Engineering the third wave of biocatalysis.

Over the past ten years, scientific and technological advances have established biocatalysis as a practical and environmentally friendly alternative to traditional metallo- and organocatalysis in chemical synthesis, both in the laboratory and on an industrial scale. Key advances in DNA sequencing and gene synthesis are at the base of tremendous progress in tailoring biocatalysts by protein engineering and design, and the ability to reorganize enzymes into new biosynthetic pathways. To highlight these achievements, here we discuss applications of protein-engineered biocatalysts ranging from commodity chemicals to advanced pharmaceutical intermediates that use enzyme catalysis as a key step.

Efficacy of geoengineering to limit 21st century sea-level rise.

Geoengineering has been proposed as a feasible way of mitigating anthropogenic climate change, especially increasing global temperatures in the 21st century. The two main geoengineering options are limiting incoming solar radiation, or modifying the carbon cycle. Here we examine the impact of five geoengineering approaches on sea level; SO(2) aerosol injection into the stratosphere, mirrors in space, afforestation, biochar, and bioenergy with carbon sequestration. Sea level responds mainly at centennial time scales to temperature change, and has been largely driven by anthropogenic forcing since 1850. Making use a model of sea-level rise as a function of time-varying climate forcing factors (solar radiation, volcanism, and greenhouse gas emissions) we find that sea-level rise by 2100 will likely be 30 cm higher than 2000 levels despite all but the most aggressive geoengineering under all except the most stringent greenhouse gas emissions scenarios. The least risky and most desirable way of limiting sea-level rise is bioenergy with carbon sequestration. However aerosol injection or a space mirror system reducing insolation at an accelerating rate of 1 W m(-2) per decade from now to 2100 could limit or reduce sea levels. Aerosol injection delivering a constant 4 W m(-2) reduction in radiative forcing (similar to a 1991 Pinatubo eruption every 18 months) could delay sea-level rise by 40-80 years. Aerosol injection appears to fail cost-benefit analysis unless it can be maintained continuously, and damage caused by the climate response to the aerosols is less than about 0.6% Global World Product.

From nature to industry: Harnessing enzymes for biocatalysis.

Biocatalysis harnesses enzymes to make valuable products. This green technology is used in countless applications from bench scale to industrial production and allows practitioners to access complex organic molecules, often with fewer synthetic steps and reduced waste. The last decade has seen an explosion in the development of experimental and computational tools to tailor enzymatic properties, equipping enzyme engineers with the ability to create biocatalysts that perform reactions not present in nature. By using (chemo)-enzymatic synthesis routes or orchestrating intricate enzyme cascades, scientists can synthesize elaborate targets ranging from DNA and complex pharmaceuticals to starch made in vitro from CO2-derived methanol. In addition, new chemistries have emerged through the combination of biocatalysis with transition metal catalysis, photocatalysis, and electrocatalysis. This review highlights recent key developments, identifies current limitations, and provides a future prospect for this rapidly developing technology.

Ustekinumab associated thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura (TTP) is a rare disorder. Plasma exchange therapy has been shown to significantly reduce mortality in patients with TTP. Here, we report a case of TTP associated with ustekinumab therapy after a period of 2-3 years. Ustekinumab, a monoclonal antibody that inhibits interleukin 12 and interleukin 23, is one of the newer treatments for psoriasis. Although our patient experienced a prolonged course of TTP requiring 1 month of daily plasma exchange therapy, he recovered and remains in remission after 6 months.

A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development.

In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.

A structural transition in class II major histocompatibility complex proteins at mildly acidic pH.

Peptide binding by class II major histocompatibility complex proteins is generally enhanced at low pH in the range of hydrogen ion concentrations found in the endosomal compartments of antigen-presenting cells. We and others have proposed that class II molecules undergo a reversible conformational change at low pH that is associated with enhanced peptide loading. However, no one has previously provided direct evidence for a structural change in class II proteins in the mildly acidic pH conditions in which enhanced peptide binding is observed. In this study, susceptibility to denaturation induced by sodium dodecyl sulfate (SDS) detergent or heat was used to probe the conformation of class II at different hydrogen ion concentrations. Class II molecules became sensitive to denaturation at pH 5.5-6.5 depending on the allele and experimental conditions. The observed structural transition was fully reversible if acidic pH was neutralized before exposure to SDS or heat. Experiments with the environment-sensitive fluorescent probe ANS (8-anilino-1-naphthalene-sulfonic acid) provided further evidence for a reversible structural transition at mildly acidic pH associated with an increase in exposed hydrophobicity in class II molecules. IAd conformation was found to change at a higher pH than IEd, IEk, or IAk, which correlates with the different pH optimal for peptide binding by these molecules. We conclude that pH regulates peptide binding by influencing the structure of class II molecules.

An insulin peptide that binds an alternative site in class II major histocompatibility complex.

We report that a peptide from the B chain of insulin, B(10-30), binds with high affinity to multiple class II proteins, including IAb,d,k, IEd,k, and DR1. The ability of B(10-30) to inhibit the binding of other peptide antigens to class II does not correlate with its affinity for class II. B(10-30) only weakly inhibits the binding of antigenic peptides. Conversely, peptides with high affinity for the peptide-binding groove of various class II proteins do not inhibit B(10-30) binding. The rate of association of B(10-30) with class II is unusually rapid, approaching saturation in 1-2 h compared with 1-2 d for classical peptide antigens in the same conditions. The dissociation rate is also relatively rapid. The B(10-30) peptide inhibits the binding of the super-antigen staphylococcal enterotoxin B (SEB) to IAk. It also inhibits SEB-mediated T cell activation. These observations support the conclusion that B(10-30) binds to a site outside the peptide-binding groove. Our findings indicate that short-lived peptide-class II complexes can be formed through interactions involving the SEB-binding site and raise the possibility that alternative complexes may serve as T cell receptor ligands.

Membrane interactions influence the peptide binding behavior of DR1.

We analyzed the binding of an influenza matrix protein-derived peptide, MAT(17-31), to cell surface and purified DR1. The pH dependence of peptide binding was dramatically influenced by the membrane environment. Cell surface binding was enhanced at low pH, with little or no binding detected at neutral pH and optimal binding at pH 4. By contrast, hydrogen ion concentration had minimal effect on peptide binding to purified DR1. Exposure to low pH in the absence of peptide did not affect the peptide binding capacity of cell-associated DR1. Purified DR1 was stable at low pH, excluding the possibility that enhanced binding was offset by a competing denaturation event at low pH. The striking effect of pH on peptide binding characteristic of cell surface DR1 was recovered after reconstitution of purified DR1 in B cell membranes by detergent dialysis. This behavior was partially recovered by reconstitution of full-length, but not truncated DR1 in vesicles containing purified lipid. Our results demonstrate that interactions involving membrane components influence the peptide-binding behavior of DR1.

Observational evidence for volcanic impact on sea level and the global water cycle.

It has previously been noted that there are drops in global sea level (GSL) after some major volcanic eruptions. However, observational evidence has not been convincing because there is substantial variability in the global sea level record over periods similar to those at which we expect volcanoes to have an impact. To quantify the impact of volcanic eruptions we average monthly GSL data from 830 tide gauge records around five major volcanic eruptions. Surprisingly, we find that the initial response to a volcanic eruption is a significant rise in sea level of 9 +/- 3 mm in the first year after the eruption. This rise is followed by a drop of 7 +/- 3 mm in the period 2-3 years after the eruption relative to preeruption sea level. These results are statistically robust and no particular volcanic eruption or ocean region dominates the signature we find. Neither the drop nor especially the rise in GSL can be explained by models of lower oceanic heat content. We suggest that the mechanism is a transient disturbance of the water cycle with a delayed response of land river runoff relative to ocean evaporation and global precipitation that affects global sea level. The volcanic impact on the water cycle and sea levels is comparable in magnitude to that of a large El Niño-La Niña cycle, amounting to approximately 5% of global land precipitation.

Determination of the effects of blood depth in the dermis on skin colour in a novel skin phantom using digital imaging.

A phantom of human port wine stain (PWS) skin, previously described by the authors, that takes into account its light propagation and scattering properties, was used to model varying depths of blood within PWS skin. Digital images of these phantoms were then acquired under controlled conditions, and the colour information was abstracted with a digital image processing suite. These colour data were analysed quantitatively for each depth of blood, and the relationship between depth of blood and colour was defined. A linear relationship was observed between depth of blood within the phantom and hue, hue being an intuitive measure of how colour is perceived by the human eye. As PWS clearance by laser treatment is dependant, to a large degree, on vessel depth within the skin, the ability to abstract colour data from PWS or, in fact, any vascular lesion within the skin, may help predict the degree of clearance before treatment is actually instigated. In the future, the comparison of phantom colour data with data from actual digital images of affected PWS skin, combined with a knowledge of laser light penetration depths, may provide a useful adjunct to clinical judgement in the prediction of PWS treatment outcomes.

Uplifted trench sediments: southwestern alaska-bering shelf edge.

Cretaceous turbidites are discontinuously exposed for 1700 kilometers along the continental margin of southwestern Alaska and the Bering shelf edge. Paleocurrent flow parallel to exposure patterns and the abundance of primary andesitic volcanic detritus suggest deposition in an oceanic trench. This Cretaceous trench bordering the continent was superseded by the Aleutian arc-trench in the earliest Tertiary.

Slaty cleavage: incipient occurrences in the deep sea.

Highly deformed Pleistocene mudstones from the inner wall of the Aleutian Trench and from the continental rise of the Gulf of Mexico show incipient slaty cleavage defined by the orientation of platy and elongate detrital minerals parallel to the axial surfaces of folds.

Energetics, patterns of interaction strengths, and stability in real ecosystems.

Ecologists have long been studying stability in ecosystems by looking at the structuring and the strengths of trophic interactions in community food webs. In a series of real food webs from native and agricultural soils, the strengths of the interactions were found to be patterned in a way that is important to ecosystem stability. The patterning consisted of the simultaneous occurrence of strong "top down" effects at lower trophic levels and strong "bottom up" effects at higher trophic levels. As the patterning resulted directly from the energetic organization of the food webs, the results show that energetics and community structure govern ecosystem stability by imposing stabilizing patterns of interaction strengths.

Influence of productivity on the stability of real and model ecosystems.

The lengths of food chains within ecosystems have been thought to be limited either by the productivity of the ecosystem or by the resilience of that ecosystem after perturbation. Models based on ecological energetics that follow the form of Lotka-Volterra equations and equations that include material (detritus) recycling show that productivity and resilience are inextricably interrelated. The models were initialized with data from 5-to 10-year studies of actual soil food webs. Estimates indicate that most ecological production worldwide is from ecosystems that are themselves sufficiently productive to recover from minor perturbations.

Oregon subduction zone: venting, fauna, and carbonates.

Transects of the submersible Alvin across rock outcrops in the Oregon subduction zone have furnished information on the structural and stratigraphic framework of this accretionary complex. Communities of clams and tube worms, and authigenic carbonate mineral precipitates, are associated with venting sites of cool fluids located on a fault-bend anticline at a water depth of 2036 meters. The distribution of animals and carbonates suggests up-dip migration of fluids from both shallow and deep sources along permeable strata or fault zones within these clastic deposits. Methane is enriched in the water column over one vent site, and carbonate minerals and animal tissues are highly enriched in carbon-12. The animals use methane as an energy and food source in symbiosis with microorganisms. Oxidized methane is also the carbon source for the authigenic carbonates that cement the sediments of the accretionary complex. The animal communities and carbonates observed in the Oregon subduction zone occur in strata as old as 2.0 million years and provide criteria for identifying other localities where modern and ancient accreted deposits have vented methane, hydrocarbons, and other nutrient-bearing fluids.

Outbreeding and possibly inbreeding depression in a pollinating fig wasp with a mixed mating system.

Mixed mating systems are somewhat of an enigma as most models predict that organisms should either inbreed when inbreeding depression is low, or outbreed when inbreeding depression is high. Many wasps mix routine inbreeding with a little random mating. This random mating is most common when all local sibmating opportunities are exhausted and dispersal is the only way males can further increase their fitness. The males of the pollinating fig wasp, Platyscapa awekei, are slightly different in that they disperse before all sibmating opportunities have been exhausted. To see if this is a response to inbreeding depression we quantify inbreeding depression by comparing females' life time reproductive success to their heterozygosity at multiple microsatellite loci. We find that a female wasp's heterozygosity is an accurate predictor of her inbreeding coefficient and that P. awekei females actually seem to suffer from outbreeding depression and possibly from a little inbreeding depression. Male dispersal is thus not a means to effect the optimal mating system, but more likely a mechanism to reduce competition among brothers. The number of mature offspring a female produces depends on her own heterozygosity and not on that of the offspring, and may be determined by egg and gall quality.

Eliminating side products and increasing succinate yields in engineered strains of Escherichia coli C.

Derivatives of Escherichia coli C were previously described for succinate production by combining the deletion of genes that disrupt fermentation pathways for alternative products (ldhA::FRT, adhE::FRT, ackA::FRT, focA-pflB::FRT, mgsA, poxB) with growth-based selection for increased ATP production. The resulting strain, KJ073, produced 1.2 mol of succinate per mol glucose in mineral salts medium with acetate, malate, and pyruvate as significant co-products. KJ073 has been further improved by removing residual recombinase sites (FRT sites) from the chromosomal regions of gene deletion to create a strain devoid of foreign DNA, strain KJ091(DeltaldhA DeltaadhE DeltaackA DeltafocA-pflB DeltamgsA DeltapoxB). KJ091 was further engineered for improvements in succinate production. Deletion of the threonine decarboxylase (tdcD; acetate kinase homologue) and 2-ketobutyrate formate-lyase (tdcE; pyruvate formate-lyase homologue) reduced the acetate level by 50% and increased succinate yield (1.3 mol mol(-1) glucose) by almost 10% as compared to KJ091 and KJ073. Deletion of two genes involved in oxaloacetate metabolism, aspartate aminotransferase (aspC) and the NAD(+)-linked malic enzyme (sfcA) (KJ122) significantly increased succinate yield (1.5 mol mol(-1) glucose), succinate titer (700 mM), and average volumetric productivity (0.9 g L(-1) h(-1)). Residual pyruvate and acetate were substantially reduced by further deletion of pta encoding phosphotransacetylase to produce KJ134 (DeltaldhA DeltaadhE DeltafocA-pflB DeltamgsA DeltapoxB DeltatdcDE DeltacitF DeltaaspC DeltasfcA Deltapta-ackA). Strains KJ122 and KJ134 produced near theoretical yields of succinate during simple, anaerobic, batch fermentations using mineral salts medium. Both may be useful as biocatalysts for the commercial production of succinate.

Combining metabolic engineering and metabolic evolution to develop nonrecombinant strains of Escherichia coli C that produce succinate and malate.

Derivatives of Escherichia coli C were engineered to produce primarily succinate or malate in mineral salts media using simple fermentations (anaerobic stirred batch with pH control) without the addition of plasmids or foreign genes. This was done by a combination of gene deletions (genetic engineering) and metabolic evolution with over 2,000 generations of growth-based selection. After deletion of the central anaerobic fermentation genes (ldhA, adhE, ackA), the pathway for malate and succinate production remained as the primary route for the regeneration of NAD+. Under anaerobic conditions, ATP production for growth was obligately coupled to malate dehydrogenase and fumarate reductase by the requirement for NADH oxidation. Selecting strains for improved growth co-selected increased production of these dicarboxylic acids. Additional deletions were introduced as further improvements (focA, pflB, poxB, mgsA). The best succinate biocatalysts, strains KJ060(ldhA, adhE, ackA, focA, pflB) and KJ073(ldhA, adhE, ackA, focA, pflB, mgsA, poxB), produce 622-733 mM of succinate with molar yields of 1.2-1.6 per mole of metabolized glucose. The best malate biocatalyst, strain KJ071(ldhA, adhE, ackA, focA, pflB, mgsA), produced 516 mM malate with molar yields of 1.4 per mole of glucose metabolized.

Cellular immune responses to platelet factor 4 and heparin complexes in patients with heparin-induced thrombocytopenia.

Essentials The immunogenesis of Heparin-induced thrombocytopenia (HIT) is not well understood. Immunization to platelet factor 4 (PF4)-heparin occurs early in life, before any heparin exposure. PF4 and PF4-heparin complexes induce the proliferation of CD14+ cells. Reduced levels of regulatory cytokines contribute to immune dysregulation in HIT. SUMMARY: Background Heparin-induced thrombocytopenia (HIT) is an adverse reaction to heparin characterized by thrombocytopenia and thrombotic complications. HIT is caused by pathogenic antibodies that bind to complexes of platelet factor 4 (PF4) and heparin, leading to platelet activation and inducing a hypercoagulable state. Previous studies have shown immunity to PF4-heparin complexes occurs early in life, even before heparin exposure; however, the immunogenesis of HIT is not well characterized. Objectives To investigate cellular proliferation in response to PF4-heparin complexes in patients with HIT. Patients/Methods Peripheral blood mononuclear cells (PBMCs) from healthy controls (n = 30), postoperative cardiac surgery patients who had undergone cardiopulmonary bypass (CPB) (n = 17) and patients with confirmed HIT (n = 41) were cultured with PF4 and PF4-heparin complexes. Cellular proliferation was assessed by [3 H]thymidine uptake and 5-ethynyl-2'-deoxyuridine detection. Results and Conclusions PBMCs proliferated in the presence of PF4, and this was enhanced by the addition of heparin in all study groups. CPB and HIT patients showed significantly greater proliferative responses than healthy controls. PBMC proliferation was antigen-specific, depended on the presence of platelets, and only CD14+ cells were identified as proliferating cells. Culture supernatants were tested for the levels of regulatory cytokines, and both CPB and HIT patients produced significantly lower levels of interleukin-10 and transforming growth factor-ß1 than healthy controls. These findings further demonstrate cellular immune sensitization to PF4-heparin complexes occurs before heparin exposure, and suggests immune dysregulation can contribute to HIT.

Directed evolution of a para-nitrobenzyl esterase for aqueous-organic solvents.

Through sequential generations of random mutagenesis and screening, we have directed the evolution of an esterase for deprotection of an antibiotic p-nitrobenzyl ester in aqueous-organic solvents. Because rapid screening directly on the desired antibiotic (loracarbef) nucleus p-nitrobenzyl ester was not feasible, the p-nitrophenyl ester was employed. Catalytic performance on the screening substrate was shown to reasonably mimic enzyme activity toward the desired ester. One p-nitrobenzyl esterase variant performs as well in 30% dimethylformamide as the wildtype enzyme in water, reflecting a 16-fold increase in esterase activity. Random pairwise gene recombination of two positive variants led to a further two-fold improvement in activity. Considering also the increased expression level achieved during these experiments, the net result of four sequential generations of random mutagenesis and the one recombination step is a 50-60-fold increase in total activity. Although the contributions of individual effective amino acid substitutions to enhanced activity are small (< 2-fold increases), the accumulation of multiple mutations by directed evolution allows significant improvement of the biocatalyst for reactions on substrates and under conditions not already optimized in nature. The positions of the effective amino acid substitutions have been identified in a pNB esterase structural model developed based on its homology to acetylcholinesterase and triacylglycerol lipase. None appear to interact directly with the antibiotic substrate, further underscoring the difficulty of predicting their effects in a 'rational' design effort.