An integrated approach for the analysis of biological pathways using mixed models

Gene class, ontology, or pathway testing analysis has become increasingly popular in microarray data analysis. Such approaches allow the integration of gene annotation databases, such as Gene Ontology and KEGG Pathway, to formally test for subtle but coordinated changes at a system level. Higher power in gene class testing is gained by combining weak signals from a number of individual genes in each pathway. We propose an alternative approach for gene-class testing based on mixed models, a class of statistical models that: a) provides the ability to model and borrow strength across genes that are both up and down in a pathway, b) operates within a well-established statistical framework amenable to direct control of false positive or false discovery rates, c) exhibits improved power over widely used methods under normal location-based alternative hypotheses, and d) handles complex experimental designs for which permutation resampling is difficult. We compare the properties of this mixed models approach with nonparametric method GSEA and parametric method PAGE using a simulation study, and illustrate its application with a diabetes data set and a dose-response data set.     

百合不定芽的快速诱导

为获得麝香百合组织培养最优外植体及探讨蔗糖、6-BA和NAA的最佳用量,本文以MS固体培养基为基础培养基,通过4因素3水平(L9 (34)) 3次重复正交试验,研究不同花器官及不同浓度的蔗糖、6-BA (6 苄基嘌呤)和NAA (α-萘乙酸)对麝香百合组织培养诱导不定芽的影响.结果表明,花托为诱导不定芽最优外植体,诱导不定芽最佳培养基为MS+6-BA 1.0 mg/L+NAA 0.3 mg/L+蔗糖90 g/L.同时经过分析,可以判断本试验中设计的4个试验因素对百合组培的影响程度依次为外植体>蔗糖>奈乙酸和6-苄氨基嘌呤.本研究结果将为百合优良品种的生产提供重要的技术支撑和资源保证.     

Mitochondrial PKC‐ε deficiency promotes I/R‐mediated myocardial injury via GSK3β‐dependent mitochondrial permeability transition pore opening

Mitochondrial fission is critically involved in cardiomyocyte apoptosis, which has been considered as one of the leading causes of ischaemia/reperfusion (I/R)‐induced myocardial injury. In our previous works, we demonstrate that aldehyde dehydrogenase‐2 (ALDH2) deficiency aggravates cardiomyocyte apoptosis and cardiac dysfunction. The aim of this study was to elucidate whether ALDH2 deficiency promotes mitochondrial injury and cardiomyocyte death in response to I/R stress and the underlying mechanism. I/R injury was induced by aortic cross‐clamping for 45 min. followed by unclamping for 24 hrs in ALDH2 knockout (ALDH2^?/?) and wild‐type (WT) mice. Then myocardial infarct size, cell apoptosis and cardiac function were examined. The protein kinase C (PKC) isoform expressions and their mitochondrial translocation, the activity of dynamin‐related protein 1 (Drp1), caspase9 and caspase3 were determined by Western blot. The effects of N‐acetylcysteine (NAC) or PKC‐δ shRNA treatment on glycogen synthase kinase‐3β (GSK‐3β) activity and mitochondrial permeability transition pore (mPTP) opening were also detected. The results showed that ALDH2^?/? mice exhibited increased myocardial infarct size and cardiomyocyte apoptosis, enhanced levels of cleaved caspase9, caspase3 and phosphorylated Drp1. Mitochondrial PKC‐ε translocation was lower in ALDH2^?/? mice than in WT mice, and PKC‐δ was the opposite. Further data showed that mitochondrial PKC isoform ratio was regulated by cellular reactive oxygen species (ROS) level, which could be reversed by NAC pre‐treatment under I/R injury. In addition, PKC‐ε inhibition caused activation of caspase9, caspase3 and Drp1Ser⁶¹⁶ in response to I/R stress. Importantly, expression of phosphorylated GSK‐3β (inactive form) was lower in ALDH2^?/? mice than in WT mice, and both were increased by NAC pre‐treatment. I/R‐induced mitochondrial translocation of GSK‐3β was inhibited by PKC‐δ shRNA or NAC pre‐treatment. In addition, mitochondrial membrane potential (?Ψ_m) was reduced in ALDH2^?/? mice after I/R, which was partly reversed by the GSK‐3β inhibitor (SB216763) or PKC‐δ shRNA. Collectively, our data provide the evidence that abnormal PKC‐ε/PKC‐δ ratio promotes the activation of Drp1 signalling, caspase cascades and GSK‐3β‐dependent mPTP opening, which results in mitochondrial injury‐triggered cardiomyocyte apoptosis and myocardial dysfuction in ALDH2^?/? mice following I/R stress.     

Hyperactivation of Mammalian Target of Rapamycin Complex 1 (mTORC1) Promotes Breast Cancer Progression through Enhancing Glucose Starvation-induced Autophagy and Akt Signaling

The mammalian target of rapamycin complex 1 (mTORC1) is a master regulator of cell growth and proliferation. Recent studies have suggested that constitutive activation of mTORC1 in normal cells could lead to malignant tumor development in several tissues. However, the mechanisms of mTORC1 hyperactivation to promote the growth and metastasis of breast or other cancers are still not well characterized. Here, using a new inducible deletion system, we show that deletion of Tsc1 in mouse primary mammary tumor cells, either before or after their transplantation, significantly increased their growth in vivo. The increase in tumor growth was completely rescued by rapamycin treatment, suggesting a major contribution from mTORC1 hyperactivation. Interestingly, glucose starvation-induced autophagy, but not amino acid starvation-induced autophagy, was increased significantly in Tsc1-null tumor cells. Further analysis of these cells also showed an increased Akt activation but no significant changes in Erk signaling. Together, these results provide insights into the mechanism by which hyperactivation of mTORC1 promotes breast cancer progression through increasing autophagy and Akt activation in vivo.     

Defluviitalea phaphyphila sp. nov., a Novel Thermophilic Bacterium That Degrades Brown Algae

Brown algae are one of the largest groups of oceanic primary producers for CO₂ removal and carbon sinks for coastal regions. However, the mechanism for brown alga assimilation remains largely unknown in thermophilic microorganisms. In this work, a thermophilic alginolytic community was enriched from coastal sediment, from which an obligate anaerobic and thermophilic bacterial strain, designated Alg1, was isolated. Alg1 shared a 16S rRNA gene identity of 94.6% with Defluviitalea saccharophila LIND6LT2^T. Phenotypic, chemotaxonomic, and phylogenetic studies suggested strain Alg1 represented a novel species of the genus Defluviitalea, for which the name Defluviitalea phaphyphila sp. nov. is proposed. Alg1 exhibited an intriguing ability to convert carbohydrates of brown algae, including alginate, laminarin, and mannitol, to ethanol and acetic acid. Three gene clusters participating in this process were predicted to be in the genome, and candidate enzymes were successfully expressed, purified, and characterized. Six alginate lyases were demonstrated to synergistically deconstruct alginate into unsaturated monosaccharide, followed by one uronic acid reductase and two 2-keto-3-deoxy-d-gluconate (KDG) kinases to produce pyruvate. A nonclassical mannitol 1-phosphate dehydrogenase, catalyzing d-mannitol 1-phosphate to fructose 1-phosphate in the presence of NAD⁺, and one laminarase also were disclosed. This work revealed that a thermophilic brown alga-decomposing system containing numerous novel thermophilic alginate lyases and a unique mannitol 1-phosphate dehydrogenase was adopted by the natural ethanologenic strain Alg1 during the process of evolution in hostile habitats.     

PRIMARY STRUCTURE OF ALLATOSTATIN 4, A NEVROPEPTIDE INHIBITOR OF JUVENILE HORMONE SYNTHESIS

Abstract Isolation and identification of allatostatin 4 (AST 4) from crude brain extracts of female Diploptera punctata are described. Synthetic analogues of allatostatin 4 truncated from the N-terminal were evaluated to gain some knowledge of structure-activity correlation. AST 4 reversibly inhibits the biosynthesis of JH in vitro . AST 4 has the following sequence: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH₂. This neurohormone (AST 4) has no sequence similarity with any known neuropeptide from other organisms. Synthetic AST 4 as well as truncation fragments, including two with the five and six amino acids terminal residues deleted showed in vitro activity distinguishable from that of the native allatostatin.     

Chitosan enhanced gene delivery of cationic liposome via non-covalent conjugation

Two new types of stable ternary complexes were formed by mixing chitosan with DOTAP/pDNA lipoplex and DOTAP with chitosan/pDNA polyplex via non-covalent conjugation for the efficient delivery of plasmid DNA. They were characterized by atomic force microscopy, gel retarding, and dynamic light scattering. The DOTAP/CTS/pDNA complexes were in compacted spheroids and irregular lump of larger aggregates in structure, while the short rod- and toroid-like and donut shapes were found in CTS/DOTAP/pDNA complexes. The transfection efficiency of the lipopolyplexes showed higher GFP gene expression than DOTAP/pDNA and CTS/pDNA controls in Hep-2 and Hela cells, and luciferase gene expression 2–3-fold than DOTAP/pDNA control and 70–120-fold than CTS/pDNA control in Hep-2 cells. The intracellular trafficking was examined by confocal laser scanning microscopy. Rapid pDNA delivery to the nucleus enchanced by chitosan was achieved after 4 h transfection.     

A male sterility-associated cytotoxic protein ORF288 in Brassica juncea causes aborted pollen development

Cytoplasmic male sterility (CMS) is a widespread phenomenon in higher plants, and several studies have established that this maternally inherited defect is often associated with a mitochondrial mutant. Approximately 10 chimeric genes have been identified as being associated with corresponding CMS systems in the family Brassicaceae, but there is little direct evidence that these genes cause male sterility. In this study, a novel chimeric gene (named orf288) was found to be located downstream of the atp6 gene and co-transcribed with this gene in the hau CMS sterile line. Western blotting analysis showed that this predicted open reading frame (ORF) was translated in the mitochondria of male-sterile plants. Furthermore, the growth of Escherichia coli was significantly repressed in the presence of ORF288, which indicated that this protein is toxic to the E. coli host cells. To confirm further the function of orf288 in male sterility, the gene was fused to a mitochondrial-targeting pre-sequence under the control of the Arabidopsis APETALA3 promoter and introduced into Arabidopsis thaliana. Almost 80% of transgenic plants with orf288 failed to develop anthers. It was also found that the independent expression of orf288 caused male sterility in transgenic plants, even without the transit pre-sequence. Furthermore, transient expression of orf288 and green fluorescent protein (GFP) as a fused protein in A. thaliana protoplasts showed that ORF288 was able to anchor to mitochondria even without the external mitochondrial-targeting peptide. These observations provide important evidence that orf288 is responsible for the male sterility of hau CMS in Brassica juncea.     

Ultrathin hydrogel films for rapid optical biosensing

Novel biosensors have been designed by reporting an analyte-induced (de)swelling of a stimuli-responsive hydrogel (usually in a form of thin film) with a suitable optical transducer. These simple, inexpensive hydrogel biosensors are highly desirable, however, their practical applications have been hindered, largely because of their slow response. Here we show that quick response hydrogel sensors can be designed from ultrathin hydrogel films. By the adoption of layer-by-layer assembly, a simple but versatile approach, glucose-sensitive hydrogel films with thickness on submicrometer or micrometer scale, which is 2 orders of magnitude thinner than films used in ordinary hydrogel sensors, can be facilely fabricated. The hydrogel films can not only respond to the variation in glucose concentration, but also report the event via the shift of Fabry-Perot fringes using the thin film itself as Fabry-Perot cavity. The response is linear and reversible. More importantly, the response is quite fast, making it possible to be used for continuous glucose monitoring.