Modulating the import of medium-chain alkanes in E. coli through tuned expression of FadL

BACKGROUND: In recent years, there have been intensive efforts to develop synthetic microbial platforms for the production, biosensing and bio-remediation of fossil fuel constituents such as alkanes. Building predictable engineered systems for these applications will require the ability to tightly control and modulate the rate of import of alkanes into the host cell. The native components responsible for the import of alkanes within these systems have yet to be elucidated. To shed further insights on this, we used the AlkBGT alkane monooxygenase complex from Pseudomonas putida GPo1 as a reporter system for assessing alkane import in Escherichia coli. Two native E. coli transporters, FadL and OmpW, were evaluated for octane import given their proven functionality in the uptake of fatty acids along with their structural similarity to the P. putida GPo1 alkane importer, AlkL. RESULTS: Octane import was removed with deletion of fadL, but was restored by complementation with a fadL-encoding plasmid. Furthermore, tuned overexpression of FadL increased the rate of alkane import by up to 4.5- fold. A FadL deletion strain displayed a small but significant degree of tolerance toward hexane and octane relative to the wild type, while the responsiveness of the well-known alkane biosensor, AlkS, toward octane and decane was strongly reduced by 2.7- and 2.9-fold, respectively. CONCLUSIONS: We unequivocally show for the first time that FadL serves as the major route for medium-chain alkane import in E. coli. The experimental approaches used within this study, which include an enzyme-based reporter system and a fluorescent alkane biosensor for quantification and real-time monitoring of alkane import, could be employed as part of an engineering toolkit for optimizing biological systems that depend on the uptake of alkanes. Thus, the findings will be particularly useful for biological applications such as bioremediation and biomanufacturing

High Density Microarray Analysis Reveals New Insights into Genetic Footprints of Listeria monocytogenes Strains Involved in Listeriosis Outbreaks

Listeria monocytogenes, a foodborne bacterial pathogen, causes invasive and febrile gastroenteritis forms of listeriosis in humans. Both invasive and febrile gastroenteritis listeriosis is caused mostly by serotypes 1/2a, 1/2b and 4b strains. The outbreak strains of serotype 1/2a and 4b could be further classified into several epidemic clones but the genetic bases for the diverse pathophysiology have been unsuccessful. DNA microarray provides an important tool to scan the entire genome for genetic signatures that may distinguish the L. monocytogenes strains belonging to different outbreaks. We have designed a pan-genomic microarray chip (Listeria GeneChip) containing sequences from 24 L. monocytogenes strains. The chip was designed to identify the presence/absence of genomic sequences, analyze transcription profiles and identify SNPs. Analysis of the genomic profiles of 38 outbreak strains representing 1/2a, 1/2b and 4b serotypes, revealed that the strains formed distinct genetic clusters adhering to their serotypes and epidemic clone types. Although serologically 1/2a and 1/b strains share common antigenic markers microarray analysis revealed that 1/2a strains are further apart from the closely related 1/2b and 4b strains. Within any given serotype and epidemic clone type the febrile gastroenteritis and invasive strains can be further distinguished based on several genetic markers including large numbers of phage genome, and intergenic sequences. Our results showed that the microarray-based data can be an important tool in characterization of L. monocytogenes strains involved in both invasive and gastroenteritis outbreaks. The results for the first time showed that the serotypes and epidemic clones are based on extensive pan-genomic variability and the 1/2b and 4bstrains are more closely related to each other than the 1/2a strains. The data also supported the hypothesis that the strains causing these two diverse outbreaks are genotypically different and this finding might be important in understanding the pathophysiology of this organism

Cycles of Police Reform in Latin America.

yesOver the last quarter century post-conflict and post-authoritarian transitions in Latin America have been accompanied by a surge in social violence, acquisitive crime, and insecurity. These phenomena have been driven by an expanding international narcotics trade, by the long-term effects of civil war and counter-insurgency (resulting in, inter alia, an increased availability of small arms and a pervasive grammar of violence), and by structural stresses on society (unemployment, hyper-inflation, widening income inequality). Local police forces proved to be generally ineffective in preventing, resolving, or detecting such crime and forms of “new violence”3 due to corruption, frequent complicity in criminal networks, poor training and low pay, and the routine use of excessive force without due sanction. Why, then, have governments been slow to prioritize police reform and why have reform efforts borne largely “limited or nonexistent” long-term results? This chapter highlights a number of lessons suggested by various efforts to reform the police in Latin America over the period 1995-2010 . It focuses on two clusters of countries in Latin America. One is Brazil and the Southern Cone countries (Chile, Argentina, and Uruguay), which made the transition to democracy from prolonged military authoritarian rule in the mid- to late 1980s. The other is Central America and the Andean region (principally El Salvador, Guatemala, Honduras, Peru, and Colombia), which emerged/have been emerging from armed conflict since the mid- 1990s. The chapter examines first the long history of international involvement in police and security sector reform in order to identify long-run tropes and path dependencies. It then focuses on a number of recurring themes: cycles of de- and re-militarization of the policing function; the “security gap” and “democratization dilemmas” involved in structural reforms; the opportunities offered by decentralization for more community-oriented police; and police capacity to resist reform and undermine accountability mechanisms

Establishing an infrastructure for collaboration in primate cognition research

Inferring the evolutionary history of cognitive abilities requires large and diverse samples. However, such samples are often beyond the reach of individual researchers or institutions, and studies are often limited to small numbers of species. Consequently, methodological and site-specific-differences across studies can limit comparisons between species. Here we introduce the ManyPrimates project, which addresses these challenges by providing a large-scale collaborative framework for comparative studies in primate cognition. To demonstrate the viability of the project we conducted a case study of short-term memory. In this initial study, we were able to include 176 individuals from 12 primate species housed at 11 sites across Africa, Asia, North America and Europe. All subjects were tested in a delayed-response task using consistent methodology across sites. Individuals could access food rewards by remembering the position of the hidden reward after a 0, 15, or 30-second delay. Overall, individuals performed better with shorter delays, as predicted by previous studies. Phylogenetic analysis revealed a strong phylogenetic signal for short-term memory. Although, with only 12 species, the validity of this analysis is limited, our initial results demonstrate the feasibility of a large, collaborative open-science project. We present the ManyPrimates project as an exciting opportunity to address open questions in primate cognition and behaviour with large, diverse datasets

Can Monkeys Make Investments Based on Maximized Pay-off?

Animals can maximize benefits but it is not known if they adjust their investment according to expected pay-offs. We investigated whether monkeys can use different investment strategies in an exchange task. We tested eight capuchin monkeys (Cebus apella) and thirteen macaques (Macaca fascicularis, Macaca tonkeana) in an experiment where they could adapt their investment to the food amounts proposed by two different experimenters. One, the doubling partner, returned a reward that was twice the amount given by the subject, whereas the other, the fixed partner, always returned a constant amount regardless of the amount given. To maximize pay-offs, subjects should invest a maximal amount with the first partner and a minimal amount with the second. When tested with the fixed partner only, one third of monkeys learned to remove a maximal amount of food for immediate consumption before investing a minimal one. With both partners, most subjects failed to maximize pay-offs by using different decision rules with each partner' quality. A single Tonkean macaque succeeded in investing a maximal amount to one experimenter and a minimal amount to the other. The fact that only one of over 21 subjects learned to maximize benefits in adapting investment according to experimenters' quality indicates that such a task is difficult for monkeys, albeit not impossible

Efficient oligonucleotide probe selection for pan-genomic tiling arrays

Background: Array comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in advance. For the accurate analysis of uncharacterized bacteria, an array must query a fully representative set of sequences from the species' pan-genome. Prior microarrays have included only a single strain per array or the conserved sequences of gene families. These arrays omit potentially important genes and sequence variants from the pan-genome. Results: This paper presents a new probe selection algorithm (PanArray) that can tile multiple whole genomes using a minimal number of probes. Unlike arrays built on clustered gene families, PanArray uses an unbiased, probe-centric approach that does not rely on annotations, gene clustering, or multi-alignments. Instead, probes are evenly tiled across all sequences of the pangenome at a consistent level of coverage. To minimize the required number of probes, probes conserved across multiple strains in the pan-genome are selected first, and additional probes are used only where necessary to span polymorphic regions of the genome. The viability of the algorithm is demonstrated by array designs for seven different bacterial pan-genomes and, in particular, the design of a 385,000 probe array that fully tiles the genomes of 20 different Listeria monocytogenes strains with overlapping probes at greater than twofold coverage. Conclusion: PanArray is an oligonucleotide probe selection algorithm for tiling multiple genome sequences using a minimal number of probes. It is capable of fully tiling all genomes of a species on a single microarray chip. These unique pan-genome tiling arrays provide maximum flexibility for the analysis of both known and uncharacterized strains.https://doi.org/10.1186/1471-2105-10-29

Preliminary Findings of a Randomized Trial of Non-Pharmaceutical Interventions to Prevent Influenza Transmission in Households

Background: There are sparse data on whether non-pharmaceutical interventions can reduce the spread of influenza. We implemented a study of the feasibility and efficacy of face masks and hand hygiene to reduce influenza transmission among Hong Kong household members. Methodology/Principal Findings: We conducted a cluster randomized controlled trial of households (composed of at least 3 members) where an index subject presented with influenza-like-illness of <48 hours duration. After influenza was confirmed in an index case by the QuickVue Influenza A+B rapid test, the household of the index subject was randomised to 1) control or 2) surgical face masks or 3) hand hygiene. Households were visited within 36 hours, and 3, 6 and 9 days later. Nose and throat swabs were collected from index subjects and all household contact at each home visit and tested by viral culture. The primary outcome measure was laboratory culture confirmed influenza in a household contact; the secondary outcome was clinically diagnosed influenza (by self-reported symptoms). We randomized 198 households and completed follow up home visits in 128; the index cases in 122 of those households had laboratory-confirmed influenza. There were 21 household contacts with laboratory confirmed influenza corresponding to a secondary attack ratio of 6%. Clinical secondary attack ratios varied from 5% to 18% depending on case definitions. The laboratory-based or clinical secondary attack ratios old not significantly differ across the intervention arms. Adherence to interventions was variable. Conclusions/Significance: The secondary attack ratios were lower than anticipated, and lower than reported in other countries, perhaps due to differing patterns of susceptibility, lack of significant antigenic drift in circulating influenza virus strains recently, and/or issues related to the symptomatic recruitment design. Lessons learn from this pilot have informed changes for the main study in 2008.published_or_final_versio

Monomeric and Dimeric CXCL8 Are Both Essential for In Vivo Neutrophil Recruitment

Rapid mobilization of neutrophils from vasculature to the site of bacterial/viral infections and tissue injury is a critical step in successful resolution of inflammation. The chemokine CXCL8 plays a central role in recruiting neutrophils. A characteristic feature of CXCL8 is its ability to reversibly exist as both monomers and dimers, but whether both forms exist in vivo, and if so, the relevance of each form for in vivo function is not known. In this study, using a ‘trapped’ non-associating monomer and a non-dissociating dimer, we show that (i) wild type (WT) CXCL8 exists as both monomers and dimers, (ii) the in vivo recruitment profiles of the monomer, dimer, and WT are distinctly different, and (iii) the dimer is essential for initial robust recruitment and the WT is most active for sustained recruitment. Using a microfluidic device, we also observe that recruitment is not only dependent on the total amount of CXCL8 but also on the steepness of the gradient, and the gradients created by different CXCL8 variants elicit different neutrophil migratory responses. CXCL8 mediates its function by binding to CXCR2 receptor on neutrophils and glycosaminoglycans (GAGs) on endothelial cells. On the basis of our data, we propose that dynamic equilibrium between CXCL8 monomers and dimers and their differential binding to CXCR2 and GAGs mediates and regulates in vivo neutrophil recruitment. Our finding that both CXCL8 monomer and dimer are functional in vivo is novel, and indicates that the CXCL8 monomer-dimer equilibrium and neutrophil recruitment are intimately linked in health and disease

Regeneration of Soft Tissues Is Promoted by MMP1 Treatment after Digit Amputation in Mice

The ratio of matrix metalloproteinases (MMPs) to the tissue inhibitors of metalloproteinases (TIMPs) in wounded tissues strictly control the protease activity of MMPs, and therefore regulate the progress of wound closure, tissue regeneration and scar formation. Some amphibians (i.e. axolotl/newt) demonstrate complete regeneration of missing or wounded digits and even limbs; MMPs play a critical role during amphibian regeneration. Conversely, mammalian wound healing re-establishes tissue integrity, but at the expense of scar tissue formation. The differences between amphibian regeneration and mammalian wound healing can be attributed to the greater ratio of MMPs to TIMPs in amphibian tissue. Previous studies have demonstrated the ability of MMP1 to effectively promote skeletal muscle regeneration by favoring extracellular matrix (ECM) remodeling to enhance cell proliferation and migration. In this study, MMP1 was administered to the digits amputated at the mid-second phalanx of adult mice to observe its effect on digit regeneration. Results indicated that the regeneration of soft tissue and the rate of wound closure were significantly improved by MMP1 administration, but the elongation of the skeletal tissue was insignificantly affected. During digit regeneration, more mutipotent progenitor cells, capillary vasculature and neuromuscular-related tissues were observed in MMP1 treated tissues; moreover, there was less fibrotic tissue formed in treated digits. In summary, MMP1 was found to be effective in promoting wound healing in amputated digits of adult mice. © 2013 Mu et al