Anna M. Carnerup

Morphology: an ambiguous indicator of biogenicity.

This paper deals with the difficulty of decoding the origins of natural structures through the study of their morphological features. We focus on the case of primitive life detection, where it is clear that the principles of comparative anatomy cannot be applied. A range of inorganic processes are described that result in morphologies emulating biological shapes, with particular emphasis on geochemically plausible processes. In particular, the formation of inorganic biomorphs in alkaline silica-rich environments are described in detail.

Condensing DNA with poly(amido amine) dendrimers of different generations: means of controlling aggregate morphology

The morphology of the aggregates formed between DNA and poly(amido amine) (PAMAM) dendrimers depends on the dendrimer generation as previously reported in separate studies at high dendrimer/DNA charge ratios (>1). This has lead to substantial work on dendrimers as possible transfection agents. Inspired by these studies, we here present novel results from a coherent and systematic study using cryo-TEM, dynamic light scattering (DLS) and fluorescence spectroscopy to reveal how the size, composition and morphology of aggregates formed between DNA (4331 base pairs) and PAMAM dendrimers, are affected by dendrimer size and charge at low charge ratios (<1) in dilute solutions. At such conditions the process is cooperative and kinetically controlled and well-defined structured aggregates are formed for lower dendrimer generations. The smaller sized dendrimers (generation 1 and 2), which have a lower total charge per molecule, allow the formation of well-structured rods and toroids. In contrast, globular and less defined aggregates, which are less stable against precipitation, are formed with higher generation dendrimers. We were also able to directly visualise the cooperative nature of the condensation process as cryo-TEM and DLS show that dendrimer/DNA aggregates, containing condensed DNA, coexist with free extended DNA chains. In fact, the apparent hydrodynamic radii of the dendrimer/DNA aggregates, obtained using DLS, are found to be almost constant for charge ratios ≤1. The fluorescence study shows that the number of dendrimers bound per DNA chain decreases with the dendrimer generation but is independent of the charge ratio.

Watching DNA condensation induced by poly(amido amine) dendrimers with time-resolved cryo-TEM

The condensation of DNA by poly(amido amine) dendrimers of generation 1, 2, and 4 has been followed by time-resolved cryogenic transmission electron microscopy (cryo-TEM). The recorded images show that significant morphological rearrangement occurs for DNA condensed with the lower generation dendrimers leading to the formation of toroidal aggregates. Higher charge density dendrimers, on the other hand, give rise to globular aggregates, where no transient morphologies are observed. We suggest that the dendrimers in this case are kinetically trapped as soon as they bind to the DNA strand.

Solubilization of Poly{1,4-phenylene-[9,9-bis(4-phenoxy-butylsulfonate)]fluorene-2,7-diyl} in Water by Nonionic Amphiphiles

In the presence of the nonionic alkyloxyethylene surfactant n-dodecylpentaoxyethylene glycol ether (C12E5), the anionic conjugated polyelectrolyte (CPE) poly{1,4-phenylene-[9,9-bis(4-phenoxy-butylsulfonate)]fluorene-2,7-diyl} (PBS-PFP) dissolves in water, leading to a blue shift in fluorescence and dramatic increases in fluorescence quantum yields above the surfactant critical micelle concentration (cmc). No significant changes were seen with a poly(ethylene oxide) of similar size to the surfactant headgroup, confirming that specific surfactant-polyelectrolyte interactions are important. From UV-visible and fluorescence spectroscopy, dynamic light scattering (DLS), small-angle X-ray scattering (SAXS), cryogenic transmission electron microscopy (cryo-TEM), and electrical conductivity, together with our published NMR and small-angle neutron scattering (SANS) results, we provide a coherent model for this behavior in terms of breakup of PBS-PFP clusters through polymer-surfactant association leading to cylindrical aggregates containing isolated polymer chains. This is supported by molecular dynamics simulations, which indicate stable polymer-surfactant structures and also provide indications of the tendency of C12E5 to break up polymer clusters to form these mixed polymer-surfactant aggregates. Radial electron density profiles of the cylindrical cross section obtained from SAXS results reveal the internal structure of such inhomogeneous species. DLS and cryo-TEM results show that at higher surfactant concentrations the micelles start to grow, possibly partially due to formation of long, threadlike species. Other alkyloxyethylene surfactants, together with poly(propylene glycol) and hydrophobically modified poly(ethylene glycol), also solubilize this polymer in water, and it is suggested that this results from a balance between electrostatic (or ion-dipole), hydrophilic, and hydrophobic interactions. There is a small, but significant, dependence of the emission maximum on the local environment.

Condensation of DNA using poly(amido amine) dendrimers: effect of salt concentration on aggregate morphology

The condensation of DNA and poly(amido amine) dendrimers of generation 1, 2, 4, 6, and 8 has been studied as a function of salt concentration in order to reveal the forces that control the aggregate size and morphology. For the lower generation dendrimers (1, 2, and 4) a dramatic increase in aggregate size occurs as a result of an increase in salt concentration. Toroidal aggregates having an outer diameter of up to several hundreds of nm are observed. For the higher generation 6 dendrimers, the size of the condensed DNA aggregates does not change, however, an alteration in morphology is seen at high salt concentration, as more rod-like aggregates are observed. The size and morphology of generation 8 dendrimers are seemingly insensitive to salt concentration. It is believed that the effective neutralisation of the dendrimer and DNA charge in the aggregate is the reason for the observed effects. It is further shown that the 2D hexagonal lattice spacing observed in toroids is close to constant irrespective of the size of the cation responsible for the DNA condensation.

Stevensite in the modern thrombolites of Lake Clifton, Western Australia: A missing link in microbialite mineralization?

Microbialites form the earliest macroscopic evidence of life, and have always been important in particular aquatic ecosystems. They demonstrate the remarkable ability of microorganisms to provide the foundation for structures that can rival coral reefs in size. Microbialites are generally assumed to form by microbial trapping and binding of detrital grains, by carbonate organomineralization of microbial biofi lms, or by inorganic mineralization around microbial templates. Here we present a signifi cant discovery that modern thrombolitic microbialites in Lake Clifton, Western Australia, gain their initial structural rigidity from biofi lm mineralization by the trioctahedral smectite mineral stevensite. This nucleates in and around microbial fi lament walls when biological processes suppress carbon and Ca activities, leaving Mg to bind with silica and form a microporous framework that replaces and infi lls the fi lament web. After microbial materials are entombed, local carbon and Ca activities rise suffi ciently for aragonite microcrystals to grow within the stevensite matrix and perhaps replace it entirely, with eradication of biogenic textural features. This may explain why many ancient microbialite carbonates lack clear evidence for biogenicity. Stevensite may provide the missing link between microbial organomineralization and subsequent abiotic calcifi cation.

Lipid based nanovectors containing ruthenium complexes: a potential route in cancer therapy.

Ruthenium complexes offer new perspectives in cancer therapy; towards this aim we have synthesized a new amphiphilic unimer able to coordinate ruthenium complexes and to form liposomes.

DNA compaction induced by a cationic polymer or surfactant impact gene expression and DNA degradation.

There is an increasing interest in achieving gene regulation in biotechnological and biomedical applications by using synthetic DNA-binding agents. Most studies have so far focused on synthetic sequence-specific DNA-binding agents. Such approaches are relatively complicated and cost intensive and their level of sophistication is not always required, in particular for biotechnological application. Our study is inspired by in vivo data that suggest that DNA compaction might contribute to gene regulation. This study exploits the potential of using synthetic DNA compacting agents that are not sequence-specific to achieve gene regulation for in vitro systems. The semi-synthetic in vitro system we use include common cationic DNA-compacting agents, poly(amido amine) (PAMAM) dendrimers and the surfactant hexadecyltrimethylammonium bromide (CTAB), which we apply to linearized plasmid DNA encoding for the luciferase reporter gene. We show that complexing the DNA with either of the cationic agents leads to gene expression inhibition in a manner that depends on the extent of compaction. This is demonstrated by using a coupled in vitro transcription-translation system. We show that compaction can also protect DNA against degradation in a dose-dependent manner. Furthermore, our study shows that these effects are reversible and DNA can be released from the complexes. Release of DNA leads to restoration of gene expression and makes the DNA susceptible to degradation by Dnase. A highly charged polyelectrolyte, heparin, is needed to release DNA from dendrimers, while DNA complexed with CTAB dissociates with the non-ionic surfactant C12E5. Our results demonstrate the relation between DNA compaction by non-specific DNA-binding agents and gene expression and gene regulation can be achieved in vitro systems in a reliable dose-dependent and reversible manner.

Phase Behavior and Coassembly of DNA and Lysozyme in Dilute Aqueous Mixtures: A Model Investigation of DNA−Protein Interactions

Results from an experimental investigation of the phase behavior of an aqueous system of DNA from salmon testes and the protein lysozyme are presented. At very low concentrations of either or both of the macromolecular components, wormlike assemblies with a width of the order of 10 nm are formed. There are strong indications that direct interactions between the protein units are instrumental both in driving the phase separation and in controlling the morphology of the formed assemblies.

Phage display and kinetic selection of antibodies that specifically inhibit amyloid self-replication

Significance The promise of antibody-based strategies to combat Alzheimer’s disease by inhibiting the aggregation of the amyloid β peptide (Aβ) has not yet been realized in clinical trials. In part, this situation has arisen because the antibodies explored so far have been developed without the benefit of the mechanistic information needed to block specific steps in Aβ aggregation. We introduce a strategy to target specifically fibril-dependent secondary nucleation, the microscopic step found to generate most neurotoxic Aβ assemblies. Phage display libraries are screened to find antibodies that bind to Aβ fibrils, followed by affinity ranking and chemical kinetic analysis. Our results suggest that such antibodies may serve as starting points for the development of effective therapeutics for neurodegenerative diseases. The aggregation of the amyloid β peptide (Aβ) into amyloid fibrils is a defining characteristic of Alzheimer’s disease. Because of the complexity of this aggregation process, effective therapeutic inhibitors will need to target the specific microscopic steps that lead to the production of neurotoxic species. We introduce a strategy for generating fibril-specific antibodies that selectively suppress fibril-dependent secondary nucleation of the 42-residue form of Aβ (Aβ42). We target this step because it has been shown to produce the majority of neurotoxic species during aggregation of Aβ42. Starting from large phage display libraries of single-chain antibody fragments (scFvs), the three-stage approach that we describe includes (i) selection of scFvs with high affinity for Aβ42 fibrils after removal of scFvs that bind Aβ42 in its monomeric form; (ii) ranking, by surface plasmon resonance affinity measurements, of the resulting candidate scFvs that bind to the Aβ42 fibrils; and (iii) kinetic screening and analysis to find the scFvs that inhibit selectively the fibril-catalyzed secondary nucleation process in Aβ42 aggregation. By applying this approach, we have identified four scFvs that inhibit specifically the fibril-dependent secondary nucleation process. Our method also makes it possible to discard antibodies that inhibit elongation, an important factor because the suppression of elongation does not target directly the production of toxic oligomers and may even lead to its increase. On the basis of our results, we suggest that the method described here could form the basis for rationally designed immunotherapy strategies to combat Alzheimer’s and related neurodegenerative diseases.