irradiation, and not to that of the centrifuged controls. These modified results were then compared with those for Smittia embryos.     

Irreversibility and Interatomic Potentials in One-dimensional Lattice Models

Abstract

Since the computational work on the relaxation process toward the equilibrium state by Fermi, Pasta, and Ulam (FPU) [1], the stochastic behaviors in one dimensional lattice models have been studied by many physicists numerically and theoretically. The objective of those studies is to understand the origin of thermodynamical properties from the microscopic particle motions which are determined by the deterministic equations of motion. In the FPU work, since the given energy was too small and anharmonicity was too weak, thermodynamic irreversibility phenomena, which lead to the equipartition of energy among modes, were not observed. Many studies after this work confirmed the existence of the threshold for the chaotic motions [2] [3]. However, the relation of the chaotic motion in the system with many degrees of freedom to the thermodynamical properties is not well understood [4]. The steady lattice thermal conduction in one dimensional lattice poses an interesting problem in this; respect. A clear linear internal temperature gradient was not observed in the FPU model [5], while it was observed in the the ding-a-ling model by Casati et al. [6] and the diatomic Toda lattice (DTL) by Mokross and Büttner [7]. Recently, the system size dependence of the coefficient of thermal conductivity has been studied [8][9] and the Fourier's law of heat conduction is begining to be confirmed in the DTL.