Cell-free biology: exploiting the interface between synthetic biology and synthetic chemistry

Just as synthetic organic chemistry once revolutionized the ability of chemists to build molecules (including those that did not exist in nature) following a basic set of design rules, cell-free synthetic biology is beginning to provide an improved toolbox and faster process for not only harnessing but also expanding the chemistry of life. At the interface between chemistry and biology, research in cell-free synthetic systems is proceeding in two different directions: using synthetic biology for synthetic chemistry and using synthetic chemistry to reprogram or mimic biology. In the coming years, the impact of advances inspired by these approaches will make possible the synthesis of nonbiological polymers having new backbone compositions, new chemical properties, new structures, and new functions.     

Nearshore macrobenthos of northern Kotzebue Sound,Alaska, with reference to local sewage disposal

Macrobenthos of the shallow (<10 m) nearshore marine waters of northern Kotzebue Sound was examined in 2002–2004 to (1) determine nearshore community structure and (2) assess the influence of sewage disposal. A variable number of benthic stations were sampled during three summers, with extensive effort at the disposal zone in 2003. The benthic community structure is similar to other nearshore Arctic locations, and was similar to a previous benthic study done in 1986–1987. The potential of sewage impact was assessed at the request of the community, because sewage is occasionally discharged into the Sound, in volumes of up to 38 million liters, typically through the ice in early spring. Only minimal effects of disposal on the benthos were evident and the effects could not be separated from the impacts of low salinity and relatively high water pigments. Low diversity (H′) and species richness (d) and high biomass characterized stations in the sewage area. Parameters often associated with extreme sewage pollution, particularly hypoxic and/or anoxic conditions and high abundance of opportunistic taxa, were not observed. Local traditional ecological knowledge was solicited throughout the study, and was used to help define the area potentially affected by sewage disposal.     

Large predators and biogeochemical hotspots: brown bear (Ursus arctos) predation on salmon alters nitrogen cycling in riparian soils

Two important themes in ecology include the understanding of how interactions among species control ecosystem processes, and how habitats can be connected through transfers of nutrients and energy by mobile organisms. An impressive example of both is the large influx of nutrients and organic matter that anadromous salmon supply to inland aquatic and terrestrial ecosystems and the role of predation by brown bears (Ursus arctos) in transferring these marine-derived nutrients (MDN) from freshwater to riparian habitats. In spite of the recognition that salmon-bear interactions likely play an important role in controlling the flux of MDN from aquatic to riparian habitats, few studies have linked bear predation on salmon to processes such as nitrogen (N) or carbon (C) cycling. We combine landscape-level survey data and a replicated bear-exclosure experiment to test how bear foraging on salmon affects nitrous oxide (N₂O) flux, carbon dioxide (CO₂) flux, and nutrient concentrations of riparian soils. Our results show that bears feeding on salmon increased soil ammonium (NH₄ ⁺) concentrations three-fold and N₂O flux by 32-fold. Soil CO₂ flux, nitrate (NO₃ ⁻), and N transformation differences were negligible in areas where bears fed on salmon. Reference areas without concentrated bear activity showed no detectable change in soil N cycling after the arrival of salmon to streams. Exclosure experiments showed that bear effects on soil nutrient cycles were transient, and soil N processing returned to background conditions within 1 year after bears were removed from the system. These results suggest that recipient ecosystems do not show uniform responses to MDN inputs and highlight the importance of large mobile consumers in generating landscape heterogeneity in nutrient cycles.     

Chemopreventive and renal protective effects for docosahexaenoic acid (DHA): implications of CRP and lipid peroxides

Background

The fish oil-derived ω-3 fatty acids, like docosahexanoic (DHA), claim a plethora of health benefits. We currently evaluated the antitumor effects of DHA, alone or in combination with cisplatin (CP) in the EAC solid tumor mice model, and monitored concomitant changes in serum levels of C-reactive protein (CRP), lipid peroxidation (measured as malondialdehyde; MDA) and leukocytic count (LC). Further, we verified the capacity of DHA to ameliorate the lethal, CP-induced nephrotoxicity in rats and the molecular mechanisms involved therein.

Results

EAC-bearing mice exhibited markedly elevated LC (2-fold), CRP (11-fold) and MDA levels (2.7-fold). DHA (125, 250 mg/kg) elicited significant, dose-dependent reductions in tumor size (38%, 79%; respectively), as well as in LC, CRP and MDA levels. These effects for CP were appreciably lower than those of DHA (250 mg/kg). Interestingly, DHA (125 mg/kg) markedly enhanced the chemopreventive effects of CP and boosted its ability to reduce serum CRP and MDA levels. Correlation studies revealed a high degree of positive association between tumor growth and each of CRP (r = 0.85) and leukocytosis (r = 0.89), thus attesting to a diagnostic/prognostic role for CRP. On the other hand, a single CP dose (10 mg/kg) induced nephrotoxicity in rats that was evidenced by proteinuria, deterioration of glomerular filtration rate (GFR, -4-fold), a rise in serum creatinine/urea levels (2–5-fold) after 4 days, and globally-induced animal fatalities after 7 days. Kidney-homogenates from CP-treated rats displayed significantly elevated MDA- and TNF-α-, but reduced GSH-, levels. Rats treated with DHA (250 mg/kg, but not 125 mg/kg) survived the lethal effects of CP, and showed a significant recovery of GFR; while their homogenates had markedly-reduced MDA- and TNF-α-, but -increased GSH-levels. Significant association was detected between creatinine level and those of MDA (r = 0.81), TNF-α ) r = 0.92) and GSH (r = -0.82); implying causal relationships.

Conclusion

DHA elicited prominent chemopreventive effects on its own, and appreciably augmented those of CP as well. The extent of tumor progression in various mouse groups was highly reflected by CRP levels (thus implying a diagnostic/prognostic role for CRP). Further, this study is the first to reveal that DHA can obliterate the lethal CP-induced nephrotoxicity and renal tissue injury. At the molecular level, DHA appears to act by reducing leukocytosis, systemic inflammation, and oxidative stress.     

lacZ Reporter System for Use in Borrelia burgdorferi

Regulation of gene expression is critical for the ability of Borrelia burgdorferi to adapt to different environments during its natural infectious cycle. Reporter genes have been used successfully to study gene regulation in multiple organisms. We have introduced a lacZ gene into B. burgdorferi, and we show that B. burgdorferi produces a protein with detectable β-galactosidase activity in both liquid and solid media when lacZ is expressed from a constitutive promoter. Furthermore, when lacZ is expressed from the ospC promoter, β-galactosidase activity is detected only in B. burgdorferi clones that express ospC, and it accurately monitors endogenous gene expression. The addition of lacZ to the repertoire of genetic tools available for use in B. burgdorferi should contribute to a better understanding of how B. burgdorferi gene expression is regulated during the infectious cycle.Borrelia burgdorferi sensu lato, the pathogen that causes Lyme disease (7), alternates between two distinct environments, an arthropod vector and a vertebrate host. As B. burgdorferi moves from one milieu to the other, its ability to adapt and survive requires dramatic changes in gene expression. Many studies have shown that different B. burgdorferi gene products are upregulated or downregulated at specific times during the infectious cycle (19, 31) and in response to host and environmental signals (6, 8a, 15, 24, 25). Although it is clear that B. burgdorferi alters gene expression to adapt to different environments, the genetic tools for studying gene regulation in B. burgdorferi are limited.Within the last 2 decades, the complete genomic sequence of B. burgdorferi strain B31 was published (10, 14) and techniques for basic genetic manipulation of B. burgdorferi became available (5, 11, 13, 27-29, 36). A chloramphenicol acetyltransferase (CAT) gene was the first reporter gene that was fused to B. burgdorferi promoters for analysis of promoter strength (33). The development of luciferase (4) and multiple fluorescent proteins (9, 11, 30) as reporter systems in B. burgdorferi followed. Although these systems have value, there are limitations with each. β-Galactosidase, encoded by lacZ, has been used extensively as a convenient reporter gene in Escherichia coli and is still applicable to a broad range of organisms, both prokaryotic and eukaryotic, but has not yet been used with B. burgdorferi. β-Galactosidase activity can be monitored easily and quickly by simple colorimetric assays in both liquid and solid media, neither of which require expensive or specialized equipment. Additionally, a wide variety of substrates for β-galactosidase allow for different levels of sensitivity in either in vitro or in vivo detection formats (17). Having lacZ available as a genetic tool for B. burgdorferi would enhance investigation of the complex regulatory events that are integral to the spirochete''s infectious cycle. To this end, we developed lacZ as a reporter gene in B. burgdorferi and demonstrated its utility.     

感染球孢白僵菌后红火蚁体内蛋白质含量的变化

为了解球孢白僵菌Beauveria bassiana成功感染红火蚁Solenopsis invicta的生理生化机制, 本研究在室内条件下测定了经球孢白僵菌感染后红火蚁工蚁、蛹和幼蚁体内蛋白质含量的变化。结果表明: 红火蚁各虫态感染球孢白僵菌后体内蛋白质含量变化存在差异。在接种后12-72 h, 工蚁体内蛋白质含量在24 h时最高, 为47.12 mg/g, 显著低于对照的56.40 mg/g(P＜0.05), 此后呈下降趋势, 72 h时蛋白质含量下降至25.16 mg/g。幼蚁在接种36 h时体内蛋白质含量最高, 为24.13 mg/g, 此后呈下降趋势, 72 h时蛋白质含量为8.95 mg/g, 显著低于对照的24.80 mg/g。蛹感染球孢白僵菌后体内蛋白质含量一直呈下降趋势, 从12 h的36.57 mg/g降到72 h的10.42 mg/g, 与对照的38.61 mg/g相比差异显著(P＜0.05)。结果显示球孢白僵菌感染能显著降低红火蚁各虫态体内蛋白质含量。     

Cicada ear geometry: species and sex effects

Many insect species rely on their sense of audition to find a mate, to localize prey or to escape from a predator. Cicadas are particularly known for their loud call and the conspicuous tympanal hearing system located in their abdomen. The vibration pattern of the tympanal membrane (TM) has been investigated recently revealing mechanical properties specific to species and sex. Although TM size and shape is likely to affect these patterns, the geometry of the cicada ear has never been examined per se. Focusing on three Mediterranean cicada species, namely Cicadatra atra, Cicada orni and Lyristes plebejus, we investigated the structure of TM shape variation at two levels, within and across species. Applying an elliptic Fourier analysis to the outlines of both male and female TMs, we estimated sexual dimorphism and species effects. Cicadatra atra showed a large TM compared with its small size, probably as a result of selective constraints related to the role of the TM in sound production. Sexual dimorphism seemed to be greater than interspecific variation, indicating that constraints operating on sex might be more selective than those acting on species identification. In addition, C. orni appeared to be significantly different from the two other species. This morphological peculiarity could be related to the unique vibrational pattern of its membrane. This would establish for the first time a direct link between the shape and mechanism of a hearing organ. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 922–934.     

Sequence periodicity and secondary structure propensity in model proteins

We explore the question of whether local effects (originating from the amino acids intrinsic secondary structure propensities) or nonlocal effects (reflecting the sequence of amino acids as a whole) play a larger role in determining the fold of globular proteins. Earlier circular dichroism studies have shown that the pattern of polar, non polar amino acids (nonlocal effect) dominates over the amino acid intrinsic propensity (local effect) in determining the secondary structure of oligomeric peptides. In this article, we present a coarse grained computational model that allows us to quantitatively estimate the role of local and nonlocal factors in determining both the secondary and tertiary structure of small, globular proteins. The amino acid intrinsic secondary structure propensity is modeled by a dihedral potential term. This dihedral potential is parametrized to match with experimental measurements of secondary structure propensity. Similarly, the magnitude of the attraction between hydrophobic residues is parametrized to match the experimental transfer free energies of hydrophobic amino acids. Under these parametrization conditions, we systematically explore the degree of frustration a given polar, non polar pattern can tolerate when the secondary structure intrinsic propensities are in opposition to it. When the parameters are in the biophysically relevant range, we observe that the fold of small, globular proteins is determined by the pattern of polar, non polar amino acids regardless of their instrinsic secondary structure propensities. Our simulations shed new light on previous observations that tertiary interactions are more influential in determining protein structure than secondary structure propensity. The fact that this can be inferred using a simple polymer model that lacks most of the biochemical details points to the fundamental importance of binary patterning in governing folding.     

Phylogeography of the mottled spinefoot Siganus fuscescens: Pleistocene divergence and limited genetic connectivity across the Philippine archipelago

Historical isolation during Pleistocene low sea level periods is thought to have contributed to divergence among marine basin populations across the Coral Triangle. In the Philippine archipelago, populations in the South China Sea, Sulu Sea–inland seas, and Philippine Sea‐Celebes Sea basins might have been partially isolated. Meanwhile, present‐day broadscale oceanographic circulation patterns suggest connectivity between these basins. To evaluate hypotheses regarding the influence of historical and contemporary factors on genetic structure, phylogeographic patterns based on mitochondrial control region sequences for a reef‐associated fish, Siganus fuscescens, were analysed. Three distinct lineages were recovered. One lineage was identified as the morphologically similar species Siganus canaliculatus, while two lineages are monophyletic with S. fuscescens. Clade divergence and demographic expansion in S. fuscescens occurred during the Pleistocene. A strong signal of latitudinal structure was detected (Φ_CT = 0.188), driven by marked differences in clade distribution: one clade is widely distributed (clade A), while a second clade (clade B) has a restricted northern distribution. Regional structure of clade A is consistent with the basin isolation hypothesis (Φ_CT = 0.040) and suggests isolation of the South China Sea (Φ_CT = 0.091). Fine‐scale structure was observed in the South China Sea and south Philippine Sea, while Sulu Sea and inland seas were unstructured. Genetic structure across multiple spatial scales (archipelagic, regional, and fine‐scale within basins) suggests the influence of vicariant barriers and contemporary limits to gene flow in S. fuscescens that may be influenced by oceanographic circulation, geographical distance between available habitats, and latitudinal temperature differences.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">ex vivo</Emphasis> effect of hyaluronic acid on erythrocyte flow properties

Background  

Hyaluronic acid (HA) is present in many tissues; its presence in serum may be related to certain inflammatory conditions, tissue damage, sepsis, liver malfunction and some malignancies. In the present work, our goal was to investigate the significance of hyaluronic acid effect on erythrocyte flow properties. Therefore we performed in vitro experiments incubating red blood cells (RBCs) with several HA concentrations. Afterwards, in order to corroborate the pathophysiological significance of the results obtained, we replicated the in vitro experiment with ex vivo RBCs from diagnosed rheumatoid arthritis (RA) patients, a serum HA-increasing pathology.