High Levels of Genetic Diversity in Salix viminalis of the Czech Republic as Revealed by Microsatellite Markers

Willows (Salix spp.) grown as short rotation coppice are recognised as an important bioenergy crop, and breeding programmes are underway in several countries, including the Czech Republic. The basket willow Salix viminalis is one of the few willow species that is widespread in the Czech Republic and thus a potential source of diversity, but the most extensive germplasm collection available shows evidence of redundancy. To investigate levels of variation in natural populations of this species for use in crop improvement programmes, a set of 38 microsatellite markers was used to assess genetic diversity and population structure among 84 S. viminalis individuals collected from seven Czech rivers (the Odra, Be?va, Morava, Dyje, Jihlava, Sázava and Vltava), covering a wide geographic distribution. The markers detected 6.95 alleles per locus on average with 92?% of the sampled individuals having a unique multilocus genotype giving a high clonal richness measure among all samples (R?=?0.952). Three sets of putative clones (with identical genotypes as determined by the markers used here) were also identified. Significant levels of genetic diversity were revealed within all sampling sites. With the exception of sites on the Odra and Morava, pairwise F _ST (0.02?C0.1) values indicated moderate differentiation between sites. Principal coordinates analysis revealed some separation of the Dyje individuals from all others. This was in agreement with the population structure results derived from Bayesian analyses using STRUCTURE software. These results provide the first evidence that potentially useful levels of genotypic variation are present within natural S. viminalis populations in the Czech Republic.     

Portage connectivity does not predict establishment success of canoe-mediated dispersal for crustacean zooplankton

Although community structure may be largely determined by local abiotic and biotic conditions under moderate levels of dispersal, anthropogenic activities can enhance dispersal rates far beyond what would otherwise occur in natural systems. We investigated the potential impact of recreational canoeing on crustacean zooplankton community structure in Killarney Provincial Park, Canada, where canoes that are transported between lakes via portage routes may enhance zooplankton community connectivity by providing a dispersal “short-cut.” We conducted a study to (1) quantify zooplankton attachment to canoe hulls after paddling through a lake and assess the importance of canoes to overall seasonal dispersal within a lake relative to other means of dispersal, (2) test the prediction that zooplankton survivorship is negatively correlated with portage duration using a mesocosm experiment, and (3) test whether variation in lake community composition was better explained by models based on reduced portage-corrected distances or true edge-to-edge distances between lakes along popular canoe routes. Here, we report the findings that canoes have the potential to act as frequent dispersal vectors, but appear to have little impact on community structure in portage-connected lakes. Substantial numbers of adult zooplankton became attached to canoe hulls and were able to establish viable populations even after exposure to portage conditions for 30 min. However, canoe-mediated dispersal only accounted for a very small proportion (<1% in this case) of overall seasonal dispersal. Moreover, environmental variables explained the greatest amount of variation in community composition among park lakes. Nevertheless, this study indicates that canoe dispersal could be more effective for specific species such as Sida crystallina than is evident by analysis of entire communities and could facilitate the spread of invasive species amenable to attaching to boat hulls. Thus, the debate about whether community composition is more strongly influenced by local environmental conditions or regional dispersal may vary depending on the scale of consideration (i.e., individual species vs. whole community).     

Birth control vaccine targeting leukemia inhibitory factor

The population explosion and unintended pregnancies resulting in elective abortions continue to impose major public health issues. This calls for a better method of contraception. Immunocontraception has been proposed as a valuable alternative that can fulfill most, if not all, of the properties of an ideal contraceptive. There are several targets that are being explored for contraceptive vaccine development. Leukemia inhibitory factor (LIF), a member of interleukin‐6 family, is required for embryo development and successful blastocyst implantation in several mammalian species. The present study was conducted to examine if LIF can be a target for the development of a birth control vaccine. Three sequences from LIF and two sequences from LIF‐receptor (LIF‐R) that span the regions involved in ligand‐receptor binding were delineated, and peptides were synthesized based upon these sequences. Antibodies raised against these five peptides reduced LIF bioactivity in an in vitro culture assay using BA/F3 mLIF‐R‐mpg130 cells. Vaccines were prepared by conjugating these peptides to various carrier proteins. Immunization of female mice with these peptide vaccines induced a long‐lasting, circulating as well as local antibody response in various parts of the genital tract, and resulted in a significant (P ≤ 0.05) inhibition in fertility in all the three trials; the LIF‐R peptide vaccines proved to be a better vaccine target. The data indicate that LIF/LIF‐R is an excellent target for the development of a birth control vaccine. This is the first study, to our knowledge, that examined LIF/LIF‐R as a target for immunocontraception. The findings of this study can be easily translated to humans since LIF/LIF‐R is also important for implantation and pregnancy in women. Mol. Reprod. Dev. 79:97–106, 2012. © 2011 Wiley Periodicals, Inc.     

M13 bacteriophage display framework that allows sortase-mediated modification of surface-accessible phage proteins

We exploit bacterial sortases to attach a variety of moieties to the capsid proteins of M13 bacteriophage. We show that pIII, pIX, and pVIII can be functionalized with entities ranging from small molecules (e.g., fluorophores, biotin) to correctly folded proteins (e.g., GFP, antibodies, streptavidin) in a site-specific manner, and with yields that surpass those of any reported using phage display technology. A case in point is modification of pVIII. While a phage vector limits the size of the insert into pVIII to a few amino acids, a phagemid system limits the number of copies actually displayed at the surface of M13. Using sortase-based reactions, a 100-fold increase in the efficiency of display of GFP onto pVIII is achieved. Taking advantage of orthogonal sortases, we can simultaneously target two distinct capsid proteins in the same phage particle and maintain excellent specificity of labeling. As demonstrated in this work, this is a simple and effective method for creating a variety of structures, thus expanding the use of M13 for materials science applications and as a biological tool.     

Efficacy of two different stretch training programs (passive vs. proprioceptive neuromuscular facilitation) on shoulder and hip range of motion in older people

The aim of this study was to determine the influence of 2 methods of stretch training (passive and proprioceptive neuromuscular facilitation [PNF]) on range of motion (ROM) in older people between the age of 60 and 70 years over a period of 13 weeks. Fifty-four participants (39 women and 15 men) were divided into 3 groups: passive (n = 17; 66.5 ± 6.5 years), PNF (n = 17; age, 64.7 ± 4.0 years old), and control (n = 17; age, 66.4 ± 4.5 years). The subjects trained 2 times per week on nonconsecutive days for 13 weeks. Each training session included 2 flexibility exercises focused on the shoulder and hip joints. The PNF group performed 6 seconds of passive stretching, 3 seconds of muscular contractions, and 2 seconds of relaxation. The passive group performed 10 seconds of stretching and 5 seconds of relaxation. This sequence was repeated 3 times by each group. The control group did not perform any stretching. In the PNF group, there was an increase in hip ROM (p < 0.001) between pretest and posttest in the passive group and an improvement (p < 0.001) was observed between pretest and posttest, whereas in the control group, there was a significant decrease (p < 0.01) in hip ROM between pretest and posttest. In shoulder ROM, there was an increase (p < 0.001) between pretest and posttest in the passive group and an improvement (p < 0.001) was observed between pretest and posttest in the PNF group. There were no changes in shoulder ROM between pretest and posttest in the control group. The analysis of variance showed significant differences in hip and shoulder ROM between passive and control groups and PNF and control groups, but no significant differences were found between passive and PNF. The main finding was that the ability of physically active older people to increase ROM in response to stretching techniques is similar for both passive and PNF techniques.     

Chemical–Physical Changes in Cell Membrane Microdomains of Breast Cancer Cells After Omega-3 PUFA Incorporation

Epidemiologic and experimental studies suggest that dietary fatty acids influence the development and progression of breast cancer. However, no clear data are present in literature that could demonstrate how n?-?3 PUFA can interfere with breast cancer growth. It is suggested that these fatty acids might change the structure of cell membrane, especially of lipid rafts. During this study we treated MCF-7 and MDA-MB-231 cells with AA, EPA, and DHA to assess if they are incorporated in lipid raft phospholipids and are able to change chemical and physical properties of these structures. Our data demonstrate that PUFA and their metabolites are inserted with different yield in cell membrane microdomains and are able to alter fatty acid composition without decreasing the total percentage of saturated fatty acids that characterize these structures. In particular in MDA-MB-231 cells, that displays the highest content of Chol and saturated fatty acids, we observed the lowest incorporation of DHA, probably for sterical reasons; nevertheless DHA was able to decrease Chol and SM content. Moreover, PUFA are incorporated in breast cancer lipid rafts with different specificity for the phospholipid moiety, in particular PUFA are incorporated in PI, PS, and PC phospholipids that may be relevant to the formation of PUFA metabolites (prostaglandins, prostacyclins, leukotrienes, resolvines, and protectines) of phospholipids deriving second messengers and signal transduction activation. The bio-physical changes after n?-?3 PUFA incubation have also been highlighted by atomic force microscopy. In particular, for both cell lines the DHA treatment produced a decrease of the lipid rafts in the order of about 20-30?%. It is worth noticing that after DHA incorporation lipid rafts exhibit two different height ranges. In fact, some lipid rafts have a higher height of 6-6.5?nm. In conclusion n?-?3 PUFA are able to modify lipid raft biochemical and biophysical features leading to decrease of breast cancer cell proliferation probably through different mechanisms related to acyl chain length and unsaturation. While EPA may contribute to cell apoptosis mainly through decrease of AA concentration in lipid raft phospholipids, DHA may change the biophysical properties of lipid rafts decreasing the content of cholesterol and probably the distribution of key proteins.     

Configuration of a high-content imaging platform for hit identification and pharmacological assessment of JMJD3 demethylase enzyme inhibitors

The biological complexity associated with the regulation of histone demethylases makes it desirable to configure a cellular mechanistic assay format that simultaneously encompasses as many of the relevant cellular processes as possible. In this report, the authors describe the configuration of a JMJD3 high-content cellular mechanistic imaging assay that uses single-cell multiparameter measurements to accurately assess cellular viability and the enzyme-dependent demethylation of the H3K27(Me)3 mark by exogenously expressed JMJD3. This approach couples robust statistical analyses with the spatial resolving power of cellular imaging. This enables segregation of expressing and nonexpressing cells into discrete subpopulations and consequently pharmacological quantification of compounds of interest in the expressing population at varying JMJD3 expression levels. Moreover, the authors demonstrate the utility of this hit identification strategy through the successful prosecution of a medium-throughput focused campaign of an 87 500-compound file, which has enabled the identification of JMJD3 cellular-active chemotypes. This study represents the first report of a demethylase high-content imaging assay with the ability to capture a repertoire of pharmacological tools, which are likely both to inform our mechanistic understanding of how JMJD3 is modulated and, more important, to contribute to the identification of novel therapeutic modalities for this demethylase enzyme.