Toll‐like receptor 9 protects non‐immune cells from stress by modulating mitochondrial ATP synthesis through the inhibition of SERCA2

Toll‐like receptor 9 (TLR9) has a key role in the recognition of pathogen DNA in the context of infection and cellular DNA that is released from damaged cells. Pro‐inflammatory TLR9 signalling pathways in immune cells have been well investigated, but we have recently discovered an alternative pathway in which TLR9 temporarily reduces energy substrates to induce cellular protection from stress in cardiomyocytes and neurons. However, the mechanism by which TLR9 stimulation reduces energy substrates remained unknown. Here, we identify the calcium‐transporting ATPase, SERCA2 (also known as Atp2a2), as a key molecule for the alternative TLR9 signalling pathway. TLR9 stimulation reduces SERCA2 activity, modulating Ca²⁺ handling between the SR/ER and mitochondria, which leads to a decrease in mitochondrial ATP levels and the activation of cellular protective machinery. These findings reveal how distinct innate responses can be elicited in immune and non‐immune cells—including cardiomyocytes—using the same ligand‐receptor system.     

Physical and genetic structure of the maize genome reflects its complex evolutionary history

Maize (Zea mays L.) is one of the most important cereal crops and a model for the study of genetics, evolution, and domestication. To better understand maize genome organization and to build a framework for genome sequencing, we constructed a sequence-ready fingerprinted contig-based physical map that covers 93.5% of the genome, of which 86.1% is aligned to the genetic map. The fingerprinted contig map contains 25,908 genic markers that enabled us to align nearly 73% of the anchored maize genome to the rice genome. The distribution pattern of expressed sequence tags correlates to that of recombination. In collinear regions, 1 kb in rice corresponds to an average of 3.2 kb in maize, yet maize has a 6-fold genome size expansion. This can be explained by the fact that most rice regions correspond to two regions in maize as a result of its recent polyploid origin. Inversions account for the majority of chromosome structural variations during subsequent maize diploidization. We also find clear evidence of ancient genome duplication predating the divergence of the progenitors of maize and rice. Reconstructing the paleoethnobotany of the maize genome indicates that the progenitors of modern maize contained ten chromosomes.     

C-type lectin-like molecule-1 (CLL1)-targeted TRAIL augments the tumoricidal activity of granulocytes and potentiates therapeutic antibody-dependent cell-mediated cytotoxicity

The therapeutic effect of anti-cancer monoclonal antibodies stems from their capacity to opsonize targeted cancer cells with subsequent phagocytic removal, induction of antibody-dependent cell-mediated cytotoxicity (ADCC) or induction of complement-mediated cytotoxicity (CDC). The major immune effector cells involved in these processes are natural killer (NK) cells and granulocytes. The latter and most prevalent blood cell population contributes to phagocytosis, but is not effective in inducing ADCC. Here, we report that targeted delivery of the tumoricidal protein tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) to granulocyte marker C-type lectin-like molecule-1 (CLL1), using fusion protein CLL1:TRAIL, equips granulocytes with high levels of TRAIL. Upon CLL1-selective binding of this fusion protein, granulocytes acquire additional TRAIL-mediated cytotoxic activity that, importantly, potentiates antibody-mediated cytotoxicity of clinically used therapeutic antibodies (e.g., rituximab, cetuximab). Thus, CLL1:TRAIL could be used as an adjuvant to optimize the clinical potential of anticancer antibody therapy by augmenting tumoricidal activity of granulocytes.     

Activation of γ-aminobutyric acid insensitive chloride channels in mouse brain synaptic vesicles by avermectin B1a

The interaction of avermectin B_1a (AVMB_1a) with mouse brain chloride channels was characterized using a radiochloride efflux assay. The loss of intravesicular chloride from synaptoneurosomes preloaded with ³⁶Cl involved an initial rapid phase followed by a slower phase that approached equilibrium within 10 min. AVMB_1a stimulated a 30% loss of intravesicular chloride within the first 2 s of exposure; however, AVMB_1a had no effect on the rate of the slower phase of chloride loss. Experiments with lysed synaptoneurosomes showed that both chloride loading and basal and AVMB_1a-stimulated chloride release required the presence of intact vesicles. The efflux of ³⁶Cl from mouse brain synaptosomes and the stimulation of efflux by AVMB_1a were qualitatively similar to the results obtained with synaptoneurosomes but involved much lower overall levels of chloride loading and release. AVMB_1a produced halfmaximal stimulation of chloride efflux from synaptoneurosomes at a concentration of 2.1 ± 0.3 μM and a 35.4 ± 1.4% maximal loss of intravesicular chloride at saturating concentrations. γ-Aminobutyric acid (GABA), bicuculline, or the chloride channel blockers picrotoxinin, t-butylbicyclophosphorothionate (TBPS) 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), and anthracene 9-carboxylic acid (9-CA) had little or no effect on the loss of chloride from synaptoneurosomes either in the presence or the absence of AVMB_1a. However, the chlorinated cycloalkane insecticides dieldrin and lindane were equally effective as inhibitors of GABA-dependent chloride uptake and AVMB_1a-stimulated chloride efflux. These data demonstrate that AVMB_1a-stimulated chloride efflux from mouse brain synaptic vesicles results from the activation of GABA-insensitive chloride channels and that this action is distinct from their previously documented effects on GABA-gated chloride channels in mouse brain preparations. Our findings imply that both GABA-gated and GABA-insensitive chloride channels may be toxicologically significant targets for the action of avermectins.     

SyMAP v3.4: a turnkey synteny system with application to plant genomes

SyMAP (Synteny Mapping and Analysis Program) was originally developed to compute synteny blocks between a sequenced genome and a FPC map, and has been extended to support pairs of sequenced genomes. SyMAP uses MUMmer to compute the raw hits between the two genomes, which are then clustered and filtered using the optional gene annotation. The filtered hits are input to the synteny algorithm, which was designed to discover duplicated regions and form larger-scale synteny blocks, where intervening micro-rearrangements are allowed. SyMAP provides extensive interactive Java displays at all levels of resolution along with simultaneous displays of multiple aligned pairs. The synteny blocks from multiple chromosomes may be displayed in a high-level dot plot or three-dimensional view, and the user may then drill down to see the details of a region, including the alignments of the hits to the gene annotation. These capabilities are illustrated by showing their application to the study of genome duplication, differential gene loss and transitive homology between sorghum, maize and rice. The software may be used from a website or standalone for the best performance. A project manager is provided to organize and automate the analysis of multi-genome groups. The software is freely distributed at http://www.agcol.arizona.edu/software/symap.     

Cloning and functional characterization of a putative sodium channel auxiliary subunit gene from the house fly (Musca domestica)

The functional expression of cloned Drosophila melanogaster and house fly (Musca domestica) voltage-sensitive sodium channels in Xenopus oocytes is enhanced, and the inactivation kinetics of the expressed channels are accelerated, by coexpression with the tipE protein, a putative sodium channel auxiliary subunit encoded by the tipE gene of D. melanogaster. These results predict the existence of a tipE ortholog in the house fly. Using a PCR-based homology probing approach, we isolated cDNA clones encoding an ortholog of tipE (designated Vssc beta) from adult house fly heads. Clones comprising 3444 bp of cDNA sequence contained a 1317 bp open-reading frame encoding a 438 amino acid protein. The predicted Vssc beta protein exhibited 72% amino acid sequence identity to the entire D. melanogaster tipE protein sequence and 97% identity within the two hydrophobic segments identified as probable transmembrane domains. Coexpression of Vssc beta with the house fly sodium channel alpha subunit (Vssc1) in oocytes enhanced the level of sodium current expression five-fold and accelerated the rate of sodium current inactivation 2.2-fold. Both of these effects were significantly larger in magnitude than the corresponding effects of the D. melanogaster tipE protein on the expression and kinetics of Vssc1 sodium channels. These results identify a second example of a putative sodium channel auxiliary subunit from an insect having functional but not structural homology to vertebrate sodium channel beta subunits.     

妊娠对于父源性皮肤移植存活时间的影响

母胎耐受机制的阐明,将为器官移植免疫耐受方案的研究提供重要启示.本研究旨在阐明妊娠状态对父系来源移植皮片的存活是否有保护作用.2月龄雌性C57BL/6小鼠和2～4月龄BALB/c雄性小鼠同笼受孕.采用流式细胞技术确定妊娠过程中调节性T细胞（Treg）比例的时间变化规律.以单向混合淋巴细胞反应（MLR）手段比较研究妊娠对于父系来源脾细胞刺激后产生的增殖反应的影响.通过同种异体小鼠全厚皮片移植模型,观察妊娠对于父系来源移植皮片的存活是否具有保护作用.并用分子生物学技术研究此种效能的可能机制.结果显示,C57BL/6小鼠妊娠过程中,Treg占CD4＋T细胞的比例从妊娠前的4.2%逐渐上升,受孕8天左右达到高峰值（6.8%）,此后开始下降并逐渐回复至基线水平.MLR结果表明,针对父系来源脾细胞的刺激,妊娠组较对照组呈现显著的低反应性,其平均刺激指数分别是7.8和13.6（P〈0.05）.定量PCR研究表明,血红素加氧酶-1和吲哚胺2,3双加氧酶mRNA在胎盘高表达,在脾脏低表达（P〈0.05）.父系来源的移植皮片的平均存活时间在妊娠组和非妊娠组分别是7.67和7.08天,无统计学差异（P〉0.05）.由此认为,在小鼠妊娠过程中,尽管出现了具有免疫抑制功能的Treg的比例增加,尽管有针对父系来源刺激细胞的较低的MLR反应性,但是单次妊娠对于父系来源的移植皮片的存活,在本研究条件下,未能显示具有统计学意义的保护作用.     

大熊猫9个BAC的测序、注释和进化分析

本文构建了相当于大熊猫10倍基因组覆盖度的BAC文库, 并随机挑选了其中9个BAC进行测序和组装, 9个BAC的选择满足更多基因更少重复序列的原则. 这9个BAC的组装将为评估基于新一代Illumina GA测序技术的大熊猫全基因组测序及组装的准确性提供有效资源. 运用同源比对和从头预测的方法, 对9个BAC, 共约878 kb的序列进行了基因和重复序列的注释以及进化分析. 一共预测到12个蛋白编码基因, 其中, 7个基因匹配到同源基因的功能注释. 这7个基因平均大小约41 kb, 编码区平均大小约1.2 kb, 每个基因平均约含6个外显子. 同时预测到7个tRNA基因. 大约27%的序列被注释为重复序列. 同时, 基于邻接法, 构建了包含人、小鼠、狗、猫以及大熊猫5个物种的物种进化树, 结果显示狗的基因与其他4个物种相比距大熊猫最近. 本实验结果提供了大熊猫9个BAC的详细序列及注释信息, 为对大熊猫的研究提供了数据资源.