Anchor ice and benthic disturbance in shallow Antarctic waters: interspecific variation in initiation and propagation of ice crystals

Sea ice typically forms at the ocean's surface, but given a source of supercooled water, an unusual form of ice--anchor ice--can grow on objects in the water column or at the seafloor. For several decades, ecologists have considered anchor ice to be an important agent of disturbance in the shallow-water benthic communities of McMurdo Sound, Antarctica, and potentially elsewhere in polar seas. Divers have documented anchor ice in the McMurdo communities, and its presence coincides with reduced abundance of the sponge Homaxinella balfourensis, which provides habitat for a diverse assemblage of benthic organisms. However, the mechanism of this disturbance has not been explored. Here we show interspecific differences in anchor-ice formation and propagation characteristics for Antarctic benthic organisms. The sponges H. balfourensis and Suberites caminatus show increased incidence of formation and accelerated spread of ice crystals compared to urchins and sea stars. Anchor ice also forms readily on sediments, from which it can grow and adhere to organisms. Our results are consistent with, and provide a potential first step toward, an explanation for disturbance patterns observed in shallow polar benthic communities. Interspecific differences in ice formation raise questions about how surface tissue characteristics such as surface area, rugosity, and mucus coating affect ice formation on invertebrates.     

Computer-aided high-throughput cloning of bacteria in liquid medium

Bacterial cloning was first introduced over a century ago and has since become one of the most useful procedures in biological research, perhaps paralleled in its ubiquity only by PCR and DNA sequencing. However, unlike PCR and sequencing, cloning has generally remained a manual, labor-intensive, low-throughput procedure. Here we address this issue by developing an automated, computer-aided bacterial cloning method using liquid medium that is based on the principles of (i) limiting dilution of bacteria, (ii) inference of colony forming units (CFUs) based on optical density (OD) readings, and (iii) verification of monoclonality using a mixture of differently colored fluorescently labeled bacteria for transformation. We demonstrate the high-throughput utility of this method by employing it as a cloning platform for a DNA synthesis process.     

Variations in the unstructured C-terminal tail of interferons contribute to differential receptor binding and biological activity

Type I interferons (IFNs) elicit antiviral, antiproliferative and immunomodulatory properties in cells. All of them bind to the same receptor proteins, IFNAR1 and IFNAR2, with different affinities. While the 13 known IFNalphas are highly conserved, the C-terminal unstructured tail was found to have large variation in its net charge, from neutral to +4. This led us to speculate that the tail may have a role in modulation of the IFN biological activity, through fine-tuning the binding to IFNAR2. To evaluate this hypothesis, we replaced the tail of IFNalpha2 with that of IFNalpha8 and IFNbeta tails, or deleted the last five residues of this segment. Mutations to the more positively charged tail of IFNalpha8 resulted in a 20-fold higher affinity to IFNAR2, which results in a higher antiviral and antiproliferative activity. Double and multiple mutant cycle analysis placed the tail near a negatively charged loop on IFNAR2, comprising of residues Glu 132-134. Deleting the tail resulted in only twofold reduction in binding compared to the wild-type. Next, we modeled the location of the tail using a two-step procedure: first we generated 200 models of the tail docked on IFNAR2 using HADDOCK, second the models were scored according to the fit between experimentally determined rates of association of nine mutant complexes, and their calculated rates using the PARE software. From the results we suggest that the unstructured tail of IFNalpha is gaining a specific structure in the bound state, binding to a groove below the 132-134 loop in IFNAR2.     

Processing DNA molecules as text

Polymerase Chain Reaction (PCR) is the DNA-equivalent of Gutenberg’s movable type printing, both allowing large-scale replication of a piece of text. De novo DNA synthesis is the DNA-equivalent of mechanical typesetting, both ease the setting of text for replication. What is the DNA-equivalent of the word processor? Biology labs engage daily in DNA processing—the creation of variations and combinations of existing DNA—using a plethora of manual labor-intensive methods such as site-directed mutagenesis, error-prone PCR, assembly PCR, overlap extension PCR, cleavage and ligation, homologous recombination, and others. So far no universal method for DNA processing has been proposed and, consequently, no engineering discipline that could eliminate this manual labor has emerged. Here we present a novel operation on DNA molecules, called Y, which joins two DNA fragments into one, and show that it provides a foundation for DNA processing as it can implement all basic text processing operations on DNA molecules including insert, delete, replace, cut and paste and copy and paste. In addition, complicated DNA processing tasks such as the creation of libraries of DNA variants, chimeras and extensions can be accomplished with DNA processing plans consisting of multiple Y operations, which can be executed automatically under computer control. The resulting DNA processing system, which incorporates our earlier work on recursive DNA composition and error correction, is the first demonstration of a unified approach to DNA synthesis, editing, and library construction.

Electronic supplementary material

The online version of this article (doi:10.1007/s11693-010-9059-y) contains supplementary material, which is available to authorized users.     

A synthetic biology approach for evaluating the functional contribution of designer cellulosome components to deconstruction of cellulosic substrates

Background

Select cellulolytic bacteria produce multi-enzymatic cellulosome complexes that bind to the plant cell wall and catalyze its efficient degradation. The multi-modular interconnecting cellulosomal subunits comprise dockerin-containing enzymes that bind cohesively to cohesin-containing scaffoldins. The organization of the modules into functional polypeptides is achieved by intermodular linkers of different lengths and composition, which provide flexibility to the complex and determine its overall architecture.

Results

Using a synthetic biology approach, we systematically investigated the spatial organization of the scaffoldin subunit and its effect on cellulose hydrolysis by designing a combinatorial library of recombinant trivalent designer scaffoldins, which contain a carbohydrate-binding module (CBM) and 3 divergent cohesin modules. The positions of the individual modules were shuffled into 24 different arrangements of chimaeric scaffoldins. This basic set was further extended into three sub-sets for each arrangement with intermodular linkers ranging from zero (no linkers), 5 (short linkers) and native linkers of 27–35 amino acids (long linkers). Of the 72 possible scaffoldins, 56 were successfully cloned and 45 of them expressed, representing 14 full sets of chimaeric scaffoldins. The resultant 42-component scaffoldin library was used to assemble designer cellulosomes, comprising three model C. thermocellum cellulases. Activities were examined using Avicel as a pure microcrystalline cellulose substrate and pretreated cellulose-enriched wheat straw as a model substrate derived from a native source. All scaffoldin combinations yielded active trivalent designer cellulosome assemblies on both substrates that exceeded the levels of the free enzyme systems. A preferred modular arrangement for the trivalent designer scaffoldin was not observed for the three enzymes used in this study, indicating that they could be integrated at any position in the designer cellulosome without significant effect on cellulose-degrading activity. Designer cellulosomes assembled with the long-linker scaffoldins achieved higher levels of activity, compared to those assembled with short-and no-linker scaffoldins.

Conclusions

The results demonstrate the robustness of the cellulosome system. Long intermodular scaffoldin linkers are preferable, thus leading to enhanced degradation of cellulosic substrates, presumably due to the increased flexibility and spatial positioning of the attached enzymes in the complex. These findings provide a general basis for improved designer cellulosome systems as a platform for bioethanol production.

     

Fluid dynamics of establishing left-right patterning in development

How does the clockwise motion of tens of monocilia drive a leftward flow in the node? And, as the observed flow is leftward, how is the fluid recirculating within the node, as it must, because the node is a closed structure? How does the nodal flow lead to left–right symmetry breaking in the embryo? These questions are within the realm of fluid physics, whose application to the problem of left–right symmetry breaking in vertebrates has led to important advances in the field. Birth Defects Research (Part C) 84:95–101, 2008. © 2008 Wiley‐Liss, Inc.     

Endothelial ICAM-1 protein induction is regulated by cytosolic phospholipase A2α via both NF-κB and CREB transcription factors

The regulated expression of ICAM-1 plays an important role in inflammatory processes and immune responses. The present study aimed to determine the in vivo involvement of cytosolic phospholipase A(2)α (cPLA(2)α) in ICAM-1 overexpression during inflammation and to elucidate the cPLA(2)α-specific role in signal events leading to ICAM-1 upregulation in endothelial cells. cPLA(2)α and ICAM-1 upregulation were detected in inflamed paws of mice with collagen-induced arthritis and in periepididymal adipose tissue of mice fed a high-fat diet. Intravenous injection of 2 mg/kg oligonucleotide antisense against cPLA(2)α (AS) that reduced cPLA(2)α upregulation also decreased ICAM-1 overexpression, suggesting a key role of cPLA(2)α in ICAM-1 upregulation during inflammation. Preincubation of endothelial ECV-304 cells that express ICAM-1 and of HUVEC that express ICAM-1 and VCAM-1 with 1 μM AS prevented cPLA(2)α and the adhesion molecule upregulation induced by TNF-α and inhibited their adherence to phagocyte like-PLB cells. Whereas AS did not inhibit NADPH oxidase 4-NADPH oxidase activity, inhibition of oxidase activity attenuated cPLA(2)α activation, suggesting that NADPH oxidase acts upstream to cPLA(2)α. Attenuating cPLA(2)α activation by AS or diphenylene iodonium prevented the induction of cyclooxygenase-2 and the production of PGE(2) that were essential for ICAM-1 upregulation. Inhibition of cPLA(2)α activity by AS inhibited the phosphorylation of both p65 NF-κB on Ser(536) and protein kinase A-dependent CREB. To our knowledge, our results are the first to show that CREB activation is involved in ICAM-1 upregulation and suggest that cPLA(2)α activated by NADPH oxidase is required for sequential phosphorylation of NF-κB by an undefined kinase and CREB activation by PGE(2)-mediated protein kinase A.     

De novo DNA synthesis using single molecule PCR

The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.