Oligo pools as an affordable source of synthetic DNA for cost-effective library construction in protein- and metabolic pathway engineering

The construction of custom libraries is critical for rational protein engineering and directed evolution. Array-synthesized oligo pools of thousands of user-defined sequences (up to ~350 bases in length) have emerged as a low-cost commercially available source of DNA. These pools cost ≤10% (depending on error rate and length) of other commercial sources of custom DNA, and this significant cost difference can determine whether an enzyme engineering project can be realized on a given research budget. However, while being cheap, oligo pools do suffer from a low concentration of individual oligos and relatively high error rates. Several powerful techniques that specifically make use of oligo pools have been developed and proven valuable or even essential for next-generation protein and pathway engineering strategies, such as sequence-function mapping, enzyme minimization, or de-novo design. Here we consolidate the knowledge on these techniques and their applications to facilitate the use of oligo pools within the protein engineering community

UN Peace operations and conflicting legitimacies

Analyses of UN peacekeeping increasingly consider legitimacy a key factor for success, conceiving of it as a resource that operations should seek and use in the pursuit of their goals. However, these analyses rarely break down legitimacy by source. Because the UN is an organization with multiple identities and duties however, different legitimacy sources—in particular output and procedural legitimacy—and the UN’s corresponding legitimation practices come into conflict in the context of peacekeeping. Drawing on a range of examples and a specific case of the UN mission in Congo, this article argues that looking at different legitimacy sources and linking them to the institutional identity of the UN is thus critical and it shows how the UN’s in contradictory legitimation practices can reduce overall legitimacy perceptions

In vitro antibacterial activity of some plant essential oils

BACKGROUND: To evaluate the antibacterial activity of 21 plant essential oils against six bacterial species. METHODS: The selected essential oils were screened against four gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Proteus vulgaris) and two gram-positive bacteria Bacillus subtilis and Staphylococcus aureus at four different concentrations (1:1, 1:5, 1:10 and 1:20) using disc diffusion method. The MIC of the active essential oils were tested using two fold agar dilution method at concentrations ranging from 0.2 to 25.6 mg/ml. RESULTS: Out of 21 essential oils tested, 19 oils showed antibacterial activity against one or more strains. Cinnamon, clove, geranium, lemon, lime, orange and rosemary oils exhibited significant inhibitory effect. Cinnamon oil showed promising inhibitory activity even at low concentration, whereas aniseed, eucalyptus and camphor oils were least active against the tested bacteria. In general, B. subtilis was the most susceptible. On the other hand, K. pneumoniae exhibited low degree of sensitivity. CONCLUSION: Majority of the oils showed antibacterial activity against the tested strains. However Cinnamon, clove and lime oils were found to be inhibiting both gram-positive and gram-negative bacteria. Cinnamon oil can be a good source of antibacterial agents

Heterogeneity of Multifunctional IL-17A Producing S. Typhi-Specific CD8+ T Cells in Volunteers following Ty21a Typhoid Immunization

Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, continues to cause significant morbidity and mortality world-wide. CD8+ T cells are an important component of the cell mediated immune (CMI) response against S. Typhi. Recently, interleukin (IL)-17A has been shown to contribute to mucosal immunity and protection against intracellular pathogens. To investigate multifunctional IL-17A responses against S. Typhi antigens in T memory subsets, we developed multiparametric flow cytometry methods to detect up to 6 cytokines/chemokines (IL-10, IL-17A, IL-2, interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and macrophage inflammatory protein-1β (MIP-1β)) simultaneously. Five volunteers were immunized with a 4 dose regimen of live-attenuated S. Typhi vaccine (Ty21a), peripheral blood mononuclear cells (PBMC) were isolated before and at 11 time points after immunization, and CMI responses were evaluated. Of the 5 immunized volunteers studied, 3 produced detectable CD8+ T cell responses following stimulation with S. Typhi-infected autologous B lymphoblastoid cell lines (B-LCL). Additionally, 2 volunteers had detectable levels of intracellular cytokines in response to stimulation with S. Typhi-infected HLA-E restricted cells. Although the kinetics of the responses differed among volunteers, all of the responses were bi- or tri-phasic and included multifunctional CD8+ T cells. Virtually all of the IL-17A detected was derived from multifunctional CD8+ T cells. The presence of these multifunctional IL-17A+ CD8+ T cells was confirmed using an unsupervised analysis program, flow cytometry clustering without K (FLOCK). This is the first report of IL-17A production in response to S. Typhi in humans, indicating the presence of a Tc17 response which may be important in protection. The presence of IL-17A in multifunctional cells co-producing Tc1 cytokines (IL-2, IFN-γ and TNF-α) may also indicate that the distinction between Tc17 and Tc1 responses in humans is not as clearly delineated as suggested by in vitro experiments and animal models

Microphytobenthos of Arctic Kongsfjorden (Svalbard, Norway): biomass and potential primary production along the shore line

During summer 2007, Arctic microphytobenthic potential primary production was measured at several stations around the coastline of Kongsfjorden (Svalbard, Norway) at ?5 m water depth and at two stations at five different water depths (5, 10, 15, 20, 30 m). Oxygen planar optode sensor spots were used ex situ to determine oxygen exchange in the overlying water of intact sediment cores under controlled light (ca. 100 ?mol photons m?2 s?1) and temperature (2–4°C) conditions. Patches of microalgae (mainly diatoms) covering sandy sediments at water depths down to 30 m showed high biomass of up to 317 mg chl a m?2. In spite of increasing water depth, no significant trend in “photoautotrophic active biomass” (chl a, ratio living/dead cells, cell sizes) and, thus, in primary production was measured at both stations. All sites from ?5 to 30 m water depth exhibited variable rates of net production from ?19 to +40 mg O2 m?2 h?1 (?168 to +360 mg C m?2 day?1) and gross production of about 2–62 mg O2 m?2 h?1 (17–554 mg C m?2 day?1), which is comparable to other polar as well as temperate regions. No relation between photoautotrophic biomass and gross/net production values was found. Microphytobenthos demonstrated significant rates of primary production that is comparable to pelagic production of Kongsfjorden and, hence, emphasised the importance as C source for the zoobenthos

Compensatory Development and Costs of Plasticity: Larval Responses to Desiccated Conspecifics

Understanding constraints on phenotypic plasticity is central to explaining its evolution and the evolution of phenotypes in general, yet there is an ongoing debate on the classification and relationships among types of constraints. Since plasticity is often a developmental process, studies that consider the ontogeny of traits and their developmental mechanisms are beneficial. We manipulated the timing and reliability of cues perceived by fire salamander larvae for the future desiccation of their ephemeral pools to determine whether flexibility in developmental rates is constrained to early ontogeny. We hypothesized that higher rates of development, and particularly compensation for contradictory cues, would incur greater endogenous costs. We found that larvae respond early in ontogeny to dried conspecifics as a cue for future desiccation, but can fully compensate for this response in case more reliable but contradictory cues are later perceived. Patterns of mortality suggested that endogenous costs may depend on instantaneous rates of development, and revealed asymmetrical costs of compensatory development between false positive and false negative early information. Based on the results, we suggest a simple model of costs of development that implies a tradeoff between production costs of plasticity and phenotype-environment mismatch costs, which may potentially underlie the phenomenon of ontogenetic windows constraining plasticity

Limited genetic variation and structure in softshell clams (Mya arenaria) across their native and introduced range

Author Posting. © Springer, 2009. This is the author's version of the work. It is posted here by permission of Springer for personal use, not for redistribution. The definitive version was published in Conservation Genetics 10 (2009): 803-814, doi:10.1007/s10592-008-9641-y.To offset declines in commercial landings of the softshell clam, Mya arenaria, resource managers are engaged in extensive stocking of seed clams throughout its range in the northwest Atlantic. Because a mixture of native and introduced stocks can disrupt locally adapted genotypes, we investigated genetic structure in M. arenaria populations across its current distribution to test for patterns of regional differentiation. We sequenced mitochondrial cytochrome oxidase I (COI) for a total of 212 individuals from 12 sites in the northwest Atlantic (NW Atlantic), as well as two introduced sites, the northeast Pacific (NE Pacific) and the North Sea and Europe (NS Europe). Populations exhibited extremely low genetic variation, with one haplotype dominating (65-100%) at all sites sampled. Despite being introduced in the last 150-400 years, both NE Pacific and NS Europe populations had higher diversity measures than those in the NW Atlantic and both contained private haplotypes at frequencies of 10% to 27% consistent with their geographic isolation. While significant genetic structure (FST = 0.159, p<0.001) was observed between NW Atlantic and NS Europe, there was no evidence for genetic structure across the pronounced environmental clines of the NW Atlantic. Reduced genetic diversity in mtDNA combined with previous studies reporting reduced genetic diversity in nuclear markers strongly suggests a recent population expansion in the NW Atlantic, a pattern that may result from the retreat of ice sheets during Pleistocene glacial periods. Lack of genetic diversity and regional genetic differentiation suggests that present management strategies for the commercially important softshell clam are unlikely to have a significant impact on the regional distribution of genetic variation, although the possibility of disrupting locally adapted stocks cannot be excluded.This work was supported by NSF grants OCE-0326734 and OCE-0215905 to L. Mullineaux and OCE- 0349177 (Biological Oceanography) to PHB

A microRNA profile of human CD8(+) regulatory T cells and characterization of the effects of microRNAs on Treg cell-associated genes.

Recently, regulatory T (Treg) cells have gained interest in the fields of immunopathology, transplantation and oncoimmunology. Here, we investigated the microRNA expression profile of human natural CD8(+)CD25(+) Treg cells and the impact of microRNAs on molecules associated with immune regulation. We purified human natural CD8(+) Treg cells and assessed the expression of FOXP3 and CTLA-4 by flow cytometry. We have also tested the ex vivo suppressive capacity of these cells in mixed leukocyte reactions. Using TaqMan low-density arrays and microRNA qPCR for validation, we could identify a microRNA 'signature' for CD8(+)CD25(+)FOXP3(+)CTLA-4(+) natural Treg cells. We used the 'TargetScan' and 'miRBase' bioinformatics programs to identify potential target sites for these microRNAs in the 3'-UTR of important Treg cell-associated genes. The human CD8(+)CD25(+) natural Treg cell microRNA signature includes 10 differentially expressed microRNAs. We demonstrated an impact of this signature on Treg cell biology by showing specific regulation of FOXP3, CTLA-4 and GARP gene expression by microRNA using site-directed mutagenesis and a dual-luciferase reporter assay. Furthermore, we used microRNA transduction experiments to demonstrate that these microRNAs impacted their target genes in human primary Treg cells ex vivo. We are examining the biological relevance of this 'signature' by studying its impact on other important Treg cell-associated genes. These efforts could result in a better understanding of the regulation of Treg cell function and might reveal new targets for immunotherapy in immune disorders and cancer