核酸探针与酶免疫反应板定向结合的方法

为了制备一种可供寡核苷酸探针定向结合的酶免疫反应板, 利用部分水解的尼龙包被酶免疫反应板,通过尼龙所含的氨基基团与寡核苷酸探针所含的5′磷酸基团,在水溶性碳二亚胺的催化下,与酶免疫反应板发生5′端特异性定向共价结合, 并对肺炎支原体的PCR扩增产物进行了重复检测,结果重复性好,方法稳定.利用该方法制备的酶免疫反应板可用于核酸探针的临床检测.     

石羊河流域自然植被对生态系统服务的约束效应

石羊河流域生态环境退化,生态屏障功能减弱,提高该地区生态系统的服务功能具有重要意义。植被的生长状况是影响生态系统服务供给的重要因素,定量的探讨分析植被与生态系统服务之间的关系十分必要。以石羊河流域为研究区,采用约束线法,分析了植被对产水量、土壤保持及固碳3个生态系统服务的约束效应;同时,计算了2015年这3个生态系统服务潜力的空间分布及石羊河流域综合潜力分布格局。结果表明:(1)植被覆盖度与产水量、土壤保持之间的约束线呈开口向下抛物线型,即随着植被覆盖度的增加,约束效应逐渐减小,后逐渐增大;与固碳服务呈正凸型,即:植被覆盖度能有效的约束固碳,且成比例增加约束效应;(2)植被覆盖度对产水量、土壤保持及固碳服务的约束效应存在时空异质性;(3)石羊河流域三种服务的提升潜力均呈西南高东北低、高服务值与高潜力值并存的分布特征,其中东大河、西大河、西营河及古浪河是流域生态服务的主要供给者和服务潜力提升的主要贡献者。     

不同土地利用方式下酚酸物质与土壤微生物群落的关系

酚酸物质是影响微生物生物量和群落结构的重要因子之一,研究酚酸物质在不同土地利用方式下的变化规律及其与微生物群落结构的关系,有助于更好地理解不同土地利用方式下微生物群落变化的作用机制。以山河屯林业局奋斗林场次生林(SF)、落叶松人工林(LP)、农田地(FL)和撂荒地(AL)为研究对象,测定不同土地利用方式下0—5 cm、5—10 cm和10—20 cm层的土壤总酚、复合态酚、水溶性酚和9种酚酸物质,并采用磷脂脂肪酸法(PLFA)测定这4种土地利用方式的土壤微生物群落。结果表明,各土层的落叶松人工林土壤总酚含量显著高于其他三种土地利用方式。在0—5 cm和5—10 cm土层中,落叶松人工林土壤水溶性酚含量最高,而在10—20 cm土层中,则是次生林显著高于其余三种土地利用方式(P0.05)。在0—5 cm土层中,次生林土壤的总PLFA、真菌含量比农田地和撂荒地分别高14.61%、80.91%和55.63%、156.55%,同时,次生林的土壤真菌∶细菌(F∶B)显著高于落叶松人工林、农田地和撂荒地(P0.05)。0—5 cm层和5—10 cm层的土壤总酚与微生物群落(细菌、真菌)分别呈正相关和负相关关系,而在10—20 cm层,三种土壤酚类物质与微生物群落均未达到显著相关(P0.05)。冗余分析表明,0—5 cm层土壤中的阿魏酸、2,4-二羟基苯甲酸和β-谷甾醇均对真菌群落和F∶B有显著影响(P0.05),而在10—20 cm层中,只有β-谷甾醇影响了微生物群落的生长。土地利用方式的变化改变了表层土壤的酚酸物质含量和微生物群落结构,酚酸物质对表层土壤各类群的微生物量有明显的促进作用,但抑制了深层土壤微生物的生长。     

沉积再悬浮颗粒物对马氏珠母贝摄食生理影响的室内模拟

采用实验生态学方法室内模拟研究了不同浓度沉积再悬浮颗粒物对马氏珠母贝清滤率、摄食率、吸收率的影响。结果表明:(1)水体中总悬浮颗粒物对马氏珠母贝清滤率的影响极显著(P<0.01)。总悬浮颗粒物由低浓度(12.6 mg/L)趋高浓度(500 mg/L)时,马氏珠母贝的清滤率呈现峰值变化规律。与总悬浮颗粒物浓度50 mg/L时的最大清滤率(1.12 L.个体-1.h-1)比较,悬浮颗粒物浓度为500 mg/L时,清滤率达最小值(0.17 L.个体-1.h-1)),其清滤率降幅达85%。这表明在高浓度悬浮颗粒物的水环境下,贝类受到环境胁迫,其生理和自身摄食机制受到限制,引起摄食减少和机体损伤。马氏珠母贝类的清滤率(CR)与总悬浮颗粒物浓度(TPM)之间的关系可表达为:CR=-0.701+1.627×TPM-0.463×TPM2+0.036×TPM3(R2=0.928)。(2)水体中总悬浮颗粒物对马氏珠母贝摄食率的影响极显著(P<0.01)。马氏珠母贝的摄食率随着总悬浮颗粒物浓度的升高而增加,在50 mg/L时达最大值(38.28 mg/h),当总悬浮颗粒物浓度超过50 mg/L时,摄食率反而下降,在总悬浮颗粒物浓度为500 mg/L时,降为最小值(16.22 mg/h),摄食率降幅为58%。随着悬浮颗粒物浓度的增加,马氏珠母贝摄食率受到的影响小于清滤率受到的影响。马氏珠母贝类的摄食率(IR)与总悬浮颗粒物浓度(TPM)之间的关系可表达为:IR=-46.631+70.957×TPM-18.385×TPM2+1.367×TPM3(R2=0.907)。(3)水体中总悬浮颗粒物对马氏珠母贝吸收率影响极显著(P<0.01)。总悬浮颗粒物由低浓度(12.6 mg/L)趋高浓度(500 mg/L)时,马氏珠母贝的吸收率呈逐渐下降趋势,在总悬浮颗粒物12.6 mg/L时,马氏珠母贝的吸收率最大(48.57%),而总悬浮颗粒物500 mg/L时,马氏珠母贝的吸收率最小(8.56%)。马氏珠母贝的吸收率(AE)与总悬浮颗粒物浓度(TPM)之间的关系可表达为:AE=52.189+0.132×TPM-3.111×TPM2+0.316×TPM3(R2=0.976)。     

Engineering a thermostable β-1,3-1,4-glucanase from Paecilomyces thermophila to improve catalytic efficiency at acidic pH

To fulfill the need for acid-tolerant and thermostable β-1,3-1,4-glucanases, an error-prone PCR and DNA-shuffling approach was employed to enhance the activity of thermostable β-1,3-1,4-glucanases from Paecilomyces thermophila (PtLic16A) at acidic pH. Mutant PtLic16AM2 was selected and characterized, and showed optimal activity at pH 5.0, corresponding to an acidic shift of 2.0 pH units relative to the wild-type enzyme. Other properties of PtLic16A such as temperature optimum and substrate specificity that are beneficial for industrial applications did not change. Based on the substituted residues of PtLic16AM2, three site-directed mutations, D56G, D221G and C263S, were designed to study these residues' roles. The amino acid residues at positions 56 and 263 were found to be important in determining optimal pH activity. Activity of the D221G variant showed no significant difference from the wild-type. Thus, it appears that the change in optimal pH for PtLic16AM2 was mainly caused by the combination of substitutions D56G and C263S. This study provides a β-1,3-1,4-glucanase (PtLic16AM2) with high potential for industrial applications.     

Gibel carp Carassius auratus gut microbiota after oral administration of trimethoprim/ sulfamethoxazole

Trimethoprim/sulfamethoxazole is widely used in the treatment of infectious diseases caused by bacterial pathogens in aquaculture. However, the practice of antibiotic administration can promote the emergence of resistant strains of bacteria and result in a wane in efficacy over time. The objective of this study was to assess the effect of oral treatment with trimethoprim/sulfamethoxazole on the gastrointestinal (GI) microbiota of healthy gibel carp and those affected with bacterial enteritis. By using denaturing gradient gel electrophoresis (DGGE), the changes in the predominant bacterial communities were directly depicted for the first time. The main findings were (1) Actinobacteria, Firmicutes and Proteobacteria were the predominant phyla in the healthy gibel carp intestine; (2) administration of antibiotics had a more profound impact on the intestinal microflora of healthy fish than of the diseased ones; and (3) Enterobacteriaceae might be one of the major drug-resistant bacteria in the gibel carp intestine. This study provides an insight into the effect of antibiotic treatment on the establishment and colonization of fish GI microbiota and speculates on some possible drug-resistant bacteria.     

Molecular Characterization, Mapping, and Haplotype Analysis of Porcine Matrix Metalloproteinase Genes MMP1 and MMP10

Matrix metalloproteinases (MMPs) are a family of enzymes that cleave protein components of the extracellular matrix, such as collagens, laminin, fibronectin, and proteoglycans, playing a role in degradation of the matrix of the uterus and in embryo implantation. We report the identification of two members of the MMP gene family in swine. The porcine MMP1 and MMP10 genes comprise 10 exons and 9 introns spanning approximately 8,460 and 7,030 bp. Of 28 potential single nucleotide polymorphisms found in the genomic region, five polymorphic positions were analyzed using PCR-RFLP. Allelic frequencies and haplotypes were analyzed in five pig breeds (n = 280). The AC haplotype of MMP1 and ATG haplotype of MMP10 were not detected in two foreign pig breeds. Association analysis in a French Large White population (n = 164, total four traits) showed no association between haplotypes and reproduction performance. In addition, porcine MMP1 and MMP10 were mapped on SSC9p¹³ and SSC2q²¹, respectively, in agreement with comparative mapping data.     

Effects of the antibiotic growth promoters flavomycin and florfenicol on the autochthonous intestinal microbiota of hybrid tilapia (Oreochromis niloticus ♀ × O. aureus ♂)

The 16S rDNA PCR-DGGE and rpoB quantitative PCR (RQ-PCR) techniques were used to evaluate the effects of dietary flavomycin and florfenicol on the autochthonous intestinal microbiota of hybrid tilapia. The fish were fed four diets: control, dietary flavomycin, florfenicol and their combination. After 8 weeks of feeding, 6 fish from each cage were randomly chosen for the analysis. The total number of intestinal bacteria was determined by RQ-PCR. The results showed that dietary antibiotics significantly influenced the intestinal microbiota and dramatically reduced the intensity of total intestinal bacterial counts. The intensity of some phylotypes (EU563257, EU563262 and EU563255) were reduced to non-detectable levels by both dietary antibiotics, while supplementation of florfenicol to the diet also reduced the intensity of the phylotypes EU563242 and EU563262, uncultured Mycobacterium sp.-like, uncultured Cyanobacterium-like and uncultured Cyanobacterium (EU563246). Dietary flavomycin only reduced the OTU intensity of one phylotype, identified as a member of the phylum Fusobacteria. The antibiotic combination only reduced the phylotypes EU563242 and EU563262. Based on our results, we conclude that the reduced effect of florfenicol on intestinal microbiota was stronger than that of flavomycin, and when flavomycin and florfenicol were added in combination, the effect of florfenicol overshadowed that of flavomycin.     

ReCGiP,a database of reproduction candidate genes in pigs based on bibliomics

Background  

Reproduction in pigs is one of the most economically important traits. To improve the reproductive performances, numerous studies have focused on the identification of candidate genes. However, it is hard for one to read all literatures thoroughly to get information. So we have developed a database providing candidate genes for reproductive researches in pig by mining and processing existing biological literatures in human and pigs, named as ReCGiP.     

Constitutively Active Inflammasome in Human Melanoma Cells Mediating Autoinflammation via Caspase-1 Processing and Secretion of Interleukin-1��

Interleukin-1β (IL-1β) is a pleiotropic cytokine promoting inflammation, angiogenesis, and tissue remodeling as well as regulation of immune responses. Although IL-1β contributes to growth and metastatic spread in experimental and human cancers, the molecular mechanisms regulating the conversion of the inactive IL-1β precursor to a secreted and active cytokine remains unclear. Here we demonstrate that NALP3 inflammasome is constitutively assembled and activated with cleavage of caspase-1 in human melanoma cells. Late stage human melanoma cells spontaneously secrete active IL-1β via constitutive activation of the NALP3 inflammasome and IL-1 receptor signaling, exhibiting a feature of autoinflammatory diseases. Unlike human blood monocytes, these melanoma cells require no exogenous stimulation. In contrast, NALP3 functionality in intermediate stage melanoma cells requires activation of the IL-1 receptor to secrete active IL-1β; cells from an early stage of melanoma require stimulation of the IL-1 receptor plus the co-stimulant muramyl dipeptide. The spontaneous secretion of IL-1β from melanoma cells was reduced by inhibition of caspase-1 or the use of small interfering RNA directed against ASC. Supernatants from melanoma cell cultures enhanced macrophage chemotaxis and promoted in vitro angiogenesis, both prevented by pretreating melanoma cells with inhibitors of caspases-1 and -5 or IL-1 receptor blockade. These findings implicate IL-1-mediated autoinflammation as contributing to the development and progression of human melanoma and suggest that inhibiting the inflammasome pathway or reducing IL-1 activity can be a therapeutic option for melanoma patients.