Engineering bacteria to solve the Burnt Pancake Problem

<p>Abstract</p> <p>Background</p> <p>We investigated the possibility of executing DNA-based computation in living cells by engineering <it>Escherichia coli </it>to address a classic mathematical puzzle called the Burnt Pancake Problem (BPP). The BPP is solved by sorting a stack of distinct objects (pancakes) into proper order and orientation using the minimum number of manipulations. Each manipulation reverses the order and orientation of one or more adjacent objects in the stack. We have designed a system that uses site-specific DNA recombination to mediate inversions of genetic elements that represent pancakes within plasmid DNA.</p> <p>Results</p> <p>Inversions (or "flips") of the DNA fragment pancakes are driven by the <it>Salmonella typhimurium </it>Hin/<it>hix </it>DNA recombinase system that we reconstituted as a collection of modular genetic elements for use in <it>E. coli</it>. Our system sorts DNA segments by inversions to produce different permutations of a promoter and a tetracycline resistance coding region; <it>E. coli </it>cells become antibiotic resistant when the segments are properly sorted. Hin recombinase can mediate all possible inversion operations on adjacent flippable DNA fragments. Mathematical modeling predicts that the system reaches equilibrium after very few flips, where equal numbers of permutations are randomly sorted and unsorted. Semiquantitative PCR analysis of <it>in vivo </it>flipping suggests that inversion products accumulate on a time scale of hours or days rather than minutes.</p> <p>Conclusion</p> <p>The Hin/<it>hix </it>system is a proof-of-concept demonstration of <it>in vivo </it>computation with the potential to be scaled up to accommodate larger and more challenging problems. Hin/<it>hix </it>may provide a flexible new tool for manipulating transgenic DNA <it>in vivo</it>.</p

Role of Serine Proteases in the Regulation of Interleukin-8₇₇ during the Development of Bronchopulmonary Dysplasia in Preterm Ventilated Infants

<div><p>Rationale</p><p>The chemokine interleukin-8 is implicated in the development of bronchopulmonary dysplasia in preterm infants. The 77-amino acid isoform of interleukin-8 (interleukin-8₇₇) is a less potent chemoattractant than other shorter isoforms. Although interleukin-8₇₇ is abundant in the preterm circulation, its regulation in the preterm lung is unknown.</p><p>Objectives</p><p>To study expression and processing of pulmonary interleukin-8₇₇ in preterm infants who did and did not develop bronchopulmonary dysplasia.</p><p>Methods</p><p>Total interleukin-8 and interleukin-8₇₇ were measured in bronchoalveolar lavage fluid from preterm infants by immunoassay. Neutrophil serine proteases were used to assess processing. Neutrophil chemotaxis assays and degranulation of neutrophil matrix metalloproteinase-9 were used to assess interleukin-8 function.</p><p>Main Results</p><p>Peak total interleukin-8 and interleukin-8₇₇ concentrations were increased in infants who developed bronchopulmonary dysplasia compared to those who did not. Shorter forms of interleukin-8 predominated in the preterm lung (96.3% No-bronchopulmonary dysplasia vs 97.1% bronchopulmonary dysplasia, p>0.05). Preterm bronchoalveolar lavage fluid significantly converted exogenously added interleukin-8₇₇ to shorter isoforms (p<0.001). Conversion was greater in bronchopulmonary dysplasia infants (p<0.05). This conversion was inhibited by α-1 antitrypsin and antithrombin III (p<0.01). Purified neutrophil serine proteases efficiently converted interleukin-8₇₇ to shorter isoforms in a time- and dose-dependent fashion; shorter interleukin-8 isoforms were primarily responsible for neutrophil chemotaxis (p<0.001). Conversion by proteinase-3 resulted in significantly increased interleukin-8 activity <i>in vitro</i> (p<0.01).</p><p>Conclusions</p><p>Shorter, potent, isoforms interleukin-8 predominate in the preterm lung, and are increased in infants developing bronchopulmonary dysplasia, due to conversion of interleukin-8₇₇ by neutrophil serine proteases and thrombin. Processing of interleukin-8 provides an attractive therapeutic target to prevent development of bronchopulmonary dysplasia.</p></div

Hematopoietic stem cell aging and self-renewal

A functional decline of the immune system occurs during organismal aging that is attributable, in large part, to changes in the hematopoietic stem cell (HSC) compartment. In the mouse, several hallmark age-dependent changes in the HSC compartment have been identified, including an increase in HSC numbers, a decrease in homing efficiency, and a myeloid skewing of differentiation potential. Whether these changes are caused by gradual intrinsic changes within individual HSCs or by changes in the cellular composition of the HSC compartment remains unclear. However, of note, many of the aging properties of HSCs are highly dependent on their genetic background. In particular, the widely used C57Bl/6 strain appears to have unique HSC aging characteristics compared with those of other mouse strains. These differences can be exploited by using recombinant inbred strains to further our understanding of the genetic basis for HSC aging. The mechanism(s) responsible for HSC aging have only begun to be elucidated. Recent studies have reported co-ordinated variation in gene expression of HSCs with age, possibly as a result of epigenetic changes. In addition, an accumulation of DNA damage, in concert with an increase in intracellular reactive oxygen species, has been associated with aged HSCs. Nevertheless, whether age-related changes in HSCs are programmed to occur in a certain predictable fashion, or whether they are simply an accumulation of random changes over time remains unclear. Further, whether the genetic dysregulation observed in old HSCs is a cause or an effect of cellular aging is unknown