Development of highly polymorphic SNP markers from the complexity reduced portion of maize [Zea mays L.] genome for use in marker-assisted breeding

The duplicated and the highly repetitive nature of the maize genome has historically impeded the development of true single nucleotide polymorphism (SNP) markers in this crop. Recent advances in genome complexity reduction methods coupled with sequencing-by-synthesis technologies permit the implementation of efficient genome-wide SNP discovery in maize. In this study, we have applied Complexity Reduction of Polymorphic Sequences technology (Keygene N.V., Wageningen, The Netherlands) for the identification of informative SNPs between two genetically distinct maize inbred lines of North and South American origins. This approach resulted in the discovery of 1,123 putative SNPs representing low and single copy loci. In silico and experimental (Illumina GoldenGate (GG) assay) validation of putative SNPs resulted in mapping of 604 markers, out of which 188 SNPs represented 43 haplotype blocks distributed across all ten chromosomes. We have determined and clearly stated a specific combination of stringent criteria (>0.3 minor allele frequency, >0.8 GenTrainScore and >0.5 Chi_test100 score) necessary for the identification of highly polymorphic and genetically stable SNP markers. Due to these criteria, we identified a subset of 120 high-quality SNP markers to leverage in GG assay-based marker-assisted selection projects. A total of 32 high-quality SNPs represented 21 haplotypes out of 43 identified in this study. The information on the selection criteria of highly polymorphic SNPs in a complex genome such as maize and the public availability of these SNP assays will be of great value for the maize molecular genetics and breeding community.     

Different protective roles in vitro of alpha- and beta-domains of growth inhibitory factor (GIF) on neuron injuries caused by oxygen free radicals.

It was well known that beta-amyloid (Abeta) and tau protein play an important role in pathological procedure of Alzheimer's disease (AD), a senile dementia. The growth inhibitory factor (GIF, also named metallothionein-3, MT-3) had been demonstrated to inhibit the outgrowth of cortex neurons in the medium with extract of the AD patient brain. In our experiments, it was found that the neurons of cortex and the PC12 (pheochromocytoma) cells could be protected from the cytotoxicity of beta-amyloid 25-35 in presence of GIF and its domains. Additionally, GIF can scavenge the hydroxyl radical efficiently in CytC-VitC radical producing system and its alpha-domain shown more effective potentials than its beta-domain. The electron paramagnetic resonance spectra also show that the alpha-domain has more potential ability for eliminating reactive oxygen free radicals than its beta-domain. The results suggest that GIF could act as an efficient scavenger against free radicals in vitro and the alpha-domain in GIF molecule shows more potential in protecting against reactive oxygen species injury than the beta-domain.     

Organization and possible origin of the Bari-1 cluster in the heterochromatic h39 region of Drosophila melanogaster

The molecular organization of the heterochromatic h39 region of the Drosophila melanogaster second chromosome has been investigated by studying two BAC clones identified both by Southern blotting and by FISH experiments as containing tandem arrays of Bari1, a transposable element present only in this region. Such BAC clones appear to contain different portions of the h39 region since they differ in the DNA sequences flanking the Bari1 repeats on both sides. Thus, the 80 Bari1 copies estimated to be present in the h39 region are split into at least two separated subregions. On the basis of the analysis of the flanking sequences a possible mechanism depending on an aberrant activity of the Bari1 transposase is proposed for the genesis of the heterochromatic tandem arrays of the element.     

Development of mechanistically based model for simulating soluble microbial products generation in an aerated/non-aerated SBR

Soluble microbial products (SMPs) are considered as the main organic components in wastewater treatment plant effluent from biological wastewater treatment systems. To investigate and explore SMP metabolism pathway for further treatment and control, two innovative mechanistically based activated sludge models were developed by extension of activated sludge model no.3 (ASM3). One was the model by combining SMP formation and degradation (ASM3-SMP model) processes with ASM3, and the other by combining both SMP and simultaneous substrate storage and growth (SSSG) mechanisms with ASM3 (SSSG-ASM3-SMP model). The detailed schematic modification and process supplements were introduced for comprehensively understanding all the mechanisms involved in the activated sludge process. The evaluations of these two models were demonstrated by a laboratory-scale sequencing batch reactor (SBR) operated under aerated/non-aerated conditions. The simulated and measured results indicated that SMP comprised about 83% of total soluble chemical oxygen demand (SCOD) in which biomass-associated products (BAPs) were predominant compared with utilization-associated products (UAPs). It also elucidated that there should be a minimum SMP value as the reactive time increases continuously and this conclusion could be used to optimize effluent SCOD in activated sludge processes. The comparative results among ASM3, ASM3-SMP and SSSG-ASM3-SMP models and the experimental measurements (SCOD, ammonia and nitrate nitrogen) showed clearly the best agreement with SSSG-ASM3-SMP simulation values (R = 0.993), strongly suggesting that both SMP formation and degradation and SSSG mechanisms are necessary in biologically activated sludge modeling for municipal wastewater treatment.     

Engineering mouse apolipoprotein A-I into a monomeric, active protein useful for structural determination

Apolipoprotein AI (apoAI), the major protein component of HDL, is one of the best predictors of coronary artery disease (CAD), with high apoAI and HDL levels being correlated with low occurrences of CAD. The primary function of apoAI is to recruit phospholipid and cholesterol for assembly of HDL particles. Like other exchangeable apolipoproteins, lipid-free apoAI forms a mixture of different oligomers even at 1.0 mg/mL. This self-association property of the exchangeable apolipoproteins is closely associated with the lipoprotein-binding activity of this protein family. It is unclear if the self-association property of apolipoprotein is required for its lipoprotein-binding activity. We developed a novel method for engineering an oligomeric protein to a monomeric, biologically active protein. Using this method, we generated a monomeric mouse apoAI mutant that is active. This mutant contains the first 216 residues of mouse apoAI and replaces six hydrophobic residues with either polar or smaller hydrophobic residues at the defined positions (V118A/A119S/L121Q/T191S/T195S/T199S). Cross-linking results show that this mutant is greater than 90% monomeric at 8 mg/mL. CD, DSC, and NMR results indicate that the mutant maintains an identical secondary, tertiary structure and stability as those of the wild-type mouse apoAI. Lipid-binding assays suggest that the mutant shares an equal lipoprotein-binding activity as that of the wild-type apoAI. In addition, both the monomeric mutant and the wild-type protein make nearly identical rHDL particles. With this monomeric mouse apoAI, high-quality NMR data has been collected, allowing for the NMR structural determination of lipid-free apoAI. On the basis of these results, we conclude that this apoAI mutant is a monomeric, active apoAI useful for structural determination.     

Wnt/β-catenin信号通路在博来霉素诱导的系统性硬化症小鼠模型血管重塑中的作用

系统性硬化症(systemic sclerosis,SSc)是一种慢性可累及全身多脏器的自身免疫性疾病,以广泛的血管病变及皮肤和内脏的纤维化为特征,但其机制迄今尚不明确。已有研究证实,Wnt通路参与了SSc纤维化,但其在血管病变中的病理作用尚未见报道。本研究拟采用博来霉素(bleomycin,BLM)诱导的SSc小鼠模型,探讨Wnt通路在SSc皮肤血管病变中的作用。将18只Balb/C小鼠随机平均分为3组,分别设为对照组(于小鼠背部皮下注射PBS 100 μL/d)、模型组(于小鼠背部皮下注射浓度为 1 mg/mL 博来霉素BLM 100 μL/d)和治疗组(于小鼠背部皮下注射 1 mg/mL BLM 100 μL/d,同时腹腔注射Wnt及β-catenin的抑制剂 iCRT3 5 mg/kg·d),于造模第28 d处死小鼠。小鼠皮肤取材后,通过HE染色及Masson染色观察到经BLM诱导的模型组小鼠背真皮、表皮厚度较对照组皮肤均明显增加(P<0.05),同时模型组的皮脂腺、毛囊等皮肤附属器明显减少,脂肪层厚度变薄并被纤维组织包绕,模型组皮肤胶原沉积较对照组增加;通过免疫组织化学染色在组织学层面鉴定α-SMA表达情况,发现模型组及治疗组α-SMA在皮肤组织中均高表达,α-SMA阳性表达在血管周围较对照组明显增加;通过ELISA方法检测出模型组小鼠血清中IL-6及IL-17表达量较对照组明显升高(P<0.05),治疗组小鼠血清中IL-6及IL-17的表达量较模型组明显下降(P<0.05);提取皮肤微血管片段,通过q-PCR检测到模型组及治疗组小鼠皮肤微血管中β-联蛋白的mRNA基因表达水平较正常组升高;通过Western印迹检测皮肤微血管Wnt5A、β-联蛋白、α-SMA、col1A1的蛋白质表达情况,发现纤维化相关蛋白质α-SMA及col1A1在模型组表达升高,较对照组有统计学差异(P<0.05),治疗组较模型组表达下降(P<0.05),Wnt通路相关蛋白质β-联蛋白及Wnt5A在模型组表达明显升高,较之对照组有统计学差异(P<0.05)。本研究提示,BLM能成功诱导小鼠系统性硬化症皮肤表型,Wnt通路的异常激活参与了BLM诱导的硬皮病小鼠皮肤微血管病变,特异性Wnt通路抑制剂iCRT3可能通过直接或间接的方式下调细胞因子IL-6及IL-17,从而降低BLM诱导的小鼠皮肤微血管中的α-SMA及col1A1蛋白质表达,改善小鼠皮肤微血管病变,干预BLM诱导的小鼠血管病变的进展。