Caspase-1 and -3 mRNAs are differentially upregulated in motor neurons and glial cells in mutant SOD1 transgenic mouse spinal cord: a study using laser microdissection and real-time RT-PCR

Amyotrophic lateral sclerosis is characterized by selective motor neuron degeneration. An apoptotic pathway is thought to be involved. It is difficult, however, to analyze the molecular pathogenic mechanism in single motor neurons because of complexity in the neural tissue, which consists of multiple lineages of cells neighboring motor neurons. We quantified the caspase-1 and -3 mRNA in single motor neurons and neighboring glial cells isolated from the spinal ventral horn of mutant SOD1 transgenic (Tg) mice and littermates. Motor neurons and neighboring glial cells were isolated from spinal sections by laser microdissection, and the mRNAs were quantified by RT-PCR. In the Tg mice, caspase-1 mRNA was first upregulated in motor neurons and second in glial cells. The caspase-3 mRNA was increased in motor neurons following the caspase-1 mRNA. These results indicated that caspase-1 and -3 mRNAs are differentially upregulated in motor neurons and glial cells of the Tg mice, and that mRNAs in isolated cells can be accurately assessed using our procedures.     

Nucleotide modifications in three functionally important regions of the Saccharomyces cerevisiae ribosome affect translation accuracy

Important regions of rRNA are rich in nucleotide modifications that can have strong effects on ribosome biogenesis and translation efficiency. Here, we examine the influence of pseudouridylation and 2′-O-methylation on translation accuracy in yeast, by deleting the corresponding guide snoRNAs. The regions analyzed were: the decoding centre (eight modifications), and two intersubunit bridge domains—the A-site finger and Helix 69 (six and five modifications). Results show that a number of modifications influence accuracy with effects ranging from 0.3- to 2.4-fold of wild-type activity. Blocking subsets of modifications, especially from the decoding region, impairs stop codon termination and reading frame maintenance. Unexpectedly, several Helix 69 mutants possess ribosomes with increased fidelity. Consistent with strong positional and synergistic effects is the finding that single deletions can have a more pronounced phenotype than multiple deficiencies in the same region. Altogether, the results demonstrate that rRNA modifications have significant roles in translation accuracy.     

Amazonian Anthrosols Support Similar Microbial Communities that Differ Distinctly from Those Extant in Adjacent, Unmodified Soils of the Same Mineralogy

We compared the microbial community composition in soils from the Brazilian Amazon with two contrasting histories; anthrosols and their adjacent non-anthrosol soils of the same mineralogy. The anthrosols, also known as the Amazonian Dark Earths or terra preta, were managed by the indigenous pre-Colombian Indians between 500 and 8,700 years before present and are characterized by unusually high cation exchange capacity, phosphorus (P), and calcium (Ca) contents, and soil carbon pools that contain a high proportion of incompletely combusted biomass as biochar or black carbon (BC). We sampled paired anthrosol and unmodified soils from four locations in the Manaus, Brazil, region that differed in their current land use and soil type. Community DNA was extracted from sampled soils and characterized by use of denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism. DNA bands of interest from Bacteria and Archaea DGGE gels were cloned and sequenced. In cluster analyses of the DNA fingerprints, microbial communities from the anthrosols grouped together regardless of current land use or soil type and were distinct from those in their respective, paired adjacent soils. For the Archaea, the anthrosol communities diverged from the adjacent soils by over 90%. A greater overall richness was observed for Bacteria sequences as compared with those of the Archaea. Most of the sequences obtained were novel and matched those in databases at less than 98% similarity. Several sequences obtained only from the anthrosols grouped at 93% similarity with the Verrucomicrobia, a genus commonly found in rice paddies in the tropics. Sequences closely related to Proteobacteria and Cyanobacteria sp. were recovered only from adjacent soil samples. Sequences related to Pseudomonas, Acidobacteria, and Flexibacter sp. were recovered from both anthrosols and adjacent soils. The strong similarities among the microbial communities present in the anthrosols for both the Bacteria and Archaea suggests that the microbial community composition in these soils is controlled more strongly by their historical soil management than by soil type or current land use. The anthrosols had consistently higher concentrations of incompletely combusted organic black carbon material (BC), higher soil pH, and higher concentrations of P and Ca compared to their respective adjacent soils. Such characteristics may help to explain the longevity and distinctiveness of the anthrosols in the Amazonian landscape and guide us in recreating soils with sustained high fertility in otherwise nutrient-poor soils in modern times.     

水稻幼芽细胞生物膜上的赤霉素结合蛋白的结合特性

在水稻 (Oryza sativa)幼芽中存在膜结合的赤霉素结合蛋白 ,其与 GA3 结合的平衡解离常数(Kd)为 6.5× 1 0 -8mol/ L,总浓度为 0 .3 pmol· mg-1 蛋白质。结合蛋白与 GA3 结合活力在 0℃时比 2 5℃时高 1 4 0 %。它与 GA3 结合的最适 p H为 5。 GA3 与此结合蛋白的结合量随反应时间延长而增加 ,1 h达最大值 ,以后又逐渐下降。 IAA、ABA可与 GA3 竞争赤霉素结合蛋白。     

Beclin1-binding UVRAG targets the class C Vps complex to coordinate autophagosome maturation and endocytic trafficking

Autophagic and endocytic pathways are tightly regulated membrane rearrangement processes that are crucial for homeostasis, development and disease. Autophagic cargo is delivered from autophagosomes to lysosomes for degradation through a complex process that topologically resembles endosomal maturation. Here, we report that a Beclin1-binding autophagic tumour suppressor, UVRAG, interacts with the class C Vps complex, a key component of the endosomal fusion machinery. This interaction stimulates Rab7 GTPase activity and autophagosome fusion with late endosomes/lysosomes, thereby enhancing delivery and degradation of autophagic cargo. Furthermore, the UVRAG-class-C-Vps complex accelerates endosome-endosome fusion, resulting in rapid degradation of endocytic cargo. Remarkably, autophagosome/endosome maturation mediated by the UVRAG-class-C-Vps complex is genetically separable from UVRAG-Beclin1-mediated autophagosome formation. This result indicates that UVRAG functions as a multivalent trafficking effector that regulates not only two important steps of autophagy - autophagosome formation and maturation - but also endosomal fusion, which concomitantly promotes transport of autophagic and endocytic cargo to the degradative compartments.     

Large-scale expansion of Vγ9Vδ2 T cells with engineered K562 feeder cells in G-Rex vessels and their use as chimeric antigen receptor–modified effector cells

Vγ9Vδ2 T cells are a minor subset of lymphocytes in the peripheral blood that has been extensively investigated for their tolerability, safety and anticancer efficacy. A hindrance to the broad application of these cells for adoptive cellular immunotherapy has been attaining clinically appropriate numbers of Vγ9Vδ2 T cells. Furthermore, Vγ9Vδ2 T cells exist at low frequencies among cancer patients. We, therefore, sought to conceive an economical method that allows for a quick and robust large-scale expansion of Vγ9Vδ2 T cells. A two-step protocol was developed, in which peripheral blood mononuclear cells (PBMCs) from healthy donors or cancer patients were activated with Zometa and interleukin (IL)-2, followed by co-culturing with gamma-irradiated, CD64-, CD86- and CD137L-expressing K562 artificial antigen-presenting cells (aAPCs) in the presence of the anti-CD3 antibody OKT3. We optimized the co-culture ratio of K562 aAPCs to immune cells, and migrated this method to a G-Rex cell growth platform to derive clinically relevant cell numbers in a Good Manufacturing Practice (GMP)-compliant manner. We further include a depletion step to selectively remove αβ T lymphocytes. The method exhibited high expansion folds and a specific enrichment of Vγ9Vδ2 T cells. Expanded Vγ9Vδ2 T cells displayed an effector memory phenotype with a concomitant down-regulated expression of inhibitory immune checkpoint receptors. Finally, we ascertained the cytotoxic activity of these expanded cells by using nonmodified and chimeric antigen receptor (CAR)–engrafted Vγ9Vδ2 T cells against a panel of solid tumor cells. Overall, we report an efficient approach to generate highly functional Vγ9Vδ2 T cells in massive numbers suitable for clinical application in an allogeneic setting.     

Comparative analysis of complete chloroplast genome sequences of two subtropical trees, <Emphasis Type="Italic">Phoebe sheareri</Emphasis> and <Emphasis Type="Italic">Phoebe omeiensis</Emphasis> (Lauraceae)

Phoebe is an economically important genus from the family Lauraceae. It is widely distributed in tropical and subtropical Asia, but systematics of the genus is unclear, and currently there is no species-level phylogeny. Here, we determined the complete chloroplast genome sequences of two species with long-range PCR and next genome sequencing technologies, and identified mutation sites and highly variable regions. These highly variable sites were used to reconstruct the phylogeny. The plastomes of Phoebe sheareri and P. omeiensis were 152, 876, and 152, 855 bp, respectively. Comparative genomic analysis indicated that there are 222 mutation sites including 146 substitutions, 73 indels, and 3 microinversions in both plastomes. Fifty-six single-nucleotide changes were identified in gene-coding regions, and 45 microsatellite sites were found for use in species identification. Fourteen divergence hotspots of 38 variable regions were located. Phylogeny was reconstructed using a Bayesian and maximum likelihood approach for 12 Phoebe species and other five related Lauraceae based on 15 of the highly variable regions including accD-psaI, atpB-rbcL, ndhC-trnV, ndhF-rpl32, petA-psbJ, psaA, psbA-trnH, rbcL, rps8-rpl14, rps16-trnQ, rpl32-trnL, trnC-petN, trnL-trnF, trnS-trnG, and ycf1 indicated that variability in the chloroplast regions proposed as variable is enough to detect divergence events among 12 taxa of Phoebe, and that maybe also useful to help to elucidate further relationships among other taxa of the genus.     

Plasma metabonomics as a novel diagnostic approach for major depressive disorder

Major depressive disorder (MDD) is a socially detrimental psychiatric disorder, contributing to increased healthcare expenditures and suicide rates. However, no empirical laboratory-based tests are available to support the diagnosis of MDD. In this study, a NMR-based plasma metabonomic method for the diagnosis of MDD was tested. Proton nuclear magnetic resonance ((1)H NMR) spectra of plasma sampled from first-episode drug-na??ve depressed patients (n = 58) and healthy controls (n = 42) were recorded and analyzed by orthogonal partial least-squares discriminant analysis (OPLS-DA). The OPLS-DA score plots of the spectra demonstrated that the depressed patient group was significantly distinguishable from the healthy control group. Moreover, the method accurately diagnosed blinded samples (n = 26) in an independent replication cohort with a sensitivity and specificity of 92.8% and 83.3%, respectively. Taken together, NMR-based plasma metabonomics may offer an accurate empirical laboratory-based method applicable to the diagnosis of MDD.     

峨眉髭蟾精子形态结构及分类学意义

应用透射电镜、扫描电镜和光学显微镜对峨眉髭蟾(Vibrissaphoraboringii)精子的形态和超微结构研究的结果表明:峨眉髭蟾的精子具角蟾科物种精子基本的形态和结构特征,即精子头部呈螺旋状,尾部呈弯曲状;精子具锥形的顶体、纤维束构成的穿孔器、平行排列的中心粒和双轴丝;线粒体位于尾部;精子核窝不明显、无轴纤维和波动膜等特征。此外,对已有报道的角蟾科和无尾类物种精子的特征进行分析比较表明:(1)角蟾科精子细胞核呈螺旋状,中心粒平行排列,尾部具双轴丝等结构不同于无尾类其他科精子的结构,具有明显的科间差别;(2)角蟾科精子各部的量度,尾部线粒体的分布和数量,以及轴丝的排列等特征在属间和种间表现出明显的差异;(3)峨眉髭蟾和东南亚拟髭蟾指名亚种精子的形态和超微结构存在明显的差异。