Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies

In this report we investigated, within a group of closely related single domain camelid antibodies (V_HHs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast‐acting toxin and biothreat agent. The V1C7‐like V_HHs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin‐neutralizing activities. Using the X‐ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta‐based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1_R29G mutant was largely devoid of toxin‐neutralizing activity (TNA). However, the TNA of the V1C7_G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen‐deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function.     

Induction,purification, and characterization of a thermo and pH stable laccase from Abortiporus biennis J2 and its application on the clarification of litchi juice

A fungus J2 producing laccase with high yield was screened in soils and identified as Abortiporus biennis. The production of laccase was induced by 0.1 mM Cu²⁺, 0.1 mM tannic acid, and 0.5 M ethanol. The laccase from Abortiporus biennis J2 was purified to electrophoretic homogeneity by a couple of steps. The N-terminal amino acid sequence of the enzyme was AIGPTADLNISNADI. The properties of the purified laccase were investigated. The result showed the laccase from Abortiporus biennis J2 is a thermo and pH stable enzyme. The laccase activity was inhibited by Hg²⁺, Cd²⁺, Fe²⁺, Ag⁺, Cu²⁺, and Zn²⁺, while promoted by Mg²⁺, Mn²⁺ at 10 mM level. Purified laccase was used to the clarification of litchi juice. After treatment with this laccase, the phenolic content of litchi juice had been found to be greatly reduced along with an increase in the clarity of the juice. The result indicated the potential of this laccase for application in juice procession.     

玉米蛋白粉替代鱼粉对大黄鱼生长、血清生化指标及肝脏组织学的影响

为探讨玉米蛋白粉替代鱼粉对大黄鱼(Larimichthys crocea) 幼鱼生长、血清生化指标及肝脏组织形态的影响, 进行了为期56d的养殖试验, 探索玉米蛋白粉替代大黄鱼幼鱼饲料鱼粉的适当比例。以初始体重为(10.49±0.03) g的大黄鱼幼鱼为研究对象, 用玉米蛋白粉替代基础饲料(含40%鱼粉)0、15%、30%、45%、60%和75%的鱼粉来配制6种等氮(粗蛋白含量45%)等脂(粗脂肪含量10%)的实验饲料, 分别标记为C0、C15、C30、C45、C60、C75组。除C0以外的替代组分别添加了适量的晶体氨基酸(赖氨酸和蛋氨酸)。结果表明, 玉米蛋白粉替代水平对大黄鱼幼鱼存活率、试验鱼特定生长率、饲料系数均无显著性差异(P>0.05)。C45和C60组肌肉粗蛋白含量显著高于C0组(P<0.05), 肌肉粗脂肪含量C45组显著高于C0、C15和C75组 (P<0.05), C45、C60和C75组肌肉水分显著低于C0组(P<0.05), 全鱼粗蛋白、粗脂肪、水分含量无显著差异(P>0.05); 灰分含量有上升趋势, C75组显著高于其他组(P<0.05)。各替代组的血清白蛋白、白球比、谷草转氨酶、葡萄糖均无显著性差异(P>0.05); 随着替代比例的升高, 总胆固醇有降低的趋势, 除C15组与C0无显著性差异外(P>0.05), 4组均显著低于CO组(P<0.05); 实验探讨替代后对鱼的影响, C75组血清总蛋白和球蛋白含量显著低于C0组(P<0.05); C75组血清谷草转氨酶含量显著高于C0(P<0.05)。各处理组总抗氧化能力、过氧化氢酶、谷胱甘肽过氧化氢酶均没有显著性影响(P>0.05), 但替代组总抗氧化能力含量均高于C0; 丙二醛在C75组显著高于C0组(P<0.05)。通过肝组织学观察表明, 当替代比例高于45%时, 呈现肝细胞核偏位、胞浆内脂肪滴较多、细胞透明空泡化等症状。综上所述, 在实验条件下, 研究认为玉米蛋白粉替代鱼粉对大黄鱼幼鱼的适宜添加量为45%。     

石榴木质素合成相关基因PgMYB308的克隆和表达分析

该研究以‘红玉石籽’(Punica granatumcv.Hongyushizi)石榴为试验材料,采用RACE和RT-PCR方法,获得与木质素的合成相关基因PgMYB308。PgMYB308基因cDNA全长792bp,编码263个氨基酸。分子量为29.56kD,理论等电点为9.07。序列比对和功能域分析发现,PgMYB308包含R2和R3保守域,以及C1、C2、C4和锌指基序。系统进化树分析显示,PgMYB308与其他物种起源相同,而与桉树EgMYB308亲缘关系最近。荧光定量PCR分析表明,PgMYB308在茎、叶和种子等组织中均有表达,其中茎中表达量最高,叶片中表达量最低;PgMYB308在‘突尼斯软籽’中表达最高,而在‘红玉石籽’和‘白玉石籽’中表达量较低;PgMYB308在‘红玉石籽’籽粒的不同时期均有表达,但在花后20d相对表达量最高,以后随发育进程呈现逐渐下降的趋势。     

Phage-host associations in a full-scale activated sludge plant during sludge bulking

Sludge bulking, a notorious microbial issue in activated sludge plants, is always accompanied by dramatic changes in the bacterial community. Despite large numbers of phages in sludge systems, their responses to sludge bulking and phage-host associations during bulking are unknown. In this study, high-throughput sequencing of viral metagenomes and bacterial 16S rRNA genes were employed to characterize viral and bacterial communities in a sludge plant under different sludge conditions (sludge volume index (SVI) of 180, 132, and 73 ml/g). Bulking sludges (SVI > 125 ml/g) taken about 10 months apart exhibited similar bacterial and viral composition. This reflects ecological resilience of the sludge microbial community and indicates that changes in viral and bacterial populations correlate closely with each other. Overgrowth of “Candidatus Microthrix parvicella” led to filamentous bulking, but few corresponding viral genotypes were identified. In contrast, sludge viromes were characterized by numerous contigs associated with “Candidatus Accumulibacter phosphatis,” suggesting an abundance of corresponding phages in the sludge viral community. Notably, while nitrifiers (mainly Nitrosomonadaceae and Nitrospiraceae) declined significantly along with sludge bulking, their corresponding viral contigs were identified more frequently and with greater abundance in the bulking viromes, implying that phage-mediated lysis might contribute to the loss of autotrophic nitrifiers under bulking conditions.     

Molecular characterization of <Emphasis Type="Italic">HOXC8</Emphasis> gene and methylation status analysis of its exon 1 associated with the length of cashmere fiber in Liaoning cashmere goat

Homeobox protein Hox-C8 (HOXC8) is a member of Hox family. It is expressed in the dermal papilla of the skin and is thought to be associated with the hair inductive capacity of dermal papilla cells. In the present study, we isolated and characterized a full-length open reading frame of HOXC8 cDNA from the skin tissue of Liaoning cashmere goat, as well as, established a phylogenetic relationship of goat HOXC8 with that of other species. Also, we investigated the effect of methylation status of HOXC8 exon 1 at anagen secondary hair follicle on the cashmere fiber traits in Liaoning cashmere goat. The sequence analysis indicated that the obtained cDNA was 1134-bp in length containing a complete ORF of 729-bp. It encoded a peptide of 242 amino acid residues in length. The structural analysis indicated that goat HOXC8 contained a typical homeobox domain. The phylogenetic analysis revealed that Capra hircus HOXC8 had a closer genetic relationship with that of Ovis aries, followed by Bos Taurus and Bubalus bubalis. The methylation analysis suggested that the methylation degree of HOXC8 exon 1 in anagen secondary hair follicle might be involved in regulating the growth of cashmere fiber in Liaoning cashmere goat. Our results provide new evidence for understanding the molecular structural and evolutionary characteristics of HOXC8 in Liaoning cashmere goat, as well as, for further insight into the role of methylation degree of HOXC8 exon 1 regulates the growth of cashmere fiber in goat.     

Bone marrow mesenchymal stem cell transplantation improves radiation-induced heart injury through DNA damage repair in rat model

Radiotherapy is an effective form of therapy for most thoracic malignant tumors. However, myocardial injury resulting from the high doses of radiation is a severe complication. Here we aimed to study the possibility of reducing radiation-induced myocardial injury with mesenchymal stem cell (MSC) transplantation. We used MSCs extracted from bone marrow (BMSCs) to transplant via the tail vein into a radiation-induced heart injury (RIHI) rat model. The rats were divided into six groups: a Sham group, an IRR (irradiation) group, and four IRR + BMSCs transplantation groups obtained at different time points. After irradiation, BMSC transplantation significantly enhanced the cardiac function in rats. By analyzing the expression of PPAR-α, PPAR-γ, TGF-β, IL-6, and IL-8, we found that BMSC transplantation alleviated radiation-induced myocardial fibrosis and decreased the inflammatory reaction. Furthermore, we found that expression of γ-H2AX, XRCC4, DNA ligase4, and TP53BP1, which are associated with DNA repair, was up-regulated, along with increased secretion of growth factors SDF-1, CXCR4, VEGF, and IGF in rat myocardium in the IRR + BMSCs transplantation groups compared with the IRR group. Thus, BMSC transplantation has the potential to improve RIHI via DNA repair and be a new therapeutic approach for patients with myocardial injury.     

Effects of potassium deficiency on root morphology,ultrastructure and antioxidant enzyme system in sweet potato (<Emphasis Type="Italic">Ipomoea batatas</Emphasis> [L.] Lam.) during early growth

Potassium (K⁺) deficiency is an important abiotic stress which has severe influence on the growth and development of sweet potato. To investigate the difference on root morphology, ultrastructure and antioxidant enzyme system at early growth stage, two representative sweet potato cultivars (Ipomoea batatas [L.] Lam.) with different K⁺ deficiency tolerance capacities were hydroponically cultivated under normal K⁺ (control) and K⁺ deficiency (?K) treatments. The results showed that after 14 days of treatment, the root length, surface area, volume, and average diameter of Ningzishu 1 (sensitive to K⁺ deficiency) were significantly decreased under ?K treatment, by comparison, those corresponding decreases of Xushu 32 (tolerant to K⁺ deficiency) were lower and not significant (except for root length). In addition, the proportion of fine roots (diameter <0.5 mm) and thick root (d > 1.0 mm) of Xushu 32 seedlings increased significantly under condition of K⁺ deficiency. Through transmission electron microscopy observations, it can be found that Xushu 32 seedlings showed a more complete root cellular structure and slighter oxidative damage than Ningzishu 1 under ?K treatment. Moreover, the hydrogen peroxide content and malondialdehyde content in the root of Xushu 32 seedlings was significantly lower than that of Ningzishu 1, and the variations of superoxide dismutase, peroxidase, and catalase activities were pronouncedly less than those of Ningzishu 1. These differences between the two cultivars indicated that the stronger root morphology and nutrients absorption ability, and the better root cellular structure and physiological role of Xushu 32 seedlings could alleviate the damage of K⁺ deficiency stress. Thus, it might be a potential mechanism for cultivar tolerance to low K⁺ in sweet potato.     

Exogenous spermidine alleviates fruit granulation in a <Emphasis Type="Italic">Citrus</Emphasis> cultivar (<Emphasis Type="Italic">Huangguogan</Emphasis>) through the antioxidant pathway

The role of exogenous spermidine (Spd) in alleviating fruit granulation in the grafted seedlings of a Citrus cultivar (Huangguogan) was investigated. Granulation resulted in increased electrical conductivity, cell membrane permeability, and total pectin, soluble pectin, cellulose, and lignin contents. However, it decreased the activities of superoxide dismutase, peroxidase, and catalase, as well as the (Spd + Spm):Put ratio. The application of exogenous Spd onto Huangguogan seedlings significantly increased proline and ascorbate contents, but decreased the H₂O₂ and O ₂ ^?· levels, which suggested that exogenous Spd provided some protection from oxidative damage. In addition, exogenous Spd decreased cell membrane permeability and MDA content, and increased the (Spd + Spm):Put ratio. The activities of antioxidant enzymes, such as catalase, peroxidase, and superoxide dismutase, were increased in Spd-treated seedlings affected by fruit granulation, resulting in a decrease in oxidative stress levels. The protective effects of Spd were reflected by a decrease in superoxide levels through osmoregulation, increased proline and ascorbate contents, and increased antioxidant activities. Our observations reveal the importance of exogenous Spd in alleviating citrus fruit granulation.