珍稀濒危植物翅果油树表皮毛的微形态观察研究

利用体视显微镜、扫描电镜和光学显微镜,对珍稀濒危植物翅果油树(Elaeagnus mollis Diels)的茎、叶、果、休眠芽等多种器官表面覆盖的表皮毛,进行了详细观察．发现其表皮毛的形态和结构具有多样性,各器官有差异,据此将它们分为分支状毛、星状毛、盾状-星状毛、盾状毛四类,以及一些特殊的表皮毛,如：分支状毛与星状毛或盾状-星状毛的过渡类型称为类星状毛和类盾状-星状毛．叶片上表面的表皮毛,为分枝状表皮毛,且稀疏散布．叶片下表面的白色表皮毛,也多为分枝状表皮毛,但分布较密,叶主脉处最为稠密多为星状毛．叶柄的表皮毛形态呈现一定的梯度变化,远轴端(靠近叶片端)为类星状毛,近轴端(靠近茎的一端)为盾状-星状表皮毛,中间段为星状毛介于前二者之间．茎表面灰色的或褐色的表皮毛,以盾状表皮毛为主,夹杂少量盾状-星状表皮毛,因茎的老嫩其表皮毛形态略有不同．外果皮沟槽内的表皮毛多为盾状表皮毛,嵴部则密集星状或分支状表皮毛．休眠芽的鳞片多覆盖盾状表皮毛,只在鳞片的尖端有少量星状毛和分枝状毛．翅果油树多种器官表面多种表皮毛的存在是其适应高山(海拔800～1300m)环境的一种形态特征．表皮毛具有反射阳光、阻止水分过度散失、保温、防止机械损伤等功能．为探究翅果油树耐早、耐寒、耐高温机理以及组织培养过程中外植体易污染等问题,提供了形态学依据,也为进一步进行表皮毛的发育和分子生物学研究打下了一定的基础。     

Reverse-vaccinology strategy for designing T-cell epitope candidates for Staphylococcus aureus endocarditis vaccine

Staphylococcus aureus is an opportunistic pathogen causing various inflammatory diseases from skin and tissue local infections, to serious life threatening infections including endocarditis. Experimental models for endocarditis demonstrated that virulence factors of S. aureus, that are very important in infection of heart vegetations, are surface proteins which promote bacterial adherence. Until now, efforts to develop effective vaccines against S. aureus were unsuccessful, partly due to the fact that different vaccine formulations have targeted mainly B-cell immunity. Reverse vaccinology is applied here, in order to identify potential vaccine epitope candidates. The basic epitopes prediction strategy relied on detection of a common antigenic 9-mer epitope meant to be able to stimulate both the B-cell and T-cell mediated immunity. Ten surface exposed proteins were chosen for antigenicity testing. Using a web-based system, five T-cell epitopes corresponding to fibronectin binding protein A (FDFTLSNNV and YVDGYIETI), collagen adhesin (FSINYKTKI), serine-rich adhesin for platelets (LTFDSTNNT) and elastin binding protein (FAMDKSHPE) were selected as potential vaccine candidates. Epitopes sequences were found to be conserved among the different S. aureus genomes screened from NCBI GenBank. In vitro and in vivo immunological tests will be performed in order to validate the suitability of the epitopes for vaccine development.     

Capacity of a model immune network

The capacity of a model immune network in terms of the number of different antigens that can be vaccinated against without any memory lost is computed and tested by numerical simulations. We also investigate memory loss and failure to vaccinate due to overcrowding the network with too many antigens. The computations are done for two different strategies for proliferation, one implying all the antigen specific clones and the second one being more thrifty.     

Somatic Embryogenesis and Analysis of Peroxidase in Cultured Lettuce (Lactuca sativa L.) Cotyledons

Somatic embryos were induced in lettuce cotyledons culturedon Murashige and Skoog's (MS) medium containing either 2 mgl^–1 6-benzylaminopurine (BA) and 0.2 mg l^–1 naphthaleneaceticacid (NAA) or 0.2 mg l^–1 BA and 2 mg l^–1 NAA. Bothcombinations induced a frequency of over 70%. The explants culturedonly in the presence of 2,4-dichlorphenoxyacetic acid (2,4-D)did not produce somatic embryos. The development of the embryoidswas studied histologically and by scanning electron microscopy.Peroxidase activity was assayed and the isoenzyme pattern ofcalluses was determined by polyacrylamide gel electrophoresis.Callus from an embryogenic line showed a much higher peroxidaseactivity than that from a non-embryogenic line, one extra peroxidaseisozyme band being present and typical of the embryogenic callus.No qualitative differences were detectable between the embryogeniccalluses. Lactuca sativa L, lettuce, somatic embryogenesis, peroxidases, isoenzymes     

The targeting of somatic hypermutation closely resembles that of meiotic mutation

We have compared the microsequence specificity of mutations introduced during somatic hypermutation (SH) and those introduced meiotically during neutral evolution. We have minimized the effects of selection by studying nonproductive (hence unselected) Ig V region genes for somatic mutations and processed pseudogenes for meiotic mutations. We find that the two sets of patterns are very similar: the mutabilities of nucleotide triplets are positively correlated between the somatic and meiotic sets. The major differences that do exist fall into three distinct categories: 1) The mutability is sharply higher at CG dinucleotides under meiotic but not somatic mutation. 2) The complementary triplets AGC and GCT are much more mutable under somatic than under meiotic mutation. 3) Triplets of the form WAN (W = T or A) are uniformly more mutable under somatic than under meiotic mutation. Nevertheless, the relative mutabilities both within this set and within the SAN (S = G or C) triplets are highly correlated with those under meiotic mutation. We also find that the somatic triplet specificity is strongly symmetric under strand exchange for A/T triplets as well as for G/C triplets in spite of the strong predominance of A over T mutations. Thus, we suggest that somatic mutation has at least two distinct components: one that specifically targets AGC/GCT triplets and another that acts as true catalysis of meiotic mutation.     

Systems chemical biology

The increasing availability of data related to genes, proteins and their modulation by small molecules has provided a vast amount of biological information leading to the emergence of systems biology and the broad use of simulation tools for data analysis. However, there is a critical need to develop cheminformatics tools that can integrate chemical knowledge with these biological databases and simulation approaches, with the goal of creating systems chemical biology.     

Biomolecular screening of formylpeptide receptor ligands with a sensitive, quantitative, high-throughput flow cytometry platform

The formylpeptide receptor (FPR) family of G protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here we describe a high-throughput flow cytometry screening approach that has successfully identified multiple families of previously unknown FPR ligands. The assay detects active structures that block the binding of a fluorescent ligand to membrane FPR of intact cells, thus detecting both agonists and antagonists. It is homogeneous in that assay reagents are added in sequence and the wells are subsequently analyzed without intervening wash steps. Microplate wells are routinely processed at a rate of 40 wells per minute, requiring a volume of only 2 microl to be sampled from each. This screening approach has recently been extended to identify a high-affinity, selective agonist for the intracellular estrogen-binding G protein-coupled receptor GPR30. With the development of appropriate assay reagents, it may be generally adaptable to a wide range of receptors. The total time required for the assay ranges between 1.5 and 2.5 h. The time required for flow cytometry analysis of a 96-well plate at the end of the procedure is less than 2.5 min. By comparison, manual processing of 96 samples will typically require 40-50 min, and a fast commercial automated sampler processes 96-well plates in less than 15 min, requiring the aspiration of 22 microl per sample for an analysis volume of 2 microl.     

High-throughput screening with HyperCyt flow cytometry to detect small molecule formylpeptide receptor ligands

High-throughput flow cytometry (HTFC), enabled by faster automated sample processing, represents a promising high- content approach for compound library screening. HyperCyt is a recently developed automated HTFC analysis system by which cell samples are rapidly aspirated from microplate wells and delivered to the flow cytometer. The formylpeptide receptor (FPR) family of G protein-coupled receptors contributes to the localization and activation of tissue-damaging leukocytes at sites of chronic inflammation. Here, the authors describe development and application of an HTFC screening approach to detect potential anti-inflammatory compounds that block ligand binding to FPR. Using a homogeneous no-wash assay, samples were routinely processed at 1.5 s/well (approximately 2500 cells analyzed/sample), allowing a 96-well plate to be processed in less than 2.5 min. Assay sensitivity and accuracy were validated by detection of a previously documented active compound with relatively low FPR affinity (sulfinpyrazone, inhibition constant [K(i)]=14 microM) from among a collection of 880 compounds in the Prestwick Chemical Library. The HyperCyt system was therefore demonstrated to be a robust, sensitive, and highly quantitative method with which to screen lead compound libraries in a 96-well format.     

Temperature-Assisted Cyclic Hybridization (TACH): An Improved Method for Supercoiled DNA Hybridization

Accurate hybridization is dependent on the ratio between sequence-specific and unspecific binding. Dissociation of unspecifically bound, while maintaining specifically hybridized, nucleic acids are key steps to obtain a well-defined complex. We have developed a new method, temperature-assisted, cyclic hybridization (TACH), which increases cognate binding at the expense of unspecific hybridization. The method was used for optimizing binding of peptide nucleic acid (PNA) to supercoiled plasmids and has several advantages over previous methods: (1) it reduces the required amount of bis-PNA by three- to fourfold; (2) it results in less unspecific binding; (3) it extends cooperative hybridization, from 3 bp to 5 bp between two adjacent binding sites; and (4) it decreases the aggregation of bis-PNA. This method might be extended to other forms of hybridizations including the use of additional nucleic acids analogs, such as locked nucleic acid (LNA) and, also, to other areas where PNAs are used such as fluorescence in situ hybridization (FISH), microarrays, or in vivo plasmid delivery.     

Temporal changes in the potential geographic distribution of Histiotus velatus (Chiroptera,Vespertilionidae), the “decade effect”

We investigated how the potential distribution of Histiotus velatus is affected by the addition of new records over decades (decade effect). Assuming that (1: hypothesis of the effect of the decade) the addition of new occurrence records over time increases the potential size of the species distribution; and (2: Wallacean distance hypothesis) over the years, the new points added are increasingly distant from the research centers. Considering the geographic knowledge gap of this species, our objective is to report a new record of this species and estimate its potential distribution in South America through environment niche models (ENMs). For this, we compiled records of occurrence of species, selected from 1900 to 2015. We used 19 bioclimatic variables available in the WorldClim database to estimate the potential distribution of the species, and we used three modeling algorithms: Maximum Entropy (MXT), Random Forest (RDF), and Support Vector Machine. To test the Wallacean distance hypothesis, we calculated the Euclidian distance from occurrences to bat research centers in Brazil, located using a national researchers’ information dataset (“Plataforma Lattes”). To test the hypothesis of the decade effect, we used the beta regression analysis, taking conservative and non‐conservative approaches. The results showed that the predicted area expanded and retracted with the addition of new occurrences over the decades, with an improvement in the accuracy of models. Most records are located in the southeastern region of Brazil, but algorithms predicted areas in regions where there are no records. Only the conservative approach has had a positive relationship over the decades. The distance from new points does not increase over the years of research centers.