Hybridization-based antibody cDNA recovery for the production of recombinant antibodies identified by repertoire sequencing

High-throughput sequencing of the antibody repertoire is enabling a thorough analysis of B cell diversity and clonal selection, which may improve the novel antibody discovery process. Theoretically, an adequate bioinformatic analysis could allow identification of candidate antigen-specific antibodies, requiring their recombinant production for experimental validation of their specificity. Gene synthesis is commonly used for the generation of recombinant antibodies identified in silico. Novel strategies that bypass gene synthesis could offer more accessible antibody identification and validation alternatives. We developed a hybridization-based recovery strategy that targets the complementarity-determining region 3 (CDRH3) for the enrichment of cDNA of candidate antigen-specific antibody sequences. Ten clonal groups of interest were identified through bioinformatic analysis of the heavy chain antibody repertoire of mice immunized with hen egg white lysozyme (HEL). cDNA from eight of the targeted clonal groups was recovered efficiently, leading to the generation of recombinant antibodies. One representative heavy chain sequence from each clonal group recovered was paired with previously reported anti-HEL light chains to generate full antibodies, later tested for HEL-binding capacity. The recovery process proposed represents a simple and scalable molecular strategy that could enhance antibody identification and specificity assessment, enabling a more cost-efficient generation of recombinant antibodies.     

Selection and Identification of Aptamers Against B Cell Activating Factor

Systemic lupus erythematosus (SLE) is the most common autoimmune disease in China. B cell activating factor (BAFF) is an important target for the treatment and detection of SLE. It is of great significance to develop novel molecular recognition elements with high affinity for BAFF. In this study, artificial nucleic acid aptamers against BAFF were screened from a 78 nt single-stranded DNA random library by systematic evolution of ligands exponential enrichment (SELEX) in vitro based on several selection and amplification steps. Through ten rounds of selection, the aptamers with high specificity and affinity for BAFF were identified. After high-throughput sequencing, several aptamers were selected and further examined for binding affinity and specificity. The investigation by dot blotting, Eastern blotting analyses and enzyme-linked oligonucleotide assay (ELONA) showed that the aptamers Apt 7 and Apt 12 with dissociation constants of 241.00±19.75 nmol/L and 413.51±46.94 nmol/L were able to recognize BAFF specifically. After molecular docking analysis, Apt 7 was truncated to Apt 7～1, and the dissociation constant was 192.10±28.61 nmol/L. A sandwich ELONA using Apt 7～1 and BAFF antibodies was established to detect BAFF. The detection limit was estimated to be 0.227 nmol/L. This study provides new molecular recognition elements for the detection of BAFF and the study of antagonists.     

Genetic analysis of somatic embryogenesis in carrot cell culture: Initial characterization of six classes of temperature-sensitive variants

Cell cultures of the carrot Daucus carota are a useful experimental system for studying the genetic regulation of plant embryogenesis. A modified filtration-enrichment procedure was used to isolate 21 temperature-sensitive variants in somatic embryogenesis; the variants display normal embryo development at the permissive temperature (24°C) and altered development at the restrictive temperature (33°C). Temperature-shift experiments were performed on these variants to determine the timing of gene action for the putative temperature-sensitive alleles. According to their phenotypes at the restrictive temperature, these variants can be divided into six classes: No Growth, Callus Proliferation, Globularstage Block, Oblong-stage Block, Lateral Growth, and Root Formation. Although many variants exhibit lengthy temperature-sensitive periods, the temperature sensitivity of some variants is restricted to one or two embryonic stages. These results plus those in the literature are incorporated into a preliminary model concerning the genetic regulation of carrot embryogenesis.     

Identification of MYH6 as the potential gene for human ischaemic cardiomyopathy

The present study aimed to explore the potential hub genes and pathways of ischaemic cardiomyopathy (ICM) and to investigate the possible associated mechanisms. Two microarray data sets ( GSE5406 and GSE57338 ) were downloaded from the Gene Expression Omnibus (GEO) database. The limma package was used to analyse the differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, Disease Ontology (DO) and Gene Ontology (GO) annotation analyses were performed. A protein-protein interaction (PPI) network was set up using Cytoscape software. Significant modules and hub genes were identified by the Molecular Complex Detection (MCODE) app. Then, further functional validation of hub genes in other microarrays and survival analysis were performed to judge the prognosis. A total of 1065 genes were matched, with an adjusted p < 0.05, and 17 were upregulated and 25 were downregulated with|log₂ (fold change)|≥1.2. After removing the lengthy entries, GO identified 12 items, and 8 pathways were enriched at adjusted p < 0.05 (false discovery rate, FDR set at <0.05). Three modules with a score >8 after MCODE analysis and MYH6 were ultimately identified. When validated in GSE23561 , MYH6 expression was lower in patients with CAD than in healthy controls (p < 0.05). GSE60993 data suggested that MYH6 expression was also lower in AMI patients (p < 0.05). In the GSE59867 data set, MYH6 expression was lower in CAD patients than in AMI patients and lower in heart failure (HF) patients than in non-HF patients. However, there was no difference at different periods within half a year, and HF was increased when MYH6 expression was low (p < 0.05–0.01). We performed an integrated analysis and validation and found that MYH6 expression was closely related to ICM and HF. However, whether this marker can be used as a predictor in blood samples needs further experimental verification.     

Long-term studies at a benthic station off the coast of Northumberland

Biannual sampling (March and September) has been carried out over the period 1972 to 1985 at a muddy sand station, 55 m depth, with fauna belonging to the deeper offshore edge of Petersen's Amphiura filiformis community.During the period 1974 to 1980 the community exhibited a high degree of persistence stability. This stability was lost between 1980 and 1983, with rising total numbers and biomass and changes in species ranking. There is some evidence of a downward reversal between 1984–85.Evidence suggests that the principal stabilising process is density dependent mortality mediated by competition in a food limited environment. The principal destabilising process appears to be periodic fluctuations in the organic flux to the bottom. A secondary destabilising process is clearly concerned with fluctuating winter temperature. In competitive terms, cold winters favour increased survival in the dominant species at the expense of the lesser ranked species. This process is, however, more ephemeral and subject to adjustment within the time scale of a year.     

The effect of urea on growth of moulds and biomass yield in solid-state cultivation

Urea, added at 2 to 20 mg/g in solid bran medium supporting growth of Aspergillus oryzae and Rhizopus oligosporus, led to a higher concentration of NH₄ ⁺ and pH but a decrease in biomass production.The authors are with the Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 61000 Ljubljana, Slovenia.     

微藻降解废水污染物的研究进展

微藻在废水处理方面有着很好的开发潜能和极大的优势,在去除氮、磷、富集重金属和降解有毒有机物质的同时,还能作为食品添加原料和养殖饲料等。重点针对微藻的特点及类型、水处理体系的优缺点、废水中的应用及氮磷的去除、重金属离子的富集和难降解有机物的降解机理进行了介绍,并阐述了微藻处理废水中存在的问题及展望了该领域今后的研究方向。     

茶树CsCCD1和CsCCD4基因的克隆和表达分析

该研究以‘铁观音’茶树为材料,同源克隆了茶树类胡萝卜素裂解双加氧酶基因CsCCD1和CsCCD4 (NCBI登录号分别为MH119136和MH119137)全长cDNA。序列分析结果显示,CsCCD1序列全长1 766 bp,包含1 641 bp开放阅读框(ORF),编码545个氨基酸,定位于细胞质中,不存在跨膜结构和信号肽;CsCCD4序列全长1 942 bp,包含1 842 bp ORF,编码612个氨基酸,不存在跨膜结构,但在N端具有叶绿体转运肽,定位于质体中。进化树分析显示,CsCCD1和CsCCD4与杜鹃聚为一类。荧光定量检测显示,CsCCD1和CsCCD4在叶、茎和花中表达量较高。在乌龙茶做青过程中,CsCCD1和CsCCD4基因都是从鲜叶到晒青表达量下调,而CsCCD1在一摇和二摇显著上调表达,在三摇后下降;CsCCD4只在一摇后上调表达,之后表达量迅速降低。推测CsCCD1和CsCCD4基因可能与乌龙茶做青香气品质形成关系密切。     

Characterization of metal(loid)s in indoor and outdoor PM2.5 of an office in winter period

The characterization of indoor (a naturally ventilated office) and outdoor (adjacent courtyard) metals in PM_2.5 during a winter period in Xi'an, China were carried out. The results indicated that the average mass concentrations of PM_2.5 in indoor and outdoor environments all exceeded the daily average limit of 75 µg m^–3 set by the Chinese government. The dominant metals in PM_2.5 were Ca, Al, Zn, Mg, Fe, and Pb in both indoor and outdoor air. Concentration of As was much higher than the standard of 6 ng m^–3 issued by the government. Enrichment factor analysis showed that anthropogenic emissions might be the primary sources of As, Cd, Pb, and Zn, while crust was the main origin of Co. A majority of indoor-to-outdoor concentration ratios of metal were lower than 1 indicating mostly the contribution of outdoor sources rather than indoor ones. As and Cr in both indoor and outdoor air posed the highest noncarcinogenic and carcinogenic risks, respectively. The noncarcinogenic and carcinogenic risks were 2.74 and 2.54 × 10^?4 indoor and 4.04 and 3.87 × 10^?4 outdoor, which suggested that possible adverse health effects should be of concern.     

Experiential Key Species for Nature-disconnected Generations: An Expert-based Framework for Their A-priori Selection

In urbanized contexts, a progressive disconnection from nature, mainly experienced by the youngest generation, is evident. In this sense, detection of species more suitable for a nature-related experience becomes of primary importance to increase pro-environmental behaviors in children. Here, we propose a simple, quick, and explorative procedure for selecting a set of Experiential Key Species (EKS). Two practical examples are provided for two contexts (a urban park and a suburban wetland) where a large amount of data on vertebrate animal species are available. Depending on their density and eco-behavioral features, each species stimulates different experiences, here considered as a product of intensity and frequency of occurrence. Therefore, for each species we calculated an Experiential Magnitude score on a three-level scale with an expert-based method. We obtained 14 EKS in the urban park site (18% of total) and 18 EKS in the suburban wetland site (9.4%). We observed that in both sites EKS mainly include: (i) heterotherm species (mainly, fishes, amphibians, and water-related reptiles) and (ii) non-native species (about 36% in the urban park and about 17% in the suburban wetland). This information suggests that (i) following a conservation zoning approach, lakes, streams, and their shores may be considered the best experiential sites for children, and (ii) in conservation measures aimed to improve experience and pro-environmental behaviors in younger generations, the role of non-native species (that are of low or null conservation interest) might be relevant. In-deed, individuals of these species (i) are easily detectable, and (ii) may be collected, handled, and farmed with a limited (or absent) impact on biodiversity. In many anthropized contexts, these are the only species that can provide nature experience for younger generations. Based on our observations, we recommend practitioners (i) to adopt the procedure of EKS selection in critical contexts, also adding further regime attributes (area utilized, seasonal duration) to calculate the experiential magnitude score, and (ii) to test the effectiveness of this selection in conservation education projects.